Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Circulating markers of extracellular matrix remodelling in severe COVID-19 patients.

BACKGROUND: Abnormal remodelling of the extracellular matrix (ECM) has generally been linked to pulmonary inflammation and fibrosis and may also play a role in the pathogenesis of severe COVID-19. To further elucidate the role of ECM remodelling and excessive fibrogenesis in severe COVID-19, we examined circulating levels of mediators involved in various aspects of these processes in COVID-19 patients. METHODS: Serial blood samples were obtained from two cohorts of hospitalised COVID-19 patients (n = 414). Circulating levels of ECM remodelling mediators were quantified by enzyme immunoassays in samples collected during hospitalisation and at 3-month follow-up. Samples were related to disease severity (respiratory failure and/or treatment at the intensive care unit), 60-day total mortality and pulmonary pathology after 3-months. We also evaluated the direct effect of inactivated SARS-CoV-2 on the release of the different ECM mediators in relevant cell lines. RESULTS: Several of the measured markers were associated with adverse outcomes, notably osteopontin (OPN), S100 calcium-binding protein A12 and YKL-40 were associated with disease severity and mortality. High levels of ECM mediators during hospitalisation were associated with computed tomography thorax pathology after 3-months. Some markers (i.e. growth differential factor 15, galectin 3 and matrix metalloproteinase 9) were released from various relevant cell lines (i.e. macrophages and lung cell lines) in vitro after exposure to inactivated SARS-CoV-2 suggesting a direct link between these mediators and the causal agent of COVID-19. CONCLUSION: Our findings highlight changes to ECM remodelling and particularly a possible role of OPN, S100A12 and YKL-40 in the pathogenesis of severe COVID-19.

Scoping Review: Neurocognitive Outcome Assessments After Critical Illness in Children.

OBJECTIVES: To map the literature regarding assessment of neurocognitive outcomes in PICU survivors. Secondary objectives were to identify literature gaps and to provide data for development of a Core Outcome Measures Set in the domain. METHODS: Planned, a priori analysis was performed of data from an over-all scoping review of Post-Intensive Care Syndrome-pediatrics (PICS-p) functional outcomes. English-language databases and registries from 1970 to 2017 were searched by a medical librarian to identify manuscripts reporting on Post Intensive Care Syndrome-pediatrics (PICS-p). Further, detailed data extraction for neurocognitive outcomes was performed focusing on study characteristics, instruments used, and populations. RESULTS: 114 instruments evaluated neurocognitive function in 183 manuscripts. 83% of manuscripts were published after 2000. Median of 3 (IQR 2-5) neurocognitive instruments per manuscript were reported. Wechsler Scales (45%), clinical neurologic evaluations (21%), Pediatric Cerebral Performance Category (20%), Bayley Scales of Infant Development (16%), and Vineland Adaptive Behavior Scales (11%) were the most commonly used instruments. Median sample size was 65 (IQR 32-129) subjects. Most (63%) assessments were conducted in-person and parents/guardians (40%) provided the information. Patients with congenital heart disease and traumatic brain injury were most commonly evaluated (31% and 24% of manuscripts, respectively). Adolescents were the most commonly studied age group (34%). Baseline function was infrequently assessed (11% of manuscripts); most studies assessed patients at only one time point after PICU discharge. Within studies, neurocognitive assessments were often combined with others - especially social (18%) and physical (8%). CONCLUSIONS: 183 manuscripts studied the neurocognitive domain of PICS-p. Studies were quantitative and tended to focus on populations with anticipated cognitive impairment. Considerable variability exists among the chosen 114 instruments used; however, 4 instruments were frequently chosen with focus on intelligence, cerebral functioning, and developmental and adaptive behavior. The literature is marked by lack of agreement on methodologies but reflects the burgeoning interest in studying PICS-p neurocognitive outcomes.

Exploring the relationship between breastfeeding and the incidence of infant illnesses in Ireland: evidence from a nationally representative prospective cohort study.

