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R A Insel

Publications and source records attributed to R A Insel.

At least 37 records · Page 2Linked to original sources
PubMed1994

Human herpesvirus-6 infection in children. A prospective study of complications and reactivation.

BACKGROUND: Infection with human herpesvirus-6 (HHV-6) is nearly universal in infancy or early childhood. However, the course of this infection, its complications, and its potential for persistence or reactivation remain unclear. METHODS: We studied infants and children under the age of three years who presented to our emergency department with acute illnesses. Infants and young children without acute illness were studied as controls. HHV-6 infection was identified by blood-mononuclear-cell culture, serologic testing, and the polymerase chain reaction (PCR). RESULTS: No primary HHV-6 infection was found among 582 infants and young children with acute nonfebrile illnesses or among 352 controls without acute illness. Of 1653 infants and young children with acute febrile illnesses, 160 (9.7 percent) had primary HHV-6 infection, as documented by viremia and seroconversion. They ranged in age from 2 weeks to 25 months; 23 percent were under the age of 6 months. HHV-6 infections accounted for 20 percent of 365 visits to the emergency department for febrile illnesses among children 6 to 12 months old. Of the 160 infants and young children with acute HHV-6 infections, 21 (13 percent) were hospitalized, and 21 had seizures. Often the seizures appeared late and were prolonged or recurrent. HHV-6 infections accounted for one third of all febrile seizures in children up to the age of two years. In follow-up studies over a period of one to two years, the HHV-6 genome persisted in blood mononuclear cells after primary infection in 37 of 56 children (66 percent). Reactivation, sometimes with febrile illnesses, was suggested by subsequent increases in antibody titers in 16 percent (30 of 187) and by PCR in 6 percent (17 of 278). No recurrent viremia was detected. Of 41 healthy newborns studied, 12 (29 percent) had the HHV-6 genome in their blood mononuclear cells; nevertheless, 6 of these newborns subsequently had primary HHV-6 infections. CONCLUSIONS: In infants and young children HHV-6 infection is a major cause of visits to the emergency department, febrile seizures, and hospitalizations. Perinatal transmission may occur, with possible asymptomatic, transient, or persistent neonatal infection.

Acute Disease↗
PubMed1994

Human monoclonal lambda light chain protein exhibits specific binding to the variable region of monoclonal anti-IgE antibody.

A monoclonal heterohybridoma cell line (B11-12) was developed by fusing EBV-transformed lymphocytes from a patient with hyper-IgE syndrome and SHM-D33 heteromyeloma cells. The resultant heterohybridoma secreted only human lambda L chains exhibiting specific binding to mouse monoclonal Ab to human IgE (mAb-algE). B11-12 bound to four separate mAb-algE, but not to mAb of the same isotypes directed to other antigens. The mAb-algE differed in isotype (IgG1; IgG2b) demonstrating that B11-12 was recognizing an idiotypic, but not an isotypic determinant on the mAb-algE. Human IgE did not inhibit B11-12 binding to mAb-algE, suggesting the interaction between B11-12 and mAb-algE involved an idiotope outside the combining site of the mAb-algE. Human sera, including serum from the donor of the lymphocytes used to produce heterohybridoma B11-12, demonstrated the capacity to inhibit B11-12 binding to mAb-algE. This, along with the inability of these same sera to directly bind B11-12, suggests that the serum contained reactivity similar to B11-12. We propose that B11-12 demonstrates specificity resembling an anti-Id to anti-human IgE Ab.

Animals↗
PubMed1994

Human splenic IgM immunoglobulin transcripts are mutated at high frequency.

Human spleen immunoglobulin gene rearrangements that used the VH6 gene and were expressed with IgM were characterized for their frequency of somatic hypermutation from PCR amplified spleen cDNA. A high frequency of rearrangements that were somatically mutated was demonstrated by restriction endonuclease analysis and sequencing of cloned rearrangements. The 24 rearrangements cloned from three different spleens had an overall mutation frequency of 3.1% mutations/bp sequenced and ranged from 0.4 to 6.0%. These mutations appeared to have been antigenically selected based on both the high frequency and high amino acid replacement to silent (R/S) ratios in the complementarity determining regions. Five clones that arose from two different rearrangements showed evidence of intraclonal diversification with both shared and unique mutations. The mutated clones of one spleen donor were lower in frequency and were not concentrated in the CDR, which suggested these mutations had not been antigenically selected. These findings support the dissociation of somatic mutation and isotype switching and the possibility that IgM-expressing B cells may serve as human memory B cells.

