OCCURRENCE CHARACTERISTIC OF KASHIN-BECK DISEASE BASED ON NUCLEAR FAMILY PEDIGREES

Objective The occurrence characteristic of Kashin Beck Disease (KBD) in pedigrees ascertained on the basis of one proband was estimated. Methods A total of 255 individuals in 40 pedigrees were collected from areas in the Shaanxi Province. Results ① Parents and siblings of index cases have a 3-4 times higher risk than a random unrelated individual. The odds ratio for disease is higher in mothers than in fathers of index cases; ② Prevalence in relatives of index cases (K r= 59.2% ) greatly exceeds population prevalence (K= 17.5% ); ③ K r increases with sibship size; ④ There is no significant difference of K r for male and female siblings of index cases. Also, population prevalence is not sex specific. Conclusion In conjunction with environmental agents, genetics may play an important role in KBD etiology.

In vitro effects of sodium hyaluronate on the proliferation and the apoptosis in chondrocytes from patients with Kashin-Beck disease and osteoarthritis

Objective：To identify the in vitro effects of sodium hyaluronate（HA） on the proliferation and the apoptosis of chondrocytes from patients with Kashin-Beck disease（KBD） and osteoarthritis（OA）. Methods：Samples of articular cartilages from KBD and OA patients, as well as healthy volunteers（6 subjects in each of the 3 groups） were dissected, digested with collagenase and the cells cultured in monolayers. Chondrocytes from each sample were assigned to an untreated group and two HA-treated groups： H0（no HA）, H100（HA, 0.1 g/L） and H500（HA, 0.5 g/L）. The first passage chondrocytes were used to observe proliferation using the MTT assay, and apoptosis by flow cytometry through Annexin V/PI staining. Results：HA promoted proliferation of chondrocytes in all the three groups, and.in KBD and OA groups, for cells cultured for 4 and 6 days, H500 significantly promoted the cell proliferation. The apoptotic rates of both KBD and OA group chondrocytes were in the order H500 〈 HA100 〈 H0. Conclusion：Sodium hyaluronate administration has a dosedependent in vitro effect to promote proliferation and inhibit apoptosis of chondrocytes from patients with KBD and OA.

POSSIBLE HYDROGEOCHEMICAL PRECONDITIONS FOR KASHIN－BECK DISEASE IN TRANSBAIKALIA

In the basin of the Amur River at the territory of Russia, in the northern regions of China and Korea the osteal-articulate disease of people and animals, well known as Kashin-Beck disease is spread. About 20 hypotheses had been suggested for explaining the disease causes. some of them are briefly introduced. The hydrogeochemical researches in Transbaikalia. Russia, showed that natural water of the endemic areas have several features in chemical composition.some of them are considered by existing hypotheses as the causes of the disease.others do not concern the possible etiologic factors of the disease for the present. The author examines these features respectively.

A STUDY ON RELATIONSHIP BETWEEN ORGANIC SUBSTANCES IN WATER AND PREVALENCE OF KASHIN BECK DISEASE

This study is to acquire the relationship between organic substances in water and prevalence of Kashin Beck disease (KBD). We sampled in three endemic areas and their corresponding neighboring non endemic areas, determined the contents of organic substances and examined the children. The results showed that:①humus type B exited only in endemic areas; ②the content of humus type B is proportional to the state of the illness. KBD may be related to the humus type B in drinking water.

STUDIES ON BLOOD GLUTATHIONE PEROXIDASE PROTEIN LEVEL OF CHILDREN IN KESHAN DISEASE AND KASHIN-BECK DISEASE AREAS

Objective To oberve the change in blood glutathione peroxidase (GSH-Px) protein levels of residents in the low-selenium (Se) area by contrasting the blood GSH-Px protein level of the children in the Keshan disease area with those in the Kashin-Beck disease and non-endemic areas. Methods GSH-Px protein levels were measured by enzyme-linked immunosorbent assays (ELISA). The Se content and GSH-Px activity were assayed by the 2,3-diaminonaphthalene spectrofluorimetric method and glutathione reductase-coupled method respectively. Results ①The blood Se content and GSH-Px protein level of children in Keshan disease area (Moding) were significantly lower than those in Xi’an non-endemic area, however, there was no significant difference when compared with the low-Se non-endemic area; ②The blood Se content, GSH-Px activity and GSH-Px protein level of children in the Kashin-Beck disease area (Yulin) were significantly lower than those of children in two non-endemic areas and in the Keshan disease area; ③The blood Se content and GSH-Px activity were positively correlated to the GSH-Px protein level respectively. Conclusion These results indicate that the blood GSH-Px protein level is decreased in the low-Se residents. The Se status not only affects the GSH-Px activity but also regulate the GSH-Px protein level.

