Involvement of the Inflammasome and Th17 Cells in Skin Lesions of Human Cutaneous Leishmaniasis Caused by Leishmania (Viannia) panamensis.

Localized cutaneous leishmaniasis (LCL) caused by Leishmania (Viannia) panamensis is an endemic disease in Panama. This condition causes ulcerated skin lesions characterized by a mixed Th1/Th2 immune response that is responsible for disease pathology. However, the maintenance of the in situ inflammatory process involves other elements, such as Th17 and inflammasome responses. Although these processes are associated with parasite elimination, their role in the increase in disease pathology cannot be discarded. Thus, the role in Leishmania infection is still unclear. In this sense, the present study aimed at characterizing the Th17 and inflammasome responses in the skin lesions of patients with LCL caused by L. (V.) panamensis to help elucidate the pathogenesis of this disease in Panama. Th17 and inflammasome responses were evaluated by immunohistochemistry (IHQ) in 46 skin biopsies from patients with LCL caused by L. (V.) panamensis. The Th17 immune response was assessed using CD3, CD4, RoRγt, IL-17, IL-6, IL-23, and TGF-ß1 antibodies, and the inflammasome response was assessed by IL-1ß, IL-18, and caspase-1 antibodies. The presence of the Th17 and inflammasome responses was evidenced by a positive reaction for all immunological markers in the skin lesions. An inverse correlation between the density of amastigotes and the density of RoRγt+, IL-17+, IL-1ß +, and caspase-1+ cells was observed, but no correlation between Th17 and the inflammasome response with evolutionary disease pathology was reported. These data showed the participation of Th17 cells and the inflammasome in the inflammatory response of the skin lesions of LCL caused by L. (V.) panamensis infection. These results suggest a role in the control of tissue parasitism of IL-17 and the activation of the NLRP3 inflammasome dependent on IL-1ß but cannot exclude their role in the development of disease pathology.

Differential Recruitment of Dendritic Cells Subsets to Lymph Nodes Correlates with a Protective or Permissive T-Cell Response during Leishmania (Viannia) Braziliensis or Leishmania (Leishmania) Amazonensis Infection.

Leishmania (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a large clinical and immunopathological spectrum in human disease; while La may be responsible for anergic disease, Lb infection leads to cellular hypersensitivity. To better understand the dichotomy in the immune response caused by these Leishmania species, we evaluated subsets of dendritic cells (DCs) and T lymphocyte in draining lymph nodes during the course of La and Lb infection in BALB/c mice. Our results demonstrated a high involvement of DCs in La infection, which was characterized by the greater accumulation of Langerhans cells (LCs); conversely, Lb infection led to an increase in dermal DCs (dDCs) throughout the infection. Considering the T lymphocyte response, an increase of effector, activated, and memory CD4(+) T-cells was observed in Lb infection. Interleukin- (IL-) 4- and IL-10-producing CD4(+)and CD8(+) T-cells were present in both La and Lb infection; however, interferon- (IFN-) γ-producing CD4(+)and CD8(+) T-cells were detected only in Lb infection. The results suggest that during Lb infection, the dDCs were the predominant subset of DCs that in turn was associated with the development of Th1 immune response; in contrast La infection was associated with a preferential accumulation of LCs and total blockage of the development of Th1 immune response.

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison.

Multiple-locus variable-number tandem-repeat analysis of Francisella tularensis from Quebec, Canada.

