Mast cells derived from human induced pluripotent stem cells are useful for allergen tests

Background

Several methods have been developed to detect allergen-specific IgE in sera. The passive IgE sensitization assay using human IgE receptor-expressing rat cell line RBL-2H3 is a powerful tool to detect biologically active allergen-specific IgE in serum samples. However, one disadvantage is that RBL-2H3 cells are vulnerable to high concentrations of human sera. Only a few human cultured cell lines are easily applicable to the passive IgE sensitization assay. However, the use of human induced pluripotent stem cells (iPSCs) to generate human mast cells (MCs) has not yet been reported.

Lack of detection of human retrovirus‐5 proviral DNA in synovial tissue and blood specimens from individuals with rheumatoid arthritis or osteoarthritis

Objective

Prior studies have suggested an association of human retrovirus 5 with rheumatoid arthritis. The purpose of this study was to determine if human retrovirus‐5 proviral DNA is present in synovial tissue and blood specimens from patients with rheumatoid arthritis or osteoarthritis, or those without joint disease.

Methods

Synovial tissue and whole blood from 75 patients with rheumatoid arthritis, 75 patients with osteoarthritis, and 50 patients without a primary arthritis diagnosis were assayed by real‐time quantitative polymerase chain reaction (PCR) using primers that amplify a 186‐bp fragment of human retrovirus‐5 proviral DNA.

Results

A total of 200 tissue specimens, 200 mononuclear cells, and 196 of 200 granulocyte specimens tested negative for human retrovirus‐5 proviral DNA. No association between human retrovirus 5 and rheumatoid arthritis or osteoarthritis (P = 0.516) was identified. Granulocyte specimens from 4 patients, 2 with rheumatoid arthritis and 2 with osteoarthritis, yielded a low positive human retrovirus‐5 proviral DNA signal (83–1,365 copies of human retrovirus‐5 proviral DNA/ml blood).

Conclusion

Contrary to prior reports, we did not find an association between human retrovirus 5 and rheumatoid arthritis or osteoarthritis using a real‐time PCR assay. Our findings are consistent with the recent finding that human retrovirus 5 is actually rabbit endogenous retrovirus H.

     

Generation and characterization of LymphoStat‐B,a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator

Objective

To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor–related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases.

Methods

A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat‐B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat‐B to cynomolgus monkeys.

Results

LymphoStat‐B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF‐R. LymphoStat‐B potently inhibited BLyS‐induced proliferation of B cells in vitro, and administration of LymphoStat‐B to mice prevented human BLyS‐induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat‐B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes.

Conclusion

A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.

     

Type II collagen levels correlate with mineralization by articular cartilage vesicles

Objective

Pathologic mineralization is common in osteoarthritic (OA) cartilage and may be mediated by extracellular organelles known as articular cartilage vesicles (ACVs). Paradoxically, ACVs isolated from OA human cartilage mineralize poorly in vitro compared with those isolated from normal porcine cartilage. We recently showed that collagens regulate ACV mineralization. We sought to determine differences between collagens and collagen receptors on human and porcine ACVs as a potential explanation of their different mineralization behaviors.

Methods

ACVs were enzymatically released from old and young human and porcine hyaline articular cartilage. Western blotting was used to determine the presence of types I, II, VI, and X collagen and various collagen receptors on ACVs. Type II collagen was quantified by enzyme‐linked immunosorbent assay. Biomineralization was assessed by measuring the uptake of ⁴⁵Ca by isolated ACVs in agarose gels and by ACVs in situ in freeze‐thawed cartilage.

Results

As previously shown, isolated human ACVs mineralized poorly in response to ATP compared with porcine ACVs, but human and porcine ACVs mineralized similarly in situ in freeze‐thawed cartilage. Type II collagen levels were 100‐fold higher in isolated human ACVs than in porcine ACVs. Type II collagen in human ACVs was of high molecular weight. Transglutaminase‐crosslinked type II collagen showed increased resistance to collagenase, suggesting a possible explanation for residual collagen on human ACVs. Expression of other collagens and collagen receptors was similar on human and porcine ACVs.

Conclusion

Higher levels of type II collagen in human ACV preparations, perhaps mediated by increased transglutaminase crosslinking, may contribute to the decreased mineralization observed in isolated human ACVs in vitro.