EFFECT OF SELENIUM ON IMMUNE FUNCTION OF ERYTHROCYTE IN KASHIN-BECK DISEASE

Objective To investigate the relationship between erythrocyte immune function and selenium (Se) level. Methods Forty-nine Kashin-Beck patients in endemic area aged 13-16 years were divided into two groups and were orally given either selenized yeast or sodium selenite to provide 200 μ g selenium per day for 12 weeks. Erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells complement receptor typeⅠ(CR1), the immune function of red blood cells, and circulating immune complexes(CIC) were determined. Results After supplementing with selenium for 12 weeks, erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells CR1 were significantly increased. But the difference in rosette formation rates of IC and CIC content was not significant between before and after Se supplementation. Conclusion The increase of the immune function of the erythrocyte by selenium-supplement may be one of the effective mechanisms for the prevention of Kashin-Beck disease.

STUDIES ON LYMPHOCYTE SUBSETS, HUMORAL IMMUNITY AND THEIR RELATIONSHIP WITH SELENIUM IN PATIENTS WITH KASHIN-BECK DISEASE

Objective To study the humoral immunity status and distribution pattern of lymphocyte subgroups of peripheral blood mononuclear cell (PBMC) in patients with Kashin-Beck Disease (KBD), and their relationship with erythrocyte selenium. Methods 23 X-ray diagnosed patients, 22 age- and sex- matched healthy children in KBD affected area (KAA), and 25 in KBD non-affected area (KNAA) were randomly selected. Immunohistochemistry with monoclonal antibodies anti-CD4, anti-CD8, anti-CD20 was conducted to analyze the lymphocyte subsets. Serum IgM, IgA, IgG, Complement C3 and C4 were assayed using rate nephelometry (Array 360 System, USA). The contents of erythrocyte selenium was determined by 2,3-diaminonaphthalene fluorescence assay. Results CD4+ and CD8+ cells percentage in PBMCs and serum IgA were significantly lower in KAA than those in KNAA(P< 0.05). CD20+ percentage in KAA displayed a decreasing trend compared to KNAA, although not statistically significantly. No statistical differences were found in CD4/CD8 ratio, serum IgG, IgM, C3 and C4 levels. Erythrocyte selenium level in KAA still showed a pronounced decrease compared to that in KNAA. Correlation analysis showed that erythrocyte selenium contents had a strong association with the CD4 cell percentage (r= 0.625, P< 0.05), as well as serum IgA (r= 0.462, P< 0.05). In addition, a moderate correlation between the serum IgA and CD4+ percentage (r= 0.130, P> 0.05) was found. Conclusion These results suggested that children in KAA had a comparably low cellular immunity level and their humoral immunity status was also in a state of moderate immune suppression. Of this immune disorder in Kashin-Beck disease patients, selenium deficiency probably played a critical role via affecting the distribution pattern of peripheral blood lymphocyte. Selenium-deficiency and immune impairment maybe both have something to do with the cause-effect chain of KBD.

EFFECTS OF CEREALS FROM KASHIN－BECK DISEASE ENDEMIC AREA ON FIBRILLOGENESIS IN VITRO OF CARTILAGE COLLAGEN TYPE Ⅱ IN RATS

In this feeding trial rats were fed on diets of cereals from Kashin-Beck Disease (KBD) endemic area, Se-supplemented cereals from the above area, and cereals from Non-KBD endemic area. The purpose of this paper was to investigate the effects of cereals from KBD endemic area and Se on the formation kinetics of cartilage type Ⅱ collagen fibril, the stability and ultrastructure of fibrils formed in vitro. The results indicated that low-selenium cereals from KBD endemic area resulted in a decelerated rate and extant of forming the type Ⅱ collagen fibril, the fibril stability reduced, fibril diameters diminished, and fibril banding periods increased or decreased in vitro. Sesupplemented cereals from KBD endemic area partially rectified the pathologic changes mentioned above. These data are important for further studying the etiology and pathology of KBD.

