The role of HLA in Balkan endemic nephropathy.

Balkan endemic nephropathy (BEN), a progressive chronic tubulointerstitial disease, occurs in the endemic focus of Croatia in a population of about 10,000 inhabitants. One of its most peculiar characteristics is a strong association with upper tract urothelial carcinoma (UTUC). Despite a high number of studies, currently there are insufficient data about the association of BEN and HLA genes. The aim of this study was to investigate the polymorphism of HLA-A, -B, and -DRB1 alleles and haplotypes among BEN patients and to determine whether an association between HLA and BEN exists. In this study, we investigated HLA-A, -B, and -DRB1 alleles and haplotypes in a population of patients with BEN (N = 111) and matched healthy controls (N = 190). All individuals were tested by PCR-SSO and PCR-SSP methods to assess the possible contribution of HLA alleles and haplotypes to the development of/protection from BEN. Our results showed a positive association between the presence of HLA-B*35:02 and DRB1*04:02 alleles and BEN (P = 0.0179 and P = 0.0151, respectively) in contrast to the protective effect of HLA-A*01:01, B*27:05 and B*57:01 alleles (P = 0.0111, P = 0.0330 and P = 0.0318, respectively). Moreover, when BEN patients' HLA haplotypes were compared to controls, two haplotypes were associated with BEN susceptibility among Croatians (HLA-A*02:01~B*08:01~DRB1*03:01 and HLA-A*02:01~B*27:02~DRB1*16:01, P = 0.0064 and P = 0.0023, respectively), while haplotypes HLA-A*02:01~B*27:05~DRB1*01:01 and HLA-A*02:01~B*38:01~DRB1*13:01 each showed a possible protective effect (P = 0.0495). Our results point toward genetic susceptibility to BEN and observed differences in both susceptible/protective HLA profiles indicate the necessity of further studies in order to elucidate the pathogenesis of this disease.

Immunological studies in Balkan Endemic Nephropathy.

AIM: To re-examine serum complement and immunoglobulin levels in patients with Balkan Endemic Nephropathy (BEN) in the early stage of the disease; to study autoantibodies (anti-thyroid, anti-smooth muscle, anti-mitochondrial, anti-parietal, anti-nuclear, and anti-DNA) in these patients; and, finally, to re-asses the immunohistology in kidney biopsies from BEN patients. A review of the immunological studies in BEN will be presented. METHODS: Forty-five BEN patients in the early stage of disease, from the South Morava Region, were included in this study. Fifty-five apparently healthy controls, matched for age and sex, from the neighbouring control villages were selected. Serum complement and immunoglobulin levels were determined by the Immunochemistry system of Beckman. Auto-antibodies were detected in the sera by the indirect fluorescence method or by radio-immunoassay (anti-single stranded DNA). Immunofluorescent microscopy was used for kidney samples treated with FITC conjugated antisera for IgG, IgM, IgA, C3, C1q, C4 and fibrin/fibrinogen. RESULTS: Serum C3 was decreased in the sera of patients with BEN compared to healthy controls (p < 0.001), serum IgM was also decreased (p < 0.05). Anti-thyroid antibodies were detected in 5/45 BEN patients, anti-parietal in 7/45 patients and ANA in 2 BEN patients. No anti-smooth muscle, anti-mitochondrial, or anti-DNA antibodies were detected in any BEN patient. Immunofluorescent studies of 10 kidney biopsy specimens showed rare, unspecific, focal, mesangial deposits of C3 and IgM in some glomeruli, and rare deposits, mostly of C3, in tubuli and extraglomerular vessels. CONCLUSION: Humoral immune mechanisms do not appear to play a pathogenetic role in BEN. A few studies on cell-mediated immunity in BEN were performed, and further studies are needed on patients in the early stage of the disease.

Urinary neopterin concentrations in patients with Balkan endemic nephropathy (BEN).

BACKGROUND: Balkan endemic nephropathy (BEN) is of great clinical importance in restricted areas of Bulgaria, Serbia, Croatia, Bosnia, Herzegovina, and Romania, since the etiology of BEN is still unknown. METHODS: In urine samples from 48 patients (41 females and 7 males, aged 65.6 +/- 6.87 years) with BEN living in an endemic area of Vratza district, Bulgaria, neopterin concentrations were measured by high-pressure liquid chromatography (HPLC) and compared with other clinical and laboratory investigations, including creatinine, hemoglobin, and erythrocyte sedimentation rates (ESRs). RESULTS: Urinary neopterin concentrations were 263 +/- 128 (mean +/- SD; range, 78 to 786 micromol/mol creatinine), 24 (50%) of BEN patients presented with increased concentrations as compared to the established normal ranges. Average ESRs were increased (1 hour, 29.0 +/- 14.7 mm/hour) and hemoglobin was decreased (109.3 +/- 16.4 g/L). Hemoglobin correlated inversely with ESRs (rs = -0.787 and -0.780) and creatinine concentrations (r = -0.690, all P < 0.001), but not with neopterin concentrations. Neopterin concentrations also did not correlate with serum creatinine levels. There existed an age relationship of ESR, creatinine, and hemoglobin, but not of neopterin. Neopterin concentrations were slightly lower in five females with low titers of antibodies against local B1 hantavirus strain (P < 0.05). CONCLUSION: The findings can support an immune-mediated inflammatory process in the pathogenesis of BEN only in a subgroup of patients.

