Progressive histopathological changes and ß-cell loss in the pancreas of a new spontaneous rat model of type 2 diabetes.

The eSMT rat is a new spontaneous model of type 2 diabetes that develops a progressive diabetic syndrome with a stronger incidence in males than in females. We decide to investigate the progression of the pancreatic histopathological changes during the lifespan of the eSMT rat, especially those associated with islet cell populations. Besides that, some plasmatic parameters were evaluated in order to correlate them with the morphological findings. Male eSMT and Sprague-Dawley control rats were used. The results showed a dramatic decrease of the volume density (VD) of endocrine tissue in the eSMT rats without evidence of insulitis. Islets became fragmented structures with strong presence of interstitial fibrosis. Consequently, plasma insulin levels showed a significant decrease, while plasma glucose, cholesterol and triglyceride levels were increased. Normal rats showed no significant changes in the VD of endocrine tissue, except for the older animals, where the VD of ß-cell population was increased. Early derangements observed in islets, together with the progressive decrease of endocrine tissue and the metabolic disorders described, would be responsible for an irreversible pathologic condition which avoids the animal survival beyond about 18 months of age. However, there is still a need to investigate the causes of endocrine tissue decrease and its possible association with an inflammatory process that it could be associated with the development and progression of fibrosis.

Allatotropin expression during the development of the fourth instar larvae of the kissing-bug Triatoma infestans (Klüg).

Allatotropin (AT) is a neuropeptide originally isolated from the brain of Manduca sexta and then characterized in several insect species. It acts as a neurohormone, as well as a neuromodulator. While it was primarily characterized on the basis of its ability to stimulate the secretion of juvenile hormones, it was also found that it acts as a cardioaccelerator and myostimulator. The presence of AT in IV instar larvae of T. infestans was previously described at the level of the Malpighian tubules. In the present study we report the presence of the peptide at the level of the brain, retrocerebral complex, as well as in the anterior midgut and aorta. The presence of AT at the corpora allata suggests that the peptide is acting on the gland during the first days of the moulting cycle. Neural processes at the level of the aorta and the anterior midgut suggests that, like in adults, the hormone is acting as a cardioaccelerator and myostimulator. The peptide was also found in open-type cells of the midgut. Finally the presence of allatotropic neurons in the optical lobe of the brain suggests that as in other species, the peptide is related with the control of circadian rhythms.

Mosquito larvae soluble fractions induce DNA synthesis alterations on human mononuclear cells.

Mosquito larvae soluble fractions obtained by molecular exclusion chromatography altered the mitotic rate of several epithelial cell populations in hepatectomised mice, as well as the proliferation of human mononuclear cells (MNC), stimulating or inhibiting them depending on the fraction and dose applied. The effect was also thermolabile, suggesting a proteic nature of the compounds involved. Analysis of cell viability after culture indicated that the extract did not have lethal toxic effects. One fraction with a molecular weight ranging between 12-80 kDa caused only an inhibitory effect. In the present study, we performed further characterisation of this fraction by assaying the effect of new fractions obtained from this one, by the use of a column with a lower molecular weight exclusion range. Assays were performed on the proliferation of adult human MNCs. Our results showed that two out of four of the sub-fractions analysed, with a MW of about 70 and 17 kDa, caused a dose-dependent response, either inhibiting or stimulating MNC proliferation respectively.

Thyroid-stimulating hormone and growth hormone release alterations induced by mosquito larvae proteins on pituitary cells.

Mosquito larvae crude extract has shown to modulate cell proliferation of different mouse epithelial as well as human mononuclear cell populations in vivo and in vitro. A soluble fraction of the extract, with a molecular weight ranging from 12 to 80 kD, also showed an inhibitory effect on the proliferation of mouse hepatocytes. This effect disappeared after heating the extract at 90 degrees C for 60 min, suggesting that some proteinaceous molecule is involved. We report the effect of dialysed extract (MW >12 kD) on the concentration of both thyroid-stimulating hormone (TSH) and growth hormone (GH) in an incubation medium of pituitary cells from normal and oestrogenised rats. Time- and dose-dependent response of both hormones resulted in increasing TSH levels. Concentrations of GH were lower in the treated than in control pituitary cells. The time elapsed until the finding of differences suggests the presence in the mosquito extract of some protein binding the hormone. The differences were not due to lethal toxic effects since the Trypan blue viability test showed no differences between control and treated cells. Furthermore, the effect disappeared when the extract had previously been heated at 90 degrees C for 60 min. Finally, our results suggest the presence of some proteins in the mosquito Culex pipiens L. larvae, which would act as a pituitary hormone regulator.

Immunohistochemical detection of a very high density lipoprotein (VHDL) in ovarian follicles of Triatoma infestans.

