Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes.

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron.

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log10, and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

Intratumoral CD4+ T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer.

Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8+ T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4+ T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4+ T cell states that are clonally expanded. These CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4+ T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1.

Immune Activation Profiles Elicited by Distinct, Repeated TLR Agonist Infusions in Rhesus Macaques.

TLR agonists are a promising class of immune system stimulants investigated for immunomodulatory applications in cancer immunotherapy and viral diseases. In this study, we sought to characterize the safety and immune activation achieved by different TLR agonists in rhesus macaques (Macaca mulatta), a useful preclinical model of complex immune interactions. Macaques received one of three TLR agonists, followed by plasma cytokine, immune cell subset representation, and blood cell activation measurements. The TLR4 agonist LPS administered i.v. induced very transient immune activation, including TNF-α expression and monocyte activation. The TLR7/8 agonist 2BXy elicited more persistent cytokine expression, including type I IFN, IL-1RA, and the proinflammatory IL-6, along with T cell and monocyte activation. Delivery of 2BXy i.v. and i.m. achieved comparable immune activation, which increased with escalating dose. Finally, i.v. bacillus Calmette-Guérin (BCG) vaccination (which activates multiple TLRs, especially TLR2/4) elicited the most pronounced and persistent innate and adaptive immune response, including strong induction of IFN-γ, IL-6, and IL-1RA. Strikingly, monocyte, T cell, and NK cell expression of the proliferation marker Ki67 increased dramatically following BCG vaccination. This aligned with a large increase in total and BCG-specific cells measured in the lung. Principal component analysis of the combined cytokine expression and cellular activation responses separated animals by treatment group, indicating distinct immune activation profiles induced by each agent. In sum, we report safe, effective doses and routes of administration for three TLR agonists that exhibit discrete immunomodulatory properties in primates and may be leveraged in future immunotherapeutic strategies.

Tourniquet Use in the Prehospital Setting.

PURPOSE: Tourniquets are a mainstay of life-saving hemorrhage control. The US military has documented the safety and effectiveness of tourniquet use in combat settings. In civilian settings, events such as the Boston Marathon bombing and mass shootings show that tourniquets are necessary and life-saving entities that must be used correctly and whenever indicated. Much less research has been done on tourniquet use in civilian settings compared to military settings. The purpose of this study is to describe the prehospital use of tourniquets in a regional EMS system served by a single trauma center. METHODS: All documented cases of prehospital tourniquet use from 2015 to 2020 were identified via a search of EMS, emergency department, and inpatient records, and reviewed by the lead investigator. The primary outcomes were duration of tourniquet placement, success of hemorrhage control, and complications; secondary outcomes included time of day (by EMS arrival time), transport interval, extremity involved, who placed/removed the tourniquet, and mechanism of injury. RESULTS: Of 182 patients with 185 tourniquets applied, duration of application was available for 52, with a median (IQR) of 43 (56) minutes. Hemorrhage control was achieved in all but two cases (96%). Three cases (5.8%) required more than one tourniquet. Complications included five cases of temporary paresthesia, one case of ecchymosis, two cases of fasciotomy, and two cases of compression nerve injury. The serious complication rate was 7.7% (4/52). Time of day was daytime (08:01-16:00) = 15 (31.9%), evening (16:01-00:00) = 27 (57.4%), and night (00:01- 08:00) = 5 (10.6%). The median transport interval was 22 (IQR 5] minutes. The limbs most often injured were the left and right upper extremities (15 each). EMS clinicians and police officers were most often the tourniquet placers. Common mechanisms of injury included gunshot wounds, motorcycle accidents, and glass injuries. CONCLUSION: Tourniquets used in the prehospital setting have a high rate of hemorrhage control and a low rate of complications.

[S073]-The impact of interval appendectomy timing on follow-up adverse outcomes.

