Veterinary Immunology and Immunopathology 180 (2016) 15–20

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Veterinary Immunology and Immunopathology

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Short communication

Comparison of cytokine responses between dogs with sepsis and dogs

with immune-mediated hemolytic anemia
Valerie Johnson a,∗ , Brandy Burgess b , Paul Morley b , Ryan Bragg b,1 , Anne Avery a ,
Steven Dow a,b,∗
a
Department of Microbiology, Immunology and Pathology, Colorado State University, Ft. Collins, CO, United States
b
Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft. Collins, CO, United States

a r t i c l e i n f o a b s t r a c t

Article history: Cytokine abnormalities have been described previously in dogs with varied immune mediated and inflam-
Received 12 January 2016 matory conditions such as IMHA and sepsis. The purpose of this study was to establish references ranges
Received in revised form 16 August 2016 for cytokine levels in dogs and to compare cytokine levels in normal dogs and dogs with two common
Accepted 16 August 2016
inflammatory diseases (sepsis and IMHA). We hypothesized that cytokine response profiles in dogs with
sepsis would be significantly different from those in dogs with IMHA due to the very different etiologies
Keywords:
of the two diseases.
Infection
Concentrations of 14 different cytokines in serum were measured and values grouped according to
Chemokine
Host response cytokine function. Serum from clinically normal dogs was used to establish cytokine concentration ref-
Bacteria erence ranges. Rank values for each of the 4 cytokine groups were then compared statistically between
Autoimmunity healthy control, septic and IMHA dogs.
This analysis revealed differences in cytokine groups between dogs with sepsis and IMHA when
compared to healthy control dogs but no difference between dogs with either of these conditions. In
conclustion, dogs in the early stage of sepsis and IMHA have similar circulating cytokines despite differ-
ent etiologies suggesting activation of common immunologic pathways. This may have implications for
immunotherapy of immune mediated diseases in dogs of varying etiology.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction (Andaluz-Ojeda et al., 2012) (DeClue et al., 2012) (Karlsson et al.,

The immunopathogenesis of sepsis, systemic inflammatory
response syndrome (SIRS), and immune mediated hemolytic ane-
mia (IMHA) in dogs is thought to resemble that of the analogous
diseases in humans. For example, cytokine dysregulation is known
to be important to the immune dysfunction characterized by sep-
sis and SIRS in both dogs and humans. (Loisa et al., 2003) (Ulloa
and Tracey, 2005) (Bozza et al., 2007) (Duffy et al., 2010; Gebhardt
et al., 2009) (Wu et al., 2009) (Song et al., 2012) (Yu et al., 2012)
Abbreviations: SIRS, systemic inflammatory response syndrome; MODS, mul-
tiple organ dysfunction syndrome; IMHA, immune mediated hemolytic anemia;
LPS, lipopolysaccharide; IL, interleukin; TNF␣, tumor necrosis factor alpha; TGF␤,
transforming growth factor beta; IFN␥, interferon gamma; MCP-1, monocyte
chemoattractant protein 1; GM-CSF, granulocyte monocyte colony stimulating fac-
tor.
∗ Corresponding author at: Department of Microbiology, Immunology and Pathol-
ogy, Colorado State University, Ft. Collins, CO, United States.
E-mail address: valerie.johnson@colostate.edu (S. Dow).
1
Permanent address: Blue Pearl Veterinary Partners, Overland Park, KS.
2013) (Floras et al., 2014) Cytokine dysregulation has been doc-
umented in IMHA in humans, and similar cytokine abnormalities
were reported recently in dogs with IMHA. (Barcellini et al., 2000)
(Fagiolo and Toriani-Terenzi, 2002) (Kjelgaard-Hansen et al., 2011;
Wu et al., 2009) (Xu et al., 2012)
Immune mediated hemolytic anemia (IMHA) is a devastating
disease of dogs characterized by an auto-inflammatory disease pro-
cess leading to immune-mediated destruction of erythrocytes by
anti-erythrocyte antibodies. (Morley et al., 2008) (Harkin et al.,
2012) This disease carries a guarded prognosis in dogs and high
mortality rates are reported. (Balch and Mackin, 2007b; Carr et al.,
2002; Goggs et al., 2015; Swann and Skelly, 2013; Weinkle et al.,
2005) Various underlying etiologies have been proposed including
vaccination, drugs, viral or bacterial infection and neoplastic pro-
cesses, although there is limited evidence that any of events are
causative (Balch and Mackin, 2007a).
IMHA in dogs differs clinically from the analogous disease in
humans, autoimmune hemolytic anemia (AIHA). For example, AIHA
in humans is a rare disease and most cases are secondary to other
diseases such as lymphoproliferative disorders or other autoim-

