Loss of heterozygosity in normal breast epithelial tissue and benign breast lesions in BRCA1/2 carriers with breast cancer

Loss of heterozygosity (LOH) of the wild-type BRCA1/2 allele is a reproducible event in breast tumors of BRCA1/2 mutation carriers, but it is unknown if this allelic loss occurs only in association with recognizable histopathologic abnormalities. We evaluated the early genomic changes that occur in the mammary glands of patients with increased predisposition to breast cancer due to germline mutations in the BRCA1/2 genes. We tested the hypothesis that these genomic changes may be detected, not only in histologically abnormal and malignant breast tissues, but also in morphologically normal tissues and in areas with pathologically benign changes. Samples were obtained from five breast cancer patients: four BRCA1 carriers and one BRCA2 carrier. In each case, nontumor tissue areas surrounding the tumor or from other locations of the breast were isolated using laser capture microdissection. We evaluated 29 areas showing normal terminal ductal lobular units (TDLUs) or histopathologically benign changes (in particular, sclerosing adenosis), using a panel of polymorphic dinucleotide microsatellite markers for the BRCA1 gene and other chromosome 17 loci, for the BRCA2 gene and other chromosome 13 loci, and for the FHIT gene on 3p14.2. Overall, we analyzed a total of 105 samples of nontumor tissues; LOH was detected in 59 of the 105 (56%). In the normal TDLUs, 15 of 30 samples (50%) showed LOH; in the tissues with benign proliferative changes, such as sclerosing adenosis, 44 of 75 samples showed LOH (59%). Our results suggest that there is a field effect of early genetic events preceding morphologic changes in the mammary glands of BRCA mutation carriers.     

A Vero cell-attenuated Goatpox virus provides protection against virulent virus challenge

An Indian isolate of Goatpox virus (GTPV) was adapted and propagated in Vero cells for development of an attenuated virus. The virus was initially passaged in primary lamb testes cells and subsequently in Vero cells. At the 55th passage, the virus showed evidence of attenuation when tested for safety in seronegative goats. At this stage, the virus was found to be completely non-pathogenic. The virus was passaged further and the 60th passage was used for testing its immunogenicity in goats. The latter were inoculated with 10, 100 and 1000 TCID50 of the attenuated virus by intradermal (i.d.) route and challenged after 28 days with virulent GTPV. The attenuated virus produced no adverse reaction even at the highest dose and conferred complete protection even at the lowest dose against challenge with a high dose (2 x 10(6) of 50% skin-reactive dose SRD50) of virulent virus. Increased levels of virus-specific serum antibodies could be demonstrated by both indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test in all the immunized goats. No horizontal transmission of the virus from the immunized to in-contact animals took place. Our results suggest that this attenuated virus could be a safe, immunogenic and potent candidate for developing a vaccine against goatpox.     

Glial cell and fibroblast cytotoxicity study on 4026-cyclotene photosensitive benzocyclobutene (BCB) polymer films

Photosensitive benzocyclobutene (photo-BCB) is a class of polymers with the trade name Cyclotene. The photoimagable property of Cyclotene makes it suitable for the manufacture of microelectronic devices. The motivation behind this study is that we see an exciting application of photo-BCB as substrates in implantable microelectronic biomedical devices due to several desirable properties distinctive from other polymer materials. To our knowledge, however, photo-BCB has never been tested for biomedical implant applications, as evidenced by the lack reported data on its biocompatibility. This study takes the first step towards assessing photo-BCB biocompatibility by evaluating the cytotoxicity and cell adhesion behavior of Cyclotene 4026 coatings exposed to monolayers of glial and fibroblast cells in vitro. It can be concluded from these studies that photo-BCB films deposited on silicon wafers using microfabrication processes did not adversely affect 3T3 fibroblast and T98-G glial cell function in vitro. We also successfully rendered photo-BCB films non-adhesive (no significant fibroblast or glial cell adhesion) with surface immobilized dextran using methods developed for other biomaterials and applications. Future work will further develop prototype photo-BCB microelectrode devices for chronic neural implant applications.     

Alteration in plasma glucose levels in Japanese encephalitis patients

A unique factor, human T cell hypoglycaemic factor (hTCHF), has been shown to produce hypoglycaemia during the convalescent stage in the plasma of patients with Japanese encephalitis virus (JEV) infection. The present study was undertaken to investigate the ability of T cells from fresh peripheral blood mononuclear cells (PBMC) of such patients to produce hTCHF. The PBMC, as well as the individual subpopulations, were cultured for 24 h and the culture supernatants (CS) were assayed for hypoglycaemic activity. The activity was observed in the CD8+ T cells. The hypoglycaemia in JE-confirmed patients coincided with the gradual rise in circulating glucagon level, with no significant alterations in insulin, growth hormone and cortisol levels. The hTCHF was purified by ion exchange chromatography and the purified protein was observed as a approximately 25 kDa band on SDS-PAGE. Secretory hTCHF in the sera of patients and T cell CS was present in 88% of convalescent serum samples. We conclude that during the convalescent stage of JEV infection, a unique factor, hTCHF, is secreted by activated CD8+ T cells from patients and that this is responsible for the development of hypoglycaemia.     

