Opa+ and Opa? Isolates of Neisseria meningitidis and Neisseria gonorrhoeae Induce Sustained Proliferative Responses in Human CD4+ T Cells

T cells may interact with a number of bacterial surface antigens, an encounter which has the potential to downmodulate host immune responses. Neisseria meningitidis, a human colonizer and an agent of septicemia and meningitis, expresses Opa proteins which interact with the CEACAM1 receptor expressed on activated T cells. Since CEACAM1 can act as an inhibitory receptor and T cells in subepithelial tissues may encounter whole bacteria, which often express Opa proteins in vivo, this study assessed primarily if Opa proteins expressed on meningococci affect T-cell functions. In addition, Opa-containing outer membrane vesicles (OMV) have been used as vaccine antigens, and therefore Opa⁺ and Opa⁻ OMV were also studied. While Opa⁺ bacteria adhered to CEACAM-expressing T cells, both the Opa⁺ and Opa⁻ phenotypes induced no to a small transient depression, followed by a prolonged increase in proliferation as well as cytokine production. Such responses were also observed with heat-killed bacteria or OMV. In addition, while anti-CEACAM antibodies alone inhibited proliferation, on coincubation of T cells with bacteria and the antibodies, bacterial effects predominated and were Opa independent. Thus, while Opa proteins of N. meningitidis can bind to T-cell-expressed CEACAM1, this is not sufficient to overcome the T-cell recognition of bacterial factors, which results in a proliferative and cytokine response, an observation consistent with the ability of the host to establish lasting immunity to Opa-expressing meningococci that it frequently encounters. The data also imply that Opa-proficient vaccine preparations may not necessarily inhibit T-cell functions via CEACAM1 binding.Neisseria meningitidis (meningococci) and Neisseria gonorrhoeae (gonococci), which are highly related at the genetic and antigenic levels, are human-specific mucosal bacteria capable of causing localized or systemic infection. N. meningitidis may colonize the human respiratory mucosal tissue of 3 to 30% of healthy individuals asymptomatically but, in some situations, may penetrate into deeper tissues to cause invasive diseases, such as septicemia and meningitis (5). N. gonorrhoeae may also be carried asymptomatically in a few individuals (13), but in most cases it causes localized infections of urogenital mucosa, and in a few untreated gonorrhea patients, disseminated infection may develop (20).Immune responses to mucosal bacteria are initiated at mucosa-associated lymphoid tissue, where CD4⁺ T-cell priming occurs and results in the generation of effector and memory T cells (12). Bacterial colonization, and the subsequent disease process in susceptible individuals, begins with adhesion to specific receptors on human mucosal epithelial cells. N. meningitidis and N. gonorrhoeae express colony opacity-associated (Opa) proteins in vitro and in vivo that enable them to attach to human cells. It is now well established that the major receptors targeted by the Opa proteins belong to the CEACAM (carcinoembryonic antigen-related cell adhesion molecule) family of receptors (6, 44, 45). CEACAM1 is one of several related molecules expressed on human epithelial cells, endothelial cells, and leukocytes, but CEACAM1 is the only member of the family expressed on T cells (19, 28). CEACAM1 is a transmembrane molecule with either a long (L) or a short (S) cytoplasmic tail. CEACAM1-L, with a long cytoplasmic tail, contains two tyrosine residues which form part of modified immunoreceptor tyrosine-based activation/inhibition motifs (ITAM/ITIM motifs) (16). The relative abundance of the isoforms, which may be present simultaneously in CEACAM1-expressing tissues, may dictate the signaling outcomes of CEACAM1 ligation (35).In addition, Opa structural variations may also affect bacterial specificity and affinity for distinct CEACAMs (10, 40, 44). N. meningitidis and N. gonorrhoeae possess multiple complete copies of opa genes (up to 4 and 11 genes, respectively), with the consequence that distinct isolates may express structurally variant Opa proteins. Variations within the Opa family of transmembrane proteins occur in three of the four surface-exposed loops. It has been shown for strains of distinct serogroups of N. meningitidis that these variations influence the specificity of Opa proteins for different members of the CEACAM family (10, 40). Different meningococcal isolates further possess a wide range of opa alleles, variable regions, and repertoires. Particular Opa repertoires appear to correlate with hyperinvasiveness and disease but not with the severity of meningococcal disease (4). The host cell interface where Opa proteins exert such an influence remains to be defined, and while several studies have assessed the potential of Opa proteins to influence meningococcal interactions with human epithelial cells (15, 44), a limited number of studies have examined their effect on T cells (23), and none have studied the potential of live Opa-expressing meningococci to influence T-cell functions.Previous studies have shown that a number of neisserial outer membrane proteins can modulate T-cell function. Of these, TspA (T-cell-stimulating protein A), IgA1 protease, pili, and porins can induce a proliferative response in T cells (27, 31, 34, 38). In contrast, an interaction of Opa⁺ N. gonorrhoeae with CEACAM1 inhibited immune responses of CD4⁺ T cells (2) and B cells (26). In the case of T cells, the inhibitory signal delivered by the N. gonorrhoeae Opa-CEACAM1 interaction was reported to involve the phosphotyrosine phosphatases 1 and 2 (SHP-1 and SHP-2) that interact with ITIM (2, 24). Interestingly, engagement of Opa⁺ N. gonorrhoeae with CEACAM1 on B cells occurred independently of ITIM involvement (26). Overall, the above reports highlight the following two important points: (i) with respect to cellular activation, the end product of neisseria-target cell interactions may be determined by a number of distinct bacterial and host cell characteristics; and (ii) in the context of the consequences of bacterial engagement with CEACAM1, such an interaction may not always bring into play the expected consequences of its ITIM-like motifs. Other notable observations include the following. Outer membrane vesicles (OMV) of some Opa-expressing N. meningitidis strains have been reported to inhibit CD4⁺ T-cell function (23), which is in line with CD4⁺ T-cell-inhibiting effects of Opa⁺ N. gonorrhoeae (2). However, N. meningitidis carriage is regarded as an immunizing event and has been shown to induce lasting T-cell memory (7, 8).Collectively, these previous reports highlight the need for comprehensive studies to define the consequences of meningococcal interactions with cells of the human immune system, particularly as mucosal bacteria are increasingly being shown to reside in subepithelial tissues, where they may encounter T cells. This may occur, and since no comprehensive studies are yet available on T-cell responses that may ensue upon encountering live N. meningitidis, and particularly on the influence of Opa proteins in general when presented in whole bacteria, the focus of this study was to assess the immune responses of CD4⁺ T cells to well-characterized phenotypes of live N. meningitidis and to compare these with responses to Escherichia coli cells either expressing or lacking meningococcal Opa proteins. In addition, we compared the effects of heat-killed N. meningitidis and OMV derived from a meningococcal serogroup B strain on T-cell proliferation. The latter preparations are likely to be used as vaccine preparations and therefore, in our view, warranted such studies. In order to assess the T-cell responses to other CEACAM-binding agents and to study the effects of bacterial presence when Opa-CEACAM interactions are inhibited, the studies included cross-linking antireceptor antibodies as well as a recombinant molecule, rD-7, which carries the CEACAM-binding motif of Moraxella catarrhalis UspA1 adhesin (17) and has the potential to block bacterial binding without inducing a signaling cascade in T cells because the recombinant peptide may not cross-link CEACAM1 on T cells.In our studies, frequently, but not invariably, an early Opa-independent transient decrease in T-cell proliferation was observed. This phase was followed by a profound stimulatory effect on T-cell immune functions, as assessed by proliferation assays and cytokine responses. In contrast, using anti-CEACAM1 antibodies in analogous assays, a significant inhibition of T-cell proliferation was observed. Overall, these data show that a certain surface component(s) of pathogenic Neisseria, whose precise identity remains to be determined, can exert either mild inhibitory or strong stimulatory effects on CD4⁺ T cells and that, most importantly, the latter predominate. Thus, it appears that human CD4⁺ T cells respond positively to one or more bacterial antigens to overcome any inhibition that may be induced via the engagement of CEACAM1, perhaps representing an advanced counterstrategy of the host.     