The ability of Triatoma infestans ovarian follicles to synthesize a very high-density lipoprotein (VHDL) has been examined by immunohistochemical methods. This kind of lipoprotein can be envisaged as a storage hexameric protein present in the hemolymph of some insect species. VHDL immunoreactivity is observed in oocytes at different stages of maturation. The antigen is present in the oocyte cytoplasm as well as in the follicular epithelial cells. The immunopositive reaction in the apical surface of follicle cells suggests both a VHDL synthesis and a secretion process. Furthermore, VHDL seems to be stored into oocyte in yolk granules. On the contrary, no immunopositive reaction is observed in the intracellular spaces between follicle cells, suggesting that VHDL is not incorporated from hemolymph into the oocyte.

Immunohistochemical detection of a very high density lipoprotein (VHDL) in ovarian follicles of Triatoma infestans

The ability of Triatoma infestans ovarian follicles to synthesize a very high-density lipoprotein (VHDL) has been examined by immunohistochemical methods. This kind of lipoprotein can be envisaged as a storage hexameric protein present in the hemolymph of some insect species. VHDL immunoreactivity is observed in oocytes at different stages of maturation. The antigen is present in the oocyte cytoplasm as well as in the follicular epithelial cells. The immunopositive reaction in the apical surface of follicle cells suggests both a VHDL synthesis and a secretion process. Furthermore, VHDL seems to be stored into oocyte in yolk granules. On the contrary, no immunopositive reaction is observed in the intracellular spaces between follicle cells, suggesting that VHDL is not incorporated from hemolymph into the oocyte.

Immunohistochemical detection of a very high density lipoprotein (VHDL) in ovarian follicles of Triatoma infestans

The ability of Triatoma infestans ovarian follicles to synthesize a very high-density lipoprotein (VHDL) has been examined by immunohistochemical methods. This kind of lipoprotein can be envisaged as a storage hexameric protein present in the hemolymph of some insect species. VHDL immunoreactivity is observed in oocytes at different stages of maturation. The antigen is present in the oocyte cytoplasm as well as in the follicular epithelial cells. The immunopositive reaction in the apical surface of follicle cells suggests both a VHDL synthesis and a secretion process. Furthermore, VHDL seems to be stored into oocyte in yolk granules. On the contrary, no immunopositive reaction is observed in the intracellular spaces between follicle cells, suggesting that VHDL is not incorporated from hemolymph into the oocyte.(AU)

Mosquito larvae proteins modulate luteinizing hormone and prolactin release in pituitary cells of normal and estrogenized rats.

Whilst looking for vertebrate growth factor homologues in insects, we found that a soluble fraction of a 12-80 kDa molecular weight band peaking at 25 kDa, isolated from mosquito larvae extracts by gel permeation chromatography, had a modulatory effect on mouse hepatocytes and adult human mononuclear cell proliferation. The effect disappeared after heating the extract at 90 degrees C for 30 min, suggesting that the active factor may be a protein. In order to determine the activity of the extract on cell function, we assessed the effect of the extract on pituitary hormone secretion in vitro. We assayed a dialyzed fraction (MW greater than 12 kDa) of mosquito larvae for its effect on the release of luteinizing hormone (LH) and prolactin (PRL) from dispersed rat pituitary cells. In normal anterior pituitary (AP) cells we found that the extract had a stimulatory effect on LH release but an inhibitory action on prolactin secretion. In AP cells obtained from estrogen-induced hyperplasia, the extract had an inhibitory effect on prolactin secretion. In all cases the effects were time- and dose-dependent. Interference of the mosquito proteins with the radioimmunoassay was checked and found to be negligible. After a 60 min incubation, cell viability was comparable in control and treated cells. Furthermore, the biological effect of the extract was thermally unstable. Our results suggest that mosquito larvae may share common factors with mammals, probably peptidic in nature, which are able to modulate cell function.

Immunohistochemical detection of a very high density lipoprotein (VHDL) in ovarian follicles of Triatoma infestans.

The ability of Triatoma infestans ovarian follicles to synthesize a very high-density lipoprotein (VHDL) has been examined by immunohistochemical methods. This kind of lipoprotein can be envisaged as a storage hexameric protein present in the hemolymph of some insect species. VHDL immunoreactivity is observed in oocytes at different stages of maturation. The antigen is present in the oocyte cytoplasm as well as in the follicular epithelial cells. The immunopositive reaction in the apical surface of follicle cells suggests both a VHDL synthesis and a secretion process. Furthermore, VHDL seems to be stored into oocyte in yolk granules. On the contrary, no immunopositive reaction is observed in the intracellular spaces between follicle cells, suggesting that VHDL is not incorporated from hemolymph into the oocyte.

Partial characterization of mosquito larvae extract inducing dna synthesis alterations on human mononuclear cells.

