Naturally occurring virulence-attenuated isolates of Listeria monocytogenes capable of inducing long term protection against infection by virulent strains of homologous and heterologous serotypes

Abstract Experimental infections of mice with strains of Listeria spp. isolated from contaminated food sources allowed discrimination of strains into those either exhibiting high, attenuated or low virulence. Compared to the highly virulent L. monocytogenes strain EGD, an attenuated strain such as L99 persisted for shorter times (5 versus 10 days) in the infected host. Using a tissue culture cell model of infection, we found that, although strain L99 was capable of accumulatinn actin like its virulent counterpart following invasion, it was unable to generate the polarized actin tails required for intracellular and cell-to-cell movement. Immunoblot analysis using specific antiserum to the ActA polypeptide, a molecule that is necessary for movement of the bacterium within the eucaryotic cell, indicated that a slightly truncated form of this polypeptide was produced in the L99 strain. Despite its reduced virulence, the attenuated strain L99 was just as effective in generating protection in immune mice as the highly virulent strains, albeit with a 1000-fold higher infective dose. Based on the results obtained from this study, we suggest that one of the mechanisms accounting for widespread resistance in humans to infection by Listeria may be due to asymptomatic infections by naturally occurring strains attenuated for virulence.     

Naturally occurring structural isomers in serum IgA1 o-glycosylation

IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of O-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr(225), Thr(228), Ser(230), Ser(232) and Thr(236) have been identified as the predominant sites of O-glycan attachment. The range and distribution of O-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 O-glycan heterogeneity by use of high resolution LC-MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 O-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the O-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable O-glycan chains attached to Ser(230), Thr(233) or Thr(236) produce the predominant positional isomers, including O-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 O-glycoforms, define the single-site heterogeneity for all O-glycan sites in a single sample, and have implications for defining epitopes based on clustered O-glycan variability.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum.

Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.     

An endotoxin induced serum factor that causes enhancement of antibody response to heterologous erythrocytes.

The sera obtained from blood of the mice, which had been intravenously injected with LPS several hours in advance, contained some active substance capable of enhancing anti-sheep red blood cell (SRBC) antibody responses in mice. Activity of the sera was still retained after passage through a rabbit anti-LPS antibody-coated Sepharose 4B column, but greatly reduced by passage through a rabbit anti-mouse thymocyte antibody-coated Sepharose 4B column. The active substance in the sera was eluted through a Sephadex G-200 column at the same position as the serum albumin. The addition of this substance to B cell rich spleen cell cultures in vitro in the presence of SRBC generated tremendous numbers of antibody forming cells 4 days after the incubation, suggesting that this substance was able to take over the helper function of T cells in thymus dependent antibody responses. However, this substance was not capable of stimulating 3H-thymidine-uptake into cultured spleen cells. The possible role of this substance in the adjuvant effect of LPS is discussed.     

Effects of anti-Ureaplasma urease antibody on homologous and heterologous urease activities

Among organisms in the class Mollicutes only Ureaplasma species possess urease. Antiserum to urease of U. urealyticum strain T960 (CX8) was used to examine the cross-reactivity of urease from other Ureaplasma species, as well as urease of jack bean and several urease-possessing walled bacteria. Immunological cross reactivity was used to establish phylogenetic relationships between various antigens. The ability of monospecific anti-urease antibody to inhibit urease activity was examined. The antiserum inhibited urease activity of the homologous strain the least of any Ureaplasma tested. It is postulated that urease possesses a minimum of two sets of epitopes. Binding of antibody to one epitope causes inhibition of enzyme activity; this epitope is common to urease of all Ureaplasma species. Binding of antibody to the other epitope prevents binding to the inhibition epitope; this epitope is specific to U. urealyticum strain T960 (CX8). No inhibition was observed with urease from jack bean or several walled bacteria.     

The monoclonal antibody AE-2 modulates fetal bovine serum acetylcholinesterase substrate hydrolysis

The monoclonal antibody (mAb) AE-2 decreases the rate of hydrolysis of acetylthiocholine (ATC) by fetal bovine serum acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) (FBS-AChE) (Doctor, B.P. et al. (1989) Proc. 32nd Oholo Conf., Eilat, Israel, in press), but increases the rate of hydrolysis (Vmax) of the nonpolar substrate, indophenyl acetate (IPA) approx. 15-fold. The affinity (Km) of FBS-AChE for IPA changes minimally in comparison with the increase in the rate of hydrolysis. The complex is dissociated, and the modulation of substrate hydrolysis is reversed by the active-center ligand, 1-methyl-2-hydroxyiminomethylpyridinium chloride (2-PAM).     

Naturally occurring plasmodium-specific IgA antibody in humans from a malaria endemic area

Blood samples collected from individuals belonging to an endemic area in Uttar Pradesh, were tested for plasmodial antigen specific immunoglobulin A (IgA) by enzyme immuno assay using soluble extract ofPlasmodium falciparum from culture. Among 773 (20.18%,P < 0.0001) samples 156 sera demonstrated a detectable seropositivity for antigen specific IgA. IgA levels were higher among individuals who experienced repeated attacks of malaria compared to acute infected patients. Among seropositive individuals the IgA titers were found increased with the age. Immunoglobulin isolated from sera having high level of IgA showed growth inhibitory effect inPlasmodium falciparum in vitro. A group of sera with high IgA antibody againstPlasmodium falciparum crude antigen showed seronegativity with specific peptides. Statistically, no positive or negative correlations were observed between antigen specific IgG and IgA. However, there was a tendency towards negative correlation between IgA and IgM. Mechanisms for the parasite specific IgA production remain to be established.     

