Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

Background

Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood.

Methodology/Principal findings

Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone.We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus.

Conclusions/significance

The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.     

Prognostic factors in canine appendicular osteosarcoma - a meta-analysis

ABSTRACT: BACKGROUND: Appendicular osteosarcoma is the most common malignant primary canine bone tumor. When treated by amputation or tumor removal alone, median survival times (MST) do not exceed 5 months, with the majority of dogs suffering from metastatic disease. This period can be extended with adequate local intervention and adjuvant chemotherapy, which has become common practice. Several prognostic factors have been reported in many different studies, e.g. age, breed, weight, sex, neuter status, location of tumor, serum alkaline phosphatase (SALP), bone alkaline phosphatase (BALP), infection, percentage of bone length affected, histological grade or histological subtype of tumor. Most of these factors are, however, only reported as confounding factors in larger studies. Insight in truly significant prognostic factors at time of diagnosis may contribute to tailoring adjuvant therapy for individual dogs suffering from osteosarcoma.The objective of this study was to systematically review the prognostic factors that are described for canine appendicular osteosarcoma and validate their scientific importance. RESULTS: A literature review was performed on selected studies and eligible data were extracted. Meta-analyses were done for two of the three selected possible prognostic factors (SALP and location), looking at both survival time (ST) and disease free interval (DFI). The third factor (age) was studied in a qualitative manner. Both elevated SALP level and the (proximal) humerus as location of the primary tumor are significant negative prognostic factors for both ST and DFI in dogs with appendicular osteosarcoma. Increasing age was associated with shorter ST and DFI, however, was not statistically significant because information of this factor was available in only a limited number of papers. CONCLUSIONS: Elevated SALP and proximal humeral location are significant negative prognosticators for canine osteosarcoma.     

The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.Hepatitis C virus (HCV)² is the main contributing agent of acute and chronic liver diseases worldwide (1). Primary infection is often asymptomatic or associated with mild symptoms. However, persistently infected individuals develop high risks for chronic liver diseases such as hepatocellular carcinoma and liver cirrhosis (1). The combination of IFNα and ribavirin that serves as current therapy for chronically HCV-infected patients not only has a low success rate (about 50%) (2) but is often associated with serious side effects (2). There is thus an urgent need for the development of novel anti-HCV treatments (2).The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV (3). Controlled trials showed that a combination of CsA with IFNα is more effective than IFNα alone, especially in patients with a high viral load (4, 5). Moreover, recent in vitro studies provided evidence that CsA prevents both HCV RNA replication and HCV protein production in an IFNα-independent manner (6–10). CsA exerts this anti-HCV activity independently of its immunosuppressive activity because the nonimmunosuppressive Cyp inhibitors such as Debio 025, NIM811, and SCY-635 also block HCV RNA and protein production (9, 11–14). Unlike CsA, these molecules do not display calcineurin affinity and specifically inhibit the peptidyl-prolyl cis-trans-isomerase (PPIase) Cyps. Most importantly, recent clinical data demonstrated that Debio 025 dramatically decreased HCV viral load (3.6 log decrease) in patients coinfected with HCV and HIV (15). This 14-day Debio 025 treatment (1200 mg orally administered twice daily) was effective against the three genotypes (genotypes 1, 3, and 4) represented in the study. More recently, the anti HCV effect of Debio 025 in combination with peginterferon α 2a (peg-IFNα2a) was investigated in treatment-inexperienced patients with chronic hepatitis C. Debio 025 (600 mg administered once daily) in combination with peg-IFNα2a (180 μg/week) for 4 weeks induced a continuous decay in viral load that reached −4.61 ± 1.88 IU/ml in patients with genotypes 1 and 4 and −5.91 ± 1.11 IU/ml in patients with genotypes 2 and 3 at week 4 (16). The Debio 025 findings are critical because they suggest that Cyp inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. The fact that several recent studies using small RNA interference knockdown approaches suggest that Cyps are critical for the HCV life cycle (9, 17, 18) strongly implies that there is a direct or indirect link between the CsA- and CsA derivative-mediated inhibitory effect on HCV replication and host Cyps.The discovery 20 years ago of the first cellular protein showing PPIase activity (19) was entirely unrelated to the discovery of CypA as an intracellular protein possessing a high affinity for CsA (20). It is only a few years later that Fischer et al. (21) demonstrated that the 18-kDa protein with PPIase activity and CypA represent a single unique protein. All Cyps contain a common domain of 109 amino acids, called the Cyp-like domain, which is surrounded by domains specific to each Cyp members and which dictates their cellular compartmentalization and function (22). Bacteria, fungi, insects, plants, and mammals contain Cyps, which all have PPIase activity and are structurally conserved (22). To date, 16 Cyp members have been identified, and 7 of them are found in humans: CypA, CypB, CypC, CypD, CypE, Cyp40, and CypNK (22).Although there is a growing body of evidence that Cyps control HCV replication in human hepatocytes, a major disagreement currently exists on the respective roles of Cyp members in HCV replication. One study suggests that CypB, but not CypA, is critical for HCV replication (17), another suggests that CypA, but not CypB and CypC, is critical for HCV replication (18), and a third study suggests that three Cyps, CypA, B, and C, are all required for HCV replication (9). Thus, although it becomes evident that Cyps serve as HCV co-factors, their respective contributions and roles in the HCV life cycle remain to be determined. An understanding of the mechanisms that control the Cyp inhibitor-mediated anti-HCV effect is imperative because it will provide new alternate anti-HCV therapies and shed light on the still poorly understood early and late steps of the HCV life cycle.     

