Day-restricted feeding during pregnancy and lactation programs glucose intolerance and impaired insulin secretion in male rat offspring.

AIM: The maternal environment during pregnancy and lactation plays a determining role in programming energy metabolism in offspring. Among a myriad of maternal factors, disruptions in the light/dark cycle during pregnancy can program glucose intolerance in offspring. Out-of-phase feeding has recently been reported to influence metabolism in adult humans and rodents; however, it is not known whether this environmental factor impacts offspring metabolism when applied during pregnancy and lactation. This study aims to determine whether maternal day-restricted feeding (DF) influences energy metabolism in offspring. METHODS: Pregnant and lactating Wistar rats were subjected to ad libitum (AL) or DF during pregnancy and lactation. The offspring born to the AL and DF dams were intra- and interfostered, which resulted in 4 group types. RESULTS: The male offspring born to and breastfed by the DF dams (DF/DF off) were glucose intolerant, but without parallel insulin resistance as adults. Experiments with isolated pancreatic islets demonstrated that the male DF/DF off rats had reduced insulin secretion with no parallel disruption in calcium handling. However, this reduction in insulin secretion was accompanied by increased miRNA-29a and miRNA34a expression and decreased syntaxin 1a protein levels. CONCLUSION: We conclude that out-of-phase feeding during pregnancy and lactation can lead to glucose intolerance in male offspring, which is caused by a disruption in insulin secretion capacity. This metabolic programming is possibly caused by mechanisms dependent on miRNA modulation of syntaxin 1a.

Annexin A1 mimetic peptide controls the inflammatory and fibrotic effects of silica particles in mice.

BACKGROUND AND PURPOSE: Endogenous glucocorticoids are pro-resolving mediators, an example of which is the endogenous glucocorticoid-regulated protein annexin A1 (ANXA1). Because silicosis is an occupational lung disease characterized by unabated inflammation and fibrosis, in this study we tested the therapeutic properties of the N-terminal ANXA1-derived peptide annexin 1-(2-26) (Ac2-26) on experimental silicosis. EXPERIMENTAL APPROACH: Swiss-Webster mice were administered silica particles intranasally and were subsequently treated with intranasal peptide Ac2-26 (200 µg per mouse) or dexamethasone (25 µg per mouse) for 7 days, starting 6 h post-challenge. Ac2-26 abolished the leukocyte infiltration, collagen deposition, granuloma formation and generation of pro-inflammatory cytokines evoked by silica; these variables were only partially inhibited by dexamethasone. KEY RESULTS: A clear exacerbation of the silica-induced pathological changes was observed in ANXA1 knockout mice as compared with their wild-type (WT) littermate controls. Incubation of lung fibroblasts from WT mice with Ac2-26 in vitro reduced IL-13 or TGF-ß-induced production of CCL2 (MCP-1) and collagen, but this peptide did not affect the production of CCL2 (MCP-1) by stimulated fibroblasts from formyl peptide receptor type 1 (FPR1) knockout mice. Ac2-26 also inhibited the production of CCL2 (MCP-1) from fibroblasts of FPR2 knockout mice. CONCLUSIONS AND IMPLICATIONS: Collectively, our findings reveal novel protective properties of the ANXA1 derived peptide Ac2-26 on the inflammatory and fibrotic responses induced by silica, and suggest that ANXA1 mimetic agents might be a promising strategy as innovative anti-fibrotic approaches for the treatment of silicosis.

Lidocaine-derivative JMF2-1 prevents ovalbumin-induced airway inflammation by regulating the function and survival of T cells.

BACKGROUND: Inhalation of the local anaesthetic lidocaine has been suggested to be beneficial for asthmatics, but airway anaesthesia is unpleasant and may exacerbate bronchoconstriction. Our previous study showed that inhalation of the lidocaine analogue JMF2-1 can elicit the anti-inflammatory properties of lidocaine without anaesthesia. This prompted further research on the mechanism of action and putative therapeutic application of JMF2-1. OBJECTIVE: We tested the hypothesis that JMF2-1 would prevent allergen-induced lung inflammation and airway hyperresponsiveness (AHR) by modulating T cell function in vivo and in vitro. Methods Local and systemic changes in leucocyte levels, cytokine production and lung mechanics were examined in a murine model of lung inflammation. JMF2-1 (0.05-2%) or saline was aerosolized twice a day during the ovalbumin (OVA)-provocation period (19-21 days post-sensitization). Analyses were performed 24 h after the final challenge. Primary cultured lymph node cells were used to assess the effects of JMF2-1 (100-600 µm) at the cellular level. RESULTS: OVA challenge resulted in lung recruitment of CD4(+) T cells and eosinophils, increased generation of inflammatory cytokines and AHR to inhaled methacholine within 24 h. These changes were prevented by JMF2-1 nebulization, and occurred in parallel with an increase in the number of apoptotic cells in the lung. JMF2-1 treatment did not alter levels of CD4(+) or CD8(+) T cells in the thymus or lymph nodes of naïve mice, although it inhibited OVA-induced IL-13 production and the lymphocyte proliferative response in vitro. It also induced apoptosis of OVA-activated lymphocytes in a mechanism sensitive to z-VAD, indicating that JMF2-1 mediates caspase-dependent apoptosis. CONCLUSION: Inhalation of JMF2-1 prevents the cardinal features of asthma by reducing T(H) 2 cytokine generation and lung eosinophilic inflammatory infiltrates via local inhibition of T cell function and survival. JMF2-1 may represent a novel therapeutic alternative for asthma control with distinct advantages over local anaesthetics.

