Immune responses against major histocompatibility complex and interleukin-2 gene transfected K1735 murine melanoma: Potential vaccines for the immunotherapy of cancer

Tumor specific immunity is mediated by cytotoxic T lymphocytes (CTL) that recognize peptide antigen (Ag) in the context of major histocompatibility complex (MHC) class I molecules and by helper T (Th) lymphocytes that recognize peptide Ag in the context of MHC class II molecules. The purpose of this study is (1) to induce or augment the immunogenicity of nonimmunogenic or weakly immunogenic tumors by genetic modification of tumor cells, and (2) to use these genetically altered cells in cancer immunotherapy. To study this, I transfected a highly tumorigenic murine melanoma cell line (K1735) that did not express constitutively either MHC class I or II molecules with syngeneic cloned MHC class I and/or class II genes, and then determined the tumorigenicity of transfected cells in normal C3H mice. K1735 transfectants expressing either $\rm K\sp{k}$ or $\rm A\sp{k}$ molecules alone produced tumors in normal C3H mice, whereas most transfectants that expressed both molecules were rejected in normal C3H mice but produced tumors in nude mice. The rejection of K1735 transfectants expressing $\rm K\sp{k}$ and $\rm A\sp{k}$ Ag in normal C3H mice required both $\rm CD4\sp+$ and $\rm CD8\sp+$ T cells. Interestingly, the $\rm A\sp{k}$ requirement can be substituted by IL-2 because transfection of $\rm K\sp{k}$-positive/A$\sp{\rm k}$-negative K1735 cells with the IL-2 gene also resulted in abrogation of tumorigenicity in normal C3H mice but not in nude mice. In addition, 1735 $(\rm I\sp+II\sp+)$ transfected cells can function as antigen presenting cells (APC) since they could process and present native hen egg lysozyme (HEL) to HEL specific T cell hybridomas. Furthermore, the transplantation immunity induced by K1735 transfectants expressing both $\rm K\sp{k}$ and $\rm A\sp{k}$ molecules completely cross-protected mice against challenge with $\rm K\sp{k}$-positive transfectants but weakly protected them against challenge with parental K1735 cells or $\rm A\sp{k}$-positive transfectants. Finally, I demonstrated that MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells can function as anti-cancer vaccines since they can abrogate the growth of established tumors and metastasis.^ In summary, my results indicate that expression of either MHC class I or II molecule alone is insufficient to cause the rejection of K1735 melanoma in syngeneic hosts and that both molecules are necessary. In addition, my data suggest that the failure of $\rm K\sp{k}$-positive K1735 cells to induce a primary tumor-rejection response in normal C3H mice may be due to their inability to induce the helper arm of the anti-tumor immune response. Finally, the ability of MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells to prevent growth of established tumors or metastasis suggests that these cell lines can serve as potential vaccines for the immunotherapy of cancer. (Abstract shortened by UMI.) ^

Influenza vaccination and cardiovascular risk in patients with recent TIA and stroke

OBJECTIVES: To determine whether current influenza vaccination is associated with reduced risk of major vascular events in patients with recent ischemic stroke or TIA of mainly atherothrombotic origin. METHODS: Data were pooled from 2 prospective cohort studies, the OPTIC Registry (n = 3,635) and the AMISTAD Study (n = 618), and from the randomized PERFORM Trial (n = 19,120), all of which included patients with recent ischemic stroke or TIA. Influenza vaccination status was determined in 23,110 patients. The primary outcome was a composite of nonfatal myocardial infarction, nonfatal stroke, or vascular death up to 2 years. Secondary outcomes were myocardial infarction and stroke separately. RESULTS: Influenza vaccination had no association with the primary outcome in the propensity score-matched cohort (hazard ratio 0.97, 95% confidence interval [CI] 0.85-1.11; p = 0.67) or in the propensity score-adjusted cohort (hazard ratio 1.00, 95% CI 0.89-1.12; p = 0.99). Similarly, the risk of stroke and myocardial infarction did not differ between the vaccinated group and the unvaccinated group; in the matched cohort, the hazard ratio was 1.01 (95% CI 0.88-1.17; p = 0.89) for stroke and 0.84 (95% CI 0.59-1.18; p = 0.30) for myocardial infarction. CONCLUSIONS: Influenza vaccination was not associated with reduced outcome events in patients with recent atherothrombotic ischemic stroke after considering all baseline characteristics (including concomitant medications) associated with influenza vaccination.

Incomplete Distal Renal Tubular Acidosis from a Heterozygous Mutation of the V-ATPase B1 Subunit

Congenital distal renal tubular acidosis (dRTA) from mutations of the B1 subunit of the V-ATPase is considered an autosomal recessive disease. We analyzed a dRTA kindred with a truncation-mutation of B1 (p.Phe468fsX487) previously shown to have failure of assembly into the V1 domain of the V-ATPase. All heterozygous carriers in this kindred have normal plasma bicarbonate concentrations, thus evaded the diagnosis of RTA. However, inappropriately high urine pH, hypocitraturia, and hypercalciuria are present either individually or in combination in the heterozygotes at baseline. Two of the heterozygotes studied also have inappropriate urinary acidification with acute ammonium chloride loading and impaired urine-blood pCO2 gradient during bicarbonaturia indicating presence of H+ gradient and flux defects. In normal human renal papillae, wild type B1 is located primarily on the plasma membrane but papilla from one of the heterozygote who had kidney stones had renal tissue secured from surgery showed B1 in both plasma membrane as well as a diffuse intracellular staining. Titrating increasing amounts of the mutant B1 subunit did not exhibit negative dominance over the expression, cellular distribution, or H+-pump activity of the wild type B1 in mammalian HEK293 cells and in V-ATPase-deficient S. cerevisiae. This is the first demonstration of renal acidification defects and nephrolithiasis in heterozygous carriers of mutant B1 subunit; which cannot be attributable to negative dominance. We propose that heterozygosity may lead to mild real acidification defects due to haploinsufficiency. B1 heterozygosity should be considered in patients with calcium nephrolithiasis and urinary abnormalities such as alkalinuria or hypocitraturia.

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules (1). Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii (2). The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production (1). (1) Gebetsberger J. and Polacek N. (2013), RNA Biol. 10:1798-1808 (2) Gebetsberger J. et. al. (2012), Archaea, Article ID 260909

Severity assessment tools in ICU patients with 2009 influenza A (H1N1) pneumonia.

The aim of this study was to determine if severity assessment tools (general severity of illness and community-acquired pneumonia specific scores) can be used to guide decisions for patients admitted to the intensive care unit (ICU) due to pandemic influenza A pneumonia. A prospective, observational, multicentre study included 265 patients with a mean age of 42 (±16.1) years and an ICU mortality of 31.7%. On admission to the ICU, the mean pneumonia severity index (PSI) score was 103.2 ± 43.2 points, the CURB-65 score was 1.7 ± 1.1 points and the PIRO-CAP score was 3.2 ± 1.5 points. None of the scores had a good predictive ability: area under the ROC for PSI, 0.72 (95% CI, 0.65-0.78); CURB-65, 0.67 (95% CI, 0.59-0.74); and PIRO-CAP, 0.64 (95% CI, 0.56-0.71). The PSI score (OR, 1.022 (1.009-1.034), p 0.001) was independently associated with ICU mortality; however, none of the three scores, when used at ICU admission, were able to reliably detect a low-risk group of patients. Low risk for mortality was identified in 27.5% of patients using PIRO-CAP, but above 40% when using PSI (I-III) or CURB65 (<2). Observed mortality was 13.7%, 13.5% and 19.4%, respectively. Pneumonia-specific scores undervalued severity and should not be used as instruments to guide decisions in the ICU.

Use of early corticosteroid therapy on ICU admission in patients affected by severe pandemic (H1N1)v influenza A infection.

INTRODUCTION Early use of corticosteroids in patients affected by pandemic (H1N1)v influenza A infection, although relatively common, remains controversial. METHODS Prospective, observational, multicenter study from 23 June 2009 through 11 February 2010, reported in the European Society of Intensive Care Medicine (ESICM) H1N1 registry. RESULTS Two hundred twenty patients admitted to an intensive care unit (ICU) with completed outcome data were analyzed. Invasive mechanical ventilation was used in 155 (70.5%). Sixty-seven (30.5%) of the patients died in ICU and 75 (34.1%) whilst in hospital. One hundred twenty-six (57.3%) patients received corticosteroid therapy on admission to ICU. Patients who received corticosteroids were significantly older and were more likely to have coexisting asthma, chronic obstructive pulmonary disease (COPD), and chronic steroid use. These patients receiving corticosteroids had increased likelihood of developing hospital-acquired pneumonia (HAP) [26.2% versus 13.8%, p < 0.05; odds ratio (OR) 2.2, confidence interval (CI) 1.1-4.5]. Patients who received corticosteroids had significantly higher ICU mortality than patients who did not (46.0% versus 18.1%, p < 0.01; OR 3.8, CI 2.1-7.2). Cox regression analysis adjusted for severity and potential confounding factors identified that early use of corticosteroids was not significantly associated with mortality [hazard ratio (HR) 1.3, 95% CI 0.7-2.4, p = 0.4] but was still associated with an increased rate of HAP (OR 2.2, 95% CI 1.0-4.8, p < 0.05). When only patients developing acute respiratory distress syndrome (ARDS) were analyzed, similar results were observed. CONCLUSIONS Early use of corticosteroids in patients affected by pandemic (H1N1)v influenza A infection did not result in better outcomes and was associated with increased risk of superinfections.

