Mutational analysis of the cystathionine beta-synthase gene: A splicing mutation, two missense mutations and an insertion in patients with homocystinuria

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G-->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G-->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA. (C) 1998 Wiley-Liss, Inc.

Acute intermittent porphyria: alternative splicing of hydroxymethylbilane synthase mRNA excludes exons 3 and 12

The hydroxymethylbilane synthase (HMBS) mRNAs from 44 control individuals and 30 patients suffering from acute intermittent porphyria (AIP), were screened for length differences by reverse transcriptase polymerase chain reaction (RT-PCR) and any abnormalities were characterized by direct sequencing. Examination of the mRNAs extracted from the peripheral blood lymphocytes of the samples revealed varying degrees of alternative splicing, involving the removal of exons 3 and 12. Approximately 10-50% of the mRNA molecules were affected, despite the absence of genomic splice site mutations or any major deviance from consensus splice sequence values. The preliminary data obtained from this study suggest that this event is a normal occurrence in peripheral blood lymphocytes, and may not be associated with the molecular pathology responsible for AIP. (C) 1998 Academic Press Limited.

Field evidence for mechanical transmission of rabbit haemorrhagic disease virus (RHDV) by flies (Diptera : Calliphoridae) among wild rabbits in Australia

Field collected flies were screened for the presence of rabbit haemorrhagic disease virus (RHDV) by applying reverse transcriptase PCR (RT-PCR) in which primers specific to the capsid protein of the virus were used. The virus was detected in flies from locations where rabbit haemorrhagic disease (RHD) was reported and also soon after the release of RHDV in a 'clean' area. Oral and/or anal excretions of flies (flyspots) were found to contain viable virus and oral inoculation of rabbits revealed that a single flyspot was able to cause RHD. We conclude that flyspots are a major potential source of the virus for oral or conjunctival transmission of the virus to rabbits. (C) 1998 Elsevier Science B.V. All rights reserved.

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Sequence analysis of rabbit haemorrhagic disease virus (RHDV) in Australia: alterations after its release

Liver samples from rabbits killed by RHDV, collected from five States in Australia in 1996 and 1997 were analysed by RT-PCR. A 398 bp fragment of the capsid protein (VP60) gene was amplified by PCR and directly sequenced. The alignment of the nucleotide and amino acid sequences and their comparison with the original strain of the virus released in Australia indicated genetic changes after two years have been small with 98.2% to 100% identity. The constructed phylogenetic tree suggests slight differences in nucleotide substitutions in various States but there is no clear evidence of clustering of sequences according to their geographic origin. In practical terms, sequencing of viral RNA provides a means of testing the efficacy of further releases and subsequent spread of the virus if such a strategy is employed as a means of enhancing RHD as a biological control of the wild rabbit in Australia.

Lentiviral vector-mediated tyro sinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs), which play a vital role in primary immune responses. Introducing genes into DCs will allow constitutive expression of the encoded proteins and thus prolong the presentation of the antigens derived therefrom. In addition, multiple and unidentified epitopes encoded by the entire tumor-associated antigen (TAA) gene may enhance T cell activation. This study demonstrated that an HIV-1-based lentiviral vector conferred efficient gene transfer to DCs. The transgene, murine tyrosinase-related protein 2 (mTRP-2), encodes a clinically relevant melanoma-associated antigen (MAA), which has been found to be a tumor rejection antigen for B16 melanoma. The transfer and proper processing of mTRP-2 in DCs, in terms of RNA transcription activity and protein expression, were verified by RT-PCR and specific antibody, respectively. Administration of mTRP-2 gene-modified DCs (DC-HR'CmT2) to C57BL/6 mice evoked strong protection against tumor challenge, for which the presence of CD4(+) and CD8(+) cells during both the priming and challenge phase was essential. In a therapy model, our results showed that four of seven mice with preestablished tumor remained tumor free for 80 days after therapeutic vaccination. Given the results shown in this study, mTRP-2 gene transfer to DCs provides a potential therapeutic strategy for the management of melanoma, especially in the early stage of the disease.

Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration

Background: T lymphocytes and mast cells infiltrate the lamina propria in oral lichen planus (OLP). Chemokines and their receptors are involved in T cell and mast cell migration and accumulation during the inflammatory process. Methods: In the present study, we investigated the role of RANTES and its receptors in OLP using immunohistochemistry, RT-PCR and an in vitro chemotaxis assay. Results: RANTES and CCR1 were expressed on T cells and mast cells in OLP, while OLP lesional T cell supernatants stimulated CCR1 mRNA expression in a human leukemia mast cell line (HMC-1). TNF-alpha stimulated CCR1, CCR4 and CCR5 mRNA expression in the same cell line. OLP lesional T cell supernatants stimulated HMC-1 migration, which was partly inhibited by anti-RANTES antibody. Conclusions: The present study shows, for the first time, the distribution of RANTES and CCR1 in OLR It is hypothesized that RANTES and CCR1 may play important roles in mast cell trafficking and related events in OLP.

