Neuroendocrine gene subsets are uniquely dysregulated in prostate adenocarcinoma.

Prostate cancer has heterogeneous growth patterns, and its prognosis is the poorest when it progresses to a neuroendocrine phenotype. Using bioinformatic analysis, we evaluated RNA expression of neuroendocrine genes in a panel of five different cancer types: prostate adenocarcinoma, breast cancer, kidney chromophobe, kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. Our results show that specific neuroendocrine genes are significantly dysregulated in these tumors, suggesting that they play an active role in cancer progression. Among others, synaptophysin (SYP), a conventional neuroendocrine marker, is upregulated in prostate adenocarcinoma (PRAD) and breast cancer (BRCA). Our analysis shows that SYP is enriched in small extracellular vesicles (sEVs) derived from plasma of PRAD patients, but it is absent in sEVs derived from plasma of healthy donors. Similarly, classical sEV markers are enriched in sEVs derived from plasma of prostate cancer patients, but weakly detectable in sEVs derived from plasma of healthy donors. Overall, our results pave the way to explore new strategies to diagnose these diseases based on the neuroendocrine gene expression in patient tumors or plasma sEVs.

NorA efflux pump mediates Staphylococcus aureus response to Pseudomonas aeruginosa pyocyanin toxicity.

Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.

Targeting the αVß3/NgR2 pathway in neuroendocrine prostate cancer.

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVß3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVß3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVß3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVß3 ligands. Moreover, we describe for the first time co-fractionation of αVß3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.

Delivery Models and Health Economics of Supportive Care Services in England: A Multicentre Analysis.

AIMS: Improvements in cancer treatment have led to more people living with and beyond cancer. These patients have symptom and support needs unmet by current services. The development of enhanced supportive care (ESC) services may meet the longitudinal care needs of these patients, including at the end of life. This study aimed to determine the impact and health economic benefits of ESC for patients living with treatable but not curable cancer. MATERIALS AND METHODS: A prospective observational evaluation was undertaken over 12 months across eight cancer centres in England. ESC service design and costs were recorded. Data relating to patients' symptom burden were collected using the Integrated Palliative Care Outcome Scale (IPOS). For patients in the last year of life, secondary care use was compared against an NHS England published benchmark. RESULTS: In total, 4594 patients were seen by ESC services, of whom 1061 died during follow-up. Mean IPOS scores improved across all tumour groups. In total, £1,676,044 was spent delivering ESC across the eight centres. Reductions in secondary care usage for the 1061 patients who died saved a total of £8,490,581. CONCLUSIONS: People living with cancer suffer with complex and unmet needs. ESC services appear to be effective at supporting these vulnerable people and significantly reduce the costs of their care.

Autoantibodies Drive Heart Damage Caused by Concomitant Radiation and PD-1 Blockade.

Concurrent PD-1 blockade and thoracic radiotherapy is being investigated in clinical trials for locally advanced, non-small cell lung cancer and small cell lung cancer, despite a potential overlapping risk of cardiotoxicity. Our prior studies demonstrate that cardiotoxicity from concurrent cardiac irradiation and anti-PD-1 administration in a mouse model is CD8+ T-cell dependent. The objective of this study was to determine whether humoral immunity contributed to the observed cardiac tissue damage, as measured by creatine kinase MB and cardiac troponin 1 release and decline in cardiac function. In the current study, we demonstrate the presence of cardiac autoantibodies, which were essential for the occurrence of cardiotoxicity from the combined therapy. Mice subjected to cardiac irradiation, while being treated with anti-PD-1, developed high levels of antibodies that reacted with cardiac tissues in vivo and cardiac antigens in vitro. Moreover, mice deficient in B cells were protected against cardiotoxicity, whereas the transfer of autoantibody-containing sera from mice that had received combined treatment reproduced the same pathologic phenotype in mice exposed to cardiac irradiation but was not observed in normal recipients. The cardiotoxic effect of the sera, which associated with CD8+ T-cell accumulation in cardiac tissue, was limited by IgG depletion. In conclusion, concurrent cardiac irradiation and PD-1 blockade leads to production of cardiac autoantibodies, likely due to antigen exposure within the irradiated cardiac tissues, which play a key role in the resulting cardiotoxicity.

