Expression, immunochemical characterization and localization of the Mycobacterium tuberculosis protein p27.

Rv2108 is a gene of the PPE family of Mycobacterium tuberculosis specific for this bacterial complex and that may encode a putative protein p27. This gene was amplified, inserted into bacterial vectors, sequenced, and expressed as a recombinant protein. Specific antibodies to this protein were generated and used for immunochemical characterization and cellular localization. Mass spectrometric analysis of the expressed protein revealed a molecule that corresponded to the p27 putative protein. The expressed protein was immunologically active, and reacted with antibodies from tuberculosis patient sera. Specific immunoblot analysis confirmed the presence of the p27 antigen in Mycobacterium bovis BCG strain and in human clinical isolates of M. tuberculosis, but not in other mycobacteria tested. Western blot and immunoelectron microscopic analysis of BCG strain indicated that the p27 protein is localized in the membrane of the cell. The specific expression of the p27 protein in the M. tuberculosis complex could provide a novel specific complimentary diagnostic test for the presence of and infection with M. tuberculosis.

Passage of human T-cell leukemia virus type-1 during progression to cutaneous T-cell lymphoma results in myelopathic disease in an HTLV-1 infection model.

Studies comparing functional differences in human T-cell leukemia virus type 1 (HTLV-1) clones that mediate distinct outcomes in experimentally infected rabbits, resulted in a dermatopathic smoldering adult T-cell leukemia/lymphoma following chronic infection with HTLV-1 strain RH/K34. During the 3.5 years' follow-up, HTLV-1 skin disease progressed to cutaneous T-cell lymphoma. When infection was passed to several naive rabbits, progressive paraparesis due to myelopathic neurodegeneration, analogous to HTLV-associated myelopathy, resulted in one of 4 transfusion recipients. Similar proviral loads were detected in the two diseases, regardless of stage of progression or tissue compartment of infection. Complete proviral sequences obtained from the donor and affected recipient aligned identically with each other and with the inoculated virus clone. Existence of disparate pathogenic outcomes following infectious transmission further extends the analogy of using rabbits to model human infection and disease. Although the experimental outcomes shown are limited by numbers of animals affected, they mimic the infrequency of HTLV-1 disease and authenticate epidemiological evidence of virus sequence stability regardless of disease phenotype. The findings suggest that further investigation of a possible role for HTLV-1 in some forms of cutaneous T-cell lymphoma is warranted.

Evidence for humoral and cellular reactivity against keratin and thyroglobulin in HTLV-I infected rabbits.

Human T cell leukemia virus type I (HTLV-I) infection was initially associated with T cell leukemia and a progressive neurologic disease but has since been linked to an increasing number of autoimmune disorders, including Sjogren's syndrome, uveitis, and polyarthritis. A survey of serum samples from a rabbit model of HTLV-I infection revealed that all had antibodies against keratin and thyroglobulin. Sera from several infected rabbits also reacted with collagen, while antibody reactions with other autoantigens tested, including DNA, were rare and sporadic. In addition to antibodies, cellular reactivity to keratin, but not thyroglobulin, was demonstrated by cellular proliferation in presence of IL-2 and keratin. Expanded cell cultures were positive for T cell activation markers and CD8. Association of the auto-reactivity with HTLV-I infection rather than random anti-cellular responses was supported by the fact that no antikeratin or antithyroglobulin was seen in uninfected controls, including that inoculated with uninfected lymphocytes. Finding autoantibodies in rabbits infected using naked HTLV-I DNA clones provided further assurance that infection induced the autoimmune reactions detected.

Cellular distribution of a mixed MHC class II heterodimer between DRalpha and a chimeric DObeta chain.