The level of interleukin-2 and of soluble interleukin-2 receptor in patients with Balkan nephropathy.

Balkan Nephropathy (BN) is defined as a clinical entity with unknown etiology. The involvement of immune system in pathogenesis of BN is not well defined yet. The aim of this study was to gain more insight into the cellular immune mechanisms in BN. We determined some factors implied in cellular immunity, such as the serum level of IL-2 and of IL-2 soluble receptor (sIL-2R), and the presence of IL-2 receptor alpha chain (CD25) on T cells membrane. The study was performed on 15 patients with BN, 15 patients with Chronic Pyelonephritis (CPN), and 10 healthy controls from a non-endemic area. Our study showed no significant differences between IL-2 level and CD25+ cells percentage in CPN compared to controls, but a significantly increased level of sIL-2R. The BN sIL-2R is significantly lower than sIL-2R in CPN, and associates an important T cell activation (high CD25+ presence, elevated IL-2 level) compared to CPN. Our conclusion is that while the high sIL-2R level could down modulate T cell activity in CPN, BN sIL-2R level is ineffective in limiting the activation effects of IL-2 on T cells. The results suggest that cellular immunity could have a role in the pathogenesis of N.

Isolation of a coronavirus from kidney biopsies of endemic Balkan nephropathy patients.

Endemic Balkan nephropathy (EBN) is a kidney disease of unknown etiology limited to Bulgaria, Rumania and former Yugoslavia. Primary kidney tissue cultures were established as explants from tissue obtained at operations from 5 EBN patients with urinary tract tumors. Four out of the five biopsy specimens on extended culture incubation at 33 degrees C yielded a coronavirus virus (EBNV) which was cytopathogenic for human fibroblast and Vero cells. In cells inoculated with EBNV, cytoplasmic immunofluorescence was found using antisera for human coronaviruses OC43 and 229E as well as the porcine transmissible gastroenteric virus and avian (chicken) bronchitis virus. In neutralization tests, EBNV failed to react with antisera to these viruses. Using hyperimmune serum raised with EBNV, positive cytoplasmic immunofluorescence was seen with cells infected with OC43, 229E, TGV and significantly with the kidney tissue of the biopsy specimens from the EBN patients. A screen for neutralizing antibody using the EBN virus revealed that 87.2% of EBN patients on dialysis were positive, also 74% of people from an endemic area were also positive, while only 13.5% from outside were positive. It is suggested that a coronavirus is involved in the etiology of the disease and that humans are an incidental host of a coronavirus zoonosis.

Identification of a novel subset of T lymphocytes in patients with Balkanic nephropathy.

Balkanic Nephropathy (BN) is characterized by: an incidence limited to a geographic area: a familial character and a slow progressive evoluation towards chronic renal failure associated with the symmetrical reduction of the kidney size. The etiology of BN is unknown. The aim of our study was to find out the immune alterations in BN pathology. In the BN patients we identified a novel subset of the CD3+ CD16+ and CD56+ T cells expressing the phenotypic characteristics of both T lymphocytes and NK cells. The analysis of various subpopulations of lymphocytes, however, showed no quantitative differences in comparison with healthy subjects and healthy subjects from the endemic area.

CD3+, CD16+, CD56+ cells: a novel subset of T lymphocytes from patients with Balkanic nephropathy.

Balkanic Nephropathy (BN) is characterized by: an incidence limited to a geographic area; a familial character and a slow progressive evolution towards chronic renal failure associated with the symmetrical reduction of the kidney size. The aim of our study was to find out the immune alterations in BN pathology. The analysis of various subpopulations of peripheral lymphocytes obtained from patients with BN showed no quantitative differences in comparison with healthy subjects and healthy subjects from the endemic area. In the BN patients we identified a novel subset of the CD3+, CD16+, CD56+ cells expressing the phenotypic characteristics of both T lymphocytes and NK cells.

Circulating inhibitor of interleukin 1 activity in patients with chronic renal diseases.

The effects of sera derived from patients suffering from chronic renal diseases (endemic Balkan nephropathy, glomerulonephritis and pyelonephritis) on T cell proliferative response was studied. It was found that these sera contained factors which affect interleukin 1 (IL-1) dependent events in T cell proliferative response. The factors prevent costimulatory effects of IL-1 on T cells but do not bind to IL-1, nor do they affect interleukin 2 (IL-2) dependent T cell proliferation. These findings indicate that immuno-suppression observed in some kidney disorders may be partially due to serum immunoinhibitory factors affecting IL-1 activity.

Renal function, protein excretion, and pathology of Balkan endemic nephropathy. IV. Immunohistology.

