Ochratoxin A and beta2-microglobulinuria in healthy individuals and in chronic interstitial nephropathy patients in the centre of Tunisia: a hot spot of Ochratoxin A exposure

Ochratoxin A (OTA) is a nephrotoxic mycotoxin considered to be the causal agent of the Balkan endemic nephropathy (BEN). In Tunisia, a chronic interstitial nephropathy (CIN) of unknown aetiology, resembling BEN, has been characterised wherein OTA seems to be implicated too. However, despite the considerable number of investigations conducted so far, the role of OTA in the outcome of this human nephropathy is still uncertain. In this study, an attempt is being made to consolidate the link between OTA and the Tunisian CIN of unknown aetiology. Blood OTA and beta(2)-microglobulinuria levels were measured in several groups of healthy individuals and patients having different renal diseases of known and unknown aetiologies (100 nephropathy patients and 40 healthy subjects). The high blood OTA and beta(2)-microglobulinuria levels seem to be strongly associated to the CIN of unknown aetiology. Our results support the involvement of this nephrotoxic agent in the outcome of this particular human nephropathy and underline furthermore the importance of beta(2)-microglobulinuria in the characterization of this disease.     

Comparative h NMR metabolomic urinalysis of people diagnosed with balkan endemic nephropathy, and healthy subjects, in romania and bulgaria: a pilot study

(1)H NMR spectroscopy of urine has been applied to exploring metabolomic differences between people diagnosed with Balkan endemic nephropathy (BEN), and treated by haemodialysis, and those without overt renal disease in Romania and Bulgaria. Convenience sampling was made from patients receiving haemodialysis in hospital and healthy controls in their village. Principal component analysis clustered healthy controls from both countries together. Bulgarian BEN patients clustered separately from controls, though in the same space. However, Romanian BEN patients not only also clustered away from controls but also clustered separately from the BEN patients in Bulgaria. Notably, the urinary metabolomic data of two people sampled as Romanian controls clustered within the Romanian BEN group. One of these had been suspected of incipient symptoms of BEN at the time of selection as a 'healthy' control. This implies, at first sight, that metabolomic analysis can be predictive of impending morbidity before conventional criteria can diagnose BEN. Separate clustering of BEN patients from Romania and Bulgaria could indicate difference in aetiology of this particular silent renal atrophy in different geographic foci across the Balkans.     

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.     

Epstein-Barr virus (EBV). V. Incidence of EBV antibodies in patients with rheumatic diseases

Serum samples from 95 patients with rheumatoid arthritis, 24 patients with other various rheumatic diseases, 50 patients with diabetes mellitus, 34 patients with acute viral infections, 6 patients with infectious mononucleosis, 77 patients with lymphomas and leukemia and 110 blood donors and 24 healthy subjects as normal controls, respectively, were tested by indirect immunofluorescence (IF) reaction for the presence of specific antibodies against Epstein-Barr virus determined viral capsid antigen (anti-VCA) and Epstein-Barr active viral infection. The IF test carried out in acetone-fixed smears of EB-3 cell line revealed EB antibodies anti-VCA in 83.3% of infectious mononucleosis, 61.0% lymphomas and leukemia, 58.0% diabetic patients. The frequency of anti-VCA antibodies in rheumatic patients was 31.4%, and 3.6% and 25% in sera from blood donors and healthy subjects, respectively. Incidence of active EBV infection was 5.7% of rheumatic diseases, 17.7% of acute virus infections, 50.0% of infectious mononucleosis, and 31.1% of lymphomas and leukemia patients. Active EBV infection was not found out in blood donors (0/110) and healthy subjects (0/24) groups as control. Rheumatoid arthritis with or without rheumatoid factor patients had serological evidence of active EBV infection 6/26 and 4/26 respectively.     

BK antibody and virus-specific IgM responses in renal transplant recipients, patients with malignant disease, and healthy people.

