Gender as a factor affecting NK cell activity in patients successfully treated for chronic hepatitis C with direct-acting antivirals.

Chronic hepatitis C (CHC) affects the activity of natural killer (NK) cells, but successful interferon- free treatment partially restores it. The goal of this study was to assess whether gender influences NK functionality. We examined 21 post-menopausal women and 24 men with CHC who were treated with direct-acting antivirals (DAA) and 33 healthy volunteers. Using flow cytometry, we analysed KIR2DS4, NKG2D, NKp30, KIR2DL2/DL3, NKG2A and TRAIL on the surface of NK cells. Intracellular granzyme B was also assessed and serum CXCL10 was quantified via ELISA. Overall, patients with CHC had higher expression of KIR2DS4, NKG2A, and NKp30 relative to the control group. Further, CHC patients had a lower percentage of NK cells among lymphocytes relative to the control group. After treatment, KIR2DS4, KIR2DL2/DL, NKG2A, TRAIL and NKp30 on NK cells were decreased whilst the percentage of NK cells and the expression of granzyme B and NKG2D increased. Prior to treatment, serum CXCL10 was elevated, but it was inhibited post-treatment. We observed gender-specific differences in the expression of KIR2DL2/DL3 (higher in women) and NKp30 (elevated in men) compared to CHC/control groups. After treatment, KIR2DL2/DL3, NKp30 and CXCL10 dropped only in the female group while granzyme B increased in the male group. In conclusion, the response of NK cells among men and women of post-menopausal ages with CHC differs. Our research may lead to more studies on the different nature of female and male immune systems in the context of HCV infection and treatment.

A case of bizarre posttransplant anti-glomerular basement membrane disease.

The non-collagenous (NC1) domain of α3 and α5 chains of type IV collagen are eminent targets of abnormal immune response in anti-glomerular basement membrane (anti-GBM) disease, which can be diagnosed by the presence of strong linear IgG staining along GBM detected by direct immunofluorescence. The presence of linear GBM fixation in renal allograft is a rare finding. We observed a 33-year-old male with de novo renal failure in a kidney transplant. An examination of a kidney biopsy specimen revealed, in light microscopy, mild mesangial hypercellularity together with mild focal interstitial fibrosis and sparse inflammatory infiltrate. In immunofluorescence microscopy strong linear IgG staining along the capillary walls was seen. Serum anti-GBM antibodies were negative and no mutation in exons coding NC1 domains of α3 and α5 chains of type IV collagen were detected. We described a rare case of a patient with atypical anti-GBM disease in renal allograft, caused probably by the same process which affected the native kidneys.

Higher expression of inhibitory CD158b and CD158e NK cell receptor and age predicts treatment response in children with chronic hepatitis C.

Treatment with pegylated interferon-α and ribavirin (PEG-IFN/RBV) is the only choice for chronic hepatitis C (CHC) in children. Natural killer (NK) cells were described to play a vital role in CHC. The aim of this study was to analyze the expression of peripheral blood NK cell receptors in their relation to PEG-IFN/RBV treatment response. Study included 26 children with CHC-13 boys, age range 13.42 ± 3.28 years. Blood for biochemical, virological and cytometric testing was taken for evaluation prior to the antiviral treatment. NK cell receptors were detected by flow cytometry and the results were presented as proportion of cells and mean fluorescence intensity (MFI). Therapy consisted of PEG-IFNα-2b (60 µg/m2 s.c 1×/week) and RBV (15 mg/kg p.o. daily). Treatment duration was response-related and varied from 12 to 72 weeks. Rapid virological response (RVR) was evaluated in the 4th week and sustained virological response (SVR) 6 months after completion of the therapy. RVR children were younger (11.67 ± 3.74 vs 15.35 ± 2.42; p = 0.001) and displayed higher CD158b (3.58 ± 0.16 vs 3.45 ± 0.13; p = 0.038) and CD158e expression (4.33 ± 0.21 vs 4.03 ± 0.16; p = 0.039). Density of CD158b (logMFI = 3.68 ± 0.22 vs 3.36 ± 0.16; p = 0.036) and CD158e expression was significantly higher (4.37 ± 0.14 vs 4.12 ± 0.21; p = 0.046) and NKG2D expression significantly lower (97.50 ± 3.46 vs 94.92 ± 5.93; p = 0.049) in SVR children. SVR children were also significantly younger (12.40 ± 3.66 vs 15.13 ± 2.83; p = 0.003). Significance of the age of patients, and expression of CD158b and CD158e were confirmed in univariate and multivariate analysis. Age of patients is negatively related to RVR and SVR. NK cell phenotype with higher expression density of CD158b and CD158e receptor was a positive predictor of SVR.