Amino Acid Sequence↗
PubMed1994

Maternal immunization to prevent infectious diseases in the neonate or infant.

The approach of providing passive protection to young infants by immunizing pregnant women can bypass the problems of immunological immaturity in the neonate, avoid or delay active immunization of the infant in the first year of life, and prevent transmission of an infection from the mother to the neonate. Optimal vaccines for this approach should induce high immunoglobulin G antibody titers that quickly reach their maximum level after immunization and persist at protective levels for several years, thus providing passive protection in subsequent pregnancies. Specific applications of this approach include the worldwide practice of maternal immunization with tetanus toxoid vaccine and ongoing studies of maternal immunization to prevent Haemophilus influenzae type b, group B streptococcal, pneumococcal, meningococcal, and human immunodeficiency virus infection in the infant. Addressing the cultural, sociological, and legal aspects of maternal immunization will be required to ensure the success of this approach.

Breast Feeding↗
PubMed1994

Bias in somatic hypermutation of human VH genes.

Translationally silent mutations, which are not antigen selected, of human VH6 Ig gene rearrangements isolated from human spleen were analyzed for bias to gain insight into intrinsic features of the mutation process. Sixty-three clones representing 38 VH6DJ rearrangements had an overall mutation frequency of 4.5%, a replacement/silent (R/S) mutation ratio of 2.1 and 167 unique silent mutations. The silent mutations showed bias in: (i) targeting to CDR1 and CDR2, (ii) an increased frequency of mutations of A compared to T nucleotide bases on the coding strand, and (iii) an increased frequency of transitions versus transversions. Bias of C-->G over C-->A, of G-->C over G-->T and of A-->C over A-->T transversions was also present. Hot spots of mutation were observed, some which corresponded to potential sites of stem-loop formation. The results suggest that the somatic mutation process in man may be targeted to the complementarity determining region for some V genes, exhibits specific base substitutions favoring transitions and specific types of transversions, and may be occurring on only one DNA strand.

B-Lymphocytes↗
PubMed1993

Use of the most JH-proximal human Ig H chain V region gene, VH6, in the expressed immune repertoire.

VH6, the most 3' human H chain V region gene, is preferentially expressed in the preimmune repertoire of the developing human fetus. To determine whether VH6 contributes to the immune repertoire, we amplified and sequenced from human spleen RNA VH6 rearrangements that were isotype-switched to IgG. Eighteen distinct in-frame VH6 clones were sequenced. Definitive assignment to C gamma 1 could be made in the majority of clones, and all had undergone extensive somatic hypermutation in the V region with an average of 14 (range, 2 to 25) mutations/281 bp in the VH6 segment. The overall frequency of somatic mutation was 5.2% of the total nucleotides sequenced. Replacement somatic mutations were targeted to the complementarity determining region, and the complementarity determining region had higher replacement to silent mutation ratios, consistent with antigenic selection. Silent somatic mutations were also significantly more frequent in the complementarity determining region compared to the framework region. Five clones were derived from the same VH6Dxp'1JH6 rearrangement, each containing unique and shared mutations. All DH segments used by independently arising clones were unique, and all DH reading frames were seen. N segments were lacking at either the 5'DH-JH junction or the 3'DH-JH junction in 7 of 13 independent rearrangements. DH-DH recombinations were frequently observed, and there was biased usage of JH3b and JH4b in the clones. The data imply that a high degree of diversity arises from use of this 3'-VH gene in the immune repertoire.

Amino Acid Sequence↗
PubMed1993

Severe combined immunodeficiency of reduced severity due to homozygosity for an adenosine deaminase missense mutation (Arg253Pro).

