The alpha-L-(1----2)-trirhamnopyranoside epitope on the group-specific polysaccharide of group B streptococci.

A number of epitope specificities associated with the group antigen (group B polysaccharide) of group B streptococci have been identified in a polyclonal antiserum induced in rabbits by a nonencapsulated variant strain of group B streptococci. This was achieved by using a series of oligosaccharide inhibitors, obtained by both synthetic and degradative procedures, to inhibit the binding of the group B polysaccharide to the polyclonal antiserum. While the dominant epitope expressed in the antiserum was alpha-L-Rhap(1----2)alpha-L-Rhap(1----2)alpha-L-Rhap, specificities associated with alpha-L-Rhap and alpha-L-Rhap(1----3)alpha-D-Galp(1----3)beta-D-Glcp-NAc(1----4)alp ha-L-Rhap were also identified. The dominant expression of the former epitope is consistent with its terminal location on the group antigen and also with highly branched multiantennary structure of this antigen. Antibodies specific for the alpha-L-trirhamnopyranoside epitope were purified by affinity chromatography, using the synthetic trisaccharide glucitol as the hapten. Oligosaccharide inhibition studies indicate that the specificity of these antibodies is identical to that of a murine monoclonal antibody induced by the same nonencapsulated strain of group B streptococci.     

Lethal Myocardial Ischemic Injury

The biologic changes occurring in severely ischemic myocytes in vivo as the affected cells pass through the phase of reversible to the phase of lethal or irreversible injury are reviewed with special emphasis on the effect of ischemia on the production and utilization of highenergy phosphate, the destruction of the adenine nucleotide pool, and the appearance of signs of damage to the plasma membrane of the sarcolemma. Evidence is presented that indicates that the events occurring in severe ischemia in vivo are essentially identical to those found in total ischemia in vitro except that the biologic changes of ischemia develop more slowly in total ischemia in vitro than in severe ischemia in vivo. The slower time course of injury, together with the uniformity of injury provided by total ischemia in vitro, may allow for more precise identification of potential lethal cellular events in ischemic injury. The production of highenergy phosphates (HEP) from anaerobic glycolysis have been estimated in both in vivo and in vitro ischemia by the measurement of lactate accumulation, and total HEP utilization has been estimated from the depletion of stores of preformed HEP. The results show that between 80% and 90% of the HEP utilized by ischemic dog left ventricle is produced by anaerobic glycolysis. The onset of irreversibility is associated with marked depletion of the HEP and adenine nucleotide pools of the tissue and the cessation of energy production via glycolysis. The cessation of anaerobic glycolysis may be caused by the low sarcoplasmic, adenosine triphosphate (ATP) concentration of the dying myocyte. In addition to the foregoing changes, irreversibly injured tissue exhibits both ultrastructural and functional evidence of disruption of the plasmalemma of the sarcolemma. The possible relationships, causal and otherwise, between severe HEP depletion and membrane damage are discussed. Both HEP depletion (ATP < 3-8% of control) and membrane damage are considered to be objective signs of the presence of irreversible myocardial ischemic injury. However, at the present time, there is no proof that these changes are causally related either to each other or to cell death in severe in vivo ischemia.     

Nitric oxide participates in the intestinal pathology associated with murine syngeneic graft-versus-host disease

Syngeneic graft-versus-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a short course of cyclosporin A (CsA) therapy. The disease is characterized by the development of a T helper cell type 1-like cytokine response [interleukin (IL)-12, interferon-gamma (IFN-gamma), and tumor necrosis factor alpha], and macrophage activation is central to development of the syndrome. It has been shown that nitric oxide (NO) participates significantly in the development of allogeneic GVHD. Studies were initiated to determine if NO participates in the pathology associated with SGVHD. Significant increases in inducible NO synthase (iNOS) mRNA and circulating NO were found in the tissues of SGVHD versus control animals. Treatment of SGVHD animals with the iNOS inhibitor aminoguanidine (AG) reversed the pathology associated with this disease. Furthermore, AG treatment reduced the production of IL-12 and IFN-gamma mRNA in the colons of CsA-treated mice. These studies demonstrate that NO participates in the pathological processes that are associated with the development of murine SGVHD.     

Conjugation of meningococcal lipopolysaccharide R-type oligosaccharides to tetanus toxoid as route to a potential vaccine against group B Neisseria meningitidis.

Oligosaccharides were obtained by the mild acid hydrolysis of the lipopolysaccharides from a number of different strains of Neisseria meningitidis, serotypes L2, L3, L4, L5, and L10. The dephosphorylated oligosaccharides were conjugated to tetanus toxoid as their 2-(4-isothiocyanatophenyl)-ethylamine derivatives, which resulted in the incorporation of from 18 to 38 oligosaccharides per molecule of tetanus toxoid. When injected in rabbits, the conjugates produced oligosaccharide-specific antibodies which were predominantly serologically specific but which also exhibited some cross-reactivity. These serological results can be attributed to regions of structural dissimilarity and similarity within the oligosaccharides. The oligosaccharide-specific antibodies were also lipopolysaccharide serotype specific, thus indicating that the oligosaccharides are the determinants associated with this serotype specificity. Consistent with the serological results, the conjugate antisera were bactericidal for the homologous serotype meningococcal organisms and in some cases for heterologous serotype organisms.     

