Increased levels of IL‐21 responses are associated with the severity of liver injury in patients with chronic active hepatitis B

Interleukin‐21 (IL‐21) participates in tissue damage in various immune‐mediated diseases. Its role in the pathogenesis of chronic active hepatitis B (CAHB) has not been clarified. The frequency of circulating IL‐21⁺ T cells and the levels of serum and intrahepatic IL‐21 have been characterized in 70 CAHB patients, 32 inactive carrier (IC), 18 chronic hepatitis C (CHC) and 20 healthy controls (HC). Their potential association with liver injury was analysed. The percentages of IL‐21⁺CD3⁺CD8⁻ and IL‐21⁺CD3⁺CD8⁺ T cells and the levels of serum IL‐21 in CAHB patients were significantly higher than that in the IC, CHC patients and HC (P < 0.001) and were correlated positively with the levels of serum alanine aminotransferase (ALT, r = 0.424, P < 0.001; r = 0.392, P = 0.001) and aspartate aminotransferase (AST, r = 0.388, P = 0.001; r = 0.329, P = 0.005) in CAHB patients, respectively. The levels of IL‐21 expression in the liver tissues were associated significantly with increased degrees of inflammation and fibrosis in CAHB patients (P < 0.01 or P < 0.05). Our findings suggest that aberrant IL‐21 responses may be associated with the progression of CHB.     

Selective T‐cell depletion targeting CD45RA reduces viremia and enhances early T‐cell recovery compared with CD3‐targeted T‐cell depletion

1 Background

T‐cell depletion (TCD) effectively reduces severe graft‐versus‐host disease in recipients of HLA‐mismatched allografts. However, TCD is associated with delayed immune recovery and increased infections. We hypothesized that specific depletion of CD45RA+ naive T cells, rather than broad depletion of CD3+ T cells, can preserve memory‐immunity in the allografts and confer protection against important viral infections in the early post‐transplant period.

2 Methods

Sixty‐seven patients who received TCD haploidentical donor transplantation for hematologic malignancy on 3 consecutive trials were analyzed.

3 Results

Patients receiving CD45RA‐depleted donor grafts had 2000‐fold more donor T cells infused, significantly higher T‐cell counts at Day +30 post transplant (550/μL vs 10/μL; P < .001), and higher T‐cell diversity by Vbeta spectratyping at Day +100 (P < .001). Importantly, these recipients experienced a significant reduction in both the incidence (P = .002) and duration (P = .02) of any viremia (cytomegalovirus, Epstein‐Barr virus, or adenovirus) in the first 6 months post transplant. Specifically, recipients of CD3‐depleted grafts were more likely to experience adenovirus viremia (27% vs 4%, P = .02).

4 Conclusion

CD45RA‐depletion provided a large number of donor memory T cells to the recipients and was associated with enhanced early T‐cell recovery and protection against viremia.     

HIV-infected long-term nonprogressors display a unique correlative pattern between the interleukin-7/interleukin-7 receptor circuit and T-cell homeostasis

Background

We hypothesized that there may be a correlation between the interleukin‐7 (IL‐7)/IL‐7 receptor (IL‐7R) regulatory system and parameters of T‐cell homeostasis in HIV‐infected long‐term nonprogressors (LTNPs) as compared with patients with disease progression.

Methods

The possibility of a correlation between T‐cell homeostatic parameters and IL‐7/IL‐7R was investigated in 22 LTNPs (CD4 count ≥500 cells/μL for >10 years) vs. HIV‐positive patients at different disease stages [12 early: CD4 count ≥400 cells/μL; 15 late (AIDS‐presenters): CD4 count ≤150 cells/μL].

