Cold weather and Kashin-Beck disease

Kashin-Beck disease(KBD)is an endemic osteoarthropathy.Its distribution region covers a long and narrow belt on the Pacific side and belongs to continental climate with short summer,long frost period,and large temperature differences between day and night.In particular,KBD patients are typically scattered in the rural areas with seasonal features such as cold winters and rainy autumns.Etiological studies have demonstrated that the carrier of pathogenic factors is the grains produced in endemic areas.Risk factors for KBD include fungal contamination of grains due to poor storage conditions associated with cold weather.The epidemiological characteristics of KBD include agricultural area,early age of onset,gender equality,family aggregation,regional differences,and annual fluctuations.A series of preventive measures have been successfully taken in the past decades.National surveillance data indicate that the annual incidence of KBD is gradually declining.

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.

Evaluation of the Sensitivity and Specificity of the New Clinical Diagnostic and Classification Criteria for Kashin-Beck Disease,an Endemic Osteoarthritis,in China

This study aimed to evaluate the sensitivity and specificity of the new clinical diagnostic and classification criteria for Kashin-Beck disease （KBD） using six clinical markers： flexion of the distal part of fingers, deformed fingers, enlarged finger joints, shortened fingers, squat down, and dwarfism. One-third of the total population in Linyou County was sampled by stratified random sampling.

Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips

To understand how differentially methylated genes（DMGs）might affect the pathogenesis of Kashin-Beck disease（KBD）.Genome-wide methylation profiling of whole blood from 12matched KBD and controls pairs was performed using a high-resolution Infinium 450 K methylation array.In total,97 CpG sites were differentially

Changing Grains for the Prevention and Treatment of Kashin-Beck Disease in Children: a Meta-analysis

To evaluate the efficacy of changing grains on the prevention and treatment of Kashin-Beck Disease （KBD） in children, community-based trials were acquired from seven electronic databases （up to July 2014）. As a result, the methodological quality of the six trials that have been included into our analysis was low. The pooled ORs favoring the prevention and treatment effects of changing grains were 0.15 （95% CI： 0.03-0.70） and 2.13 （95% CI： 1.44-3.16） respectively by meta-analysis. Subgroup analysis demonstrated the pooled OR favoring treatment effect of exchanging grains rather than drying grains both compared with endemic grains. The results showed that changing grains had obvious effects on the prevention and treatment of KBD in children. However, the evidences were limited by the potential biases and confounders. Large and well-designed trials are still needed.

Crosstalk between CpG Methylation and Polymorphisms (CpG-SNPs) in the Promotor Region of DIO2 in Kashin-Beck Disease

Objective This study was designed to determine the methylation profile of four CpGs and the genotypes of two CpG-SNPs located in promoter region of DIO2 in patients with Kashin-Beck disease(KBD).We also analyzed the interaction between the CpGs methylations and CpG-SNPs.Methods Whole blood specimens were collected from 16 KBD patients and 16 healthy subjects.Four CpGs and two CpG-SNPs in the promoter regions of DIO2 were detected using matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS).The CpGs methylation levels were compared between samples from KBD patients and healthy subjects.The methylation levels were also analyzed in KBD patients with different CpG-SNP genotypes.Results The mRNA expression of DIO2 in whole blood of KBD patients was significnatly lower than in healthy controls(P<0.05).The methylation levels of DIO2-1_CpG_3 in KBD patients were significantly higher than those in healthy controls(P<0.05).The methylation levels of four CpGs were not significantly different between KBD patients and healthy controls.The methylation level of DIO2-1_CpG_3 in the promoter region of DIO2 in KBD patients with GA/AA genotype was significantly higher than that of KBD patients with GG genotype(P<0.05).Conclusion The methylation level of DIO2 increases in KBD patients.Similar trends exist in KBD carriers of variant genotypes of CpG-SNPs DIO2 rs955849187.

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.

Serum Metabolomic Indicates Potential Biomarkers and Metabolic Pathways of Pediatric Kashin-Beck Disease

Objective To explore potential serum biomarkers of children with Kashin-Beck Disease(KBD)and the metabolic pathways to which the biomarkers belong.Methods A two-stage metabolomic study was employed.The discovery cohort included 56 patients,51 internal controls,and 50 external controls.The metabolites were determined by HPLC-(Q-TOF)-MS and confirmed by Human Metabolome Databases(HMDB)and Metlin databases.MetaboAnalyst 3.0 and the Kyoto Encyclopedia of Genes and Genomes(KEGG)database were used to analyze the metabolic pathways of the candidate metabolites.The use of HPLC-(Q-TRAP)-MS enabled quantitative detection of the target metabolites which were chosen using the discovery study and verified in another independent verification cohort of 31 patients,41 internal controls,and 50 external controls.Results Eight candidate metabolites were identified out in the discovery study,namely kynurenic acid,N-α-acetylarginine,6-hydroxymelatonin,sphinganine,ceramide,sphingosine-1 P,spermidine,and glycine.These metabolites exist in sphingolipid,glutathione,and tryptophan metabolic pathways.In the second-stage study,five candidate metabolites were validated,including kynurenic acid,N-α-acetylarginine,sphinganine,spermidine,and sphingosine-1 P.Except for spermidine,all substances exhibited low expression in the case group compared with the external control group,and the difference in levels of sphinganine,spermidine,and sphingosine-1 P was statistically significant.Conclusion The direction of change of levels of sphinganine,spermidine,and sphingosine-1 P in the two-stage study cohorts was completely consistent,and the differences were statistically significant.Therefore,these substances can be used as potential biomarkers of KBD.Furthermore,these results raise the possibility that sphingolipid metabolic pathways may be closely related to KBD.

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.

Hyaluronic acid inhibited the upregulation of heat-shock protein 70 in human chondrocytes from osteoarthritis and Kashin-Beck disease

This study aimed to investigate the effect of hyaluronic acid(HA)on the expression of heat-shock protein 70(HSP70)in chondrocytes isolated from patients with osteoarthritis(OA)and Kashin-Beck disease(KBD).The chondrocytes were collected from OA and KBD patients,and chondrocytes isolated from patients of accident injuries were used as the control.The chondrocytes were treated with HA at different doses.HSP70 expression in chondrocytes at both mRNA and protein levels was tested by PCR and Western blot analysis.Compared with control,both mRNA and protein levels of HSP70 were higher in chondrocytes from KBD and OA.However,HA at the dose of 500μg/mL significantly inhibited HSP70 expression levels in both KBD and OA groups(P<0.05).In conclusion,HSP70 is highly expressed in chondrocytes of patients of OA and KBD.HA intervention inhibits the upregulation of HSP70 in chondrocytes of OA and KBD patients and could be a promising agent for treatment of OA and KBD.

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.

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.

Perturbations in Amino Acid Metabolism in Children with Kaschin-Beck Disease: A Study of Urinary Target Metabolomics

Kaschin-Beck disease (KBD) is an endemic and chronic osteoarthropathy characterized by pathological aspects including chondrocyte degeneration, necrosis, progressive loss of articular cartilage, and secondary degenerative osteoarthrosis of epiphyseal cartilage, epiphyseal plate cartilage, and articular cartilage, during puberty[1]. The main clinical symptoms are limb joint pain, thickening, deformation, limited movement, muscle atrophy, and in case of more severely affected patients, short fingers (toes), short limbs, and even short stature[1].

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.

