Subcutaneous (s.c.) immunization of rats with the human 60-kDa heat shock protein (HSP)-derived peptide 336-351 induced clinical and/or histological uveitis in 80 % of rats. Subsequent experiments to prevent the development of uveitis by oral or nasal administration of the peptide have failed. Instead, uveitis was induced in 74.6 % of rats given the peptide orally (5 times), in 75 % given the peptide nasally (5 times) or 91.7 % of those administered the peptide by both routes (10 times). Histological examination showed that any one route of administration of the peptide elicited iridocyclitis in 42.2 % but loss of photoreceptors only in 4.9 % of rats. In contrast, sequential administrations of the peptide by a combined mucosal-s.c. route resulted in iridocyclitis in only 25 % but loss of photoreceptors in 40 % of animals. Examination of mRNA from CD4-enriched splenic cells by reverse transcription-PCR failed to yield significant differences in Th1 or Th2 cytokines. Treatment with monoclonal antibody (mAb) to CD4 yielded a dose-dependent decrease in uveitis from 82 % to 25 %. Similarly, treatment with IL-4 significantly decreased the development of uveitis from 68 % to 30.4 %. Conversely, treatment of the rats with mAb to CD8 greatly enhanced the onset of uveitis (from about 22 days in the controls to 11 days) and all the rats developed uveitis by day 24. Thus, CD4 + cells mediate, whereas CD8 + cells suppress the development of uveitis. We suggest that this novel experimental mucosal model of induction of uveitis by the human 60-kDa HSP-derived peptide 336-351, which is specific in stimulating T cell responses in Behcet's disease, is consistent with the oro-genital onset of this disease and the development of uvei-tis. Received 17/2/98 Revised 9/4/98 Accepted 24/4/98 [I 18049] Abbreviations: HSP: Heat shock protein SAg: Retinal soluble antigen IRBB: Interphotoreceptor binding protein MLN: Mesenteric lymph node(s)
The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.
No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. Here we combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Our results showed that complete single donor STR profiles could be successfully obtained from sperm/epithelial cell and sperm mixtures from two contributors. For unbalanced sperm/epithelial cells and sperm cells mixtures, sensitivity results revealed that target cells could be detected at as low as 1:32 and 1:8 mixed ratios, respectively. Although highly relies on cell number and blood types or secretor status of the individuals, this procedure would still be useful tools for forensic DNA analysis of multi-suspect sexual assault cases by the combined use of FACS and MACS based on sperm-specific AKAP3 antigen and human blood type antigen.
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