Bruton's tyrosine kinase is required for activation of IkappaB kinase and nuclear factor kappaB in response to B cell receptor engagement.

Mutations in the gene encoding Bruton's tyrosine kinase (btk) cause the B cell deficiency diseases X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. In vivo and in vitro studies indicate that the BTK protein is essential for B cell survival, cell cycle progression, and proliferation in response to B cell antigen receptor (BCR) stimulation. BCR stimulation leads to the activation of transcription factor nuclear factor (NF)-kappaB, which in turn regulates genes controlling B cell growth. We now demonstrate that a null mutation in btk known to cause the xid phenotype prevents BCR-induced activation of NF-kappaB. This defect can be rescued by reconstitution with wild-type BTK. This mutation also interferes with BCR-directed activation of IkappaB kinase (IKK), which normally targets the NF-kappaB inhibitor IkappaBalpha for degradation. Taken together, these findings indicate that BTK couples IKK and NF-kappaB to the BCR. Interference with this coupling mechanism may contribute to the B cell deficiencies observed in XLA and xid.

Independent and opposing roles for Btk and lyn in B and myeloid signaling pathways.

Transphosphorylation by Src family kinases is required for the activation of Bruton's tyrosine kinase (Btk). Differences in the phenotypes of Btk-/- and lyn-/- mice suggest that these kinases may also have independent or opposing functions. B cell development and function were examined in Btk-/-lyn-/- mice to better understand the functional interaction of Btk and Lyn in vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk-/-lyn-/- mice. However, Btk-/-lyn-/- animals had a more severe immunodeficiency than Btk-/- mice. B cell numbers and response to T cell-dependent antigens were reduced. Btk and Lyn therefore play independent or partially redundant roles in the maintenance and function of peripheral B cells. Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn-/- mice. A transgene expressing Btk at approximately 25% of endogenous levels (Btklo) was crossed onto Btk-/- and Btk-/-lyn-/- backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.

Involvement of Bruton's tyrosine kinase in FcepsilonRI-dependent mast cell degranulation and cytokine production.

We investigated the role of Bruton's tyrosine kinase (Btk) in FcepsilonRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcepsilonRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcepsilonRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcepsilonRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed by the improvement in the ability of btk mutant mast cells to degranulate and to secrete cytokines after the retroviral transfer of wild-type btk cDNA, but not of vector or kinase-dead btk cDNA. Retroviral transfer of Emt (= Itk/Tsk), Btk's closest relative, also partially improved the ability of btk mutant mast cells to secrete mediators. Taken together, these results demonstrate an important role for Btk in the full expression of FcepsilonRI signal transduction in mast cells.

An Autoimmune Basis for Raynaud's Phenomenon: Murine Model and Human Disease.

OBJECTIVE: Raynaud's phenomenon (RP) is common in anti-RNP-positive patients with rheumatic diseases but is not itself known to be caused by autoimmunity. The aim of this study was to assess autoantibodies that could mediate this process. METHODS: Antibodies derived from patient sera and from murine models of anti-RNP autoimmunity were screened for the ability to induce RP-like tissue ischemia and endothelial cell apoptosis in murine models and in vitro systems. RESULTS: RNP-positive sera from RP patients and murine sera from RNP-positive B cell adoptive transfer recipients induced RP-like tissue ischemia and endothelial cell apoptosis. Proteomic analysis identified cytokeratin 10 (K10) as a candidate autoantigen in RP. Monoclonal anti-K10 antibodies reproduced patterns of ischemic tissue loss and endothelial cell apoptosis; K10 knockout or depletion of anti-K10 activity in serum was protective. Cold exposure enhanced K10 expression and in vivo tissue loss. CONCLUSION: Anti-K10 antibodies are sufficient to mediate RP-like ischemia in murine models and are implicated in the pathogenesis of RP in patients with anti-RNP autoimmunity.

Cloning and functional characterization of the early-lymphocyte-specific Pb99 gene.

The Pb99 gene is specifically expressed in pre-B cells and thymocytes and not in mature B and T cells or nonlymphoid tissues, implying that it may function in early lymphoid development. We have previously described the cloning of an incomplete cDNA for Pb99. Here we report the isolation of full-length cDNAs and genomic clones for the murine Pb99 gene and the mapping of its location to mouse chromosome 8. Sequence analyses of different Pb99 cDNA clones suggest that there may be at least three forms of the Pb99 protein generated by differential processing of the Pb99 transcript. The cDNA with the longest open reading frame encodes a putative protein that has seven hydrophobic domains similar to those of seven membrane-spanning proteins, such as the classical G protein-coupled receptors. To directly address the role of the Pb99 protein in lymphoid development, Pb99-deficient mice were generated by gene targeting, and lymphocyte development in these mice was analyzed.