Glioblastoma Contains Topologically Distinct Proliferative and Metabolically Defined Subpopulations of Nestin- and Glut1-Expressing Cells.

The difficulty in treatment of glioblastoma is a consequence of its natural infiltrative growth and the existence of a population of therapy-resistant glioma cells that contribute to growth and recurrence. To identify cells more likely to have these properties, we examined the expression in tumor specimens of several protein markers important for glioma progression including the intermediate filament protein, Nestin (NES), a glucose transporter (Glut1/SLC2A1), the glial lineage marker, glial fibrillary acidic protein, and the proliferative indicator, Ki-67. We also examined the expression of von Willebrand factor, a marker for endothelial cells as well as the macrophage/myeloid markers CD163 and CD15. Using a multicolor immunofluorescence and hematoxylin and eosin staining approach with archival formalin-fixed, paraffin embedded tissue from primary, recurrent, and autopsy IDH1 wildtype specimens combined with high-resolution tissue image analysis, we have identified highly proliferative NES(+)/Glut1(-) cells that are preferentially perivascular. In contrast, Glut1(+)/NES(-) cells are distant from blood vessels, show low proliferation, and are preferentially located at the borders of pseudopalisading necrosis. We hypothesize that Glut1(+)/NES(-) cells would be naturally resistant to conventional chemotherapy and radiation due to their low proliferative capacity and may act as a reservoir for tumor recurrence.

Broad Spectrum Polyphenol Supplementation from Tart Cherry Extract on Markers of Recovery from Intense Resistance Exercise.

BACKGROUND: Tart cherry supplementation has been shown to enhance recovery from strenuous exercise due to its antioxidant properties. The majority of these studies used tart cherry juice, with a significant calorie content. The primary purpose of this study was to assess whether powdered tart cherry extract with minimal calorie content reduces oxidative stress and enhances recovery following intense resistance exercise. METHODS: Thirteen men (mean age: 26.2 ± 5.3 years; height: 184.3 ± 8.2 cm; weight: 92.9 ± 15.6 kg) performed a demanding resistance exercise protocol consisting of 6 sets of 10 repetitions of barbell back squat with 80% 1RM. The protocol was performed once following 7 days of 500 mg of tart cherry extract and once following placebo. Serum protein carbonyl (PC) content, creatine kinase activity (CK) and creatine kinase myocardial band content (CK-MB) were used to assess oxidative stress, skeletal and cardiac muscle damage respectively. Muscle soreness was assessed by visual analog scale. Physical performance was measured by countermovement jump power and handgrip dynamometer strength. RESULTS: There was a significant increase in PC in the placebo (PL) condition when compared to the Tart Cherry (TC) condition at Immediate Post (IP) (PL: 0.4 ± 0.3 vs. TC: - 0.4 ± 0.2 nmol∙mg- 1; p < 0.001), 1 h (PL: 0.3 ± 0.3 vs. TC: - 0.7 ± 0.3 nmol∙mg- 1; p < 0.001) and 24 h (PL: 0.1 ± 0.4 vs. TC: - 0.3 ± 0.5 nmol∙mg- 1; p = 0.010). There was a significant increase in CK activity in PL when compared to the TC at IP (PL: 491.1 ± 280 vs. TC: 296.3 ± 178 U∙L- 1; p = 0.008) and 3 h (PL: - 87 ± 123 vs. TC: 43.1 ± 105.3 U∙L- 1; p = 0.006). There was a significant (p = 0.003) increase in CKMB concentration in PL when compared to the TC (PL: 21.6 ± 12.4 vs. TC: - 0.3 ± 11.8 ng∙ml- 1; p = 0.006) at 1 h post. There was a significant increase in handgrip strength in TC when compared to PL (PL: - 2 ± 5.1 vs. TC: 1.7 ± 3 kg; p = 0.017) at 24 h post. CONCLUSIONS: This study demonstrated that tart cherry extract reduced oxidative stress and markers of muscle and cardiac damage following intense resistance exercise. This occurred along with a prevention of the decrease in handgrip strength seen following the intense exercise protocol, indicating a potential reduction in central fatigue. These benefits were seen with minimal energy intake.

Systemic Immune Bias Delineates Malignant Astrocytoma Survival Cohorts.

