Jacek Malejczyk

CD4+ CD25+ FOXP3+ regulatory T cells in peripheral blood and peritoneal fluid of patients with endometriosis

STUDY QUESTION Is endometriosis associated with changes in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg cells)? SUMMARY ANSWER Endometriosis is associated with disturbed compartmentalization of CD25(high) FOXP3⁺ Treg cells. WHAT IS KNOWN ALREADY Endometriosis is associated with an abrogated immune response and displays some features of an autoimmune disorder. Treg cells play a part in the development of autoimmune reactions; however, their role in pathogenesis of endometriosis is still poorly recognized. STUDY DESIGN, SIZE AND DURATION Case-control study comparing 17 women with laparoscopically and histopathologically confirmed ovarian endometriosis with 15 control women without visible endometriosis foci, pelvic inflammation or related pathology who were subjected to laparoscopic surgery between 2010 and 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS Peripheral blood and peritoneal fluid were collected during laparoscopy and T cell subpopulations were analysed by flow cytometry using specific monoclonal antibodies recognizing CD4⁺, CD25⁺ and FOXP3⁺ markers. MAIN RESULTS The percentage of CD25(high) FOXP3⁺ Treg cells was significantly decreased in the peripheral blood of women with ovarian endometriosis compared with control women. On the other hand, the proportion of these cells was significantly increased in the peritoneal fluid of women with endometriosis. LIMITATIONS, REASONS FOR CAUTION The present study is limited to patients with ovarian endometrioma and further investigations are needed, including patients with lower grade of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS The present results suggest that Treg cells may play a part in immunopathogenesis of endometriosis being responsible for abrogated local cellular immune responses and facilitation and development of autoimmune reactions. Treg cells may be thus a potential target in the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by 1M15/N/2011 and NK1W grants from the I Faculty of Medicine, Warsaw Medical University. None of the authors has any competing interests to declare.

Association of leptin with inflammatory cytokines and lymphocyte subpopulations in peritoneal fluid of patients with endometriosis

INTRODUCTION Endometriosis is a common, complex and chronic disease related to ectopic implantation and growth of endometrial tissue that may manifest by pelvic inflammatory reactions, chronic pelvic pain and subfertility. Endometriosis may be associated with increased peritoneal fluid leptin levels. Leptin is known to exert immunomodulatory effects; however, an association between leptin and inflammatory reactions in endometriosis has not been documented. Therefore, the aim of this study was to investigate a relationship between leptin concentrations in peritoneal fluid and the levels of peritoneal fluid inflammatory cytokines and mononuclear leukocyte subpopulations. MATERIALS AND METHODS Peritoneal fluid was aspirated by laparoscopy from 46 women in whom endometriosis had been confirmed by clinical and histopathological examinations and from 10 control women qualified for ART in whom pelvic pathology has been excluded. Concentrations of leptin and inflammatory cytokines (IL-1beta, IL-6, IFN-gamma and TNF) in peritoneal fluid were evaluated by specific ELISAs. Percentage of peritoneal leukocyte subpopulations (CD3+, CD4+, CD8+ and CD14+) was analyzed by FACS using specific monoclonal antibodies. RESULTS Leptin concentrations in peritoneal fluid correlated negatively with concentrations of IL-1beta and IFN-gamma (r(s)=-0.38, p=0.01 and r(s)=-0.31, p=0.03, respectively) and correlated positively with the percentage of CD3+ pan-T cells (r(s)=0.69, p=0.009) and CD4+ T helper cells (r(s)=0.74, p=0.036). CONCLUSIONS Increased leptin levels in peritoneal fluid from endometriosis patients may affect local inflammatory/immune reactions, especially infiltration of CD4+ T helper cells. Thus, leptin may play an important role in the immunopathogenesis of endometriosis.

