Pregnancy and birth rate outcomes in alpacas (Vicugna pacos) inseminated with frozen semen using two commercial extenders.

The aim of this study was to evaluate alpaca pregnancy outcomes and birth rates of females inseminated with frozen semen using two commercial extenders. A total of 18 ejaculates from 8 adult alpaca males were obtained with artificial vagina, and macroscopic and microscopic semen characteristics were assessed. Afterwards, samples were divided into two aliquots, diluted with Biladyl® B or AndroMed®, and cooled for 2 h at 5°C. At that moment, sperm motility was evaluated, and samples were frozen through a gradual descent of temperature using a liquid nitrogen tank. To analyse frozen sperm quality, samples were thawed at 38°C for 30 s. Even though a significant decrease in sperm motility and viability was detected when thawed (p < .05), no superiority was found between the two commercial extenders (Biladyl® B vs. AndroMed®). A total of 36 alpaca females were artificially inseminated (AI) between 30 and 34 h post-injection of a GnRH analogue, administered when a growing dominant follicle was detected through transrectal palpation and ultrasonography. Obtained pregnancy rates were similar between Biladyl® B (33.3%, 6/18) and AndroMed® (22.2%, 4/18). No significant differences were detected in birth rates between the two tested extenders, obtaining 4 and 3 births for Biladyl® and AndroMed®, respectively. In conclusion, alpaca pregnancies and alive offspring can be obtained through AI with frozen semen at similar efficiency rates using commercial diluents, Biladyl® B or AndroMed®.

Effects of direct-fed microorganisms and enzyme blend co-administration on intestinal bacteria in broilers fed diets with or without antibiotics.

Direct-fed microorganisms (DFM) and exogenous enzymes have been demonstrated to improve growth performance in poultry and are potentially important alternatives to antibiotic growth promoters (AGP). We investigated the administration of a feed additive composed of a DFM product containing spores of 3 Bacillus amyloliquefaciens strains and an enzyme blend of endo-xylanase, α-amylase, and serine-protease in diets with or without sub-therapeutic antibiotics in broiler chickens over a 42-d growth period. Evaluation of growth performance determined feed efficiency of broiler chickens which were administered the feed additive was comparable to those fed a diet containing AGPs. Characterization of the gastrointestinal microbiota using culture-dependent methods determined administration of the feed additive increased counts of total Lactic Acid Bacteria (LAB) relative to a negative control and reduced Clostridium perfringens to levels similar to antibiotic administration. Additionally, greater counts of total LAB were observed to be significantly associated with reduced feed conversion ratio, whereas greater counts of C. perfringens were observed to be significantly associated with increased feed conversion ratio. Our results suggest the co-administration of DFMs and exogenous enzymes may be an important component of antibiotic free poultry production programs and LAB and C. perfringens may be important targets in the development of alternatives to AGPs in poultry production.

The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils.

AIM: Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. METHODS: Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. RESULTS: We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1(-/-) mice are significantly less effective at recruiting neutrophils into the site of inflammation. CONCLUSIONS: These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions.

Africanized honey bees (Apis mellifera) have low infestation levels of the mite Varroa destructor in different ecological regions in Mexico.

Honey bee (Apis mellifera) colonies of African and European descent were compared for levels of Varroa destructor infestation in 3 different ecological regions in Mexico. The 300 colonies that were studied were located in subtropical, temperate sub-humid, and temperate dry climates. The morphotype and mitotype of adult bees as well as their rates of infestation by varroa mites were determined. Additionally, the number of combs with brood and covered with bees was recorded for each colony. The highest frequency of colonies that were classified as African-derived was found in the subtropical environment, whereas the lowest occurred in the temperate dry region. Overall, the colonies of African genotype had significantly lower mite infestation rates (3.5±0.34%) than the colonies of European genotype (4.7±0.49%) regardless of the region sampled. Significant effects of genotype and region on Varroa infestation rates were evident, and there were no differences in bee population or capped brood between genotypes. Mite infestation levels were significantly lower in the colonies of the temperate dry region than in the colonies of the other 2 regions. These results are discussed within the context of results from studies that were previously conducted in Brazil. This is the first study that demonstrates the effects of Africanization and ecological environment on V. destructor infestation rates in honey bee colonies in North America.

