Mesenchymal-like Tumor Cells and Myofibroblastic Cancer-Associated Fibroblasts Are Associated with Progression and Immunotherapy Response of Clear Cell Renal Cell Carcinoma.

Immune checkpoint inhibitors (ICI) represent the cornerstone for the treatment of patients with metastatic clear cell renal cell carcinoma (ccRCC). Despite a favorable response for a subset of patients, others experience primary progressive disease, highlighting the need to precisely understand the plasticity of cancer cells and their cross-talk with the microenvironment to better predict therapeutic response and personalize treatment. Single-cell RNA sequencing of ccRCC at different disease stages and normal adjacent tissue (NAT) from patients identified 46 cell populations, including 5 tumor subpopulations, characterized by distinct transcriptional signatures representing an epithelial-to-mesenchymal transition gradient and a novel inflamed state. Deconvolution of the tumor and microenvironment signatures in public data sets and data from the BIONIKK clinical trial (NCT02960906) revealed a strong correlation between mesenchymal-like ccRCC cells and myofibroblastic cancer-associated fibroblasts (myCAF), which are both enriched in metastases and correlate with poor patient survival. Spatial transcriptomics and multiplex immune staining uncovered the spatial proximity of mesenchymal-like ccRCC cells and myCAFs at the tumor-NAT interface. Moreover, enrichment in myCAFs was associated with primary resistance to ICI therapy in the BIONIKK clinical trial. These data highlight the epithelial-mesenchymal plasticity of ccRCC cancer cells and their relationship with myCAFs, a critical component of the microenvironment associated with poor outcome and ICI resistance. SIGNIFICANCE: Single-cell and spatial transcriptomics reveal the proximity of mesenchymal tumor cells to myofibroblastic cancer-associated fibroblasts and their association with disease outcome and immune checkpoint inhibitor response in clear cell renal cell carcinoma.

Accuracy of Tongue Swab Testing Using Xpert MTB-RIF Ultra for Tuberculosis Diagnosis.

Tongue dorsum swabs have shown promise as alternatives to sputum for detecting Mycobacterium tuberculosis (MTB) in patients with pulmonary tuberculosis (TB). Some of the most encouraging results have come from studies that used manual quantitative PCR (qPCR) to analyze swabs. Studies using the automated Cepheid Xpert MTB/RIF Ultra qPCR test (Xpert Ultra) have exhibited less sensitivity with tongue swabs, possibly because Xpert Ultra is optimized for testing sputum, not tongue swab samples. Using two new sample preprocessing methods that demonstrated good sensitivity in preliminary experiments, we assessed diagnostic accuracy and semi-quantitative signals of Xpert Ultra performed on tongue swabs collected from 183 adults with presumed TB in Kampala, Uganda. Relative to a sputum Xpert Ultra reference standard, the sensitivity of tongue swab Xpert Ultra was 77.8% (95% confidence interval [CI] 64.4-88.0) and specificity was 100.0% (95% CI, 97.2-100.0). When compared to a microbiological reference standard (MRS) incorporating both sputum Xpert Ultra and sputum mycobacterial culture, sensitivity was 72.4% (95% CI, 59.1-83.3) and specificity remained the same. Semi-quantitative Xpert Ultra results were generally lower with tongue swabs than with sputum, and cycle threshold values were higher. None of the eight sputum Xpert Ultra "trace" or "very low" results were detected using tongue swabs. Tongue swabs should be considered when sputum cannot be collected for Xpert Ultra testing, or in certain mass-screening settings. Further optimization of tongue swab analysis is needed to achieve parity with sputum-based molecular testing for TB.

Using surveillance data for action: lessons learnt from the second generation HIV/AIDS surveillance project in Pakistan.

Although many countries are now collecting useful, high-quality HIV surveillance data, more efforts are put into the collection of data than into ensuring it is deployed effectively. The Canada-Pakistan HIV/AIDS surveillance project has strengthened and expanded the existing national AIDS control programme surveillance system in Pakistan through a comprehensive estimate of the size and location of some of the most at-risk populations (sex workers and injection drug users) and annual assessments of their sociodemographic characteristics, behaviours and HIV prevalence. The country now uses second generation surveillance data at a broad level to lobby for policy change, mobilize resources, improve programming and measure the success of prevention through an integrated national effort. This article aims to share the experiences and lessons learnt in the development of a second generation surveillance system for HIV/AIDS in Pakistan.

Chlorhexidine vaginal and neonatal wipes in home births in Pakistan: a randomized controlled trial.

OBJECTIVE: To assess tolerance and safety of 0.6% chlorhexidine vaginal and neonatal wipes to improve perinatal outcomes in home deliveries in Pakistan and the ability of traditional birth attendants and project staff to perform a randomized trial of this intervention. METHODS: Focus groups of pregnant and nonpregnant women and in-depth interviews of traditional birth attendants explored barriers to the use of chlorhexidine wipes. Then, a study was performed of women delivering at home attended by traditional birth attendants. Consenting women were randomly assigned to receive either 0.6% chlorhexidine or saline vaginal and neonatal wipes. Women and their infants were followed up on postpartum days 7, 14, and 28. Acceptability and tolerance of vaginal and neonatal wipes, as well as maternal and neonatal outcomes, were assessed. RESULTS: The focus groups and interviews indicated that the chlorhexidine intervention would be acceptable to women and their providers. Of the 213 eligible pregnant women approached, 203 (95%) gave informed consent and were enrolled and allocated to groups. Traditional birth attendants had no difficulty administering chlorhexidine vaginal and neonatal wipes in a home setting. Of the 203 births, 103 (51%) of whom received 0.6% chlorhexidine, there were no allergic reactions, vaginal itching, burning, or requests for study termination. Follow-up at 28 days postpartum was more than 95%. Although this study was not powered to show significant differences in neonatal outcomes between treatment groups, the lower rates of some neonatal adverse clinical outcomes in the chlorhexidine group were encouraging. CONCLUSION: Use of 0.6% chlorhexidine vaginal and neonatal wipes for the prevention of neonatal infection is well-tolerated and seems safe. A trial of this intervention by traditional birth attendants in a home-delivery setting is feasible. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00121394 LEVEL OF EVIDENCE: I.

Distinct bacterial dissemination and disease outcome in mice subcutaneously infected with Borrelia burgdorferi in the midline of the back and the footpad.

Subcutaneous inoculation of mice with Borrelia burgdorferi, the causative agent of Lyme disease, results in established infection and the development of acute arthritis and carditis, hallmarks of human disease. Because conflicting results may originate from the site of subcutaneous inoculation, we addressed the dissemination capacity of spirochetes injected in the shoulder region versus the footpad. Spirochetes inoculated in the footpad disseminated to a lesser extent to distant organs, such as the ear and the heart. This resulted in distinct degrees of joint and cardiac inflammation at the peak of the disease. The differences eventually leveled out. These results suggest that caution must be exercised in the interpretation of results obtained with routes of inoculation that do not closely represent the natural site of infection.

Delivery of the immunosuppressive antigen Salp15 to antigen-presenting cells by Salmonella enterica serovar Typhimurium aroA mutants.

A Salmonella enterica serovar Typhimurium aroA-deficient delivery system was used to target the immunosuppressive protein Salp15 to antigen-presenting cells. In vitro and in vivo infections with Salp15-containing Salmonella resulted in an impaired CD4(+)-T-cell activation, suggesting that the protein was produced by antigen-presenting cells in a physiologically active form.

E2F3 contributes both to the inappropriate proliferation and to the apoptosis arising in Rb mutant embryos.

The E2F transcription factors are thought to be key downstream targets of the retinoblastoma protein (pRB) tumor suppressor. It is widely believed that E2F1, E2F2, and E2F3 can all activate cellular proliferation but that E2F1 is the specific inducer of apoptosis. Here we show that the E2f3 mutation completely suppresses both the inappropriate proliferation and the p53-dependent apoptosis arising in the Rb mutant embryos. Through the analysis of Rb(-/-);E2f3(+/-) embryos, we have been able to separate E2F3's role in the induction of apoptosis from its ability to induce proliferation. Thus, contrary to the prevailing view of E2F action, E2F3 makes a major contribution to the apoptosis resulting from pRB loss.

Effects of neurotensin administered into the ventral tegmental area on prepulse inhibition of startle.

Prepulse inhibition (PPI) of the acoustic startle reflex is an operational measure of sensorimotor gating. Both locomotor activity and PPI are regulated by mesolimbic dopamine activity. Neurotensin is a neuropeptide, which coexists with dopamine in mesolimbic neurons. Neurotensin receptors have been identified in the nucleus accumbens (NAC) and ventral tegmental area (VTA). Previous studies have shown that neurotensin administered into the NAC differentially modulates PPI and locomotor activity. In this study we tested the effects of neurotensin administered into the VTA on PPI and locomotor activity. Consistent with previous studies, intra-VTA administered neurotensin significantly increased spontaneous locomotor activity. However, intra-VTA administered neurotensin did not have any significant effect on PPI. These results suggest that PPI and locomotor activity may have dissociable mesolimbic substrates and that neurotensin in the VTA does not play an important role in regulating PPI.

Oxytocin modulates psychotomimetic-induced deficits in sensorimotor gating.

Oxytocin plays an important role in the regulation of normal cognitive functions and behaviors, which are disturbed in schizophrenia. Several studies suggest that oxytocinergic function is abnormal in schizophrenia patients. Thus, oxytocin may be involved in the pathophysiology associated with this disorder. This study investigated the regulatory effects of oxytocin on deficits in prepulse inhibition (PPI) associated with schizophrenia. Prepulse inhibition (PPI) is an operational measure of sensorimotor gating which can be measured across many species. PPI is the normal suppression of the startle reflex when the intense startling stimulus ("pulse") is immediately preceded by a weaker stimulus ("prepulse"). Subcutaneously administered oxytocin (0.04-1.0 mg/kg) dose-dependently restored PPI that had been reduced in rats by dizocilpine, a non-competitive NMDA antagonist, and by amphetamine, an indirect dopamine agonist. Oxytocin did not produce a significant effect on baseline PPI or PPI decreased by the direct dopamine agonist, apomorphine. The underlying startle response amplitude was also not significantly altered by oxytocin. These results suggest that oxytocin may play an important role in the modulation of dopaminergic and glutamatergic regulation of PPI, and that it may act as a novel endogenous antipsychotic.

Novel antipsychotic-like effects on prepulse inhibition of startle produced by a neurotensin agonist.

Agonists of the neuropeptide neurotensin have been proposed as potential novel antipsychotics based on their ability to modulate neurotransmission in brain regions associated with schizophrenia. To test this hypothesis, we examined the effects of a neurotensin mimetic with improved metabolic stability in an animal model with strong predictive validity for antipsychotic activity. Subcutaneous injections of PD149163, a reduced amide neurotensin(8-13) mimetic, significantly antagonized the reduction of prepulse inhibition (PPI) of the rat startle reflex produced by amphetamine and by the phencyclidine analog dizocilpine. PD149163 had no significant effect on baseline PPI or on baseline startle amplitude and did not antagonize the reduction of PPI produced by the direct dopamine agonist apomorphine. These findings suggest that PD149163 has novel antipsychotic-like properties that are distinct from known members of both the "typical" and "atypical" families of antipsychotics.

Antipsychotic potential of CCK-based treatments: an assessment using the prepulse inhibition model of psychosis.

Systemic injections of cholecystokinin (CCK), a "gut-brain" peptide, have been shown to modulate brain dopamine function and produce neuroleptic-like effects on such dopamine-regulated behaviors as locomotor activity. However, clinical trials of CCK agonists in schizophrenia patients showed mixed results. To re-examine the antipsychotic potential of CCK-based treatments, we examined systemic injections of CCK analogs in an animal model with strong face and construct validity for sensorimotor-gating deficits seen in schizophrenia patients and with strong predictive validity for antipsychotic drug activity. Prepulse inhibition (PPI) occurs when a weak acoustic lead stimulus ("prepulse") inhibits the startle response to a sudden loud sound ("pulse"). PPI is significantly reduced in schizophrenia patients and rats treated with dopamine agonists. Antipsychotics reverse decreased PPI in rats to a degree highly correlated with their clinical efficacy. Subcutaneous (s.c.) injections of caerulein (10 micrograms/kg) a mixed CCKA/B agonist, partially reversed amphetamine-induced reduction of PPI; whereas, s.c. haloperidol (0.5 mg/kg) totally reversed amphetamine-induced disruption of PPI. Caerulein's effect on PPI was blocked by pretreatment with a CCKA antagonist (devazepide) but not a CCKB antagonist (L-365,260). CCK-4, a preferential CCKB agonist, had no significant effect on PPI. These results suggest that caerulein produces a weak neuroleptic-like effect on PPI that is mediated by stimulation of CCKA receptors. Possible circuities in this effect are discussed. In a separate experiment, s.c. caerulein produced to a more potent neuroleptic-like profile on amphetamine-induced hyperlocomotion, suggesting that selection of preclinical paradigms may be important in evaluating the antipsychotic potential of CCK-based treatments.

Pro-dopamine effects of neurotensin on sensorimotor gating deficits.

Neurotensin is a neuropeptide which coexists with mesolimbic dopamine and has exhibited neuroleptic-like activity in the nucleus accumbens. This study examined the effects of neurotensin infused into the nucleus accumbens on prepulse inhibition (PPI) of the rat's acoustic startle reflex, a measure which is relevant to the sensorimotor gating deficits seen in schizophrenia. Neurotensin (5 micrograms) had no effect on the amplitude of the acoustic startle reflex nor on baseline PPI, but it potentiated the disruption of PPI produced by amphetamine and apomorphine. This is the first report of a pro-dopamine action for intra-accumbens neurotensin, and suggests that a complex behavioral pharmacology is associated with this neuropeptide.