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Objective: As the global village becomes a reality, there is an increasing need to conduct international collaborative studies in family practice. A workshop at the WONCA meeting in Hong Kong used international attendees to produce a set of guidelines for international research.Methods: At the workshop four completed international projects, each using a different strategy, were presented so that common themes might become apparent. The themes were then discussed and guidelines emerged from the process.Results: Seven guidelines emerged for consideration before embarking on an international collaborative research project in family medicine. The guidelines deal with the characteristics of the research question and the importance of communication. The need for simple, brief methods of data collection, funding and pilot testing were identified.Conclusion: The question must be relevant to all participants to maintain interest and measurement tools must be validated to understand the impact of cultural differences in understanding. [ABSTRACT FROM AUTHOR]

Objective: To evalute emotional response towards perceived loss of activities of daily living in Chinese elders with dementia. Patients and Methods: Eighty-one elderly people with a clinical diagnosis of dementia were recruited from residential homes and social cnetre for the elderly in Hong Kong. A purpose-designed questionnaire on subjective evaluation of ability and emotional reactions towards functional deterioration was derived. The association between the subjective evaluation of ability, emotional reactions, and acutal activities of daily living performance measured by the Chinese version of Disability Assessment for Dementia were evaluated. Resutls: There were no significant correlations between subjective evaluation of ability and the emotional reactions towards functional impairment. Subjects reported greater higher emotional distress over possible loss of basic activities of daily living than instrumental activities of daily living (t=3.04,p=0.003). Subjects with better basic activities of daily living abilities were likely to report greater distress if their instrumental activities of daily living were impaired (Spearman's rho=0.30,p=0.01). Conclusion: Although elderly people with dementia may have compromised congnitive abilities, attention to functional training is an important means of improving their emotional well-being. [ABSTRACT FROM AUTHOR]

Personalized dosing approaches play important roles in clinical practices to improve benefit: risk profiles. Whereas this is also important for drug development, especially in the context of drugs with narrow therapeutic windows, such approaches have not been fully evaluated during clinical development. Fazpilodemab (BFKB8488A) is an agonistic bispecific antibody which was being developed for the treatment of nonalcoholic steatohepatitis. The objective of this study was to characterize the exposure-response relationships of fazpilodemab with the purpose of guiding dose selection for a phase II study, as well as to evaluate various personalized dosing strategies to optimize the treatment benefit. Fazpilodemab exhibited clear exposure-response relationships for a pharmacodynamic (PD) biomarker and gastrointestinal adverse events (GIAEs), such as nausea and vomiting. Static exposure-response analysis, as well as longitudinal adverse event (AE) analysis using discrete-time Markov model, were performed to characterize the observations. Clinical trial simulations were performed based on the developed exposure-response models to evaluate probability of achieving target PD response and the frequency of GIAEs to inform phase II dose selection. Dynamic simulation of personalized dosing strategies demonstrated that the AE-based personalized dosing is the most effective approach for optimizing the benefit-risk profiles. The approach presented here can be a useful framework for quantifying the benefit of personalized dosing for drugs with narrow therapeutic windows., (© 2024 Genentech, Inc. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

