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Genetic analyses indicate that genes within the major histocompatibility complex (MHC) can be involved in susceptibility to autoimmune disease. To investigate the role of the tumour necrosis factor beta (TNFB) gene in myasthenia gravis (MG) susceptibility, we analysed an NcoI polymorphism within the TNFB gene in 63 MG patients and 93 healthy individuals. When patients were subdivided according to thymic pathology, we found differences between MG patients with thymic hyperplasia and thymoma versus controls. In MG patients with thymic hyperplasia we found a positive association with the TNFB*1 allele [Relative risk (RR): 2.6; P<0.001] and phenotype (RR: 1.8; P<0.005) and a negative association with the TNFB*2/2 genotype (RR: 0.2; P<0.001) when compared to the controls. On the other hand, in MG patients with thymoma we found a positive association with the TNFB*2/2 genotype (RR: 5.6; P<0.01) and a negative association with the TNFB*1 allele (RR: 0.3; P<0.05) and *1/2 genotype (RR: 0.2; P<0.01). These data suggest that the two different forms of MG can have different pathogenesis and that the TNFB gene could influence susceptibility to MG. [ABSTRACT FROM AUTHOR]

Background: To determine whether an imbalanced interaction between proapototic and antiapoptotic signals may account for the loss of the normal cell growth control in benign prostatic hyperplasia (BPH), the expression of some apoptosis-regulating genes (bcl-2, bax, c-myc, fas) was investigated., Patients and Methods: BPH specimens were obtained from 20 patients who underwent trans-urethral resection of the prostate (TURP) or adenomectomy. Gene expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and its correlation with age and serum PSA level was also investigated., Results: Genes were found to be differentially expressed in BPH tissues. In particular, the antiapoptotic gene bcl-2, which was found in 18/20 samples, gave the weakest signal (p < 0.05 - p < 0.001, Wilcoxon's signed rank test), whereas the cell cycle regulator c-myc was detected in all the specimens and was the most highly expressed (p < 0.001). A positive relationship between the expression of bcl-2 and that of the two proapoptotic genes bax and fas was observed (p < 0.05, Spearman's rank correlation test), as well as between c-myc and fas levels (p < 0.005). Moreover, bax expression positively correlated with age and PSA (p < 0.02), which have also been shown to directly correlate (p < 0.01)., Conclusion: The higher expression of the oncogene c-myc suggests the activation of mitogenic signals within hyperplastic prostate tissue which a relatively high expression of the proapoptotic genes bax and fas fails to counterbalance.

It is well established that certain subpopulations of human adult stem cells can generate hepatocyte-like cells when transplanted into adult immunosuppressed mice. In the present study, we wanted to explore whether xeno-transplantation of human cord blood CD34(+) (hCBCD34(+)) cells during pre-immune stages of development in immunocompetent mice might also lead to human-mouse liver chimerism. Freshly isolated hCBCD34(+) cells were xeno-transplanted into non-immunosuppressed mice by both intra-blastocyst and intra-fetal injections. One and four weeks after birth, immunostaining for different human-specific hepatocyte markers: human hepatocyte-specific antigen, human serum albumin, and human alpha-1-antitrypsin indicated the presence of human hepatocyte-like cells in the livers of transplanted animals. Detection of human albumin mRNA further corroborated the development of pre-immune human-mouse chimeras. The current report, besides providing new evidence of the potential of hCBCD34(+) cells to generate human hepatocyte-like cells, suggests novel strategies for generating immunocompetent mice harboring humanized liver.

