Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance

Therapeutic application of broadly reactive anti-T cell antibodies can lead not only to potent immunosuppression but also to profound and long-lived T cell depletion. We reasoned that a strategy that almost exclusively targets activated cytopathic donor reactive T cells and spares immunoregulatory networks might prove to be an exceptionally potent and highly selective means of producing long-term engraftment and tolerance. Herein we show that the combined administration of rapamycin and agonist IL-2- and antagonist IL-15-related cytolytic fusion proteins provides for long-term engraftment/tolerance in exceptionally stringent allotransplant models by (1) limiting the early expansion of activated T cells, (2) preserving and even exaggerating their subsequent apoptotic clearance, and (3) further amplifying the depletion of these activated T cells by antibody-dependent mechanisms, while (4) preserving CD4+CD25+ T cell-dependent immunoregulatory networks.     

Interdependent effect of angiotensin-converting enzyme and platelet-activating factor acetylhydrolase gene polymorphisms on the progression of immunoglobulin A nephropathy

In order to investigate the interdependent action of the insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene and polymorphism in exon 11 (C1136-->T; Ala379Val) of the platelet-activating factor acetylhydrolase (PAF-AH) gene, which encodes a functional antagonist of PAF, on the progression of immunoglobulin A (IgA) nephropathy, we analysed both polymorphisms in patients with primary IgA nephropathy, who were followed-up for longer than 3 years. During the follow-up (87.3 +/- 50.0 months), the disease progressed in 38 of the 191 patients (19.9%). The D allele of the ACE gene in the absence of the T allele of the PAF-AH gene did not affect the prognosis [odds ratio (OR), 3.6; 95% confidence interval (CI), 0.8-16.4] and neither did the T allele in the absence of the D allele (OR, 3.0; 95% CI, 0.4-24.2). However, the presence of both was a significant prognostic factor (OR, 6.6; 95% CI, 1.4-31.3). After adjusting for other risk factors, the presence of both proved to be an independent risk factor (OR, 4.5; 95% CI, 1.6-12.7). These results suggest that the interdependent effects of ACE and PAF-AH polymorphisms on the progression of IgA nephropathy might be more important than the effect of the individual polymorphisms.     

A mouse model of galactose-induced cataracts

Galactokinase (GK; EC 2.7.1.6) is the first enzyme in the metabolism of galactose. In humans, GK deficiency results in congenital cataracts due to an accumulation of galactitol within the lens. In an attempt to make a galactosemic animal model, we cloned the mouse GK gene (Glk1) and disrupted it by gene targeting. As expected, galactose was very poorly metabolized in GK-deficient mice. In addition, both galactose and galactitol accumulated in tissues of GK-deficient mice. Surprisingly, the GK-deficient animals did not form cataracts even when fed a high galactose diet. However, the introduction of a human aldose reductase transgene into a GK-deficient background resulted in cataract formation within the first postnatal day. This mouse represents the first mouse model for congenital galactosemic cataract.     

Molecular cloning and expression of a novel alternative splice variant of BRDT gene

In this study, a human adult testis cDNA microarray was constructed and hybridized with (33)P-labeled human adult testis, embryo testis and sperm cDNA probes, respectively. A novel alternative splice variant of BRDT gene, named BRDT-NY, presumably involved in testicular function was cloned. It was expressed 3.96-fold more in human adult than embryo testis and also expressed in human spermatozoa. Similarly, RT-PCR revealed a differential expression pattern of this gene in human adult testes and fetal testes. The full length of BRDT-NY was 3438 bp and contained a 2883 bp open reading frame, encoding a 960-amino-acid protein. Sequence analysis showed that it has two bromodomains in N-terminal of the protein. Multiple tissue RT-PCR results showed that BRDT-NY was exclusively expressed in testis. mRNA expression of BRDT-NY gene was deleted in some azoospermic patients' testes. These experiments suggested that BRDT-NY gene may have an important role in the process of spermatogenesis and may be correlated with male infertility.     

The MDR1 polymorphisms at exons 21 and 26 predict steroid weaning in pediatric heart transplant patients

Various polymorphisms of the MDR1 gene that encodes for P-glycoprotein (P-gp), a transmembrane pump, have been identified. A silent mutation C3435T in exon 26 and a G2677T mutation in exon 21 have been correlated with P-gp expression and function in humans. The objectives of this study were (a) to determine whether the MDR1 exon 21 and exon 26 polymorphisms were related to steroid weaning in a pediatric heart transplant (HTx) population, and (b) to determine whether an association exist between the MDR1 exon 21 and exon 26 polymorphisms in these patients. Sixty-nine pediatric HTx patients were studied. MDR1 genotyping was determined by polymerase chain reaction amplification, sequencing the DNA, and sequence evaluation using Polyphred software (University of Washington) to identify genotypes. The steroid dose at 1 year post-transplantation was recorded. For steroid weaning at one year post-HTx for MDR1 C3435T, 12 of 18 (67%) patients in the CC genotype were still on prednisone, whereas only 18 of 47 (38%) of the CT/TT group were still receiving prednisone (p = 0.04). Similar results were observed for the MDR1 G2677T genotyping and steroid weaning. Forty-three of 46 patients (93.5%) who have MDR1 C3435T allele also have a mutant G2677T allele (p < 0.001). We conclude that (a) a significantly larger number of MDR1 3435 CC HTx patients remain on steroids at 1 year after transplantation, and (b) the MDR1 C3435T genotype is associated with the G2677 genotype in pediatric HTx patients.     

Arachidonic acid, but not its metabolites, is essential for FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcgamma receptor (FcgammaR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcgammaR-mediated killing process. The production of O-2 by monocytes upon FcgammaR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcgammaR cross-linking. Furthermore, FcgammaR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcgammaR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcgammaR-stimulated intracellular killing of S. aureus by monocytes.     

Microsatellite instability and alteration of the expression of hMLH1 and hMSH2 in ovarian clear cell carcinoma

Microsatellite instability (MSI) is commonly seen in tumors associated with the hereditary nonpolyposis colorectal cancer syndrome and is caused by defects in the DNA mismatch repair genes. MSI has also been observed in various sporadic cancers, including colorectal, gastric, and endometrial. The role and incidence of MSI in ovarian clear cell carcinoma remain unknown. This study was conducted to evaluate the frequency of MSI in ovarian clear cell carcinomas and to evaluate the sensitivity and specificity of immunohistochemistry in predicting mismatch-repair gene deficiency. A total of 42 ovarian clear cell carcinomas were analyzed for MSI using a panel of 5 microsatellite markers (BAT25, BAT26, D5S346, D2S123, and D17S250). Alterations in the expression of hMLH1 and hMSH2 proteins in these tumors were examined. Of the 42 ovarian clear cell tumors analyzed, 6 demonstrated a high level of MSI (MSI-H), 3 demonstrated a low level of MSI (MSI-L), and the remaining 33 exhibited microsatellite stability (MSS). No correlation was found between MSI level and patient age or tumor stage or size (P >0.05). Loss of expression of either hMLH1 or hMSH2 was observed in 4 of the 6 (67.7%) MSI-H tumors, whereas 34 of the 36 (94.4%) MSI-L or MSS tumors expressed both the hMLH1 and hMSH2 gene products. Our results indicate that MSI-H is involved in the development of a subset of ovarian clear cell carcinomas. A strong correlation exists between alterations in the expression of hMLH1 and hMSH2 and the presence of MSI-H in these tumors. However, immunohistochemical testing alone may miss a small fraction of cases with MSI-H.