Expression of Natural Killer Cell Inhibitory Receptors is Associated with Significant Liver Injury in Chronic Hepatitis C in Children.

INTRODUCTION AND AIM: Natural Killer (NK) cells play an important role in innate immune response to viral infections and their high proportion is situated in the liver. The aim of this study was to analyze possible relation between the expression of NK cell receptors and varied intensity of liver lesions in chronic hepatitis C (CHC) in children. MATERIAL AND METHODS: Study included 105 children with CHC - 54 boys and 51 girls, age 13.62 ± 3.48 years. Blood specimens were taken at the day of the liver biopsy. Histological evaluation was performed according to METAVIR scoring system. Circulating NK cells were evaluated by flow cytometry. The results were shown as a proportion of cells expressing evaluated receptor and its' mean fluorescent intensity (MFI). RESULTS: In 58 children with CHC (55.2%) significant liver fibrosis was observed ( ≥F2). Higher proportion of cells expressing CD158e inhibitory receptors was observed in the group of children with ALT > 2UNL (21.11 ± 14.60 vs. 12.22 ± 8.99%; p = 0.037). While higher proportion of cells expressing inhibitory CD158b receptor was observed in children with significant fibrosis (F ≥ 2) compared to minimal fibrosis (F < 2) - (34.14 ± 12.44 vs. 27.48 ± 8.71%; p = 0.049). Children with advanced fibrosis (F ≥ 3) had higher MFI of NK cell CD 158b receptor than children with fibrosis scored F < 3 - (5344.20 ± 3407.49 vs. 2979.67 ± 1190.64; p = 0.049). Proportion of NK cells expressing CD158b was found a predictor of significant fibrosis in univariate analysis - [OR 1.065; 95%CI (1.07-1.15); p = 0.046]. CONCLUSIONS: Higher proportion of NK cells expressing inhibitory CD158b and CD158e receptors is associated with significant liver injury.

Patterns of glomerular disease based on 4-year kidney biopsy material analyzed by light microscopy and immunofluorescence: a retrospective single-center analysis in Poland.

The aim of this study was to assess the epidemiology of different patterns of chronic glomerular diseases based on clinical, histopathological and immunofluorescent findings of glomerulonephritis patients hospitalized in the Department of Nephrology, Transplantology and Internal Diseases in Poznan between January 2009 and December 2012. We retrospectively studied 418 patients who had been subjected to renal biopsies. Data on serum creatinine concentration, 24 h proteinuria, arterial hypertension, diabetes mellitus, and histological and immunofluorescent findings were collected. The patients' mean age was 42 ±15. The male sex prevailed (53.1%). Immunoglobulin A nephropathy was the most common finding (18.9%), followed by focal segmental glomerulosclerosis (16.3%), membranous glomerulonephritis (10.1%), lupus nephritis (8.4%), extracapillary glomerulonephritis (3.3%) and membranoproliferative glomerulonephritis (2.6%). In 69 (16.5%) patients the biopsy was non-informative or non-diagnostic. Patients with membranous nephropathy presented the highest frequency of nephrotic syndrome (71.4%), followed by membranoproliferative glomerulonephritis and focal segmental glomerulosclerosis. Combined analysis of the clinical, histopathological and immunofluorescent findings in glomerulonephritis patients based on a single center's data can provide important epidemiological findings.

RNA-HCV viral load in serum, peripheral blood mononuclear cells and liver in children with chronic hepatitis C.

