Investigation of the effect of the adsorbent DAV131A on the propensity of moxifloxacin to induce simulated Clostridioides (Clostridium) difficile infection (CDI) in an in vitro human gut model.

BACKGROUND: Clostridioides difficile infection (CDI) remains a high burden worldwide. DAV131A, a novel adsorbent, reduces residual gut antimicrobial levels, reducing CDI risk in animal models. OBJECTIVES: We used a validated human gut model to investigate the efficacy of DAV131A in preventing moxifloxacin-induced CDI. METHODS: C. difficile (CD) spores were inoculated into two models populated with pooled human faeces. Moxifloxacin was instilled (43 mg/L, once daily, 7 days) alongside DAV131A (5 g in 18 mL PBS, three times daily, 14 days, Model A), or PBS (18 mL, three times daily, 14 days, Model B). Selected gut microbiota populations, CD total counts, spore counts, cytotoxin titre and antimicrobial concentrations (HPLC) were monitored daily. We monitored for reduced susceptibility of CD to moxifloxacin. Growth of CD in faecal filtrate and medium in the presence/absence of DAV131A, or in medium pre-treated with DAV131A, was also investigated. RESULTS: DAV131A instillation reduced active moxifloxacin levels to below the limit of detection (50 ng/mL), and prevented microbiota disruption, excepting Bacteroides fragilis group populations, which declined by ∼3 log10 cfu/mL. DAV131A delayed onset of simulated CDI by ∼2 weeks, but did not prevent CD germination and toxin production. DAV131A prevented emergence of reduced susceptibility of CD to moxifloxacin. In batch culture, DAV131A had minor effects on CD vegetative growth, but significantly reduced toxin/spores (P < 0.005). CONCLUSIONS: DAV131A reduced moxifloxacin-induced microbiota disruption and emergence of antibiotic-resistant CD. Delayed onset of CD germination and toxin production indicates further investigations are warranted to understand the clinical benefits of DAV131A in CDI prevention.

Cerebral phaeohyphomycosis due to Cladophialophora bantiana in a French Guianese child.

We report a case of cerebral phaeohyphomycosis, a fungal brain infection due to a dark (dematiaceous) fungi in a 6-year-old French Guyanese boy. The child presented fever and drowsiness due to several paraventricular brain abscesses. Neurological surgeries were performed to reduce intracranial hypertension and to obtain abscess biopsies. Mycological cultures of intraoperative samples led to the diagnosis of cerebral phaeohyphomycosis due to Cladophialophora bantiana. The patient neurological status deteriorated and remained critical after several weeks of combination antifungal therapy with voriconazole 8mg/kg/day, liposomal amphotericin B 10mg/kg/day and flucytosine 200mg/kg/day. A complete surgical resection was not possible because of multiple small abscesses. A multidisciplinary ethical staff decided on home medical care with palliative ventriculoperitoneal shunt, nasogastric feeding and analgesics. One year later, the patient's neurological condition had improved and cerebral lesions had regressed, while he had not received any antifungal treatment but only traditional medicines. Cerebral phaeohyphomycosis are rare diseases affecting immunocompromised but also apparently non-immunocompromised patients, as in this case. A complete surgical resection is not always possible and mortality rates are high in spite of treatments with a combination of antifungals. The diagnosis may be difficult because of these dematiaceous fungi's slowly growing and their potential pathogenicity for laboratory staff.

[Clustered cases of histoplasmosis due to Histoplasma capsulatum var. capsulatum in Martinique: Case reports and environmental investigation]. / Cas groupés d'histoplasmoses à Histoplasma capsulatum var. capsulatum à la Martinique : description des cas et enquête environnementale.

Between the 24th of June and the 6th of July 2005, nine men came to Fort-de-France emergency department (Martinique, French West Indies) with more or less pronounced pulmonary symptoms associated in two cases with skin lesions. Three weeks before these nine men performed work in a deserted house. The diagnosis of histoplasmosis was based on pulmonary sample mycological analysis (direct examination and culture), molecular biology and serological tests. Interrogatory and environmental investigations on the presumed place of exposition to H. capsulatum var. capsulatum spores allowed confirming how and where contamination took place.

Phaeohyphomycosis Caused by a Novel Species, Pseudochaetosphaeronema martinelli.

Among the opportunistic mycoses that are emerging in patients with immunosuppression or severe underlying illness, many isolates lack of characteristic sporulation and until recently could not be identified. Clinical signs are mostly nonspecific and therefore such infections have often been disregarded. In the present paper we describe a novel, nonsporulating fungal species causing subcutaneous phaeohyphomycosis in two patients of different origin. One is a 73-year-old female from Martinique who suffered from rheumatoid arthritis, while the other case concerns a 72-year-old male from Mexico who had a history of type 2 diabetes mellitus. Sequencing of the partial ribosomal operon revealed that in both cases a member of the order Pleosporales was concerned which could not be affiliated to any family within this order. Multilocus analysis revealed that the fungus was related to another, unaffiliated agent of human mycetoma, Pseudochaetosphaeronema larense, and therefore the name Pseudochaetosphaeronema martinelli was introduced.