GPKPDviz is a Shiny application (app) dedicated to real-time simulation, visualization, and assessment of the pharmacokinetic/pharmacodynamic (PK/PD) models. Within the app, gPKPDviz is capable of generating virtual populations and complex dosing and sampling scenarios, which, together with the streamlined workflow, is designed to efficiently assess the impact of covariates and dosing regimens on PK/PD end points. The actual population data from clinical trials can be loaded into the app for simulation if desired. The app-generated dosing regimens include single or multiple dosing, and more complex regimens, such as loading doses or intermittent dosing. When necessary, the dosing regimens can be defined externally and loaded to the app for simulation. Using mrgsolve as the simulation engine, gPKPDviz is typically used for population simulation, however, with a slight modification of the mrgsolve model, gPKPDviz is capable of performing individual simulations with individual post hoc parameters, individual dosing logs, and individual sampling timepoints through an external dataset. A built-in text editor has a debugging feature for the mrgsolve model, providing the same error messages as model compilation in R. GPKPDviz has had stringent validation by comparing simulation results between the app and using mrgsolve in R. GPKPDviz is a member of the suite of Modeling and Simulation Shiny apps developed at Genentech to facilitate the typical modeling work in Clinical Pharmacology. For broader access to the Pharmacometric community, gPKPDviz has been published as an open-source application in GitHub under the terms of GNU General Public License., (© 2023 Genentech, Inc. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Over two-thirds of pregnant women in the U.S. have insufficient 25(OH)D (Vitamin D) concentrations, which can adversely impact fetal health. Several pollutants have been associated with 25(OH)D, but have not been considered in the context of chemical co-exposures. We aimed to determine associations between a broad mixture of prenatal environmental chemical exposures and 25(OH)D concentrations in mid-pregnancy. Stored mid-pregnancy serum samples were assayed from 421 women delivering live births in Southern California in 2000-2003. 25(OH)D, six BFRs, eleven polychlorinated biphenyls (PCBs), six per- and polyfluoroalkyl substances, and two organochlorine pesticides were detected in ≥60% of specimens. Gestational exposures to airborne particulate matter ≤ 10 μm (PM 10 ) and ≤ 2.5 μm (PM 2.5 ), nitrogen monoxide (NO), nitrogen dioxide (NO 2 ), and ozone concentrations were derived from monitoring station data. Bayesian Hierarchical Modeling (BHM) and Bayesian Kernel Machine Regression (BKMR) analyses estimated overall mixture and individual chemical associations accounting for co-exposures and covariates with mean 25(OH)D levels, and BHM was used to estimate associations with insufficient (<75 nMol/L) 25(OH)D levels. Non-mixture associations for each chemical were estimated with linear and logistic models. PM 10 [BHM estimate: -0.133 nmol/l 95% Credible Interval (-0.240, -0.026)] was associated with lower 25(OH)D in BHM and BKMR. Higher quantiles of combined exposures were associated with lower 25(OH)D, though with wide credible intervals. In non-mixture models, PM 10 , PM 2.5 , NO, and NO 2 were associated with lower concentrations, while O 3 and PBDE153 were associated with higher 25(OH)D and/or lower insufficiency. While some chemicals were associated with increased and others with decreased 25(OH)D concentrations, the overall mixture was associated with lower concentrations. Mixture analyses differed from non-mixture regressions, highlighting the importance of mixtures approaches for estimating real-world associations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: To report the safety and efficacy of ipatasertib (AKT inhibitor) combined with rucaparib (PARP inhibitor) in patients with metastatic castration-resistant prostate cancer (mCRPC) previously treated with second-generation androgen receptor inhibitors., Patients and Methods: In this two-part phase Ib trial (NCT03840200), patients with advanced prostate, breast, or ovarian cancer received ipatasertib (300 or 400 mg daily) plus rucaparib (400 or 600 mg twice daily) to assess safety and identify a recommended phase II dose (RP2D). A part 1 dose-escalation phase was followed by a part 2 dose-expansion phase in which only patients with mCRPC received the RP2D. The primary efficacy endpoint was prostate-specific antigen (PSA) response (≥50% reduction) in patients with mCRPC. Patients were not selected on the basis of tumor mutational status., Results: Fifty-one patients were enrolled (part 1 = 21; part 2 = 30). Ipatasertib 400 mg daily plus rucaparib 400 mg twice daily was the selected RP2D, received by 37 patients with mCRPC. Grade 3/4 adverse events occurred in 46% (17/37) of patients, with one grade 4 adverse event (anemia, deemed related to rucaparib) and no deaths. Adverse events leading to treatment modification occurred in 70% (26/37). The PSA response rate was 26% (9/35), and the objective response rate per Response Criteria in Solid Tumors (RECIST) 1.1 was 10% (2/21). Median radiographic progression-free survival per Prostate Cancer Working Group 3 criteria was 5.8 months [95% confidence interval (CI), 4.0-8.1], and median overall survival was 13.3 months (95% CI, 10.9-not evaluable)., Conclusions: Ipatasertib plus rucaparib was manageable with dose modification but did not demonstrate synergistic or additive antitumor activity in previously treated patients with mCRPC., (©2023 American Association for Cancer Research.)

