Cytokines and growth factors in a biologic product obtained from patients' urine as immune-modulators to treat autoimmune and allergic diseases.

At "Instituto de Alergias y Autoinmunidad Dr. Maximiliano Ruiz Castañeda, A.C." in Mexico City, a non-traditional health care center focused on the treatment of autoimmune and allergic diseases using personalized medicine, an alternative treatment referred to as an "immune-modulator" has been developed. In this study, we will refer to this treatment substance as the "immune-modulator." In brief, a urine sample is collected from the patient and processed to obtain the peptide fraction, which is conditioned and then administered sublingually to the patient. Sample processing involves multiple steps aimed at the removal of toxic compounds and enrichment for cytokines, growth factors, and other immune peptides that may contribute to the function of the immune-modulator. This treatment has been administered for many years, and patients testify that it is useful and reliable. Despite the benefits of this treatment, the molecular mechanisms underlying its effects have not been thoroughly investigated. Therefore, this study aims to identify immunoregulatory peptides, such as cytokines and growth factors, in the immune-modulator. Urine and immune-modulator concentrations of cytokines and growth factors were assessed using a Luminex assay. Twenty-one cytokines and growth factors were identified in immune-modulator samples. MCP-1 was identified in 100% of the samples; MIP-1ß, IL-8, RANTES, INF-γ, and IP-10 were identified in approximately 65-70% of samples; IL5, IL-1B, and IL-17 in 50-60%; eotaxin, VEGF, IL-6, and FGF in about 40%; MIP-1α, IL-9, GM-CSF, G-CSF, IL-12, and IL-15 in about 20-30%; and IL-13 and PDGF-bb were identified in <6% of samples. Additionally, patients exhibited significant changes in IL-1ß, IFN-γ, and MCP-1 concentrations after treatment with the immune-modulator, whereas healthy individuals showed no significant change in response to the treatment. The immune-modulator is an alternative treatment based on the administration of cytokines and growth factors obtained from the urine of patients. In this study, its composition was characterized. The isolated products could be responsible for the effects of the immune-modulator. Further trials are required to evaluate the effective delivery of these molecules by the administration route described.

Effect of milk minerals on calf gains and sex differences in mineral composition of milk from Iberian red deer (Cervus elaphus hispanicus).

Milk mineral content has received little attention in studies focusing on milk nutrient effects on offspring growth. This study examines calf growth in Iberian deer and compares the influence of milk minerals, other nutrients, and lactation variables relevant for growth to discern the relative weight of each factor. In addition, because Iberian deer hinds are the first mammal found to produce different milk for sons and daughters, the present study examines whether there are also sex differences in milk mineral composition. Concentrations and yields of Ca, P, Mg, Na, K, Fe, and Zn in milk of 46 red deer hinds were monitored through 18 weeks of lactation. Calf growth was influenced by Ca and P percent, and total Fe production. Milk for males had a lower content in Ca and P, a greater content of K, and Mg, whereas no sex effects were found in Na, Fe, or Zn percentages. Higher percentages in Ca and P for daughters might constitute a compensatory response, as daily production was not biased towards females in Ca or P, whereas in the latter and all the other minerals daily production was greater for heavier calves, which are usually males. In conclusion, milk mineral content and production influence calf growth even after controlling for other important lactation variables and nutrients, and they show effects and interactions more complicated than expected.

Seasonal changes in plasma leptin concentration related to antler cycle in Iberian red deer stags.

The aim of this study is to describe the leptin cycle in male Iberian red deer (Cervus elaphus hispanicus) and relate it to antler and testosterone cycles. An additional aim is to assess the relationship between the plasma leptin concentration during antlers' growth and their final size. Therefore, blood from 21 Iberian red deer males was sampled monthly to analyse leptin and testosterone. At the same time the deer were weighed and their body condition was assessed. The length of antlers was measured every 2 weeks and, after casting, their final length and perimeters were taken. Leptin showed a seasonal cycle, with a peak in June that decreased as testosterone increased. Low values were observed in autumn, winter and early spring. The relationship observed between leptin and body mass or body condition score was different in spring, when plasma testosterone concentration is low, than in autumn, when testosterone increases. Leptin peak amplitude was positively related to final antler size. In conclusion, the relationship between leptin and body mass and body condition score changes through the year, possibly due to the influence of androgens and photoperiod. There was a positive relationship between plasma concentration of leptin during antler growth and final antler length.

E-cadherin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in rats.

An alteration in the expression of E-cadherin has been observed in many epithelial neoplasms. No data exist, however, for the expression of this protein in an animal model for urinary bladder cancer. The present study investigated the expression of E-cadherin in rat urothelial preneoplastic lesions and tumours induced by oral administration of N-butyl-N-(4-hydroxybutyl) nitrosamine, during 10, 15 and 20 weeks. Simple hyperplasia and squamous metaplasia showed a similar E-cadherin pattern when compared with normal urothelium, with its expression confined to cell membrane. Thirty eight percent of the nodular hyperplasia, 41.4% of the dysplasia and 100% of the papillomas showed a weak E-cadherin expression. All papillary neoplasm of low malignant potential, low-and high-grade papillary carcinoma, and invasive carcinoma revealed an abnormal staining pattern with an increase in cytoplasm reactivity and discontinuous cell membrane positivity. The loss of expression for low-grade papillary carcinoma versus simple hyperplasia, nodular hyperplasia and dysplasia was statistically significant (p = 0.0001, p = 0.007 and p=0.008, respectively). There was a similar decrease in E-cadherin expression for papillary neoplasm of low malignant potential versus simple hyperplasia, nodular hyperplasia and dysplasia (p = 0.0001; p = 0.001 and p=0.0001, respectively). These results suggest that alteration in the expression of this adhesion molecule in rat may be indicative of tumour progression in N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder cancer.

Leptin, reproduction and sex steroids.

Leptin is a hormone secreted mainly by the adipose cells with a primary role in the regulation of body weight by establishing a feedback loop between the energy reserves and the hypothalamic centers that control food intake. Recent data suggest that, in addition, leptin interacts with other endocrine systems to provide critical information about the size of the fat stores, acting as a permissive factor that allows the triggering of energy-demanding situations, as the onset of puberty and the reproduction, only when the size of the fuel reserve is large enough to guarantee its success. In addition, leptin appears to play a role during pregnancy and lactation, as it is produced by the placenta and is present in maternal milk. The fact that leptin levels are always higher in females, even after correcting for body fat content, suggests that the interaction between the adipose tissue and the reproductive system is modulated in a different way in males and females by androgenic and estrogenic hormones. In fact, adipose tissue samples taken from male donors are completely refractory in vitro to the action of both estrogens and androgens. On the contrary, dihydrotestosterone, androstenedione and dehydroepiandrosterone-S are potent inhibitors of leptin secretion, while estradiol induces a strong stimulation in adipose tissue taken from women. Testosterone is devoid of activity in either gender.

Dual effect of insulin on in vitro leptin secretion by adipose tissue.

Although it is widely accepted that insulin stimulates leptin secretion, a dual action was observed using a validated in vitro system, i.e., an early (less than 48 h) inhibitory action, followed later (48-96 h) by a clear-cut stimulation. While the inhibitory phase was observed at every glucose concentration tested (from 1 to 25 mM), the stimulatory phase required the presence of physiological or supraphysiological glucose concentrations. In fact, leptin secretion was virtually eliminated in the presence of glucose uptake inhibitors. This dual effect of insulin was not due to modifications of the ob mRNA levels, suggesting that it depends entirely on posttranslational mechanisms. In conclusion, insulin appears to induce an early inhibition of leptin secretion by the adipose cell, followed later by a stimulatory effect secondary to the metabolic changes triggered by the insulin-induced increase in glucose uptake.

GHRP-6 in heifer and cow adenohypophisial cells separated by elutriation.

Previous studies have reported that the growth hormone (GH)-releasing peptide (GHRP-6), a synthetic Met-enkephalin peptide analog, stimulates GH release in vivo in a variety of species, including bovine. In the present study, the in vitro effects of GHRP-6 on bovine somatotropes separated by elutriation were analyzed as well as its interactions with the GH-releasing hormone (GHRH). The administration of GHRP-6 at doses from 10(-8) M to 10(-5) M stimulated GH release, and also 10(-9) M in cow pituitary cells, and produced maximal stimulation at 10(-6) M. The effects of GHRP-6 (10(-6) M) on GH release were shown at 1, 2, 3 and 4-h incubation (p < 0.05), except for heifer pituitary cells at 1-h incubation (p > 0.05). The GH releasing effects of either GHRH alone or GHRH+GHRP-6 were significantly more potent than that of GHRP-6 alone (p < 0.05). Contrary to what occurred in rat pituitary cells, the combined administration of 10(-6) M GHRP-6 with 10(-8) M GHRH did not result in a synergist action of GH release. Although the additive effect was significant when compared with GHRH alone (p < 0.05). The results demonstrate the existence of differences in the effect of GHRH+GHRP-6 on bovine somatotropes. These differences may reflect the physiological importance of distinct cell subpopulation, like the mammosomatotroph cells.

Effect of growth hormone-releasing peptide 1-6 on GH secretion-stimulated by GHRH and pyridostigmine in lambs.

The GHRP-6 seems to act at a pituitary site, activating different intracellular messenger pathways from those utilized by GHRH, and at the hypothalamic level where receptors for GHRP-6 have been demonstrated. This study examines the effect of GHRP-6 on GH secretion in vitro and in vivo. Lamb adenohypophysial cell cultures were subjected to a challenge with 1) 10 nM GHRH-1-29; 2) 1 microM GHRP-6; and 3) 10 nM GHRH plus 1 microM GHRP-6. Both peptides released GH, GHRP-6 being less potent than GHRH, and the GH response to GHRH and GHRP-6 not being synergistic, without statistically significant differences between GHRH+GHRP-6 and GHRH alone. In in vivo studies, six lambs received 15 microg/kg GHRH (1-29) or 15 microg/kg GHRH plus 10 microg/kg GHRP-6 and six other lambs received 100 microg/kg GHRP-6 or 3 mg/kg pyridostigmine plus 100 microg/kg GHRP-6. The results have shown that the combination of GHRH plus GHRP-6, at low doses, causes higher GH peak (p < 0.05) and higher GH area under curve (p < 0.05) with respect to administration of GHRH alone. The administration of GHRP-6 plus pyridostigmine produced in a stronger GH response to GHRP-6 (100 microg/kg) in the amplitude of the GH peak (p < 0.001) and in the area under the GH response curve (p < 0.001). The complementary interactions of GHRP-6 with GHRH or pyridostigmine in releasing GH seem to indicate independent actions of these compounds. These results suggest that GHRP-6 potentiates the GH secretion stimulated by GHRH at the hypothalamic level, in these animals.

Simultaneous localization of Pit-1 protein and gonadotropins on the same cell type in the anterior pituitary glands of the rat.

Pit-1 is a prototypic member of the POU transcription factor family and plays a critical role in pituitary-specific action of growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) beta-subunit genes. The purpose of the present study was to elucidate the changes in the expression of the Pit-1 product in the pituitary of pregnant rats employing an improved double-immunohistochemical method. The positive cells showed nuclear immunoreactivity and Pit-1 protein was frequently observed in the nuclei of many cells which were also immunopositive for GH, PRL, and betaTSH. Unexpectedly, a significant number of pituitary cells containing both Pit-1 and gonadotropins were also observed. These cells were usually distributed near blood vessels that supply the pituitary. While a prominent increase in the percentage of Pit-1/PRL, Pit-1/beta-luteinizing hormone and Pit-1/beta-follicle-stimulating hormone immunoreactive cells was observed in pregnant rats, the percentage of Pit-1/GH immunoreactive cells was strongly decreased. In contrast, no significant differences in the percentage of Pit-1/betaTSH doubly immunolabeled cells were noticed. Our findings strongly support the hypothesis that PRL could coexist in the Pit-1 immunopositive gonadotropes. Although Pit-1 protein was not detected in the nuclei of corticotropes, the existence of these cells in the rat pituitary cannot be excluded.

Effects of acute administration of growth hormone-releasing hormone (GHRH) and oxytocin on somatotroph cells in sheep: morphometric study and growth hormone (GH) secretion.

In this work, the combined use of the morphometric study of somatotroph cells and plasma GH levels provided new data for the interpretation of the role played by OT and GHRH on GH cells. GHRH 1-29 (15 micrograms/kg), oxytocin (2.5 IU animal) or 1 ml saline solution were administered to male lambs. The GH plasma concentration was measured by RIA and for the morphometric study the cellular area, nuclear area and volume density of the somatotroph cells were measured in 1 micron semi-thin sections immunolabeled with avidin-biotin technique (ABC). The area under the GH response curve for 3 hours after injection was similar in both saline and OT-treated animals (24.8 +/- 9.1 and 31.4 +/- 14.7 micrograms/ml, respectively) and much lower than that observed in GHRH-treated animals (445.5 +/- 126.7 micrograms/ml). The cell area of somatotrophs was smaller in the GHRH-treated lambs and larger in the OT-treated lambs than in the control lambs (71.47 +/- 1.56, 91.42 +/- 1.72 and 83.1 +/- 1.74 microns 2, respectively). A similar change was observed in the nuclear area; it decreased in the GHRH-treated lambs (21.61 +/- 0.52 microns 2) and increased in the OT-treated lambs (25.45 +/- 0.68 microns 2) with respect to the control group (23.75 +/- 0.44 microns 2). No significant differences were found in volume density.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of specific saxitoxin binding to rat mast cells.

We studied whether or not mast cells are endowed with specific sodium channels, by using tritiated saxitoxin which binds to site 1 of sodium channels on excitable tissues. Our results suggest that rat pleural and peritoneal mast cells lack specific sodium channels.

Changes in rat mast cell responses in sodium-free media. Lack of demonstrable sodium channel activity.

Exposure of rat mast cells to isotonic sucrose (employed as a sodium free medium) increased several-fold the sensitivity to calcium, which itself became a stimulus for exocytosis. Concentrations of the cation as low as 25 microM permitted maximal histamine release. Preincubation of cells in sucrose to allow sodium efflux before adding the ionophore A23187 led to a slower release of histamine. We postulate that sodium efflux can generate a membrane potential that causes the increased sensitivity to calcium and the delay in response after preincubation. The response to A23187 is somewhat unspecific since the ionophore can release histamine from internal calcium reservoirs. Saxitoxin or veratridine did not affect cell responses, so that sodium activity is not mediated through defined sodium channels.

[Effect of isoproterenol on the liberation of histamine from rat lung mast cells]. / Efecto del isoproterenol sobre la liberación de histamina en mastocitos de pulmón de rata.

Rat lung mast cells were stimulated with drugs with distinct mechanisms of action, namely concanavalin A, compound 48/80 ionophore A23187, in the presence of the beta adrenergic agonist (-)isoproterenol. Cells show a high response when they are stimulated with FNa-calcium. Isoproterenol does not inhibit histamine release induced by any stimuli, but enhances the response to concanavalin A and compound 48/80. Results point to the lack of beta activity on rat lung mast cells.

[Inhibition of histamine liberation in mast cells from the lung and the intestine of the dog by isoproterenol]. / Inhibición de la liberación de histamina en mastocitos de pulmón y de intestino de perro por el isoproterenol.

Enzymatically isolated dog lung and gut mast cells were stimulated with compound 48/80, ionophore A23187, concanavalin A and FNa-Ca. Cell response elicited by A23187, concanavalin A or 48/80 is almost completely inhibited by isoproterenol. Concanavalin A induced histamine release on gut mast cells is high, indicating an elevated degree of sensitization of these cells. Results point to the existence of beta adrenergic inhibitory activity on dog lung and gut mast cells.

Developmental changes induced by glucocorticoids treatment in breeder quail (Coturnix coturnix japonica).

The effects produced in offspring by corticosterone treatment to breeder quail were investigated. The breeders received corticosterone orally every day during 7 days. The total dose administered was 3.15 mg/quail/7 days. The quail from treated breeders have a decreased rate of body growth during the experimental period. The studied metabolic and nutritives parameters were smaller than the control group, however, the relative indices were similar. The breeders treated with corticosterone produce quail smaller than controls, but the pattern during the growth period is similar, there it can be observed by the same relative indices.

[Changes in thyroid function caused by continuous treatment with thiouracil in rats]. / Variaciones de la función tiroidea por tratamiento continuado de tiouracilo en ratas.

Serum concentration evolution of thyroxine and triiodothyronine, and thyrotropin (TSH), have been studied in rats while they were given 6-propylthiouracil (PTU) as antithyroid drug, and during the recovery period after suppression of treatment. In the same way thyroid hypertrophy and plasmatic levels of thyrotropin were correlated. Animals received orally a daily dose of 1 mg/100 g body weight during thirty-five days and had a two week recovery period. Thyroid hormone concentrations in plasma were determined by immunoenzymatic assay ELISA with peroxidase as labelled enzyme. From the results obtained, it can be stated that chronic administration of PTU implies a continuous decrease in thyroid hormone concentrations in plasma, reaching nearly zero values, while after treatment, levels recover their normal values in a week's time. A parallelism exists between thyroid hypertrophy and pituitary TSH hypersecretion, due to a decrease in thyroid hormone levels.

[Comparative study of the effect of glucocorticoids on nitrogen metabolism in adult male and non-laying female quail (Coturnix coturnix japonica)]. / Estudio comparativo del efecto de los glucocorticoides sobre el metabolismo nitrogenado en codornices adultas (Coturnix coturnix japonica) machos y hembras sin puesta.

The effects of glucocorticoids on nitrogen metabolism have been studied in three different groups of quail: a) control animals; b) quail treated with cortisol; c) quail treated with corticosterone. All the experiments were carried out in adult male and non-laying female animals. During the 7 day period that the experiments lasted, hormones were administered daily by via oral at 4 mg/100 g body weight/7 day doses. While body weight, food intake, nitrogen balance and liver weight, in the treated animals stayed under the values of the control group, in both males and females, their uric acid excretion and hepatic glycogen were always higher. Values, in general, were similar for both males and females.