Antibody inhibition of HeLa cell invasion by Yersinia enterocolitica

Antisera were prepared in rabbits against formalized and heat-killed bacteria of Yersinia enterocolitica serotype O:5,27 and against formalized bacteria of serotype O:8. Both strains used for immunization demonstrated adhesion to and invasion of HeLa cells. Coating of the bacteria with antibody did not greatly alter adhesion (i.e., extracellular attachment) to HeLa cells; however, antibody against formalized bacteria of both serotypes inhibited HeLa cell invasion by the homologous and heterologous strains. The Fab fragments from purified immunoglobulins also demonstrated cross-reacting inhibition of HeLa cell invasion. Antibody against heat-killed bacteria of serotype O:5,27 had no inhibitory activity. Adsorption of the antiserum against formalized bacteria of serotype O:5,27 with lipopolysaccharide from the homologous strain removed anti-lipopolysaccharide antibody but did not remove the inhibitory activity. The antiserum against formalized bacteria of serotype O:8 showed no antibody against lipopolysaccharide from serotype O:5,27 and no agglutinins against heat-killed bacteria of this strain. From these results, it is tentatively suggested that protein structures are important in mediating epithelial cell invasion by Y. enterocolitica.     

Lymphocytes from children and infants mediate natural cell mediated cytotoxicity, (cytotoxicity)

Summary Lymphocytes from infants and young children were tested for natural cell mediated cytotoxicity (NCMC) against K562 and CCRF-CEM. NCMC by lymphocytes from pediatric donors of all ages was equivalent to that mediated by lymphocytes from adults. Since it has been suggested that the biological function of NCMC is to effect immunological surveillance against cancer, the appearance of NCMC effector cells early in development is consistent with early mobilization of the policing mechanism.This work was supported by Grant CA 25765 from the National Cancer InstitutePartially supported by a Grant from the Concern Foundation.     

Iso- and Anisoasymmetry Testing in Pre-post Rating Designs

It is suggested to test against anisoasymmetry rather than against marginal homogeneity if (1) a pre-post treatment matched pairs contingency table is ordinally scaled by clinical ratings and if (2) the alternative to marginal homogeneity is a shift in location from pre- to post treatment ratings. Testing against anisoasymmetry may detect treatment effects which are not manifest in marginal inhomogeneities.     

胶州湾放线菌的分离及其抗病原微生物的活性检测

采用涂布法,从胶州湾海泥样品中分离到224株放线菌菌株,并且从海水样品中分离到32株放线菌菌株。根据形态学特性,菌株被分成了7个组。同时利用杯碟法测定了它们的抗菌活性,其中约7％对金黄色葡萄球菌有活性,11％对八叠球菌有效,10％对大肠杆菌有效,11％对绿脓杆菌有效,2％对白色念珠菌有效,5％对隐球菌有效,6％对绿色产色链霉菌有效和6％对米赫毛霉有效。分离到的22％放线菌对所测定的病原微生物有抗性作用。我们的结果表明胶州湾放线菌能够产生对病原微生物有抗性作用的不同代谢物,这些代谢物可以作为筛选新颖天然产物的独特和丰富的资源。     

Production and partial characterization of anti-cord factor (trehalose-6,6'-dimycolate) IgG antibody in rabbits recognizing mycolic acid subclasses of Mycobacterium tuberculosis or Mycobacterium avium

An ELISA with cord factor (trehalose-6,6'-dimycolate) is useful for the serodiagnosis of tuberculosis. To clarify the exact antigenic epitope in cord factor, recognized by a rabbit anti-cord factor IgG antibody, and to ascertain the most sensitive and specific diagnostic test antigen, rabbits were immunized with two kinds of cord factors isolated from Mycobacterium tuberculosis or Mycobacterium avium and the reactivities of the sera were tested against cord factors or the component mycolic acid methyl esters by ELISA. The serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against M. tuberculosis cord factor, but less reactive against M. avium cord factor. In contrast, the serum from rabbits immunized with M. avium cord factor was highly reactive against M. avium cord factor but less reactive against M. tuberculosis cord factor. Moreover, the serum from rabbits immunized with M. tuberculosis cord factor reacted against mycolic acid methyl esters, especially methoxy mycolic acid methyl ester. On the other hand, the serum from rabbits immunized with M. tuberculosis cord factor was less reactive against trehalose-6-monomycolate and not reactive against sulfolipid (2,3,6,6'-tetraacyl trehalose 2'-sulfate). From these results, it was concluded that the anti-cord factor IgG antibody, produced experimentally in rabbits, recognized the differences in the cord factor structures, i.e. the hydrophobic moiety rather than the carbohydrate moiety. It was also noted that the serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against methoxy mycolic acid as an epitope. This paper is the first to describe how the anti-cord factor IgG antibody can recognize the mycolic acid subclasses, which differ according to the species of mycobacteria.     

Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies

DNA delivery of tumor antigens can activate specific immune attack on cancer cells. However, antigens may be weak, and immune capacity can be compromised. Fusion of genes encoding activating sequences to the tumor antigen sequence facilitates promotion and manipulation of effector pathways. Idiotypic determinants of B-cell tumors, encoded by the variable region genes, are clone-specific tumor antigens. When assembled as single-chain Fv (scFv) alone in a DNA vaccine, immunogenicity is low. Previously, we found that fusion of a sequence from tetanus toxin (fragment C; FrC) promoted anti-idiotypic protection against lymphoma and myeloma. We have now investigated an alternative fusion gene derived from a plant virus, potato virus X coat protein, a primary antigen in humans. When fused to scFv, the self-aggregating protein generates protection against lymphoma and myeloma. In contrast to scFv-FrC, protection against lymphoma is mediated by CD4+ T cells, as is protection against myeloma. Plant viral proteins offer new opportunities to activate immunity against linked T-cell epitopes to attack cancer.     

2007年重庆市流脑流行季节前健康人群抗体水平和带菌率调查

为了解重庆市流脑流行季节前健康人群免疫水平和带菌状况,预测发病趋势,提出新的免疫策略,提供有效的预防措施。按照全国流脑监测方案并结合重庆市2006年流脑疫情情况,选择重庆市北碚区、长寿区和万盛区为2007年流脑监测点,采集0~、5~、10~、15~、25~、35~及45岁以上共7个年龄组人群的静脉血和咽拭子,分别进行抗体水平和带菌率检测。结果显示,重庆市健康人群A群流脑IgG抗体的阳性率为76.02%(485/638),C群流脑IgG抗体的阳性率为47.65%(304/638),健康人群脑膜炎奈瑟菌(简称Nm)带菌率为0.47%(3/638)。重庆市健康人群A群流脑IgG抗体阳性率维持在较高水平,而C群流脑IgG抗体的阳性率较低,提示该市大部分人群对A群流脑有免疫作用,但C群抗体水平较低,有引起局部流脑爆发的风险。     

Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye)

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

Response of human fetal lymphocytes in xenogeneic mixed leukocyte culture: Phylogenetic and ontogenetic aspects

Cells from liver, thymus, and spleen of human fetuses at different stages of development were capable of a proliferation response against xenogeneic and allogeneic lymphocytes. The kinetics of fetal responses against rat lymphocytes were identical to those of fetal and adult responses against allogeneic cells. With all of the cell types studied, including adult lymphocytes, allogeneic responses were stronger than xenogeneic. Xenogeneic responses against lymphocytes from rat, mouse, or sheep were stronger than those against lymphocytes from rabbit, chicken, snake, or frog. These results are interpreted to indicate that recognition of foreign lymphocytes by human lymphocytes depends on the phylogenetic position of the species used as a source of stimulating cells. The degree of recognition decreases as the phylogenetic distance increases. Specific elimination of responding cells and restimulation with another cell population was used to study the specificity of proliferation responses against mouse and rat lymphocytes. Responses by prethymic liver cells from human fetuses were not due to the existence of specifically recognizing subpopulations. Thymus and spleen at 16 weeks' gestation contained specific subpopulations capable of differentiating between xenogeneic and allogeneic cells, as well as between xenogeneic cells with different intraspecies histocompatibility patterns. Generation of receptor diversity on T lymphocytes is discussed briefly in the light of these findings.     

Thermomonospora sp. T-SA-125 and its production of a growth promoting antibiotic

Thermomonospora sp. T-SA-125 is a true thermophilic actinomycete isolated from a soil sample collected from the Saudi Arabian desert. It is characterized by the formation of single spores at the tips of dichotomously branched aerial mycelium and differs from Thermomonospora curvata and T. viridis in certain aspects. It produces a basic water-soluble antibiotic which is active against Gram-positive bacteria, moderately active against Gram-negative bacteria and inactive against fungi. At high concentrations, this antibiotic, stimulated the growth of both Hordeum coleoptile and lettuce hypocotyl.     