Disease Caused by Mutations in NaV-β Subunit Genes

NaV-b subunits associate with the NaV-a or pore-forming subunit of the voltage-dependent sodium channel and play critical roles in channel expression, voltage dependence of the channel gating, cell adhesion, signal transduction, and channel pharmacology. Five NaV-b subunits have been identified in humans, all of them implicated in many primary arrhythmia syndromes that cause sudden death or neurologic disorders, including long QT syndrome, Brugada syndrome, cardiac conduction disorders, idiopathic ventricular fibrillation, epilepsy, neurodegenerative diseases, and neuropsychiatric disorders.

Persistent and compartmentalised disruption of dendritic cell subpopulations in the lung following influenza A virus infection.

Immunological homeostasis in the respiratory tract is thought to require balanced interactions between networks of dendritic cell (DC) subsets in lung microenvironments in order to regulate tolerance or immunity to inhaled antigens and pathogens. Influenza A virus (IAV) poses a serious threat of long-term disruption to this balance through its potent pro-inflammatory activities. In this study, we have used a BALB/c mouse model of A/PR8/34 H1N1 Influenza Type A Virus infection to examine the effects of IAV on respiratory tissue DC subsets during the recovery phase following clearance of the virus. In adult mice, we found differences in the kinetics and activation states of DC residing in the airway mucosa (AMDC) compared to those in the parenchymal lung (PLDC) compartments. A significant depletion in the percentage of AMDC was observed at day 4 post-infection that was associated with a change in steady-state CD11b+ and CD11b- AMDC subset frequencies and significantly elevated CD40 and CD80 expression and that returned to baseline by day 14 post-infection. In contrast, percentages and total numbers of PLDC were significantly elevated at day 14 and remained so until day 21 post-infection. Accompanying this was a change in CD11b+and CD11b- PLDC subset frequencies and significant increase in CD40 and CD80 expression at these time points. Furthermore, mice infected with IAV at 4 weeks of age showed a significant increase in total numbers of PLDC, and increased CD40 expression on both AMDC and PLDC, when analysed as adults 35 days later. These data suggest that the rate of recovery of DC populations following IAV infection differs in the mucosal and parenchymal compartments of the lung and that DC populations can remain disrupted and activated for a prolonged period following viral clearance, into adulthood if infection occurred early in life.

Relative positioning of classical benzodiazepines to the γ2-subunit of GABAA receptors.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60, and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors.

Influenza A(H1N1)pdm09 and cystic fibrosis lung disease: a systematic meta-analysis.

BACKGROUND To systematically assess the literature published on the clinical impact of Influenza A(H1N1)pdm09 on cystic fibrosis (CF) patients. METHODS An online search in PUBMED database was conducted. Original articles on CF patients with Influenza A(H1N1)pdm09 infection were included. We analyzed incidence, symptoms, clinical course and treatment. RESULTS Four surveys with a total of 202 CF patients infected by Influenza A(H1N1)pdm09 were included. The meta-analysis showed that hospitalisation rates were higher in CF patients compared to the general population. While general disease symptoms were comparable, the clinical course was more severe and case fatality rate (CFR) was higher in CF patients compared to asthmatics and the general population. CONCLUSIONS Evidence so far suggests that CF patients infected with Influenza A(H1N1)pdm09 show increased morbidity and a higher CFR compared to patients with other chronic respiratory diseases and healthy controls. Particularly, CF patients with advanced stage disease seem to be more susceptible to severe lung disease. Accordingly, early antiviral and antibiotic treatment strategies are essential in CF patients. Preventive measures, including vaccination as well as hygiene measures during the influenza season, should be reinforced and improved in CF patients.

An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses

Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.