Retrospective analysis of local injection site adverse reactions associated with 230 allogenic administrations of bone marrow-derived mesenchymal stem cells in 164 horses.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) are frequently used in the treatment of musculoskeletal injuries. Fully characterised cells that are readily available for use is optimum. Allogenic BM-MSCs can satisfy the need for rapid treatment, however, their safety has been questioned. OBJECTIVES: Objectives were to characterise BM-MSCs from an adult donor horse, in vitro, and to identify and describe adverse reactions that occurred following their injection into other horses. We hypothesised that BM-MSCs capable of proliferation, differentiation and lacking MHC II from one donor could be implanted into another individual without significant adverse reactions and the frequency of adverse reactions in clinical cases would be similar to that previously reported for autologous BM-MSCs. STUDY DESIGN: Retrospective clinical study. METHODS: BM-MSCs were proliferated and characterised from one donor and cryopreserved for clinical use. Medical records for horses injected with allogenic BM-MSCs from this donor at a single hospital were used. After routine lameness exam, lesions were identified using diagnostic ultrasound or MRI. Post injection reaction was defined as increased pain, swelling, or heat at or near injection site, or increased lameness. Treatments required for each reaction were noted. RESULTS: BM-MSCs proliferated and underwent differentiation. Cells were found to be negative for MHC-II (<2%) and were viable after cryopreservation and shipping. Ten of 230 (4.35%) injections were noted to be associated with an adverse reaction. Adverse reactions occurred in synovial structures (n = 3) and in soft tissues (n = 7). MAIN LIMITATIONS: This investigation could underestimate the number and severity of reactions. Mild reactions, such as synovitis, may have been missed. Also, anti-inflammatory drugs could overshadow mild reactions, making them less likely to be detected. CONCLUSIONS: Fully characterised allogenic BM-MSCs originating from a single donor horse can be administered to horses with soft tissue injuries with a low rate of adverse reaction. The Summary is available in Portuguese - see Supporting Information.

Effects of pro-inflammatory cytokines on chondrogenesis of equine mesenchymal stromal cells derived from bone marrow or synovial fluid.

Mesenchymal stromal cells (MSCs) have the capacity to differentiate into cells of mesenchymal lineage, such as chondrocytes, and have potential for use in regeneration of equine articular cartilage. MSCs instilled intra-articularly would be exposed to the inflamed environment associated with equine osteoarthritis (OA), which may compromise their function and ability to heal a cartilaginous defect. The aim of this study was to assess the ability of equine adult MSCs to differentiate into chondrocytes when stimulated with pro-inflammatory cytokines. MSCs derived from equine bone marrow (BM) and from synovial fluid (SF) were cultured in chondrogenic induction medium containing transforming growth factor (TGF)-ß1. BM-derived MSCs (BMMSCs) and SF-derived MSCs (SFMSCs) were stimulated with 100 ng/mL interferon (IFN)-γ and 10 ng/mL tumor necrosis factor (TNF)-α. Chondrogenic differentiation was measured quantitatively with the glycosaminoglycan (GAG) assay and qualitatively by immunofluorescence (IF) for SOX-9, TGF-ß1, aggrecan and collagen II. The viability of equine MSCs was maintained in the presence of IFN-γ and TNF-α, but production of GAGs from both types of MSCs was decreased in stimulated medium. Exposure of BMMSCs to pro-inflammatory cytokines reduced the levels of SOX-9, TGF-ß1, aggrecan and collagen II, whereas exposure of SFMSCs to these cytokines reduced the levels of aggrecan only. These data suggest that pro-inflammatory cytokines do not affect proliferation of MSCs, but could inhibit chondrogenesis of MSCs.

Detection of Yersinia enterocolitica in food: an overview.

Yersinia enterocolitica is a gastrointestinal pathogen which causes yersiniosis, an illness characterized by diarrhea, ileitis, and mesenteric lymphadenitis. Y. enterocolitica is transmitted via the feco-oral route by the consumption of contaminated food or water. Several phenotypic and genotypic methods have been developed to reliably detect Y. enterocolitica in food. However, the source of infection of many recently reported foodborne outbreaks remains obscure. The detection of this pathogen in food is a challenging task, since it shares similarities with other enteric bacteria. The presence of other microorganisms in the food samples makes it even more difficult to identify this slow-growing pathogen. Therefore, the present-day emphasis is on the development of sensitive, easily automated methods suitable for in-situ detection, allowing quick and cost-effective characterization of food samples. This review summarizes and compares the currently available cultural, immunological, and molecular methods, particularly in relation to their specific merits or demerits when implemented for the detection of Y. enterocolitica in food.

In vitro analysis of equine, bone marrow-derived mesenchymal stem cells demonstrates differences within age- and gender-matched horses.

