Innate immunity: Looking beyond T-cells in radiation and immunotherapy combinations.

Radiation therapy is an established and effective anti-cancer treatment modality. Extensive pre-clinical experimentation has demonstrated that the pro-inflammatory properties of irradiation may be synergistic with checkpoint immunotherapy. Radiation induces double-stranded DNA breaks (dsDNA). Sensing of the dsDNA activates the cGAS/STING pathway, producing Type 1 interferons essential to recruiting antigen-presenting cells (APCs). Radiation promotes cytotoxic CD8 T-cell recruitment by releasing tumour-associated antigens captured and cross-presented by surveying antigen-presenting cells. Radiation-induced vascular normalisation may further promote T-cell trafficking and drug delivery. Radiation is also immunosuppressive. Recruitment of regulatory T cells (Tregs) and innate cells such as myeloid-derived suppressive cells (m-MDSCs) all counteract the immunostimulatory properties of radiation. Many innate immune cell types operate at the interface of the adaptive immune response. Innate immune cells, such as m-MDSCs, can exert their immunosuppressive effects by expressing immune checkpoints such as PD-L1, further highlighting the potential of combined radiation and checkpoint immunotherapy. Several early-phase clinical studies investigating the combination of radiation and immunotherapy have been disappointing. A greater appreciation of radiotherapy's impact on the innate immune system is essential to optimise radioimmunotherapy combinations. This review will summarise the impact of radiotherapy on crucial cells of the innate immune system and vital immunosuppressive cytokines.

Mucosal-associated invariant T-cell activation and accumulation after in vivo infection depends on microbial riboflavin synthesis and co-stimulatory signals.

Despite recent breakthroughs in identifying mucosal-associated invariant T (MAIT) cell antigens (Ags), the precise requirements for in vivo MAIT cell responses to infection remain unclear. Using major histocompatibility complex-related protein 1 (MR1) tetramers, the MAIT cell response was investigated in a model of bacterial lung infection employing riboflavin gene-competent and -deficient bacteria. MAIT cells were rapidly enriched in the lungs of C57BL/6 mice infected with Salmonella Typhimurium, comprising up to 50% of αß-T cells after 1 week. MAIT cell accumulation was MR1-dependent, required Ag derived from the microbial riboflavin synthesis pathway, and did not occur in response to synthetic Ag, unless accompanied by a Toll-like receptor agonist or by co-infection with riboflavin pathway-deficient S. Typhimurium. The MAIT cell response was associated with their long-term accumulation in the lungs, draining lymph nodes and spleen. Lung MAIT cells from infected mice displayed an activated/memory phenotype, and most expressed the transcription factor retinoic acid-related orphan receptor γt. T-bet expression increased following infection. The majority produced interleukin-17 while smaller subsets produced interferon-γ or tumor necrosis factor, detected directly ex vivo. Thus the activation and expansion of MAIT cells coupled with their pro-inflammatory cytokine production occurred in response to Ags derived from microbial riboflavin synthesis and was augmented by co-stimulatory signals.

The value of ultrasonography in the diagnosis of appendicitis.

INTRODUCTION: Ultrasonography is a commonly used investigation in the UK for patients with right iliac fossa pain where the diagnosis of appendicitis is unclear. The published sensitivity and specificity of ultrasonography is higher than the results observed by clinicians in every day practice. The aim of this study was to elucidate the real-world value of ultrasonography in the diagnosis of appendicitis, and its impact on negative appendicectomy rates (NAR). METHODS: A retrospective multicentre audit was conducted at three UK hospitals over a twelve month period in 2012. RESULTS: 573 patients underwent ultrasonography prior to appendicectomy. The appendix was not visualised in 45% of scans. The sensitivity and specificity of ultrasonography for the diagnosis of appendicitis was 51.8% and 81.4%. The mean NAR was 26.7%, or 18.3% after a positive ultrasound scan. CONCLUSION: In clinical practice at UK centres, ultrasonography commonly does not visualise the appendix, and has a low sensitivity for appendicitis. To reduce the NAR, management options include a return to observation and serial examination, increased use of CT or a commitment to improving the performance of ultrasonography.

High-frequency electrical stimulation of the subthalamic nucleus excites target structures in a model using c-fos immunohistochemistry.