The liver is the major site of hepatitis C virus (HCV) infection and replication. However, HCV may infect and replicate in extrahepatic sites as well. Several investigators have demonstrated that peripheral blood mononuclear cells (PBMCs) are the major extrahepatic milieu of infection and viral replication. The aim of the study was to investigate the correlation between RNA-HCV level in serum. PBMCs and liver in children with chronic viral hepatitis C (CHC). The impact of RNA-HCV level on the sustained virological response (SVR) after therapy was also determined. Study was carried out in the group of 10 children with CHC, age 8 to 17 years. Antiviral therapy was implemented in all patients with pegylated interferon alpha (Peg-lFNalpha) 2a or 2b and ribavirin during 48 weeks. The following tests were performed prior the therapy: basic laboratory parameters, histology of liver biopsy, RNA-HCV viral load in serum, PBMCs and in liver. The behavior of HCV-RNA viral load in serum, PBMCs and liver in children with CHC did not present strict mutual relations. However, the positive correlation between serum and PBMCs viral load (r = 0.47) and negative correlation between PBMCs and liver viral load (r = -0.47) was demonstrated. Although no statistically significant results were found, some trends of relationship in viral load between various body compartments were present. Given the aforementioned results, it is clear that more data are needed, mostly more numerous groups of patients, especially those whose influence of RNA-HCV viral load had a major impact on the antiviral treatment.

Treg cells in the course of chronic hepatitis C virus infection partially normalize in longitudinal observation after successful DAA treatment regardless of hepatic fibrosis stage.

AIM OF THE STUDY: Elevated circulating CD4+ CD25+ Foxp3+ regulatory T cells in patients with chronic hepatitis C (CHC) play an unspecified role in liver fibrosis development. This study aimed to determine whether Treg cells diminish after successful treatment with directacting antivirals (DAA) in patients at different liver fibrosis stages. MATERIAL AND METHODS: We examined 44 patients with CHC (including 29 with liver cirrhosis) seven days before DAA treatment (T0), six months later (T1) and then 22 of them were examined one year (T2) after the first dose. Subsequently, these were compared with 28 volunteers without hepatitis C virus (HCV) (15 with excessive alcohol intake). We assessed the degree of liver fibrosis with FibroScan, aspartate transaminase (AST) to platelet ratio index (APRI), FibroIndex, the Forns index and Fib-4. Circulating Treg cells were measured using flow cytometry. RESULTS: All patients achieved a sustained virological response (SVR). After the treatment, all liver fibrosis indicators decreased significantly. The number of circulating Tregs was lower in healthy controls than in patients with CHC (0.0066 × 103 cells/µl and 0.0084 × 103 cells/µl, respectively, p = 0.048). After the treatment we observed an insignificant change to 0.0047 × 103 cells/µl for T1 (p > 0.05) and a significant fall to 0.0041 × 103 cells/µl for T2 (p = 0.03). There was no correlation between the degree of hepatic fibrosis and number of Tregs or post-treatment dynamics. CONCLUSIONS: Our study shows that Treg cells normalize gradually over a prolonged period of time after a successful DAA treatment. Their number and dynamics remain independent of liver fibrosis degree. The correlation of this revelation with metabolic disorders, increased susceptibility to infections or persistent risk of HCC remains unclear.

Immunofluorescent evaluation of renal biopsy: current point of view.

Immunomorphologic assessment of percutaneous renal biopsy became a standard procedure for establishing diagnosis in kidney disease in parallel with routine haematoxylin and eosin stained paraffin sections. Among various immunomorphologic techniques, direct immunofluorescence of cryosections with a panel of fluorochrome labelled polyclonal or monoclonal antibodies to various serum proteins turned out to be the most reliable and rapid diagnostic procedure. The panel of antibodies may be expanded to include those to microbial or tumour antigens, when needed. The authors specify major advantages of immunofluorescence for such task and potential pitfalls in the case of nonspecific staining. In the next step, various types of fluorescence within renal structures are confronted with particular kidney disorders. Special attention is paid to various types of glomerulonephritis. Lesions in transplanted kidney are also discussed and the role of deposition of C4d complement component along peritubular capillaries is underlined as the evidence of humoral anti-graft reaction. The article is supplemented with a detailed technical procedure for performing of immunofluorescent reaction and evaluation of kidney biopsy, including several control steps.

Pattern Recognition Receptors: Significance of Expression in the Liver.