OBJECTIVE: This study aims to compare the timing of interval appendectomy (IA) and its impact on post-operative outcomes. METHODS: A retrospective analysis was performed for adult patients diagnosed with appendicitis between 2006 and 2017. IA was defined as a follow-up appendectomy > 1 week and < 2 years after the initial presentation. Time intervals were divided into 4 groups based on patient quartiles: 1-6 weeks, 7-9 weeks, 10-15 weeks, and > 15 weeks. The primary outcome measure was length of stay (LOS). Secondary outcomes included 30-day readmission and IA post-operative complications. Tertiary outcomes included 30-day mortality and colonoscopy suggesting neoplasm or Inflammatory Bowel Disease. RESULTS: A total of 5069 patients' records whose interval appendectomy fell > 1 week and < 2 years after initial presentation were analyzed. Among them, 1006 (19.85%) underwent an initial percutaneous abscess drainage at diagnosis. The median timing for IA was 9.2 weeks. Patients with IA at 1-6 weeks were more likely to have longer LOS when compared to 7-9 weeks (ratio 1.33, 95% CI 1.2-1.48) and 10-15 weeks (ratio 1.38, 95% CI 1.25-1.52). IA between 7 and 9 weeks (ratio 0.81, 95% CI 0.73-0.89) and 10-15 weeks (ratio 0.78, 95% CI 0.71-0.86) was associated with significantly shorter LOS compared to those receiving the operation after 15 weeks. Further, patients requiring abscess drainage (ratio 1.2, 95% CI 1.13-1.34) or those with comorbidities (ratio 1.51, 95% CI 1.39-1.63) were more likely to have longer LOS at IA. Socioeconomic and demographic differences including Black, Hispanic, and those with Medicare and Medicaid insurance had a greater LOS after their IA. CONCLUSION: LOS remains lowest among patients undergoing IA between 7-9 weeks and 10-15 weeks after initial appendicitis presentation. Patients with lower socioeconomic status or from racial minorities had a longer LOS after IA.

Safety and feasibility of destination care for bariatric surgery: a single institution retrospective study.

INTRODUCTION: Increasing emphasis on value-based healthcare has prompted both employers and healthcare organizations to develop innovative strategies to supply high quality care to patients. One such strategy is through the bundled care payment model (BCPM). Through this model, our institution partnered with employers from across the country to provide quality care for their members. Patients traveling greater than 2 h driving time from the bariatric center were considered "destination" patients. To properly care for our destination patients, our institution created a "destination bariatric program." We sought to investigate comparative outcomes for the first 100 patients who completed the program. We hypothesized that there would be no difference in patient outcomes or complications between destination and local patient groups undergoing sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB). METHODS AND PROCEDURES: A retrospective cohort analysis of patients undergoing bariatric surgery at a MBSAQIP-accredited bariatric surgery center between May 2019 and October 2021 was conducted. Patients were divided into destination or local patient groups based on participation in the established destination surgery program. Patient demographics, perioperative clinical outcomes, and complications were compared and statistically analyzed using two-sample t-tests, Chi-square tests, Fisher's exact tests, and univariate logistic regressions. RESULTS: This study identified 296 patients, which consisted of destination (n = 110) and local (n = 186) patient cohorts. Patients in the destination group had higher rates of diabetes mellitus (29.1% vs 24.2%, p = 0.029), but otherwise cohorts had similar basic demographics and comorbidities. Outcomes revealed no statistically significant associations between patient cohort (destination versus local) and ED admission (p = 0.305), hospital readmission (p = 0.893), surgical reintervention (p = 0.974), endoscopic-reintervention (p = 0.714), and patient complications in the postoperative period (30 days). CONCLUSION: Participation in destination care programs for bariatric surgery was found to be both safe and feasible. These destination programs represent an opportunity to provide a broader patient population access to complex surgical care.

The impact of interval cholecystectomy timing after percutaneous transhepatic cholecystostomy on post-operative adverse outcomes.

OBJECTIVE: This study aims to explore how timing of interval of cholecystectomy (IC) after percutaneous transhepatic cholecystostomy tube (PTC) placement impacts post-operative outcomes. METHODS: A retrospective database analysis of New York State SPARCs database of IC between 2005 and 2015. The timing for IC ranged between > 1 week and < 2 years. Patients undergoing this procedure were further divided into quartiles using 4-time intervals; 1-5 weeks (Q1), 5-8 weeks (Q2), 8-12 weeks(Q3), and > 12 weeks(Q4). The study's primary outcome was hospital length of stay (LOS). Secondary outcomes included discharge status, 30-day readmission, 30-day ED visit, and 90-day reoperation, surgery type, complication, and bile duct injury. Multivariable regression models were used to compare patients across the four-time intervals after adjusting for confounding factors. RESULTS: A total of 1038 patients with a history of PTC followed by IC between > 1 week and < 2 years were included in the final analysis. The median time to IC was 7.7 weeks. Q2 and Q3 both had a significantly higher median LOS of 3 days versus Q1 and Q4 at median of 5 days (p < 0.0001). Patients from racial and ethnic minorities (e.g., African Americans and Hispanics) were more likely to get their IC after 12 weeks (p < 0.05). Further, Black patients had a significantly higher median LOS than White, non-Hispanic patients (8 days vs 4 days, p < 0.0001) and were more likely to have open procedure. Multivariable regression analysis identified shorter LOS during Q2 (Ratio, 0.76, 95%, 0.67-0.87, p < 0.0001), and Q3 (Ratio 0.75, 95% CI, 065-0.86, p < 0.0001) compared to those who got their IC in Q4. Similar findings exist when comparing Q2 and Q3 to those receiving treatment during Q1. CONCLUSION: A time interval of 5-12 weeks between PTC and IC was associated with a decreased LOS. This study also suggests the persistence of racial disparities among these patients.