http://dx.doi.org/10.1016/j.vetimm.2016.08.010
0165-2427/© 2016 Elsevier B.V. All rights reserved.
16 V. Johnson et al. / Veterinary Immunology and Immunopathology 180 (2016) 15–20
mune diseases. (Gehrs and Friedberg, 2002; Semple and Freedman,
2005; Valent and Lechner, 2008) The disease is often chronic with
patients experiencing nonspecific symptoms which can increase
and decrease in intensity. In pediatric patients, AIHA is most often
associated with preceding viral illness and subsequent mortality
rates are very low. (Aladjidi et al., 2011; Gehrs and Friedberg, 2002)
(Teachy, 2013) (Fan et al., 2015; Vagace et al., 2014) Experiments
in animal models suggest that inflammation secondary to infection
or other causes, may play a more significant role in stimulation of
AIHA than is currently recognized. (Murakami et al., 1997).
The pathogenesis of sepsis in dogs appears to parallel the disease
process in humans (Otto, 2007) with similar reported mortality
rates in dogs. (de Laforcade et al., 2003) (Conti-Patara et al., 2012;
Greiner et al., 2008; Keir and Kellum, 2015) (Keir and Dickinson,
2015) Sepsis is defined as an infection accompanied by a sys-
temic inflammatory response and as such it encompasses a wide
range of diseases and physiologic responses. Evaluation of cytokine
profiles in people with sepsis has demonstrated an initial exu-
berant pro-inflammatory response followed by a compensatory
anti-inflammatory response which results in immune suppression.
Evaluation of cytokines in dogs with sepsis has demonstrated sim-
ilar trends. (Duffy et al., 2010; Floras et al., 2014; Gebhardt et al.,
2009; Otto, 2007; Song et al., 2012) (Yu et al., 2012).
Few studies have documented the cytokine abnormalities
present in dogs with IMHA or dogs with sepsis. (Duffy et al., 2010;
Gebhardt et al., 2009; Rau et al., 2007; Yu et al., 2012) (DeClue
et al., 2012; Song and Wang, 2011) (Karlsson et al., 2013) (Song
et al., 2012) Documentation of cytokine abnormalities in dogs is
further complicated by the lack of published normal ranges for
cytokine concentrations in healthy animals. Therefore, we con-
ducted a cross-sectional study to establish a normal range for
cytokine values in a population of normal dogs and to compare
the cytokine response profiles of dogs with sepsis and dogs with
primary IMHA. The study reported here was also done to charac-
terize cytokine profiles in dogs with sepsis and IMHA and compare
these two immune mediated disease processes.
2. Materials and methods
2.1. Study animals
Healthy control adult dogs were obtained from staff owned
dogs (n = 47) and dogs owned by veterinary students undergoing
heartworm testing (n = 15) at Colorado State University Veterinary
Teaching Hospital (CSU-VTH). Criteria for selection of healthy dogs
included the absence of known medical problems or prior seri-
ous illness in the past 6 months, normal physical examination, and
absence of treatment with any therapeutic medications. Consent
was obtained from owners prior to enlisting in the study. Com-
plete blood count and serum biochemical analysis was performed
on the 47 staff-owned dogs. Sixteen dogs were excluded from this
group based on history of illness, current medical therapy or CBC
or serum biochemical abnormalities.
The 15 healthy dogs participating in the heartworm study
received a complete blood count only. Two dogs were excluded
from this group based on abnormalities found on the complete
blood count. Dogs from the two groups of healthy dogs were ran-
domly assigned to be included in the group used to establish normal
cytokine reference ranges (n = 40) or to the group used for compar-
ison to dogs with IMHA and sepsis (n = 22).
Dogs with primary IMHA evaluated at the CSU-VTH were
enrolled in the study based on the presence of anemia (packed
cell volume PCV < 30%), and either a positive saline slide aggluti-
