The histamine H4 receptor antagonist 1-[(5-chloro-2,3-dihydro-1-benzofuran-2-yl)methyl]-4-methyl-piperazine(LINS01007) prevents the development of DSS-induced colitis in mice.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with growing incidence worldwide. Our group reported the compound 5-choro-1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazine (LINS01007) as H4R antagonist (pKi 6.2) and therefore the effects and pharmacological efficacy on a DSS-induced mice model of UC were assessed in this work. Experimental acute colitis was induced in male BALB/c mice (n = 5-10) by administering 3 % DSS in the drinking water for six days. The test compound LINS01007 was administered daily i.p. (5 mg/kg) and compared to control group without treatment. Body weight, water and food consumption, and the presence of fecal blood were monitored during 7-day treatment period. The levels of inflammatory markers (PGE2, COX-2, IL-6, NF-κB and STAT3) were also analyzed. Animals subjected to the acute colitis protocol showed a reduction in water and food intake from the fourth day (p < 0.05) and these events were prevented by LINS01007. Histological signs of edema, hyperplasia and disorganized intestinal crypts, as well as neutrophilic infiltrations, were found in control mice while these findings were significantly reduced in animals treated with LINS01007. Significant reductions in the levels of PGE2, COX-2, IL-6, NF-κB and STAT3 were observed in the serum and tissue of treated animals. The results demonstrated the significant effects of LINS01007 against DSS-induced colitis, highlighting the potential of H4R antagonism as promising treatment for this condition.

Intrauterine growth restriction leads to a high-corticosterone producing offspring: An implication for pulmonary infection susceptibility.

AIMS: Although intrauterine growth restriction (IUGR) impairs immune system homeostasis and lung development, its relationship with the susceptibility to pulmonary infections remains unclear. Thus, this study aimed to investigate the impact of IUGR on acute lung inflammatory response induced by bacterial stimulus. MATERIALS AND METHODS: Pregnant female Wistar rats were subjected to 50% caloric-protein food restriction during gestation. To mimic bacterial lung infection, adult male offspring (12 weeks old) were challenged with a single lipopolysaccharide (LPS) intranasal instillation, and 6 h later, we assessed the acute inflammatory response. Normal birth weight (NBW) animals represent the control group. KEY FINDINGS: LPS instillation increased the protein levels in the airways of both the NBW and low birth weight (LBW) groups, indicating vascular leakage. LBW animals exhibited a lower number of neutrophils, reduced production of interleukin-6 and macrophage-inflammatory protein-2 and decreased upregulation of intercellular adhesion molecule-1 gene expression in lung tissues. Further analysis revealed that the LBW group produced lower levels of prostaglandin-E2 and failed to secrete leukotriene-B4 upon LPS stimulation, which correlated with impaired cyclooxygenase-2 and 5-lipoxygenase expression. These results were probably associated with their inability to upregulate the expression of Toll-like receptor-4 and downstream signaling proteins, such as nuclear factor kappa-B, in the lungs. The LBW group also exhibited abnormal airway thickening and high corticosterone levels under basal conditions. SIGNIFICANCE: This study suggests that IUGR-induced foetal programming in LBW offspring threatens HPA axis physiology and corticosterone biodisponibility, and impairs the innate response to bacterial antigens, increasing future susceptibility to pulmonary infection.

The Novel H4R Antagonist 1-[(5-Chloro-2,3-Dihydro-1-Benzofuran-2-Yl)Methyl]-4-Methyl-Piperazine (LINS01007) Attenuates Several Symptoms in Murine Allergic Asthma.

BACKGROUND/AIMS: Histamine is an important chemical transmitter involved in inflammatory processes, including asthma and other chronic inflammatory diseases. Its inflammatory effects involve mainly the histamine H4 receptor (H4R), whose role in several studies has already been demonstrated. Our group have explored the effects of 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazines as antagonists of H4R, and herein the compounds LINS01005 and LINS01007 were studied with more details, considering the different affinity profile on H4R and the anti-inflammatory potential of both compounds. METHODS: We carried out a more focused evaluation of the modulatory effects of LINS01005 and LINS01007 in a murine asthma model. The compounds were given i.p. (1-7 mg/kg) to ovalbumin sensitized BALB/c male mice (12 weeks old) 30 min before the antigen challenging, and after 24 h the cell analysis from the bronchoalveolar lavage fluid (BALF) was performed. The lung tissue was used for evaluation by western blot (COX-2, 5-LO, NF-κB and STAT3 expressions) and histological analysis. RESULTS: Treatment with the more potent H4R antagonist LINS01007 significantly decreased the total cell count and eosinophils in BALF at lower doses when compared to LINS01005. The expression of COX-2, 5-LO, NF-κB and STAT3 in lung tissue was significantly reduced after treatment with LINS01007. Morphophysiological changes such as mucus and collagen production and airway wall thickening were significantly reduced after treatment with LINS01007. CONCLUSION: These results show important down regulatory effect of novel H4R antagonist (LINS01007) on allergic lung inflammation.