Human MHC class II antigens include HLA-DR, -DQ, and -DP molecules that present antigens to CD4+ T cells, as well as the non-classical molecules HLA-DM and -DO. HLA-DM promotes peptide binding to class II molecules in endocytic compartments and HLA-DO, which is physically associated with HLA-DM in B lymphocytes, regulates HLA-DM function. Antibodies specific for the DObeta chain were obtained by immunization of mice with a heterodimer consisting of a chimeric DObeta chain (DR/DObeta), containing 18 N-terminal residues of DRbeta, paired with the DRalpha chain and isolated from transfected murine fibroblasts. The specificity of this serum for the DObeta chain and the lysosomal expression of the HLA-DO protein was confirmed using mutant human B cell lines lacking DR or DO molecules. The lysosomal localization of HLA-DO in human B cells contrasts with the cell surface expression of the mixed pair in transfected murine fibroblasts and raises questions concerning the role of the putative targeting motifs in HLA-DO. Transfection of the chimeric DR/DObeta chain along with DRalpha into human epithelial HeLa cells resulted in high levels of expression of the mixed isotypic pair at the surface of transfectants as well as in lysosomes. The same pattern was observed in HeLa cells transfected with the DObeta chimera and a DRa chain lacking the cytoplasmic tail. Taken together, these results suggest that functional sorting motifs exist in the DObeta chain but that the tight compartmentalization of HLA-DO observed inside B lymphocytes is controlled by the HLA-DOalpha chain and HLA-DM.

Mapping of staphylococcal enterotoxin A functional binding sites and presentation by monoclonal antibodies and fusion proteins.

Staphylococal enterotoxins (SE) bind with high affinity to major histocompatibility complex (MHC) class II proteins and stimulate large number of T cells via the Vbeta region of the T-cell receptor (TCR). To map the epitopes of SE type A (SEA) involved in MHC binding and cell proliferation, 20 specific anti-SEA monoclonal antibodies (MAbs) and two large glutathione S-transferase fusion proteins corresponding to the amino and carboxy termini, respectively, of SEA were used. The functionality of these antibodies was tested, by MHC binding inhibition, interleukin-2 production, and T-cell proliferation assays. Moreover, I studied the ability of the MAbs to present SEA in vitro to human and murine cells and their reactivity with the two fusion proteins. This study showed that all of the MAbs have a defined effect on one or both immunological properties of SEA and were able to present SEA to human and murine cells. However, one MAb (4H8) recognized SEA but without any interference with its biological activities. When the MAbs were tested to react with the two fusion proteins representing the SEA molecule, all of the MAbs were negative except for two. These results confirmed the presence of two functionally different binding sites of SEA with MHC class II molecules and the importance of the disulfide loop for the mitogenic activity of SEA. I further demonstrated that MAbs can present SEA to immune cells independent of the site recognized by the antibody and that the integrity of the SEA molecule is very important for its functions.

Genes in the pX region of human T cell leukemia virus I influence Vav phosphorylation in T cells.

Human T cell leukemia virus I (HTLV-I) causes acute leukemic disease in a low percentage of infected individuals through obscure mechanisms. Our studies compare two rabbit HTLV-I-infected T cell lines: one, RH/K34, causes lethal experimental leukemia and the other, RH/K30, mediates asymptomatic infection. We show herein that the product of the protooncogene vav is constitutively Tyr-phosphorylated in RH/K34 but not in RH/K30. A role for the retrovirus in phosphorylation of Vav was assigned by transfection experiments with molecular clones of HTLV-I derived from the two lines. The HTLV-I molecular clone from RH/K30, but not that from RH/K34, down-regulates Vav phosphorylation in a Herpesvirus ateles-transformed T cell line. Use of recombinant virus clones revealed that a pX region sequence differing by two nucleotides between the two clones mediates this down-regulation. Because Vav is involved in T cell signaling and Vav phosphorylation occurs upon activation of T cells, control of the activation state of Vav by viral proteins may relate to the leukemogenic potential of certain HTLV-I-infected cells.

Agonist-specific tyrosine phosphorylation of Cbl in human neutrophils.