Anti-anaerobic activity of a new ß-lactamase inhibitor NXL104 in combination with ß-lactams and metronidazole.

NXL104 is a new ß-lactamase inhibitor (BLI) that inhibits class A and class C ß-lactamase enzymes. In this study, the activity of NXL104 in combination with the third-generation cephalosporins ceftazidime (CAZ) and ceftriaxone (CRO) or with piperacillin (PIP) was evaluated against 316 anaerobic bacteria. Minimum inhibitory concentrations (MICs) were determined using an agar dilution method. The BLIs NXL104 or tazobactam (TAZ) were added to the ß-lactams at a fixed concentration of 4 mg/L. A triple combination of NXL104 with an 8:1 ratio of CAZ and metronidazole (MTZ) was also tested. The activities of CAZ, CRO and PIP in combination with NXL104 were enhanced against many of the bacteria. MIC(50) values (MIC for 50% of the organisms) for CAZ+NXL104 were 8-16-fold lower than those of CAZ against Gram-negative anaerobes. Antibiotic resistance rates against all anaerobic strains were: CAZ, 37.7%; CRO, 31%; CAZ+NXL104, 15.2%; CRO+NXL104, 5.4%; and MTZ, 4.1%. No resistant strains could be observed with PIP+TAZ, PIP+NXL104 or the triple combination MTZ+CAZ+NXL104. In conclusion, the triple combination of MTZ+CAZ+NXL104 demonstrated potent antibacterial activity against anaerobes representing most clinical species. It appears appropriate for the treatment of polymicrobial infections, since CAZ+NXL104 also exhibits potent activity against ß-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa. It is currently being tested in phase 2 clinical trials for the treatment of complicated intra-abdominal infections.

Enzymatic activity of two caspases related to interleukin-1beta-converting enzyme.

Interleukin-1beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occurred within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1beta. TX cleaved the same synthetic substrates as ICE (Km of 90 microM and k(cat) of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 microM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1beta cleavage.

Evidence for CPP32 activation in the absence of apoptosis during T lymphocyte stimulation.

Cysteine proteases of the interleukin-1beta-converting enzyme family have been implicated in the effector process of apoptosis in several systems. Among these, CPP32 has been shown to be processed to active enzyme at the onset of apoptosis. Here, we show that CPP32 precursor is cleaved into its active form during phytohaemaglutinin A activation of T lymphocytes. Maximal processing is observed between day 3 and day 4 following addition of mitogen and is a transient process. Precursor cleavage is associated with the appearance of a CPP32-like enzymatic activity in cell lysates. At this time in the culture, almost no apoptotic cell and no dead cell can be detected, and T lymphocytes are actively proliferating. CPP32 processing also occurs when lymphocytes are stimulated through an allogeneic primary mixed lymphocyte reaction. Our results suggest that proteolytic activation of CPP32 could be a physiological step during T lymphocyte activation. In addition, these data indicate that CPP32 activation can occur independently of programmed cell death in T lymphocytes.

Evidence for an interleukin-1beta converting enzyme (ICE)-like activity distinct from ICE and responsible for ICE and CPP32 cleavage during apoptosis.

IL-1beta converting enzyme (ICE) and ICE-related proteases (IRPs) have been suggested to play a central role in apoptosis. We report the use of peptidic ICE inhibitors to reassess the role of this enzyme in the apoptosis induced by Fas or TNFalpha receptor ligation in Jurkat cells, U937 cells or monocytes. Our results show that inhibition of IL-1beta processing can be dissociated from inhibition of apoptosis. Indeed, two out of three com-pounds active on ICE are not inhibitory for apoptosis. This shows that ICE is not required for progression in the apoptotic pathway, but that one or several IRPs are necessary. In addition, Western blot analysis of cell lysates shows that both ICE and CPP32 precursors disappear rapidly after apoptosis induction, while ICH-1L precursor remains intact. Concomitant appearance of cleavage products can be visualized for CPP32, but not for ICE, suggesting that the former is proteolytically activated. In addition, this precursor cleavage can be blocked by an ICE inhibitor active on apoptosis. Altogether, our data support the hypothesis that one or several IRPs are necessary for apoptosis and are responsible for ICE and CPP32 cleavage during this process.

Use of monoclonal antibodies to study interleukin-1 beta-converting enzyme expression: only precursor forms are detected in interleukin-1 beta-secreting cells.

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1 beta-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67,000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1 beta was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1 beta cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.