nation test or the presence of spherocytosis on a blood smear, or
both. Patients were excluded from the study if any underlying pro-
cess was identified that could be a potential cause of secondary
IMHA, including neoplasia, rickettsial infection, recent medication
or vaccination. This group initially consisted of 14 dogs, of which 4
were determined to be ineligible due to other concurrent disease
processes.
Dogs selected for inclusion in the bacterial sepsis group were
based on clinical evidence of a systemic inflammatory response
and documented evidence of bacterial infection. Diagnosis of sys-
temic inflammatory response syndrome (SIRS) was determined by
the presence of 3 out of 4 of the following criteria: heart rate> 120
beats per minute, respiratory rate >40 breaths per minute, tempera-
ture <100.4 ◦ F or >104 ◦ F, or white blood cell count >18,000 cells/␮l,
<5000 cells/␮l or >10% band neutrophils. (Brady and Otto, 2001)
Bacterial infection was confirmed with a positive bacterial culture
from a body cavity or an open wound or detection of the presence
of intracellular bacteria during cytologic evaluation of an effusion.
All of the 11 dogs initially evaluated for potential enrollment were
determined to be eligible for inclusion.
2.2. Laboratory analysis
Blood samples were obtained and centrifuged within 1 h of
collection. Serum was frozen at −80 ◦ C and cytokine analysis per-
formed on multiple samples simultaneously.
Multiplex cytokine analysis was performed using a canine
Luminex multiplex bead assay (Millipore). This assay has been
used previously to quantitate cytokine concentrations in dogs
(Kjelgaard-Hansen et al., 2011; Barthelemy et al., 2015). The 13
cytokines measured by this assay include IL-2, IL-4, IL-6, IL-
7, IL-8, IL-10, IL-15, IL-18, interferon inducible protein (IP)-10,
tumor necrosis factor (TNF)-␣, granulocyte macrophage colony-
stimulating factor (GM-CSF), keratinocyte chemoattractant (KC),
and monocyte chemotactic protein (MCP)-1. The assays were
performed according to the manufacturer’s instructions. IFN-␥
concentrations were not included in the results as the Luminex
multiplex assay was incapable of detecting canine IFN-␥ in serum
(data not shown). Serum concentrations of TGF-␤ were determined
using a commercial human TGF-␤ ELISA kit (R&D Systems), accord-
ing to manufacturer directions. Canine and human TGF-␤ have
been reported previously to be identical at the amino acid level.
(Prud’homme and Piccirillo, 2000).
Cytokine multiplex analysis was performed according to manu-
facturer’s instructions to generate a standard curve from provided
reagents of known concentrations for each of the 13 cytokines pro-
vided in the kit. Standard curves were generated using the cytokine
standards with serum matrix added to the standard wells. An addi-
tional standard curve was generated using the cytokine standards
with the addition of normal dog serum in place of the serum matrix
provided with the kit. These curves were compared for all cytokines
and were found to be approximately equivalent for all the 13
cytokines measured.
2.3. Statistical analysis
2.3.1. Determination of normal ranges
Healthy dogs (n = 62) were selected as described above for use in
this study. To derive normal ranges for each cytokine of interest, 40
of these normal dogs were randomly selected using a random num-
ber generator, reserving the remaining 22 dogs for comparison to
the study population of dogs with IMHA and sepsis. As the data was
not normally distributed, normal ranges were based on percentiles
with all values less than or equal to the 90th percentile being
deemed normal for the purposes of this study. (Table 1) The 90th
percentile represents the cut point which minimized misclassifying
normal dogs and minimally affected the classification of abnormal
 V. Johnson et al. / Veterinary Immunology and Immunopathology 180 (2016) 15–20 17