Patients with grade III anaplastic astrocytomas (AA) separate into survival cohorts based on the presence or absence of mutations in isocitrate dehydrogenase (IDH). Progression to glioblastoma (GBM), morphologically distinguishable by elevated microvascular proliferation, necrosis, and cell division in tumor tissues, is considerably more rapid in IDH wild-type tumors such that their diagnosis as AA is relatively rare. More often initially presenting as GBM, these contain higher numbers of tumor-associated macrophages (TAMs) than most AA, and GBM patients also have higher levels of circulating M2 monocytes. TAM and M2 monocytes share functional properties inhibitory for antitumor immunity. Yet, although there is a wealth of data implicating TAM in tumor-immune evasion, there has been limited analysis of the impact of the circulating M2 monocytes. In the current study, immune parameters in sera, circulating cells, and tumor tissues from patients with primary gliomas morphologically diagnosed as AA were assessed. Profound differences in serum cytokines, glioma extracellular vesicle cross-reactive Abs, and gene expression by circulating cells identified two distinct patient cohorts. Evidence of type 2-immune bias was most often seen in patients with IDH wild-type AA, whereas a type 1 bias was common in patients with tumors expressing the IDH1R132H mutation. Nevertheless, a patient's immune profile was better correlated with the extent of tumor vascular enhancement on magnetic resonance imaging than IDH mutational status. Regardless of IDH genotype, AA progression appears to be associated with a switch in systemic immune bias from type 1 to type 2 and the loss of tumor vasculature integrity.

Staphylococcus aureus Tet38 Efflux Pump Structural Modeling and Roles of Essential Residues in Drug Efflux and Host Cell Internalization.

The Staphylococcus aureus Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F411P412G413, in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in S. aureus internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D38D39L40, in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R106 and G151 (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.

Phase Ib Clinical Trial of IGV-001 for Patients with Newly Diagnosed Glioblastoma.

PURPOSE: Despite standard of care (SOC) established by Stupp, glioblastoma remains a uniformly poor prognosis. We evaluated IGV-001, which combines autologous glioblastoma tumor cells and an antisense oligonucleotide against IGF type 1 receptor (IMV-001), in newly diagnosed glioblastoma. PATIENTS AND METHODS: This open-label protocol was approved by the Institutional Review Board at Thomas Jefferson University. Tumor cells collected during resection were treated ex vivo with IMV-001, encapsulated in biodiffusion chambers with additional IMV-001, irradiated, then implanted in abdominal acceptor sites. Patients were randomized to four exposure levels, and SOC was initiated 4-6 weeks later. On the basis of clinical improvements, randomization was halted after patient 23, and subsequent patients received only the highest exposure. Safety and tumor progression were primary and secondary objectives, respectively. Time-to-event outcomes were compared with the SOC arms of published studies. RESULTS: Thirty-three patients were enrolled, and median follow-up was 3.1 years. Six patients had adverse events (grade ≤3) possibly related to IGV-001. Median progression-free survival (PFS) was 9.8 months in the intent-to-treat population (vs. SOC, 6.5 months; P = 0.0003). In IGV-001-treated patients who met Stupp-eligible criteria, PFS was 11.6 months overall (n = 22; P = 0.001) and 17.1 months at the highest exposure (n = 10; P = 0.0025). The greatest overall survival was observed in Stupp-eligible patients receiving the highest exposure (median, 38.2 months; P = 0.044). Stupp-eligible patients with methylated O6-methylguanine-DNA methyltransferase promoter (n = 10) demonstrated median PFS of 38.4 months (P = 0.0008). Evidence of immune activation was noted. CONCLUSIONS: IGV-001 was well tolerated, PFS compared favorably with SOC, and evidence suggested an immune-mediated mechanism (ClinicalTrials.gov: NCT02507583).

Brain tissue-resident immune memory cells are required for long-term protection against CNS infection with rabies virus.

Immune memory cells residing in previously infected, nonlymphoid tissues play a role in immune surveillance. In the event that circulating antibodies fail to prevent virus spread to the tissues in a secondary infection, these memory cells provide an essential defense against tissue reinfection. CNS tissues are isolated from circulating immune cells and antibodies by the blood-brain barrier, making the presence of tissue-resident immune memory cells particularly needed to combat recurrent infection by neurotropic viruses. Wild-type and laboratory-engineered rabies viruses are neurotropic, differ in pathogenicity, and have varying effects on BBB functions. These viruses have proven invaluable tools in demonstrating the importance of tissue-resident immune memory cells in the reinfection of CNS tissues. Only Type 1 immune memory is effective at therapeutically clearing a secondary infection with wild-type rabies viruses from the CNS and does so despite the maintenance of blood-brain barrier integrity.