Pattern recognition receptors (PRRs) are a pivotal part of the immune system. They are distributed in almost every site of higher organisms, able to recognize foreign pathogens or unwanted remnants of metabolism and mount innate immune response. Moreover, PRRs create bridging signaling to initiate adaptive immunity. The liver being the largest organ of the body, exposed to myriads of foreign substances often being immunogenic, is well equipped with PRRs. They act as sentinels of the organ, both in health and disease. In viral hepatitis C at least two of them, RIG-1 and TLR3 sense HCV, induce protective interferon production and create proinflammatory status. The hepatitis B virus is apparently invisible to PRRs, which has recently been denied. Besides, they are active in the course of infection. In liver injury and hepatic fibrogenesis Toll-like receptors (TLRs), predominantly TLR4, TLR3 and TLR9 are associated with gut microflora-related products and DNA from dying hepatocytes, lead to the activation of hepatic stellate cells. The latter initiate production of fibrillar collagens, the main agents forming hepatic fibrosis. Tumor cells of primary liver cancer also express PRRs, mainly TLRs. In concert with non-resolving liver inflammation, they are considered pivotal factors leading to carcinogenesis.

Impact of chronic HCV treatment on quality of life of patients with metabolic disorders in context of immunological disturbances.

Chronic viral hepatitis C (CHC) and its complications have a negative effect on patient's quality of life. We evaluated the impact of a successful interferon-free treatment on the quality of life of patients with obesity and metabolic disorders in the context of immunological disturbances. Twenty overweight or obese (BMI > 25) patients with CHC were tested before the therapy and after a successful treatment regimen. After the therapy, patient's emotional well-being improved (p = 0.02), while physical well-being remained unchanged. There was a decrease of patient's liver fibrosis and an increase of steatosis along with body mass. Among HCV-infected individuals, the expression of toll-like receptor 3 (TLR3) on lymphocytes was higher than in the control group (p = 0.03), but it decreased (p = 0.001) after the treatment. There was also a decrease of the intensity of immunofluorescence of FoxP3+ after the treatment (p = 0.04). Our study showed an improvement in mental aspects of patient's quality of life after the treatment. Unfortunately, probably due to rapid immunological changes, patient's BMI, serum cholesterol levels and hepatic steatosis have a tendency to increase and may lead to cardiovascular and other complications, like hepatocellular carcinoma.

[Anti-HCV and HCV RNA in patients with the primary Sjögren syndrome]. / Anty-HCV oraz HCV-RNA u chorych z pierwotnym zespolem Sjögrena.

The subject of study were 104 patients with the primary Sjögren Syndrome (p. Sj. s.) in whom markers of hepatitis C infection were investigated. All the patients fulfilled the criteria of the European Expert Group of the Sjögren Syndrome. Antibodies anti-HCV were found in 20 patients (19.2%) and HCV-RNA found in 5 patients (4.8%). These data were compared with those observed in several European countries and Japan. The following percentages of anti-HCV were observed until now in p.Sj.s. patients: Swedish--2%, Hungarian--6%, Japanese--12%, French--17%, Polish--19% and Spanish--26%. Our patients in whom liver data were available, showed only minor elevations of ALT and AST. International team of experts postulated the delineation of the disease entity: 'HCV-related primary Sjögren syndrome', separate from the p.Sj.s. itself. If this will be substantiated, we can put forward the hypotesis that 'HCV-related p.Sj.s.' may develop in a special subgroup of persons, perhaps genetically predisposed, and is a part of extrahepatic manifestations of HCV infection.

Correction to: Significance and Role of Pattern Recognition Receptors in Malignancy.

The authors would like to correct the following error.

Significance and Role of Pattern Recognition Receptors in Malignancy.

Pattern recognition receptors (PRRs) are members of innate immunity, playing pivotal role in several immunological reactions. They are known to act as a bridge between innate and adaptive immunity. They are expressed on several normal cell types but have been shown with increasing frequency on/in tumor cells. Significance of this phenomenon is largely unknown, but it has been shown by several authors that they, predominantly Toll-like receptors (TLRs), act in the interest of tumor, by promotion of its growth and spreading. Preparation of artificial of TLRs ligands (agonists) paved the way to use them as a therapeutic agents for cancer, so far in a limited scale. Agonists may be combined with conventional anti-cancer modalities with apparently promising results. PRRs recognizing nucleic acids such as RIG-1 like receptors (sensing RNA) and STING (sensing DNA) constitute a novel promising approach for cancer immunotherapy.