Does the KIR2DS5 Gene Protect from Some Human Diseases

Background KIR2DS5 gene encodes an activating natural killer cell receptor whose ligand is not known. It was recently reported to affect the outcome of hematopoietic stem cell transplantation. Methodology/Principal Findings In our studies on KIR2DS5 gene associations with human diseases, we compared the frequencies of this gene in patients and relevant controls. Typing for KIR2DS5 gene was performed by either individual or multiplex polymerase chain reactions which, when compared in the same samples, gave concordant results. We noted an apparently protective effect of KIR2DS5 gene presence in several clinical conditions, but not in others. Namely, this effect was observed in ankylosing spondylitis (p = 0.003, odds ratio [OR] = 0.47, confidence interval [CI] = 0.28–0.79), endometriosis (p = 0.03, OR = 0.25, CI = 0.07–0.82) and acute rejection of kidney graft (p = 0.0056, OR = 0.44, CI = 0.24–0.80), but not in non-small-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (all p>0.05). In addition, the simultaneous presence of KIR2DS5 gene and HLA-C C1 allotype exhibited an even stronger protective effect on ankylosing spondylitis (p = 0.0003, OR = 0.35, CI = 0.19–0.65), whereas a lack of KIR2DS5 and the presence of the HLA-C C2 allotype was associated with ankylosing spondylitis (p = 0.0017, OR = 1.92, CI = 1.28–2.89), whereas a lack of KIR2DS5 and presence of C1 allotype was associated with rheumatoid arthritis (p = 0.005, OR = 1.47, CI = 1.13–1.92). The presence of both KIR2DS5 and C1 seemed to protect from acute kidney graft rejection (p = 0.017, OR = 0.47, CI = 0.25–0.89), whereas lack of KIR2DS5 and presence of C2 seemed to favor rejection (p = 0.0015, OR = 2.13, CI = 1.34–3.37). Conclusions/Significance Our results suggest that KIR2DS5 may protect from endometriosis, ankylosing spondylitis, and acute rejection of kidney graft.

Peritoneal cytokines and adhesion formation in endometriosis: an inverse association with vascular endothelial growth factor concentration

OBJECTIVE To evaluate inflammatory/angiogenic cytokines-interleukin-1β (IL-1β), IL-6, IL-8, IL-12, interferon-γ (IFN-γ), tumor necrosis factor (TNF), and vascular endothelial growth factor A (VEGF-A)-in the peritoneal fluid of patients with endometriosis in relation to the occurrence and severity of pelvic adhesions and in control women without pelvic pathology. DESIGN Case-control study. SETTING University research institution and hospital. PATIENT(S) Sixty-five women with laparoscopically and histopathologically confirmed endometriosis, including 40 women with pelvic adhesions, and 37 control women without pelvic pathology. INTERVENTION(S) Peritoneal fluid aspirated during routine diagnostic laparoscopic examination. MAIN OUTCOME MEASURE(S) Cytokines evaluated in the peritoneal fluid via specific enzyme-linked immunosorbent assays. RESULT(S) Endometriosis and the revised American Fertility Society score of this disease were associated with statistically significantly increased levels of peritoneal IL-6 and IL-8 whereas the incidence and score of endometriosis-related pelvic adhesions were negatively associated with increased levels of VEGF-A. Notably, the concentration of VEGF-A predicted adhesion development and severity after adjustment for endometriosis severity. The adhesion score also correlated with increased levels of IL-6; however, after adjustment for endometriosis severity, the effect of this cytokine was no longer statistically significant. CONCLUSION(S) Increased levels of VEGF-A may be associated with a decreased rate of pelvic adhesion formation in the course of endometriosis.

N-acetylcysteine inhibits IL-8 and MMP-9 release and ICAM-1 expression by bronchoalveolar cells from interstitial lung disease patients

N-acetylcysteine (NAC), owing to its antioxidant, mucolytic and anti-inflammatory properties, is used in the treatment of various pulmonary disorders. However, the direct effects of NAC on bronchoalveolar lavage (BAL) cells from patients suffering from interstitial lung diseases have not yet been studied. Therefore, the aim of the present work was to evaluate the effect of NAC on interleukin-8 (IL-8) and matrix metalloproteinase-9 (MMP-9) production as well as intercellular cell adhesion molecule-1 (ICAM-1) expression by BAL cells from interstitial lung diseases. The study was performed on BAL cells from nine patients with interstitial lung disease: four patients with idiopathic pulmonary fibrosis (IPF) and five patients with sarcoidosis. Cultured unstimulated BAL cells were treated with increasing doses of NAC (1-30 mM). Production of IL-8 and MMP-9 was evaluated by specific enzyme-linked immuno-sorbent assays and ICAM-1 expression was studied by immunohistochemistry. NAC exerted a dose-dependent inhibitory effect on IL-8 and MMP-9 release and ICAM- expression by BAL macrophages and lymphocytes from patients with IPF and sarcoidosis. In conclusion, NAC inhibits production of factors playing a key role in the etiopathogenesis of interstitial lung diseases, thus suggesting its possible therapeutic potency in the treatment of these disorders.