Results

Compared with early‐stage HIV‐positive patients, LTNPs displayed a higher circulating IL‐7 concentration (P=0.05), which was positively associated with higher IL‐7Rα expression and a higher T‐cell receptor excision circle (TREC) content specifically within CD4 cells (P<0.05). Compared with late‐stage disease patients, early‐stage disease patients displayed a lower IL‐7 concentration (P<0.01) and higher percentages of IL‐7Rα⁺ CD4 and CD8 cells (P=0.05). IL‐7 was positively correlated with the percentage of TREC⁺ CD4 cells (P<0.01), which translated into a higher percentage of naïve CD4 cells in early‐stage disease patients than in late‐stage disease patients; however, the CD4 cells in early‐stage disease patients were less enriched in recent thymic emigrants (RTEs) compared with LTNPs (P<0.05). In late‐stage AIDS‐developing patients, substantially increased IL‐7 was correlated with a decreased percentage of IL‐7Rα⁺ CD4 cells (P=0.01), which resulted in these patients having a significantly lower percentage of naïve T cells (P<0.01) and a significantly lower content of TREC (P<0.01) than the other patients.

Conclusions

The maintenance of high CD4 cell counts in LTNPs was associated with a specific IL‐7/IL‐7R pattern characterized by increased IL‐7 and highest IL‐7Rα‐expressing CD4 cells relative to other patients. Compared with patients with late‐stage disease, LTNPs displayed a phenotypically naïve, less activated CD4 cell pool highly enriched in RTEs, suggesting the existence of a compensatory IL‐7‐mediated pathway specifically sustaining peripheral CD4 counts.

     

Bone marrow characterization in sickle cell disease: inflammation and stress erythropoiesis lead to suboptimal CD34 recovery

Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, ex vivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non-SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin-A (GPA) expression on HSPCs after CD34⁺ selection were analysed by conventional and imaging flow cytometry. Compared to non-SCD BM, multiple markers of inflammation, contamination (red cells, P < 0·01; platelets, P < 0·01) and aggregates (platelet/granulocytes, P < 0·01; mononuclear/red cells, P < 0·01) were higher in SCD BM. Total CD34⁺ cell count was lower in SCD BM (P < 0·05), however CD34⁺ count was higher in SCD BM when collected in acid citrate dextrose-A (ACDA) versus heparin (P < 0·05). Greater than 50% of CD34⁺ HSPCs from SCD BM are CD34^dim due to higher erythroid lineage expression (P < 0·01) as single cell CD34⁺CD45⁺GPA⁺ (P < 0·01) and CD34⁺CD45⁻GPA⁺ (P < 0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non-SCD BM.     

The cardioprotective effect of melatonin and exendin‐4 treatment in a rat model of cardiorenal syndrome

We investigated the cardioprotective effect of melatonin (Mel) and exendin‐4 (Ex4) treatment in a rat model of cardiorenal syndrome (CRS). Adult male SD rats (n=48) were randomly and equally divided into sham control (SC), dilated cardiomyopathy (DCM) (doxorubicin 7 mg/kg i.p. every five days/4 doses), CRS (defined as DCM+CKD) only, CRS‐Mel (20 mg/kg/d), CRS‐Ex4 (10 μg/kg/d), and CRS‐Mel‐Ex4 groups. In vitro results showed protein expressions of oxidative stress (NOX‐1/NOX‐2/oxidized protein), DNA/mitochondrial damage (γ‐H2AX/cytosolic cytochrome c), apoptosis (cleaved caspase‐3/PARP), and senescence (β‐galactosidase cells) biomarkers were upregulated, whereas mitochondrial ATP level was decreased in doxorubicin/p‐cresol‐treated H9c2 cells that were revised by Mel and Ex4 treatments (all P<.001). By day 60, LVEF was highest in the SC and lowest in the CRS, significantly lower in the DCM than in other treatment groups, lower in the CRS‐Mel and CRS‐Ex4 than in the CRS‐Mel‐Ex4, and lower in the CRS‐Mel than in the CRS‐Ex4, whereas LV chamber size and histopathology score showed a pattern opposite to that of LVEF among all groups (all P<.001). Plasma creatinine level was highest in the CRS and lowest in the SC and progressively decreased from the CRS‐Mel, CRS‐Ex4, CRS‐Mel‐Ex4 to DCM (P<.0001). Protein expressions of inflammation (TNF‐α/NF‐κB/MMP‐2/MMP‐9/IL‐1β), apoptosis/DNA damage (Bax/c‐caspase‐3/c‐PARP/γ‐H2AX), fibrosis (Smad3/TGF‐β), oxidative stress (NOX‐1/NOX‐2/NOX‐4/oxidized protein), cardiac hypertrophy/pressure overload (BNP/β‐MHC), and cardiac integrity (Cx43/α‐MHC) biomarkers in LV myocardium showed an opposite pattern compared to that of LVEF among all groups (all P<.001). Fibrotic area, DNA damage (γ‐H2AX⁺/53BP1⁺CD90⁺/XRCC1⁺CD90⁺), and inflammation (CD14⁺/CD68⁺) biomarkers in LV myocardium displayed a pattern opposite to that of LVEF among all groups (all P<.001). Combined melatonin and exendin‐4 treatment suppressed CRS‐induced deterioration of LVEF and LV remodeling.     

Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis

Objective

To analyze the frequency, surface phenotype, and cytokine secretion of CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with both healthy control subjects and patients with rheumatoid arthritis (RA).

Methods

Eight‐color flow cytometry was used to analyze the surface phenotype and cytokine production of PBMCs from 20 patients with AS, 12 patients with RA, and 16 healthy control subjects, following stimulation ex vivo with phorbol myristate acetate and ionomycin for 5 hours. Secretion of interleukin‐17 (IL‐17) by PBMCs was measured by enzyme‐linked immunosorbent assay, following stimulation with anti‐CD3/CD28 for 4 days.

Results

The percentages of IL‐17–positive CD4+ T cells and IL‐22–positive CD4+ T cells were increased in the PBMCs of both patients with AS and patients with RA compared with healthy control subjects, whereas there were no differences in the percentages of interferon‐γ (IFNγ)–positive or IL‐10–positive CD4+ T cells. Likewise, concentrations of IL‐17 in supernatants from patients with AS were significantly higher compared with those from healthy control subjects. In patients with RA, the concentrations of IL‐17 were increased but not significantly. There was a correlation between the percentages of IL‐17–positive CD4+ T cells detected in PBMCs and the amounts of IL‐17 in culture supernatants (r = 0.414, P = 0.0034). All IL‐17–producing cells were CD4+CD45RO+; most expressed both CCR6 and CCR4, but only 50% expressed the IL‐23 receptor (IL‐23R). Nevertheless, there was a positive relationship between the percentage of IL‐23R–positive CD4+ T cells and the frequency of IL‐17–positive CD4+ T cells or IL‐22–positive CD4+ T cells (r = 0.57, P < 0.0001 and r = 0.46, P = 0.001, respectively). A significant proportion of cells that produced IL‐17 also produced IL‐22 and IFNγ, but none produced IL‐10.

Conclusion

The frequencies of IL‐17–positive and IL‐22–positive CD4+ T cells were increased in PBMCs from patients with AS and patients with RA, resulting in secretion of higher quantities of IL‐17 by PBMCs following stimulation. These data support the hypothesis that Th17 cells, particularly when present in excess of IL‐10–producing cells, are involved in the pathogenesis of inflammatory arthritis.

     

In vivo effects of the anti–interleukin‐6 receptor inhibitor tocilizumab on the B cell compartment

Objective

Interleukin‐6 (IL‐6) receptor inhibition by tocilizumab was recently licensed for the treatment of rheumatoid arthritis (RA). IL‐6 induces in vitro differentiation of B cells into antibody‐forming cells; however, the in vivo effects of IL‐6 inhibition on the B cell compartment are currently not known. The purpose of this study was to examine this feature.

Methods

Sixteen patients with active RA were treated in an open‐label study with tocilizumab (8 mg/kg every 4 weeks). Immunophenotyping was performed at baseline, week 12, and week 24.

