Cytokine production of stimulated whole blood cultures in rheumatoid arthritis patients receiving short-term infliximab therapy

Patients with rheumatoid arthritis (RA) treated with anti-tumor necrosis factor (TNF) strategies have an increased susceptibility to infections, especially those caused by intracellular pathogens. In this study we assessed the cytokine production capacity in patients with RA and we further investigated whether anti-TNF therapy modulates the production of pro-inflammatory cytokines involved in the resistance against infections. Whole blood cultures from 10 RA patients and 10 healthy controls were stimulated with heat-killed Candida albicans, Salmonella typhimurium, Staphyloccocus aureus, Aspergillus fumigatus or Mycobacterium tuberculosis and production of interleukin (IL)-1beta, IL-6, IL-10, interferon (IFN)-gamma and TNF-alpha was measured. Before anti-TNF therapy, whole blood cultures from RA patients released significantly less IFN-gamma than healthy controls after stimulation with all tested microorganisms. Short-term anti-TNF therapy did not have an inhibitory effect on the release of the cytokines tested. We conclude that cells of patients with RA have a strongly reduced production capacity of IFN-gamma after bacterial challenge. Although short-term therapy with anti-TNF agents did not further decrease the release of other proinflammatory cytokines, the combination of defective IFN-gamma production in basal conditions and TNF neutralization during anti-TNF therapy is likely to be responsible for the higher susceptibility to infections in patients with RA.     

Computational analysis of small RNA cloning data

Cloning and sequencing is the method of choice for small regulatory RNA identification. Using deep sequencing technologies one can now obtain up to a billion nucleotides--and tens of millions of small RNAs--from a single library. Careful computational analyses of such libraries enabled the discovery of miRNAs, rasiRNAs, piRNAs, and 21U RNAs. Given the large number of sequences that can be obtained from each individual sample, deep sequencing may soon become an alternative to oligonucleotide microarray technology for mRNA expression profiling. In this report we present the methods that we developed for the annotation and expression profiling of small RNAs obtained through large-scale sequencing. These include a fast algorithm for finding nearly perfect matches of small RNAs in sequence databases, a web-accessible software system for the annotation of small RNA libraries, and a Bayesian method for comparing small RNA expression across samples.     

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

Patient-specific induced pluripotent stem cells as“disease-in-adish”models for inherited cardiomyopathies and channelopathies–15 years of research

Among inherited cardiac conditions,a special place is kept by cardiomyopathies(CMPs)and channelopathies(CNPs),which pose a substantial healthcare burden due to the complexity of the therapeutic management and cause early mortality.Like other inherited cardiac conditions,genetic CMPs and CNPs exhibit incomplete penetrance and variable expressivity even within carriers of the same pathogenic deoxyribonucleic acid variant,challenging our understanding of the underlying pathogenic mechanisms.Until recently,the lack of accurate physiological preclinical models hindered the investigation of fundamental cellular and molecular mechanisms.The advent of induced pluripotent stem cell(iPSC)technology,along with advances in gene editing,offered unprecedented opportunities to explore hereditary CMPs and CNPs.Hallmark features of iPSCs include the ability to differentiate into unlimited numbers of cells from any of the three germ layers,genetic identity with the subject from whom they were derived,and ease of gene editing,all of which were used to generate“disease-in-a-dish”models of monogenic cardiac conditions.Functionally,iPSC-derived cardiomyocytes that faithfully recapitulate the patient-specific phenotype,allowed the study of disease mechanisms in an individual-/allele-specific manner,as well as the customization of therapeutic regimen.This review provides a synopsis of the most important iPSC-based models of CMPs and CNPs and the potential use for modeling disease mechanisms,personalized therapy and deoxyribonucleic acid variant functional annotation.     

In vitro differentiation of human mesenchymal stem cells to epithelial lineage

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.     

A reference-quality,fully annotated genome from a Puerto Rican individual

Until 2019, the human genome was available in only one fully annotated version, GRCh38, which was the result of 18 years of continuous improvement and revision. Despite dramatic improvements in sequencing technology, no other genome was available as an annotated reference until 2019, when the genome of an Ashkenazi individual, Ash1, was released. In this study, we describe the assembly and annotation of a second individual genome, from a Puerto Rican individual whose DNA was collected as part of the Human Pangenome project. The new genome, called PR1, is the first true reference genome created from an individual of African descent. Due to recent improvements in both sequencing and assembly technology, and particularly to the use of the recently completed CHM13 human genome as a guide to assembly, PR1 is more complete and more contiguous than either GRCh38 or Ash1. Annotation revealed 37,755 genes (of which 19,999 are protein coding), including 12 additional gene copies that are present in PR1 and missing from CHM13. Fifty-seven genes have fewer copies in PR1 than in CHM13, 9 map only partially, and 3 genes (all noncoding) from CHM13 are entirely missing from PR1.     

Palm tree peroxidase-based biosensor with unique characteristics for hydrogen peroxide monitoring

Three amperometric enzyme electrodes have been constructed by adsorbing anionic royal palm tree peroxidase (RPTP), anionic sweet potato peroxidase (SPP), or cationic horseradish peroxidase (HRP-C) on spectroscopic graphite electrodes. The resulting H(2)O(2)-sensitive biosensors were characterized both in a flow injection system and in batch mode to evaluate their main bioelectrochemical parameters, such as pH dependency, I(max), K(M)(app), detection limit, linear range, operational and storage stability. The obtained results showed a distinctly different behavior for the plant peroxidase electrodes, demonstrating uniquely superior characteristics of the RPTP-based sensors. The broader linear range observed for the RPTP-based biosensor is explained by a high stability of this enzyme in presence of H(2)O(2). The higher storage and operational stability of RPTP-based biosensor as well as its capability to measure hydrogen peroxide under acidic conditions connect with an extremely high thermal and pH-stability of RPTP.     

The link between the electrogenic and redox functions of the plasmalemma and the energy metabolism of the cell

A dependence of the plasmalemma redox activity, determined by the reduction of external electron acceptors (ferricyanide, nitro-blue tetrazolium), on the energy state of the cell, which was modified by light conditions or introduction of glucose into the media, was shown on leaves of Elodea canadensis Rich. Glucose (10 m M ) and light (40 W m^-2) caused hyperpolarization of the membrane potential and stimulated the redox activity of the plasmalemma. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) completely inhibited the light activation of electrogenic and redox functions of the plasmalemma. The light saturation intensity for membrane potential and ferricyanide reductase activity was 10–30% of the light saturation of photosynthesis. Membrane potential, K⁺ transport and plasmalemma redox activity changed in parallel in response to light and darkness and when DCMU was added. Ferricyanide reductase activity is suggested to be a simple parameter for characterizing the energy state of the cell. The functional significance of the light-induced hyperpolarization of the membrane potential is discussed.