Streptococcus pneumoniae: new tools for an old pathogen

The pneumococcus is one of the longest-known pathogens. It has been instrumental to our understanding of biology in many ways, such as in the discovery of the Gram strain and the identification of nucleic acid as the hereditary material. Despite major advances in our understanding of pneumococcal pathogenesis, the need for vaccines and antibiotics to combat this pathogen is still vital. Genomics is beginning to uncover new virulence factors to advance this process, and it is enabling the development of DNA chip technology, which will permit the analysis of gene expression in specific tissues and in virulence regulatory circuits.     

Molecular and immunogenetic analysis of major histocompatibility haplotypes in northern bobwhite enable direct identification of corresponding haplotypes in an endangered subspecies,the masked bobwhite

The major histocompatibility complex (MHC) is a group of genetic loci coding for haplotypes that have been associated with fitness traits in mammals and birds. Such associations suggest that MHC diversity may be an indicator of overall genetic fitness of endangered or threatened species. The MHC haplotypes of a captive population of 12 families of northern bobwhites (Colinus virginianus) were identified using a combination of immunogenetic and molecular techniques. Alloantisera were produced within families of northern bobwhites and were then tested for differential agglutination of erythrocytes of all members of each family. The pattern of reactions determined from testing these alloantisera identified a single genetic system of alloantigens in the northern bobwhites, resulting in the assignment of a tentative genotype to each individual within the quail families. Restriction fragment patterns of the DNA of each bird were determined using the chicken MHC B‐G cDNA probe bg11. The concordance between the restriction fragment patterns and the alloantisera reactions showed that the alloantisera had identified the MHC of the northern bobwhite and supported the tentative genotype assignments, identifying at least 12 northern bobwhite MHC haplotypes. Eighteen northern bobwhite alloantisera were then used to detect a minimum of 17 masked bobwhite MHC haplotypes. Subsequent restriction fragment pattern analyses using cDNA probes for chicken MHC genes were in agreement with agglutination patterns displayed by the antisera, showing that the immunogenetically identified alloantigen system constituted the MHC of the masked bobwhite. These data demonstrate that a non‐endangered species may be used to provide antisera for differentiating MHC haplotypes in a closely related endangered species, thus providing a practical basis for long‐range monitoring of MHC haplotypes of birds surviving in their native habitats. Zoo Biol 18:279–294, 1999. © 1999 Wiley‐Liss, Inc.     

Association between theRfp-Y haplotype and the incidence of Marek's disease in chickens

 Certain haplotypes at the major histocompatibility (B) complex (Mhc) of the chicken provide an easily demonstrated influence on tumor formation following infections with Marek’s disease virus (MDV). Recognition that there is a second histocompatibility complex of genes in the chicken, Rfp-Y, comprised of Mhc class I and class II genes, some of which are at least transcribed, evokes the question of whether this gene complex might also influence the outcome of MDV infections. To test this hypothesis, pedigree-hatched chicks in families from the original Rfp-Y-defining stock in which three Rfp-Y and two B system haplotypes are segregating were challenged with the RB1B strain of MDV. Birds with the Y ³ /Y ³ genotype were found to have 2.3 times the risk of developing a tumor compared with birds with other Rfp-Y genotypes combined (P <0.02). Additionally, birds carrying the B ^R9 /B ¹¹ genotype had 2.3 times the risk of tumor formation, relative to birds with the B ¹¹ /B ¹¹ genotype (P <0.02). We found no evidence for an interaction between genotypes within the B and Rfp-Y systems. These data provide evidence that Rfp-Y haplotypes, as well as B haplotypes, can significantly influence the outcome of infection with MDV. Received: 15 February 1996 / Revised: 23 April 1996     

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.      

Membrane Lipoteichoic Acid Is Not a Precursor to Wall Teichoic Acid in Pneumococci

The distribution of a pulse of teichoic acid-specific radiolabel between wall and membrane teichoic acids in pneumococci was constant over a subsequent chase period, suggesting that wall and membrane teichoic acids are biosynthesized simultaneously and independently.     

Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

The mouse heavy chain variable region marker, J606-GAC, is not restricted to particular B cell isotypes or subsets

The heavy chain variable region (VH) marker J606-GAC, which is expressed on a subset of mouse heavy chain variable region group III antibodies, is expressed at similar frequencies on antibodies with mu, gamma 3, gamma 1, gamma 2, and alpha heavy chains. We have previously shown the J606-GAC determinant to be present on all anti-inulin and on the majority of anti-group-A-carbohydrate (GAC) antibodies examined. The responses to these two antigens are designated thymus-independent type 2 (TI-2) and thymus-dependent type 2 (TD-2), respectively, and have been shown previously to be largely restricted to the mu and gamma 3 heavy chain classes. TI-2 and TD-2 antigens are distinguished from other antigens such as T-independent type 1 (TI-1) and other thymus-dependent (TD) antigens, in part because they are virtually not immunogenic in CBA/N mice which express the x-linked immunodefiency (xid) allele. Surprisingly, we found no difference in the percentage of J606-GAC determinant-bearing plasma cells in the spleens of xid vs normal mice.     

Monoclonal antibodies to Salmonella lipopolysaccharide: anti-O-polysaccharide antibodies protect C3H mice against challenge with virulent Salmonella typhimurium

The present investigation reports the production of monoclonal antibodies to antigenic determinants of the O-polysaccharide of Salmonella typhimurium lipopolysaccharide (LPS), and assesses the effectiveness of these antibodies in protecting C3H mice against the lethal effects of Salmonella infection. Hybridomas were generated by fusing spleen cells from (BALB/c X A/J)F1 (CAF1) mice hyperimmunized by i.v. injection with acetone-killed S. typhimurium SR-11 with X63-Ag8.653 murine myeloma cells. Hybridomas producing antibodies reactive with S. typhimurium SR-11 whole cells were subcloned, and seven of the resulting clones as well as one previously described clone were selected for use in the studies reported here. Monoclonal antibodies from these eight clones were of the IgG1 (1), IgG3 (6), or IgM (1) isotype and were specific for the O-polysaccharide region of Salmonella LPS, reacting with LPS from smooth S. typhimurium SR-11 and LT-2, but not with LPS from rough S. minnesota R60 (Ra), R345 (Rb), or R595 (Re). The effectiveness of each monoclonal antibody in protecting C3H/HeN and C3H/HeJ mice against the lethal effects of Salmonella infection was evaluated by comparing the median length of survival of groups of mice given antibody by i.p. injection before i.p. challenge with virulent S. typhimurium SR-11 to that of animals that received no antibody. Three out of eight monoclonal anti-O-polysaccharide antibodies, ST-1 (IgM), 10-5-47 (IgG3), and 10-5-6 (IgG3), provided significant (p less than 0.01) protection to C3H/HeN mice challenged with approximately 10(4) LD100 of Salmonella. Only antibodies ST-1 and 10-5-6, however, extended the median length of survival of C3H/HeJ mice beyond that of infected controls. Mouse antiserum prepared against S. typhimurium SR-11 was equally protective in C3H/HeJ mice. In an attempt to understand the contribution of antibody specificity to the relative differences in the protective capacities of the monoclonal antibodies, their reactivities with several Salmonella reference strains of different classical serotypes were examined. Although some differences in reactivity against the different strains were apparent, this approach was not adequate for defining the fine specificity of these monoclonal antibodies. The results of this study provide evidence that monoclonal antibodies with specificity to the O-polysaccharide region of Salmonella LPS can protect C3H mice against challenge with the homologous bacterial strain.     

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.