Increased levels of human neutrophil peptides 1, 2, and 3 in peritoneal fluid of patients with endometriosis: association with neutrophils, T cells and IL-8

Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside the uterine cavity. This disease is associated with pelvic inflammation and displays some features of autoimmune disorder. Human neutrophil peptides 1, 2, and 3 (HNP 1-3) belonging to α-defensin family play a crucial role in innate immunity against infections and may exert immunoregulatory effects. They may play a role in various inflammatory reactions; however, their role in endometriosis has not been studied. Therefore, the aim of the present study was to evaluate HNP 1-3 in the peritoneal fluid of 67 patients with endometriosis and 16 healthy control women in relation to peritoneal leukocyte subpopulations (neutrophils, T cells, and macrophages) and inflammatory cytokines (IL-6 and IL-8). HNP 1-3, IL-6 and IL-8 were evaluated in the peritoneal fluid by specific enzyme-linked immunosorbent assays (ELISA), and peritoneal leukocyte subpopulations were evaluated by flow cytometry. We found that the levels of HNP 1-3 were significantly increased in the peritoneal fluid of endometriosis patients, compared with control women, and correlated with severity of the disease. Endometriosis was also associated with increased concentrations of peritoneal neutrophils. In endometriosis the levels of HNP 1-3 strongly correlated with concentrations of neutrophils, T cells and IL-8. HNP 1-3 levels were not associated with peritoneal IL-6 or macrophages. These data suggest that HNP 1-3 and neutrophils might play a role in immunopathogenesis of endometriosis and may be worth evaluating as targets for anti-endometriosis therapy.

PTPN22/LYP 1858C>T gene polymorphism and susceptibility to endometriosis in a Polish population.

Endometriosis is a common gynaecological disorder due to ectopic implantation of endometrial tissue. It is manifested by pelvic inflammation and abrogation of cell-mediated immunity and may be also characterised by autoantibody production, thus suggesting that endometriosis might be an autoimmune disorder. Genetic factors also play a role in the aetiopathogenesis of this disease. Therefore, the present study was aimed at testing the association of endometriosis with the PTPN22/LYP 1858C> T gene polymorphism, which appears to be related to the development of a variety of autoimmune disorders. The study included 171 Polish patients of Caucasian origin with laparoscopically and histopathologically confirmed endometriosis and 310 unrelated, ethnically matched control individuals. DNA and serum were isolated from the peripheral blood. The PTPN22/LYP 1858C> T polymorphism was typed using a PCR-RFLP method. Anti-nuclear (ANA) and anti-cardiolipin (ACA) autoantibodies were detected by the Hep-2 indirect immunofluorescence test and a specific ELISA respectively. No statistically significant differences were found in distribution of C and T PTPN22/LYP alleles and genotypes in patients with endometriosis compared with the control population. However, on exploratory analyses we noted that the PTPN22/LYB T allele and the TT genotype may be associated with the prevalence of double positivity for ANA and ACA (p=0.003 by chi(2) test for trend and p=0.009 by Fishers exact test respectively). The results of the present study show that endometriosis in a Polish population is not associated with the PTPN22/LYP 1858C> T gene polymorphism. The putative effect of the PTPN22/LYP genotype on the development of autoantibodies is potentially interesting, but it should be verified in further studies.