The effects of soluble and particulate agonists on the tyrosine phosphorylation levels of the proto-oncogene Cbl in human neutrophils were examined. Experimental conditions allowing the maintenance of Cbl as well as of its tyrosine phosphorylation status were first established. Their use allowed us to observe that Cbl was tyrosine phosphorylated in response to some (FcgammaRII ligation, opsonized bacteria and zymosan, granulocyte-macrophage colony-stimulating factor, monosodium urate, and calcium pyrophosphate microcrystals), but not all (fMet-Leu-Phe, interleukin-8) neutrophil agonists. Cbl was also shown to account for a varying proportion of the 120-kDa phosphoprotein(s) observed in response to the above stimuli. These data establish that Cbl is present in human neutrophils and that its level of tyrosine phosphorylation is modulated by some of these cells' agonists, and in particular by phagocytic particles. Furthermore, the signaling pathways activated by chemotactic factors and the other neutrophil stimuli tested in this investigation diverge at or downstream from the tyrosine phosphorylation of Cbl.

Physical association of Gi2alpha with interleukin-8 receptors.

Interleukin-8 (IL-8), one of the major mediators of the inflammatory response, belongs to a family of chemokines that includes NAP-2 (neutrophil-activating peptide-2) and Gro-alpha and whose biological activities are directed to a great extent toward neutrophils. Two distinct receptors have been described with overlapping, but not identical, binding affinities for IL-8, NAP-2, and Gro-alpha. This study was designed to examine the intracellular pathways activated upon the occupation of each of the IL-8 receptors (IL-8R). The formation of a physical coupling between IL-8 receptors and the alpha-subunit of heterotrimeric G proteins was tested in neutrophils by examining the presence of the former in anti-Galpha immune precipitates. The addition of IL-8 to a suspension of human neutrophils led to a time-dependent detection of IL-8 in anti-Gi2alpha (raised against amino acids 159-168 (LERIAQSDYI) of Gi2alpha) and anti-Gtalpha (raised against the COOH-terminal 10 amino acids (KENLKDCGLF) of Gtalpha), but not anti-Gq, immunoprecipitates. Similar results were obtained in human 293 cells stably transfected with IL-8RA or IL-8RB. The peptide derived from the COOH-terminal sequence of Gt inhibited the co-immunoprecipitation of IL-8R and Gi observed in response to the anti-Gtalpha and anti-Gi2alpha antibodies. On the other hand, the Gi2alpha peptide only inhibited the immunoprecipitation induced by the anti-Gi2alpha antibody. Peptides derived from Gi1alpha or Gi3alpha had no effect in this assay. The introduction of the anti-Gi2alpha or anti-Gtalpha antibodies or their neutralizing peptides, but not the Gi1alpha or Gi3alpha peptides, into 293 IL-8RA or 293 IL-8RB cells completely blocked the calcium responses obtained upon stimulation with IL-8. These results demonstrate that the occupation of either type of IL-8 receptor leads to a physical coupling to the alpha-subunit of Gi2. In addition, the use of the subunit-specific peptides identified two functionally important but distinct regions of Gialpha, one involved in receptor/Gialpha interaction (KENLKDCGLF) and the other mediating downstream signal transmission (LERIAQSDYI). Finally, the results of this study also validate the use of the transfected 293 cell line as a model for the study of the signal transduction pathway(s) initiated by IL-8.

A natural mutation of the amino acid residue at position 60 destroys staphylococcal enterotoxin A murine T-cell mitogenicity.

A variety of techniques have been used to identify the amino acid residues of bacterial superantigens involved in their interactions with major histocompatibility complex (MHC) class II and T-cell receptor (TCR). In this study, we isolated a naturally mutated staphylococcal enterotoxin A (SEA) from three different Staphylococcus aureus strains, in which the amino acid at position 60 has been changed from aspartic acid (D) to asparagine (N). We then studied the influence of this change on the immunological activities of SEA. Our results demonstrated that this mutation does not affect the capacity of SEA to bind MHC class II molecules and consequently activates human monocytes and peripheral blood lymphocytes. In contrast, mutated SEA failed to stimulate the proliferation of murine splenic lymphocytes of two different strains, and when presented by human MHC class II molecules, it also failed to activate murine cell line 3DT, which expresses the SEA-specific TCR V beta element (V beta 1). These results indicate that this mutation alters the interaction between SEA and murine TCR. The reactivity patterns of the mutated SEA with two specific anti-SEA monoclonal antibodies suggested that the observed effect of the isolated mutation in the murine system might be due to certain conformational changes in the SEA molecule introduced upon changing the D at position 60 to N. Site-directed mutagenesis of the N residue to D or to glycine reconstituted the ability of SEA to stimulate murine splenic lymphocytes. The different effects of this natural mutation at position 60 on the immunological activities of SEA with murine and human cells highlight the relevance of the affinity and avidity in SEA-TCR interactions in the function of different species or may reflect a difference in epitope specificity.