Deep-brain stimulation at high frequencies (HFS) directed to the subthalamic nucleus (STN) is used increasingly to treat patients with Parkinson's disease. However, the mechanism of action by which HFS of the STN achieves its therapeutic effects remains unresolved. Insofar as lesions of the STN have similar therapeutic benefit, a favored hypothesis is that HFS acts by suppressing neural activity in the STN. The purpose of the present study was to exploit prior observations that exposure to ether anesthesia in a rodent model evokes c-fos expression (a marker of neural activation) in the STN and its efferent structures, the globus pallidus, entopeduncular nucleus and substantia nigra. We showed first that exposure to ether induced a profound oscillatory pattern of neural activity in the STN and SNr, which could explain the marked induction of c-fos immunoreactivity in these structures. Secondly, inhibition of the STN by local injections of the GABA agonist, muscimol, suppressed ether-evoked c-fos expression in all target structures. This showed that excitation of target structures in the ether model originated, at least in part, from the STN. Thirdly, and contrary to expectation, HFS of the STN increased further the expression of c-fos in the STN target structures of animals treated with ether. Finally, we demonstrated, in the absence of ether treatment, that HFS and chemical stimulation of the STN with local injections of kainic acid both induced c-fos expression in the globus pallidus, entopeduncular nucleus and substantia nigra. Together these results suggest that the principal action of STN stimulation at high frequencies is to excite rather than inhibit its efferent targets. Given that Parkinsonism has been associated with increased levels of inhibitory output activity from the basal ganglia, it is unlikely that excitation of output structures revealed in this study provides a basis for deep-brain stimulation's therapeutic action.

Streptomycete endophytes from anti-diabetic medicinal plants of the Western Ghats inhibit alpha-amylase and promote glucose uptake.

UNLABELLED: α-amylase inhibitor retards the liberation of glucose from dietary complex carbohydrates and delays the absorption of glucose. The purpose of the study was to isolate and select α-amylase inhibitor-producing endophytic actinomycetes from the leaves and stems of Leucas ciliata and Rauwolfia densiflora, two of the well-known medicinal plants used in the treatment for diabetes. Sterilized plant samples were inoculated on the actinomycete isolation agar medium containing 50 ppm cycloheximide and incubated for 4-8 weeks at room temperature. The actinomycetes were isolated on agar medium and identified on the basis of 16S rRNA sequences, the isolates exhibiting >99% similarities were submitted to NCBI, and gene accession numbers were obtained. They were inoculated to International Streptomyces Project 1 medium (ISP 1) for fermentation. The extracts obtained were tested for the anti-diabetic potential by the inhibition of alpha-amylase by colorimetric assay and glucose uptake in the porcine hemidiaphragm. Streptomyces longisporoflavus (JX965948) isolated from the stem fragments of L. ciliata exhibited alpha-amylase inhibitory activity (IC50 values = 162.3 ± 1.05 µg ml⁻¹) in comparison with the standard Acarbose™ (IC50 value = 73.1 ± 1.12 µg ml⁻¹). Extract of Streptomyces sp. (JQ926174) from R. densiflora indicated glucose uptake in the porcine hemidiaphragm. Results indicate for the first time the potential of endophytic streptomycete extracts with anti-diabetic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic actinomycetes were isolated from two medicinal species of the Western Ghats, a biodiversity 'hotspot' in southern India and screened for the anti-diabetic potential for inhibition of α-amylase and improved glucose uptake in the porcine hemidiaphragm. Results indicate the inhibition of α-amylase by Streptomyces longisporoflavus extract with IC50 values of 162.3 ± 1.05 µg ml⁻¹ in comparison with the standard inhibitor Acarbose™ with IC50 value 73.1 ± 1.12 µg ml⁻¹. Further, extract from Streptomyces sp. showed increased glucose uptake by hemidiaphragm. The present investigation implicates the potential of endophytic actinomycetes as sources of anti-diabetic agents.

Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts.

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.

A phase I/II dose-escalation trial of vitamin D3 and calcium in multiple sclerosis.

