The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study demonstrated that intensive glucose control reduced the risk of developing diabetic peripheral neuropathy (DPN) and cardiovascular autonomic neuropathy (CAN). We evaluated multiple risk factors and phenotypes associated with DPN and CAN in this large, well-characterized cohort of participants with type 1 diabetes, followed for >23 years. DPN was defined by symptoms, signs, and nerve conduction study abnormalities in ≥2 nerves; CAN was assessed using standardized cardiovascular reflex tests. Generalized estimating equation models assessed the association of DPN and CAN with individual risk factors measured repeatedly. During DCCT/EDIC, 33% of participants developed DPN and 44% CAN. Higher mean HbA_1c was the most significant risk factor for DPN, followed by older age, longer duration, greater height, macroalbuminuria, higher mean pulse rate, β-blocker use, and sustained albuminuria. The most significant risk factor for CAN was older age, followed by higher mean HbA_1c, sustained albuminuria, longer duration of type 1 diabetes, higher mean pulse rate, higher mean systolic blood pressure, β-blocker use, estimated glomerular filtration rate <60 mL/min/1.73 m², higher most recent pulse rate, and cigarette smoking. These findings identify risk factors and phenotypes of participants with diabetic neuropathy that can be used in the design of new interventional trials and for personalized approaches to neuropathy prevention.

Approximately 40% of patients with diabetic macular edema (DME) are resistant to anti–vascular endothelial growth factor (VEGF) therapy (rDME). Here, we demonstrate that significant correlations between inflammatory cytokines and VEGF, as observed in naive DME, are lost in patients with rDME. VEGF overexpression in the mouse retina caused delayed inflammatory cytokine upregulation, monocyte/macrophage infiltration (CD11b⁺ Ly6C⁺ CCR2⁺ cells), macrophage/microglia activation (CD11b⁺ CD80⁺ cells), and blood-retinal barrier disruption due to claudin-5 redistribution, which did not recover with VEGF blockade alone. Phosphorylated protein analysis of VEGF-overexpressed retinas revealed rho-associated coiled-coil–containing protein kinase (ROCK) activation. Administration of ripasudil, a selective ROCK inhibitor, attenuated retinal inflammation and claudin-5 redistribution. Ripasudil also contributed to the stability of claudin-5 expression by both transcriptional enhancement and degradation suppression in inflammatory cytokine–stimulated endothelium. Notably, the anti-VEGF agent and the ROCK inhibitor were synergic in suppressing cytokine upregulation, monocyte/macrophage infiltration, macrophage/microglia activation, and claudin-5 redistribution. Furthermore, in vitro analysis confirmed that claudin-5 redistribution depends on ROCK2 but not on ROCK1. This synergistic effect was also confirmed in human rDME cases. Our results suggest that ROCK-mediated claudin-5 redistribution by inflammation is a key mechanism in the anti-VEGF resistance of DME.

Genes of the Notch signaling pathway are expressed in different cell types and organs at different time points during embryonic development and adulthood. The Notch ligand Delta-like 1 (DLL1) controls the decision between endocrine and exocrine fates of multipotent progenitors in the developing pancreas, and loss of Dll1 leads to premature endocrine differentiation. However, the role of Delta-Notch signaling in adult tissue homeostasis is not well understood. Here, we describe the spatial expression pattern of Notch pathway components in adult murine pancreatic islets and show that DLL1 and DLL4 are specifically expressed in β-cells, whereas JAGGED1 is expressed in α-cells. We show that mice lacking both DLL1 and DLL4 in adult β-cells display improved glucose tolerance, increased glucose-stimulated insulin secretion, and hyperglucagonemia. In contrast, overexpression of the intracellular domain of DLL1 in adult murine pancreatic β-cells results in impaired glucose tolerance and reduced insulin secretion, both in vitro and in vivo. These results suggest that Notch ligands play specific roles in the adult pancreas and highlight a novel function of the Delta/Notch pathway in β-cell insulin secretion.