Genetic deficiency of adenosine deaminase (ADA) results in varying degrees of immunodeficiency, including neonatal onset severe combined immunodeficiency (ADA- SCID) and milder, later onset immunodeficiency. We have determined the molecular basis of disease in a child from a consanguineous mating with ADA- SCID of clinically and biochemically reduced severity, diagnosed at 15 months of age and characterized by retention of more immunologic function than is typical of the fulminant neonatal onset type. The course was notable for an early predominance of bacterial infections and eosinophilia. In contrast to its absence in most ADA- SCIDs, residual ADA activity (1-2% of normal) could be detected in EBV-transformed B cells. Consistent with the increased residual ADA, excretion of the substrate deoxyadenosine and accumulation of the toxic metabolite deoxyATP were less than seen in ADA- SCID patients with fulminant disease. Sequence analysis of cDNA revealed a G853C transversion, predicting a substitution of proline for arginine at codon 253 (Arg253Pro). The parents were heterozygous and the child was homozygous for the mutation, as shown by sequence analysis of amplified genomic DNA. Transient expression of mutant cDNA in Cos cells revealed an electrophoretically abnormal, more negatively charged ADA with 1-2% of normal activity. These observations are consistent with replacement of positively charged arginine by proline, the lower accumulation of toxic metabolites, and the milder phenotype. By contrast, transient expression of a Gly216Arg mutant cDNA, associated, when homozygous, with neonatal onset ADA-SCID, did not reveal ADA activity. Mutations such as Arg253Pro, which retain residual activity of monomeric ADA, should be dominant for ameliorating the phenotype in patients carrying two different allelic mutations. Identification of additional similar mutations may be significant in evaluating the goals for and efficacy of current trials of gene and gene product replacement.

Adenosine Deaminase↗
PubMed1993

Isolation of germinal centerlike events from human spleen RNA. Somatic hypermutation of a clonally related VH6DJH rearrangement expressed with IgM, IgG, and IgA.

12 rearranged human VH6 immunoglobulin heavy chain genes arising from the same rearrangement were isolated without preselection from the RNA of a fragment of human spleen. The 12 clones were isolated from a pool of 31 unique VH6 clones arising from 18 unique rearrangements. 2 of the 12 related clones were expressed with IgM, 2 with IgG, and 8 with IgA1. All the clones, including those expressing IgM, showed extensive somatic mutation of germline bases (5.6%), which was consistent with antigen-driven activation of these VH6-expressing clones with recruitment into the immune repertoire. On the basis of significant sharing of somatic mutations between the IgM clones and clones expressing the other isotypes (six mutations shared with IgG clones and eight mutations shared with IgA clones), it was apparent that the IgM-expressing precursor in this diversified family had undergone extensive antigen-driven somatic mutation prior to isotype switching. This family of related clones suggests that a germinal centerlike event had been sampled. The highly mutated IgM clones suggest that there may exist memory B cells capable of further somatic mutation and differential isotype-switching depending on the specific antigenic stimulus.

B-Lymphocytes↗
PubMed1993

Restricted immunoglobulin VH usage and VDJ combinations in the human response to Haemophilus influenzae type b capsular polysaccharide. Nucleotide sequences of monospecific anti-Haemophilus antibodies and polyspecific antibodies cross-reacting with self antigens.

To examine the human antibody repertoire generated against a biologically significant antigen we have obtained sequences of heavy chain variable region genes (IgVH) from 15 monoclonal antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). All VH segments are members of the VH3 family and 9 of 15 are members of the smaller VH3b subfamily. Restriction is evident by the shared use of certain VDJ joints in independent hybridomas from different subjects. Two hybridomas generated from the same subject demonstrate identical heavy chain variable region gene sequences but differ in isotype and rearrange alternative light chain variable region genes (IgVL), suggesting that in a normal immune response, a single pre-B cell clone may use different light chain rearrangements and give rise to progeny capable of reacting with antigen. Using a polymerase chain reaction assay optimized to detect base pair differences among VH genes we demonstrate that at least a portion of expressed anti-Hib PS VH genes have undergone somatic mutation. Anti-Hib PS heavy chain genes are homologous to VH segments encoding autoantibodies and two hybridomas secrete anti-Hib PS antibody that cross-reacts with self antigens (double-stranded DNA and single-stranded DNA). Comparison of VH regions of self-reactive and monospecific anti-Hib PS Ab demonstrates no consistent structural feature correlating with fine antigen specificity. These data demonstrate significant restriction in VH usage and VDJ recombination in the anti-Hib PS response and confirm that autoantibodies may be elicited during normal immune responses.

