Seronegative Healthy Controls Research Articles

Evaluation of Host Constitutive and Ex Vivo Coccidioidal Antigen-Stimulated Immune Response in Dogs with Naturally Acquired Coccidioidomycosis.

The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105-310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations (p = 0.02) and serum CRP concentrations compared to controls (p < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination (p = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α (p = 0.0003), interleukin (IL)-6 (p = 0.04), interferon (IFN)-γ (p = 0.03), monocyte chemoattractant protein (MCP)-1 (p = 0.02), IL-10 (p = 0.02), and lower IL-8 (p = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis.

Herpes simplex virus 1 infection on grey matter and general intelligence in severe mental illness

Schizophrenia and bipolar disorder are severe mental illnesses (SMI) linked to both genetic and environmental factors. Herpes simplex virus 1 (HSV1) is a common neurotropic pathogen which after the primary infection establishes latency with periodic reactivations. We hypothesized that the latent HSV1 infection is associated with brain structural abnormalities and cognitive impairment, especially in SMI. We included 420 adult patients with SMI (schizophrenia or bipolar spectrum) and 481 healthy controls. Circulating HSV1 immunoglobulin G concentrations were measured with immunoassays. We measured the total grey matter volume (TGMV), cortical, subcortical, cerebellar and regional cortical volumes based on T1-weighted MRI scans processed in FreeSurfer v6.0.0. Intelligence quotient (IQ) was assessed with the Wechsler Abbreviated Scale of Intelligence. Seropositive patients had significantly smaller TGMV than seronegative patients (642 cm3 and 654 cm3, respectively; p = 0.019) and lower IQ (104 and 107, respectively; p = 0.018). No TGMV or IQ differences were found between seropositive and seronegative healthy controls. Post-hoc analysis showed that (a) in both schizophrenia and bipolar spectrum, seropositive patients had similarly smaller TGMV than seronegative patients, whereas the HSV1-IQ association was driven by the schizophrenia spectrum group, and (b) among all patients, seropositivity was associated with smaller total cortical (p = 0.016), but not subcortical or cerebellar grey matter volumes, and with smaller left caudal middle frontal, precentral, lingual, middle temporal and banks of superior temporal sulcus regional cortical grey matter volumes. The results of this cross-sectional study indicate that HSV1 may be an environmental factor associated with brain structural abnormalities and cognitive impairment in SMI.

Endothelial dysfunction and altered endothelial biomarkers in patients with post-COVID-19 syndrome and chronic fatigue syndrome (ME/CFS)

BackgroundFatigue, exertion intolerance and post-exertional malaise are among the most frequent symptoms of Post-COVID Syndrome (PCS), with a subset of patients fulfilling criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). As SARS-CoV-2 infects endothelial cells, causing endotheliitis and damaging the endothelium, we investigated endothelial dysfunction (ED) and endothelial biomarkers in patients with PCS.MethodsWe studied the endothelial function in 30 PCS patients with persistent fatigue and exertion intolerance as well as in 15 age- and sex matched seronegative healthy controls (HCs). 14 patients fulfilled the diagnostic criteria for ME/CFS. The other patients were considered to have PCS. Peripheral endothelial function was assessed by the reactive hyperaemia index (RHI) using peripheral arterial tonometry (PAT) in patients and HCs. In a larger cohort of patients and HCs, including post-COVID reconvalescents (PCHCs), Endothelin-1 (ET-1), Angiopoietin-2 (Ang-2), Endocan (ESM-1), IL-8, Angiotensin-Converting Enzyme (ACE) and ACE2 were analysed as endothelial biomarkers.ResultsFive of the 14 post-COVID ME/CFS patients and five of the 16 PCS patients showed ED defined by a diminished RHI (< 1.67), but none of HCs exhibited this finding. A paradoxical positive correlation of RHI with age, blood pressure and BMI was found in PCS but not ME/CFS patients. The ET-1 concentration was significantly elevated in both ME/CFS and PCS patients compared to HCs and PCHCs. The serum Ang-2 concentration was lower in both PCS patients and PCHCs compared to HCs.ConclusionA subset of PCS patients display evidence for ED shown by a diminished RHI and altered endothelial biomarkers. Different associations of the RHI with clinical parameters as well as varying biomarker profiles may suggest distinct pathomechanisms among patient subgroups.

