Mutational analysis of hemoglobin binding and heme utilization by a bacterial hemoglobin receptor.

Iron is an essential nutrient for most living organisms. To acquire iron from their environment, Gram-negative bacteria use TonB-dependent transporters that bind host proteins at the bacterial surface and transport iron or heme to the periplasm via the Ton machinery. TonB-dependent transporters are barrel-shaped outer membrane proteins with 22 transmembrane domains, 11 surface-exposed loops, and a plug domain that occludes the pore. To identify key residues of TonB-dependent transporters involved in hemoglobin binding and heme transport and thereby locate putative protective epitopes, the hemoglobin receptor of Haemophilus ducreyi HgbA was used as a model of iron/heme acquisition from hemoglobin. Although all extracellular loops of HgbA are required by H. ducreyi to use hemoglobin as a source of iron/heme, we previously demonstrated that hemoglobin binding by HgbA only involves loops 5 and 7. Using deletion, substitution, and site-directed mutagenesis, we were able to differentiate hemoglobin binding and heme acquisition by HgbA. Deletion or substitution of the GYEAYNRQWWA region of loop 5 and alanine replacement of selected histidines affected hemoglobin binding by HgbA. Conversely, mutation of the phenylalanine in the loop 7 FRAP domain or substitution of the NRQWWA motif of loop 5 significantly abrogated utilization of heme from hemoglobin. Our findings show that hemoglobin binding and heme utilization by a bacterial hemoglobin receptor involve specific motifs of HgbA.

[Filling the thymic space after treatment of Hodgkin's disease: rebound or relapse]. / Comblement de la loge thymique après traitement d'une maladie de Hodgkin: rebond ou rechute?

We report the case of a 36-year-old women with Hodgkin's disease treated with polychemotherapy and bone marrow autograft. Progressive growth of a thymic mass suggested possible relapse four months after treatment withdrawal. This mass did not exhibit gallium-67 uptake but showed strong affinity for 18-FDG (SUV=6.8). Surgical biopsy ruled out recurrence of Hodgkin's disease of the thymus and led to the diagnosis of thymic rebound. The aspect of the thymic compartment returned to normal spontaneously at one year.

Cloning, overexpression, purification, and immunobiology of an 85-kilodalton outer membrane protein from Haemophilus ducreyi.

We have identified an 85-kDa outer membrane protein that is expressed by all tested strains of Haemophilus ducreyi. Studies of related proteins from other pathogenic bacteria, including Haemophilus influenzae, Pasteurella multocida, Neisseria gonorrhoeae, Neisseria meningitidis, and Shigella dysenteriae, suggested a role for these proteins in pathogenesis and immunity. In keeping with the first such described protein from Haemophilus influenzae type B, we termed the H. ducreyi protein D15. The gene encoding the H. ducreyi D15 protein was cloned and sequenced, and the deduced amino acid sequence was found to be most similar to sequences of the D15-related proteins from other Pasteurella spp. The arrangement of the flanking genes was similar to that of H. influenzae Rd and suggested that D15 was part of a multigene operon. Attempts to make a null mutation of the D15 gene were unsuccessful, paralleling results in other D15 gene studies. Overexpression of H. ducreyi D15 in Escherichia coli resulted in a source of recombinant D15 (rD15) from which it was readily purified. rD15 was immunogenic, and it was found that immunization of rabbits with an rD15 vaccine preparation conferred partial protection against a virulent challenge infection. Antisera to an N-terminal peptide recognized all tested strains of H. ducreyi.

Dexamethasone inhibits apoptosis of eosinophils isolated from hypereosinophilic patients.

Glucocorticoids reduce in vivo the number of eosinophils and are effective in treatment of blood and tissue hypereosinophilia. Dexamethasone is a synthetic glucocorticoid known to induce in vitro apoptosis of eosinophils of healthy donors, and apoptosis may be a mechanism induced by glucocorticoids to reduce eosinophilia. Here we confirm that dexamethasone exerts a pro-apoptotic effect on eosinophils isolated from healthy subjects but that dexamethasone is not always a pro-apoptotic agent towards eosinophils from hypereosinophilic patients. Implications of these results in the resistance of some patients to a treatment by glucocorticoids are discussed.

