Biomedical subjects

S A Perera

Publications and source records attributed to S A Perera.

7 recordsLinked to original sources

PubMed2002

Reactivity and isotype profiling of monoclonal antibodies using multiple antigenic peptides.

The characterisation of monoclonal antibodies (MAbs) is essential for the development of assay systems particularly where antigens have been developed using synthetic peptides. Indeed some peptide-carrier conjugates fail to induce immune responses and may not generate antibodies that bind to native protein. As an alternative to peptide-carrier conjugates, multiple antigenic peptides (MAPs) have been used for immunization strategies, but with little regard to the characteristics of the MAbs produced. In this study, we used 3 MAPs of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) to immunise BALB/c mice. Overall, the polyclonal antibody responses from tail bleeds showed that MAPs evoked B-cell responses. However, on screening 144 hybridomas, 24 MAb supernatants exhibited weak to moderate reactivity in enzyme-linked immunosorbant assay (ELISA) and against cell cytospin preparations (B95.8 and AG876 LCL), respectively. Isotype profiling of hybridoma supernatants also showed that 11 out of 24 were IgM. Further characterization of 6 MAbs in Western blotting showed reactivity to recombinant LMP1 and only one MAb (B28D) showed weak reactivity to the malignant cells (Hodgkin/Reed-Sternberg; HRS cells) of an EBV+ Hodgkin's lymphoma using paraffin-embedded tissue. It is probable that these MAPs failed to augment T-cell help and contributed to the production of low affinity (IgM) antibodies. These observations may be of importance to future immunization strategies, where MAPs are used in the production of monoclonal reagents.

Animals↗

PubMed1999

Polarized distribution of Bcr-Abl in migrating myeloid cells and co-localization of Bcr-Abl and its target proteins.

Bcr-Abl plays a critical role in the pathogenesis of Philadelphia chromosome-positive leukemia. Although a large number of substrates and interacting proteins of Bcr-Abl have been identified, it remains unclear whether Bcr-Abl assembles multi-protein complexes and if it does where these complexes are within cells. We have investigated the localization of Bcr-Abl in 32D myeloid cells attached to the extracellular matrix. We have found that Bcr-Abl displays a polarized distribution, colocalizing with a subset of filamentous actin at trailing portions of migrating 32D cells, and localizes on the cortical F-actin and on vesicle-like structures in resting 32D cells. Deletion of the actin binding domain of Bcr-Abl (Bcr-AbI-AD) dramatically enhances the localization of Bcr-Abl on the vesicle-like structures. These distinct localization patterns of Bcr-Abl and Bcr-Abl-AD enabled us to examine the localization of Bcr-Abl substrate and interacting proteins in relation to Bcr-Abl. We found that a subset of biochemically defined target proteins of Bcr-Abl redistributed and co-localized with Bcr-Abl on F-actin and on vesicle-like structures. The co-localization of signaling proteins with Bcr-Abl at its sites of localization supports the idea that Bcr-Abl forms a multi-protein signaling complex, while the polarized distribution and vesicle-like localization of Bcr-Abl may play a role in leukemogenesis.

3T3 Cells↗

PubMed1999

Molecular investigations implicate human endogenous retroviruses as mediators of anti-retroviral antibodies in autoimmune rheumatic disease.

Polymerase chain reaction using specific primers, failed to detect HTLV-I amplicons in patients with rheumatic diseases previously shown to possess antibodies to retroviral products. However, by employing broad spectrum oligonucleotide primers, 135 bp amplicons were generated from peripheral blood mononuclear cells and synovial fluid cells. Subsequent cloning and DNA sequencing revealed homology to a number of exogenous and human endogenous retroviruses (HERVs). Furthermore, in combining the presence of type B and C related endogenous retroviruses, a significant association (p=0.014) was apparent for chronic autoimmune rheumatic diseases as compared to controls. Reverse transcription polymerase chain reaction of RNA derived from patients, healthy controls and cell lines (U937, BJAB, human endothelial lung fibroblasts) demonstrated ubiquitous expression of HERV-K10 and RTVL-H2. Furthermore messenger RNA expression of HERV-K10 was enhanced in fibroblasts infected with human cytomegalovirus. It is plausible that subsequent production of HERV peptides could explain the presence of anti-retroviral antibodies in cohorts of patients with autoimmune rheumatic diseases.

Antibodies, Viral↗

PubMed1997

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Amino Acid Sequence↗

PubMed1995

Atrial natriuretic peptide replacement therapy in rats subjected to biatrial appendectomy.

The postoperative fluid retention found in some patients after the Cox maze procedure has been attributed to surgically induced loss of atrial natriuretic peptide. We postulated that exogenous atrial natriuretic peptide could reverse this antidiuresis. A rat model was used to investigate this hypothesis. In group I, the sham group, the atrial appendages were left intact and the animals were then subjected to a fluid challenge equivalent to 1% of the animal's body weight. In group II, after biatrial appendectomy, the animals were subjected to a fluid challenge similar to that in group I. Animals in group III underwent the same protocol as that for group II plus intravenous administration of atriopeptin III at varying concentrations. Urine output and plasma atrial natriuretic peptide levels were significantly decreased after biatrial appendectomies (p < or = 0.01). Urine output returned to control levels after biatrial appendectomies with low-dose atrial natriuretic peptide infusion (0.5 pmol/min = 25.5 pg/min), although circulating atrial natriuretic peptide levels were lower. Urine output and plasma atrial natriuretic peptide levels increased with atrial natriuretic peptide infusions between 0.5 and 50 pmol/min. Heart rate and mean blood pressure did not vary significantly with atrial natriuretic peptide infusions. Thus atrial natriuretic peptide can be used effectively in low doses to induce a diuresis after biatrial appendectomies. Atrial natriuretic peptide may have clinical application after the Cox maze procedure.

Animals↗

PubMed1987

Leucocyte esterase test as rapid screen for non-gonococcal urethritis.

The two standard tests for the initial diagnosis of non-gonococcal urethritis (NGU), microscopic examination of gram stained urethral smears and the two glass urine test, have the disadvantage of being insensitive and subjective. The leucocyte esterase test detects enzymes specific to polymorphonuclear leucocytes and can therefore be used as a sensitive indicator of pyuria. This study sought to evaluate its use as a rapid, sensitive, and non-subjective method of screening for NGU. Of the 81 men with urethral symptoms in the study group, 26 had more than 5 polymorphonuclear leucocytes per high power field (x 1000) and all 26 were leucocyte esterase test positive; whereas 55 had fewer than 5 polymorphonuclear leucocytes per high power field, but 29 (53%) of them had a positive leucocyte esterase test result. In addition, 25 patients in the study group yielded Chlamydia trachomatis on culture. Of these 25, 24 (96%) were leucocyte esterase test positive, whereas only 11 (44%) were Gram stain positive. All 40 patients in the control group (without urethral symptoms or signs) were leucocyte esterase test negative. The leucocyte esterase test is thus a rapid, sensitive, and non-subjective screening aid in the diagnosis of NGU.

Adolescent↗

PubMedDate not supplied

Use of Kova-Slide II with grid and uncentrifuged segmented urine specimens in the diagnosis of nongonococcal urethritis: a quantitative technique.

An attempt was made to use uncentrifuged segmented urine specimens and Kova-Slide II with grid as a quantitative technique for the diagnosis of nongonococcal urethritis. Of the 100 men with urethral symptoms, 54 had fewer than five polymorphonuclear leukocytes per high-power field (x1000) in their gram-stained urethral smear. On the basis of examination of segmented urine and Kova-Slide test, 41 of these 54 patients were diagnosed as having nongonococcal urethritis. None of the 13 patients considered to be free of urethritis on the basis of segmented urine tests had a Chlamydia trachomatis--positive culture, and all became asymptomatic without treatment. All 46 patients who had more than five polymorphonuclear leukocytes per high-power field (x1000) also had positive segmented urine tests. The rate of isolation of C. trachomatis was similar for both groups of tests. This study revealed that patients with lower polymorphonuclear leukocyte counts in the first 10 ml of urine passed (voided-bladder urine 1) also had cultures positive for C. trachomatis. None of the 50 patients in the control group had polymorphonuclear leukocytes in either voided-bladder urine 1 or in the midstream specimen (voided-bladder urine 2). All controls had cultures negative for C. trachomatis.

Bacteriological Techniques↗