Factors associated with subclinical atherosclerosis in HIV infected patients from northeast of Brazil

Introduction: AIDS has changed its morbidity curve, rising cardiovascular diseases. HIV-infected patients have increased cardiovascular event rates but data on the prevalence of subclinical atherosclerosis are not uniform. Methods: HIV-infected patients underwent to coronary tomography for CACs assessement. We performed a comparison between 97 HIV-infected patients and 129 seronegative healthy controls. The univariable analysis matched the association of HIV infection, cardiovascular risk profile, and HIV-related factors with subclinical atherosclerosis. Results: HIV-infected patients with CACs above zero were older (54.8±7.0 vs. 43.3.5±11.0 years; p&lt;0.001) and more likely to have hypertension (36.7% vs. 12.5%; p=0.07) than HIV(-) CACs zero ones. Factors associated with altered CACs in unadjusted hazard ratio were age (HR=1.13; 95%CI=1.07-1.20; p&lt;0.0001) and hypertension (HR=4.05; 95%CI=1.42-11.60; p=0.0009). When adjusted hazard ratio was constructed age, male gender and protease inhibitors (PI) use appeared as factors associated with coronary calcification. HIV-infected patients were less likely to have hypertension (20.2% vs 50.4%; p&lt;0.001) and diabetes (5.3% vs 23.3%; p&lt;0.001) than HIV uninfected ones. Conversely, both groups have same CACs level. Among HIV-infected patients altered CACs was 30.9%, vs 42.3% among control. Most of HIV-infected patients showed undetectable viremia and high CD4+ count, in parallel with lipid profile disturbances. Conclusion: Increased CAC incidence was associated with age, male gender and PI use among HIV-infected patients. Despite younger, fewer traditional risk factors and with controlled disease, the PLHIV had similar CAC scores compared with controls. Besides viruses itself, antiretroviral drugs play a role, mainly because control viruses at expense of worsening in lipid profile.

Circulating NKG2C + NK cell expressing CD107a/LAMP-1 subsets at the onset of CMV reactivation in seropositive kidney transplant recipients

Cytomegalovirus (CMV) infection contributes to morbidity and mortality among kidney transplant recipients. Natural killer (NK) cells can battle against CMV in kidney transplant recipients (KTRs). This study aimed to analyze the association between CMV reactivation and the proportion of NK cell subsets and their activity. In a cross-sectional study, ten CMV reactivated KTRs, and ten non- CMV reactivated ones were recruited. Ten matched healthy controls were also included in this cohort. The presence of anti-CMV-IgG Ab in both KTR subgroups from seronegative donors and healthy controls was determined. The frequency of distinct subsets of memory-like NK cells was analyzed through NKG2C, NKG2A, and CD57 using flow cytometry. The activity of NK cells was evaluated after stimulation via coculture with K562 cell line and then assessment of the frequency of CD107a and granzyme B. The mRNA levels of transcription factors, including T-bet, EAT, and inflammatory proteins, including IFN-γ and perforin contributing to NK cell activation, were also evaluated.Results showed a significantly lower frequency of NKG2C + NKG2A-CD57+ NK cell population in CMV-reactivated KTRs compared to non-reactivated ones (P-value:0.003). NKG2C+ NK cells expressing CD107a/LAMP-1 significantly was increased in CMV-reactivated KTRs compared to non-reactivated ones (P-value: 0.0002). The mRNA level of IFN-γ had a significant increase in the CMV-reactivated KTRs vs. nonreactive ones (P-value: 0.004). Finally, evaluation of the NK cells' cytotoxicity and activity through assessment of CD107a/LAMP-1 expression and IFN-γ secretion may be helpful for the identification of the risk of CMV reactivation in KTRs.

T cell and antibody responses to SARS-CoV-2: Experience from a French transplantation and hemodialysis center during the COVID-19 pandemic.