Cosmic-ray antinuclei as messengers of new physics: status and outlook for the new decade.

The precise measurement of cosmic-ray antinuclei serves as an important means for identifying the nature of dark matter and other new astrophysical phenomena, and could be used with other cosmic-ray species to understand cosmic-ray production and propagation in the Galaxy. For instance, low-energy antideuterons would provide a "smoking gun" signature of dark matter annihilation or decay, essentially free of astrophysical background. Studies in recent years have emphasized that models for cosmic-ray antideuterons must be considered together with the abundant cosmic antiprotons and any potential observation of antihelium. Therefore, a second dedicated Antideuteron Workshop was organized at UCLA in March 2019, bringing together a community of theorists and experimentalists to review the status of current observations of cosmic-ray antinuclei, the theoretical work towards understanding these signatures, and the potential of upcoming measurements to illuminate ongoing controversies. This review aims to synthesize this recent work and present implications for the upcoming decade of antinuclei observations and searches. This includes discussion of a possible dark matter signature in the AMS-02 antiproton spectrum, the most recent limits from BESS Polar-II on the cosmic antideuteron flux, and reports of candidate antihelium events by AMS-02; recent collider and cosmic-ray measurements relevant for antinuclei production models; the state of cosmic-ray transport models in light of AMS-02 and Voyager data; and the prospects for upcoming experiments, such as GAPS. This provides a roadmap for progress on cosmic antinuclei signatures of dark matter in the coming years.

Potential role of CSF cytokine profiles in discriminating infectious from non-infectious CNS disorders.

Current laboratory testing of cerebrospinal fluid (CSF) does not consistently discriminate between different central nervous system (CNS) disease states. Rapidly distinguishing CNS infections from other brain and spinal cord disorders that share a similar clinical presentation is critical. New approaches focusing on aspects of disease biology, such as immune response profiles that can have stimulus-specific attributes, may be helpful. We undertook this preliminary proof-of-concept study using multiplex ELISA to measure CSF cytokine levels in various CNS disorders (infections, autoimmune/demyelinating diseases, lymphomas, and gliomas) to determine the potential utility of cytokine patterns in differentiating CNS infections from other CNS diseases. Both agglomerative hierarchical clustering and mixture discriminant analyses revealed grouping of CNS disease types based on cytokine expression. To further investigate the ability of CSF cytokine levels to distinguish various CNS disease states, non-parametric statistical analysis was performed. Mann-Whitney test analysis demonstrated that CNS infections are characterized by significantly higher CSF lP-10/CXCL10 levels than the pooled non-infectious CNS disorders (p = 0.0001). Within the infection group, elevated levels of MDC/CCL22 distinguished non-viral from viral infections (p = 0.0048). Each disease group of the non-infectious CNS disorders independently showed IP-10/CXCL10 levels that are significantly lower than the infection group [(autoimmune /demyelinating disorders (p = 0.0005), lymphomas (p = 0.0487), gliomas (p = 0.0294), and controls (p = 0.0001)]. Additionally, of the non-infectious diseases, gliomas can be distinguished from lymphomas by higher levels of GRO/CXCL1 (p = 0.0476), IL-7 (p = 0.0119), and IL-8 (p = 0.0460). Gliomas can also be distinguished from autoimmune/demyelinating disorders by higher levels of GRO/CXCL1 (p = 0.0044), IL-7 (p = 0.0035) and IL-8 (p = 0.0176). Elevated CSF levels of PDGF-AA distinguish lymphomas from autoimmune/demyelinating cases (p = 0.0130). Interrogation of the above comparisons using receiver operator characteristic analysis demonstrated area under the curve (AUC) values (ranging from 0.8636-1.0) that signify good to excellent utility as potential diagnostic discriminators. In conclusion, our work indicates that upon formal validation, measurement of CSF cytokine levels may have clinical utility in both identifying a CNS disorder as infectious in etiology and, furthermore, in distinguishing viral from non-viral CNS infections.

