The cytoplasmic protein-tyrosine kinase BTK plays an essential role for differentiation and survival of B-lineage cells and, hence, represents a suitable drug target. The number of BTK inhibitors (BTKis) in the clinic has increased considerably and currently amounts to at least 22. First-in-class was ibrutinib, an irreversible binder forming a covalent bond to a cysteine in the catalytic region of the kinase, for which we have identified 228 active trials listed at ClinicalTrials.gov. Next-generation inhibitors, acalabrutinib and zanubrutinib, are approved both in the United States and in Europe, and zanubrutinib also in China, while tirabrutinib is currently only registered in Japan. In most cases, these compounds have been used for the treatment of B-lymphocyte tumors. However, an increasing number of trials instead addresses autoimmunity and inflammation in multiple sclerosis, rheumatoid arthritis, pemphigus and systemic lupus erythematosus with the use of either irreversibly binding inhibitors, e.g., evobrutinib and tolebrutinib, or reversibly binding inhibitors, like fenebrutinib. Adverse effects (AEs) have predominantly implicated inhibition of other kinases with a BTKi-binding cysteine in their catalytic domain. Analysis of the reported AEs suggests that ibrutinib-associated atrial fibrillation is caused by binding to ERBB2/HER2 and ERBB4/HER4. However, the binding pattern of BTKis to various additional kinases does not correlate with the common assumption that skin manifestations and diarrhoeas are off-target effects related to EGF receptor inhibition. Moreover, dermatological toxicities, diarrhoea, bleedings and invasive fungal infections often develop early after BTKi treatment initiation and subsequently subside. Conversely, cardiovascular AEs, like hypertension and various forms of heart disease, often persist.

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the “gatekeeper” residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application.

Pharmacological inhibitors of Bruton tyrosine kinase (BTK) have revolutionized treatment of B-lymphocyte malignancies and show great promise for dampening autoimmunity. The predominant BTK inhibitors tether irreversibly by covalently binding to cysteine 481 in the BTK catalytic domain. Substitution of cysteine 481 for serine (C481S) is the most common mechanism for acquired drug resistance. We generated a novel C481S knock-in mouse model and, using a battery of tests, no overt B-lymphocyte phenotype was found. B lymphocytes from C481S animals were resistant to irreversible, but sensitive to reversible, BTK inhibitors. In contrast, irreversible inhibitors equally impaired T-lymphocyte activation in mice, mimicking the effect of treatment in patients. This demonstrates that T-lymphocyte blockage is independent of BTK. We suggest that the C481S knock-in mouse can serve as a useful tool for the study of BTK-independent effects of irreversible inhibitors, allowing for the identification of novel therapeutic targets and pinpointing potential side effects.

Ibrutinib is a covalently binding inhibitor of the B-cell receptor signaling-mediator Bruton's tyrosine kinase (BTK) with great efficacy in chronic lymphocytic leukemia (CLL). Common side effects like atrial fibrillation (AF), bleeding and infections might be caused by ibrutinib's inhibition of other kinases in non-B cells. Five-year follow-up of plasma biomarkers by proximity extension assay and immune cell numbers by flow cytometry during ibrutinib treatment revealed that 86 of the 265 investigated plasma biomarkers significantly changed during treatment, 74 of which decreased. Among the 12 markers that increased, 6 are associated with cardiovascular diseases and therefore potentially involved in ibrutinib-induced AF. Comparison between healthy donors and X-linked agammaglobulinemia (XLA) patients, who have nonfunctional BTK and essentially lack B cells, showed indicative changes in 53 of the 265 biomarkers while none differed significantly. Hence, neither B cells nor BTK-dependent pathways in other cells seem to influence the levels of the studied plasma biomarkers in healthy donors. Regarding immune cells, the absolute number of T cells, including subsets, decreased, paralleling the decreasing tumor burden. T helper 1 (Th1) cell numbers dropped strongly, while Th2 cells remained relatively stable, causing Th2-skewing. Thus, long-term ibrutinib treatment has a profound impact on the plasma proteome and immune cells in patients with CLL.

SUMMARY:In spite of its importance as an experimental model, information on the renal artery in pigs is scarce. The objective of this work was to determine the morphological characteristics of the renal artery (RA) and its branches in pigs. One hundred and twenty one (121) pairs of kidneys of pigs destined to slaughter by stunning were studied. The RA and its branches were perfused with polyester resin (Palatal 85 % t Styrene 15 %) and subjected to KOH infusion (potassium hydroxide) for total corrosion. Calibers were measured, and trajectories and relations with adjacent arterial structures were evaluated. Continuous variables were assessed with Student's t test, and discrete variables with Pearson's chi square test. The significance level used was p<0.05. The origin of the RA was symmetric in 57 specimens (50.4 %), with the right renal artery being more cranial in 55 % of the cases. Single renal arteries were seen in 98.4 %, without significant side differences (p=0.31). The caliber and length of the RA were 5.11 mm (SD 1.35) and 30.1 mm (SD 8.63), respectively. A pattern of RA expression that causes one cranial and one caudal polar branch (Type I) was observed in 97.1 % of the cases. The distribution of the RA in cranial and caudal polar branches was found to be higher than what is described in the literature, since these morphometric features have not been reported in prior studies. These findings contribute to a better knowledge in the field of the compared anatomy of the kidney, and allow for applying the pig model in procedural and hemodynamic applications.

Purpose The aim of this study was to characterize clinical effects and biomarkers in three patients with chronic mucocutaneous candidiasis (CMC) caused by gain-of-function (GOF) mutations in the STAT1 gene during treatment with Janus kinase (JAK) inhibitors. Methods Mass cytometry (CyTOF) was used to characterize mononuclear leukocyte populations and Olink assay to quantify 265 plasma proteins. Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) was used to quantify the reactivity against Candida albicans. Results Overall, JAK inhibitors improved clinical symptoms of CMC, but caused side effects in two patients. Absolute numbers of neutrophils, T cells, B cells, and NK cells were sustained during baricitinib treatment. Detailed analysis of cellular subsets, using CyTOF, revealed increased expression of CD45, CD52, and CD99 in NK cells, reflecting a more functional phenotype. Conversely, monocytes and eosinophils downregulated CD16, consistent with reduced inflammation. Moreover, T and B cells showed increased expression of activation markers during treatment. In one patient with a remarkable clinical effect of baricitinib treatment, the immune response to C. albicans increased after 7 weeks of treatment. Alterations in plasma biomarkers involved downregulation of cellular markers CXCL10, annexin A1, granzyme B, granzyme H, and oncostatin M, whereas FGF21 was the only upregulated marker after 7 weeks. After 3 months, IFN-ɣ and CXCL10 were downregulated. Conclusions The clinical effect of JAK inhibitor treatment of CMC is promising. Several biological variables were altered during baricitinib treatment demonstrating that lymphocytes, NK cells, monocytes, and eosinophils were affected. In parallel, cellular reactivity against C. albicans was enhanced.

(Folia Morphol 2017; 76, 2: 262-268)

ABSTRACT. The objective of this research was to characterise morphologically the right coronary artery and its branches in the horse. The right coronary arteries of 120 horse hearts were perfused with semi-synthetic resin (85% Palatal GP40L; 15% styrene) and mineral red dye. The morphological and biometric characteristics of the right coronary artery and its branches (digital calibrator) were assessed. The diameter of the right coronary artery was 6.72 ± 2.58 mm. The interventricular subsinusal branch ended at the apex in 94 specimens (78.4%). The right circumflex branch originated at the site of intersection of the subsinnusal interventricular sulcus and the atrioventricular septum, extended along the coronary sulcus with a convoluted trajectory, ended at the obtuse edge of the heart or even at the anterior aspect of the left ventricle in 62 hearts (52.5%), and at the middle segment of the left ventricle in 42 cases (35.6%), whereas in 14 samples (11.9%) it ended at the adjacent surface of the left ventricle. The right conus branch was found in 98 specimens (81.6%) and in 2 of them (1.7%) it emerged directly from the right aortic sinus (third coronary artery). The hearts exhibited right coronary dominance in 118 specimens (98.3%) and in 2 specimens (1.7%) the coronary dominance was balanced. No myocardial bridges were observed. The high incidence of right coronary dominance observed in this study is consistent with previous studies. Due to its similarity with the human heart, we may ratify the equine model for procedural and hemodynamic applications.Key words: horse, coronary artery, dominance, heart.RESUMEN. El objetivo de esta investigación fue caracterizar morfológicamente la arteria coronaria derecha del caballo y sus ramas. En 120 corazones de caballo se perfundieron las arterias coronarias derechas con resina semisintética (palatal GP40L al 85%; estireno al 15%) y color rojo mineral. Se evaluó las características morfológicas y biométricas de la arteria coronaria derecha y sus ramas (calibrador digital). El diámetro de la arteria coronaria derecha fue 6,72 +/-2,58 mm. La rama interventricular subsinusal finalizó en el ápex en 94 especímenes (78,4%). La rama circunfleja derecha se originó a nivel del sitio de cruce del surco interventricular subsinusal y el septum atrioventricular, se extendió a lo largo del surco coronario con una trayectoria contorneada y finalizando en el borde obtuso del corazón o incluso en la cara anterior del ventrículo izquierdo en 62 corazones (52,5%) y en el segmento medio del ventrículo izquierdo en 42 casos (35,6%), mientras que en 14 muestras (11,9%) esta finalizó en la superficie adyacente del ventrículo izquierdo. La rama derecha del cono, se encontró en 98 especímenes (81,6%) y en 2 corazones (1,7%) emergió directamente del seno aórtico derecho (tercera coronaria). Los corazones presentaron dominancia coronaria derecha en 118 muestras (98,3%) y en 2 casos (1,7%) la dominancia coronaria fue balanceada. No se observó puentes miocárdicos. La alta incidencia de dominancia coron...