Naturally occurring subclinical Corynebacterium kutscheri infection in laboratory rats: strain and age related antibody response

Naturally occurring subclinical Corynebacterium kutscheri infection was analyzed by antibody response related to the strain of rats. Wistar-Lewis, Wistar and Spraque-Dawley rats were high responders in seroconversion rates and antibody titers, while Brown Norway and Fischer rats were low responders. The antibody response was related to age also. Some young rats had maternal antibody to C. kutscheri, but antibody disappeared before 8 weeks of age. Rats were antibody-negative for several months thereafter and became antibody-positive after 6 months of age. The antibody response was highest at 8 to 9 months of age in subclinical C. kutscheri infection. This antibody response was very late, compared to the antibody response to Sendai virus and Mycoplasma infections.     

Naturally occurring glucosinolates and isothiocyanates as a weapon against chronic pain: potentials and limits

Phytochemistry Reviews - Investigation into glucosinolates (GLs) therapeutic effects boasts a long history, which began with the evidence that their hydrolysis-derived isothiocyanates (ITCs) could...     

Goat serum: an alternative to fetal bovine serum in biomedical research

Serum is frequently added to the defined basal medium as a source of certain nutritional and macromolecular growth factors essential for cell growth. Although a number of synthetic media have been prepared serum continues to be used in cell culture by many investigators. The best supplementation to a basal medium is fetal bovine serum (FBS) that is most frequently used for all types of cell cultures. During last four decades National Institute of Virology, Pune, has been working on isolation and identification of viruses from clinical specimens, employing tissue culture. Initially FBS was used for this purpose. However, due to its prohibitive cost and uncertain supply an alternative was sought. Commercially available sera from newborn calf, sheep, horse, human and serum obtained from goat blood (available from local abattoir) were tried. Goat serum (GS) was found to be suitable for most of the cell lines and primary cultures. Primary cultures from guinea pig embryo, monkey kidney, chick embryo, mouse peritoneal macrophages, and established cell lines were prepared and grown in growth media supplemented with GS. These cultures were studied for their morphology and growth in comparison with cultures grown in FBS containing media, and were used for mass cultivation of cells, quantitation and susceptibility of various virus strains, studies on effects of different nutrients and natural substances on cellular metabolism and virus replication, epitope analysis of various strains of Japanese encephalitis (JE) virus, strain differentiation studies, studies on antibody dependent plaque enhancement, assay of murine migration inhibition factor. Monoclonal antibodies against JE virus adapted to GS were characterised for their retention of functionalities. The results were comparable to those of cell cultures grown in FBS containing media. Similar results on chromosome studies were obtained from patient's whole blood cultures prepared in GS and FBS containing growth media. Organ cultures from mammalian, reptile and avian hosts; successfully grown in GS supplemented growth media, were used for different virological studies. Growth media supplemented with GS were used for in vitro cultivation of malarial parasites. Thus since the last three decades many scientists are using GS in place of FBS, in various fields of biomedical research. The present article reviews an account of the same.     

Characteristics of antibodies against 17 beta-oestradiol in homologous and heterologous systems of radioimmunoassay

Directly iodinated oestradiol-2(4)-iodo-[125I] and oestradiol-6-O-(CMO)-[125I]iodohistamine were prepared and used in conjunction with anti-oestradiol-2(4)-azo and anti-oestradiol-6-O-(CMO) sera for the development of various radioimmunoassay (RIA) systems which showed marked differences in sensitivity and relatively small differences in specificity. Whereas the heterologous combination of oestradiol-6-O-(CMO)-[125I]iodohistamine and anti-oestradiol-2(4)-azo-serum showed a sensitivity expressed in femtograms, the homologous combination using oestradiol-6-O-(CMO)-[125I]iodohistamine radioligand exhibited a sensitivity two orders lower. The specificity of both the heterologous and homologous system did not differ significantly from the RIA system using tritiated radioligand. The combination of directly iodinated oestradiol and anti-oestradiol-2(4)-azo-serum showed a higher sensitivity and a lower specificity as compared with tritiated radioligand.     

Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes

We have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. We found sequence identify or probable identity in 111 out of 112 residues when we compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated.     

Experimental vaccine protection against homologous and heterologous strains of feline immunodeficiency virus.

More than 90% of cats immunized with inactivated whole infected-cell or cell-free feline immunodeficiency virus (FIV) vaccines were protected against intraperitoneal infection with 10 50% animal infectious doses of either homologous FIV Petaluma (28 of 30 cats) or heterologous FIV Dixon strain (27 of 28 cats). All 15 control cats were readily infected with either strain of FIV. Protection appears to correlate with antiviral envelope antibody levels by a mechanism yet to be determined.