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an “advanced unmixing process”, identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.     

Automated system for on-line desorption of dried blood spots applied to LC/MS/MS pharmacokinetic study of flurbiprofen and its metabolite

This paper illustrates the development of an automated system for the on-line bioanalysis of dried blood spots (on-line DBS). In this way, a prototype was designed for integration into a conventional LC/MS/MS, allowing the successive extraction of 30 DBS toward the analytical system without any sample pretreatment.     

Multiparity and Breast Cancer Risk Factor among Women in Burkina Faso

The relative lack of information on breast cancer etiology in Burkina Faso led us to undertake the present work to highlight risk factors. This prospective study was conducted using a questionnaire between January 2015 and February 2016 on women admitted to Yalgado OUEDRAOGO hospital, for consultation or supervision. The characteristics of multiparous breast cancer patients (n = 44) were compared with their non-multiparous counterparts (n = 36). The study found that increased risk of breast cancer among non-multiparous cases was related to body mass index (BMI) (p <0.001), age at menopause (p <0.004) and use of oral contraception (p <0.021) while abortion (p <0.002) was a risk factor among multiparous cases. These results suggest that even if multiparity is associated with a decreased risk in some women, avoidance of abortion during reproductive life should be recommended. The results provide preliminary information, which now need to be supplemented by survey of a larger sample in the national territory.     

Laser assisted periodontics: A review of the literature

Over the years, the use of the laser within health field and more particularly dentistry has been increasing and improving. The application of laser in the periodontal treatment takes part of a non-surgical and surgical approaches, is used for the decontamination of periodontal pockets due to its bactericidal effect, and the removal of granulation tissues, inflamed and diseased epithelium lining, bacterial deposits and subgingival calculus. However in spite of all the marketing surrounding, the use of laser highlighting its beneficial effect, the capacity of laser to replace the conventional treatment for chronic periodontitis is still debatable. In fact there is no evidence that any laser system adds substantial clinical value above conventional treatments of chronic periodontitis. Some studies showed a significant positive effect on clinical attachment level gain and probing depth reduction. In the other hand, several articles demonstrated no evidence of the superior effectiveness of laser therapy compared to root planing and scaling. Our aims is to review the literature on the capacity of erbium:Yttrium-aluminum-garnet and neodymium:Yttrium-aluminium-garnet laser to either replace or complete conventional mechanical/surgical periodontal treatments.     

Pediatric gastrointestinal motility--future directions and challenges

The study of gastrointestinal motility has evolved to a sophisticated diagnostic technique that is widely used clinically to further guide management of children with complex gastrointestinal problems. Thorough comprehension requires a multidisciplinary approach with the integration of molecular and cell biology, organ physiology, and clinical observations. During the past decade there has been a dramatic increase in our knowledge of the enteric neuromuscular system. Continued exploration of targeted gene mutations in animal models has the potential of enhancing our understanding of congenital disorders of gastrointestinal motility. Experiments studying polymorphisms in serotonin transporter gene (SERT) and different therapeutic responses to serotonergic agents in adults with irritable bowel syndrome need to be carried out in children with functional bowel disorders. Additional considerations that need to be addressed if advances are to continue include increasing the number of specialists interest in motility disorders and identifying funding sources to support the establishment of research consortiums among pediatric centers.