REASONS FOR PERFORMING THE STUDY: Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. OBJECTIVE: To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. STUDY DESIGN: Descriptive study of stem cell characteristics. METHODS: Equine BMMSCs from 6 horses were cultured in vitro and evaluated for viability, proliferation, osteogenesis, chondrogenesis, adipogenesis, cluster-of-differentiation markers and gene expression. RESULTS: Equine BMMSCs from all 6 donors demonstrated fibroblastic, cellular morphology, adherence to plastic and expression of cluster-of-differentiation markers. They varied in their rate of proliferation and trilineage differentiation. The equine BMMSCs of one of 6 donors demonstrated a higher rate of proliferation, enhanced ability for cell passaging and a more robust in vitro differentiation. Comparatively, equine BMMSCs from 2 donors demonstrated a lower rate of proliferation and lack of osteogenic and chondrogenic differentiation. CONCLUSION: The results of this study confirm that donor-to-donor variation in equine BMMSCs exists and this variation can be documented using in vitro assays. Subjective assessment suggests that the rate of proliferation tends to correlate with differentiation potential.

Transient Silencing of a Type IV P-Type ATPase, Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes.

Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs), p38, and p44/42. Additionally, glucose transporter-1 (GLUT1) was significantly upregulated; no changes in glucose transporter-4 (GLUT4) expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

Point mutations in the murine fumarylacetoacetate hydrolase gene: Animal models for the human genetic disorder hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive metabolic disease associated with point mutations in the human fumarylacetoacetate hydrolase (FAH) gene that disrupt tyrosine catabolism. An acute form of HT1 results in death during the first months of life because of hepatic failure, whereas a chronic form leads to gradual development of liver disease often accompanied by renal dysfunction, childhood rickets, neurological crisis, and hepatocellular carcinoma. Mice homozygous for certain chromosome 7 deletions of the albino Tyr; c locus that also include Fah die perinatally as a result of liver dysfunction and exhibit a complex syndrome characterized by structural abnormalities and alterations in gene expression in the liver and kidney. Here we report that two independent, postnatally lethal mutations induced by N-ethyl-N-nitrosourea and mapped near Tyr are alleles of Fah. The Fah(6287SB) allele is a missense mutation in exon 6, and Fah(5961SB) is a splice mutation causing loss of exon 7, a subsequent frameshift in the resulting mRNA, and a severe reduction of Fah mRNA levels. Increased levels of the diagnostic metabolite succinylacetone in the urine of the Fah(6287SB) and Fah(5961SB) mutants indicate that these mutations cause a decrease in Fah enzymatic activity. Thus, the neonatal phenotype present in both mutants is due to a deficiency in Fah caused by a point mutation, and we propose Fah(5961SB) and Fah(6287SB) as mouse models for acute and chronic forms of human HT1, respectively.

Parental alleles of an imprinted mouse transgene replicate synchronously.

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.

Localization of a new ferritin heavy chain sequence present in human brain mRNA to chromosome 11.

Two types of ferritin heavy (H) chain clones have been isolated from cDNA libraries of human fetal and adult brain: one corresponds to the ferritin H chain mRNA that is abundant in liver and is called "liver-like" brain cDNA; the other contains an additional 279 nucleotide (nt) sequence in the 3' untranslated region and is called brain ferritin H chain cDNA. To map the 279-nt sequence, polymerase chain reaction (PCR) amplification was carried out using DNA from rodent x human hybrid cell lines containing single human chromosomes as templates, and oligomeric primers homologous to the 3' end of the 279-nt sequence (primer A) and to a coding sequence just 5' to the 279-nt sequence. Significant PCR product of the size expected from analysis of the brain ferritin H chain cDNA clones and a genomic ferritin H chain clone (487 bp) was generated only from hybrid-cell DNA containing human chromosome 11. This PCR product and the "liver-like" brain cDNA (lacking the 279-nt sequence) both hybridized to chromosome 11 fragments that are known to define the well-characterized functional liver ferritin H chain gene and a putative pseudogene. Preliminary data indicate that primer A (and thus the 279-nt sequence) maps to the functional ferritin H chain gene fragments, but binding to the pseudogene has not been ruled out.

Dictyostelium discoideum contains a single-copy gene encoding a unique subtype of histone H1.

A Dictyostelium discoideum genomic library was screened using a degenerate oligodeoxyribonucleotide derived from the peptide, GPKAPT, obtained from the N terminus of purified histone H1. Two identical H1 clones were isolated. Comparative sequence data reveal a typical H1 three-domain structure with considerable homology to the globular domain of higher eukaryotic H1 histones, especially to plant H1 histones. Southern blot analysis shows that this gene is probably a single-copy gene, and suggests that any other H1 gene(s), if present, must be very different in sequence. Amino acid (aa) sequence comparison of the globular core of D. discoideum H1 to the consensus globular core reveals the absence of a 6-aa motif, GXGXXG, from D. discoideum. This motif matches the consensus for a putative nucleotide-binding loop, which is also absent in plant H1 histones like Arabidopsis thaliana, pea and wheat.