Regulation of B lymphocyte development and activation by Bruton's tyrosine kinase.

The generation and maintenance of B lymphocytes is controlled by biochemical signals transmitted by the B cell antigen receptor(BCR) complex. These signals are transduced by multiple cytoplasmic protein tyrosine kinases (PTKs) including Lyn, Syk, and Bruton's tyrosine kinase (BTK). Upon BCR engagement, these PTKs activate downstream effectors, including transcription factors that modulate gene expression. In turn, activation of downstream effectors is critical for B cell survival, cell cycle progression, and antibody production. Our studies focus on the role of BTK in these biological responses. We have discovered that BTK is required for activation of the BCR-responsive transcription factor, NF-kappaB. Furthermore, BTK-dependent activation of NF-kappaB is essential for reprogramming the expression of genes that control B cell survival and proliferation. The biochemical mechanisms by which BTK regulates signaling components that activate NF-kappaB, and the identification of BTK-responsive genes are under investigation. Elucidation of these regulatory mechanisms is expected to reveal new therapeutic targets for B cell pathologies involving defects in BTK, including X-linked agammaglobulinemia (XLA).

Ceftazidime in the treatment of meningitis in infants and children over one month of age.

Ceftazidime, a new beta-lactamase-resistant cephalosporin, was compared with a combination of ampicillin and chloramphenicol for the treatment of meningitis in 100 infants and children aged one month to 15 years. In this open, randomized trial conducted in the Dominican Republic, 61 patients received 50 mg/kg of ceftazidime intravenously every eight hours; 39 received ampicillin plus chloramphenicol in conventional dosages. Seventy-eight of the patients had discernible isolates in samples from cerebrospinal fluid, six had a positive diagnostic Directogen result, and the remainder either had miscellaneous pathogens evident in samples of cerebrospinal fluid, bacteriologic growth in cultures of blood samples only, or no bacteriologic growth in cultures of either cerebrospinal fluid or blood. Among patients with discernible etiologic agents in samples of cerebrospinal fluid, 11 of 57 (19 percent) ceftazidime-treated patients died, and five of 27 (19 percent) patients treated with the combination died. Mortality by pathogen was as follows for patients who received ceftazidime or ampicillin plus chloramphenicol, respectively: Hemophilus influenzae, two of 27 (7 percent) and one of 15 (6 percent); Streptococcus pneumoniae, six of 12 (50 percent) and two of five (40 percent); Neisseria meningitidis, none of 11 (0 percent) and one of six (17 percent); and Salmonella, neither of two (0 percent) and one of one (100 percent). Overall mortality in the ceftazidime group was 20 percent versus 21 percent in the combination group. No significant toxicities were noted in the patients treated with ceftazidime.

Treatment of Pseudomonas meningitis with ceftazidime with or without concurrent therapy.

In ongoing studies in Europe and the United States, 10 pediatric patients with bacterial meningitis caused by Pseudomonas species were treated with ceftazidime. Pseudomonas aeruginosa was isolated from the CSF of 7 patients and other Pseudomonas species from the remaining 3. Eight of the 10 patients had received previous antimicrobial treatment which included aminoglycosides in 6, along with ticarcillin and ureidopenicillins in 3. Ceftazidime was administered 10 to 42 days in dosages ranging from 109 to 300 mg/kg/day. Seven of the 10 patients received ceftazidime only for 10 to 42 days. The other 3 patients received amikacin in 2 and gentamicin and tobramycin in the other. Seven patients were cured clinically and 3 died; 9 were cured bacteriologically and one who was presumed cured on the basis of clinical response subsequently died. Sterilization of the cerebrospinal fluid occurred at 48 hours to 12 days. Ceftazidime appears useful in treating bacterial meningitis caused by Pseudomonas species.

Prospective, randomized, investigator-blinded study of the efficacy and safety of meropenem vs. cefotaxime therapy in bacterial meningitis in children. Meropenem Meningitis Study Group.

