Potential tumor imaging radiopharmaceutical agents have been prepared by attaching a cisplatin derivative to a ligand capable of forming a stable complex with 99mTc. Three new organometallic compounds, with iminodiacetic acid as the 99mTc chelating group and 2,3-diaminopropionamide as the platinum complexing group, have been prepared and characterized. Preliminary biodistribution studies in tumor bearing mice support the utility of this approach.
A cytotoxic T lymphocyte (CTL) response to dengue virus-infected target cells is described. Effector cells were generated in an in vitro secondary culture and appeared to be T cells possessing both the Lyt 1.1 and Lyt 2.1 surface antigens. A stronger CTL response was noted with the H-2k haplotype compared to H-2d, and H-2 compatibility was required between CTL and target cells. CTL generated showed some cross-reactivity with target cells infected with Japanese encephalitis virus (JEV), another flavivirus, but not with target cells infected with an alphavirus, Sindbis. The significance and importance of these findings are discussed.
A monoclonal antibody raised against Haemophilus ducreyi was tested for its sensitivity and specificity as an immunofluorescence (IF) reagent using simulated vaginal smears containing H. ducreyi, smears taken from skin lesions of mice infected with H. ducreyi and patients from South Africa, Thailand and Malaysia with clinically diagnosed chancroid. The IF test was more sensitive than culture or Gram staining in the simulated smears, theoretically detecting less than 4 organisms/sample. It detected H. ducreyi in 95% of the animal lesions compared with 14% detected by culture. Immunofluorescence testing identified over 90% of culture-positive cases of chancroid but also detected organisms in some culture-negative cases where clinical evidence for the diagnosis was strong. These results suggest that this antibody may provide a simple, rapid and sensitive means of detecting H. ducreyi in cases of chancroid.
The Sarawak strain of Japanese encephalitis virus (JE-Sar) is virulent in 3-week-old mice when inoculated intraperitoneally. The nucleotide sequence for the envelope glycoprotein (E) of this virus was determined and compared with the published sequences of four other strains. There were several silent nucleotide differences and five codon changes. Monoclonal antibodies (MAbs) against the E protein of JE-Sar virus were prepared and characterized. MAb-resistant mutants of JE-Sar were selected to determine if mutations in the E protein gene could affect its virulence for mice. Eight mutants were isolated using five different MAbs that identified virus-specific or group-reactive epitopes on the E protein. The mutants lost either complete or partial reactivity with selecting MAb. Several showed decreased virulence in 3-week-old mice after intraperitoneal inoculation. Two (r27 and r30) also showed reduced virulence in 2-week-old mice. JE-Sar and the derived mutants were comparable in their virulence for mice, when inoculated intracranially. Mutant r30 but not r27 induced protective immunity in adult mice against intracranial challenge with parent virus. However, r27-2 did induce protective immunity against itself. Nucleotide sequencing of the E coding region for the mutants revealed single base changes in both r30 and r27 resulting in a predicted change from isoleucine to serine at position 270 in r30 and from glycine to aspartic acid at position 333 in r27. The altered capacity of the mutants to induce protective immunity is consistent with the immunogenicity changes predicted by computer analysis using the Protean II program.
Four homologous single chain phospholipases A2 (Pa-1G, Pa-5, Pa-12C and Pa-15) were tested for neuromuscular effects on chick biventer cervicis and mouse hemidiaphragm nerve-muscle preparations. The four isozymes blocked directly elicited (mouse hemidiaphragm) and indirectly elicited (mouse and chick nerve-muscle preparations) twitch responses in concentrations of 1-30 micrograms/ml. The order of potency seen in both types of preparations was Pa-1G = Pa-5 greater than Pa-12C much greater than Pa-15. All four isozymes caused slow-onset, sustained contractures and reduction of muscle membrane potentials. In the chick preparation, responses to acetylcholine, carbachol and KCl were reduced by exposure to the toxins. It is concluded that the toxins act primarily postsynaptically to depress muscle contractility, perhaps by directly damaging muscle fibres. The order of potency agrees with their phospholipase A2 activity. Pa-1G is unusual because it is an acidic molecule, most toxic phospholipases being basic.