BACKGROUND: Ireland has one of the lowest BF rates in the world. This study investigates the association between breastfeeding and infant health in Ireland. METHODS: A cross-sectional, secondary analysis of data collected from Growing Up in Ireland (GUI): the National Longitudinal Study of Children was conducted. The average morbidity for 2212. infants exclusively breastfed for at least 90 days (EBF90days) was compared to data for 3987 infants in the non-breastfed (Non-BF) group. Data were weighted using entropy balancing to ensure the comparability of groups. Sensitivity analyses considered alternative definitions of the breastfeeding group. RESULTS: Infants who were EBF90days were significantly less likely to be admitted to hospital (CI: - 0.06 to - 0.03), spent less nights in hospital (CI: - 0.37 to - 0.11), and were less likely to develop respiratory diseases including asthma (CI: - 0.03 to - 0.01), chest infections (CI: - 0.12 to - 0.08), snuffles/common colds (CI: - 0.07 to - 0.02), ear infections (CI: - 0.08 to - 0.04), eczema (CI: - 0.08 to - 0.04), skin problems (CI: - 0.04 to - 0.00), wheezing or asthma (CI: - 0.06 to - 0.03), vomiting (CI: - 0.03 to - 0.00), and colic (CI: - 0.04 to - 0.01). Further outcomes such as current health of the infant at time of interview (CI: - 0.04 to - 0.00), feeding problems (CI: - 0.04 to - 0.02) and sleeping problems (CI: - 0.02 to - 0.00) indicated a protective effect of EBF90days versus Non-BF. However, these infants were also more likely to fail to gain weight (CI: 0.01 to 0.02) and were at a slightly higher risk of developing nappy rash (CI: 0.00 to 0.02). CONCLUSION: Exclusive breastfeeding for 90+ days is associated with protection against childhood morbidity. Given the protective effect of breastfeeding on adverse health effects in infants, policy makers should prioritise policies that support, promote and protect exclusive breastfeeding.

Low-cost, on-farm intervention to reduce spores in bulk tank raw milk benefits producers, processors, and consumers.

Bacterial spores, which are found in raw milk, can survive harsh processing conditions encountered in dairy manufacturing, including pasteurization and drying. Low-spore raw milk is desirable for dairy industry stakeholders, especially those who want to extend the shelf life of their product, expand their distribution channels, or reduce product spoilage. A recent previous study showed that an on-farm intervention that included washing towels with chlorine bleach and drying them completely, as well as training milking parlor employees to focus on teat end cleaning, significantly reduced spore levels in bulk tank raw milk. As a follow up to that previous study, here we calculate the costs associated with that previously described intervention as ranging from $9.49 to $13.35 per cow per year, depending on farm size. A Monte Carlo model was used to predict the shelf life of high temperature, short time fluid milk processed from raw milk before and after this low-cost intervention was applied, based on experimental data collected in a previous study. The model predicted that 18.24% of half-gallon containers of fluid milk processed from raw milk receiving no spore intervention would exceed the pasteurized milk ordinance limit of 20,000 cfu/mL by 17 d after pasteurization, while only 16.99% of containers processed from raw milk receiving the spore intervention would reach this level 17 d after pasteurization (a reduction of 1.25 percentage points and a 6.85% reduction). Finally, a survey of consumer milk use was conducted to determine how many consumers regularly consume fluid milk near or past the date printed on the package (i.e., code date), which revealed that over 50% of fluid milk consumers surveyed continue to consume fluid milk after this date, indicating that a considerable proportion of consumers are exposed to fluid milk that is likely to have high levels spore-forming bacterial growth and possibly associated quality defects (e.g., flavor or odor defects). This further highlights the importance of reducing spore levels in raw milk to extend pasteurized fluid milk shelf life and thereby reducing the risk of adverse consumer experiences. Processors who are interested in extending fluid milk shelf life by controlling the levels of spores in the raw milk supply should consider incentivizing low-spore raw milk through premium payments to producers.

