Mario Mariano

Immune complex induced pancreatitis: Effect of BN 52021, a selective antagonist of platelet-activating factor

A model of acute pancreatitis was developed by induction of an immune complex mediated hypersensitivity reaction in rats. This acute inflammatory reaction was characterized by intense interstitial edema, neutrophil infiltration and margination, and congestion of small vessels whereas serum amylase levels remained unchanged. Microscopic examination of the pancreatic tissue revealed the presence of immune complex deposition around blood vessels and ducts. Vascular permeability, as measured by Evans blue extravasation increased by 6 fold. In addition, circulating platelets dropped to 50% of normal levels. Injection of platelet-activating factor (PAF) in the peritoneal cavity of rats also produced an increase in vascular permeability in the pancreas. A selective PAF-antagonist, BN 52021 reduced by approximately 50% the increase in vascular permeability produced by immune complex in the pancreas as well as that elicited by intraperitoneal injection of PAF. These results suggest that PAF plays a role in the pathological manifestations of immune complex-mediated pancreatitis.

A new murine model of pulmonary eosinophilic hypersensitivity: Contribution to experimental asthma

BACKGROUNDnWe have recently described a model of hypersensitivity reaction in the mouse paw, which induces a typical late-phase reaction with a marked eosinophilic infiltrate.nnnOBJECTIVEnIn the search for a murine model of asthma, this model was adapted to the lungs and compared with other models of pulmonary hypersensitivity.nnnMETHODSnA fragment of heat-coagulated hens egg white was implanted subcutaneously, and 14 days later, the mice were challenged intratracheally with aggregated ovalbumin. Comparison was made with a group that received subcutaneous injection of soluble ovalbumin in alumen, challenged as described above and with four additional protocols of immunization and challenge.nnnRESULTSnForty-eight hours after challenge, the percentage of eosinophils was higher in the egg white implant group (35%) than in the group immunized with ovalbumin in alumen (10.4%). The eosinophil peroxidase activity in lung homogenates of the first group was also significantly higher (529 ng/ml) than that of the second group (43 ng/ml). These results were reproduced in five different mouse strains. Compared with five different models of lung hypersensitivity, the egg white implant model was unique in terms of persistence of the pulmonary eosinophilia. Histopathologic analysis of the lungs of mice immunized with egg white implant showed peribronchial, perivascular, and intraepithelial eosinophil infiltration; morphologic characteristics of bronchoconstriction; and patchy epithelial shedding. At 21 days, in addition to persistence of eosinophil infiltrate, enlarged alveoli, reflecting air trapping, were observed.nnnCONCLUSIONnOn the basis of the characteristics of the model described here, we propose it as a suitable murine model of asthma.

Histoarchitecture of schistosomal granuloma development and involution: morphogenetic and biomechanical approaches

The authors present morphogenetic and biomechanical approaches on the concept of the Schistosoma mansoni granulomas, considering them as organoid structures that depend on cellular adhesion and sorting, forming rearrangement into hierarchical concentric layers, creating tension-dependent structures, aiming to acquire round form, since this is the minimal energy form, in which opposing forces pull in equally from all directions and are in balance. From the morphogenetic point of view, the granulomas function as little organs, presenting maturative and involutional stages in their development with final disappearance (pre-granulomatous stages, subdivided in: weakly and/or initial reactive and exudative; granulomatous stages: exudative-productive, productive and involutional). A model for the development of granulomas was suggested, according to the following stages: encapsulating, focal histolysis, fiber production, orientation and compacting and involution and disintegration. The authors concluded that schistosomal granuloma is not a tangled web of individual cells and fibers, but an organized structure composed by host and parasite components, which is not formed to attack the miracidia, but functions as an hybrid interface between two different phylogenetic beings.

Protein malnutrition: Effect on myeloid cell production and mobilization into inflammatory reactions in mice

The effect of a low protein diet on mobilization of inflammatory cells into injured tissues was studied using the experimental murine model of subcutaneous glass implants. We observed an impaired mobilization of mononuclear and polymorphonuclear phagocytes into the injury site in undernourished mice, reflecting apparently a low availability of inflammatory cells at the systemic level. Analysis of bone marrow and blood leukograms has shown that the mobilization of blood leukocytes into the lesions was not compensated by their production and release from the bone marrow. Cultures of bone marrow cells in the presence of plasma obtained from undernourished animals showed that the stimulatory factors for proliferation of early precursors of myeloid lineages were present in normal or increased levels. The full maturation and release of myeloid cell lineages was delayed. The impaired production of myeloid cells in undernourished animals does not appear to be a consequence of a general insufficient protein availability, but reflects specific modifications of the stimulatory factors that control proliferation, differentiation and release of leukocytes from the bone marrow.