Immunosuppressed organ‐transplanted patients are considered at risk for severe forms of COVID‐19. Moreover, exaggerated innate and adaptive immune responses might be involved in severe progression of the disease. However, no data on the immune response to SARS‐CoV‐2 in transplanted patients are currently available. Here, we report the first assessment of antibody and T cell responses to SARS‐CoV‐2 in 11 kidney‐transplanted patients recovered from RT‐PCR–confirmed (n = 5) or initially suspected (n = 6) COVID‐19. After reduction of immunosuppressive therapy, RT‐PCR–confirmed COVID‐19 transplant patients were able to mount vigorous antiviral T cell and antibody responses, as efficiently as two nontherapeutically immunosuppressed COVID‐19 patients on hemodialysis. By contrast, six RT‐PCR–negative patients displayed no antibody response. Among them, three showed very low numbers of SARS‐CoV‐2–reactive T cells, whereas no T cell response was detected in the other three, potentially ruling out COVID‐19 diagnosis. Low levels of T cell reactivity to SARS‐CoV‐2 were also detected in seronegative healthy controls without known exposure to the virus. These results suggest that during COVID‐19, monitoring both T cell and serological immunity might be helpful for the differential diagnosis of COVID‐19 but are also needed to evaluate a potential role of antiviral T cells in the development of severe forms of the disease.

Frequent Recurrences of Genital Herpes Are Associated with Enhanced Systemic HSV-Specific T Cell Response.

Objectives Genital herpes simplex virus (HSV) infection is controlled by HSV-specific T cells in the genital tract, and the role of systemic T cell responses is not fully understood. Thus, we analysed T cell responses in patients with recurrent genital herpes (GH). Methods T cell responses to HSV-1 and HSV-2 native antigens and the expression of HLA-DR and CD38 molecules on circulating CD8+ T cells were analysed in adults with high frequency of GH recurrences (19 patients) and low frequency of GH recurrences (7 patients) and 12 HSV-2 seronegative healthy controls. The study utilized the interferon-γ Elispot assay for measurement of spot-forming cells (SFC) after ex vivo stimulation with HSV antigens and flow cytometry for analysis of the expression of activation markers in unstimulated T cells. Results The patients with high frequency of GH recurrences (mean number of recurrences of 13.3 per year) had significantly enhanced HSV-specific T cell responses than the HSV-2 seronegative healthy controls. Moreover, a trend of higher numbers of SFC was observed in these patients when compared with those with low frequency of GH recurrences (mean number of recurrences of 3.3 per year). Additionally, no differences in CD38 and HLA-DR expression on circulating CD8+ T cells were found among the study groups. Conclusions Frequency of GH recurrences positively correlates with high numbers of systemic HSV-specific T cells.

Annexin-A5 resistance and non-criteria antibodies for the diagnosis of seronegative antiphospholipid syndrome.

In this study, we aimed to analyze the value of annexin-A5 anticoagulant ratio (A5R) and non-criteria antibodies for the diagnosis of APS in patients with clinicalseronegative APS. Three groups were defined, including 21 seronegative APS patients with unexplained obstetrical adverse events or thrombosis history, 15 confirmed APS patients with triple aPL positivity, and a control group of 20 healthy patients without any history of thrombosis or pregnancy complications. Seronegative APS patients have similar levels of A5R in comparison to healthy controls (202% [171%-238%] versus 191% [178%-221%]; p = 0.65), whereas triple-positive APS patients have significantly more reduced A5R in comparison to both seronegative and healthy patients (149% [138%-158%] versus 202% [171%-238%] and 191% [178%-221%], respectively, p < 0.001). The non-criteria aPL were found in 24% of seronegative APS: anti-PE IgM in 3 cases (14%) and anti-PS/PT IgG and anti-PS/PT IgM in 1 (5%) case each. The frequency of non-criteria APL was significantly more frequent in comparison to healthy controls (p = 0.048). All triple-positive APS patients have at least one non-criteria aPL, and the non-criteria aPL were significantly more frequent in these patients compared to seronegative APS and healthy controls (p < 0.001). Whereas A5R levels do not allow to discriminate seronegative APS from healthy controls, our results demonstrate that non-criteria aPL can helpto APS diagnosis in clinicalseronegative APS.Key points• Annexin-A5 resistance testing does not help for the diagnosis of seronegative APS.• The non-criteria antiphospholipid antibodies can contribute to APS diagnosis in patients without conventional antibodies.

