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Advancements in phosphodiesterase (PDE)-targeted therapies have shown promise in recent years for treating patients with a variety

of autoimmune diseases. This review summarizes the development of PDE4 inhibitors and the associated literature with a focus on treatments for autoimmune diseases. After the initial investigations of the prototypic PDE inhibitor, rolipram, more selective inhibitors targeting the PDE4 isozyme have been developed. With phase II and phase III clinical trials currently underway to evaluate the safety and efficacy of the latest generation of PDE4 inhibitors, namely apremilast, a new class of treatments may be around the corner for patients suffering from chronic, autoimmune diseases.

Autoimmune; Apremilast; Crohn’s; Dermatitis; PDE4; Psoriasis; SLE

Our earliest understanding of phosphodiesterase (PDE) inhibitors began with a series of publications by Sutherland and Rall in the 1950s, describing the properties of cyclic adenosine monophosphate (cAMP). Various cellular pathways and inflammatory responses are mediated by cAMP, an essential intracellular second messenger made up of phosphodiester bonds. Evidence showed formation of cAMP was induced by substances such as epinephrine and glucagon, and the suppression of the enzymes hydrolyzing cAMP, including PDEs, by sodium fluoride and caffeine[1,2]. By the 1960s, the role of cyclic nucleotide second messengers, such as cAMP, in cell signaling and homeostasis was established, and regulation of this pathway by PDE inhibitors arose as a field of considerable interest. However, the immunomodulatory properties of cAMP and the anti-inflammatory potential of PDE inhibitors were not demonstrated until the early 1970s [3-5]. Additional research would later demonstrate the expression of the PDE isoenzyme PDE4 almost exclusively within inflammatory cells [6]. Inhibition of PDE4 leads to the reduction of intracellular cAMP levels, and decreases in T cell and monocyte derived cytokine and chemokines including tumor necrosis factor (TNF) α [7-11]. Targeting PDE4 has enormous clinical potential because it targets a central pathogenic process that bypasses complex antigen receptor-specific immunoregulatory mechanisms. Indeed, selective PDE4 inhibitors have generated substantial interest as a treatment for several autoimmune conditions including ankylosing spondylitis, Alzheimer’s disease, psoriasis, psoriatic arthritis, sarcoidosis, systemic lupus erythematosus, inflammatory bowel disease, atopic dermatitis, rheumatoid arthritis, and multiple sclerosis.

Mechanism of action
PDEs are a family of enzymes responsible for the hydrolysis and subsequent inactivation of cyclic nucleotides, and have been organized into at least 11 families based on sequence homogeneity, inhibitor sensitivity, and biochemical properties [12]. Each enzyme within the PDE4 family specifically targets cAMP for degradation and consists of four subtypes (PDE4A to PDE4D). These enzymes are located within brain and immunocompetent cells such as neutrophils, T lymphocytes, macrophages and eosinophils [13]. PDE4 inhibition results in the accumulation of the intracellular second messenger cAMP, downstream activation of protein kinase A (PKA), and subsequent phosphorylation of the transcription factor cAMP-response element binding protein (CREB). Activation of this pathway modulates gene transcription of numerous cytokines, and results in suppression of TNF α production and eventual inhibition of their proinflammatory and destructive properties [14].

The newest and most promising of the PDE4 inhibitors, apremilast, has been evaluated for its pharmacokinetic properties and disposition following oral administration. Multiple daily doses showed rapid absorption (T max = 2 h) and a moderately long half-life (8.2 h) [15]. A separate study monitored healthy male subjects following a single, 20 mg, oral dose and found that apremilast was extensively metabolized via multiple pathways, with unchanged drug representing 45% of the circulating radioactivity and <7% of the excreted radioactivity. Analysis of total radioactivity suggests rapid absorption, with plasma T max values also at 2 h. Mean Cmax and area under the curve (AUC) values in plasma were 333 ng/ml and 1,970 ng*h/ml, respectively. Metabolic clearance of apremilast was the major route of elimination with the key metabolites demonstrating at least 50-fold less pharmacologic activity than apremilast [16]. Man et al. optimized the structures of a series of 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dialkoxyphenyl) propionic acid analogues to enhance PDE4 and TNFα inhibitory activity. So far, oral and intravenous administration of these analogues in female rats has shown good pharmacokinetics with low clearance, a moderate volume of distribution, and a 64% oral bioavailability [17].

Adverse effects
PDE4 is also one of the major phosphodiesterase isoenzymes expressed in the central nervous system, and therefore nausea and emesis are common adverse effects of drug administration[18]. Early PDE4 inhibitors actually failed in clinical trials due to the high prevalence of nausea and emesis [19]. Other adverse effects associated with repeated administration of PDE4 inhibitors include headache, diarrhea, fatigue, dyspepsia, nasopharyngitis, and gastroenteritis [20]. Mesenteric vasculitis is a more worrisome toxicity

that may be associated with the PDE4 inhibitors. Studies performed in rodents have demonstrated medial necrosis of the mesenteric arteries after administration of the second generation PDE4 inhibitor cilomilast. However at a meeting convened by the US Food and Drug Administration (FDA) in 2003 to discuss cilomilast in phase III studies, the committee unanimously agreed that the risk of mesenteric vasculitis is not a safety concern based on human studies [21]. The newer PDE4 inhibitor, apremilast, has been well tolerated with few side effects in phase I and II studies. Phase III clinical trials are currently underway and will provide more insight into its dosing and side effect profile. The most frequently reported adverse events have been headache, nausea and pharyngitis (15,34). Researchers used a recognized pharmacophore from the PDE4 inhibitors rolipram and roflumilast in the development of apremilast, and added it to a series of thalidomide analogs in efforts to optimize activity and reduce side effects classically seen with earlier PDE4 inhibitors [22].

The discovery of the anti-inflammatory actions of PDE4 inhibitors arose from early studies with the prototypic PDE4 inhibitor, rolipram. This was the first selective PDE4 inhibitor investigated, and has been used numerous times for drug comparator studies [23]. Rolipram was also studied as an antidepressant several years before the discovery of its potent PDE4 inhibitory activity [24,25]. Despite its potent anti-inflammatory effects in vitro, clinical trials were associated with unacceptably high rates of adverse events, specifically nausea and vomiting [26].

Roflumilast was the first, and currently only drug in the PDE4 inhibitor class to be FDA approved. In several countries, this highly selective PDE4 inhibitor is licensed for oral, once-daily treatment of severe chronic obstructive pulmonary disease (COPD). In a pooled analysis of over 6,000 patients receiving roflumilast, higher rates of diarrhea, weight loss, nausea, headache, back pain, insomnia, decreased appetite, and dizziness were reported as compared to those receiving placebo. However the overall rate of adverse events was comparable to that among patients receiving placebo [27]. Pooled results from the pivotal COPD studies M2-124 and M2-125 showed that the weight loss for those in the roflumilast group was generally small (<3% of baseline weight) and typically occurred in the first 6 months of treatment. By the end of the 6 months, almost two-thirds of the weight loss could be attributed to a reduction in fat mass [28]. Prior investigations have revealed a link between PDE4 and lipolysis, possibly through regulation of cAMP pools in human adipocytes and increased plasma glucagon-like peptide 1 (GLP-1) concentrations in rats [29,30].

Many flavonoids have been reported to inhibit PDE4 [31,32], and also demonstrate additional anti-inflammatory effects through other pathways. For example, in addition to PDE4 inhibition, the flavonoid dioclein suppresses the production of the inflammatory mediators interleukin (IL)-6, TNFα, a chemokine ligand (CXCL)1/KC, CCL2/JE (monocyte chemotactic protein 1) and nitric oxide (NO), and acts as a scavenger of reactive oxygen species [33]. Studies on dioclein have also demonstrated a synergistic antiinflammatory effect by targeting multiple pathways [34]. However in contrast to rolipram, dioclein does not selectively inhibit PDE4 with additional activity against PDE1, which may lead to unwanted side effects [35].

Psoriasis is a chronic inflammatory autoimmune disorder characterized by inflammatory cell infiltration into the dermis and epidermis accompanied by keratinocyte hyperproliferation [36]. Current treatments, including biological therapies, downregulate cytokine cascades and chemokine production. While these interventions can be highly efficacious, limitations include side effects, intravenous or subcutaneous administration, quality control, and production costs. AN-2728 is a topically administered, boron-containing compound developed for the treatment of psoriasis. This compound was found to reduce cytokine production, such as TNFα and interferon (IFN)y, and demonstrated activity against the PDE4 enzyme [37]. Several clinical trials of AN-2728 have reported significant effects on markers of efficacy, such as TNFα, in addition to being well tolerated [38]. The determination of cytokines in skin homogenates revealed that both T helper (Th)1 as well as Th2 cytokines are suppressed by PDE4 inhibitors, further proving its utility in the treatment of T cell mediated diseases, such as psoriasis [39]. An open-label, single-arm pilot study investigated the biological and clinical effects of oral apremilast 20 mg once daily in patients with severe plaque-type psoriasis. Of the 19 patients enrolled, 17 completed the study. Of the 19 subjects, 14 (73.7%) showed improvement in their Psoriasis Area and Severity Index (PASI) scores following a 29-day treatment phase. Among these responders, T cells were reduced by 28.8% and 42.6% in the dermis and epidermis, respectively. Epidermal thickness was also reduced by a mean of 20.5% from baseline [15]. More recently, a phase IIb, randomized, multicenter, placebo-controlled, dose-ranging trial for the treatment of plaque-type psoriasis with oral apremilast was completed. Patients were randomly assigned to apremilast 10 mg twice daily, apremilast 20 mg twice daily, apremilast 30 mg twice daily, or placebo. By week 16, a 75% improvement in PASI scores (PASI75) was achieved in 6% (5/88) assigned placebo, 11% (10/89) assigned apremilast 10 mg, 29% (25/87) assigned 20 mg, and 41% (36/88) assigned 30 mg. Significant differences from placebo were seen with apremilast 20 mg and 30 mg (P <0.0001), but not 10 mg. Reported adverse

Nevertheless. in addition to a 4year extension phase [40]. the 90% of subjects enrolled were white and therefore the study may lack generalizability.49]. and phase III clinical trials are currently underway. The apremilast group also saw a significant change from baseline (mean percentage) in levels of receptor activator of nuclear factor κB (NFκB) ligand (RANKL) and sclerostin [50]. the degree of clinical response with treatment may also correlate with fluctuations in biomarkers [45-47].events were most frequently mild to moderate and included nausea. These trials include a 52 week randomized. The primary endpoint was the proportion of subjects achieving the American College of Rheumatology criteria for 20% improvement (ACR20) at week 12. results from a small pilot study were presented showing that apremilast may be efficacious in patients with longstanding AS. fatigue. PALACE 3 (NCT01212770). apremilast 40 mg once daily. blinded. and PALACE 4 (NCT01307423)) [40]. at the 2011 ACR meeting. 17 received apremilast 30 mg twice daily compared to 19 who received placebo. Results of this study are encouraging. 44% of actively treated patients achieved the primary endpoint of ACR20 compared with 12% of the placebo cohort (P <0. Given the pathophysiologic characteristics of RA. Moreover. Rheumatoid arthritis Rheumatoid arthritis (RA) is another chronic. leaving patients with limited treatment options. placebo controlled phase II unpowered pilot study included 36 subjects with longstanding AS who had not sufficiently responded to NSAIDs over 12 weeks. as well as cellular activation and articular destruction in RA [53]. or placebo. headache. prior systemic therapy for PsA may alter the efficacy of apremilast and was not examined in this study. with primary endpoints measured at week 16. double-blind. TNFα has been shown to promote cytokine and chemokine production. randomized. After completing the initial 12-week phase.52]. This double-blind.001). Following the treatment phase in both groups. and new bone formation. However most events were mild to moderate and no clinically relevant laboratory or electrocardiographic abnormalities were reported [41]. Healthy joint architecture was also maintained in a dose-dependent manner. double-blind. PALACE 2 (NCT01212757). subjects receiving placebo were given a 12-week course of apremilast. nasopharyngitis. Subjects were randomized to 20 mg apremilast twice daily. In total. and dyspepsia. a study was performed to assess the anti-inflammatory effects of apremilast in human synovial cells collected from rheumatoid arthritis patients. both murine models showed a significant reduction in arthritis clinical scores over a ten-day treatment period with apremilast. Lastly. placebo-controlled. Additionally. diarrhea. upper respiratory tract infection. Psoriatic arthritis A phase II. none were judged to be related to apremilast [40]. Therapeutic response centers on patient-reported outcomes such as pain. The efficacy and tolerability of apremilast in patients with psoriatic arthritis are now being studied in four independent phase III studies (PALACE 1 (NCT01172938). updated management guidelines published by the Assessment of SpondyloArthritis (ASAS) and the European League Against Rheumatism (EULAR) report that there is no evidence for the efficacy of disease-modifying antirheumatic drugs (DMARDs) such as methotrexate and sulfasalazine for the treatment of axial disease. headache. multicenter. fat infiltration. These studies include both patients who have received disease-modifying antirheumatic drugs and those who have not. subjects participated in a 4-week observation phase. and eventual erosion of cartilage and bone [51. Local production of cytokines and chemokines leads to leukocyte infiltration. Additionally. Ankylosing spondylitis Manifestations of ankylosing spondylitis (AS) include axial and peripheral skeletal inflammation. and nasopharyngitis with 84. Of these subjects. and new bone formation that can be visualized by magnetic resonance imaging (MRI) and conventional radiography [42-44]. inflammatory autoimmune disease and primarily targets the synovial tissues of joints. multicenter trials (ESTEEM 1 (NCT01194219) and ESTEEM2 (NCT01232283)) investigating the use of oral apremilast 30 mg in adults with moderate to severe plaque psoriasis. Recently. Of the eight serious adverse events. The two classes of drugs that have been shown to reduce the signs and symptoms of AS include non-steroidal anti-inflammatory drugs (NSAIDs) and TNFα blockers [48. Results from this study showed that apremilast led to a dose-dependent inhibition of spontaneous production of TNFα from human rheumatoid synovial membrane cultures. gastroenteritis. placebo-controlled phase.3% of subjects in the treatment phase reporting at least one AE. The study revealed promising results for the treatment of PsA with oral apremilast. Currently there are two phase III. These synovial cells were cultured in the presence of increasing concentrations of apremilast for 48 h and enzyme-linked immunosorbent assay (ELISA) was used to analyze spontaneous TNFα production. Unlike . as well as two murine models of arthritis. The most common adverse events (AEs) were diarrhea. mobility and function as well as objective measures such as inflammation. placebo-controlled study enrolled 168 subjects with psoriatic arthritis (PsA) during a 12-week treatment phase. nausea. but was limited by the relatively short duration and unclear long-term efficacy and safety data.

TNFα is thought to damage the gut via upregulation of matrix metalloproteinase (MMP) production by gut myofibroblasts. Only two patients needed dosage reduction due to jitteriness in one patient and nausea in the other. Results showed that both the survival time and the appearance of proteinuria of NCS 613-treated mice are significantly delayed. although these results were not significant statistically. randomized. double-blind. sarcoidosis. pentoxifylline. possibly due to enhanced selectivity of apremilast [54]. However.the first generation PDE4 inhibitor rolipram. Given the suppression of TNFα and NFκB associated with a variety of PDE4 inhibitors. and ulcer formation [65].017). apremilast did not demonstrate any adverse effects in treatment-naïve mice. Existing anti-inflammatory agents such as 5-aminosalicyclates and other immunosuppressants have limitations due to adverse drug reactions. placebo-controlled. perpetuating further inflammation and disease processes [66]. Of note. and fever caused by inflammation in the gut. NFκB activation works in a positive feedback to induce TNFα. While study size was limited. tissue damage.58]. or lack of response in some patients [63]. By week 8. Results showed a significant reduction in SASI induration scores at weeks 4 and 12. Systemic lupus erythematosus Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can affect a variety of organs and is predominantly seen in women. 5 much worse). three patients developed significant worsening of their cutaneous lesions within 3 months after discontinuation of apremilast[61].005 [59]. A small study evaluated the use of apremilast in 15 subjects who had failed systemic therapies for sarcoidosis. as well as TNFα upregulation. and nuclear translocation of the proinflammatory transcription factor complex NFκB [64]. with a reduction to once daily dosing following the onset of any adverse event. leading to breakdown of extracellular matrix. the inflammation in inflammatory bowel disease (IBD) has been linked to upregulation of proinflammatory cytokines. 9 and 13 weeks with one of ethanol. parallel-arm. the results demonstrate potential for the use of PDE4 inhibitors in patients with SLE. such as TNFα. two phase III clinical trials (FACT I and FACT II) were designed. with good inter-reader consistency. abdominal pain. Sarcoidosis Previous reports have described the use of PDE4 inhibitors in patients with the systemic inflammatory disease. The long-term therapeutic goal for these patients focuses on inducing and maintaining remission of symptoms to improve patient quality of life [62]. Both Crohn’s disease and ulcerative colitis (UC) present with diarrhea. in treating moderately severe UC. Tetomilast was not found to have a significant effect on individual symptom or sigmoidoscopy scores. Four groups of female MRL/lpr mice were injected at 5. denbufylline. the efficacy scores had improved for those patients receiving tetomilast (with or without concomitant 5-aminosalicylic acid) compared to those on placebo. dose comparison studies of tetomilast in subjects with active UC. The quantity of NFκB activation has also been shown to correlate with the degree of mucosal inflammation and disease activity. One potential reason for the lack of difference in the therapeutic . While pentoxifylline has been shown to be effective. Both studies were multicenter. Photographs were presented in random order to three assessors and were scored from 1 to 5 (1 much better. tetomilast. A recent study targeted increased PDE4 activity in lupus conditions using MRL/lpr mice (a mouse model developing severe lupus disease). bleeding. An interim analysis of the data from a phase II pilot study investigating the use of apremilast in combination with methotrexate reported that the primary endpoint of ACR20 was not reached [55]. Inflammatory bowel disease The group of inflammatory conditions affecting the colon and small bowel known as inflammatory bowel disease can present in patients with life-altering symptoms lasting weeks to months at a time. there was a trend for tetomilast to improve severity of rectal bleeding from baseline when compared to placebo ( P = 0. The normalized mean score given by the assessors after 12 weeks of therapy was 2/5 (somewhat better after therapy). loss of therapeutic response. both with P values of 0. fecal urgency and incontinence. Like many autoimmune diseases. Active lesions were assessed during 12 weeks of therapy with the Sarcoidosis Activity and Severity Index (SASI) as well as with photographs taken at baseline and at week 12. Treatment is focused on controlling symptoms and often involves the use of corticosteroids and other systemic immunosuppressant therapies [57. No further adverse events were reported following this change in dose. A placebo-controlled phase II trial using apremilast as monotherapy for RA is currently underway[56]. or NCS 613 (a novel PDE4 inhibitor). 7. Patients received oral apremilast 20 mg twice daily. These studies investigated the safety and efficacy of the PDE4 inhibitor. use of this drug is limited by associated adverse events [60].

including MMP-9 [79]. High levels of PDE4 activity are also found in leukocytes of these patients [68]. Improvement was seen during both the initial presentation of disease as well as subsequent relapses [77]. These positive findings may be at least partially attributed to the blockade of inflammatory responses and apoptosis mediated by cAMP/CREB signaling. Accumulation of amyloid beta (Aβ) peptides has been shown to produce inflammatory responses[73]. cilomilast. KF66490 also produced less potent emetic effects [70]. When rats primed to EAE were treated with rolipram. increased levels of pCREB were reported in the hippocampus following treatment with rolipram. Similar responses were induced by infusion of aged Ab25-35 into the hippocampus. eosinophils. with elevations of biomarkers seen even i n early stages of disease [72]. respectively. Compared to the first generation PDE4 inhibitor. and impair memory [75]. Inflammatory infiltrates in these cutaneous lesions consist of T lymphocytes. and EASI (P = 0. suppressed myeloperoxidase (MPO) activity. efficacy. Both 25 and 50 mg of tetomilast were generally well tolerated in subjects with active UC with no major adverse events [63]. KF66490 significantly inhibited increases in ear thickness. Alzheimer’s disease PDE4 inhibitors have also been investigated in the treatment of patients with Alzheimer’s disease. statistically significant improvement was seen in all outcomes in cohort 2. Nausea. a significant reduction of itch from baseline (VAS) and improvement in quality of life (assessed via Dermatology Life Quality Index (DLQI) score) was seen in cohort 1 ( P = 0. the high levels of NFκB activation in freshly obtained cells were prevented. a quantitative index of neutrophils accumulating in skin associated with chronic inflammation. Participants in the study were required to remain on triamcinolone acetonide 0. After 18 days of treatment. neutrophils. After 3 months of treatment. such as MS [78]. and possible mechanism of action of apremilast in AD. More recently. The neuropathological chan ges seen in Alzheimer’s are closely linked to chronic inflammation and apoptosis. while Eczema Area and Severity Index (EASI) and quality of life (DLQI) scores improved in cohort 2 ( P = 0. the most common adverse event.008 and P = 0. IL-4 and IL-1B levels. inhibition was also seen after incubation of encephalitogenic cells with rolipram. and rolipram on induced dermatitis in mice models. These results may suggest a potential role for the use of PDE4 inhibitors in treatment of memory loss in patients with Alzheimer’s [75]. macrophages. inhibit hippocampal synaptic plasticity. and proliferation of fibroblasts and CD3-positive T cells. monocytes. respectively). rolipram. Atopic dermatitis Atopic dermatitis is a chronic inflammatory disease characterized by eczematous lesions and intense itching. Cilomilast.03).1% for 2 weeks prior to the start of the study as well as throughout the trial. was rated as mild and improved over the course of the study in all patients. while the second cohort consisted of ten subjects treated with apremilast 30 mg twice daily for 6 months. especially in the earlier stages of treatment [69]. roflumilast. activate the apoptotic pathway [74]. Multiple sclerosis The demyelinating autoimmune disease. A subsequent study by Harada et al.03). and to a lesser extent rolipram. Inhibition of NFκB was also accompanied by a decrease in MMP-9 gene expression [80]. more selective inhibitors targeting the PDE4 isozyme have been developed.02 and P = 0.002) [71]. with reversal of memory deficits seen following repeated treatment with rolipram. With phase II and phase III clinical trials currently underway to evaluate the safety and . This effect was more potent than the 25% recovery seen with cyclosporine A.response may be the very high placebo response rates seen in patients with IBD. At 6 months. including VAS ( P = 0.01. indicating that interference with NFκB activation is a direct effect of the drug. and mast cells [67].003. Furthermore. roflumilast. cilomilast and roflumilast showed a 47 and 36% recovery in skin severity score. multiple sclerosis (MS). has often been studied in animal models by inducing experimental autoimmune encephalomyelitis (EAE) in genetically susceptible animals [76]. One study demonstrated a reduction in the clinical signs of EAE in mice models during the administration of rolipram. respectively). used the PDE4B inhibitor. In fact. A study in Japan looked at the effects of PDE4 inhibitors. an open-label prospective trial of apremilast in 16 patients with moderate to severe AD was conducted to assess the safety. The EAE model also mimics the relapsing-remitting presentation of MS seen in humans. a key regulator of inflammatory processes and gene expression related to EAE and MS. Conclusions Advancements in phosphodiesterase-targeted therapies have shown promise in recent years for treating patients with a variety of autoimmune diseases. After the initial investigations of the prototypic PDE inhibitor rolipram. DLQI (P = 0. One cohort consisted of six subjects treated with apremilast 20 mg twice daily for 3 months. Matrix metalloproteinases (MMPs) are a gene family of zinc-dependent endopeptidases involved in the proteolytic modeling of the extracellular matrix as well as the pathogenesis of several autoimmune disorders of the peripheral and central nervous systems. KF66490 to treat induced AD in mouse models. In vitromodels have shown that rolipram inhibits NFκB.

Celgene Corporation. MPO: Myeloperoxidase.. VAS: Visual analog scale.. Janssen-Ortho Inc. MS: Multiple sclerosis. More direct causes of plaque rupture include pro-inflammatory cytokines. and Teva. Studying generation-to-generation changes of human morphology that occurred in historical times. COPD: Chronic obstructive pulmonary disease. cAMP: Cyclic adenosine monophosphate. EAE: Experimental autoimmune encephalomyelitis. PsA: Psoriatic arthritis. EASI: Eczema area and severity index. ELISA: Enzyme-linked immunosorbent assay. Pfizer Inc. one mechanism being anti-inflammatory.. ABG holds an investigator role for Abbott Laboratories. Keywords: . ABG has received an institutional grant from Amgen. PASI: Psoriasis area and severity index. and UCB. Heat shock proteins could be one major target for atherogenic immune reactions. Belersdorf Inc. and still occur in present-day populations under the forces of evolution. Also. NFκB: Nuclear factor κB. PDE: Phosphodiesterase. helps to explain medical conditions and warns clinicians that their current practices may influence future humans. CXCL: Chemokine C-X-C motif ligand. the majority of EM studies have used pure theories of hominin macroevolution to explain the present-day state of human health. MMP: Matrix metalloproteinase. Amgen. Novo Nordisk A/S. Actelion. a new class of treatments may be around the corner for patients suffering from chronic. Amgen. Competing interests ABG is on the advisory boards of Abbott Laboratories. but it has been difficult to find direct evidence. Canfite. IFN: Interferon... RANKL: Receptor activator of NFκB ligand. To date. Immune Control. Incyte Corporation. UC: Ulcerative colitis. NO: Nitric oxide. Here.. SLE: Systemic lupus erythematosus. current and future microevolutionary changes of human structure.. ABG is a consultant to Abbott Laboratories. Lilly ICOS LLC. Antibodies against PC (anti-PC) may be atheroprotective.. and their coadaptations.. TNF: Tumor necrosis factor. Pfizer Inc. The potential causes of immune reactions and inflammation in atherosclerosis and how inflammation can be targeted therapeutically to provide novel treatments for CVD are reviewed. Celgene Corporation. DMARD: Disease-modifying antirheumatic drug. functions and pathologies. AE: Adverse event. To prove that inflammation is a cause of atherosclerosis and CVD. Astellas Pharma US Inc. and honoraria from DermiPsor. Novartis Pharmaceuticals Corp. Evolutionary medicine (EM) is a growing field focusing on the evolutionary basis of human diseases and their changes through time. Celgene Corporation. DLQI: Dermatology life quality index. Abbreviations Aβ: Amyloid beta. and UCB. SASI: Sarcoidosis activity and severity index. NSAID: Non-steroidal anti-inflammatory drug. Authors’ contributions Abstract (provisional) Atherosclerosis. analyzing historic tissue specimens such as mummies is crucial in order to address the molecular evolution of pathogens. BMS. Lilly ICOS LLC. Inc. the major cause of cardiovascular disease (CVD).efficacy of the latest generation of PDE4 inhibitors. GLP-1: Glucagon-like peptide. NKu. we propose a different approach by addressing more empirical and health-oriented research concerning past. is a chronic inflammatory condition with immune competent cells in lesions producing mainly pro-inflammatory cytokines. chemokines. namely apremilast. clinical studies with anti-inflammatory and/or immune-modulatory treatment are needed. Dead cells and oxidized forms of low density lipoproteins (oxLDL) are abundant. and lipid mediators. ASAS: Assessment of spondyloarthritis. Novo Nordisk A/S. CREB: cAMP-response element binding protein. IL: Interleukin. PKA: Protein kinase A. Bacteria and virus have been discussed. Janssen-Ortho Inc. EULAR: European League Against Rheumatism. Merck & Co. Coronado Biosciences. The major direct cause of CVD appears to be rupture of atherosclerotic plaques. of the human genome. AMG and NKi have no competing interests. AS: Ankylosing spondylitis. oxLDL has proinflammatory and immune-stimulatory properties. RA: Rheumatoid arthritis. Novartis Pharmaceuticals Corp. ACR20: American college of rheumatology criteria for 20% improvement. and antibiotic trials have not been successful. autoimmune diseases. causes cell death at higher concentrations and contains inflammatory phospholipids with phosphorylcholine (PC) as an interesting epitope.

based on their causes as explained earlier. Microevolution is observable as a process of changes occurring in phenotypes of successive generations. Although classic descriptions of evolutionary processes refer to long time spans. physiology and pathophysiology. there is no reason to expect that a change in gene frequencies may not occur during the time span of a century. such alterations may occur due to environmental changes such as a reduction of chewing effort in the processing of food. They have occurred in anatomical structures. Theoretically. mutations and selection. while true microevolution involves change of gene frequencies. The platonic. They can only occur through the process of reproduction that requires the genetic endowment of one generation to be passed on to the next generation. the minimum time span required for microevolutionary change of the gene pool is that of two generations. secular trends Definition. a better understanding of the etiology of present-day human ailments can be achieved. for 'a generation') or microevolutionary alterations. each providing an opportunity for change of gene frequencies. Microevolutionary changes in human lineages during historical times are clearly understandable in the evolution of immunity to diseases. under the operation of forces of evolution. It has been the topic of several textbooks [3-5] and also recently a major scientific colloquium [6]. history of evolutionary medicine research and present situation Evolutionary medicine (EM). gene flow. The average age of parents at the time they produce offspring is approximately 20 to 40 years and thus on average three generations can be turned over during a century. once formed. These alterations are all likely to be at least partially the result of structural reductions in response to technology diminishing the need for the use of physical strength and introducing extraoral food processing. Aside from genetic changes. Secular changes. drift. For example. It espouses approaches to population biology that do not deal with individuals. These changes may result from changing. or Darwinian medicine as it is sometimes called. generations are widely overlapping in living populations.2]. investigates human disease vulnerability and disease etiologies (genetics. Human microevolution has recently accelerated due to the rapid growth of human population numbers facilitated through cultural development and technologies [17]. reductions in the size and number of teeth [15] and spinal morphology alterations [16]. remains the same biological entity throughout the centuries is patently incorrect. or from adaptive phenotypic responses to changing living conditions. in terms of anatomical structures and physiological processes as well as disease patterns and prevalence. essentialist view that Homo sapiens. Early applications of poorly understood Darwinian concepts to human biology led to eugenic theories [7. like in the case of accumulating mutations. the distinction between phenotypic adaptive trends and true microevolution . However. changes in human genes and phenotypes from generation to generation do occur. Irrespective of the disparate views on the origin of humans held by adherents to different religions and scientific theories. but also in the appearance of new metabolic processes such as lactose tolerance [9] or in the widespread acquisition of genetic variations in the capacity to process ethanol [10]. such significant changes in morphological characteristics include: decrease in the robusticity of the musculoskeletal apparatus (gracilization) [11. but only within the limits of genetically determined plasticity of individual responses to environmental stimuli. while living conditions in modern economies change rapidly. mortality. Since. EM is a biomedical scientific concept of increasing interest since the 1990s [1. and so on) from an evolutionary perspective. EM (as we strongly emphasize here) does not deal with eugenic approaches. A distinction can be made between secular changes and microevolutionary alterations. The intellectual beginnings of evolutionary medicine stemmed from the recognition that past evolutionary events can explain present-day conditions of the human body. Since for many morphological and physiological characters the exact mode of inheritance is not known. but with intergenerational manifestations of biological processes that have no value attached to them. gene frequencies. due to a long human fertile lifespan of some 30 years. physiological processes and anatomical structures. empirical. pathology. microevolution. such as increases in stature or weight are usually alterations of phenotypic expression of genetic potential without any changes in gene frequencies. pathogens. Phenotypic manifestations of these changes are sometimes referred to as secular (derived from the Latin term saeculum. Humans still evolve. too. environment. while there is no constraint on the minimum time span for an adaptive phenotypic change. evolutionary medicine. behavior. weight and height [13]. The changes of gene frequencies are a part of the general evolutionary process involving mating systems. it is not always easy to distinguish between a truly evolutionary change and a phenotypic secular trend if the specific genetic determination of changing functions or structures is not known. During that process frequencies of alleles or of genotypes can be altered leading to permanent alterations of immune responses. This change may be rapid if a particular force of evolution operates strongly. by applying the concept of nature's evolution to human morphology. Phenotypic adaptive responses are modifiable through alteration of living conditions during the lifespan of one generation.anatomical variation. Thus.8].12]. gene flow resulting from mass migration may profoundly alter the gene pool of a given geographic region within several decades. microcranialization and brachycephalization (reduction in braincase size and change of its shape) [14]. leading to a mechanically caused reduction in jaw size.

the majority of natural selection pressures were relaxed to the apparent benefit of all of us. there was a very significant opportunity for the process of natural selection (Figure 1). explanations of psychiatric diseases such as depression. it discusses present-day public health activities and biomedical practices from a perspective of future generations. many were linked to varying physical strength. where the time periods investigated extend over a number of centuries or even a few millennia. while fertility became dependent on the conscious decisions of individuals and couples in both the sense of avoiding births and giving birth by infertile couples. and its impact on human morphology and pathology. At the genetic level. predisposition to type I diabetes mellitus) may not be considered maladaptive in an environment where there is an effective treatment for diabetes. of the human spine [28]. evolutionary genetics [23]. This situation has changed drastically during the last approximately 150 years with the most welcome advent of sanitation and generally available medical treatments. metabolic disorders such as iron deficiency [32] or nutritionbased pathological effects [33. Until the mid-19th century infant and child mortality was so high that the survivorship to age 15 years was around 50% or somewhat less. since they must reflect the changing genetic endowment of successive generations. more than 90% of newborns had an opportunity to fully participate in the reproduction of the next generation [36]. as well as the possible impact of EM on academic curricula. This might not be true for psychiatric disorders.27]. Although some deaths happened without a link to individual genetic endowment. There is also evidence that. on the changes in infectious diseases through time [3]. The increase in variability of heritable traits is a predictable outcome of such a relaxation of selection as its stabilizing effects are diminished [40]. The value of EM has been recognized particularly for clinical research [26. at least for some disorders.34]. thus EM research specifically uses historic samples. and personality disorders [29-31]. even in countries presently considered to be 'developed' [36. metabolic disorders (for example. The value of such studies of ancient tissues has become more and more accepted even for clinical research. Most microevolutionary alterations have medical implications for individual patients (for example. for example. eliminating the other half [35]. the value of ancient tissue samples such as mummified bodies and archaeological bones and teeth to study recent evolution of human disease is addressed. they assume it by interaction with the rest of the genome and the epigenetics determines their Darwinian fitness [42]. It mostly occurred due to high levels of differential mortality that allowed less than half of individuals born to pass on their genes to next generations. at the end of the 20th century. alleles do not have an absolute adaptive or maladaptive value. Studies of microevolutionary changes require time depths of at least a few generations. .37]. knowledge of current anatomical variations for surgeons) as well as at the population level (for example. Current EM research Major fields of primarily non-clinical EM research to date have included aspects of demography[22]. understood as differential reproductive success of carriers of different genes. vision defects [38] and less common congenital defects. levels of immunity. Thus. particularly as a crucial reservoir to study the evolution of infectious diseases [18-21]. anxiety disorders. The opportunity for natural selection through differential mortality has been so severely reduced that. For the first time in the evolution of humanity. EM concepts have been applied in clinical settings with a major focus on disease-provoking morphology. schizophrenia. sex [24]. phenylketonuria). sociospecific public health measures). Possible approaches in future EM studies Relaxed natural selection and microevolution of human morphology One major field for future evolutionary research with a particular biomedical perspective is the study of alterations of natural selection. Differential fertility contributed much less to the overall opportunity for selection since there was little genetic variation in this characteristic [39]. Over the entire evolution of humankind. where social pressure may still influence reproductive success [31].may be made by observing whether the magnitude of a particular change exceeds the range of adaptive phenotypic responses of the same genetic potential. A gene producing pathological effects in the past (for example. Presently. type 1 diabetes. psychiatric disorders may be linked to allele variations that predispose to differential susceptibility and adverse effects in terms of developing a disorder [41]. The aim of this review is to highlight the potential of novel directions in EM empirical research for current and future biological and medical applications rather than discuss pure theoretical understanding of the origin of humans. If generation-to-generation changes exceed full phenotypic expression of the same genetic potential they can be considered microevolutionary ones. and socioanthropological issues [25]. Furthermore.

The response. Figure 2. has affected body proportions since most of the stature increase. The lower the index value the greater the opportunity for natural selection. skeletal robustness or bone density) have also been shown. xenoestrogens or endocrine disruptors) into the food chain [58]. sx = reproductive value of a person of age x (for example. for example. It is evident that our biological properties are changing even within very short historical time frames. More research . while at the beginning of the 20th century it was present in only around 10% of individuals [43]) (Figure 2). and to what extent microevolution of human growth regulation has occurred. where it occurred.0011 (z test for linear trends in proportions)). Sexual maturity accelerated. such as Paget's disease. an increase in the range of human biological variation has been already documented for a plethora of anatomical structures. seems to be harmful and needs to be treated by interventions based on the understanding of human heritable adaptations to past diets. It remains to be determined to what extent acceleration of sexual maturation and increases in peak growth velocity are the result of alterations in socioeconomic conditions. obesity is linked to increased mortality and morbidity and thus any short-term alteration in obesity rates will have huge public health implications. Although it can be argued that short-term changes in body height and body weight are not the result of changes in gene frequencies. This change has become most obvious in the adolescent period [56]. Many other rather short-term changes of body morphology. One can multiply such clinically relevant examples of relaxed natural selection. For further data see [71]. especially in the case of increasing body weight. In the skeletal system. while the rates of growth at puberty became much higher than before. P <0. Besides direct economic costs. Changes in the Biological State Index over the last 15.70]. non-heritable responses to changing living conditions. too [47]. Some 'anomalous' arteries have more than doubled their prevalence (for example. was due to the growth of the lower extremities [51]. Finally. opening of the sacral canal (spina bifida occulta) became more common in cohorts born in the second half of the 20th century than it was before [46]. even within short time periods disorders of unknown cause. the ability of the human body to respond to such changes is a product of its earlier evolution. and the introduction of biologically active substances (for example.000 years of human evolution. The widely reported secular increase in stature.94.001 z = 3. Trend of increase in the incidence is significant (χ2(1) = 11. modern diabetes-causing genes were advantageous in the past) [53]. Regulation of postnatal growth and development has undergone a significant transformation during the last century. and the thyroidea ima branch of the aortic arch had disappeared completely by the end of the 20th century [44]. the median artery of the forearm is now present in around 30% of individuals in different populations. Also. The range of variations of hormones regulating human appetite. Frequencies of individuals with median arteries of forearms by date of birth in a sample of 284 South African dissection cadavers. resulting in problems in adolescence [56]. but simply adaptive. The economic impact of such body shape alterations on 'biological standards of living' has been addressed previously [54].90. while s15 = 1). Skeletal pathologies such as ossification of the posterior longitudinal ligament of the spine have increased [48] as have diffuse idiopathic skeletal hyperostosis [49]. For further explanation and data see [35. which occurred with varying speeds (from 0 to over 150 mm per century [50]) in various populations. Studies of the occurrence of skeletal manifestations such as hyperostosis frontalis interna. may possibly further elucidate the recent evolution of the human endocrine system [57]. Climatic factors have been proposed to influence the altered prevalence of the internal thoracic artery [45]. Thus. For example. and tarsal coalitions appear more often in more modern times. thus adding to the evolutionary explanation for part of the obesity problem. The index value is a probability that an average person will be able to fully participate in reproduction of the next generation. s65 = 0. the current epidemic of obesity may in part result from increasing variation in the size of the body frame that reflects a greater variation in the size of the gastrointestinal tract [52] rather than just caloric imbalance. can show a notable yet etiologically enigmatic alteration in prevalence [59]. Other examples might be the alterations in prevalence and the etiology of metabolic syndrome.Figure 1. Finally. such as alterations in body dimensions and proportions (for example. leptin and ghrelin and enzymes regulating carbohydrate and fat metabolism in past and present populations may differ. it is also not clear if the entirety of body height increases that occurred during the 20th century are adaptive rather than of a genetic nature [55]. recording these secular alterations is the very first step to explore possible environmental cofactors of such disorders. the so called thrifty genotype hypothesis debate (for example. body mass index. P <0. Labels in the formula are: d x = a fraction of dying people of age x.

Studies have discussed the impact of recent genetic sweeps such as the positive selection of Tay-Sachs disease-affected people versus tuberculosis [62]. not applying EM principles may restrict the true scientific impact and applicability of a particular research result. This needs to be realized and communicated accordingly (for example. Epigenetics is another field for future EM research. the imminent conflict of our short-term and long-term evolutionary genetic endowment is etiologically linked to the major causes of death in first-world countries such as cardiovascular or oncological disease. Thus. Many attempts at explaining host-pathogen coevolution in relation to major infectious diseases such as leishmaniasis or plague have been made [63. by general practitioners to their patients). future clinical studies in EM should focus particularly on the genomic evolution of bacterial and viral diseases and responses in the evolution of human immune system. human biological traits still evolve. Since it has been suggested that micro-RNA is linked with human pathologies such as cancer. Competing interests FJR has received research funding from the Mäxi Foundation. The same is true for any biomedical researcher. the introduction of EM topics into medical (and science) curricula is recommended. which was substantially expanded and modified by FJR. biological compromises in a rapidly changing environment. systems of public health and ways of handling the recent resurgence of treatment resistant forms of the disease. Abbreviations EM: evolutionary medicine. with what intensity and to predict their biomedical consequences is needed. We are not simply 'stone age bodies in a modern world'. Therefore. A 'morphological anomaly' may become more frequent or even 'normal' in a given population and. Authors' contributions FJR conceived the idea for this paper and discussed it with MH.64]. as well as in the general community. the lively and still continuing debate about the origin of syphilis [66. we are now only at the edge of an entirely new era that will allow us to unravel the mysteries of human disease evolution. Thus. Accepting variation as normal is an important issue in clinical medicine. molecular evolutionary studies may solve certain etiological enigmas. but without any evolutionary knowledge he/she certainly will not be able to provide the best. To summarize. even though meta-analytic standards known from clinical studies can hardly ever be met. No other relevant conflicts of interest exist. including maternal adversity [65]. As highlighted previously [68]. Issues such as viral pandemics or evolution of strain-dependent virulence can be explored by using a temporal and thus historical perspective. but we are both at the same time adapted and adapting. which attempts to describe diseases in the past and to trace changes of those diseases in response to the historical development of humans. DNA viruses are easier to extract than RNA viruses. especially during the last several thousand years. For the latter. sex differences in genetic vulnerability to cancer or arteriosclerosis can be addressed by EM research. Both authors contributed examples from their earlier studies and incorporated them into the text. new technological advances will allow more sensitive and specified research. With the prospect of improved ancient DNA and proteomic analyses. the incorporation of principles of human evolution and its forces into the knowledge of future medical practitioners is needed. MH has no conflicts of interest. For genomic studies in particular. medicosocial advice and prescribe optimal personal treatment [69]. individualized diagnosis. it should be no reason for concern for a particular individual.elucidating what changes occur. Epigenetic factors mediating gene expression such as early-in-life stress ('fetal programming') would be one such example. any progress in fighting disease based on evolutionary insights would be most welcome in the medical community. the discipline has evolved into palaeoepidemiological studies [60. has stimulated closer scrutiny of pathogens. thus. MH produced the first draft. Another example.61]. Outlook Medically oriented empirical research with an evolutionary focus may help to redesign public health policies and public awareness of science. . Poor intrauterine conditions are predictive for somatic and psychiatric disorders. Both authors edited the text and approved the final manuscript. From diagnoses of individual cases observed in ancient skeletons and in mummies. with the latter also being full of coevolving pathogens. Also. Furthermore. A general practitioner may not directly heal a patient using only EM principles. the study of their impact on the health of populations.67]. and should be a major future field of EM research. Lessons from paleopathology: Evolution of diseases and genomic studies Of special importance to EM is the subdiscipline of paleopathology.

Modern lifestyle and medicine have influenced nutritional and infectious constraints on puberty. with the latter defined as the life-history stage of reproduction (Table 1). While acknowledging heritable individual differences in pubertal timing. Developmental tasks for adolescents and young adults [67]. In consequence. youth Introduction Evolutionary development biology (evo-devo) is concerned with how developmental systems evolved. both accelerating and delaying it. We explore implications of this evolutionary-developmentalendocrinological-anthropological framework for theory building. Indeed. Conceptual foundations Life-history theory . social needs of adolescence and maturation toward youth and adulthood. Here we explore implications of an evolutionary-developmental-endocrinological-anthropological framework for theory building. A central claim will be that the transition from the preceding stage of juvenility to adolescence entails adaptive developmental response to energy resources. early maturation affords a potential fitness advantage for females more than males.and not just in a singular direction . puberty. in the last decades. social needs of adolescence and maturation toward youth and adulthood. In girls. while probing the consequences of these historically established systems for organismal evolution [1]. more than in boys. Evolutionary analysis highlights the fact that it is the female who is reproductively constrained in terms of the maximum number of offspring she can generate over her reproductive years. we emphasize developmental plasticity and the role of the environment in regulating pubertal timing in the service of fitness goals. allowing more time to reproduce. we show that familial psychosocial stress fosters a fast life history and reproductive strategy rather than early maturation being just a risk factor for aggression and delinquency.92 years in 1991 to 11. evolutionary life-history thinking challenges the prevailing notion that early puberty is exclusively or primarily pathological in origin. we examine adolescence as an evolutionary life-history stage in its developmental context. Utilizing evolutionary development biology (evo-devo). other environmental cues. as we hope to show. evolutionary developmental genetics. The plasticity which we argue characterizes adolescence is regulated by hormonal processes. Evidence that the timing of boys' somatic maturation is changing has recently been reviewed and remains inconclusive [3]. much variation remains to be explained. leaving room for the influence of environmental factors to adjust its phenotypic trajectory in the service of fitness goals.References Despite substantial heritability in pubertal development. resulting in the secular trend in pubertal development over the past 150 years. We challenge the pathological view by advancing an evolutionary perspective on the issue of juvenile transition and timing of pubertal development. viewing it rather as an adaptive response to changing life conditions. Table 1. and experimental epigenetics. Thus. and the mechanism for the 'epidemic' has been much debated. other environmental cues. as drawn from life-history theory.66 years in 2008) [4]. while illuminating new directions for research. evidence indicates that since the emergence of homo sapiens there has been much change in the timing of pubertal maturation . pointing fingers at toxins and perhaps other chemical products. To our way of thinking.and a variety of contextual factors appear to regulate pubertal development. juvenility. Using the evolutionary theory of socialization. We show that the transition from the preceding stage of juvenility entails adaptive plasticity in response to energy resources. while illuminating new directions for research. evolution. the rate of precocious sexual maturation in girls has been high and increasing. it is a mistake to focus only on environmental toxins or even simply cast changes in pubertal timing in disease terms. Here we examine adolescence from an evo-devo perspective. Toward this end we consider the anthropological record. Keywords: adolescence. the change in pubertal age has been intriguing. showing that adolescence as a stage was a new development in primate life history and that over time there have been many changes in pubertal timing. Research in evo-devo has formed around comparative embryology and morphology. though one Danish study documented a 3-month acceleration in male pubertal onset across a 15-year period (from 11. using hormonal and developmental mechanisms. treating this life-history stage of rapid growth and maturation in its ecological and developmental context [2].

