Efeito radioprotetor da L-Glutamina na parede da bexiga de ratos submetidos à irradiação pélvica

A radioterapia é frequentemente utilizada no tratamento de tumores da próstata, porém durante esse procedimento a bexiga sadia usualmente sofre efeitos colaterais. Através do uso de um modelo animal para irradiação pélvica, avaliamos se a suplementação nutricional com L-glutamina poderia prevenir possíveis danos na parede da bexiga, especialmente em suas camadas mais superficiais. Ratos Wistar adultos machos com idade entre 3 e 4 meses foram separados em grupos de 8 animais: grupo controle que não recebeu a irradiação; grupos somente irradiados que foram mortos 7 (R7) e 15 dias (R15) após a irradiação (dose única de 10 Gy na região pélvico-abdominal); grupos irradiados e suplementados com L-glutamina (0,65g/kg de peso por dia), que foram mortos 7 (RG7) ou 15 após a irradiação. Células e vasos sanguíneos da lâmina própria, bem como o urotélio, foram avaliados com métodos histológicos. No urotélio foram feitas análises da altura e densidade nuclear e na lâmina própria densidade celular, densidade vascular e o número de mastócitos. Os resultados mostraram que em R7, a altura e densidade nuclear do urotélio e a densidade celular da lâmina própria não foram alterados significativamente. Entretanto a densidade dos vasos sanguíneos foi reduzida em 48% (p<0,05) e essa alteração foi evitada pela glutamina (p <0,02). No grupo R15, a densidade celular do epitélio aumentou em 35% (p<0,02). A densidade celular da lâmina própria não apresentou diferença estatística entre os grupos. Os mastócitos na lâmina própria foram reduzidos em R7 e R15. Apesar de ainda reduzidos em RG7 em RG15 houve aumento no número desse tipo celular o que sugere uma ação positiva da glutamina. Células α-actina positivas na lâmina própria formam uma camada suburotelial e foram identificadas como miofibroblastos. A espessura dessa camada aumentou em R7, mas foi semelhante ao controle em RG7, enquanto alterações em R15 e RG15 foram menos evidentes. Esses resultados mostraram que a utilização da L-glutamina antes e após a radioterapia deve ser considerada para uso humano na proteção da bexiga contra os efeitos da radiação.

Efeitos tardios no tecido ovariano de ratas Wistar após tratamento com Docetaxel e Ciclofosfamida

O câncer de mama (CM) é o segundo tipo de câncer mais comum no mundo. Sabe-se que a maior incidência de CM ocorre nas mulheres pós-menopausa, entretanto é crescente o número de mulheres jovens acometidas por esta doença. O tratamento do CM pode incluir: quimioterapia, radioterapia e/ou hormonioterapia. A quimioterapia, por se ser um tratamento sistêmico, pode causar importantes efeitos colaterais, entre eles a falência ovariana induzida por quimioterapia (FOIQ). As principais consequências da FOIQ são a infertilidade, além de complicações tardias relacionadas à diminuição do estrogênio, como a osteoporose e doenças cardiovasculares. O regime quimioterápico TC, adota a associação do docetaxel com a ciclofosfamida, como uma opção por fármacos que resultem numa taxa de sobrevida livre do câncer, e menores efeitos colaterais. Este trabalho teve como objetivo estudar os efeitos tardios no ovário causados pelo tratamento com a associação dos quimioterápicos docetaxel e ciclofosfamida (TC), em modelo animal com ratos Wistar. Para verificar o sinergismo desses quimioterápicos e assim analisar o efeito da administração conjunta, ratos Wistar fêmeas foram divididos em dois grupos: um grupo controle e um grupo que recebeu quimioterapia (TC). Os animais foram submetidos a eutanasia cinco meses após o fim do tratamento, e foram recolhidos o plasma e os ovários. Foram observadas alterações importantes. O nível de estradiol no plasma foi significativamente reduzido no grupo de TC em comparação com o grupo controle. Além disso, o número de núcleos apoptóticos foi maior no grupo TC. O papel da resposta inflamatória no desenvolvimento da lesão ovariana foi também investigado, e notou-se um aumento do número de mastócitos, e aumento da expressão de Fator de Necrose Tumoral-α (TNF-α) no grupo TC. O envolvimento de fibrose nesse processo, foi também investigado. Os resultados mostraram que níveis de expressão de Fator de Crescimento Tumoral-β1 (TGF-β1), Colágeno Tipo I (Col-I) e Colágeno Tipo III (Col-III) estavam maiores no grupo TC em comparação com o grupo de controle. A análise ultraestrutural revelou a presença de feixes de colágeno no grupo tratado, e mostrou que a arquitetura do tecido do ovário estava mais desorganizada neste grupo comparado ao grupo controle. Os resultados obtidos neste trabalho indicam que a combinação de ciclofosfamida e docetaxel, um recente regime quimioterápico proposto para o tratamento do CM, pode levar a importantes alterações no ovário. O processo inflamatório, desencadeado pela administração dos quimioterápicos, estimula a apoptose e liberação de TGF-β no estroma ovariano, que induz a produção de matriz extracelular e subsequente, substituição do tecido sadio por tecido fibrótico. A principal consequência deste processo é a diminuição, ou perda, da função ovariana, levando à menopausa precoce e possível infertilidade. É importante compreender os mecanismos envolvidos na infertilidade provocada pelo regime TC, a fim de estudar novos métodos que evitem este efeito indesejável em mulheres submetidas a tratamento do CM.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens), This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions, pLR was synthesized and elicited rapid, noncytolytic histamine release that had a a-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin, pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action, pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i,e. mature neutrophils, pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Antioxidants and oxidative stress in BAL fluid of atopic asthmatic children