Exosomal αvß6 integrin is required for monocyte M2 polarization in prostate cancer.

Therapeutic approaches aimed at curing prostate cancer are only partially successful given the occurrence of highly metastatic resistant phenotypes that frequently develop in response to therapies. Recently, we have described αvß6, a surface receptor of the integrin family as a novel therapeutic target for prostate cancer; this epithelial-specific molecule is an ideal target since, unlike other integrins, it is found in different types of cancer but not in normal tissues. We describe a novel αvß6-mediated signaling pathway that has profound effects on the microenvironment. We show that αvß6 is transferred from cancer cells to monocytes, including ß6-null monocytes, by exosomes and that monocytes from prostate cancer patients, but not from healthy volunteers, express αvß6. Cancer cell exosomes, purified via density gradients, promote M2 polarization, whereas αvß6 down-regulation in exosomes inhibits M2 polarization in recipient monocytes. Also, as evaluated by our proteomic analysis, αvß6 down-regulation causes a significant increase in donor cancer cells, and their exosomes, of two molecules that have a tumor suppressive role, STAT1 and MX1/2. Finally, using the Ptenpc-/- prostate cancer mouse model, which carries a prostate epithelial-specific Pten deletion, we demonstrate that αvß6 inhibition in vivo causes up-regulation of STAT1 in cancer cells. Our results provide evidence of a novel mechanism that regulates M2 polarization and prostate cancer progression through transfer of αvß6 from cancer cells to monocytes through exosomes.

Type 1 Immune Mechanisms Driven by the Response to Infection with Attenuated Rabies Virus Result in Changes in the Immune Bias of the Tumor Microenvironment and Necrosis of Mouse GL261 Brain Tumors.

Immunotherapeutic strategies for malignant glioma have to overcome the immunomodulatory activities of M2 monocytes that appear in the circulation and as tumor-associated macrophages (TAMs). M2 cell products contribute to the growth-promoting attributes of the tumor microenvironment (TME) and bias immunity toward type 2, away from the type 1 mechanisms with antitumor properties. To drive type 1 immunity in CNS tissues, we infected GL261 tumor-bearing mice with attenuated rabies virus (RABV). These neurotropic viruses spread to CNS tissues trans-axonally, where they induce a strong type 1 immune response that involves Th1, CD8, and B cell entry across the blood-brain barrier and virus clearance in the absence of overt sequelae. Intranasal infection with attenuated RABV prolonged the survival of mice bearing established GL261 brain tumors. Despite the failure of virus spread to the tumor, infection resulted in significantly enhanced tumor necrosis, extensive CD4 T cell accumulation, and high levels of the proinflammatory factors IFN-γ, TNF-α, and inducible NO synthase in the TME merely 4 d postinfection, before significant virus spread or the appearance of RABV-specific immune mechanisms in CNS tissues. Although the majority of infiltrating CD4 cells appeared functionally inactive, the proinflammatory changes in the TME later resulted in the loss of accumulating M2 and increased M1 TAMs. Mice deficient in the Th1 transcription factor T-bet did not gain any survival advantage from RABV infection, exhibiting only limited tumor necrosis and no change in TME cytokines or TAM phenotype and highlighting the importance of type 1 mechanisms in this process.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes.

Immune biomarkers of treatment failure for a patient on a phase I clinical trial of pembrolizumab plus radiotherapy.