The Pim-1 kinase stimulates maturation of TCRbeta-deficient T cell progenitors: implications for the mechanism of Pim-1 action.

We demonstrate that overexpression of Pim-1, a cytoplasmic serine/threonine kinase of poorly defined function, results in the development of substantial numbers of CD4(+)CD8(+) double-positive thymocytes in two independent knock-out mouse models (i.e. the RAG-1-deficient and TCRbeta gene enhancer-deleted mice) in which production of a functionally rearranged TCRbeta gene (hence the pre-TCR) is impaired. This activity of Pim-1, however, does not affect signaling through the Ras/Raf/MAP kinase cascade nor signaling which mediates suppression of TCRbeta gene recombination (i.e. allelic exclusion). While overexpression of Pim-1 positively affects cell cycle progression in selected CD4(-)CD8(-) double-negative precursors, it did not affect expression of components of the cell cycle machinery, with the exception of the G(1)-specific phosphatase Cdc25A upon antigen receptor stimulation. We propose that Pim-1 acts downstream, or in parallel, to pre-TCR-mediated selection as one factor involved in the proliferative expansion of beta-selected pre-T cells.

Plasminogen activator inhibitor 1, transforming growth factor-beta1, and BMI are closely associated in human adipose tissue during morbid obesity.

In adipose tissue from both obese mice and humans, plasminogen activator inhibitor 1 (PAI-1) expression has been reported to be upregulated to levels of increased plasma PAI-1. This elevated expression has been shown to be partly controlled by tumor necrosis factor (TNF)-alpha in mice. In humans, increased PAI-1 expression is associated with insulin resistance characterized by visceral fat accumulation. Therefore, the aim of this study was to investigate the expression pattern of PAI-1 and TNF-alpha (antigen and mRNA) in visceral human adipose fat in comparison with subcutaneous (SC) fat. Because transforming growth factor (TGF)-beta1 is a potent inducer of PAI-1 synthesis and has been shown to influence adipocyte metabolism, this work was extended to TGF-beta1 quantification. A total of 32 obese individuals (BMI 42 +/- 6.8 kg/m2) were investigated. Freshly collected visceral adipose tissue did not exhibit a higher content of PAI-1 or TGF-beta1 than did SC tissue. Although most of the TNF-alpha values were at the detection limit of the methods, TNF-alpha antigen was 3-fold higher and TNF-alpha mRNA was 1.2-fold higher in visceral fat. The levels of tissue TGF-beta1 antigen correlated well with those of PAI-1 antigen, regardless of the fat depot studied (SC tissue: n = 21, r = 0.72, P = 0.0006; visceral tissue: n = 20, r = 0.49, P < 0.03), and they were both significantly associated with BMI. Conversely, no relationship was observed between the levels of TNF-alpha and PAI-1 or TNF-alpha and BMI. Tissue PAI-1 levels were also significantly correlated with those of circulating PAI-1. These results describe, in severe obesity, a proportional increase in tissue PAI-1 and TGF-beta1 in visceral and SC tissues. This increased PAI-1 expression could be the result of tissue cytokine disturbances, such as elevated TGF-beta1 expression.

T cell development in TCR beta enhancer-deleted mice: implications for alpha beta T cell lineage commitment and differentiation.