Effect of applying selenium fertilizer to improve soil and increase selenium level in food for prevention and treatment of Kaschin-Beck disease

For prevention and treatment of Kaschin-Beck disease by applying Se fertilizer to soil to increase Se up to normal level for the wheat grains is introduced in this paper. After use this measure the intake of Se by the residents in the exemplary area increased from 10.4μg to 33.6 μg per day in average. After supplementing Se to human body one year, there is no new patient found among 300 healthy children in the exemplary area but 4 patients found among 264 healthy children in the control area. It is shown that by applying Se fertilizer to KBD area is a effective way for preventing and cure this disease.

Study on correlation between humic acid and selenium in Kaschin-Beck disease areas

The relationship between humic acid and selenium content was studied in this research. The results of calculation showed that there is more humic acid and less selenium in Kaschin-Beck disease areas in China. Fulvic acid in soil could increase selenium level in plants. The chemical bonding between humic acid and selenium was observed with an I.R. spectroscope. It was remarkable for the changes of absorption peak and frequency in 1100-1000 cm-1, 900-700 cm-1.

A new hypothesis for the etiology of Kaschin-Beck disease

Kaschin-Beck disease (KBD) could be exogeneous free radical induced endemic osteoarthropathy under conditions of lacking necessary antioxidant defences.especially selenium-cored defence in Chinese eco-en-vironment. Experimental evidences have been provided for the internal relations among previously suggested pathologic factors, i.e., selenium deficiency, organic matters of drinking waterand mycotoxins from contaminated grains, based on the facts that the former is a preventory factor while the others are exogenous freeradical carriers. ESR spectrum shows that these exogenous free radical carriers have a property of semi-quinone compounds and are toxic in in-vitro cartilage cell culture and animal tests in inducing lipid peroxidation process.

The epidemic characteristics of Kaschin-Beck disease (KBD) in different eco-environments

Epidemiological survey of 573 families, clinical examination of 2593 persons, and X-ray examination of 1136 persons (in 16 typical endemic villages) were conducted, based on the classification of endemiology for KBD disease areas, i.e, mountain type, loess plateau type, plateau type, and flatlands type. It was revealed that the KBD disease areas exhibited the regular pattern of corming-into-being, development and passing-away and the characteristics of growth and decline. Further, it was found clinically that the disease areas may be divided into 4 types, i.e., recent onset, developing, stable, and historical. This division is simple and easy, practical, scientific, and reliable and can be applied by medical personnel at different levels.

Role of interaction between selenium and organic matters on causes of Kaschin-Beck disease

Influences of organic matters of soil and water on speciation and biological utilization of selenium are discussed. Humic acid is found to be the main pool of selenium in soil and affects the bio-availability of the element in soil-plant system. Fulvic acid from drinking water inhibits the absorption of selenium taken from diet. The relationship between high content of organic matters of water and selenium deficiency on the causes of KBD could be reflected in the limited synthesis of glutathione peroxidase, which is generally stimulated by fulvic acid from drinking water.

Humic acid and free radical in environment of Kaschin-Beck disease areas

The humic acid contents in drinking water and soil in Kaschin-Beck disease areas were found more than that of non-disease areas in this research. Changes of free radical concentration in drinking water were agreed with that of humic acid contents in drinking water of Kaschin-Beck disease areas. A positive correlation of free radical concentration and humic acid content in drinking water has been shown (r=0.913) . The structure of I. R. spectra of humic acid under ultraviolet light has been changed. Thus it indicated that free radical was resulted from benzoqiunonyl groups of humic acid in environment.

