Protein and chemotherapy profiling of extracellular vesicles harvested from therapeutic induced senescent triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is an aggressive subtype with relatively poor clinical outcomes and limited treatment options. Chemotherapy, while killing cancer cells, can result in the generation of highly chemoresistant therapeutic induced senescent (TIS) cells that potentially form stem cell niches resulting in metastases. Intriguingly, senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Our aim was to profile EVs harvested from TIS TNBC cells compared with control cells to identify a potential mechanism by which TIS TNBC cells maintain survival in the face of chemotherapy. TIS was induced and confirmed in Cal51 TNBC cells using the chemotherapeutic paclitaxel (PTX) (Taxol). Mass spectrometry (MS) analysis of EVs harvested from TIS compared with control Cal51 cells was performed using Ingenuity Pathway Analysis and InnateDB programs. We demonstrate that TIS Cal51 cells treated with 75 nM PTX for 7 days became senescent (senescence-associated ß-galactosidase (SA-ß-Gal) positive, Ki67-negative, increased p21 and p16, G2/M cell cycle arrest) and released significantly more EVs (P=0.0002) and exosomes (P=0.0007) than non-senescent control cells. Moreover, TIS cells displayed an increased expression of the multidrug resistance protein 1/p-glycoprotein. MS analysis demonstrated that EVs derived from senescent Cal51 cells contained 142 proteins with a significant increased fold change compared with control EVs. Key proteins included ATPases, annexins, tubulins, integrins, Rabs and insoluble senescence-associated secretory phenotype (SASP) factors. A fluorescent analogue of PTX (Flutax-2) allowed appreciation of the removal of chemotherapy in EVs from senescent cells. Treatment of TIS cells with the exosome biogenesis inhibitor GW4869 resulted in reduced SA-ß-Gal staining (P=0.04). In summary, this study demonstrates that TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, apoptosis and the SASP. These findings may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge.

Investigation of the possible functions of PACAP in human trophoblast cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the female reproductive system. Both the peptide and its receptors have been shown in the placenta but its role in placental growth, especially its human aspects, remains unknown. The aim of the present study was to investigate the effects of PACAP on invasion, proliferation, cell survival, and angiogenesis of trophoblast cells. Furthermore, cytokine production was investigated in human decidual and peripheral blood mononuclear cells. For in vitro studies, human invasive proliferative extravillous cytotrophoblast (HIPEC) cells and HTR-8/SVneo human trophoblast cells were used. Both cell types were used for testing the effects of PACAP on invasion and cell survival in order to investigate whether the effects of PACAP in trophoblasts depend on the examined cell type. Invasion was studied by standardized invasion assay. PACAP increased proliferation in HIPEC cells, but not in HTR-8 cells. Cell viability was examined using MTT test, WST-1 assay, and annexin V/propidium iodide flow cytometry assay. Survival of HTR-8/SVneo cells was studied under oxidative stress conditions induced by hydrogen peroxide. PACAP as pretreatment, but not as co-treatment, significantly increased the number of surviving HTR-8 cells. Viability of HIPEC cells was investigated using methotrexate (MTX) toxicity, but PACAP1-38 could not counteract its toxic effect. Angiogenic molecules were determined both in the supernatant and the cell lysate by angiogenesis array. In the supernatant, we found that PACAP decreased the secretion of various angiogenic markers, such as angiopoietin, angiogenin, activin, endoglin, ADAMTS-1, and VEGF. For the cytokine assay, human decidual and peripheral blood lymphocytes were separated and treated with PACAP1-38. Th1 and Th2 cytokines were analyzed with CBA assay and the results showed that there were no significant differences in control and PACAP-treated cells. In summary, PACAP seems to play various roles in human trophoblast cells, depending on the cell type and microenvironmental influences.

Progesterone-induced blocking factor (PIBF) and trophoblast invasiveness.