XmAb24306 is a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein currently under clinical investigation as an immunotherapeutic agent for cancer treatment. XmAb24306 contains mutations in IL-15 that attenuate its affinity to the heterodimeric IL-15 receptor βγ (IL-15R). We observe substantially prolonged pharmacokinetics (PK) (half-life ∼ 2.5 to 4.5 days) in single- and repeat-dose cynomolgus monkey (cyno) studies compared to wild-type IL-15 (half-life ∼ 1 hour), leading to increased exposure and enhanced and durable expansion of NK cells, CD8+ T cells and CD4-CD8- (double negative [DN]) T cells. Drug clearance varied with dose level and time post-dose, and PK exposure decreased upon repeated dosing, which we attribute to increased target-mediated drug disposition (TMDD) resulting from drug-induced lymphocyte expansion (i.e., pharmacodynamic (PD)-enhanced TMDD). We developed a quantitative systems pharmacology (QSP) model to quantify the complex PKPD behaviors due to the interactions of XmAb24306 with multiple cell types (CD8+, CD4+, DN T cells, and NK cells) in the peripheral blood (PB) and lymphoid tissues. The model, which includes nonspecific drug clearance, binding to and TMDD by IL15R differentially expressed on lymphocyte subsets, and resultant lymphocyte margination/migration out of PB, expansion in lymphoid tissues, and redistribution to the blood, successfully describes the systemic PK and lymphocyte kinetics observed in the cyno studies. Results suggest that after 3 doses of every-two-week (Q2W) doses up to 70 days, the relative contributions of each elimination pathway to XmAb24306 clearance are: DN T cells > NK cells > CD8+ T cells > nonspecific clearance > CD4+ T cells. Modeling suggests that observed cellular expansion in blood results from the influx of cells expanded by the drug in lymphoid tissues. The model is used to predict lymphoid tissue expansion and to simulate PK-PD for different dose regimens. Thus, the model provides insight into the mechanisms underlying the observed PK-PD behavior of an engineered cytokine and can serve as a framework for the rapid integration and analysis of data that emerges from ongoing clinical studies in cancer patients as single-agent or given in combination., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The pharmacokinetic (PK) characteristics of omalizumab and its pharmacodynamic (PD) effect in patients has yet to be fully characterized in chronic spontaneous urticaria, which could elucidate its pathogenesis and treatment response. This study has two objectives; (1) characterize the population PK of omalizumab and its PD effect on IgE, and (2) develop a drug effect model of omalizumab in urticaria (via change in weekly itch severity score). The target-mediated population of PK/PD model incorporating omalizumab-IgE binding and turnover adequately described PK and PD of omalizumab. The effect compartment model and linear drug effect and additive placebo response adequately described placebo and treatment effects of omalizumab. Several baseline covariates were identified for PK/PD and drug effect models. The developed model has the potential to aid in understanding variability in PK/PD as well as response to omalizumab treatment., (© 2023 Genentech, Inc and QuanTx Consulting. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Tryptase, a protease implicated in asthma pathology, is secreted from mast cells upon activation during an inflammatory allergic response. MTPS9579A is a novel monoclonal antibody that inhibits tryptase activity by irreversibly dissociating the active tetramer into inactive monomers. This study assessed the relationship between MTPS9579A concentrations in healthy subjects and tryptase levels in serum and nasal mucosal lining fluid from healthy subjects and patients with moderate-to-severe asthma. These data were used to develop a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model that quantitatively inter-relates MTPS9579A exposure and inhibition of active tryptase in the airway of patients with asthma. From initial estimates of airway tryptase levels and drug partitioning, the PK/PD model predicted almost complete neutralization of active tryptase in the airway of patients with asthma with MTPS9579A doses of 900 mg and greater, administered intravenously (i.v.) once every 4 weeks (q4w). Suppression of active tryptase during an asthma exacerbation event was also evaluated using the model by simulating the administration of MTPS9579A during a 100-fold increase in tryptase secretion in the local tissue. The PK/PD model predicted that 1800 mg MTPS9579A i.v. q4w results in 95.7% suppression of active tryptase at the steady-state trough concentration. Understanding how the exposure-response relationship of MTPS9579A in healthy subjects translates to patients with asthma is critical for future clinical studies assessing tryptase inhibition in the airway of patients with moderate-to-severe asthma., (© 2023 Genentech, Inc. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