Toxoplasma gondii in cancer patients receiving chemotherapy: seroprevalence and interferon gamma level.

Toxoplasma gondii is an opportunistic parasite causing life-threatening diseases in immune-compromised patients. The purpose of the study is to determine the seroprevalence of Toxoplasma gondii in chemotherapy receiving cancer patients in relation to different types of malignancies, and to estimate the level of interferon gamma in Toxoplasma seropositive and seronegative cancer patients and healthy controls. Anti-Toxoplasma IgG and IgM antibodies, and interferon gamma were analyzed in 120 cancer patients receiving chemotherapy (60 having hematological malignancies and 60 with solid organ tumors) and 60 healthy controls using ELISA method. Toxoplasma (IgG and IgM) were determined in (66.7% and 9.2%) of the cancer group compared to (33.3% and 6.7%) of the control group with statistical significance only in IgG seropositivity (p < 0.001, OR = 4). Patients with hematological malignancies had higher IgG seropositivity than solid organ tumors (40% vs 26.7%). The difference between the groups was statistically significant (p = 0.002, OR = 3.5). Median level of interferon gamma was in the same range between cancer patients and control group. However, it was highly elevated in Toxoplasma seropositive (76pg/ml) than seronegative (44.5pg/ml) cases with statistical significance (p < 0.001). T. gondii infection remains a major threat to cancer patients and still needs proper screening, diagnosis and treatment.

Extracellular vesicles are associated with the systemic inflammation of patients with seropositive rheumatoid arthritis

Patients with rheumatoid arthritis (RA) and autoantibodies, such as rheumatoid factor and those against cyclic citrullinated peptides, are designated as seropositive and have a more severe disease with worse prognosis than seronegative RA patients. Understanding the factors that participate in systemic inflammation, in addition to articular commitment, would allow better treatment approaches for prevention of RA comorbidities and disease reactivation. We evaluated whether monocyte subsets and extracellular vesicles (EVs) could contribute to this phenomenon. Seropositive patients had higher levels of proinflammatory cytokines than those of seronegative patients and healthy controls (HCs); however, this systemic inflammatory profile was unrelated to disease activity. High frequencies of circulating EVs positive for IgG, IgM, CD41a, and citrulline, together with altered counts and receptor expression of intermediate monocytes, were associated with systemic inflammation in seropositive patients; these alterations were not observed in seronegative patients, which seem to be more similar to HCs. Additionally, the EVs from seropositive patients were able to activate mononuclear phagocytes in vitro, and induced proinflammatory cytokines that were comparable to the inflammatory response observed at the systemic level in seropositive RA patients; therefore, all of these factors may contribute to the greater disease severity that has been described in these patients.

Distinct monocyte subset phenotypes in patients with different clinical forms of chronic Chagas disease and seronegative dilated cardiomyopathy.

BackgroundChronic infection with Trypanosoma cruzi leads to a constant stimulation of the host immune system. Monocytes, which are recruited in response to inflammatory signals, are divided into classical CD14hiCD16—, non-classical CD14loCD16+ and intermediate CD14hiCD16+ subsets. In this study, we evaluated the frequencies of monocyte subsets in the different clinical stages of chronic Chagas disease in comparison with the monocyte profile of seronegative heart failure subjects and seronegative healthy controls. The effect of the anti-parasite drug therapy benznidazole on monocyte subsets was also explored.Methodology/Principal findingsThe frequencies of the different monocyte subsets and their phenotypes were measured by flow cytometry. Trypanosoma cruzi-specific antibodies were quantified by conventional serological tests. T. cruzi-infected subjects with mild or no signs of cardiac disease and patients suffering from dilated cardiomyopathy unrelated to T. cruzi infection showed increased levels of non-classical CD14loCD16+ monocytes compared with healthy controls. In contrast, the monocyte profile in T. cruzi-infected subjects with severe cardiomyopathy was skewed towards the classical and intermediate subsets. After benznidazole treatment, non-classical monocytes CD14loCD16+ decreased while classical monocytes CD14hiCD16—increased.Conclusions/SignificanceThe different clinical stages of chronic Chagas disease display distinct monocyte profiles that are restored after anti-parasite drug therapy. T. cruzi-infected subjects with severe cardiac disease displayed a profile of monocytes subsets suggestive of a more pronounced inflammatory environment compared with subjects suffering from heart failure not related to T. cruzi infection, supporting that parasite persistence might also alter cell components of the innate immune system.

