Gleydes G. Parreira

Mycobacterium bovis Bacillus Calmette-Guérin Induces TLR2-Mediated Formation of Lipid Bodies: Intracellular Domains for Eicosanoid Synthesis In Vivo

Differentiation of macrophages into foamy (lipid-laden) macrophages is a common pathological observation in tuberculous granulomas both in experimental settings as well as in clinical conditions; however, the mechanisms that regulate intracellular lipid accumulation in the course of mycobacterial infection and their significance to pathophysiology of tuberculosis are not well understood. In this study, we investigated the mechanisms of formation and function of lipid-laden macrophages in a murine model of tuberculosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG), but not Mycobacterium smegmatis, induced a dose- and time-dependent increase in lipid body-inducible nonmembrane-bound cytoplasmic lipid domain size and numbers. Lipid body formation was drastically inhibited in TLR2-, but not in TLR4-deficient mice, indicating a role for TLR2 in BCG recognition and signaling to form lipid bodies. Increase in lipid bodies during infection correlated with increased generation of PGE2 and localization of cyclooxygenase-2 within lipid bodies. Moreover, we demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid that lipid bodies were the predominant sites of PGE2 synthesis in activated macrophages. Our findings demonstrated that BCG-induced lipid body formation is TLR2 mediated and these structures function as signaling platforms in inflammatory mediator production, because compartmentalization of substrate and key enzymes within lipid bodies has impact on the capacity of activated leukocytes to generate increased amounts of eicosanoids during experimental infection by BCG.

Lipid bodies: Structural markers of inflammatory macrophages in innate immunity.

Abstract.Objective and designThe correlation between innate immune responses and formation of cytoplasmic lipid bodies (LBs) was investigated in vivo in inflammatory macrophages from rats infected with Trypanosoma cruzi, the intracellular parasite which causes Chagas’ disease.Material and methodsWe used an experimental model of high-dose irradiation prior to infection, which depletes the humoral and cellular immune responses except for the phagocytic activity of macrophages. Rats, irradiated or not, were infected with T. cruzi and macrophages from different origins (peritoneum, heart, uterus) were studied by transmission electron microscopy (TEM).ResultsAs documented by quantitative TEM, innate immune responses induced prominent formation, structural changes and intracellular interactions of LBs. LBs significantly increased their size and changed their osmiophilia in response to both infection alone and when macrophages were challenged with irradiation-induced increased parasite load. Remarkably, a consistent LB-phagolysosome association was identified. LBs were surrounding, attached to or internalized by phagolysosomes.ConclusionsWe demonstrated that LBs are dynamic organelles notably involved in the host response to acute T. cruzi infection, an event that may be important for pathogen control during innate immunity. Our findings highlight LBs as structural markers of the innate immune responses in phagocytic cells.

Type 2 iodothyronine deiodinase is highly expressed in germ cells of adult rat testis

The testis has been classically described as a thyroid hormone unresponsive tissue, but recent studies indicate that these hormones might play an important role in developing testes. We have previously demonstrated that type 2 iodothyronine deiodinase (D2), a thyroid hormone-activating enzyme, is expressed in adult rodent testis and that its activity is induced by hypothyroidism. Nevertheless, the precise location of D2 in testis is not known. The aim of the present work was to determine the testicular cell types in which D2 is expressed using real-time PCR analysis, in situ hybridization histochemistry, and determination of D2 activity in cell fractions isolated from adult euthyroid and/or hypothyroid rat testis. The D2 mRNA levels in germ cells were higher than those from somatic cells (6.94 +/- 1.49 vs 2.32 +/- 0.79 arbitrary units (au); P = 0.017). Hypothyroidism increased D2 expression in germ cells (6.94 +/- 1.49 vs 8.78 +/- 5.43 au, P = 0.002) but did not change D2 transcripts in somatic cells significantly (2.12 +/- 0.79 vs 2.88 +/- 1.39 au, P = 0.50). In situ hybridization analysis showed that D2 mRNA is specifically present in elongated spermatids undergoing differentiation, whereas other germ cell types and Sertoli cells of seminiferous epithelium and the interstitial cells were virtually negative for this enzyme. The enzyme activity measured in germ and somatic isolated cell fractions (0.23 +/- 0.003 vs 0.02 +/- 0.013 fmol/min per mg protein respectively; P < 0.001) further confirmed the real-time PCR and in situ hybridization results. Hence, our findings demonstrated that D2 is predominantly expressed in elongated spermatids, suggesting that thyroid hormone might have a direct effect on spermatogenesis in the adult rats.