UNLABELLED: Francisella tularensis is ubiquitous in the Northern Hemisphere. Yet, little is known about the disease and its ecology within Canada as few serological studies have shown exposure to the disease and fewer case studies have been reported. This report is the first to describe the molecular subtyping of F. tularensis isolates within eastern Canada using multiple-locus variable-number tandem-repeat analysis. From 1998 to 2011, a total of 73 specimens were isolated from unique human and animal sources. As expected, F. tularensis subsp. tularensis AI and F. tularensis subsp. holarctica subtypes were observed, corresponding to the known geographical division within this species. The majority of human isolates (78%) and all animal (hare) isolates were of the more virulent, AI type. Half of the B isolates were isolated from patients living in a region of Quebec where muskrat densities are known to be high. A relatively high level of marker diversity was found, suggestive of multiple introductions of the organism to the region, or more likely ongoing endemicity. There was no evidence of ongoing outbreaks or transmission, and the bulk of cases were likely due to interaction between human activity and the environment (e.g. hunting/trapping activities). SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals the diversity of Francisella tularensis in eastern Canada using multiple-locus variable-number tandem-repeat analysis. It was initiated to further the understanding of the species within North America as previous studies elucidating the diversity and phylogeography of the species have consisted mostly of specimens from the United States. Type A tularaemia, the most life-threatening subtype of the species and a Category A biothreat agent, is restricted to North America, and this study serves to broaden the knowledge of the epidemiology and diversity of the organism.

Two novel toxin variants revealed by whole-genome sequencing of 175 Clostridium botulinum type E strains.

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay.

Whole-genome sequencing drug susceptibility testing is associated with positive MDR-TB treatment response.

Until recently, multidrug-resistant TB (MDR-TB) was treated with lengthy and toxic regimens. New three-drug anti-TB regimens raise the question of whether they are sufficiently active for MDR-TB in Central Asia, an MDR-TB hotspot region.In a cohort of rifampicin-resistant (RR) and MDR-TB patients in the Kyrgyz Republic, we investigated the impact of the number of drugs that were tested susceptible by whole-genome sequencing (WGS) and conventional drug susceptibility testing (DST) and used for treatment on the treatment response, defined as 'matches'. Logistic regressions were performed to assess the effect of having ≥ 4 susceptible drugs in a regimen at baseline and at Month 2 on the treatment response.The study included 227 participants with RR/MDR-TB (30.8% female; median age 30.4 years). The age- and sex-adjusted analysis showed an association between a regimen with ≥ 4 WGS matches at baseline (adjusted odds ratio [aOR] 2.10, 95% CI 1.00-4.41). No association was found when using conventional DST to define matches.Our study confirms that the inclusion of four efficacious anti-TB drugs in an MDR-TB regimen increases the chances of a positive treatment response. Susceptibility of at least four drugs in WGS-DST predicts a positive treatment response..

Operator training requirements and diagnostic accuracy of Fibroscan in routine clinical practice.

BACKGROUND: Fibroscan is a quick, non-invasive technique used to measure liver stiffness (kPa), which correlates with fibrosis. To achieve a valid liver stiffness evaluation (LSE) the operator must obtain all the following three criteria: (1) ≥10 successful liver stiffness measurements; (2) IQR/median ratio <0.30 and (3) ≥60% measurement success rate. OBJECTIVES: To assess the operator training requirements and the importance of adhering to the LSE validity criteria in routine clinical practice. METHODS: We retrospectively analysed the LSE validity rates of 2311 Fibroscans performed (1 August 2008 to 31 July 2011) in our tertiary liver outpatients department at the University Hospital Birmingham, UK. The diagnostic accuracy of Fibroscan was assessed in 153 patients, by comparing LSE (valid and invalid) with the modified Ishak fibrosis stage on liver biopsy. RESULTS: Learning curve analysis highlighted that the greatest improvement in validity of LSE rates occurs in the operator's first 10 Fibroscans, reaching 64.7% validity by the 50th Fibroscan. The correlation between LSE and the fibrosis stage on liver biopsy was superior in patients with a valid LSE (n=97) compared with those with an invalid LSE (n=56) (rs 0.577 vs 0.259; p=0.022). Area under receiving operating characteristics for significant fibrosis was greater when LSE was valid (0.83 vs 0.66; p=0.048). Using an LSE cut-off of 8 kPa, the negative predictive value of valid LSE was superior to invalid LSE for the detection of significant (84% vs 71%) and advanced fibrosis (100% vs 93%). CONCLUSIONS: Fibroscan requires minimal operator training (≥10 observed on patients), and when a valid LSE is obtained, it is an accurate tool for excluding advanced liver fibrosis. To ensure the diagnostic accuracy of Fibroscan it is essential that the recommended LSE validity criteria are adhered to in routine clinical practice.

Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model.

The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4(+) and CD8(+) immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207(+), CD11c(+), CD4(+), CD8(+), iNOS(+) cellular densities. Cytokine (IFN-γ, IL-4 and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207(+) and CD11c(+) were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4(+) and CD8(+) were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS(+) was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-γ was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response.

Secondary amyloidosis in Indigenous Australians.

Secondary amyloidosis (AA) is an established consequence of many chronic inflammatory conditions. In the developed world, it is most often the result of rheumatological disease. However, the relative frequency of underlying causes may be different in indigenous populations. We present a case series of three remote-living, Indigenous Australians found to have pathologically confirmed amyloidosis and renal impairment at diagnosis. The presence of an underlying inflammatory condition was unclear in two cases. The remaining case had established bronchiectasis and suffered rapidly progressive renal impairment at a young age.

Development of real-time PCR assays for detection of the Streptococcus milleri group from cystic fibrosis clinical specimens by targeting the cpn60 and 16S rRNA genes.

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.

Boomslang (Dispholidus typus) envenomation in a patient on warfarin therapy.

The venom of the boomslang (Dispholidus typus) has potent effects on the coagulation system. It is known to produce a venom-induced consumptive coagulopathy (VICC) through the proposed activation of clotting factor II (prothrombin), factor X, and possibly factor IX. Warfarin, an anticoagulant medication, decreases the circulating vitamin K-dependent clotting factors II, VII, IX and X. We report a unique case of a boomslang bite in a patient on warfarin therapy. During the patient's hospital stay he developed abnormal clotting profiles indicating an underlying VICC, but without major bleeding. He received monovalent antivenom and recovered with no complications. We discuss two possible outcomes of a boomslang bite in a patient on warfarin therapy, exploring the underlying pathophysiology that could lead to the presentation of a reduced risk of overall bleeding or, alternatively, that the bleeding could be compounded and exacerbated. It is possible that in our case the anticoagulant effect of warfarin was wholly obscured by the VICC of the boomslang venom. The composition of the snake venom may have been a contributory factor in the reduced clinical bleeding observed.

Effects of salivary gland homogenate from wild-caught and laboratory-reared Lutzomyia longipalpis on the evolution and immunomodulation of Leishmania (Leishmania) amazonensis infection.

We investigated the effects of Lutzomyia longipalpis salivary glands homogenate of wild-caught and laboratory-reared vectors on the lesion evolution and immunomodulation of the infection caused by Leishmania (Leishmania) amazonensis. To compare the effect of both salivary glands homogenate (SGH), C57BL/6 mice were inoculated subcutaneously into the hind footpads or into the ear dermis with 10(6) promastigotes in the presence or not of SGH from wild-caught and laboratory-colonized sand flies. Comparing SGH groups, the lesion size was lower in mice co-inoculated with wild-caught SGH, as the parasitism and the infiltration of macrophages at the inoculation site. Wild-caught SGH also determined lower production of IL-4 and IL-10 but higher IL-12 levels compared with laboratory-reared SGH. Our findings address a probable bias by using SGH from laboratory-colonized sand flies instead of wild-caught vector SGH in studies concerning saliva effects. A possible mild influence of sand fly saliva in natural infections caused by Leishmania is also speculated, as infection is transmitted by wild and not by laboratory-reared vectors.

Immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis in American cutaneous leishmaniasis.

The immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis were reviewed in the light of more recent features found in the clinical and immunopathological spectrum of American cutaneous leishmaniasis. It was shown a dichotomy in the interaction between these Leishmania species and human T-cell immune response; while L. (V.) braziliensis shows a clear tendency to lead infection from the localized cutaneous leishmaniasis (LCL), a moderate T-cell hypersensitivity form at the centre of the spectrum, toward to the mucocutaneous leishmaniasis (MCL) at the T-cell hypersensitivity pole and with a prominent Th1-type immune response, L. (L.) amazonensis shows an opposite tendency, leading infection to the anergic diffuse cutaneous leishmaniasis (ADCL) at the T-cell hyposensitivity pole and with a marked Th2-type immune response. Between the central LCL and the two polar MCL and ADCL, the infection can present an intermediary form known as borderline disseminated cutaneous leishmaniasis, characterized by an incomplete inhibition of T-cell hypersensitivity but with a evident supremacy of Th1 over Th2 immune response (Th1 > or = Th2). These are probably the main immunopathogenic competences of L. (V.) braziliensis and L. (L.) amazonensis regarding the immune response dichotomy that modulates human infection outcome by these Leishmania parasites.

Experience gained by basic surgical trainees in varicose vein surgery over a 15 year period - has it changed?

OBJECTIVE: The aim of this study was to see if the training provided for Basic Surgical Trainees (BST's) by one consultant vascular surgeon has changed over a 15-year period. METHOD: From a computerized database we have a 15-year record of varicose vein operations identifying the first and second surgeon. We have analysed cases involving the BST and those in which the consultant operated alone. In such instances training opportunities were lost. RESULTS: No change was found in the total number of cases performed, the number of operations carried by the BST as the first surgeon, or the cases performed by the consultant operating alone. The number of operations performed by a BST annually correlated positively with the total number of cases. A BST was named as first surgeon in 39% of cases (632/1622). CONCLUSION: This study has shown no definite evidence to support the view that training in varicose vein surgery has deteriorated.

First Results and Experience with PRRT in South Africa.

Neuroendocrine tumors (NETs) are a diverse group of tumors that often present late due to nonspecific symptoms. These tumors frequently express somatostatin receptors (SSRs), which allows for positron emission tomography/computed tomography (PET/CT) imaging with Ga-68-DOTATATE. In eligible patients, this may then be followed by peptide receptor radionuclide therapy (PRRT). Here, we report our initial results and experience with PRRT in a developing country, as one of the first groups to provide this therapy in South Africa. Eligible patients with confirmed inoperable NETs were recruited prospectively and treated with Lu-177-DOTATATE. Baseline imaging was performed with either single-photon emission CT- or PET-based SSR analogs, whereas follow-up was performed with 68Ga-DOTATATE PET/CT 6 months post treatment completion. Interim treatment response evaluation was based on post therapy imaging of Lu-177-DOTATATE. A total of 48 patients with a mean age of 58 years were treated with PRRT, of whom 22 (46%) demonstrated stable disease, 20 (42%) demonstrated a partial response, and 6 (12%) demonstrated progressive disease. The median progression-free survival (PFS) was 20 months with an interquartile range (IQR)25%-75% of 4.5-30 months. The median freedom from progression duration was 32 months with an IQR25%-75% of 25-40 months, and the median overall survival was 10 months with an (IQR)25%-75% of 5-24 months. Our subgroup analysis demonstrated an inverse association between metabolic tumor volume with PFS, which requires further validation. In conclusion, PRRT with Lu-177-DOTATATE resulted in a median PFS of 20 months in patients with inoperable NETs in the absence of significant side effects.

Anisotropy and Strain Localization in Dynamic Impact Experiments of Tantalum Single Crystals.

Deformation mechanisms in bcc metals, especially in dynamic regimes, show unusual complexity, which complicates their use in high-reliability applications. Here, we employ novel, high-velocity cylinder impact experiments to explore plastic anisotropy in single crystal specimens under high-rate loading. The bcc tantalum single crystals exhibit unusually high deformation localization and strong plastic anisotropy when compared to polycrystalline samples. Several impact orientations - [100], [110], [111] and [[Formula: see text]] - are characterized over a range of impact velocities to examine orientation-dependent mechanical behavior versus strain rate. Moreover, the anisotropy and localized plastic strain seen in the recovered cylinders exhibit strong axial symmetries which differed according to lattice orientation. Two-, three-, and four-fold symmetries are observed. We propose a simple crystallographic argument, based on the Schmid law, to understand the observed symmetries. These tests are the first to explore the role of single-crystal orientation in Taylor impact tests and they clearly demonstrate the importance of crystallography in high strain rate and temperature deformation regimes. These results provide critical data to allow dramatically improved high-rate crystal plasticity models and will spur renewed interest in the role of crystallography to deformation in dynamics regimes.

Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection.

In the last decades, many regional and country-wide control programmes for Johne's disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn's disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of cattle and restrict within-farm and farm-to-farm transmission. Although JD control programmes have been implemented in most developed countries, lessons learned from JD prevention and control programmes are underreported. Also, JD control programmes are typically evaluated in a limited number of herds and the duration of the study is less than 5 year, making it difficult to adequately assess the efficacy of control programmes. In this manuscript, we identify the most important gaps in knowledge hampering JD prevention and control programmes, including vaccination and diagnostics. Secondly, we discuss directions that research should take to address those knowledge gaps.

In vitro behavior of human intestinal mucosa. The influence of acetyl choline on ion transport.

The possibility that the autonomic nervous system may influence the function of intestinal mucosa was investigated by assessing the effect of acetyl choline on ion transport in human intestine. Isolated pieces of stripped ileal mucosa were mounted in Perspex flux-chambers and bathed in isotonic glucose Ringer's solution. Acetyl choline caused a rise in mean potential difference (8.8-12.3 mV, P less than 0.002) and short circuit current (287.7-417.2 muA-cm-2, P less than 0.01) (n = 12), observable at a concentration of 0.01 mM and maximal at 0.1 mM. This effect was enhanced by neostigmine and blocked by atropine. Isotopic flux determinations revealed a change from a small mean net Cl absorption (58) to a net Cl secretion (-4.3mueq-cm-2-h-1P less than 0.001) due predominantly to an increase in the serosal to mucosal unidirectional flux of Cl (10.63-14.35 mueq-cm-2-h-1P less than 0.05) and a smaller reduction in the mucosal to serosal flux (11.22 to 10.02 mueq-cm-2-h-1P less than 0.05). Unidirectional and net Na transport was unaffected. A similar electrical and ion transport response was observed in a single study of two pieces of jejunal mucosa. In the absence of glucose net chloride secretion was produced and again an insignificant effect on net sodium transport was noted. Acetyl choline did not provoke a sustained effect on mucosal cyclic adenine nucleotide levels although a short-lived cyclic adenine nucleotide response was seen in some tissues 20-30 s after drug addition. These studies demonstrate that acetyl choline does influence human intestinal ion transport by stimulating chloride secretion and suggest a possible mechanism by which the parasympathetic nervous system could be concerned in the control of ion transport.

Soluble signals from mechanically disrupted lung tissue induce lung-related gene expression in bone marrow-derived cells in vitro.

Differentiation of bone marrow (BM)-derived cells into lung epithelial cells has been reported in vivo in animal models of lung injury. Most studies have used cytokeratin or surfactant protein expression as markers of BM-to-lung cell differentiation. However, concerns as to whether fusion rather than differentiation is the mechanism involved, verification of BM-derived lung cells, and inconsistent findings with different injury models mean that the differentiation potential of BM-derived cells remains unclear. We used a co-culture system, in which BM cell-lung cell fusion is prevented, to examine the ability of 'damage' signals released from mechanically disrupted lung tissue to induce expression of lung-related genes in BM-derived cells in vitro. BM-derived hematopoietic progenitor cells (BM-HPCs) were co-cultured with mechanically disrupted lung tissue. Liver tissue and medium-only co-cultures were also studied as controls. BM-HPCs differentiated into myeloid cells in culture. BM-HPCs proliferated in response to soluble lung damage signals and differentiated into suspension and adherent populations with dendritic cell and Langerhans cell-like characteristics, respectively. Cytokeratins 7 and 18 and surfactant protein B mRNA expression was either induced or upregulated in the dendritic cell (DC)-like population in lung co-cultures. In contrast, these genes were not induced or up-regulated in medium only or liver co-cultures. Up-regulation of E-cadherin mRNA and protein expression also occurred in response to lung damage signals. These results confirm that signals released from damaged lung tissue can induce lung-related gene expression in BM-derived DC-like cells in the absence of cell fusion.