Modulation of equine articular chondrocyte messenger RNA levels following brief exposures to recombinant equine interleukin-1beta.

The effect of recombinant equine IL-1beta (EqIL-1beta) on steady-state mRNA levels of equine articular chondrocytes in high-density monolayer culture was investigated using a customized cDNA array analysis. Total RNA samples isolated from chondrocytes cultured in media alone or with the addition of 1 ng/ml EqIL-1beta for 1-, 3-, and 6-h durations of exposure were reverse transcribed, radiolabeled, and hybridized to a customized 380-target cDNA array. Means of duplicate log base 2 transformed hybridization signals were normalized to equine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mean signal intensities. Differentially expressed transcripts were identified using a two-stage mixed linear analysis of variance model (Statistical Analysis Software, Cary, NC). A time-dependent pattern was observed in the number of transcripts increased > or =two-fold in response to EqIL-1beta after 1, 3 and 6h (1, 2 and 109 transcripts, respectively). At 6 h of EqIL-1beta stimulation, signal intensities for 88 cDNA targets with purported function in processes related to cell cycle, intracellular signaling, transcription, translation, extracellular matrix turnover, and inflammation, as well as a number of cDNAs lacking homology to previously reported cDNA sequences, were increased >two-fold and were associated with p<0.05. Principal component analysis identified a vector component ( approximately 10% of the total variation) corresponding to a potential EqIL-1beta co-regulation of cell cycle associated gene transcription. These results support and expand our existing comprehension of the complex role of IL-1 in modulated chondrocyte gene expression and suggest the involvement of specific target gene up-regulation and activation of downstream inflammatory cascade mediators. This study adds to the current understanding of the molecular events associated with an IL-1 induced inflammation and pathobiologic processes that may be associated with the development of equine osteoarthritis.

Reduced birth defects caused by maternal immune stimulation in diabetic ICR mice: lack of correlation with placental gene expression.

Diabetes mellitus alters placental structure and function, events that may be related to embryopathy. Three different methods of maternal immune stimulation that modulate placental function and that result in approximately equal reduction of diabetic embryopathy were studied: footpad injection with complete Freund's adjuvant, intraperitoneal injection with granulocyte-macrophage colony stimulating factor (GM-CSF), or intraperitoneal injection with interferon-gamma (IFN-gamma). A gene microarray was then used to examine expression of 151 placental genes. We hypothesized that maternal immune stimulation may overcome an embryopathy-inducing effect of diabetes on placenta, that might be detected by a shared profile of placental gene expression changes induced by the different immune stimulation procedures. However, the immune stimulation that caused the greatest reduction in birth defect incidence, IFN-gamma, did not change the placental gene expression profile as compared to control or diabetes. Complete Freund's adjuvant and GM-CSF significantly changed placental gene expression relative to control or diabetes, but differentially affected such genes. No common pattern of improved cytokine, cell-cycle, apoptotic, transcription factor, or other gene expression was identified, that might explain the ability of this procedure to reduce birth defects. These data suggest that maternal immune stimulation reduces birth defects in diabetic mice by a mechanism independent of placenta.

Reduced birth defects caused by maternal immune stimulation may involve increased expression of growth promoting genes and cytokine GM-CSF in the spleen of diabetic ICR mice.

Maternal immune stimulation in mice decreases fetal abnormalities caused by diverse etiologies. Growth factors produced by activated immune cells were proposed to be key mediators that may exert their effects on placenta or embryo. Diabetes disrupts the secretion of cytokines, which may associate with diabetic embryopathy. Three different methods of maternal immune stimulation that result in approximately equal reduction of diabetic embryopathy were used in the present studies: footpad injection with complete Freund's adjuvant (CFA), intraperitoneal (i.p.) injection with granulocyte-macrophage colony-stimulating factor (GM-CSF), or i.p. injection with interferon-gamma (IFN-gamma). A gene microarray was then used to examine expression of a selected gene panel in splenic leukocytes. We hypothesized that maternal immune stimulation may act by overcoming altered gene expression patterns of immune cells in the diabetic mice, which partially mitigates the teratogenic effect of diabetes. It further seemed likely that a shared profile of splenic gene expression changes induced by the different immune stimulation procedures may be identified and related to reduced teratogenesis. The three procedures produced a common altered gene expression profile. Significantly affected genes included apoptotic and anti-apoptotic genes, and genes controlling cellular proliferation, and likely reflect a state of immune activation. The GM-CSF gene was up-regulated by all three immune stimulation procedures. The protein product of this gene regulates placental development, and was recently associated with reduced cleft palate in immune-stimulated pregnant mice after exposure to urethane. These data suggest that further studies of GM-CSF as mediator of reduced birth defects in teratogen-challenged, immune-stimulated mice are warranted.

Maternal immune stimulation reduces both placental morphologic damage and down-regulated placental growth-factor and cell cycle gene expression caused by urethane: are these events related to reduced teratogenesis?

Activation of the maternal immune system in mice decreased cleft palate caused by the chemical teratogen, urethane. Direct and indirect mechanisms for this phenomenon have been suggested, including maternal macrophages that cross the placenta to find and eliminate pre-teratogenic cells, or maternal immune proteins (cytokines) that cross placenta to alleviate or partially alleviate toxicant-mediated effects in the developing fetus. A third mechanism to explain improved fetal developmental outcome in teratogen-challenged pregnant mice might involve beneficial effects of immune stimulation on the placenta. In the present experiments, urethane treatment altered placental morphology and impaired placental function, the latter indicated by down-regulated activity of cell cycle genes and of genes encoding cytokines and growth factors. Maternal immune stimulation with either Freund's complete adjuvant (FCA) or interferon-gamma (IFNgamma) reduced morphologic damage to the placenta caused by urethane and normalized expression of several genes that were down-regulated by urethane. Urethane treatment also shifted placental cytokine gene expression toward a T cell helper 1 (Th1) profile, while immunostimulation tended to restore a Th2 profile that may be more beneficial to pregnancy and fetal development. These data suggest that the beneficial effects of maternal immune stimulation on fetal development in teratogen-exposed mice may, in part, result from improved placental structure and function.

Immune stimulation in urethane-exposed pregnant mice increases expression level of spleen leukocyte genes for TGFbeta3 GM-CSF and other cytokines that may play a role in reduced chemical-induced birth defects.

For unknown reasons, activation of the maternal immune system in mice reduces morphologic defects caused by diverse teratogenic agents. Such immune stimulation of the maternal animal has been correlated with altered cytokine mRNA transcripts in the placenta (e.g., TGFbeta2) as well as in fetal target tissues of the teratogen (e.g., TNFalpha in fetal heads of cyclophosphamide-exposed pregnant mice). The teratogen urethane was reported to down-regulate cell cycle and apoptotic regulatory genes in fetal mouse heads that displayed cleft palate, an effect that was also reversed by maternal immune stimulation. The molecular mediators of the above phenomena have not been identified, however proteins synthesized and released by activated maternal immune cells have been suggested. The present studies therefore evaluated the effects of maternal immune stimulation in urethane-exposed mice on thymus and spleen leukocyte populations, in an attempt to identify events that may correlate with protection against birth defects. Immune stimulation did not change the hypocellularity of the thymus nor the altered T cell differentiation caused by urethane. A limited and transient increase in splenic leukocyte number, including increased T and B lymphocytes and macrophages, was caused by immune stimulation and was not felt to play a significant role in reduced morphologic defects. Urethane treatment caused down-regulated expression of numerous genes involved in cell-cycle control, while maternal immune stimulation caused comparative up-regulation of many of these genes. Coordinate shifts in gene expression by treatment were evaluated using principal component analysis, which identified several growth factor genes that were differentially expressed in mice receiving urethane alone as compared to urethane plus immune stimulation. Up-regulated expression of TGFbeta3 and GM-CSF genes, in particular, was observed in leukocytes of urethane-exposed mice receiving immunostimulation. Interestingly, the cytokine products of these two genes were recently suggested as growth factors that may be related to reduction of fetal defects caused by teratogens. Genes for growth factors IGF-I, IGF-II and IL-2 were also identified as differentially expressed in urethane vs. urethane+immune stimulation mice, suggesting that these proteins should be considered for a potential contributing effect to reduced birth defects caused by immunostimulation.

Maternal immune stimulation in mice decreases fetal malformations caused by teratogens.

For unknown reasons, non-specific stimulation of the maternal immune system in pregnant mice has what appears to be a broad-spectrum efficacy for reducing birth defects. Immune stimulation by diverse procedures has proven effective, including footpad injection with Freund's complete adjuvant (FCA), intraperitoneal (IP) injection with inert particles to activate resident macrophages, IP injection with attenuated Bacillus Calmette-Guerin (BCG), and intrauterine injection with allogeneic or zenogeneic lymphocytes. Morphologic lesions that were significantly reduced included cleft palate and associated craniofacial defects, digit and limb defects, tail malformations, and neural tube defect (NTD). Teratogenic stimuli to induce these lesions included chemical agents (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], cyclophosphamide [CP], and valproic acid [VA]), physical agents (X-rays, hyperthermia), and streptozocin (STZ)-induced diabetes mellitus. Limited information is available regarding mechanisms by which such immune stimulation reduced fetal dysmorphogenesis. The collective literature suggests the possibility that immunoregulatory cytokines of maternal origin may be the effector molecules in this phenomenon.

Functional and ultrastructural cell pathology induced by fuel oil in cultured dolphin renal cells.

Investigations were undertaken to elucidate in a marine mammal renal cell culture system the toxicity and some of the mechanisms of cytopathology in a standardized preparation following exposure to No. 1 fuel oil. Cell survivability of a cultured SP1K renal cell line from the Atlantic spotted dolphin Stenella plagiodon was reduced in a dose-dependent manner after a 12-h exposure to fuel oil. Early morphologic changes reflecting cytotoxicity, as revealed by transmission electron microscopy, included enlarged rough endoplasmic reticula, cytoplasmic vacuolization, and degenerative cytoplasmic inclusions, but mitochondria remained resistant. Assessment of extracellular proton loss by microphysiometry of cultured cells revealed fuel oil-induced enhancement of proton loss that was dependent upon both protein kinase C and renal epithelial Na(+)/H(+) counter-transport functioning, as the specific inhibitors H-7 and amiloride reduced this stimulatory petroleum effect. Cell cycle progression and apoptosis (programmed cell death) were studied in dolphin renal cells exposed to fuel oil for 12, 24, and 48 hours. The toxicant increased the percentage of cells in GO/GI phase and decreased the percentage of cells in S phase starting after 24 hours. The number of cells undergoing early apoptosis was also increased after 24 hours.

[A preclinical study of the embryotoxic properties of cucumarioside and its effect on adult generative function]. / Doklinicheskoe issledovanie émbriotoksicheskikh svoistv kukumariozida i ego vliianiia na generativnuiu funktsiiu vzroslykh osobei.

The preclinical study of embryotoxicity of a new immunomodulator cucumariosid was carried out. Cucumariosid at long-term use exerts no effect on the generative function of rats, the general condition of pregnant females and the postnatal development of the offspring, possesses no teratogenic action. The use of the drug in the preimplantation period of pregnancy and the implantation period produces the contraceptive effect.

[Dependence of the embryonic development of Rana temporaria on incubation conditions in the period from insemination to the first cleavage division]. / Zavisimost' émbrional'nogo razvitiia Rana temporaria ot uslovii inkubatsii v period ot osemeneniia do pervogo deleniia drobleniia.

The R. temporaria embryos at stages from insemination to the 1st cleavage division were incubated for 1 tau 0 in different saline solutions. The subsequent development in the control and after all kinds of treatment proceeded in the same conditions (in settled tap water and at 19-20 degrees). After incubation in the solutions containing potassium and sodium chlorides, bromides or iodides in a concentration of 30 mM, which decreased the amplitude of activation potential and the value of transmembrane currents, the morphogenetic movement during neurulation and, more rarely, gastrulation were accelerated (coefficient of relative velocity 1.16-1.38). After treatment with calcium chloride, gluconate and choline chloride in the same concentration, the morphogenetic processes were delayed (0.85-0.95).

[Effect of local anesthetics and phorbol ester on intracellular pH and rate of development of sea urchin embryos]. / Vliianie lokal'nykh anestetikov i forbolovogo éfira na vnutrikletochnyi pH i temp razvitiia zarodyshei morskikh ezhei.

The unfertilized eggs of sand-dollars (Scaphechinus mirabilis) were treated with local anesthetics (novocaine, lidocaine, trimecaine) or phorbol ester for 10 min. The treatment with the local anesthetics increased pH of the cytoplasm in the unfertilized eggs (pHc) and changed the value of the pHc increment in response to fertilization, as compared with the control eggs. In the treated embryos, the onset of immigration of the primary mesenchyme cells was delayed but the hatching was accelerated. The phorbol ester treatment rapidly accelerated development from gastrulation on and practically did not affect the preceding stages.

[Development of isolated loach blastoderm during cultivation in different salt media]. / Razvitie izolirovannykh blastoderm v'iuna pri kul'tivirovanii v raznykh solevykh sredakh.

The loach blastoderms were isolated from the yolk during the periods of cleavage and blastulation (5--14.5 tau0) and incubated in various isotonic saline media which differed by the content of K+ and Na+. The survival of blastoderms upon the short-term incubation (up to 60 tau0) varied insignificantly in all the media and upon the longer one (up to 120 tau0) was the maximal in the medium with isotonic content of K+ and Na+ and the minimal in the medium with the low content of K+. The cell proliferation upon incubation in the medium with the low content of K+ was inhibited when the blastoderms were isolated at the stage of 8 tau0 and suffered no changes at the stage of 14 tau0. The ability of differentiation manifested itself earlier and the percentage of differentiated embryos was higher in the media with the high content of K+. The content of K+ in the blastoderms isolated at the stage of 7 tau0 changes with its content in the incubation medium; upon isolation at the stages of 8--9 tau0 to blastoderms accumulate K+ irrespective of its content in the medium and at the stage of 10 tau0 maintain the constant level of K+. Hence, the development of isolated blastoderms depends on the content of K+ in the medium and this dependence reduces with the age.