A novel human protease similar to the interleukin-1 beta converting enzyme induces apoptosis in transfected cells.

We have identified a novel cDNA encoding a protein (named TX) with > 50% overall sequence identity with the interleukin-1 beta converting enzyme (ICE) and approximately 30% sequence identity with the ICE homologs NEDD-2/ICH-1L and CED-3. A computer homology model of TX was constructed based on the X-ray coordinates of the ICE crystal recently published. This model suggests that TX is a cysteine protease, with the P1 aspartic acid substrate specificity retained. Transfection experiments demonstrate that TX is a protease which is able to cleave itself and the p30 ICE precursor, but not to generate mature IL-1 beta from pro-IL-1 beta. In addition, this protein induces apoptosis in transfected COS cells. TX therefore delineates a new member of the growing Ice/ced-3 gene family coding for proteases with cytokine processing activity or involved in programmed cell death.

Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.

The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3-expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.

CD3.TCR1, a human CD3 epitope expressed on viable gamma/delta lymphocytes exclusively.

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.

Molecular characterization of human T cell receptor alpha chains including a V delta 1-encoded variable segment.

Previously we have shown that a small fraction of human peripheral T cells expresses a surface receptor recognized both by the BMA031 mAb, specific for a TcR alpha/beta framework epitope, and by the A13 mAb, putatively specific for an epitope encoded by the V delta 1 gene segment. An interleukin 2-dependent polyclonal cell line (termed T2) was derived from such A13+BMA031+ circulating lymphocytes. The molecular characterization of the TcR chains expressed by T2 cells demonstrated indeed that the V delta 1 gene (one of the two major V delta genes) was transcribed with the C alpha gene segment. In the T2 polyclonal cell line, distinct V delta 1/C alpha transcripts were all found to include the same J alpha segment suggesting the existence of "hybrid" TcR alpha/delta chains encoded by unique V delta 1/J alpha rearrangements. The present study was designed to characterize further the V delta 1/J alpha rearranged genes expressed in A13+BMA031+ cells. Three additional cell lines were generated from peripheral blood of distinct adult healthy donors. Using the anchored polymerase chain reaction, it was found that 17 different J alpha segments were used in the 20 V delta 1J alpha C alpha transcripts which have been studied. Together, these data indicate that V delta 1 is a "mixed" (i.e. alpha/delta) TcR V segment which can join with most (if not all) J segments in the alpha/delta locus. In addition, it can be definitely concluded that the A13 mAb recognizes a V delta 1-encoded antigenic determinant and not a V delta 1J epitope (i.e. it can be defined and used as an anti-V delta 1 mAb, as opposed to reagents such as for example delta-TCS-1).

T cell target 1 (TCT.1): a novel target molecule for human non-major histocompatibility complex-restricted T lymphocytes.

We have studied two gamma/delta T cell clones, E102 and E117, generated in a mixed lymphocyte culture using an allogeneic Epstein-Barr virus-transformed B cell line, E418. These clones were both found to express a molecular form of T cell receptor (TCR) infrequent in human peripheral blood, associating a V1-J1-C delta chain and a V3-JP2-C2 gamma chain. Functionally, they appeared as cytotoxic T lymphocytes (CTL) with non-major histocompatibility complex (MHC) (class I and II) requiring cytotoxicity, able to kill both the immunizing (i.e., E418) and unrelated (e.g., K562, REX, F601, and KAS) target cells. A monoclonal antibody, anti-10H3, able to selectively inhibit the cytotoxic activity of the clones has been produced. This reagent defines a 43-kD molecule, designated TCT.1, with broad distribution in the hematopoietic system, that appears to be distinct from class I MHC gene products. A series of functional experiments using various effector/target cell combinations strongly suggested that TCT.1 may represent a unique TCR ligand involved in the interaction between these particular CTL clones and certain of the target cells tested, while others were likely to be recognized and killed through a TCR-independent natural killer-like pathway. Although further experimentation will be needed to strengthen our interpretation of the present data, this study provides additional evidence that some T lymphocytes, in particular of the gamma/delta type, may interact specifically with target cells in a non-MHC class I/II-requiring fashion.

Further analysis of the T cell receptor gamma/delta+ peripheral lymphocyte subset. The V delta 1 gene segment is expressed with either C alpha or C delta.