[The role of NF-kappaB transcription factor in chronic viral hepatitis C and B]. / Rola czynnika transkrypcyjnego NF-kappaB w przewleklych wirusowych zapaleniach watroby typu B i C.

Essential data pertaining to the structure and function of selected transcription factors such as NFAT, AP-1 and NF-kappaB in particular, are presented in the relation to viral hepatitis C and B. In chronic hepatitis C the activation of NF-kappaB is markedly modulated by viral proteins such as core protein and nonstructural ones, particularly NS5A and NS3. In hepatitis B the major factor influencing NF-kappaB function appears to be HBx protein. Effects of viral proteins on NF-kappaB function in relation to the course of hepatitis are complex. They participate in the perpetuation of inflammatory state in the liver, inhibit apoptosis of hepatocytes, as well as the differentiation of antigen-presenting cells (dendritic ones). The latter effect has deleterious impact on the formation of specific immune response to viral peptides. It seems that both viruses, C and B acquired the ability to modify NF-KB function in advantage for the pathogens in question.

[Expression of toll-like receptors on blood leucocytes of children with chronic viral hepatitis C]. / Ekspresja receptorów toll-podobnych na krwinkach bialych krwi obwodowej u dzieci z przewleklym wirusowym zapaleniem watroby typu C.

OBJECTIVE: Assessment of the effect of anti-viral therapy in children affected by chronic hepatitis C on the expression of Toll-like receptors (TLR) on blood leucocytes METHODS: A cohort of children (n=24) infected with C virus with completed anti-viral therapy with interferon-alpha + ribavirin and another group of children HCV+ (n=23) awaiting for treatment entered this study. Three ml of blood was drawn from each child, divided on 100 ml aliquots and incubated with the panel offluorochrome labelled monoclonal antibodies versus differentiation antigens of leucocytes, anti - TLR2 and TLR4. After washing samples were subjected to acquisition in flow cytometer FACScan (Becton Dickinson). Data were analyzed by means of BD FACS Diva version 4.1.2 software. RESULTS: It was found that blood leucocytes of HCV+ children which completed anti-viral therapy, show significantly higher expression of TLR2 and TLR4. This rise was evident not only in percentage of cells, but also in mean fluorescence intensity (MFI). It was true in the case of granulocytes, T, B lymphocytes and monocytes, but the latter did not differ significantly from their counterparts derived from untreated children. CONCLUSION: Anti-viral treatment of children infected with C virus results in heightened expression of Toll-like receptors on blood leucocytes.

[The assessment of intravital staining of parotic gland using methylene blue in the surgery of benign gland tumors--preliminary report]. / Ocena przyzyciowego barwienia slinianki przyusznej blekitem metylenowym w operacjach lagodnych guzów gruczolu--doniesienie wstepne.

INTRODUCTION: The operation of parotic gland tumors are challenge for the surgeon. The main aim of these operations is tumor removal and facial nerve preservation. Despite of imaging examinations, showing the localization of the tumor, it's important for surgeon to have the opportunity to discriminate between tumor and normal parotic gland tissue during the operation, e.g. in color difference. Methylene blue (MB) has been used in intravital staining of various tissues for a long time. MATERIAL AND METHODS: The aim of the study was an attempt of intravital staining of parotic gland, using MB during the surgery of five benign gland tumors. RESULTS: In all cases parenchyma of the parotic gland was dark-blue stained and in all cases, while the tumor didn't show any staining. CONCLUSIONS: Using of MB may be useful in the surgery of parotic gland.

Genetic (KIR, HLA-C) and Some Clinical Parameters Influencing the Level of Liver Enzymes and Early Virologic Response in Patients with Chronic Hepatitis C.