Cellular immune responses of patients with juvenile chronic arthritis to retinal antigens and their synthetic peptides

The objective of this study was to determine the proliferative responses of peripheral blood lymphocytes of ocular antigens like retinal S-antigen, peptides M and G of S-antigen, yeast histone H3 peptide 106–121 homologous to peptide M and peptide R16 of interphotoreceptor retinoid binding protein (IRBP) in children with juvenile chronic arthritis (JCA). We have studied the in vitro proliferative response of peripheral blood lymphocytes from 41 patients with JCA (10 with and 31 without uveitis) and 23 healthy controls against the above antigens. The responders were retested after 1 or 6 months. Fifty (5/10) and 9.7% (3/31) of JCA patients with and without uveitis, respectively, responded (stimulation index >3) to S-antigen or one of its peptide listed above or yeast histone H3 peptide or R16 of IRBP. None of the healthy controls responded to any of these antigens. The difference in the frequency of responders (SI>3) between JCA associated with uveitis and healthy controls was statistically significant (p=0.001). Similarly, the difference between JCA with and without uveitis was also significant (p=0.013). Our findings suggest that these antigens may have a role in the pathogenesis of uveitis in a subset of patients with JCA.     

CD1d deficiency exacerbates inflammatory dermatitis in MRL-lpr/lpr mice

Mechanisms responsible for the development of autoimmune skin disease in humans and animal models with lupus remain poorly understood. In this study, we have investigated the role of CD1d, an antigen-presenting molecule known to activate natural killer T cells, in the development of inflammatory dermatitis in lupus-susceptible MRL-lpr/lpr mice. In particular, we have established MRL-lpr/lpr mice carrying a germ-line deletion of the CD1d genes. We demonstrate that CD1d-deficient MRL-lpr/lpr mice, as compared with wild-type littermates, have more frequent and more severe skin disease, with increased local infiltration with mast cells, lymphocytes and dendritic cells, including Langerhans cells. CD1d-deficient MRL-lpr/lpr mice had increased prevalence of CD4(+) T cells in the spleen and liver and of TCR alpha beta (+)B220(+) cells in lymph nodes. Furthermore, CD1d deficiency was associated with decreased T cell production of type 2 cytokines and increased or unchanged type 1 cytokines. These findings indicate a regulatory role of CD1d in inflammatory dermatitis. Understanding the mechanisms by which CD1d deficiency results in splenic T cell expansion and cytokine alterations, with increased dermal infiltration of dendritic cells and lymphocytes in MRL-lpr/lpr mice, will have implications for the pathogenesis of inflammatory skin diseases.     

Effect of TNF priming of murine peritoneal macrophages on their activation to a tumoricidal state

Murine peritoneal macrophages, on treatment with TNF (10 U/ml) for various durations, showed enhanced cyototoxicity against tumor target cells. The macrophage-mediated cytotoxicity was significantly enhanced when TNF-primed macrophages were treated with cisplatin, LPS, FK-565 or interferon-gamma for 24 h, compared to unprimed and treated macrophages. TNF-primed macrophages also showed enhanced expression of interleukin-1 and tumor necrosis factor activities on activation with different biological response modifiers.     

EGPred: prediction of eukaryotic genes using ab initio methods after combining with sequence similarity approaches

EGPred is a Web-based server that combines ab initio methods and similarity searches to predict genes, particularly exon regions, with high accuracy. The EGPred program proceeds in the following steps: (1) an initial BLASTX search of genomic sequence against the RefSeq database is used to identify protein hits with an E-value <1; (2) a second BLASTX search of genomic sequence against the hits from the previous run with relaxed parameters (E-values <10) helps to retrieve all probable coding exon regions; (3) a BLASTN search of genomic sequence against the intron database is then used to detect probable intron regions; (4) the probable intron and exon regions are compared to filter/remove wrong exons; (5) the NNSPLICE program is then used to reassign splicing signal site positions in the remaining probable coding exons; and (6) finally ab initio predictions are combined with exons derived from the fifth step based on the relative strength of start/stop and splice signal sites as obtained from ab initio and similarity search. The combination method increases the exon level performance of five different ab initio programs by 4%-10% when evaluated on the HMR195 data set. Similar improvement is observed when ab initio programs are evaluated on the Burset/Guigo data set. Finally, EGPred is demonstrated on an approximately 95-Mbp fragment of human chromosome 13. The list of predicted genes from this analysis are available in the supplementary material. The EGPred program is computationally intensive due to multiple BLAST runs during each analysis. The EGPred server is available at http://www.imtech.res.in/raghava/egpred/.