OBJECTIVES: To compare the efficacy and safety of meropenem with cefotaxime for the treatment of infants and children with bacterial meningitis. METHODS: Infants and children with strongly suspected or documented bacterial meningitis were randomly assigned in a prospective multicenter study to receive either meropenem or cefotaxime. Patients were assessed at the end of therapy and at 5 to 7 weeks and 5 to 7 months after the end of treatment for the presence of neurologic and sensory neural sequelae. RESULTS: A total of 258 children were randomized to either treatment group. A further 8 patients with suspected pneumococcal meningitis were treated with meropenem without randomization. Of the randomized patients 154 were fully evaluable, 79 in the meropenem group and 75 in the cefotaxime group. At the end of treatment there were no significant differences in clinical outcome between the two treatment groups. Clinical cure with or without sequelae was achieved in 97 and 96% of the meropenem- and cefotaxime-treated patients, respectively. At the end of treatment and at 5 to 7 weeks, 46 and 54% of meropenem patients were cured with no sequelae, respectively. Corresponding results for cefotaxime patients were 56 and 58%. All pathogens were eradicated. In total 37 patients had seizures during treatment, 15 (12%) in the meropenem and 22 (17%) in the cefotaxime group. None of the seizures was considered to be drug-related. CONCLUSIONS: This trial shows that meropenem is suitable therapy for bacterial meningitis in infants and children and that it offers an efficacy and safety profile similar to that of cefotaxime.

Streptococcus pneumoniae resistant to penicillin: incidence and potential therapeutic options.

Streptococcus pneumoniae was recovered from 12 (50%) samples of middle ear fluid of 24 consecutive patients with AOME and in mixed culture of middle ear pathogens from one (4%) additional specimen. Two (15.3%) isolates had intermediate resistance to penicillin (minimal inhibitory concentration (MIC) 0.125 and 1.0 micrograms/mL). The antimicrobial susceptibility to various antimicrobials of 30 S pneumoniae strains recovered from patients seen in the last 12 months was also determined. One of the patients with AOME developed bacteremia that resolved uneventfully, whereas the other developed meningitis. MIC90 was determined from penicillin (2 micrograms/mL), erythromycin (> 32 micrograms/mL), cefaclor (32 micrograms/mL), loracarbef (> or = 64 micrograms/mL), cefixime (16 micrograms/mL), ceftibuten (> 64 micrograms/mL), chloramphenicol (16 micrograms/mL), cefpodoxime (4 micrograms/mL), ciprofloxacin (2 micrograms/mL), cephalexin (> or = micrograms/mL), augmentin (2 micrograms/mL), cefprozil (8 micrograms/mL), clindamycin (64 micrograms/mL), TMP-SXT (> 64 micrograms/mL), clarithromycin (32 micrograms/mL), rifampin (0.06 micrograms/mL), cefuroxime (2 micrograms/mL), cefotaxime (0.25 micrograms/mL), vancomycin (0.25 micrograms/mL), and imipenem (0.5 micrograms/mL). Cefprozil, vancomycin, and rifampin inhibited all strains, whereas cefpodoxime, cefuroxime, clindamycin, and clarithromycin exhibited very good activity.

Persistent purulent otitis media.

Of 429 children with acute otitis media who returned for follow-up evaluation, 49 (11%) were unresponsive to a 10- to 14-day course of ampicillin, amoxicillin, or erythromycin/sulfisoxazole. Patients with persistent purulent otitis media were noted to have immobile, bulging, yellow or grey, abscessed tympanic membranes at the follow-up visit. A myringotomy was performed on 45 children. Cultures of middle-ear exudate yielded ampicillin-resistant Haemophilus influenzae in 14 (31%), ampicillin-susceptible pathogens (H. influenzae or Streptococcus pneumoniae) in 23 (51%), and no growth in 8 (18%).

Evaluation of cefaclor in acute otitis media caused by ampicillin-resistant H. influenzae.

Cefaclor was used to treat 13 children with acute otitis media caused by ampicillin-resistant strains of Hemophilus influenzae. The children were re-examined on days 4, 10, and 28. Pharmacologic compliance was assessed by means of a bioinhibition assay on a urine specimen obtained on days 4, 7, and 10. Nine of the 13 children had evidence by tympanogram of residual otitis media with effusion (secretory otitis media). Of these, three were noted to have a bulging, yellow or grey eardrum, which suggested an ongoing acute process. A second middle ear culture was obtained from two of the 3 children, but no pathogens were recovered. Two others had recurrence of otitis shortly after cessation of therapy. Cefaclor is an acceptable antibiotic for the treatment of ampicillin-resistant acute otitis media; but, after cefaclor therapy, some children continued to have a bulging eardrum and sterile pus in the middle ear or had recurrences of otitis soon after cessation of therapy.

Biosynthesis of carcinoembryonic antigen (CEA) gene family members expressed in human tumor cell lines: evidence for cleavage of the glycosyl phosphatidyl inositol (GPI) anchor by GPI-PLC and GPI-PLD.

Three Carcinoembryonic Antigen (CEA) gene family members: CEA, Non-specific cross-reactive antigen 50/90 (NCA) and biliary glycoprotein (BGP) were expressed in the colon carcinoma cell lines LS174T and HT29. The CEA, NCA50/90 and four alternatively spliced BGP transcripts (BGP a-d) were identified. The molecular weights of the mature glycoproteins were: CEA, 180kD; NCA50/90, 70-100 kD; BGP, 85, 120 and 140 kD. Pulse chase experiments demonstrated that CEA first appears as a 165 kD high mannose precursor which is trimmed to a 160 kD intermediate and finally transformed into the mature 180 kD glycoprotein. The precursor form of NCA had a molecular weight of 50 kD. CEA and NCA50/90, but not BGP, were linked to the cell membrane via glycosyl phosphatidyl inositol and could be released from the intact tumor cells by glycosyl phosphatidyl inositol-specific phospholipase C. CEA on isolated membranes and in cell lysates, but not on intact cells, was also cleaved by fresh human serum or purified glycosyl phosphatidyl inositol-specific phospholipase D.

Identification of a new carcinoembryonic antigen (CEA) family member in human fetal liver--cloning and sequence determination of pregnancy-specific glycoprotein 7.

The carcinoembryonic antigen gene family consists of the CEA- and the Pregnancy-Specific Glycoprotein- (PSG) subfamilies. Human fetal liver express several PSGs. Here we report cloning and sequencing of a new PSG subfamily member, PSG7. It is the fifth type of PSG found in fetal liver. PSG7 has the N-A1-A2-B2-C domain arrangement. Unlike other PSGs the N-terminal of PSG7 is unblocked. PSG7 has a cysteine in the C-terminal domain, which may allow dimerization. Variability analysis according to Wu and Kabat reveals that the region in the N-domain corresponding to complementarity determining region 3 of immunoglobulin is different between PSG subfamily members. Many members, including PSG7, contain the RGD sequence in this region. The CD2 region as well as two other short sequences (in N and A1 domains respectively) also show some variability. The function of PSGs is probably linked to the N-domain and the CDR2- and CD3-like regions are most likely responsible for ligand binding.

Carcinoembryonic antigen gene family: molecular cloning of cDNA for a PS beta G/FL-NCA glycoprotein with a novel domain arrangement.

Human fetal liver contains a family of carcinoembryonic antigen related glycoproteins called the pregnancy specific beta 1 glycoprotein/fetal liver non specific cross-reactive antigen (PS beta G/FL-NCA) glycoprotein family. The sequence for the major FL-NCA species has been reported [Khan et al., Proc. Natl. Acad. Sci. USA 1989, 86 in press]. Two additional CEA-related fetal liver glycoproteins FL-NCA-2 and 3 were cloned, sequenced and expressed. FL-NCA-3 is a new gene family member. It has a unique domain arrangement (N-A2-B2-T) and contains a hydrophobic tail. FL-NCA-3 has a molecular weight of approximately 54 kD and is released from the transfected cells. Like three other members of the family, FL-NCA-3 contains the Arg-Gly-Asp sequence in a position in the N-domain corresponding to complementarity determining region 3 of immunoglobulin. FL-NCA-2 is identical in structure to PS beta G-D previously found in placenta. The PS beta G/FL-NCA glycoprotein family may be involved in processes related to cell adhesion and cell differentiation.

Phospholipase C-gamma 2 couples Bruton's tyrosine kinase to the NF-kappaB signaling pathway in B lymphocytes.

Mutations in the gene encoding Bruton's tyrosine kinase (BTK) interfere with B cell proliferation and lead to an X-linked immunodeficiency in mice characterized by reduced B cell numbers. Recent studies have established that BTK transmits signals from the B cell antigen receptor (BCR) to transcription factor NF-kappaB, which in turn reprograms a set of genes required for normal B cell growth. We now demonstrate that induction of NF-kappaB via this pathway requires the intermediate action of the -gamma2 isoform of phospholipase C (PLC-gamma2), a potential phosphorylation substrate of BTK. Specifically, pharmacologic agents that block the action of either PLC-gamma2 or its second messengers prevent BCR-induced activation of IkappaB kinase. Moreover, activation of NF-kappaB in response to BCR signaling is completely abolished in B cells deficient for PLC-gamma2. Taken together, these findings strongly suggest that PLC-gamma2 functions as an integral component of the BTK/NF-kappaB axis following BCR ligation. Interference with this NF-kappaB cascade may account for some of the B cell defects reported for plc-gamma2(-/-) mice, which develop an X-linked immunodeficiency-like phenotype.