Online Forums as a Tool for Broader Inclusion of Voices on Health Care Communication Experiences and Serious Illness Care: Mixed Methods Study.

BACKGROUND: Existing health care research, including serious illness research, often underrepresents individuals from historically marginalized communities. Capturing the nuanced perspectives of individuals around their health care communication experiences is difficult. New research strategies are needed that increase engagement of individuals from diverse backgrounds. OBJECTIVE: The aim of this study was to develop a mixed methods approach with qualitative online forums to better understand health communication experiences of individuals, including people from groups historically marginalized such as Black and Latino individuals; older adults; and people with low income, disability, or serious illness. METHODS: We used a multiphase mixed methods, community-informed research approach to design study instruments and engage participants. We engaged a diverse group of collaborators with lived experience of navigating the health care system who provided feedback on instruments, added concepts for testing, and offered guidance on creating a safe experience for participants (phase 1). We conducted a national quantitative survey between April and May 2021 across intrapersonal, interpersonal, and systems-level domains, with particular focus on interpersonal communication between patients and clinicians (phase 2). We conducted two asynchronous, qualitative online forums, a technique used in market research, between June and August 2021, which allowed us to contextualize the learnings and test concepts and messages (phase 3). Using online forums allowed us to probe more deeply into results and hypotheses from the survey to better understand the "whys" and "whats" that surfaced and to test public messages to encourage action around health. RESULTS: We engaged 46 community partners, including patients and clinicians from a Federally Qualified Health Center, to inform study instrument design. In the quantitative survey, 1854 adults responded, including 50.5% women, 25.2% individuals over 65 years old, and 51.9% individuals with low income. Nearly two-thirds identified as non-Hispanic white (65.7%), 10.4% identified as non-Hispanic Black, and 15.5% identified as Hispanic/Latino. An additional 580 individuals participated in online forums, including 60.7% women, 17.4% individuals over 65 years old, and 49.0% individuals with low income. Among the participants, 70.3% identified as non-Hispanic white, 16.0% as non-Hispanic Black, and 9.5% as Hispanic/Latino. We received rich, diverse input from our online forum participants, and they highlighted satisfaction and increased knowledge with engagement in the forums. CONCLUSIONS: We achieved modest overrepresentation of people who were over 65 years old, identified as non-Hispanic Black, and had low income in our online forums. The size of the online forums (N=580) reflected the voices of 93 Black and 55 Hispanic/Latino participants. Individuals who identify as Hispanic/Latino remained underrepresented, likely because the online forums were offered only in English. Overall, our findings demonstrate the feasibility of using the online forum qualitative approach in a mixed methods study to contextualize, clarify, and expound on quantitative findings when designing public health and clinical communications interventions.

Experience with cultured thymus tissue in 105 children.

BACKGROUND: Currently, there are no approved therapies to treat congenital athymia, a condition of immune deficiency resulting in high early mortality due to infection and immune dysregulation. Multiple syndromic conditions, such as complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE (coloboma, heart defects, choanal atresia, growth or mental retardation, genital hypoplasia, and ear anomalies and/or deafness) syndrome, diabetic embryopathy, other genetic variants, and FOXN1 deficiency, are associated with congenital athymia. OBJECTIVE: Our aims were to study 105 patients treated with cultured thymus tissue (CTT), and in this report, to focus on the outcomes of 95 patients with treatment-naive congenital athymia. METHODS: A total of 10 prospective, single-arm open-label studies with patient enrollment from 1993 to 2020 form the basis of this data set. Patients were tested after administration of CTT for T-cell development; all adverse events and infections were recorded. RESULTS: A total of 105 patients were enrolled and received CTT (the full analysis set). Of those patients, 10 had diagnoses other than congenital athymia and/or received prior treatments. Of those 105 patients, 95 patients with treatment-naive congenital athymia were included in the efficacy analysis set (EAS). The Kaplan-Meier estimated survival rates at year 1 and year 2 after administration of CTT in the EAS were 77% (95% CI = 0.670-0.844) and 76% (95% CI = 0.657-0.834), respectively. In all, 21 patients died in the first year before developing naive T cells and 1 died in the second year after receipt of CTT; 3 subsequent deaths were not related to immunodeficiency. A few patients developed alopecia, autoimmune hepatitis, psoriasis, and psoriatic arthritis after year 1. The rates of infections, autologous graft-versus-host-disease manifestations, and autoimmune cytopenias all decreased approximately 1 year after administration of CTT. CONCLUSION: Treatment with CTT led to development of naive T cells with a 1-year survival rate of 77% and a median follow-up time of 7.6 years. Immune reconstitution sufficient to prevent infections and support survival typically develops 6 to12 months after administration of CTT.

Laparoscopic Heller Myotomy and Dor Fundoplication.

INTRODUCTION: Achalasia is an esophageal motility disorder characterized by disordered esophageal peristalsis with failed relaxation of the lower esophageal sphincter resulting in a functional obstruction.Treatment can include medical, endoscopic, or surgical interventions. Although none of these are curative, they each offer methods to create esophageal outflow. MATERIALS AND METHODS: This article discusses our preferred surgical technique used for laparoscopic Heller myotomy with Dor fundoplication. This technique has been developed over the author's career. CONCLUSION: The technique discussed provides a safe and effective strategy to manage achalasia.

Prolonged Hyperglycemia Causes Visual and Cognitive Deficits in Danio rerio.

The present study induced prolonged hyperglycemia (a hallmark symptom of Type 2 diabetes [T2DM]) in Danio rerio (zebrafish) for eight or twelve weeks. The goal of this research was to study cognitive decline as well as vision loss in hyperglycemic zebrafish. Fish were submerged in glucose for eight or twelve weeks, after which they were assessed with both a cognitive assay (three-chamber choice) and a visual assay (optomotor response (OMR)). Zebrafish were also studied during recovery from hyperglycemia. Here, fish were removed from the hyperglycemic environment for 4 weeks after either 4 or 8 weeks in glucose, and cognition and vision was again assessed. The 8- and 12-week cognitive results revealed that water-treated fish showed evidence of learning while glucose- and mannitol-treated fish did not within the three-day testing period. OMR results identified an osmotic effect with glucose-treated fish having significantly fewer positive rotations than water-treated fish but comparable rotations to mannitol-treated fish. The 8- and 12-week recovery results showed that 4 weeks was not enough time to fully recovery from the hyperglycemic insult sustained.

Care of Children with DiGeorge Before and After Cultured Thymus Tissue Implantation.

BACKGROUND: Children with complete DiGeorge anomaly (cDGA) have congenital athymia plus a myriad of other challenging clinical conditions. The term cDGA encompasses children with congenital athymia secondary to 22q11.2DS, CHARGE syndrome (coloboma, heart defects, choanal atresia, growth or mental retardation, genital abnormalities, and ear abnormalities and/or deafness), and other genetic abnormalities. Some children have no known genetic defects. Since 1993, more than 100 children with congenital athymia have been treated with cultured thymus tissue implantation (CTTI). Naïve T cells develop approximately 6 to 12 months after CTTI. Most of the children had significant comorbidities such as heart disease, hypoparathyroidism, and infections requiring complex clinical care post cultured thymus tissue implantation (CTTI). OBJECTIVE: The purpose of this guidance is to assist multidisciplinary teams in caring for children with cDGA both before and after CTTI. METHODS: Thirty-one specialists, in addition to the authors, were asked to share their experience in caring for children with cDGA at Duke University Health System, before and after CTTI. These specialists included physicians, nurses, dentists, therapists, and dieticians. RESULTS: The goal of a multidisciplinary approach is to have children in the best possible condition for receiving CTTI and provide optimal care post CTTI through development of naïve T cells and beyond. The CTT (cultured thymus tissue) must be protected from high doses of steroids which can damage CTT. Organs must be protected from adverse effects of immunosuppression. CONCLUSION: Creating a multidisciplinary team and a detailed plan of care for children with cDGA is important for optimal outcomes.

Extracellular vesicle-associated miRNAs are an adaptive response to gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is a serious public health issue affecting 9-15% of all pregnancies worldwide. Recently, it has been suggested that extracellular vesicles (EVs) play a role throughout gestation, including mediating a placental response to hyperglycaemia. Here, we investigated the EV-associated miRNA profile across gestation in GDM, assessed their utility in developing accurate, multivariate classification models, and determined the signaling pathways in skeletal muscle proteome associated with the changes in the EV miRNA profile. METHODS: Discovery: A retrospective, case-control study design was used to identify EV-associated miRNAs that vary across pregnancy and clinical status (i.e. GDM or Normal Glucose Tolerance, NGT). EVs were isolated from maternal plasma obtained at early, mid and late gestation (n = 29) and small RNA sequencing was performed. Validation: A longitudinal study design was used to quantify expression of selected miRNAs. EV miRNAs were quantified by real-time PCR (cases = 8, control = 14, samples at three times during pregnancy) and their individual and combined classification efficiencies were evaluated. Quantitative, data-independent acquisition mass spectrometry was use to establish the protein profile in skeletal muscle biopsies from normal and GDM. RESULTS: A total of 2822 miRNAs were analyzed using a small RNA library, and a total of 563 miRNAs that significantly changed (p < 0.05) across gestation and 101 miRNAs were significantly changed between NGT and GDM. Analysis of the miRNA changes in NGT and GDM separately identified a total of 256 (NGT-group), and 302 (GDM-group) miRNAs that change across gestation. A multivariate classification model was developed, based on the quantitative expression of EV-associated miRNAs, and the accuracy to correctly assign samples was > 90%. We identified a set of proteins in skeletal muscle biopsies from women with GDM associated with JAK-STAT signaling which could be targeted by the miRNA-92a-3p within circulating EVs. Interestingly, overexpression of miRNA-92a-3p in primary skeletal muscle cells increase insulin-stimulated glucose uptake. CONCLUSIONS: During early pregnancy, differently-expressed, EV-associated miRNAs may be of clinical utility in identifying presymptomatic women who will subsequently develop GDM later in gestation. We suggest that miRNA-92a-3p within EVs might be a protected mechanism to increase skeletal muscle insulin sensitivity in GDM.

Obstetric and perinatal outcomes for women with pre-existing diabetes in rural compared to metropolitan settings in Victoria, Australia.

BACKGROUND: Pre-existing diabetes in pregnancy is associated with an increased risk of complications. Likewise, living in rural, regional and remote Victoria, Australia, is also associated with poorer health outcomes. There is a gap in the literature with regard to whether Victorian women with pre-existing diabetes experience a greater risk of adverse pregnancy outcomes compared to their metropolitan counterparts. AIM: Our objective is to compare obstetric and perinatal outcomes for women with pre-existing diabetes delivering in rural vs metropolitan hospitals in Victoria, Australia. MATERIALS AND METHODS: Retrospective population-based study using routinely collected state-based data of singleton births to women with type 1 and type 2 diabetes who delivered in metropolitan (n = 3233) and rural hospitals (n = 693) in Victoria, Australia, between 2006-2015. Pearson's χ2 test, Fisher's exact test and MannWhitney U-test were used to compare obstetric and perinatal outcomes between metropolitan and rural locations. RESULTS: Delivery in a rural hospital was associated with higher rates of stillbirth (2.3% vs 1.1%, P = 0.027), macrosomia (25.9% vs 16.9%, P < 0.001), shoulder dystocia (8.4% vs 3.5%, P < 0.001) and admission to the neonatal intensive care unit/special care nursery (73.2% vs 59.3%, P < 0.001). Smoking (18.0% vs 8.9%, P < 0.001), overweight/obesity (P = 0.047) and socioeconomic disadvantage (P < 0.001) were more common in rural women. CONCLUSIONS: Women with pre-existing diabetes who deliver in rural hospitals experience a greater risk of adverse perinatal outcomes and present with increased maternal risk factors. These results suggest a need to improve care for women with pre-existing diabetes in rural Victoria.

Escherichia coli K1 utilizes host macropinocytic pathways for invasion of brain microvascular endothelial cells.

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.

Early consequences of allopolyploidy alter floral evolution in Nicotiana (Solanaceae).