Comparison of in vitro and in vivo alpha/beta ratios for prostate cancer

Parallel in vitro and in vivo studies provide insight into the relationship between clinical response and intrinsic cellular radiosensitivity and may aid in the development of predictive assays. Compilations of radiosensitivity parameters from in vitro experiments can also be used to examine the potential effectiveness of alternative or new treatment plan designs until enough clinical data become available to directly estimate the requisite radiosensitivity parameters. In this work, survival data for six prostate cancer cell lines (ten datasets total) have been extracted from the literature and re-analysed using the linear-quadratic (LQ) survival model. The paired bootstrap technique for regression is used to compute 95% confidence intervals for the estimated radiosensitivity parameters. LQ radiosensitivity parameters derived from the in vitro data are then compared to radiosensitivity parameters derived from clinical data for prostate cancer. Estimates of alpha range from 0.09 to 0.35 Gy(-1) (all cell lines), and the alpha/beta ratio ranges from 1.09 to 6.29 Gy (all cell lines). Point estimates of the repair half-time (PPC-1, TSU-Pr1, PC-3 and DU-145 cell lines) range from 5.7 to 8.9 h (95% confidence interval from 0.26 h to 10.7 h). Differences in the radiosensitivity parameters determined from the data reported by different laboratories are as large as or larger than the differences in radiosensitivity parameters observed among the various prostate cell lines. The reported studies demonstrate that even seemingly small corrections for dose rate effects, such as those expected in high dose rate (HDR) experiments, can sometimes have a significant impact on estimates of alpha and alpha/beta. By neglecting dose rate effects in the analysis of HDR experiments, estimates of the alpha/beta, ratio may be too high by factors as large as 1.3 to 6.2. The half-time for repair derived from the in vitro experiments appears significantly larger (slower repair rate) than estimates derived from the clinical data. However, the prostate radiosensitivity parameters alpha and alpha/beta may be approximately the same in vitro and in vivo. Most of the in vitro data are consistent with an alpha/beta ratio for prostate cancer less than 3 or 4 Gy.     

Characterization of the progeny of precursor-T (pre-T) cells maintained in vitro by interleukin-3 (IL-3). Development of T-cell function in vivo.

We have recently described a bone marrow culture system which is able to maintain a portion of the precursor-T (pre-T) cell compartment of adult murine marrow in vitro, in the presence of interleukin-3 (IL-3), for at least 2 weeks. However, because growth in IL-3 might also induce the differentiation of the pre-T cells, it is necessary to determine the extent to which the developmental potential of the pre-T cells is altered during their residency in vitro. Previously, we analysed the progeny of cultured pre-T cells and compared their intrathymic development, their appearance in the periphery, and their V beta gene utilization to that of the progeny of fresh pre-T cells. Within these parameters, the cells derived from cultured marrow cells did not differ significantly from cells derived from fresh marrow cells. However, these studies did not allow us to determine the functional status of the T-cell progeny of cultured marrow. In the work presented here, we analysed the functional potential of T cells which were derived either from fresh pre-T cells or pre-T cells which had been maintained for 1 week in vitro. The T-cell mediated functions analysed included mitogen- and alloantigen-induced proliferation, IL-2 production, and generation of cytotoxic T cells. We found that the cultured pre-T cells were capable of giving rise to mature, immunocompetent T cells which did not differ significantly from the progeny of fresh pre-T cells in their functional potential.     

Immunisation with phage displaying peptides representing single epitopes of the glycoprotein G can give rise to partial protective immunity to HSV-2

Filamentous phage displaying peptides representing single epitopes of the glycoprotein G of HSV-2 (gG2) were used as immunogens via the subcutaneous route in Balb/c mice without additional adjuvant. The phage were isolated from a random phage peptide display library and contain 15-mer peptide inserts that mimic epitopes of gG2. In each case, an antibody response to gG2 was generated that was dependent on the dose of phage administered and on the presence of the peptide insert. Phage displaying epitopes of gG2, which map to amino acids 551-570, were the most immunogenic; interestingly, this region of gG2 is frequently recognised by patients infected with HSV-2. The data also provide interesting information as regards choice of peptide mimics for use as immunogens because, surprisingly, the most antigenic of the individual clones was the least immunogenic. In two of the experiments, mice immunised with phage displaying a single epitope of gG2 were protected against challenge with a lethal dose of whole HSV-2. This suggests a possible role for phage-displayed peptides in inducing protective immunity against pathogens and provides a model system for investigating the underlying mechanisms.     

Function and phenotype of immature CD4+ lymphocytes in healthy infants and early lymphocyte activation in uninfected infants of human immunodeficiency virus-infected mothers.

The function and phenotypes of CD4+ lymphocytes in infants are different than in adults and are modulated by maturational changes and exposure to environmental antigens. Infants of non-human immunodeficiency virus (HIV)-infected mothers and uninfected infants of HIV-infected mothers, 0 to 6 months of age, were examined for CD4+ lymphocyte function by in vitro interleukin-2 (IL-2) production and for CD4+ phenotypes by three-color flow cytometry. A minority of these uninfected infants (28%) had functional responses similar to those of healthy adult women (IL-2 production in response to anti-CD3, alloantigen, and mitogen), while the remainder were capable of responding to alloantigen and mitogen but not to anti-CD3. We did demonstrate reduced phytohemagglutinin-stimulated IL-2 production in uninfected infants born to HIV-seropositive mothers compared to that in infants from seronegative mothers. The proportions of CD3+ CD4+, CD4+ HLA-DR- CD38+, and CD4+ CD45RA+ RO- (naive) lymphocytes were much higher in infants than in adults, and the proportions of CD4+ CD45RA- RO+ (memory) and CD4+ CD25+ (IL-2 receptor-bearing) lymphocytes were lower in infants than in adults. The proportions of activated (CD4+ HLA-DR+ CD38+) and memory (CD4+ CD45RA- RO+) lymphocytes were increased in uninfected infants of HIV-infected mothers compared to infants of uninfected mothers. Therefore, T-helper-cell function is immature in many infants, but the CD4+ lymphocytes of some HIV-exposed, uninfected infants have been stimulated by antigen at an early age.