Evaluating the utility of pre-operative airway assessment for intubation management in difficult airway patients.

OBJECTIVE: To assess intubation management in difficult airway patients by performing a multidisciplinary pre-operative examination of the airway using a flexible fibre-optic laryngoscope. METHODS: Patients with a known but stable difficult airway were evaluated prior to surgery in the pre-operative holding suite by both an ENT surgeon and an anaesthesiologist via a fibre-optic laryngeal examination. RESULTS: Performing a pre-operative fibre-optic examination of the difficult airway led to a change in intubation strategy in 6 out of 12 cases. Intubation 'first-pass' success occurred in 9 out of 12 (75 per cent) of our patients. CONCLUSION: By performing a multidisciplinary airway examination immediately prior to surgery, a safe plan to intubate on the initial attempt was developed. This resulted in improved first-pass success at intubation compared to historical data.

Clinical evaluation of the marginal gingiva as a donor tissue to augment the width of keratinized gingiva: Series of 2 cases with 3-year follow-up.

The indications to increase the width of keratinized gingiva have not been proven beyond doubt; however it becomes indispensable in certain clinical situations. Inspite of frequently encountered complications, palate is considered most preferred area to harvest the free gingival graft (FGG). This procedure aimed at investigating the potential of buccal marginal gingiva as a donor to augment keratinized gingiva. To the best of our knowledge, no such cases have been documented in the literature. FGG harvested from maxillary buccal marginal gingiva was used to augment gingiva in the mandibular anterior region for two patients. This not only improved plaque control but also resulted in acceptable esthetic results over 3 years. Furthermore, gingiva at donor sites gained its normal form and was in harmony with the neighboring teeth. It may be concluded that buccal marginal gingiva may provide a predictable substitute to other donor tissues to augment gingiva.

TL1A/TNFSF15 directly induces proinflammatory cytokines, including TNFα, from CD3+CD161+ T cells to exacerbate gut inflammation.

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A)/TNF superfamily member 15 (TNFSF15) is a proinflammatory cytokine and TNFα superfamily member that is linked preclinically and clinically to inflammatory bowel disease (IBD). By homology and function, TNFα is its closest family member. In this study, we investigated the mechanism of TL1A-induced inflammation in CD4+ T cells and compared it with the TNFα pathway. We found that TL1A induces proinflammatory cytokines, including TNFα, from isolated human CD4+CD161+ T cells, whereas these cells were resistant to TNFα treatment. Anti-TNFα failed to block TL1A-induced cytokine production, indicating that the effects of TL1A are direct. Lastly, CD161 and TL1A expression were significantly and selectively increased in gut tissue biopsies, but not in the peripheral blood, from IBD patients. Thus, TLIA not only functions upstream of TNFα, driving its expression from CD161+ T cells, but is also independent of TNFα. These findings may have therapeutic IBD implications.

A study on clinical attachment loss and gingival inflammation as etiologic factors in pathologic tooth migration.

BACKGROUND: Several etiologic factors have been listed for pathologic migration of periodontally involved teeth based mainly on clinical observations with scarce scientific evidence. Present study was carried out to find out relationship of clinical attachment loss and gingival inflammation with pathologic tooth migration. MATERIALS AND METHODS: A total of 37 patients having 50 pairs of migrated and non-migrated contralateral teeth were taken into consideration. RESULTS: Mean total attachment loss per tooth in migrated and non migrated tooth is 13.32 ± 0.74 S.E. and 8.34 ± 0.58 S.E., respectively (P < 0.001), which reveals a positive correlation. There seems to be an association between frequency of migration and severity of attachment loss since highest percentage of migrations were seen in maximum total attachment loss group. Relationship could not be established between severity of attachment loss and severity of migration for which more data may be required. Also, it was seen that gingival index was significantly higher in migrated group. CONCLUSION: Findings suggest that there exists a direct relationship between pathologic migration and clinical attachment loss as well as gingival inflammation. CLINICAL RELEVANCE: Results emphasize the importance of early treatment of periodontitis to curb inflammation, which seems to be more important since it is completely reversible, and attachment loss also in order to prevent unaesthetic complications. Moreover bleeding along with recent change in position of teeth should be considered as important sign of active, moderate to severe periodontal disease by general dentists and hygienists so that they can refer for specialist consultation.

Unilateral gingival fibromatosis with localized aggressive periodontitis (involving first molars): An unusual case report.

An atypical and rare case report is presented here of a 16 years old female patient who presented with severe, unilateral, gingival enlargement along with aggressive periodontitis around first molars that was confined to the left side of her mouth. A careful recording of the case history and results of clinical examination, laboratory blood analysis, radiological findings, and microbiological and histopathological investigations were noted and a critical review of similar conditions was taken into account to arrive at the said diagnosis.

Inferior mesenteric artery aneurysm with occlusion of the superior mesenteric artery, coeliac trunk and right renal artery.

Inferior mesenteric artery aneurysms are amongst the rarest of visceral aneurysms. We present here a case associated with occlusion of the superior mesenteric artery, coeliac trunk and right renal artery. Operative treatment was resection of the aneurysm, with end-to-end anastomosis. This is the first description of this condition from the UK, with only nine other reports worldwide. Such pathology may be caused by a "jet disorder" phenomenon, with increased flow through the inferior mesenteric artery due to chronic mesenteric occlusive disease.

Protein phosphorylation and signal transduction modulation: chemistry perspectives for small-molecule drug discovery.

Protein phosphorylation has been exploited by Nature in profound ways to control various aspects of cell proliferation, differentiation, metabolism, survival, motility and gene transcription. Cellular signal transduction pathways involve protein kinases, protein phosphatases, and phosphoprotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3-3) containing cellular proteins to provide multidimensional, dynamic and reversible regulation of many biological activities. Knowledge of cellular signal transduction pathways has led to the identification of promising therapeutic targets amongst these superfamilies of enzymes and adapter proteins which have been linked to various cancers as well as inflammatory, immune, metabolic and bone diseases. This review focuses on protein kinase, protein phosphatase and phosphoprotein-interacting cellular protein therapeutic targets with an emphasis on small-molecule drug discovery from a chemistry perspective. Noteworthy studies related to molecular genetics, signal transduction pathways, structural biology, and drug design for several of these therapeutic targets are highlighted. Some exemplary proof-of-concept lead compounds, clinical candidates and/or breakthrough medicines are further detailed to illustrate achievements as well as challenges in the generation, optimization and development of small-molecule inhibitors of protein kinases, protein phosphatases or phosphoprotein-interacting domain containing cellular proteins.

Deciduous dentition and enamel defects.

Two hundred eighty children including wellnourished, malnourished and infants with intrauterine growth retardation (IUGR) were examined for dental eruption and enamel hypoplasia. In malnourished and IUGR children eruption of teeth was delayed. The prevalence of enamel hypoplsia in wellnourished children was 20% being significantly higher in females as compared to males in age group 1-2 years. Enamel hypoplasia was seen in 36.6% malnourished subjects. Breast-feeding was protective against enamel hypoplasia.

Type 1 T regulatory cells.

Suppression by T regulatory (Tr) cells is essential for induction of tolerance. Many types of Tr cells have been described in a number of systems, and their biology has been the subject of intensive investigation. Although many aspects of the mechanisms by which these cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell-to-cell interactions and/or the production of interleukin (IL)-10 and transforming growth factor (TGF)-beta. Type-1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-beta. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL-10 in vitro. Tr1 cells have a low proliferative capacity, which can be overcome by IL-15. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL-10 and TGF-beta. Further characterisation of Tr1 cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance, can be foreseen.

Human B cell-attracting chemokine 1 (BCA-1; CXCL13) is an agonist for the human CXCR3 receptor.

The CXC chemokine CXCL13, known as BCA-1 (B cell-attracting chemokine 1) or BLC (B-lymphocyte chemoattractant), has been identified as an efficacious attractant selective for B lymphocytes. The chemokine receptor BLR1 (Burkitt's lymphoma receptor 1)/CXCR5 expressed by all mature B cells has to date been identified as the only known receptor for BCA-1. As the loss of the BLR1/CXCR5 receptor is sufficient to disrupt organization of follicles in spleen and Peyer's patches, BCA-1 may act as a B cell homing chemokine. Nonetheless, BCA-1 has not been tested against all known chemokine receptors. In this study, we report that human BCA-1 competes with radiolabeled interferon gamma (IFN-gamma) inducible protein 10 (IP-10) for binding to the human CXCR3 receptor expressed in Ba/F3 and 293EBNA cell lines. Furthermore, human BCA-1 is an efficacious attractant for human CXCR3 transfected cells; BCA-1-induced chemotaxis is inhibited by a monoclonal antibody against human CXCR3. In these cells, as in human B lymphocytes expressing CXCR5, BCA-1 does not induce a calcium flux. Indeed, BCA-1 attenuates the calcium flux induced by IP-10. In addition, human BCA-1 is an agonist in stimulating GTP gamma S binding. Together these data suggest that human BCA-1 is a specific and functional G-protein-linked chemotactic ligand for the human CXCR3 receptor. The biological significance of this new finding is supported by our recent observation that human BCA-1 induces chemotaxis of activated T cells and the BCA-1-induced chemotaxis is inhibited by a monoclonal antibody against human CXCR3.

Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility, and premature death.

p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-alpha and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.

Selective inhibition of Src SH2 by a novel thiol-targeting tricarbonyl-modified inhibitor and mechanistic analysis by (1)H/(13)C NMR spectroscopy.

Detailed analysis of Src SH2 binding by peptides containing a novel tricarbonyl-modified pTyr moiety is described. We envisaged that Src SH2 selectivity might be obtained by exploiting the thiol group of Cys188 present in the pTyr binding pocket of the protein at the betaC3 position. Peptidyl as well as non-peptidyl compounds 1-4 possessing a 4-alpha,beta-diketoester-modified pTyr mimic exhibited micromolar affinity to Src SH2. Furthermore, these tricarbonyl compounds were selective for Src SH2 to the extent they showed no significant affinity for either Cys188Ser or Cys188Ala Src SH2 mutants. Upon closer examination of the binding of these tricarbonyls to Src SH2 using NMR of 13C-labeled compounds (6a, 6b, and 6c), we found that after the initial binding event the molecule disproportionated in a 'retro-Claisen' fashion to provide benzoic acid 16 and, following hydrolysis of the methyl ester 17, the hemiketal adduct of glyoxalic acid 18.

Expression and isolation of antimicrobial small molecules from soil DNA libraries.

Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

Aberrant in vivo T helper type 2 cell response and impaired eosinophil recruitment in CC chemokine receptor 8 knockout mice.

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

Human interferon-inducible 10-kDa protein and human interferon-inducible T cell alpha chemoattractant are allotopic ligands for human CXCR3: differential binding to receptor states.

The human CXC chemokines IP-10 (10-kDa interferon-inducible protein), MIG (monokine induced by human interferon-gamma), and I-TAC (interferon-inducible T cell alpha chemoattractant) attract lymphocytes through activation of CXCR3. In the studies presented here, we examined interaction of these chemokines with human CXCR3 expressed in recombinant cells and human peripheral blood lymphocytes (PBL). IP-10, MIG, and I-TAC were agonists in stimulating [(35)S]GTP gamma S binding in recombinant cell and PBL membranes but had no effect in the absence of hCXCR3 expression. (125)I-IP-10 and (125)I-I-TAC bound hCXCR3 with high affinity, although the (125)I-I-TAC B(max) value in saturation bindings was 7- to 13-fold higher than that measured with (125)I-IP-10. Coincubation with unlabeled chemokines decreased (125)I-IP-10 binding with a single discernible affinity. However, with (125)I-I-TAC, competition with IP-10 or MIG was incomplete, and multiple binding affinities were evident. Moreover, in contrast to I-TAC, IP-10 and MIG binding IC(50) values did not increase predictably with increased (125)I-I-TAC concentration in competition bindings, suggesting that these chemokines are noncompetitive (i.e., allotopic) ligands. Uncoupling of hCXCR3 eliminated (125)I-IP-10 binding but only decreased (125)I-I-TAC binding 30 to 80%, indicating that unlike IP-10, I-TAC binds with high affinity to uncoupled (R) and coupled (R*) hCXCR3. To examine chemokine binding to R*, we tested the effect of anti-hCXCR3 antibody on I-TAC- and IP-10-stimulated [(35)S]GTP gamma S binding. The antibody attenuated [(35)S]GTP gamma S binding in response to IP-10 but not to I-TAC, suggesting that the two chemokines bind differently to R*. Moreover, increased occupancy of R* with a >75-fold increase in (125)I -IP-10 concentration did not increase the I-TAC binding IC(50) value, and I-TAC increased the dissociation rate of (125)I-IP-10. From these data, we conclude that the binding of IP-10 and I-TAC to the R* state of hCXCR3 is allotopic.

Altered T-cell receptor + CD28-mediated signaling and blocked cell cycle progression in interleukin 10 and transforming growth factor-beta-treated alloreactive T cells that do not induce graft-versus-host disease.

