Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen.

The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes.

Characterization of a recombinant factor IX molecule fused to coagulation factor XIII-B subunit.

INTRODUCTION AND AIM: Severe haemophilia B (HB) is characterized by spontaneous bleeding episodes, mostly into joints. Recurrent bleeds lead to progressive joint destruction called haemophilic arthropathy. The current concept of prophylaxis aims at maintaining the FIX level >3-5 IU/dL, which is effective at reducing the incidence of haemophilic arthropathy. Extended half-life FIX molecules make it easier to achieve these target trough levels compared to standard FIX concentrates. We previously reported that the fusion of a recombinant FIX (rFIX) to factor XIII-B (FXIIIB) subunit prolonged the half-life of the rFIX-LXa-FXIIIB fusion molecule in mice and rats 3.9- and 2.2-fold, respectively, compared with rFIX-WT. However, the mechanism behind the extended half-life was not known. MATERIALS AND METHODS: Mass spectrometry and ITC were used to study interactions of rFIX-LXa-FXIIIB with albumin. Pharmacokinetic analyses in fibrinogen-KO and FcRn-KO mice were performed to evaluate the effect of albumin and fibrinogen on in-vivo half-life of rFIX-LXa-FXIIIB. Finally saphenous vein bleeding model was used to assess in-vivo haemostatic activity of rFIX-LXa-FXIIIB. RESULTS AND CONCLUSION: We report here the key interactions that rFIX-LXa-FXIIIB may have in plasma are with fibrinogen and albumin which may mediate its prolonged half-life. In addition, using the saphenous vein bleeding model, we demonstrate that rFIX-FXIIIB elicits functional clot formation that is indistinguishable from that of rFIX-WT.

GGCX variants leading to biallelic deficiency to γ-carboxylate GRP cause skin laxity in VKCFD1 patients.

γ-Glutamyl carboxylase (GGCX) catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-hemorrhagic phenotypes remain elusive. Therefore, we have analyzed 20 pathogenic GGCX variants on their ability to γ-carboxylate six non-hemostatic VKD proteins in an in vitro assay, where GGCX variants were expressed in GGCX-/- cells and levels of γ-carboxylated co-expressed VKD proteins were detected by a functional ELISA. We observed that GGCX variants causing markedly reduced γ-carboxylation of Gla rich protein (GRP) in vitro were reported in patients with skin laxity. Reduced levels of γ-carboxylated Matrix gla protein (MGP) are not exclusive for causing skeletal dysmorphologies in VKCFD1 patients. In silico docking of vitamin K hydroquinone on a GGCX model revealed a binding site, which was validated by in vitro assays. GGCX variants affecting this site result in disability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to understand the development of non-hemorrhagic phenotypes and hence improve treatment in VKCFD1 patients.

Endothelial Nitric Oxide Synthase Gene Polymorphisms Increase Risk of Deep Vein Thrombosis by Altering Homocysteine Levels.

BACKGROUND: Deep Vein Thrombosis (DVT) is a multicausal disease involving both acquired as well as genetic factors. Nitric oxide is an influential endogenous factor having its role in the development of deep vein thrombosis. It maintains the vascular integrity and any alterations in its levels may lead to a thrombotic event. It may also modulate homocysteine metabolism to cause hyperhomocysteinemia, which is a prominent risk factor for thrombosis. The objective of the study was to study if endothelial nitric oxide gene polymorphisms, 894G/T, and 2479G/A alter the plasma nitric oxide and homocysteine levels which may eventually increase the risk of deep vein thrombosis. METHODS: One hundred Doppler ultrasonography and computerized tomography confirmed (for cerebral venous thrombosis), non-related DVT patients (M:F = 58:42; age range = 18 to 61 years) served as the study population. Two hundred hospital staff and their relatives or unrelated attendants of the patients served as the controls. Nitric oxide levels were determined by measuring its metabolites (NOx), and EIA was used to measure homocysteine levels. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for detecting the eNOS polymorphisms 894G/T and 2479G/A. RESULTS: In total, DVT subjects have 25% higher plasma levels of homocysteine and 37% lower levels of NOx in their circulation when compared to controls. In tertile analysis of nitric oxide and homocysteine levels, 894G/T and 2479G/A polymorphisms were associated with plasma nitric oxide and homocysteine levels. The increased risk of deep vein thrombosis was associated with endothelial nitric oxide gene polymorphisms and nitric oxide levels, but homocysteine levels were not a risk for deep vein thrombosis. CONCLUSION: The present study demonstrates that 894G/T and 2479G/A polymorphisms interact with lower levels of nitric oxide and higher levels of homocysteine that may possess the risk of deep vein thrombosis.