Fenofibrate prevents orotic acid—Induced hepatic steatosis in rats

The experiments performed in this report were designed to investigate the mechanisms involved in the metabolic alterations associated with orotic acid-induced hepatic steatosis and the effect of fenofibrate, a stimulant of peroxisome proliferators-activated receptor alpha (PPARalpha), on these alterations. Male Wistar rats were divided into three experimental groups: 1) fed a balanced diet (C); 2) fed a balanced diet supplemented with 1% orotic acid (OA); 3) fed OA diet containing 100 mg.kg(-1) bw.day(-1) fenofibrate (OA+F), for 9 days. Administration of OA to rats induced significant increase in the hepatic total lipids content, marked microvesicular steatosis and decrease in plasma lipids concentrations compared to control group. Fenofibrate treatment prevented fatty liver induction, caused an additional reduction on plasma lipids concentrations and caused a 40% decrease in the lipogenic rate in adipose tissue. The results also showed a 40% increase in lipoprotein lipase (LPL) activity in adipose tissue from OA treated group and fenofibrate administration induced a 50% decrease in LPL activity. The liver mRNA expression of PPARalpha and ACO (acyl CoA oxidase) were 85% and 68% decreased in OA group when compared to control, respectively. Fenofibrate treatment increased the PPARalpha and ACO expressions whereas the CPT-1 (carnitine palmitoyl transferase-1) expression was not altered. Our results have shown that fenofibrate treatment decreases the hepatic lipid content induced by OA which is mediated by an important increase in fatty acid oxidation consequent to an increase in hepatic mRNA expression of PPARalpha and ACO.

Relationship of Sertoli-Sertoli Tight Junctions to Ectoplasmic Specialization in Conventional and En Face Views

Abstract Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. At sites between inter-Sertoli cell attachments, near the base of the cells, the sites are also related to tight junctions. We studied the characteristics of ectoplasmic specializations from six species using conventional views in which thin sections were perpendicular to the plane of the membranes, we used rare views in which the sections were in the plane of the membrane (en face views), and we also used the freeze-fracture technique. Tissues postfixed by osmium ferrocyanide showed junctional strands (fusion points between membranes) and actin bundles, actin sheets, or both, which could be visualized simultaneously. En face views demonstrated that the majority of tight junctional strands ran parallel to actin filament bundles. Usually, two tight junctional strands were associated with each actin filament bundle. Parallel tight junctions were occasionally extremely close together (∼12 nm apart). Tight junctional strands were sometimes present without an apparent association with organized actin bundles or they were tangential to actin bundles. En face views showed that gap junctions were commonly observed intercalated with tight junction strands. The results taken together suggest a relationship of organized actin with tight junction complexes. However, the occasional examples of tight junction complexes being not perfectly aligned with actin filament bundles suggest that a precise and rigidly organized actin-tight junction relationship described above is not absolutely mandatory for the presence or maintenance of tight junctions. Species variations in tight junction organization are also presented.

In vitro activity of labdane diterpene from Alomia myriadenia (Asteraceae): immunosuppression via induction of apoptosis in monocytes

A screening program in Brazilian flora was carried out to detect the presence of immunosuppressive compounds by using the in vitro phytohemagglutinin A (PHA)-induced human peripheral blood mononuclear cell (PBMC) proliferation assay. In this screening, we isolated from Alomia myriadenia Schultz-Bip. ex. Baker (Asteraceae), a labdane-type diterpene named myriadenolide. Incubation of human PBMC with this compound reduced significantly the percentage of CD14(+) cells, but it has no effect on the relative amount of CD3(+)CD4(-)CD8(+) and CD3(+)CD4(+)CD8(-) T lymphocyte subpopulations. Neither viability nor proliferative competence of T lymphocytes was significantly affected by myriadenolide. The toxic effect on monocytes (CD14(+) cells) may explain the inhibitory effect observed on PHA-induced lymphocyte proliferation. The cytotoxic effect of myriadenolide on monocytes was determined by measuring the percentage of hypodiploid nuclei content by propidium iodide staining, electron microscopy and simultaneous detection of CD14 and annexin V binding by flow cytometry. The results showed that myriadenolide induces a dose-dependent apoptosis in monocytes and thus explain the immunosuppressive effect observed.