Intrauterine Malnutrition Reduced Long Leptin Receptor Isoform Expression and Proinflammatory Cytokine Production in Male Rat Pulmonary Endothelial Cells Stimulated by Lipopolysaccharide.

BACKGROUND/AIMS: We have previously shown that low birth weight (LBW) rats exposed to intrauterine malnutrition have an impaired lung inflammatory response and reduced levels of inflammatory mediators; however, circulating leptin levels were not increased. We evaluated long leptin receptor isoform (ObRb) expression in lung endothelial cells from low birth weight rats and examined its role in the production of lipid mediators and cytokines. METHODS: Lung endothelial cells were obtained from normal birth weight (NBW) rats or LBW rats subjected to intrauterine malnutrition. These cells were stimulated with leptin (10 ng/mL), LPS (lipopolysaccharide, 1 µg/mL), or leptin plus LPS. Six hours after stimulation, the production of inflammatory mediators (PGE2, LTB4, IL-1ß, and IL-6) was evaluated using commercial ELISA kits, and Western blotting was performed to investigate p38MAPK, NF-κB, and ObRb expression. RESULTS: Leptin increased IL-1ß levels in only cells from the NBW group, whereas LPS increased PGE2 and LTB4 levels in cells from both groups; leptin addition potentiated lipid mediator production induced by LPS in the NBW group. LPS enhanced the production of IL-1ß and IL-6 in only endothelial cells from NBW rats. Leptin receptor expression was decreased (63%) in endothelial cells from LBW rats. None of the stimuli increased NF-κB or p38 signaling pathway expression in cells from LBW rats. CONCLUSION: These results suggest that intrauterine malnutrition compromises leptin receptor expression and cytokine production in pulmonary endothelial cells stimulated by LPS; these effects seem to involve the NF-κB and p38MAPK signaling pathways.

A comparative study of pathophysiological alterations in scorpionism induced by Tityus serrulatus and Tityus bahiensis venoms.

Scorpionism is a relevant public health problem in several countries in tropical and subtropical regions. In Brazil, Tityus serrulatus sting can induce acute lung injury in part as a consequence of inflammation. Despite the occurrence of other scorpions of Tityus genus in Brazilian scorpiofauna, the knowledge regarding pulmonary alterations is related to T. serrulatus venom (Tsv). Here we studied, comparatively, the pathophysiological changes in the rat airways envenomed by Tsv or T. bahiensis venom (Tbv), since both scorpions are involved in human accidents but with severe envenomations occurring when victims are stung by T. serrulatus. After intravenous injection of the venoms (200 µg/kg), both were able to induce Evans blue extravasation (in 30 min) into airways and increased protein extravasation into lungs at 4 and 24 h, but the magnitude of such events was higher in Tsv group. Hemorrhage (in 60 min) in the lungs was higher in Tbv group, while IL-1ß (at 1 h) and IL-6 (at 1 and 4 h) in lung homogenates were detected only in Tsv group. Four and 24 h after envenomation, myeloperoxidase activity in lung was equally augmented in the envenomed groups, as well as an increased in polymorphonuclear cell numbers in bronchoalveolar lavage fluid. At 4 h blood leukogram showed increased leukocyte values with the highest neutrophilia in Tsv group. The numbers of leukocytes and neutrophils remained higher than control at 24 h in Tsv and Tbv groups, and it was accompanied by lympho (envenomed groups) and monocytosis (Tsv group). In conclusion, although Tbv was capable of inducing acute lung injury and blood leukocyte mobilization, most of the evaluated parameters were more affected by the Tsv. These results could help to explain the pathophysiology of the scorpionism and the clinical data arguing toward the greatest severity associated with T. serrulatus stings.

Leptin downregulates LPS-induced lung injury: role of corticosterone and insulin.

