Incremento de las citoquinas proteína quimiotáctica de monocitos-1 (MCP-1) y proteína inflamatoria macrofágica-1beta (MIP-1beta) en líquido cefalorraquídeo de pacientes con esclerosis lateral amiotrófica / Increased cerebrospinal fluid levels of cytokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1Beta (MIP-1Beta) in patients with amyotrophic lateral sclerosis

INTRODUCCIÓN: En la esclerosis lateral amiotrófica (ELA) se ha descrito recientemente la presencia de neuroinflamación. Sin embargo, no se ha definido el rol de citoquinas proinflamatrorias como la proteína quimiotáctica de monocitos-1 (MCP-1) y la proteína inflamatoria macrofágica-1beta (MIP-1beta) en ELA. En este estudio evaluamos niveles de MCP-1 y MIP-1beta en líquido cefalorraquídeo (LCR), analizando su participación en la duración y gravedad de la ELA. MÉTODOS: En 77 pacientes con ELA definida y 13 sujetos controles, se comparó el nivel de citoquinas MCP-1 y MIP-1beta en LCR. Se analizaron estos niveles con relación a la duración de la ELA (< 12 meses vs. > 12 meses) y a la gravedad de esta determinada mediante el puntaje obtenido al ingreso en la escala funcional estratificada de la ELA (< 30 puntos vs. > 30 puntos). RESULTADOS: En los 77 pacientes con ELA, se encontraron aumentados los niveles de MIP-1beta (4,69 pg/ml vs.10,68 pg/ml, p < 0,0001) y MCP-1 (160,95 pg/ml vs.234,89 pg/ml, p = 0,011) en comparación con sujetos controles. No se observó diferencia de los niveles de estas citoquinas con la duración o la gravedad de la enfermedad. Sin embargo, observamos una correlación positiva significativa entre MIP-1beta y MCP-1 en pacientes con ELA. CONCLUSIONES: El aumento de MIP-1beta y MCP-1 sugiere que estas citoquinas parecen tener un efecto sinérgico en la patogénesis de la ELA. Sin embargo, en nuestra cohorte no se asociaron con la duración o la gravedad de la ELA

INTRODUCTION: Neuroinflammation has recently been described in amyotrophic lateral sclerosis (ALS). However, the precise role of such proinflammatory cytokines as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1beta (MIP-1Beta) in ALS has not yet been determined. In this study, we determined cerebrospinal fluid (CSF) MCP-1 and MIP-1beta levels and assessed their association with the duration and severity of ALS. METHODS: Concentrations of MCP-1 and MIP-1beta were determined in the CSF of 77 patients diagnosed with ALS and 13 controls. Cytokine levels were analysed in relation to ALS duration (< 12 months vs. > 12 months) and severity (< 30 points vs. > 30 points on the ALS Functional Rating Scale administered at hospital admission). RESULTS: Higher CSF MIP-1Beta (10.68 pg/mL vs.4.69 pg/mL, P < .0001) and MCP-1 (234.89 pg/mL vs.160.95 pg/mL, P = .011) levels were found in the 77 patients with ALS compared to controls. There were no differences in levels of either cytokine in relation to disease duration or severity. However, we did observe a significant positive correlation between MIP-1Beta and MCP-1 in patients with ALS. CONCLUSIONS: The increase in MIP-1Beta and MCP-1 levels suggests that these cytokines may have a synergistic effect on ALS pathogenesis. However, in our cohort, no association was found with either the duration or the clinical severity of the disease

Increased cerebrospinal fluid levels of cytokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1ß (MIP-1ß) in patients with amyotrophic lateral sclerosis. / Incremento de las citoquinas proteína quimiotáctica de monocitos-1 (MCP-1) y proteína inflamatoria macrofágica-1ß (MIP-1ß) en líquido cefalorraquídeo de pacientes con esclerosis lateral amiotrófica.

INTRODUCTION: Neuroinflammation has recently been described in amyotrophic lateral sclerosis (ALS). However, the precise role of such proinflammatory cytokines as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1ß (MIP-1ß) in ALS has not yet been determined. In this study, we determined cerebrospinal fluid (CSF) MCP-1 and MIP-1ß levels and assessed their association with the duration and severity of ALS. METHODS: Concentrations of MCP-1 and MIP-1ß were determined in the CSF of 77 patients diagnosed with ALS and 13 controls. Cytokine levels were analysed in relation to ALS duration (<12months vs. >12months) and severity (<30points vs. >30points on the ALS Functional Rating Scale administered at hospital admission). RESULTS: Higher CSF MIP-1ß (10.68pg/mL vs. 4.69pg/mL, P<.0001) and MCP-1 (234.89pg/mL vs. 160.95pg/mL, P=.011) levels were found in the 77 patients with ALS compared to controls. There were no differences in levels of either cytokine in relation to disease duration or severity. However, we did observe a significant positive correlation between MIP-1ß and MCP-1 in patients with ALS. CONCLUSIONS: The increase in MIP-1ß and MCP-1 levels suggests that these cytokines may have a synergistic effect on ALS pathogenesis. However, in our cohort, no association was found with either the duration or the clinical severity of the disease.

Human hematopoietic progenitor cells grow faster under rotational laminar flows.

We report significant and reproducible growth acceleration of human progenitor cells when exposed to rotational flow when compared with stationary conditions. Nonenriched CD34+ umbilical cord derived human hematopoietic progenitor cells were cultured in Petri dishes located at different radial distances with respect to the central axis of a rotating platform. Growth dynamics under 3 or 5 rpm agitation was compared against that observed under typical stationary conditions. Cells cultured at 3 or 5 rpm exhibited (a) the absence of a latency phase, (b) an increase in final cell concentrations by 54-58.5%, and (c) reduced doubling time in their exponential phase by 12-16% in comparison with stationary culture. Cells grown under rotational agitation were confirmed to remain CD34+ by PCR. These results document a significant positive effect of exposure to laminar flow fields on the growth of human hematopoietic progenitor cells.

Estrogen mitogenic action. I. Demonstration of estrogen-dependent MTW9/PL2 carcinogen-induced rat mammary tumor cell growth in serum-supplemented culture and technical implications.

The MTW9/PL cell line was established by our laboratory in culture from the carcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a Wistar-Furth (W/Fu) rat. This tumor formed estrogen, androgen, and progesterone responsive tumors in W/Fu rats (Sirbasku, D. A., Cancer Res. 38:1154-1165; 1978). It was later used to derive the MTW9/PL2 cell population which was also estrogen-responsive in vivo (Danielpour, D., et al., In Vitro Cell. Dev. Biol. 24:42-52; 1988). In the study presented here, we describe serum-supplemented culture conditions in which the MTW9/PL2 cells demonstrate > or = 80-fold steroid hormone growth responses. All sera used were steroid hormone-depleted by charcoal-dextran treatment at 34 degrees C. The studies were done with horse serum as well as serum from other mammalian species. The growth of the MTW9/PL2 cells was biphasic in response to hormone-depleted serum. Concentrations of < or = 5% (v/v) promoted optimum growth. Above this concentration, serum was inhibitory. Concentrations > or = 40% (v/v) inhibited growth altogether. Addition of 1.0 x 10(-13)-1.0 x 10(-8) M 17beta,-estradiol (E2) reversed the inhibition completely. At 1.0 x 10(-8) M, estrone, estriol and diethylstilbestrol promoted growth as well as E2. Testosterone and dihydrotestosterone promoted growth only at > or = 10(-7) M. Progesterone was effective only at > or = 10(-6) M. Cortisol was ineffective. Labeled-hormone-binding analysis and Western immunoblotting documented that MTW9/PL2 cells had estrogen and progesterone receptors but not androgen or cortisol receptors. Estrogen treatment of MTW9/PL2 cells induced a concentration and time dependent increase in progesterone receptors. We conclude (1) the MTW9/PL2 population is the first highly steroid hormone-responsive rat mammary tumor cell line to be established in culture from a carcinogen-induced tumor, and (2) sera from a number of species including horse, rat and human contain an inhibitor which mediates estrogen sensitive MTW9/PL2 cell growth in culture.

Estrogen mitogenic action. III. is phenol red a "red herring"?

The reported estrogenic action of phenol red and/or its lipophilic contaminants has led to the widespread use of indicator-free culture medium to conduct endocrine studies in vitro. Because we have recently developed methods to measure large-magnitude estrogen effects in the tissue culture medium containing phenol red, we concluded that the indicator issue required further evaluation. To do this, we selected nine estrogen receptor positive (ER+) cell lines representing four target tissues and three species. We investigated phenol red using five different experimental protocols. First, 17beta-estradiol (E2) responsive growth of all nine ER+ cells lines was compared in the medium with and without the indicator. Second, using representative lines we asked if phenol red was mitogenic in the indicator-free medium. The dose-response effects of phenol red were compared directly to those of E2. Third, we asked if tamoxifen-inhibited growth equally in phenol red-containing and indicator-free medium. This study was based on a report indicating that antiestrogen effects should be seen only in phenol red-containing medium. Fourth, we asked if phenol red displaced the binding of 3H-E2 using ERK intact human breast cancer cells. Fifth, we compared E2 and phenol red as inducers of the progesterone receptor using a human breast cancer cell line. All the experiments presented in this report support the conclusion that the concentration of phenol red contaminants in a standard culture medium available today is not sufficient to cause estrogenic effects. In brief, our studies indicate that the real issue of how to demonstrate estrogenic effects in culture resides elsewhere than phenol red. We have found that the demonstration of sex steroid hormone-mitogenic effects in culture depends upon conditions that maximize the effects of a serum-borne inhibitor(s). When the effects of the inhibitor are optimized, the presence or absence of phenol red makes no everyday difference to the demonstration of estrogen mitogenic effects with several target cell types from diverse species.

Estrogen mitogenic action. ii. negative regulation of the steroid hormone-responsive growth of cell lines derived from human and rodent target tissue tumors and conceptual implications.

In an accompanying report (Moreno-Cuevas, J. E.; Sirbasku, D. A., In Vitro Cell. Dev. Biol.; 2000), we demonstrated 80-fold estrogen mitogenic effects with MTW9/PL2 rat mammary tumor cells in cultures supplemented with charcoal-dextran-treated serum. All sera tested contained an estrogen reversible inhibitor(s). The purpose of this report is to extend those observations to additional sex steroid-responsive human and rodent cell lines. Every line tested showed a biphasic response to hormone-depleted serum. Concentrations of < or = 10% (v/v) promoted substantive growth. At higher concentrations, serum was progressively inhibitory. With estrogen receptor-positive (ER+) human breast cancer cells, rat pituitary tumor cells, and Syrian hamster kidney tumor cells, 50% (v/v) serum caused significant inhibition, which was reversed by very low physiologic concentrations of estrogens. This same pattern was observed with the steroid hormone-responsive LNCaP human prostatic carcinoma cells. Because steroid hormone mitogenic effects are now easily demonstrable using our new methods, the identification of positive results has nullified our original endocrine estromedin hypothesis. We also evaluated autocrine/paracrine growth factor models of estrogen-responsive growth. We asked if insulin-like growth factors I and II, insulin, transforming growth factor alpha, or epidermal growth factor substituted for the positive effects of estrogens. Growth factors did not reverse the serum-caused inhibition. We asked also if transforming growth factor beta (TGFP) substituted for the serum-borne inhibitor. TGFbeta did not substitute. Altogether, our results are most consistent with the concept of a unique serum-borne inhibitor as has been proposed in the estrocolyone model. However, the aspect of the estrocolyone model related to steroid hormone mechanism of action requires more evaluation. The effects of sex steroids at picomolar concentrations may reflect mediation via inhibitor "activated" intracellular signaling pathways.

Epidermal growth factor receptors in rat ovarian tissue.

Epidermal growth factor is known to have mitogenic effects in the ovary. To determine whether this effect is receptor-mediated, and whether the receptor number changes during the estrus cycle, in this study we have evaluated the total and occupied EGF receptor (EGFr) concentrations and receptor binding characteristics in adult rat ovaries collected at diestrus and early estrus. These represent stages before and after the pituitary LH/FSH surges of the estrus cycle. The 100,000 g plasma membrane fractions were isolated and EGFr concentrations evaluated with and without 1 mM EDTA treatment at pH 4.5 in order to release receptor-bound ligand and therefore assess the unoccupied and total receptor concentrations. EGF receptor affinity and number were analyzed by Scatchard analysis. No statistically significant difference between unoccupied and total receptor concentration in the diestrus phase ovaries was found. The percentage of occupied receptors (total-unoccupied) in diestrus membranes was 7.1%. In early estrus ovaries, a significant increase (P < 0.05) was observed in total receptor concentration when compared with the number of unoccupied receptors. The percentage of occupied receptors in early estrus membranes was 35.7%. Comparison between the ovaries from the two phases indicated an almost twofold increase in total EGF receptor number in early estrus membranes when compared with diestrus membranes. Furthermore, the percentage of occupancy between the two studied groups indicate a significant increase (P < 0.05) in the number of occupied receptors in early estrus when compared to diestrus. Thus, we have demonstrated that not only EGF receptor concentrations are modulated through the rat estrus cycle, but also the concentration of EGF-like substances bound to such receptors.