BACKGROUND: Pembrolizumab is a monoclonal antibody that is designed against programmed cell death protein 1 (PD-1). Pembrolizumab and other immunocheckpoint-blocking monoclonal antibodies work by modulating a patient's own immune system to increase anti-tumor activity. While immunocheckpoint blockade has shown promising results, only 20-40 % of patients experience objective clinical benefit. Differences in individual tumor biology and the presence multiple immune checkpoints present a challenge for treatment. Because radiotherapy has immunomodulatory effects on the tumor microenvironment, it has the potential to synergize with immunotherapy and augment tumor response. NCT02318771 is a phase 1 clinical trial designed to investigate the immunomodulatory effects of radiation therapy in combination with pembrolizumab. CASE PRESENTATION: The patient is a 64-year-old male with metastatic clear cell renal cell carcinoma, Fuhrman grade 4, pathologically staged as T3 N0. Metastatic disease was well controlled for several years with sunitinib. Following disease progression, he was switched to axitinib. When disease progression continued, the patient was enrolled in NCT02318771, a phase 1 clinical trial combining radiotherapy and pembrolizumab. The patient experienced unusually rapid disease progression during treatment, which was confirmed by repeated CT scans to rule out pseudoprogression. Tissue biopsies and peripheral blood draws were obtained before, during, and after treatment. Samples were analyzed to provide plausible rationale for rapid treatment failure. CONCLUSIONS: Biomarker analysis demonstrated an absence of TILs, which may be a cause of treatment failure as pembrolizumab works through T cell-dependent mechanisms. Furthermore, the presence of other non-redundant immune checkpoints in the periphery and tumor microenvironment presents a treatment challenge. Additionally, the radiation dose and fractionation schedule may have played a role in treatment failure as these factors play a role in the effect radiotherapy on the tumor microenvironment as well as the potential for synergy with immunotherapy. TRIAL REGISTRATION: An Exploratory Study to Investigate the Immunomodulatory Activity of Radiation Therapy (RT) in Combination With MK-3475 in Patients With Recurrent/Metastatic Head and Neck, Renal Cell Cancer, Melanoma and Lung Cancer, NCT02318771 .

Postpartum Granulomatous Hypophysitis: A Case Study, Review of the Literature, and Discussion of Pathogenesis.

Hypophysitis is a rare inflammatory condition of the pituitary gland that has three main histologic subtypes: lymphocytic hypophysitis (LH), granulomatous hypophysitis (GH), and xanthomatous hypophysitis (XH). Among these, LH is the most common and is strongly associated with the postpartum state, while XH is the least common. Many hypophysitis cases have been reported in the literature but only a few cases of postpartum GH have been discussed. Here, we describe a case of GH in a 24-year-old female presenting eleven days postpartum. We also review the current literature on postpartum GH and discuss the possible alterations in the immune environment during and after pregnancy that could explain this phenomenon. With more cases of GH being reported, the commonalities of female predominance, postpartum time of presentation, and occasional spontaneous resolution between LH and GH lend support to the theory that these two diseases likely represent spectrums of a single immunologic disorder.

Serum exosomes and cytokines promote a T-helper cell type 2 environment in the peripheral blood of glioblastoma patients.

BACKGROUND: Glioblastoma (GBM) is an aggressive infiltrative brain tumor with a particularly poor prognosis that is characterized by microvascular proliferation, necrotic tissue, and significant infiltration of M2-like monocytes. Compromised barrier function in tumor vasculature might be expected to permit communication between the tumor microenvironment and peripheral blood. METHODS: To ascertain whether tumor-derived vesicles and/or factors might reach the bloodstream and what effects these molecules have on the peripheral compartment, we analyzed blood samples collected from primary GBM patients. RESULTS: Notably, a significant number of patient sera samples contained tumor exosome-reactive immunoglobulin (Ig)G2 and IgG4 antibody isotypes, which are consistent with Th2 immunity. M2-like monocytes expressing CD14+ and CD163+, another indicator of Th2 bias, are elevated in GBM patient blood and associated with high serum concentrations of colony-stimulating factor 2 and 3, as well as interleukin-2, -4, and -13, the latter 2 cytokines being hallmarks of Th2 immunity. GBM patient sera samples induce high levels of CD163 expression when added to normal monocytes, providing mechanistic evidence of a basis for Th2 bias. Fractionation of GBM patient sera into samples enriched for exosomes or soluble factors proved that both fractions are capable of inducing CD163 expression in normal monocytes. CONCLUSIONS: The results of the current study indicate a Th2 bias in the periphery of GBM patients, likely as a result of products elaborated by the tumor. Consequentially, through immune modulation these brain tumors exert systemic effects beyond the confines of the CNS.

Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells.

We previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ΔtetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its Δtet38, ΔtetR21, and ΔmgrA mutants, and a Δtet38 ΔtetR21 double mutant. After 2 h of contact, the Δtet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrA mutant. The growth patterns of RN6390 and the ΔmgrA and ΔtetR21 mutants within A549 cells were similar, while no growth was observed for the Δtet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells.