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ABSTRACTThe transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of channels, has a major role in different types of pain. TRPA1 is primarily localized to a subpopulation of primary sensory neurons of the trigeminal, vagal, and dorsal root ganglia. This subset of nociceptors produces and releases the neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP), which mediate neurogenic inflammation. TRPA1 is characterized by unique sensitivity for an unprecedented number of reactive byproducts of oxidative, nitrative, and carbonylic stress and to be activated by several chemically heterogenous, exogenous, and endogenous compounds. Recent preclinical evidence has revealed that expression of TRPA1 is not limited to neurons, but its functional role has been reported in central and peripheral glial cells. In particular, Schwann cell TRPA1 was recently implicated in sustaining mechanical and thermal (cold) hypersensitivity in mouse models of macrophage-dependent and macrophage-independent inflammatory, neuropathic, cancer, and migraine pain. Some analgesics and herbal medicines/natural products widely used for the acute treatment of pain and headache have shown some inhibitory action at TRPA1. A series of high affinity and selective TRPA1 antagonists have been developed and are currently being tested in phase I and phase II clinical trials for different diseases with a prominent pain component.Abbreviations:4-HNE, 4-hydroxynonenal; ADH-2, alcohol dehydrogenase-2; AITC, allyl isothiocyanate; ANKTD, ankyrin-like protein with transmembrane domains protein 1; B2 receptor, bradykinin 2 receptor; CIPN, chemotherapeutic-induced peripheral neuropathy; CGRP, calcitonin gene related peptide; CRISPR, clustered regularly interspaced short palindromic repeats; CNS, central nervous system; COOH, carboxylic terminal; CpG, C-phosphate-G; DRG, dorsal root ganglia; EP, prostaglandins; GPCR, G-protein-coupled receptors; GTN, glyceryl trinitrate; MAPK, mitogen-activated protein kinase; M-CSF, macrophage-colony stimulating factor; NAPQI, N-Acetyl parabenzoquinone-imine; NGF, nerve growth factor; NH2, amino terminal; NKA, neurokinin A; NO, nitric oxide; NRS, numerical rating scale; PAR2, protease-activated receptor 2; PMA, periorbital mechanical allodynia; PLC, phospholipase C; PKC, protein kinase C; pSNL, partial sciatic nerve ligation; RCS, reactive carbonyl species; ROS, reactive oxygen species; RNS, nitrogen oxygen species; SP, substance P; TG, trigeminal ganglion; THC, Δ9-tetrahydrocannabinol; TrkA, neurotrophic receptor tyrosine kinase A; TRP, transient receptor potential; TRPC, TRP canonical; TRPM, TRP melastatin; TRPP, TRP polycystin; TRPM, TRP mucolipin; TRPA, TRP ankyrin; TRPV, TRP vanilloid; VG, vagal ganglion;

ABSTRACTIntroductionChronic pain affects approximatively 30–50% of the population globally. Pathologies such as migraine, diabetic neuropathy, nerve injury and treatment with chemotherapeutic agents, can induce chronic pain. Members of the transient receptor potential (TRP) channels, including the TRP ankyrin 1 (TRPA1), have a major role in pain.Areas coveredWe focus on TRPA1 as a therapeutic target for pain relief. The structure, localization, and activation of the channel and its implication in different pathways to signal pain are described. This paper underlines the role of pharmacological interventions on TRPA1 to reduce pain in numerous pain conditions. We conducted a literature search in PubMed up to and including July 2020.Expert opinionOur understanding of the molecular mechanisms underlying the sensitization of central and peripheral nociceptive pathways is limited. Preclinical evidence indicates that, in murine models of pain diseases, numerous mechanisms converge on the pathway that encompasses oxidative stress and Schwann cell TRPA1 to sustain chronic pain. Programs to identify and develop treatments to attenuate TRPA1-mediated chronic pain have emerged from this knowledge. Antagonists explored as a novel class of analgesics have a new and promising target in the TRPA1 expressed by peripheral glial cells.

Background: Mycobacterium Tuberculosis (Mtb) is the causative pathogen of Tuberculosis (TB) and outbreaks are more common among immunosuppressed persons infected with HIV. The current treatment regimens are lengthy and toxic, yet the therapy has remained unchanged for many decades, so there is a need to find new structures with selective mechanism of action. Moreover, the increased incidence of severe disseminated infections produced by undiagnosed Multidrug-resistant (MDR), worsen clinical treatment and contribute the spread of the disease. Objective: The aim of our study was to evaluate the potential of imidazole and triazole moieties for antimycobacterial activity, by synthesizing some 1-(1-(aryl)-2-(2,6-dichlorophenyl)hydrazono)ethyl- 1H-imidazole and 1H-1,2,4-triazole derivatives 2a-l. Methods: The title compounds were obtained via classical organic synthesis. The antimicrobial activity was evaluated using the method of microdilution and the cytotoxicity assay was performed by MTT method. Results: The results indicated that the presence of both the imidazole ring and that of the 2,6- dichlorosubstituted phenyl moiety, is more relevant for inhibitory activity against Mtb than the triazole nucleus and the unsubstituted phenyl ring. Among the series, (E)-1-(2-(5-chlorothiophen-2-yl)-2-(2- (2,6-dichlorophenyl)hydrazono)ethyl)-1H-imidazole derivative 2f and (Z)-1-(2-([1,1’-biphenyl]-4-yl)- 2-(2-(2,6-dichlorophenyl)hydrazono)ethyl]-1H-imidazole derivatives 2e exhibited a promising antimycobacterial property and the latter also displayed a safe cytotoxic profile. Conclusion: The synthesized compounds were studied for their antitubercular activity. Among the series, the compounds 2e and 2f appeared to be the most promising agents and, according to the docking assessment, the compounds could be CYP51 inhibitors. These evidences could be useful for the future development of new antimycobacterial derivatives targeting CYP51 with more specificity for the mycobacterial cell enzyme.

Tumor microenvironment plays a crucial role in primary cutaneous melanoma (CM) progression. Although the role of tumor-infiltrating lymphocyte (TIL) density has been known for a long time, its spatial distribution and impact with or without tumor-associated macrophages (TAMs) remain controversial. Herein, we investigated spatial proximity between tumor cells and immune cells in 113 primary CM and its correlation with disease-free (DFS) and overall survival (OS). The study cohort included clinical stage II (n = 79) and stage III (n = 34) primary CM with a Breslow thickness of >2 mm (with a median age of 64 years, including 72 men and 41 women). In univariate models, patients with SOX10+ melanoma cells with high proximity to CD8+ TILs in a 20 μm radius showed longer DFS (hazard ratio [HR], 0.58; 95% CI, 0.36–0.93; P = .025) and OS (HR, 0.55; 95% CI, 0.32–0.92; P = .023). Furthermore, at multivariate combined analysis, patients with SOX10+ melanoma cells with high proximity to CD8+ TILs or low proximity to CD163+ TAMs in a 20 μm radius showed an increased OS (aHR, 0.37; 95% CI, 0.14–0.96; P = .04) compared with melanoma patients with low proximity to CD8+ TILs or high proximity to CD163+ TAMs. In a subgroup analysis including 92 patients, a significant negative impact on DFS (aHR, 4.49; 95% CI, 1.73–11.64; P = .002) and OS (aHR, 3.97; 95% CI, 1.37–11.49; P = .01) was observed in sentinel lymph node (SLN)-negative patients with a high proximity of CD163+ TAMs to CD8+ TILs. These findings could help identify high-risk patients in the context of thick melanoma and a negative SLN. Our study suggests the importance of quantifying not only the density of immune cells but also the individual and combined relative spatial distributions of tumor cells and immune cells for clinical outcomes in SLN-negative primary CM patients.

Treatment of metastatic melanoma was radically changed by the introduction of inhibitors of BRAF, an oncogene mutated in 40–50% of patients. Another area of advancement was the use of immunotherapy, and specifically, immune checkpoint inhibitors. There is compelling evidence that oncogenic BRAF, in addition to driving melanoma proliferation, differentiation and survival, induces T-cell suppression directly through the secretion of inhibitory cytokines or through membrane expression of co-inhibitory molecules such as the PD-1 ligands PD-L1 or PD-L2. Furthermore, the presence of oncogenic BRAF leads to an immune suppressive phenotype characterized by the presence of inhibitory immune cells such as regulatory T cells, myeloid-derived suppressor cells, or tumor-associated macrophages, which can in turn inhibit the function of tumor-infiltrating T cells. Growing evidence suggests that, in addition to their established molecular mechanism of action, the therapeutic efficacy of BRAF inhibitors and MEK inhibitors relies on additional factors that affect the tumor–host interactions, including the enhancement of melanoma antigen expression and the increase in immune response against tumor cells. Focus of the present review is to summarize the off target mechanisms of response to BRAF inhibitors and MEK inhibitors and the synergy between targeted therapy and immunotherapy as the biological source to open a window of strategic opportunities for the design of new exciting clinical trials.

