Artur P. Águas

Diabetes-prone NOD mice are resistant to Mycobacterium avium and the infection prevents autoimmune disease.

It was recently proposed that the diabetes genes of non‐obese diabetic (NOD) mice are linked to the Bcg gene that is associated with resistance to infection by mycobacteria; however, it has not been established whether NOD mice are resistant or susceptible to the infection, although there are previous investigations on response of NOD mice to other intracellular parasites (e.g. Kaye et al., Eur. J. Immunol.22: 357–364). We have investigated here this question, as well as the consequences of mycobacterial infection on the natural history of murine diabetes. Female NOD mice were intraperitoneally infected with 108 viable bacilli of Mycobacterium avium at 2 months of age, i.e. before the mice show diabetes; they were studied up to the sixth month of age (when more than half of untreated female NOD mice show glycosuria). To determine whether NOD mice were susceptible or resistant to M. avium infection, we have compared the kinetics of bacterial growths in liver and spleen of the mice with those determined in M. avium‐susceptible (BALB/c) and resistant (C3H) strains of mice. NOD mice were able to control the M. avium infection, following a pattern similar to that observed in infected C3H mice. The mycobacterial infection prevented the expression of diabetes in all of the infected NOD mice and it also decreased the incidence of proteinuria in the treated mice. The infected NOD mice showed a marked enhancement in antibodies against the 65 000 mycobacterial antigen (heat‐shock protein (hsp) 65) up to the second month of infection and these elevated titres slowly decreased in the following months; anti‐hsp 65 antibodies were not detected in age‐matched controls. This is the first demonstration that NOD mice are naturally resistant to mycobacterial infection, and we reinforce evidence on the role of immune response triggered by mycobacteria and its hsp 65 antigen in prevention of diabetes in NOD mice.

Evaluation of the effect of the degree of acetylation on the inflammatory response to 3D porous chitosan scaffolds

The effect of the degree of acetylation (DA) of 3D chitosan (Ch) scaffolds on the inflammatory reaction was investigated. Chitosan porous scaffolds with DAs of 4 and 15% were implanted using a subcutaneous air-pouch model of inflammation. The initial acute inflammatory response was evaluated 24 and 48 h after implantation. To characterize the initial response, the recruitment and adhesion of inflammatory cells to the implant site was studied. The fibrous capsule formation and the infiltration of inflammatory cells within the scaffolds were evaluated for longer implantation times (2 and 4 weeks). Chitosan with DA 15% attracted the highest number of leukocytes to the implant site. High numbers of adherent inflammatory cells were also observed in this material. For longer implantation periods Ch scaffolds with a DA of 15% induced the formation of a thick fibrous capsule and a high infiltration of inflammatory cells within the scaffold. Our results indicate that the biological response to implanted Ch scaffolds was influenced by the DA. Chitosan with a DA of 15% induce a more intense inflammatory response when compared with DA 4% Ch. Because inflammation and healing are interrelated, this result may provide clues for the relative importance of acetyl and amine functional groups in tissue repair and regeneration.

Macrophage polarization following chitosan implantation

Macrophages are a key cell in the host response to implants and can be polarized into different phenotypes capable of inducing both detrimental and beneficial outcomes in tissue repair and remodeling, being important in tissue engineering and regenerative medicine. The objective of this study was to evaluate the macrophage response to 3D porous chitosan (Ch) scaffolds with different degrees of acetylation (DA, 5% and 15%). The M1/M2 phenotypic polarization profile of macrophages was investigated in vivo using a rodent air-pouch model. Our results show that the DA affects the macrophage response. Ch scaffolds with DA 5% induced the adhesion of lower numbers of inflammatory cells, being the M2 the predominant phenotypic profile among the adherent macrophages. In the inflammatory exudates F4/80(+)/CD206(+) cells (M2 macrophages) appeared in higher numbers then F4/80(+)/CCR7(+) cells (M1 macrophages), in addition, lower levels of pro-inflammatory cytokines together with higher levels of anti-inflammatory cytokines were found. Ch scaffolds with DA 15% showed opposite results, since M1 were the predominant macrophages both adherent to the scaffold and in the exudates, together with high levels of pro-inflammatory cytokines. In conclusion, Ch scaffolds with DA 5% induced a benign M2 anti-inflammatory macrophage response, whereas Ch scaffolds with DA 15% caused a macrophage M1 pro-inflammatory response.

Mechanisms of Mycobacterium avium-induced resistance against insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice: role of Fas and Th1 cells

NOD mice spontaneously develop autoimmune diabetes. One of the manipulations that prevent diabetes in NOD mice is infection with mycobacteria or immunization of mice with mycobacteria‐containing adjuvant. Infection of NOD mice with Mycobacterium avium, done before the mice show overt diabetes, results in permanent protection of the animals from diabetes and this protective effect is associated with increased numbers of CD4+ T cells and B220+ B cells. Here, we investigate whether the M. avium‐induced protection of NOD mice from diabetes was associated with changes in the expression of Fas (CD95) and FasL by immune cells, as well as alterations in cytotoxic activity, interferon‐gamma (IFN‐γ) and IL‐4 production and activation of T cells of infected animals. Our data indicate that protection of NOD mice from diabetes is a Th1‐type response that is mediated by up‐regulation of the Fas–FasL pathway and involves an increase in the cytotoxicity of T cells. These changes are consistent with induction by the infection of regulatory T cells with the ability of triggering deletion or anergy of peripheral self‐reactive lymphocytes that cause the autoimmune disease of NOD mice.

A role for CD45RBlow CD38+ T cells and costimulatory pathways of T‐cell activation in protection of non‐obese diabetic (NOD) mice from diabetes

Non‐obese diabetic (NOD) mice spontaneously develop autoimmune insulin‐dependent diabetes mellitus (IDDM). Infection of the animals with mycobacteria, or immunization with mycobacteria‐containing adjuvant, results in permanent protection of NOD mice from diabetes and we have recently reported that the phenomenon is associated with increased numbers of interferon‐γ‐producing T cells, possessing increased cytotoxic activity, and also with augmented numbers of activated immunoglobulin M‐positive (IgM+) B cells. Here, we have investigated whether protection of NOD mice from IDDM was associated with changes on costimulatory pathways of T and B cells, namely CD28/CTLA‐4–B7 and CD40–CD40 ligand (CD40L) and we also further characterized protective T helper (Th) cells with regards to the expression of the differentiation markers CD45RB and CD38. We report that Th cells involved in diabetes vaccination of NOD mice by mycobacterial infection seem to belong to CD45RBlo CD38+ phenotype. The protective effect of Mycobacterium avium infection is also associated with increased CD40L and CTLA‐4‐ expressing Th cells and with the generation of a CD40− IgG+ B cells. Our data are consistent with induction by mycobacterial infection of regulatory CD45RBlo CD38+ Th cells with the ability to trigger deletion or anergy of peripheral self‐reactive lymphocytes, with shutting down of IgG+ B‐cell response. They also implicate a role for IgG+ B cells in the autoimmune aggression of the endocrine pancreas of NOD mice.

High-resolution localization of lactoferrin in human neutrophils: labeling of secondary granules and cell heterogeneity.

We studied by electron microscopy the topography of the iron‐binding protein lactoferrin in human peripheral blood neutrophils by immunogold labeling of thin‐sectioned cells. We show that fixation in 0.3% glutararaldehyde, supplemented by 2% formaldehyde, and combined with embedding in the hydrophilic resin LR White results in excellent preservation of the lactoferrin antigenicity and of the neutrophil microanatomy. Most of the immunogold marking was seen on the specific (secondary) granules of polymorphonuclear leukocytes, thus strengthening prevailing views on the subcellular distribution of lactoferrin in neutrophils. We document that a minority (8‐10%) of neutrophils showed significant amounts of lactoferrin associated with a flocculent, protein‐like material seen inside large cytoplasmic vacuoles. We show that circulating neutrophils comprise 6‐10% of cells that are virtually devoid of lactoferrin labeling. This finding provides specific cytochemical support for the concept that human polimorphonuclear leukocytes are made up of a heterogeneous population of cells.

Prostate cancer cell proliferation and angiogenesis in different obese mice models

Obesity has been associated with increased incidence and aggressiveness of prostate cancer. Although controversial, several studies suggest that leptin could influence tumour cell growth and proliferation. The main goal of this study was to assess cellular growth of prostate adenocarcinoma cells in obese mice with different endogenous hormonal environments in what relates to leptin circulating levels and sensitivity. Four groups of mice (n = 6/group) were used, namely obese mice with congenital non‐functioning leptin receptor OBR (db/db), obese mice with congenital leptin deficiency (ob/ob), mice with diet induced obesity (DIO) and normal weight C57BL/6J mice (control). All groups of mice were injected subcutaneously with 3.0 × 105 RM1 cells/500 μl PBS (murine prostate carcinoma androgen insensitive cells) and tumour growth and angiogenesis were evaluated 14 days after inoculation. The tumours induced in ob/ob and DIO mice were significantly larger (P < 0.001) while those induced in db/db mice were significantly smaller (P = 0.047), when compared with controls. Morphometric analysis revealed that mitotic index and Ki‐67 positive nuclear density, both cell proliferation markers, were also significantly lower in the tumours of db/db mice (P < 0.001) when compared to controls. An inverse correlation was observed between leptin plasma levels and tumour weight (r = −0.642, P < 0.001), mitotic index (r = −0.646, P < 0.01) and Ki‐67 positive nuclear density (r = −0.795, P < 0.001). These results suggest that high leptin concentrations are not favourable to RM1 cell growth and proliferation. On the contrary, high plasma leptin levels were associated with less cellular proliferation and angiogenesis in vivo.