Controlled trophoblast invasion is a key process during human placentation and a prerequisite for successful pregnancy. Progesterone is one of the factors to regulate trophoblast invasiveness. Progesterone-induced blocking factor (PIBF) is a progesterone-induced molecule expressed by the trophoblast, and also by tumors. The distribution of PIBF within the first-trimester decidua coincides with sites of trophoblast invasion. Another molecule that has been implicated in the control of trophoblast invasiveness is placental leptin. Leptin inhibits the secretion of progesterone by cytotrophoblast. The aim of this work was to investigate the possible interaction of PIBF and leptins in regulating trophoblast invasion. Paraffin-embedded sections from normal first-trimester placentae, partial moles, complete moles, and choriocarcinomas were reacted with PIBF, leptin, and leptin receptor specific antibodies. PIBF-deficient trophoblast cells were generated using siRNA and leptin receptor was detected on Western blot analysis. The lysates of PIBF-treated cells were used for detecting leptin expression in a protein array. PIBF was expressed in both normal first-trimester villous trophoblast and in partial mole. Compared with this, PIBF expression was markedly decreased in complete mole and absent in choriocarcinoma. Neither leptinR nor leptin were detected in partial mole, whereas both of these molecules were present in complete mole and choriocarcinoma. Leptin receptor expression was upregulated in PIBF-deficient cells, while leptin expression was decreased in PIBF-treated cells. These data suggest that PIBF affects the expression of leptin and its receptor, and that PIBF expression is inversely related to trophoblast invasiveness.

Influence of the flocculating agent in sedimentation and performance of a non flocculent strain of Zymomonas mobilis in the ethanol production process.

The influence of the flocculating agent was studied in the performance (measured by microbial growth and ethanol production) of a non flocculent strain of Zymomonas mobilis, as well as the potentiality of the sedimentation process in the separation of the biomass from the fermentation broth. Among the flocculating agents studied, it was verified that both tannin and the polyelectrolyte yielded good results with regard to cellular performance. However, with regard to sedimentation tannin is more adequate to be used in processes involving Zymomonas mobilis.

Specific sensitization to Dermatophagoides pteronyssinus and cutaneous reactivity to histamine in Brazilian children.

The immediate hypersensitivity cutaneous test is a recognized method for etiologic diagnosis of allergic diseases. It is easily conducted, painless and can be quickly interpreted, making it deal for use with children. However, its low positivity in infants limits its use this population. The purpose of our study was to evaluate the skin reactivity to atopic and non-atopic children of different ages to increasing concentrations of histamine and to an extract of Dermatophagoides pteronyssinus (Dpt). Atopic children (A, n = 217) and non-atopic children (C, n = 198), under 15 years of age were grouped by age and administered skin prick tests. Increasing concentrations of histamine (1, 9, 81 and 243 mg/ml) and Dpt (10,000 AU/ml) were applied to the forearm. Skin reactions were recorded after 15 minutes and expressed as the mean of the largest wheal and its midpoint perpendicular diameter. Cutaneous reactions were observed in the early phases of life with a progressive increase observed in both groups. Significant differences were noted between the two groups starting at 48 months of life. As the concentration of histamine was increased, we observed a higher level of cutaneous reactivity in younger children. Comparative analysis of cutaneous reactivity to the histamine solution (1 mg/ml) and the Dpt extract showed parallelism between them and wheals with diameters larger than 3 mm after 12 months of age. We concluded that the skin prick test with Dpt is reliable in atopic children after 1 year of age. Reactivity to higher concentrations of histamine was observed after 3 months of age for all children. However, considering all age groups, the skin reactivity in atopic children to histamine (1 mg/ml) was more precocious when compared to controls.

Suspected adverse drug reactions in the Hungarian veterinary practice between 1982 and 1992.