Redox potential E_H [Se(VI)/Se(IV)] of aqueous extractof soils from Yongshou Kaschin-Beck diseaseregion and the influence by humic acid

The relation between EH [Se(VI)/Se(IV)] and pH of soil aqueous extract in Kaschin-Beck disease region and the effects of natural redox agents, namely humic substances, MnO2 and Fe2+, on the redox property of the system were studied. The results indicated that both humic acid and Fe2+ could enhance the reducibility of Se(VI) and MnO2, a limited oxidizability for Se(IV). Fe2+ showed a weak reducibility only at low pH value. The reducibilities of three sulfur-containing compounds for Se(VI) were in following order:thioglycollic acid > L-cysteine > sulfide

A retrospective survey on the effect of Kaschin-Beck disease prophylaxis by a change of water sources

In order to provide further references for studing on the causes of Kaschin-Beck discase (KBD) and measuring for its prevention and treatment from a macroscopic view, we analyzed the natural growth and declineof KBD and the effects of selenium and humic acid on its occurrence from an epidemiologic angle. In this article through a retrospective survey on the spots of disease areas by comparison between a change in water sources and that without. It was proved that a change in water sources was an effective measure for the prevention of KBD occurrence, and the pathogenic factor of KBD was one (or several kinds) of organic compounds or active radicals related to water.

The roles of organic degradation products andselenium in the pathogenesis ofKaschin-Beck disease

An investigation on the organic degradation products and selenium in the pathogenesis of Kaschin-Beck disease was carried out. The results demonstrated that the organic degradation products existed in food and drinking water were the pathogens of the KBD; their leading pathological process of cells would be 'membrane injury' due to peroxide. As GSH-Px is a selenium contained enzyme, therefore insufficient selenium would be one of the most important conditions to cause KBD.

Research on organic matters in the drinking water of Kaschin-Beck disease area

Organic matters in drinking water of Kaschin-Beck disease areas were extracted. Then analyses and characterization were performed by means of multiply chemical and physical methods. The results did not show the obvious difference in the frame structure of humic substances and the structure of rmcromolecular compounds in the drinking water of disease and non-disease areas, but the difference in the contents of some micromolecular compounds and radicals. The investigation also includes the preliminary research on the photoreaction of drinking water from disease and non-disease areas and the accumulation of natural organic matter in the bone of tested animals.

Study on the action and mechanism of humic acids in Kaschin-Beck disease

Humic acids, especially FA in fractions, contain more oxygen functional groups. In this experiment, on the basis of confirming the action of humic acids on KBD, what was studied is the biological effects of one of main oxygen functional groups, hydroxy group (-OH). The results indicate that inducing pathologic process of KBD, obviously decrease the GSH-Px activity and induce peroxidation membrane injury of tissue. The SOD activity increase in the tissues caused by oxygen functional groups showed that enhancing of free radical reaction should not be neglected.

Immunology of Kaschin-Beck Disease:Studies on lymphocyte subsets, humoral immunity and their relationship with selenium

Objective To study the humoral immunity status and distribution pattern of lymphocyte subgroups of peripheral blood mononuclear cell (PBMC) in patients with Kaschin-Beck Disease (KBD), and their relationship with erythrocyte selenium.Methods 23 X-ray diagnosed patients, 22 age- and sex- matched healthy children in KBD affected area (KAA), And 25 in KBD non-affected area (KNAA) were randomly selected. Immunohistochemistry with monoclonal antibodies anti-CD4, anti-CD8, anti-CD20 was conducted to analyze the lymphocyte subsets. Serum IgM, IgA, IgG, Complement C3 and C4 were assayed using rate nephelometry (Array 360 System, USA). The contents of erythrocyte selenium was determined by 2,3-diaminonaphthalene fluorescence assay. Results CD4+ and CD8+ cells percentage in PBMCs and serum IgA were significantly lower in KAA than those in KNAA(P<0.05). CD20+ percentage in KAA displayed a decreasing trend compared to KNAA, although not statistically significantly. No statistical differences were found in CD4/CD8 ratio, serum IgG, IgM, C3 and C4 levels. Erythrocyte selenium level in KAA still showed a pronounced decrease compared to that in KNAA. Correlation analysis showed that erythrocyte selenium contents had a strong association with the CD4 cell percentage (r=0.625,P<0.05), and also a close relationship with serum IgA (r=0.462,P<0.05). In addition, we detected a moderate correlation between the serum IgA and CD4+ percentage (r=0.130, P>0.05).Conclusion Taken together, our results suggested that children in KAA had a comparably low cellular immunity level manifested by the marked depression of CD4 and CD8 cells percentage, and their humoral immunity status was also in a state of moderate immune suppression. Of this immune disorder in KBD patients, selenium deficiency probably played a critical role via affecting the distribution pattern of peripheral blood lymphocyte. Selenium-deficiency and immune impairment maybe both have something to do with the cause-effect chain of KBD.