Prediction of tumor-reactive T cell receptors from scRNA-seq data for personalized T cell therapy.

The identification of patient-derived, tumor-reactive T cell receptors (TCRs) as a basis for personalized transgenic T cell therapies remains a time- and cost-intensive endeavor. Current approaches to identify tumor-reactive TCRs analyze tumor mutations to predict T cell activating (neo)antigens and use these to either enrich tumor infiltrating lymphocyte (TIL) cultures or validate individual TCRs for transgenic autologous therapies. Here we combined high-throughput TCR cloning and reactivity validation to train predicTCR, a machine learning classifier that identifies individual tumor-reactive TILs in an antigen-agnostic manner based on single-TIL RNA sequencing. PredicTCR identifies tumor-reactive TCRs in TILs from diverse cancers better than previous gene set enrichment-based approaches, increasing specificity and sensitivity (geometric mean) from 0.38 to 0.74. By predicting tumor-reactive TCRs in a matter of days, TCR clonotypes can be prioritized to accelerate the manufacture of personalized T cell therapies.

Endothelial Indoleamine-2,3-Dioxygenase-1 is not Critically Involved in Regulating Antitumor Immunity in the Central Nervous System.

The vascular niche of malignant gliomas is a key compartment that shapes the immunosuppressive brain tumor microenvironment (TME). The blood-brain-barrier (BBB) consisting of specialized endothelial cells (ECs) and perivascular cells forms a tight anatomical and functional barrier critically controlling transmigration and effector function of immune cells. During neuroinflammation and tumor progression, the metabolism of the essential amino acid tryptophan (Trp) to metabolites such as kynurenine has long been identified as an important metabolic pathway suppressing immune responses. Previous studies have demonstrated that indoleamine-2,3-dioxygenase-1 (IDO1), a key rate-limiting enzyme in tryptophan catabolism, is expressed within the TME of high-grade gliomas. Here, we investigate the role of endothelial IDO1 (eIDO1) expression for brain tumor immunity. Single-cell RNA sequencing data revealed that in human glioma tissue, IDO1 is predominantly expressed by activated ECs showing a JAK/STAT signaling pathway-related CXCL11+ gene expression signature. In a syngeneic experimental glioma model, eIDO1 is induced by low-dose tumor irradiation. However, cell type-specific ablation of eIDO1 in experimental gliomas did not alter frequency and phenotype of tumor-infiltrating T cells nor tumor growth. Taken together these data argue against a dominant role of eIDO1 for brain tumor immunity.

Explaining Osteomyelitis and Prosthetic Joint Infections (PJI) in terms of Biofilm - A Review.

Osteomyelitis is a chronic infection of bones. Eradication of bone infection is usually with antibiotics and debridement, but it is slow and the infection can recur even after many years. It is now established that osteomyelitis is due to biofilm and a better understanding of the process is required. We review the development of biofilm and apply it to osteomyelitis management. The planktonic microbes' response to adverse conditions is the formation of biofilm. Bacterial infections in planktonic forms cause infections that can be controlled with antibiotics and immunisation, however the same microbe when its phenotype becomes biofilm is more resilient. The understanding of how planktonic bacteria convert to biofilm is one of the aims set out for this article.

Vision loss in an immunocompetent child post varicella infection: A case report.

Chickenpox may lead to several neurological complications. Optic neuritis is one of the complications which has rarely been described, especially in immunocompetent individuals. We report a case of an 11-year-old immunocompetent girl who presented with sudden onset bilateral vision loss three weeks after varicella eruption. Ophthalmic examination revealed bilateral optic disc edema. Diagnosis of bilateral optic neuritis secondary to varicella was established based upon the preceding medical history, supported with clinical and radiological findings.

Cells with ganglionic differentiation frequently stain for VE1 antibody: a potential pitfall.