Results

Memory B cell subsets declined significantly during tocilizumab therapy. Preswitch memory B cells decreased from a median of 19.6% to 12.3% at week 24 and postswitch memory B cells declined from a median of 18.6% to 15.0% at week 24 (P = 0.04). In parallel, CD19+IgA+ and CD19+IgG+ B cells decreased significantly. The proportion of IgA‐expressing B cells fell from a median of 9.2% at baseline to 4.3% at week 12 and to 3.6% at week 24 (P = 0.01). IgG+ B cells declined from a median of 6.7% at baseline to 4.9% at week 12 (P = 0.007) and 2.8% at week 24 (P = 0.01). In parallel, serum levels of IgA and IgG were significantly diminished at week 24 (P < 0.05). There was a good correlation between relative and absolute numbers of IgA+ B cells with serum IgA at week 24.

Conclusion

Tocilizumab induced a significant reduction in the frequency of peripheral preswitch and postswitch memory B cells. In addition, the number of IgG+ and IgA+ B cells declined and correlated well with reduced serum immunoglobulin levels. The data indicate that IL‐6 blockade affects the B cell hyperreactivity in RA patients.

     

Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV‐coinfected patients

Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV‐infected and HIV/HCV‐coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy‐derived liver‐infiltrating lymphocytes from 26 HIV/HCV‐coinfected, 10 chronic HCV‐infected and 10 HIV‐infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T‐cell frequency by flow cytometry. CD8⁺ T cells expressing the exhaustion marker PD‐1 were increased in HCV‐infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4⁺CD25⁺Foxp3⁺ T cells, as well as CD4⁺CD25⁺PD1⁺ T cells, were more frequent in HIV/HCV‐coinfected than in HCV‐monoinfected samples (P < 0.001). HCV was associated with increased regulatory T cells, PD‐1⁺ T cells and decreased memory B cells, regardless of HIV infection (P ≤ 0.005 for all). Low CD8⁺ expression was observed only in PD‐1⁺CD8⁺ T cells from HCV‐infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8⁺ T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.     

Regulatory B‐cell compartment in transfused alloimmunized and non‐alloimmunized patients with sickle cell disease

Transfusion therapy is a life‐sustaining treatment for patients with sickle cell disease (SCD), but can cause serious complications including alloimmunization. We previously reported diminished regulatory T cells (Tregs) and skewed Th2 responses in alloimmunized SCD patients. We hypothesized that the B cell regulatory (Breg) compartment, which controls Treg and Th differentiation, may also be compromised in allosensitized SCD patients. Phenotypically, we did not find differences in the frequency or numbers of CD24^hiCD38^hi and CD24^hiCD27⁺ B cell subsets, both previously identified as human Bregs, between alloimmunized and non‐alloimmunized SCD patients on regular transfusions. However, at the functional level, CD19+ B cells from alloimmunized SCD patients expressed lower levels of IL‐10 following stimulation as compared with non‐alloimmunized patients (P < 0.05), and had reduced ability in inhibiting autologous CD14+ monocyte TNF‐α expression (P < 0.05). These findings suggest that Bregs from alloimmunized and non‐alloimmunized SCD patients differ in their ability to produce IL‐10 and dampen monocyte activation, all consistent with an altered immunoregulatory state in alloimmunized SCD patients. Am. J. Hematol. 88:736–740, 2013. © 2013 Wiley Periodicals, Inc.     

Lack of isoprenoid products raises ex vivo interleukin‐1β secretion in hyperimmunoglobulinemia D and periodic fever syndrome

Objective

To investigate whether the increased interleukin‐1β (IL‐1β) secretion in hyperimmunoglobulinemia D and periodic fever syndrome is due to the accumulation of mevalonate kinase (MK), the substrate of the deficient enzyme, or the lack of its products, the isoprenoid compounds.