T cell differentiation in the mouse thymus is an intricate, highly coordinated process that requires the assembly of TCR complexes from individual components, including those produced by the precisely timed V(D)J recombination of TCR genes. Mice carrying a homozygous deletion of the TCR beta transcriptional enhancer (E beta) demonstrate an inhibition of V(D)J recombination at the targeted TCR beta locus and a block in alpha beta T cell differentiation. In this study, we have characterized the T cell developmental defects resulting from the E beta-/- mutation, in light of previously reported results of the analyses of TCR beta-deficient (TCR beta-/-) mice. Similar to the latter mice, production of TCR beta-chains is abolished in the E beta-/- animals, and under these conditions differentiation into cell-surface TCR-, CD4+CD8+ double positive (DP) thymocytes depends essentially on the cell-autonomous expression of TCR delta-chains and, most likely, TCR gamma-chains. However, contrary to previous reports using TCR beta-/- mice, a minor population of TCR gamma delta+ DP thymocytes was found within the E beta-/- thymi, which differ in terms of T cell-specific gene expression and V(D)J recombinase activity, from the majority of TCR-, alpha beta lineage-committed DP thymocytes. We discuss these data with respect to the functional role of E beta in driving alpha beta T cell differentiation and the mechanism of alpha beta T lineage commitment.

[Autoimmune hepatitis and lupus syndrome associated with minocycline]. / Hépatite auto-immune et syndrome lupique secondaires à la prise de minocyline.

INTRODUCTION: Among several adverse effects following treatment with minocycline, certain cases of autoimmune hepatitis, associated with lupus erythematosus, have been described. The possibility of hepatic damage, although rare, is important to keep in mind because of its delicate diagnostic. EXEGESIS: We report one case of autoimmune hepatitis following treatment with minocycline for acne, in a 25-year-old woman. This autoimmune hepatitis was associated with induced lupus syndrome. Usual causes of hepatitis were eliminated. Evolution was spontaneously favorable upon minocycline treatment interruption, with the disappearance of clinical symptoms and normalization of hepatic and immunologic biological values. CONCLUSION: The possibility of hepatic damage and lupus syndrome, following treatment with minocycline, should be recalled and verified in cases of long-term prescription. This observation stresses the difficulties of anamnesis in internal medicine. For those who know how to listen cautiously and rigorously, anamnesis may prove more helpful than many complementary examinations.

Polyarthritis following intravesical BCG immunotherapy. Report of a case and review of 26 cases in the literature.

OBJECTIVE: To delineate the characteristics of aseptic arthritis induced by intravesical BCG immunotherapy. METHODS: Review of a personal case and 26 cases from the literature. RESULTS: Mean number of intravesical BCG instillations at arthritis onset was five. Arthritis onset was within two weeks of the last instillation in 90% of cases. Half the patients had fever and half had conjunctivitis or uveitis. Symmetric polyarthritis was the most common pattern (n = 19), followed by oligoarthritis (n = 7). One patient had monoarthritis. The main targets were the knees (81%), ankles (48%), and wrists (40%). Twenty-six percent of patients reported back pain and 11% had sacroiliitis manifesting as pain or radiological changes. Mean erythrocyte sedimentation rate was 89 mm/h and mean C-reactive protein was greater than 70 mg/l. HLA B27 was positive in 56% of cases. Joint fluid usually exhibited inflammatory properties with polymorphonuclear neutrophils as the predominant cell type. Synovial membrane biopsy showed nonspecific synovitis in the six patients who had this investigation. Nonsteroidal antiinflammatory therapy was effective in 75% of cases. Three of the six patients given isoniazid and/or rifampin responded to this treatment. CONCLUSION: Although arthritis induced by intravesical BCG immunotherapy is more often polyarticular than oligoarticular, it shares many features with reactive arthritis.

Membrane isoforms of human immunoglobulins of the A1 and A2 isotypes: structural and functional study.

As for IgM, human IgA occurs either as soluble molecules in plasma and various other body fluids, or as membrane-bound molecules on differentiated B cells, where they are part of the B-cell receptor for antigen (BCR). We studied the structure of transcripts encoding the membrane-anchored alpha-chain of the human BCR alpha, which may be present in two different forms resulting from alternate splicing of the alpha-chain mRNA (type I or type II). The ratio of type I versus type II did not vary upon stimulation of a B-cell line with various cytokines. Rather, it differed strikingly in cells expressing either the IgA1 or IgA2 isotype of the BCR alpha, with virtually no type II alpha-chain in the latter. Co-modulation experiments also yielded different results for both isotypes, since they demonstrated a physical association of both membrane (m)IgA1 and mIgA2 with CD79b, the beta component of the BCR Ig alpha/Ig beta heterodimer, but only of mIgA1 with CD19. Whatever the isotype, the BCR of the IgA class was able to carry out signal transduction upon cross-linking by specific monoclonal antibodies but, in contrast to mIgM, it relied mainly on the entry of extracellular Ca2+ rather than on the release of intracellular stocks.