Treatment of metastatic melanoma was radically changed by the introduction of inhibitors of BRAF, an oncogene mutated in 40–50% of patients. Another area of advancement was the use of immunotherapy, and specifically, immune checkpoint inhibitors. There is compelling evidence that oncogenic BRAF, in addition to driving melanoma proliferation, differentiation and survival, induces T-cell suppression directly through the secretion of inhibitory cytokines or through membrane expression of co-inhibitory molecules such as the PD-1 ligands PD-L1 or PD-L2. Furthermore, the presence of oncogenic BRAF leads to an immune suppressive phenotype characterized by the presence of inhibitory immune cells such as regulatory T cells, myeloid-derived suppressor cells, or tumor-associated macrophages, which can in turn inhibit the function of tumor-infiltrating T cells. Growing evidence suggests that, in addition to their established molecular mechanism of action, the therapeutic efficacy of BRAF inhibitors and MEK inhibitors relies on additional factors that affect the tumor–host interactions, including the enhancement of melanoma antigen expression and the increase in immune response against tumor cells. Focus of the present review is to summarize the off target mechanisms of response to BRAF inhibitors and MEK inhibitors and the synergy between targeted therapy and immunotherapy as the biological source to open a window of strategic opportunities for the design of new exciting clinical trials.

Growing evidence indicates that transient receptor potential (TRP) channels contribute to different forms of pruritus. However, the endogenous mediators that cause itch through transient receptor potential channels signaling are poorly understood. In this study, we show that genetic deletion or pharmacological antagonism of TRPV4 attenuated itch in a mouse model of psoriasis induced by topical application of imiquimod. Human psoriatic lesions showed increased expression of several microRNAs, including the miR-203b-3p, which induced a calcium ion response in rodent dorsal root ganglion neurons and scratching behavior in mice through 5-HTR2B activation and the protein kinase C‒dependent phosphorylation of TRPV4. Computer simulation revealed that the miR-203b-3p core sequence (GUUAAGAA) that causes 5-HTR2B/TRPV4-dependent itch targets the extracellular side of 5-HTR2B by interacting with a portion of the receptor pocket consistent with its activation. Overall, we reveal the unconventional pathophysiological role of an extracellular microRNA that can behave as an itch promoter through 5-HTR2B and TRPV4.

The presence of tumor-infiltrating lymphocytes (TILs) has been associated with a favorable prognosis of primary melanoma (PM). The recent development of the artificial intelligence (AI)-based approach in digital pathology was proposed for the standardized assessment of TILs on hematoxylin and eosin–stained images [whole slide images (WSI)]. Here, we applied a new convolution neural network (CNN) analysis of PM WSI to automatically assess the infiltration of TILs and extract a TIL score. A CNN was trained and validated in a retrospective cohort of 307 PMs including a training set (237 WSI, 57,758 patches) and an independent testing set (70 WSI, 29,533 patches). After the classification of tumor patches by the presence or absence of TILs, we identified an AI-based TIL density index (AI-TIL). The proposed CNN showed high performance in recognizing TILs in PM WSI, showing specificity and sensitivity of 100% on the testing set. We showed that the AI-based TIL index correlated with conventional TIL evaluation and clinical outcome. The AI-TIL index was an independent prognostic marker associated directly with a favorable prognosis. A fully automated and standardized AI-TIL appears to be superior to conventional methods at differentiating the PM clinical outcome. Further studies are required to develop an easy-to-use tool to assist pathologists to assess TILs in the clinical evaluation of solid tumors.

Actually, autologous fat grafts have many clinical applications in breast surgery, facial rejuvenation, buttock augmentation, and Romberg syndrome as well as a treatment of liposuction sequelae.

ABSTRACTThe 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosisresiding within macrophages as well as against Mycobacterium aviumand other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovisBCG, and M. tuberculosisH37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [14C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.