Amino Acid Sequence↗
PubMed1992

Primary human herpesvirus 6 infection in young children.

BACKGROUND: Human herpesvirus 6 (HHV-6) is a recently discovered virus that, on the basis of serologic evidence, appears to infect most children by the age of three years. However, the clinical manifestations of primary HHV-6 infection have not been well defined. METHODS: We studied consecutive children two years old or younger who presented to an emergency ward with febrile illnesses. Our evaluation included the isolation of HHV-6 from peripheral-blood mononuclear cells, an immunofluorescent-antibody assay, the detection of HHV-6 by the polymerase chain reaction (PCR), and restriction-endonuclease-fragment profiles of HHV-6 isolates. RESULTS: HHV-6 was isolated from 34 of 243 acutely ill children (14 percent). The children with viremia had irritability, high temperatures (mean, 39.7 degrees C), and inflammation of tympanic membranes (in 21), but few other localizing signs. Two children were hospitalized, but all 34 recovered after an average of four days of fever. The rash characteristic of roseola, which has been associated with HHV-6 infection, was noted in only three children. In 29 children (85 percent), serum samples obtained during convalescence had at least a fourfold increase in IgG antibody titers; 4 infants less than three months old who presumably had maternal antibody did not have this increase. HHV-6 was isolated from blood obtained during convalescence in only one child, but in two thirds of the children the virus could be detected by PCR. The isolates had genomic heterogeneity, indicating the presence of multiple strains. CONCLUSIONS: Primary infection with HHV-6 is a major cause of acute febrile illness in young children. Such infection is associated with varied clinical manifestations, viremia, and the frequent persistence of the viral genome in mononuclear cells.

Antibodies, Viral↗
PubMed1992

Immunoglobulin light chain variable region gene sequences for human antibodies to Haemophilus influenzae type b capsular polysaccharide are dominated by a limited number of V kappa and V lambda segments and VJ combinations.

The immune repertoire to Haemophilus influenzae type b capsular polysaccharide (Hib PS) appears to be dominated by certain light chain variable region genes (IgVL). In order to examine the molecular basis underlying light chain bias, IgVL genes have been cloned from a panel of heterohybridomas secreting human anti-Hib PS (antibody) (anti-Hib PS Ab). One hybridoma, representative of the predominant serum clonotype of anti-Hib PS Ab in older children and adults following immunization or Hib infection, uses a V kappa II segment identical to the germline gene A2, and a JK3 segment. A second kappa hybridoma uses a member of the V kappa I family and a JK4 segment. Four lambda antibodies, all cross-reactive with the structurally related antigen Escherichia coli K100 PS, use V lambda VII segments which are 96-98% homologous to one another, and may originate from a single germline gene. Two additional lambda antibodies, not K100-cross-reactive, are encoded by members of the V lambda II family. All lambda antibodies use highly homologous J lambda 2 or J lambda 3 segments. The VJ joints of all lambda antibodies and the V kappa II-encoded antibody are notable for the presence of an arginine codon, suggesting an important role in antigen binding. Although more complex than heavy chain variable region gene usage, a significant portion of serum anti-Hib PS Ab is likely to be encoded by a limited number of V kappa and V lambda segments and VJ combinations, which may be selectively expressed during development, or following antigen exposure.

Adult↗
PubMed1992

The repertoire of human antibody to the Haemophilus influenzae type b capsular polysaccharide.