A rat model of restrictive bariatric surgery with gastric banding.

Background: Gastric banding is a well established weight reduction operation that is effective in the treatment of severe obesity. Its metabolic and endocrine mechanisms of action, however, remain unclear. The aim of this study was to establish a rat model of gastric banding that would replicate the procedure performed in human obese patients. Methods: Male Wistar rats were submitted either to gastric banding (n=5) or sham gastric banding (n=4), and were followed for 21 days. Detailed description on how to perform gastric banding in rats are herein described. Results: The Wistar rats submitted to gastric banding showed a decrease in weight gain and food intake when compared to sham-operated rats. The cumulative weight gain during the 21 days after the surgical procedure was 143 ± 2.58 g for the gastric banded rats and 162 ± 2.48 g for the sham-operated animals (P=0.001). The cumulative food intake was 329 ± 0.53 g for the gastric banded rats and 380 ± 15.22 g for the sham-operated animals, also statistically significant (P=0.025). Conclusion: A rat model to study gastric banding is described. This model can now be used for experimental investigation of biochemical and molecular mechanisms of weight loss resulting from this type of surgery.

Liver enzymes and ultrastructure in rabbit haemorrhagic disease (RHD)

Rabbit haemorrhagic disease (RHD) is caused by a calicivirus infection that kills most adult rabbits 24–72 h after viral inoculation. Two liver enzymes (AST, aspartate aminotransferase, and ALT, alanine aminotransferase) were monitored in blood samples of calicivirus-infected rabbits during the short course of RHD. Values of AST were used to differentiate three stages of hepatocellular degeneration in RHD: mild (up to 20-fold increase in AST), moderate (150–200-fold elevation of AST) and severe (more than 1000-fold elevation in AST). Liver samples of rabbits from these three biochemical stages of hepatocellular degeneration of RHD were studied by transmission electron microscopy to define the fine structure of the hepatocytes. In the mild hepatocellular degeneration there was proliferation (microvesiculation) of the smooth endoplasmic reticulum and swelling of mitochondria into spheroid bodies with loss of cristae. In moderate hepatocellular degeneration, vacuolization of cytoplasm and mitochondrial damage continued to be present, and there was also formation of autophagic vesicles. In the severe hepatocellular degeneration of RHD, the altered mitochondria also showed loss of density of their matrix; rupture of cytoplasmic vacuoles led to the formation of large vesicles. Marked depletion of liver glycogen was also found in this late stage of RHD. These data offer a correlation between biochemical and cytological features of the liver during the hepatocellular degeneration of RHD.

Inflammatory response of the lung to tungsten particles: An experimental study in mice submitted to intratracheal instillation of a calcium tungstate powder

Tungsten has been implicated as a cause of a severe form of pneumoconiosis in humans, the so-called “hard metal” lung disease. We have investigated the effect of intratracheal instillation of a powder of calcium tungstate on the pulmonary tissue of CD-1 mice. The tungsten-induced alterations were studied using 3 microanatomical methods: cytologic study of exudates obtained by bronchoalveolar lavage (BAL); histologic examination of paraffin-embedded sections of the lung; and scanning electron microscopic (SEM) examination of lung samples using x-ray microanalysis to detect tungsten in situ. The animals were sacrificed 1, 3, 7, 14, and 21 days after a single intratracheal instillation of 250 µg calcium tungstate particles suspended in 100 µl of saline. We found that the metal particles induced a marked inflammatory response in the bronchoalveolar space characterized by a biphasic attraction of leukocytes with cellular peaks observed at day 1 and 14. More than 50% of the BAL macrophages showed ingested tungsten. In the lung parenchyma, the inflammatory infiltrates were predominantly located at the periphery of the bronchiolar walls. From 7 days on after the tungsten deposition, large inflammatory exudates were seen invading focal areas of the alveolar domain of the lung. SEM views revealed that the tungsten particles could be inside alveolar macrophages, in cells making up the alveolar wall, or inside periacinar lymphatics. Our data document that tungsten particles cause a marked inflammatory response in the lung tissue and that the leukocyte exudates may invade alveolar areas of the lung. This strong inflammatory response may correspond to the early stages of the tungsten-induced “hard-metal” lung disease previously reported in humans.