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Biomedical subjects

R B Lal

Publications and source records attributed to R B Lal.

At least 145 records · Page 8Linked to original sources
PubMed1991

Synthetic peptide-based immunoassays for distinguishing between human T-cell lymphotropic virus type I and type II infections in seropositive individuals.

Until now, serologic tests that distinguish the closely related human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) infections have not been available. Synthetic peptide assays, employing peptides derived from the core and envelope proteins of HTLV-I and HTLV-II (SynthEIA and Select-HTLV tests), were evaluated for the ability to serologically discriminate HTLV-I and HTLV-II infections. Of 32 HTLV-I- and 57 HTLV-II-positive serum specimens from individuals whose infections were confirmed by polymerase chain reaction, the SynthEIA test categorized 29 (91%) as HTLV-I and 50 (88%) as HTLV-II, and 10 (11%) were nontypeable. In contrast, the Select-HTLV test categorized 32 (100%) as HTLV-I and 55 (96%) as HTLV-II, and 2 (2%) were nontypeable. The specificity of both the assays in seropositive serum specimens was 100% in that none of the specimens were incorrectly classified. Additional serum specimens obtained from clinically diseased patients from the United States (n = 8) and asymptomatic carriers and patients from Japan (an endemic population for HTLV-I; n = 40) were categorized as HTLV-I by at least one of the assays, while serum specimens from Guaymi Indians from Panama (an endemic population for HTLV-II; n = 13) were categorized as HTLV-II. Thus, peptide enzyme immunoassays appear to represent a simple technique employing chemically synthesized antigens for discrimination between antibodies of HTLV-I and HTLV-II.

Antibodies, Viral↗
PubMed1991

Evaluation of a recombinant human T-cell lymphotropic virus type I (HTLV-I) p21E antibody detection enzyme immunoassay as a supplementary test in HTLV-I/II antibody testing algorithms.

To evaluate the usefulness of a human T-cell lymphotropic virus type I (HTLV-I) recombinant p21E immunoassay as a supplementary test in HTLV-I/II serologic testing algorithms, we used this assay to test 378 serum samples previously categorized as positive, indeterminant, or negative for HTLV-I/II antibody, as defined by U.S. Public Health Service guidelines. We found this test to be highly sensitive for detecting antibody to HTLV-I/II env (99.4%) but slightly less specific (96.0%), particularly among samples from intravenous drug users. Our data suggest that this test is most appropriately used to confirm the absence of env antibody in samples which are repeatably reactive in an HTLV-I/II screening assay and gag reactive only by immunoblotting. Because of the high sensitivity of this recombinant p21E test, a negative result in this context could preclude radioimmunoprecipitation testing. However, pending further evaluation of the specificity of this assay, samples testing positive for p21 env antibody may require confirmation by radioimmunoprecipitation, particularly in situations in which the results will be used for diagnostic purposes or blood donor counseling.

Algorithms↗
PubMed1991

Characterization of immunodominant epitopes of gag and pol gene-encoded proteins of human T-cell lymphotropic virus type I.

A series of synthetic peptides derived from the corresponding regions of the gag, pol, and env proteins of human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) were used in an enzyme immunoassay to map the immunodominant epitopes of HTLV. Serum specimens from 79 of 87 (91%) HTLV-I-infected patients reacted with the synthetic peptide Gag-1a (amino acids [a.a.] 102 to 117) derived from the C terminus of the p19gag protein of HTLV-I. Minimal cross-reactivity (11%) was observed with serum specimens from HTLV-II-infected patients. Peptide Pol-3, encoded by the pol region of HTLV-I (a.a. 487 to 502), reacted with serum specimens from both HTLV-I- and HTLV-II-infected patients (94 and 86%, respectively). The antibody levels to Pol-3 were significantly higher (P less than 0.01) in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in either adult T-cell leukemia patients or HTLV-I-positive asymptomatic carriers. None of the other peptides studied demonstrated significant binding to serum specimens obtained from HTLV-I- or HTLV-II-infected individuals. While Gag-1a did not react with serum specimens from normal controls, Pol-3 demonstrated some reaction with specimens from seronegative individuals (11.4%). The antibodies to Gag-1a and Pol-3 in serum specimens from HTLV-I-infected patients could be specifically inhibited by the corresponding synthetic peptides and by a crude HTLV-I antigen preparation, indicating that these peptides mimic native epitopes present in HTLV-I proteins that are recognized by serum antibodies from HTLV-I- and -II-infected individuals.