陕西省大骨节病患者关节活动度及肌肉萎缩相关调查

目的描述陕西地区大骨节病(Kashin-Beck disease,KBD)患者膝、踝关节活动度(range of motion,ROM)和肌肉萎缩相关指标的分布特征,探究膝、踝ROM和肌肉萎缩指标的相关性,为KBD患者临床特征分析提供可参考依据。方法对来自陕西省KBD病区在册KBD患者进行抽样调查。采集KBD患者一般人口学资料,测量患者膝关节(屈曲、伸展),踝关节(背屈、跖屈)活动度以及患者上臂围、大腿围、小腿围和握力等肌肉萎缩相关指标。按照人群特征[性别、年龄、体质量指数(BMI)]和KBD分度分组,报告KBD患者ROM和肌肉萎缩指标的中位数和四分位数,分析不同组间各指标的差异。控制性别、年龄和BMI等变量后,采用偏相关分析探索各指标间的相关性。结果本研究共调查KBD患者480例,其中男性249(51.9%)例,女性231(48.1%)例,平均年龄(63.10±7.32)岁,平均BMI(23.49±8.90)kg/m^(2)。结果显示,KBD患者膝关节屈曲活动度[105.0(95.0,120.0)]°,膝关节伸展活动度[0.0(-15.0,0.0)]°,踝关节背屈活动度[5.0(0.0,15.0)]°,踝关节跖屈活动度[20.0(15.0,30.0)]°,左侧大腿围[43.0(40.0,47.0)]cm,右侧大腿围[43.0(39.0,47.0)]cm,左侧小腿围[29.0(27.0,32.0)]cm,右侧小腿围[29.5(27.0,32.0)]cm,左侧和右侧上臂围均为[27.0(25.0,30.0)]cm,左手握力[13.4(9.5,18.4)]kg,右手握力[13.9(9.8,18.2)]kg。随着年龄的增加,KBD患者两侧膝关节伸展活动度呈现降低趋势(H=31.499、31.847;P均<0.001)。BMI正常组两侧膝关节屈曲活动度高于超重或肥胖组,差异均有统计学意义(H=7.753、12.333;P=0.021、0.002)。不同KBD分度下,两侧膝关节屈曲、大腿围和小腿围均呈现降低趋势(H=14.345、17.256、8.000、8.462、8.558、9.633;P均<0.05)。相关性分析显示,KBD患者膝关节屈曲活动度与大腿围、小腿围和握力呈正相关关系(P均<0.05)。KBD患者膝关节伸展活动度与大腿围呈正相关关系(P均<0.01)。结论陕西省KBD患者ROM受损和肌肉萎缩较为严重,且ROM与肌肉萎缩存在相关关系。

大骨节病病因病理学研究

目的寻找并认定大骨节病(Kashin-Beck disease,KBD)的病因。方法复查本所KBD剖检档案16例的骨切片。光镜观察KBD患儿末梢血涂片中白细胞的病变,电镜观察患儿白细胞的超微病变。观察KBD患儿血浆引发培养兔软骨细胞损伤的实验结果。结果KBD剖检例的软骨细胞、骨髓血细胞及KBD患儿的白细胞,这三类细胞病变的特征及其全过程完全一致,均显示特异的凝固性坏死,细胞核增大,核内出现由少到多、由小到大的嗜酸性(红色)包涵体,伴随以细胞核染色质的溶解减少;若病变持续恶化进展,则整个细胞核可转化成一个红染的大团块;其后出现该包涵体的后续性系列变化;细胞体缩小,细胞质红染。患儿血浆引发培养兔软骨细胞复制出等同于剖检例软骨细胞特异坏死病变的细胞模型,且在其细胞核内检可见病毒核衣壳。在患儿白细胞的核内亦可见相同的病毒核衣壳。在剖检例的骨髓内可见充血、水肿、纤维素渗出,造血主质和骨小梁的灶性坏死以及骨髓纤维化等。结论在KBD剖检例病变的软骨细胞和骨髓血细胞的核内及KBD病患儿末梢血白细胞的核内均显示相同的包涵体形成;包涵体形成是病毒杀细胞性感染最为熟知的病理形态学结果;特别是以KBD患儿血浆作用于培养软骨细胞所出现的阳性实验结果,均提示该三类细胞坏死的病因理应是两次检出的同一病毒,该病毒就应是KBD的病因。剖检例的骨髓内有急性骨髓炎伴发传染性迟发型超敏反应。KBD是该病毒引起的全身性传染病;软骨病变、骨髓病变和血液病变都仅是其全身病变的一部分。

