Fibronectin enhances the opsonic and protective activity of monoclonal and polyclonal antibody against group B streptococci.

We have investigated the opsonic and protective effects of fibronectin (FN) against type III group B streptococci. When used by itself, the FN failed to promote actual internalization of group B organisms. The addition of FN to group B streptococci that had been preopsonized in an immunoglobulin preparation modified for intravenous use ( IgIV ) or a type-specific, murine monoclonal antibody of IgG isotype markedly enhanced interaction with human polymorphonuclear leukocytes (PMN). A similar enhanced effect was observed when the FN was combined with type-specific monoclonal antibody preparations of IgM and, surprisingly, IgA isotype. Preincubation experiments indicated that the major effect was upon the PMN rather than directly on the bacteria, but we could not demonstrate an effect of FN on cell surface receptors for the Fc fragment of Ig or C3b using rosetting techniques. In addition to enhancing the in vitro opsonic activity of Ig, the FN significantly increased the protective effect of the polyclonal and monoclonal Ig preparations in an animal model of neonatal group B streptococcal disease. Thus, FN appears to have a critical role in the host defense mechanisms against group B streptococci.

Assessment of group B streptococcal opsonins in human and rabbit serum by neutrophil chemiluminescence.

The factors important in host defense against group B streptococci are not well understood. The role of antibody and complement in the prevention of serious infection by these organisms is not known because, to date, a reliable measure of functional opsonic activity has not been developed. Recently, it has been shown that neutrophils produce a chemiluminescence after ingestion of particulate matter, and that this event can be detected and quantitated in a liquid scintillation system. We have adapted the chemiluminescence procedure to examine rabbit hyperimmune and human serum for the presence of group B streptococcal opsonins. Group B streptococci of types Ia, II, and III that were opsonized in homologous but not heterologous type serum produced a peak in chemiluminescence when added to normal human neutrophils. Such activity was correlated, in each instance, with ingestion of bacteria by neutrophils and deposition of immunoglobulin and C3 on the bacterial surface as detected by indirect immunofluorescence. With this assay, we have examined sera from colonized and diseased patients for the presence of opsonins to types Ia, II, and III group B streptococci. Maternal sera often contained type-specific opsonins which resided in the IgG fraction and which crossed the placenta to appear in paired cord specimens. 63% of patients colonized with group B streptococci had serum opsonins to their colonizing type of organism. In contrast, none of the 15 patients with sepsis or meningitis had opsonins directed against their infecting strain. These data suggest that the lack of type-specific opsonins to group B streptococci may be one of the important factors in determining host susceptibility to systemic infection with strains of this group.

A single strand conformation polymorphism study of CD40 ligand. Efficient mutation analysis and carrier detection for X-linked hyper IgM syndrome.

Mutations in the gene for CD40 ligand are responsible for the X-linked form of hyper IgM syndrome. However, no clinical or laboratory findings that reliably distinguish X-linked disease from other forms of hyper IgM syndrome have been reported, nor are there tests available that can be used to confidently provide carrier detection. To identify efficiently mutations in the gene for CD40 ligand, eight pairs of PCR primers that could be used to screen genomic DNA by single strand conformation polymorphism (SSCP) were designed. 11 different mutations were found in DNA from all 13 patients whose activated T cells failed to bind a recombinant CD40 construct. The exact nature of four of these mutations, a deletion and three splice defects, could not be determined by cDNA sequencing. In addition, SSCP analysis permitted rapid carrier detection in two families in whom the source of the mutation was most likely a male with gonadal chimerism who passed the disorder on to some but not all of his daughters. These studies document the utility of SSCP analysis for both mutation detection and carrier detection in X-linked hyper IgM syndrome.

Comparative opsonic activity of intravenous gamma globulin preparations for common bacterial pathogens.

Immune globulin intravenous is a reduced and alkylated preparation of gamma globulin that is stabilized in 10 percent maltose and 0.1 M glycine at pH 6.8. Recently, a modified immune globulin intravenous preparation was developed that is identical to the standard preparation except that it does not contain glycine and the pH has been lowered to 5.25. The effect of these modifications has resulted in a higher IgG monomer content in the preparation. In the present studies the opsonic activity against several common bacterial pathogens was assessed in the standard (pH 6.8) versus the more acidic immune globulin intravenous (pH 5.25). Opsonic activity was detected in each preparation for Staphylococcus aureus, group B streptococci, Pseudomonas aeruginosa, Escherichia coli, and Serratia marcescens. With all of the organisms except S. marcescens, an intact complement system was required for optimal uptake and killing with each preparation. In general, the opsonic activity of the pH 5.25 immune globulin intravenous was equivalent to the standard pH 6.8 preparation. With several organisms, however, the more acidic preparation had greater activity than the standard one. An immune globulin intravenous preparation with increased antibody titers to P. aeruginosa was also prepared from selected donors and tested for opsonic activity against six of the seven Pseudomonas immunotypes. This preparation was found to have strikingly increased opsonic titers for most of the Pseudomonas immunotypes when compared with the standard immune globulin intravenous. These studies indicate that changes in donor selection or minor modifications in production techniques may markedly affect the biologic activity of intravenous gamma globulin.

Di George anomaly and velocardiofacial syndrome.

The velocardiofacial syndrome is an autosomal dominant disorder characterized by cleft palate, cardiac anomalies, characteristic facies, and learning disabilities. The Di George anomaly involves developmental defects of the third and fourth pharyngeal pouches, resulting in thymic and parathyroid hypoplasia and cardiac defects. The cases of individuals in two families help substantiate the notion that the Di George anomaly occurs as a feature of the velocardiofacial syndrome. The proband in family 1 was a male infant with persistent hypocalcemia and cardiac defects consisting of truncus arteriosus, atrial septal defect, ventricular septal defect, and abnormal aortic arch vessels. Autopsy revealed absence of thymic and parathyroid tissue, and the Di George anomaly was diagnosed. His father had a submucous cleft palate, T cell dysfunction, and facial features consistent with the velocardiofacial syndrome. This is the third case of male-to-male transmission of velocardiofacial syndrome. The proband of family 2 was a 4-year-old girl with developmental delay, persistent neonatal hypocalcemia, ventricular septal defect, T cell dysfunction, and facial features of the velocardiofacial syndrome. The Di George anomaly has been reported to occur in at least 18 different disorders. The observation that the Di George anomaly is a component manifestation of the velocardiofacial syndrome in these two families provides further evidence that the Di George anomaly is not a distinct syndrome of a single origin but rather a heterogeneous developmental field defect. It is proposed that all previously reported cases of autosomal dominant Di George anomaly are examples of the velocardiofacial syndrome.

Neonatal cellular and humoral immunity to group B streptococci.

The mechanisms of host resistance to group B streptococci have not been defined precisely. In the studies reported here we have assessed the contributions of both humoral and cellular factors in protection against strains of this group. With assays of specific opsonic activity based upon the production of polymorphonuclear leukocyte chemiluminescence and radiolabeled bacterial uptake, we have demonstrated that specific heat-stable antibody and the classic complement pathway are major factors in opsonization of these organisms. In the absence of specific antibody, fresh serum resulted in markedly reduced bacterial uptake indicating, at best, a minor role for the alternative complement pathway. Additional studies have indicated that strain-specific antiphagocytic factors as well as type-specific ones may play a role in the virulence of these organisms. Neonates who developed group B streptococcal sepsis usually lacked opsonic activity in their infecting strain. In addition, polymorphonuclear leukocytes from normal term and stressed neonates showed impaired metabolic activation as measured in the chemiluminescence assay following exposure to opsonized group B streptococci. These results suggest that neonates who develop group B streptococcal disease may have defects in both the humoral and cellular aspects of their acute inflammatory response which may contribute to the high mortality observed in this most fulminant of bacterial infections.

Growth failure, intracranial calcifications, acquired pancytopenia, and unusual humoral immunodeficiency: a genetic syndrome?

We report on two children who may represent a novel syndrome consisting of a deficiency of immunoglobulin-bearing B lymphocytes and serum antibody, deficient intrauterine and/or postnatal growth, intracranial calcifications, and acquired pancytopenia. Poor growth, intracranial calcifications, developmental delay, and hematological abnormalities are common manifestations of congenital infection. However, humoral immunodeficiency is not characteristic in these infections, and no infection was found on extensive evaluation. Rare genetic syndromes may mimic intrauterine infections and may also include immunodeficiency. However the children reported here lack important characteristics or share distinctive manifestations not described in these disorders. Infants presenting with apparent congenital infections in whom a specific infectious cause cannot be identified should be followed carefully with immunological evaluations since this disorder may be progressive and considerable morbidity is attributable to hematological and immunological manifestations.

Recurrent Pseudomonas infection associated with neutrophil dysfunction.

A 72-year-old male is described with a history of 4 episodes of Pseudomonas aeruginosa sepsis and chronic otitis media caused by pseudomonas species. In vitro testing of the patient's polymorphonuclear leukocytes (PMNs) revealed profoundly abnormal chemotactic responses and defective intracellular killing of Ps. aeruginosa, Staphylococcus aureus and Escherichia coli. Chemiluminescence production by the patient's PMNs in response to opsonized zymosan as well as endotoxin stimulated nitroblue tetrazolium dye reduction were markedly depressed. These data indicate the presence of a profound, apparently acquired, defect in PMN function in an elderly male. Detailed evaluation of adult patients with recurrent infections may reveal similar, apparently acquired defects in PMN function.

Strain specificity of opsonins for group B streptococci types II and III.

Strains of types II and III group B streptococci do not appear to be uniformly susceptible to opsonization by antibody-containing human sera, as studied using both a chemiluminescence and a radiolabeled bacterial uptake technique. We could not demonstrate a correlation of serum-sensitive or resistant strains with capsular antigen quantities, although serum absorption studies with whole organisms and HCl, trichloroacetic acid, and saline extracts indicated that the antibody to type-specific capsular polysaccharide is important in opsonizing both serum-resistant and serum-sensitive strains. Since trypsin treatment produced significantly enhanced opsonization of serum-resistant and serum-sensitive strains, proteins present on some group B streptococci may be important antiphagocytic factors.

Functional analysis of neutrophil granulocytes from healthy, infected, and stressed neonates.

Neutrophil granulocyte function was assessed in 17 well term infants, 14 stressed infants, and eight infants with group B streptococcal infection. Chemiluminescence production elicited by opsonized zymosan or by a wild strain of type III group B streptococci, as well as phagocytosis and killing of streptococci, were measured. Chemiluminescence production by PMNs of term neonates in response to opsonized zymosan or group B streptococci was equal to that of adult controls. In contrast, six of nine stressed or infected neonates had depressed CL responses upon zymosan challenge. When opsonized type III group B streptococci were used to elicit CL, seven of ten stressed or infected infants had markedly depressed responses. Phagocytosis, as determined by a radiolabeled bacterial uptake technique, was normal in the healthy and stressed neonates. Depressed CL production by the PMNs of stressed or infected neonates was associated with impaired intracellular bactericidal activity, however. These studies indicate that stressed or infected neonates have impaired leukocyte metabolic activation that may be associated with depressed bactericidal activity. Such impairment may contribute to the morbidity and mortality observed in serious neonatal infections.

Functional leukocyte administration in protection against experimental neonatal infection.

Studies have shown that human neonates who develop group B streptococcal sepsis usually lack opsonic antibody (Ab) to their infecting strain and that these neonates may also have impaired polymorphonuclear leukocyte (PMN) function. The present study was designed to determine the efficacy of administration of PMNs or opsonic Ab-containing serum in protecting against group B streptococcal infection in a newborn rat model. After intraperitoneal (IP) injection of congruent to 5 X 10(6) streptococci, animals received separate IP injections of saline, serum lacking opsonic antibody (Ab negative), Ab positive serum or washed adult human PMNs (2 X 10(6)). The mortality rate in 55 neonatal rats infected with group B streptococci who received saline or Ab negative serum was 91%. In contrast, 40 animals who received adult human PMNs at the time of inoculation had a survival rate of 50% (P less than 0.001). Human serum containing opsonic antibody also provided significant protection against mortality in this model (survival rate 51%, P less than 0.001). Leukocytes from normal term neonates, stressed neonates, or ones pretreated with cytochalasin B offered les protection than did functional adult human cell (P less than 0.001).

Serum opsonic activity and peripheral neutrophil counts before and after exchange transfusion in infants with early onset group B streptococcal septicemia.

Serum opsonic antibody activity and peripheral neutrophil counts were measured in 41 infants who received an exchange transfusion of fresh whole blood for early onset group B streptococcal septicemia. Immature and mature neutrophils increased significantly in the peripheral blood post-exchange transfusion among survivors. Increases in opsonic antibody levels did not reach statistical significance following exchange transfusion. It is possible that exchange transfusion with fresh whole blood may enhance mobilization of neutrophils in infants with adequate bone marrow storage pools. A prospective controlled study is required to evaluate properly the role of exchange transfusion in infants with group B streptococcal sepsis.

Blood-transfusion in group-B streptococcal sepsis.

Transfusion of fresh whole blood was evaluated as a means of supplying opsonins and lessening the high mortality of group-B streptococcal sepsis in neonates. Pre-transfusion and post-transfusion sera from 22 infants were examined for the presence of opsonins against group-B organisms. Opsonic activity rose only when donor blood containing heat-stable antibody was administered in high volume (greater than 40% of blood-volume). 9 of 9 infants transfused with blood containing antibody to their infecting strain survived septic episodes. 3 of 6 who received blood lacking antibody to their infecting strain died.

Neutrophil mobilization induced by complement fragments during experimental group B streptococcal (GBS) infection.

Degradation products of the third component of complement have been reported to have the ability to mobilize leukocytes from the marrow and induce leukocytosis. The effect of C3d,g preparations on neutrophil responses in a neonatal rat model of group B streptococcal infection in which neutrophil mobilization from the marrow is inadequate has been evaluated. Dimeric and monomeric fragments of C3d,g were isolated from human serum; the identity of the C3d,g preparations was confirmed by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing. Uninfected neonatal rats responded to intraperitoneal injection of C3d,g with a peripheral blood neutrophilia at 30 minutes and 4 hours after inoculation. C3d,g, which lacks intrinsic chemotactic activity, enhanced the local accumulation of neutrophils in the peritoneal cavity of infected, but not uninfected, neonatal rats. In addition, myeloid cell release from the marrow of isolated femurs of neonatal rats receiving C3d,g was significantly enhanced. Thus, the effect of C3d,g in this model was to mobilize marrow cells and induce peripheral leukocytosis. Chemotactic factors released at the site of infection then resulted in the local accumulation of these inflammatory cells. Complement-derived components capable of releasing marrow myeloid elements may play a major role in determining the outcome of bacterial infection in the immature host.

Absolute requirement for complement in monoclonal IgM antibody-mediated protection against experimental infection with type III group B streptococci.

The role of complement in the protective and opsonic activity of monoclonal IgM antibody to type III group B streptococci (GBS) was examined in a neonatal rat model of infection and in vitro with human sera as the complement source. C3 levels in uninfected neonatal rats were less than 50% of those in adult rats, similar to the low complement levels observed in human neonates. The monoclonal type III-specific IgM antibody provided protection to neonatal rats (with unaltered complement levels) that were infected intra-peritoneally or intranasally with type III GBS. In contrast, neonatal rats depleted of complement by administration of cobra venom factor were not protected by IgM antibody. In vitro, classical complement pathway activity was adequate in sera from well, term neonates and GBS-infected neonates in the presence of higher concentrations of the monoclonal IgM antibody. At lower IgM levels, however, the alternative complement pathway was less efficient in both neonatal sera and adult sera.

Protective efficacy of hybridoma type-specific antibody against experimental infection with group-B Streptococcus.

Murine monoclonal antibodies to group-B Streptococcus type III were developed by fusion of splenic lymphocytes from immunized BALB/c mice with the mouse-myeloma cell line SP 2/0. The four type III-specific antibodies, which were of the IgM class, protected neonatal rats against intraperitoneal infection with homologous-type group-B streptococci; survival rates were 95%-100% for protected rats but only 17% for unprotected rats. One antibody preparation offered excellent protection against any of five type-III strains employed. Monoclonal antibody provided protection when administered 4, 8, or 12 hr after infection, although delayed administration was less efficacious against more virulent strains. Monoclonal IgM also protected against intranasal inoculation of bacteria. These results indicate the potential therapeutic efficacy of monoclonal antibody in neonatal group-B streptococcal disease, but further laboratory investigations must precede clinical investigation of monoclonal or polyclonal antibody therapy in humans.

Circulating and storage neutrophil changes in experimental type II group B streptococcal sepsis.

The availibility of neutrophils is an important factor in host resistance to bacterial infection. Therefore, circulating and storage neutrophil quantification was carried out on groups of neonatal rats intranasally inoculated with type II group B streptococci. The dose of type II group B streptococci used produced 56% mortality in 48 neonatal rats with death being due to pneumonia and sepsis. Neutropenia (1300 +/- 150/mm3 versus 2300 +/- 170/mm3; mean +/- S.D.; P < 0.01) and an elevation in band/polymorphonuclear ratio (0.98 +/- 0.04 versus 0.30 +/- 0.04; P < 0.01) were observed in infected neonatal rats 24 hr following inoculation. Femoral marrow as well as splenic and hepatic neutrophil storage compartment quantification revealed dimunition of postmitotic (polymorphonuclear, band, and metamyelocyte) neutrophils in the infected group (P < 0.01) with sparing of the proliferative neutrophils (myeloblasts, promyelocytes, and myelocytes). Repletion of the myeloid but not the splenic or hepatic neutrophil storage compartments with normalization of the neutrophil count and band/polymorphonuclear ratio occurred in animals surviving 72 hr. These studies establish that neutropenia and neutrophil storage pool depletion are prominent features of experimental type II group B streptococci infection in neonates.