Few controversial observations on the deposition of immunoglobulins (Igs) and complement components in kidney biopsies of Balkan endemic nephropathy (BEN) patients have been reported. In the present study, direct or indirect immunofluorescence testing of the deposition of IgA, IgG, IgM, C3, C1q, C4 fibrin/fibrinogen, albumin, B2-microglobulin (beta 2m) and Tamm-Horsfall glycoprotein (THG) on frozen renal tissue sections was performed in 52 BEN patients. Glomerular findings were negative or mostly insignificant, with mild or moderate mesangial deposition of IgM in 16, IgA in 11, IgG in three, C3 in 15, C1q in two, C4 in one and fibrin/fibrinogen in two cases, respectively. The predominance of mesangial IgA deposits in five cases suggested IgA glomerulonephritis (GN) concomitant with BEN. Homogeneous lumpy or granular deposits in small extraglomerular vessels contained IgM in nine, C3 in 45, C1q in three, and C4 in one case. Focal linear or granular C3 was noted along the tubular basement membrane in eight cases. Resorptive droplets in tubular epithelial cells contained Igs and albumin, while complement, fibrin/fibrinogen and THG were found in tubular casts. There was no positive reaction with anti-beta 2m and anti-THG antibodies. According to these results, humoral immune mechanisms would not appear to play a pathogenetic role in BEN. However, immunohistologic examinations are important in recognition of possible concomitant immune complex-mediated GN.

[Immunosuppressive factors in the blood in patients during the development of various kidney diseases]. / Imunospresivni cinioci u serumu bolesnika tokom evolucije razlicitih bubreznih oboljenja.

Sera of patients suffering from Balkan nephropathy, pyelonephritis and glomerulonephritis inhibit lectin-induced T-cell proliferation in vitro. Immunosuppressive factors were registered in the patients' sera during the early stage of the existing disease, and their activity was not in correlation with the degree of renal insufficiency. Serum inhibitors revealed their activity during the early phase of T-cell activation and had no effect on T-cell proliferation and DNK synthesis. Inhibitor activity was registered even if sera were not present in cell culture continuously but only 6 hours of preincubation; then their action was irretrievable.

Immunological investigations in inbred mice chronically infected with cytopathogenic agents isolated from human cases of Balkan endemic nephropathy.

An experimental chronic infection was induced in inbred A2G and CBA mice by repeated administration of four cytopathogenic agents isolated from human cases of Balkan endemic nephropathy (BEN). The slight humoral and cell-mediated immune responses to the BEN agents recorded in A2G mice were associated with autoimmunity and hypersensitivity phenomena. In contrast, the normal, intense immune response observed in CBA mice was not accompanied by any immunopathological changes. In both mouse lines the chronic infection with human BEN agents led to a secondary immune deficiency against sheep red blood cells or tuberculin.

Virological and immunological study of 20 patients with Balkan endemic nephropathy.

A virological and immunological study was carried out in 20 cases of Balkan endemic nephropathy (BEN) belonging to two distinct geographical areas of a Romanian county. Cytopathogenic agents could be isolated in the BHK-21 cell line from 14 urine samples and from one kidney biopsy specimen. Sera from BEN patients gave autologous and heterologous neutralization reactions against several cytopathogenic agents, within the same or from the other geographical area. Humoral and cell-mediated immunity tests against standard antigens and one of the cytopathogenic agents revealed a general preservation of immune reactivity in BEN patients.

Serum antibodies to papova viruses (BK and SV 40) in subjects from the area with Balkan endemic nephropathy.

The presence of antibodies to BK virus and SV40 was investigated in 63 patients with Balkan endemic nephropathy (BEN) and in 83 apparently healthy subjects from the endemic area. Serum antibodies to BK virus were detected in 95.2% of the former and in 74.7% of the latter, high antibody levels being prevalent in the age groups 41-60 years. Antibodies to SV40 were absent in the BEN patients and their frequency in the healthy subjects (27.7%) was much lower than that previously recorded in healthy persons from other zones of Romania (40%). The results obtained plead for a prevalence of BK virus infection in the endemic area with BEN.

Immunologic studies in patients with glomerulonephritis.

Blood levels of E-rosette forming cells, HbsAg, C3 and immunoglobulins G, A and M were determined in a series of 101 patients with different forms of glomerulonephritis. Cell-mediated immunity to glomerular basement membrane (GBM) antigens and renal tubular epithelial (RTE) antigen was studied in 39 patients with glomerulonephritis and in 53 patients with Balkan nephropathy (BEN). The patients with glomerulonephritis had statistically higher serum levels of IgG, IgA and IgM than normal individuals. Decreased serum C3 values were recorded in 40.6 per cent of the patients and circulating immune complexes (CIC) were detected in 47.5 per cent; 6.9 per cent of the patients were HBsAg positive. The patients' levels of E-rosette forming cells were significantly lower than those of healthy controls. Cell-mediated immunity to GBM was recorded in 53.8 per cent of the patients with chronic glomerulonephritis and to RTE-antigen in 58.5 per cent of the patients with BEN. These changes are interpreted as being a manifestation of an abnormal immune response, characterized by disorders of T lymphocyte function and activation of B lymphocytes. Evidence for this includes the presence of CICs, the low C3 levels the high number of positive tests for HBsAg.