Amino Acid Sequence↗
PubMed1991

Lack of IgG4 antibody response to carbohydrate antigens in patients with lymphatic filariasis.

It has been suggested that humans are genetically restricted from making IgG4 antibody responses to carbohydrate antigens. To test this hypothesis we examined sera from 35 patients with bancroftian filariasis (an infection known to induce very high levels of IgG4 antibodies to the parasite and known to be associated with repeated streptococcal infections) as well as from 15 normal individuals for their IgG and IgG subclass responses to streptococcal protein [streptolysin-O (SO), deoxyribonuclease B (DB)] and carbohydrate [group A carbohydrate (GAC)] antigens. Levels of IgG antibodies to all three antigens were found to be significantly higher in the filariasis patients compared to normals (P less than 0.01), and the subclass composition of these antibodies proved heterogenous. Although responses to all three antigens included IgG1, IgG2 and IgG3 antibodies and although IgG4 responses to the proteins SO and DB were significantly higher in the filariasis patients than in normals (P less than 0.001), more importantly there were no detectable anti-GAC IgG4 antibodies in either study group. These observations, coupled with our earlier finding of the absence of IgG4 responses to phosphocholine (PC) in patients with lymphatic filariasis, suggest that even the chronic antigenic stimulation of filarial helminth infection, which leads to very prominent IgG4 responses to protein antigens, cannot overcome the genetic restriction in humans for making IgG4 antibodies to carbohydrate antigens, whether of parasite or non-parasite origin.

Adolescent↗
PubMed1990

Elevated levels of soluble CD8 molecule in patients with lymphatic filariasis.

The serum levels of soluble forms of suppressor/cytotoxic cells (sCD8) and interleukin-2 receptor (sCD25) were analyzed in 67 patients with lymphatic filariasis and 28 normal controls. Our results show that patients with lymphatic filariasis have significantly higher levels of sCD8 (p less than 0.05) than the control groups, whereas no such difference was observed for sCD25. Within the patient group, however, those in the chronic lymphatic obstruction (CP) had significantly higher levels of both sCD8 (491 +/- 52 U/ml, p less than 0.001) and sCD25 (293 +/- 36 U/ml; p less than 0.02) than those with asymptomatic microfilaremia (sCD8 344 +/- 32 U/ml; sCD25 190 +/- 10 U/ml, respectively). No statistically significant correlation was observed between the serological levels of sCD8 and the percentage of CD8 on peripheral blood T lymphocytes in any of the patient groups. Evaluation of the activation state of B lymphocyte did not reveal any difference in the cellular expression of B7, or serum levels of soluble CD23 in any of the groups studied. Thus the selective increase of sCD8 in patients with filariasis suggests a possible pathogenic role of the cells involved in the release of this molecule.

Antibodies, Monoclonal↗
PubMed1990

Long-term preservation of whole blood samples for flow cytometry analysis in normal and HIV-infected individuals from Africa.

A whole blood method requiring less than 4 ml of heparinized blood was developed to assess the practicality of preparing whole blood samples that could be easily stored, transported and readily used to determine the lymphocyte phenotypes and proliferation responses of individuals from remote areas who are infected with the human immunodeficiency virus. Minor modifications in standard whole blood procedure for lymphocyte phenotyping have significantly increased the stability of light scatter and fluorescence intensity of the cells for subsequent flow cytometry (FC) analysis. These changes include removal of lysis solution prior to fixation, fixation of monoclonal antibody-stained cells in 1% paraformaldehyde for 30 minutes and storage of fixed samples in medium containing 1% bovine serum albumin. Lymphocyte subsets and their functional subsets could reliably be determined on samples stored for up to 4 weeks. Further, blood samples could be kept at room temperature for up to 96 hours or at ambient temperature during transportation from Africa before staining for FC without affecting their quantitation. While samples could be processed for FC analysis under field-laboratory conditions, proliferation assays could only be performed on samples that were transported within 48 hours of their collection. The whole blood method saves time and expense and decreases the volumes of blood required to perform phenotypic analysis and functional assays on specimens collected in remote areas.

Adult↗
PubMed1990

Aggressive pacemaker twiddler's syndrome. Dislodgement of an active fixation ventricular pacing electrode.