Earlier studies in adults have indicated that increased oxidative stress may occur in the blood and airways of asthmatic subjects. Therefore the aim of this study was to compare the concentrations of antioxidants and protein carbonyls in bronchoalveolar lavage fluid of clinically stable atopic asthmatic children (AA, n = 78) with our recently published reference intervals for nonasthmatic children (C, n = 124). Additionally, lipid peroxidation products (malondialdehyde) in bronchoalveolar lavage fluid and several antioxidants in plasma were determined. Bronchoalveolar lavage concentrations (median and interquartile range) of ascorbate [AA: 0.433 (0.294-0.678) versus C: 0.418 (0.253-0.646) micromol/L], urate [AA: 0.585 (0.412-0.996) versus C: 0.511 (0.372-0.687) micromol/L], alpha-tocopherol [AA: 0.025 (0.014-0.031) versus C: 0.017 (0.017-0.260) micromol/L], and oxidized proteins as reflected by protein carbonyls [AA: 1.222 (0.970-1.635) versus C: 1.243 (0.813-1.685) nmol/mg protein] were similar in both groups (p > 0.05 in all cases). The concentration of protein carbonyls correlated significantly with the number of eosinophils, mast cells, and macrophages in AA children only. Concentrations of oxidized proteins and lipid peroxidation products (malondialdehyde) correlated significantly in AA children (r = 0.614, n = 11, p = 0.044). Serum concentrations of ascorbate, urate, retinol, alpha-tocopherol, beta-carotene, and lycopene were similar in both groups whereas alpha-carotene was significantly reduced in asthmatics. Overall, increased bronchoalveolar lavage eosinophils indicate ongoing airway inflammation, which may increase oxidatively modified proteins as reflected by increased protein carbonyl concentrations.

The pathophysiology of eosinophilic esophagitis: Altered mechanisms of antigen detection in the esophagus

Recently, a chronic idiopathic disease of the esophagus has emerged, which is now known as eosinophilic esophagitis (EoE). Incomplete knowledge regarding the pathogenesis of EoE has limited treatment options. EoE is known to be a Th2-type immune-mediated disorder. Based on previous studies in both patients and experimental models, it is possible that an abnormal reaction to antigen mediates the pathophysiology of EoE. In this thesis, symptoms and signs unique to EoE were identified by an age-matched, case-controlled study of 326 patients with EoE and gastroesophageal reflux disease. The molecular mechanisms involved in antigen detection in the esophagus, in relation to EoE were then investigated. Esophageal epithelial cells were found, for the first time, to be capable of acting as non-professional antigen presenting cells, with the ability to engulf, process and present antigen on MHC class II to T helper lymphocytes. Antigen presentation by esophageal epithelial cells was induced by interferon-γ, which is increased in biopsies from patients with EoE. Next, it was discovered that esophageal epithelial cell lines expressed functional toll-like receptor (TLR) 2 and TLR3, but in esophageal mucosal biopsies only infiltrating immune cells (including eosinophils) expressed TLR2 and TLR3. Finally, the potential involvement of IgE in the pathogenesis of esophageal inflammation was investigated. IgE in the esophagus was found to be present on mast cells, which are increased in density in the esophageal mucosae of patients with EoE and especially those with a history of atopy. Mechanisms of antigen detection may mediate the pathophysiology of EoE in the esophagus through antigen presentation by epithelial cells, detection by TLRs on immune cells and detection through IgE on mucosal mast cells. Together, these findings demonstrate that mechanisms of antigen detection may actually contribute to the pathophysiology of EoE. Through increased understanding of the mechanisms of EoE, the results of this thesis may contribute to future therapy.