Methods

The effects of lovastatin and farnesol (FOH), geranylgeraniol (GGOH), and mevalonate on peripheral blood mononuclear cells (PBMCs) from 8 patients with MK deficiency and from 13 controls were studied. Lovastatin inhibits isoprenoid biosynthesis by reducing the production of mevalonate. FOH and GGOH restore isoprenoid biosynthesis downstream from MK. Culture supernatants were collected for cytokine analysis 48 hours after stimulation with monoclonal antibodies against CD2 + CD28.

Results

Lovastatin induced a 15‐fold rise in IL‐1β secretion by normal anti–CD2 + CD28–stimulated cells (P < 0.001). This effect could be countered by mevalonate and, to a lesser extent, by FOH and GGOH. In the absence of lovastatin, mevalonate did not change IL‐1β secretion. Stimulated MK‐deficient cells secreted 9‐fold more IL‐1β than control PBMCs (P < 0.005), rising 2.4‐fold in the presence of lovastatin. The effect of lovastatin on IL‐1β secretion was reduced by mevalonate, FOH, and GGOH. Isoprenoid biosynthesis in PBMCs from patients was impaired due to the endogenous MK deficiency. Bypassing this defect with FOH, in the absence of lovastatin, led to a 62% reduction (P < 0.02) in IL‐1β secretion by these cells.

Conclusion

In this model, shortage of isoprenoid end products contributes to increased IL‐1β secretion by MK‐deficient PBMCs, whereas excess mevalonate does not.

     

Melatonin: Antioxidant and modulatory properties in age‐related changes during Trypanosoma cruzi infection

The purpose of this study was to investigate the effects of melatonin on selected biomarkers of innate and humoral immune response as well as the antioxidant/oxidant status (superoxide dismutase—SOD and reduced glutathione levels (GSH) to understand whether age‐related changes would influence the development of acute Trypanosoma cruzi (T. cruzi) infection. Young‐ (5 weeks) and middle‐aged (18 months) Wistar rats were orally treated with melatonin (gavage) (05 mg/kg/day), 9 days after infection. A significant increase in both SOD activity and GSH levels was found in plasma from all middle‐aged melatonin‐treated animals. Melatonin triggered enhanced expression of major histocompatibility class II (MHC‐II) antigens on antigen‐presenting cell (APC) and peritoneal macrophages in all treated animals. High levels of CD4⁺CD28‐negative T cells (*P<.05) were detected in middle‐aged control animals. Melatonin induced a significant reduction (***P<.001) in CD28‐negative in CD4⁺ and CD8⁺ T cells in middle‐aged control animals. Contrarily, the same group displayed upregulated CD4⁺CD28⁺T and CD8⁺CD28⁺T cells. Melatonin also triggered an upregulation of CD80 and CD86 expression in all young‐treated groups. Significant percentages of B and spleen dendritic cells in middle‐aged infected and treated animals were observed. Our data reveal new features of melatonin action in inhibiting membrane lipid peroxidation, through the reduction in 8‐isoprostane, upregulating the antioxidant defenses and triggering an effective balance in the antioxidant/oxidant status during acute infection. The ability of melatonin to counteract the immune alterations induced by aging added further support to its use as a potential therapeutic target not only for T. cruzi infection but also for other immunocompromised states.     

Interferon and alternative activation of monocyte/macrophages in systemic sclerosis–associated pulmonary arterial hypertension

Objective

To explore the relationship between biomarkers of pulmonary arterial hypertension (PAH), interferon (IFN)–regulated gene expression, and the alternative activation pathway in systemic sclerosis (SSc).

Methods

Peripheral blood mononuclear cells (PBMCs) were purified from healthy controls, patients with idiopathic PAH, and SSc patients (classified as having diffuse cutaneous SSc, limited cutaneous SSc [lcSSc] without PAH, and lcSSc with PAH). IFN‐regulated and “PAH biomarker” genes were compared after supervised hierarchical clustering. Messenger RNA levels of selected IFN‐regulated genes (Siglec1 and MX1), biomarker genes (IL13RA1, CCR1, and JAK2), and the alternative activation marker gene (MRC1) were analyzed on PBMCs and on CD14− and CD14+ cell populations. Interleukin‐13 (IL‐13) and IL‐4 concentrations were measured in plasma by immunoassay. CD14, MRC1, and IL13RA1 surface expression was analyzed by flow cytometry.