TMEM16 proteins: the long awaited calcium-activated chloride channels?

Currents mediated by calcium-activated chloride channels (CaCCs), observed for the first time in Xenopus oocytes, have been recorded in many cells and tissues ranging from different types of neurons to epithelial and muscle cells. CaCCs play a role in the regulation of excitability in neurons including sensory receptors. In addition, they are crucial mediators of chloride movements in epithelial cells where their activity regulates electrolyte and fluid transport. The roles of CaCCs, particularly in epithelia, are briefly reviewed with emphasis on their function in secretory epithelia. The recent identification by three independent groups, using different strategies, of TMEM16A as the molecular counterpart of the CaCC is discussed. TMEM16A is part of a family that has 10 other members in mice. The discovery of the potential TMEM16 anion channel activity opens the way for the molecular investigation of the role of these anion channels in specific cells and in organ physiology and pathophysiology. The identification of TMEM16A protein as a CaCC chloride channel molecule represents a great triumph of scientific perseverance and ingenuity. The varied approaches used by the three independent research groups also augur well for the solidity of the discovery.

TMEM16 proteins: the long awaited calcium-activated chloride channels?

Currents mediated by calcium-activated chloride channels (CaCCs), observed for the first time in Xenopus oocytes, have been recorded in many cells and tissues ranging from different types of neurons to epithelial and muscle cells. CaCCs play a role in the regulation of excitability in neurons including sensory receptors. In addition, they are crucial mediators of chloride movements in epithelial cells where their activity regulates electrolyte and fluid transport. The roles of CaCCs, particularly in epithelia, are briefly reviewed with emphasis on their function in secretory epithelia. The recent identification by three independent groups, using different strategies, of TMEM16A as the molecular counterpart of the CaCC is discussed. TMEM16A is part of a family that has 10 other members in mice. The discovery of the potential TMEM16 anion channel activity opens the way for the molecular investigation of the role of these anion channels in specific cells and in organ physiology and pathophysiology. The identification of TMEM16A protein as a CaCC chloride channel molecule represents a great triumph of scientific perseverance and ingenuity. The varied approaches used by the three independent research groups also augur well for the solidity of the discovery.

A randomized controlled trial on the efficacy and safety of a food ingredient, collagen hydrolysate, for improving joint comfort.

INTRODUCTION: Current options to promote joint comfort are limited to medicines that can reduce pain but can also have adverse effects. Collagen, a major component of joint cartilage, is found in the diet, particularly in meat. Its hydrolysed form, collagen hydrolysate (CH), is well absorbed. CH may stimulate the joint matrix cells to synthesize collagen, so helping to maintain the structure of the joint and potentially to aid joint comfort. METHODS: In a randomized, double-blind, controlled multicentre trial, 250 subjects with primary osteoarthritis of the knee were given 10 g CH daily for 6 months. RESULTS: There was a significant improvement in knee joint comfort as assessed by visual analogue scales to assess pain and the Womac pain subscale. Subjects with the greatest joint deterioration, and with least intake of meat protein in their habitual diets, benefited most. CONCLUSION: CH is safe and effective and warrants further consideration as a food ingredient.

Modulation of renal kallikrein by a high potassium diet in rats with intense proteinuria.