The immunological footprint of CMV in HIV-1 patients stable on long-term ART.

BackgroundMost HIV-infected persons are cytomegalovirus (CMV) seropositive and retain latent virus that can be reactivated by immune activation. Their T cell populations express markers reflecting a late stage of differentiation, but the contributions of HIV and CMV to this profile are unclear. We investigated the immunological “footprint” of CMV in HIV patients who had a history of extreme immunodeficiency but were now stable on antiretroviral therapy (ART).ResultsTwenty CMV seropositive HIV patients >50 years old with nadir CD4 T-cell counts <200 cells/μl were studied after >12 years on ART. 16 CMV seropositive and 9 CMV seronegative healthy controls were included. CMV antibody titres were higher in HIV patients than controls (P < 0.001-0.003). Levels of soluble B-cell activating factor (sBAFF) were elevated in patients (P = 0.002) and correlated with levels of CMV antibodies (P = 0.03-0.002), with no clear relationship in controls. CD8 T-cell IFNγ responses to the IE1 peptide (VLE) remained elevated in HIV patients (P = 0.005). The CD57+CD45RA+CD27− phenotype of CD8 T-cells correlated with age (r = 0.60, P = 0.006), antibodies against CMV IE1 protein (r = 0.44, P = 0.06) and CD4 T-cell IFNγ response to CMV lysate (r = 0.45, P = 0.05).ConclusionsHumoral and T-cell responses to CMV remained elevated in HIV patients after >12 years on ART. Age and presence of CMV disease influenced CD8 T-cell phenotypes. Elevated levels of sBAFF may be a consequence of HIV disease and contribute to high titres of CMV antibody.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0041-0) contains supplementary material, which is available to authorized users.

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India.

BackgroundLeptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.Methods/Principal FindingsIn the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).ConclusionThe application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.

Sa1758 In Vitro Adalimumab Exposure Decreases RORγT Expressing and RORγT/FoxP3 Double Expressing T-Lymphocytes From Peripheral Blood of Crohn's Disease Patients: A Study of Th17 Plasticity in Response to Anti-TNF

G A A b st ra ct s recognizing E. coli OmpC. Methods: Tetramer-guided epitope mapping (TGEM) in HLADR*1501 subjects consistently identified a single T cell epitope in OmpC for use in tetramerbased assays. Direct ex vivo characterization was performed by staining with PE-labeled tetramers against OmpC and Flu peptides and subsequent enrichment with anti-PE magnetic beads. Cells were stained for surface markers and analyzed by flow cytometry. Frequency of antigen-specific T cells was calculated as the number of post-enrichment tetramer+ cells/ total pre-enrichment CD4+ cells. Results: The frequency of OmpC-specific T cells was 9.1 (+/-2)/million CD4+ T cells (n=26) compared to 13.7 (+/-2)/million Flu-specific T cells (n= 17), p=.18. The majority of both Flu and OmpC-specific T cells expressed a memory T cell phenotype (CD45RA-). There were no regulatory T cells (CD25+CD127-) identified among OmpC or Flu-specific CD4s. A high percentage of OmpC-specific T cells expressed the guthoming marker, a4b7, compared to Flu-specific T cells (44%+/-2 vs. 13.7% +/-2, p<.0001). Expression of the Th17 marker, CD161, was more frequent on OmpC than Flu-specific T cells (38.5 %+/-5 vs. 13% +/-2, p <.0001). While there was some expression of the Th1 marker, CXCR3, on OmpC-specific T cells, this was less than was expressed by Flu-specific T cells (27%+/-4 vs. 50% +/-5, p= .0015). The current study was underpowered to detect differences in frequency or phenotype among OmpC seropositive and seronegative CD subjects and healthy controls. OmpC-specific T cells identified above were isolated for future RNA transcript analyses to reveal qualitative differences in these cells between patients with and without CD. Conclusions: We have identified T cell epitopes for E.coli OmpC in HLADR *1501 subjects and generated MHC class II tetramers that identify OmpC-specific T cells. OmpC-specific T cells have a frequency similar to Flu-specific T cells and bear a distinct phenotype. They are memory T cells with gut-homing integrin a4b7 expression. They lack Treg markers and express mixed Th1 and Th17 markers. These data confirm that healthy humans and CD subjects harbor commensal-specific T cells and suggest that active suppression of T cell responses is lost in subjects with CD.