Results

Increased PBMC expression of both IFN‐regulated and biomarker genes distinguished SSc patients from healthy controls. Expression of genes in the biomarker cluster, but not in the IFN‐regulated cluster, distinguished lcSSc with PAH from lcSSc without PAH. The genes CCR1 (P < 0.001) and JAK2 (P < 0.001) were expressed more highly in lcSSc patients with PAH compared with controls and mainly by CD14+ cells. MRC1 expression was increased exclusively in lcSSc patients with PAH (P < 0.001) and correlated strongly with pulmonary artery pressure (r = 0.52, P = 0.03) and higher mortality (P = 0.02). MRC1 expression was higher in CD14+ cells and was greatly increased by stimulation with IL‐13. IL‐13 concentrations in plasma were most highly increased in lcSSc patients with PAH (P < 0.001).

Conclusion

IFN‐regulated and biomarker genes represent distinct, although related, clusters in lcSSc patients with PAH. MRC1, a marker for the effect of IL‐13 on alternative monocyte/macrophage activation, is associated with this severe complication and is related to mortality.

     

Spontaneous left atrial echo contrast,mitral annular systolic velocity,and left atrial appendage late emptying velocity in predicting improvement of left atrial function after percutaneous balloon mitral valvuloplasty

Background

Thromboembolic events are the major cause of morbidity and mortality in patients with mitral stenosis (MS). This study aims to investigate left atrial spontaneous echo contrast (LA SEC), mitral annular systolic velocity (Sa‐wave), left atrial appendage (LAA) late emptying velocity (LAAEV), LAA filling velocity (LAAFV) pre‐ and postpercutaneous balloon mitral valvuloplasty (PBMV) for MS. This also aims to study the association of LA SEC with inflammatory marker, high‐sensitivity C‐reactive protein (hs‐CRP) in MS.

Methods

The study population consisted of 100 patients with symptomatic MS with sinus rhythm who underwent PBMV. Transthoracic echo (TTE), tissue Doppler imaging (TDI), and transesophageal echo (TEE) examinations were carried out before and 14 days following PBMV. High‐sensitivity C‐reactive protein (hs‐CRP) was measured at the time of admission.

Results

The mean age was 33.2 ± 10.3 years with female preponderance (71%). There was a decrease in SEC grading, (pre‐PBMV 2.8 ± 0.9 and post‐PBMV 0.4 ± 0.1; P < .01), increase in LAAEV (pre‐PBMV 23.0 ± 7.9 cm/s and post‐PBMV 40.9 ± 8.4 cm/s; P < .01), and LAAFV (pre‐PBMV 31.8 ± 9.3 cm/s and post‐PBMV 51.2 ± 8.7 cm/s; P < .01).A significant positive correlation was present between LAAEV and Sa‐wave (r = .52, P < .01). Correlation between hs‐CRP and SEC was positive and significant (r = .33, P < .01). Optimal cutoff value of hs‐CRP for prediction of moderate to dense SEC was >2.3 mg/dL, the cutoff value of Sa‐wave was≤ 5.5 cm/s for prediction of the presence of inactive LAA (LAAEV < 25 cm/s).

Conclusion

Mitral annular systolic velocity (Sa‐wave) is an independent predictor of inactive LAA and a useful parameter in estimating inactive LAA in MS. Sa‐wave and hs‐CRP are independent predictors for SEC. PBMV improves LAA function in patients with MS.     

Somatosensory profile of patients with haemophilia

Introduction

Patients with haemophilia (PwH) suffer from an enhanced pain sensitivity due to repetitive joint bleedings. A comprehensive, quantitative examination of the somatosensory system has not been performed in this population to date.