Mitogen-activated protein kinase (MAPK) pathway plays a major role in pediatric low-grade gliomas (pLGGs). Immunohistochemistry with mutant-specific antibody, VE1, has appeared to be the most affordable and rapidly deployable method to identify tumors with aberrant MAPK signaling pathway, by highlighting tumor with BRAFV600E mutation. Nonetheless, positive staining cases but not associated with BRAFV600E mutation are also seen. We analyzed 62 pLGGs for the two commonest genetic aberrations in MAPK pathway: KIAA1549-BRAF fusion, using reverse-transcriptase polymerase chain reaction, and BRAFV600E mutation, using VE1 antibody and Sanger sequencing. We recorded a specificity and accuracy rate of 68.75% and 75%, respectively, for VE1, when strong cytoplasmic staining is observed. Interestingly, we observed that cells with ganglionic features frequently bind VE1 but not associated with BRAFV600E mutation. Such observation was also confirmed in four cases of differentiating neuroblastoma. This false positive staining may serve as an important confounder in the interpretation of VE1 immunoreactivity with major therapeutic implication. It is important to confirm the presence of BRAFV600E mutation by DNA-based method, especially in tumor entities not known to, or rarely harbor such mutations.

miR-375 inhibits the proliferation and invasion of glioblastoma by regulating Wnt5a.

The aberrant expression of microRNA-375 (miR-375) has been proved to be associated with carcinogenesis. However, the role of miR-375 in glioblastoma (GBM) remains unknown. The aim of this study was to investigate biological functions and its molecular mechanisms of miR-375 in GBM cells. In this study, real-time PCR results showed that the level of miR-375 expression in GBM tissues and GBM cell lines (U87 and U251) was decreased. Using MTT assay, Transwell migration and invasion assay, we demonstrated that miR-375 overexpression significantly suppress cell proliferation, cell migration and cell invasion capacity in U87 and U251 cells. However, downregulation of miR-375 had reverse effects on cell proliferation, migration and invasion. Targeting association analysis, dual luciferase assay, RT-PCR and western blot analysis results confirmed that miR-375 could target the 3'UTR of Wnt5a mRNA and regulated its protein expression. Further studies also find overexpression of Wnt5a could significantly reverse miR-375-mediated proliferation, migration and invasion on U87 and U251 cells. Therefore, we concluded that miR-375 inhibited the proliferation and invasion of GBM by regulating Wnt5a and might be a possible therapeutic agent for GBM.

A rapidly fatal intracranial anaplastic hemangiopericytoma with de-novo dedifferentiation: emphasis on diagnostic recognition, molecular confirmation and discussion on treatment dilemma.

Solitary fibrous tumors/ hemangiopericytomas (SFT/HPC) are mesenchymal tumors that share a common genetic aberration and very rarely undergo dedifferentiation. We report a unique case of an intracranial anaplastic SFT/HPC with de-novo dedifferentiation, which pursued a rapidly fatal clinical course in a 41-year-old lady. The dedifferentiated component comprised a focal area of glandular formation with epithelial immunophenotype acquisition. The distinct biphasic pattern of the tumor imparted great diagnostic challenges to the pathologists. An increased awareness of SFT/HPCs with a diverse morphologic spectrum or even a biphasic histologic pattern is essential in working up such cases. We first attempted gamma knife radiosurgery in treating a recurrent dedifferentiated SFT/HPC; unfortunately it was to no avail. Although it is now known that SFT/HPC is characterized by NAB2-STAT6 gene fusion, the unavailability of targeted therapy against this molecular signature still results in a treatment dilemma.

Fulvestrant inhibits the glycolysis of prolactinoma GH3 cells by downregulating IRE1/XBP1 signaling pathway.

OBJECTIVE: We aimed to evaluate the effects of fulvestrant on the glycolysis of prolactinoma GH3 cells, and reveal the potential regulatory mechanisms. MATERIALS AND METHODS: Prolactinoma cell line GH3 was treated with different concentrations of fulvestrant (0, 0.12, 0.25, 0.5 and 1 ng/ml) for 4 h. siRNAs XBP1s and XBP1u were constructed to treat GH3 cells. The expression levels of XBP1s, XBP1u, IRE1, PKM2 and GRP78 of GH3 cells were detected by Western blot. Meanwhile, the glycolytic activity of GH3 cells, including the glucose uptake, ATP/ADP, and lactate production were detected. RESULTS: The expression levels of XBP1s and XBP1u were significantly inhibited by fulvestrant in a dose-dependent manner. The glucose uptake, ATP/ADP and lactate production of GH3 cells were significantly inhibited by fulvestrant as well as siRNA XBP1s and XBP1u (p < 0.05). Western blot analysis suggested that the expression levels of IRE1, PKM2 and GRP78 were significantly decreased in GH3 cells treated by fulvestrant as well as siRNA XBP1s and XBP1u, compared with those in normal control (p < 0.05). CONCLUSIONS: Fulvestrant could inhibit the glycolysis of GH3 cells by downregulating IRE1/XBP1 signaling pathway, and this process was closely related with the downregulation of PKM2.