Haemagglutination-inhibition (HAI) antibodies to BK virus, including BK-virus-specific IgM, were determined before and after renal transplantation in 20 patients, in 57 patients with malignant disease, and in 66 healthy controls, Before transplantation 11 of the renal transplant recipients were seronegative, but eight later serocconverted, two before and six after transplantation. Twenty of the patients with malignant disease and 22 controls were also seronegative. The geometric mean titre of BK HAI antibodies was significantly higher among transplanted patients (1/180) than among controls (1/90). BK-virus-specific IgM antibody was detected in seven renal transplant recipients, six patients with malignant disease, and 13 healthy controls. In transplant recipients BK-virus-specific IgM antibody usually persisted throughout the duration of the study, and studies on controls from whom second serum samples were available suggested that they too had persistent BK-virus-specific IgM responses. The geometric mean titre of BK-virus-specific IgM HAI antibody was significantly greater in post-transplantation sera (1/223) than in control sera (1/28). The specificity of the detection of BK-virus-specific IgM HAI antibody was confirmed by direct visualisation of antibody by immune electron microscopy. The persistence of BK-virus-specific IgM suggested that BK virus continued to provide an antigenic stimulus. Nevertheless, there was no obvious association between the serological findings and any clinical features, and prospective studies will be needed to elucidate any such association.     

Studies on the presence of viral antibodies in patients with various forms of malignant neoplasia.

The study of viral antibodies in 6714 sera demonstrated that the level of antibodies to infectious viruses was the same in cancer patients as in controls. However, the patients with various forms of neoplasia showed a considerable percentage and high antibody levels to viruses with oncogenic potential for animals (adenovirus, SV40, Rous virus) or involved in human carcinogenesis (herpes virus).     

Possible relation between viruses and oromaxillofacial tumors. II. Research on the presence of the SV40 antigen and specific antibodies in patients with oromaxillofacial tumors

SV40 antigen was detected on sections through malignant oromaxillofacial tumors in 47.8% of the cases, with a higher incidence among the epithelium originating tumors. Anti-SV40 CF antibodies were detected in 83.6% of the examined subjects with cancer, but only in 57.9% of the blood donors. The titers were quite low in general. The results are discussed from the point of view of the immunologic status of the patients.     

Vaccination Strategies Against Hepatitis A in Travelers Older Than 40 Years: An Economic Evaluation

Background.  In recent years, the number of travelers aged >40 years who acquire hepatitis A while traveling has increased. Therefore, there is a need to review hepatitis A vaccination protocols in travelers. The aims of the study were to assess immunity levels to hepatitis A virus (HAV) in international travelers >40 years and to determine the least costly immunization strategy.
Methods.  A serological examination of HAV antibodies in 427 international travelers aged >40 years traveling endemic zones was carried out. The prevalence of antibodies in each age group was assessed. The costs of two preventive strategies, direct vaccination of all subjects (independent of the immune status) or screening and subsequent vaccination of susceptible subjects were compared. The critical value of prevalence (CVP) (the value at which the costs for the two strategies are equal) was calculated.
Results.  Total prevalence of HAV antibodies was 78.9% [95% confidence interval (CI): 74.8–82.5] and was 80.0% (95% CI: 73.8–85.2) in men and 77.9% (95% CI: 71.9–83.2) in women. There was a positive association with age. In the 40 to 49, 50 to 59, 60 to 69, and 70 to 95 years age groups, the prevalence rates were 62.6 (95% CI: 53.8–71.5), 76.8 (95% CI: 70.0–82.7), 91.7 (95% CI: 85.2–95.6), and 97.5% (95% CI: 87.4–99.6), respectively. The CVP was 58.4% using two doses of vaccine.
Conclusions.  The CVP was lower than the prevalence rate found in our international travelers. Therefore, we recommend systematic screening for HAV antibodies before selective vaccination of international travelers aged >40 years traveling to hepatitis A endemic zones.     

The role of lecithin cholesterol acyltransferase and organic substances from coal in the etiology of Balkan endemic nephropathy: a new hypothesis.

Balkan endemic nephropathy (BEN) occurs in Serbia, Bulgaria, Romania, Bosnia and Herzegovina, and Croatia. BEN has been characterized as a chronic, slowly progressive renal disease of unknown etiology. In this study, we examined the influence of soluble organic compounds in drinking water leached from Pliocene lignite from BEN-endemic areas on plasma lecithin-cholesterol acyltransferase (LCAT) activity. We found that changes for all samples were the most prominent for the dilution category containing 90% plasma and 10% of diluting media. Water samples from BEN villages from Serbia and Romania showed higher LCAT inhibiting activity (p=0.02) and (p=0.003), respectively, compared to deionised water and non-endemic water. A secondary LCAT deficiency could result from this inhibitory effect of the organic compounds found in endemic water supplies and provide an ethiopathogenic basis for the development of BEN in the susceptible population.     