In the present study, we have characterized the reactivity of two mAbs that are directed at the human TCR-gamma/delta. These reagents, designated anti-A13 and anti-TiV delta 2, were found to recognize antigenic determinants encoded by the TCR V delta 1 and V delta 2 gene segments, respectively. Immunofluorescence analyses performed with the antibodies confirmed that, in the TCR-gamma/delta+ cell subpopulation, the expression of V delta 2+ delta chains is largely predominant, as compared with the V delta 1+ counterparts. However, these experiments led to an apparently discrepant finding. Indeed, the total number of cells recognized by the anti-A13 plus the anti-TiV delta 2 antibodies was often greater than that detected with anti-TCR-delta 1, a reagent specific for a constant epitope of the human delta chain. Further investigation showed that the presence of a sizeable peripheral lymphocyte subset coexpressing the BMA031 and the A13 epitopes. Because the former antibody is known to recognize an invariant antigenic determinant of the TCR-alpha/beta dimer, these results suggested that the V delta 1 gene segment may be expressed with either C delta or C alpha. This hypothesis was confirmed using T2, an IL-2-dependent BMA031+ A13+ polyclonal cell line developed from peripheral blood of a healthy adult donor. Indeed, T2 cells were found to have productively rearranged the V delta 1 gene. Together, results of Northern blot analysis and cDNA cloning indicated that V delta 1 was expressed in these cells as part of a 1.6-kb full-length message including J alpha-C alpha segments.

CD1c as a target recognition structure for human T lymphocytes: analysis with peripheral blood gamma/delta cells.

It has been shown recently that one gamma/delta cell line, termed IDP2, derived from a immunodeficient patient recognizes the CD1c molecule on the surface of target cells. In light of these data, we have tested 43 cloned and 11 polyclonal gamma/delta cell lines derived from peripheral blood of 19 donors following nonspecific mitogenic stimulation. In this panel, which included lymphocytes expressing various combinations of gamma and delta chains, only one clone, termed J2B7, was found to interact with target cells via a CD1c-dependent recognition pathway. These J2B7 lymphocytes have, like IDP2, a delta chain which results from the frequent V1/J1 rearrangement while they use a distinct V gamma gene segment. The data support the view that the CD1c major histocompatibility complex "class I-like" gene product does not have a pivotal contribution to the repertoire of peripheral blood gamma/delta cells in adult individuals.

Regulatory effects of IgG-BF on hybridoma B cells. molecular characterization of variant cell lines.

Immunoglobulin G-binding factors (IgG-BF) produced by mouse T cells or hybridoma T cells (T2D4) were used to manipulate in vitro mouse hybridoma B cells. Both IgG production by, and proliferation of, these cells was inhibited by IgG-BF, or during co-cultures with IgG-BF-producing T2D4 cells. Thus, treatment of tumor B cells, besides its potential therapeutic use, represents an invaluable model for studying the regulation of Ig production by IgG-BF at a molecular level. To further analyze the molecular events induced by IgG-BF in B cell hybridomas, a set of variant clones of a hybridoma cell line (UN2) was isolated and variants were characterized for their Ig production and their Fc gamma R expression.

Cloned CD3+ TcR alpha/beta- Ti gamma A- peripheral blood lymphocytes compared to the Ti gamma A+ counterparts: structural differences of the gamma/delta receptor and functional heterogeneity.

We have assessed the organization of T cell gamma rearranging genes (TRG) in circulating TcR gamma/delta+ lymphocytes which do not express V gamma 9-encoded Ti gamma A+ gamma chain. Following purification of the minor TcR gamma/delta+ Ti gamma A- fraction, cloned cell lines were developed from peripheral blood of 5 individuals. Out of the 26 clones studied, only 3 TcR gamma/delta+ Ti gamma A- cells were found to express a disulfide-linked C1-encoded gamma chain. The remaining 23 Ti gamma A- clones with a C2-encoded nondisulfide-linked receptor were found to display rearrangements of various V genes to J2 segments on both chromosomes; there was no predominance of a unique rearrangement even though the TRG-V3 and -V4 genes belonging to subgroup I were frequently employed. Together, these findings further strengthen the hypothesis that lymphocytes with a C gamma 1 encoded chain are produced earlier in T cell ontogeny than the C gamma 2 counterparts. The "non-major histocompatibility complex (MHC) requiring" (i.e., "natural killer-like") cytotoxicity mediated by many TcR gamma/delta+ Ti gamma A- cells appeared to be very low as compared to that of Ti gamma A+ clones. Yet, treatment by the OKT3 monoclonal antibody revealed a strong lytic potential in the Ti gamma A- lymphocytes with little, if any, natural killer-like activity. Thus, with respect to the latter function, a substantial heterogeneity is found in cells expressing distinct gamma chains. In an attempt to characterize undefined specificities of Ti gamma A- lymphocytes, they were screened against a panel of Epstein-Barr virus-transformed B cell lines homozygous for HLA-DR1 to DR10 determinants; one of the clones was found to recognize DR7. In light of reports from other groups describing class I-related specificities, it is apparent that TcR gamma/delta+ lymphocytes are able, like the TcR alpha/beta+, to recognize and kill target cells through either an MHC-dependent (with involvement of either class I or class II gene products) or a non-MHC-requiring pathway.