Developing neurites from mouse basal forebrain gonadotropin-releasing hormone neurons use Sonic hedgehog to modulate their growth.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are required for fertility in all mammalian species studied to date. GnRH neuron cell bodies reside in the basal forebrain, and most extend long neurites in the caudal direction to terminate at the median eminence (ME), the site of hormone secretion. Using in vitro neurite growth assays, histological methods, and genetic deletion strategies in mice we have analysed the role of the morphogen and neurite growth and guidance molecule, Sonic hedgehog (Shh), in the growth of GnRH neurites to their target. Immunohistochemistry revealed that Shh was present in the basal forebrain, the preoptic area (POA) and mediobasal hypothalamus (MBH) at gestational day 14.5 (GD 14.5), a time when GnRH neurites grow towards the ME. Furthermore, in situ hybridization revealed that mRNA encoding the Shh receptor, Smoothened (Smo), was present in GnRH neurons from GD 15.5, when the first GnRH neurites are extending towards the MBH. In vitro neurite growth assays using hypothalamic explants from GD 15.5 fetuses in 3-D collagen gels showed that Shh was able to significantly stimulate GnRH neurite outgrowth. Finally, genetic deletion of Smo specifically from GnRH neurons in vivo, using Cre-loxP technology, resulted in a significant decrease in GnRH neurites innervating the ME. These experiments demonstrate that GnRH neurites use Shh for their neurite development, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional hypothalamic neuronal population for which Shh/Smo signaling is developmentally important.

[Surgical options for chronic pancreatitis].

Objective: Discuss the surgical options for the chronic pancreatitis on the basis of anatomical morphological changes. Methods: A retrospective review of chronic pancreatitis patients in Department of Pancreatic Surgery, West China Hospital, Sichuan University between January 2010 and December 2014 was performed. The data of medical records, image feature, surgical types and records of follow-up were collected. Total 295 patients including 275 male and 20 female aged from 14 to 74 years with median age of 51 years. The clinical symptoms included abdominal pain in 280 cases, jaundice in 3 cases, single hemorrhage in digestive tract, diarrhea or mellitus in 12 cases. The anatomical morphological changes included pancreatic fibrosis and atrophy of the main pancreatic duct lesions in 44 cases (14.9%), inflammatory mass in the pancreatic head in 69 cases (22.4%), sporadic stones with calcification in the pancreatic head in 165 cases(55.9%), hyperplasia mass of pancreatic head and body in 14 cases (4.8%), sporadic stones with calcification in whole branch ducts accompanied with different degree of hyperplasia in whole pancreas in 3 cases (1.0%). The surgical options included longitudinal pancreaticojejunostomy, duodenum-preserving pancreatic head resection, Frey/Frey+ distal pancreatectomy, total and subtotal pancreatectomy. All patients were followed-up for 3 to 6 months in the outpatient. A cross-sectional study was carried out by telephone, letters, questionnaire and outpatient from April to June 2016. Results: Among 295 patients, 267 cases were followed-up for an average time of 40 months(18 to 78 months), 28 cases were lost to followed-up(9.5%). Pain remission rate of pancreatic fibrosis and atrophy of the main pancreatic duct lesions patients was 97.0%, of inflammatory mass in the pancreatic head patients was 96.8%, of sporadic stones with calcification in the pancreatic head patients was 96.6%, of hyperplasia mass of pancreatic head and body patients was 12/13, of sporadic stones with calcification in whole branch ducts accompanied with different degree of hyperplasia in whole pancreas patients was 3/3. There were 19 cases(9.6%) with blood glucose rising. Conclusion: According to the pathological changes of chronic pancreatitis, the reasonable choice of surgical procedures can be done to maximize the removal of the cause of pain and the preservation of pancreatic tissue.

