Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.     

Prevalence of hepatitis B virus DNA in anti-HBc-positive/HBsAg-negative sera correlates with HCV but not HIV serostatus.

BACKGROUND: Hepatitis B virus (HBV) DNA often remains detectable in serum despite clinical recovery and loss of HBsAg. OBJECTIVE: To study whether coinfection with HIV and HCV influence the chance of detecting HBV DNA in sera with markers of past hepatitis B. STUDY DESIGN AND RESULTS: The test panel included 160 anti-HBc-positive/HBsAg-negative sera collected in the diagnostic setting. The following parameters were determined in the sera: anti-HIV (32% positive), anti-HCV (34% positive), HCV RNA (18% positive), and anti-HBs (37% positive). A highly sensitive PCR (90%-detection limit 100 copies/ml) amplifying the terminal protein (TP) region of HBV was established and HBV DNA was detected in 12.5% of the samples. In 70% of these samples, the HBV DNA concentration was below 500 copies/ml as measured by real-time PCR in the S gene. Logistic regression analysis revealed that the chance of detecting HBV DNA was increased by a positive HCV serostatus (odds ratio 5.0, 95%-CI 1.6-15.7), whereas HIV coinfection (odds ratio 2.0, 95%-CI 0.7-5.8), anti-HBs (odds ratio 0.9, 95%-CI 0.3-2.6), and HCV RNA status (odds ratio 0.4, 95%-CI 0.1-1.7) had no statistically significant influence. In contrast, the chance of detecting HCV RNA in the subgroup of anti-HCV-positive sera was increased by HIV coinfection (odds ratio 4.5, 95%-CI 1.2-17.4). Sequencing of the TP PCR products revealed neither a specific phylogenetic origin of the circulating HBV DNA nor clustering of uncommon mutations in the TP region. CONCLUSIONS: The prevalence of HBV DNA in serum of anti-HBc-positive/HBsAg-negative subjects correlates with HCV rather than HIV serostatus.     

Pooled protein tagging,cellular imaging,and in situ sequencing for monitoring drug action in real time

The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein–protein interactions, condensate formation, and chemical degradation.

Currently available mass-spectrometry methods (Rix and Superti-Furga 2009; Martinez Molina et al. 2013; Savitski et al. 2014; Huber et al. 2015; Drewes and Knapp 2018) for monitoring the effects of cellular perturbations on proteomes cannot be scaled efficiently to monitor time-dependent effects in high throughput. A different approach to study drug action is live-cell imaging of protein dynamics in cells expressing a protein of interest fused to a fluorescent tag. Traditionally, such reporter cells are generated either by overexpression to nonphysiologic levels, by oligonucleotide-directed homologous recombination in yeast, or by using CRISPR-Cas9 and homology-directed repair (HDR) to endogenously tag proteins in human cells (Ghaemmaghami et al. 2003; Huh et al. 2003; Chong et al. 2015; Leonetti et al. 2016). In addition to those targeted approaches, “gene trapping” or “CD-tagging” strategies, which rely on the random, viral integration of fluorescent tags as synthetic exons, have been used for analyzing dynamic changes in response to drugs (Jarvik et al. 1996; Morin et al. 2001; Cohen et al. 2008; Kang et al. 2016), but they are limited by integration site biases and require the isolation and characterization of clones before using them in an arrayed format. Recently, a strategy combining genome engineering and gene trapping using homology-independent CRISPR-Cas9 editing to place a fluorescent tag as a synthetic exon into introns of individual target genes has been described (Serebrenik et al. 2019). The strategy relies on a generic sgRNA excising a fluorescent tag flanked by splice acceptor and donor sites from a generic donor plasmid, which is coexpressed with a gene-specific intron-targeting sgRNA specifying the integration site. Here we show the scalability of that strategy to enable pooled protein tagging of more than 900 metabolic enzymes and epigenetic modifiers. Exposing the GFP-tagged cells to compounds allows us to monitor drug effects on the localization and levels of hundreds of proteins in real time in a pooled format, followed by identification of responding clones by in situ sequencing of the expressed intron-targeting sgRNA that corresponds to the tagged protein (Fig. 1A).Open in a separate windowFigure 1.Pooled GFP intron-tagging of metabolic enzymes. (A) Schematic outline of the approach. (B) Identification of targetable introns within metabolic genes. (C) FACS sorting of clones with successful GFP-tagging by signal enrichment over background mCherry intensity used as control for autofluorescence. (D) Representative image of sorted GFP-tagged cell pool. Scale bar, 25 µm. (E) Comparison of RNA-seq expression in HAP1 cells between genes for which GFP-tagged cells could be isolated and genes that were targeted in the sgRNA library but did not result in successful clone isolation.     