Table 1 (Fig. 1a–d). Individual cytokines were described for each com-
Cytokine normal ranges derived from 40 normal dogs.
parison group (healthy dogs, dogs with IMHA and dogs with
Cytokine pg/ml Reference Rangea Range among normal dogs sepsis Table 2), however individual cytokines were not compared
TNF ␣ 0 (0,0) between groups because we had insufficient power to do so after
GMCSF 0–331.9 (0,1645.2)
applying multiple comparison correction.
KC 0–855.1 (36.5,1631.6)
IP-10 0–1 (0,33.7)
In this study serum concentrations of 14 different cytokines
IL-2 0–362.6 (0,6641.2) were measured in healthy dogs and dogs with IMHA and dogs
IL-4 0 (0,1752.3) with sepsis. These cytokines were evaluated and grouped accord-
IL-6 0–133 (0,8588.8) ing to function. Cytokine panel 1 consisted of chemokines, MCP-1,
IL-7 0–507 (0,16809.8)
IL-8, KC and IP-10. Cytokine panel 2 included the pro-inflammatory
IL-8 0–3774.8 (0,5303.1)
IL-10 0–2063.7 (0,4680.9) cytokines IL-6 and IL-18. Cytokine panel 3 included the anti-
IL-15 0–578.2 (0,24294.4) inflammatory cytokines TGF␤ and IL-10. Cytokine panel 4 included
IL-18 0–637.6 (0,10570.1) the T-cell derived cytokines IL-2, IL-4, IL-7 and IL-15. Interferon ␥
MCP1 0–317.1 (0,953.3)
was not included in the analysis as it was not able to be read on
TGF 0–63195 (0,115770.0)
the canine multiplex analysis. When IFN ␥ standards supplied with
the kit were spiked into normal canine serum the cytokine became
undetectable. TNF ␣ standards were detectable in canine serum,
dogs (i.e. those with sepsis or immune mediated hemolytic anemia
however we did not detect TNF ␣ in any sample tested. GM-CSF
[IMHA]).
was not included in the panels as it did not fit into any of the panel
categories chosen and there was not a sufficient number of samples
2.3.2. Cytokine analysis to analyze each cytokine individually.
Cytokine measurements from three groups of patients (22 As expected, in dogs with either IMHA or sepsis cytokine
healthy, 10 with IMHA and 11 septic) were used in this analysis. concentrations in Panel 1 (chemokines, p < 0.0001), Panel 2 (pro-
Data were entered into a spreadsheet, validated, and explored using inflammatory cytokines p = 0.003), Panel 4 (T cell derived cytokines,
descriptive statistics. Cytokines were categorized into four panels p = 0.0004)) were elevated compared to cytokine concentrations
based on biological relevance: Panel 1 included the chemokines in healthy dogs. Surprisingly, cytokine values in Panel 3 (anti-
MCP-1, KC, IP-10 and interleukin (IL)-8; Panel 2 included the pro- inflammatory cytokines) were not significantly different between
inflammatory cytokines IL-6 and IL-18, while Panel 3 included the healthy dogs or dogs with sepsis or IMHA (p = 0.069). Even more
anti-inflammatory cytokines IL-10 and TGF-␤. Panel 4 included the unexpected, we found that cytokine profiles in all 4 Panels were
T-cell produced cytokines IL-2, IL-4, IL-7 and IL-15. A Panel Score statistically indistinguishable between dogs with IMHA and dogs
was derived for each normal dog (n = 22), for each dog with sep- with sepsis (p ≤ 0.003). (Table 3).
sis (n = 11), and for each dog with IMHA (n = 10). A Panel-Score There are several potential explanations for the nearly com-
was developed by assigning a “1” to each dog with an elevation in plete overlap in cytokine responses observed in dogs with two
cytokine concentration and a “0” to each dog with a normal concen- inflammatory diseases of dogs (IMHA and sepsis) with very differ-
tration, based on the previously derived normal ranges described ent etiologies. For one, very early in both disease process, similar
above. For each dog, these scores were added for each panel, immune responses pathways may be triggered. For example, if
thereby determining a Panel-Score for each of the 4 cytokine pan- early release of a cytokine such as TNF-␣ is a key initiating immune
els for each dog in the study. Overall differences in Panel-Scores response in both IMHA and sepsis, regardless of the inciting cause,
among groups (normal, sepsis, and IMHA) were determined using then the ensuing cytokine responses may be quite similar. Alterna-
a Kruskal-Wallis test. If there was an overall statistically signifi- tively, once the disease processes are well underway, the immune
cant difference, then pair-wise comparisons were performed using responses may converge on similar inflammatory and chemokine
a Wilcoxon-Mann-Whitney Rank Sum test. A P-value of ≤ 0.05 was response pathways. We were not able to detect TNF-␣ in any of
regarded as statistically significant. Statistical analysis was per- our samples. This may be because this cytokine is released so early
formed using SAS v9.3 software (SAS, Inc., Carey, NC). in the inflammatory process that it may have already peaked and
fallen by the time the dogs presented for treatment at our facility.
3. Results/Discussion Perhaps the biggest surprise was that T cell derived cytokine
responses were so similar in dogs with IMHA and sepsis, particu-
Eighty one dogs were included in this study; 62 healthy dogs, larly since IMHA is considered to be disease driven primarily by
10 dogs with IMHA and 11 dogs with sepsis. A wide range of breeds autoreactive T cells, while sepsis is considered a disease driven
and ages were represented with no statistical difference in ages by innate immune responses. (Brady and Otto, 2001; Fagiolo and
between groups. Toriani-Terenzi, 2002; Toriani-Terenzi and Fagiolo, 2005) Currently
Seventy-one percent of the dogs in the sepsis group (8 of 11 we can provide no convincing explanation for why T cell cytokine
dogs) died or were euthanized due to severity of disease. The responses were so similar. It is tempting to speculate that perhaps
remaining 3 dogs survived until discharge. Two of 10 dogs (20%) in IMHA in dogs is not driven by autoreactive T cells, but may instead
the IMHA group died or were euthanized prior to discharge. Eight represent an unusual manifestation of an infectious disease. For
dogs in the IMHA group survived until discharge. However, two example, there are unique features of IMHA in dogs (strong sea-
dogs with IMHA died or were euthanized within two weeks of dis- sonality, rapid disease onset, strong association with coagulation
charge due to complications associated with IMHA. All the dogs abnormalities, high mortality rate) that suggest an infectious pro-
with IMHA in this study were determined to have SIRS based on cess rather than pure autoimmunity, as noted by others. (Balch
the standard criteria noted in Materials and Methods. and Mackin, 2007a; Karagianni et al., 2012; Kidd et al., 2014) In
The normal reference range values for cytokines were based on contrast, autoimmune hemolytic anemia in humans is generally
the 10th and 90th percentile values for each cytokine. These val- considered a more chronic disease without strong systemic abnor-
ues were used for comparisons with dogs with IMHA and sepsis malities. (Gehrs and Friedberg, 2002) In dogs, if both sepsis and
(Table 1) IMHA are in fact triggered by an infectious pathogen, this might
Individual Cytokine panels (Panels 1–4) were compared in turn explain the similarities in cytokine responses in the two
between healthy dogs, dogs with IMHA and dogs with sepsis diseases.
18 V. Johnson et al. / Veterinary Immunology and Immunopathology 180 (2016) 15–20