A murine monoclonal multireactive immunoglobulin kappa light chain.

N12.12 is a monoclonal immunoglobulin (Ig) kappa light chain (KLC) secreted by a B-cell hybridoma derived from spleen cells of a normal SJA mouse. No heavy chain was detected in the culture supernatant of this hybridoma using an enzyme immunoassay (EIA) and after polyacrylamide gel electrophoresis (SDS-PAGE) of the 35S-methionin biosynthetically labelled proteins secreted by the cells. It was shown that N12.12 KLC reacted with mouse actin, trinitrophenylated bovine serum albumin (TNP25-BSA) and weakly with bovine myoglobin. The binding of the N12.12 'monoclonal antibody' to mouse actin or to TNP25-BSA was inhibited specifically by both antigens with a dissociation constant (KD) for binding to mouse actin of 10(-7) M. The results indicate that a free KLC can bind both to mouse and to non-mouse molecules, thus exhibiting binding characteristics usually attributed to natural multireactive antibodies.

Specific and natural antibody production during Salmonella typhimurium infection in genetically susceptible and resistant mice.

Genetically susceptible (C57BL/6) and resistant (CBA) mice were infected with an avirulent strain of Salmonella typhimurium and studied over a 35-day period for the production of antibodies directed against bacterial antigens including lipopolysaccharide (LPS) (specific antibodies) and antibodies directed against self antigens [natural antibodies (NAb)]. Antibodies directed against LPS and self antigens were detected by enzyme immunoassay (EIA) and those directed against other bacterial antigens by immunoblotting. We found that serum natural antibody titres in C57BL/6 and CBA mice were similar and correlated with the bacterial load in the spleen and liver. In C57BL/6 mice, anti-LPS antibodies remained polyreactive and of the IgM isotype. In contrast, CBA mice, after an early increase in polyreactive IgM anti-LPS antibodies, mounted a specific anti-LPS IgG antibody response. The immunoblotting results demonstrated that the IgM polyreactive antibodies in the resistant and susceptible mice recognized bacterial antigens of different molecular weights and that CBA, but not C57BL/6 mice, were able to produce IgG antibodies recognizing bacterial components. Our results suggest that the synthesis of antibodies directed against bacterial antigens and natural antibodies follow, at least partially, distinct pathways, but they do not allow us to determine whether these two antibody populations are produced by the same or distinct B-cell subpopulations.

Mice ascites as a source of polyclonal and monoclonal antibodies.

A large quantity of polyclonal anti-ovalbumin antibodies was obtained from mice by a simple modification of the method described by Kurpisz et al. (1988). In addition, the cells from ascitic fluid were used to produce monoclonal antibodies. Egg ovalbumin hyperimmunized BALB/c mice were injected successively with pristane, antigen and a non-antibody secreting myeloma cell line: the production of ascitic fluid containing antiovalbumin antibody activity was observed after 10-25 days. Cells from ascitic fluid were harvested, washed and fused together with polyethylene glycol to produce monoclonal antibodies. Two fusions were performed and a large number of monoclonal anti-ovalbumin antibodies was obtained. This method is simple, reproducible, allows many fusions to be obtained from one mouse, and allows the use of ascitic B cells rather than the more, frequently used splenic B cells.

Effect of microbial inoculant on quality of alfalfa hay baled at high moisture and lamb performance.