Micronuclei containing whole chromosomes harbouring the selectable gene do not lead to mutagenesis

Loss of heterozygosity is one genetic change observed in manytumours. We do not know whether the loss of chromosomal materialthrough micronucleus formation is a viable mechanism associatedwith, and possibly leading to, genetic disease. Previously,we treated L5178Y mouse lymphoma cells with four aneugens. Althoughthese aneugens induced micronuclei containing predominantlywhole chromosomes, they did not induce mutations at Tk1, theselectable gene, under the same non-toxic conditions in whichthey induced micronuclei. This suggested that the inductionof micronuclei containing whole chromosomes was not an earlyevent leading to phenotypically expressed mutations in thesecells under the conditions used. However, it is possible thatchromosome 11, on which Tk1 resides, may be under-representedin the micronucleus population. To find out the frequency ofinduction of micronuclei containing chromosome 11, we appliedfluorescence in situ hybridization using a chromosome 11 paintto micronuclei induced by colcemid and vinblastine. We foundthat the numbers of micronuclei containing chromosome 11 aremore than sufficient to be detectable as mutations if thesemicronuclei lead to viable mutants. We conclude that the formationof micronuclei containing whole chromosomes does not lead toviable, dividing mutants in this system. ⁵To whom correspondence should be addressed     

Infrared-spectrometric determination of D2O in biological fluids

The determination of D₂O in biological fluids by means of infrared spectrometry was reinvestigated. When the temperature of a solution, containing D₂O in the range from natural abundance to 5 ml·1^–1 increases, its absorbance decreases and the wavenumber of maximum absorption shifts to a higher value. Both changes are linearly related to the change in temperature. Storage for 17 d in either glass or polyethylene tubes does not affect the D₂O concentration. Purification of biological fluids by vacuum-sublimation removes all substances which also absorb at the O-D vibration band and the recovery of D₂O from plasma and urine is complete. The partition ratio of D₂O between plasma water and red cell water equals unity, and the same holds for plasma water and urine water over a wide range of urine flows and osmolalities. The arterial and urinary disappearance curves of D₂O, measured over several days, both permit the calculation of the total amount of body water (V ^bw), the daily water turn-over (F) and the half-time of water in the body (t _1/2), but the data derived from arterial disappearance curves are more precise. In 16 male mongrel dogs (25–32 kg body mass) the following results were obtained:V ^bw=626±28 ml·kg^–1,F=12.0±3.2% andt _1/2=6.21±1.78 d.     

Zur Katalyse der stereospezifischen Butadienpolymerisation mit den Katalysatorsystemen aus Nd(η3-C3H5)3 · dioxan und methylaluminoxan (MAO) sowie Hexaisobutylaluminoxan (HIBAO)

The activation of the tris(allyl)neodymium complex Nd(η³-C₃H₅)₃ · dioxane with alkylaluminoxanes (MAO or HIBAO) results in highly selective catalysts for the 1,4-cis-polymerization of butadiene (cis-selectivity up to 80%). Under standard conditions (50°C, toluene), the turnover frequency (TOF) of the catalyst/MAO system amounts to 10–15000 mol butadiene/(mol Nd · h). Molecular weight determinations indicate the formation of only one polymer chain per neodymium center as in a living polymerization reaction, and for the catalyst/HIBAO system the rate law r_p = k_p [Nd][C₄H₆] with k_p = 8,7 · 10^?2 mol/(L · s) (at 25°C) has been derived. As the catalytically active species, a cationic monobutenyl neodymium(III) complex is discussed, which is stabilized through coordinative interaction with the counter anion as well as the growing polybutadiene chain. This cationic complex reacts under insertion with butadiene in a bimolecular fashion.     