Human antibody to the Haemophilus influenzae capsular polysaccharide (Hib CP) is restricted in diversity in the individual and the population with a limited number of variable region genes encoding antibody. Antibody to the Hib CP shows restricted isoelectric focusing gel patterns and light chain usage with frequent restriction to use of only kappa light chains. Shared cross-reactive idiotypes are expressed on antibody. The heavy chain of antibody to the Hib CP is predominantly encoded by two members of the VH3 family--LSG 6.1/M85-like and VH26/30P1-like. In VH the CDR1, based on complete identity in LSG 6.1/M85-like antibodies, CDR2, based on the suggestion of mutation in this region, and CDR3, based on conserved CDR3 usage in unrelated individuals, may be important for antigen binding. Six or more different VL gene families encode antibody. The predominant antibody of the majority of individuals uses the A2-V kappa II gene in germline or near germline configuration, which encodes an idiotype designated HibId-1. Antibody can also be encoded by V kappa I, non-A2 V kappa II, V kappa III, V kappa IV, V lambda II, and V lambda VII genes. Although different VL genes can be used, unrelated individuals appear to use the same V kappa III (A27), V lambda II (V lambda 2.1 and V lambda VII (4A) genes. The VL diversity accounts for differences in fine binding specificity, with A2-V kappa II genes not encoding E. coli K100 CP cross-reactive antibodies and V lambda VII genes and some of the non-A2 V kappa genes encoding cross-reactive antibodies. The arginine in CDR3 of both antibody kappa and lambda light chains and the asparagine in CDR2 of VL sequences and in CDR1 of LSG6.1-M85 VH sequences of antibody appear to be important residues for antigen binding. A relatively limited degree of somatic mutation has occurred in the non-A2 VL genes, V lambda VII, and the VH genes. Further studies comparing the polymorphism of germline V genes to antibody-encoding V genes are needed to clarify this issue. Research comparing this repertoire to repertoires directed to other bacterial CP and to self antigens and defining structure-antigen binding relationships is in progress.

Amino Acid Sequence↗
PubMed1990

Response to a Haemophilus influenzae type b diphtheria CRM197 conjugate vaccine in children with a defect of antibody production to Haemophilus influenzae type b polysaccharide.

A defect in antibody response to immunization with Haemophilus influenzae type b (Hib) capsular polysaccharide vaccine has been reported in children with recurrent infections and normal immunoglobin levels. We identified 15 children, aged 2 to 6 years, with this defect, and we evaluated their response to immunization with an Hib capsular oligosaccharide diphtheria CRM197 protein-conjugate vaccine (HbOC). The children received a series of three vaccines: HbOC at 0 and 8 weeks, and the Hib polysaccharide vaccine at 16 weeks. Levels of antibody to the Hib capsular polysaccharide (polyribosyl ribitol phosphate, PRP) and to diphtheria toxoid were obtained before and 4 weeks after each vaccination. The geometric mean serum anti-PRP concentration was 0.17 microgram/ml before immunization and 29.3 micrograms/ml after the second HbOC immunization (week 12). All 15 children had postvaccination anti-PRP antibody levels greater than 1.0 microgram/ml after receiving the second HbOC (week 12). In addition, booster responses were observed after the second Hib conjugate in 13 of the patients and after the Hib polysaccharide in four of the patients. All patients with low preimmunization diphtheria titers had a response to the diphtheria toxoid. These results suggest that conjugation of Hib polysaccharide with diphtheria CRM197 overcomes the defective antibody response to Hib oligosaccharide in children who are initially observed with recurrent infections and inability to respond to the Hib polysaccharide vaccine.

Antibodies, Bacterial↗
PubMed1990

Selective concentration of IgD class-specific antibodies in human milk.

The participation of human IgD class antibody in local immune responses of breast tissue was studied by analysing the sera-to-milk ratios of total IgD, IgM, IgA, IgG isotypes and albumin found in matched samples, and by analysing the sera-to-milk (S/M) ratios of IgD, IgM, IgA, IgG antibodies against Haemophilus influenzae capsular polysaccharide (PRP), phosphorylcholine, tetanus and in some cases diphtheria antigens. The study group consisted of eight women immunized during pregnancy with PRP, and control, unimmunized women. Albumin, and total IgG showed high S/M ratios. IgA had a low S/M ratio as expected, consistent with reports that IgA is locally concentrated. Total IgD and IgM isotype ratio values were intermediate between IgG and IgA suggesting they were selectively concentrated in breast fluids due to local production or transport mechanisms, or both. Ratios for specific antibodies of IgA and IgM isotypes and for total IgA and IgM isotype showed parallel data. Among the IgD antibodies, those specific for PRP and phosphorylcholine suggested a higher degree of selective concentration as compared with tetanus antigen. In the group of unimmunized women, although selective concentration of total IgD was observed, specific antibody studies were inconclusive due to the low milk IgD antibody levels encountered. The results indicate that IgD (and also IgM) may participate in local immune responses of human breast tissues and fluids; possibly influenced by the nature of the antigen, the state of immunization and the hormonal environment (pregnancy).