A crude mosquito larvae and dialysed extract alters the mitotic rate of several epithelial cell populations in normal young and adult hepatectomized mice. A crude extract also showed a biphasic effect on the proliferation of human mononuclear cells (MNCs), either stimulating or inhibiting them depending on the dose applied. In the present paper, we assayed the effect of the dialysed mosquito larvae extract and two different protein fractions on human MNCs. Analysis of cell viability after culture indicated that the extract did not have toxic effects. Our results show a dual response of the MNCs to the dialysed, as well as to the protein fraction, with the highest molecular weight inhibiting or stimulating proliferation, depending on the dose applied. The protein fraction with the lowest molecular weight (range between 12-80 kDa) showed only an inhibitory effect on cell proliferation.

Partial characterization of mosquito larvae extract modulating mouse and human cell proliferation.

Mosquito larvae crude extract have been found to alter the mitotic rate of several mouse epithelial cell populations such as enterocytes and tongue keratinocytes. Also, the dialysed fraction inhibits hepatocyte proliferation in hepatectomized males. These experiments suggested an inhibitory effect on the G1/S interphase. Consequently, we suggested the presence of some molecule or molecules related to the TGF-beta superfamily. In the present paper, we have assayed the crude extract on human mononuclear cells and the dialysed fraction of the extract on tongue keratinocyte proliferation. Furthermore, different protein fractions obtained using a molecular exclusion chromatographic column were assayed on hepatocyte proliferation of hepatectomized mice. Three groups of proteins have been isolated. Results show a dose-dependent effect of crude extract on mononuclear cell proliferation and the dialysed extract caused an inhibitory effect on tongue keratinocyte proliferation. With regard to the hepatocyte mitotic rate, an inhibitory effect appeared only in animals receiving the fraction with lower molecular weight. These results suggest the presence in mosquito larvae of some peptidic molecule or molecules resembling the activity of members of the TGF-beta superfamily.

Mosquito larvae extract induces mitotic rate alterations of mice tongue keratinocytes along a circadian rhythm period.

It has been demonstrated that the crude extract of mosquito larvae alters the mitotic rate of several mouse cell populations of young growing mice (25 +/- 1 days old). Furthermore, the dialysed fraction of the extract inhibited proliferation of hepatocytes from hepatectomized adult male mice (90 days old). Sampling during the period between 16 and 24 h after treatment (when mitotic peak normally occurs) shows an inhibiting effect on the G1/S interphase, whereas evaluation during the dark phase of the circadian rhythm period (i.e. 4 to 12 h after treatment) shows an increment of the mitotic rates suggesting a probable effect at G2/M restriction point. In the present paper we report the effect of the mosquito larvae crude extract on the proliferative activity of tongue keratinocytes along a circadian rhythm period. Treatments were intraperitoneally applied at 16/00 Time of Day/Time post Injection and mice were killed at 00/08, 04/12, 08/16, 12/20 and 16/24 TD/TPI. As other cell populations previously analysed, the mitotic rate of tongue keratinocytes of extract receivers was significantly increased during night (when S phase normally occurs) and inhibited during the 08/16 to 16/24 period.

Early effect of mosquito larvae extract on mouse cells proliferation in vivo.

It has been demonstrated that mosquito larvae crude extract has an inhibiting effect on the mitotic rate of several mouse cell populations. The sampling period was 16-24 h after treatment, when mitotic peak normally occurs. The present paper reports the effect of mosquito larvae crude extract on the proliferation of hepatocytes, renocytes, Lieberkhün crypt enterocytes, and sialocytes. In this case, the sampling period covered the dark phase of the day, during the first 12 h after treatment. Colchicine-arrested metaphases were controlled at 20/04, 00/08 and 04/12 (Time of Day/Time Post Injection). The mitotic rate was significantly increased in hepatocytes and renocytes and inhibited in duodenum enterocytes. In view of the time chosen to administer the treatments and the time elapsed until sampling, we conclude a probable effect of the extract at the G2-M point of the cell cycle.

Inhibition of mouse parenchymal cells proliferation by mosquito larvae extract.

As it is known, some cell populations of mosquito Aedes aegypti larvae do not proliferate. This fact suggests the existence of some mitosis inhibiting factor. We have assayed the effect of mosquito larvae crude extract on 85 C3HS young male mice (25 +/- 1 days old) and the effect of the dialyzed fraction of the same extract on 47 partially hepatectomized C3HS adult male mice. The injection was performed at 16:00 hour (before the raising of the DNAS curve) and the controls were made at 08:00/16, 12:00/20 and 16:00/24 (Time of Day/Time Post Injection). Colchicine arrested metaphases of nephrocytes, hepatocytes, sialocytes (submaxillary glands) and enterocytes were controlled. The mitotic rate was significantly inhibited in hepatocytes, sialocytes and enterocytes in young growing mice, and in hepatocytes in partially hepatectomized adult mice. We conclude that TGF-beta, actually found in insects, or alternatively some other known or unknown factor, could be responsible for the results observed in the present experiments.