BACKGROUND/AIMS: We investigated the effects of leptin in the development of lipopolysaccharide (LPS)-induced acute lung inflammation (ALI) in lean mice. METHODS: Mice were administered leptin (1.0µg/g) or leptin (1.0µg/g) followed by LPS (1.5µg/g) intranasally. Additionally, some animals were given LPS (1.5µg/g) or saline intranasally alone, as a control. Tissue samples and fluids were collected six hours after instillation. RESULTS: We demonstrated that leptin alone did not induce any injury. Local LPS exposure resulted in significant acute lung inflammation, characterized by a substantial increase in total cells, mainly neutrophils, in bronchoalveolar lavages (BAL). We also observed a significant lymphocyte influx into the lungs associated with enhanced lung expression of chemokines and cytokines (KC, RANTES, TNF-α, IFN-γ, GM-CSF and VEGF). LPS-induced ALI was characterized by the enhanced expression of ICAM-1 and iNOS in the lungs. Mice that received LPS showed an increase in insulin levels. Leptin, when administered prior to LPS instillation, abolished all of these effects. LPS induced an increase in corticosterone levels, and leptin potentiated this event. CONCLUSION: These data suggest that exogenous leptin may promote protection during sepsis, and downregulation of the insulin levels and upregulation of corticosterone may be important mechanisms in the amelioration of LPS-induced ALI.

Acute kidney injury reduces phagocytic and microbicidal capacities of alveolar macrophages.

BACKGROUND/AIMS: Renal ischaemia-reperfusion injury (IRI) is a systemic inflammatory process in which Th1 responses predominate affecting other organs including the lungs. The present study explored the phagocytic and microbicidal capacity of macrophages in rats with lung inflammation that underwent IRI. METHODS: The alveolar macrophages of rats sensitised to OVA were evaluated for phagocytosis and bacterial killing 24h after antigen challenge in animals with or without prior submission to 60 min of renal ischaemia. RESULTS: Bronchoalveolar lavage had a high level of cellular infiltrate in immunised animals (420%) compared with control animals; IRI significantly reduced this infiltration (52%). Macrophages from animals immunised and challenged with OVA presented a 10x increase in phagocytic capacity compared to the control group, whereas immunised animals subjected to IRI showed a reduction in the phagocytic index of 68%. The killing of Klebsiella pneumoniae by macrophages from immunised animals was higher (56%) compared with the control group but reduced in animals submitted to IRI (45%). Immunised and challenged group showed an increase in gene expression levels of IL-10(450%), HO-1 (259%), INF-γ (460%) and MCP-1 (370%) compared to the immunised group subjected to IRI. CONCLUSIONS: Renal ischaemia and reperfusion injury apparently alters the phagocytic and microbicidal capacity of macrophages, reducing lung inflammation to OVA.

Generation and characterization of a spontaneously immortalized endothelial cell line from mice microcirculation.

Endothelial cells from microvasculature are directly involved in a large number of vascular diseases; however, culture of these cells is problematic, since most methodologies employ proteolytic enzymes or mechanical techniques, leading to cell damage and contamination of endothelial cultures with other cellular types. Besides, primary cultured cells have a short life span in vitro and undergo replicative senescence after 3-4 passages, limiting long-term studies. In the present work we report the generation of a spontaneously immortalized endothelial culture obtained from mice pulmonary capillaries. Firstly, primary (third passage) and immortalized (100th) cultures were established. Further, monoclonal populations were obtained by serial dilutions from immortalized cultures. Cells were analyzed according to: (1) morphological appearance, (2) expression of specific endothelial markers by fluorescent staining [von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE) and Ulex europaeus (UEA-1)] and by flow cytometry (endoglin, VE-cadherin and VCAM-1), and (3) release of nitric oxide (NO), assessed by the specific fluorescent dye DAF-2 DA, and prostacyclin (PGI2), quantified by enzyme immune assay. In both cultures cells grew in monolayers and presented cobblestone appearance at confluence. Positive staining for vWF, eNOS, ACE and UEA-1 was detected in cloned as well as in early-passage cultured cells. Similarly, cultures presented equal expressions of endoglin, VE-cadherin and VCAM-1. Values of NO and PGI2 levels did not differ between cultures. From these results we confirm that the described spontaneously immortalized endothelial cell line is capable of unlimited growth and retains typical morphological and functional properties exhibited by primary cultured cells. Therefore, the endothelial cell line described in the present study can become a suitable tool in the field of endothelium research and can be useful for the investigation of production of endothelial mediators, angiogenesis and inflammation.

Intrauterine undernourishment alters TH1/TH2 cytokine balance and attenuates lung allergic inflammation in wistar rats.

