RESUMO
Although there is evidence that cytokine gene polymorphisms are associated with varying quantities of cytokine protein production, the exact role of these polymorphisms in allograft rejection remains unclear. In a previous study, we demonstrated a significant association between high IL-10 secretion in mixed lymphocyte culture (MLC), together with HLA mismatching for at least 4-6 antigens, with the occurrence of acute rejection following renal transplantation. We, therefore, wished to ascertain whether cytokine gene polymorphisms are associated with varying levels of protein secretion and/or allograft rejection in the same group of patients. Cytokine protein secretion in MLC for IL-4, IL-6, IL-10 and IFN-gamma was measured by ELISA in 49 patient-donor pairs. Protein secretion for the above cytokines was also measured in phytohaemagglutinin (PHA) stimulated cultures in 30 normal controls. In both patient and control groups, single nucleotide polymorphism analysis for IL-4 G(-590)T, IL-6 G(-174)C, IL-10 G(-1082)A, IL-10 C(-819)T, IL-10 C(-592)A, TNF-alpha G(-308)A and microsatellite analysis for IFNG (CA repeat) was performed. No correlation was found between cytokine gene polymorphisms and cytokine protein secretion in either mitogen stimulated cultures (control group) or MLC (patient group). In addition, no correlation was demonstrated between cytokine gene polymorphisms and renal allograft rejection.
Assuntos
Citocinas/genética , Transplante de Rim , Doença Aguda , Substituição de Aminoácidos , Estudos de Coortes , Citocinas/metabolismo , Seguimentos , Predisposição Genética para Doença , Genótipo , Rejeição de Enxerto/genética , Rejeição de Enxerto/metabolismo , Análise Heteroduplex , Histocompatibilidade , Humanos , Imunossupressores/uso terapêutico , Interleucinas/genética , Interleucinas/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Repetições de Microssatélites , Fito-Hemaglutininas/farmacologia , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Resultado do Tratamento , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We have previously demonstrated significant inter-individual variations in cytokine protein secretion between normal individuals and patients prior to renal transplantation. In this study, pre-transplant patient vs. donor mixed lymphocyte cultures (MLC) were set up between 57 renal allograft patient/donor pairs, and secretion of cytokine protein (IL-2, IL-4, IL-6, IL-10 and IFN-gamma) into the culture supernatant measured by ELISA. Significant inter-individual variations in protein secretion in MLC were observed for all cytokines studied. Univariate analysis demonstrated that high levels of IFN-gamma and IL-10 in MLC and spontaneous IL-4, together with female donor sex and a high degree of HLA mismatching (especially HLA-DR) were significantly associated with rejection. However, multivariate analysis revealed the greatest risk of rejection (RR = 25.5, P = 0.003) was associated with a combination of high IL-10 secretion in MLC and mismatching for at least four HLA antigens (HLA-A, -B and -DR). It remains to be determined whether cytokine secretion in MLC is linked to cytokine gene polymorphisms. In future, assays for measuring either cytokine secretion or genetic polymorphisms may prove to be useful in aiding donor selection and tailoring immunosuppressive therapy.
Assuntos
Citocinas/metabolismo , Rejeição de Enxerto , Teste de Histocompatibilidade , Transplante de Rim/imunologia , Teste de Cultura Mista de Linfócitos , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Análise Multivariada , PrognósticoRESUMO
Aims-To develop a highly sensitive technique for the reliable detection and typing of human papillomavirus (HPV) DNA in clinical tissue.Methods-A two step, semi-nested PCR was used with primers spanning the L1 region of the HPV genome and capable of detecting HPV DNA of all known HPV types. The clinical samples were typed by digestion of the 412 base pair PCR product with Rsa I, generating unique fragments for each HPV type. Thirteen samples were screened by this method, including nine vulval carcinoma samples and four wart samples from the penis and vulva.Results-Experiments using DNA extracted from HPV DNA positive cell lines-that is, CaSki (HPV type 16) and HeLa (HPV type 18) established that the technique could detect as few as 50 HPV copies and that the predicted Rsa I fragments from HPV types 16 and 18 were generated. The predicted 412 base pair fragment was observed for all 13 clinical samples subjected to semi-nested PCR. Rsa I digestion of the product of the second round of PCR permitted the positive identification of the HPV type in most cases.Conclusions-This technique provides an effective and rapid means of detecting HPV DNA, in most cases providing the HPV type. High risk HPV types were always detected in the nine vulval carcinoma samples analysed. The amount of tissue available from the biopsy specimens was small, confirming the sensitivity of the method.