The allergic inflammatory reaction in neonatal streptozotocininduced diabetic rats: evidence of insulin resistance and microvascular dysfunction.

OBJECTIVE: To investigate the allergic reaction in neonatal streptozotocin (nSTZ)-induced diabetes mellitus. MATERIAL: Male newborn Wistar rats were made diabetic by the injection of streptozotocin (160 mg/kg, i. p.) and used 8 weeks thereafter. TREATMENT: Animals were sensitized against ovalbumin (OA, 50 microg and Al(OH)3, 5 mg, s. c.) and challenged 14 or 21 days thereafter. METHODS: OA-induced airway inflammation and OA-induced pleurisy models were used to investigate leukocyte migration (total and differential leukocyte counts) and lung vascular permeability (Evans blue dye extravasation). RESULTS: nSTZ-diabetic rats presented glucose intolerance and insulin resistance. Relative to controls, nSTZ rats exhibited a 30% to 50% reduction in lung vascular permeability. Leukocyte infiltration in both models of allergen-induced inflammation, and number of pleural mast cells did not differ between groups. CONCLUSIONS: Data suggest that the reduction of allergic inflammatory reactions in nSTZ rats is restricted to microvascular dysfunctions and associated, probably, with insulin resistance in lung microvascular endothelium.

Suppressive effects of nitric oxide-releasing prednisolone NCX-1015 on the allergic pleural eosinophil recruitment in rats.

BACKGROUND: The addition of a nitric oxide (NO)-releasing moiety to prednisolone was shown to enhance the anti-inflammatory activity of this glucocorticoid in some experimental conditions, but its effectiveness in the context of eosinophilic inflammation remains to be elucidated. OBJECTIVE: This study compared the anti-inflammatory effect of prednisolone to a NO-releasing derivative of prednisolone, NCX-1015, using a model of allergen-evoked eosinophil recruitment in rats. The efficacy of a NO-donor compound, DETA-NONOate, was also assessed for comparison. METHODS: Wistar rats were actively sensitized with Al(OH)(3) plus ovalbumin and 14 days later challenged with antigen intrapleurally. Treatments were performed locally 1 h before challenge. Cysteinyl-leucotrienes (Cys-LT) and eotaxin were measured by ELISA. RESULTS: Antigen challenge induced an eosinophil infiltration at 12 h, maximal at 24 h. It also caused an increase in the levels of Cys-LTs in the pleural exudate and in the expression of 5-lipoxygenase (5-LO) in infiltrated leucocytes at 6 h, peaking at 12 h and persisting for at least 24 h. Treatment with equimolar doses of prednisolone and NCX-1015 inhibited the late eosinophil infiltration, although the dose required to produce maximal inhibition was about one-tenth that of prednisolone. Cys-LT generation and 5-LO expression were inhibited by NCX-1015 but not by prednisolone. Treatment with prednisolone combined with the NO-donor DETA-NONOate led to a greater inhibition of the eosinophilia and Cys-LT generation as compared with either drug alone. Administration of the steroid receptor antagonist RU 486, 1 h before prednisolone and NCX-1015, abolished the inhibitory effect of the former, under conditions where it only partially affected the latter. CONCLUSIONS: Our findings indicate that NCX-1015 provided a greater anti-inflammatory effect than prednisolone on the allergic eosinophil recruitment in rats, suggesting that NO-releasing steroids can be considered as a promising therapeutic approach to allergic diseases.

Immunomodulatory pretreatment with Kalanchoe pinnata extract and its quercitrin flavonoid effectively protects mice against fatal anaphylactic shock.