Recurrent high level parvovirus B19/genotype 2 viremia in a renal transplant recipient analyzed by real-time PCR for simultaneous detection of genotypes 1 to 3

Organ transplant recipients infected with parvovirus B19 frequently develop persistent viremia associated with chronic anemia and pure red cell aplasia. In this study, a male renal transplant recipient who had been infected with parvovirus B19/genotype 2 after renal transplantation at the age of 34 years is described. The patient was repeatedly treated with high dose intravenous immunoglobulin (IVIG) that resulted in the resolvement of symptoms but not in virus eradication. During an observation period of 33 months after transplantation three phases associated with high parvovirus B19 viremia were observed. Both the first and the second viremic phases were combined with severe anemia. Parvovirus B19 specific IgM-antibodies were initially detected at the beginning of the second phase in continually rising concentrations. Initially eradication of the virus by immunoglobulin therapy was reported after the first viremic phase [Liefeldt et al. (2002): Nephrol Dial Transplant 17:1840-1842]. Retrospectively this statement has to be corrected. It was based on the use of a qualitative PCR assay specific for parvovirus B19 genotype 1 associated with reduced sensitivity for detection of genotype 2. After sequence analysis of the viral DNA and adjustment of a real-time PCR assay (TaqMan) for quantitative detection of all three B19 virus genotypes analysis of consecutive serum samples allowed the demonstration of long lasting phases with reduced viral loads following IVIG-treatment. These results demonstrate that IVIG treatment of parvovirus B19-triggered anemia in transplant recipients offers an opportunity to resolve symptoms, but does not guarantee eradication of the virus. Since reactivation of parvovirus B19 infection can result in high virus load associated with the recurrence of symptoms repeated screening for viral DNA is recommended using the TaqMan system established for quantitative detection of all three genotypes of parvovirus B19.     

Cross-reactive trinitrophenylated peptides as antigens for class II major histocompatibility complex-restricted T cells and inducers of contact sensitivity in mice. Limited T cell receptor repertoire

The induction of contact sensitivity in mice by hapten reagents such as trinitrochlorobenzene (TNCB) involves the activation of class II major histocompatibility complex (MHC)-restricted, hapten-specific, CD4⁺ T cells. Reports from different laboratories have indicated that the relevant antigenic epitopes in such reactions might include hapten-conjugated, MHC class II-associated peptides. This study for the first time directly demonstrates that hapten-peptides account for the majority of determinants recognized by trinitrophenyl (TNP)-specific CD4⁺ T lymphocytes. The sequences of those TNP carrier peptides do not have to be related to mouse proteins. Thus, we show that TNP-modified peptides derived from mouse IgG, pigeon cytochrome c or staphylococcal nuclease known to bind to I-A^b or from λ represser with specificity to I-A^d as well as TNP-proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin all create class II-restricted hapten determinants for a number of TNP-specific T cell clones and hybridomas. All of these cells were induced with cells modified by trinitrobenzene sulfonic acid (TNBS). In addition, we present arguments indicating that individual TNP-specific helper T cells may cross-react with different TNP-peptides bound to identical class II molecules. Chemical treatment of antigen-presenting cells with TNCB or TNBS may thus result in a limited number of particularly repetitive immunodominant hapten epitopes. Immunodominant epitopes were also indicated by an overrepresentation of the TCR elements Vβ2 and Vα10 in I-A^b/TNP-specific T cells. Most importantly, however, we demonstrate that TNP attached to lysine 97 in the staphylococcal nuclease peptide 93–105 (i.e. a clearly “non-self” sequence) is able to prime mice for subsequent elicitation of contact sensitivity by TNCB in the absence of foreign protein. We take this to indicate that those TNP-peptide determinants defined by us as immuno-dominant are responsible for the induction of contact sensitivity to haptens.