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

Effect of oligosaccharide chain length, exposed terminal group, and hapten loading on the antibody response of human adults and infants to vaccines consisting of Haemophilus influenzae type b capsular antigen unterminally coupled to the diphtheria protein CRM197.

Vaccines consisting of oligosaccharide (OS) derived from Haemophilus influenzae type b capsular polysaccharide and conjugated to carrier proteins had been shown capable of eliciting memory-type capsular polysaccharide of H. influenza type b antibody responses in human infants, but the structural variables governing immunogenicity were not defined. Here a series of conjugates were made with the diphtheria protein CRM197 and with uniterminally coupled OS haptens that varied in chain length, exposed terminal residue, or multiplicity of loading as defined by ribose/protein ratio. Adults were given a single injection, 1-yr-old infants were given a two-injection sequence, and capsular polysaccharide of H. influenzae type b antibody responses were assessed by radioantigen binding. Vaccines C-4r, C-6r, and C-12r, in which ribitol-ended OS of mean length 4, 6, or 12 repeat units were coupled at low hapten loading, were about equally immunogenic (geometric means 2 to 5 micrograms/ml in infants, 5 to 9 micrograms/ml in adults). Vaccine C7p was made with a higher loading of OS having mean length 7 repeat units and having mainly phosphate monoester at the exposed termini Vaccine C-7R was made from a portion of C-7p by enzymatic removal of most of the terminal phosphates. Compared to the C-4r, C-6r, and C-12r series, vaccines C-7p and C-7R induced geometric means about 10-fold higher in adults and 20-fold higher in infants. Thus OS chain length (in the range studied) and exposed terminus are less critical variables in this system than the extent of hapten loading.

Adult↗
PubMed1989

Autologous mixed lymphocyte reaction in human neonatal lymphocytes.

Human neonatal mononuclear cells were examined to determine their ability to participate in an autologous mixed lymphocyte reaction (AMLR). Stimulator cells were isolated by plastic adherence and nylon wool adherence. The nylon wool-nonadherent cells were used as responder cells. In 10 of 10 neonatal samples and 6 of 7 adult samples, a significant AMLR was present when plastic-adherent cells were used as stimulators. Neonatal blood showed a mean increase in proliferation of 7.6 (3.6-14.9), while adult cultures showed a mean stimulation index of 11.8 (1.0-39.0). When nylon wool-adherent cells were used as stimulator cells, only 2 of 7 neonatal blood samples and 1 of 5 adult blood samples showed a significant AMLR. When recombinant interleukin 2 (IL-2) was added to AMLR cultures of plastic-adherent cells and nylon wool-nonadherent cells, a mean augmentation of 12.0 was seen in the neonatal AMLR, while the adult cultures were augmented by a mean response of 4.1. Addition of IL-2 to nylon wool-nonadherent cells alone produced a 5.9-fold increase in adult cells, while neonatal cells showed an 85.8-fold mean increase in proliferation. The results suggest that autoreactive T cells are present in neonatal blood and that these cells can be activated by plastic-adherent autologous cells. However, neonatal and adult nylon wool-adherent cells do not consistently activate autoreactive T cells.

Female↗
PubMed1989

Immunoglobulin VH gene expression in human B cell lines and tumors: biased VH gene expression in chronic lymphocytic leukemia.

We have studied frequencies of VH gene utilization in a panel of monoclonal Epstein-Barr virus (EBV)-transformed B cell lines derived from human adult and fetal tissues as well as in monoclonal B cells obtained from fresh chronic lymphocytic leukemia (CLL) samples. The results show that IgM-secreting EBV cell lines from both fetal and adult tissues utilize VH genes from particular families roughly in proportion to estimated family size, suggesting that the repertoire of sigM-positive B cells in both fetal and adult organs is 'normalized' with respect to the V(H) gene family. In contrast, we find a highly biased pattern of VH gene expression in CLLs. The significance of these findings is discussed in the context of mechanisms that could be involved in normal B cell repertoire development and in the process of malignant transformation of precursors of CLL.

Alleles↗