Previously, we reported the immunosuppressive action of the aqueous extract of Kalanchoe pinnata (Kp) in mice. In the present study, we report on the protective effect of Kp in fatal anaphylactic shock, likewise a Th2-driven immunopathology, and the identification of its active component. Mice daily treated with oral Kp during hypersensitization with ovalbumin were all protected against death when challenged with the allergen, as compared with the 100% mortality in the untreated group. A single intraperitoneal dose 3 h prior to challenge was partially effective. Oral protection was accompanied by a reduced production of OVA-specific IgE antibodies, reduced eosinophilia, and impaired production of the IL-5, IL-10 and TNF-alpha cytokines. In vitro, Kp prevented antigen-induced mast cell degranulation and histamine release. Oral treatment with the quercitrin flavonoid isolated from Kp prevented fatal anaphylaxis in 75% of the animals. These findings indicate that oral treatment with Kp effectively downmodulates pro-anaphylactic inducing immune responses. Protection achieved with quercitrin, although not maximal, suggests that this flavonoid is a critical component of Kp extract against this extreme allergic reaction.

Thymus involution in alloxan diabetes: analysis of mast cells.

We previously reported that alloxan-induced diabetes results in reduction in the number and reactivity of mast cells at different body sites. In this study, the influence of diabetes on thymic mast cells was investigated. Thymuses from diabetic rats showed marked alterations including shrinkage, thymocyte depletion, and increase in the extracellular matrix network, as compared to those profiles seen in normal animals. Nevertheless, we noted that the number and reactivity of mast cells remained unchanged. These findings indicate that although diabetes leads to critical alterations in the thymus, the local mast cell population is refractory to its effect. This suggests that thymic mast cells are under a different regulation as compared to those located in other tissues.

A novel hot-plate test sensitive to hyperalgesic stimuli and non-opioid analgesics.

It is widely accepted that the classical constant-temperature hot-plate test is insensitive to cyclooxygenase inhibitors. In the current study, we developed a variant of the hot-plate test procedure (modified hot-plate (MHP) test) to measure inflammatory nociception in freely moving rats and mice. Following left and right hind paw stimulation with a phlogogen and vehicle, respectively, the animals were placed individually on a hot-plate surface at 51 degrees C and the withdrawal latency for each paw was determined simultaneously in measurements performed at 15, 60, 180, and 360 min post-challenge. Plantar stimulation of rats (250 and 500 microg/paw) and mice (125-500 microg/paw) with carrageenan led to a rapid hyperalgesic response of the ipsilateral paw that reached a plateau from 15 to 360 min after challenge. Pretreatment with indomethacin (4 mg/kg, i.p.) inhibited the phenomenon at all the times analyzed. Similarly, plantar stimulation of rats and mice with prostaglandin E2 (0.5 and 1 microg/paw) also resulted in rapid hyperalgesia which was first detected 15 min post-challenge. Finally, we observed that the MHP test was more sensitive than the classical Hargreaves' test, being able to detect about 4- and 10-fold lower doses of prostaglandin E2 and carrageenan, respectively. In conclusion, the MHP test is a simple and sensitive method for detecting peripheral hyperalgesia and analgesia in rats and mice. This test represents a low-cost alternative for the study of inflammatory pain in freely moving animals.

A novel hot-plate test sensitive to hyperalgesic stimuli and non-opioid analgesics

It is widely accepted that the classical constant-temperature hot-plate test is insensitive to cyclooxygenase inhibitors. In the current study, we developed a variant of the hot-plate test procedure (modified hot-plate (MHP) test) to measure inflammatory nociception in freely moving rats and mice. Following left and right hind paw stimulation with a phlogogen and vehicle, respectively, the animals were placed individually on a hot-plate surface at 51°C and the withdrawal latency for each paw was determined simultaneously in measurements performed at 15, 60, 180, and 360 min post-challenge. Plantar stimulation of rats (250 and 500 æg/paw) and mice (125-500 æg/paw) with carrageenan led to a rapid hyperalgesic response of the ipsilateral paw that reached a plateau from 15 to 360 min after challenge. Pretreatment with indomethacin (4 mg/kg, ip) inhibited the phenomenon at all the times analyzed. Similarly, plantar stimulation of rats and mice with prostaglandin E2 (0.5 and 1 æg/paw) also resulted in rapid hyperalgesia which was first detected 15 min post-challenge. Finally, we observed that the MHP test was more sensitive than the classical Hargreaves' test, being able to detect about 4- and 10-fold lower doses of prostaglandin E2 and carrageenan, respectively. In conclusion, the MHP test is a simple and sensitive method for detecting peripheral hyperalgesia and analgesia in rats and mice. This test represents a low-cost alternative for the study of inflammatory pain in freely moving animals.

