Characterization of the active site of group B streptococcal hyaluronan lyase.

Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.

Characterization of PepB, a group B streptococcal oligopeptidase.

Group B streptococci were recently reported to possess a cell-associated collagenase. Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase. We cloned and sequenced the gene for the enzyme (pepB). The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases. The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin.

In vitro effects of Mycoplasma pulmonis on murine natural killer cell activity.

We evaluated the effect of coculture of virulent and avirulent strains of Mycoplasma pulmonis with mononuclear cells from resistant and susceptible strains of mice on natural killer (NK) cell activity against YAC-1 cells in a standard 4-h 51Cr-release assay. Endogenous NK activity was minimal in the specific-pathogen-free-mice without an external stimulus. There was no correlation between in vivo virulence of the mycoplasmas or host resistance and the in vitro stimulation of NK cell activity. Only two of the avirulent strains of M. pulmonis tested induced significant increases in NK cell activity, and virulent M. pulmonis increased activity only in cells from susceptible C3H/HeN mice.

Cloning and expression of the gene for group B streptococcal hyaluronate lyase.

Group B streptococci (GBS) are a major cause of serious human perinatal infections. Most clinical isolates of GBS secrete hyaluronate lyase, and production of high levels of the enzyme has been associated with strain virulence. Degenerate oligonucleotide primers, designed on the basis of the amino acid sequences of tryptic peptides prepared from the purified enzyme, permitted the polymerase chain reaction amplification from GBS chromosomal DNA of a 363-base pair internal DNA fragment of the GBS hyaluronate lyase gene (hylB). This DNA fragment was used as a probe to screen a lambda phage library of GBS chromosomal DNA fragments. Sequence analysis of positive clones identified an open reading frame capable of coding for a 111-kDa protein. Since no single clone was found to contain the entire gene it was necessary to reconstruct the gene from two plasmids containing inserts with suitable overlapping sequences. When this reconstructed gene was transformed into Escherichia coli, high level expression of hyaluronate lyase activity was obtained.

Murine monoclonal antibodies to type Ib polysaccharide of group B streptococci bind to human milk oligosaccharides.

The chemical structures of the repeating units of the type Ib polysaccharide of group B streptococci and of the desialylated form of this antigen are almost identical to those of some oligosaccharides in human milk and certain fetal antigens. The structural similarities suggested that the molecules may be immunologically cross-reactive. Mouse monoclonal antibodies to the sialylated and nonsialylated forms of the type Ib polysaccharide were produced and tested for their ability to bind to immobilized human milk oligosaccharides. One antibody, SMB19, reacted specifically with the sialylated form of the type Ib polysaccharide and was also bound by an affinity column containing immobilized sialyllacto-N-tetraose a. The antibody was eluted from the affinity column with EDTA, since its binding to the antigen was calcium dependent. A second monoclonal antibody. SIbD2, bound specifically to the nonsialylated form of the type Ib polysaccharide and also to immobilized lacto-N-tetraose. The antibody was eluted from the affinity column at an acidic pH and retained immunologic activity. These results further extend our previous observations that certain antibodies raised against group B streptococci can also react with normal human glycoconjugates.

Effect of fibronectin on IgA-mediated uptake of type III group B streptococci by phagocytes.

Previous studies have shown that a type-specific IgA monoclonal antibody alone or in combination with fibronectin (Fn) enhances protective efficacy in two animal models of group B streptococcal infection. To investigate the mechanisms by which IgA mediates protection, the effects of Fn on phagocytosis of group B streptococci (GBS) opsonized with a type III-specific IgA monoclonal antibody were examined. Specific IgA alone or in combination with Fn did not promote the phagocytosis of GBS by polymorphonuclear leukocytes (PMNL). Fibronectin also had no significant effect on phagocytosis of IgA-opsonized GBS by monocytes. Specific IgA alone promoted phagocytosis of GBS by culture-derived macrophages in a dose-dependent fashion. Fibronectin enhanced macrophage uptake of the GBS opsonized in a suboptimal concentration of specific IgA (phagocytic index = 2.32 +/- 0.56 vs. 3.26 +/- 0.48 with Fn; P less than .05). These data suggest that protection against GBS in neonatal rats by a combination of Fn and specific IgA is mediated by macrophages rather than by PMNL or monocytes. Fibronectin may have a critical role in host defense at sites where IgA and macrophages predominate.

An IgA monoclonal antibody directed against type III antigen on group B streptococci acts as an opsonin.