Epstein-Barr virus (EBV). III. Incidence of EBV antibodies in patients with various tumors

Serum samples from 553 patients with various tumors, from 26 patients with different viral infections and from 78 clinically healthy subjects were tested by indirect immunofluorescence (IF) reaction for the presence of specific antibodies to Epstein-Barr virus. The test revealed antibodies to EBV in 127 patients with tumors, in 14 patients with viral infections and in 8 healthy persons.     

Epidemiology of Balkan endemic nephropathy.

The first outbreak of Balkan endemic nephropathy (BEN) was reported between 1955 and 1957, initially in Serbia and soon afterwards in Croatia and in Bosnia and Herzegovina. The disease appears to be of a focal nature. In Yugoslavia at least six foci are known, generally along major rivers of the Danubian river basin, in areas that have often been flooded in the past and even today suffer from high ground waters. The prevalence rate of the disease is reported to be between 2 and 10%. In the endemic area of Croatia, a systematic survey of 'in-the-field' cases of the disease since 1975 has shown a prevalence between 0.5 and 4.4%. When suspected cases are also included the prevalence rises to 20% or more. Specific mortality (based on official statistics) during the period 1957-1984 averaged 1.54/1000 per annum, but some studies have shown that mortality is actually more than twice as high as this figure. More women are affected than men; women also more frequently die of BEN than men. Lethality is extremely high. A striking feature of BEN is the familial occurrence of the disease. Incidence does not seem to be connected with ethnic group differences. Immigrants into the endemic area may also contract the disease. An increased incidence of malignant tumours of the urinary tract has been recorded in populations living in endemic areas. Epidemiological characteristics suggest that the disease is contracted in the domestic situation, or possibly from other family members. Factors to be considered are food, water or long close contact. It is also possible that the disease is contracted outside the house, in connection with farming activities, since the affected persons are almost exclusively farmers.     

Possible relation between viruses and oromaxillofacial tumors. III. Demonstration of the SV40 antigen and anti-SV40 antibodies in patients with tumors of the parotid gland

SV40 antigen was detected in 7 of 13 malignant tumors developed in the head and neck region. Specific complement fixing antibodies were found in all the patients with the SV40 antigen present in the parotid gland tumoral cells. Incidence of the anti-SV40 complement fixing antibodies in parotid gland tumor bearing patients was of 69.6%.     

Ochratoxin A and human chronic nephropathy in Tunisia: is the situation endemic?

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that is being increasingly considered as the main causal agent of Balkan endemic nephropathy (BEN), a fatal kidney disease associated with the end stage of urothelial tumours. However, despite the considerable amount of data, it is still controversial whether OTA plays a causative or only a subordinate role in the induction of this human nephropathy. Tunisia for years had to confront a very similar human nephropathy, which is tentatively called chronic interstitial nephropathy of unknown cause. This study tends firstly to consolidate the suspected link between this Tunisian chronic interstitial nephropathy (CIN) of unknown cause and the presence of OTA in the blood and food of such patients, and second to enlighten the endemic character of this particular nephropathy. Therefore, in four consecutive inquiries, performed within the period 1991-2000, blood and food OTA contaminations were assayed and compared for 954 nephropathy patients and 205 healthy subjects from the Tunisian general population. This survey was also designed to show that, although the whole population is likely to be exposed to OTA, specific people living in conditions showing similarities with the Balkans do have a kidney disease apparently linked to ochratoxin in food. The results showed that the highest incidences were found in patients with CIN of unknown cause. Indeed, the percentages of OTA-positive samples ranged from 93% to 100%, whereas it was only from 62% to 82% in healthy subjects. Mean OTA concentrations were also higher in patients with CIN of unknown cause than in controls (44.4 +/- 19 microg/L to 55.6 +/- 19 microg/L as opposed to 1.22 +/- 1.2 microg/L to 3.35 +/- 2.32 microg/L, respectively). This study emphasizes further the implication of OTA on this particular human nephropathy and underlines the probable causative role of OTA in the onset of this disease. It is important to note that the highest levels of food OTA contamination were found in the group presenting with CIN of unknown cause, indicating that, similar to the case in the Balkans, people are exposed to OTA essentially by their food.     

Ochratoxin A and β2-microglobulin in BEN patients and controls

Ochratoxin A (OTA) is a mycotoxin naturally occurring in different foods. OTA is arguably a risk factor for Balkan endemic nephropathy (BEN). The aims of this study are to (1) test the OTA-BEN association in BEN-groups and controls and (2) determine whether urine β2-microglobulin, a marker of impaired ability of the kidneys to re-absorb, is related to OTA. BEN patients had significantly higher OTA serum levels. Within the offspring, OTA was significantly related to higher β2-microglobulin excretion. OTA (2005/2006) was related to a higher incidence of BEN after 2008, providing further evidence that OTA is a risk factor for BEN.     

Laboratory investigations in Balkan endemic nephropathy

Serum samples from three patients with Balkan endemic nephropathy (BEN) were inoculated intraperitoneally to guinea pigs. After a very long incubation period (58-273 days) the animals developed an experimental disease characterized by apathy, tremor, convulsions and a fatal outcome. The disease could be serially propagated, the same clinical symptoms being recorded at each of the three passages performed. The morphological features of the experimental disease included glomerular and tubular lesions and the presence of interstitial fibrous and lymphoplasmocytic reactions. Different hypotheses on the etiology of BEN are discussed.     

Hodgkin's disease: in vitro susceptibility of skin fibroblasts to infection with virus and to lysis by autologous lymphocytes

Fibroblast cultures established from the skin of 16 untreated stage 1 and stage 2 Hodgkin's patients (HD) and 60 healthy controls were studied in their 3rd, 4th and 5th in vitro passage. HD cultures were similar to control cultures with respect to transformation with Simian sarcoma virus (SSV) and SV40 and with respect to interferon release. HD cultures, however, showed a depressed replication of both herpes simplex virus (HSV) type 1 and type 2. Preliminary evidence for enhanced immune interaction with fibroblasts in HD was a more frequent injury of HD than control fibroblast cultures when incubated 4 hours with autologous mononuclear leucocytes in the presence of autologous serum.     

The Role of SV40 in Malignant Mesothelioma and Other Human Malignancies

SV40 is a DNA tumor virus thrust upon human populations primarily as a contaminant in various vaccine preparations. Some estimates suggest that millions of people are currently infected with the virus. The virus causes primary brain tumors, bone tumors, lymphomas, and mesotheliomas when injected into some rodent models. It has also been detected in a similar spectrum of human tumors. However, epidemiological studies have failed to conclusively demonstrate a higher incidence of disease in affected populations. To date, over 60 reports from 49 different laboratories have shown SV40 sequences in tissues from human cancer patients. Six studies, however, have failed to detect evidence of virus in similar tissues. Some have suggested that SV40 may act as a cocarcinogen with asbestos to cause mesothelioma formation, or that it may be responsible for the 10–20% of mesotheliomas with no reported history of asbestos exposure. This report briefly covers the historical evidence for SV40 carcinogenesis and then covers experiments now underway to better understand the role of SV40 in human mesotheliomas.     

The role of SV40 in malignant mesothelioma and other human malignancies

SV40 is a DNA tumor virus thrust upon human populations primarily as a contaminant in various vaccine preparations. Some estimates suggest that millions of people are currently infected with the virus. The virus causes primary brain tumors, bone tumors, lymphomas, and mesotheliomas when injected into some rodent models. It has also been detected in a similar spectrum of human tumors. However, epidemiological studies have failed to conclusively demonstrate a higher incidence of disease in affected populations. To date, over 60 reports from 49 different laboratories have shown SV40 sequences in tissues from human cancer patients. Six studies, however, have failed to detect evidence of virus in similar tissues. Some have suggested that SV40 may act as a cocarcinogen with asbestos to cause mesothelioma formation, or that it may be responsible for the 10-20% of mesotheliomas with no reported history of asbestos exposure. This report briefly covers the historical evidence for SV40 carcinogenesis and then covers experiments now underway to better understand the role of SV40 in human mesotheliomas.     

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.