Thymus involution in alloxan diabetes: analysis of mast cells

We previously reported that alloxan-induced diabetes results in reduction in the number and reactivity of mast cells at different body sites. In this study, the influence of diabetes on thymic mast cells was investigated. Thymuses from diabetic rats showed marked alterations including shrinkage, thymocyte depletion, and increase in the extracellular matrix network, as compared to those profiles seen in normal animals. Nevertheless, we noted that the number and reactivity of mast cells remained unchanged. These findings indicate that although diabetes leads to critical alterations in the thymus, the local mast cell population is refractory to its effect. This suggests that thymic mast cells are under a different regulation as compared to those located in other tissues.

Down-regulation of mast cell activation and airway reactivity in diabetic rats: role of insulin.

Hormones play a modulating role in allergic inflammation. An inverse relationship between atopy and diabetes mellitus was reported. The mechanisms regulating this interaction are not completely understood. This study examined whether insulin influences mast cell activation following antigen challenge in rats. The experimental design included alloxan-induced diabetic rats and matching controls. Experiments were performed 30 days after alloxan injection. The animals were sensitised by s.c. injection of ovalbumin (OA) and aluminium hydroxide. OA-induced airway contraction, morphometric analysis of airway mast cells and tissue histamine quantification were evaluated in the isolated main bronchus and intrapulmonary bronchus upon exposure to antigen in vitro. Relative to controls, a reduced contraction to OA was observed in bronchial segments isolated from diabetic rats. This was accompanied by a 50% reduction in the number of degranulated mast cells and in histamine release. A complete recovery of the impaired responses was observed under the influence of insulin. In conclusion, the data suggested that insulin might modulate the controlling of mast cell degranulation; therefore, the early-phase response to antigen provocation, which represents a new insight into a better understanding of the mechanisms, accounted for the decreased risk of asthma among type-1 diabetic patients.

Kinetics of eosinophil and IgE-mast cell changes following infection with Angiostrongylus costaricensis in Wistar rats.

Human abdominal angiostrongyliasis is a severe eosinophilic disease caused by Angiostrongylus costaricensis. Previous studies have demonstrated that wild rodents are critically involved as definitive hosts to this nematode in nature. In this study, we have evaluated the susceptibility of Wistar rats (Rattus norvegicus) to A. costaricensis infection. Kinetics of parasitological and pathological changes, including the number of adult worms recovered from mesenteric arteries, and of IgE, mast cell and eosinophil levels in several compartments have been assessed. The oral inoculation of third-stage larvae (L3) into adult Wistar rats led to a marked accumulation of worms in the branches of the mesenteric arteries 25 and 50 days post-inoculation. Intense bone marrow eosinophilia ranging from 7 to 50 days was accompanied by marked accumulation of eosinophils in the blood, peritoneal and bronchoalveolar spaces. Eosinophilic periarteritis, oedema and granuloma in the intestinal and lung tissues were also histologically evident. Total serum IgE and specific anti-parasite IgE peaked at 25 days post-infection, as measured by ELISA and by the passive cutaneous anaphylaxis test, respectively. At that time point, there was a drastic reduction in the number of intact mast cells in the peritoneal effluent. These findings indicate that Wistar rats are permissive to A. costaricensis infection. IgE-mast cell activation and massive tissue eosinophil infiltration are marked features in the process and are likely to play a crucial role in the immune-response evoked by this parasite.

Histamine-potentiating activity in rat anaphylactic pleural fluid: role of serotonin

The identity of the histamine-potentiating activity detected in the rat anaphylactic pleural washing was investigated. Wistar rats of both sexes, weighing 150-200 g, were sensitized by injecting subcutaneously (sc) a mixture of ovalbumin and Al(OH)3 14 days before allergen challenge. In sensitized rats, intrapleural (ipl) injection of ovalbumin (l2 mug/cavity) caused an intense protein exudation. A single ipl administration of compound 48/80 (l2 mug/cavity) exhausted the resident mast cell population and turned the pleural cavity hyporeactive to the allergen challenge performed 5 days later. Allergen-induced exudation occurred in parallel to a dramatic decrease in the amount of cell-stored histamine (from 9.6 ñ 1.4 (N = 8) to 1.3 ñ 0.1 (N = 6) mug/cavity, P<0.001) in the pleural fluid within 10 min. The anaphylactic cell-free pleural washing obtained at this time, as well as histamine at a concentration equivalent to that stored in pleural mast cells (10 mug/cavity), did not induce pleural exudation when injected into normal rats. In contrast the combined administration of histamine and anaphylactic pleural washing led to remarkable pleural exudation, comparable to that obtained with a high dose of histamine (200 mug/cavity) alone. It is noteworthy that the anaphylactic washing from compound 48/80 pretreated rats failed to synergize with histamine. Also, synergism was not reproduced when recipient rats were pretreated with methysergide (50 mug/cavity). Consistently, serotonin (5 mug/cavity) acted synergistically with histamine (1O mug/cavity), producing a greater exudative response than observed with the sum of the effects of each vasoactive amine alone. The results indicate that serotonin accounts for the histamine-potentiating activity noted in the anaphylactic pleural washing, confirming that the synergistic interaction between these vasoactive amines plays a critical role in the rat allergic pleurisy.