     

Osteoarthritic chondrocyte–secreted morphogens induce chondrogenic differentiation of human mesenchymal stem cells

Objective

The potential of stem cells to repair compromised cartilage tissue, such as in osteoarthritis (OA), depends strongly on how transplanted cells respond to factors secreted from the residing OA chondrocytes. This study was undertaken to determine the effect of morphogenetic signals from OA chondrocytes on chondrogenic differentiation of human mesenchymal stem cells (MSCs).

Methods

The effect of OA chondrocyte–secreted morphogens on chondrogenic differentiation of human MSCs was evaluated using a coculture system involving both primary and passaged OA chondrocytes. The findings were compared against findings for human MSCs cultured in OA chondrocyte–conditioned medium. Gene expression analysis, biochemical assays, and immunofluorescence staining were used to characterize the chondrogenic differentiation of human MSCs. Mass spectrometry analysis was used to identify the soluble factors. Numerical analysis was carried out to model the concentration profile of soluble factors within the human MSC–laden hydrogels.

Results

The human MSCs cocultured with primary OA chondrocytes underwent chondrogenic differentiation even in the absence of growth factors; however, the same effect could not be mimicked using OA chondrocyte–conditioned medium or expanded cells. Additionally, the cocultured environment down‐regulated hypertrophic differentiation of human MSCs. Mass spectrometry analysis demonstrated cell–cell communication and chondrocyte phenotype–dependent effects on cell‐secreted morphogens.

Conclusion

The experimental findings, along with the results of the numerical analysis, suggest a crucial role of soluble morphogens and their local concentrations in the differentiation pattern of human MSCs in a 3‐dimensional environment. The concept of using a small number of chondrocytes to promote chondrogenic differentiation of human MSCs while preventing their hypertrophic differentiation could be of great importance in formulating effective stem cell–based cartilage repair.

     

Degradation of islet amyloid polypeptide by neprilysin

Aims/hypothesis

A progressive loss of pancreatic beta cell function, a decrease in beta cell mass and accumulation of islet amyloid is characteristic of type 2 diabetes mellitus. The main constituent of islet amyloid is islet amyloid polypeptide (IAPP). In this study, we examined the ability of the peptidase neprilysin to cleave IAPP and prevent human IAPP-induced pancreatic beta cell toxicity.

Methods

Neprilysin and a catalytically compromised neprilysin mutant were tested for their ability to inhibit human IAPP fibrillisation and human IAPP-induced pancreatic beta cell cytotoxicity. Degradation of human IAPP by neprilysin was followed by HPLC, and the degradation products were identified by MS.

Results

Neprilysin prevented IAPP fibrillisation by cleaving IAPP at Arg¹¹-Leu¹², Leu¹²-Ala¹³, Asn¹⁴-Phe¹⁵, Phe¹⁵-Leu¹⁶, Asn²²-Phe²³ and Ala²⁵-Ile²⁶. It also appears to prevent human IAPP fibrillisation through a non-catalytic interaction. Neprilysin protected against beta cell cytotoxicity induced by exogenously added or endogenously produced human IAPP.

Conclusions/interpretation

The data presented support a potential therapeutic role for neprilysin in preventing type 2 diabetes mellitus. This study supports the hypothesis that extracellular human IAPP contributes to human IAPP-induced beta cell cytotoxicity. Whether human IAPP exerts its cytotoxic effect through a totally extracellular mechanism or through a cellular reuptake mechanism is unclear at this time.     

Human protective monoclonal antibodies against the HA stem of group 2 HAs derived from an H3N2 virus-infected human

Objectives

Broadly reactive human monoclonal antibodies against the HA stem of influenza A virus are being developed as therapeutic agents as well as to understand the epitopes that are essential for a universal influenza virus vaccine.

Prognostic significance of PINCH signalling in human pancreatic ductal adenocarcinoma

Objective:

Prognostic markers for pancreatic ductal adenocarcinoma (PDA) have failed to accurately predict patient prognosis. Recently, interest has developed in the accuracy of integrin-associated PINCH protein expression in human cancers as a predictive marker of tumour status. The goal of this study was to define the expression of PINCH protein in PDA.