Yan Xing, Hongling Wei, Xiumei Xiao, Zekun Chen, Hui Liu, Xiaomei Tong, and Wei Zhou

Infants with intrauterine growth retardation (IUGR) have a high risk of developing bronchial asthma in childhood, but the underlying mechanisms remain unclear. This study aimed to disclose the role of vascular non-inflammatory molecule 1 (vannin-1, encoded by the Vnn1 gene) and its downstream signaling in IUGR asthmatic mice induced by ovalbumin. Significant histological alterations and an increase of vannin-1 expression were revealed in IUGR asthmatic mice, accompanied by elevated methylation of Vnn1 promoter regions. In IUGR asthmatic mice, we also found (i) a direct binding of HNF4α and PGC1α to Vnn1 promoter by ChIP assay; (ii) a direct interaction of HNF4α with PGC1α; (iii) upregulation of phospho-PI3K p85/p55 and phospho-Akt^Ser473 and downregulation of phospho-PTENT^yr366, and (iv) an increase in nuclear NFB p65 and a decrease in cytosolic IB-α. In primary cultured bronchial epithelial cells derived from the IUGR asthmatic mice, knockdown of Vnn1 prevented upregulation of phospho-Akt^Ser473 and an increase of reactive oxygen species (ROS) and TGF-β production. Taken together, we demonstrate that elevated vannin-1 activates the PI3K/Akt/NFB signaling pathway, leading to ROS and inflammation reactions responsible for asthma occurrence in IUGR individuals. We also disclose that interaction of PGC1α and HNF4α promotes methylation of Vnn1 promoter regions and then upregulates vannin-1 expression.

PURPOSE

Anticholinergic burden (ACB), the cumulative effect of anticholinergic medications, is associated with adverse outcomes in older people but is less studied in middle-aged populations. Numerous scales exist to quantify ACB. The aims of this study were to quantify ACB in a large cohort using the 10 most common anticholinergic scales, to assess the association of each scale with adverse outcomes, and to assess overlap in populations identified by each scale.

PURPOSE

General practitioners (GPs) are part of the US physician workforce, but little is known about who they are, what they do, and how they differ from family physicians (FPs). We describe self-identified GPs and compare them with board-certified FPs.

PURPOSE

Although cesarean delivery is the most common surgical procedure in the United States, postoperative opioid prescribing varies greatly. We hypothesized that patient characteristics, procedural characteristics, or both would be associated with high vs low opioid use after discharge. This information could help individualize prescriptions.

Background:

Unnecessary antibiotic use in the community in Canada is not well defined. Our objective was to quantify unnecessary antibiotic prescribing in a Canadian primary care setting.

Background:

Historically, some chiropractors have been critical of vaccination, and this has been the subject of recent media attention in Canada. We explored the association between media attention and public dissemination of vaccination information on Canadian chiropractors’ websites.

Background:

Deaths from respiratory tract infections (RTIs) in children are preventable through timely access to public health and medical interventions. We aimed to assess whether socioeconomic disparities in mortality related to pediatric RTI persisted after accounting for health status at birth.

Background:

The number of medical undergraduate and postgraduate students completing palliative care clinical rotations in Canadian medical schools is currently unknown. The aim of this study was to assess the proportion of Canadian medical trainees completing clinical rotations in palliative care and to determine whether changes took place between 2008 and 2018.

Dramatically rising costs in drug development are in large part because of the high failure rates in clinical phase trials. The poor correlation of animal studies to human toxicity and efficacy have led many developers to question the value of requiring animal studies in determining which drugs should enter in-human trials. Part 1 of this 2-part series examined some of the data regarding the lack of concordance between animal toxicity studies and human trials, as well as some of the potential reasons behind it. This second part of the series focuses on some alternatives to animal trials (hereafter referred to as animal research) as well as current regulatory discussions and developments regarding such alternatives.

We are already a couple of months into 2020 and I hope you had a good start to the new year. I wish you, our readers, reviewers, authors and editors, happiness, success and health in this new decade.

It is a source of frustration to many clinicians: you know what the patient's problem is, you know that effective and safe treatment is available, you've explained the disease and its causative mechanisms, the treatment and its principles, and the importance of taking the controller medication daily, you've prescribed this highly effective therapy and you've approached the patient with respect and patience, yet somehow the patient does not take the medication. When this patient has another exacerbation, you know it could have been prevented by following your advice and taking the medication.

Purpose:

There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed.

In this retrospective study, whole-genome sequencing (WGS) data generated on an Ion Torrent platform was used to predict phenotypic drug resistance profiles for first- and second-line drugs among Swedish clinical Mycobacterium tuberculosis isolates from 2016 to 2018. The accuracy was ~99% for all first-line drugs and 100% for four second-line drugs. Our analysis supports the introduction of WGS into routine diagnostics, which might, at least in Sweden, replace phenotypic drug susceptibility testing in the future.

Tigecycline serves as one of the antibiotics of last resort to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanism, Tet(X), challenges the clinical efficacy of this class of antibiotics. In this study, we detected 180 tet(X)-harboring Acinetobacter isolates (8.9%, n = 180) from 2,018 samples collected from avian farms and adjacent environments in China. Eighteen tet(X)-harboring isolates (10.0%) were found to cocarry the carbapenemase gene bla_NDM-1, mostly from waterfowl samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, tet(X) and bla_NDM-1 were found to colocalize on the same plasmids. Moreover, whole-genome sequencing (WGS) revealed a novel orthologue of tet(X) in the six isolates coharboring tet(X) and bla_NDM-1. Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be cotransferred. Sequence comparison between six tet(X)- and bla_NDM-1-coharboring plasmids showed that they shared a highly homologous plasmid backbone even though they were isolated from different Acinetobacter species (three from Acinetobacter indicus, two from Acinetobacter schindleri, and one from Acinetobacter lwoffii) from various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X)- and bla_NDM-1-coharboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

Addition of sodium bicarbonate (NaHCO₃) to standard antimicrobial susceptibility testing medium reveals certain methicillin-resistant Staphylococcus aureus (MRSA) strains to be highly susceptible to β-lactams. We investigated the prevalence of this phenotype (NaHCO₃ responsiveness) to two β-lactams among 58 clinical MRSA bloodstream isolates. Of note, ~75% and ~36% of isolates displayed the NaHCO₃ responsiveness phenotype to cefazolin (CFZ) and oxacillin (OXA), respectively. Neither intrinsic β-lactam MICs in standard Mueller-Hinton broth (MHB) nor population analysis profiles were predictive of this phenotype. Several genotypic markers (clonal complex 8 [CC8]; agr I and spa t008) were associated with NaHCO₃ responsiveness for OXA.

The rising rates of antibiotic resistance increasingly compromise empirical treatment. Knowing the antibiotic susceptibility of a pathogen’s close genetic relative(s) may improve empirical antibiotic selection. Using genomic and phenotypic data for Escherichia coli isolates from three separate clinically derived databases, we evaluated multiple genomic methods and statistical models for predicting antibiotic susceptibility, focusing on potentially rapidly available information, such as lineage or genetic distance from archived isolates. We applied these methods to derive and validate the prediction of antibiotic susceptibility to common antibiotics. We evaluated 968 separate episodes of suspected and confirmed infection with Escherichia coli from three geographically and temporally separated databases in Ontario, Canada, from 2010 to 2018. Across all approaches, model performance (area under the curve [AUC]) ranges for predicting antibiotic susceptibility were the greatest for ciprofloxacin (AUC, 0.76 to 0.97) and the lowest for trimethoprim-sulfamethoxazole (AUC, 0.51 to 0.80). When a model predicted that an isolate was susceptible, the resulting (posttest) probabilities of susceptibility were sufficient to warrant empirical therapy for most antibiotics (mean, 92%). An approach combining multiple models could permit the use of narrower-spectrum oral agents in 2 out of every 3 patients while maintaining high treatment adequacy (~90%). Methods based on genetic relatedness to archived samples of E. coli could be used to predict antibiotic resistance and improve antibiotic selection.

Antiretroviral therapy (ART) suppresses HIV-1 replication but fails to cure the infection. The presence of an extremely stable viral latent reservoir, primarily in resting memory CD4⁺ T cells, remains a major obstacle to viral eradication. The "shock and kill" strategy targets these latently infected cells and boosts immune recognition and clearance, and thus, it is a promising approach for an HIV-1 functional cure. Although some latency-reversing agents (LRAs) have been reported, no apparent clinical progress has been made, so it is still vital to seek novel and effective LRAs. Here, we report that thiostrepton (TSR), a proteasome inhibitor, reactivates latent HIV-1 effectively in cellular models and in primary CD4⁺ T cells from ART-suppressed individuals ex vivo. TSR does not induce global T cell activation, severe cytotoxicity, or CD8⁺ T cell dysfunction, making it a prospective LRA candidate. We also observed a significant synergistic effect of reactivation when TSR was combined with JQ1, prostratin, or bryostatin-1. Interestingly, six TSR analogues also show reactivation abilities that are similar to or more effective than that of TSR. We further verified that TSR upregulated expression of heat shock proteins (HSPs) in CD4⁺ T cells, which subsequently activated positive transcriptional elongation factor b (p-TEFb) and NF-B signals, leading to viral reactivation. In summary, we identify TSR as a novel LRA which could have important significance for applications to an HIV-1 functional cure in the future.

Tedizolid (TZD) and daptomycin (DAP) were assessed in a rat endocarditis model against Enterococcus faecalis, Enterococcus faecium (resistant to vancomycin and ampicillin), and Staphylococcus aureus. As a monotherapy, TZD for 5 days was not effective in a comparison with no-treatment controls, while DAP for 5 days was significantly effective against these bacteria. Step-down therapy (DAP for 3 days followed by TZD for 2 days) was as effective as DAP for 5 days and was comparable to 3 days of DAP plus ceftriaxone against all bacteria and to 3 days of DAP plus gentamicin against E. faecalis OG1RF.

Balamuthia mandrillaris is an under-reported, pathogenic free-living amoeba that causes Balamuthia amoebic encephalitis (BAE) and cutaneous skin infections. Although cutaneous infections are not typically lethal, BAE with or without cutaneous involvement is usually fatal. This is due to the lack of drugs that are both efficacious and can cross the blood-brain barrier. We aimed to discover new leads for drug discovery by screening the open-source Medicines for Malaria Venture (MMV) Malaria Box and MMV Pathogen Box, with 800 compounds total. From an initial single point screen at 1 and 10 μM, we identified 54 hits that significantly inhibited the growth of B. mandrillaris in vitro. Hits were reconfirmed in quantitative dose-response assays and 23 compounds (42.6%) were confirmed with activity greater than miltefosine, the current standard of care.

Helicobacter pylori is an important risk factor for gastric ulcers. However, antibacterial therapies increase the resistance rate and decrease the eradication rate of H. pylori. Inspired by the microaerophilic characteristics of H. pylori, we aimed at effectively establishing an oxygen-enriched environment to eradicate and prevent the recurrence of H. pylori. The effect and the mechanism of an oxygen-enriched environment in eradicating H. pylori and preventing the recurrence were explored in vitro and in vivo. During oral administration and after drug withdrawal, H. pylori counts were evaluated by Giemsa staining in animal cohorts. An oxygen-enriched environment in which H. pylori could not survive was successfully established by adding hydrogen peroxide into several solutions and rabbit gastric juice. Hydrogen peroxide effectively killed H. pylori in Columbia blood agar and special peptone broth. Minimum inhibition concentrations and minimum bactericidal concentrations of hydrogen peroxide were both relatively stable after promotion of resistance for 30 generations, indicating that hydrogen peroxide did not easily promote resistance in H. pylori. In models of Mongolian gerbils and Kunming mice, hydrogen peroxide has been shown to significantly eradicate and effectively prevent the recurrence of H. pylori without toxicity and damage to the gastric mucosa. The mechanism of hydrogen peroxide causing H. pylori death was related to the disruption of bacterial cell membranes. The oxygen-enriched environment achieved by hydrogen peroxide eradicates and prevents the recurrence of H. pylori by damaging bacterial cell membranes. Hydrogen peroxide thus provides an attractive candidate for anti-H. pylori treatment.

Etravirine (ETR) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) used in treatment-experienced individuals. Genotypic resistance test-interpretation systems can predict ETR resistance; however, genotype-based algorithms are derived primarily from HIV-1 subtype B and may not accurately predict resistance in non-B subtypes. The frequency of ETR resistance among recombinant subtype C HIV-1 and the accuracy of genotypic interpretation systems were investigated. HIV-1_LAI containing full-length RT from HIV-1 subtype C-positive individuals experiencing virologic failure (>10,000 copies/ml and >1 NNRTI resistance-associated mutation) were phenotyped for ETR susceptibility. Fold change (FC) was calculated against a composite 50% effective concentration (EC₅₀) from treatment-naive individuals and three classifications were assigned: (i) <2.9-FC, susceptible; (ii) ≥2.9- to 10-FC, partially resistant; and (iii) >10-FC, fully resistant. The Stanford HIVdb-v8.4 was used for genotype predictions merging the susceptible/potential low-level and low-level/intermediate groups for 3 x 3 comparison. Fifty-four of a hundred samples had reduced ETR susceptibility (≥2.9-FC). The FC correlated with HIVdb-v8.4 (Spearman’s rho = 0.62; P < 0.0001); however, 44% of samples were partially (1 resistance classification difference) and 4% completely discordant (2 resistance classification differences). Of the 34 samples with an FC of >10, 26 were HIVdb-v8.4 classified as low-intermediate resistant. Mutations L100I, Y181C, or M230L were present in 27/34 (79%) of samples with an FC of >10 but only in 2/46 (4%) of samples with an FC of <2.9. No other mutations were associated with ETR resistance. Viruses containing the mutation K65R were associated with reduced ETR susceptibility, but 65R reversions did not increase ETR susceptibility. Therefore, genotypic interpretation systems were found to misclassify ETR susceptibility in HIV-1 subtype C samples. Modifications to genotypic algorithms are needed to improve the prediction of ETR resistance for the HIV-1 subtype C.

We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum. First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC₅₀] ≤ 10 μM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC₅₀ of ≤1 μM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 μM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.

A fosfomycin-resistant and carbapenemase (OXA-48)-producing Klebsiella pneumoniae isolate was recovered, and whole-genome sequencing revealed ISEcp1-bla_CTX-M-14b tandemly inserted upstream of the chromosomally encoded lysR-fosA locus. Quantitative evaluation of the expression of lysR and fosA genes showed that this insertion brought a strong hybrid promoter leading to overexpression of the fosA gene, resulting in fosfomycin resistance. This work showed the concomitant acquisition of resistance to broad-spectrum cephalosporins and fosfomycin due to a single genetic event.

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.

Patients with HPV16⁺ cervical cancer and high T-cell responses to an HPV16 vaccine survived longer.

A large international study found that the composition of the intestinal microbiome can predict clinical outcomes in patients undergoing allogenic hematopoietic-cell transplant (HCT) for blood cancers. The findings may help assess patients' transplantation-related mortality risk and aid in developing interventions to prevent or mitigate microbiome changes that affect HCT outcomes.

Tumor-derived retinoic acid promotes monocyte differentiation into immunosuppressive macrophages.

Wilms tumor (WT) is the most common renal malignancy of childhood and accounts for 6% of all childhood malignancies. With current therapies, the 5-yr overall survival (OS) for children with unilateral favorable histology WT is greater than 85%. The prognosis is worse, however, for the roughly 15% of patients who relapse, with only 50%–80% OS reported in those with recurrence. Herein, we describe the extended and detailed clinical course of a rare case of a child with recurrent, pulmonary metastatic, favorable histology WT harboring a BRAF V600E mutation. The BRAF V600E mutation, commonly found in melanoma and other cancers, and previously undescribed in WT, has recently been reported by our group in a subset of epithelial-predominant WT. This patient, who was included in that series, presented with unilateral, stage 1, favorable histology WT and was treated with standard chemotherapy. Following the completion of therapy, the patient relapsed with pulmonary metastatic disease, that then again recurred despite an initial response to salvage chemotherapy and radiation. Next-generation sequencing (NGS) on the metastatic pulmonary nodule revealed a BRAF V600E mutation. After weighing the therapeutic options, a novel approach with dual BRAF/MEK inhibitor combination therapy was initiated. Complete radiographic response was observed following 4 months of therapy with dabrafenib and trametinib. At 12 months following the start of BRAF/MEK combination treatment, the patient continues with a complete response and has experienced minimal treatment-related side effects. This represents the first case, to our knowledge, of effective treatment with BRAF/MEK molecularly targeted therapy in a pediatric Wilms tumor patient.

Quality Improvement Success Stories are published by the American Diabetes Association in collaboration with the American College of Physicians, Inc. (ACP), and the National Diabetes Education Program. This series is intended to highlight best practices and strategies from programs and clinics that have successfully improved the quality of care for people with diabetes or related conditions. Each article in the series is reviewed and follows a standard format developed by the editors of Clinical Diabetes. The following article describes an initiative of the Cleveland Clinic’s internal medicine residents to improve diabetes care and outcomes within an underserved patient population at an East Cleveland, OH, health center.

Background:

Sex steroid hormones and sex hormone–binding globulin (SHBG) have been implicated in the etiology of invasive breast cancer, but their associations with risk of the precursor lesion, ductal carcinoma in situ (DCIS) of the breast, remain unclear.