[Viability of extended distal pancreatectomy for pancreatic adenocarcinoma of the body or tail].

OBJECTIVE: To analyze the viability of extended distal pancreatectomy and the associated prognostic factors. METHODS: The data of 57 patients with pancreatic adenocarcinoma who underwent standard distal pancreatectomy(DP) or extended distal pancreatectomy(EDP) from January 2011 to December 2014 were reviewed retrospectively. Thirty-five patients were performed with DP and 22 with EDP. Operation safety and survival benefit between DP and EDP were compared by t-test or χ(2) test.Cox regression analysis was used to explore the prognostic indicators. RESULTS: Compared to DP group, operation time((255±91)min vs.(208±80)min)(t=2.066, P=0.044) and ratio of blood transfusion (50.0% vs.17.1%)(χ(2)=12.836, P=0.008) were greater in EDP group, respectively.There were no significant differences in amount of intraoperative blood loss and postoperative duration of hospitalization. Delayed gastric emptying was greater in EDP(22.7% vs.2.9%)(Z=-2.251, P=0.027), while other complications had no differences. Mortality and ratio of relaparotomy also showed no differences. Median survival following DP was 13.1 months compared to 8.2 months following EDP. There was no difference in survival between DP and EDP. According to the results of multivariate analysis, tumor size(RR=1.275, P=0.03)and perioperative blood transfusions(RR=2.673, P=0.04) were independent prognostic factors. CONCLUSIONS: Though patients with pancreatic adenocarcinoma who undergo EDP have a worse pathologic staging, they will gain a comparable long-term survival to the patients undergo DP. Tumor size and perioperative blood transfusions are independent prognostic factors.

Meta-analysis of pancreaticogastrostomy versus pancreaticojejunostomy after pancreaticoduodenectomy.

BACKGROUND: Surgical reconstruction following pancreaticoduodenectomy (PD) is associated with significant morbidity and mortality. Because of great variability in definitions of specific complications, it remains unclear whether there is a difference in complication rates following the two commonest types of reconstruction, pancreaticogastrostomy (PG) and pancreaticojejunostomy (PJ). Published consensus definitions for postoperative pancreatic fistula (POPF) have led to a series of randomized clinical trials (RCTs) uniquely placed to address this question. METHODS: A literature search was carried out to identify all RCTs comparing postoperative complications of PG versus PJ reconstruction following PD published between January 1995 and December 2013. Pooled odds ratios (ORs) with 95 percent confidence intervals (c.i.) were calculated using fixed-effect or random-effects models. RESULTS: In total, seven RCTs with 1121 patients were included. Four of these trials applied definitions as published by the International Study Group on Pancreatic Fistula (ISGPF). Using ISGPF definitions, the incidence of POPF was lower in patients undergoing PG than in those having PJ (OR 0·50, 95 per cent c.i. 0·34 to 0·73; P < 0·001). Using definitions applied by each individual study, PG was associated with significantly lower rates of POPF (OR 0·51, 0·36 to 0·71; P < 0·001), intra-abdominal fluid collection (OR 0·50, 0·34 to 0·74; P < 0·001) and biliary fistula (OR 0·42, 0·18 to 0·93; P = 0·03) than PJ. CONCLUSION: Meta-analysis of four RCTs based on ISGPF criteria, and seven RCTs using non-standard criteria, revealed that PG reduced the incidence of POPF after PD compared with PJ.

Pneumocystis carinii (jirovecii) pneumonia (PCP): the most common opportunistic infection observed in HIV/AIDS cases at the University Malaya Medical Centre, Kuala Lumpur, Malaysia.