Comparative Whole-Genome Analysis of Clinical Isolates Reveals Characteristic Architecture of Mycobacterium tuberculosis Pangenome

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.     

Heat shock protein expression analysis in canine osteosarcoma reveals HSP60 as a potentially relevant therapeutic target

Heat shock proteins (HSP) are highly conserved across eukaryotic and prokaryotic species. These proteins play a role in response to cellular stressors, protecting cells from damage and facilitating recovery. In tumor cells, HSPs can have cytoprotective effects and interfere with apoptotic cascades. This study was performed to assess the prognostic and predictive values of the gene expression of HSP family members in canine osteosarcoma (OS) and their potential for targeted therapy. Gene expressions for HSP were assessed using quantitative PCR (qPCR) on 58 snap-frozen primary canine OS tumors and related to clinic-pathological parameters. A significant increased expression of HSP60 was found in relation to shorter overall survival and an osteoblastic phenotype. Therefore, the function of HSP60 was investigated in more detail. Immunohistochemical analysis revealed heterogeneous staining for HSP60 in tumors. The highest immunoreactivity was found in tumors of short surviving dogs. Next HSP expression was shown in a variety of canine and human OS cell lines by qPCR and Western blot. In two highly metastatic cell lines HSP60 expression was silenced using siRNA resulting in decreased cell proliferation and induction of apoptosis in both cell lines. It is concluded that overexpression of HSP60 is associated with a poor prognosis of OS and should be evaluated as a new target for therapy.     

Identification of ALK Gene Alterations in Urothelial Carcinoma

Background

Anaplastic lymphoma kinase (ALK) genomic alterations have emerged as a potent predictor of benefit from treatment with ALK inhibitors in several cancers. Currently, there is no information about ALK gene alterations in urothelial carcinoma (UC) and its correlation with clinical or pathologic features and outcome.

Methods

Samples from patients with advanced UC and correlative clinical data were collected. Genomic imbalances were investigated by array comparative genomic hybridization (aCGH). ALK gene status was evaluated by fluorescence in situ hybridization (FISH). ALK expression was assessed by immunohistochemistry (IHC) and high-throughput mutation analysis with Oncomap 3 platform. Next generation sequencing was performed using Illumina Genome Analyzer IIx, and Illumina HiSeq 2000 in the FISH positive case.

Results

70 of 96 patients had tissue available for all the tests performed. Arm level copy number gains at chromosome 2 were identified in 17 (24%) patients. Minor copy number alterations (CNAs) in the proximity of ALK locus were found in 3 patients by aCGH. By FISH analysis, one of these samples had a deletion of the 5′ALK. Whole genome next generation sequencing was inconclusive to confirm the deletion at the level of the ALK gene at the coverage level used. We did not observe an association between ALK CNA and overall survival, ECOG PS, or development of visceral disease.

Conclusions

ALK genomic alterations are rare and probably without prognostic implications in UC. The potential for testing ALK inhibitors in UC merits further investigation but might be restricted to the identification of an enriched population.     

Leptospirosis in pregnancy: A systematic review

IntroductionLeptospirosis is a leading zoonotic disease worldwide with more than 1 million cases in the general population per year. With leptospirosis being an emerging infectious disease and as the world’s environment changes with more floods and environmental disasters, the burden of leptospirosis is expected to increase. The objectives of the systematic review were to explore how leptospirosis affects pregnancy, its burden in this population, its effects on maternal and fetal outcomes and the evidence base surrounding treatment options.MethodsWe performed a systematic review of published and unpublished literature using automated and manual methods to screen nine electronic databases since inception, with no language restriction. Two reviewers independently screened articles, completed the data extraction and assessment of risk of bias. Due to significant heterogeneity and paucity of data, we were unable to carry out a meta-analysis, but we conducted a pooled analysis of individual patient data from the case reports and case series to examine the patient and disease characteristics, diagnostic methods, differential diagnoses, antibiotic treatments, and outcomes of leptospirosis in pregnancy. The protocol for this review was registered on the International Prospective Register of Systematic Reviews, PROSPERO: CRD42020151501.ResultsWe identified 419 records, of which we included eight observational studies, 21 case reports, three case series and identified four relevant ongoing studies. Overall the studies were with moderate bias and of ‘fair’ quality. We estimated the incidence of leptospirosis in pregnancy to be 1.3 per 10,000 in women presenting with fever or with jaundice, but this is likely to be higher in endemic areas. Adverse fetal outcomes were found to be more common in pregnant patients who presented in the second trimester compared with patients who presented in the third trimester. There is overlap between how leptospirosis presents in pregnancy and in the general population. There is also overlap between the signs, symptoms and biochemical disturbances associated with leptospirosis in pregnancy and the presentation of pregnancy associated conditions, such as Pre-Eclampsia (PET), Acute Fatty Liver of Pregnancy (AFLP) and HELLP Syndrome (Haemolysis Elevated Liver enzymes Low Platelets). In 94% of identified cases with available data, there was an indicator in the patient history regarding exposure that could have helped include leptospirosis in the clinician’s differential diagnosis. We also identified a range of suitable antibiotic therapies for treating leptospirosis in pregnancy, most commonly used were penicillins.ConclusionThis is the first systematic review of leptospirosis in pregnancy and it clearly shows the need to improve early diagnosis and treatment by asking early, treating early, and reporting well. Ask early—broaden differential diagnoses and ask early for potential leptospirosis exposures and risk factors. Treat early—increase index of suspicion in pregnant patients with fever in endemic areas and combine with rapid field diagnosis and early treatment. Report well—need for more good quality epidemiological studies on leptospirosis in pregnancy and better quality reporting of cases in literature.