Adenosine, inflammation and asthma - a review

Adenosine is a ubiquitous molecule present in every cell of the human body. It has a wide range of physiological functions mediated predominantly through specific cell surface adenosine receptors. Adenosine has both pro- and anti-inflammatory effects and acts on inflammatory and resident immune cells and antioxidant enzymes. The elevation of adenosine in the bronchoalveolar lavage (BAL) fluid of asthmatics combined with its bronchoconstrictor effect on the airways in asthmatics has led to increased research into the contribution of adenosine in the pathophysiology of inflammation and asthma. This review looks at the airway response to adenosine and at the interaction of adenosine with mast cells and basophils.

Inflammatory mediators in bronchoalveolar lavage samples from children with and without asthma

Background: We investigated whether eosinophils and mast cells, found in the airways of children with wheeze, were activated during relatively asymptomatic periods.
Methods: A nonbronchoscopic bronchoalveolar lavage (BAL) procedure was performed on children presenting for an elective surgical procedure. Eosinophil-derived (eosinophil cationic protein, ECP) and mast cell-derived (histamine/tryptase) mediator concentrations were measured in the BAL fluid. A detailed history and serum immunoglobulin E were used to classify the children into four groups: atopic with and without asthma, viral-associated wheeze and normal controls.
Results: The ECP concentrations in BAL from atopic asthmatic subjects were significantly higher than those measured in BAL from normal controls (P < 0.01), no other groups differed significantly. Histamine concentrations were elevated in both the atopic asthmatic and viral-associated wheeze groups compared with controls (P < 0.02) and additionally higher concentrations were obtained in atopics with asthma compared with atopics without asthma (P < 0.03). Tryptase concentrations did not differ between groups, although the tryptase and histamine concentrations correlated significantly (r = 0.78, P < 0.0001).
Conclusions: Elevated histamine concentrations were found in children with wheeze regardless of the aetiology, whereas ECP was only elevated in those asthmatics with atopy. This suggests that even in relatively quiescent periods, there is some on going activation of airway eosinophils in children with atopic asthma.

Investigating paediatric airways by non-bronchoscopic lavage: Normal cellular data

Background: Bronchoscopic bronchoalveolar lavage in children to investigate bronchia disorders such as asthtna has both ethical and procedural difficulties.


Objective: The aim of this study was to establish a standardized non-bronchoscopic method to perform bronchoalveolar lavage in children attending for elective surgery to obtain normal cellular data.


Methods: Bronchoalveolar lavage was performed on normal children (n= 55) by infusing saline (20 mL) through an 8 FG suction catheter passed after endotracheal intubation. Oxygen saturation, heart and respiratory rate were monitored during the bronchoalveolar lavage procedure. Cellular analysis and total protein estimation of the lavage fluid were performed. Epithelial lining fluid volume was calculated (n = 15) using the urea dilution method.


Results: The procedure was well tolerated by all children. Total cell count and differential cell count for children (macrophages 70.8 ± 2.3%, lymphocytes 3.8 ± 0.6%, neutrophils 5,7 ± 1.0%, eosinophils 0.14 ± 0.03%. epithelial cells 19.6 ± 2.1%, mast cells 0.21 ± 0.02%) were similar to those reported for adults. Age and sex comparisons revealed no differences between groups. The mean total protein recovered in the cell free supernatant was 49.72 ± 4.29 mg/L and epithelial lining fluid volume was 0.82 ± 0.11% of return lavageate.


Conclusion This method allows bronchoalveolar lavage to be performed safely and quickly on children attending for routine elective surgery. Using this method and taking the ‘window of opportunity’ of elective surgery, the presence or absence of airway inflammation could be studied in children with various patterns of asthma during relatively asymptomatic periods.

Antimicrobial peptides in the venom of the European hornet, Vespa crabro, identified as mastoparan C and crabrolin

Amphibian skin secretions are rich sources of cationic amphipathic peptides which often possess potent and broad-spectrum antimicrobial activity. However, the venoms of other animals such as hymenopteran insects, also contain peptides with these characteristics and the literature is unclear as to their antimicrobial potential. Here we subjected the venom of the European hornet, Vespa crabro, to reverse phase HPLC fractionation followed by screening of aliquots of individual fractions in bacterial zonal inhibition assays. Two major peptides possessing activity in these assays were further purified by HPLC and subjected to MALDI-TOF MS analysis and MS/MS fragmentation using an ESI mass spectrometer. The peptides were identified as mastoparan C (LNLKALLAVAKKILamide) and crabrolin (FLPLILRKIVTALamide). Replicates of both peptides were synthesised by solid-phase methodology and mean inhibitory concentrations (MICs) established against Staphylococcus aureus and Escherichia coli. Mastoparan C was found to be a potent antimicrobial with MIC values of 2 µM and 4 µM against S. aureus and E. coli, respectively. Crabrolin was found to be less potent with MIC values of > 160 µM and 40 µM for S. aureus and E. coli. Hornet venom thus contains a potent antimicrobial peptide that has been unambiguously identified as mastoparan C, a peptide that is known to affect profound histamine release from mast cells and to generally activate membrane G protein-linked receptors. It is thus highly probable that its antimicrobial effects, like those previously documented, are a result of a generalized membrane interactive and disruptive function — perhaps reflective of the authentic role of amphibian skin antimicrobials.