We have investigated the mechanisms by which a murine IgA mAb directed against the type III Ag (IgA anti-III mAb) of group B streptococci (GBS) protects neonatal rats from lethal infection with these organisms. Purified IgA anti-III mAb enhanced phagocytosis of type III GBS by rat peritoneal macrophages in vitro by fourfold compared with phagocytosis of buffer-treated GBS. In the absence of antibody, neonatal rat serum did not promote phagocytosis, but addition of neonatal rat serum to GBS opsonized with IgA anti-III led to a sevenfold increase in phagocytosis. Heat inactivation of C destroyed the ability of neonatal rat serum to enhance phagocytosis in the presence of IgA. C3 deposition was observed when GBS coated with IgA anti-III mAb were incubated in untreated neonatal rat serum or in serum treated with Mg/EGTA. This latter observation suggested that C3 deposition occurred through activation of the alternative pathway. The control IgA mAb MOPC 315 did not enhance GBS ingestion or C3 deposition on GBS. Depletion of C in vivo by using cobra venom factor abolished the protective effect of IgA anti-III mAb in the neonatal rat model. These data suggest that the ability of this IgA to activate C further enhances its opsonic activity and may be essential for its protective effect in vivo.

Specificity of monoclonal antibodies against group B streptococcus type II and inhibition of their binding by human secretions.

Mouse monoclonal antibodies against the type-specific polysaccharide antigen of type II group B Streptococcus may be divided into two general groups based on their antigen-binding properties. One group of antibodies binds to both intact and desialylated type II antigen and the binding can be inhibited by beta-galactopyranosides. The second group of antibodies react only with intact type II polysaccharide and are not inhibited by beta-galactopyranosides. The binding of antibodies of the first group can also be inhibited by carbohydrate components of normal human secretions that share structural similarities with the type II polysaccharide. The binding of antibodies of one specificity precluded the binding of antibodies of the other specificity, apparently by competing for binding sites within a combining area that includes both determinants.

Immunochemical characterization of the polysaccharide antigens of group B streptococci.

Mouse monoclonal antibodies were used in the immunochemical characterization of the polysaccharide antigens of group B streptococci (GBS). Monoclonal antibodies specific for the sialylated form of the GBS type III polysaccharide were highly protective in a mouse model of GBS type III infection, whether of IgM, IgG2a, or IgA isotypes, but monoclonal antibodies specific for the nonsialylated form of the type III antigen were not protective, regardless of isotype. Monoclonal antibodies reacting with the type II polysaccharide antigen could be divided into two general antigen binding groups on the basis of the ability of beta-methylgalactopyranoside to inhibit their binding to purified type II antigen. Various proportions of antibodies with the two specificities were observed in rabbit and human sera. Although it was previously reported that rabbit antisera could not distinguish between the sialylated and nonsialylated forms of the type Ib polysaccharide, mouse monoclonal antibodies were found to exhibit exclusive specificity for one or the other form of the antigen. Only monoclonal antibodies specific for the sialylated Ib polysaccharide were protective in a mouse model.

Rheumatoid arthritis associated with expanded populations of granular lymphocytes.

Two patients with classic rheumatoid arthritis developed severe neutropenia and increased numbers of large granular lymphocytes in the blood and bone marrow. These lymphocytes exhibited homogeneous surface membrane immunophenotypes of Leu5+, Leu11-, Leu4+, Leu3-, Leu2-, Leu7+ and Leu5+, Leu11+, Leu4+, Leu3-, Leu2+, Leu7-, respectively. In both patients, neutropenia was initially corrected with corticosteroid therapy; long-term improvement followed low-dose oral cyclophosphamide and methotrexate therapies. In these 2 patients and 12 previous patients with rheumatoid arthritis associated with expanded populations of immunophenotypically homogeneous large granular lymphocytes, neutropenia occurred in all 14, thrombocytopenia in 6, anemia in 7, and mild or moderate splenomegaly in 12. In contrast to Felty's syndrome, granular lymphocyte expansions in rheumatoid arthritis usually occur in older patients, may appear simultaneously with arthritis, and are usually associated with normal or elevated blood leukocyte counts. Mild hemocytopenias in these patients can often be managed with observation. Therapy with corticosteroids or immunosuppressive-cytotoxic drugs may be beneficial in more severe cases, but splenectomy is not recommended.

The urban-rural dimension in national economic development.

PIP: Urban growth should be evaluated less as good or bad in itself than in terms of whether it promotes the efficient and equitable performance of vital economic functions within a nation. Much urban growth in developing nations both reflects national growth and promotes it. Cities are sources of economic growth, which is their dominant characteristic. There is a strong tendency for large cities and their surrounding core regions to be the most active, rapidly growing areas of developing nations. Certain economic functions tend to be found only in cities and tend to cluster into certain cities because it is economically efficient. 3 mechanisms which make cities economically efficient are 1) internal economies of scale, 2) localization economies, and 3) agglomeration economies. Urban areas can provide support functions for rural areas and, in turn, their growth depends on the support of an agricultural base. Urban areas also provide alternative employment and income opportunities for the rural surplus population. There are 4 prominent questions often raised about possible negative effects of urbanization on national growth and development. One question is urban growth and urban bias, which the authors argue is overemphasized. Another question is diseconomies of scale in large cities; this, the authors suggest, is not a matter of size as much as operating efficiently. Third is urbanization and regional dualism, which the authors argue can be maintained through a strategy of changing a nation's mix and location of urban activity. Fourth is the question of cities and rural outmigration. The authors argue that although most people who leave rural areas are younger, more motivated, and better educated than those left behind, their departures are economically favorable. Getting economic activity located correctly along an urban-rural spectrum is important to the growth of developing countries. 6 rules that illustrate how to do this are 1) be guided by local circumstances, not theoretical models; 2) promote better management of major urban areas; 3) avoid direct controls on migration and location; 4) understand the reluctance of industry to locate outside core regions; 5) develop secondary cities with an eye to economic eficiency; and 6) be cautious about "new town" developments. The authors conclude that 1) both host governments and those involved in development assistance should realize that urban growth is a natural process; and 2) when they do intervene in the process, they should do so in ways designed to build upon economic forces already at work.^ieng

Cellular origins of human polymeric and monomeric IgA: intracellular and secreted forms of IgA.

Intracellular and secreted IgA from pokeweed mitogen (PWM)-stimulated normal peripheral blood lymphocytes, from 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated peripheral blood lymphocytes of a patient with chronic lymphocytic leukemia (CLL), or from an IgA-producing human Epstein Barr virus (EBV)-transformed lymphoblastoid cell line were analyzed by molecular-sieve chromatography, electrophoresis in sodium dodecyl sulfate, and sucrose density ultracentrifugation. Fluorochrome-labeled anti-human IgA and secretory component (SC) were used as probes for the detection of polymeric IgA in individual cells. These methods demonstrated that the majority of intracellular IgA occurred in monomeric form, even when the predominant form of secreted IgA was polymeric. Sequential analyses of the IgA secreted by PWM-stimulated normal peripheral blood lymphocytes revealed that the proportion of polymeric IgA increased with the time of culture and that polymers represented the prevalent form of secreted IgA from the fifth day of culture. Although approximately one-half of TPA-stimulated CLL cells bound fluorochrome-labeled SC, only trace amounts of extracellular and intracellular polymeric IgA were detected in both culture supernatants and lysates. Culture supernatants of an IgA-secreting EBV-transformed cell line contained predominantly polymeric IgA. However, intracellular IgA was largely represented by monomers. The predominance of intracellular monomers in polymeric IgA-secreting cells suggested that the pathway of the assembly of human IgA molecules is analogous to that described for mouse IgA synthesis.

Protection of mice from experimental infection with type III group B Streptococcus using monoclonal antibodies.

Mouse hybridoma antibodies of several major classes against group B streptococcus type III have been produced. Mice were immunized with either whole heat-killed or acid-treated organisms to obtain antibodies against both the complete (sialated) or incomplete (nonsialated) forms of the type III polysaccharide. Resulting monoclonal antibodies showed exclusive specificity for either the complete or incomplete antigen. The ability of these antibodies to protect mice from a lethal challenge of live type III organisms was tested with a mucin model that permitted use of very small inocula given intraperitoneally with antibody and mucin. Antibodies specific for the nonsialated antigen were not protective, whether of IgM, IgG2a, or IgG3 isotypes. Antibodies specific for the complete antigen were, however, highly protective, including monoclonals of IgM, IgG2a, and IgA isotypes. These mouse monoclonal antibodies against group B streptococci that are directed against either complete or incomplete antigenic determinants, and include isotypes other than IgM, should be particularly useful for studying the mechanism of protection against experimental infection.

Natural killer cells in systemic lupus erythematosus. Abnormal numbers and functional immaturity of HNK-1+ cells.

A monoclonal antibody, HNK-1, that detects a differentiation antigen on human granular lymphocytes with natural killer (NK) activity was used to enumerate this subpopulation in the peripheral blood of 14 patients with systemic lupus erythematosus (SLE). Nine patients had severely decreased numbers of HNK-1+ cells, 3 patients had elevated levels of HNK-1+ cells, and 2 patients had appropriate numbers of HNK-1+ cells compared with the levels in 112 normal controls. All SLE patients exhibited low NK killing ability against K562 target cells compared with controls. An increased proportion of the HNK-1+ cells was categorized as immature granular lymphocytes in over 50% of the SLE patients because their HNK-1+ cells coexpressed the OKT3 antigen and contained a paucity of cytoplasmic granules. The numbers of HNK-1+ cells or the HNK-1+ OKT3+ subgroup did not correlate with steroid therapy. This evidence suggests that levels of HNK-1+ lymphocytes are abnormal and functionally immature in most SLE patients. Longitudinal studies conducted over several months on a number of SLE patients demonstrated fluctuations in the ratio of mature and immature HNK-1+ cells and total HNK-1+ cells. Additional patients tested over longer periods of time will have to be studied to determine whether the proportion of mature NK cells (HNK-1+ OKT-) and immature NK cells (HNK-1+ OKT3+) will be useful in predicting the clinical course of disease.

Production of predominantly polymeric IgA by human peripheral blood lymphocytes stimulated in vitro with mitogens.

Human peripheral blood lymphocytes (PBL) were cultured for various time periods (up to 8 d) in the presence of pokeweed mitogen (PWM), lipopolysaccharide, or Epstein-Barr virus. Cell-free supernates were fractionated on a standardized ultrogel AcA 22 column and the proportion of polymeric and monomeric IgA was determined by radioimmunoassay. The results demonstrate that PBL stimulated with these mitogens produce IgM and IgG with molecular characteristics identical to those found in serum, but that the IgA produced is predominantly of the polymeric type. To prove that this IgA represented disulfide bond-linked polymers rather than aggregated monomers, we have demonstrated that the high molecular weight IgA (a) maintains its polymeric form upon treatment with acidic buffers, (b) contains J chain, a glycoprotein associated only with polymeric immunoglobulins, and (c) dissociates to the monomeric form upon reduction of disulfide bonds. After 1 wk in culture, approximately 60% of the PWM-stimulated cells that contained IgA were positive for IgA2, whereas 40% were IgA1 positive as determined by immunofluorescence. Therefore, peripheral blood contains a population of lymphocytes with the potential to display, after appropriate stimulation and differentiation, characteristics similar to IgA cells found in external secretory tissues. The demonstration of the presence of such cells in the peripheral circulation suggests that these cells are precursors of IgA-producing plasma cells with the potential to populate mucosal tissues.

Mitogen-induced plasma cell differentiation in patients with multiple sclerosis.

Cellular interactions involved in mitogen-stimulated plasma cell differentiation were investigated in eight patients with severe but stable multiple sclerosis (MS). No significant differences were detected between normals and MS patients with regard to percent of B lymphocytes and T lymphocytes in peripheral blood. Autologous and allogeneic combinations of normal and MS B and T cells were stimulated with pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS), and plasma cell differentiation was monitored after 7 days in culture. T lymphocytes from patients with MS induced 2- to 4-fold increases in plasma cell development when combined with normal B cell fractions. Allogeneic combinations on normal B and T cells did not provide enhanced plasma cell generation. B lymphocytes from MS patients exhibited poor responses to both PWM and LPS when cultured with their own or normal T cells. Such B cell fractions did not differ from normals with regard to percent monocytes or surface Ig+ B lymphocytes initially contained in these cell populations. We conclude that T cells from MS patients are able to provide excessive help for normal B cell differentiation due either to increased T helper activity or deficient T suppressor activity. B cell differentiation may be diminished in MS patients as a result of a deficiency of a population of B cells in blood that are able to be stimulated by polyclonal B cell activators.

Amino-terminal sequence of a carcinoembryonic antigen-like glycoprotein isolated from the colonic lavages of healthy individuals.

A glycoprotein has been isolated from the colonic lavages of healthy individuals that is immunologically equivalent to carcinoembryonic antigen purified from tumor tissue. The NH2-terminal sequence of the glycoprotein from normal colon lavages is Lys-Leu-Thr-lle-Glu-Ser-Thr-Pro-Phe-(Asn)-Val-Ala-Glu-Gly-Lys-Glu-Val-(Leu,lle)-(Leu,lle)-(Leu,lle)-Val-(His,Arg?)-?-(Leu,lle). This is homologous to the NH2-terminal sequence of 23 of the first 24 amino acids of carcinoembryonic antigen isolated from tumor tissue.