Exploring Diverse Coagulation Factor XIII Subunit Expression Datasets: A Bioinformatic Analysis.

Coagulation factor XIII (FXIII) circulates in plasma as a pro-transglutaminase heterotetrameric complex (FXIIIA2B2), which upon activation by thrombin and calcium covalently crosslinks preformed fibrin polymers. The heterotetrameric complex is composed of a catalytic FXIIIA2 subunit and a protective/regulatory FXIII-B2 subunit coded by F13A1 and F13B genes, respectively. The catalytic FXIIIA2 subunit is encoded by the F13A1 gene, expressed primarily in cells of mesenchymal origin, whereas the FXIIIB subunit encoded by the F13B gene is expressed and secreted from hepatocytes. The plasma FXIIIA2 subunit, which earlier was believed to be secreted from cells of megakaryocytic lineage, is now understood to result primarily from resident macrophages. The regulation of the FXIII subunits at the genetic level is still poorly understood. The current study adopts a purely bioinformatic approach to analyze the temporal, time-specific expression array-data corresponding to both the subunits in specific cell lineages, with respect to the gene promoters. We analyze the differentially expressed genes correlated with F13A1 and F13B expression levels in an array of cell types, utilizing publicly available microarray data. We attempt to understand the regulatory mechanism underlying the variable expression of FXIIIA2 subunit in macrophages (M0, M1, M2 and aortic resident macrophages). Similarly, the FXIIIB2 subunit expression data from adult, fetal hepatocytes and embryonic stem cells derived hepatoblasts (hESC-hepatoblast) was analyzed. The results suggest regulatory dependence between the two FXIII subunits at the transcript level. Our analysis also predicts the involvement of the FXIIIA2 subunit in macrophage polarization, plaque stability, and inflammation.

von Willebrand factor propeptide missense variants affect anterograde transport to Golgi resulting in ER retention.

von Willebrand disease (VWD), the most prevalent congenital bleeding disorder, arises from a deficiency in von Willebrand factor (VWF), which has crucial roles in hemostasis. The present study investigated functional consequences and underlying pathomolecular mechanisms of several VWF propeptide (VWFpp) missense variants detected in our cohort of VWD patients for the first time. Transient expression experiments in HEK293T cells demonstrated that four out of the six investigated missense variants (p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp) severely impaired secretion. Their cotransfections with the wild-type partly corrected VWF secretion, displaying loss of large/intermediate multimers. Immunostaining of the transfected HEK293 cells illustrated the endoplasmic reticulum (ER) retention of the VWF variants. Docking of the COP I and COP II cargo recruitment proteins, ADP-ribosylation factor 1 and Sec24, onto the N-terminal VWF model (D1D2D'D3) revealed that these variants occur at VWFpp putative interfaces, which can hinder VWF loading at the ER exit quality control. Furthermore, quantitative and automated morphometric exploration of the three-dimensional immunofluorescence images showed changes in the number/size of the VWF storage organelles, Weibel-Palade body (WPB)-like vesicles. The result of this study highlighted the significance of the VWFpp variants on anterograde ER-Golgi trafficking of VWF as well as the biogenesis of WPB-like vesicles.

Machine learning improves early prediction of small-for-gestational-age births and reveals nuchal fold thickness as unexpected predictor.

OBJECTIVE: To investigate the performance of the machine learning (ML) model in predicting small-for-gestational-age (SGA) at birth, using second-trimester data. METHODS: Retrospective data of 347 patients, consisting of maternal demographics and ultrasound parameters collected between the 20th and 25th gestational weeks, were studied. ML models were applied to different combinations of the parameters to predict SGA and severe SGA at birth (defined as 10th and third centile birth weight). RESULTS: Using second-trimester measurements, ML models achieved an accuracy of 70% and 73% in predicting SGA and severe SGA whereas clinical guidelines had accuracies of 64% and 48%. Uterine PI (Ut PI) was found to be an important predictor, corroborating with existing literature, but surprisingly, so was nuchal fold thickness (NF). Logistic regression showed that Ut PI and NF were significant predictors and statistical comparisons showed that these parameters were significantly different in disease. Further, including NF was found to improve ML model performance, and vice versa. CONCLUSION: ML could potentially improve the prediction of SGA at birth from second-trimester measurements, and demonstrated reduced NF to be an important predictor. Early prediction of SGA allows closer clinical monitoring, which provides an opportunity to discover any underlying diseases associated with SGA.

A reasoned approach towards administering COVID-19 vaccines to pregnant women.

There are over 50 SARS-CoV-2 candidate vaccines undergoing Phase II and III clinical trials. Several vaccines have been approved by regulatory authorities and rolled out for use in different countries. Due to concerns of potential teratogenicity or adverse effect on maternal physiology, pregnancy has been a specific exclusion criterion for most vaccine trials with only two trials not excluding pregnant women. Thus, other than limited animal studies, gradually emerging development and reproductive toxicity data, and observational data from vaccine registries, there is a paucity of reliable information to guide recommendations for the safe vaccination of pregnant women. Pregnancy is a risk factor for severe COVID-19, especially in women with comorbidities, resulting in increased rates of preterm birth and maternal morbidity. We discuss the major SARS-CoV-2 vaccines, their mechanisms of action, efficacy, safety profile and possible benefits to the maternal-fetal dyad to create a rational approach towards maternal vaccination while anticipating and mitigating vaccine-related complications. Pregnant women with high exposure risks or co-morbidities predisposing to severe COVID-19 infection should be prioritised for vaccination. Those with risk factors for adverse effects should be counselled accordingly. It is essential to support patient autonomy by shared decision-making involving a risk-benefit discussion with the pregnant woman.

NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure-Activity Relationships.

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1-Cys37) and the flexible C-terminal part (Arg38-Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure-activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.

Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at ∼0.4 gestation. The initial cohort received 1 × 1012 vector genomes (vgs) of AAV5-hFIX ( n = 5; 0.45 × 1013 vg/kg birth weight), resulting in ∼3.0% hFIX at birth and 0.6-6.8% over 19-51 mo. The next cohort received 0.2-1 × 1013 vg boluses. AAV5-hFX animals ( n = 3; 3.57 × 1013 vg/kg) expressed <1% at birth and 9.4-27.9% up to 42 mo. AAV8-hFIX recipients ( n = 3; 2.56 × 1013 vg/kg) established 4.2-41.3% expression perinatally and 9.8-25.3% over 46 mo. Expression with AAV8-hFX ( n = 6, 3.12 × 1013 vg/kg) increased from <1% perinatally to 9.8-13.4% >35 mo. Low expressers (<1%, n = 3) were postnatally challenged with 2 × 1011 vg/kg AAV5 resulting in 2.4-13.2% expression and demonstrating acquired tolerance. Linear amplification-mediated-PCR analysis demonstrated random integration of 57-88% of AAV sequences retrieved from hepatocytes with no events occurring in or near oncogenesis-associated genes. Thus, early-IUGT in macaques produces sustained curative expression related significantly to integrated AAV in the absence of clinical toxicity, supporting its therapeutic potential for early-onset monogenic disorders.-Chan, J. K. Y., Gil-Farina I., Johana, N., Rosales, C., Tan, Y. W., Ceiler, J., Mcintosh, J., Ogden, B., Waddington, S. N., Schmidt, M., Biswas, A., Choolani, M., Nathwani, A. C., Mattar, C. N. Z. Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Care of the pregnant woman with coronavirus disease 2019 in labor and delivery: anesthesia, emergency cesarean delivery, differential diagnosis in the acutely ill parturient, care of the newborn, and protection of the healthcare personnel.

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2, has been declared a pandemic by the World Health Organization. As the pandemic evolves rapidly, there are data emerging to suggest that pregnant women diagnosed as having coronavirus disease 2019 can have severe morbidities (up to 9%). This is in contrast to earlier data that showed good maternal and neonatal outcomes. Clinical manifestations of coronavirus disease 2019 include features of acute respiratory illnesses. Typical radiologic findings consists of patchy infiltrates on chest radiograph and ground glass opacities on computed tomography scan of the chest. Patients who are pregnant may present with atypical features such as the absence of fever as well as leukocytosis. Confirmation of coronavirus disease 2019 is by reverse transcriptase-polymerized chain reaction from upper airway swabs. When the reverse transcriptase-polymerized chain reaction test result is negative in suspect cases, chest imaging should be considered. A pregnant woman with coronavirus disease 2019 is at the greatest risk when she is in labor, especially if she is acutely ill. We present an algorithm of care for the acutely ill parturient and guidelines for the protection of the healthcare team who is caring for the patient. Key decisions are made based on the presence of maternal and/or fetal compromise, adequacy of maternal oxygenation (SpO2 >93%) and stability of maternal blood pressure. Although vertical transmission is unlikely, there must be measures in place to prevent neonatal infections. Routine birth processes such as delayed cord clamping and skin-to-skin bonding between mother and newborn need to be revised. Considerations can be made to allow the use of screened donated breast milk from mothers who are free of coronavirus disease 2019. We present management strategies derived from best available evidence to provide guidance in caring for the high-risk and acutely ill parturient. These include protection of the healthcare workers caring for the coronavirus disease 2019 gravida, establishing a diagnosis in symptomatic cases, deciding between reverse transcriptase-polymerized chain reaction and chest imaging, and management of the unwell parturient.

Coronavirus disease 2019 (COVID-19) pandemic and pregnancy.

The current coronavirus disease 2019 (COVID-19) pneumonia pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading globally at an accelerated rate, with a basic reproduction number (R0) of 2-2.5, indicating that 2-3 persons will be infected from an index patient. A serious public health emergency, it is particularly deadly in vulnerable populations and communities in which healthcare providers are insufficiently prepared to manage the infection. As of March 16, 2020, there are more than 180,000 confirmed cases of COVID-19 worldwide, with more than 7000 related deaths. The SARS-CoV-2 virus has been isolated from asymptomatic individuals, and affected patients continue to be infectious 2 weeks after cessation of symptoms. The substantial morbidity and socioeconomic impact have necessitated drastic measures across all continents, including nationwide lockdowns and border closures. Pregnant women and their fetuses represent a high-risk population during infectious disease outbreaks. To date, the outcomes of 55 pregnant women infected with COVID-19 and 46 neonates have been reported in the literature, with no definite evidence of vertical transmission. Physiological and mechanical changes in pregnancy increase susceptibility to infections in general, particularly when the cardiorespiratory system is affected, and encourage rapid progression to respiratory failure in the gravida. Furthermore, the pregnancy bias toward T-helper 2 (Th2) system dominance, which protects the fetus, leaves the mother vulnerable to viral infections, which are more effectively contained by the Th1 system. These unique challenges mandate an integrated approach to pregnancies affected by SARS-CoV-2. Here we present a review of COVID-19 in pregnancy, bringing together the various factors integral to the understanding of pathophysiology and susceptibility, diagnostic challenges with real-time reverse transcription polymerase chain reaction (RT-PCR) assays, therapeutic controversies, intrauterine transmission, and maternal-fetal complications. We discuss the latest options in antiviral therapy and vaccine development, including the novel use of chloroquine in the management of COVID-19. Fetal surveillance, in view of the predisposition to growth restriction and special considerations during labor and delivery, is addressed. In addition, we focus on keeping frontline obstetric care providers safe while continuing to provide essential services. Our clinical service model is built around the principles of workplace segregation, responsible social distancing, containment of cross-infection to healthcare providers, judicious use of personal protective equipment, and telemedicine. Our aim is to share a framework that can be adopted by tertiary maternity units managing pregnant women in the flux of a pandemic while maintaining the safety of the patient and healthcare provider at its core.

Identification and characterization of a novel variant in C-terminal region of Antithrombin (Ala427Thr) associated with type II AT deficiency leading to polymer formation.

Antithrombin (AT) deficiency is a rare but strong risk factor for the thrombosis development. Mutations in gene encoding AT (SERPINC1) have provided a detailed understanding of AT deficiency and subsequent development of thrombotic complications. In the present study, we describe a case of thrombotic patient with reduced AT activity and normal AT antigen levels. AT deficiency in the patient was explained by the presence of heterozygous mutation g.13397A>G (Ala427Thr) in exon 6 of SERPINC1. Reduced APTT and TT with normal PT were observed. The mutation was found to be absent in healthy controls (n = 62). In vitro purification and characterization of variant AT showed significant decrease in fluorescence emission intensity, decreased bis-ANS fluorescence emission, changes in secondary structure and presence of polymerized AT in patient's plasma as assessed by fluorescence, circular dichroism and transmission electron microscopy respectively. Furthermore, molecular dynamics simulation studies showed altered conformation due to Ala427Thr substitution. Our study shows that genetic screening should be carried out in AT deficient patients in addition to the routinely used functional assays to understand the molecular basis of disease development.

Mechanisms and evidence of vertical transmission of infections in pregnancy including SARS-CoV-2s.

There remain unanswered questions concerning mother-to-child-transmission of SARS-CoV-2. Despite reports of neonatal COVID-19, SARS-CoV-2 has not been consistently isolated in perinatal samples, thus definitive proof of transplacental infection is still lacking. To address these questions, we assessed investigative tools used to confirm maternal-fetal infection and known protective mechanisms of the placental barrier that prevent transplacental pathogen migration. Forty studies of COVID-19 pregnancies reviewed suggest a lack of consensus on diagnostic strategy for congenital infection. Although real-time polymerase chain reaction of neonatal swabs was universally performed, a wide range of clinical samples was screened including vaginal secretions (22.5%), amniotic fluid (35%), breast milk (22.5%) and umbilical cord blood. Neonatal COVID-19 was reported in eight studies, two of which were based on the detection of SARS-CoV-2 IgM in neonatal blood. Histological examination demonstrated sparse viral particles, vascular malperfusion and inflammation in the placenta from pregnant women with COVID-19. The paucity of placental co-expression of ACE-2 and TMPRSS2, two receptors involved in cytoplasmic entry of SARS-CoV-2, may explain its relative insensitivity to transplacental infection. Viral interactions may utilise membrane receptors other than ACE-2 thus, tissue susceptibility may be broader than currently known. Further spatial-temporal studies are needed to determine the true potential for transplacental migration.

Basal cell carcinomas developing independently from BAP1-tumor predisposition syndrome in a patient with bilateral uveal melanoma: Diagnostic challenges to identify patients with BAP1-TPDS.

Basal cell carcinomas (BCC) have been recently included into the spectrum of BAP1-tumor predisposition syndrome (TPDS). Uveal melanoma (UM) is also a tumor often observed in patients with this hereditary tumor syndrome, in particular bilateral UM is highly suspicious for BAP1-TPDS although no patient has been reported yet. Based on our index patient with BAP1-TPDS with bilateral UM (choroid OD, oculus dexter; iris OS, oculus sinister), several BCCs and thyroid cancer as well as a family history for cancer, this paper analyzes hints and pitfalls to diagnose this syndrome clinically and histologically. A previously undescribed germline variant, namely a heterozygous deletion of a single nucleotide on position 2001 (c.2001delG;p.[Thr668Profs*24] in exon 16 of the BAP1 gene), was identified. Structural changes in the C-terminal of the BAP1 protein were observed by in silico analysis. While the excised iris melanoma showed loss of BAP1 nuclear staining by immunohistochemical staining, the BCCs of our patient (and in the control group, n = 13) were BAP1 positive. Genetic analysis of the BCC of the ocular adnexae confirmed a remaining intact BAP1 copy. The constellation of (bilateral) UM in combination with BCC should raise suspicion for a BAP1-TPDS. As our BCCs probably developed independently from the BAP1-TPDS and UMs frequently show loss of nuclear BAP1 staining, genetic analysis is mandatory to diagnose this syndrome.

Histological, immunohistochemical, and genomic evaluation of excisional and diabetic wounds treated with human Wharton's jelly stem cells with and without a nanocarrier.

We showed in previous studies that human umbilical cord Wharton's jelly stem cells (hWJSCs) improved the healing rates of excisional and diabetic wounds in the mouse model. As an extension of those studies, we report here the more detailed quantitative histological, immunohistochemical, and genomic evaluation of biopsies from those excisional and diabetic wounds in an attempt to understand the mechanisms of the enhanced wound healing aided by hWJSCs. Bright-field microscopic observations and ImageJ software analysis on histological sections of the excisional and diabetic wound biopsies collected at different time points showed that the thickness of the epidermis and dermis, and positive picrosirius-red stained areas for collagen, were significantly greater in the presence of hWJSCs compared with controls (P < 0.05). Immunohistochemistry of the diabetic wound biopsies showed increased positive staining for the vascular endothelial marker CD31 and cell proliferation marker Ki67 in the presence of hWJSCs and its conditioned medium (hWJSC-CM). Quantitative real-time polymerase chain reaction showed upregulation of groups of genes involved in extracellular matrix regulation, collagen biosynthesis, angiogenesis, antifibrosis, granulation, and immunomodulation in the presence of hWJSCs. Taken together, the results demonstrated that hWJSCs and hWJSC-CM that contains the paracrine secretions of hWJSCs, enhance the healing of excisional and diabetic wounds via re-epithelialization, collagen deposition, angiogenesis, and immunomodulation. The inclusion of an Aloe vera-polycaprolactone (AV/PCL) nanocarrier did not significantly change the effect of the hWJSCs. However, the topical application of an AV/PCL nanocarrier impregnated with hWJSCs is convenient and less invasive than the administration of hWJSC injections into wounds.

Manufacturing of human Wharton's jelly stem cells for clinical use: selection of serum is important.

BACKGROUND: Human Wharton's jelly-derived mesenchymal stromal cells (hWJSCs) have gained considerable attention for their use in cell therapy. Many of these applications would require manufacturing of millions of hWJSCs. It is, therefore, necessary to develop a Good Manufacturing Practice (GMP)-compliant hWJSC expansion protocol, allowing the generation of a large quantity of cells to meet both clinical and regulatory requirements. Here, we compared human platelet lysate (HPL) and human serum (HS) in supporting clinical-grade hWJSC expansion. METHODS: hWJSCs were successfully isolated from six different umbilical cords using GMP-compliant dissociation enzymes. Freshly isolated hWJSCs were cultured in media supplemented with 10% of one of the following sera: fetal bovine serum (FBS), HPL and HS. Properties of the expanded hWJSCs were analyzed. RESULTS: We showed that GMP-compliant dissociation enzymes were as efficient as research-grade dissociation enzymes in isolating hWJSCs. hWJSC fresh cell yield and cell viability using HPL and HS supplementations were at greater advantages than FBS. Moreover, hWJSCs expanded in HPL and HS supplementations not only preserved classical MSCs phenotypes and differentiation potential to adipocytes, osteocytes and chondrocytes, they also enhanced the migration of skin fibroblasts. However, HS, unlike HPL, did not alter immunogenicity properties of hWJSCs. hWJSCs expanded in HS supplementation also exerted greater immunosuppressive action in inhibiting T-cell proliferation and increased extracellular matrix (ECM) gene expression, making them useful in tissue repair clinical application. CONCLUSION: Our findings indicate that HS can be considered as a promising and safer alternative to FBS, and should be recommended for clinical-grade expansion of hWJSCs.

Characterization of the mutation spectrum in a Pakistani cohort of type 3 von Willebrand disease.

INTRODUCTION: Type 3 von Willebrand disease (VWD), a severe autosomal recessive hereditary bleeding disorder, is described by the virtual absence of von Willebrand factor (VWF). In consanguineous populations, for example Pakistan, the disease is reported with a higher incidence rate than the worldwide prevalence. AIMS: This study aims to characterize molecular pathology and clinical profile of type 3 VWD cohort of Pakistani origin. METHODS: In total, 48 patients were enrolled in the current study. Initially, the index patients (IPs) were evaluated by a standardized questionnaire for recording bleeding manifestations and by performing conventional coagulation tests. The diagnosis of VWD type 3 was confirmed by VWF antigens less than 5 IU/dL. Direct sequencing of VWF gene (VWF) was carried out to identify causative gene variations. We evaluated the potential consequence of novel splice site and missense variations by predictive computational programs and in silico structural analysis. RESULTS: VWF mutations were detected in 46 out of 48 IPs (95.8%), predominantly as homozygous variants. In total, twenty-nine different gene defects were characterized in this cohort from which 10 (34.5%) are novel. The majority of the mutations were null alleles (66%; including gene conversions, nonsense, splice site variations, small deletions and insertions), and 34% of them were missense substitutions. CONCLUSION: Herein, we reported for the first time, the pattern of gene defects in Pakistani type 3 VWD cohort. We identified a wide heterogeneous mutation spectrum along with variability in the type of bleeding episodes.