Diagnosis and treatment of graft-versus-host-disease after kidney transplantation: A Case Report and Literature Review / 中华器官移植杂志

Objective To investigate the diagnosis and treatment of graft-versus-host-disease (GVHD) after solid organ transplant (SOT) and the possible mechanism.Methods In this study,we retrospectively reported a rare case of GHVD after kidney transplantation and performed a literature review.This 51 year old male patient presented with over 10-day history of sporadic skin rash on postoperative day 50.Skin biopsy examination revealed GVHD.For further clear diagnosis,patient's peripheral blood was collected immediately for short tandem repeats (STR) enriched for CD3+ cells and we also performed immunofluorescent staining for patient's skin with specific HLA-A11 antibody,one of donor specific HLA sites.Meanwhile,the decreased immunosuppression was used for treatment.Results In our report,chimerism analysis by STR revealed no chimerism in patient's peripheral blood.However,immunofluorescent staining of patient's skin demonstrated abundant donor-derived lymphocytic infiltration existed.GVHD was definitely made in this case.After treatment with decreased immunosuppression and a low dose of methylprednisolone (MP),his clinical symptom was quickly alleviated.Now the patient was discharged and the renal function was normal.Conclusion Combined with literature review and our case report,we thought that chimerism analysis by STR and immunofluorescent staining by donor-specific HLA antibody were very useful for diagnosis of GVHD after SOT.Furthermore,GVHD referred to over-immunosuppression and prognosis of GVHD after kidney transplantation is usually satisfactory.

Development of the mouse spinal cord and neuroapoptosis / 解剖学报

Objective To investigate the neural proliferation , differentiation and apoptosis of the developing spinal cord of the mouse and to discuss the mechanism of spinal cord ’ s development .Methods 5-Bromodeoxyuridine ( BrdU) assay was used to mark the proliferative neural stem cells , and the immunofluorescent stainings ( DCX, NeuN and Caspase8) were carried out to visualize the newborn neurons , mature cells and apoptotic cells in the spinal cord with 173 mice arrange from E18 to P90.Results BrdU positive neural stem cells appeared evenly in the spinal cord at early days . With age increasing , the neural stem cells differentiated into neuroglial cells and neurons .The newborn neurons in the subventricular zone migrated toward the intermediate zone ( putative gray matter ) and differentiated into mature neurons gradually .With neurons ’ concentrating towards the center , the gray matter formed an “H” shape .In the meantime , with neural differentiation , some apoptotic neurons appeared among the newborn neurons and mature neurons . Double immunostaining showed that most apoptotic neurons were newborn neurons , suggesting the neuroapoptosis more likely occurred in newborn neurons .The statistical data showed that the number of DCX , NeuN and Caspase-8 positive cells reduced with age increasing , suggesting neural differentiation and neuroapoptosis decreased during spinal cord ’ s development .Conclusion Neural proliferation , neural differentiation and neuroapoptosis occur in developing spinal cord . They work together to regulate the formation and development of the spinal cord .

A sensitive and specific ES-31 antigen detection based fluorometric assay for confirmation of Mycobacterium tuberculosis in cell culture.

Confirmation of presence of M. tuberculosis bacilli on microscopic examination is very important in diagnosis of tuberculosis. The present study was undertaken to find the usefulness of mycobacterial ES-31 serine protease as a marker to detect tuberculosis bacilli using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. This immunofluorescence method was compared with Ziehl-Neelsen and auramine-O staining methods for detection of tuberculosis bacilli. Slides were prepared for each serially diluted tuberculosis H37Ra bacilli (1×107 bacilli/ml to 5 bacilli/ml). Slides for each dilution group were stained by ZN method, auramine-O and immunostaining methods using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. ZN staining method showed efficacy for detection of M. tuberculosis H37Ra upto 1×104 bacilli/ml while auramine-O method showed upto 1×102 bacilli/ml. The presence of bacilli was indicated by green fluorescence on immunostaining using anti-ES-31 antibody conjugate and this method was effective upto 10 bacilli/ml. The slides which were negative for ZN (1×103 cells/ml) and auramine-O (100 cells/ml) method showed positivity on restaining with immunofluorescent staining method. The results of this preliminary study showed that immunofluorescent staining method using specific anti-ES-31 antibody conjugate was more sensitive for detection of tuberculosis bacilli than ZN and auramine-O methods in samples of laboratory strain. The utility of this method will be studied further in clinical specimens.