IL-4 produced by Th2 cells can block cytokine production by Th1 cells, and Th1 IFN-γ is known to counterregulate Th2 immune response, inhibiting allergic eosinophilia. As intrauterine undernutrition can attenuate lung inflammation, we investigated the influence of intrauterine undernourishment on the Th1/Th2 cytokine balance and allergic lung inflammation. Intrauterine undernourished offspring were obtained from dams fed 50% of the nourished diet of their counterparts and were immunized at 9 weeks of age. We evaluated the cell counts and cytokine protein expression in the bronchoalveolar lavage, mucus production and collagen deposition, and cytokine gene expression and transcription factors in lung tissue 21 days after ovalbumin immunization. Intrauterine undernourishment significantly reduced inflammatory cell airway infiltration, mucus secretion and collagen deposition, in rats immunized and challenged. Intrauterine undernourished rats also exhibited an altered cytokine expression profile, including higher TNF-α and IL-1ß expression and lower IL-6 expression than well-nourished rats following immunization and challenge. Furthermore, the intrauterine undernourished group showed reduced ratios of the IL-4/IFN-γ and the transcription factors GATA-3/T-Bet after immunization and challenge. We suggest that the attenuated allergic lung inflammation observed in intrauterine undernourished rats is related to an altered Th1/Th2 cytokine balance resulting from a reduced GATA-3/T-bet ratio.

Inflammatory milieu as an early marker of kidney injury in offspring rats from diabetic mothers.

The present study investigated the early presence of inflammatory response in renal tissue of young offspring from diabetic mothers. The effect of L-arginine (L-arg) supplementation was also investigated. The offspring was divided into four groups: group CO (controls); group DO (diabetic offspring); group CA (CO receiving 2% L-arg solution) and group DA (DO receiving the 2% L-arg solution). Glycemia, arterial pressure and renal function were evaluated; gene and protein expression of pro-inflammatory cytokines were also measured. Blood pressure levels were significantly increased in 2 and 6 month-old DO rats, whereas L-arg administration caused a significant decrease in the DA group, at both ages. DO rats showed a significantly blunted glycemic response to exogenous insulin. In 2 month-old DO animals, renal protein expression of pro-inflammatory molecules was significantly increased. At six months of age, we also observed an increase in gene expression of pro-inflammatory molecules, whereas L-arg supplementation prevented this increase at both ages. Our data suggest that activation of inflammatory pathways is present early in the kidney of DO rats, and that L-arg can attenuate the expression of these markers of tissue inflammation. Our results also reinforce the concept that intrauterine environmental factors are a fundamental determinant in the development of metabolic and vascular diseases later in life.

Modulation of lung allergic response by renal ischemia and reperfusion injury.

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors.

Leukotrienes produced in allergic lung inflammation activate alveolar macrophages.

It has been well-documented that leukotrienes (LTs) are released in allergic lung inflammation and that they participate in the physiopathology of asthma. A role for LTs in innate immunity has recently emerged: Cys-LTs were shown to enhance FcgammaR-mediated phagocytosis by alveolar macrophages (AMs). Thus, using a rat model of asthma, we evaluated FcgammaR-mediated phagocytosis and killing of Klebsiella pneumoniae by AMs. The effect of treatment with a cys-LT antagonist (montelukast) on macrophage function was also investigated. Male Wistar rats were immunized twice with OVA/alumen intraperitoneally and challenged with OVA aerosol. After 24 h, the animals were killed, and the AMs were obtained by bronchoalveolar lavage. Macrophages were cultured with IgG-opsonized red blood cells (50:1) or IgG-opsonized K. pneumoniae (30:1), and phagocytosis or killing was evaluated. Leukotriene C(4) and nitric oxide were quantified by the EIA and Griess methods, respectively. The results showed that AMs from sensitized and challenged rats presented a markedly increased phagocytic capacity via FcgammaR (10X compared to controls) and enhanced killing of K. pneumoniae (4X higher than controls). The increased phagocytosis was inhibited 15X and killing 3X by treatment of the rats with montelukast, as compared to the non-treated group. cys-LT addition increased phagocytosis in control AMs but had no effect on macrophages from allergic lungs. Montelukast reduced nitric oxide (39%) and LTC(4) (73%). These results suggest that LTs produced during allergic lung inflammation potentiate the capacity of AMs to phagocytose and kill K. pneumonia via FcgammaR.

Modulation of lung allergic inflammation and malnutrition.

Nutritional deficiency is commonly associated with a significantly impaired immune response, particularly in relation to cell-mediated immunity, the complement system, cytokine production and phagocyte function. However, there are few data on the consequences of nutritional deficiency in allergic diseases of the lung. In fact, malnutrition is the most common cause of immunodeficiency worldwide. Several studies have indicated that the incidence of alterations in lung functions can be associated with birth weight, specifically with maternal malnutrition, but data linking intrauterine undernutrition with allergic diseases of the lung are lacking. The purpose of this review is to associate malnutrition, including intrauterine malnutrition, with the establishment of immune responses and the development of lung allergic inflammation.