Pacemaker twiddler's syndrome is the phenomenon whereby a pacemaker patient may actively dislodge a pacing electrode by manipulating, often unconsciously, the permanent implanted pacemaker. This unusual case is remarkable in several respects: surgical precautions, originally taken to secure the pulse generator against migration, proved ineffective; the displaced lead featured an active fixation "screw-in" electrode; and the reported dislodgement occurred not in the early postimplant stage, but fully three months later. It can be concluded that even with advanced lead technology and operative precaution, electrode dislodgement cannot be absolutely prevented. Awareness of this phenomenon, especially by pacemaker follow-up care personnel, could lessen its occurrence.

Aged↗
PubMed1990

Phosphocholine-containing antigens of Brugia malayi nonspecifically suppress lymphocyte function.

The immunosuppressive effect of Brugia malayi antigen (BmA) on phytohemagglutinin (PHA) driven T cell proliferation was evaluated in patients with filariasis (n = 14) and compared to control individuals (n = 12). When peripheral blood lymphocytes were co-cultured with BmA and PHA, BmA markedly suppressed the T cell proliferative response to PHA in both filarial patients and control individuals in a dose-dependent manner. The suppression resulted neither from any direct toxicity of BmA nor from nonspecific absorption of the PHA mitogenic activity by BmA. The major suppressive component appears to be phosphocholine (PC), an immunodominant molecule present in abundance on filarial parasites and on circulating filarial antigen. Both purified PC as well as PC-containing antigens affinity purified from BmA were capable of suppressing the proliferative responses of co-cultured autologous lymphocytes to PHA. The suppressive activity was not abolished by mitomycin-C treatment and was greater in patients with filariasis than in normal controls, suggesting that levels of PC-containing antigens determines the magnitude of the suppressive effect of PC-antigen. Further, as induction of the suppressive activity was completely abrogated when antigen pre-treated cells were T cell-depleted, the suppressive effect appears to be mediated primarily by T cells.

Adult↗
PubMed1989

Lymphocyte subpopulations in Bancroftian filariasis: activated (DR+) CD8+ T cells in patients with chronic lymphatic obstruction.

To examine the relationship between lymphocyte phenotypes and states of activation in patients with Bancroftian filariasis, dual colour flow cytometry and concurrent in vitro cell culture were performed on normal individuals (NV; n = 15), and on patients with either asymptomatic microfilaraemia (MF; n = 12) or elephantiasis (CP; n = 11). In contrast to findings by others in a population with Brugian filariasis, the percentages of total B lymphocytes (CD19), T lymphocytes (CD3), helper/inducer T lymphocytes (CD4), and suppressor/cytotoxic T lymphocytes (CD8) in both patient groups were found to be within the range defined by clinically normal individuals. Furthermore, there were no differences among the groups in the expression of the IL-2 receptor (CD25) on T cells. There was, however, a significantly greater proportion (P less than 0.01) of 'activated' cytotoxic/suppressor lymphocytes (defined by co-expression of CD8 and HLA-DR) in patients with elephantiasis (16.4 +/- 8.6%) than in the MF (8.9 +/- 2.6%) or NV (8.3 +/- 2.9%) groups. Further, when the expression of this activation antigen was examined in parallel with in vitro mitogen responsiveness, an inverse correlation between the percentage of CD8+ HLA-DR+ lymphocytes and pokeweed mitogen-induced proliferation was seen (r = -0.54; P less than 0.001). These data provide further definition of the immunoregulatory abnormalities seen in human filarial infections and suggest that activated CD8+ T lymphocytes may be involved in the pathogenesis of the chronic obstructed lymphatic form of this disease.

Adult↗
PubMed1989

Antibodies to phosphocholine-bearing antigens in lymphatic filariasis and changes following treatment with diethylcarbamazine.

Sera from a total of 78 patients infected with Wuchereria bancrofti or appropriate controls were assayed for anti-phosphocholine antibodies in an enzyme-linked immunosorbent-assay (ELISA), using phosphocholine as an antigen. Anti-PC antibodies (both IgM and IgG) were observed in patients with all clinical forms of filariasis but, unexpectedly, were not significantly different from those of normal controls. Among the filariasis patients, however, individuals with patent microfilaraemia had significantly lower IgM-anti-PC titres (P less than 0.05) than did those of the other clinical groups. Competitive studies showed that 'naturally occurring' anti-PC antibodies interfere with PC antigen detection. Transient increases in the levels of circulating PC antigen were found by 3 days after treatment of microfilaraemic individuals with the filaricidal drug diethylcarbamazine. The subsequent decreases in antigen levels were generally associated with increased IgM- and IgG-anti-PC antibody levels. Such changes likely affect not only the ability to monitor the parasitological status of such individuals, but also the patient's subsequent immune interactions with filarial parasites.