BACKGROUND: Polyploidy has played a major role in angiosperm evolution. Previous studies have examined polyploid phenotypes in comparison to their extant progenitors, but not in context of predicted progenitor phenotypes at allopolyploid origin. In addition, differences in the trends of polyploid versus diploid evolution have not been investigated. We use ancestral character-state reconstructions to estimate progenitor phenotype at allopolyploid origin to determine patterns of polyploid evolution leading to morphology of the extant species. We also compare trends in diploid versus allopolyploid evolution to determine if polyploidy modifies floral evolutionary patterns. RESULTS: Predicting the ancestral phenotype of a nascent allopolyploid from reconstructions of diploid phenotypes at the time of polyploid formation generates different phenotype predictions than when extant diploid phenotypes are used, the outcome of which can alter conclusions about polyploid evolution; however, most analyses yield the same results. Using ancestral reconstructions of diploid floral phenotypes indicate that young polyploids evolve shorter, wider corolla tubes, but older polyploids and diploids do not show any detectable evolutionary trends. Lability of the traits examined (floral shape, corolla tube length, and corolla tube width) differs across young and older polyploids and diploids. Corolla length is more evolutionarily labile in older polyploids and diploids. Polyploids do not display unique suites of floral characters based on both morphological and color traits, but some suites of characters may be evolving together and seem to have arisen multiple times within Nicotiana, perhaps due to the influence of pollinators. CONCLUSIONS: Young polyploids display different trends in floral evolution (shorter, wider corolla tubes, which may result in more generalist pollination) than older polyploids and diploids, suggesting that patterns of divergence are impacted by the early consequences of allopolyploidy, perhaps arising from genomic shock and/or subsequent genome stabilization associated with diploidization. Convergent evolution in floral morphology and color in Nicotiana can be consistent with pollinator preferences, suggesting that pollinators may have shaped floral evolution in Nicotiana.

Pregnancy outcomes for women with pre-pregnancy diabetes mellitus in Australian populations, rural and metropolitan: A review.

BACKGROUND: Historically, pre-pregnancy diabetes (PPDM) is a recognised risk factor for poor pregnancy outcome. Co-existing pathology and adverse social determinants including rural-metropolitan inequities in health and healthcare access may confer additional risks. Multidisciplinary care before, during and after pregnancy can improve outcomes for women with PPDM and their infants. The extent to which rural Australian women and their families share in improved outcomes is unknown. We aimed to summarise maternal characteristics and pregnancy outcomes for women with PPDM, including women in rural settings and examine applications of existing clinical guidelines to rural Australian practice. METHODS: We sought English language population and cohort studies about PPDM using Medline, Embase, PubMed, Australian epidemiological and international clinical practice guidelines. RESULTS: Women with PPDM are changing: older, more obese, of lower parity, less likely to smoke, more likely to have type 2 rather than type 1 diabetes and shorter duration of PPDM. Women with PPDM continue to experience excess adverse pregnancy outcomes, including maternal morbidity, complicated birth, perinatal loss, congenital anomalies and mother-infant separation. On face value, clinical guidelines appear relevant to women living in rural settings but there are only a few, conflicting outcome studies for rural women with PPDM. CONCLUSIONS: PPDM is changing. A significant minority live in rural locations, and although perinatal mortality/morbidity seems to be improving, it is unclear if this is also true for rural women due to a lack of recent Australian studies. Further research is necessary to achieve excellence everywhere for women with PPDM and their babies.

Timing of diagnosis of gestational diabetes and pregnancy outcomes: A retrospective cohort.

BACKGROUND: Recent guidelines suggest screening high-risk women in early pregnancy for gestational diabetes (GDM); however, there is little evidence to support this. AIMS: To compare pregnancy outcomes associated with diabetes for women with risk factors for GDM according to gestation of diagnosis. Early GDM was defined as a positive test before 20 weeks gestation, late GDM as a positive test at 20 or more weeks and no GDM when both tests were negative. MATERIALS AND METHODS: Retrospective analysis in an Australian tertiary hospital of women who underwent a glucose tolerance test in pregnancy prior to 20 weeks gestation, and a repeat test after 20 weeks gestation if the initial test was negative. Results were adjusted for maternal demographics. RESULTS: Women with early GDM (n = 170) were no more likely to experience the obstetric composite outcome than women with late GDM (n = 171) or no GDM (n = 547) (early odds ratio (OR) 1.16, 95%CI 0.79-1.71; late OR 0.78, 95%CI 0.53-1.12). Infants of women with early GDM, but not late GDM, were more likely (early OR 1.8, 95%CI 1.15-2.92; late OR 1.4, 95%CI 0.90-2.23) to have the neonatal composite outcome than infants of women without GDM, predominantly due to an increase in neonatal hypoglycaemia. CONCLUSIONS: This result may be due to careful management of GDM, or because, after adjustment for maternal demographics, the early diagnosis of GDM does not substantially increase rates of adverse outcomes compared to GDM diagnosed in later pregnancy or no GDM in women with risk factors for GDM.