Effects of experimental diabetes on the structure and ultrastructure of the coagulating gland of C57BL/6J and NOD mice

Diabetes mellitus can lead to reproductive disorders that in turn result in weakened fertility brought about by morphofunctional changes in the testes and accessory sex glands. However, doubts persist concerning the basic biology of the secretory epithelial cells and the stroma of the coagulating gland of diabetic mice. Thus, the objective of the present study was to analyze the histological and ultrastructural changes associated with stereology of the coagulating gland of mice with alloxan-induced diabetes, and of spontaneously diabetic mice. Sixteen mice of the C57BL/6J strain, and eight non-obese diabetic (NOD) mice were used. The animals were divided into three groups: 1) control (C), 2) alloxan diabetic (AD), and 3) NOD. Thirty days after the detection of diabetic status in group 2, all of the animals were killed and then perfused with Karnovsky's solution through the left cardiac ventricle. The coagulating gland was then removed and processed for morphometric study by light microscopy and electron microscopy. The results showed thickening of the stroma, atrophy of secretory epithelial cells, and disorganization of the organelles involved in the secretory process in both NOD and alloxan-induced mice. Thus, it may be concluded that the coagulating gland suffered drastic morphological changes, and consequently impaired glandular function, in the presence of diabetes mellitus type I in both NOD and AD mice. (C) 2003 Wiley-Liss, Inc.

Enhancement of natural killer cell function by titanocenes in mice bearing Ehrlich ascites tumour

In the present work, we studied the effects of two titanocenes, biscyclopentadienyidichlorotitanium IV, (DDCT) and its derivative, biscyclopentadienylditiocianatetitanium IV (BCDT), on the activity of natural killer (NK) cells in Ehrlich ascites tumour (EAT)-bearing BALB/c mice. In order to investigate a more direct effect of these compounds on NK cell function, we performed experiments with severe combined immunodeficiency (SCID) mice, which exhibit a normal NK cell response in the absence of T and B cells. The treatment consisted of intraperitoneal (i.p.) administration of 15 mg/kg/day of DDCT for 2 days or 10 mg/kg/day of BCDT for 3 days. In addition, to verify whether the effects produced by the titanocenes were compound specific or related to a direct antitumour effect, we also investigated the effects of a 3-day treatment with 100 mg/kg of cyclophosphamide cyclophosphamide on NK cell activity. Our results demonstrated that, in BALB/c and SCID mice, NK cell function declined to subnormal levels after inoculation of the tumour. In these animals, although treatment with DDCT and BCDT significantly enhanced NK cell function, only DDCT restored NK cell activity to normal values in all stages studied. Conversely, treatment with cyclophosphamide reduced NK cell function in nontumour bearing SCID mice and was also unable to restore the decreased NK activity of tumour-bearing SCID mice, thus demonstrating that the enhancement of NK cell function by titanocenes is compound specific. The same effect of cyclophosphamide was observed with BALB/c mice. In the present study, the up-modulatory effects of these two compounds on NK cell function reveal a new aspect of the mechanism of antitumoural action of titanocenes. (C) 2003 Elsevier B.V. All rights reserved.

Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin-producing beta cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA-HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA-HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA-HSP65-injected mice. The protective effect of DNA-HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4(+) and CD8(+) T cells infiltration, appearance of CD25(+) cells influx and an increased staining for interleukin (IL)-10 in the islets. These results show that DNA-HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

EFFECT OF MACROPHAGE BLOCKADE ON THE RESISTANCE OF INBRED MICE TO PARACOCCIDIOIDES-BRASILIENSIS INFECTION

The effect of macrophage blockade on the natural resistance and on the adaptative immune response of susceptible (B10.D2/oSn) and resistant (A/Sn) mice to Paracoccidioides brasiliensis infection was investigated. B10.D2/oSn and A/Sn mice previously injected with colloidal carbon were infected ip with yeast cells to determine the 50% lethal dose, and to evaluate the anatomy and histopathology, macrophage activation, antibody production and DTH reactions. Macrophage blockade rendered both resistant and susceptible mice considerably more susceptible to infection, as evidenced by increased mortality and many disseminated lesions. P. brasiliensis infection and/or carbon treatment increased the ability of macrophages from resistant mice to spread up to 25 days after treatment. In susceptible mice the enhanced spreading capacity induced by carbon treatment was impaired at ail assayed periods except at 1 week after infection. Macrophage blockade enhanced DTH reactions in resistant mice, but did not alter these reactions in susceptible mice, which remained anergic. To the contrary, macrophage blockade enhanced specific antibody production by susceptible mice, but did nor affect the low levels produced by resistant mice. The effect of macrophage blockade confirms the natural tendency of resistant animals to mount DTH reactions in the course of the disease and the preferential antibody response developed by susceptible mice after P. brasiliensis infection. on the whole, macrophage functions appear to play a fundamental role in the natural and acquired resistance mechanisms to P. brasiliensis infection.