Sa1757 Targeting Innate Immune Receptors to Treat Inflammatory Bowel Disease: Preclinical Activity of IMO-9200, an Antagonist of TLRS 7, 8, and 9 in Mouse Models of Colitis

G A A b st ra ct s recognizing E. coli OmpC. Methods: Tetramer-guided epitope mapping (TGEM) in HLADR*1501 subjects consistently identified a single T cell epitope in OmpC for use in tetramerbased assays. Direct ex vivo characterization was performed by staining with PE-labeled tetramers against OmpC and Flu peptides and subsequent enrichment with anti-PE magnetic beads. Cells were stained for surface markers and analyzed by flow cytometry. Frequency of antigen-specific T cells was calculated as the number of post-enrichment tetramer+ cells/ total pre-enrichment CD4+ cells. Results: The frequency of OmpC-specific T cells was 9.1 (+/-2)/million CD4+ T cells (n=26) compared to 13.7 (+/-2)/million Flu-specific T cells (n= 17), p=.18. The majority of both Flu and OmpC-specific T cells expressed a memory T cell phenotype (CD45RA-). There were no regulatory T cells (CD25+CD127-) identified among OmpC or Flu-specific CD4s. A high percentage of OmpC-specific T cells expressed the guthoming marker, a4b7, compared to Flu-specific T cells (44%+/-2 vs. 13.7% +/-2, p<.0001). Expression of the Th17 marker, CD161, was more frequent on OmpC than Flu-specific T cells (38.5 %+/-5 vs. 13% +/-2, p <.0001). While there was some expression of the Th1 marker, CXCR3, on OmpC-specific T cells, this was less than was expressed by Flu-specific T cells (27%+/-4 vs. 50% +/-5, p= .0015). The current study was underpowered to detect differences in frequency or phenotype among OmpC seropositive and seronegative CD subjects and healthy controls. OmpC-specific T cells identified above were isolated for future RNA transcript analyses to reveal qualitative differences in these cells between patients with and without CD. Conclusions: We have identified T cell epitopes for E.coli OmpC in HLADR *1501 subjects and generated MHC class II tetramers that identify OmpC-specific T cells. OmpC-specific T cells have a frequency similar to Flu-specific T cells and bear a distinct phenotype. They are memory T cells with gut-homing integrin a4b7 expression. They lack Treg markers and express mixed Th1 and Th17 markers. These data confirm that healthy humans and CD subjects harbor commensal-specific T cells and suggest that active suppression of T cell responses is lost in subjects with CD.

Sa1756 The Renin-Angiotensin System Promotes Colitis Independently of Blood Pressure