Natural killer cells play an important role as effectors of innate immunity and regulators of adaptive immunity. They are important elements of the innate response to viral infections, which they detect using human leukocyte antigen (HLA) class I-binding receptors. Most polymorphic of these are killer cell immunoglobulin-like receptors (KIRs) which exist as two basic isotypes, activating or inhibitory receptors and are encoded by genes distributed differently in unrelated individuals. We searched for links between selected clinical data (including HCV viremia, liver enzymes level and liver histology parameters) and the presence of genes encoding these receptors and their ligands in hepatitis C virus-infected individuals subjected to pegylated interferon-α and ribavirin therapy. Genomic DNA samples from two hundred and ninety-two chronically infected patients were typed by polymerase chain reaction for the presence or absence of genes for KIRs and their ligands, class I HLA molecules, and clinical data of the patients were collected. Our results suggest an importance of clinical parameters and the contribution of KIR and HLA genes to the course of hepatitis C virus infection and the response to therapy. The study revealed that levels of liver enzymes before therapy were about 30% higher in patients who possessed a variant KIR2DS4 gene with 22-base pair deletion. Decrease of ALT activity after treatment was higher in HLA-C C2-positive than negative individuals. Beside it, patients demonstrated early virologic response to the therapy if the time lag before treatment was short, particularly in women.

Effect of aminoguanidine and albendazole on inducible nitric oxide synthase (iNOS) activity in T. spiralis-infected mice muscles.

The aim of this study was to provide evidence for the expression of iNOS in the cells of inflammatory infiltrates around larvae in skeletal muscles of T. spiralis infected mice. The BALB/c mice (n = 8) divided into subgroups, received either aminoguanidine (AMG)--a specific iNOS inhibitor or albendazole (ALB)--an antiparasitic drug of choice in trichinellosis treatment. Control animals (n = 2 in each subgroup) were either uninfected and treated or uninfected and untreated. Frozen sections of hind leg muscles from mice sacrificed at various time intervals after infection were cut and subjected to immunohistochemistry, using monoclonal anti-iNOS antibody. The ALB-treated mice revealed stronger iNOS staining in the infiltrating cells around larvae than the infected and untreated animals. On the contrary, in the AMG-treated animals, the infiltrating cells did not show any specific iNOS reaction. These data confirm the specificity of iNOS staining in the cellular infiltrates around T. spiralis larvae and shed some light on the role of nitric oxide during ALB treatment in experimental trichinellosis.

Flow cytometric analysis of CD55 and CD59 expression on blood cells in paroxysmal nocturnal haemoglobinuria.

PNH is a rare clonal disorder of hematopoietic stem cells, therefore all blood cells lineages are involved. The main feature is an increased sensitivity of erythrocytes to complement-mediated cell lysis due to deficiency of membrane-bound GPI (glycosylphosphatidylinositol)-anchored proteins which normally function as inhibitors of reactive hemolysis. In the present study, we performed flow cytometric analysis using monoclonal antibodies against CD55 and CD59 for the detection of PNH-type clone in the blood of 50 patients (28 females and 22 males, age range 7-67 yrs). In one patient only we found a large population (95%) of granulocytes with decreased expression of both CD55 and CD59 molecules (type I PNH) and in two others with partial loss of CD55 expression (type II PNH). The expression was determined chiefly on granulocytes which in the control group showed reliable and high expression of CD55 and CD59.

[Alterations within white blood cell subsets in children with chronic viral hepatitis C before treatment with pegylated interferon and ribavirin]. / Zmiany w obrebie subpopulacji krwinek bialych krwi obwodowej u dzieci z przewleklym zapaleniem watroby typu C przed leczeniem pegylowanym intereferonem i rybawiryna.

OBJECTIVE: Detection of differences in white blood cell subsets I children with chronic viral hepatitis type C before antiviral treatment. METHODS: A cohort of children (n = 52) with proven HCV infection were subjected to flow cytometric analysis of their white blood cells and compared to non-infected control group. MAIN OBSERVATIONS: It has been found that the hepatitis group has higher number of cells with cytotoxic potential, decreased CD4/CD8 ratio than the other one. RESULTS: Significant rise of CD8+, CD28- cells, NK cells and NKT lymphocytes was demonstrated in hepatitis group. Several correlations were noticed between various cell subsets studied in virus C infected children. CONCLUSIONS: This data show that HCV infection affects child immunity at systemic level. Cellular alterations are detectable by means of flow cytometry. Evaluation of its parameters might have predictive value in antiviral treatment.