A Cox proportional hazard (CoxPH) model is conventionally used to assess exposure-response (E-R), but its performance to uncover the ground truth when only one dose level of data is available has not been systematically evaluated. We established a simulation workflow to generate realistic E-R datasets to assess the performance of the CoxPH model in recovering the E-R ground truth in various scenarios, considering two potential reasons for the confounded E-R relationship. We found that at high doses, when the pharmacological effects are largely saturated, missing important confounders is the major reason for inferring false-positive E-R relationships. At low doses, when a positive E-R slope is the ground truth, either missing important confounders or mis-specifying the interactions can lead to inaccurate estimates of the E-R slope. This work constructed a simulation workflow generally applicable to clinical datasets to generate clinically relevant simulations and provide an in-depth interpretation on the E-R relationships with confounders inferred by the conventional CoxPH model., (© 2022 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Black women have the highest incidence of preterm birth (PTB). Upstream factors, including neighborhood context, may be key drivers of this increased risk. This study assessed the relationship between neighborhood quality, defined by the Healthy Places Index, and PTB among Black women who lived in Oakland, California, and gave birth between 2007 and 2011 (N = 5418 women, N = 107 census tracts). We found that, compared with those living in lower quality neighborhoods, women living in higher quality neighborhoods had 20-38% lower risk of PTB, independent of confounders. Findings have implications for place-based research and interventions to address racial inequities in PTB., (© 2022. The Author(s).)

SPINK6 was identified in human skin as a cellular inhibitor of serine proteases of the KLK family. Airway serine proteases are required to cleave hemagglutinin (HA) of influenza A viruses (IAVs) to initiate an infection in the human airway. We hypothesized that SPINK6 may inhibit common airway serine proteases and restrict IAV activation. We demonstrate that SPINK6 specifically suppresses the proteolytic activity of HAT and KLK5, HAT- and KLK5-mediated HA cleavage, and restricts virus maturation and replication. SPINK6 constrains the activation of progeny virions and impairs viral growth; and vice versa, blocking endogenous SPINK6 enhances HA cleavage and viral growth in physiological-relevant human airway organoids where SPINK6 is intrinsically expressed. In IAV-infected mice, SPINK6 significantly suppresses viral growth and improves mouse survival. Notably, individuals carrying the higher SPINK6 expression allele were protected from human H7N9 infection. Collectively, SPINK6 is a novel host inhibitor of serine proteases in the human airway and restricts IAV activation., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Competing Interests: The authors have no conflicts of interest to disclose.