Injury of the renal tubulointerstitial compartment is recognized to play an important role in hypertension. Its damage may in turn, impair the activity of vasodepressor systems, like the kallikrein-kinin, in blood pressure regulation. The overload proteinuria model induces tubulointerstitial injury with activation of the renin-angiotensin system, but renal kallikrein and the development of hypertension have not received special attention. Sprague-Dawley rats received seven intraperitoneal doses of bovine serum albumin (BSA) 2 g/day under normosodic diet and were hydrated ad libitum. A second group received a high potassium diet to stimulate kallikrein production during the previous four weeks and while under BSA administration. A third one received potassium and BSA in the same schedule, but with the kinin B2 receptor antagonist, HOE140, added during the protein load phase. A control group received seven saline injections. Kallikrein protein was detected by immune labeling on renal sections and enzymatic activity in the urine. The BSA group showed massive proteinuria followed by intense tubulointerstitial damage. Blood pressure increased after the third dose in BSA animals, remaining elevated throughout the experiment, associated with significant reductions in renal expression and urinary activity of kallikrein, compared with controls. An inverse correlation was found between blood pressure and immunohistochemistry and urinary activity of kallikrein. Potassium induced a significant increase in both urinary activity and renal kallikrein expression, associated with significant reduction in blood pressure. The HOE140 antagonist blunted the antihypertensive effect of kallikrein stimulation in proteinuric rats. Loss of renal kallikrein, produced by tubulointerstitial injury, may participate in the pathogenesis of the hypertension observed in this model.

No gender-associated differences of cyclosporine pharmacokinetics in stable renal transplant patients treated with diltiazem.

Cytochrome-P450 enzymes metabolize cyclosporine both in the liver and in the intestinal wall. Diltiazem, by competitive inhibition of these enzymes, may increase the absorption and the bioavailability of cyclosporine. Some evidence points to a higher activity of some specific enzymes in women, such as CYP3A, that may influence differences in cyclosporine pharmacokinetics. We examined possible gender-associated differences in pharmacokinetic profiles of cyclosporine in 19 stable renal transplant recipients cotreated with diltiazem. Ten women and nine men, chronically using diltiazem associated with cyclosporine, azathioprine, and prednisone were randomly assigned to an 8-week period of continued controlled treatment with diltiazem (10 patients) or a wash-out period discontinuing diltiazem (nine patients). At the end of this period, the time-concentration curves of cyclosporine in the first 4 hours were performed after a single dose of cyclosporine. Thereafter, a cross-over between groups was performed, and time-concentration curves repeated. A specific RIA was used to measure cyclosporine concentrations. Comparisons between male and female patients in doses of cyclosporine and other pharmacokinetics parameters (C(0), C(2), AUC(0-4)), with or without diltiazem, did not show any difference related to gender. The association of diltiazem allowed a similar degree of reduction in Neoral dosage in male and female patients (21%). No changes in serum creatinine, blood urea nitrogen, potassium, uric acid, or blood pressure, or other adverse event were observed during the study. In these groups of patients, gender was not an important factor to be considered when diltiazem is added to cyclosporine therapy.

Correlation Between C2 and AUC(0-4) in Renal Transplant Patients Treated With Diltiazem.

BACKGROUND: The area-under-the-curve (AUC) of cyclosporine (CsA) reflects exposure to the drug, but this monitoring strategy is time-consuming and not cost-effective. Recently, it has been suggested that the concentration at 2 hours after dosing (C2) shows the best correlation with AUC. The C2 has been replacing the trough measurement (C0) to monitor CsA therapy, but in patients receiving diltiazem there is not much information about this issue. We investigated the correlations between C2 and C0 with absorption AUC over the first 4 hours (AUC(0-4)) in renal stable transplant patients receiving CsA therapy with or without diltiazem. PATIENTS AND METHODS: Ten patients (five men) of ages 23 to 68 years and 6 to 84 months after transplantation, were randomly assigned to an 8-week initial period of either diltiazem washout or controlled treatment with diltiazem. Time-concentration curves of cyclosporine were performed at the end of this period using a specific RIA measurement of blood samples. Thereafter, a crossover of the groups was performed and after another 8 weeks, a second curve was obtained. Drugs that change the pharmacokinetics of cyclosporine or diltiazem were not allowed. RESULTS: The cyclosporine daily dose was lower with diltiazem (173 +/- 4 mg vs 213 +/- 4 mg, P = .002), but despite a dose reduction of only 19% +/- 1.5%, there was a trend to a larger AUC/dose (28 +/- 5 ng x h/mL x mg vs 17 +/- 2 ng x h/mL x mg, P = .1) and a trend to an increased C2 when treatment included diltiazem (1035 +/- 156 ng/mL vs 652 +/- 126 ng/mL, P = NS). Moreover, we confirmed that C2 showed the best correlation with AUC(0-4), (r = 0.7, P = .04), a correlation that improved with diltiazem (r = 0.9, P < .002). CONCLUSION: C2 is the point that correlates best with AUC(0-4) with or without diltiazem. C2 in the presence of diltiazem was associated with a stronger, more significant correlation with AUC(0-4).

Valsartan-induced hematocrit changes in renal transplant patients.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor type 1 blockers (ARB) are frequently prescribed for renal transplant patients. The main reasons for their use are that their antihypertensive and antifibrogenic effects may prevent chronic renal allograft dysfunction, potentially improving transplant survival. Furthermore, ACE and ARB have been used to reduce the hematocrit in patients with posttransplant erythrocytosis. We evaluated the effects of the ARB valsartan on the evolution of hematocrit in stable renal transplant patients treated with cyclosporine (CsA), azathioprine (Aza), and prednisone. PATIENTS AND METHODS: Twenty-six stable renal transplant patients treated with valsartan 80 mg/d orally were followed for 6 months. Evaluations were performed prior to as well as at 3 and 6 months following the initiation of valsartan. RESULTS: The hematocrit levels decreased significantly at 3 months (46.1 +/- 7.3 vs 39.9 +/- 5.8 ; P < .0001) in patients with a normal hematocrit, namely a level over 38%, with no further reduction at 6 months. In recipients with an hematocrit less than 38%, there was no significant reduction, either at 3 or 6 months follow-up. Valsartan was well tolerated without significant side effects. CONCLUSION: We postulate that inhibition of the proerythropoietic effects of angiotensin II and/or the reduction in hypoxia within the renal tubulointerstitium as well as the vasodilator effects on the efferent arterioles, represent possible mechanisms for the reduction and stabilization of the hematocrit in stable renal transplant patients.

Sex-related differences in the distribution of opioid receptor-like 1 receptor mRNA and colocalization with estrogen receptor mRNA in neurons of the spinal trigeminal nucleus caudalis in the rat.

We recently reported that exogenously applied orphanin FQ, the endogenous ligand for opioid receptor-like 1 (ORL(1)) receptor, produces sex-specific modulation of trigeminal nociception, and that estrogen contributes to these sex-related differences. Estrogen could produce these sex-related differences by altering the expression of the ORL(1)-receptor gene in the trigeminal nucleus caudalis. Utilizing in situ hybridization, we compared levels of ORL(1) receptor mRNA and investigated its colocalization with estrogen receptor mRNA in trigeminal neurons. Our results showed that in male rats, ORL(1) receptor mRNA is abundantly expressed in the rostral part of the trigeminal nucleus caudalis, and at the junction of caudalis and interpolaris (Vc/Vi). In comparison with males, levels of ORL(1) receptor mRNA were not significantly different in proestrus females, but were significantly higher in the rostral trigeminal nucleus caudalis and at the junction of Vc/Vi of diestrus females. In addition, ovariectomy raised the levels in the rostral trigeminal nucleus caudalis, and at the junction of Vc/Vi. Levels were reduced to proestrus levels in these regions following estradiol replacement. Our results also showed that ORL(1) receptor mRNA is present in majority of estrogen receptor (alpha and/or beta) mRNA-containing neurons. We conclude that there are sex-related differences in the ORL(1)-receptor gene expression in the trigeminal nucleus caudalis, which appear to be determined in part by estrogen levels.

Orphanin FQ produces gender-specific modulation of trigeminal nociception: behavioral and electrophysiological observations.

The present study aimed to determine if orphanin FQ, an endogenous ligand for the opioid receptor like-1 receptor, produces gender-specific effects in the modulation of N-methyl-D-aspartate (NMDA)-evoked responses of trigeminal nociceptive neurons, and in the NMDA-induced nociceptive behavior. Single-unit extracellular recordings were made from nociceptive-specific and wide dynamic range neurons in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in anesthetized (1.5 g/kg urethane) rats. In the proestrous female, orphanin FQ applied microiontophoretically produced facilitation of the NMDA-evoked responses in 50% (16/32) of nociceptive neurons, inhibition in 31% (10/32), and biphasic effects in 19% (6/32). In contrast, in the male, it inhibited the responses in 86% (18/21), and facilitated the responses in 14% (4/21). In ovariectomized animals, orphanin FQ inhibited the responses in 75% (9/12) of nociceptive neurons, facilitated the responses in 17% (2/12) and produced biphasic effects in 8% (1/12). In contrast, in estradiol-treated ovariectomized rats, it facilitated the responses in 46% (5/11), inhibited the responses in 36% (4/11) and produced biphasic effects in 18% (2/11). For behavioral studies, NMDA-induced scratching behavior was used to assess the effects of orphanin FQ. Twenty-eight male, ovariectomized and estradiol-treated ovariectomized rats were microinjected with NMDA (2 nmol in 10 microl) alone through a cannula implanted in the medullary region, while another 27 rats were microinjected with orphanin FQ (10 nmol in 10 microl) 10 min prior to giving NMDA. Orphanin FQ reduced the NMDA-induced nociceptive scratching behavior by 92% in the male, and by 96% in ovariectomized rats. In contrast, in estradiol-treated ovariectomized animals, orphanin FQ facilitated the NMDA-induced scratching behavior by 210%. We conclude from these studies that orphanin FQ is primarily pronociceptive in the female and primarily antinociceptive in the male. Furthermore, we suggest that estrogen is involved in generating the gender-specific effects of orphanin FQ.

Endomorphin-1 and endomorphin-2 modulate responses of trigeminal neurons evoked by N-methyl-D-aspartic acid and somatosensory stimuli.

The present study investigated the modulation of N-methyl-D-aspartate (NMDA)-evoked and peripheral cutaneous stimulus-evoked responses of trigeminal neurons by endomorphins, endogenous ligands for the mu-opioid receptor. Effects of endomorphins, administered microiontophoretically, were tested on the responses of nociceptive neurons recorded in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in anesthetized rats. Endomorphin-1 and endomorphin-2 predominantly reduced the NMDA-evoked responses, producing an inhibitory effect of 54.1 +/- 2.96% (mean +/- SE; n = 34, P < 0.001) in 92% (34/37) of neurons and 63.6 +/- 3.61% (n = 32, P < 0.001) in 91% (32/35) of neurons, respectively. The inhibitory effect of endomorphins was modality specific; noxious stimulus-evoked responses were reduced more than nonnoxious stimulus-evoked responses. Naloxone applied at iontophoretic current that blocked the inhibitory effect of [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin, reduced the peak inhibitory effect of endomorphins on the NMDA- and natural stimulus-evoked responses. We suggest that endomorphins by acting at micro-opioid receptor selectively modulate noxious stimulus-evoked responses in the medullary dorsal horn.

Bothrops lanceolatus (Fer de lance) venom induces oedema formation and increases vascular permeability in the mouse hind paw.

The ability of snake venoms to increase vascular permeability and to induce oedema through the release of pharmacologically active substances is well known. We have studied the oedema and vascular permeability induced by Bothrops lanceolatus venom in male Swiss white mice. Paw oedema was induced by the subplantar injection of B. lanceolatus venom (125-1000 ng/paw) and was quantified as the increase in paw weight. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. The oedema and the increase in vascular permeability were maximal within 2 h and had resolved after 24 h. The administration of the vasodilator iloprost (20 ng/paw) immediately after B. lanceolatus venom potentiated the oedema and the increase in vascular permeability by approximately four-fold. Pretreating the mice with indomethacin, dexamethasone, NDGA or BW A4C inhibited the venom-induced oedema and the increase in vascular permeability. In contrast, histamine, serotonin and PAF-acether antagonists (mepyramine, cyproheptadine and WEB 2086, respectively) were ineffective. Histological examination showed that B. lanceolatus venom (250 ng and 500 ng/paw) caused thickening of the inner dermal layers which was accompanied by extensive intercellular spaces indicative of oedema. In addition, there was a marked infiltration of inflammatory cells, particularly neutrophils, into the underlying muscle layer. The latter, however, remained morphologically unaffected during the 3 h of observation. Venom doses larger than 500 ng/paw produced intense haemorrhage. These results indicate that B. lanceolatus venom induces oedema and increases vascular permeability in the mouse hind paw. The principal mediators of this inflammatory response are cyclooxygenase and lipoxygenase products.

American Academy of Pediatrics. Committee on Nutrition. Calcium requirements of infants, children, and adolescents.

This statement is intended to provide pediatric caregivers with advice about the nutritional needs of calcium of infants, children, and adolescents. It will review the physiology of calcium metabolism and provide a review of the data about the relationship between calcium intake and bone growth and metabolism. In particular, it will focus on the large number of recent studies that have identified a relationship between childhood calcium intake and bone mineralization and the potential relationship of these data to fractures in adolescents and the development of osteoporosis in adulthood. The specific needs of children and adolescents with eating disorders are not considered.

Prevention of cyclophosphamide-induced hemorrhagic cystitis by glucose-mannose binding plant lectins.

PURPOSE: Neutrophils are implicated in the physiopathologic alterations of hemorrhagic cystitis (HC). Thus, we decided to test the antiinflammatory activity of glucose-mannose binding lectins extracted from D. violacea and D. guianensis seeds, which showed an inhibitory effect upon neutrophil migration in a model of rat peritonitis based on HC experimentally induced by cyclophosphamide (CYP). MATERIALS AND METHODS: Mice were treated with mesna (40 mg./kg., i.p.), lectins (1 and 10 mg./kg., i.v.) and 0.1 M of alpha-D-methyl-mannoside (alpha-CH3) or alpha-D-galactose (alpha-D-gal). The HC was induced by CYP (200 mg./kg., i.p.). The results were evaluated 12 hours after HC induction, based on the following parameters: vesical edema measurements, macroscopic and microscopic analysis of the bladders (Gray's analysis). The vesical edema was quantified either by the increase in bladder wet weight or by the determination of vesical vascular permeability (Evans' blue leakage). RESULTS: CYP-induced vesical edema was prevented by mesna and lectin treatment. The lectin effects were dose-dependent, and at the highest dose were similar to that achieved by mesna treatment. alpha-CH3 selectively reversed the lectin inhibitory effect. Histopathological analysis corroborated these findings and showed an intense reduction of leukocyte infiltration and tissue damage by lectin treatment. The bladders from the mesna-treated group showed a nearly normal histological pattern. However, differently from this group, none of the lectins abolished CYP-induced hemorrhage. CONCLUSIONS: The glucose-mannose binding lectins showed strong antiinflammatory activity in the mouse model of HC induced by CYP. As lectins mainly affected leukocyte vesical infiltration, we suggest a competitive blockage of glucosylated (mannose-glucose) selectin binding sites by lectins.

Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment.

The natural physiological ligands for selectins are oligosaccharides found in glycoprotein or glycolipid molecules in cell membranes. In order to study the role of sugar residues in the in vivo lectin anti-inflammatory effect, we tested three leguminous lectins with different carbohydrate binding affinities in the peritonitis and paw oedema models induced by carrageenin in rats. L. sericeus lectin was more anti-inflammatory than D. virgata lectin, the effects being reversed by their specific binding sugars (N-acetylglucosamine and alpha-methylmannoside, respectively). However, V. macrocarpa, a galactose-specific lectin, was not anti-inflammatory. The proposed anti-inflammatory activity of lectins could be due to a blockage of neutrophil-selectin carbohydrate ligands. Thus, according to the present data, we suggest an important role for N-acetylglucosamine residue as the major ligand for selectins on rat neutrophil membranes.

Toxicidad aguda de altas dosis de metotrexato en el tratamiento de leucemia linfoblástica aguda en niños / Acute toxicity of high close of Methotrexae in children with acute lymphoblastic leukemia

El objetivo de ese estudio fue evaluar y comparar la aparición de efectos adversos agudos luego de la infusión de metotrexato (MTX), citostático incluido en alas dosis en 2 diferentes protocolos (2g. vs. 5g/m2 por ciclo), utilizados durante la fase M del tratamiento de las leucemias linfoblásticas agudas (LLA) en el paciente pediátrico. Sesenta y seis pacientes que recibieron 264 ciclos de quimioterapia con altas dosis de MTX, fueron estudiados entre enero de 1995 y febrero de 1997. Todos ellos tenían el diagnóstico de LLA de riesgo estándar e intermedio, estaban en remisión completa y habián recibido anteriormente el protocolo I. Se anlizaron 2 tratamiento: el gurpo A) 132 ciclos con MTX a razón de 2g/m2/ciclo (n=32); grupo B 136 cilos con MTX a razón de 5g/m2/ciclo (n=34). Todos los pacientes recibieron 4 infusiones de MTX (1/10 de la dosis en 30´ y 9/10 de la dosis en el resto de las 24 hs.), con hidratación a 3000 ml/m2/día y alcalinización urinaria, en forma bimensual, junto a una punción lumbar con mediación intratecal (Profilaxis para evitar compromiso del sistema nervioso central) y 6-mercaptopurina oral diariamente durante el ciclo. No se observaron diferencias estadísticas significativas en relación a toxicidad hematológica severa: en 42 cukis (32.81 por ciento) en el grupo A y 44 cilcos (32.35 por ciento) en el grupo B; z=0.042: p=0.967. Sin embargo, la aparición de mucositis moderada y severa y valores elevados de las enzimas hepáticas se observaron con mayor frecuencia en el grupo B, mostrando una diferencia estadísticamente significativa. En nuestro estudio, no se observaron valores anormales de creatinina sérica. No hubo impacto sobre variaciones en el peso corporal en ambos protocolos. Concluimos que la dosis de MTX no influyó sobre la aparición de toxicidad hematológica, pero si sobre la aparición de efectos adversos en el tracto gastrointestinal y la función hepática.

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 ñ 0.19 and Dex = 0.35 ñ 0.13 vs saline (S) = 2.85 ñ 0.59; fMLP: Ptx = 0.43 ñ 0.09 and Dex 0.01 ñ 0.01 vs S = 1.08 ñ 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 ñ 1.4 and Dex = 3.06 ñ 0.76 vs S = 15.94 ñ 3.97; fMLP: Ptx = 3.85 ñ 0.56 and Dex = 0.40 ñ 0.16 vs S = 7.15 ñ 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 µm), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 ñ 0.38 vs S = 4.20 ñ 1.01; fMLP: Dex = 0.25 ñ 0.11 vs S = 2.20 ñ 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 ñ 2.25 vs S = 4.20 ñ 1.01; fMLP: Ptx = 4.66 ñ 1.24 vs S = 2.20 ñ 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.