Background: In this study we confirmed the ability of a Gonadotropin Releasing Hormone (GnRH) agonist, leuprorelin acetate (LA), to counteract or even suppress the 5alpha-dihydrotestosterone (DHT)-stimulated growth of androgen-sensitive prostate cancer cells (LNCaP). Since the cellular mechanisms mediating this effect are not well defined, we investigated the activity of LA, also in combination with DHT or with cyproterone acetate (CA), on the expression of genes (bcl-2, bax, c-myc) which may contribute to the proliferative behaviour of LNCaP cells. In addition, experiments aimed to evaluate the action of the analogue on apoptosis were performed., Materials and Methods: Gene expression was evaluated by RT-PCR and Western blotting on cells treated with LA (10(-11) or 10(-6) M), alone or combined with 10(-9) M DHT or 10(-7) M CA. The occurrence of apoptosis following treatment with LA (10(-11), 10(-6) or 10(-5) M), alone or combined with 10(-9) M DHT, was assessed by DNA fragmentation analysis., Results: Both the mRNA and protein of the anti-apoptotic gene bcl-2 were induced (30-125%) by DHT after 24-144 h. LA decreased bcl-2 mRNA (10-40%), while it did not unequivocally affect protein expression. The analogue always reduced (13-74%) both mRNA and protein levels obtained under DHT treatment. The mRNA and protein of the pro-apoptotic gene bax were down-regulated by DHT (15-40%), while LA generally induced bax protein but not its mRNA. LA counteracted DHT activity, even increasing bax protein levels over the controls. c-myc mRNA and protein were enhanced by DHT (15-45%) but down-regulated by LA (10-40%). Once more, the androgen effect was antagonized by LA, sometimes reducing c-myc content below the controls. CA produced the most similar effects to those triggered by DHT. The hormonal treatment did not induce any DNA fragmentation., Conclusion: In spite of gene modulation, apoptosis was not observed under LA treatment, in agreement with the lack of a cell growth effect when the analogue was used alone. Nevertheless, the observed changes in gene expression may be directly or indirectly involved in the antiproliferative effect of LA on androgen-stimulated cells.

The intracoelomic route for in utero hematopoietic stem cell transplantation has been evaluated in pre-immune fetal sheep and the engraftment characteristics defined. Twelve ovine fetuses (gestational ages: 40-45 days) received intracoelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspension of the liver, spleen, bone marrow and thymus by flow cytometry, cloning assays and polymerase chain reaction (PCR) analysis for human beta(2)-microglobulin gene. The engraftment of liver samples was also evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH) and immunohistochemistry. Four fetuses (33%) aborted shortly after intracoelomic transplantation and were not evaluable for engraftment. Engraftment was detected in 4 fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degree of engraftment in these 4 fetuses ranged from 6 to 22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (No. 435184) and 105 (Nos 915293, 037568) days and 1 fetus delivered at term, which received CD34-selected cord blood cells, had human engraftment with 10, 32, 20 and 10% CD45+ cells in bone marrow, respectively. A further check of human chimerism was done at 1 year after birth of the fetus delivered at term and 7.6% of bone marrow chimerism was detected. In 6 out of 8 fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta(2)-microglobulin which also identified human cells in brain, spinal cord, heart, lung and skeletal muscle. On liver samples, FISH and RT-PCR confirmed the xenograft of human cells and the immunohistochemical analysis detected human markers of hematopoietic and hepatic lineage of differentiation. This preliminary study indicates that intracoelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment., (Copyright 2004 S. Karger AG, Basel)

Background: We investigated the regulation of prostate-specific antigen (PSA) gene expression in prostatic tumor cells by an LH-RH analogue, Leuprorelin acetate (LH-RHa), alone or combined with dihydrotestosterone (DHT)., Materials and Methods: PSA gene expression was evaluated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in androgen-sensitive LNCaP and androgen-insensitive PC-3 cells., Results: LH-RHa at both high and low concentrations induced a diminution in the levels of PSA gene expression which reached 70% in androgen-insensitive PC-3 cells. The analogue lowered the levels of expression obtained under DHT stimulation in LNCaP cells. These effects were visible from 6-24 hours of treatment and persisted until 120 hours., Conclusion: PSA gene expression is directly regulated by LH-RHa in both LNCaP and PC-3 cells. High concentrations of the analogue are particularly effective, but low doses of the drug have a similar behaviour. Interestingly, Leuprorelin acetate is able to counteract the androgen-induced gene expression. Our results suggest that, at least in androgen-sensitive cells, the analogue may regulate PSA gene expression also by interfering with the androgen receptor machinery.