Regadenoson for the treatment of COVID-19: A five case clinical series and mouse studies.

BACKGROUND: Adenosine inhibits the activation of most immune cells and platelets. Selective adenosine A2A receptor (A2AR) agonists such as regadenoson (RA) reduce inflammation in most tissues, including lungs injured by hypoxia, ischemia, transplantation, or sickle cell anemia, principally by suppressing the activation of invariant natural killer T (iNKT) cells. The anti-inflammatory effects of RA are magnified in injured tissues due to induction in immune cells of A2ARs and ecto-enzymes CD39 and CD73 that convert ATP to adenosine in the extracellular space. Here we describe the results of a five patient study designed to evaluate RA safety and to seek evidence of reduced cytokine storm in hospitalized COVID-19 patients. METHODS AND FINDINGS: Five COVID-19 patients requiring supplemental oxygen but not intubation (WHO stages 4-5) were infused IV with a loading RA dose of 5 µg/kg/h for 0.5 h followed by a maintenance dose of 1.44 µg/kg/h for 6 hours, Vital signs and arterial oxygen saturation were recorded, and blood samples were collected before, during and after RA infusion for analysis of CRP, D-dimer, circulating iNKT cell activation state and plasma levels of 13 proinflammatory cytokines. RA was devoid of serious side effects, and within 24 hours from the start of infusion was associated with increased oxygen saturation (93.8 ± 0.58 vs 96.6 ± 1.08%, P<0.05), decreased D-dimer (754 ± 17 vs 518 ± 98 ng/ml, P<0.05), and a trend toward decreased CRP (3.80 ± 1.40 vs 1.98 ± 0.74 mg/dL, P = 0.075). Circulating iNKT cells, but not conventional T cells, were highly activated in COVID-19 patients (65% vs 5% CD69+). RA infusion for 30 minutes reduced iNKT cell activation by 50% (P<0.01). RA infusion for 30 minutes did not influence plasma cytokines, but infusion for 4.5 or 24 hours reduced levels of 11 of 13 proinflammatory cytokines. In separate mouse studies, subcutaneous RA infusion from Alzet minipumps at 1.44 µg/kg/h increased 10-day survival of SARS-CoV-2-infected K18-hACE2 mice from 10 to 40% (P<0.001). CONCLUSIONS: Infused RA is safe and produces rapid anti-inflammatory effects mediated by A2A adenosine receptors on iNKT cells and possibly in part by A2ARs on other immune cells and platelets. We speculate that iNKT cells are activated by release of injury-induced glycolipid antigens and/or alarmins such as IL-33 derived from virally infected type II epithelial cells which in turn activate iNKT cells and secondarily other immune cells. Adenosine released from hypoxic tissues, or RA infused as an anti-inflammatory agent decrease proinflammatory cytokines and may be useful for treating cytokine storm in patients with Covid-19 or other inflammatory lung diseases or trauma.

Regadenoson Reduces Soluble Receptor for Advanced Glycation End-Products in Lung Recipients.

BACKGROUND: The selective adenosine A2A receptor (A2AR) agonist regadenoson reduces inflammation due to lung ischemia-reperfusion injury (IRI). The objective of this study was to investigate molecular and cellular mechanisms by which regadenoson reduces IRI in lung transplant recipients. METHODS: Fourteen human lung transplant recipients were infused for 12 hours with regadenoson and 7 more served as untreated controls. Plasma levels of high mobility group box 1 and its soluble receptor for advanced glycation end-products (sRAGE) were measured by Luminex. Matrix metalloproteinase (MMP) 2 and 9 were measured by gelatin zymography. Tissue inhibitor of metalloproteinase 1 was measured by mass spectroscopy. A2AR expression on leukocytes was analyzed by flow cytometry. MMP-9-mediated cleavage of RAGE was evaluated using cultured macrophages in vitro. RESULTS: Regadenoson treatment during lung transplantation significantly reduced levels of MMP-9 (P < .05), but not MMP-2, and elevated levels of tissue inhibitor of metalloproteinase 1 (P < .05), an endogenous selective inhibitor of MMP-9. Regadenoson infusion significantly reduced plasma levels of sRAGE (P < .05) during lung reperfusion compared with control subjects. A2AR expression was highest on invariant natural killer T cells and higher on monocytes than other circulating immune cells (P < .05). The shedding of RAGE from cultured monocytes/macrophages was increased by MMP-9 stimulation and reduced by an MMP inhibitor or by A2AR agonists, regadenoson or ATL146e. CONCLUSIONS: In vivo and in vitro studies suggest that A2AR activation reduces sRAGE in part by inhibiting MMP-9 production by monocytes/macrophages. These results suggest a novel molecular mechanism by which A2AR agonists reduce primary graft dysfunction.

Evidence That Binding of Cyclic GMP to the Extracellular Domain of NKA (Sodium-Potassium ATPase) Mediates Natriuresis.

BACKGROUND: Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na+) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular α1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na+ transport similar to ouabain-a cardiotonic steroid. METHODS: Urine Na+ excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 µg/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis±rostafuroxin infusion. Rostafuroxin is a digitoxigenin derivative that displaces ouabain from NKA. RESULTS: Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated SrcTyr416 and Erk 1/2 (extracellular signal-regulated protein kinase 1/2)Thr202/Tyr204; these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 µM)+cGMP (10 µM) or rostafuroxin (10 µM) and 8-biotin-11-cGMP (2 µM)+ouabain (10 µM) or rostafuroxin (10 µM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N3-PET-8-biotin-11-cGMP (2 µM); 8-N3-6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N3-PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non-cross-linked samples and cross-linked or non-cross-linked 8-N3-6-biotin-10-cAMP RPTs. Ouabain (10 µM) reduced NKA in cross-linked 4-N3-PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N3-PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA. CONCLUSIONS: cGMP can bind to NKA and thereby mediate natriuresis.

Isotonic design for phase I cancer clinical trials with late-onset toxicities.

This article addresses the problem of identifying the maximum tolerated dose (MTD) in Phase I dose-finding clinical trials with late-onset toxicities. The main design challenge is how best to adaptively allocate study participants to tolerable doses when the evaluation window for the toxicity endpoint is long relative to the accrual rate of new participants. We propose a new design framework based on order-restricted statistical inference that addresses this challenge in sequential dose assignments. We illustrate the proposed method on real data from a Phase I trial of bortezomib in lymphoma patients and apply it to a Phase I trial of radiotherapy in prostate cancer patients. We conduct extensive simulation studies to compare our design's operating characteristics to existing published methods. Overall, our proposed design demonstrates good performance relative to existing methods in allocating participants at and around the MTD during the study and accurately recommending the MTD at the study conclusion.

Standardizing Prostaglandin Initiation in Prenatally Diagnosed Ductal-Dependent Neonates; A Quality Initiative.

Prostaglandin E1 (PGE) is used in patients with ductal-dependent congenital heart disease (CHD). Side effects of apnea and fever are often dose dependent and occur within 48 h after initiation. We initiated a standardized approach to PGE initiation after our institution recognized a high incidence of side effects and a wide variety of starting doses of PGE. Neonates with prenatally diagnosed ductal-dependent CHD were identified, started on a standardized protocol that started PGE at 0.01 mcg/kg/min, and evaluated for PGE related side effects. Compliance, outcomes and dose adjustments during the first 48 h post-PGE initiation were evaluated. Fifty patients were identified (25 pre-intervention; 25 post-intervention). After intervention, compliance with the protocol was 96%, and apnea or fever occurred in 28% (compared to 63% pre-intervention, p = 0.015). Dose adjustments (either increase or decrease) prior to cardiac surgery were similar in both cohorts (60%, 52%, p = 0.569). There were no mortalities or emergent procedures performed due to ductus arteriosus closure. Standardizing a protocol for initiating PGE in prenatally diagnosed ductal-dependent CHD was successful and reduced the incidence of apnea, fever, and sepsis evaluations. A starting dose of 0.01 mcg/kg/min did not cause increased adverse effects.

Ceramide nanoliposomes augment the efficacy of venetoclax and cytarabine in models of acute myeloid leukemia.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

Breast Cancer Antiestrogen Resistance 3 (BCAR3) promotes tumor growth and progression in triple-negative breast cancer.

Triple-Negative Breast Cancers (TNBCs) constitute roughly 10-20% of breast cancers and are associated with poor clinical outcomes. Previous work from our laboratory and others has determined that the cytoplasmic adaptor protein Breast Cancer Antiestrogen Resistance 3 (BCAR3) is an important promoter of cell motility and invasion of breast cancer cells. In this study, we use both in vivo and in vitro approaches to extend our understanding of BCAR3 function in TNBC. We show that BCAR3 is upregulated in ductal carcinoma in situ (DCIS) and invasive carcinomas compared to normal mammary tissue, and that survival of TNBC patients whose tumors contained elevated BCAR3 mRNA is reduced relative to individuals whose tumors had less BCAR3 mRNA. Using mouse orthotopic tumor models, we further show that BCAR3 is required for efficient TNBC tumor growth. Analysis of publicly available RNA expression databases revealed that MET receptor signaling is strongly correlated with BCAR3 mRNA expression. A functional role for BCAR3-MET coupling is supported by data showing that both proteins participate in a single pathway to control proliferation and migration of TNBC cells. Interestingly, the mechanism through which this functional interaction operates appears to differ in different genetic backgrounds of TNBC, stemming in one case from potential differences in the strength of downstream signaling by the MET receptor and in another from BCAR3-dependent activation of an autocrine loop involving the production of HGF mRNA. Together, these data open the possibility for new approaches to personalized therapy for individuals with TNBCs.

Operating characteristics are needed to properly evaluate the scientific validity of phase I protocols.

PURPOSE: Operating characteristics for proposed clinical trial designs provide insight into performance regarding safety and accuracy, allowing the study team and review entities to determine the design's suitability to achieve the study's proposed objectives. Advances in cancer therapeutics have augmented the needs of early phase clinical trial design. Additionally, advances in research on early-phase trial design have led to the availability of a wide range of methods that show vast improvement over outdated approaches. METHODS: Three trials utilizing variations of the 3 + 3 decision rule are discussed. The protocols lacked detail, including operating characteristics and guidance for decision-making that deviated from the 3 + 3 decision rule and MTD determination. We provide a discussion of the statistical issues associated with each design and operating characteristics for the proposed design compared to alternatives better suited to achieve the aims of each trial. RESULTS: Our results illustrate how operating characteristics inform a design's safety and accuracy. Operating characteristics can unmask poor behavior, such as a high percentage of particiapnts exposed to overly toxic doses, a low probability of correctly identifying the MTD, and inappropriate early study termination. CONCLUSION: Selection of early-phase trial design has significant implications on a trial's ability to meet its objectives. Operating characteristics are a necessary component in the design and review of a protocol, determining if the study's objectives can be achieved and documenting the study's scientific validity. Continued use of outdated approaches due to historical acceptance hinders scientific rigor and the effort to move effective agents through the drug development process.

Immune mechanisms orchestrate tertiary lymphoid structures in tumors via cancer-associated fibroblasts.

Tumor-associated tertiary lymphoid structures (TA-TLS) are associated with enhanced patient survival and responsiveness to cancer therapies, but the mechanisms underlying their development are unknown. We show here that TA-TLS development in murine melanoma is orchestrated by cancer-associated fibroblasts (CAF) with characteristics of lymphoid tissue organizer cells that are induced by tumor necrosis factor receptor signaling. CAF organization into reticular networks is mediated by CD8 T cells, while CAF accumulation and TA-TLS expansion depend on CXCL13-mediated recruitment of B cells expressing lymphotoxin-α1ß2. Some of these elements are also overrepresented in human TA-TLS. Additionally, we demonstrate that immunotherapy induces more and larger TA-TLS that are more often organized with discrete T and B cell zones, and that TA-TLS presence, number, and size are correlated with reduced tumor size and overall response to checkpoint immunotherapy. This work provides a platform for manipulating TA-TLS development as a cancer immunotherapy strategy.

Hospital Survival After Surgical Repair of Truncus Arteriosus with Interrupted Aortic Arch: Results from a Multi-institutional Database.

Truncus arteriosus (TA) is a major congenital cardiac malformation that requires surgical repair in the first few weeks of life. Interrupted aortic arch (IAA) is an associated malformation that significantly impacts the complexity of the TA operation. The aim of this study was to (1) define the comorbid conditions associated with TA and (2) determine the hospital survival and morbidity of patients with TA with and without an IAA. Data was collected from the Vizient Clinical Database/Resource Manager, formerly University HealthSystem Consortium, which encompasses more than 160 academic medical centers in the United States. The database was queried for patients admitted from 2002 to 2016 who were ≤ 4 months of age at initial admission, diagnosed with TA, and underwent complete surgical repair during that hospitalization. Of the 645 patients with TA who underwent surgery, 98 (15%) had TA with an interrupted aortic arch (TA-IAA). Both TA and TA-IAA were associated with a high prevalence of comorbidities, including DiGeorge syndrome, prematurity, and other congenital malformations. There was no difference in mortality between TA and TA-IAA (13.7-18.4%, p value = 0.227). No comorbid conditions were associated with an increased mortality in either group. However, patients with TA-IAA had a longer post-operative length of stay (LOS) compared to those without IAA (30 versus 40.3 days, p value = 0.001) and this effect was additive with each additional comorbid condition. In conclusion, the addition of IAA to TA is associated with an increased post-operative LOS, but does not increase in-hospital mortality.

Differential Expression of CD49a and CD49b Determines Localization and Function of Tumor-Infiltrating CD8+ T Cells.

CD8+ T-cell infiltration and effector activity in tumors are correlated with better overall survival of patients, suggesting that the ability of T cells to enter and remain in contact with tumor cells supports tumor control. CD8+ T cells express the collagen-binding integrins CD49a and CD49b, but little is known about their function or how their expression is regulated in the tumor microenvironment (TME). Here, we found that tumor-infiltrating CD8+ T cells initially expressed CD49b, gained CD49a, and then lost CD49b over the course of tumor outgrowth. This differentiation sequence was driven by antigen-independent elements in the TME, although T-cell receptor (TCR) stimulation further increased CD49a expression. Expression of exhaustion markers and CD49a associated temporally but not mechanistically. Intratumoral CD49a-expressing CD8+ T cells failed to upregulate TCR-dependent Nur77 expression, whereas CD69 was constitutively expressed, consistent with both a lack of productive antigen engagement and a tissue-resident memory-like phenotype. Imaging T cells in live tumor slices revealed that CD49a increased their motility, especially of those in close proximity to tumor cells, suggesting that it may interfere with T-cell recognition of tumor cells by distracting them from productive engagement, although we were not able to augment productive engagement by short-term CD49a blockade. CD49b also promoted relocalization of T cells at a greater distance from tumor cells. Thus, our results demonstrate that expression of these integrins affects T-cell trafficking and localization in tumors via distinct mechanisms, and suggests a new way in which the TME, and likely collagen, could promote tumor-infiltrating CD8+ T-cell dysfunction.

Adapting isotonic dose-finding to a dynamic set of drug combinations with application to a phase I leukemia trial.

BACKGROUND/AIMS: This article describes the proposed design of a phase I study evaluating the safety of ceramide nanoliposome and vinblastine among an initial set of 19 possible dose combinations in patients with relapsed/refractory acute myeloid leukemia and patients with untreated acute myeloid leukemia who are not candidates for intensive induction chemotherapy. METHODS: Extensive collaboration between statisticians and clinical investigators revealed the need to incorporate several adaptive features into the design, including the flexibility of adding or eliminating certain dose combinations based on safety criteria applied to multiple dose pairs. During the design stage, additional dose levels of vinblastine were added, increasing the dimension of the drug combination space and thus the complexity of the problem. Increased complexity made application of existing drug combination dose-finding methods unsuitable in their current form. RESULTS: Our solution to these challenges was to adapt a method based on isotonic regression to meet the research objectives of the study. Application of this adapted method is described herein, and a simulation study of the design's operating characteristics is conducted. CONCLUSION: The aim of this article is to bring to light examples of novel design applications as a means of augmenting the implementation of innovative designs in the future and to demonstrate the flexibility of adaptive designs in satisfying changing design conditions.

Adenosine A2A receptor agonist (regadenoson) in human lung transplantation.

BACKGROUND: Currently, there are no clinically approved treatments for ischemia-reperfusion injury after lung transplantation. Pre-clinical animal models have demonstrated a promising efficacy of adenosine 2A receptor (A2AR) agonists as a treatment option for reducing ischemia-reperfusion injury. The purpose of this human study, is to conduct a Phase I clinical trial for evaluating the safety of continuous infusion of an A2AR agonist in lung transplant recipients. METHODS: An adaptive, two-stage continual reassessment trial was designed to evaluate the safety of regadenoson (A2AR agonist) in the setting of lung transplantation. Continuous infusion of regadenoson was administered to lung transplant recipients that was started at the time of skin incision. Adverse events and dose-limiting toxicities, as pre-determined by a study team and assessed by a clinical team and an independent safety monitor, were the primary end-points for safety in this trial. RESULTS: Between January 2018 and March 2019, 14 recipients were enrolled in the trial. Of these, 10 received the maximum infused dose of 1.44 µg/kg/min for 12 hours. No dose-limiting toxicities were observed. The steady-state plasma regadenoson levels sampled before the reperfusion of the first lung were 0.98 ± 0.46 ng/ml. There were no mortalities within 30 days. CONCLUSIONS: Regadenoson, an A2AR agonist, can be safely infused in the setting of lung transplantation with no dose-limiting toxicities or drug-related mortality. Although not powered for the evaluation of secondary end-points, the results of this trial and the outcome of pre-clinical studies warrant further investigation with a Phase II randomized controlled trial.

Looking past PD-L1: expression of immune checkpoint TIM-3 and its ligand galectin-9 in cervical and vulvar squamous neoplasia.

Immunotherapies targeting the PD-1/PD-L1 pathway have shown some success in cervical and vulvar squamous cell carcinomas, but little is known about the potential vulnerability of these tumors to other checkpoint inhibitors. TIM-3 is a checkpoint molecule that exerts immunosuppressive function via its interaction with Gal-9. TIM-3 and Gal-9 have been identified on a variety of malignancies but have not been studied in cervical and vulvar cancers, nor has their relationship to PD-L1 been established. Sixty-three cervical and vulvar invasive (n = 34) and intraepithelial lesions (n = 29) were assessed for TIM-3, Gal-9, and PD-L1 in tumor/lesional cells and associated immune cells. Tumoral TIM-3 expression was identified in 85% of squamous cell carcinomas but only 21% of intraepithelial lesions (p < 0.0001). When immune cells were also accounted for, 97% of invasive and 41% of intraepithelial lesions had a TIM-3 combined positive score (CPS) ≥ 1 (p < 0.0001). Tumoral membranous expression of Gal-9 was seen in 82% of squamous cell carcinomas and 31% of intraepithelial lesions (p = 0.0001); nearly all cases had Gal-9-positive immune cells. Tumoral PD-L1 was seen in 71% of squamous cell carcinomas and 10% of intraepithelial lesions (p < 0.0001), while the PD-L1 CPS was ≥1 in 82 and 21%, respectively (p < 0.0001). There were no significant differences in TIM-3, GAL-9, or PD-L1 expression in cervical vs. vulvar neoplasms, nor was HPV status significantly associated with any of the three markers. Dual TIM-3/Gal-9 expression was present in the majority (86%) of PD-L1-positive cases including 100% of PD-L1-positive squamous cell carcinomas, suggesting a possible role for TIM-3 checkpoint inhibition in concert with anti-PD-1/PD-L1.

Consequences of Performing Parallel Dose Finding Trials in Heterogeneous Groups of Patients.

Patient heterogeneity, in which patients can be grouped by risk of toxicity, is a design challenge in early phase dose finding trials. Carrying out independent trials for each group is a readily available approach for dose finding. However, this often leads to dose recommendations that violate the known order of toxicity risk by group, or reversals in dose recommendation. In this manuscript, trials for partially ordered groups are simulated using four approaches: independent parallel trials using the continual reassessment method (CRM), Bayesian optimal interval design, and 3 + 3 methods, as well as CRM for partially ordered groups. Multiple group order structures are considered, allowing for varying amounts of group frailty order information. These simulations find that parallel trials in the presence of partially ordered groups display a high frequency of trials resulting in reversals. Reversals occur when dose recommendations do not follow known order of toxicity risk by group, such as recommending a higher dose level in a group of patients known to have a higher risk of toxicity. CRM for partially ordered groups eliminates the issue of reversals, and simulation results indicate improved frequency of maximum tolerated dose selection as well as treating a greater proportion of trial patients at this dose compared with parallel trials. When information is available on differences in toxicity risk by patient subgroup, methods designed to account for known group ordering should be considered to avoid reversals in dose recommendations and improve operating characteristics.

Acid ceramidase promotes drug resistance in acute myeloid leukemia through NF-κB-dependent P-glycoprotein upregulation.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of older AML patients fail to respond to cytotoxic chemotherapy, and most responders relapse with drug-resistant disease. Failure to achieve complete remission can be partly attributed to the drug resistance advantage of AML blasts that frequently express P-glycoprotein (P-gp), an ATP-binding cassette transporter. Our previous work showed that elevated acid ceramidase (AC) levels in AML contribute to blast survival. Here, we investigated P-gp expression levels in AML relative to AC. Using parental HL-60 cells and drug-resistant derivatives as our model, we found that P-gp expression and efflux activity were highly upregulated in resistant derivatives. AC overexpression in HL-60 conferred resistance to the AML chemotherapeutic drugs, cytarabine, mitoxantrone, and daunorubicin, and was linked to P-gp upregulation. Furthermore, targeting AC through pharmacologic or genetic approaches decreased P-gp levels and increased sensitivity to chemotherapeutic drugs. Mechanistically, AC overexpression increased NF-κB activation whereas NF-kB inhibitors reduced P-gp levels, indicating that the NF-kappaB pathway contributes to AC-mediated modulation of P-gp expression. Hence, our data support an important role for AC in drug resistance as well as survival and suggest that sphingolipid targeting approaches may also impact drug resistance in AML.

Reasons for Early Cessation of Breastfeeding Among Women with Low Income.

Background: Most women in the United States do not meet their breastfeeding goals, and low-income women breastfeed at lower rates than the general population. While risk factors for early cessation have been documented, specific reasons for discontinuing among this population are less understood. We examined reasons for cessation among low-income mothers to inform the development of targeted strategies to address breastfeeding disparities. Materials and Methods: We performed a secondary data analysis using prospective data collected during a randomized intervention trial of Special Supplemental Nutrition Program for Women, Infants, and Children (WIC)-eligible women interviewed in the third trimester and at 1, 3, and 6 months postpartum. We included the 221 women who initiated breastfeeding and stopped by 6 months. Women's reasons for discontinuing breastfeeding were grouped by thematic category and compared by time of breastfeeding cessation. Results: The most common reasons reported overall for breastfeeding cessation were concerns about breast milk supply and latch difficulty. Some reasons differed significantly by time of cessation. Latch difficulty was reported most often by women who breastfed for 1 month or less; supply concerns increased with increasing breastfeeding duration. Returning to work/school was uncommonly reported for those who stopped by 1 month, but more frequently reported in those with later cessation. Conclusions: We found that low-income women reported similar reasons for early breastfeeding cessation as have been reported for other populations of women. These results underscore the need for appropriately timed, culturally sensitive interventions to reduce disparities in duration of breastfeeding, specifically to address latch difficulty in the first few weeks and supply concerns as infants grow.

The Impact of Early-Phase Trial Design in the Drug Development Process.

PURPOSE: Many of the therapeutic agents that are being used currently were developed using the 3+3 decision rule for dose finding. Over the past 30 years, several dose-finding designs have been proposed and evaluated, including the "continual reassessment method" (CRM) and the "Bayesian optimal interval design" (BOIN). This research investigates the role of the choice of an early-phase design on the likelihood that drugs entering the drug development pipeline will have 2 successful phase III trials.Experimental Design: Using simulation, each agent in a population of hypothetical agents was tracked through the drug development process, from initial dose finding to 2 confirmatory phase III trials. Varying the designs of the phase I, II, and III trials allows for an assessment of the effect of the choice of designs on the proportion of agents with successful phase III trials. RESULTS: The results indicate that using the CRM or BOIN, rather than the 3+3, substantially enhances the proportion of effective agents that have successful phase III trials, with the CRM having a greater effect than BOIN. A larger phase II trial magnifies the effect of the phase I design. CONCLUSIONS: The results underscore the importance of the choice of the early-phase designs. Use of the 3+3 results in fewer agents with successful phase III trials compared with the CRM or BOIN. The difference is more pronounced among highly effective agents. In addition, the results show the importance of a sufficiently powered phase II trial.

Vitamin D pathway activation selectively deactivates signal transducer and activator of transcription (STAT) proteins and inflammatory cytokine production in natural killer leukemic large granular lymphocytes.

Calcitriol, the active form of vitamin D, has been well documented to act directly on immune cells and malignant cells. Activated T cells are one of the best characterized targets of calcitriol, with effects including decreasing inflammatory cytokine output and promoting anti-inflammatory cytokine production. However, the effects of calcitriol on natural killer (NK) cells are less clear. Reports suggest that only immature NK cell populations are affected by calcitriol treatment resulting in impaired cytotoxic function and cytokine production, while mature NK cells may have little or no response. NK cell large granular lymphocyte leukemia (NK-LGLL) is a rare leukemia with CD3-CD16+CD56+NK cell clonal expansion. The current standard treatments are immunosuppressant therapies, which are not curative. The Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway is hyperactivated in LGLL and is one pathway of interest in new drug target investigations. We previously demonstrated the ability of calcitriol to decrease STAT1 tyrosine 701 (p-STAT1) and STAT3 tyrosine 705 (p-STAT3) phosphorylation as well as inflammatory cytokine output of T cell large granular lymphocyte leukemia cells, but did not determine the effects of calcitriol on NK-LGLL. Therefore, in the present study, we investigated whether NKL cells, a model of NK-LGLL, and NK-LGLL patient peripheral blood mononuclear cells (PBMCs) are susceptible to treatment with calcitriol or seocalcitol (EB1089), a potent analog of calcitriol. NKL cells are dependent on interleukin (IL)-2 for survival and we show here for the first time that treatment with IL-2 induced tyrosine phosphorylation of STATs 1 through 6. Both calcitriol and EB1089 caused significant upregulation of the vitamin D receptor (VDR). IL-2 induction of p-STAT1 and p-STAT3 phosphorylation was significantly decreased after calcitriol or EB1089 treatment. Additionally, IL-10, interferon (IFN)-γ, and FMS-like tyrosine kinase 3 ligand (Flt-3L) extracellular output was significantly decreased at 100 nM EB1089 and intracellular IL-10 was decreased with either calcitriol or EB1089 treatment. We treated NK-LGLL patient PBMCs with calcitriol or EB1089 and found decreased p-STAT1 and p-STAT3 while VDR increased, which matched the NKL cell line data. We then measured 75 serum cytokines in NK-LGLL patients (n = 8) vs. age- and sex-matched normal healthy donors (n = 8), which is the first serum cytokine study for this LGLL subtype. We identified 15 cytokines, including IL-10 and Flt-3L, which were significantly different between normal donors and NK-LGLL patients. Overall, our results suggest that activating the vitamin D pathway could be a mechanism to decrease STAT1 and 3 activation and inflammatory cytokine output in NK-LGLL patients.