Kassorins: Novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC50 = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.

Peptide Tyrosine Arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin secretions of the dusky gopher frog, Rana sevosa

An octadecapeptide was isolated from the skin secretions of the dusky gopher frog (Rana sevosa) on the basis of histamine release from rat peritoneal mast cells. This peptide was purified to homogeneity by HPLC and found to have the following primary structure, YLKGCWTKSYPPKPCFSR, using both Edman degradation chemistry and peptide sequencing using high-resolution mass spectrometry (Q-TOF MS). The peptide, named peptide Tyrosine Arginine (pYR) shares 77.8% homology with peptide Leucine Arginine (pLR). The effects of the natural amidated peptide, non-amidated peptide and C-loop region of pYR on granulopoiesis and neutrophil apoptosis were investigated. All three analogues inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, the mature neutrophil. Thus, pYR is a novel member of an important and emerging new class of amphibian peptides with hemopoietic actions. (c) 2004 Elsevier Inc. All rights reserved.

Bronchoalveolar lavage findings in patients with chronic nonproductive cough

Mast cells and eosinophils may play a role in the pathophysiology of chronic cough in nonasthmatics. It is unknown, how;ever, whether degranulation of these cells occurs in the airways of such patients.

INTRAVASCULAR ANTI-IGE CHALLENGE IN PERFUSED LUNGS - MEDIATOR RELEASE AND VASCULAR PRESSOR-RESPONSE

Intravascular application of goat anti-rabbit immunoglobulin E (IgE) was used to stimulate parenchymal mast cells in situ in perfused rabbit lungs. Sustained pulmonary arterial pressure rise was evoked in the absence of lung vascular permeability increase and lung edema formation. Early prostaglandin (PG) D2 and histamine release into the perfusate was documented, accompanied by more sustained liberation of cysteinyl leukotrienes (LT), LTB4, and PGI2. The quantities of these inflammatory mediators displayed the following order: histamine > cysteinyl-LT > PGI2 > LTB4 > PGD2. Pressor response and inflammatory mediator release revealed corresponding bell-shaped dose dependencies. Cyclooxygenase inhibition (acetylsalicylic acid) suppressed prostanoid generation, increased LT release, and did not substantially affect pressor response and histamine liberation. BW755 C, a cyclo- and lipoxygenase inhibitor, blocked the release of cysteinyl-LT and markedly reduced the liberation of the other inflammatory mediators as well as the pressor response. The H-1-antagonist clemastine caused a moderate reduction of the anti-IgE-provoked pressure rise. We conclude that intravascular anti-IgE challenge in intact lungs provokes the release of an inflammatory mediator profile compatible with in situ lung parenchymal mast cell activation. Pulmonary hypertension represents the predominant vascular response, presumably mediated by cysteinyl-LT and, to a minor extent, histamine liberation.

Immunomodulatory molecules of Fasciola hepatica:Candidates for both vaccine and immunotherapeutic development

The liver fluke, Fasciola hepatica, causes fascioliasis in domestic animals (sheep, cattle), a global disease that is also an important infection of humans. As soon as the parasite invades the gut wall its interaction with various host immune cells (e.g. dendritic cells, macrophages and mast cells) is complex. The parasite secretes a myriad of molecules that direct the immune response towards a favourable non-protective Th2-mediate/regulatory environment. These immunomodulatory molecules, such as cathepsin L peptidase (FhCL1), are under development as the first generation of fluke vaccines. However, this peptidase and other molecules, such as peroxiredoxin (FhPrx) and helminth defence molecule (FhHDM-1), exhibit various immunomodulatory properties that could be harnessed to help treat immune-related conditions in humans and animals.

Cis interactions of immunoreceptors with MHC and non-MHC ligands

The conventional wisdom is that cell-surface receptors interact with ligands expressed on other cells to mediate cell-to-cell communication (trans interactions). Unexpectedly, it has recently been found that two classes of receptors specific for MHC class I molecules not only interact with MHC class I molecules expressed on opposing cells, but also with those on the same cell. These cis interactions are a feature of immunoreceptors that inhibit, rather than activate, cellular functions. Here, we review situations in which cis interactions have been observed, the characteristics of receptors that bind in trans and cis, and the biological roles of cis recognition.