Biomedical subjects

P L Lim

Publications and source records attributed to P L Lim.

At least 37 records · Page 2Linked to original sources

PubMed1996

Common occurrence of an antiidiotypic antibody that recognizes T14+ anti-DNA antibodies in patients with systemic lupus erythematosus.

OBJECTIVE: To investigate whether antibodies to a T14 anti-DNA antibody can be found in patients with systemic lupus erythematosus (SLE). METHODS: Seventy-six serum samples (37 from patients with SLE) were randomly selected from among sera submitted for routine antinuclear antibody testing. Short, overlapping peptides based on the partial VH (variable region of the heavy chain) sequence of the T14 antibody were synthesized on multipins and screened for reactivity with SLE sera. In addition, selected peptides from T14 and related proteins were synthesized in bulk and screened for reactivity with both SLE and control sera. A monoclonal antibody was generated to determine the prevalence of the T14 idiotype (T14+ Id) in the different study populations. RESULTS: Antibodies were detected by a peptide based on the third complementarity-determining region (CDR3) of the T14 protein in 15 (41%) of 37 patients with SLE or 15 (54%) of 28 who had anti-DNA antibodies, in 3 (9%) of 34 patients without anti-DNA antibodies (9 of whom had SLE), and in 6 (10%) of 57 healthy controls. In SLE sera, the antiidiotypic (anti-Id) responses (IgM and IgG) correlated well with the anti-DNA responses (IgG), and both responses correlated well with the T14+ Id activity in SLE sera. Control peptides based on the 18/2 (16/6+ Id) and S107 proteins detected low antibody activities in SLE sera, attributable to cross-reactivity with the T14 peptide. A peptide based on an unrelated human antibody was not reactive with these sera. CONCLUSION: Anti-Id antibodies directed to T14 VHCDR3 were found commonly in the sera of patients with SLE, and they appeared to be induced by the anti-DNA antibodies present in the sera. Based on these findings, these secondary antibodies may be pathogenic in SLE.

Adolescent↗

PubMed1996

Workshop on a detailed characterization of Trichinella spiralis antigens: a platform for future studies on antigens and antibodies to this parasite.

In order to characterize immunodominant components of T. spiralis a workshop was organized. In this the reactivity of monoclonal and polyclonal antibodies, provided by different research groups, towards total extracts from adult, new born larvae and muscle larvae as well as to excretory/secretory components of muscle larvae were tested by ELISA, Western blot and immunoprecipitation assays. As a result of this workshop T. spiralis ML antigens have been classified into eight groups (TSL-1-TSL-8) according to their recognition by monoclonal and polyclonal antibodies. Among them, TSL-1 antigens have been the most extensively characterized both biochemically and immunologically. These antigens are stage specific, originate in the muscle stichosome and are abundant in both E/S and on the larval cuticular surface. The TSL-1 antigens share an immunodominant carbohydrate epitope (tyvelose), which is unique for Trichinella and is not associated with phosphorylcholine. The data collected in this workshop has allowed both the unification of the nomenclature for T. spiralis antigens and their biochemical characterization. It also has provided a platform for further studies on the characterization of other T. spiralis antigens and indeed for other Trichinella species.

Animals↗

PubMed1994

CD4-positive cells from patients with IgA nephropathy demonstrate increased mRNA of cytokines that induce the IgA switch and differentiation.

IgA nephropathy (IgAN) is characterized by raised serum IgA1 and mesangial IgA1 deposits. We have previously shown increased T-cell activation in IgAN. Recently, transforming growth factor-beta (TGF-beta) has been shown to induce IgA isotype switch at a clonal level and interleukin 5 (IL5) promotes differentiation into IgA-bearing B cells. In the present study we have examined the TGF-beta and IL5 mRNA expression by mitogen-activated CD4-positive T cells from patients with IgAN (n = 25), patients with other primary nephritides (CGN) (n = 24), and healthy control subjects (n = 25). The cytokine genes were analysed by reverse transcription (RT)-polymerase chain reaction (PCR) and were semi-quantitated by normalizing the differences occurring during RT and PCR using a housekeeping gene, beta-actin. CD4-positive T cells from IgA nephritic patients expressed a higher level of IL5 mRNA than healthy controls (P < 0.01) and patients with CGN (P < 0.005). CD4-positive T cells from IgA nephritic patients expressed a higher level of TGF-beta mRNA than healthy controls (P < 0.01) but no difference was demonstrated on comparison with CGN patients. Elevated TGF-beta mRNA expression in patients with CGN probably reflects its other important function as a 'sclerogenic' factor involved in the glomerulosclerosis found in these nephritides. Our data suggest that there is increased expression of cytokine genes which induce the IgA isotype switch and differentiation; these immunological abnormalities may be important in the pathogenesis of IgAN.

Adult↗

PubMed1994

Structural analysis of a phosphorylcholine-binding antibody which exhibits a unique carrier specificity for Trichinella spiralis.

A phosphorylcholine (PC)-binding IgG (Mab2) antibody produced by a hybridoma derived from a BALB/c mouse which had been immunized against Trichinella spiralis was found to bind to the immunizing antigen (TSC) but not to other PC-associated antigens such as pneumococcal antigen (PNC) and PC-conjugated ovalbumin (PC-OVA). Sequence analysis of the protein revealed the presence of a heavy chain (VH) which was very similar (differing in only four amino acids) to that of the M511 myeloma protein, and a light chain (VL) which was completely identical to that of the M167 myeloma protein. Several M511/M167+ proteins, including the prototypic M511 protein and PC-binding proteins of other families (TEPC 15 and W3207), were examined in their binding to the various PC-associated antigens. These were found to be largely indiscriminate although subtle differences were observed for some antigens with some of the antibodies. A comparison of the VH sequences of Mab2 and these proteins revealed that of the differences seen, the single most important substitution in Mab2 which could contribute to the unique specificity of the molecule is the glycine residue at 49H. None of the other proteins, including other PCV-binding proteins published to-date, which utilize the same VH segment (99 in total), has this substitution.

Amino Acid Sequence↗

PubMed1994

The antigen-specific immunoglobulin G receptor is more sensitive to stimulation than the IgM receptor in transfected B cells.

A murine lymphoma cell line (M12.4) was transfected with immunoglobulin (Ig) genes encoding a T15+ (idiotype) IgM antibody or an idiotypically identical IgG antibody. Three transfectant clones of each class which showed similar (albeit distinguishable) levels of membrane expression of the transfected genes were used in the study. The response of each cell population to stimulation with anti-T15 antibodies was followed by measurement of the change in the intracellular Ca++ concentration. The IgG transfectants were found to be significantly more responsive to such stimulation than the IgM cells. In contrast, there was no difference in their response to a nonspecific reagent, the calcium ionophore A23187.

Animals↗

PubMed1994

Production of anti-phosphorylcholine antibodies of the T15 idiotype in CBA/N xid mice: investigation of the defect using a T15 immunoglobulin transgene.

A notable defect in CBA/N xid mice is their relative inability to make antibodies to phosphorylcholine (PC), particularly those of the T15 idiotype which predominate in the anti-PC responses of immunologically normal mice. To investigate the basis of this defect, we introduced functionally rearranged genes encoding a T15+ PC-binding immunoglobulin G antibody into the germline of these animals. Expression of these genes in the xid cells was observed, shown by the existence of a distinct population of T15+ cells (3 x 10(6)) in the spleen of the transgenic animals, and the presence of PC-binding T15+ IgG antibodies (1-15 micrograms/ml) in the serum. Mixed antibody molecules were also found, however, which were composed of both transgene-encoded and endogenously-derived chains. Existence of the T15+ cells in these animals seemed normal, since these were not depleted (to any great extent) and were immunocompetent as well. The latter was shown by the increased T15+ antibody production in the transgenic animals when stimulated with a PC-associated thymus-independent type 1 (TI-1) antigen and anti-idiotype antibodies, but not with the pneumococcal TI-2 antigen. This is similar to the PC-specific (T15-) responsiveness of normal CBA/N xid mice. Based on these results, we argue that a reason why T15+ antibodies are not normally made by CBA/N xid animals is because T15+ genes are not utilized or, as with any T15+ precursors present, selected for in these animals, in contrast to normal mice where the Lyb-5 or CD5 cells (which are absent in CBA/N xid animals) are known to be specially endowed to make such antibodies.

Animals↗

PubMed1994

Transgene-encoded antiphosphorylcholine (T15+) antibodies protect CBA/N (xid) mice against infection with Streptococcus pneumoniae but not Trichinella spiralis.

Immunodeficient CBA/N (xid) mice are highly susceptible to Streptococcus pneumoniae infection. Previous studies indicated that this susceptibility may be attributed to the lack of antibodies to phosphorylcholine (PC) in the circulation of these animals. We now provide direct proof that when these mice are genetically manipulated to produce significant amounts of circulating anti-PC immunoglobulin G antibodies of the T15 idiotype, they can be protected against a lethal challenge with S. pneumoniae. Transgenic mice were also used to investigate whether the transgene-encoded antibodies could protect the animals against another PC-bearing microorganism, Trichinella spiralis; in this case, there was no protection. These results were further supported by experiments with CFW mice which had been immunized to produce high levels of anti-PC antibodies but which were found to be just as susceptible to T. spiralis infection as nonimmunized animals.

Animals↗

PubMed1994

Occurrence of IgG subclass antibodies to ovalbumin, avidin, and pneumococcal polysaccharide in children.

The serum levels of the four subclasses of IgG to three different antigens--ovalbumin (OVA), pneumococcal polysaccharide (PNC), and avidin--were determined by enzyme-linked immunosorbent assay in three age groups of healthy children: 1.2-2.5 years (group A, n = 20), 6-7 years (group B, n = 21) and 15-17 years (group C, n = 20). The anti-OVA response in all groups was dominated by IgG1, IgG2, and IgG4 antibodies which generally increased in levels from group A to group B, but then declined significantly in group C. In all age groups, the IgG2 and IgG4 as well as the IgG1 and IgG3 responses were correlated; an association was also observed between IgG1 and IgG2 in groups B and C, but not in group A. The anti-PNC response was restricted to the IgG2 and IgG4 subclasses; correlation between these subclasses was seen only in group B children. The more predominant IgG2 antibodies increased dramatically in concentration from group A to group B, and then less remarkably in group C. Occurrence of the IgG1 anti-PNC antibodies was different which resembled the anti-OVA response. Responses to avidin, a protein derived from the same source as OVA, were different from the anti-OVA responses. These were low and confined to the IgG1 and IgG2 subclasses only and mainly to the younger children. A small group (n = 8) of 6- to 7-year-old atopic children who had a history of asthma or eczema appeared to have responses similar to their healthy counterparts.

Adolescent↗

PubMed1993

Regulatory elements necessary for termination of transcription within the Ig heavy chain gene locus.

Previous experiments have shown that the extent of delta gene transcription during B cell development is regulated primarily at the transcriptional level. We have shown that deletion of a sequence located between the mu and delta coding regions in the Ig heavy chain locus where transcriptional termination has been previously mapped abrogates the termination. Restoration of termination requires reintroduction of this segment as well as sequence elements within the microM poly (A) site which cannot be substituted by the microS poly (A) site. Recognition of the termination site by non-lymphoid cells suggests that initiation of delta transcription in mature B lymphocytes requires the activation of an anti-termination mechanism not yet developed in early B cells.

Animals↗

PubMed1993

Microbiology of the pericoronal pouch in mandibular third molar pericoronitis.

The microorganisms associated with mandibular third molar pericoronitis were investigated using direct microscopy and anaerobic culture method. The pericoronal pouch was sampled with paper points in A) 8 patients without mandibular third molar pericoronitis and B) 6 patients with mandibular third molar pericoronitis. Under the microscope, the microflora was found to be a complex mixture comprising gram-positive and gram-negative cocci, rods and filaments (including fusiform and curved rods), motile rods and spirochetes. Significantly higher proportions of motile, gram-negative rods were found in group B than in group A. The predominant cultivable microflora of 9 samples: A (4) and B (5) comprised several species of facultative and obligate anaerobic bacteria, namely Peptostreptococcus, Streptococcus, Actinomyces, Eubacterium, Propionibacterium, Veillonella, Porphyromonas, Prevotella, Bacteriodes, Fusobacterium, Campylobacter, Staphylococcus, Stomatococcus, Lactobacillus, Neisseria, Capnocytophaga, Haemophilus, Selenomonas and Centipeda species. The microflora in pericoronitis appeared similar to that of diseased periodontal pockets.

Adolescent↗

PubMed1993

Ultrastructural localization of the phosphorylcholine-associated antigen in Trichinella spiralis.

Two monoclonal antibodies that are specific for phosphorylcholine (PC) were used as probes in immunoelectron microscopy to locate the PC-associated antigen(s) in Trichinella spiralis. This antigen was found to be present abundantly in various internal structures in the muscle larva and the adult worm, but it was virtually absent in the fetal/newborn larva. In the muscle larva, the major pool of PC was found in the inner layer of the body cuticle and in various organelles such as the "rapid-responding" group. No PC-associated antigen, however, was seen on the surface of the cuticle or in the stichocyte granules. In the adult worm and the fetal/newborn larva, the inner layer of cuticle was not stained (or only weakly so) by the antibodies to PC, demonstrating the basic difference in structure between these cuticles and that of the muscle larva.

Animals↗

PubMed1991

Trichinella spiralis: light microscope monoclonal antibody localization and immunochemical characterization of phosphorylcholine and other antigens in the muscle larva.

A panel of monoclonal antibodies was used to examine the structure of the muscle larva of Trichinella spiralis under the light microscope. Immunofluorescence and, in some cases, immunoperoxidase staining were used. All four antibodies reacted with the cuticle of the organism, although differences in the staining pattern were observed for some of these. Interestingly, all the antibodies also reacted with the stichosome. One of the antibodies (Ts2Ab) is specific for the hapten, phosphorylcholine. In a binding assay, this antibody also reacted with extracts of Trichuris suis, Ascaris suum, and Fasciolopsis buski, but not with extracts derived from Cysticercus cellulosae, Candida albicans, Salmonella typhi, or Escherichia coli. This crossreactivity was confirmed microscopically in which the cuticle, oviduct and eggs of T. suis, the cuticle, muscle cells, and eggs of A. suum, and the cuticle and vitelline glands of F. buski were seen to be clearly stained by the antibody. In addition, Ts2Ab also reacted with the cuticle and stichosome of the adult T. spiralis worm. In Western blot analysis, Ts2Ab recognized a 43-kDa antigen from T. spiralis muscle larvae extracts, while a previously studied antibody (7C2C5Ab) identified four major antigens (48.5, 47, 43, and 39 kDa) in this preparation. Similar results were obtained when the 24-hr excretory-secretory (ES) antigens of T. spiralis were immunoblotted with the antibodies, although the reactivity shown by Ts2Ab was relatively weak. With the 72-hr ES material, on the other hand, major antigens of lower mol wt (44, 28, and 25 kDa) were revealed by 7C2C5Ab, and no reactivity was seen with Ts2Ab. However, this antigen preparation reacted well with both antibodies in an enzyme-linked immunoassay. Taken together, the findings suggest that the 72-hr ES antigens probably result from extensive degradation of material originally secreted or excreted by the worm. Similar binding studies on the 24-hr ES preparation indicated that this source may be relatively rich in 7C2C5Ab-reactive epitopes and relatively poor in the antigen identified by Ts2Ab. Other studies performed demonstrated that the antigens recognized by these two antibodies were distinct and physically unassociated.

Animals↗

PubMed1990

A tube latex test based on colour separation for the detection of IgM antibodies to either one of two different microorganisms.

A simple two stage assay was developed for the detection of IgM antibodies to either one of two microorganisms chosen arbitrarily for this study. Salmonella enteritidis and Trichinella spiralis. In the first stage, magnetic polystyrene beads (Dynabeads) coated with anti-mu (mouse) antibodies were incubated with the test material for 45 min to capture the IgM antibodies. In the second stage, indicator latex particles were incubated with the Dynabeads for 30 min and the results read following settlement of the Dynabeads under the influence of a magnet. Two types of indicator particles were used: blue-coloured (sensitized with Trichinella antigen) and red-coloured (sensitized with Salmonella antigen). These were mixed in suitable proportions to form a purple suspension. Reaction of either type of latex particles due to binding to the adsorbed IgM antibodies resulted in the settlement of that particle and hence a change of colour in the suspension to either red (if Trichinella-specific antibodies alone were present) or blue (if Salmonella-specific antibodies alone were present). When applied to the sera (used at 1/10 dilution) of both normal and immunized mice, the assay was positive for all but one (18) immune sera, and negative for all but one (9) normal sera.

Animals↗

PubMed1990

A one-step two-particle latex immunoassay for the detection of Salmonella typhi endotoxin.

A simple and rapid test (LIMM, short for latex immunoassay) is described for detecting Salmonella typhi endotoxin. It involves the simultaneous binding of the antigen by two types of reagent particles contained in a micro-tube: an indicator latex particle coated with a monoclonal antibody specific for the O-9 determinant on the endotoxin, and a magnetic bead coated with another monoclonal antibody specific for a different O-determinant. At the end of-the test, the magnetic beads are sedimented by use of a magnet, and the result is read based on the turbidity of the indicator latex suspension. Compared to a similar assay developed previously which uses only a single particle reagent (i.e., a tube agglutination system), LIMM was found to be slightly more sensitive especially when using short (less than 30 min) incubation times, and was at all times easier to read. The sensitivity of LIMM, in fact, increased with increasing time of incubation. When compared to the sensitivity (25 ng/ml) of a conventional slide latex agglutination test performed using the same indicator latex reagent, this parameter was 0-, 4.9-, 12.5- and 28.7-fold better after 5, 15, 30 and 60 min of incubation in the LIMM.

Agglutination Tests↗

PubMed1990

A thymus-independent (type 1) phosphorylcholine antigen isolated from Trichinella spiralis protects mice against pneumococcal infection.

A phosphorylcholine (PC)-containing glycoprotein of 68,000 molecular weight (MW) was isolated from Trichinella spiralis. The potential of this antigen (Tsp) as a species-specific vaccine against Streptococcus pneumoniae was studied in both immunologically deficient (CBA/N) and normal (CFW) mice. Unlike the PC determinant found in S. pneumoniae, Tsp is a type 1 thymus-independent (TI-1) antigen, as it was able to stimulate PC-specific antibody production in CBA/N animals, though less well than in CFW mice. Immunological memory to this antigen was observed in both strains of mice, and the predominant class of antibodies formed was IgM. In further studies, Tsp-immunized CFW mice were protected against a fatal challenge of S. pneumoniae type 3. Protection in these animals is probably mediated by the PC-specific antibodies present, which comprised 87.9% of antibodies reactive to S. pneumoniae, or 58.7% of total antibodies formed.

Animals↗

PubMed1989

A rapid method for detecting Trichinella spiralis larvae in pork using a monoclonal antibody-latex conjugate.

A simple, non-microscopic method was developed for the direct detection of Trichinella spiralis in pork. Samples of meat were treated with pepsin for 2 h to liberate the larvae, and sonicates were made from the deposit and used in an antigen detection system. In this system, latex particles, sensitized with a parasite-specific monoclonal antibody, were utilized in a slide or tube agglutination test. The efficiencies of these immunological methods were compared with that of trichinelloscopy in the examination of 102 pork specimens, 35 of which contained varying numbers of larvae after enzymatic digestion of 2 g of the meat. Trichinelloscopy detected 77.1% of these positive samples, but this was lower (55.5 and 41.7%) for those samples with a low larval burden (less than 50 or less than 10 larvae g-1 tissue, respectively). Better results (88.6, 76.5 and 60.0%) were obtained with the slide latex agglutination technique. The efficiency of detection was further improved (100%) using the more sensitive tube agglutination method. However, this was based on a small number (20) of samples examined and the limit of detection of this technique was subsequently found to be three larvae in total. No false-positives were seen with the latex agglutination methods from a total of 67 uninfected specimens examined.

Animals↗

PubMed1989

A two-particle turbidometric latex immunoassay for the detection of specific IgM antibodies.

A simple two stage assay using latex particles as a reaction indicator has been developed for the detection of IgM antibodies to Trichinella spiralis. In the first stage, magnetic polystyrene beads (Dynabeads) coated with T. spiralis antigen were incubated for 30 min with the test serum. After washing, in the second stage, the assay was developed for 1 h using anti-mu-coated latex particles. After sedimentation of the Dynabeads the turbidity of the resultant latex suspension was measured spectrophotometrically at a wavelength of 400 nm. A decrease in turbidity of more than 20% from that of the control, unreacted, suspension was considered positive. Using an IgM phosphorylcholine-binding monoclonal antibody which was reactive with T. spiralis, the sensitivity of the assay was determined to be 110 ng/ml of antibody. This was 20-fold less than the sensitivity achieved in an indirect enzyme-linked immunosorbent assay (ELISA). When the assay was applied to sera obtained from CBA/N or BALB/c mice, which were either normal or immunized against T. spiralis, the expected results were obtained with titers up to 1/640 observed, and confirmed (r = 0.93, P less than 0.001) in the ELISA.

Animals↗

PubMed1989

An improved latex agglutination-inhibition test for the detection of human chorionic gonadotropin in urine.

A commercially available slide latex agglutination-inhibition test for pregnancy was used in a tube modification for the detection of human chorionic gonadotropin (hCG) in urine. The tube method used 100 microliters sample and small quantities of reagents which were mixed continuously for 30 min. The results obtained, based on flocculation, were 15-30-fold better than those obtained by slide agglutination using spiked samples or urine samples from pregnant women. The lowest concentration of hCG detected in the spiked samples was 31 IU/l. When 43 clinical specimens were examined, the tube method detected four more samples than the 18 detected by the slide method. A good correlation (r = 0.96) was observed between the two methods in the determination of the hCG content in the positive samples.

Chorionic Gonadotropin↗