High Circulating Levels of the Homeostatic Chemokines CCL19 and CCL21 Predict Mortality and Disease Severity in COVID-19.

BACKGROUND: Immune dysregulation is a major factor in the development of severe coronavirus disease 2019 (COVID-19). The homeostatic chemokines CCL19 and CCL21 have been implicated as mediators of tissue inflammation, but data on their regulation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is limited. We thus investigated the levels of these chemokines in COVID-19 patients. METHODS: Serial blood samples were obtained from patients hospitalized with COVID-19 (n = 414). Circulating CCL19 and CCL21 levels during hospitalization and 3-month follow-up were analyzed. In vitro assays and analysis of RNAseq data from public repositories were performed to further explore possible regulatory mechanisms. RESULTS: A consistent increase in circulating levels of CCL19 and CCL21 was observed, with high levels correlating with disease severity measures, including respiratory failure, need for intensive care, and 60-day all-cause mortality. High levels of CCL21 at admission were associated with persisting impairment of pulmonary function at the 3-month follow-up. CONCLUSIONS: Our findings highlight CCL19 and CCL21 as markers of immune dysregulation in COVID-19. This may reflect aberrant regulation triggered by tissue inflammation, as observed in other chronic inflammatory and autoimmune conditions. Determination of the source and regulation of these chemokines and their effects on lung tissue is warranted to further clarify their role in COVID-19. CLINICAL TRIALS REGISTRATION: NCT04321616 and NCT04381819.

Persistent T-cell exhaustion in relation to prolonged pulmonary pathology and death after severe COVID-19: Results from two Norwegian cohort studies.

BACKGROUND: T-cell activation is associated with an adverse outcome in COVID-19, but whether T-cell activation and exhaustion relate to persistent respiratory dysfunction and death is unknown. OBJECTIVES: To investigate whether T-cell activation and exhaustion persist and are associated with prolonged respiratory dysfunction and death after hospitalization for COVID-19. METHODS: Plasma and serum from two Norwegian cohorts of hospitalized patients with COVID-19 (n = 414) were analyzed for soluble (s) markers of T-cell activation (sCD25) and exhaustion (sTim-3) during hospitalization and follow-up. RESULTS: Both markers were strongly associated with acute respiratory failure, but only sTim-3 was independently associated with 60-day mortality. Levels of sTim-3 remained elevated 3 and 12 months after hospitalization and were associated with pulmonary radiological pathology after 3 months. CONCLUSION: Our findings suggest prolonged T-cell exhaustion is an important immunological sequela, potentially related to long-term outcomes after severe COVID-19.

Application of blood microsampling in cynomolgus monkey and demonstration of equivalent monoclonal antibody PK parameters compared to conventional sampling.

PURPOSE: The purpose of this study was to evaluate the suitability of whole blood microsampling procedures in non-human primate (NHP) to support toxicokinetic assessments of biotherapeutics in non-human primates. METHOD: A one-month single dose intravenous pharmacokinetic (PK) study was performed in male cynomolgus monkeys with a human IgG1 control monoclonal antibody (mAb) as a surrogate monoclonal antibody biotherapeutic. In this study, both serum samples (conventional sample collection) and microsampling samples were collected. Microsampling samples were collected from two sites on cynomolgus monkey, with each site using two different devices for the whole blood collection. The drug concentrations from all sample types were determined using a quantitative ligand binding assay (LBA). The PK parameters obtained from microsampling samples and serum samples were examined using a standard PK analysis method. The comparability of key PK parameters from both sample types were analyzed statistically. RESULTS: Similar profiles of drug concentrations versus timepoints from all sampling procedures were observed. The correlations of PK concentration data obtained from serum and microsampling samples were ≥ 0.97 using Brand Alman Plot analysis. The key PK parameters obtained from microsampling samples were comparable to those obtained from serum samples (the % differences of mean PK parameters obtained from both sample types were within ±25%). CONCLUSION: This study confirmed that PK parameters obtained from samples using microsampling were comparable to that of serum samples in cynomolgus monkeys. Therefore, the microsampling procedure described can be used as a substitute for conventional sampling procedure to support PK/TK studies of biotherapeutics in non-clinical product developments.