Differences in Cartilage Formed Intramuscularly or in Joint Surface Defects by Syngeneic Rat Chondrocytes Isolated from the Articular-Epiphyseal Cartilage Complex

Syngeneic rat chondrocytes isolated from the articular-epiphyseal cartilage complex were suspended in hyaluronic acid and transplanted intramuscularly or into joint surface defects. Transplants were fixed in ruthenium hexammonium trichloride and embedded in glycol methacrylate. In cartilage nodules produced intramuscularly, chondrocyte hypertrophy and matrix calcification were observed after 2 wk. Partial ossification occurred after 4 wk and the cartilage was almost completely replaced by an ossicle after 8 wk. Only small, dispersed groups of chondrocytes remained within the ossicle. In cartilage formed in joint surface defects a superficial and a deep zone were distinguished. Chondrocytes in the superficial zone did not hypertrophy and cartilage remained unossified. In the deep zone matrix calcification and bone formation occurred. These processes were, however, retarded in comparison with intramuscular transplants. Thus, either intraarticular environment exerted an inhibitory effect on chondrocyte hypertrophy and matrix calcification or articular chondrocytes present among transplanted cells accumulated close to the joint lumen and reconstructed normal articular cartilage.

Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 as an Activator of the β4-Integrin Gene in Human Keratinocytes

ABSTRACT The papillomavirus life cycle parallels keratinocyte differentiation in stratifying epithelia. We have previously shown that the human papillomavirus type 8 (HPV8) E2 protein downregulates β4-integrin expression in normal human keratinocytes, which may trigger subsequent differentiation steps. Here, we demonstrate that the DNA binding domain of HPV8 E2 is sufficient to displace a cellular factor from the β4-integrin promoter. We identified the E2-displaceable factor as activator protein 1 (AP-1), a heteromeric transcription factor with differentiation-specific expression in the epithelium. β4-Integrin-positive epithelial cells displayed strong AP-1 binding activity. Both AP-1 binding activity and β4-integrin expression were coregulated during keratinocyte differentiation suggesting the involvement of AP-1 in β4-integrin expression. In normal human keratinocytes the AP-1 complex was composed of JunB and Fra-1 subunits. Chromatin immunoprecipitation assays confirmed that JunB/Fra-1 proteins interact in vivo with the β4-integrin promoter and that JunB/Fra-1 promoter occupancy is reduced during keratinocyte differentiation as well as in HPV8 E2 positive keratinocytes. Ectopic expression of the tethered JunB/Fra-1 heterodimer in normal human keratinocytes activated the β4-integrin promoter, while coexpression of HPV8 E2 reverted the JunB/Fra-1 effect. In summary, we identified a novel mechanism of human β4-integrin regulation that is specifically targeted by the HPV8 E2 protein mimicking transcriptional conditions of differentiation. This may explain the early steps of how HPV8 commits its host cells to the differentiation process required for the viral life cycle.

Immunological response against allogeneic chondrocytes transplanted into joint surface defects in rats

Rat chondrocytes isolated from the articular-epiphyseal cartilage complex were transplanted into defects prepared in articular cartilage and subchondral bone. Transplants were taken for examination after 3 and 8 wk. Cartilage formed by syngeneic chondrocytes did not evoke formation of infiltrations. Contrary to that, in the vicinity of cartilage produced by allogeneic chondrocytes numerous infiltrating cells were present and cartilage resorption could be observed. Cyclosporine-A (CsA) treatment of recipients of allogeneic chondrocytes only partially suppressed accumulation of infiltrating cells and matrix resorption. Antichondrocyte immune response of chondrocyte graft recipients was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocyte-chondrocyte cultures and by evaluation of antichondrocyte cytotoxic antibodies. No difference in stimulation of SMC from intact rats by syngeneic and allogeneic chondrocytes was observed. Stimulation by allogeneic chondrocytes was slightly but significantly higher in recipients of syngeneic grafts. SMC of allogenic chondrocyte recipients were strongly stimulated by allogeneic chondrocytes. This response was absent in recipients treated with CsA. Spontaneous antichondrocyte cytotoxic antibody activity was detected in intact rats and in recipients of syngeneic grafts. In recipients of allogeneic chondrocytes the antibody response against allogeneic chondrocytes was raised but was statistically not significant owing to the considerable variation in the level of spontaneously occurring antichondrocyte antibodies.