Several hybridomas were produced against antigens extracted from the latex sap of Hevea brasiliensis. One of the monoclonal antibodies (LA-E3) reacted with antigens demonstrating binding to patient sera on crossed enzyme immunoelectrophoresis. This monoclonal antibody reacted with 2 of 10 glove extracts studied and with both Malaysian and Indian latex plant extracts. The antigens purified with monoclonal antibody affinity chromatography demonstrated specific IgE in the sera of patients with latex allergy as determined by ELISA. This monoclonal antibody can thus be utilized to obtain reliable antigens useful in the diagnosis of latex sensitivity, although additional antigens will likely be necessary to enhance sensitivity and specificity.
Previous studies have found Kunjin (KUN) virus isolates from within Australia to be genetically homogenous and that the envelope protein of the type strain (MRM61C) was unglycosylated and lacked a potential glycosylation site. We investigated the extent of antigenic variation between KUN virus isolates from Australia and Sarawak using an immunoperoxidase assay and a panel of six monoclonal antibodies. The glycosylation status of the E protein of each virus was also determined by N glycosidase F (PNGase F) digestion and limited sequence analysis. The results showed that KUN viruses isolated within Australia oscillated between three antigenic types defined by two epitopes whose expression was influenced by passage history and host cell type. In contrast an isolate from Sarawak formed a stable antigenic type that was not influenced by passage history and was distinct from all Australian isolates. PNGase F digestions of KUN isolates indicated that 19 of the 33 viruses possessed a glycosylated E protein. Nucleotide sequence of the 5' third of the E gene of selected KUN isolates revealed that a single base change in PNGase F sensitive strains changed the tripeptide N-Y-F (amino acids 154-156 of the published sequence) to the potential glycosylation site N-Y-S. Further analysis revealed that passage history also had a significant influence on glycosylation.
The intraspecific variation of four laboratory-reared isolates of Taenia taeniaformis the SRN and KRN isolates from Norway rats, Rattus norvegicus, captured in Japan and Malaysia, respectively; the BMM isolated from a house mouse, Mus musculus, captured in Belgium; and the ACR isolate from a gray red-backed vole, Clethrionomys rufocanus bedfordiae, captured in Japan was examined by various criteria. Eggs of each of the four isolates were orally inoculated into several species of intermediate host. They were most infective to the rodent species from which the original metacestode of each isolate had been isolated in the field, and only the ACR isolate was infective to the gray red-backed vole. Although little difference was found between the SRN, KRN, and BMM isolates by the other criteria, including the morphology of rostellar hooks, the protein composition of the metacestode, and restriction endonuclease analysis of DNA, the ACR isolate was clearly different from the others. It was considered that the ACR isolate was independent as a strain distinct from the other three isolates.
Purified lipopolysaccharide (LPS) of Pasteurella multocida type 6:B, while toxic at higher doses, was protective at lower dose levels against experimentally-induced pasteurellosis in mice. However, the observed protection was abrogated if such LPS was digested with proteinase K prior to use in immunisation. The O-antigen polysaccharide side-chain (OS) of LPS did not appear to contribute to the observed protection as judged by the fact that immunisation of mice with purified OS or OS-protein conjugates, all of which were nontoxic, failed to confer protection against challenge with homologous virulent organisms. This was despite generation of significant levels of OS-specific antibodies, predominantly either of the IgM or IgG isotypes, in immunised mice.
Three monoclonal antibodies (McAb) were produced against soluble antigens of Legionella pneumophila serogroup 1 which was cultured on BCYE agar. The McAbs were all of the IgM isotype. The McAbs were used in the McAb-based ELISA for detection of circulating L. pneumophila antigens in 186 sera collected from patients with symptoms and signs suggestive of atypical pneumonia. The normal reference optical (OD) density value of each of the McAbs was determined using 44 sera collected from healthy blood donors. The antigen positivity rates for the McAbs 1C7.2B, 2B2.10F and 2B2.11E were 11.3%, 7.7% and 22.2% respectively. Antigen positivity of the McAb 2B2.10F was significantly higher in the younger age group (p < 0.05). There is no significant association between the antigen positivity with age and sex for all the McAbs. There was no cross-reaction demonstrated between the McAbs with other bacterial antigens.
The effect of the mannose-binding champedak (Artocarpus integer) lectin-M on the cellular proliferation of murine lymphocytes was investigated in this study. Our data demonstrated that the lectin was the main mitogenic component in the crude extract of the champedak seeds. It stimulated the proliferation of murine T cells at an optimal concentration of 2.5 microg/ml in a 3 day culture. Lectin-M appeared to be a T-cell mitogen as it does not induce significant DNA synthesis when cultured with spleen cells from the nude mouse. In the absence of T cells, the lectin was incapable of inducing resting B cells to differentiate into immunoglobulin secreting plasma cells.
This report extends the knowledge on the epizootical situation of foot-and-mouth disease in Asia. RNA from six samples of type A and five of type O virus, isolated between 1987 and 1997 in Bangladesh, Iran, Malaysia and Turkey, was subjected to reverse transcription-dependent polymerase chain reactions that amplify large parts of the capsid protein VP1 encoding genome region. The amplification products were sequenced, and the sequences aligned to each other and to published sequences. This showed the type O isolates of 1987-1997 from Bangladesh to be of same genotype and closely related to isolates of 1988 and later from Saudi Arabia, 1990 from India, 1996 from Greece and Bulgaria, and 1997 from Iran. Among the analyzed type A isolates, those of 1992 and 1996 from Turkey were of same genotype and related to previously described isolates of 1987 from Iran and of 1992 from Saudi Arabia. The isolate of 1997 from Malaysia was found to be related to isolates from Thailand of 1993 and 1996. The isolates of 1987 from Bangladesh and 1997 from Iran, however, represent different so far not described genotypes. Monoclonal antibodies, raised against the vaccine production strains A22 Iraq, Asial Shamir, O1 Kaufbeuren and O1 Manisa, and the recent type A field isolates Saudi Arabia/92 and Albania/96, were used in an ELISA to compare the reaction patterns of many of the field isolates. The monoclonal antibodies were further characterized for virus-neutralizing activity and binding to trypsinized homologous virus. The failure of neutralizing antibodies in binding to trypsinized homologous as well as to heterologous virus suggested the epitopes to reside at the major antigenic component of the virus, which is the capsid protein VP1. Two non-neutralizing antibodies that bind to trypsin-sensitive epitopes cross-reacted, however, with heterologous virus. This indicates the existence of a trypsin-sensitive antigenic site outside of VP1. In summary, the results obtained by ELISA confirm the observed sequence differences, but indicate further sequence differences at minor antigenic sites that do not reside on VP1.
Congenitally T and B cell-deficient SCID mice and T cell-deficient NUDE mice, with BALB/c mice as immunologically normal controls, were inoculated with Rhinosporidium seeberi. At 3 and 16 weeks after inoculation, no evidence of rhinosporidiosis was detected. The reasons for the failure to establish rhinosporidiosis in immunodeficient or normal mice remain obscure.
The aim of the present study was to determine whether Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) may stimulate nitric oxide (NO) production by murine spleen cells. Spleen cells derived from Balb/c mice were cultured in the presence of Pg-LPS or LPS from Salmonella Typhosa. The cell were also cultured in the presence of Pg-LPS with or without L-arginine, L-arginine plus NG-monomethyl-L-arginine (NMMA), or IFN-gamma. Furthermore, the plastic non-adherent spleen cells were stimulated with Pg-LPS and L-arginine. The results showed that Pg-LPS failed to stimulate splenic NO production by themselves. Exogenous L-arginine or IFN-gamma up-regulated the NO production of Pg-LPS-stimulated spleen cells, but the stimulatory effects of L-arginine were completely blocked by NMMA. It was also demonstrated that in the presence of Pg-LPS and L-arginine, splenic macrophages were the cellular source of NO. These results suggest, therefore, that P. gingivalis-LPS may induce murine splenic macrophages to produce NO in a L-arginine and an IFN-gamma-dependent mechanism.
Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah virus-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells. In the immune plaque assay, anti-P antibody is localised by an alkaline phosphatase-linked second antibody and the Western blot substrates 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium. A modification of the rapid immune plaque assay was also used to detect antibodies to Nipah virus in a panel of porcine field sera from Malaysia and the results showed good agreement between the immune plaque assay and a traditional serum neutralisation test. After methanol fixation, plates can be stored for up to 7 months and may be used in the immune plaque assay to complement the enzyme-linked immunosorbent assay screening of sera for antibodies to Nipah virus. At present, all enzyme-linked immunosorbent assay positive sera are subject to confirmatory serum neutralisation tests. Use of the immune plaque assay may reduce the number of sera requiring confirmatory neutralisation testing for Nipah virus antibodies under biohazard level 4 conditions by identifying those that generate false positive in the enzyme-linked immunosorbent assay.
The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.