Kinetics of B cell response during Yersinia enterocolitica infection in resistant and susceptible strains of mice

In this study we analyze the B-cell response in murine yersiniosis. To this end, we determined whether polyclonal activation of B-lymphocytes occurs during infection of susceptible (BALB/c) and resistant (C57BL/6) mice with Y. enterocolitica 0:8 and compared the immunoglobulin (Ig) isotypes produced in response to the infection by the two strains. The number of splenic cells secreting nonspecific and specific immunoglobulins was determined by ELISPOT. The presence of anti-Yersinia antibodies in serum was detected by ELISA. In both strains, the number of specific Ig-secreting cells was relatively low. Polyclonal B-cell activation was observed in both strains of mice, and the greatest activation was observed in the BALB/c mice, mainly for lgG(1)- and IgG(3)- secreting cells. The C57BL/6 mice showed a predominance of IgG(2a)-secreting cells. The peak production of anti-Yersinia IgG antibodies in the sera of BALB/c mice was seen on the 28th day after infection. The greatest increase in IgM occurred on the 14th day. A progressive increase of specific IgG antibodies was observed in C57BL/6 mice up to the 28th day after infection while IgM increased on the 21st day after infection. The production of specific IgA antibodies was not detected in either BALB/c or C57BL/6 mice. We conclude that polyclonal. activation of B lymphocytes occurs in both the Yersinia resistant and Yersinia-susceptible mice and that the more intense activation of B lymphocytes observed in the susceptible BALB/c mice does not enhance their resistance to Y. enterocolitica infection.

Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a three-dose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-1oaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-gamma in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-gamma production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis.

Distribution of I-125-labeled crotamine in mice tissues

Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration, followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with 125, using chloramine T method, and separated by get filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6) cpm/mouse) of I-125 native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain. (c) 2006 Elsevier Ltd. All rights reserved.

Evaluation of macrophage activity and antibody production in genetically selected mice infected with Leptospira serovar pomona

The aim of the present study was to evaluate the role of macrophage activity and antibody production in experimental infection with Leptospira Pomona in mice genetically selected for high (H) or low (L) humoral immune response. To evaluate macrophage activity, reactive oxygen and nitrogen intermediates were determined. Also, the production of tumor necrosis factor (TNF-alpha) and the recovery of Leptospira-specific antibodies in the kidneys and liver were assessed; histological lesions were analyzed using the hematoxylin-eosin technique, and Leptospira antigens in tissues were determined by immunohistochemistry. Results showed that recovery of microorganisms from the analyzed organs was lower in LIV-A mice. However, HIV-A animals showed total restraint since the 14th day after infection, whereas LIV-A mice still had bacteria in the liver at the 21st post-infection day. Immune response against Pomona serovar in those lineages was characterized as high production of antibodies, mainly in late periods of the infectious process. The production of reactive oxygen and nitrogen intermediates also contributed to the elimination of Leptospira Pomona in all two lineages; H2O2 production was an important factor in HIV-A mice, as well as NO production in the LIV-A animals, mainly at the latest post-inoculation periods. The same occurred regarding TNF-alpha production. Severe renal lesions were observed at periods in which larger numbers of leptospires were isolated using the culture technique. Tissue alterations persisted in LIV-A mice, even at periods in which leptospires were not recovered. Immunohistochemistry showed to be more sensitive than culturing. However, both techniques were appropriate for the agent identification in the studied lineages. Results suggest that such lineages could represent an important model to investigate pathogenesis and immune response against the varied serovars of leptospires.

Implication of the 5-HT(2A) and 5-HT(2C) (but not 5HT(1A)) receptors located within the periaqueductal gray in the elevated plus-maze test-retest paradigm in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Contrasting effects of acute and chronic treatment with imipramine and fluoxetine on inhibitory avoidance and escape responses in mice exposed to the elevated T-maze

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)