Ann O. Shigeoka

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.

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.

X-Linked Syndrome of Polyendocrinopathy, Immune Dysfunction, and Diarrhea Maps to Xp11.23-Xq13.3

We describe genetic analysis of a large pedigree with an X-linked syndrome of polyendocrinopathy, immune dysfunction, and diarrhea (XPID), which frequently results in death during infancy or childhood. Linkage analysis mapped the XPID gene to a 17-cM interval defined by markers DXS8083 and DXS8107 on the X chromosome, at Xp11. 23-Xq13.3. The maximum LOD score was 3.99 (recombination fraction0) at DXS1235. Because this interval also harbors the gene for Wiskott-Aldrich syndrome (WAS), we investigated mutations in the WASP gene, as the molecular basis of XPID. Northern blot analysis detected the same relative amount and the same-sized WASP message in patients with XPID and in a control. Analysis of the WASP coding sequence, an alternate promoter, and an untranslated upstream first exon was carried out, and no mutations were found in patients with XPID. A C-->T transition within the alternate translation start site cosegregated with the XPID phenotype in this family; however, the same transition site was detected in a normal control male. We conclude that XPID maps to Xp11.23-Xq13.3 and that mutations of WASP are not associated with XPID.

Protective efficacy of a modified immune serum globulin in experimental group B streptococcal infection

In spite of aggressive antimicrobial therapy and extensive support measures, the mortality rate in early-onset group B streptococcal infection continues to be exceedingly high. In previous studies, we have demonstrated that passive immunotherapy with fresh whole blood containing opsonic antibody-improved survival in human neonates with group B disease. Transfusion of whole blood, plasma, or other blood products has a number of drawbacks, however. In the present study, we have evaluated immune serum globulin and a preparation of ISG modified for intravenous use for levels of type-specific antibody, opsonic activity, and protective efficacy against type Ia, II, and III group B streptococci. Type-specific antibody was detected in most of the preparations tested. In general, the level in MISG was less than that in the comparison ISG lot. Opsonic activity was also detected in these preparations against the more antibody-sensitive group B strains but was not present for opsonin resistant strains of type Ia, II, and III. Both ISG and MISG provided protection in neonatal rats infected with group B streptococci; in most cases MISG was more efficacious than the ISG from which it was made. These studies suggest that passive immunotherapy with MISG may be a valuable adjunct to current regimens used in the management of early-onset group B disease. This would be especially so if donors could be selected whose serum or plasma contained high levels of opsonic and protective activity against both antibody-sensitive and antibody-resistant group B strains.

Circulating and Storage Neutrophil Changes in Experimental Type II Group B Streptococcal Sepsis

Summary: 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.Speculation: Although neutrophil storage pool depletion is rare in infected adult animals or humans, its presence can be correlated with an increased mortality. It appears that neutrophil storage pool depletion is a prominent feature of experimental neonatal type II group B streptococci infection. The resulting deficiency of phagocytic cell supply probably represents diminution in the already compromised host defense mechanism of the neonate. Thus, it is postulated that neutrophil storage pool depletion represents a pathophysiologic factor contributing to the high mortality rate observed in neonatal type II group B streptococci infection.

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.

Functional leukocyte administration in protection against experimental neonatal infection.

Summary: 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 unpaired 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 ±5 ± 105 streptococci, animals received separate IP injections of saline, serum lacking opsonic antibody (Ab negative), Ab positive serum or washed adult human PMNs (2 ± 106). 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 ≤0.001). Human serum containing opsonic antibody also provided significant protection against mortality in this model (survival rate 51%, P ≤0.001). Leukocytes from normal term neonates, stressed neonates, or ones pretreated with cytochalasin B offered less protection than did functional adult human cell (P ≤0.001).Speculation: This study provides evidence for polymorphonuclear leukocyte (PMN) dysfunction as another risk factor in group B streptococcal disease and suggests that fully functional PMNs are required for protection. Furthermore, this model involving the administration of human PMNs to an animal appears to have the potential for the in viw evaluation of PMN function in neonates and other compromised hosts.

Nocardia pneumonia in a neonate with chronic granulomatous disease.

We present an unusual case of 1-month-old male infant with Nocardia asteroides pneumonia that led to the diagnosis of chronic granulomatous disease (CGD). This is one of the earliest presentations of Nocardia reported in the literature and illustrates the importance of investigating immunodeficiency in such patients

Evaluation of group B streptococcal opsonins by radiolabeled bacterial uptake

Abstract The incidence of neonatal group B streptococcal sepsis and meningitis has increased in the last decade. Antibiotic therapy has proved inadequate in controlling this problem, and in developing immunological methods of disease prevention and control it is important to define the critical bacterial and host factors involved in the immune response. We have used a radiolabeled bacterial uptake method to assess the presence of functional opsonins for a strain of type III group B streptococci. Bacteria, labeled with tritiated leucine, were opsonized in fresh, heated or Mg-EGTA treated serum. The opsonized streptococci were added to monolayers of adult human phagocytes 90% adherent to glass coverslips. At 10-min intervals for 60 min the coverslips were washed free of uningested bacteria and placed in a scintillation counter to determine the degree of phagocytosis. Maximum opsonic activity was obtained with specific antibody containing serum. No uptake of unopsonized bacteria was observed. Markedly reduced activity with heated and Mg-EGTA treated serum indicated a major role for the classical complement pathway in host defense against group B streptococci. Serum lacking specific antibody demonstrated almost no uptake indicating a minor role for the alternative pathway in opsonizing these organisms. Reduced levels of phagocytosis were also measured in every case where sera from patients with known type III infections were used for opsonization. These results agree with previous data from our laboratory using a chemiluminescence assay of opsonic activity.

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.