Soil selenium concentration and Kashin-Beck disease prevalence in Tibet,China

In order to investigate the correlation between the prevalence of Kashin-Back disease(KBD)and Se concentrations,natural soil samples and cultivated soil samples were collected from southeastern Tibet,China;and the soil Se concentrations were measured by atomic fluorescent spectrophotometer.It was found that the mean Se concentrations of natural soil samples in KBD areas,from the first layer to the third layer,were 0.17 mg/kg,0.11 mg/kg,and 0.10 mg/kg,respectively,and in nondisease areas were 0.21 mg/kg,0.24 mg/kg,and 0.13mg/kg,respectively.The mean Se concentrations of cultivated soil samples were 0.10 mg/kg in KBD areas and 0.23 mg/kg in non-disease areas,respectively.Soil Se concentrations in KBD areas were lower than that in non-disease areas,and the mean concentrations of soil Se in Tibet were lower than the average of China(0.29 mg/kg).Therefore,there is a close relationship between soil Se concentrations and KBD in Tibet.More studies should be concentrated on the impacts of Se deficiency in soils and its relationship with Se concentrations in food-grain and the human body in Tibet areas.

X-ray image characteristics and related measurements in the ankles of 118 adult patients with Kashin-Beck disease

Background Kashin-Beck disease （KBD） is a chronic,degenerative osteoarthropathy that causes severe skeletal deformation.Although many researchers have proven that almost all KBD patients who showed an increaseing proximo-distal gradient had radiographic abnormalities of the ankle,few detailed description of radiographic changes in the ankles of patients with KBD has been reported,especially for variable measurements of ankle changes.The purpose of this study was to demonstrate the radiographic characteristics of the ankles of adult KBD patients.Methods One hundred and eighteen adult KBD patients from september to October 2010 in Rongtang county in China were examined with lateral radiographs of the right ankle.The morphological abnormalities in the talus,calcaneus,navicular bone,distal tibia,and joint space were analyzed,and the calcaneus length,height,length-height ratio,tuber angle,front angle,plantar angle,and distal tibia anteroposterior （AP） length were measured using Riepert＇s method.Results Eighty-one patients （68.6％） had abnormal ankle radiographs; 72 （88.9％） patients had talus changes,69 （85.2％） patients had calcaneus changes,28 （34.6％） patients had navicular bone changes,and 48 （59.2％） patients had distal tibia changes.For 118 KBD patients,the average calcaneus length was 7.4 cm,height was 4.3 cm,and the length-height ratio was 1.7.The calcaneus tuber angle was 28.2°,front angle was 38.0° and the plantar angle was 74.2°.The distal tibia anteroposterior length was 4.05 cm.Compared with 50 normal adults （control group）,significant differences were found for the calcaneus length,the calcaneus length-height ratio,and the distal tibia AP length.Conclusions Patients with KBD have characteristic abnormalities on ankle radiographs; talus depression and deformity,calcaneus shortening deformity,and distal tibia deformity with AP length widening were the most typical changes.

Relationship between selenium concentration in child hair and the distribution of Kashin-Beck Disease in Tibet, China

The focus of this study is to determine the relationship between selenium(Se)concentrations in child hair and the distribution of Kashin-Beck disease(KBD)in Tibet.The hair of children in typical KBD-affected areas and non-disease areas was collected,and its Se concentrations were measured by atomic fluorescent spectrometry.The Se concentrations in child hair were(0.13±0.04)and(0.18±0.07)μg/g respectively in KBD areas and non-disease areas respectively,which are significantly different.In areas supplemented with Se in Tibet,the Se concentration of hair in KBD-affected areas was higher than that in non-diseased areas,being(0.28±0.03)and(0.18±0.04)μg/g respectively.There is a close relationship between the Se concentrations in hair and KBD distribution.Se supplement increase the Se concentrations in the human body.Appropriate Se supplementation is important to prevent KBD in Tibet.