Neurological Involvement in COVID-19 and Potential Mechanisms: A Review.

As the current understanding of COVID-19 continues to evolve, a synthesis of the literature on the neurological impact of this novel virus may help inform clinical management and highlight potentially important avenues of investigation. Additionally, understanding the potential mechanisms of neurologic injury may guide efforts to better detect and ameliorate these complications. In this review, we synthesize a range of clinical observations and initial case series describing potential neurologic manifestations of COVID-19 and place these observations in the context of coronavirus neuro-pathophysiology as it may relate to SARS-CoV-2 infection. Reported nervous system manifestations range from anosmia and ageusia, to cerebral hemorrhage and infarction. While the volume of COVID-19-related case studies continues to grow, previous work examining related viruses suggests potential mechanisms through which the novel coronavirus may impact the CNS and result in neurological complications. Namely, animal studies examining the SARS-CoV have implicated the angiotensin-converting-enzyme-2 receptor as a mediator of coronavirus-related neuronal damage and have shown that SARS-CoV can infect cerebrovascular endothelium and brain parenchyma, the latter predominantly in the medial temporal lobe, resulting in apoptosis and necrosis. Human postmortem brain studies indicate that human coronavirus variants and SARS-CoV can infect neurons and glia, implying SARS-CoV-2 may have similar neurovirulence. Additionally, studies have demonstrated an increase in cytokine serum levels as a result of SARS-CoV infection, consistent with the notion that cytokine overproduction and toxicity may be a relevant potential mechanism of neurologic injury, paralleling a known pathway of pulmonary injury. We also discuss evidence that suggests that SARS-CoV-2 may be a vasculotropic and neurotropic virus. Early reports suggest COVID-19 may be associated with severe neurologic complications, and several plausible mechanisms exist to account for these observations. A heightened awareness of the potential for neurologic involvement and further investigation into the relevant pathophysiology will be necessary to understand and ultimately mitigate SARS-CoV-2-associated neurologic injury.

Standardized Volume Dosing Protocol of 23.4% Hypertonic Saline for Pediatric Critical Care: Initial Experience.

Background: Standardized volume dosing of 23.4% hypertonic saline (HTS) exists for adults, but the concentration, dosing and administration of HTS in pediatrics is variable. With emerging pediatric experience of 23.4% HTS, a standard volume dose approach may be helpful. Objective: To describe initial experience with a standardized 23.4% HTS weight-based volume dosing protocol of 10, 20, or 30 mL in the pediatric intensive care unit. Methods: Standard volume doses of 23.4% HTS were developed from weight dosing equivalents of 3% HTS. Pre and post sodium and intracranial pressure (ICP) measurements were compared with paired t-test or Wilcoxon rank-sum test. The site of administration and complications were noted. Results: A total of 16 pediatric patients received 37 doses of 23.4% HTS, with the smallest patient weighing 11 kg. For protocol compliance, 17 doses (46%) followed recommended dosing, 19 were less volume than recommended (51%), and 1 dose (3%) was more than recommended. Mean increase in sodium was 3.5 mEq/L (95% CI = 2-5 mEq/L); P < 0.0001. The median decrease in ICP was 10.5 mm Hg (interquartile range [IQR] 8.3-19.5) for a 37% (IQR 25%-64%) reduction. Most doses were administered through central venous access, although peripheral intravenous administrations occurred in 4 patients without complication. Conclusion and Relevance: Three standard-volume dose options of 23.4% HTS based on weight increases sodium and reduces ICP in pediatric patients. Standard-volume doses may simplify weight-based dosing, storage and administration for pediatric emergencies, although the optimum dose, and safety of 23.4% HTS in children remains unknown.

