Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.     

实时定量RT-PCR的原理及方法

实时定量RT-PCR广泛应用于定量检测mRAN表达水平,为基础研究、分子药物学和生物技术研究提供了一种有力的方法。该法具有易操作、高通量、敏感性高和特异性强的特点,随着新酶、新探针和新仪器的发展而得到快速的发展。本文将对实时定量RT-PCR的定量原理、仪器应用、探针种类的研究进展以及细胞因子mRNA表达水平检测上的应用作一综述。     

The molecular basis of classic Ehlers-Danlos syndrome: a comprehensive study of biochemical and molecular findings in 48 unrelated patients

Classic Ehlers-Danlos syndrome (EDS) is characterized by fragile and hyperextensible skin, atrophic scarring, and joint hypermobility. Mutations in the COL5A1 and the COL5A2 gene encoding the alpha1(V) and the alpha2(V) chains, respectively, of type V collagen have been shown to cause the disorder, but it is unknown what proportion of classic EDS patients carries a mutation in these genes. We studied fibroblast cultures from 48 patients with classic EDS by SDS-PAGE for the presence of type V collagen defects. An abnormal collagen pattern was detected in only 2 out of 48 cell lines, making this a poor method for routine diagnostic evaluation. A total of 42 out of 48 (88%) patients were heterozygous for an expressed polymorphic variant in COL5A1. cDNA from 18 (43%) of them expressed only one COL5A1 allele. In 37 patients, the COL5A1/A2 genes were then analyzed by SSCP and conformation sensitive gel electrophoresis (CSGE). A total of 26 patients that were mutation-negative after SSCP/CSGE screening were reanalyzed by dHPLC. In addition, 11 other patients were analyzed by dHPLC only. In total, 17 mutations leading to a premature stop codon and five structural mutations were identified in the COL5A1 and the COL5A2 genes. In three patients with a positive COL5A1 null-allele test, no causal mutation was found. Overall, in 25 out of 48 patients (52%) with classic EDS, an abnormality in type V collagen was confirmed. Variability in severity of the phenotype was observed, but no significant genotype-phenotype correlations emerged. The relatively low mutation detection rate suggests that other genes are involved in classic EDS. We excluded the COL1A1, COL1A2, and DCN gene as major candidate genes for classic EDS, since no causal mutation in these genes was found in a number of patients who tested negative for COL5A1 and COL5A2.     

Mapping location of excitation during magnetic stimulation: Effects of coil position

We determined the location of excitation for different positions of a round and butterfly coil duringin vitro magnetic stimulation of cut peripheral nerves. We analyzed the conditions under which excitation occurs, either at the termination or at the peak of the field gradients (first spatial derivative of the electric field). These results were then compared to predictions about the location of excitation sites from a theoretical model of magnetic stimulation of finite neuronal structures. Excitation along a straight nerve occurred at terminations when 1) a coil was positioned close to the end of a nerve (at least one diameter length from the end), 2) a nerve ended in a finite terminating impedance much greater than the axial resistance of the nerve, 3) the induced electric field was of sufficient magnitude, pointing in a direction away from the axis of a nerve. Excitation occurred at the negative peak of the field gradients along a nerve when 1) a coil was positioned far away from the ends of a nerve, 2) there were no geometric or volume conductor inhomogeneities around a nerve, and 3) it was of sufficient magnitude. Threshold strengths for excitation at terminations were significantly lower than that for field gradient excitation and comparable to that due to geometric and volume conductor inhomogeneities.     

In-vitro oocyte maturation: some questions concerning the initiation and prevention of this process in humans

Human oocytes isolated from healthy non-preovulatory antralfollicles which had not been stimulated spontaneously resumedmeiosis at a low rate (31%) when compared with those from atreticfollicles (73%). Human follicular fluid did not inhibit spontaneousmaturation of rat oocytes in vitro, regardless of the qualityor maturity of the follicle or the treatment of the patient.In the human, oocyte maturation may involve a stimulatory processas well as a removal of inhibition.     

Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis

Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.     

Postpartum copulation and induction of pregnancy in roof rats (Rattus rattus)

Two groups of 10 female roof rats were mated either in cycling or postpartum estrus to provide a quantitative comparison of copulatory behavior and to compare the stimulus requirements for the initiation of pregnancy. Animals in both estrous conditions were mated to one ejaculation, three ejaculations and to sexual satiety in repeated tests. The results indicate there were no differences between estrous conditions in the qualitative patterns of copulation but that males achieved more intromissions in the first series of postpartum tests than in cycling estrous tests. The stimulus requirements for pregnancy initiation were not found to vary as a function of estrous condition, however it was found that postpartum matings resulted in more pups per litter than did cycling estrous matings. The results were discussed with regard to the functional significance of species differences in copulatory behavior and in stimulus requirements for pregnancy.