Investigating the efficacy of amnion-derived compared with bone marrow–derived mesenchymal stromal cells in equine tendon and ligament injuries

Background aimsThis is the first study to compare the treatment of horse tendon and ligament injuries with the use of mesenchymal stromal cells (MSCs) obtained from two different sources: amniotic membrane (AMSCs) and bone marrow (BM-MSCs). The objective was to prove the ability of AMSCs to exert beneficial effects in vivo.MethodsFive million allogeneic frozen-thawed AMSCs or autologous fresh BM-MSCs were injected intralesionally in horses belonging to group A (51 horses) and group B (44 horses). The interval lesion/implantation was of 6–15 days for the AMSCs and 16–35 days for the BM-MSCs. Healing was assessed clinically and ultrasonographically. Follow-up was monitored for 2 further years from return to full work.ResultsNo significant adverse effects after MSCs treatment were seen in any of the horses studied, independent of the type of stromal cell implanted. All animals belonging to group A resumed their activities between 4–5 months after treatment, whereas animals of group B resumed their activities after 4–12 months. The rate of re-injury in horses treated with AMSCs is lower (4.00%) compared with the average observed when horses were treated with BM-MSCs (23.08%).ConclusionsThe possibility to inject allogeneic AMSCs in real time, before any ultrasonographic change occurs within the injured tendon and ligament, together with the higher plasticity and proliferative capacity of these cells compared with BM-MSCs, represents the main features of interest for this novel approach for the treatment of equine tendon diseases. An obvious active proliferative healing in the area injected with AMSCs makes these cells more effective than BM-MSCs.     

Differential biodiversity responses between kingdoms (plants,fungi, bacteria and metazoa) along an Alpine succession gradient

Biological diversities of multiple kingdoms potentially respond in similar ways to environmental changes. However, studies either compare details of microbial diversity across general vegetation or land use classes or relate details of plant community diversity with the extent of microbially governed soil processes, via physiological profiling. Here, we test the hypothesis of shared responses of plant and rhizosphere bacterial, fungal and metazoan biodiversities (especially across‐habitat β‐diversity patterns) along a disturbance gradient encompassing grazed to abandoned Alpine pasture, on acid soil in the European Central Alps. Rhizosphere biological diversity was inferred from eDNA fractions specific to bacteria, fungi and metazoans from contrasting plant habitats indicative of different disturbance levels. We found that soil β‐diversity patterns were weakly correlated with plant diversity measures and similarly ordinated along an evident edaphic (pH, C:N, assimilable P) and disturbance gradient but, contrary to our hypothesis, did not demonstrate the same diversity patterns. While plant communities were well separated along the disturbance gradient, correlating with fungal diversity, the majority of bacterial taxa were shared between disturbance levels (75% of bacteria were ubiquitous, cf. 29% plant species). Metazoa exhibited an intermediate response, with communities at the lowest levels of disturbance partially overlapping. Thus, plant and soil biological diversities were only loosely dependent and did not exhibit strictly linked environmental responses. This probably reflects the different spatial scales of organisms (and their habitats) and capacity to invest resources in persistent multicellular tissues, suggesting that vegetation responses to environmental change are unreliable indicators of below‐ground biodiversity responses.     

The effect of dexamethasone treatment on the expression of the regulatory genes of ketogenesis in intestine and liver of suckling rats

The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT 1) and mitochondrial HMG-CoA synthase. Dexamethasone produced a 2 fold increase in mRNA and activity of CPT I in intestine, but led to a decrease in mitt HMG-CoA synthase. In liver the mRNA levels and activity of both CPT I and mitt HMG-CoA synthase decreased. Comparison of these values with the ketogenic rate of both tissues following dexamethasone treatment suggests that mitt HMG-CoA synthase could be the main gene responsible for the regulation of ketogenesis in suckling rats. The changes produced in serum ketone bodies by dexamethasone, with a profile that is more similar to the ketogenic rate in the liver than that in the intestine, indicate that liver contributes more to ketone body synthesis in suckling rats. Two day treatment with dexamethasone produced no change in mRNA or activity levels for CPT I in liver or intestine. While mRNA levels for mitt HMG-CoA synthase changed little, the enzyme activity is decreased in both tissues.     

False-Positive Anti-Toxoplasma Fluorescent-Antibody Tests in Patients with Antinuclear Antibodies

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.     

Comparative Analysis of a Mitochondrial DNA Control Region Fragment Amplified from Three Adriatic Flatfish Species and Molecular Phylogenesis of Pleuronectiformes

The 5′-end of the mitochondrial control region of three Pleuronectiformes from the Adriatic Sea, Platichthys flesus italicus (Adriatic flounder), Solea vulgaris (common sole), and Solea kleini (Klein's sole), was sequenced and compared with that of six other flatfish species from the families Pleuronectidae and Bothidae. The sequence structures of all flatfishes appear very similar and consist of alternate short segments with low, medium, and high rates of nucleotide substitution. Four conserved 19-bp repeats occur at the beginning of the European and Adriatic flounder sequences. The common occurrence of tandem arrays in fish control regions could be related to a stable secondary structure. Molecular phylogenetic relationships among Pleuronectiformes agree well with previous morphologic data at all taxonomic levels. Molecular analyses could therefore contribute to resolving phylogenetic and taxonomic debates within the Pleuronectiformes. Received December 1, 1997; accepted June 30, 1998.     

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation,proteolytic activity,and cell surface localization

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.     

Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone

Glucocorticoids inhibit the proliferation, but induce the differentiation, of bone marrow stromal cells into osteoblast-like cells. The mechanisms, however, are still conjectural. Since insulin-like growth factors (IGFs) have profound effects on osteoblast growth and differentiation, it is possible that glucocorticoids exert their effects on bone marrow stromal cells in part via regulation of IGFs. Therefore, we analyzed the effects of dexamethasone (Dex) on the expression of IGF I and IGF II in cultured preosteoblastic normal human bone marrow stromal cells (HBMSC). Whereas Dex decreased the concentration of IGF I in the conditioned medium since early in the treatment, the concentration of IGF II was increased progressively as culture period lengthened. As the activities of IGF I and IGF II are regulated by the IGF binding proteins (IGFBPs), we analyzed the effects of Dex on the expression of IGFBPs. Dex increased IGFBP-2 in a time-dependent manner. The increase in IGFBP-2, however, was only to the same extent as that of IGF II at most, depending on the length of treatment. Therefore, the increase in IGFBP-2 would dampen, but not eliminate, the increased IGF II activities. By contrast, Dex decreased IGFBP-3 levels, the latter increasing the bioavailability of IGF II. Although IGFBP-4 mRNA levels were stimulated by Dex, IGFBP-4 concentration in the conditioned medium was unchanged as measured by RIA. IGFBP-5 and IGFBP-6 mRNA levels were decreased by Dex in a time-dependent fashion. IGFBP-5 protein level was also decreased 1–4 days after Dex treatment. IGFBP-1 mRNA was not detectable in HBMSC. These accumulated data indicate that Dex regulates IGF I and IGF II and their binding proteins differentially in normal human bone marrow stromal cells. The progressive increase in IGF II may contribute to Dex-induced cell differentiation. J. Cell. Biochem. 71:449–458, 1998. © 1998 Wiley-Liss, Inc.