Adolescent↗
PubMed1988

Characterization of stage-specific antigens of infective larvae of the filarial parasite Brugia malayi.

Three stages of the filarial parasite Brugia malayi (infective third stage larvae, adult worms, and microfilariae) were analyzed for differences in their protein composition by two-dimensional gel electrophoresis. Comparison of protein profiles of the different stages showed both identical polypeptides (reflecting common proteins) and polypeptides specific for each stage. Three polypeptides present only in infective stage larvae were seen at 72 kDa at an isoelectric point (pI) of 4.98 (p72), 30 kDa at pI 5.5 (p30), and 22 kDa at pI 4.75 (p22). p72 could be labeled chemically with 125I by either chloramine T or IODO-GEN and biosynthetically with [3H]leucine and [35S]methionine during in vitro culture of live larvae; thus, p72 is most likely a surface protein of parasite origin. The antigenic composition of these polypeptides was elucidated by immunoblot analysis. Both p72 and p22 were recognized by hyperimmune rabbit sera to infective larvae; sera from rabbits immunized with adult worms, however, did not recognize any of these Ag. Sera from humans infected with the related Wuchereria bancrofti filaria recognized only p72 and not the other two polypeptides. It therefore appears that p72 is a stage-specific but not genus-specific Ag that is immunogenic in the infected host. p22 also appears to be stage specific and, because it is not recognized by W. bancrofti-infected sera, it may be either a species-specific Ag or a poorly immunogenic molecule of the parasite. With mAb raised to p72, p30, and p22, these proteins were shown to share several antigenic determinants when analyzed by immunoblotting. The shared epitopes were present on numerous molecules with a wide range of apparent m.w. in each of the different parasite stages. Thus, despite the apparent larval stage specificity of these molecules themselves, they must contain certain epitopes shared by molecules from other stages as well. The identification of the p72 polypeptide as a molecule with epitopes exposed on the surface of infective larvae provides a candidate Ag for testing as a protective immunogen.

Animals↗
PubMed1988

Fixation and long-term storage of human lymphocytes for surface marker analysis by flow cytometry.

A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4 degrees C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analyzing lymphocytes that express T-cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.

Antibodies, Monoclonal↗
PubMed1988

Enhanced diagnostic specificity in human filariasis by IgG4 antibody assessment.

Phosphocholine (PC), an immunodominant molecule present on a wide range of organisms, including filariae, evokes antibody responses that lead to false-positive reactions in routine serological assays. Humans essentially do not respond to PC in the IgG4 subclass; therefore, by configuring an enzyme immunoassay to assess antibodies of the IgG4 subclass only, we were able to eliminate serological false-positive results in 32 of 34 cross-reactive sera from patients with non-filarial parasitic infections. Specificity was thus greatly enhanced with minimal loss of sensitivity. Because preadsorption of these cross-reactive sera to remove antibodies to PC eliminated only approximately 50% of the original cross-reactivity, other shared epitopes (perhaps similar to PC) are also likely to be restricted by IgG subclass.

Animals↗
PubMed1987

Brugia malayi antigens associated with lymphocyte activation in filariasis.

Filarial parasites induce immune response in humans which are still poorly characterized. To define the antigens responsible for inducing lymphocyte responses, fast protein liquid chromatography was used to fractionate the antigens of Brugia malayi adult worms which were then tested on lymphocytes from patients with filariasis and normal controls. From an anion exchange column (Mono Q), three peaks of lymphocyte-stimulating activity were eluted which were further fractionated by gel filtration (Superose-12). Peak I induced both a proliferative response as well as the production of filaria-specific antibody in patient lymphocytes. Peak II, capable of inducing only a proliferative response (without antibody production) in patient lymphocytes, was a glycoprotein with phosphocholine as one of the antigenic determinants. Peak III induced proliferative responses in both patient and normal lymphocytes and thus appears to be mitogenic. Two-dimensional gel electrophoresis was then used to identify changes in the major cellular proteins associated with the activation of patient lymphocytes by these partially purified antigens. Stimulation of patient lymphocytes with peak I resulted in increased synthesis of immunoglobulin heavy, light, and J chains. Further, these were the only major secreted proteins found in the culture supernatants. Peak II resulted in quantitative changes in proteins associated with T and not B lymphocyte stimulation. Further analysis of these antigens should help to elucidate the mechanism of host-parasite interaction at both the cellular and molecular levels.