Pharmacological characterization of polycation-induced rat hind-paw oedema.

1 The inflammatory response induced by poly‐l‐arginine in the rat hind‐paw was studied both by measuring paw oedema and histologically. 2 The paw volume was measured with a hydroplethysmometer at 0.5, 1, 2, 4, 6 and 18 h after the subplantar injection of the polycation. Protein extravasation was evaluated with Evans blue and the histology studied by light microscopy. 3 Poly‐l‐arginine (12, 24, 43 and 115 kD) caused dose‐ and molecular weight‐dependent oedema which had a rapid onset and long duration. Evans blue extravasation paralleled the oedema induced by poly‐l‐arginine. Microscopic examination of the paws at early stages of oedema formation showed exuberant liquid exudate with no inflammatory cells. After 18 h, a cellular infiltrate was present, consisting mainly of mononuclear cells. 4 Indomethacin, dexamethasone, BW755c or the PAF‐antagonist WEB 2086 caused no significant inhibition of the poly‐l‐arginine‐induced oedema. Cyproheptadine had inhibitory effects only on the early stages of the polycation‐induced oedema. Similar results were observed with rats depleted of histamine and 5‐hydroxytryptamine. 5 Heparin, a polyanion, injected in the rat paw caused a marked inhibition of the polycation‐induced oedema. NG‐monomethyl‐l‐arginine (LNMMA), an inhibitor of EDRF synthesis, injected locally also produced a marked inhibition, but this inhibition was reversed by iloprost. 6 These results suggest that the oedema induced by polycations was due to their cationic charge. The inhibitory effect of LNMMA is probably due to a decrease in vascular flow rather than a decrease in vascular permeability.

Inflammatory responses induced by poly-L-arginine in rat lungs in vivo.

The inflammatory responses induced by the synthetic polycation poly-l-arginine injected either into the pleural cavity or into the trachea in rats have been investigated. Poly-l-arginine (4–40 nmol/rat) injected intrapleurally induced exudate formation and leucocyte migration (mainly polymorphonuclear cells). The exudate formation (but not cell migration) was dependent on the molecular weight of the poly-l-arginine used (24 and 115 kD). The poly-l-arginine-induced pleurisy was mainly dependent on activation of mast cells since it was significantly reduced either in rats depleted of their stores of histamine and serotonin or in rats previously treated with the serotonin receptor antagonist methysergide. The polyanions heparin and dermatan sulphate when administered intrapleurally with the polycation markedly reduced the exudate formation. Poly-l-arginine (115 kD, 8.5 nmol/rat) injected intratracheally caused lung oedema, increased leucocyte number and protein content of bronchoalveolar lavage, respiratory insufficiency and 60% mortality in 6 h. Depletion of histamine and serotonin stores or of circulating neutrophils decreased the leucocytes in bronchoalveolar lavage but did not increase survival rate, whereas the polyanion dermatan sulphate prevented the mortality completely. These results suggest that the inflammatory changes caused by poly-l-arginine are dependent on mast cell activation but that the lethality after intratracheal administration is due to electrostatic interactions of the polycation with anionic surfaces present in the pulmonary epithelium.

Keloid heterograft in the hamster (Mesocricetus auratus) cheek pouch

PURPOSEnTo study the integration of keloid heterograft in hamster (Mesocricetus auratus) cheek pouch.nnnMETHODSnThe sample is formed by 18 male hamsters, heterogenic ones, aged between 10 and 14 weeks. Keloid fragments were obtained from keloid scars of the breast region of adult female mulatto patient. Each hamster received keloid fragments into both of its pouches, in a total of 36 grafted fragments. Animals were distributed into 6 groups for having their grafts assessed in the days 5, 12, 21,42, 84, and 168. A macroscopic assessment is performed by comparing the pouch containing the grafted fragment, at each time point, with the same pouch in the immediate post surgical moment through a comparison of standardized photographs. Under microscope, the presence of blood vases is considered within the conjunctive tissue of the grafted fragment, as a criterion of its integration. Other events, as keratin secretion, the presence of cellular infiltrated, epithelium and keloid collagen fibers aspects are also analyzed.nnnRESULTSnMacroscopy reveals intensive vascularization of the pouch up to 12 days from the transplantation and the presence of constant dark brown pigmentation on the grafted keloid fragments. In microscopy, the integration of keloid fragments is considered by the presence of blood capillary vases within conjunctive tissue. The presence of intensive cellular inflammatory type infiltrated up to 12 days is also observed, as well as the remaining of keloid epithelium up to 21 days, and the appearing of melanocytes from the day 42.nnnCONCLUSIONnHamster cheek pouch represents, a priori, an experimental model for the investigation of keloid.

Minisuíno (minipig) na pesquisa biomédica experimental: o Minipig br1

Different species of vertebrate and invertebrate animals have been used in experimental investigation. In this report a brief review on the use of swine in biomedical research and the similarities of the anatomy, physiology, biochemistry and immunology of this specie with those of man is presented. It is also reviewed the development in the 60th of the minipig and the achievements obtained with this animal model in distinct areas of experimental medicine. Finally, the basic characteristics of a colony of minipigs developed by the author are described. The conditions these animals are feed, housed, vaccinated and breed are described. They weight from 10-15 kilos when six months of age and from 30-37 kilos with 11 months of age. The possible contribution of this animal model for the development of experimental surgery and other areas of animal experimentation in this country is discussed.

Role of Lymphatic Drainage on the Development of Calmette-Guérin Bacillus-Induced Granulomas in the Hamster

The influence of lymphatic drainage on Calmette-Guérin bacillus (BCG)-induced granulomas was investigated by comparing the time course of granuloma formation in two sites: the hamster footpad and the check pouch, an area deprived of lymphatic vessels. Typical epithelioid granulomas developed in both sites. Whereas in the footpad the size of granulomas increased and the volume of the lesion persisted, in the pouch the lesion decreased in volume. The inoculation of BCG into the footpad of animal with granulomas in the pouch, reactivated the pouch lesions. T lymphocytes were detected by an immunocytochemical technique at the edge of these lesions. Inoculation of the bacteria into the pouch induces suppressive mechanisms which hold down the volume of the lesions induced in the footpad. The cutaneous purified protein derivative (PPD) test, positive in animals inoculated in the footpad, was always negative in animals with granulomas in the pouch. The number of bacteria per microscopic field in pouch granulomas increased from 10 to 1,000 after PPD injection. Evidence that PPD has a direct effect on pouch granuloma cells is given.

A RAT MODEL PRESENTING EOSINOPHILIA IN THE AIRWAYS, LUNG EOSINOPHIL ACTIVATION, AND PULMONARY HYPERREACTIVITY

The aim of this study was to examine antigen-induced lung cell migration, eosinophil activation, and pulmonary reactivity of Wistar rats exposed to a new sensitization technique. The animals were sensitized with a single subcutaneous implant of a fragment of heat coagulated hen egg white and challenged 21 days later with an intratracheal injection of heat-aggregated ovalbumin (EWI). For comparison, another group of rats were sensitized by an intraperitoneal injection of ovalbumin in alum as adjuvant, with one booster on day 14 and challenge on day 21 post immunization (OVA/AL). Twenty-four hours after antigen challenge, the EWI group presented a higher number of eosinophils in the bronchoalveolar lavage (BAL) (4.85 +/- 1.43 x 10(6)) than the OVA/AL group (0.2 +/- 0.06 x 10(6)) or the control group, where the level of eosinophils were essentially undetectable. Levels of eosinophil peroxidase activity were increased in the cell-free BAL and homogenates of lung tissue in the EWI group (12.10 +/- 2.97 mg/mL and 36.14 +/- 7.21 ng/mg, respectively), but not in the OVA/AL group (4.83 +/- 1.4 ng/mL and 11.95 +/- 2.54 ng/mg, respectively), as compared with controls (5.16 +/- 1.65 ng/mL and 12.13 +/- 1.74 ng/mg, respectively). Thromboxane B2 levels were also increased in the BAL of EWI group (2.89 +/- 0.54 ng/mL) but not the OVA/AL group (1.13 +/- 0.23 ng/mL) as compared with controls (1.14 +/- 0.19 ng/mL). In contrast, the levels of prostaglandin E2 in the BAL were increased in both groups (456.4 +/- 11.8 pg/mL in the EWI group and 303.5 +/- 31.7 pg/mL in the OVA/AL group) as compared with controls (205.7 +/- 29.7 ng/mL). Moreover, only the EWI group developed increased pulmonary reactivity to serotonin (around two-fold), 24 hours after antigen challenge. The extent of lung eosinophil migration and activation and the pulmonary hyperreactivity induced by this novel sensitization procedure without adjuvants represents a significant improvement over existing experimental models of asthma.