Cytokines in the oral mucosa of mice infected with Candida albicans

Oropharyngeal candidiasis is associated with defects in cell-mediated immunity, and is commonly seen in immunocompromised patients. We have previously shown that T-cell-deficient BALB/c nude (nu/nu) mice are extremely susceptible to oropharyngeal candidiasis, and that recovery from a chronic infection is dependent on CD4 T lymphocytes. In this study we describe the local tissue cytokine profile in lymphocyte-reconstituted immunodeficient mice and their euthymic counterparts. Mice were infected orally with 10(8) cells of the yeast Candida albicans , and oral tissues sampled on days 0, 4, 8, and 14. Nude mice were reconstituted with 3 x 10(7) naive lymphocytes following oral inoculation. Interleukin (IL)-6, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were identified in the oral tissues of infected euthymic mice recovering from oral infection, as well as naive controls. TNF-alpha was identified in nude oral tissue on days 4 and 8, but only after lymphocyte reconstitution. No IL-2, IL-4 or IL-10 was detected in either euthymic or athymic mice at any time-point throughout the experiment. This study confirms the functional activity of T lymphocytes in reconstituted nude mice, and suggests that TNF-alpha may be an important mediator in the recovery from oropharyngeal candidiasis.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Alterations in cardiac dihydropyridine receptors and calcium channel function in mdx mice

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular condition affecting approximately one in 3500 live male births resulting from the lack of the myocyte protein dystrophin. The absence of dystrophin in cardiac myocytes is associated with calcium overload which in turn activates calcium-dependent proteolytic enzymes contributing to congestive heart failure, muscle necrosis and fibrosis. To date, the basis for the calcium overload has not been determined. Since L-type calcium channels are a major mediator of calcium influx we determined their potential contribution to the calcium overload. Male muscular dystrophy (mdx) mice and control C57BL10ScSn (C57) mice aged 12– 16 weeks were used in all experiments. In tissue bath studies, isolated contracting left atria from mdx revealed a reduced potency to the dihydropyridine (DHP) agonist BayK8644 and antagonist nifedipine (P < 0.05). Similarly, radioligand binding studies using the DHP antagonist [3H]-PN 200-110 showed a reduced potency (P < 0.05) in isolated membranes, associated with an increased receptor density (P < 0.05). The increased receptor density was supported by RT-PCR experiments revealing increased RNAfor the DHP receptor. Patch clamp studies revealed the presence of a diltiazem sensitive calcium current that showed delayed inactivation in isolated mdx myocytes (P < 0.01). In conclusion, the increased number of DHP binding sites and the delay in L-type current inactivation may both contribute to increased calcium influx and hence calcium overload in the dystrophin deficient mdx cardiac myocytes.

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24 h elicited a marked increase in mRNA expression for IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway.. although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

ERCC1 protein, mRNA expression and T19007C polymorphism as prognostic markers in head and neck squamous cell carcinoma patients treated with surgery and adjuvant cisplatin-based chemoradiation

Adjuvant cisplatin-based chemoradiation improves survival in HNSCC patients presenting with risk features. ERCC1 (excision repair cross-complementation group 1) is associated with resistance to chemo- and radiation therapy and may have a prognostic value in HNSCC patients. Here we studied ERCC1 expression and the polymorphism T19007C as prognostic markers in these patients. This is a retrospective and translational analysis, where ERCC1 protein expression was evaluated by immunohistochemistry, using an H-score, and mRNA expression was determined by RT-PCR. T 19007C genotypes were detected by PCR-RFLP carried out using DNA template extracted from normal lymph nodes. A high H-score was seen in 32 patients (54%), who presented better 5-year overall survival (5-y OS: 50% vs. 18%, HR 0.43, p=0.026). Fifteen out of 45 patients (33%), with high mRNA expression, presented better 5-year overall survival (OS) (86% vs. 30%, HR 0.26, p=0.052). No OS difference was detected among T 19007C genotypes. High H-score and mRNA expression remained significant as favorable prognostic factors in a multivariate analysis. Collectively, our results suggest that high ERCC1 expression seems to be associated with better OS rates in HNSCC patients submitted to adjuvant cisplatin-based chemoradiation.

The therapeutic potential of recombinant BCG expressing the antigen S1PT in the intravesical treatment of bladder cancer

Purpose: Bacillus Calmette-Guerin (BCG) continues to be employed as the most effective immunotherapy against superficial bladder cancer. We have developed an rBCG-S1PT strain that induces a stronger cellular immune response than BCG. This preclinical study was designed to test the potential of rBCG-S1PT as an immunotherapeutic agent for intravesical bladder cancer therapy. Materials and methods: A tumor was induced in C57BL/6 mice after chemical cauterization of the bladder and inoculation of the tumor cell line MB49. Next, mice were treated by intravesical instillation with BCG, rBCG-S1PT, or PBS once a week for 4 weeks. After 35 days, the bladders were removed and weighed, Th1 (IL-2, IL-12, INOS, INF-gamma, TNF-alpha), and Th2 (IL-5, IL-6, IL-10, TGF-beta) cytokine mRNA responses in individual mice bladders were measured by quantitative real time PCR, and the viability of MB49 cells in 18-hour coculture with splenocytes from treated mice was assessed. In an equivalent experiment, animals were observed for 60 days to quantify their survival. Results: Both BCG and rBCG-S1PT immunotherapy resulted in bladder weight reduction, and rBCG-S1PT increased survival time compared with the control group. There were increases in TNF-alpha in the BCG treated group, as well as increases in TNF-alpha and IL-10 mRNA in the rBCG-S1PT group. The viability of MB49 cells cocultured with splenocytes from rBCG-S1PT-treated mice was lower than in both the BCG and control groups. Conclusions: rBCG-S1PT therapy improved outcomes and lengthened survival times. These results indicate that rBCG could serve as a useful substitute for wild-type BCG. (C) 2010 Elsevier Inc. All rights reserved.

The role of interleukin-6, endothelins, and apoptotic genes in small bowel transplantation, in a swine model of ischemia and reperfusion injury

IRI is closely related to sepsis in ITx setting. Complete understanding of the mechanisms involved in IRI development may improve outcomes. Ortothopic ITx without immunosuppression was performed in order to characterize IRI-associated mucosal damage. Twenty pigs underwent ITx. Two groups were assigned to different CI times: G1: 90 min and, G2: 180 min. Euro-Collins was used as preservation solution. Jejunal fragments were collected at donor laparotomy, 30 min, and 3 days after reperfusion. IRI assessment involved: histopathologic analysis, quantification of MPO-positive cells through immunohistochemical studies, quantification of epithelial apoptotic cells using TUNEL staining, and quantification of IL-6, ET-1, Bak, and Bcl-XL genes expression by RT-PCR. Neutrophilic infiltration increased in a similar fashion in both groups, but lasted longer in G2. Apoptosis detected by TUNEL staining increased and anti-apoptotic gene Bcl-XL expression decreased significantly in G1, 3 days after surgery. Endothelin-1 and IL-6 genes expression increased 30 min after the procedure and returned to baseline 3 days after surgery. In conclusion, IL-6 and ET-1 are involved precociously in the development of intestinal IRI. Apoptosis was more frequently detected in G1 grafts by TUNEL-staining and by RT-PCR.

Direct evidence for the expression of multiple endogenous retroviruses in the synovial compartment in rheumatoid arthritis

Objective. Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases, The aim of the present study was to obtain direct evidence of RV gene expression in rheumatoid arthritis (RA). Methods. Synovial samples were obtained from patients with RA, patients with osteoarthritis (OA), and normal control subjects, Reverse transcription-polymerase chain reaction (RT-PCR) was performed using synovial RNA and primers to conserved sequences in the polymerase (pol) genes of known RVs. Results. PCR products (n = 857) were cloned and sequenced, Multiple pol transcripts, many with open reading frames, were expressed in every sample, Sequences were aligned and classified into 6 families (F1-F6) that contained 33 groups of known and unknown endogenous RVs (ERVs), each distinguished by a specific, deduced peptide motif, The frequency of sequences in each family was similar between RA, OA, and normal synovial tissue, but differed significantly in RA synovial fluid cells, F1 sequences (undefined, but related to murine and primate type C RVs) were lower in frequency, F2 (ERV-9-related), F4 (HERV-K-related), and F6 (HERV-L-related) sequences were higher in frequency, and F3 (RTVL-H-related) sequences were not detected, in the RA synovial fluid cells compared with the RA synovial tissues. Conclusion. Multiple ERVs are expressed in normal and diseased synovial compartments, but specific transcripts can be differentially expressed in RA.