and a youth stage. In addition to being a period of rapid growth.800. Testosterone.Evolutionary life-history theory deals with the strategic allocation of an organism's energy toward growth.before the onset of reproduction [2]. estradiol levels increase during this period also [11]. followed by adolescence. and the attainment of biosocial skills needed for successful reproduction. including dependency to sexual maturity. From this time forward. The collective endpoint of the adolescence package is the socially and reproductively mature adult. Relative to other species. allowing them to practice reproductive skills without conceiving [12]. hormonal and mental maturations are intimately coupled through iterative transactions between the nervous system and endocrine systems. Before and during adolescence .000 to 4. The onset of puberty in girls is generally considered to take place when breast buds erupt (thelarche). human life-history strategy includes a long period of postnatal growth. while avoiding death [2. maintenance and reproduction.5 years of infancy. Adolescence as a unique life-history stage As late as 3. The perception of fertility in girls facilitates their entry into the social-economic-sexual world of adult women. yet are not fertile. sexual selection and fertility fitness. pre-adult lifehistory stages. and menarche about a year after the peak height velocity. but perceived by adults as mature. about two years after their peak height velocity. girls have an apparent womanly body form. buttocks and breasts. lasting 30 to 36 months. These enable her to get through periods of scarcity. voice and facial features. rapid adolescent growth and delayed reproduction [5]. 5 years of juvenility and 2 years of youth . psychological and behavioral mechanisms that make strategic tradeoffs involved in the allocation of energetic resources to influence the three foundations of natural selection: survival. childhood.000 years ago hominids may have had a pattern of growth indicative of an adolescent stage of development [8]. adolescence is a time of subcutaneous fat deposition. Table 2. Boys and girls coming of age Boys and girls embark on different come-of-age strategies to achieve their fertility goals (Table 2). The uniquely human adolescent growth spurt is often regarded by physical anthropologists and ape biologists as the operational definition of adolescence [7]. including raising offspring to independence. lasting an additional 2 to 4 years. and much later than the onset of genital changes in boys. pubertal tempo. Boys show a pattern of gradual maturation of the hypothalamic-pituitary-gonadal axis similar to that of girls. appears to be important for activation of the courtship behavior that leads to the formation of sexual pairing bonds [14]. but even during childhood and juvenility girls have active ovaries that generate estrogens [10]. and social aspects in learning. the package we call adolescence includes such pubertal development plus the growth spurt. whose gonads are assessed via direct palpation and show testicular growth before sex-steroid concentrations increase. which lasts an average of 4 years [2]. even though it starts before the emergence of secondary sexual characteristics in girls. Whereas subcutaneous fat is evenly spread over the female chimpanzee's body.13]. signal sexual maturity and facilitate sexual attraction of a mate. body size. Whereas puberty refers to the activation of the neuroendocrine hypothalamic-pituitary-gonadal axis that culminates in gonadal maturation and the biological effects of sex steroids. just like the chimpanzee . Boys will learn their adult social-economic-sexual roles while already sexually mature but not yet perceived as such by adults. A matter of definition The terms puberty and adolescence are often used interchangeably and thus incorrectly. a juvenility stage of 3 to 4 years of semi-independence. But they are still young in outward development. which lasts 3 to 5 years. they will develop an adult cycle of ovulation and adult size of the birth canal much later. the ovaries start to grow discretely about two years before thelarche. becoming fertile at 14 to 15 years on average. Adolescence in boys and girls manifests differently with regard to their actual fertility. development of new relationships. Growth acceleration in the girl occurs some 6 months before the budding of breasts. ultimate size and cognitive targets. It is now recognized that thelarche is not the first sign of maturation of the female hypothalamic-pituitary-gonadal axis. intensification of pre-existing friendships.5]. and allows others to continuously monitor her nutritional state[9]. This allows them to interact and learn from older adolescents and adults without seeming to compete for status and other important resources.000 years ago the early homininae Australopithecus afarensis had only three postnatal. which plays a central role in male peak growth velocity. childhood and adolescence were inserted as new life-history stages ofHomo sapiens [7]: infancy. Consideration of intermediate growth stages and the transitions between them from a life-history perspective affords insight into strategic objectives that include the age of pubertal onset. During the evolution of the Hominidae. especially in girls.000. Very much like boys.000. there is some suggestive evidence that 1. with the latter involving gonadal steroid hormones [6]. including fertile females [7. the human adolescent has striking fat deposits in the thighs. intimacy and mutual support. With obvious limitations as to what can be inferred from fossilized skeletal remains. around 18 years of age. There is no evidence for such a human-like adolescent growth spurt in any living ape. They gain knowledge of their adult social roles while still infertile. cognitive and brain maturation. To promote reproductive and parenting success in the service of reproductive fitness. even if she is thin overall. It predicts that selection will promote fitnessenhancing physiological.

Comparison with the African apes suggests that the timing of adrenarche and the sex difference in chimpanzees timing of transition from infancy to juvenility may be similar to that of humans moving from childhood to juvenility [ part by assaying one's social status and standing in the competitive world of peers [17]. but not other family relationships. while working out their social standing among age-mates. characterizing it in terms of endocrine and body composition changes resulting in changes to social assignments and psychological maturation [16. Coinciding with participation in adult social activities. 'the five-to-seven-year shift' and 'the age of reason and responsibility' [18]: the brain reaches its final size . Alternatively. he argues that sex differences in attachment relationships emerge in middle childhood and have adaptive significance for sexually selected life-history strategies. whereas the human/chimp enzyme differs from the baboon or rhesus enzyme by 25 to 27 residues (95% identity) [22]. Serum DHEA and DHEAS rise progressively throughout juvenility [23]. Emphasizing fitness goals. with effects on a wide variety of physiological systems. Transition from childhood to juvenility is marked by the onset of adrenal androgen generation (adrenarche). Evolution of the hominidae life history during the first 20 years of life. Detailed accounts of these stages are given in[2]. The chimpanzee serves as a living representative of the assumed Australopithecus afarensis life history. During evolution. Del Giudice contends that juvenility (adrenarche) represents a 'switch point'. Thus. increases in body size evident among Homo erectus imply increases in lifespan and delayed adrenarche and reproductive maturation. We and others have defined juvenility as a distinct life-history stage in humans. adiposity rebound. Whereas humans and chimpanzees exhibit adrenarche. so may prove especially adaptive under conditions of ecological risk (provided nutrients are sufficient to foster maturation).and primates. Developmental psychologists refer to this period as 'middle childhood'. and the eruption of the first molar teeth [16]. affecting neurological functions and modulating mood [27.20-lyase. and the adrenals of most other mammals produce little or no DHEA [22].17]. juveniles develop a strong odor during the juvenile period. intriguingly. schedule and efficiency [31]. a time when the environment can reprogram nascent reproductive strategies established earlier in life [17.Preceding adolescence . Accelerated pubertal development also reduces generation time. DHEA in humans operates as a neurosteroid. The age at transition from childhood to juvenility has been remarkably constant. early transition to adolescence and the abbreviation of its duration increase the likelihood of reproduction before death. other primates such as the baboon and rhesus monkey do not. and as such are a natural point at which to consider the potential role of delayed transition from childhood to adrenarche in human evolution. In social terms. As childhood emerged and prolonged. Indeed. equipped with adult molars. while potentially lengthening reproductive lifespan. The human and chimp DHEA-generating enzymes. whereas insecure females tend to adopt an anxious/ambivalent strategy (which maximize investment from kin and mates).the juvenile stage All mammals. In modern societies the transition to juvenility coincides with the age when children go to school and compete to some extent with adults for food and space.28]. as well as somatic growth and development [21. may be similar to that in humans. Early psychosocial stress and insecure attachment during juvenility direct development towards mating-oriented reproductive strategies. infancy has gradually become shorter.26]. including neurological [24]and immune [25]. Figure 1. Strategies such as those involving bearing few or many offspring are passed to future generations [17]. forage independently for food and care for themselves. insecure males tend to adopt avoidant reproductive strategy. Campbell argued that the increased brain size and extended lifespan of humans relative to the great apes imply changes in the timing and impact of adrenarche [15]. including the great apes. adrenarche is a recent evolutionary event. presumably for incest avoidance [19]. 17. especially when compared to the plasticity that characterizes other life-history features such as age of sexual maturation [29]. differ at only two amino acids. olfactory aversion emerges in the case of father-daughter and brother-sister.even if neuronal development is not complete . as measured by adrenarche. deceleration of growth [21]. juvenility offers opportunities to prepare for the social complexities of adolescence youth and adulthood .30]. Assuming an important role for adrenarche in human brain maturation. Comparison with African apes suggests that the timing of the transition to juvenility. late transition into adolescence lengthens preadolescent growth and the opportunity to embody or internalize . and the latest introduced adolescence came at the expense of a shorter juvenility. transit directly from infancy to juvenility without passing through the childhood stage except humans (Figure 1). Thus. although the full course of age-related changes in dehydroepiandrosterone sulfate (DHEAS) and their relationship to reproductive and brain maturation are not clear [15].20]. childhood and adolescence have been added as new life-history stages and compared with apes and the presumably early hominidae. Transition from juvenility to adolescence Age and size at adolescence have strong effects on an individual's fitness because they affect reproductive potential.

thereby. At the same time. resulting in a U-shape relationship [39. Such population growth increased opportunity for genetic mutations.and. This claim is consistent with data on the Aeta of the Philippines. The age of menarche gradually increased until the recent secular trend's decline. when animals are raised under ideal conditions that promote rapid growth. pubertal development is accelerated. which may be compensated for by continuing parental care. Later maturation to adulthood was a tradeoff to adapt to poor nutrition as well as the increasing complexity of being an adult in a society engaged in agriculture. thereby increasing the likelihood of reproduction before death or disability. thereby accelerating the pace of human evolution[35]. This suggests that menarche in Neolithic times occurred even earlier than observed in modern Western countries [36]. human development . who reproduce as early as age 10 to 14 years [38]. Nutritional cues have clear temporal influence. Figure 2. When immature animals experience severe environmental stresses such as malnutrition or disease. Ultimately. when animals are raised under ideal conditions that promote rapid growth. When immature animals experience severe environmental stresses such as malnutrition or disease. Adaptive developmental plasticity The changing age of adolescence Substantial physiological variation of some 4 to 5 years is evident for age at onset of sexual maturation across individuals under varying life conditions [32]. Domestication of animals and agriculture altered the human environment . including adaptive changes in the onset of puberty. Data from [36]. settlement and population aggregation.000 years ago .was 7 to 13 years.000 years ago) occurred at age 9 to 14 years (Figure 2) [36]. and that full reproductive competence in Neolithic females (New Stone Age.000 to 12. social or psychological. pubertal development is accelerated. be those resources the beginning of the agrarian period . Menarche age over the last 12.5 years. A relatively sedentary lifestyle increased local human densities. by medieval times. thereby increasing the likelihood of reproduction before death or disability.000 years 4-year gap between menarche and reproductive competence [37].the various resources to which the individual is exposed. facilitated the spread of infectious diseases and was associated with recurrent several ways. This crucial point is further discussed below under 'Evolutionary Theory of Socialization'.34]. Collectively.000 years. since for any given growth rate earlier maturation implies smaller size at transition. with a world population of several thousands. By contrast. Thus. individuals face a tradeoff between maturing to reproduction young and small and maturing at large body size. to that of several billion today. which themselves led to an overall increase in the mean age of menarche. with a trend towards earlier maturation among those whose average body mass early in life is lower or higher than average. But when contextual stress is not so great as to challenge survival. But when contextual stress is not so great as to challenge survival. Increased differentiation of social tasks and the creation of societal hierarchies in wealth-accumulating agrarian societies resulted in variation in nutritional status and family conditions. Developmental and maturational tempo is flexible and responsive to environmental conditions in a presumptively adaptive manner. The human population has grown exponentially from a brink of extinction 80. much variation remains to be explained [34]. leaving room for the influence of environmental factors to adjust the phenotype in the service of fitness goals [32]. internal checkpoints ensure that maturation does not occur until juvenile development is complete. Figure 3. This would place menarche at 7 to 12 years. delayed maturation prolongs the preadolescent hazard period. The U-shaped link between contextual risk and nutritional cues as they predict pubertal development. maturation is often delayed until conditions improve and normal growth can resume. assuming a 2. yet later among those with a poor childhood weight gain [32].000 to 5. Despite substantial heritability in pubertal development [33. as shown in Figure 3. 12. these phenomena highlight a U-shaped link between contextual risk and nutritional cues as they predict pubertal development (Figure 3). By contrast. internal checkpoints ensure that maturation does not occur until juvenile development is complete. including the timing of juvenility and adolescence. as it remains today among underprivileged adolescents in developing countries [32]. maturation is often delayed until conditions improve and normal growth can resume. the average age of menarche was deferred to 16.40]. . Estimates indicate that the age for menarche around 20.

5 years in such societies [44]. it can hardly explain the secular trend in the age of menarche over the last 150 years. resulting in the secular trend in pubertal development over the past 150 years (see below). The Philippine Aeta provide unique insight into the strategic importance of the age of adolescence. viewing it rather as an adaptive response to improving life conditions. . [20] and Gluckman and Hanson [36]. The evolutionary reasoning was that early maturation would be selected under conditions of emotional risk and uncertainty. the popular explanation has been that this phenomenon results from environmental endocrine disruptors that accelerate hypothalamic maturation [43]. Thus it seems that early fecundity evolved to adapt to high-risk.the age of menarche has fallen by a full 4 years in the industrialized West (Figure 4). With life expectancy of 16 and 27 years at birth and adulthood. unemployment). first genital petting and first sexual intercourse. father absence). The latter would make evolutionary sense for reproductive fitness in a secure world. their first reproduction occurs at age 10. Declining age of menarche in Western societies from 1840 to 2000. Thus. first kissing. maturing early and breeding promiscuously would enhance reproductive fitness more than delaying development. 0. mating cautiously and investing heavily in parenting. marital conflict. against beginning early reproduction and increasing the number of reproductive cycles. Natural selection favors accelerated development when early life experiences suggest an insecure world in which intimate relations are not enduring [47]. with shortness of stature resulting from a short period of preadolescent growth [38]. in line with the attachment theory of Bowlby [45.50]. however. and higher rates of adolescent pregnancy.42]. we explicitly challenge the concept that this has been a disease process. respectively.S. boys adopted from developing to industrial countries show accelerated sexual development[41. both these attributes correlate with higher birth rates. thereby improving the odds for successful pregnancy while also risking death before sexual maturation. The secular trend in puberty. along with lesser parental investment. Whereas endocrine disruptors may have a bearing on the earlier age of thelarche. The line does not show a saturation point. which under such conditions is 18 years. to a lesser extent. As much as the secular trend in human size has been an adaptive response to a nutritionally rich environment. Following Belsky et al. early pubertal spurt and early growth termination [38]. as perceived by the young child and juvenile [48]. Early reproduction minimizes the likelihood of death before reproduction. thereby setting the stage for earlier sexual debut.46]. proposing that accelerated pubertal development reflects contextually regulated reproductive and life-history strategies. evolutionary life-history thinking challenges the notion that earlier puberty is the result of a hypothalamic control malfunction. The ever-younger age of girls' thelarche and menarche may have more than a single justification. The secular trend in pubertal maturation The secular trend provides compelling evidence that pubertal development is developmentally plastic. itself induced by extrafamilial ecological stress (for example. showing an early juvenile deceleration. A later first birth allows for a longer period of adolescent weight gain. highmortality life of a short duration. more promiscuous mating and the bearing of more offspring. slower physical maturation would risk lowering reproductive fitness and survival: in an insecure world. [20] advanced an evolutionary theory of socialization stipulating that familial psychosocial stress (for example. and heavier women in traditional societies are more fertile. Such theorizing is certainly consistent with evidence that earlier pubertal development is associated with greater sexual risk taking. Data from [68]. as reviewed[49. similar to that witnessed in the case of the secular trend. Thus. Women face a tradeoff between spending a long time accumulating resources through childhood growth.01th percentile. and it is females more than males who may enjoy a fitness advantage from early maturation. In the last decade. Evolutionary theory of socialization Belsky et al. This tradeoff has been used to model the optimal age at first birth. which is hereby expanded (Figure 5). the age at transition from juvenility to adolescence in humans has a variety of physical and social correlates. The greater tendency of adopted girls to respond with pubertal onset to a changing environment is in line with female preponderance of idiopathic central precocious puberty. They claimed that pubertal maturation played a previously unappreciated role in linking early rearing experiences with subsequent mating and parenting. Their growth deviates from the U. the trend is expected to continue. limited income. As note earlier. Figure 4. earlier age of menarche is associated with earlier age of first dating. harsh parenting. it is the female who has intrinsic constraints on the number of offspring she can generate over her reproductive years. which is a recent trend. Indeed. near the observed mean of 17. fosters a fast life-history and reproductive strategy. the receding age of adolescence and pubertal development has been an adaptive response to positive environmental cues in terms of energy balance. Over the past 150 years without any documented change in gene frequencies .Modern hygiene and medicine have influenced nutritional and infectious constraints on puberty. Further evidence to this effect would seem to come from research showing that girls and.

sexual behavior and parenting [56]. appeared. sexual behavior. thereby. an abundance of evidence consistent with its critical pubertal-timing prediction has emerged (for review see [51]). Thus.57-62]. The transition from juvenility to adolescence is deferred when food supply is short. greater sexual 'risk taking' in adolescence [53]. somatic and behavioral development. pair bonding and eventual childbearing and parental investment. whereas the vast majority of early puberty probably reflects normal physiological and adaptive developmental plasticity [36]. each assigned with its own domain. In a recent geneenvironment interaction study. negative family relationships) is associated with females' advanced adrenarche and early puberty [52]. Developmental and maturational traits that respond to environmental cues enhance fecunditysurvival schedules and behavioral strategies that yield the highest fitness in a given environment. preceded by a juvenile phase and followed by a youth phase leading to such a delayed reproduction? Like other organisms. thereby. . authoritarian parenting. In line with the above.Figure 5. programming at the same time for later fecundity and fertility and for longevity [65. pubertal timing.66]. the term precocious puberty is to be reserved for those few with anatomical or genetic defects. More and less supportive family contexts (and broader ecologies) influence the quality and quantity of parental investment. Such findings suggest that population estimates of environmental influences on pubertal development do not necessarily reflect individual response. Based on [20]. the gradual secular trend in industrial societies over the last 150 years is not. and that harsh parenting in early childhood predicts earlier age of menarche and. and that girls evacuated from their homeland during World War II and sent to live in Sweden and Denmark reached menarche at a younger age and even bore more children than members of the same birth cohort who remained at home [55]. Important also are data showing that younger sisters with greater earlier exposure to an absent father as a result of divorce or separation matured earlier than did their older sisters [54]. humans evolved to withstand environmental hardships by responding in ways that maintain evolutionary fitness. Individual differences in developmental plasticity Some individuals are more plastic and responsive to environmental cues and others less so. The faster strategy enables the individual to reduce the risk of dying before reproducing and reflects the fact that the individual's capacity to attract and maintain a high-quality mate and provide resources to their own (eventual) offspring will be limited. as reviewed [51]. The means to do this is a series of predictive adaptive responses that utilize the sensitive times of transitions from one life-history stage to the next. even if submaximal. Faster and slower reproductive strategies: Reproductive strategies develop in different contexts and are characterized by diverging patterns of psychological. whereas the slower strategy fits a world in which resources are predictably available and sufficient. without a precise definition in the present context. which in turn influences psychological and behavioral development. Collectively these forces regulate the timing of pubertal development and. The slower strategy reflects the opposite. The faster strategy fits a world in which risk and uncertainty is high. given the short timescale. Whereas the gradual tendency to mature late over Homo's evolution is genetic. Why is it that we have a unique adolescence phase. Children who were more physiologically reactive in terms of cortisol response to a psychological challenge were more responsive to family forces in accelerating pubertal development [63]. Consider in this regard findings from longitudinal research indicating that limited family support during childhood (for instance. adopting a more fixed developmental trajectory for reproductive strategy [50. an allelic variation in the estrogen-receptor gene determined which girls' menarche age was accelerated by high levels of family conflict [64]. Conclusions This review uses an evo-devo approach and life-history theory for understanding human adolescence and especially variation in the timing of reproductive maturation. experimental manipulation of licking and grooming of newborn rats by their mothers illuminates the role of epigenetic processes in regulating the stress-response system. The more recent tendency for earlier puberty reflects the overall quality of modern environments. This term implies pathology. The timing of puberty sheds light on the relationship between phenotypic adaptive plasticity and adaptive genetic changes. In the two or more decades since the adaptive-predictive-response theory of human development by Belsky et al. allowing females to approach the extremes of their genetic range of reaction. Such evo-devo thinking demands reconsideration of the notion of 'precocious puberty'.

and at present they are used in conjunction with. and result in both mental and somatic consequences. Diffusion-weighted MRI. Perhaps the most fundamental question raised by the life-history approach to adolescence concerns the uniqueness of each child in her given genetic background and current environment as they best serve her reproductive fitness. diffusion-weighted MRI and positron emission tomography (PET)-CT allows characterization of tumor physiology and potential genotypic activity. Further validation of these methods is required however. DHEAS: dehydroepiandrosterone-sulfate. This framework is quite distinct from the disease perspective that fails to appreciate the evolutionary wisdom of maturing early and behaving in opportunistic. social isolation. Virtual cystoscopy Introduction Bladder cancer is a common disease with significant associated morbidity and mortality. The use of multidetector 64-slice computed tomography (CT) and magnetic resonance imaging (MRI) remain standard staging modalities. and (iii) metastatic disease [3]. In an American study.The implications of disconnection of the mental and somatic components of human adolescence are underappreciated. Multidetector computed tomography. conventional imaging techniques. how do epigenetic events influence the transition from one life-history stage to the next. that early life experience during infancy and childhood will be associated with a change in maturational tempo. including perhaps aggression. Epigenetic mechanisms potentially play an important role. we have to better understand how these interact with or via endocrine mechanisms to generate signals that affect life history and adolescence. Staging. were more likely to be depressed and to have many sexual partners in young adulthood compared with their counterparts. early sexual behavior and psychiatric problems [67]. approximately 20% at presentation. the insulin-like growth factors and the sex hormones. early-maturing girls displayed higher levels of self-reported criminality. Globally it is the ninth most common cause of cancer related death in men [1. These hormones drive the rate of growth and development. Keywords: Bladder cancer. The development of functional imaging such as dynamic contrast-enhanced MRI. delinquency and promiscuity because these responses were selected to promote fitness. but their relative contribution to the phenotypic outcome and the extent of stochastic epigenetic reprogramming that is required to alter human phenotypes is not known because few data are available [69]. Rather. Competing interests The authors declare that they have no competing interests. If the environment can influence developmental and maturational trajectories during pre-adult life-history stages. Abbreviations DHEA: dehydroepiandrosterone. approximately 70% to 80% of patients. but also mental and social behaviors. is markedly different from the widespread assertion that early maturation is a risk factor for aggression and delinquency [68]. particularly those with a history of adolescent conduct disorder. PET-CT. but it is unclear how the environment shapes the timing of the different phases of developmental events. however. permitting early identification of response and allowing timely change in treatment. drug abuse. growth and puberty at the molecular level? Growth and puberty are regulated by insulin. Early-maturing girls. The claim we make. The clinical spectrum at presentation can be divided in to those with (i) superficial or nonmuscle-invasive bladder cancer (NMIBC). rather than as an alternative to. (ii) muscle-invasive bladder cancer (MIBC). Given the evidence on the strong influence of socioeconomic conditions early in life. such that harsh rearing conditions predict earlier age maturation and associated behavioral phenotypes.2]. Among them are the obesity and polycystic ovary syndrome epidemics. to help stratify and inform future patient management. advantagetaking ways under certain contextual conditions. we contend here. growth hormone. Hereditary. The challenge . Authors' contributions The purpose of this article is to review the imaging techniques that have changed and are anticipated to change bladder cancer evaluation. They open up the possibility of tumor mapping and individualized treatment solutions. to mention a few of the control factors. Ultrasound. Early puberty may thus represent a social pathology in industrialized societies. environmental and stochastic factors regulate puberty in a unique environment.

Three-dimensional CE US may also have future role in assessing treatment response of the primary tumor to guide bladder-sparing approaches.6%[18]. allows views in multiple planes to be obtained and improves the rate of primary bladder tumor diagnosis (88. Transurethral resection of bladder tumor (TURBT) also understages tumors. Therefore.9% with two-dimensional US versus 100% with threedimensional US) when identifying T3b disease [20]. It is however an important diagnostic tool in the investigation of hematuria in particular to assess large renal masses/upper renal tracts. carcinoma in situ(CIS). plaque like tumors are present. inflammation.15]. .4% versus 72.11. predict early response to treatment and provide non-invasive alternatives to cystoscopy for those requiring long-term surveillance. conventional imaging modalities are unable to identify microscopic disease and can be inaccurate in identifying macroscopic disease. allowing signal from the tumor to be highlighted by suppressing signal from adjacent surrounding normal tissue [5. approximately 30% to 50% patients are understaged at the time of cystectomy [6-10]. extent of loco regional disease and to determine the presence of metastases. Three-dimensional CE US uses enhanced images in three orthogonal planes and reflections of microbubbles to depict blood vessels. This has been shown to be clinically useful in differentiating MIBC and NMIBC [21]. The objective of any staging modality is to achieve adequate visualization of the primary tumor. which has made it an important staging modality for bladder cancer. its precise role in diagnosing bladder cancer is controversial. The volume data can be retrieved and manipulated as if in real time. It is limited by interobserver variability and inability to distinguish the muscle layers of the bladder[5. although the presence of hydronephrosis is suggestive of MIBC. Two-dimensional transabdominal and transvaginal US have been investigated with regards to aiding bladder cancer staging but transurethral ultrasound has demonstrated the best ability to visualize depth of tumor penetration through the bladder wall [16.5]. Magnetic resonance imaging (MRI) MRI has excellent soft tissue resolution and multiplanar capabilities. To address this newer imaging techniques are being explored to improve pretreatment staging. systemic chemotherapy and intravesical drugs also interfere with interpretation [5.17]. However. Flat lesions. Prior to TURBT CE US has a reported accuracy of 88. It is also unreliable in determining deeply infiltrating disease and nodal involvement [5].23. Computed tomography (CT) Multidetector (64-slice) CT scanning has provided the mainstay in radiological assessment. It has a reported sensitivity of 85% and specificity of 94% for the diagnosis of bladder cancers [11]. On T2-weighted images the bladder tumor is usually more conspicuous.Anatomically the bladder is divided into several layers. Its use in assessing treatment has been evaluated other solid tumors including primary liver cancer following local therapy [22]. which increases objectivity. Fundamental to its importance in local staging is the ability to manipulate image contrast by using different sequences. for lesions less than 5 mm sensitivity is reduced to 20% with a negative predictive value of 28. are more likely to be falsely negative [5. The disadvantage of this technique is that the entire tumor is not visualized and detection is particularly difficult when flat. In those with MIBC this has been shown to be inaccurate in 23% to 50% of cases [4. Contrast-enhanced US (CE US) has been shown to be superior to conventional US in differentiating non-invasive and invasive bladder tumors [18. Detection is dependent on the morphology and size of the tumor. there is coexistent calcification. bladder cancer evaluation.12]. Previous biopsy.1% compared to standard two-dimensional US. tumors less than 1 cm and in those whom recent resection has been performed. Although recent developments in US techniques overcome many of the limitations of two-dimensional US. and are anticipated to change. Accurate local staging with imaging is dependent on reliably distinguishing these layers.13]. Ultrasound (US) US is at present not used routinely in clinical practice for the assessment of known bladder cancer. This article will review the development of imaging techniques that have changed. with 94. they remain under investigation and have yet not translated in to widespread clinical use. Use of two-dimensional US is often limited by the subjectivity and expertise of the examiner.19].7% sensitivity for tumors greater than 5 mm. Bladder tumors may be visualized by ultrasound but a negative test does not exclude the presence of bladder cancer. Three-dimensional US has been developed to provide reconstruction of the actual tumor with visualization of the bladder wall layers. The signal from perivesical fat can be suppressed using short tau inversion recovery (STIR) sequences. accurate radiological correlation is important to help guide patient management. CT remains the modality of choice for investigating hematuria and has replaced intravenous urogram (IVU) at most centers. Although a 64-slice multidetector CT provides high spatial resolution allowing visualization of extravesical spread it is not reliable in determining the extent of locoregional disease [14]. the abdominal wall is rigid or the patient has central obesity [21].24]. Clinical staging of the primary tumor is with bimanual examination under anesthesia.

6% and negative predictive value 71. Dynamic contrast-enhanced MRI (DCE-MRI) enables in vivo assessment of tumor blood flow and permeability using paramagnetic contrast agents. Intrinsic-susceptibility-weighted or blood-oxygenation-level-dependent (BOLD) MRI. The clearest advantage of MRI over CT is the ability to determine the presence of muscle-invasive and extravesicle disease. There is also evidence that the ADC value may help identify high-grade tumors. Visualization of tumor blood flow can be used to identify areas of hypoxia and subsequently be used to predict treatment response [27].37-39]. However. False positives are due to reactive hyperplasia.43]. Nodal enhancement is dependent on the tumor burden. enlarged reactive. Nanoparticle-enhanced MRI Involved lymph node detection by convention is governed by size and shape. This has the potential to provide both qualitative and quantitative information to aid tumor assessment. Consistent with other studies higher ADC was associated with unfavorable response [40]. Diffusion-weighted MRI (DW MRI) is a functional imaging technique dependent on the inhibitory effect of cell membranes to the random motion of water molecules (Brownian motion) to generate image contrast by applying two equally sized but opposite diffusion sensitizing gradients. which phagocytose the USPIO causing them to accumulate within the node. characterized by their b-values. Preliminary results for lymph node evaluation using DW MRI do not appear to be accurate (sensitivity 76. However. specificity of 67%.14].28]. the Response Evaluation Criteria In Solid Tumors (RECIST) criteria). Deoxyhemoglobin is a paramagnetic molecule that allows it to act as an intrinsic contrast agent. specificity and accuracy of 92%. 90% and 91% respectively. exploits the difference in magnetic susceptibility of oxyhemoglobin and deoxyhemoglobin. positive predictive value 86. It is an effective biomarker in predicting pathological complete response in those receiving primary chemotherapy for breast cancer and chemoradiotherapy for rectal cancer[23. reflected in the low mean apparent diffusion coefficient value (ADC)). BOLD MRI image acquisition during high oxygen concentration inhalation (carbogen.In conjunction with gadolinium-containing contrast (Gd-Ca). Failure to detect microscopic foci of metastatic disease in very small lymph nodes leads to false negative results. As tumors have greater cellularity than normal tissue they demonstrate higher signal intensity (that is. restricted diffusion on MRI.1%) and higher stage tumors (92.7% versus 15. with a reported sensitivity of 98. 95% oxygen. and is our preferred modality of local staging prior to definitive radical treatment in MIBC. Conventional assessment of local response is with cystoscopy. DW MRI is also more accurate than T2-weighted MRI in staging both organ confined (≤pT2) (69. or where evaluation of morphological change is difficult to interpret [34.4%. with a false negative rate of 21% [5. This causes a drop in signal intensity on T2-weighted images.1% and positive predictive value of 100% [35]. After intravenous administration. It demonstrated that the ADC was the only significant. 5% carbon dioxide) reflects improved tumor oxygenation and blood flow and may help identify patients more likely to benefit from carbogen radiosensitization [29]. Techniques that evaluate nodal function rather than morphology are aimed to more accurately characterize nodal disease. nonmalignant lymph nodes can mimic metastatic involvement with these modalities. Sinerem®). with low ADC (<1 × 10-3 mm2/s) suggesting G3 disease[36].1%).5% versus 80. Following treatment the ADC value increases. Gd-Ca should be avoided in those with renal impairment (estimated glomerular filtration rate (GFR) <60 ml/minute). MRI has an accuracy of 85% in differentiating NMIBC from MIBC and 82% accuracy in distinguishing organ confined disease from non-organ confined disease [25]. independent predictor of chemoradiotherapy response with a sensitivity. the USPIO particles reach the lymph nodes via the lymphatics. Nodes greater than 1 cm are considered malignant on CT with a sensitivity of 85%. Benign nodes have functioning macrophages. specificity 89.4%. DW MRI therefore has the potential for monitoring treatment response to chemotherapy or radiotherapy with identification of early non-responders who may benefit from change in treatment approach. These nodal regions retain their signal intensity on T2-weighted images allowing detection on post-contrast imaging [42. reflecting decreased cellularity. localized nodal lipomatosis and insufficient . The use of functional MRI imaging to provide biological information of tumor characteristics is under investigation. as there is an increased risk of developing nephrogenic systemic fibrosis [26]. Metastatic lymph nodes that are partially or completely infiltrated are unable to take up these particles effectively. Lymphotropic nanoparticle enhanced MRI exploits nodal macrophage function to detect metastases using ultra-small superparamagnetic particles of iron oxide (USPIO) (ferumoxtran-10. One study has explored the role of DW MRI to assess response to chemoradiotherapy in 23 patients with MIBC. The clinical use of functional MRI in bladder cancer assessment is not yet clearly defined but work to date suggests it likely to provide important information that may help guide treatment selection. Histogram analysis of ADC through the entire tumor volume captures the diffusivity microenvironment and may aid identification of the heterogeneity known to exist within tumors that may have prognostic and predictive value [30-34]. consistent with response.4%) [41]. In other tumor types change in ADC has been used to identify and quantify early treatment response that may occur before conventional assessment of response is seen (for example.

9% respectively. locally recurrent and perivesical nodal disease has been difficult because the interference caused by the urinary excretion of the isotope.46]. In a meta-analysis of 3084 patients from 26 studies to determine the validity of VC by CT. Despite these limitations. specificity of 95%. 90. flat lesions and CIS [24. MRI or US. (f) axial DW MRI at b-value 750. The use of 18-FDG-PET in staging primary bladder disease. Figure 1. the pooled sensitivity for bladder cancer detection using CT virtual cystoscopy. In those whom FDG-urine washout was encouraged by diuretic injection. with sensitivity of 96%. it cannot distinguish between increased metabolic rate occurring as a result of infection. A number of techniques encouraging adequate washout of 18-FDG from the urinary tract have been investigated to overcome this. Radiotracers are used to identify the altered metabolic activity occurring within tumors.8% and 96. Although VC is unlikely at present to replace conventional cystoscopy it may be considered in conjunction allowing the possibility of minimally invasive follow-up. positive predictive value of 89% and a negative predictive value of 98% [44]. inflammation or the normal physiological activity in some organs[24]. The patient first moves through a spiral CT then a gamma camera in a single investigation. Metabolic change detectable by PET may precede anatomical changes on CT or MRI leading to greater sensitivity as compared to conventional axial imaging alone. USPIO are no longer readily available. Patient with known T2 N0 M0 bladder cancer (left bladder wall): (a) contrast-enhanced computed tomography (CT) scan. These include elective voiding.7% and 100% respectively for detecting recurrent disease within the bladder [56].1%. bladder irrigation. The advantage of this technique is that it is non-invasive. (e) axial DW MRI at b-value 100. and continuous bladder irrigation during image acquisition increases staff exposure to radiation [58]. 18  F-Flurodeoxyglucose (18-FDG) is currently the most commonly used PET tracer in oncological imaging and has an established role in the initial staging. (d) axial diffusion-weighted (DW) MRI at b-value 0. VC is performed on a dedicated workstation using a variety of computer algorithms [45-48].9%. and forced diuresis with intravenous frusemide prior to delayed image acquisition[54-57]. 94. response assessment and recurrence detection of many cancer types [50-53]. catheterization. and (g) magnetic resonance virtual cystoscopy (MRVC) of same tumor with threedimensional reconstruction of tumor bed showing opening into adjacent diverticulum.8% and 77. The pooled specificity for bladder cancer detection was 98. This method has been used to assess a number of other hollow organs including the colon and bronchus. Previous VC studies have focused on the potential diagnostic capability of evaluating hematuria.2% respectively [49].USPIO [24]. These measures are invasive. the reported accuracy when evaluated prospectively in 58 patients prior to surgery is 95%. Its use is dependent on the increase glucose metabolism occurring within the tumor. Uniform radiotracer distribution throughout the body produces a SUV of 1 [24]. The disadvantages include inability to obtain pathology and low sensitivity in identifying smaller tumors (<1 cm). However. allowing indirect visualization of the mucosa and simulation of endoscopic evaluation. This uptake is quantified using the standardized uptake value (SUV). Further investigation is necessary however to evaluate the impact of radiotherapy. However. Once source images are obtained. which limits the scope for further investigation and clinical application. Figure 1illustrates appearance of tumor as seen on VC and on CT and MRI. oral hydration and voiding. the sensitivity and specificity for FDG-PET CT was 86. (b) axial T2-weighted image performed on a 3 T magnetic resonance imaging (MRI) unit showing a hypointense lesion. making them less acceptable to patients. Catheterization and irrigation prior to FDG-PET imaging has a reported 40% false positive rate for detection of recurrent or residual bladder cancer [57]. Virtual cystoscopy (VC) Three-dimensional surface modeling is possible using cross sectional data obtained from CT or MRI. Positron emission tomography (PET) PET/CT: metabolic tracers Composite PET/CT images provide three-dimensional whole body structural and functional information. magnetic resonance virtual cystoscopy and US was 93. It also allows visualization of areas that are difficult to access such as the bladder neck and mucosa within diverticulae. endoscopic intervention and intravesical chemotherapy on FDG-PET . making it a potential alternative for those unable to tolerate conventional cystoscopy. (c) corresponding T1 image.

Although there is evidence that FDG PET-CT has a diagnostic role for identifying metastatic bladder disease. Uptake was seen in all primary transitional cell carcinomas (mean SUV 7. Non-FDG tracers are not in widespread clinical use partly because of the lack of robust evidence supporting clinical benefit but also because of their cost and limited availability. PET/CT: receptor specific radiopharmaceuticals Imaging biomarkers using PET-CT and radioimmunotherapy opens the possibility of an individualized therapeutic and imaging approach. 11 C-Choline has also been used to detect residual disease after TURBT. or the HER2 gene is found in bladder cancers [72. significant urinary excretion occurs as compared to 11C-choline [67]. Therefore clinical use is restricted predominantly to those centers with an on-site cyclotron. 11C-choline PET was comparable to CT alone for detecting residual cancer after TURBT but appeared to be superior for detecting nodal involvement.92 [60]. 11C-choline was highly positive for primary and metastatic bladder cancer.2 SD). It therefore represents a potential target for both molecular imaging and therapy in those with known HER2positive disease [61]. Compared to 18-FDG PET in identifying primary tumors within the bladder. Malignant tumors have a high turnover of cellular membranes representing their increased proliferation rate [66]. hypoxia and growth factors are also under investigation [61. however. The sensitivity and specificity of 18-FDG PET or PET/CT for staging or restaging (metastatic lesions) of bladder cancer was 82% and 89% respectively.54. estimation of radiation dose distribution prior to therapy. 11C-Choline and 11C-methionine are not excreted in the urine and may have role in future imaging of bladder cancer [24. with a reported sensitivity of 78% in tumors greater than 1 cm but its value in local staging is not superior to conventional imaging [54]. 18 F-choline analogs with greater half-lives have been developed to overcome this. In a meta-analysis of the overall diagnostic accuracy of 18-FDG PET in bladder cancer. and angiogenesis. 90Y-rituximab (Zevalin®) to target the CD20 antigen on B cells for the treatment of lymphoma [70. however. It has been shown to have increased diagnostic accuracy as compared to technetium-99 m-methylene diphosphonate (99mTc-MDP) planar or single photon emission computed tomography (SPECT) in other solid tumors [68. When imaging is performed after chemotherapy the sensitivity decreases to 50% and therefore 18FDG PET results should be interpreted with caution following systemic treatment [59]. The global measure of accuracy was 0. trastuzumab labeled with 18 F.71]. There is however limited data at present to support routine clinical use. . The selective accumulation within the target tissue can then be visualized on PET. These imaging techniques have the potential to permit an ‘image and t reat approach’ by allowing tumor staging.4% for contrastenhanced CT alone [65].interpretation within the bladder. This represents a disadvantage for pelvic imaging as previously discussed unless adequate urinary washout can be encouraged. Choline is an essential component of cell membranes. 18  F Fluoride is a bone-seeking radiopharmaceutical that accumulates at sites of increased bone formation reflecting increased osteoblastic activity occurring within metastases. In the preoperative staging of 18 patients. 11 C-Methionine is a radiolabeled amino acid and is a potential tracer for visualizing protein metabolism.3 ± 3. three studies were retrospective in nature and only two studies assessed detection of the primary tumor. and early monitoring of treatment response [70]. The rationale is that a tumor specific target is combined with a therapeutic radioactive agent. of those. In a prospective study of 27 patients prior to radical surgery. Overexpression and amplification of epidermal growth factor receptor (EGFR) (HER1 or ErdB1) and.77]. it provides important contributory information when CT or MRI alone raises uncertainty regarding staging. uptake is proportional to tumor stage.5% and 100% versus 50% and 68. Monoclonal antibodies for example. allows in vivo monitoring of HER2 expression by PET as well as assessing change in HER2 expression with therapy [74.62]. Commonly used nuclides in other tumors types include β emitters such as 131I and 90Y. apoptosis.63-65]. 11 C-Choline has a short half-life of approximately 20 minutes.76. Alternative radiotracers that are dependent on cell proliferation. In certain circumstances. 6 studies involving 203 patients were assessed. In six patients.75]. with reported sensitivity and specificity of 62. The normal bladder has low uptake with 11C-choline [63]. Trastuzumab has also been labeled with nucleotides suitable for therapy with the future possibility of treating metastatic disease and improving the outcome of those with HER2 bladder cancer[70.73]. cellular proliferation and amino acid transport. four proceeded to surgery and three had pathological conformation of nodal disease [64]. They are attached to somatostatin receptor binding agents such as 90Y-DOTA-d-Phe(1)-Tyr(3)-octreotide (90Y-DOTATOC) for the treatment of neuroendocrine tumors and to antibodies in 131I-tositumomab (Bexxar®). The limitations accepted by the authors include variation in the imaging technique used. in our clinical practice it is not used as principal staging modality because of the limitations discussed above. 11C-choline uptake was seen in lymph nodes as small as 5 mm.69]. one study used PET alone which meant anatomical accuracy because of the poor spatial resolution was lost.

A summary of imaging modalities and their current clinical role in staging known MIBC is presented in Table 1 and Figure 2.asp#site. where a two-dimensional US. available athttp://www. Table webcite andhttp://www. webcite Patient perspectives Image acquisition time and tolerability of any proposed scan is important. In addition to allergy. in view of the evidence supporting MRI (as discussed above) our preferred practice is to stage local disease using this modality. Use of MRI and US are free from ionizing radiation as compared to CT and PET/CT. In the future we anticipate it will be routine to tailor a patient’s treatment plan to both the physiological and p hysical characteristics of their disease. CT and MRI remain central to bladder cancer staging. and additionally from the patient’s perspective scanning time is significantly longer. Authors’ contributions . PET/CT is associated with exposure of 14 mSv (PET component 8 mSv. *Diagnostic investigations for haematuria differ from imaging to determine extent of local and distant disease in confirmed muscle invasive bladder cancer. to monitor effectiveness of the intervention allowing a more dynamic approach to treatment. This means there is loss of temporal correlation.uroweb. Conclusions Accurate staging is important in determining prognostic information and identifying appropriate treatment options.PET/MRI Image acquisition with PET/CT occurs sequentially rather than simultaneously. Anticipated clinical pathway for staging of confirmed muscle invasive bladder cancer. The role of PET-CT using current tracers in staging and guiding management of bladder cancer remains to be defined. high-grade NMIBC or suspicion of an upper tract lesion. Technology is rapidly changing so the ability to use this information to identify the most effective intervention for patients is critical. The possibility of assessing different fun ctional parameters using PET. The clinical significance of these values in terms of inducing second malignancy is small in the context of the overall poor prognosis from muscle-invasive and metastatic bladder cancer at present. At our institution a 64-slice multidetector CT scan of the chest. 6 mSv from a rapid image acquisition CT). Most patients in the UK will present to urology teams via a ‘one-stop hematuria clinic’. This can impact on patient compliance and predispose results to motion artifacts.nccn. On the basis of these results a TURBT will be performed if intravenous contrast is also omitted for CT scanning in the presence of significant renal impairment. A summary of imaging modalities and their current clinical role(s) in staging known muscle-invasive bladder cancer Figure 2. CT image acquisition is usually within minutes but some research MRI protocols may take up to 1 h. Competing interests The authors declare that they have no competing interests. Future developments in functional imaging are likely to be important in predicting treatment response allowing timely identification of non-responders to guide appropriate change in treatment but further studies are required to determine which techniques or combination of techniques will optimize patient care. However. urine cytology and flexible cystoscopy will be carried out. abdomen and pelvis is associated with radiation exposure of 16 mSv during imaging. **Based on European Association of Urology guidelines 2012. DW MRI and combining that data with high-resolution anatomical information from MRI may provides new opportunities to study pathological and biochemical processes in vivo[78-81]. A CT scan will be undertaken where there is evidence of MIBC. MRI scanning also does not require the use of iodine contrast agents that can induce reactions potentially anaphylactic in some individuals.

SH drafted the manuscript. SH and RH .