Significant impact of antibiotic exposure on GI-GVHD, NRM, and GRFS following allogeneic HCT with non-myeloablative Flu-TBI conditioning.

BACKGROUND: Acute gastro-intestinal graft-versus-host disease (GI-GVHD) and non-relapse mortality (NRM) after allogeneic HCT are closely related to loss of microbial diversity and intestinal dominance by single taxa resulting from the use of antibiotics, dietary changes, and mucosal barrier injury. There is a paucity of data on the impact of use of antibiotics in HCT after Flu-TBI-based non-myeloablative (NMA) conditioning where there is absence of mucositis and limited malnutrition. METHODS: We did a retrospective single-center analysis of patients receiving Flu-TBI-based NMA HCT for a high-grade myeloid malignancy, mostly AML, and MDS, or acute lymphoblastic leukemia (ALL). We analyzed the impact of pre-engraftment antibiotic exposure, prophylactic ciprofloxacin, and or treatment with broad-spectrum cephalosporin/carbapenem, on HCT outcomes, with a focus on the incidence of acute GI-GVHD by day 180 and NRM at 1 year. RESULTS: A total of 150 patients were evaluable with a median age of 62 years. Antibiotics were used in 90 patients; 60 prophylactic use only and 30 therapeutic use with or without previous prophylaxis. Antibiotic use resulted in a significant higher incidence of GI-GVHD Stage 1-4; 29% (26/90) versus 5% (3/60) in those not receiving antibiotics (OR 8.1 (95% CI 2.3-28.3), p = 0.001). Use of antibiotics resulted in higher 1-year NRM (19% (17/90) versus 10% (6/60), HR 2.3, p = 0.06), and decreased 2-year GRFS (42% (38/90) versus 55% (33/60), HR 1.7, p = 0.04), but did not impact RFS or OS. CONCLUSIONS: Use of antibiotics was related to the occurrence of GI-GVHD, NRM, and GRFS in patients receiving truly NMA HCT. Therefore, in the absence of mucositis and low incidence of bacteremia, antibiotics can and should be used restrictively in this setting.

SARS-CoV-2 whole-genome sequencing using reverse complement PCR: For easy, fast and accurate outbreak and variant analysis.

During the course of the SARS-CoV-2 pandemic reports of mutations with effects on spreading and vaccine effectiveness emerged. Large scale mutation analysis using rapid SARS-CoV-2 Whole Genome Sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring transmission and rising levels of mutant strains. Here we report a novel WGS technique for SARS-CoV-2, the EasySeq™ RC-PCR SARS-CoV-2 WGS kit. By applying a reverse complement polymerase chain reaction (RC-PCR), an Illumina library preparation is obtained in a single PCR, thereby saving time, resources and facilitating high-throughput screening. Using this WGS technique, we evaluated SARS-CoV-2 diversity and possible transmission within a group of 173 patients and healthcare workers (HCW) of the Radboud university medical center during 2020. Due to the emergence of variants of concern, we screened SARS-CoV-2 positive samples in 2021 for identification of mutations and lineages. With use of EasySeq™ RC-PCR SARS-CoV-2 WGS kit we were able to obtain reliable results to confirm outbreak clusters and additionally identify new previously unassociated links in a considerably easier workaround compared to current methods. Furthermore, various SARS-CoV-2 variants of interest were detected among samples and validated against an Oxford Nanopore sequencing amplicon strategy which illustrates this technique is suitable for surveillance and monitoring current circulating variants.

Anakinra: efficacy in the management of fever during neutropenia and mucositis in autologous stem cell transplantation (AFFECT-2)-study protocol for a multicenter randomized double-blind placebo-controlled trial.

BACKGROUND: Since decades, fever and infections have been the most important complications of intensive chemotherapy and hematopoietic stem cell transplantation (HSCT) in the treatment of hematologic malignancies. Neutropenia has long been considered to be the most important risk factor for these complications. However, recent studies have shown that not neutropenia, but the development of mucositis is the most important cause of these complications. Currently, limited options for the prevention and treatment of mucositis are available, of which most are only supportive. The pro-inflammatory cytokine interleukin-1 (IL-1) plays a crucial role in the pathogenesis of mucositis. Pre-clinical studies of chemotherapy-induced mucositis have shown that recombinant human IL-1 receptor antagonist anakinra significantly ameliorated intestinal mucositis. In our pilot study AFFECT-1, we examined the safety and maximal tolerated dose of anakinra in patients with multiple myeloma, treated with high-dose melphalan (HDM) and autologous HSCT, selecting a dose of 300 mg daily for the phase IIb trial. The aim of the AFFECT-2 study is to determine the efficacy of anakinra in preventing fever during neutropenia (FN) and mucositis in this study population. METHODS/DESIGN: A multicenter, randomized, placebo-controlled, double-blind phase IIb trial will be conducted. Ninety patients with multiple myeloma scheduled for treatment with HDM and autologous HSCT will be included. Patients will be randomized between intravenous treatment with anakinra (300 mg) or placebo. Each group will be treated from day - 2 (day of HDM; day 0 is HSCT) up until day + 12. Outcome measures will be assessed at baseline, during admission, at discharge or day + 30, at day + 90, and + 1 year. The primary outcome will be reduction of FN. Secondary outcome measures include mucositis scores, bloodstream infections, citrulline levels, quality of life, and fatigue severity. DISCUSSION: The AFFECT-2 trial will examine the efficacy of anakinra in the management of fever during neutropenia and mucositis in patients with multiple myeloma treated with HDM and autologous HSCT. The results of this study may provide a new treatment option for these important complications. Also, this study will give us more insight in the pathophysiology of mucositis, including the role of IL-1 and the role of the microbiota in mucositis. TRIAL REGISTRATION: Clinicaltrials.gov NCT04099901 . Registered on September 23, 2019. EudraCT: 2018-005046-10.

Cyclosporine A trough concentrations are associated with acute GvHD after non-myeloablative allogeneic hematopoietic cell transplantation.

Low plasma CsA concentrations (<300-350 ng/mL) early following allogeneic hematopoietic stem cell transplantation (HSCT) is associated with an increased risk of developing acute graft-versus-host disease (aGvHD). Nevertheless, the current optimal target trough concentration for CsA following HSCT is considered to be 200-400 ng/mL. Here, we performed a retrospective analysis of a homogeneous group of 129 patients who received HSCT after non-myeloablative conditioning, and we analyzed the impact of CsA trough concentration measured during the first four weeks (CsA W1-4) on the incidence aGvHD, relapse-free survival (RFS), non-relapse mortality (NRM), overall survival (OS), and toxicity. The 180-day incidence of grade II-IV aGvHD was 25% (32/129 patients). In multivariate analysis the incidence of grade II-IV aGvHD was significantly lower among patients with a CsA W1-4 concentration ≥350 ng/mL compared to patients with a concentration <350 ng/mL (18% versus 38%, respectively; P = 0.007), with a hazard ration (HR) of 0.38 (95% CI: 0.19-0.77). In contrast, we found no correlation between CsA trough concentration and RFS, NRM, or OS. Moreover, we found an increased incidence of hypomagnesemia at higher CsA concentrations, but no difference in the incidence of acute renal toxicity, hepatic toxicity, or electrolyte imbalance. Interestingly, 30% of patients experienced hyponatremia with no apparent cause other than the use of CsA, with urinalysis suggesting SIADH as the underlying cause. Our findings suggest that a CsA trough concentration of 350-500 ng/mL might be more appropriate in the first month following non-myeloablative HSCT.

Phase I/II Trial of a Combination of Anti-CD3/CD7 Immunotoxins for Steroid-Refractory Acute Graft-versus-Host Disease.

Effective therapies for treating patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD), particularly strategies that reduce the duration of immunosuppression following remission, are urgently needed. The investigated immunotoxin combination consists of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT), which induces in vivo depletion of T cells and natural killer (NK) cells and suppresses T cell receptor activation. We conducted a phase I/II trial to examine the safety and efficacy of CD3/CD7-IT in 20 patients with SR-aGVHD; 17 of these patients (85%) had severe SR-aGVHD, and all 20 patients had visceral organ involvement, including 18 (90%) with gastrointestinal (GI) involvement and 5 (25%) with liver involvement. A validated 2-biomarker algorithm classified the majority of patients (11 of 20) as high risk. On day 28 after the start of CD3/CD7-IT therapy, the overall response rate was 60% (12 of 20), with 10 patients (50%) achieving a complete response. The 6-month overall survival rate was 60% (12 of 20), including 64% (7 of 11) classified as high risk by biomarkers. The 1-week course of treatment with CD3/CD7-IT caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse TCR Vß repertoire, and preservation of Epstein-Barr virus- and cytomegalovirus-specific T cell clones. Furthermore, our results indicate that CD3/CD7-IT appeared to be safe and well tolerated, with a relatively low prevalence of manageable and reversible adverse events, primarily worsening of hypoalbuminemia, microangiopathy, and thrombocytopenia. These encouraging results suggest that CD3/CD7-IT may improve patient outcomes in patients with SR-aGVHD.

High-Dose, Diazoxide-Mediated Insulin Suppression Boosts Weight Loss Induced by Lifestyle Intervention.

Context: Obesity-related hyperinsulinism may impede lifestyle-initiated weight loss. Objective: Proof-of-concept study to investigate the amplifying effects of diazoxide (DZX)-mediated insulin suppression on lifestyle-induced weight loss in nondiabetic, hyperinsulinemic, obese men. Design: Twelve-month study comprising an initial 6-month, double-blind trial, followed by a partially de-blinded 6-month extension in men with obesity with a body mass index of 30 to 37.5 kg/m2 and a fasting serum C-peptide level >1.00 nM. Patients were randomized into three treatment groups: DZX + placebo (DZX + PL), DZX + metformin (DZX + MTF), and double PL (PL + PL). Results: At 6 months, DZX treatment was associated with a 6.1-kg PL-subtracted decline in fat mass (FM), and at 12 months, FM had decreased by a total of 15.7 ± 2.5 kg. Twelve months of DZX treatment was also associated with a significant decline in systolic (-6.6%) and diastolic (-8.6%) blood pressure and low-density lipoprotein-cholesterol (-18%) and triglycerides (-43%) and a 39% rise in high-density lipoprotein-cholesterol. These effects were achieved at the cost of a small rise in fasting glucose (95% CI: 0.2 to 1.0 mM) and hemoglobin A1c (95% CI: -0.08% to 0.44%). There were no differences between DZX monotherapy and the combination of DZX + MTF. Conclusion: High-dose DZX treatment of 1 year resulted in a substantial decrease in FM, blood pressure, and lipid levels at the cost of a small rise in blood glucose levels.

Effects of Diazoxide-Mediated Insulin Suppression on Glucose and Lipid Metabolism in Nondiabetic Obese Men.

Context: It has been suggested that stimulation of lipolysis by diazoxide (DZX)-mediated insulin suppression may be useful in treating obesity. However, the optimal dose to promote lipolysis without causing hyperglycemia is unknown. Objective: To assess the effects of DZX in nondiabetic obese men on lipid and glucose metabolism. Design: Double-blind, placebo (PL)-controlled, 6-month trial in men with a body mass index of 30 to 37.5 kg/m2 treated with a combination of caloric restriction, a standardized exercise program, and DZX or PL dose escalation. Results: The mean maximal tolerated dose of DZX was 422 ± 44 mg/d (range, 200 to 700 mg/d). Dose-limiting events were edema (n = 11), hyperglycemia (n = 6), and nausea (n = 2). After dose reduction to a level free of clinical side effects, DZX treatment was associated with a markedly greater decrease in fasting insulin levels than PL (-72.3 ± 3.5% vs -23.0 ± 12.6%; P < 0.001) and a significant improvement of blood pressure and plasma lipid levels. The decline in insulin levels occurred at the cost of a small increase in plasma glucose (0.6 ± 0.2 mmol/L vs -0.1 ± 0.1 mmol/L; P = 0.04) and hemoglobin A1C (0.2 ± 0.1% vs 0.0 ± 0.1%; P = 0.17). Conclusion: In nondiabetic obese men, insulin levels can be reduced up to 70% without major metabolic side effects. The marked intersubject variation in maximal tolerated dose indicates that DZX dose titration needs to be individualized.

Combination Therapy with Inolimomab and Etanercept for Severe Steroid-Refractory Acute Graft-versus-Host Disease.

Steroid-refractory acute graft-versus-host disease (aGVHD) remains an important cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). A protocol on the management of aGVHD was introduced in our center that incorporated a prospective study on combination therapy with inolimomab (anti-IL-2Rα) and etanercept (anti-tumor necrosis factor-α) for steroid-refractory aGVHD. We evaluated the efficacy and safety in 21 consecutively treated patients. The patients had developed refractory aGVHD after SCT (n = 16) or donor lymphocyte infusion (n = 5), and aGVHD was classified as severe in all patients, mostly due to gastrointestinal involvement stages 2 to 4. No drug-related side effects were observed apart from the infections expected to occur in these severely immunocompromised patients. Overall response at day 28 of second-line therapy was 48% (10/21), with 6 and 4 patients achieving a complete and partial response, respectively. Eventually, 19 patients died (90%), with early mortality (<6 months) predominantly resulting from refractory aGVHD and secondary infections and late mortality resulting from relapse of the underlying disease. With a median follow-up of 55 days, the estimated rates of 6-month and 2-year overall survival were dismal, 29% and 10%, respectively. In conclusion, the combination of inolimomab and etanercept for steroid-refractory aGVHD failed to improve the dismal prognosis of severe steroid-refractory aGVHD.

Vitamin D absorption: consequences of gastric bypass surgery.

BACKGROUND: Severe vitamin D deficiency is a common finding in morbid obesity, and the incidence increases markedly after RYGB. Normalization of vitamin D levels after RYGB is difficult to achieve because the degree of surgery-induced malabsorption is not known. OBJECTIVE: To develop a test that quantifies the changes in intestinal cholecalciferol absorption induced by Roux-en-Y gastric bypass (RYGB) surgery. METHODS: Absorption characteristics of cholecalciferol were studied in 14 morbidly obese, premenopausal women before and 4 weeks after laparoscopic RYGB. Serum cholecalciferol levels were measured at baseline and 1, 2, 3, and 14 days after a single oral dose of 50 000 IU solubilized cholecalciferol. RESULTS: Peak serum cholecalciferol levels were observed on day 1 in all patients. They were 26.6±3.7% lower after RYGB (P=0.02). Inter-individual variability was high. CONCLUSION: Peak cholecalciferol levels are reduced by about 25% after RYGB. Further analysis suggested that the timing of sampling in the current study was not optimal. This might have caused an underestimation of the true decrease in cholecalciferol absorption induced by RYGB.

Cholecalciferol loading dose guideline for vitamin D-deficient adults.

INTRODUCTION: Severe vitamin D deficiency is very common. Evidence-based guidelines for rapid correction with high-dose oral cholecalciferol are not yet available. OBJECTIVE: To develop a practical cholecalciferol loading dose regimen. MATERIALS AND METHODS: A total of 208 vitamin D-deficient subjects (serum 25-hydroxyvitamin D(3) (25-OHD(3)) level <50 nmol/l), aged 18-88 years, were treated with solubilized cholecalciferol, 50,000 IU/ml. They received either 25,000 IU every fortnight for 8 weeks (total dose 100,000 IU), 25,000 IU every week for 6 weeks (total dose 150,000 IU), or 25 000 IU every week for 8 weeks (total dose 200,000 IU). Blood samples were collected at baseline and 10 days after the final dose of cholecalciferol. Results Most patients were severely vitamin D deficient: 76% had a serum 25-OHD(3) level <30 nmol/l at baseline. Cholecalciferol in a cumulative dose of 100,000, 150,000, and 200,000 IU increased mean serum 25-OHD(3) level by 29 nmol/l (95% confidence interval (CI): 23-35 nmol/l), 43 nmol/l (95% CI: 36-50 nmol/l), and 69 nmol/l (95% CI: 64-75 nmol/l) respectively. The change in 25-OHD(3) (Delta25-OHD(3)) was related to the dose per kilogram body weight (R(2)=0.38, P<0.0001), and is described by the equation: Delta25-OHD(3)=0.025x(dose per kg body weight). CONCLUSION: The cholecalciferol loading dose required to reach the serum 25-OHD(3) target level of 75 nmol/l can be calculated as follows: dose (IU)=40x(75-serum 25-OHD(3))xbody weight.

Which physiological mechanism is responsible for the increase in blood pressure during leg crossing?

OBJECTIVE: To determine which physiological mechanism is responsible for the blood pressure increase during leg crossing at knee-level in the sitting position. METHODS: Finger blood pressure was measured with the Finometer in 102 participants (47 men) before and during leg crossing: 24 treated hypertensive patients, 50 diabetic individuals (25 with and 25 without antihypertensive medication) and 28 healthy volunteers. Mean age, 53 +/- 15 years (range 21-82 years). All participants crossed their legs at knee-level, with the upper part of the popliteal fossa on the suprapatellar bursa, in the sitting position. Differences in mean blood pressure, cardiac output, stroke volume, heart rate and total peripheral resistance were assessed with legs crossed versus legs uncrossed. RESULTS: Mean blood pressure [+3.3 +/- 5.5 mmHg; 95% confidence interval (CI) = 2.7-3.8], stroke volume (+7.6 +/- 5.4 ml; 95% CI = 6.7-8.6) and cardiac output (+0.4 +/- 0.9 l/min; 95% CI = 0.3-0.5) were significantly higher with legs crossed than in the uncrossed position, while the heart rate (-1.8 +/- 3.9 beats/min; 95% CI = -2.2 to -1.4) was significantly lower. Total peripheral resistance did not differ significantly (-0.01 +/- 0.16 AU; 95% CI = -0.03 to 0.00). The largest differences occurred in the hypertensive diabetic individuals, the smallest in the healthy volunteers. The changes were similar in men and women. There were no significant correlations in the total group between the differences of the hemodynamic variables and sex, age, body mass index or leg circumference. CONCLUSION: The study shows that higher blood pressure with legs in the crossed position is due to higher cardiac output and not to a higher total peripheral resistance.