By Prof. Dr.

Mona El Samahy Head of The Diabetes Clinic Children’s Hospital, Ain Shams University

Diabetes Mellitus (DM)
• A group of metabolic diseases characterized by hyperglycemia, resulting from defects in insulin secretion, action or both.

• The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction and failure of various organs, especially the eyes, kidneys, nerves, heart and blood vessels. • It is the most common endocrine – metabolic disorder of childhood and adolescence.

Etiology and Pathogenesis

Pathogenesis of type 1 diabetes

The clinical and biological characteristics of different subtypes of type 1 diabetes
Type 1a
Signs of anti-islet autoimmunity Duration of symptoms Before diagnosis Ketosis, ketoacidosis at diagnosis Blood glucose levels at diagnosis + 8 months frequent ↑↑

Type 1b
7 months frequent ↑↑

Type 1c
< 1 week constant ↑↑↑

HbAlc at diagnosis

↑↑

↑↑

Normal or slightly elevated

The war on Diabetes Mellitus

The war on DM
• Starts by attempts to identify environmental determinants of DM in the young which focus to identify dietary, infectious, and other environmental triggers of type 1a DM in genetically at risk children.

• Identification of such triggers could lead to the study and implementation of primary prevention.

1- Primary Prevention
Through

Avoidance of dietary offender

Immunization against an infectious trigger

TRIGR STUDY
• The trial to reduce IDDM in the genetically at risk is ongoing. • Its focus is to determine whether delayed exposure to or avoidance of certain food proteins will reduce the risk of developing type 1 DM in infants at

Genetic Risk.

• Type 1a DM is the autoimmune form whose major genetic
risk traits are defined on certain HLA DR/DQ alleles.

• The genes mutation will not be correctable by avoidance of dietary or infectious agents.
• However, insights gained from these mutations may facilitate ultimate prevention strategies such as immunization with specific segments or epitopes of the insulin molecule to limit autoimmune self aggression.

The results of primary prevention trials are still unknown (June, 2008)

2- Secondary Prevention
• Diabetes Prevention Trial (DPT-1) for type 1 DM used Insulin as a possible means to suppress the immune response. • Other intervention studies (Blocking progression of autoimmunity) by: • CTLA-4Ig : block T-cell activation • Rituximab (anti-CD20) : block B-cell from presenting antigens • GAD vaccine : interfers with presenting islet cell antigens • Anti-CD3 : deletes activate T-cells

2- Secondary Prevention
Even if these studies identify one or several magic bullets for arresting or preventing the progression of DM, we would still be confronted with the problem of curing or reversing established disease.

Insulin has been the gold standard therapy for DM since its discovery. It remains the only pharmacologic therapy for type 1DM

Before Insulin….What’s After?
Before insulin was discovered in 1921, everyone with type 1 diabetes died within weeks to years of its onset
JL on 12/15/22 and 2 mos later

Insulin Therapy in Type 1 Diabetes

is a must………

Ideal Insulin Regimens Type 1 DM
The challenge is to Come as close as possible to normoglycemia and reduce hypoglycemia.

Minimizes nocturnal hypos

Optimize FBS

Minimizes late morning and afternoon hypos

Basal-Bolus Regimen

Although molecularly produced insulin administered 4 or 5 times per day can provide a physiologic regulation, yet it is not able to prevent diabetic complications that account for the morbidity and mortality of diabetic patients.

Also insulin does not eliminate the type1 DM hallmark : BETA- CELL SPECIFIC AUTOIMMUNITY.

In other words

Insulin is not a cure

A successful cure must meet the following criteria:
1. Either replace or maintain the functional integrity of the natural insulinproducing tissue. 2. Must at least control the autoimmunity or eliminate it altogether. 3. Easy to apply to a large number of patients

Criterion 1
Has been partially realized by allogenic islet transplantation.

Development of Insulin-Secreting Pancreatic-Like Cells From Mouse Embryonic Stem Cells.

Criterion 2 Has been partially realized using monoclonal antibodies specific for T-cell surface proteins.

Criterion 3 Has yet to be realized

New Approach to
(fulfill the 3 criteria)

A non-insulin based therapy, With a focus on Cell-Based Immunomodulation

Hoping for cure

All proposals of treatment in the past and recently are depending on discoveries in understanding the immune pathophysiology of destruction of pancreatic β cells that results in DM.

Nature of autoimmunity in type 1a DM
• The major affectors of β-cell destruction are T cells reactive to β-cell-specific antigens. • A strong genetic predisposition is the key for genetic susceptibility loci that affect genesis, function and survival of immune cell subsets including T cells and dendritic cells.

Type 1 Diabetes as immune destruction of beta cells
T-cell T-cell

Inflammatory cell

APC
(IL1, TNF, NO)

Fas Cytotoxic T-cell
Cytokines Th1 (IL2, IFN)

Beta cell antigens

Beta cell
Beta cell death

B-cell
Antibodies (ICA, IAA, IA2, GAD65) Type 1 diabetes

The epitope spreading phenomenon i.e. expansion of newly recognized antigens observed in the islet inflammation is due to:

Islet-reactive T cells that were generated in the thymus early in ontogeny

Generation and survival of T cells activated in the periphery by these new antigens

There is a pathologic vicious circle of continuous presentation of old and new antigens, collected by the dendritic cells (DC) from the newly destroyed β-cells to naive T cells in the pancreatic lymph nodes  eventually go back to the pancreas to kill other β-cells.

This vicious circle does not allow the recovery of the insulin secreting cells, even when the physiologic homeostasis process tries to substitute the lost cells with new cells.

Therefore, an approach developed to stop autoimmunity in already diabetic patients, could possibly facilitate the recovery of autologous insulin production. So, safe induction of an autoimmunity – free status might became a new promising therapy for type 1a DM.

In the past 20 years, researches have made many promises to cure type 1 DM. Only recently it has been possible to clinically implement a limited number of successes.

Autologous Bone Marrow Transplantation.
• Autologous transplantation of hematopoietic stem cell enriched BM was used to treat type 1 DM. • The effects were limited to simple postponement of DM recurrence, i.e.: just delayed by the time necessary for the transplanted BM to reorganize itself and to reestablish all of its immunocompetent cell subpopulations.

Autologous Bone Marrow Transplantation.
• The autologous BM did not change the patient's genetic characteristics under which tolerance for the insulin – producing beta cells was not achieved in the first place.

So, Autoimmunity easily recurred.

BMT can encourage beta cell regeneration
Regeneration of beta cells in diabetic NOD mice A) Infiltration of T cells destroys beta cells B) After BMT infusion, signs of autoimmune destruction diminish C) Insulin-producing cells start to appear (red) D) 4 mo after BMT infusion, see proliferation of insulin-producing cells (red)

Another approach was developed to modify the genetic characteristics of a patient BM-derived stem cells in vitro which will result in T-cells or (APC) that cannot initiate an autoimmune response.
(maybe transferable to clinical trials in the near future)

This was achieved by: • Transduction of BM-derived stem cells with
protective (non diabetogenic) HLA transgenes.
• Infusion of the engineered cells will culminate in repopulation of the thymus with protective HLA-expressing APCs. • This in turn will decrease the probability that T – cells with diabetogenic T cell receptors mature and exit into the periphery.

• It is postulated that the Rest period from the autoimmune destructive process permits regeneration of endogenous β cells from pancreatic stem cells within the pancreatic ducts or islets. • The suppression of the immune environment by several means was conclusively shown to be the mechanism that resulted in recovery from type 1a DM in NOD mouse.

Two novel recent mechanisms involving cell based immunomodulation aimed at producing a cure for type 1a DM :
1. Diabetes-suppressive autologous DC.

2. Diabetes-suppressive microsphere vaccine

1. Diabetes-suppressive autologous DC.
The first clinically adapted immunoregulatory cell therapeutic

Why dendritic cells?
• DC are the body's sentinels largely responsible for host surveillance against micro-environmental anomalies including pathogen invasion, infection, and damaged tissue architecture, while coordinating the mechanisms of self tolerance.

Why dendritic cells?
• DC continously traffic throughout all body tissues sampling molecules from their surroundings, where it is believed they maintain potentially autoreactive immune cells in quiescence either directly or via indirect regulatory immune cell networks.

When DC encounter local disruption of tissue architecture & elevated proinflammatory signal from infected cells:
Undergo maturation through a series of internal changes. Migrate away from the site of danger and into the closest LNs.

Within the LNs, the DC, as a powerful APC, initially interacts using its class I or class II MHC / peptide complex with the TCR present on a naïve T cell. This will constitute the so-called first signal for T-cell activation.

• To

bring a T cell to full activation, a subsequent contact between receptors on both APC & T cell (Co receptors) is necessary.

• Co-stimulatory molecules present on the APC with their counterparts on the T cell interact to further stabilize the signal of activation between the two cells, thus providing the second signal.

• Absence of this co-stimulatory molecule binding
and consequently lack of secondary signal generation has been shown to lead to impaired activation of the responding T cell, eventually bring it to functional anergy or apoptosis. • This is indeed the outcome of many immunosuppressive strategies aimed at costimulation blockade.

• Many investigations support the concept that functionally immature DC (characterized by low to absent co-stimulation) lead to Immune hyporesponsiveness.
• Exogenous administration of functionally immature DC achieves long term and stable allograft survival in a variety of mouse and rat models and prevents a number of autoimmune diseases.

Recently

In vitro

administration of nuclear factor-kappa β (NFκB) decays to DC as well as direct targeting of CD40, CD80 and CD86 with antisense oligodeoxyribonucleotides (AS-ODN) reduces co-stimulatory molecule levels producing functionally immature DC capable of preventing or reversing new-onset DM in the NOD mouse.

• Numerous clinical trials have safely used DC-based treatments for cancer therapy providing the basis for clinical adaptation of DC administration for Type 1aDM ttt.

• A National institutes of Health funded protocol approved by FDA is currently underway phase I clinical trial with an adult (18 yr or older) cohort documented with Type 1aDM of at least 15-yr duration. • Leukocytes are obtained from the patient by apheresis and DC are generated in vitro and engineered in Good manufacturing practice facilities with the addition of AS-ODN.

• These DC which express low levels of CD40, CD80 and CD86 are injected into the patient by ID route at an anatomical site proximal to the pancreas.
• DC will migrate to the nearest LNs where they will start to interrupt the vicious circle that maintains isletspecific inflammation i.e., insulitis. • This therapeutic approach should be more successful when DC injections start close to the clinical onset of DM.

IN THE PANCREAS
• DC acquire β-cell specific antigens from apoptotic cells, leading to the eventual display of these antigens to T cells in the pancreasdraining LNs.

• The lack of co-stimulatory molecules will result in anergizing signal to the T cells, and interrupt the T cell mediated anti-beta-cell epitope spreading phenomenon.

• The cessation of the autoimmune diabetogenic insult should be sufficient to:
 Promote rescue of still present insulin producing β cells and/or  Promote neogenesis of other insulin producing cells in the pancreas, even after the disease onset. • This trial is underway at (6/2008) & once safety has been demonstrated, a phase II efficacy trial will start, involving new onset diabetic patients.

2. Diabetes Suppressive microsphere vaccine

Diabetes Suppressive microsphere vaccine
• In spite of the promise of the previous study, many cumbersome logistical requirements to generate these diabetes-suppressive DC have been encountered.
• This may limit the future enrollment of new onset diabetic children in the efficacy phase of the trial.

Difficulties:
• Leukopheresis takes 2 or 3 hours to provide sufficient precursors cells to generate the number of DC necessary for six to eight injections.
• The obtained DC should be exposed to AS-ODN in GMP facilities in which the laboratory practices are frequently difficult to reproduce. GMP facilities are frequently located far away from the clinic where the patients are treated. • Many DC are lost during the freezing/thawing procedures.

• In an effort to avoid these steps, an alternative method to stabilize DC immaturity directly in vivo using microparticle carriers of immunomodulating agents like AS-ODN (microsphere delivery system) have been done.

• This microparticle carriers (microsphere delivery system) is directed against 3 key costimulatory molecules: CD40 CD80 CD86

Essential for normal immune response

• When injected in vivo, these microspheres attract DC, which phagocytose the microsphere particles  the microsphere particles are deformulated inside the DC  AS-ODN release  block the expression of the genes to which they are targeted.

• This proof technique reverse or prevent type 1aDM in mice

• This microsphere delivery system in which AS-ODN have been incorporated, is named Baxter Healthcare's PROMAXX®. • The inert PROMAXX microsphere technology has been shown to be safe and effective in human trials.

• More importantly, when administered in vivo, this technology is neutral with respect to DC maturation state compared with the known immunostimulatory properties of other microsphere formulations.

• Other polyplex formulations have an inherent capacity to induce the upregulation of costimulatory proteins at the DC surface, whereas the PROMAXX technology does not. • This neutrality on DC maturation is a critical criterion in adopting microsphere chemistry for immunosuppressive objectives where DC are involved as mediators.

• They predict that once all preclinical studies are completed, a phase I/II trial can be initiated.
• The microspheres are simple to manufacture to clinical grade on a large scale and do not involve the cumbersome logistics for DC based therapy.

This novel microsphere formulation represents the first diabetes suppressive and reversing nucleic acid vaccine that confers an immunoregulatory phenotype to endogenous DC.

• How long is a single course of microsphere particles effective?

• Does response wane with repeated injection?

These questions remain to be answered. But, if it's effective NEW ERA in treating type 1 DM will appear.

The light at the end of the tunnel for

Diabetes Cure

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