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Background and Objectives: Passive immunization by the infusion of convalescent plasma (CP) obtained from patients who have recently recovered from COVID-19, thus having antibodies to severe acute respiratory syndrome coronavirus 2, is a potential strategy to reduce the severity of illness. A high prevalence of antiphospholipid antibodies (APLA) in patients with COVID-19 has been reported during the pandemic, raising a concern whether the use of CP could increase the risk of thrombosis in transfused patients. We aimed to evaluate the prevalence of APLA in COVID-19 CP (CCP) in order to assess the potential prothrombotic influence of transfused CCP to COVID-19 patients., Materials and Methods: We studied the prevalence of APLA in 122 CCP samples collected from healthy donors who recovered from mild-COVID-19 at two time periods: September 2020-January 2021 (defined as 'early period' samples) and April-May 2021 (defined as 'late period' samples). Thirty-four healthy subjects unexposed to COVID-19 were used as controls., Results: APLA were present in 7 of 122 (6%) CCP samples. One donor had anti-β2-glycoprotein 1(anti-β2GP1) IgG, one had anti-β2GP1 IgM and five had lupus anticoagulant (LAC) using silica clotting time (SCT), all in 'late period' donors. In the control group, one subject had anti-β2GP1 IgG, two had LAC using dilute Russell viper venom time (dRVVT) and four had LAC SCT (both LAC SCT and LAC dRVVT in one subject)., Conclusion: The low prevalence of APLA in CCP donors reassures the safety of CCP administration to patients with severe COVID-19., (© 2023 International Society of Blood Transfusion.)

Background : Platelet distribution width (PDW) has demonstrated clinical significance in populations with specific disorders; its prognostic significance in internal medicine wards has not been investigated. Methods : Demographic, clinical and laboratory data were collected prospectively for 1036 internal medicine inpatients. The primary outcome was 90-day mortality, secondary outcomes were: treatment with mechanical ventilation, prolonged hospital stay, in-hospital death, and all-cause mortality following discharge. Data were assessed according to PDW values on admission ≤16.7% (group A) and >16.7% (group B). Results : Compared to group A patients (n = 273), group B patients (n = 763) were more likely to be older, admitted for cardio-cerebrovascular disorder, to present with comorbidities, to be mechanically ventilated, to have prolonged hospital stay and to die during the current hospitalization. The respective 90-day and total (median follow-up of 5 months) mortality rates were significantly higher in group B (13.2% and 16.3%) than in group A (6.6% and 9.5%), P < 0.01. On multivariate analysis, higher PDW values on admission predicted 90-day mortality and shortened survival (relative risks 1.58 and 1.26; 95% confidence intervals 0.89 - 2.78 and 0.97-1.64, respectively). Conclusion : Higher PDW values on admission to internal medicine wards are associated with a more severe clinical profile and increased risk of 90-day mortality., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of Greater Baltimore Medical Center.)

Introduction: In patients treated with direct oral anti activated factor X (anti-FXa) anticoagulants such as apixaban and rivaroxaban, there are several emergency and non-emergency conditions in which anticoagulation activity should be measured. The validity of the common global clotting tests, prothrombin time and international normalized ratio (PT/INR) for determination of blood levels of these drugs, has been widely investigated. As the anticoagulation activity evaluation "calibrated anti-FXa" of these drugs is relatively more expensive and less available, we aimed to build a prediction model for anticoagulation activity assessment based on INR values. Methods and Findings: One hundred sixty samples from 80 hospitalized patients treated with apixaban or rivaroxaban were tested using PT/INR and Anti-FXa chromogenic assay. Two blood samples, trough and peak, were collected from each subject. Participants were randomly divided into two equal groups. One group (n = 40) was used to build the model, which was validated by the second group (n = 40). There was a strong correlation between anti-FXa concentrations and INR in rivaroxaban treated patients (r = 0.899, p < 0.001). Therefore, we were able to build a formula for rivaroxaban patient group which reliably represent the relationship between these two parameters. The correlation in apixaban treated patients was less predictive (r = 0.798, p < 0.001) and the formula suggested could not be validated. Conclusions: In our study, we developed a formula that estimates the anticoagulant activity of rivaroxaban by obtaining INR values. Where anti-FXa assay is unavailable, our proposed formula may be considered as a screening test for rivaroxaban., (Copyright © 2019 Ofek, Barchel, Perets, Ziv-Baran, Mahajna, Filipovich-Rimon, Garach-Jehoshua, Berlin and Berkovitch.)

Patients with multiple myeloma (MM) have an increased thrombotic risk, but pathogenesis remains uncertain. Low levels of Protein Z (PZ), a vitamin K-dependent plasma protein, are associated with venous as well as arterial thrombosis. The purpose of this study was to analyze PZ levels in patients with plasma cell neoplasms., Patients and Methods: The study consisted of 64 plasma cells neoplasm patients and 42 healthy individuals. Clinical investigations included measurement of plasma PZ and IL-6 levels., Results: PZ levels in patients with plasma cell neoplasms were significantly lower compared to healthy controls in the entire cohort (1392±659 vs.2010±603ng/mL, P<0.01), as well as in specific disease subgroups; symptomatic MM (1428±652ng/mL, p<0.01), smoldering MM (1437±883ng/mL, p=0.045) and monoclonal gammopathy of undetermined significance (MGUS) (1247±593ng/mL, p=0.01). PZ was negatively correlated with IL-6 levels in MM patients (r=-0.7, P<0.01). There was no significant difference in PZ levels between patients with or without thrombotic event., Conclusion: Plasma cell neoplasm patients have low levels of PZ. This is presumably related to the increased IL-6 production by the bone marrow microenvironment, and could have a potential role in the increased thrombotic tendency in those patients., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In recording the changes acquired in gene expression profile during culture of fresh bone marrow samples from patients with multiple myeloma or acute myeloid leukemia, the most remarkable finding in both instances was widespread downregulation of mitotic and transcriptional genes (e.g. MKI67, CCNB1, ASPM, SGOL1, DLGAP5, CENPF, BUB1, KIF23, KIF18a, KIF11, KIF14, KIF4, NUF2, KIF1, AE2FB, TOP2A, NCAPG, TTK, CDC20, and AURKB), which could account for the ensuing proliferation arrest. Many of these genes were also underexpressed in leukemic cells from the blood or myeloma cells from an extramedullary site compared with their expression in the aspirates. Taken together, our results exhibited mitotic and transcriptional gene subsets where their expression appears to be coordinated and niche dependent. In addition, the genes induced during culture specified a variety of angiogenic factors (e.g. interleukin-8 and CXCL-5) and extracellular matrix proteins (e.g. osteopontin and fibronectin) probably released by the tumor cells while generating their favored microenvironment.

In a comparison of gene expression profile in unsorted bone marrow (BM) samples from patients with multiple myeloma (MM), acute leukemia, and diffuse large B-cell lymphoma infiltrating the BM, the leading myeloma distinguishing gene was GPRC5D. This gene was highly expressed in BM samples from the 10 MM cases examined as opposed to minimal expression in samples from the eight cases with other hematological malignancies. Moreover, following antimyeloma treatment the expression of GPRC5D decreased several folds. The strong and selective expression of GPRC5D in MM cells makes this gene and its encoded surface protein as promising markers for monitoring the tumor load and hopefully also as targets for antimyeloma antibodies.

Bone marrow (BM) cells depend on their niche for growth and survival. However, the genes modulated by niche stimuli have not been discriminated yet. For this purpose, we investigated BM aspirations from patients with various hematological malignancies. Each aspirate was fractionated, and the various samples were fixed at different time points and analyzed by microarray. Identification of niche-modulated genes relied on sustained change in expression following loss of niche regulation. Compared with the reference ('authentic') samples, which were fixed immediately following aspiration, the BM samples fixed after longer stay out-of-niche acquired numerous changes in gene-expression profile (GEP). The overall genes modulated included a common subset of functionally diverse genes displaying prompt and sustained 'switch' in expression irrespective of the tumor type. Interestingly, the 'switch' in GEP was reversible and turned 'off-and-on' again in culture conditions, resuming cell-cell-matrix contact versus respread into suspension, respectively. Moreover, the resuming of contact prolonged the survival of tumor cells out-of-niche, and the regression of the 'contactless switch' was followed by induction of a new set of genes, this time mainly encoding extracellular proteins including angiogenic factors and extracellular matrix proteins. Our data set, being unique in authentic expression design, uncovered niche-modulated and niche-modulating genes capable of controlling homing, expansion and angiogenesis.

Lupus anticoagulants (LAC) are antibodies which are detected by a prolongation of phospholipid-dependent coagulation assays, and are associated with thrombotic events and pregnancy complications in patients with the antiphospholipid syndrome. The antiphospholipid syndrome is defined by arterial or venous thrombosis and/or pregnancy morbidity and by laboratory diagnosis of antiphospholipid antibodies. The laboratory diagnosis is based on LAC and/or anticardiolipin and/or anti-beta2-glycoprotein I antibodies present in plasma, on two or more occasions at least 12 weeks apart. ALthough the presence of LAC correlates best with thrombosis, the Laboratory testing of LAC is not well standardized. In this article, the Laboratory evaluation of LAC will be explained, including the different tests that are recommended by the Israeli Sub-committee of Thrombosis and Hemostasis Laboratories, the possibility to evaluate LAC in patients treated with antithrombotic therapy, and how to report and interpret the results.

Objectives: Central retinal vein occlusion (CRVO) and central retinal artery occlusion (CRAO) are common disorders associated with risk factors for atherosclerosis. Protein Z is a cofactor for the inactivation of activated factor X (Xa) by the protein Z dependent protease inhibitor. Protein Z deficiency was recently linked to increased risk of arterial thrombosis. We investigated whether CRVO and CRAO are associated with low protein Z levels., Patients and Methods: Patients with CRVO, CRAO or recurrent branch retinal vein occlusion were recruited to the study. Protein Z level, lupus anticoagulant (LAC), anticardiolipin antibodies (ACA) and activated protein C resistance (APCR) were determined in plasma from patients (n = 36) and healthy controls (n = 42)., Results: Thirty patients in the study group had traditional risk factors for retinal vessel occlusion and six patients had none. There was no significant difference in protein Z levels between the whole study group patients and controls (1995 +/- 810 vs. 2010 +/- 603 ng/mL, P = 0.922). However, patients with no risk factors for retinal vessel occlusion had significantly lower protein Z levels than controls (1379 +/- 682 vs. 2010 +/- 603 ng/mL, P = 0.022). Positive LAC was found in six patients and one control subject (P = 0.04). There were three patients and one control subject with abnormal APCR (P = 0.3) and none with positive ACA. Low protein Z level (lower than fifth percentile of control) was not associated with the presence of LAC or APCR., Conclusion: Low protein Z level may be another risk factor for retinal vessel occlusion in patients without traditional risk factors for these disorders.

Topical treatment of normal skin with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or its synthetic analogs results in enhanced keratinocyte proliferation. Autocrine growth factors belonging to the epidermal growth factor (EGF) family play a major role in controlling keratinocyte proliferation. 1,25-(OH)2D3 enhanced the autonomous proliferation of HaCaT human keratinocytes in the absence of exogenous growth factors. Autonomous and 1,25-(OH)2D3-stimulated proliferations were inhibited by a specific inhibitor of EGF receptor (EGFR) tyrosine kinase, an EGFR-neutralizing antibody, heparin, the heparin antagonist hexadimethrine, and the proteoglycan sulfation inhibitor chlorate. These results indicate the involvement of proteoglycan-dependent EGFR ligands. The initial events in EGFR (i.e. ErbB1) mitogenic signal transduction are dimer formation with another ErbB protein and tyrosine cross-phosphorylation. By immunoprecipitation followed by Western blotting we showed that ErbB1/ErbB3 heterodimers are the major mitogenic signaling entity in 1,25-(OH)2D3-stimulated cells. 1,25-(OH)2D3 did not affect the levels of the proteoglycan-dependent EGFR ligands amphiregulin and heparin-binding EGF nor the synthesis of proteoglycans, as assessed by 35S labeling and ion exchange chromatography. 1,25-(OH)2D3 caused a marked increase in the cellular contents of ErbB1, ErbB2, and ErbB3 proteins. The increase in ErbB proteins that mediates signal transduction by EGFR ligands can account for the stimulatory effect of 1,25-(OH)2D3 on autonomous keratinocyte proliferation.

Calpain is a ubiquitous neutral calcium-activated thiol protease that is implicated in various cellular functions including exocytosis, cell fusion, apoptosis and proliferation. The calpain system is composed of the enzymes mu-calpain and m-calpain and their endogenous inhibitor, calpastatin. We employed the spontaneously immortalized human HaCaT keratinocytes, which retain their ability to differentiate in vitro and in vivo, to study the modulation of the calpain system during keratinocyte differentiation. The cellular levels of keratinocyte differentiation markers and of the components of the calpain system were monitored by immunoblotting. Three established differentiation stimuli: increase in cell density as a function of time in culture, elevation of extracellular calcium concentration and exposure to 1,25-dihydroxyvitamin D3 enhanced the expression of the three keratinocyte differentiation markers keratin 10, involucrin and transglutaminase. The differentiation of HaCaT cells was accompanied by elevation of the components of the calpain system, although the pattern of increase varied according to the specific differentiation stimulus. A higher increase in calpains as compared with the increase in calpastatin suggests an increase in net calpain activity during differentiation. Such an increase may play a part in the differentiation process itself and/or in the regulation of key events in differentiating keratinocyte metabolism.

mu-Calpain is a calcium-dependent neutral thiol protease activated by micromolar concentrations of calcium. mu-Calpain is implicated in various cellular functions regulated by calcium including exocytosis, cell fusion, apoptosis and control of cell proliferation. We studied the effect of 1,25-(OH)2D3 on mu-calpain levels in the human renal cell carcinoma line SK-RC-29 using monoclonal antibodies to the 80 kDa subunit of mu-calpain. Exposure of low density cultures (15000 cells/cm2) to 1,25-(OH)2D3 (100nM) for 48 hours resulted in 1.5-3 fold increase of mu-calpain cell content. The effect was not observed in higher density cultures (40000 cells/cm2). mu-Calpain content of high density cultures was higher than that of low density cultures and similar to that in low density cultures treated by 1,25-(OH)2D3. The cellular content of two other calcium binding proteins, annexin II and annexin VI was not affected by the hormone. 1,25-(OH)2D3 did not affect cell number or viability therefore its effect on mu-calpain is not secondary to changes in cell density. The effect of 1,25-(OH)2D3 was dose-dependent apparent already at 1nM and was not observed with 24,25-(OH)2D3. Increase in mu-calpain content may underlie some of the actions of 1,25-(OH)2D3 on classical and non classical target cells.

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the cytotoxic action of TNF on MCF-7 human breast cancer cells and on adult bovine aortic endothelial cells. 1,25(OH)2D3 increased the effect of TNF on MCF-7 cells but not on endothelial cells over a wide TNF concentration range. At a suboptimal concentration (1 ng/ml) the potentiation was twofold. The effect of 1,25(OH)2D3 was specific, dose-dependent and apparent at a physiological concentration (0.1 nM) of the hormone. The potentiating effect of 1,25(OH)2D3 on TNF action was abolished by cycloheximide indicating that their interaction requires protein synthesis. Addition of 1,25(OH)2D3 13 h after TNF in a 28-h assay was sufficient to induce its full potentiating effect indicating that the hormone modulates a late event in the cytokine's action. These data suggest that some of the in vivo antitumor effects of 1,25(OH)2D3 may be due to an increase in the anticancer activity of the immune system.