The most characteristic adverse drug reactions observed after the use of home-manufactured or imported veterinary medicinal products in Hungary between 1982 and 1992 included toxicosis, severe local reaction, lack of efficacy, and presence of residues in the edible tissues of food animals. The causes of adverse drug reactions comprised manufacturing defects, lack of chemical or microbiological stability, misuse or extra label use, and neglecting the warnings in the directions for use. Collection and analysis of data relating to adverse drug reactions are indispensable for the prevention of similar cases. The authorities can facilitate data collection by supplying veterinary practitioners with the necessary report forms.

Dynamics of the site visit. Laboratory for the small group study of scientific administration.

Site visits are conducted by National Institutes of Health review groups when obstacles arise in peer appraisal of research projects. Because such visits provide a window into the state of a scientific community and present highly charged group dynamics that revolve about significant scientific issues, examples from the 1970s and 1980s were subjected to behavioral analysis. Selye's general adaptation syndrome was used to model the forms of coping behavior manifested by investigators targeted for a site visit. A Lewinian approach was taken to analyze the course and outcome of site visit team deliberations. The perspective was that of the NIH official responsible for the operation of the review process.

Comparative pharmacokinetic studies on dogs following systemic and intraportal gentamycin infusion.

Our results, which confirmed on the basis of our experiment that Gentamycin in the bile is extracted by the liver only after prolonged systemic infusion and only in low concentration, contrasted with intraportal infusion of the drug resulting in doses above MIC level after 30 minutes and the difference is highly significant after 60 minutes.

Fluctuations of the bile oxytetracyclin level on regional administration of the antibiotic.

The elimination of oxytetracyclin on regional administration was studied. The time course of the appearance of the antibiotic in different body fluids determined by a microbological assay method. On the data reported here it is assumed that regional antibiotic treatment can efficiently applied in bacterial cholangitis therapy.

Oxytetracycline concentration of bile after systemic and regional administration.

Oxytetracycline concentration of bile was determined after systemic and regional (intraportal) infusions. A single dose of 3.60 mg/kg of oxytetracycline was infused into the brachial vein of 8 dogs and into the portal vein of 9 dogs in 120 minutes. Bile samples taken at 30, 60, 120 and 180 minutes showed that the liver excretes into the bile higher concentrations of oxytetracycline in the case of intraportal infusion. The results suggest that treatment of inflammatory diseases of bacterial origin of the biliary tree might be more effective when the antibiotic is applied in intraportal infusion.

Body size and neonatal hypoglycemia in intrauterine growth retardation.

The relationship between blood glucose and body physique of 233 (42 hypo-and 191 normologycemic) intrauterine growth-retarded neonates was analyzed using different body measurements and indices of body proportions. Classification by combination of weight and length deficit for fetal age revealed that the disproportionately retarded infants (deficit in weight for age > 30%; deficit in length for age less than equal to 15%) were particularly prone to hypoglycemia. The lowest incidence of hypoglycemia was observed in the group with severe proportionate retardation (weight deficit for age > 30%; length deficit for age > 15%). Among the indices of body proportions ponderal index (W/L3), and percentage deviation from the expected weight for length turned out to be a sensitive predictor of the risk of hypoglycemia. The majority of hypoglycemia neonates were underweight for length and a considerable number of normoglycemic infants were overweight for length. These findings point to the significance of soft tissue wasting rather than low birth weight for gestational age itself, in the development and diagnosis of neonatal hypoglycemia. The significance of anthropometry in the classification of different types of intrauterine growth impairment, as well as in predicting specific hazards after birth is discussed.

Hallucinogen-tranquilizer interaction: its nature.

Study of the competition between hallucinogens and tranquilizers at cerebral synapses and on behavior in various species of animals indicates a continuum of effects from protection to dominance of tranquilizer toxicity as the dose of tranquilizer increases. Data on cat and monkey behavior, supplementing that on the rat, show that it is possible to arrive at a tranquilizer dose that can aggravate instead of protect, in accord with the competitive inhibitory nature of the interaction of hallucinogen and tranquilizer.