Nancy H. Augustine

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs(∗)7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853(∗)), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.

Effects of Group B Streptococci on Cord and Adult Mononuclear Cell Interleukin-12 and Interferon-γ mRNA Accumulation and Protein Secretion

Group B streptococci (GBS) are a major cause of early-onset infection in neonates. Neonates, who have defects in neutrophil function that likely contribute to susceptibility to GBS infection, are deficient in the production of the phagocyte activator interferon (IFN)-gamma. GBS-stimulated mRNA accumulation and protein secretion of IFN-gamma and interleukin (IL)-12, a major enhancer of IFN-gamma production, by mixed mononuclear cells (MMCs) from umbilical cord and adult peripheral blood was examined. GBS-exposed cord blood MMCs secreted lower concentrations of both IL-12 and IFN-gamma proteins than did MMCs from adults. IL-12 and IFN-gamma mRNA accumulation was examined by use of comparative reverse transcriptase-polymerase chain reaction. Cord blood MMCs accumulated less mRNA for both IL-12 and IFN-gamma than did adult blood MMC. The deficiency in cord blood cell production of IL-12 may have a role in inadequate IFN-gamma production, which contributes to the unique susceptibility of neonates to GBS infections.

Defective monocyte chemotactic responses in diabetes mellitus

A variety of polymorphonuclear leukocyte (PMN) defects has been described in diabetics, but to date no investigations of monocyte (MN) function have been reported in diabetics. In the present studies, we have employed an underagarose chemotactic assay to examine directed and random motility of MNs from diabetic individuals. Among 45 diabetics and 25 controls there was no significant difference in the maximal distance migrated by the leading front of cells to zymosan-activated serum (chemotaxis) or medium alone (random migration). In contrast, many fewer diabetic MNs [293±28 (SE)] moved toward the attractant than did control MNs (446±21;P<0.001). Random motility of the diabetic MNs (121±14 SE) was also decreased compared to that of the controls (225±24;P<0.0004).In vitro incubation of the diabetic monocytes with insulin (25, 75, and 100 µU/ml) failed to improve chemotactic function. Treatment of diabetic MNs with 2,3-dihydroxybenzoic acid, a scavenger of H2O2 and O2−, increased both random motility and directed movement, however. In addition,in vivo therapy of 7 diabetic patients with α-tocopherol (vitamin E) resulted in a marked increase in the number of MNs moving toward the chemoattractant (pretherapy, 118±12; posttherapy, 246±31;P<0.05). These results indicate that diabetic individuals have defective monocyte chemotactic function which can be corrected, in part, byin vitro orin vivo treatment with antioxidants. Thus, defective function may be, at least partly, the result of autooxidative membrane damage.

Intracellular and Extracellular Cytokine Production by Human Mixed Mononuclear Cells in Response to Group B Streptococci

ABSTRACT Group B streptococci (GBS) are a major cause of severe infection in newborns, pregnant females, and other immunocompromised hosts. Infection often includes septicemia, shock, pneumonia, and respiratory failure. In previous studies, we have reported that GBS induce marked production of tumor necrosis factor alpha (TNF-α) by human mononuclear cells. The present study was designed to measure the production of TNF-α as well as additional cytokines, including interleukin 1β (IL-1β), IL-6, IL-8, IL-12, and gamma interferon (IFN-γ) but also to determine from what cells and at what time point during incubation with GBS that these cytokines are produced. Mixed mononuclear cells were incubated with heat-killed GBS, media alone, or 1 μg of Escherichia coli lipopolysaccharide (LPS). Brefeldin A was added to each sample prior to staining, which prevented the export of cytokines by the Golgi apparatus. The cells were then stained with the appropriate conjugated antibodies and analyzed by using a flow cytometer. Results indicate that intracellular cytokines appear, in almost all cases, simultaneous to or before secreted proteins are detected. In contrast to the response to LPS, where TNF-α, IL-1β, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to GBS results in the production of TNF-α but delayed appearance of IL-1β, IL-6, and IL-8. The lymphocyte response to GBS was also strikingly different from that to LPS in that both secreted IFN-γ and IL-12 was detected, while LPS failed to induce production of these critical cytokines. This suggests an important role for TNF-α, IFN-γ, and IL-12 in GBS pathogenesis and/or immunity.