The activities of six derivatives of a new class of isonicotinoylhydrazones were investigated in vitro against Mycobacterium tuberculosis H37Rv ATCC 27294, isoniazid-resistant M. tuberculosis ATCC 35822, rifampicin-resistant ATCC 35838, pyrazinamide-resistant ATCC 35828, streptomycin-resistant ATCC 35820 and 16 clinical isolates of M. tuberculosis. Several compounds showed interesting antimycobacterial activity against both ATCC strains and clinical isolates, but were less active against isoniazid-resistant M. tuberculosis. Combinations of five isonicotinoylhydrazone derivatives and rifampicin, ethambutol, para-aminosalicylic acid, isoniazid and clofazimine were also investigated against M. tuberculosis H37Rv ATCC 27294 and against ATCC drug-resistant strains. Addition of sub-MICs of some isonicotinoylhydrazone derivatives resulted in a four- to 16-fold reduction in MICs of ethambutol, para-aminosalicylic acid and rifampicin with fractional inhibitory concentrations (FICs) ranging between 0.17 and 0.37, suggesting a synergic interaction against M. tuberculosis H37Rv. Increased activity was also observed with other combinations (FICs 0.53-0.75), including isoniazid, and a synergic interaction between one of the isonicotinoylhydrazone derivatives and isoniazid (FIC 0.26) was shown against isoniazid-resistant M. tuberculosis ATCC 35822, whereas no effects were observed on combining the isonicotinoylhydrazones with clofazimine. The ability of isonicotinoylhydrazones to inhibit specifically the growth of M. tuberculosis, the high selectivity index and their ability to enhance the activity of standard antituberculous drugs in vitro indicate that they may serve as promising lead compounds for future drug development for the treatment of M. tuberculosis infections.

Antibodies and antibody combinations are often evaluated only by their potency in inactivating a known quantity of virus in dose–effect assays. However, a crucial additional parameter is the rate at which neutralization takes place, or kinetics. Synergism of certain antibody combinations in dose–effect assays has been previously demonstrated. In the present report, using a battery of murine monoclonal antibodies to herpes simplex virus (HSV), we investigated whether antiviral antibodies can also synergize in neutralization kinetics. To determine whether synergism in dose–effect assays can predict synergism in neutralization rate, the ability of neutralizing antibodies to synergize in neutralization rate (kinetics) was compared to their ability to synergize in dose–effect assays (potency) in cell-free assays. Although certain antibody combinations synergized in both neutralization rate and potency, combinations that did not clearly synergize in potency could still significantly synergize in neutralization rate. Weak neutralizing antibodies could also greatly increase the neutralization rate of more potent antibodies. These results suggest that evaluating antibody combinations in dose–effect assays but not in neutralization kinetics provides a partial picture of neutralizing antibody dynamic interactions and may prevent the identification of certain favorable antibody combinations. These findings also support the importance of establishing defined antibody cocktails for prophylactic and therapeutic purposes. A simple strategy to evaluate antibody interactions in neutralization kinetics is proposed in which a quantitative prediction of additivity is made on the basis of the neutralization rate constants of the individual antibodies in the combination.

Specific anti-tumor and anti-viral activities can be conferred on lymphocytic and myeloid effector cells by retargeting them with bispecific antibodies. These are antibodies which possess an anti-target binding region and a region capable of binding specific effector cell surface markers. For the rapid evaluation of recombinant human Fabs as bispecific antibodies, we have constructed a vector that allows for the conversion of Fabs into protein A fusion proteins. These can be used to generate bispecific antibodies when complexed to appropriate anti-effector cell immunoglobulins. As a model system, a protein A fusion derivative of a human recombinant anti-herpes simplex virus (HSV) Fab was constructed and complexed to OKT3, a T cell-activating antibody specific for CD3. This complex reduced HSV-2 yields in infected cells by about three logs relative to controls when incubated on HSV-2-infected cell monolayers in the presence of IL-2-actiyated lymphocytes. The system described allows for the rapid evaluation of recombinant human Fabs as bispecific antibodies for therapeutic applications. In addition, Fab-protein A fusion proteins can be used in ELISA and other immuno-assays with increased sensitivity.

Herpes simplex viral disease is an important cause of morbidity and mortality in man. Although the development of very effective nucleoside analogs with a high therapeutic index has greatly improved the clinical management of herpetic infections, the emergence of drug-resistant viral strains has become a cause of serious concern both because of its clinical implications and in terms of viral ecology. The present report is the first demonstration of thein vivoprotective activity of a type-common human recombinant monoclonal antibody derived from a combinatorial antibody library. Athymic nude mice were infected with HSV type 1 either intracutaneously in the flank or by corneal scarification. Beside reducing mortality rates when administered before infection, the antibody dramatically and significantly prolonged survival times (P< 0.0001) when administered up to 24 hr postinfection, a time when the virus had already reached the peripheral nervous system. This suggests that the antibody may act, at least in part, by interfering with axonal transport of the virus and/or with viral expression. These results indicate that human recombinant antibodies isolated by antigen selection from combinatorial libraries can be effectivein vivo.Such antibodies could complement antiviral chemotherapy and represent valuable tools for the prophylaxis of infections by the herpes simplex viruses.

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

ABSTRACTWe report the characterization of a type-common human recombinant monoclonal antibody previously isolated by antigen selection from a phage-displayed combinatorial antibody library established from a herpes simplex virus (HSV)-seropositive individual. Competition with well-characterized murine monoclonal antibodies and immunodetection of gD truncations revealed that this antibody recognizes the group Ib antigenic site of glycoprotein D, a highly conserved and protective type-common determinant. To our knowledge, this is the first human group Ib monoclonal antibody ever described. The antibody also displayed first-order neutralization kinetics and a high neutralization rate constant, was capable of completely inhibiting syncytium formation by a fusogenic strain of HSV type 1, and efficiently neutralized low-passage clinical isolates of both HSV serotypes. Taken together with our earlier observations of the in vivo antiviral activities of this human recombinant antibody in animal models of HSV infection, the present results support the high therapeutic potential of this antibody.

Review: 36 refs.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

&lt;it>Objectives&lt;/it>: The aim of this study was to investigate the &lt;it>in vitro&lt;/it> antifungal activity of an isothiosemicarbazone cyclic analogue against isolates of &lt;it>Candida&lt;/it> spp. including fluconazole-resistant &lt;it>Candida albicans&lt;/it>. &lt;it>Methods&lt;/it>: We investigated the activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole (EM-01D2) against 114 clinical isolates of &lt;it>Candida&lt;/it> spp., representing five different species, by microdilution, according to the NCCLS method 27-A. The activity against &lt;it>C. albicans&lt;/it> biofilms was also investigated. Toxicity &lt;it>in vitro&lt;/it> was evaluated by MTT reduction assay. &lt;it>Results&lt;/it>: EM-01D2 demonstrated low toxicity, broad spectrum, fungicidal activity and was active against &lt;it>C. albicans&lt;/it> and &lt;it>Candida krusei&lt;/it> at concentrations lower than those shown by amphotericin B and fluconazole (&lt;it>P&lt;/it> &lt; 0.05). It maintained potent &lt;it>in vitro&lt;/it> activity against fluconazole-resistant &lt;it>C. albicans&lt;/it> isolates. Fungicidal activity occurred at concentrations 1–2 doubling dilutions greater than the corresponding MICs, and time–kill analysis indicated that a 99.9% loss of &lt;it>C. albicans&lt;/it> viability occurred after 6 h of incubation in the presence of EM-01D2 at concentrations equal to four times the MIC. EM-01D2 was also active in inhibiting the growth of &lt;it>C. albicans&lt;/it> ATCC 10231 biofilms, even though such inhibition occurred at concentrations higher than the MICs determined under planktonic growth conditions. However, when &lt;it>C. albicans&lt;/it> biofilms were pre-exposed to subinhibitory concentrations of EM-01D2, a reduction of MIC&lt;inf>50&lt;/inf> of amphotericin B was observed. &lt;it>Conclusions&lt;/it>: Based on these results, EM-01D2 could represent a template for the development of novel fungicidal agents.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The imidic acid esters (I) react with the arylhydrazones (II) to give mixtures of the carbohydrazonamides (III) and carbohydrazonic acid ethyl esters (IV).

The ethoxyaminoacrylate (I) is coupled with the piperazines (II) to give the amidines (III) which undergo cyclocondensation with the bromoacetophenones (IV), yielding the pyrroles (V).

The thiosemicarbazones (IV), prepared by successive reaction of the thiosemicarbazide (I) with the alkyl bromides (II) and the benzaldehyde (III), are cyclized with the bromoacetophenone (V) to produce the imidazoles (VI).

Reports on the presence of pirates who plunder cargo ships plying the Brazilian waters. Use of scuba gear and submachine guns to raid cargo ships anchored in ports; Brazil's reputation as a haven of sea pirates; Concerns of shippers about the potential impact of piracy on trade.