The induction of anergy in T cells, although widely accepted as critical for the maintenance of tolerance, is still poorly understood at the molecular level. Recent evidence demonstrates that in addition to blockade of costimulation using monoclonal antibodies (mAbs) directed against cell surface determinants, treatment of mixed lymphocyte reaction (MLR) cultures with interleukin 10 (IL-10) and transforming growth factor-beta (TGF-beta) results in induction of tolerance, rendering alloreactive murine CD4(+) T cells incapable of inducing graft-versus-host disease (GVHD) after in vivo transfer to histoincompatible recipients. The present study, using these cells prior to adoptive transfer, determined that IL-10 + TGF-beta-tolerant CD4(+) T cells exhibit an altered pattern of T-cell receptor (TCR) + CD28-mediated signaling and are incapable of progressing out of the G(1) phase of the cell cycle during stimulation with HLA class II disparate antigen-presenting cells. TGFbeta + IL-10-tolerant cells were incapable of phosphorylating TCR-zeta, or activating ZAP-70, Ras, and MAPK, similarly to T-cell tolerized by blockade of B7/CD28 and CD40/CD40L pathways. Moreover, these cells were incapable of clonal expansion due to defective synthesis of cyclin D3 and cyclin A, and defective activation of cyclin-dependent kinase (cdk)4, cdk6, and cdk2. These cells also exhibited defective down-regulation of p27(kip1) cdk inhibitor and lack of cyclin D2-cdk4 activation, Rb hyperphosphorylation, and progression to the S phase of the cell cycle. These data link anergy-specific proximal biochemical alterations and the downstream nuclear pathways that control T-cell expansion and provide a biochemical profile of IL-10 + TGF-beta-tolerant alloreactive T cells that do not induce GVHD when transferred into MHC class II disparate recipients in vivo.

Structure-based design of an osteoclast-selective, nonpeptide src homology 2 inhibitor with in vivo antiresorptive activity.

Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3',4'-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC(50) = 0.30 microM for AP22408 and 5.5 microM for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model.

CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue.

Chemokine-directed migration of leukocyte subsets may contribute to the qualitative differences between systemic and mucosal immunity. Here, we demonstrate that in mice lacking the chemokine receptor CCR6, dendritic cells expressing CD11c and CD11b are absent from the subepithelial dome of Peyer's patches. These mice also have an impaired humoral immune response to orally administered antigen and to the enteropathic virus rotavirus. In addition, CCR6(-/-) mice have a 2-fold to 15-fold increase in cells of select T lymphocyte populations within the mucosa, including CD4+ and CD8+ alphabeta-TCR T cells. By contrast, systemic immune responses to subcutaneous antigens in CCR6(-/-) mice are normal. These findings demonstrate that CCR6 is a mucosa-specific regulator of humoral immunity and lymphocyte homeostasis in the intestinal mucosa.

Signaling by covalent heterodimers of interferon-gamma. Evidence for one-sided signaling in the active tetrameric receptor complex.

Interferon-gamma (IFN-gamma) and its receptor complex are dimeric and bilaterally symmetric. We created mutants of IFN-gamma that bind only one IFN-gammaR1 chain per dimer molecule (called a monovalent IFN-gamma) to see if the interaction of IFN-gamma with one-half of the receptor complex is sufficient for bioactivity. Mutating a receptor-binding sequence in either AB loop of a covalent dimer of IFN-gamma yielded two monovalent IFN-gammas, gamma(m)-gamma and gamma-gamma(m), which cross-link to only a single soluble IFN-gammaR1 molecule in solution and on the cell surface. Monovalent IFN-gamma competes fully with wild type IFN-gamma for binding to U937 cells but only at a greater than 100-fold higher concentration than wild type IFN-gamma. Monovalent IFN-gamma had anti-vesicular stomatitis virus activity and antiproliferative activity, and it induced major histocompatibility complex class I and class II (HLA-DR) expression. In contrast, the maximal levels of activated Stat1alpha produced by monovalent IFN-gammas after 15 min were never more than half of those produced by either wild type or covalent IFN-gammas in human cell lines. These data indicate that while monovalent IFN-gamma activates only one-half of a four-chain receptor complex, this is sufficient for Stat1alpha activation, major histocompatibility complex class I surface antigen induction, and antiviral and antiproliferative activities. Thus, while interaction with both halves of the receptor complex is required for high affinity binding of IFN-gamma and efficient signal transduction, interaction with only one-half of the receptor complex is sufficient to initiate signal transduction.