Effect of extracorporeal shock wave on proliferation and differentiation of equine adipose tissue-derived mesenchymal stem cells in vitro.

Mesenchymal stem cells are regarded as common cellular precursors of the musculoskeletal tissue and are responsible for tissue regeneration in the course of musculoskeletal disorders. In equine veterinary medicine extracorporeal shock wave therapy (ESWT) is used to optimize healing processes of bone, tendon and cartilage. Nevertheless, little is known about the effects of the shock waves on cells and tissues. Thus, the aim of this study was to investigate the influence of focused ESWT on the viability, proliferation, and differentiation capacity of adipose tissue-derived mesenchymal stem cells (ASCs) and to explore its effects on gap junctional communication and the activation of signalling cascades associated with cell proliferation and differentiation. ASCs were treated with different pulses of focused ESWT. Treated cells showed increased proliferation and expression of Cx43, as detected by means of qRT-PCR, histological staining, immunocytochemistry and western blot. At the same time, cells responded to ESWT by significant activation (phosphorylation) of Erk1/2, detected in western blots. No significant effects on the differentiation potential of the ASCs were evident. Taken together, the present results show significant effects of shock waves on stem cells in vitro.

Activity of CuDIPS against mammary cancer in C3H HeNCR mice.

A copper complex with superoxide dismutase mimetic activity, Cu(II)2 (3,5-diisopropylsalicylate)4 (CuDIPS), was tested for activity against mammary cancer in female C3H/HeNCR mice. Initiation of treatment at 20 or at 30 weeks of age had a limited beneficial effect on survival rates. Further, a small, but not statistically significant, difference in tumorigenesis was observed in comparisons between experimental and control groups of animals. Implications of these findings are discussed.

Evaluation of the anticancer activities of Tweens 20, 40 and 60 in SJL/J mice.

Tween 20 (polyoxyethylenesorbitan monolaurate), Tween 40 (polyoxyethylenesorbitan monopalmitate), and Tween 60 (polyoxyethylenesorbitan monostearate) were found to have little activity against development of reticulum cell sarcoma in SJL/J mice. These findings resulted in studies to explain the previous observation that Tween 80 (polyoxyethylenesorbitan monooleate) has anticancer activity in the SJL/J tumor system. Evidence is presented to suggest that the apparent difference may be related to the fatty acid component (saturated or unsaturated) of the Tween.

Combination therapy with CuDIPS, Tween 80 and cyclophosphamide: effects on survival and tumor incidence in SJL/J mice.

Combinations of Cu(II)2(3,5-diisopropylsalicylate)4 (CuDIPS), polyoxyethylenesorbitan monooleate (Tween 80) and cyclophosphamide (CY) were tested for their long-term effects on survival rate and incidence of reticulum cell sarcoma (RCS) in female SJL/J mice. Animals treated with CuDIPS + Tween 80 (72 hours later) exhibited a reduction in survival rate and an accelerated rate of tumorigenesis; the combination of CuDIPS + saline (24 or 72 hours later) proved of no value. Reductions in tumor incidence were achieved by treatment with Tween 80 + CY (72 hours later), Tween 80 + polyvinyl alcohol (PVA) or saline (72 hours later), or CY + PVA or saline (24 or 72 hours later); one of these combinations (CY + PVA or saline) also had a beneficial effect on survival rate. Results were most impressive among mice receiving the combination of CuDIPS + CY (24 hours later). Survival rate was not enhanced, but incidence of RCS was reduced to the lowest level (38%) of all experimental groups. Implications of these findings are discussed.

Pharmacokinetics of CuDIPS in mice.

Pharmacokinetic evaluation of Cu(II)2(3,5-diisopropylsalicylate)4(H2O)2 (CuDIPS), a copper complex with anticancer activity in mice, showed rapid absorption into the circulation after subcutaneous (SC) injection of a 0.50mumol, 0.75 mumol or 1.0 mumol dose. A direct relationship was observed between peak plasma copper concentration (one or two hours) and dose. Rate of plasma release also appeared to be dose related. The lowest dose of CuDIPS had no effect on plasma zinc levels; higher doses produced significant increases, but only at one hour. Rapid absorption into the blood (apparent peak in concentration, Tmax, at 0.5 hours) was also found in studies to determine distribution of 67Cu after SC injection of 67Cu and 14C double-labeled CuDIPS (50 mumol/kg body weight). Distribution to other tissues and organs occurred less rapidly with apparent peak 67Cu concentrations at three hours after administration in femur (bone marrow) and intestine, and at six hours in spleen and thymus.

Evaluation of combinations of CuDIPS, Tween 80 and cyclophosphamide as therapy for reticulum cell sarcoma in SJL/J mice.

Three agents, shown previously to have anticancer activity in the SJL/J tumor system, were tested in combinations for effects on survival rate and incidence of reticulum cell sarcoma at 52 weeks of age. Combined treatment with Cu(II)2(3,5-diisopropylsalicylate)4 (CuDIPS) and polyoxyethylenesorbitan monooleate (Tween 80) proved of no value. Indeed, inhibitory influence on tumorigenesis was diminished when Tween 80 was injected weekly at 24 hours or at 72 hours after weekly injections of CuDIPS. However, tumor incidence was reduced significantly, 90% (controls) to 7%, in mice treated weekly with cyclophosphamide (CY) at 24 hours after weekly injection of CuDIPS. Further, weekly administration of CY at 72 hours after weekly injection of Tween 80 resulted in a significant decrease in tumor incidence, 85% (controls) to 0%, and an increase in survival rate, 75% (controls) to 100%. Implications of these findings are discussed.

Treatment of reticulum cell sarcoma in SJL/J mice with Tween 80.

Tween 80 (polyoxyethylenesorbitan monooleate) was found to have anticancer activity in the SJL/J tumor system. Investigations to evaluate variables of importance in treatment revealed a loss of inhibitory influence when Tween 80 was administered by the subcutaneous rather than the intraperitoneal route of injection. Age at the onset of treatment was also a factor with significant reductions in incidences of reticulum cell sarcoma when treatment was initiated at 30 or at 40 weeks, but not at 20 weeks of age. Optimal inhibitory effects were achieved with concentrations of 5% Tween 80 (0.2 ml/mouse) or 10% Tween 80 (0.1 ml/mouse). No differences in inhibitory influence on SJL/J tumorigenesis were observed with variation in the frequency of treatment (biweekly, weekly or twice weekly injections).

Evaluation of the anticancer activity of CuDIPS in SJL/J mice.

A copper complex, Cu(II)2(3,5-diisopropylsalicylate)4 (CuDIPS) was found to have anticancer activity in the SJL/J tumor system. Toxic effects were eliminated by use of the subcutaneous rather than the intraperitoneal route of administration and, unlike Tween 80, polyvinyl alcohol was shown to have no effect on development of reticulum cell sarcoma when used as the carrier. Age at the onset of treatment was a factor of importance with significant reductions in tumor incidences when therapy was initiated at 20 or at 40 weeks, but not at 30 weeks of age. Maximal inhibitory effects on tumor incidence were achieved by CUDIPS concentrations of 0.35 mumol/mouse and 0.50 mumol/mouse. No difference in inhibitory influence was observed when mice were injected weekly rather than biweekly with the complex.

Effects of CuDIPS and tween 80 on SJL/J tumorigenesis.

Plasma copper and zinc levels were measured in SJL/J mice, an inbred strain characterized by a high, spontaneous incidence of reticulum cell sarcoma (RCS). The changes with age in mean concentrations of these metals were consistent with a physiological response that is required for remission of neoplasia. Treatment of SJL/J mice with a copper complex, Cu(II)(3,4-diisopropylsalicylate)2 (Cu 3,5-DIPS), dissolved in a 10% Tween 80-saline solution revealed a decrease in survival and decline in the incidence of RCS at 52 wk of age. The toxic effects of Cu 3,5-DIPS therapy appeared to be related to the intraperitoneal route of administration and to extracellular deposition of collagen. The inhibitory effect on tumor development was not related to Cu 3,5-DIPS. Rather, Tween 80 was found to be the factor of importance.

Effect of cyclophosphamide on development of reticulum cell sarcoma in SJL/J mice.

Studies to determine the effect of cyclophosphamide (CY) on the development of reticulum cell sarcoma (RCS) in SJL/J mice indicated a dependence on the duration of the test period. Age also appeared a factor of importance. Thus, a comparison of tumor incidences at 52 weeks of age showed maximal inhibition when CY was administered at 40 weeks, minimal inhibition when the drug was given at 30 weeks, and intermediate inhibition when treatment was initiated at 10 and 20 weeks. Consistent with these findings, long-term treatment of 40-week-old SJL/J mice with low doses of CY resulted in an increase in the mean survival time and in a reduction in the incidence of RCS.

Detection and characterization of a lymphocyte proliferating factor (LPF) in plasma of SJL/J mice infected with the LDH virus.

Infection of 25- to 30-week-old SJL/J mice with the lactate dehydrogenase (LDH) virus resulted in a transitory T-cell lymphocytopenia. However, in 40- to 45-week-old animals, the initial lymphocytopenia was followed by persistent elevations in the peripheral leukocyte counts (B and T lymphocytes). Studies indicated that this difference had its basis in a plasma factor, termed lymphocyte proliferating factor (LPF), which was induced by infection of older SJL/J mice with the LDH virus. Further experimentation revealed that plasma LPF was sensitive to storage at -60 degrees C, to heating at 90 degrees C, and to treatment with DNase. These results suggested an identification of LPF as DNA. This possibility was supported by the demonstration of LPF activity in association with DNA extracts (blood, spleen and lymph nodes from LDH virus-infected mice) which was inactivated by DNase.

Adenosine deaminase activity in peripheral blood cells from SJL/J mice.

Adenosine deaminase (ADA) activities were measured in peripheral blood cells of SJL/J mice, and compared with those of C57BL/6J animals. No association was observed between the levels of lymphocyte or erythrocyte ADA and immunologic abnormalities in SJL/J mice nor was evidence obtained to suggest a relationship between ADA activity and tumorigenesis in this strain.

Preparation of lymphocytes from small volumes of peripheral mouse blood.

A method was developed which allowed for the isolation of lymphocytes (95% of leukocytes in the final suspensions) from small volumes of peripheral mouse blood (38% recovery). The method proved of value for studies of murine lymphocytes which required biochemical analysis of samples from individual animals on a serial basis.