A B
40
40
* *
* 30

A verag e R an k
30
A verag e R an k
*
20
20

10
10

0
0
Normal IMHA Sepsis
Normal IMHA Sepsis

C D

30
40

A vera ge R ank 30
*
*
A ve r a g e R an k

20
20

10
10

0 0
Normal IMHA Sepsis Normal IMHA Sepsis
Fig 1. Average rank of panel score comparison for cytokines grouped according to function into 4 groups: Panel 1: Pro-inflammatory cytokines IL-6 and IL-18 (A), Panel 2:
Chemokines IL-8, IP-10, MCP-1,KC (B); Panel 3: Anti-inflammatory cytokines TGB␤ and IL-10 (C); Panel 4: T cell derived cytokines IL-2, IL-4, IL-7 and IL-15 (D). (*p < 0.05).

Table 2
Cytokine profiles of normal, septic, and AIHA study dogs.

Panel Cytokinea Normal Group (n = 22) AIHA Group (n = 10) Sepsis Group (n = 11)

Median Range Median Range Median Range

Chemokines IL8 1340 (0, 3989) 7066 (0, 40000) 1719 (0, 11117)
IP10 0 (0, 22) 0 (0, 49) 0 (0, 66)
KC 206 (74, 353) 3320 (281, 7469) 2547 (76, 10326)
MCP1 0 (0, 554) 135 (74, 1087) 614 (0, 9903)
Pro-Inflammatory IL6 0 (0, 45) 0 (0, 103) 22 (0, 311)
IL18 42 (0, 286) 14 (0, 945) 0 (0, 144)
Anti-Inflammatory IL10 0 (0, 3989) 10 (0, 327) 0 (0, 3200)
TGF 41400 (0, 113430) 599 (39, 1533) 1104 (0, 2817)
T-cell Associated IL2 0 (0, 208) 0 (0, 663) 0 (0, 138)
IL4 0 (0, 0) 0 (0, 97) 0 (0, 2229)
IL7 6 (0, 217) 0 (0, 368) 0 (0, 26)
IL15 0 (0, 330) 0 (0, 965) 0 (0, 57)
a
All cytokine values in pg/ml.

Table 3
Comparison of cytokine panels.

Panels Cytokines evaluated Different from normal* Different between sepsis and IMHA*

1 Chemokines MCP-1, KC, IL-8, IP-10 Yes No

2 Pro-inflammatory IL-6, IL-18 Yes No
3 Anti-inflammatory TGF␤, IL-10 No No
4 T-cell derived IL-2, IL-4, IL-7,IL-15 Yes No
*
p < 0.05.
 V. Johnson et al. / Veterinary Immunology and Immunopathology 180 (2016) 15–20
There are also numerous clinical similarities between dogs
with primary IMHA and dogs with sepsis. These include signifi-
cant immune dysregulation, the presence of multiple coagulation
abnormalities, a rapid onset of severe clinical illness, fever, signif-
icant neutrophil and monocyte abnormalities, and high mortality
rates. While not conclusive, this convergence of clinical signs sug-
gests the possibility that the two diseases may both reflect a similar
pathogenesis. Thus, it cannot be fully excluded at present that
IMHA may represent an unusual manifestation of an acute systemic
inflammatory process triggered by an unknown pathogen.
Cytokine concentrations have been extensively examined in
human septic patients and in animal models, both as prognos-
tic indicators and as potential targets for immunotherapy. (Bozza
et al., 2007; Ulloa and Tracey, 2005) The recent development of
multiplex cytokine analysis enables simultaneous rapid determi-
nation of multiple cytokines on small samples of blood. (Khan et al.,
2004) It has been demonstrated in humans that different cytokine
profiles are present in patients with septic shock than in patients
with severe sepsis. (Wu et al., 2009) The ability to monitor serial
cytokine profiles in critically-ill dogs could lead to opportunities
for individualized immunotherapy as well as providing prognostic
information.
Anti-inflammatory cytokines have been demonstrated to play
a role in the pathogenesis of both immune mediated anemia and
sepsis in humans, with increased concentrations of IL-10 being
observed in both diseases. (Surbatovic et al., 2015; Toriani-Terenzi
and Fagiolo, 2005) Therefore, we had expected to detect increased
concentrations of IL-10 (and TGF-␤) in dogs with IMHA and dogs
with sepsis. However, significant differences in Panel 3 cytokines
were not detected between any of the 3 study groups (healthy,
IMHA, sepsis) in the study. This finding might be explained if serum
samples were collected for cytokine evaluation early in the disease
process, prior to the development of counter-regulatory immune
responses. Alternatively, dogs may mount a weaker immune
counter-regulatory response than other species, or responses dom-
inated by other cytokines (eg, IL-13, IL-1Ra) not measured in our
multiplex array.
A recent paper by Kjelgaard-Hansen et al. described cytokine
concentrations in dogs with IMHA and found concentrations
of MCP-1 and IL-18 to be associated with increased mortality
(Kjelgaard-Hansen et al., 2011). Their study also reported eleva-
tions in IL-2, IL-10, KC and MCP-1 in IMHA dogs when compared to
normal dogs. Our study also documented increased concentrations
in the panels that included IL-2, KC and MCP-1 in dogs with IMHA.
However, increased concentrations of IL-10 were not identified in
our study. These minor differences in study findings may reflect our
decision to compare groups of cytokines of similar function, rather
than to compare pair-wise all cytokines. Limitations to the cur-
rent study included small sample sizes, which precluded pair-wise
analysis of all cytokines. The chances of making a type I error when
making multiple pairwise comparisons with such a small sample
group are large, which would have increased the risk of report-
ing a difference that did not exist. Therefore, we elected to group
cytokines according to function. In addition, assumptions regarding
classification of cytokines into groups could bias the results of the
statistical analyses we performed. Many of these cytokines have
overlapping or multiple functions. For example IL-8 functions to
attract neutrophils and so could be considered an inflammatory
cytokine in addition to a chemokine. (Andaluz-Ojeda et al., 2012)
Therefore IL-8 could have been placed in either Panel 1 or Panel 2.
This could confound interpretation of cytokine panels with respect
to disease pathogenesis. The time of sample collection relative to
the onset of the disease process in IMHA and sepsis may also have
affected cytokine concentrations. However, for both dogs with sep-
sis and IMHA, samples were collected as soon as possible once the
disease was diagnosed, typically within 24 h of diagnosis.
19
To further elucidate the immunopathogenesis of IMHA, addi-
tional studies may be informative. For example, it would be
interesting to compare cytokine profiles of dogs with IMHA to
dogs with other immune mediated condition such as immune-
mediated thrombocytopenia or immune-mediated polyarthritis.
It would also be helpful to compare dogs with IMHA to a more
standardized group of dogs with sepsis, for example dogs with
pyometra or dogs with bacterial pneumonia. Finally, comparison
of dogs with IMHA to dogs with non-infectious systemic inflam-
mation would further complete the comparison of IMHA to other
pro-inflammatory conditions provoking cytokine abnormalities.
Conflicts of interest
None.
Acknowledgments
NIH T32 Fellowship Training Grant (VJ) Shipley Foundation
Grant (SD).
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