Animals↗
PubMed1987

Proteins associated with B lymphocyte hyperactivity in New Zealand black mice.

New Zealand Black (NZB) mice exhibit polyclonal B cell activation and elevated immunoglobulin production, an abnormality associated with the spontaneous autoimmune disease that affects this strain. To further our understanding of this abnormality of B cell differentiation and maturation, we have employed two-dimensional polyacrylamide gel electrophoresis to analyze the proteins synthesized by lymphocytes of several strains. Two proteins were produced by lymphocytes from NZB mice but not those from normal strains. One was a 16 kd protein with a pI of 5.1, and the other was a 27.5 kd protein with a pI of 4.5. The presence of the xid gene on the NZB background suppressed production of both proteins. They were synthesized by spleen cells but not by bone marrow or lymph node cells, and production was restricted to enlarged B lymphocytes. p16 was synthesized by normal mouse strain B cells upon stimulation with LPS. The 27.5 kd protein was shown to be secreted. On the basis of partial amino acid sequence determination of proteins eluted from gels, p27.5 was identified as J chain and p16 as the C terminal fragment of mu-chain. The synthesis of two other proteins, 13 kd and 18 kd in size, was elevated in NZB spleen lymphocytes. The 18 kd protein was identified as translation initiation factor eIf-4D. The increased level of this protein may be related to the upregulation of immunoglobulin synthesis.

Amino Acid Sequence↗
PubMed1987

Circulating parasite antigen(s) in lymphatic filariasis: use of monoclonal antibodies to phosphocholine for immunodiagnosis.

Hybridoma cell lines producing monoclonal antibodies (MAb) against a 200 kD antigen found circulating in the sera of microfilaremic patients infected with Wuchereria bancrofti were obtained by immunizing mice with a partially purified antigen preparation. A sensitive MAb (CA101)-based ELISA for measuring circulating parasite antigen was capable of detecting antigen in the sera of 93% of patients with microfilaremia, 46% of those with lymphatic obstruction, and 56% of patients with tropical pulmonary eosinophilia syndrome. Circulating antigen was absent from sera of normal controls, and "false positives" were recorded in only two of 17 patients with nonfilarial helminth infections. By ELISA and immunoblot analysis, it was shown that three of the monoclonals raised to this 200 kD antigen were directed to epitopes of phosphocholine (PC). Two MAb (CA86, CA101) were identified as having the T15 idiotype previously associated with antibodies to the PC of pneumococcal teichoic acid; one was untypeable. All three of these anti-PC MAb reacted with adult, microfilaria, and larval antigen preparations, and by immunoblotting showed multiple banding patterns that indicated the presence of PC determinants on many different antigenic molecules. On the other hand, target antigens of CA101 which were found in the circulation of infected patients were limited to three species with apparent m.w. of 200, 160, and 78 kD. The 200 kD antigen was seen more frequently than the other two antigens. Other T15 anti-PC MAb derived from mice not immunized with filarial antigen showed similar patterns of reactivity with circulating filarial antigen.

Animals↗
PubMed1987

Dietary copper deficiency and autoimmunity in the NZB mouse.

NZB mice were exposed from birth to a diet either adequate or deficient in copper. By age 6 wk the mice exposed to the copper-deficient diet showed symptoms characteristic of copper deficiency (anemia, hypoceruloplasminemia, and achromatrichia). The splenic lymphocytes from the copper-deficient group had reduced numbers of cells expressing the following surface markers: Ly-5, Ly-1, B-220, and sIg. Less than 10% of the splenic lymphocytes in this group were cycling, as determined by flow cytometry analysis. The spontaneous 96-h anti-ss-DNA levels in the copper-deficient group were lower than those in the control group. The exogenous colony-forming units (CFUs) were significantly enhanced in the copper-deficient mice. The decreased splenic lymphoid populations, decreased anti-ss-DNA titers, and increased exogenous CFUs in the copper-deficient mice appear to be due to an increase in erythropoiesis at the expense of lymphopoiesis.

Animals↗