OBJECTIVE: Low vitamin D status has been associated with multiple sclerosis (MS) prevalence and risk, but the therapeutic potential of vitamin D in established MS has not been explored. Our aim was to assess the tolerability of high-dose oral vitamin D and its impact on biochemical, immunologic, and clinical outcomes in patients with MS prospectively. METHODS: An open-label randomized prospective controlled 52-week trial matched patients with MS for demographic and disease characteristics, with randomization to treatment or control groups. Treatment patients received escalating vitamin D doses up to 40,000 IU/day over 28 weeks to raise serum 25-hydroxyvitamin D [25(OH)D] rapidly and assess tolerability, followed by 10,000 IU/day (12 weeks), and further downtitrated to 0 IU/day. Calcium (1,200 mg/day) was given throughout the trial. Primary endpoints were mean change in serum calcium at each vitamin D dose and a comparison of serum calcium between groups. Secondary endpoints included 25(OH)D and other biochemical measures, immunologic biomarkers, relapse events, and Expanded Disability Status Scale (EDSS) score. RESULTS: Forty-nine patients (25 treatment, 24 control) were enrolled [mean age 40.5 years, EDSS 1.34, and 25(OH)D 78 nmol/L]. All calcium-related measures within and between groups were normal. Despite a mean peak 25(OH)D of 413 nmol/L, no significant adverse events occurred. Although there may have been confounding variables in clinical outcomes, treatment group patients appeared to have fewer relapse events and a persistent reduction in T-cell proliferation compared to controls. CONCLUSIONS: High-dose vitamin D (approximately 10,000 IU/day) in multiple sclerosis is safe, with evidence of immunomodulatory effects. CLASSIFICATION OF EVIDENCE: This trial provides Class II evidence that high-dose vitamin D use for 52 weeks in patients with multiple sclerosis does not significantly increase serum calcium levels when compared to patients not on high-dose supplementation. The trial, however, lacked statistical precision and the design requirements to adequately assess changes in clinical disease measures (relapses and Expanded Disability Status Scale scores), providing only Class level IV evidence for these outcomes.

An overview on nucleases (DNase, RNase, and phosphodiesterase) in snake venoms.

In this review, we have compiled the data on pharmacological activities associated with endogenous purine release related enzymes-nucleases (DNases, RNases, and phosphodiesterases). The results of studies on toxic effects of these enzymes, emphasizing the future directions in this field, are summarized. One of the major problems facing toxicologists is the identification and characterization of specific venom nucleases since they share similar substrate specificities and biochemical properties. In this review, we have attempted to clarify some of the discrepancies about these enzymes. Further, we have tried to correlate the existence of nuclease enzymes in relation to endogenous release of purines, a multitoxin, during snake envenomation, and we also discuss the possible actions of purines. We hope that this review will stimulate renewed interest among toxicologists to biologically characterize these enzymes and elucidate their role in envenomation.

Vanillic acid as a novel specific inhibitor of snake venom 5'-nucleotidase: a pharmacological tool in evaluating the role of the enzyme in snake envenomation.

Vanillic acid has been investigated for its inhibitory effect on Naja naja, Daboia russellii, and Trimeresurus malabaricus venom 5'-nucleotidase activity. Trimeresurus malabaricus venom 5'-nucleotidase activity was 1.3- and 8.0-fold higher than that of N. naja and D. russellii venoms, respectively. Substrate specificity studies showed that for all the venoms tested, 5'-AMP was the preferred substrate for 5'-nucleotidase. This indicates the central role of adenosine in snake envenomation. Vanillic acid selectively and specifically inhibited 5'-nucleotidase activity among several enzymes present in the three venoms tested. The inhibitor was competitive, as the inhibition was relieved by increased substrate concentration. It appears that the COOH group in vanillic acid is the determining factor for inhibition as vanillin, a structurally similar compound with respect to vanillic acid, had no inhibitory activity. This study for the first time exemplifies vanillic acid as a pharmacological tool in evaluating the role of 5'-nucleotidase in snake envenomation.

Mesocolic hernia: an unusual internal hernia.

Internal hernia may be either congenital or acquired. Its incidence has been reported to be 1-2%. Herniation may be persistent or intermittent. Internal hernia is a rare cause of small bowel obstruction with a reported incidence of 0.2-0.9%. The most common type is paraduodenal. Less common types include mesocolic hernia, which occurs following abdominal surgery. We report mesocolic hernias in two young patients, which presented as small bowel obstruction without any prior abdominal surgery.

Management of psychosis in Parkinson's disease.

Psychosis is one of the most disabling complications associated with Parkinson's disease (PD) and occurs in up to 30% of PD patients treated chronically with antiparkinsonian drugs. Visual hallucinations, with or without delirium and paranoid delusions, are the most frequent symptoms. Psychosis complicating PD can be more disabling than the motor symptoms of PD; it frequently poses a serious threat to the patient's ability to maintain independence and is the single greatest risk factor for nursing home placement. Choosing an antipsychotic drug for a PD patient is a common clinical dilemma. The conventional antipsychotic drugs are poorly tolerated in PD because of their predictable and at times profound worsening in parkinsonian motor symptoms. The recent availability of atypical antipsychotic drugs that can control psychotic symptoms without compromising motor function has led to significant advances in therapeutic strategies in the management of PD psychosis in the community. This article reviews data on the use of atypical antipsychotics in patients with PD and the current recommendations on their use in the management of PD psychosis.

Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans.