G A A b st ra ct s recognizing E. coli OmpC. Methods: Tetramer-guided epitope mapping (TGEM) in HLADR*1501 subjects consistently identified a single T cell epitope in OmpC for use in tetramerbased assays. Direct ex vivo characterization was performed by staining with PE-labeled tetramers against OmpC and Flu peptides and subsequent enrichment with anti-PE magnetic beads. Cells were stained for surface markers and analyzed by flow cytometry. Frequency of antigen-specific T cells was calculated as the number of post-enrichment tetramer+ cells/ total pre-enrichment CD4+ cells. Results: The frequency of OmpC-specific T cells was 9.1 (+/-2)/million CD4+ T cells (n=26) compared to 13.7 (+/-2)/million Flu-specific T cells (n= 17), p=.18. The majority of both Flu and OmpC-specific T cells expressed a memory T cell phenotype (CD45RA-). There were no regulatory T cells (CD25+CD127-) identified among OmpC or Flu-specific CD4s. A high percentage of OmpC-specific T cells expressed the guthoming marker, a4b7, compared to Flu-specific T cells (44%+/-2 vs. 13.7% +/-2, p<.0001). Expression of the Th17 marker, CD161, was more frequent on OmpC than Flu-specific T cells (38.5 %+/-5 vs. 13% +/-2, p <.0001). While there was some expression of the Th1 marker, CXCR3, on OmpC-specific T cells, this was less than was expressed by Flu-specific T cells (27%+/-4 vs. 50% +/-5, p= .0015). The current study was underpowered to detect differences in frequency or phenotype among OmpC seropositive and seronegative CD subjects and healthy controls. OmpC-specific T cells identified above were isolated for future RNA transcript analyses to reveal qualitative differences in these cells between patients with and without CD. Conclusions: We have identified T cell epitopes for E.coli OmpC in HLADR *1501 subjects and generated MHC class II tetramers that identify OmpC-specific T cells. OmpC-specific T cells have a frequency similar to Flu-specific T cells and bear a distinct phenotype. They are memory T cells with gut-homing integrin a4b7 expression. They lack Treg markers and express mixed Th1 and Th17 markers. These data confirm that healthy humans and CD subjects harbor commensal-specific T cells and suggest that active suppression of T cell responses is lost in subjects with CD.

Differential regulation of cytotoxicity pathway discriminating between HIV, HCV mono- and co-infection identified by transcriptome profiling of PBMCs.

BackgroundDespite the easy accessibility and diagnostic utility of PBMCs and their potential to show distinct expression patterns associated with the accelerated disease progression in HIV/HCV co-infection, there has not been a systematic study focusing on the global dysregulations of the biological pathways in PBMCs from HIV, HCV mono- and co-infected individuals. This study aimed at identifying the transcriptome distinctions of PBMCs between these patient groups.MethodsGenome-wide transcriptomes of PBMCs from 10 HIV/HCV co-infected patients, 7 HIV+ patients, 5 HCV+ patients, and 5 HIV/HCV sero-negative healthy controls were analyzed using Illumina microarray. Pairwise comparisons were performed to identify differentially expressed genes (DEGs), followed by gene set enrichment analysis (GSEA) to detect the global dysregulations of the biological pathways between HIV, HCV mono- and co-infection.ResultsForty-one, 262, and 44 DEGs with fold change > 1.5 and FDR (false discovery rate) <0.05 for the comparisons of HCV versus co-infection, HIV versus co-infection, and HIV versus HCV were identified, respectively. Significantly altered pathways (FDR < 0.05), featured by those involved in immune system, signaling transduction, and cell cycle, were detected. Notably, the differential regulation of cytotoxicity pathway discriminated between HIV, HCV mono- and co-infection (up-regulated in the former versus the latter group: co-infection versus HIV or HCV, HIV versus HCV; FDR <0.001 ~ 0.019). Conversely, the cytokine-cytokine receptor interaction pathway was down-regulated in co-infection versus either HCV (FDR = 0.003) or HIV (FDR = 0.028). For the comparison of HIV versus HCV, the cell cycle (FDR = 0.016) and WNT signaling (FDR = 0.006) pathways were up- and down-regulated in HIV, respectively.ConclusionsOur study is the first to identify the differential regulation of cytotoxicity pathway discriminating between HIV, HCV mono- and co-infection, which may reflect the distinct patterns of virus-host cell interactions underlying disease progression. Further inspection of cytotoxicity pathway has pinned down to the expression of the KIR genes to be associated with specific patterns of particular virus-host interactions. Between HIV and HCV, the altered cell cycle and WNT signaling pathways may suggest the different impact of HIV and HCV on cell proliferation and differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-014-0236-6) contains supplementary material, which is available to authorized users.

Interleukin-4 receptor alpha T1432C and A1652G polymorphisms are associated with risk of visceral leishmaniasis.

Background:Immune responses play significant roles in protection against leishmaniasis. Polymorphisms within the interleukin 4 receptor alpha chain (IL-4Rα) gene affect the production of cytokines, which is important for the clearance of many pathogens. The aim of the current study was to identify the relationship between visceral leishmaniasis (VL) infection and polymorphisms at positions T1432C and A1652G of IL-4Rα in an Iranian population.Materials and Methods:This cross-sectional study was performed during 2004–2012 and included three groups of participants: VL patients (Group 1, n = 124), seropositive healthy controls (Group 2, n = 101), and seronegative healthy controls (Group 3, n = 55). The IL-4Rα T1432C and A1652G polymorphisms were evaluated using a polymerase chain reaction-restriction fragment length polymorphism technique, and anti-Leishmania antibody titers were determined by using immunofluorescence technique. Alleles and genotypes were compared between groups of the study as well as Groups 1 and 2 based on the titer of antibodies. The validity of the data was analyzed using Hardy–Weinberg equilibrium and one-way analysis of variance, as well as χ2 tests.Results:The polymorphisms at IL-4Rα positions T1432C and A1652G were significantly associated with active VL infection. These results demonstrated that the IL-4Rα T1432C and A1652G polymorphisms were not associated with anti-Leishmania antibody production.Conclusion:Our results indicate that these IL-4Rα polymorphisms may be risk factors for the development of VL.

Echocardiographic study of left ventricular function in HIV-infected Nigerians

Context: Left ventricular function in HIV/AIDS patients from South-East Nigeria has not been reported. Aims: We sought to determine the prevalence and spectrum of left ventricular function abnormalities in patients with HIV infection in the University of Nigeria Teaching Hospital (UNTH), Enugu, South-East Nigeria. Settings and Design: This was a descriptive, cross-sectional study of patients with HIV/AIDS at UNTH, Enugu from September 2006 to July 2007. Methods and Material: Sixty-six HIV-infected patients being managed at the antiretroviral therapy clinic and who had no other cardiovascular risk factor other than HIV infection were consecutively recruited. They were matched for sex, and age with seronegative healthy controls. Clinical and echocardiographic evaluation was carried out to assess the left ventricular function. Statistical Analysis Used: The findings were analyzed with statistical package for social sciences (SPSS) version 10.0. Results: The patients consisted of 29 males and 37 females aged between 23 and 62 years. Left ventricular systolic dysfunction was identified in 9 (13.6%) of 66 HIV-infected patients and 1 (4.3%) of 23 controls ( P = 0.201) while left ventricular diastolic dysfunction (reversed fi lling pattern) was identifi ed in 19 (28.8%) and 3 (13.0%) of the HIV-infected patients and controls, respectively (0.021). Left ventricular hypertrophy was seen in 2 (3.0%) patients but in only 1 (4.3%) control ( P = 0.647), while left ventricular dilatation was absent in all the patients and controls. Conclusions: Systolic function does not vary significantly between our patients with HIV/AIDS and sero-negative controls. This calls for further investigation of cardiac function in Nigerian HIV/AIDS patients.

HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis Is Not Associated with SNP rs12979860 of the IL-28B Gene.

The present study investigated the association between the rs12979860 polymorphism in the IL-28B gene and HTLV-1 infection as well as the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1-infected patients (26 HAM/TSP symptomatic and 53 asymptomatic) and 300 seronegative healthy controls were investigated. Plasma levels of the cytokines TNF-α, TNF-β, IL-8, IL-10, IL-6, and IFN-γ from infected patients were measured using an indirect enzyme-linked immunosorbent assay. The HTLV proviral load was measured using a real-time PCR assay, and T-cell subset counts were determined by flow cytometry. Real-time PCR was used to genotype the rs12979860 SNP. The allelic and genotypic distributions displayed no significant differences among the investigated groups. No significant association between the serum cytokine levels and the presence of the rs12979860 SNP in symptomatic and asymptomatic subjects was observed. A positive correlation (p = 0.0015) between TNF-β and IFN-γ was observed in the asymptomatic group, but a positive correlation was only observed (p = 0.0180) between TNF-α and IL-6 in the HAM/TSP group. The proviral load was significantly higher in HAM/TSP patients than in asymptomatic subjects. The present results do not support a previous report indicating an association between the SNP rs12979860 and HAM/TSP outcome.