Transient Humoral Protection against H5N1 Challenge after Seasonal Influenza Vaccination of Humans

Current influenza vaccines are believed to confer protection against a narrow range of virus strains. The identification of broadly influenza neutralizing antibodies (bnAbs) has triggered efforts to develop vaccines providing ‘universal’ protection against influenza. Several bnAbs were isolated from humans recently vaccinated with conventional influenza vaccines, suggesting that such vaccines could, in principle, be broadly protective. Assessing the breadth-of-protection conferred to humans by influenza vaccines is hampered by the lack of in vitro correlates for broad protection. We designed and employed a novel human-to-mouse serum transfer and challenge model to analyze protective responses in serum samples from clinical trial subjects. One dose of seasonal vaccine induces humoral protection not only against vaccine-homologous H1N1 challenge, but also against H5N1 challenge. This heterosubtypic protection is neither detected, nor accurately predicted by in vitro immunogenicity assays. Moreover, heterosubtypic protection is transient and not boosted by repeated inoculations. Strategies to increase the breadth and duration of the protective response against influenza are required to obtain ‘universal’ protection against influenza by vaccination. In the absence of known correlates of protection for broadly protective vaccines, the human-to-mouse serum transfer and challenge model described here may aid the development of such vaccines.     

Serum of normal subjects contains IgG with antibody-like specificity for phospholipids

Using counterimmunoelectrophoresis, sera from normal subjects display precipitin activity against liposomes made by phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine and cardiolipin. No activity is detected by using liposomes made with phosphatidyl choline, sphingomyelin and phosphatidyl inositol. The same pattern of precipitation is obtained when pure IgG from normal subjects is tested against phospholipids. No precipitin activity is found by using pure IgM. The precipitin reaction is prevented by preincubating liposomes with Fab obtained from normal IgG.     

Avian influenza H9N2 seroprevalence among poultry workers in Pune, India, 2010

Avian influenza (AI) H9N2 has been reported from poultry in India. A seroepidemiological study was undertaken among poultry workers to understand the prevalence of antibodies against AI H9N2 in Pune, Maharashtra, India. A total of 338 poultry workers were sampled. Serum samples were tested for presence of antibodies against AI H9N2 virus by hemagglutination inhibition (HI) and microneutralization (MN) assays. A total of 249 baseline sera from general population from Pune were tested for antibodies against AI H9N2 and were negative by HI assay using ≥40 cut-off antibody titre. Overall 21 subjects (21/338?=?6.2%) were positive for antibodies against AI H9N2 by either HI or MN assays using ≥40 cut-off antibody titre. A total of 4.7% and 3.8% poultry workers were positive for antibodies against AI H9N2 by HI and MN assay respectively using 40 as cut-off antibody titre. This is the first report of seroprevalence of antibodies against AI H9N2 among poultry workers in India.     

Autologous melanoma-induced activation of regulatory T cells that suppress cytotoxic response

The host immune response toward autologous human cancer is subject to regulation by the immunoregulatory network. We show that certain CD4+ T cell clones, derived from melanoma involved lymph node lymphocytes and from PBL stimulated by autologous melanoma cells, selectively down-regulated the induction of cytotoxic immune response of PBL against the respective autologous melanoma cells in two autologous systems. In both systems, only the generation of cytotoxic response against the autologous melanoma cells were suppressed. Cytotoxic response against EBV-infected autologous lymphoblastoid cell line in one case and cytotoxic responses against allogeneic targets in the other were not affected. In addition to suppressor activity selectively expressed against the autologous melanoma cells, the T cell clones up-regulated their Tac receptors when cocultured with the autologous melanoma cells and APC. These results support the existence of a putative tumor Ag-driven activation of regulatory T cells that affect cytotoxic immune response, in vitro, against autologous human melanoma.     

Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco

Protease inhibitors provide a promising means of engineering plant resistance against attack by insects and pathogens. Sporamin (trypsin inhibitor) from sweet potato and CeCPI (phytocystatin) from taro were stacked in a binary vector, using pMSPOA (a modified sporamin promoter) to drive both genes. Transgenic tobacco lines of T0 and T1 generation with varied inhibitory activity against trypsin and papain showed resistance to both insects and phytopathogens. Larvae of Helicoverpa armigera that ingested tobacco leaves either died or showed delayed growth and development relative to control larvae. Transgenic tobacco-overexpressing the stacked genes also exhibited strong resistance against bacterial soft rot disease caused by Erwinia carotovora and damping-off disease caused by Pythium aphanidermatum. Thus, stacking protease-inhibitor genes, driven by the wound and pathogen responsive pMSPOA promoter, is an effective strategy for engineering crops to resistance against insects and phytopathogens.     