Influence of age on the development of immunological lung response in intrauterine undernourishment.

OBJECTIVE: We investigated the effect of intrauterine undernourishment on some features of asthma using a model of allergic lung inflammation in rats. The effects of age at which the rats were challenged (5 and 9 wk) were also evaluated. METHODS: Intrauterine undernourished offspring were obtained from dams that were fed 50% of the nourished diet of counterparts and were immunized at 5 and 9 wk of age. They were tested for immunoglobulin E anti-ova titers (by passive cutaneous anaphylaxis), cell count in the bronchoalveolar fluid, leukotriene concentration, airway reactivity, mucus production, and blood corticosterone and leptin concentrations 21 d after immunologic challenge. RESULTS: Intrauterine undernourishment significantly reduced the antigen-specific immunoglobulin E production, inflammatory cell infiltration into airways, mucus secretion, and production of leukotrienes B(4)/C(4) in the lungs in both age groups compared with respective nourished rats. The increased reactivity to methacholine that follows antigen challenge was not affected by intrauterine undernourishment. Corticosterone levels increased with age in the undernourished rats' offspring, but not in the nourished rats' offspring. Undernourished offspring already presented high levels of corticosterone before inflammatory stimulus and were not modified by antigen challenge. Leptin levels increased with challenge in the nourished rats but not in the undernourished rats and could not be related to corticosterone levels in the undernourished rats. CONCLUSION: Intrauterine undernourishment has a striking and age-dependent effect on the offspring, reducing lung allergic inflammation.

Mechanisms involved in the reduced leukocyte migration in intrauterine undernourishment.

OBJECTIVE: We investigated factors that may be involved in the reduced leukocyte migration observed in intrauterine undernourished rats. METHODS: Male Wistar rat offspring (8-9 wk of age) of dams fed during pregnancy with 50% less food than control dams were used to measure L-selectin expression (by flow cytometry), bone marrow cell count, blood cell count, laminin and type IV collagen in the basal membrane of venules of the spermatic fascia (by immunohistochemistry), total protein level and serum albumin, and the production of leukotriene B4 after stimulation with tumor necrosis factor-alpha and corticosterone plasma levels (by enzyme-linked immunosorbent assay). RESULTS: Hypocellularity in bone marrow and peripheral blood and reduced L-selectin expression were found in the undernourished rat offspring (UR) compared with nourished offspring (NR; P < 0.05). Type IV collagen in the basal membrane of the venules of the spermatic fascia was less in UR than in NR (P < 0.05). The total protein levels and serum albumin did not differ between the two groups. Leukotriene B4 production after stimulation with tumor necrosis factor-alpha was lower in UR (P < 0.05). These differences could not be attributed to circulating glucocorticoids levels, which were not different in the NR and UR groups. CONCLUSION: Our data suggest that all observed differences contribute to reduced leukocyte migration in undernourishment.

Intrauterine undernutrition in rats interferes with leukocyte migration, decreasing adhesion molecule expression in leukocytes and endothelial cells.

Experimental and epidemiologic data have shown that malnutrition predisposes individuals to infections. Immune responses are compromised, particularly in undernourished children. Therefore, we investigated the migratory capacity of leukocytes, using the intravital microscopy technique, in male Wistar rats (8-9 wk of age) that were undernourished in utero after their dams were fed 50% less food than the amount consumed by control dams. The number of leukocytes rolling along the venular endothelium, sticking after stimulation with leukotriene B4, tumor necrosis factor-alpha (TNF-alpha) or zymosan-activated plasma, or migrating after TNF-alpha stimulation was significantly reduced in the undernourished rat offspring. Compared with nourished rat offspring, undernourished offspring had significantly reduced numbers of circulating leukocytes, higher blood pressure, and higher leukocyte rolling velocity (V(WBC)), as well as a higher ratio between V(WBC) and RBC velocity (V(RBC)). Endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1) expression, analyzed by immunohistochemistry, and basal leukocyte L-selectin expression, analyzed by flow cytometry, were significantly reduced in the undernourished rat offspring. Because the groups did not differ in leukocyte CD11/18 expression, endothelial expression of platelet-endothelial cell adhesion molecule-1, or venular blood flow velocity and, consequently, venular shear rate, we conclude that intrauterine undernutrition in rats reduces leukocyte migration, downregulates endothelial expression of P-selectin and ICAM-1, as well as leukocyte expression of L-selectin, while reducing leukocyte counts. The higher V(WBC) and V(WBC)/V(RBC) ratio may also play a role in this reduced leukocyte migration. Our data suggest that this phenomenon is involved in the increased predisposition to infections in undernourished subjects.