Material and methods

Thirty patients with moderate or severe haemophilia A or B and 30 healthy controls were examined by means of Quantitative Sensory Testing to assess the function of the somatosensory system. Detection (DT) and pain thresholds (PT) were determined, amounting to a total of 13 parameters. Both knee joints and the hand as reference were examined in order to assess both joint‐specific as well as general changes in the somatosensory profile.

Results

Analysing DT and PT, a significant main effect was found for group × stimulus interaction (P ≤ .001). Post hoc tests revealed significant differences in DT between PwH and controls for thermal stimuli across both knees (cold DT: P < .001; warm DT: P < .01) and the hand (cold DT: P < .01; warm DT: P < .05). Mechanical DT was increased in PwH at both knee joints (left knee: P ≤ .05; right knee: P ≤ .01). Furthermore, pressure PT was decreased in PwH at both knees (P ≤ .001).

Conclusion

Haemophilic arthropathy leads to alterations of the somatosensory profile in PwH. Our results reveal initial evidence of a combination of peripheral sensitization, indicated by decreased pressure PT and mechanical DT at the knee joints, as well as general changes of the somatosensory system, shown by reduced thermal DT at affected sites and remote from these. Therefore, both mechanisms have to be considered regarding the pain management in PwH.     

IL28B genetic variation and hepatitis C virus–specific CD4+ T‐cell responses in anti‐HCV‐positive blood donors

Summary. Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome‐wide association studies have shown a strong correlation between single‐nucleotide polymorphisms (SNPs) near the interleukin‐28B (IL28B) gene and spontaneous or treatment‐induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV‐specific T‐cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti‐HCV‐positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV‐specific CD4⁺ T‐cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon‐γ ELISpot assay. The rs12979860‐CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2%vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4–25.3, P < 0.001). HCV‐specific CD4⁺ T‐cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T‐cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T‐cell responses were only observed among those with non‐CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4⁺ T‐cell responses towards NS3 were only evident among those with non‐CC haplotypes.     

Melatonin treatment further improves adipose‐derived mesenchymal stem cell therapy for acute interstitial cystitis in rat

This study tests the hypothesis that combined melatonin and adipose‐derived mesenchymal stem cell (ADMSC, 1.2 × 10⁶ given intravenously) treatment offer superior protection against cyclophosphamide (CYP 150 mg/kg)‐induced acute interstitial cystitis (AIC) in rats. Male adult Sprague‐Dawley rats were treated as follows: sham controls, AIC alone, AIC + melatonin, AIC + ADMSC, and AIC + melatonin +ADMSC. When melatonin was used, it was given as follows: 20 mg/kg at 30 min after CYP and 50 mg/kg at 6 and 18 hr after CYP. Twenty‐four‐hour urine volume, urine albumin level, and severity of hematuria were highest in AIC rats and lowest in the controls; likewise urine volume was higher in AIC + melatonin rats than in AIC + ADMSC and AIC + melatonin + ADMSC treated rats; in all cases, P < 0.001. The numbers of CD14+, CD74+, CD68+, MIP+, Cox‐2+, substance P+, cells and protein expression of IL‐6, IL‐12, RANTES, TNF‐α, NF‐κB, MMP‐9, iNOS (i.e. inflammatory biomarkers), glycosaminoglycan level, expression of oxidized protein, and protein expression of reactive oxygen species (NOX‐1, NOX‐2, NOX‐4) in the bladder tissue exhibited an identical pattern compared with that of hematuria among the five groups (all P < 0.0001). The integrity of epithelial layer and area of collagen deposition displayed an opposite pattern compared to that of hematuria among all groups (P < 0.0001). The cellular expressions of antioxidants (GR, GPx, HO‐1, NQO 1) showed a significant progressive increase form controls to AIC + melatonin + ADMSC (all P < 0.0001). Combined regimen of melatonin and ADMSC was superior to either alone in protecting against CYP‐induced AIC.     

