Enhanced<Emphasis Type="Italic">In vitro</Emphasis> chondrogenic differentiation of murine embryonic stem cells

Current approaches have focused on deriving ESCs differentiation into chondrocytes from a cell source of spontaneously formed intact mesoderm in EB formation, resulting in limited yield. Our study aimed at upregulating chondrogenic differentiation of murine ESCs by enhancing mesoderm formation. Specifically, culture of mESCs with conditioned medium from a human hepatocarcinoma cell line resulted in a cell population with a gene expression pattern similar to that of primitive streak/nascent mesoderm, including up-regulation of brachyury, goosecoid, nodal, and cripto. From this cell population, reducing the embryoid body formation time resulted in enhancement of chondrogenic differentiation as evidenced by larger Alcian blue-stained cartilage nodules, higher production of sulfated glycosaminoglycan matrix, the presence of well-organised type II collagen and type II collagen, aggrecan and sox-9 gene expression. In conclusion, we present here a new approach to the generation of chondrocytes from mESCs that enhances yields and, thus, could have widespread applications in cartilage tissue engineering.     

Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm

A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H₂O₂), sodium azide (NaN₃), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H₂O₂, BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN₃ in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames’ test, as well as that of the cytosolic enzyme GST.A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3 mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3 mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed.     

Patients with early rheumatoid arthritis exhibit elevated autoantibody titers against mildly oxidized low-density lipoprotein and exhibit decreased activity of the lipoprotein-associated phospholipase A<Subscript>2</Subscript>

Rheumatoid arthritis is a chronic inflammatory disease, associated with an excess of cardiovascular morbidity and mortality due to accelerated atherosclerosis. Oxidized low-density lipoprotein (oxLDL), the antibodies against oxLDL and the lipoprotein-associated phospholipase A₂ (Lp-PLA₂) may play important roles in inflammation and atherosclerosis. We investigated the plasma levels of oxLDL and Lp-PLA₂ activity as well as the autoantibody titers against mildly oxLDL in patients with early rheumatoid arthritis (ERA). The long-term effects of immunointervention on these parameters in patients with active disease were also determined. Fifty-eight ERA patients who met the American College of Rheumatology criteria were included in the study. Patients were treated with methotrexate and prednisone. Sixty-three apparently healthy volunteers also participated in the study and served as controls. Three different types of mildly oxLDL were prepared at the end of the lag, propagation and decomposition phases of oxidation. The serum autoantibody titers of the IgG type against all types of oxLDL were determined by an ELISA method. The plasma levels of oxLDL and the Lp-PLA₂ activity were determined by an ELISA method and by the trichloroacetic acid precipitation procedure, respectively. At baseline, ERA patients exhibited elevated autoantibody titers against all types of mildly oxLDL as well as low activity of the total plasma Lp-PLA₂ and the Lp-PLA₂ associated with the high-density lipoprotein, compared with controls. Multivariate regression analysis showed that the elevated autoantibody titers towards oxLDL at the end of the decomposition phase of oxidation and the low plasma Lp-PLA₂ activity are independently associated with ERA. After immunointervention autoantibody titers against all types of oxLDL were decreased in parallel to the increase in high-density lipoprotein-cholesterol and high-density lipoprotein-Lp-PLA₂ activity. We conclude that elevated autoantibody titers against oxLDL at the end of the decomposition phase of oxidation and low plasma Lp-PLA₂ activity are feature characteristics of patients with ERA, suggesting an important role of these parameters in the pathophysiology of ERA as well as in the accelerated atherosclerosis observed in these patients.     

Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity

The therapeutic application of siRNA shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the nonviral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line showed that siRNA conjugated to cholesterol, TAT(48-60), and penetratin, but not siRNA alone, achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localization within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6 h. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown, while penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression, while the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA-mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.     

Comparative analysis of peripheral and localised cytokine secretion in glioblastoma patients

BACKGROUND: Malignant gliomas are the most common primary brain tumours of both children and adults. The unique aspects of their biology and anatomic site render them refractory to conventional therapeutic strategies such as surgery and chemotherapy. Significant attention has been given, recently, to immunotherapy which, although promising in preclinical studies, has not yet enhanced the survival of patients with glioblastomas. METHODS: To further understand the immunobiology of glioblastomas in clinical settings, we examined the secretion of four main cytokines in the peripheral blood and in primary cell cultures of 33 human glioblastoma patients. An ELISPOT methodology was used for the first time to examine Th1, and Th2 cytokine secretion from both peripheral lymphocytes and glioma tumour cells. RESULTS: Th1 cytokines (tumour necrosis factor (TNF-alpha), interferon (IFN-gamma) were markedly reduced compared to control levels (P=0.01 and P<0.001, respectively), whereas in contrast, Th2 (interleukin (IL)-4 and IL-10) were strongly expressed in both peripheral lymphocytes and glioma cell cultures (P=0.05 and P<0.001, respectively). CONCLUSION: This pattern indicates an 'immunosuppressive status' in glioblastomas which is related to their origination and the evasion of glioma cells from immune surveillance and could account for the failure of immunotherapy in such tumours. Furthermore, ELISPOT methodology can be used for monitoring of cytokine secretion from tumour cells, in addition to the well-established peripheral cytokine secretion.     

The effects of three months of aerobic and strength training on selected performance- and fitness-related parameters in modern dance students

The purpose of the present study was to assess the effects of a 12-week aerobic and muscular strength training program on selected dance performance and fitness-related parameters in modern dance students. The sample consisted of 32 men and women (age 19 +/- 2.2 years) who were randomly assigned into exercise (n = 19) and control (n = 13) groups. Anthropometric and flexibility assessments, treadmill ergometry, strength measurements, and- on a separate day-a dance technique test were conducted pre- and postexercise training in both groups. After the end of the program, the exercise group revealed significant increases in dance (p < 0.02), VO(2)max (p < 0.04), flexibility (p < 0.01), and leg strength (p < 0.001) tests compared to controls. It is concluded that in modern dance students (a) a 3-month aerobic and strength training program has positive effects on selected dance performance and fitness-related parameters, (b) aerobic capacity and leg strength improvements do not hinder dance performance as studied herein, and (c) the dance-only approach does not provide enough scope for physical fitness enhancements.     

A synthetic method for diversification of the P1' substituent in phosphinic dipeptides as a tool for exploration of the specificity of the S1' binding pockets of leucine aminopeptidases

A novel, general, and versatile method of diversification of the P1' position in phosphinic pseudodipeptides, presumable inhibitors of proteolytic enzymes, was elaborated. The procedure was based on parallel derivatization of the amino group in the suitably protected phosphinate building blocks with appropriate alkyl and aryl halides. This synthetic strategy represents an original approach to phosphinic dipeptide chemistry. Its usefulness was confirmed by obtaining a series of P1' modified phosphinic dipeptides, inhibitors of cytosolic leucine aminopeptidase, through computer-aided design basing on the structure of homophenylalanyl-phenylalanine analogue (hPheP[CH(2)]Phe) bound in the enzyme active site as a lead structure. In this approach novel interactions between inhibitor P1' fragment and the S1' region of the enzyme, particularly hydrogen bonding involving Asn330 and Asp332 enzyme residues, were predicted. The details of the design, synthesis, and activity evaluation toward cytosolic leucine aminopeptidase and aminopeptidase N are discussed. Although the potency of the lead compound has not been improved, marked selectivity of the synthesized inhibitors toward both studied enzymes was observed.     

Mechanical stress induces DNA synthesis in PDL fibroblasts by a mechanism unrelated to autocrine growth factor action

Periodontal regeneration is thought to require the proliferation of stress-sensitive periodontal ligament (PDL) fibroblast cells. The influence of physiological amounts of mechanical stretching on the DNA synthesis potential of human PDL fibroblasts was examined by means of an established, simple in vitro system of stretch application. A significant increase in the relative levels of incorporation of tritiated thymidine was observed in cultures stretched for 1–6 h. Neutralising antibodies for platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-β) did not blunt the DNA synthesis induction. This mitogenic response to stretch appears to be independent of an autocrine mechanism involving growth factors in general, because stretch-conditioned medium, when transferred to non-stretched fibroblasts, did not mimic the mitogenic effect of stretch.