This retrospective study was conducted among 59 HIV/AIDS patients with opportunistic infections admitted to the University Malaya Medical Centre between 2000 and 2009. Fifty-five point nine percent of cases were Chinese, 25.4% were Malays, 11.9% were Indians and 6.8% were of unknown ethnic origin. The male:female ratio was 2.9:1 (44 males and 15 females). The highest prevalence (38.9%) occurred in the 30-39 year old age group. Men comprised 47.7% and women 53.3%; the majority of both were married. The majority of cases were Malaysians (89.8%) and the rest (10.2%) were immigrants. Most of the patients (18.6%) were non-laborers, followed by laborers (11.9%), the unemployed (5.1%) and housewives (3.4%). The most common risk factor was unprotected sexual activity (20.3%). The two most common HIV/AIDS related opportunistic infections were Pneumocystis carinii (jirovecii) pneumonia (PCP) (62.7%) and toxoplasmosis (28.8%). Seventy-two point nine percent of patients had a CD4 count <200 cells/microl and 5.1% had a CD4 count >500 cells/microl. Eleven point nine percent of cases died during study period. A low CD4 count had a greater association with opportunistic infections. Most of the patients presented with fever (44.1%), cough (42.4%) and shortness of breath (28.8%). Detection of the etiologic pathogens aids clinicians in choosing appropriate management strategies.

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

BACKGROUND: Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. METHODS: A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. RESULTS: Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. CONCLUSION: Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

Ministry of health clinical practice guidelines: screening of cardiovascular disease and risk factors.

The Ministry of Health (MOH) publishes clinical practice guidelines on Screening of Cardiovascular Disease and Risk Factors to provide doctors and patients in Singapore with evidence-based guidance on the screening of cardiovascular disease and risk factors. This article reproduces the introduction and executive summary (with recommendations from the guidelines) from the MOH clinical practice guidelines on Screening of Cardiovascular Disease and Risk Factors, for the information of readers of the Singapore Medical Journal. Page numbers mentioned in the reproduced extract refer to the full text of the guidelines, which are available from the Ministry of Health website (http://www.moh.gov.sg/mohcorp/publications.aspx?id=25776). The recommendations should be used with reference to the full text of the guidelines. Following this article are multiple choice questions based on the full text of the guidelines.

Singapore Armed Forces Medical Corps-Ministry of Health clinical practice guidelines: management of heat injury.

The Singapore Armed Forces (SAF) Medical Corps and the Ministry of Health (MOH) have published clinical practice guidelines on Management of Heat Injury to provide doctors and patients in Singapore with evidence-based guidance on the prevention and clinical management of exertional heat injuries. This article reproduces the introduction and executive summary (with recommendations from the guidelines) from the SAF Medical Corps-MOH clinical practice guidelines on Management of Heat Injury, for the information of readers of the Singapore Medical Journal. Chapters and page numbers mentioned in the reproduced extract refer to the full text of the guidelines, which are available from the Ministry of Health website: http://www.moh.gov.sg/mohcorp/publications.aspx?id=25178. The recommendations should be used with reference to the full text of the guidelines. Following this article are multiple choice questions based on the full text of the guidelines.

Nutritional supplement use among university athletes in Singapore.

INTRODUCTION: Nutritional supplements used by athletes can be classified as sports food, dietary supplements and ergogenic aids. The aim of this study was to examine the use of such supplements among university athletes in Singapore. METHODS: 82 athletes from 16 sport disciplines completed a questionnaire which sought information on demographical parameters, type of supplements, frequency of use, motivations, knowledge, expenditure and side effects. RESULTS: The prevalence of supplement use was 76.8 percent and 20 different products were used. Each individual consumed a mean and standard deviation of 3.18 +/- 1.7 products over a 12-month period. The mean number of products consumed daily was 2.1 +/- 1.2. Popular products included sports drinks, vitamin C, multivitamins and traditional/herbal preparations such as essence of chicken, bird's nest and ginseng. Before using a product, 65.9 percent sought information, usually from the media, the Internet, coaches and fellow athletes. However, many did not know where to obtain reliable information and 86.4 percent were also unaware that supplementation can have adverse effects. CONCLUSION: Although there is a high prevalence of supplement use in our study population, many do not have accurate information about these products. Hence, there is an urgent need to provide athletes with education and access to scientific and unbiased information.