A glycine transporter SLC6A5 frameshift mutation causes startle disease in Spanish greyhounds.

Startle disease, or hyperekplexia, is a glycinergic disorder characterized by hypertonia and apnea that is triggered by noise and/or touch. Mutations in five genes have been associated with startle disease in humans, dogs, cattle, and mice. We identified a novel recessive startle disease in a family of Spanish greyhounds. Whole genome resequencing of an affected dog revealed a homozygous two base pair deletion in the ninth exon of SLC6A5, encoding the presynaptic glycine transporter. The deletion is predicted to cause a frameshift, p.S460FfsX47, leading to a premature stop codon that truncates over a third of the protein. Family members were genotyped for the deletion, and findings were consistent with an autosomal recessive inheritance pattern. The pathogenic variant was absent from 34 unrelated greyhounds, 659 domestic dogs of pure and mixed breeds, and 54 wild canids, suggesting it occurred recently and may be private to the family. The findings of this study can be used to inform future breeding decisions and prevent dissemination of the deleterious allele in greyhounds.

Variable impact by ambient temperature on fatigue resistance of heat-treated nickel titanium instruments.

OBJECTIVES: The purpose of this study was to evaluate the effect of different ambient temperatures on cyclic fatigue (CF) life of two NiTi rotary systems and correlate the results with martensitic transformation temperatures. MATERIALS AND METHODS: Heat-treated NiTi Vortex Blue (VB) and EdgeSequel Sapphire (SP) instruments (tip sizes no. 20, 25, 30, 35, 40) were tested for CF resistance at room and body temperature (n = 20 each group) in a simulated canal (angle of curvature 60°; radius 3 mm; center from instrument tip 4.5 mm) with a motor controlled by an electric circuit. Mean half-life, beta and eta Weibull parameters were determined and compared. Two further instruments of each brand were subjected to differential scanning calorimetry (DSC). RESULTS: Temperature had an effect on fatigue behavior: all instruments lasted significantly longer at room than at body temperature. All VB significantly outlasted those of SP at body temperature; while smaller diameters of VB (size no. 20) were also significantly more resistant than SP when tested at room temperature; SP with larger diameters (sizes no. 30, no. 35, and no. 40) lasted significantly longer than VB did. CONCLUSIONS: Immersion in water at body temperature was associated with a marked decrease in the fatigue life of all rotary instruments tested. VB instruments were significantly more CF resistant at body temperature and showed the highest predictability in terms of fracture resistance. CLINICAL RELEVANCE: Rotary instruments manufactured with different post-machining heat treatment responded differently to changed ambient temperatures. DSC assessment of martensitic conversion temperatures helps to predict the behavior of nickel titanium rotaries in different environments.

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains.

Advances in membrane cell biology are hampered by the relatively high proportion of proteins with no known function. Such proteins are largely or entirely devoid of structurally significant domain annotations. Structural bioinformaticians have developed profile-profile tools such as HHsearch (online version called HHpred), which can detect remote homologies that are missed by tools used to annotate databases. Here we have applied HHsearch to study a single structural fold in a single model organism as proof of principle. In the entire clan of protein domains sharing the pleckstrin homology domain fold in yeast, systematic application of HHsearch accurately identified known PH-like domains. It also predicted 16 new domains in 13 yeast proteins many of which are implicated in intracellular traffic. One of these was Vps13p, where we confirmed the functional importance of the predicted PH-like domain. Even though such predictions require considerable work to be corroborated, they are useful first steps. HHsearch should be applied more widely, particularly across entire proteomes of model organisms, to significantly improve database annotations.

Pediatric Neurocritical Care: Evolution of a New Clinical Service in PICUs Across the United States.

