Effects of Lianhuaqingwen Capsules in adults with mild-to-moderate coronavirus disease 2019: an international, multicenter, double-blind, randomized controlled trial.

BACKGROUND: In a randomized trial, Lianhuaqingwen (LHQW) capsule was effective for accelerating symptom recovery among patients with coronavirus disease 2019 (COVID-19). However, the lack of blinding and limited sample sizes decreased the level of clinical evidence. OBJECTIVES: To evaluate the efficacy and safety of LHQW capsule in adults with mild-to-moderate COVID-19. METHODS: We conducted a double-blind randomized controlled trial in adults with mild-to-moderate COVID-19 (17 sites from China, Thailand, Philippine and Vietnam). Patients received standard-of-care alone or plus LHQW capsules (4 capsules, thrice daily) for 14 days. The primary endpoint was the median time to sustained clinical improvement or resolution of nine major symptoms. RESULTS: The full-analysis set consisted of 410 patients in LHQW capsules and 405 in placebo group. LHQW significantly shortened the primary endpoint in the full-analysis set (4.0 vs. 6.7 days, hazards ratio: 1.63, 95% confidence interval: 1.39-1.90). LHQW capsules shortened the median time to sustained clinical improvement or resolution of stuffy or runny nose (2.8 vs. 3.7 days), sore throat (2.0 vs. 2.6 days), cough (3.2 vs. 4.9 days), feeling hot or feverish (1.0 vs. 1.3 days), low energy or tiredness (1.3 vs. 1.9 days), and myalgia (1.5 vs. 2.0 days). The duration to sustained clinical improvement or resolution of shortness of breath, headache, and chills or shivering did not differ significantly between the two groups. Safety was comparable between the two groups. No serious adverse events were reported. INTERPRETATION: LHQW capsules promote recovery of mild-to-moderate COVID-19 via accelerating symptom resolution and were well tolerated. Trial registration ChiCTR2200056727 .

[Intraoperative autologous based blood conservation strategies in mitral valve replacement].

OBJECTIVE: To evaluate whether intraoperative autologous donation (IAD) can reduce perioperative blood transfusion for patients underwent mitral valve replacement (MVR). METHODS: A total of 318 patients received implementation of IAD from January 2011 to December 2013 were analyzed retrospectively, and compared with 517 patients of the previous 36-month period (from January 2008 to December 2012). The method of small-volume retrograde autologous priming, strict blood transfusion standard along with IAD together constituted a progressive blood-saving strategy. Statistical methods including Students' t-test, Pearson's χ(2) test, Kruskal-Wallis analysis and multivariate Logistic regression model were used for comparisons of the data. RESULTS: There were no significant difference between IAD group and non-IAD group considering preoperative patient demographics, characteristics and preoperative comorbidities. However, IAD group significantly reduced number of patients transfused with intra/post-operative packed red-blood cell (PRBC) (55(17.0%) vs. 215 (42.1%), χ(2)=53.0, P=0.000), and had significantly reduced postoperative chest tube output (150(380) ml vs. 700(660) ml, H=195.648, P=0.000), length of stay ((16±6) d vs. (20±8)d, t=9.60, P=0.000). But hematocrit were lower in IAD group (30%±5% vs.33%±4% at end of operation, t=7.76, P=0.000; 30%±4% vs. 32%±5% at discharge, P=0.000, t=3.86). Multivariate logistic aggression analysis revealed that age, IAD and smoking history were factors influencing the probability of intra or postoperative blood transfusion. CONCLUSION: Implementation of blood conservation strategies based on intraoperative autologous donation in mitral valve replacement surgery can significantly reduce intra/postoperative blood transfusion as well as postoperative complications.

Initial Characterization of Osteoblast Differentiation and Loss of RUNX2 Stability in the Newly Established SK11 Human Embryonic Stem Cell-Derived Cell Line.

We describe a novel model for investigation of genetically normal human osteoblasts in culture. SK11 is a clonal progenitor cell line derived from human embryonic stem cells. Initially selected based on the expression of chondrogenic markers when differentiated in micromass culture, SK11 cells display typical mRNA expression patterns of bone phenotypic genes under osteogenic conditions. These include osterix, α1(I) collagen, alkaline phosphatase, osteonectin, osteopontin, and osteocalcin. Similar to well-characterized murine osteoblast cultures, the osteoblast master regulator RUNX2 was present during the first few days after plating, but the protein disappeared during the first week of culture. Loss of RUNX2 expression is considered an important regulatory feature for osteoblast maturation. Indeed, following ∼2 weeks of differentiation, SK11 cultures exhibited robust calcium deposition, evidenced by alizarin red staining. We also introduced a lentiviral vector encoding doxycycline (dox)-inducible FLAG-tagged RUNX2 into SK11 cells. Dox-mediated enhancement of RUNX2 expression resulted in accelerated mineralization, which was further increased by co-treatment with BMP-2. Like the endogenous RUNX2, expression of the virally coded FLAG-RUNX2 was lost during the first week of culture despite persistent dox treatment. By following RUNX2 decay after dox withdrawal from day-5 versus day-3 cultures, we demonstrated a developmentally regulated decrease in RUNX2 stability. Availability of culture models for molecular investigation of genetically normal human osteoblasts is important because differences between murine and human osteoblasts, demonstrated here by the regulation of matrix Gla Protein, may have significant biomedical implications.

New Self-Expanding Transcatheter Valve for Off-Pump Transatrial Mitral Valve-In-Ring Implantation.

OBJECTIVES: To validate a self-expanding transcatheter valve for off-pump transatrial mitral valve-in-ring (VIR) implantation via a left thoracotomy. METHODS: Mitral valve annuloplasty was performed via sternotomy during cardiopulmonary bypass on 9 pigs. After successful weaning from extracorporal circulation, the custom-made, self-expanding transcatheter VIR device was deployed under fluoroscopic guidance within the annuloplasty ring via a left thoracotomy. Hemodynamic data before and after the implantation were recorded. Mitral annulus diameter and valve area were measured by echocardiography. Transvalvular and left-ventricular outflow-tract pressure gradient were measured invasively. RESULTS: Eight successful implantations were performed. Implantation failed in 1 pig because of difficulty with technical delivery of the sheath. Mean transatrial procedure time was 12.6 ± 1.7 min. Hemodynamic status during transatrial implantation was stable, and differences were not statistically significant. Mean mitral annulus diameter and mean mitral orifice area were 2.32 ± 0.2 and 3.84 ± 0.55 cm2, respectively. Mild regurgitation was detected in 7 animals and moderate regurgitation in 1. Mean gradients were 6.1 ± 5.0 mm Hg across the device. Postmortem examination confirmed adequate positioning of devices within the annuloplasty ring. CONCLUSIONS: This custom-made transcatheter device allows for safe and reproducible off-pump transatrial mitral VIR implantations. Transatrial access is a promising route to facilitate VIR implantations. Our custom-made stent-valve may be suitable for VIR procedures.

Clonal derivation of white and brown adipocyte progenitor cell lines from human pluripotent stem cells.

BACKGROUND: The role of brown fat in non-shivering thermogenesis and the discovery of brown fat depots in adult humans has made it the subject of intense research interest. A renewable source of brown adipocyte (BA) progenitors would be highly valuable for research and therapy. Directed differentiation of human pluripotent stem (hPS) cells to white or brown adipocytes is limited by lack of cell purity and scalability. Here we describe an alternative approach involving the identification of clonal self-renewing human embryonic progenitor (hEP) cell lines following partial hPS cell differentiation and selection of scalable clones. METHODS: We screened a diverse panel of hPS cell-derived clonal hEP cell lines for adipocyte markers following growth in adipocyte differentiation medium. The transcriptome of the human hES-derived clonal embryonic progenitor cell lines E3, C4ELS5.1, NP88, and NP110 representing three class of definitive adipocyte progenitors were compared to the relatively non-adipogenic line E85 and adult-derived BAT and SAT-derived cells using gene expression microarrays, RT-qPCR, metabolic analysis and immunocytochemistry. Differentiation conditions were optimized for maximal UCP1 expression. RESULTS: Many of the differentiated hEP cell lines expressed the adipocyte marker, FAPB4, but only a small subset expressed definitive adipocyte markers including brown adipocyte marker, UCP1. Class I cells (i.e., E3) expressed CITED1, ADIPOQ, and C19orf80 but little to no UCP1. Class II (i.e., C4ELS5.1) expressed CITED1 and UCP1 but little ADIPOQ and LIPASIN. Class III (i.e., NP88, NP110) expressed CITED1, ADIPOQ, C19orf80, and UCP1 in a similar manner as fetal BAT-derived (fBAT) cells. Differentiated NP88 and NP110 lines were closest to fBAT cells morphologically in adiponectin and uncoupling protein expression. But they were more metabolically active than fBAT cells, had higher levels of 3-hydroxybutyrate, and lacked expression of fetal/adult marker, COX7A1. The hEP BA progenitor lines were scalable to 17 passages without loss of differentiation capacity and could be readily rederived. CONCLUSIONS: Taken together, these data demonstrate that self-renewing adipocyte progenitor cells can be derived from hES cells and that they are functionally like BAT cells but with unique properties that might be advantageous for basic research and for development of cell-based treatments for metabolic diseases.

Blood conservation strategies in cardiac valve replacement: A retrospective analysis of 1645 patients.

We aimed to evaluate whether blood conservation strategies including intraoperative autologous donation (IAD) could reduce perioperative blood transfusion for patients undergoing cardiac valve replacement including mitral valve replacement, aortic valve replacement (AVR), and double valve replacement (DVR).A total of 726 patients were studied over a 3-year period (2011-2013) after the implementation of IAD and were compared with 919 patients during the previous 36-month period (January 2008-December 2010). The method of small-volume retrograde autologous priming, strict blood transfusion standard together with IAD constituted a progressive blood-saving strategy.Baseline characteristics and preoperative information showed no statistically significant difference between IAD group and non-IAD group. Most of the postoperative morbidities are statistically the same in the 2 groups. Chest tube output (415.2 vs 1029.8 mL, P < 0.001) and postoperative respiratory failure (5.9% vs 8.6%, P = 0.039) favored the IAD group, whereas hematocrit levels were more favorable in the non-IAD group (30.3% vs 33.0% at the end of the operation, P < 0.001; 30.4% vs 31.5% at the time of discharge). The use of blood product transfusion was higher in the non-IAD group (22.6% vs 43.3%, P < 0.001). Binary multivariate logistic regression analysis showed that high age, non-IAD, DVR surgery, and absent smoking history are associated with a higher risk of intra-/postoperative blood transfusion.Blood conservation is effective and safe in cardiac valve replacement surgeries. The use of intraoperative autologous donation can lead to improved outcomes including a significantly lower rate of intra-/postoperative blood transfusion and postoperative complications.

The antibody response to bacteriophage is linked to the lymphopenia gene in congenic BioBreeding rats.

Congenic BioBreeding (BB) rats, homozygous for the autosomal lymphopenia (Lyp) gene (Lyp/Lyp), heterozygous (Lyp/+), or wild-type (+/+), were immunized with the T cell-dependent bacteriophage PhiX174 to determine effects of Lyp on primary and secondary antibody responses. The primary PhiX174 antibody response did not differ between the three different genotypes. In contrast, the secondary immune response, expressed as the peak neutralizing titer, was markedly reduced in Lyp/Lyp (9.9+/-3.2; mean value+/-S.E.M. for seven rats) compared to both Lyp/+ (51+/-12; n=13; P=0.006) and +/+ (100+/-20; n=7; P=0.004) BB rats. We suggest that the secondary antibody response to the T cell-dependent neoantigen PhiX174 is linked in a recessive manner to genetic factor(s) in the Lyp gene region.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation.

AIMS: The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFß3, BMP4, SCF and HyStem-C matrices. MATERIALS & METHODS: The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. RESULTS: In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFß3, SCF, and SCF and TGFß3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFß3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFß3. CONCLUSION: The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates.

AIM: The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS & METHODS: The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. RESULTS: In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-ß3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. CONCLUSION: The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

Generation of insulin-producing islet-like clusters from human embryonic stem cells.

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to beta cells. Generation of glucose-responsive, insulin-producing beta cells from self-renewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mug of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner. Disclosure of potential conflicts of interest is found at the end of this article.

Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium.

Previous studies have shown that prolonged propagation of undifferentiated human embryonic stem cells (hESCs) requires conditioned medium from mouse embryonic feeders (MEF-CM) as well as matrix components. Because hESCs express growth factor receptors, including those for basic fibroblast growth factor (bFGF), stem cell factor (SCF), and fetal liver tyrosine kinase-3 ligand (Flt3L), we evaluated these and other growth factors for their ability to maintain undifferentiated hESCs in the absence of conditioned medium. We found cultures maintained in bFGF alone or in combination with other factors showed characteristics similar to MEF-CM control cultures, including morphology, surface marker and transcription factor expression, telomerase activity, differentiation, and karyotypic stability. In contrast, cells in media containing Flt-3L, thrombopoietin, and SCF, individually or in combination, showed almost complete differentiation after 6 weeks in culture. These data demonstrate that hESCs can be maintained in nonconditioned medium using growth factors.

Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth.

Human embryonic stem cells (hESCs) have the potential to generate multiple cell types and hold promise for future therapeutic applications. Although undifferentiated hESCs can proliferate indefinitely, hESC derivatives significantly downregulate telomerase and have limited replication potential. In this study we examine whether the replicative lifespan of hESC derivatives can be extended by ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex. To this end, we have derived HEF1 cells, a fibroblast-like cell type, differentiated from hESCs. Infection of HEF1 cells with a retrovirus expressing hTERT extends their replicative capacity, resulting in immortal human HEF1-hTERT cells. HEF1-hTERT cells can be used to produce conditioned medium (CM) capable of supporting hESC growth under feeder-free conditions. Cultures maintained in HEF1-CM show characteristics similar to mouse embryonic fibroblast CM control cultures, including morphology, surface marker and transcription factor expression, telomerase activity, differentiation, and karyotypic stability. In addition, HEF1-hTERT cells have the capacity to differentiate into cells of the osteogenic lineage. These results suggest that immortalized cell lines can be generated from hESCs and that cells derived from hESCs can be used to support their own growth, creating a genotypically homogeneous system for the culture of hESCs.

Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene.

The BB (BioBreeding) rat is one of the best models of spontaneous autoimmune diabetes and is used to study non-MHC loci contributing to Type 1 diabetes. Type 1 diabetes in the diabetes-prone BB (BBDP) rat is polygenic, dependent upon mutations at several loci. Iddm1, on chromosome 4, is responsible for a lymphopenia (lyp) phenotype and is essential to diabetes. In this study, we report the positional cloning of the Iddm1/lyp locus. We show that lymphopenia is due to a frameshift deletion in a novel member (Ian5) of the Immune-Associated Nucleotide (IAN)-related gene family, resulting in truncation of a significant portion of the protein. This mutation was absent in 37 other inbred rat strains that are nonlymphopenic and nondiabetic. The IAN gene family, lying within a tight cluster on rat chromosome 4, mouse chromosome 6, and human chromosome 7, is poorly characterized. Some members of the family have been shown to be expressed in mature T cells and switched on during thymic T-cell development, suggesting that Ian5 may be a key factor in T-cell development. The lymphopenia mutation may thus be useful not only to elucidate Type 1 diabetes, but also in the function of the Ian gene family as a whole.