Detection and quantitation of the novel ferritin heavy chain message in human tissues.

We reported that in the fetal brain use of an alternate polyadenylation site in the premRNA for ferritin heavy chain generates two mature mRNAs of different lengths. The larger mRNA contains an additional 279 nucleotide sequence at the 3' untranslated region. Here we use Northern blot analysis and show that this mRNA is also present in other human tissues. Its relative concentration is in the order: brain > kidney > lung > skeletal muscle > pancreas > heart = placenta = liver.

Inaccessibility of the Euplotes telomere binding protein.

The telomere binding protein (TP) from the macronucleus of the ciliate Euplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequenced. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augmented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.

Differential processing of the ferritin heavy chain mRNA in human liver and adult human brain.

Northern blot analyses of the poly(A)+ RNAs from human brain and liver, using a human brain ferritin heavy chain (FTH) cDNA as the probe, shows the presence of two transcripts of 1.4 and 1.1 kb. The larger, 1.4-kb RNA, is expressed predominantly in the brain, whereas the smaller, 1.1 kb, is expressed abundantly in the liver. Screening of two normal human brain cDNA libraries yielded two types of human brain FTH cDNAs. One type corresponds to the previously characterized 1.1-kb RNA from liver and lymphocytes. The other is also identical to the previously characterized FTH cDNA except that it contains an additional 279-bp sequence at the 3' untranslated region. This additional sequence shows 94.1%, 62.5%, and 58.9% identity to the 3' flanking sequence of the human liver and mouse and rat FTH genomic clones, respectively. A fragment of a genomic clone containing the 279-bp sequence was also isolated and sequenced. These data suggest that differential processing of the primary transcript for the FTH mRNA in human brain and liver could generate two mature mRNAs of 1.4 and 1.1 kb. This could be due to the use of alternative polyadenylation sites in the pre-mRNA.

The human and bovine 14-3-3 eta protein mRNAs are highly conserved in both their translated and untranslated regions.

14-3-3 proteins form a highly conserved protein family whose members have been shown to activate tyrosine and tryptophan hydroxylases, inhibit protein kinase C and possess phospholipase A2 activity in vitro. We have isolated and analyzed a 14-3-3 protein cDNA clone (H14-3-3) from a human fetal brain cDNA library and found it to possess a high level of sequence identity with the bovine 14-3-3 eta protein cDNA in both the translated and untranslated regions, suggesting the presence of cis-regulatory elements in the untranslated regions of these mRNAs. The proteins encoded by these two cDNAs are 98.4% identical. Two different sized RNA species, approx. 1.9 and 3.5 kb in size that are expressed in a variety of tissues hybridize with this cDNA. However, only the 1.9 kb RNA is detected in the fetal brain. Northern blot analysis of poly(A)+ RNA isolated from eight different human tissues shows that 14-3-3 protein mRNAs are expressed in many tissues in the body. In agreement with previous reports, the highest abundance of RNA hybridizing with this cDNA is seen in the brain.

Predominance and tissue specificity of adenine methylation in rice.

Using 'A' and 'C' methylation-specific restriction enzymes, namely, MboI, Sau3AI, DpnI, MspI, and HpaII, total rice cv Basmati 370 DNA, repetitive DNAs, and a specific repeat sequence indicated an abundance of adenine methylation. Although cytosine methylation in 5'-CCGG-3' sequences suggested more CpC methylation than CpG, the 'C' methylation in sequence 5'-GATC-3' was comparatively less than 'A' methylation. Furthermore, the presence of adenine methylation was tissue specific; it was predominant in rice shoot DNA as compared to embryo DNA. This pattern was also observed in two other cultivars of rice, i.e., R-24 and Sona, and was again confirmed using a cloned probe of a specific repeat sequence. Besides the changes in adenine methylation, there was also a qualitative change in 5mC from CpG to CpC dinucleotides in these two tissue systems.

Identification of a dispersed MboI repeat family in five higher plant genomes.

Digestion of nuclear DNAs of five plants, namely Cucurbita maxima (red gourd), Trichosanthes anguina (snake gourd), Cucumis sativus (cucumber), Cajanus cajan (pigeon pea) and Phaseolus vulgaris (french bean) with the restriction endonuclease MboI yielded discrete size classes with molecular weights in the range of 0.5 to 5 kbp. The MboI digestion pattern of Cot 0.1 DNA in french bean is comparable with that of total DNA, indicating that these bands represented highly repeated DNA sequences. Cleavage of the DNAs with varying amounts of MboI indicated the dispersed nature of the repeat families. Southern hybridization studies using french bean highly repetitive DNA as a probe indicated more homology with repeats of pigeon pea and less homology with red gourd, snake gourd and cucumber repeats.