[Non-bacterial thrombosing endocarditis. Apropos of 2 cases]. / Endocardite thrombosante non bactérienne. A propos de deux cas.

The diagnosis of nonbacterial thrombosing endocarditis or marasmic endocarditis must be considered in patients presenting with a combination of cancer and systemic embolism. The pathophysiological mechanisms of this entity are unclear and purely hypothetical. However, hypercoagulability appears to play an essential role in the pathogenesis of this endocarditis, which could be the cardiac expression of a coagulopathy involving the entire vascular system. The authors report two cases of marasmic endocarditis which emphasize the value of transthoracic and transoesophageal echocardiography in the difficult diagnosis of this disease.

Regulatory elements of the mb-1 gene encoding the Ig-alpha component of the human B-cell antigen receptor.

The mb-1 gene encodes the Ig-alpha component of the B-cell antigen receptor. It is specifically expressed in pre-B and mature B cells but not in plasma cells losing membrane Ig (mIg) expression. We looked for transcriptional regulatory elements within a 12 kb genomic fragment. A strong promoter activity was found in a 591 bp fragment harboring consensus binding sites for known transcription factors including Ets, EBF/BlyF, LyF1/micro B and Spl. It was able to drive transcription of a reporter gene in the absence of any additional enhancer and was mostly active in B lymphocytes not in plasma cells or T cells. Although no fragment from the mb-1 gene displayed enhancer activity in combination with either the SV40, a Ig VH or a Ig VL promoter, a 1078 bp fragment corresponding to the 5' part of the gene behaved as a strong enhancer in either orientation in constructs driven by the mb-1 promoter itself. Deletions within this fragment allowed to delineate shorter sequences with enhancer activity upstream the first exon. The tissue-restricted, promoter-restricted and stage-specific activity of this 5' flanking region suggests that it is the main regulatory element of the mb-1 gene.

Involvement of a tyrosine kinase pathway in the growth-promoting effects of angiotensin II on aortic smooth muscle cells.

Angiotensin II (AII) is a growth factor that stimulates protein synthesis and induces cellular hypertrophy in aortic smooth muscle cells (SMC). This trophic effect is mediated by the AT1 subtype of AII receptors. However, very little is known about the cellular signaling pathways involved in this response. In the present study, we examined the role of protein tyrosine phosphorylation in the growth-promoting effects of AII on rat aortic SMC. The addition of AII to quiescent aortic SMC induced tyrosine phosphorylation of multiple substrates, as revealed by antiphosphotyrosine immunoblotting. This response was blocked by preincubation with the AT1-selective antagonist losartan. To explore the functional role of this signaling pathway, we performed experiments with two mechanistically distinct tyrosine kinase inhibitors. Treatment of quiescent aortic SMC with genistein and herbimycin A abolished the stimulatory effect of AII on overall protein tyrosine phosphorylation. Similarly, the two inhibitors prevented AII-induced tyrosine phosphorylation of the cytoskeletal protein paxillin. Under the same conditions, incubation with genistein or herbimycin A did not interfere with AII binding to the AT1 receptor and did not significantly affect AII-stimulated inositol-1,4,5-trisphosphate production and Ca2+ mobilization. In parallel to their selective action on tyrosine phosphorylation, both genistein and herbimycin A completely inhibited AII-stimulated protein synthesis in a dose-dependent manner. In contrast, the two inhibitors were much less potent in preventing the trophic effect of phorbol-12-myristate 13-acetate in these cells. We further demonstrate that genistein and herbimycin A did not prevent mitogen-activated protein kinase activation and c-fos gene induction, which is consistent with the notion that these downstream effectors do not link AII-induced tyrosine phosphorylation to protein synthesis. These results provide evidence that tyrosine phosphorylation has a critical role in cellular hypertrophy and is involved in AII action in vascular SMC.