Expression and Distribution of BDNF (Brain Derived Neurotrophic Factor) in the Rat Hypothalamus / 대한해부학회지

BDNF belongs to the neurotrophin family and important molecular mediator of functional and structural plasticity. The highest levels of BDNF are found in the hippocampus and hypothalamus of the adult rat. Hypothalamus is important because of its high degree of plasticity, but little is known about distribution of BDNF in hypothalamic nuclei. Therefore, it is necessary to study distribution and expression pattern of BDNF in each hypothalamic nuclei to understand changes of BDNF through various neural damages including spinal cord injury. Through this experiment, we found specific BDNF expression pattern in some regions of hypothalamus and the results are as follows. 1) BDNF expressions were found in median eminence, arcuate nucleus, supraoptic nucleus, and periventricular nucleus of rat hypothalamus. 2) BDNF immunoreactive cells and nerve fibers were of various shapes and sizes. 3) Glial cells also express BDNF in certain hypothalamic nuclei. These results seem to be useful for future investigations of neurochemical changes in the hypothalamus induced by various neural trauma or degenerative changes

Preparation and Characterization of Polyclonal Antibody Against Xenopus PAPC / 生物化学与生物物理进展

Xenopus Paraxial Protocadherin (PAPC), which was initially identified in a screen for genes present in the Spemann organizer of Xenopus embryos, is required for gastrulation, somitogenesis and otic vesicle formation. In order to investigate its function in various developmental events, an antibody was prepared which could specifically recognize Xenopus PAPC. Glutathione S transferase (GST) expression system was used to express the fusion protein GST-PAPC. Rabbits were immunized with GST-PAPC Western blotting analysis of FL-PAPC transfected HEK 293T cells lysates, which could be specifically blocked by pre-adsorption of prokaryotic expressed GST-PAPC fusion protein. Furthermore, by using immunofluorescence analysis the polyclonal antibody recognized membrane-bound PAPC in FL-PAPC transfected 293T cells and Xenopus animal cap cells. By Western blotting analysis,the endogenous 150 ku PAPC protein was detected in Xenopus embryos using the anti-PAPC antibody. Take together it could be concluded that a polyclonal antibody specifically against Xenopus PAPC was developed.

Expression of protease activated receptors in mast cells / 临床检验杂志

Objective To investigate the expression of protease activated receptors(PARs)in mast cells.Methods Reverse transcription polymerase chain reaction(RT-PCR),flowcytometry and immunofluorescent cell staining were used to detect the expression of PARs in mast cell lines P815 and MC/9 at the levels of protein and mRNA.Results Both the P815 and MC/9 of mast cell lines expressed PAR-1,PAR-2,PAR-3 and PAR-4 at either protein or mRNA level.Conclusion The expression of all the four PARs in mast cells were detectable,which may be of significance for the further study on the function of PARs in mast cells.

Development of the Enzyme Immunoassay for the Detection of Anti-HSV-2 Antibody with HSV-2 Specific Monoclonal Antibody / 감염

BACKGROUND: The serological diagnosis of herpes simplex virus type 2 (HSV-2) infection has pitfalls, in that most of the antibodies against HSV-2 cross-react with HSV-1 and the prevalence of HSV-1 infection is high, especially in Korea. In this study, we tried to establish the serological diagnostic method, which could detect and measure the specific antibodies against HSV- 2 by competitive immunofluorescent staining method as well as competitive ELISA based on the specific monoclonal antibody, MH2-7. METHODS: Immunofluorescent staining and western blot analysis were used to characterize the antigens recognized by MH2-7. Competitive immunofluorescent staining (IF), competitive enzyme immunoassay (ELISA), and western blot analysis were used to detect specific antibodies against HSV-2 in patients' sera. RESULTS: In western blot analysis, the sera from two of six patients clinically diagnosed as genital herpes showed characteristic band patterns, which have been known to be compatible with HSV-2 infection. In competitive immunofluorescent staining, only the sera from the two patients clinically diagnosed as genital herpes and with characteristic band pattern showed competition with MH2-7 monoclonal antibody. The dilution range of the serum showing specific competition was between 1:10 and 1:80. Competitive ELISA was also performed and evaluated as the diagnostic efficacy as ELISA has been known to be advantageous over IF staining in mass screening. The result showed linear dose-response relationship for the patient's sera in inhibition of the reactivity of MH2-7. CONCLUSION: We suggest that the competitive immunofluorescent staining method and competitive ELISA based on the specific monoclonal antibody MH2-7 is a simple, accurate, and precise method, which can be used in serological diagnosis of HSV-2 infection.

Comparison of Direct Immunofluorescent Staining, Shell Vial Culture and Tube Culture for the Detection of Varicella-Zoster Virus in Skin Lesions / 대한임상병리학회지

BACKGROUND: For the diagnosis of varicella-zoster virus (VZV) infection, virus culture has been considered the reference method, but it gives delayed results and needs cell culture facilities. Shell vial culture is more rapid, but it also takes 2 days or more and needs cell culture. Immunofluorescent (IF) method has known to be rapid and sensitive. We compared the tube culture, shell vial culture, and direct IF method to find the most efficient diagnostic method. In addition, the MRC-5 cells were compared with A549 cells for the recovery of VZV in culture. METHODS: A total of 48 specimens were obtained from skin vesicles of patients with clinical herpes zoster. The vesicle smears were stained with FITC-conjugated monoclonal antibody. The vesicle aspirates obtained in 2 mL of viral transport media were inoculated into shell vials and tubes containing MRC-5 and A549 cell monolayers. After 48 h of incubation at 36degrees C the shell vials were stained with VZV-specific monoclonal antibody. The tubes were stained with the same antibody after 3 weeks or when the monolayer showed cytopathic effect. RESULTS: The positive rates of direct IF, shell vial culture, and tube culture were 67.5%, 87.5%, and 72.5% respectively. The positive rate of direct IF was increased to 96.4% when inadequate specimens for the direct IF were excluded. The MRC-5 and A549 cells showed no significant difference in the isolation rates of VZV in both shell vial and tube culture. CONCLUSIONS: Direct IF is the most rapid and practical method for the laboratory confirmation of VZV infection when the swab specimen is adequately obtained. The MRC-5 cells are recommended for the tube culture and A549 cells for the shell vial culture.

EXPRESSION OF GST-SNAIL FUSION PROTEIN IN PROKARYOTIC CELLS AND PREPARATION OF POLYCLONAL ANTIBODY AGAINST SNAIL / 解剖学报

Objective The aim of this study was to prepare anti-snail polyclonal antibody and make it widely useful in snail detection. Methods The DNA fragment encoding human full length 264 amino acid of snail was obtained by PCR from cDNA library of human umbilical vein epithelial cells and was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST).The GST-snail fusion protein was expressed by E.coli BL21 after IPTG induction and purified from total proteins of BL21 transformed by the recombinant plasmid pGEX-4T-1/snail.The New Zealand rabbit was immunized with the purified fusion protein to prepare polyclonal antiserum.The antiserum was identified by western blotting and immunofluorescent staining. Results The prokaryotic expression plasmid pGEX-4T-1/ snail was successfully constructed,and the fusion protein GST-snail was expressed efficiently.The polyclonal antibody raised in the rabbit could react specifically with snail in human cells.Conclusion The snail antiserum was of good purity with high titer and specificity which could satisfy the requirement for studying immuno-analysis on snail protein.