Cholesterol crystal binding of biliary immunoglobulin A: visualization by fluorescence light microscopy

AIM: To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA. METHODS: Crystal binding biliary proteins were extracted from human gallbladder bile using lectin affinity chromatography.Biliary IgA was isolated from the bound protein fraction by immunoaffinity chromatography. Pure cholesterol monohydrate crystals were incubated with biliary IgA and fluoresceine isothiocyanate (FITC)conjugated anti IgA at 37 degree. Samples were examined under polarizing and fluorescence light microscopy with digital image processing. RESULTS: Binding of biliary IgA to cholesterol monohydrate crystals could be visualized with FITC conjugated anti IgA antibodies.Peak fluorescence occurred at crystal edges and dislocations. Controls without biliary IgA or with biliary IgG showed no significant fluorescence. CONCLUSION: Fluorescence light microscopy provided evidence for cholesterol crystal binding of biliary IgA. Cholesterol crystal binding proteins like IgA might be important mediators of crystal agglomeration and growth of cholesterol gallstones by modifying the evolving crystal structures in vivo.

Evaluation of a Direct Rapid Immunohistochemical Test (dRIT) for Rapid Diagnosis of Rabies in Animals and Humans

Presently the gold standard diagnostic technique for rabies is the direct immunofluorescence assay （dFA） which is very expensive and requires a high level of expertise. There is a need for more economical and user friendly tests, particularly for use in developing countries. We have established one such test called the direct rapid immunohistochemical test （dRIT） for diagnosis of rabies using brain tissue. The test is based on capture of rabies nucleoprotein （N） antigen in brain smears using a cocktail of biotinylated monoclonal antibodies specific for the N protein and color development by streptavidin peroxidase-amino ethyl carbazole and counter staining with haematoxollin. The test was done in parallel with standard FAT dFA using 400 brain samples from different animals and humans. The rabies virus N protein appears under fight microscope as reddish brown particles against a light blue background. There was 100 % correlation between the results obtained by the two tests. Also, interpretation of results by dRIT was easier and only required a light microscope. To conclude, this newly developed dRIT technique promises to be a simple, cost effective diagnostic tool for rabies and will have applicability in field conditions prevalent in developing countries.

Preconditioning crush increases the survival rate of motor neurons after spinal root avulsion

In a previous study, heat shock protein 27 was persistently upregulated in ventral motor neurons following nerve root avulsion or crush. Here, we examined whether the upregulation of heat shock protein 27 would increase the survival rate of motor neurons. Rats were divided into two groups： an avulsion-only group （avtflsion of the L4 lumbar nerve root only） and a crush-avulsion group （the L4 lumbar nerve root was crushed 1 week prior to the avulsion）. Immunofluores- cent staining revealed that the survival rate of motor neurons was significantly greater in the crush-avulsion group than in the avulsion-only group, and this difference remained for at least 5 weeks after avulsion. The higher neuronal survival rate may be explained by the upregulation of heat shock protein 27 expression in motor neurons in the crush-avulsion group. Further- more, preconditioning crush greatly attenuated the expression of nitric oxide synthase in the motor neurons. Our findings indicate that the neuroprotective action of preconditioning crush is mediated through the upregulation of heat shock protein 27 expression and the attenuation of neuronal nitric oxide synthase upregulation following avulsion.

Diagnosis,fetal risk and treatment of pemphigoid gestationis in pregnancy:A case report

BACKGROUND Pemphigoid gestationis(PG)is a rare autoimmune blistering disease that usually presents in the second or third trimester,with an incidence of 1 per 50000 pregnancies.PG tends to recur with an earlier onset and a more severe course in subsequent pregnancies.Skin biopsy markers can be confirmed by direct immunofluorescence staining.CASE SUMMARY Our patient was diagnosed with PG at 8 mo of gestation with fresh bullous lesion marks on the abdomen and limbs.Termination of the pregnancy was performed by cesarean section at 37+4 wk of gestation.The patient delivered an infant weighing 3620 gm.The infant had urticaria-like and vesicular skin lesions and was diagnosed with PG.The patient was discharged on prednisolone and in a satisfactory condition.The infant was discharged after anti-inflammatory therapy for one week.CONCLUSION PG is a rarely reported disease,and 10%of newborns develop mild clinical symptoms consisting of urticaria-like or vesicular skin lesions.We intend to remind clinicians to consider this condition when a patient presents with such lesions so that treatment can be started early and neonatal morbidity can be taken into account.

Changes in gap junctional intercellular communication in rabbits lens epithelial cells induced by low power density microwave radiation

OBJECTIVE: To demonstrate the changes in gap junctional intercellular communication (GJIC) mediated by low power density microwave radiation in rabbits lens epithelial cells (LECs) and its mechanisms. METHODS: Rabbits' eyes were exposed to 5 mW/cm(2) and 10 mW/cm(2) power densities of microwave radiation for 3 hours. The fluorescence-recovery-after-photobleaching (FRAP) method was used to determine the GJIC. The localization and function of connexin 43 in LECs was detected by laser scanning confocal microscopy. RESULTS: The GJIC of rabbits LECs was inhibited by microwave radiation especially in the 10 mW/cm(2) irradiated samples. A decrease in connexin 43-positive staining was seen in 5 mW/cm(2) x 3 h treated LECs. Intracellular space accumulation and cytoplasmic internalization were clearly demonstrated in 10 mW/cm(2) group. CONCLUSIONS: Low power densities microwave radiation (5 mW/cm(2) and 10 mW/cm(2)) induces damage to connexin 43 and inhibits the GJIC of rabbits LECs. These changes result in an osmotic imbalance within the lens and induce early cataract. 5 mW/cm(2) or 10 mW/cm(2) microwave radiation is cataractogenic.

Detection of Epstein-Barr virus infection subtype in patients with multiple sclerosis by indirect immunofluorescence assay

Aim:The aim was to investigate the infectious conditions of Epstein-Barr virus(EBV)in patients with multiple sclerosis(MS).Methods:Cerebrospinal fluid(CSF)of 20 patients with MS and 20 with other neurological diseases(OND)were tested with indirect immunofluorescence for anti-EBV capsid antigen(EBV-CA)immunoglobulin G(IgG),IgG affinity for anti-EBV-CA,anti-EBV-CA immunoglobulin M(IgM),anti-EBV early antigen(EBV-EA)IgG and anti-EBV nuclear antigen(EBNA)IgG.According to the pattern of antibodies in CSF,infection rates of acute,chronic,primary,recurrent,and past infections were analyzed in the two groups of patients.Results:There were no significant differences in anti-EBV-CA,anti-EBC-EA,and anti-EBNA antigen IgG in CSF between MS and OND patients(P>0.05).The positive rate of low affinity for anti-EBV-CA IgG in MS patients was significantly higher than that for OND patients(75%vs.40%,P<0.05).Furthermore,significant differences in the positive rate of anti-EBV-CA IgM were found between MS and OND patients(70%vs.25%,P<0.05).Of the MS patients,75%were in an EBV acute infection state compared with 40%of OND patients(P<0.05).Conclusion:Acute infection of EBV closely correlates with the occurrence of MS.