Expansion and Activation Kinetics of Immune Cells during Early Phase of GVHD in Mouse Model Based on Chemotherapy Conditioning

In the present paper, we have investigated early pathophysiological events in graft-versus-host disease (GVHD), a major complication to hematopoietic stem cell transplantation (HSCT). BLLB/c female mice conditioned with busulfan/cyclophosphamide (Bu-Cy) were transplanted with allogeneic male C57BL/6. Control group consisted of syngeneic transplanted Balb/c mice. In allogeneic settings, significant expansion and maturation of donor dendritic cells (DCs) were observed at day +3, while donor T-cells CD8+ were increased at day +5 (230%) compared to syngeneic HSCT. Highest levels of inflammatory cytokines IL-2, IFN-gamma, and TNF-alfa at day +5 matched T-cell activation. Concomitantly naïve T-cells gain effecr-memory phenotype and migrated from spleen to peripheral lymphoid organs. Thus, in the very early phase of GHVD following Bu-Cy conditioning donor, DCs play an important role in the activation of donor T cells. Subsequently, donor naïve T-cells gain effector-memory phenotype and initiate GVHD

Increasing risk of breakthrough COVID-19 in outbreaks with high attack rates in European long-term care facilities, July to October 2021

We collected data from 10 EU/EEA countries on 240 COVID-19 outbreaks occurring from July-October 2021 in long-term care facilities with high vaccination coverage. Among 17,268 residents, 3,832 (22.2%) COVID-19 cases were reported. Median attack rate was 18.9% (country range: 2.8-52.4%), 17.4% of cases were hospitalised, 10.2% died. In fully vaccinated residents, adjusted relative risk for COVID-19 increased with outbreak attack rate. Findings highlight the importance of early outbreak detection and rapid containment through effective infection prevention and control measures.S

Busulphan-Cyclophosphamide Cause Endothelial Injury, Remodeling of Resistance Arteries and Enhanced Expression of Endothelial Nitric Oxide Synthase

Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late effects on vascular system

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure &lt; 100 mmHg (n = 1127), estimated glomerular filtration rate &lt; 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Studies on mechanisms of busulphan cytotoxicity and pharmacokinetics: With special reference to liposomal busulphan

Busulphan is an alkylating agent currently used in conditioning regimen prior to stem cell transplantation (SCT). High dose therapy with busulphan has been shown to contribute to transplantation-related toxicities, such as veno-occlusive disease (VOID) and interstitial pneumonia, Previous pharmacodynamic studies have attempted to define a target systemic exposure to busulphan, and thresholds for different side effects. Pharmacokinetic studies have shown a wide inter-patient variation in the systemic exposure to busulphan after a fixed dose. The wide variation may be due to bioavailability, age, underlying disease, drug-drug interaction and circadian rhythm. The aim of the first part of this thesis was to develop and evaluate an intravenous formulation of busulphan encapsulated in liposomes. Small, uncharged liposomes containing cholesterol suspended in 5% glucose were selected for the preparation of liposomal busulphan (LBu). The distribution of LBu was assessed with 14C labeled busulphan in a rat model, and compared with a solution of free drug. The distribution of LBu was higher to bone marrow and spleen, and lower to brain, lung and heart, compared with free drug, while the distribution to liver was similar. The myelosuppressive effect of LBu was stable and reproducible in a mouse model. Pharmacokinetics of liposomal busulphan was studied in man after either low dose or high dose of LBu. A linear relation between the systemic exposure and the dose was found. The pharmacokinetic parameters were in agreement with those previously reported for orally administered drug. It was feasible to use liposomal busulphan for high dose therapy. The advantageous distribution and good myelosuppressive effect in animals, and the more predictable pharmacokinetics in man have motivated the launching of a phase 1/11 clinical trial. The aim of the second part of the thesis was to investigate mechanisms of busulphan-induced cytotoxicity, and the role of glutathione (GSH) in this process. The myeloid P39 cell line was used in vitro as a model for induction of differentiation and/or apoptosis with the differentiating agent all-trans retinoic acid and the cytostatic agent etoposide. Both agents induced apoptosis that was mediated through caspase activation, but different pathways were involved. These pathways diverged in kinetics, preceding maturation, cleavage of BcI-2 and actin, and rescue from apoptosis by granulocyte colonystimulating factor (G-CSF). Busulphan- and etoposide-induced apoptosis shared common features, but differed in kinetics. The P39 cells were arrested in G2 phase of the cell cycle before apoptotic morphology developed. Proliferation and clonogenic capacity of the P39 cells showed an inverse linear relation to the exposure to busulphan expressed as AUC. Also in busulphan-treated human CD34+ hematopoietic progenitors, clonogenic capacity was inversely and linearly related to the exposure to busulphan expressed as AUC. Myeloid progenitors were more sensitive than erythroid progenitors (AUCs completely inhibiting colony formation were 69 ± 7.5 µg.hr/ml and 140 ± 36 µg.hr/ml for CFUGM and erythroid colonies, respectively). Neither an increase (induced by N-acetylcysteine), nor a decrease (induced by buthionine sulfoximine) of the cellular content of GSH affected busulphan-induced cytotoxicity in human CD34+ hernatopoietic progenitors in vitro, or in murine bone marrow cells in vivo. In conclusion, liposomal busulphan is a suitable formulation for high dose treatment in conditioning regimen prior to stem cell transplantation. N-acetylcysteine does not decrease busulphan-induced cytotoxicity in hernatopoietic progenitor cells, and may thus serve as a potential prophylactic agent against transplantation-related hepatotoxicity

The Role of Pharmacokinetics and Pharmacodynamics in Early Drug Development with reference to the Cyclin-dependent Kinase (Cdk) Inhibitor - Roscovitine

Pharmacokinetics, pharmacodynamics and pharmacogenetics play an important role in drug discovery and contribute to treatment success. This is an essential issue in cancer treatment due to its high toxicity. During the last decade, cyclin-dependent kinase inhibitors were recognised as a new class of compounds that was introduced for the treatment of several diseases including cancer. Cyclin-dependent kinases (Cdks) play a key role in the regulation of cell cycle progression and ribonucleic acid transcription. Deregulation of Cdks has been associated with several malignancies, neurodegenerative disorders, viral and protozoa infections, glomerulonephritis and inflammatory diseases. (R)-roscovitine is a synthetic tri-substituted purine that inhibits selectively Cdk1, 2, 5, 7 and 9. Roscovitine has shown promising cytotoxicity in cell lines and tumor xenografts. In this paper, we present several aspects of pharmacokinetics (PK) and pharmacodynamics (PD) of roscovitine. We present also some of our investigations including bioanalysis, haematotoxicity, age dependent kinetics, PK and effects on Cdks in the brain. Unfavourable kinetic parameters in combination with poor distribution to the bone marrow compartment could explain the absence of myelosuppression in vivo despite the efficacy in vitro. Higher plasma and brain exposure and longer elimination half-life found in rat pups compared to adult rats may indicate that roscovitine can be a potential candidate for the treatment of brain tumours in children. Cdk5 inhibition and Erk1/2 activation that was detected in brain of rat pups may suggest the use of roscovitine in neurodegenerative diseases. Early pharmacokinetic/pharmacodynamic studies are important issues in drug discovery and may affect further development of promising drug candidates

Dynamics of early histopathological changes in GVHD after busulphan/cyclophosphamide conditioning regimen

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for otherwise incurable diseases. Conditioning regimen is an important part of HSCT and consists of chemotherapy with or without irradiation. Conditioning exerts myelosuppressive, immunosuppressive and antitumor effects, but also contributes to HSCT-related complications including graft-versus-host disease (GVHD). Since almost 50% of the transplanted patients are conditioned with cytostatics without irradiation, we developed and characterized a GVHD mouse model following conditioning with busulphan and cyclophosphamide. Recipient Balb/c female mice were treated with busulphan (20 mg/kg/day for 4 days) and cyclophosphamide (100 mg/kg/day for two days). After one day of rest, recipient mice were transplanted with 2×10(7) bone marrow and 3×10(7) spleen cells from male C57BL/6 (allogeneic group) or female Balb/c (syngeneic/control group) mice. The allogeneic, but not syngeneic transplanted mice developed GVHD. Histopathology of the major internal organs (liver, pancreas, spleen, lungs, heart and kidney) was examined before conditioning start, after conditioning's end and 5, 7 and 21 days after transplantation using hematoxylin-eosin staining. Decreased spleen cellularity and diminished glycogen content in the liver were observed after conditioning regimen. Histopathological changes such as vasculitis, inflammation and apoptotic cell forms in liver, spleen, pancreas, lungs and heart were observed in allogeneic transplanted mice, however, only hypocellular spleen and extramedullar hematopoiesis were detected in syngeneic transplanted animals. No morphological changes were observed in kidney in either HSCT setting. This is the first study describing early histopathological changes after conditioning regimen with busulphan/cyclophosphamide and dynamics of GVHD development in several major internal organs