Objective: The benefits of aerobic exercise early after stroke are well known, but concerns about cardiovascular risk are a barrier to clinical implementation. Symptom-limited exercise testing with electrocardiography (ECG) is recommended but not always feasible. The purpose of this study was to determine the frequency of and corresponding exercise intensities at which ECG abnormalities occurred during submaximal exercise testing that would limit safe exercise prescription beyond those intensities., Methods: This study was a retrospective analysis of ECGs from 195 patients who completed submaximal exercise testing during stroke rehabilitation. A graded submaximal exercise test was conducted with a 5- or 12-lead ECG and was terminated on the basis of predetermined endpoint criteria (heart rate, perceived exertion, signs, or symptoms). ECGs were retrospectively reviewed for exercise-induced abnormalities and their associated heart rates., Results: The peak heart rate achieved was 65.4% (SD = 10.5%) of the predicted maximum heart rate or 29.1% (SD = 15.5%) of the heart rate reserve (adjusted for beta-blocker medications). The test was terminated more often because of perceived exertion (93/195) than because of heart rate limits (60/195). Four patients (2.1%) exhibited exercise-induced horizontal or downsloping ST segment depression of ≥1 mm. Except for 1 patient, the heart rate at test termination was comparable with the heart rate associated with the onset of the ECG abnormality., Conclusion: A graded submaximal exercise test without ECG but with symptom monitoring and conservative heart rate and perceived exertion endpoints may facilitate safe exercise intensities early after stroke. Symptom-limited exercise testing with ECG is still recommended when progressing to higher intensity exercise., Impact: Concerns about cardiovascular risk are a barrier to physical therapists implementing aerobic exercise in stroke rehabilitation. This study showed that, in the absence of access to exercise testing with ECG, submaximal testing with conservative heart rate and perceived exertion endpoints and symptom monitoring can support physical therapists in the safe prescription of aerobic exercise early after stroke., Lay Summary: It is recommended that people with stroke participate in aerobic exercise as early as possible during their rehabilitation. A submaximal exercise test with monitoring of heart rate, perceived exertion, blood pressure, and symptoms can support physical therapists in safely prescribing that exercise., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Physical Therapy Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Vitamin D is essential for several physiological functions and biological processes. Increasing levels of maternal vitamin D are required throughout pregnancy as a unique source of vitamin D for the fetus, and consequently maternal vitamin D deficiency may result in several adverse outcomes in newborns. However, the genetic regulation of vitamin D in pregnancy and at birth is not yet well understood. We performed genome-wide association studies of maternal midgestational serum-derived and neonatal blood-spot-derived total 25-hydroxyvitamin D from a case-control study of autism spectrum disorder (ASD). We identified one fetal locus (rs4588) significantly associated with neonatal vitamin D levels in the GC gene, encoding the binding protein for the transport and function of vitamin D. We also found suggestive cross-associated loci for neonatal and maternal vitamin D near immune genes, such as CXCL6-IL8 and ACKR1 We found no interactions with ASD. However, when including a set of cases with intellectual disability but not ASD ( N = 179), we observed a suggestive interaction between decreased levels of neonatal vitamin D and a specific maternal genotype near the PKN2 gene. Our results suggest that genetic variation influences total vitamin D levels during pregnancy and at birth via proteins in the vitamin D pathway, but also potentially via distinct mechanisms involving loci with known roles in immune function that might be involved in vitamin D pathophysiology in pregnancy., (Copyright © 2020 by the Genetics Society of America.)

The CXCL12 gene produces a series of transcript variants through alternative splicing at the 3' end of its pre-mRNA. This study explores the biological activities of these alternative transcripts and the mechanisms involved in the regulation of CXCL12 transcription and RNA splicing. We identified a "GA" insertion mutation in the region of CXCL12α DNA encoding the conserved 3'UTR. This variant transcript was named CXCL12-3'GA+. The mutation occurred at a frequency of 13.2% in healthy Chinese individuals. However, its frequency in healthy Caucasians was 22.6%, significantly higher than what was observed in the Chinese. Genomic analysis indicated that the GA+ mutation likely encodes a G-quadruplex structure in close proximity to a cluster of important AU-rich elements (AREs) that are well-established regulators of mRNA stability at the 3'UTR. Experiments using molecular constructs encoding the 3'UTR of CXCL12 revealed that the GA+ allele can significantly increase gene expression compared to the WT allele. Further studies uncovered that the WT allele was associated with the production of a 225-bp minor transcript isoform (MTI) through alternative splicing resulting in the deletion of exon 2. ARMS-PCR using samples collected from cultured PBMCs of WT/GA+ genotype carriers indicated that the GA+ allele was preferentially transcribed compared to the WT allele. In summary, the study demonstrates that a GA insertion in the region encoding the 3'UTR of CXCL12α may affect gene expression through alternative mRNA splicing. This finding provides a basis for understanding how multiple elements in the sequence encoding the 3'UTR of the CXCL12 gene regulates its transcription and may lead to insights about diseases involving abnormal CXCL12α expression., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We evaluated whether low-grade albuminuria or black race modulates ambulatory blood pressure (BP) or nocturnal BP response to the DASH diet. Among 202 adults enrolled in the DASH multicenter trial who were fed the DASH or control diet for 8 weeks, reductions in 24-hour daytime and nighttime SBP and DBP were significantly larger for DASH compared to control. Median changes in nocturnal BP dipping were not significant. Compared to urine albumin excretion of <7 mg/d, ≥7 mg/d was associated with larger significant median reductions in 24-hour SBP (-7.3 vs -3.1 mm Hg), all measures of DBP (24-hour: -5.9 vs -1.8 mm Hg; daytime: -9.9 vs -4.0 mm Hg; nighttime -9.0 vs -2.0 mm Hg), and with increased nocturnal SBP dipping (2.3% vs -0.5%). Black race was associated with larger median reduction in 24-hour SBP only (-5.5 vs -2.4 mm Hg). This analysis suggests greater effect of DASH on ambulatory BP in the presence of low-grade albuminuria., (©2018 Wiley Periodicals, Inc.)

People with stroke do not achieve adequate levels of physical exercise following discharge from rehabilitation. We developed a group exercise and self-management program (PROPEL), delivered during stroke rehabilitation, to promote uptake of physical activity after discharge. This study aimed to establish the feasibility of a larger study to evaluate the effect of this program on participation in self-directed physical activity. Participants with subacute stroke were recruited at discharge from one of three rehabilitation hospitals; one hospital offered the PROPEL program whereas the other two did not (comparison group; COMP). A high proportion (11/16) of eligible PROPEL program participants consented to the study. Fifteen COMP participants were also recruited. Compliance with wearing an accelerometer for 6 weeks continuously and completing physical activity questionnaires was high (>80%), whereas only 34% of daily heart rate data were available. Individuals who completed the PROPEL program seemed to have higher outcome expectations for exercise, fewer barriers to physical activity, and higher participation in physical activity than COMP participants (Hedge's g ≥ 0.5). The PROPEL program delivered during stroke rehabilitation shows promise for reducing barriers to exercise and increasing participation in physical activity after discharge. This study supports feasibility of a larger randomized trial to evaluate this program.

To discern virulent from innocuous microbes, the innate immune system senses events associated with bacterial access to immunoprivileged sites such as the host cell cytosol. One such pathway is triggered by the cytosolic delivery of flagellin, the major subunit of the flagellum, by bacterial secretion systems. This leads to inflammasome activation and subsequent proinflammatory cell death (pyroptosis) of the infected phagocyte. In this study, we demonstrate that the causative agent of typhoid fever, Salmonella enterica serovar Typhi, can partially subvert this critical innate immune recognition event. The transcriptional regulator TviA, which is absent from Salmonella serovars associated with human gastroenteritis, repressed the expression of flagellin during infection of human macrophage-like (THP-1) cells. This mechanism allowed S. Typhi to dampen inflammasome activation, leading to reduced interleukin-1β (IL-1β) secretion and diminished cell death. Likewise, the introduction of the tviA gene in nontyphoidal Salmonella enterica serovar Typhimurium reduced flagellin-induced pyroptosis. These data suggest that gene regulation of virulence factors enables S. Typhi to evade innate immune recognition by concealing a pathogen-induced process from being sensed by the inflammasome., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Delivery of microbial products into the mammalian cell cytosol by bacterial secretion systems is a strong stimulus for triggering pro-inflammatory host responses. Here we show that Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, tightly regulates expression of the invasion-associated type III secretion system (T3SS-1) and thus fails to activate these innate immune signaling pathways. The S. Typhi regulatory protein TviA rapidly repressed T3SS-1 expression, thereby preventing RAC1-dependent, RIP2-dependent activation of NF-κB in epithelial cells. Heterologous expression of TviA in S. enterica serovar Typhimurium (S. Typhimurium) suppressed T3SS-1-dependent inflammatory responses generated early after infection in animal models of gastroenteritis. These results suggest that S. Typhi reduces intestinal inflammation by limiting the induction of pathogen-induced processes through regulation of virulence gene expression.

Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns. However, these are produced by all microbes, regardless of their pathogenic potential. To distinguish virulent microbes from those with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that NOD1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of RAC1 and CDC42 by bacterial delivery or ectopic expression of SopE, a virulence factor of the enteric pathogen Salmonella, triggered the NOD1 signalling pathway, with consequent RIP2 (also known as RIPK2)-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the NOD1 signalling pathway by peptidoglycan required RAC1 activity. Furthermore, constitutively active forms of RAC1, CDC42 and RHOA activated the NOD1 signalling pathway. Our data identify the activation of small Rho GTPases as a pathogen-induced process sensed through the NOD1 signalling pathway.

Information on how emerging pathogens can invade and persist and spread within host populations remains sparse. In the 1980s, a multidrug-resistant Salmonella enterica serotype Typhimurium clone lysogenized by a bacteriophage carrying the sopE virulence gene caused an epidemic among cattle and humans in Europe. Here we show that phage-mediated horizontal transfer of the sopE gene enhances the production of host-derived nitrate, an energetically highly valuable electron acceptor, in a mouse colitis model. In turn, nitrate fuels a bloom of S. Typhimurium in the gut lumen through anaerobic nitrate respiration while suppressing genes for the utilization of energetically inferior electron acceptors such as tetrathionate. Through this mechanism, horizontal transfer of sopE can enhance the fitness of S. Typhimurium, resulting in its significantly increased abundance in the feces. IMPORTANCE During gastroenteritis, Salmonella enterica serotype Typhimurium can use tetrathionate respiration to edge out competing microbes in the gut lumen. However, the concept that tetrathionate respiration confers a growth benefit in the inflamed gut is not broadly applicable to other host-pathogen combinations because tetrathionate respiration is a signature trait used to differentiate Salmonella serotypes from most other members of the family Enterobacteriaceae. Here we show that by acquiring the phage-carried sopE gene, S. Typhimurium can drive the host to generate an additional respiratory electron acceptor, nitrate. Nitrate suppresses genes for the utilization of energetically inferior electron acceptors such as tetrathionate while enhancing the luminal growth of S. Typhimurium through anaerobic nitrate respiration. Pathways for anaerobic nitrate respiration are widely conserved among members of the family Enterobacteriaceae, thereby making our observations relevant to other enteric pathogens whose relative abundance in the intestinal lumen increases during infection.

Activation of CRH transcription requires phosphorylation of cAMP response element-binding protein (CREB) and translocation of the CREB coactivator, transducer of regulated CREB activity (TORC) from cytoplasm to nucleus. In basal conditions, transcription is low because TORC remains in the cytoplasm, inactivated by phosphorylation through Ser/Thr protein kinases of the AMP-dependent protein kinases (AMPK) family, including salt-inducible kinase (SIK). To determine which kinase is responsible for TORC phosphorylation in CRH neurons, we measured SIK1 and SIK2 mRNA in the hypothalamic paraventricular nucleus of rats by in situ hybridization. In basal conditions, low mRNA levels of the two kinases were found in the dorsomedial paraventricular nucleus, consistent with location in CRH neurons. One hour of restraint stress increased SIK1 mRNA levels, whereas SIK2 mRNA showed only minor increases. In 4B hypothalamic neurons, or primary cultures, SIK1 mRNA (but not SIK2 mRNA) was inducible by the cAMP stimulator, forskolin. Overexpression of either SIK1 or SIK2 in 4B cells reduced nuclear TORC2 levels (Western blot) and inhibited forskolin-stimulated CRH transcription (luciferase assay). Conversely, the nonselective SIK inhibitor, staurosporine, increased nuclear TORC2 content and stimulated CRH transcription in 4Bcells and primary neuronal cultures (heteronuclear RNA). Unexpectedly, in 4B cells specific short hairpin RNA knockdown of endogenous SIK2 but not SIK1 induced nuclear translocation of TORC2 and CRH transcription, suggesting that SIK2 mediates TORC inactivation in basal conditions, whereas induction of SIK1 limits transcriptional activation. The study provides evidence that SIK represses CRH transcription by inactivating TORC, providing a potential mechanism for rapid on/off control of CRH transcription.

Resistance of pathogens to antimicrobial therapeutics has become a widespread problem. Resistance can emerge naturally, but it can also be engineered intentionally, which is an important consideration in designing therapeutics for bioterrorism agents. Blocking host receptors used by pathogens represents a powerful strategy to overcome this problem, because extensive alterations to the pathogen may be required to enable it to switch to a new receptor that can still support pathogenesis. Here, we demonstrate a facile method for producing potent receptor-directed antitoxins. We used phage display to identify a peptide that binds both anthrax-toxin receptors and attached this peptide to a synthetic scaffold. Polyvalency increased the potency of these peptides by >50,000-fold in vitro and enabled the neutralization of anthrax toxin in vivo. This work demonstrates a receptor-directed anthrax-toxin inhibitor and represents a promising strategy to combat a variety of viral and bacterial diseases.

Numerous biological processes involve the recognition of a specific pattern of binding sites on a target protein or surface. Although ligands displayed by disordered scaffolds form stochastic rather than specific patterns, theoretical models predict that recognition will occur between patterns that are characterized by similar or "matched" statistics. Endowing synthetic biomimetic structures with statistical pattern matching capabilities may improve the specificity of sensors and resolution of separation processes. We demonstrate that statistical pattern matching enhances the potency of polyvalent therapeutics. We functionalized liposomes with an inhibitory peptide at different densities and observed a transition in potency at an interpeptide separation that matches the distance between ligand-binding sites on the heptameric component of anthrax toxin. Pattern-matched polyvalent liposomes inhibited anthrax toxin in vitro at concentrations four orders of magnitude lower than the corresponding monovalent peptide, and neutralized this toxin in vivo. Statistical pattern matching also enhanced the potency of polyvalent inhibitors of cholera toxin. This facile strategy should be broadly applicable to the detection and neutralization of toxins and pathogens.

Insulin increases glucose uptake into muscle via glucose transporter-4 (GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol (PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed the following: (a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. (b) Insulin caused greater immunological availability of the C terminus than myc epitopes, suggesting that C terminus unmasking had occurred. Delivering phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) to intact cells significantly increased lawn-associated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate (PI3P) increased the detection of C terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion, and C terminus unmasking, through distinct phosphoinositides. PI(3,4,5)P(3) causes arrival and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.

Angiomatoid vascular changes in lungs and pulmonary hemorrhage are described in a woman in whom mitomycin C-associated hemolytic-uremiclike syndrome developed. These changes represent part of the spectrum of vascular damage that may complicate mitomycin therapy. Patients receiving mitomycin C chemotherapy require careful monitoring for possible development of these complications.

Platelet-leukocyte interaction was observed in an asymptomatic woman. After incubation in the patient's EDTA-plasma, autologous and allogeneic platelets adhered to the surfaces of neutrophils, monocytes, macrophages and, rarely, eosinophils. Monocytes, macrophages, and occasionally neutrophils phagocytosed platelets. Degranulation of peroxidase-positive lysosomes into the platelet-containing phagosome was demonstrated ultrastructurally. Bone marrow studies indicated that bands and earlier neutrophilic precursors did not participate in the reaction, and that neutrophils adhered to, and were rarely engulfed by megakaryocytes. Sequential exposure of the patient's EDTA-plasma to platelets and leukocytes indicated that a nondialyzable factor(s) was first absorbed by platelets which then interacted with leukocytes. The reaction proceeded best in the presence of EDTA at 21 degrees C, and was inhibited or dissociated by divalent cations or at 37 degrees C. Metabolic integrity of both platelets and leukocytes was also essential for the reaction since each was inhibited by formalin fixation and partially inhibited by the metabolic inhibitor 2-deoxyglucose. Formalin-treated platelets continued to absorb the plasma factor(s). The plasma and the cell fractions were inactivated by periodate and nonspecific protease. Treatment of the platelets with trypsin or the leukocytes with neuraminidase diminished the interaction by 50%. The reaction was also interfered with by concanavalin A. Immunofluorescent and immunoabsorption studies failed to identify an immune component of this interaction. These findings indicate that the plasma factor(s) and the cell surface receptors are nonimmune glycoconjugates and consequently differ from previously documented cases of platelet-leukocyte interaction.