Methods:

Human PDA samples and orthotopic tumours from a murine model were analysed by immunohistochemistry for PINCH expression. In the animal model, PINCH expression was compared between primary and metastatic tumours. In the human samples, PINCH expression was correlated with stage, nodal involvement, margin status and overall survival.

Results:

In the murine model, there was greater PINCH expression in metastatic tumours than in primary tumours. In the human PDA samples, greater staining for PINCH in the tumour cells was correlated with higher T status. Additionally, high PINCH expression in the stroma was associated with decreased overall survival.

Conclusions:

Findings of increased PINCH protein in more advanced stages of human PDA, as well as in metastatic tumours in the animal model, support the hypothesis that PINCH is an important controller of cell survival and migration. Additionally, the importance of the differential expression of PINCH in the human tumour and stroma warrants further evaluation.     

Quantifying the composition of human skin for glucose sensor development

Background

Glucose is heterogeneously distributed within human skin. In order to develop a glucose measurement method for human skin, both a good quantification of the different compartments of human skin and an understanding of glucose transport processes are essential. This study focused on the composition of human skin. In addition, the extent to which intersubject variability in skin composition alters glucose dynamics in human skin was investigated.

Methods

To quantify the composition of the three layers of human skin—epidermis, dermis, and adipose tissue—cell and blood vessel volumes were calculated from skin biopsies. These results were combined with data from the literature. The composition was applied as input for a previously developed computational model that calculates spatiotemporal glucose dynamics in human skin. The model was used to predict the physiological effects of intersubject variability in skin composition on glucose profiles in human skin.

Results

According to the model, the lag time of glucose dynamics in the epidermis was sensitive to variation in the volumes of interstitial fluid, cells, and blood of all layers. Data showed most variation/uncertainty in the volume composition of the adipose tissue. This variability mainly influences the dynamics in the adipose tissue.

Conclusions

This study identified the intersubject variability in human skin composition. The study shows that this variability has significant influence on the glucose dynamics in human skin. In addition, it was determined which volumes are most critical for the quantification and interpretation of measurements in the different layers.     

A role for ultra-high resolution three-dimensional printed human heart models

Introduction

Echocardiography is essential for diagnosing and assessing the severity of perioperative structural and functional heart disease. Yet, educational opportunities to better understand echocardiography-based cardiac anatomy remain limited by the two-dimensional display, lack of anatomic details, variability of heart models, and costs and global access of training.

Methods

We performed micro computed tomography of human heart specimens not suitable for orthotopic transplantation. We created high-resolution computational 3D models of different human hearts, sliced them in the different recommended American Society of Echocardiography views, and 3D printed them using different materials.

Results

We scanned, 3D modeled, and 3D printed a variety of human hearts both healthy and diseased. We have made the models available in the cardiac operating rooms and routinely use them for teaching anesthesia residents and cardiothoracic anesthesia fellows about basic and advanced echocardiographic views, cardiopulmonary bypass cannulation strategies, and valvular pathology and planned interventions.

Conclusion

We have generated a library of 3D printed hearts to display the recommended echocardiographic views as a unique educational tool designed to safely accelerate the understanding of absolute and relative human cardiac anatomy and pathology, especially related to gaining advanced appreciation of clinically employed perioperative echocardiography.     

Chimeric antibodies with extended half‐life in ferrets

Background

Ferrets have long been used as a disease model for the study of influenza vaccines, but a more recent use has been for the study of human monoclonal antibodies directed against influenza viruses. Published data suggest that human antibodies are cleared unusually quickly from the ferret and that immune responses may be partially responsible. This immunogenicity increases variability within groups and may present an obstacle to long-term studies.

Objective

Our aim was to identify an antibody design with reduced immunogenicity and longer circulating half-life in ferrets.

Methods

The constant region coding sequences for ferret immunoglobulin G were cloned, and chimeric human/ferret antibodies were expressed and purified. Some of the chimeric antibodies included substitutions that have been shown to extend the half-life of human IgG antibodies. These chimeric antibodies were tested for binding to recombinant ferret FcRn receptor and then evaluated in pharmacokinetic studies in ferrets.

Results

A one-residue substitution in the ferret Fc domain, S252Y, was identified that increased binding affinity to the ferret neonatal receptor by 24-fold and extended half-life from 65 ± 27 to 206 ± 28 hours or ∼9 days. Ferrets dosed twice with this surrogate antibody showed no indications of an immune response.

Conclusion

Expressing the variable region of a candidate human therapeutic antibody with ferret constant regions containing the S252Y substitution can offer long half-life and limit immunogenicity.     

Fluoxetine and citalopram exhibit potent antiinflammatory activity in human and murine models of rheumatoid arthritis and inhibit toll‐like receptors

Objective

Selective serotonin reuptake inhibitors (SSRIs), in addition to their antidepressant effects, have been reported to have antiinflammatory effects. The aim of this study was to assess the antiarthritic potential of 2 SSRIs, fluoxetine and citalopram, in murine collagen‐induced arthritis (CIA) and in a human ex vivo disease model of rheumatoid arthritis (RA).

Methods

Following therapeutic administration of SSRIs, paw swelling was assessed and clinical scores were determined daily in DBA/1 mice with CIA. Joint architecture was examined histologically at the end of the treatment period. Cultures of human RA synovial membranes were treated with SSRIs, and cytokine production was measured. Toll‐like receptor (TLR) function was examined in murine and human macrophages, human B cells, and human fibroblast‐like synovial cells treated with SSRIs.

Results

Both SSRIs significantly inhibited disease progression in mice with CIA, with fluoxetine showing the greatest degree of efficacy at the clinical and histologic levels. In addition, both drugs significantly inhibited the spontaneous production of tumor necrosis factor, interleukin‐6, and interferon‐γ–inducible protein 10 in human RA synovial membrane cultures. Fluoxetine and citalopram treatment also inhibited the signaling of TLRs 3, 7, 8, and 9, providing a potential mechanism for their antiinflammatory action.

Conclusion

Fluoxetine and citalopram treatment selectively inhibit endosomal TLR signaling, ameliorate disease in CIA, and suppress inflammatory cytokine production in human RA tissue. These data highlight the antiarthritic potential of the SSRI drug family and provide further evidence of the involvement of TLRs in the pathogenesis of RA. The SSRIs may provide a template for potential antiarthritic drug development.

     

Genome Editing and Induced Pluripotent Stem Cell Technologies for Personalized Study of Cardiovascular Diseases

Purpose of Review

The goal of this review is to highlight the potential of induced pluripotent stem cell (iPSC)-based modeling as a tool for studying human cardiovascular diseases. We present some of the current cardiovascular disease models utilizing genome editing and patient-derived iPSCs.

Recent Findings

The incorporation of genome-editing and iPSC technologies provides an innovative research platform, providing novel insight into human cardiovascular disease at molecular, cellular, and functional level. In addition, genome editing in diseased iPSC lines holds potential for personalized regenerative therapies.

Summary

The study of human cardiovascular disease has been revolutionized by cellular reprogramming and genome editing discoveries. These exceptional technologies provide an opportunity to generate human cell cardiovascular disease models and enable therapeutic strategy development in a dish. We anticipate these technologies to improve our understanding of cardiovascular disease pathophysiology leading to optimal treatment for heart diseases in the future.

     

A genomic-based approach identifies FXYD domain containing ion transport regulator 2 (FXYD2)γa as a pancreatic beta cell-specific biomarker

Aims/hypothesis

Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers.

Methods

We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays.

Results

After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na⁺–K⁺-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2γa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2γa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2γa and loss of insulin-positive cells.

Conclusions/interpretation

We propose human FXYD2γa as a novel beta cell-specific biomarker.     

Treatment of Syphilis-Associated Cerebral Vasculitis: Reappearance of an Old Question

Background

Cerebral vasculitis caused by neurosyphilis is a re-emerging problem with diagnostic and treatment issues, especially for human immunodeficiency virus patients.

Co-circulation of classic and novel astrovirus strains in patients with acute gastroenteritis in Germany

Objectives

In order to analyze the molecular epidemiology of human astroviruses (HAstV) in Germany, a retrospective long-term study was performed to characterize circulating human astrovirus in patients with acute gastroenteritis in Germany.

Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine

Background

Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine.

Design and Methods

We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin.

Results

We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form.

Conclusions

Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients.