Risk factors for community-associated Clostridioides difficile infection in young children.

The majority of paediatric Clostridioides difficile infections (CDI) are community-associated (CA), but few data exist regarding associated risk factors. We conducted a case-control study to evaluate CA-CDI risk factors in young children. Participants were enrolled from eight US sites during October 2014-February 2016. Case-patients were defined as children aged 1-5 years with a positive C. difficile specimen collected as an outpatient or ⩽3 days of hospital admission, who had no healthcare facility admission in the prior 12 weeks and no history of CDI. Each case-patient was matched to one control. Caregivers were interviewed regarding relevant exposures. Multivariable conditional logistic regression was performed. Of 68 pairs, 44.1% were female. More case-patients than controls had a comorbidity (33.3% vs. 12.1%; P = 0.01); recent higher-risk outpatient exposures (34.9% vs. 17.7%; P = 0.03); recent antibiotic use (54.4% vs. 19.4%; P < 0.0001); or recent exposure to a household member with diarrhoea (41.3% vs. 21.5%; P = 0.04). In multivariable analysis, antibiotic exposure in the preceding 12 weeks was significantly associated with CA-CDI (adjusted matched odds ratio, 6.25; 95% CI 2.18-17.96). Improved antibiotic prescribing might reduce CA-CDI in this population. Further evaluation of the potential role of outpatient healthcare and household exposures in C. difficile transmission is needed.

Augmented expression of a myeloid-specific protein tyrosine kinase gene (hck) after macrophage activation.

Protein tyrosine kinases are thought to participate in signal transduction pathways in a variety of cell types. Recent studies have identified a new src family protein tyrosine kinase (hck) that is preferentially expressed in myeloid cells. To examine the hypothesis that this kinase may regulate myeloid cell activity, antisera were generated that define the 59-kD product of the hck gene. Functional activation of human cultured macrophages with LPS augmented the expression of hck transcripts and of p59hck, but decreased the level of transcripts encoded by the closely related c-fgr protooncogene. Thus these two structurally similar src family kinases almost certainly subserve distinct functions. Reasoning from the known properties of the src family protein tyrosine kinases, it is likely that the products of these two protooncogenes assist in regulating the behavior of activated phagocytes.

Diversity in the germline antibody repertoire. Molecular evolution of the T15 VN gene family.

The T15 heavy chain variable region (VH) gene family in BALB/c mice includes four elements each greater than 88% homologous with the other. One of these elements, V1, encodes virtually all of the VH regions in BALB/c antiphosphorylcholine antibodies, while another element, V3, is a pseudogene and cannot be transcribed or translated. We have examined the structural features of this VH gene family in other mouse strains and, in particular, have cloned and sequenced the alleles of these gene segments present in B10.P mice. Each of the four B10.P sequences can be matched with its allelic counterpart in BALB/c mice. This represents the first successful analysis of allelism in antibody variable region gene segments. The V1B10.P allele, like its BALB/c counterpart, encodes most of the known phosphorylcholine binding heavy chains from C37BL/6 mice. Similarly, the V3B10.P gene segment is a pseudogene like V3BALB, although only two of four abnormalities present in the BALB/c allele are also present in the B10.P allele. Careful analysis of the specific substitutions observed in the T15 VH gene family suggests that environmental selection for functional combining regions contributes significantly to the pattern of variation in the germline antibody repertoire. In addition, evidence is presented supporting frequent gene conversion events in the divergence of antibody genes.

Delayed thymocyte development induced by augmented expression of p56lck.

Accumulating evidence supports the contention that CD4 and CD8 receptor molecules play a critical signaling role during thymocyte development. The lymphocyte-specific protein tyrosine kinase (p56lck), by virtue of its physical association with these surface components, provides a likely candidate for the biochemical signal transducing element required for these effects. To investigate the function of p56lck in T lymphocytes, transgenic mice were produced that carry either the wild-type lck gene or a mutated lck gene encoding a constitutively activated form of p56lck (p56lckF505). Both transgenes were expressed in thymocytes under the control of the lck proximal promoter element. A large set of founder animals was obtained in which steady-state accumulation of lck transgene mRNA directly correlated with transgene copy number, suggesting that this transgene contains a dominant control region. Progeny of these founders exhibited a transgene-dependent dose-related decrease in the production of thymocytes bearing functional antigen receptors. This effect was strictly dependent on p56lck activity, in that both wild-type and mutated versions of the genes induced similar effects with differing efficiencies. Remarkably, even a twofold increase in p56lck abundance was sufficient to substantially disrupt the appearance of functional thymocytes. These results indicate that thymocyte maturation is regulated in part by signals derived from p56lck.

The protein tyrosine kinase p56lck regulates thymocyte development independently of its interaction with CD4 and CD8 coreceptors [corrected].

The lck gene encodes a lymphocyte-specific protein tyrosine kinase of the nonreceptor type that is implicated in signal transduction pathways emanating from the CD4 and CD8 coreceptors. Previous studies also support a role for p56lck in regulating T cell receptor beta gene rearrangements and, more generally, thymocyte development. Here we report that a mutant form of p56lck, which is incapable of interacting with CD4 or CD8, behaves indistinguishably from association-competent p56lck with respect to its ability to affect thymocyte maturation. The effects of p56lck remained specific in that the closely related src-family kinase p59hck was incapable of substituting for p56lck in arresting beta locus gene rearrangements. These data support the view that src-family kinases perform highly specialized and often nonoverlapping functions in hematopoietic cells, and that p56lck acts independently of its association with CD4 and CD8 to regulate thymocyte development.

Replacement of pre-T cell receptor signaling functions by the CD4 coreceptor.

An important checkpoint in early thymocyte development ensures that only thymocytes with an in-frame T cell receptor for antigen beta (TCR-beta) gene rearrangement will continue to mature. Proper assembly of the TCR-beta chain into the pre-TCR complex delivers signals through the src-family protein tyrosine kinase p56lck that stimulate thymocyte proliferation and differentiation to the CD4+CD8+ stage. However, the biochemical mechanisms governing p56lck activation remain poorly understood. In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation. To study the effect of synchronously inducing p56lck activation in immature CD4-CD8- thymocytes, we generated mice expressing a CD4 transgene in Rag2-/- thymocytes. Remarkably, without further experimental manipulation, the CD4 transgene drives maturation of Rag2-/- thymocytes in vivo. We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules. Together these results indicate that binding of MHC class II molecules to CD4 can deliver a biologically relevant, Lck-dependent activation signal to thymocytes in the absence of the TCR-alpha or -beta chain.

Requirement for p56lck tyrosine kinase activation in T cell receptor-mediated thymic selection.

The nonreceptor protein tyrosine kinase p56lck (Lck) serves as a fundamental regulator of thymocyte development by delivering signals from the pre-T cell receptor (pre-TCR) that permit subsequent maturation. However, considerable evidence supports the view that Lck also participates in signal transduction from the mature TCR. We have tested this conjecture by expressing a dominant-negative form of Lck under the control of a promoter element (the distal lck promoter) that directs high expression in CD4+CD8+ thymocytes, mature thymocytes, and peripheral T cells, thereby avoiding, complications that result from the well-documented ability of dominant-negative Lck to block very early events in thymocyte maturation. Here we report that expression of the catalytically inactive Lck protein at twice normal concentrations inhibits thymocyte positive selection by as much as 80%, while leaving other aspects of cell maturation intact. This effect was studied in more detail in mice simultaneously bearing the male-specific H-Y alpha/beta TCR transgene and ovalbumin-specific DO10 alpha/beta TCR transgene, where even equimolar expression of the dominant-negative Lck protein substantially vitiated the positive selection process. Although deletion of H-Y alpha/beta thymocytes proceeded normally in male mice despite the presence of catalytically inactive Lck, modest inhibition of superantigen-mediated deletion was in some cases observed. These data further implicate Lck in the propagation of all TCR-derived signals, and indicate that even very modest deficiencies in the representation of functional Lck molecules could in humans, profoundly alter the character of the peripheral TCR repertoire.

Positive and negative selection invoke distinct signaling pathways.

During T cell development, interaction of the T cell receptor (TCR) with cognate ligands in the thymus may result in either maturation (positive selection) or death (negative selection). The intracellular pathways that control these opposed outcomes are not well characterized. We have generated mice expressing dominant-negative Ras (dnRas) and Mek-1 (dMek) transgenes simultaneously, either in otherwise normal animals, or in animals expressing a transgenic TCR, thereby permitting a comprehensive analysis of peptide-specific selection. In this system, thymocyte maturation beyond the CD4+8+ stage is blocked almost completely, whereas negative selection, assessed using an in vitro deletion protocol, is quantitatively intact. This suggests that activation of the mitogen-activated protein kinase (MAPK) cascade is necessary for positive selection, but irrelevant for negative selection. Generation of gamma/delta and of CD4-8- alpha/beta T cells proceeds normally despite blockade of the MAPK cascade. Hence, only cells that mature via conventional, TCR-mediated repertoire selection require activation of the MAPK pathway to complete their maturation.

Ig heavy chain variable region gene complex of lupus mice exhibits normal restriction fragment length polymorphism.

B cell hyperactivity, hypergammaglobulinemia, and autoantibody expression, the hallmarks of systemic lupus erythematosus, might be associated with structural abnormalities within the Ig heavy chain variable region (Igh-V) gene complex. The Igh-V loci from several lupus-prone mouse strains, their ancestors, and other nonautoimmune mice were therefore analyzed by restriction fragment length polymorphisms with DNA probes corresponding to seven VH gene families. These seven families comprise the majority of the known polymorphic murine VH gene repertoire, including some involved in autoantibody generation. Our study showed that the Igh-V loci from lupus and haplotype-matched nonlupus mice resulted in essentially identical restriction fragment patterns, a finding which suggests that the Igh-V gene complex does not carry a primary defect responsible for autoimmune disease.

Regulation of L-selectin-mediated rolling through receptor dimerization.

L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin-GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by approximately 700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors.

Control of lymphopoiesis by p50csk, a regulatory protein tyrosine kinase.

The csk gene encodes a nonreceptor protein tyrosine kinase that acts in part by regulating the activity of src-family protein tyrosine kinases. Since the src-family kinases p56lck and p59fyn play pivotal roles during lymphocyte development, it seemed plausible that p50csk might contribute to these regulatory circuits. Using a gene targeting approach, mouse embryonic stem cell lines lacking functional csk genes were generated. These csknull embryonic stem cells proved capable of contributing to many adult tissues, notably heart and brain. However, although csknull progenitors colonized the developing thymus, T and B cell differentiation were both blocked at very early stages. This represented a relatively selective interdiction of lymphocyte maturation, since csknull hematopoietic progenitors supported the development of normal-appearing MAC-1+ blood leukocytes, and the successful maturation of granulocyte/macrophage-colony-forming units from fetal liver progenitors. We conclude that p50csk regulates normal lymphocyte differentiation, but that it almost certainly does so by acting on targets other than p56lck and p59fyn.

Immunoglobulin subclass-specific immunodeficiency in mice with an X-linked B-lymphocyte defect.

CBA/N mice express an X-linked deficiency in their antibody response to many bacterial carbohydrates; we have shown recently that these antigens normally elicit antibody responses predominantly of the IgM and IgG3 isotypes. Here we demonstrate that mice, with the CBA/N phenotype have perferential deficiencies of IgM and IgG3 immunoglobulin expression, both when measured in serum and in cells secreting these isotypes, and that this deficiency is only partially corrected by polyclonal activation of B cells. This suggests that CBA/N mice may lack a subpopulation of B cells that contain most of the IgG3 precursors.

Multiple VH gene segments encode murine antistreptococcal antibodies.

Most mouse strains are able to mount a diverse antibody response against group A streptococcal carbohydrate (GAC). We have previously reported that murine anti-GAC antibodies are for the most part restricted to IgM and IgG3 subclasses. In addition, despite extensive heterogeneity in their isoelectric focusing patterns, greater than 50% of A/J anti-GAC antibodies share a common light chain defined by spectrotypic and idiotypic (VK1GAC) criteria. We have used protein and DNA sequencing strategies to examine the genetic basis of diversity in murine anti-GAC antibodies. In particular, we report that, (a) multiple, closely homologous VH gene segments contribute to the generation of anti-GAC antibodies, (b) a common framework sequence, related to the VK27 subgroup, probably defines VK1GAC, and (c) the A/J anti-GAC VH regions and BALB/c anti-inulin VH sequences are 95% homologous at the protein level and are likely encoded by overlapping VH gene families. Lastly, we discuss the genetic mechanisms that might permit the evolution of multiple, closely homologous germline VH gene segments in the context of highly divergent flanking region sequences.

Molecular dissection of the murine antibody response to streptococcal group A carbohydrate.

Monoclonal antibodies (mAb) to streptococcal group A carbohydrate (GAC) are encoded by a minimum of two VH, four JH, four V kappa, three J kappa, one V lambda, and one J lambda gene segments. The IdX, IdI-1, and Id5 idiotypic determinants are expressed by anti-GAC mAb and are found on free kappa chains. Each pattern of these determinants is encoded by a distinct V kappa gene segment, apparently without the requirement for a particular J kappa, VH, or JH gene segment, or somatic mutation. In contrast, the binding site-associated idiotypic determinant IdI-3a does not correlate with any single V or J gene segment.

Interleukin 4 expressed in situ selectively alters thymocyte development.

Using a transgenic mouse model we show that increased intrathymic expression of interleukin 4 (IL-4) significantly perturbs the development of thymocytes. Transgenic double-positive (CD4+CD8+) thymocytes, which are present in dramatically reduced numbers, exhibit increased T cell receptor (TCR) expression and increased mobilization of calcium mediated by these receptors. In contrast, transgenic single-positive (CD4+CD8- and CD4-CD8+) thymocytes and peripheral T cells exhibit decreased TCR-mediated calcium mobilization. The development of CD4-CD8+ thymocytes is significantly perturbed by IL-4 expressed in vivo; only peripheral CD4+ T cells are found in significant numbers in transgenic mice, while CD4-CD8+ thymocytes are present in increased numbers, apparently because of their failure to emigrate to the periphery. In contrast to these selective effects on T cell development, no significant differences in the numbers of B cells or mast cells, or in the plasma levels of IgE and IgG1 are observed between transgenic and control mice. These observations suggest that IL-4 in vivo exerts its major effects locally rather than systemically, even when its expression is constitutively increased.

Developmental regulation of lck gene expression in T lymphocytes.

In the mouse and human, mRNA transcripts encoding the lymphocyte-specific protein tyrosine kinase p56lck are derived from two separate promoters resulting in heterogeneity in the 5' untranslated region sequence. The proximal promoter lies just 5' to the coding region for the gene and is active only in thymocytes. In contrast, the distal promoter lies 34 kilobases (kb) 5' in the human, and is active both in thymocytes and mature peripheral T cells. As previously reported, transgenic mice bearing functional proximal promoter sequence juxtaposed with the SV40 large T antigen gene invariably develop lymphoid tumors confined to the thymus. In the current work, transgenic mice bearing a 2.6-kb fragment of the human distal promoter fused to the SV40 large T antigen gene express large T antigen in thymocytes and in peripheral lymphoid cells, and develop tumors of both the thymus and the peripheral lymphoid organs. The ability of the human distal promoter to function appropriately in transgenic mice is consistent with the strong similarity observed between the mouse and human distal promoter sequences. With the exception of a single short interval that serves as a target for binding of nuclear factors, significant sequence similarity is not seen when the distal and proximal promoter sequences are compared. Hence, developmentally regulated, lineage-specific transcription of the lck gene is mediated by distinct promoter sequences that appear to be capable of functioning independently.

Involvement of p59fynT in interleukin-5 receptor signaling.

Previous studies implicate the nonreceptor protein tyrosine kinase (PTK) p59fyn in the propagation of signals from the B cell antigen receptor. To elucidate the functions of this kinase, we examined B cell responsiveness in mice engineered to lack the hematopoietic isoform of p59fyn. Remarkably, antigen receptor signaling was only modestly defective in fynTnull B cells. In contrast, signaling from the interleukin (IL)-5 receptor which ordinarily provides a comitogenic stimulus with antiimmunoglobulin, was completely blocked. Our results document the importance of p59fynT in IL-5 responses in B cells, and they support a general model for cytokine receptor signal transduction involving the simultaneous recruitment of at least three families of PTK.

Early restriction of the human antibody repertoire.

Diversification of the antibody repertoire in mammals results from a series of apparently random somatically propagated gene rearrangement and mutational events. Nevertheless, it is well known that the adult repertoire of antibody specificities is acquired in a developmentally programmed fashion. As previously shown, rearrangement of the gene segments encoding the heavy-chain variable regions (VH) of mouse antibodies is also developmentally ordered: the number of VH gene segments rearranged in B lymphocytes of fetal mice is small but increased progressively after birth. In this report, human fetal B-lineage cells were also shown to rearrange a highly restricted set of VH gene segments. In a sample of heavy-chain transcripts from a 130-day human fetus the most frequently expressed human VH element proved to be closely related to the VH element most frequently expressed in murine fetal B-lineage cells. These observations are important in understanding the development of immunocompetence.

Developmentally controlled expression of immunoglobulin VH genes.

Although antibody diversity arises mainly from apparently random combinatorial and somatic mutational mechanisms acting upon a limited number of germline antibody genes, the antibody repertoire develops in an ordered fashion during mammalian ontogeny. A series of early pre-B and B-lymphocyte cell lines were examined to determine whether an ordered rearrangement of gene families of the variable region of immunoglobulin heavy chains (VH) may be the basis for the programmed development of the antibody response. The results indicated that the VH repertoire of fetal B-lineage cells is largely restricted to the VH 7183 gene family and that subsequent recruitment of additional VH gene families occurs during neonatal development. These results have important implications in understanding the ontogeny of immune function.

Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association.

In the interleukin-2 (IL-2) system, intracellular signal transduction is triggered by the beta chain of the IL-2 receptor (IL-2R beta); however, the responsible signaling mechanism remains unidentified. Evidence for the formation of a stable complex of IL-2R beta and the lymphocyte-specific protein tyrosine kinase p56lck is presented. Specific association sites were identified in the tyrosine kinase catalytic domain of p56lck and in the cytoplasmic domain of IL-2R beta. As a result of interaction, IL-2R beta became phosphorylated in vitro by p56lck. Treatment of T lymphocytes with IL-2 promotes p56lck kinase activity. These data suggest the participation of p56lck as a critical signaling molecule downstream of IL-2R via a novel interaction.