Increasing Sequence Search Sensitivity with Transitive Alignments

Sequence alignment is an important bioinformatics tool for identifying homology, but searching against the full set of available sequences is likely to result in many hits to poorly annotated sequences providing very little information. Consequently, we often want alignments against a specific subset of sequences: for instance, we are looking for sequences from a particular species, sequences that have known 3d-structures, sequences that have a reliable (curated) function annotation, and so on. Although such subset databases are readily available, they only represent a small fraction of all sequences. Thus, the likelihood of finding close homologs for query sequences is smaller, and the alignments will in general have lower scores. This makes it difficult to distinguish hits to homologous sequences from random hits to unrelated sequences. Here, we propose a method that addresses this problem by first aligning query sequences against a large database representing the corpus of known sequences, and then constructing indirect (or transitive) alignments by combining the results with alignments from the large database against the desired target database. We compare the results to direct pairwise alignments, and show that our method gives us higher sensitivity alignments against the target database.     

Rank relations among sisters in semi-free-ranging Barbary macaques (Macaca sylvanus)

Although semi-free-ranging Barbary macaque females are able to outrank older females from lower-ranking matrilines (matrilineal rank acquisition), they do not systematically outrank their older sisters, as is known to be the case for semi-free-ranging rhesus monkeys (Macaca mulatta) and Japanese macaques (Macaca fuscata). We test the hypothesis that differences in the support received by younger sisters against their older sisters and against older lower-ranking females might account for this interspecific difference. Thirty-one sister dyads, members of a group of 109 Barbary macaques living at La Montagne des Singes, France, were observed during 16 months. The results indicate that (1) all females were dominant to their younger sisters, and the latter were never observed to challenge their older sisters; (2) younger sisters received as much kin support against their older sisters as against older lower-ranking females; (3) only very young females received support from their kin against their older sisters; (4) younger sisters received much more support from nonkin females against lower-ranking females than against their older sisters; and (5) Barbary macaque females appear to be supported against their older sisters less frequently than rhesus macaque females are. We conclude that the lack of nonkin support is the main factor accounting for the failure of younger sisters to outrank their older sisters in Barbary macaques. Initially this might result from kin support not being sufficient to induce younger sisters to challenge and to solicit support against their older sisters.     

An inhibitor of collagenase from human amniotic fluid. Purification, characterization and action on metalloproteinases.

1. An inhibitor of collagenase of apparent mol.wt. 28000 was isolated from term human amniotic fluid. 2. It is active against mammalian collagenases from a number of species and tissues as well as other mammalian metalloproteinases, but has no activity against bacterial metalloproteinases. 3. Activity is destroyed by treatment with either trypsin or 4-aminophenylmercuric acetate, by heat, and by reduction and carboxymethylation. 4. All the properties observed suggest that it is similar to the synthesized tissue inhibitor of metalloproteinases.     

Protection of macaques against intrarectal infection by a combination immunization regimen with recombinant simian immunodeficiency virus SIVmne gp160 vaccines

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against intravenous challenge by the cloned homologous virus E11S but that this protection was only partially effective against the uncloned virus, SIVmne. In the present study, we examine the protective efficacy of this immunization regimen against infection by a mucosal route. We found that the same gp160-based vaccines were highly effective against intrarectal infection not only with the E11S clone but also with the uncloned SIVmne. Protection against mucosal infection is therefore achievable by parenteral immunization with recombinant envelope vaccines. Protection appears to correlate with high levels of SIV-specific antibodies and, in animals protected against the uncloned virus, the presence of serum-neutralizing activities. To understand the basis for the differential efficacies against the uncloned virus by the intravenous versus the intrarectal routes, we examined viral sequences recovered from the peripheral blood mononuclear cells of animals early after infection by both routes. We previously showed that the majority (85%) of the uncloned SIVmne challenge stock contained V1 sequences homologous to the molecular clone from which the vaccines were made (E11S type), with the remainder (15%) containing multiple conserved changes (the variant types). In contrast to intravenously infected animals, from which either E11S-type or the variant type V1 sequences could be recovered in significant proportions, animals infected intrarectally had predominantly E11S-type sequences. Preferential transmission or amplification of the E11S-type viruses may therefore account in part for the enhanced efficacy of the recombinant gp160 vaccines against the uncloned virus challenge by the intrarectal route compared with the intravenous route.