OBJECTIVES: Pediatric neurocritical care as a conceptual service is relatively new, and implementation of such specialized services may improve outcomes for children with disorders of the brain or spinal cord. How many pediatric neurocritical care services currently exist in the United States, and attitudes about such a service are unknown. DESIGN: Web-based survey, distributed by e-mail. SETTING: Survey was sent to PICU Medical Directors and Program Directors of Pediatric Neurosurgery fellowship and Child Neurology residency programs. PATIENTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 378 surveys were distributed; 161 respondents representing 128 distinct hospitals completed the survey (43% response rate). Thirty-five percent (45/128) reported having a pediatric neurocritical care service. The most common type of service used a consultation model (82%; 32/39 responses). Other types of services were intensivist-led teams in the PICU (five hospitals) and dedicated PICU beds (two hospitals). Hospital characteristics associated with availability of pediatric neurocritical care services were level 1 trauma status (p = 0.017), greater numbers of PICU beds (χ [6, n = 128] = 136.84; p < 0.01), and greater volume of children with pediatric neurocritical care conditions (χ [3, n = 128] = 20.16; p < 0.01). The most common reasons for not having a pediatric neurocritical care service were low patient volume (34/119 responses), lack of subspecialists (30/119 responses), and lack of interest by PICU faculty (25/119 responses). The positive impacts of a pediatric neurocritical care service were improved interdisciplinary education/training (16/45 responses), dedicated expertise (13/45 responses), improved interservice communication (9/45 responses), and development/implementation of guidelines and protocols (9/45 responses). The negative impacts of a pediatric neurocritical care service were disagreement among consultants (2/45 responses) and splitting of the PICU population (2/45 responses). CONCLUSIONS: At least 45 specialized pediatric neurocritical care services exist in the United States. Eighty percent of these services are a consultation service to the PICU. Hospitals with level 1 trauma status, greater numbers of PICU beds, and greater numbers of patients with pediatric neurocritical care conditions were associated with the existence of pediatric neurocritical care as a clinical service.

VAP, a Versatile Access Point for the Endoplasmic Reticulum: Review and analysis of FFAT-like motifs in the VAPome.

Dysfunction of VAMP-associated protein (VAP) is associated with neurodegeneration, both Amyotrophic Lateral Sclerosis and Parkinson's disease. Here we summarize what is known about the intracellular interactions of VAP in humans and model organisms. VAP is a simple, small and highly conserved protein on the cytoplasmic face of the endoplasmic reticulum (ER). It is the sole protein on that large organelle that acts as a receptor for cytoplasmic proteins. This may explain the extremely wide range of interacting partners of VAP, with components of many cellular pathways binding it to access the ER. Many proteins that bind VAP also target other intracellular membranes, so VAP is a component of multiple molecular bridges at membrane contact sites between the ER and other organelles. So far approximately 100 proteins have been identified in the VAP interactome (VAPome), of which a small minority have a "two phenylalanines in an acidic tract" (FFAT) motif as it was originally defined. We have analyzed the entire VAPome in humans and yeast using a simple algorithm that identifies many more FFAT-like motifs. We show that approximately 50% of the VAPome binds directly or indirectly via the VAP-FFAT interaction. We also review evidence on pathogenesis in genetic disorders of VAP, which appear to arise from reduced overall VAP levels, leading to ER stress. It is not possible to identify one single interaction that underlies disease. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Delirium in Critically Ill Children: An International Point Prevalence Study.