Ascaris suum infection modulates inflammation: Implication of CD4+ CD25high Foxp3+ T cells and IL‐10

Helminth infections have the ability to modulate host's immune response through mechanisms that allow the chronic persistence of the worms in the host. Here, we investigated the mechanisms involved on the suppressive effect of Ascaris suum infection using a murine experimental model of LPS‐induced inflammation. We found that infection with A. suum markedly inhibited leucocyte influx induced by LPS into air pouches, suppressed secretion of pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐6) and induced high levels of IL‐10 and TGF‐β. Augmented frequency of CD4⁺ CD25^high Foxp3⁺ T cells was observed in the mesenteric lymph nodes of infected mice. Adoptive transfer of purified CD4⁺ CD25⁺ T cells to recipient uninfected mice demonstrated that these cells were able to induce a suppressive effect in the LPS‐induced inflammation in air pouch model. In addition, adoptive transfer of CD4⁺ CD25⁺ T cells derived from IL‐10 knockout mice suggests that this suppressive effect of A. suum infection involves IL‐10 cytokine. In conclusion, our results demonstrated that A. suum experimental infection was capable of suppressing LPS‐induced inflammation by mechanisms, which seem to be dependent on responses of CD4⁺ CD25⁺ T cells and secretion of IL‐10 cytokine.     

Down‐regulation of the nonspecific and antigen‐specific T cell cytokine response in ankylosing spondylitis during treatment with infliximab

Objective

Treatment of active ankylosing spondylitis (AS) with the monoclonal tumor necrosis factor α (TNFα) antibody infliximab is highly clinically effective. This study was undertaken to investigate the precise mechanism of action of anti‐TNFα treatment in AS.

Methods

Cytokine expression of CD4+ and CD8+ T cells was investigated before and 6 and 12 weeks after the start of treatment in 10 patients treated with infliximab, and before and after 6 weeks of treatment and 6 weeks after placebo was switched to infliximab in 10 patients treated initially with placebo. Peripheral blood mononuclear cells (PBMCs) were stimulated for 6 hours either nonspecifically with phorbol myristate acetate (PMA)/ionomycin or antigen specifically with a pool of 46 overlapping 18‐mer peptides derived from the G1 domain of aggrecan. Cells were stained for T cell surface markers CD4 and CD8 and for the intracellular cytokines interferon‐γ (IFNγ), TNFα, interleukin‐4 (IL‐4), and IL‐10. Positive cells were quantified by flow cytometry. For monocyte‐derived cytokines, PBMCs were stimulated with lipopolysaccharide (LPS) for 18 hours and TNFα and IL‐10 in the supernatant were measured by enzyme‐linked immunosorbent assay.

Results

Compared with baseline, infliximab treatment induced a significant decrease at 12 weeks in the number of CD4+ and CD8+ T cells that were positive for IFNγ and TNFα upon PMA/ionomycin stimulation (P = 0.005). A significant reduction had already begun to occur at 6 weeks. No change in the percent IFNγ or TNFα positivity among CD4+ and CD8+ subpopulations was observed after 6 weeks in patients treated with placebo. However, when these patients began infliximab treatment after 6 weeks of receiving placebo, there was a similar significant decrease in IFNγ and TNFα production by CD4+ and CD8+ T cells (P < 0.05). Furthermore, infliximab treatment induced a significant reduction in the number of IFNγ+ and TNFα+ CD8+ T cells (P = 0.005 at week 6 and week 12) after antigen‐specific in vitro stimulation with G1‐derived peptides. Between‐group analysis showed that the change in the expression of IFNγ and TNFα in both CD4+ and CD8+ T cells was significantly different between the infliximab and placebo groups (P = 0.001 for all variables). There was no change in the number of IL‐10+ or IL‐4+ T cells during treatment. No significant change in the production of TNFα and IL‐10 upon in vitro stimulation of PBMCs with LPS was detectable during infliximab treatment.

Conclusion

Infliximab down‐regulates both IFNγ and TNFα secreted by T cells but does not induce a change in cytokines produced by monocytes during 3 months of treatment. This is likely to be a relevant mechanism for the clinical efficacy of this therapy.