The effectiveness of a microbial hay inoculant in high moisture alfalfa hay was evaluated. Alfalfa (third cutting) was baled at 72% DM without or with inoculant and at 82% DM without inoculant during yr 1. In yr 2, alfalfa (second cutting) was baled at 75% DM without or with inoculant and at 82% DM without inoculant. Application rate of inoculant was 3.8 L/.98 tonne each year. At this application rate, 90 billion cfu were applied per .98 tonne of forage. Hays were core sampled at 0, 14, 30, and 60 d after baling to determine chemical composition. By d 30, all hays had DM content of 89%. In yr 2, 12 wether lambs were assigned to three treatments in a replicated 3 x 3 Latin square. Treatments were chopped, low moisture hay plus corn; chopped, inoculated high moisture hay plus corn; and chopped, high moisture hay plus corn. All diets contained 63% alfalfa hay, 35% ground corn, and 2% minerals and vitamins. In yr 1, inoculated and low moisture hays were not different in chemical composition but were higher in CP and lower in NDF than high moisture hay. Neither NDF nor CP were different among the three hays in yr 2. Average daily gain was not different on the three diets. The feed to gain ratio was lowest for the inoculated hay, intermediate for the low moisture hay, and highest for the high moisture hay diet. Daily gain and feed to gain ratio were not different for lambs fed the inoculated hay baled at 75% DM compared with lambs fed untreated hay baled at 82% DM.

Common antigenic properties of a g-type (goose) and a c-type (duck) egg white lysozyme: antibody responses in rabbits and mice.

Embden goose (GEWL) and Barbary duck (DEWL) egg white lysozymes possess different amino acid sequences corresponding to the g-type and c-type, respectively. GEWL was shown to be a better immunogen than DEWL in both rabbits and mice. The antigenicity of the two lysozymes was tested using different techniques (i.e. indirect ELISA, inhibition tests and immunoabsorption experiments). Injection of either GEWL or DEWL into rabbits and mice induced both specific antibodies and cross-reacting antibodies. Moreover, anti-GEWL antibodies, in contrast to anti-DEWL antibodies, did not cross-react with hen egg white lysozyme (HEWL), a c-type lysozyme. While the structure of GEWL was not modified after binding to plastic, DEWL was denaturated, but it did keep some native epitopes. It was concluded that g-type and c-type lysozymes, which have different amino acid sequences, exhibit strong common antigenic properties.

Immunochemical probes for food proteins after heat processing.

While better hygiene controls and vaccinations have diminished the occurrence of infectious diseases in humans, food-borne diseases have increased. Thus sterilization of food products is of prime importance. The introduction of new technologies applied to food has necessitated new methods for the control of food safety and food quality. This review aims to point out the importance of immunochemistry in the identification of structural changes induced in food proteins during food processing. New technologies have introduced the use of additives in food products, therefore it is important to identify and quantify such additives, even after complete hear denaturation. Toxic chemicals, toxins and pesticides which can contaminate food products before or during processing should also be identified. Finally, the use of immunochemical tests as a control of sterilization procedures in heterogeneous foodstuffs is discussed.

Syngeneic albumin can be used as a carrier in neonatal mice.

Free native horseradish peroxidase (PO) or PO coupled to either syngeneic mouse serum albumin (PO-MSA) or to xenogeneic bovine serum albumin (PO-BSA) in sterile phosphate buffered saline (PBS), were injected repeatedly into newborn BALB/c mice. Serum antibody titres were evaluated by enzyme immunoassay on 30 and 60 of age and on d 75 and 88 after 1 or 2 booster injections respectively. The response to PO was found in all sera from neonatal immunized mice with all forms of PO, but only in control adult mice immunized by PO-BSA. Immunization with either PO-MSA or PO-BSA induced mainly IgG anti-PO antibodies of high avidity while immunization with free PO resulted in the induction of both IgM and IgG anti-PO antibodies of low avidity. A large number of hybridomas with anti-PO specificity were obtained from the spleen of mice injected with PO-MSA. The results indicate that neonatal immunization of mice with PO is an effective procedure, which is probably applicable to other proteins.

[Immunochemical analysis of structural changes in ovalbumin caused by heat: possible application to the quick control of food sterilization]. / Analyse immunochimique des modifications structurales de l'ovalbumine par la chaleur: application possible au contrôle rapide de la stérilisation des aliments.

Bacteriological methods, to determine proper hygiene control for food for human consumption suffers from inordinate delay (1-4 days) in the methodology before an accurate answer can be given regarding the food safety. In contrast by immunochemistry results can be obtained in 15 to 30 minutes. As it is known that during heating processing protein structure is modified, a careful identification of epitopic changes by monoclonal antibodies could be of help. It was found that after heat treatment, ovalbumin structure modified depending upon the temperature of heating. Monoclonal antibodies were raised against either native or heat denatured ovalbumin. Such monoclonal antibodies bound to plastic can be used for the immunocapture of ovalbumin in solution which is then revealed by polyclonal antibodies. With five different monoclonal antibodies it was possible to identify either native ovalbumin or heat denatured ovalbumin between 50 degrees C and 65 degrees C or heat denatured ovalbumin between 85 degrees C and 100 degrees C. Work is in progress to recognize epitopes specific of temperature and time of heating.

Studies on active immunization with self antigens. I. Production of antibody to unmodified proteins by neonatal immunization.

Newborn BALB/c mice were repeatedly injected either with syngeneic (BALB/c) or xenogeneic (bovine) myosin, albumin, or actin in sterile physiological saline. The serum antibody response was evaluated by enzyme immunoassay 1 and 2 months after birth and after two booster injections. At 1 month, higher antibody titres were found in the sera of mice injected with syngeneic than with xenogeneic antigens. At 2 months and after boosting, anti-syngeneic actin antibodies were present in equal or higher amounts, anti-syngeneic albumin antibodies were not detected, and anti-syngeneic myosin antibodies were considerably decreased. Antibodies produced after booster injections of syngeneic actin were found to be highly specific and to belong mainly to the IgG isotype. These results suggest that newborn mice are better able than adult mice to respond to stimulation with self antigens, and that administration of self proteins during neonatal life may lead to the induction of immunological memory. They also indicate that one of the primary functions of the immune system in newborn mice is the recognition of self antigens.

Studies on active immunization with self antigens. II. Production of antibody related to hapten substitution.

BALB/c mice were injected during neonatal life with conjugates in buffered physiological saline, prepared by coupling trinitrophenyl groups (TNP) at various densities to either syngeneic mouse serum albumin (TNP-MSA) or xenogeneic bovine serum albumin (TNP-BSA). Serum samples were obtained on days 30 and 60 after birth, on days 75 and 88 after two booster injections, and monoclonal antibodies were prepared from spleens of neonatally treated mice. The antibody titres, isotypes, and specificities were evaluated by enzyme-immunoassay. It was found that the extent of the anti-TNP immune response to TNP-MSA conjugates depends on the degree of hapten substitution, which is not the case for the anti-TNP-BSA. All the TNP-MSA conjugates induced mainly IgG and only a few IgM antibodies. These antibodies reacted essentially with the TNP group but seemed to have a higher avidity for the TNP-protein conjugate used in their induction. During the course of the immunization, decreasing quantities of TNP-MSA conjugates were needed to inhibit antibody binding. A large amount of monoclonal anti-TNP antibodies was found in hybridomas obtained after neonatal treatment either with TNP-MSA or TNP-BSA. Therefore, it appears that the anti-TNP immune response obtained after antigenic stimulation with sufficiently substituted TNP-MSA conjugates possesses all the characteristics of a normally occurring humoral immune response.

Regulation of the humoral immune response by polyspecific natural autoantibodies.

Two different BALB/c IgMk polyspecific monoclonal natural autoantibodies E7 and D23 were administered to neonatal BALB/c mice. When adults, these mice were immunized and challenged with calf myosin, BALB/c actin, human transferrin, calf thymus DNA or TNP-coupled bovine serum albumin (TNP/BSA), in complete Freund's adjuvant. The levels of serum antibody were evaluated by enzyme immunoassay. No differences in anti-actin, anti-transferrin and anti-DNA antibody titres were noted between control and antibody-treated mice. However, anti-myosin antibody titres significantly increased in mice treated with either the E7 or D23 antibody, and anti-TNP antibody titres significantly decreased in mice treated with E7 but not with D23. These differences persisted after antigenic challenge and involved only the IgG response of treated mice. These results suggest that polyspecific natural autoantibodies may be involved in the regulation of the humoral immune response.