Angiotensin II stimulates tyrosine phosphorylation of the focal adhesion-associated protein paxillin in aortic smooth muscle cells.

Treatment of vascular smooth muscle cells (SMC) with angiotensin II (AII) leads to an increase in the tyrosine phosphorylation of multiple cellular substrates. Here, we have demonstrated that AII stimulates tyrosine phosphorylation of the focal adhesion-associated protein paxillin in rat aortic SMC. AII-induced phosphorylation of paxillin was detectable within 1 min and was sustained up to 60 min. Preincubation with the AT1-selective antagonist losartan abolished this response. The stimulatory effect of AII on paxillin tyrosine phosphorylation was observed only in aortic SMC and not in other target cells such as adrenal zona glomerulosa cells, chromaffin cells, or hepatocytes. The effect of AII was dependent on the activation of phospholipase C. Chelation of intracellular calcium completely inhibited the ability of AII to stimulate paxillin tyrosine phosphorylation, while selective inhibition of protein kinase C partially attenuated the response. In contrast, treatment of the cells with pertussis toxin had no effect on AII-induced paxillin tyrosine phosphorylation. These findings identify paxillin as a new substrate for AII-stimulated tyrosine phosphorylation and suggest a role for cytoskeleton-associated proteins in the growth response of aortic SMC.

A human myeloma IgA with a hybrid heavy chain resulting from putative somatic gene conversion.

A human monoclonal IgA1-IgA2 lambda hybrid molecule was detected in a myeloma patient homozygous for the A2m(1) allotype during a systematic study of monoclonal IgA with subclass-specific monoclonal antibodies (mAb) and the lectin jacalin. This monoclonal immunoglobulin (GAU) reacted with both, although not with all, anti-alpha 1 and anti-alpha 2 mAb. Its heavy (H) chain contained an alpha 1 hinge region as shown by jacalin reactivity, the presence of disulfide-linked H and light chains in spite of its belonging to the IgA2m(1) allotype and amino acid composition of the isolated hinge region. The complete sequence of the H chain was deduced from that of complementary DNA clones from bone marrow cells. The CH1 domain, hinge region and beginning of the CH2 domain and the membrane peptide were encoded by the alpha 1 gene, with an insertion of an alpha 2m(1) gene sequence accounting for the end of the CH2 and part or all of the CH3 domain (sequence identity between the two normal genes precludes a precise definition of breakpoints). The region of the 5' recombination site included a repeat of a six base pair sequence which might play a role in the genetic exchange. The GAU hybrid alpha gene was undetectable in the patient's genomic DNA from polymorphonuclear cells. The genetic event which occurred at the level of the proliferating plasma cell clone is most likely to be a gene conversion.

Structure and expression of the mb-1 transcript in human lymphoid cells.

The mb-1 gene encodes a protein associated with membrane-bound immunoglobulins (mIg) and it has been suggested that it may play a role in signal transduction. Using a murine probe, we cloned a human complementary DNA homologous to the murine mb-1 sequence. Its complete sequence is in full agreement with a recently published human cDNA sequence but differs from a previously reported partial sequence. We studied mb-1 expression in human cells at various maturation stages. Large amounts of a single 1.4 kb transcript were detectable in B cell lines carrying mIg of the mu, gamma, alpha 1 or alpha 2 isotype. The mb-1 mRNA was also expressed in pre-B cells and in cells expressing truncated membrane mu chains devoid of associated light chains. Only trace amounts of mb-1 mRNA were found in tissue samples containing numerous plasma cells, while no mRNA was detected in several plasmacytoma cell lines, indicating that expression is shut down in plasma cells. No signal was found in T cells or in non-lymphoid tissues. Altogether, these results show that the human mb-1 gene is expressed in pre-B cells and B lymphocytes, regardless of the isotype of the mIg heavy chain and is no longer expressed in plasma cells.