Endotoxin induces eosinophil accumalation in the rat pleural cavity

Changes in eosinophil counts after intrathoracic (it) injection of endotoxin (LPS) were investigated in Wistar rats (150 - 180 g). Increasing doses of endotoxin (62.5 - 500 ng/cavity) induced a dose-dependent increase in the number of eosinophils recovered from the rat pleural cavity 24 h later. The eosinophilia was apparent within 24 h, peaked within 48 h (from 0.76 ñ 0.12 to 3.68 ñ 0.51 eosinophils x 10**6/cavity, P < 0.001) and returned to basal levels 120h after the it inection of endotoxin (250 ng/cavity). Endotoxin (3 ng - 4 µg/ml) failed to attract eosinophilis in vitro under conditions in which PAF-acether induced a dose-related response. These findings indicate that endotoxin-induced eosinophil migration in vivo is mediated by a secondary mechanism

Pleural eosinophil infiltration induced by PAF-acether is not a desensitizable phenomenon

This study investigated the effect of successive daily intrathoracic (it) injections of PAF-acether upon its demonstrated ability to generate eosinochemotaxin(s). Repeated administration of PAF-acether led to a selective state of desensitization, characterized by a gradual reduction of its ability to induce exudation. Concomitantly, however, there was a progressive pleural accumulation of eosinophilis leading to a 7-fold increase in the eosinophil counts after the 4th restimulation. The generation of eosinochemotaxin(s) elicited by PAF-acether was not modified by desensitization, as detected by transferring the cell-free pleural fluid from donor to recipient animals. We conclude that, in contrast to exudation, eosinophil tissue infiltration induced by PAF-acether is not a desensitizable phenomenon

Kinetics of pleural exudation and cellular alterations induced by antigen in actively sensitized rats

This study was undertaken to characteize the different phases of the allergic pleurisy induced by ovalbumin in actively sinsitized rats. The reaction was triggered by the intrathoracic injection of ovalbumin (12 microng/cavity) into animals sensitized 14 days before. The challenge caused, at 30 mjin, a drastic mast cell degranulation and exudation which peaked within 4h. At this time, an intense pleural leucocyte recruitment also occurred, accounted for by an increase in the mononuclear cell counts and by a predominant influyx of neutrophils. After 24h, the mast cell counts stated to reover, accompanied by a long-lasting (96 h) accumaltion of pleural eosinophils. Forty-eight hour later, the exudation and neutrophils were at basal levels, whereas mast cell counts increased progressively to reach control values at 120 h. This study describes the time course of the exudatory and cellular alterations observed during pleural inflammation induced by low antigen concentrations

Late eosinophil mobilization induced by PAF-acether in the pleural cavity of rats

In view of the rapid degradation of PAF in biological fluids, this study was designed to determine if late eosinophil infiltration induced in rats by PAF was deriived from its direct chemotatic action. A significant and selective 5-fold increase in the pleural eosinophil counts was detected 24 h after intrapleural PAF injection. The transfer of 6-h PAF washings to the pleural cavity of recipient rats also induced a 3-fold selective accumulation of eosinophils. The protein synthesis inhibitor cycloheximide abolished the pleural eosinophil migration and the generation of transferable chemotactic activity when administered to donor but not to recipient rats. These findings suggest that a secondary protein mediator accounts for the late eosinophil mobilization induced by PAF

Adrenalectomy exacerbates paw edema without interfering with desensitization induced by PAF-acether in rats

Subcutaneous injection of PAF-acether into rat's paw to a local inflammatory response (edema) followed by desensitization after repeated injections of the substance. The desensitizing effect was not modified by previous adrenalectomy, whereas the inflammatory response observed after the first injection of PAF-acether was exacerbated. This finding suggests that adrenal hormones may act as modulators of PAF-induced inflammatory reactions. Because LY 171883, an LTD4 blocker, inhibited the edema which follows the first injection of PAF-acether, we suggest that leukotrienes may play an important role in the phenomenon