OBJECTIVES: To determine prevalence of delirium in critically ill children and explore associated risk factors. DESIGN: Multi-institutional point prevalence study. SETTING: Twenty-five pediatric critical care units in the United States, the Netherlands, New Zealand, Australia, and Saudi Arabia. PATIENTS: All children admitted to the pediatric critical care units on designated study days (n = 994). INTERVENTION: Children were screened for delirium using the Cornell Assessment of Pediatric Delirium by the bedside nurse. Demographic and treatment-related variables were collected. MEASUREMENTS AND MAIN RESULTS: Primary study outcome measure was prevalence of delirium. In 159 children, a final determination of mental status could not be ascertained. Of the 835 remaining subjects, 25% screened positive for delirium, 13% were classified as comatose, and 62% were delirium-free and coma-free. Delirium prevalence rates varied significantly with reason for ICU admission, with highest delirium rates found in children admitted with an infectious or inflammatory disorder. For children who were in the PICU for 6 or more days, delirium prevalence rate was 38%. In a multivariate model, risk factors independently associated with development of delirium included age less than 2 years, mechanical ventilation, benzodiazepines, narcotics, use of physical restraints, and exposure to vasopressors and antiepileptics. CONCLUSIONS: Delirium is a prevalent complication of critical illness in children, with identifiable risk factors. Further multi-institutional, longitudinal studies are required to investigate effect of delirium on long-term outcomes and possible preventive and treatment measures. Universal delirium screening is practical and can be implemented in pediatric critical care units.

The PI3K/mTOR inhibitor PF-04691502 induces apoptosis and inhibits microenvironmental signaling in CLL and the Eµ-TCL1 mouse model.

Current treatment strategies for chronic lymphocytic leukemia (CLL) involve a combination of conventional chemotherapeutics, monoclonal antibodies, and targeted signaling inhibitors. However, CLL remains largely incurable, with drug resistance and treatment relapse a common occurrence, leading to the search for novel treatments. Mechanistic target of rapamycin (mTOR)-specific inhibitors have been previously assessed but their efficacy is limited due to a positive feedback loop via mTOR complex 2 (mTORC2), resulting in activation of prosurvival signaling. In this study, we show that the dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor PF-04691502 does not induce an mTORC2 positive feedback loop similar to other PI3K inhibitors but does induce substantial antitumor effects. PF-04691502 significantly reduced survival coincident with the induction of Noxa and Puma, independently of immunoglobulin heavy chain variable region mutational status, CD38, and ZAP-70 expression. PF-04691502 inhibited both anti-immunoglobulin M-induced signaling and overcame stroma-induced survival signals and migratory stimuli from CXCL12. Equivalent in vitro activity was seen in the Eµ-TCL1 murine model of CLL. In vivo, PF-04691502 treatment of tumor-bearing animals resulted in a transient lymphocytosis, followed by a clear reduction in tumor in the blood, bone marrow, spleen, and lymph nodes. These data indicate that PF-04691502 or other dual PI3K/mTOR inhibitors in development may prove efficacious for the treatment of CLL, increasing our armamentarium to successfully manage this disease.

A 100-Year Review: Microbiology and safety of milk handling.

Microbes that may be present in milk can include pathogens, spoilage organisms, organisms that may be conditionally beneficial (e.g., lactic acid bacteria), and those that have not been linked to either beneficial or detrimental effects on product quality or human health. Although milk can contain a full range of organisms classified as microbes (i.e., bacteria, viruses, fungi, and protozoans), with few exceptions (e.g., phages that affect fermentations, fungal spoilage organisms, and, to a lesser extent, the protozoan pathogens Cryptosporidium and Giardia) dairy microbiology to date has focused predominantly on bacteria. Between 1917 and 2017, our understanding of the microbes present in milk and the tools available for studying those microbes have changed dramatically. Improved microbiological tools have enabled enhanced detection of known microbes in milk and dairy products and have facilitated better identification of pathogens and spoilage organisms that were not known or well recognized in the early 20th century. Starting before 1917, gradual introduction and refinement of pasteurization methods throughout the United States and many other parts of the world have improved the safety and quality of milk and dairy products. In parallel to pasteurization, others strategies for reducing microbial contamination throughout the dairy chain (e.g., improved dairy herd health, raw milk tests, clean-in-place technologies) also played an important role in improving microbial milk quality and safety. Despite tremendous advances in reducing microbial food safety hazards and spoilage issues, the dairy industry still faces important challenges, including but not limited to the need for improved science-based strategies for safety of raw milk cheeses, control of postprocessing contamination, and control of sporeforming pathogens and spoilage organisms.