Regulation of decidual cell chemokine production by group B streptococci and purified bacterial cell wall components.

OBJECTIVE Our purpose was to determine whether cultured human decidual cells produce chemokines in response to different strains of group B streptococci and purified bacterial cell wall components. STUDY DESIGN Human decidual cells were cultured from term placentas by standard techniques. Different strains of group B streptococci were isolated from neonates with early-onset group B streptococci sepsis. Confluent cell monolayers were incubated with these different strains of group B streptococci and various concentrations of purified bacterial cell wall components (including lipoteichoic acid, sialic acid, lipopolysaccharide, and lipid A) for 16 hours at 37 degrees C. Culture supernatants were collected and assayed for macrophage inflammatory protein-1 alpha and interleukin-8. Statistical analysis was by analysis of variance. RESULTS We found that cultured human decidual cells produced significant amounts of the two chemokines macrophage inflammatory protein-1 alpha and interleukin-8 in a strain-specific fashion to the various different strains of group B streptococci tested, from 215% to 421% over baseline production (p < 0.05 by analysis of variance). Also, we found that incubation of decidual cells with various concentrations of lipoteichoic acid, sialic acid, lipopolysaccharide, and lipid A resulted in significant concentration-dependent increases in decidual cell macrophage inflammatory protein-1 alpha and interleukin-8 production (p < 0.05.) CONCLUSIONS Decidual cells produced significant amounts of the chemokines macrophage inflammatory protein-1 alpha and interleukin-8 in response to intact group B streptococci in a strain-specific fashion and in response to various concentrations of different bacterial cell wall components. Because chemokines are important mediators signaling migration of different immune effector cells into areas of inflammation, we suggest that decidual cell chemokine production in response to bacteria and bacterial cell wall components may be a key early event in the pathogenesis of infection-associated preterm labor.

Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Identification of Patients with RAG Mutations Previously Diagnosed with Common Variable Immunodeficiency Disorders

PurposeCombined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients.MethodsTwo different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients.ResultsTwo young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection.ConclusionAstute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.

Comprehensive analysis of antibody responses to streptococcal and tissue antigens in patients with acute rheumatic fever

Acute rheumatic fever (ARF) is an autoimmune disease occurring in individuals following untreated group A streptococcal infection believed to be triggered by antibodies to bacterial components that cross-react with human tissues. We developed a multiplexed immunoassay for the simultaneous quantitation of antibodies to nine streptococcal-related antigens including streptolysin O (SLO), DNase B, collagen I and IV, fibronectin, myosin, group A carbohydrate, M6 protein and streptococcal C5a peptidase. Utilizing this method, we examined serum from 49 ARF, 58 pharyngitis patients and age- and sex-matched controls in samples collected at initial disease onset, and at 4 weeks, 6 months and 1 year after diagnosis. Antibody responses were significantly higher for SLO, DNase B, M6 protein, group A carbohydrate and the cross-reactive antigens collagen I and myosin in ARF compared with pharyngitis patients (P <or= 0.05). Moreover, we found significantly elevated antibody responses in the ARF patients with rheumatic heart disease to fibronectin and collagen I compared with ARF patients without heart disease. The major differences between the ARF patients with and without carditis appear to be in the immune response to the putative heart valve components, collagen I and fibronectin.

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.

Diffuse Large B Cell Lymphoma in Hyper-IgE Syndrome Due To STAT3 Mutation

The Job or hyper-immunoglobulinemia E syndrome is a primary immunodeficiency that is usually inherited in an autosomal dominant fashion. With the discovery of mutations in the STAT3 gene in the majority of autosomal dominant cases, it is now possible to make a molecular diagnosis of hyper-IgE syndrome. Both primary and secondary immunodeficiencies, including hyper-IgE syndrome, may predispose for malignancies, especially lymphomas, mainly mature B cell lymphomas, and classical Hodgkin lymphoma. Here, we report of a 48-year-old male with hyper-IgE syndrome who developed a primary parotid gland diffuse large B cell lymphoma. Analysis for STAT3 mutations demonstrated that the causal mutation of hyper-IgE syndrome, R382Q, arose de novo in the patient and it was transmitted to three of his five children, all three of whom are clinically affected. We review the literature regarding lymphoma in hyper-IgE syndrome and the possible etiologic relationship with STAT3 mutations.