Glutamine synthetase (GS), EC 6.3.1.2, is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the Aspergillus nidulans glnA gene encoding GS and have shown that glnA encodes a highly expressed but not highly regulated mRNA. Inactivation of glnA results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functional glnA gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic Anabaena glnA gene from the A. nidulans alcA promoter allowed full complementation of the A. nidulans glnADelta mutation. However, the A. nidulans fluG gene, which encodes a protein with similarity to prokaryotic GS, did not replace A. nidulans glnA function when similarly expressed. Our studies with the glnADelta mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.

Treatment of aneurysmal subarachnoid haemorrhage in elderly patients.

Spontaneous subarachnoid haemorrhage (SAH) due to ruptured saccular aneurysm is the fourth most frequent cerebrovascular disorder following atherosclerosis, embolism, and primary intracerebral haemorrhage (1). SAH is a common and often devastating condition, which is a significant cause of world-wide morbidity and mortality (2). The aim of this article is to review the epidemiology, pathophysiology and current management of SAH.

Characterization of the role of the FluG protein in asexual development of Aspergillus nidulans.

We showed previously that a DeltafluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG ( approximately 400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of DeltafluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from DeltaflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

Humoral hypercalcemia of malignancy: severe combined immunodeficient/beige mouse model of adult T-cell lymphoma independent of human T-cell lymphotropic virus type-1 tax expression.

The majority of patients with adult T-cell leukemia/lymphoma (ATL) resulting from human T-cell lymphotropic virus type-1 (HTLV-1) infection develop humoral hypercalcemia of malignancy (HHM). We used an animal model using severe combined immunodeficient (SCID)/beige mice to study the pathogenesis of HHM. SCID/beige mice were inoculated intraperitoneally with a human ATL line (RV-ATL) and were euthanized 20 to 32 days after inoculation. SCID/beige mice with engrafted RV-ATL cells developed lymphoma in the mesentery, liver, thymus, lungs, and spleen. The lymphomas stained positively for human CD45RO surface receptor and normal mouse lymphocytes stained negatively confirming the human origin of the tumors. The ATL cells were immunohistochemically positive for parathyroid hormone-related protein (PTHrP). In addition, PTHrP mRNA was highly expressed in lymphomas when compared to MT-2 cells (HTLV-1-positive cell line). Mice with lymphoma developed severe hypercalcemia. Plasma PTHrP concentrations were markedly increased in mice with hypercalcemia, and correlated with the increase in plasma calcium concentrations. Bone densitometry and histomorphometry in lymphoma-bearing mice revealed significant bone loss because of a marked increase in osteoclastic bone resorption. RV-ATL cells contained 1.5 HTLV-1 proviral copies of the tax gene as determined by quantitative real-time polymerase chain reaction (PCR). However, tax expression was not detected by Western blot or reverse transcriptase (RT)-PCR in RV-ATL cells, which suggests that factors other than Tax are modulators of PTHrP gene expression. The SCID/beige mouse model mimics HHM as it occurs in ATL patients, and will be useful to investigate the regulation of PTHrP expression by ATL cells in vivo.

Conserved cAMP signaling cascades regulate fungal development and virulence.

Two well characterized signal transduction cascades regulating fungal development and virulence are the MAP kinase and cAMP signaling cascades. Here we review the current state of knowledge on cAMP signaling cascades in fungi. While the processes regulated by cAMP signaling in fungi are as diverse as the fungi themselves, the components involved in signal transduction are remarkably conserved. Fungal cAMP signaling cascades are also quite versatile, which is apparent from the differential regulation of similar biological processes. In this review we compare and contrast cAMP signaling pathways that regulate development in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and differentiation and virulence in the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis. We also present examples of interaction between the cAMP and MAP kinase signaling cascades in the regulation of fungal development and virulence.

Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

CD8- and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis.

The role of CD8 T lymphocytes in the immune response to Mycobacterium tuberculosis infection remains enigmatic, with persuasive reports of both cytolytic and noncytolytic (cytokine-mediated) responses to infection. To address the importance of the cytolytic mechanisms, mice with targeted disruptions for CD8 and perforin or with gene mutations in the CD95/ CD95L signaling pathway were exposed to pulmonary infection. All mice tested showed no differences in their ability to contain the growth of infection during the early phase of disease. As the chronic phase of the disease ensued, however, both CD8- and CD95/CD95L-deficient mice gradually lost their ability to limit bacterial growth. This was associated with a tendency toward pyogenic inflammation in the lung. This tendency was not seen in the perforin gene-disrupted mice. In CD8 gene-disrupted mice, the ability to generate interferon-gamma secreting T cells was unimpaired. Although these cells were capable of entering the lung they were unable to influence the increasing bacterial load in this organ.