Spermatozoon and its relationship with the ovarian lamellae in the internally inseminating catfish Trachelyopterus galeatus.

We examined the spermatozoa and their relationship with the ovarian lamellae in the catfish Trachelyopterus galeatus by classical light microscopy, high‐resolution light microscopy, and transmission electron microscopy. Trachelyopterus galeatus is an internally inseminating species the spermatozoon of which presented an elongated cylindrical head (12.3 ± 1.5 μm), elongated midpiece (5.0 ± 0.7 μm), and flagellum (23.9 ± 2.8 μm). Fertilized eggs or embryos were not found in its ovaries. Spermatozeugmata were demonstrated for the first time in this species. At the ultrastructural level, the anterior region of the head was devoid of chromatin with its shape being rounded with a hyaline tip in frontal sections and flattened in sagittal sections. The proximal centriole and most of the distal centriole were contained within a nuclear fossa. Mitochondria with lamellar cristae, as well as glycogen granules, were located just caudal to the nuclear fossa and distally in the midpiece. A single row of accessory microtubules ran peripherally in the midpiece. The flagellar axoneme had the typical 9 + 2 arrangement, having electron‐dense and electron‐lucent A‐tubules at different points along the flagellum; flagellar fins were lacking. The ovarian lamellae were covered by a simple cuboidal epithelium. In maturing/mature females, spermatozoa were free in the ovarian lumen or inserted in pits of the lamellar epithelial cells. Tight junctions and desmosomes were seen between the epithelial cells. In addition to nourishment of the spermatozoon, the lamellar epithelial cells may play a role in protecting the spermatozoa against the female immune system. Microsc. Res. Tech., 2009.

Glycol Methacrylate Embedding for Improved Morphological, Morphometrical, and Immunohistochemical Investigations Under Light Microscopy: Testes as a Model

Glycol methacrylate (GMA), a water and ethanol miscible plastic resin, is a medium handy to use for light microscopy embedding that has a number of advantages than paraffin embedding. The GMA improves the histological, morphometrical, and immunohistochemical evaluations, mainly due to the accurate assessment of cytological details. This chapter focuses on our experience in the GMA processing and describes in detail the fixation, embedding, and staining methods that we have been using for testes evaluations.

The Intriguing Ultrastructure of Lipid Body Organelles Within Activated Macrophages

Macrophages are widely distributed immune system cells with essential functions in tissue homeostasis, apoptotic cell clearance, and first defense in infections. A distinguishing feature of activated macrophages participating in different situations such as inflammatory and metabolic diseases is the presence of increased numbers of lipid-rich organelles, termed lipid bodies (LBs) or lipid droplets, in their cytoplasm. LBs are considered structural markers of activated macrophages and are involved in different functions such as lipid metabolism, intracellular trafficking, and synthesis of inflammatory mediators. In this review, we revisit the distinct morphology of LB organelles actively formed within macrophages in response to infections and cell clearance, taking into account new insights provided by ultrastructural studies. We also discuss the LB interactions within macrophages, revealed by transmission electron microscopy, with a focus on the remarkable LB-phagosome association and discuss potential links between structural aspects and function.

Rat models to investigate host macrophage defense against Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of Chagas’ disease, an infection with a great impact on public health in Latin America. One of the challenges to understand Chagas’ disease lies on the complex host-parasite interaction. The understanding of this interaction requires the use of appropriate experimental models that mimic the human disease. Here, we have used two lineages of rats (Wistar and Holtzman) to comparatively evaluate the course of the acute infection (Y strain). Infection was monitored by parasitemia, cardiac and skeletal muscle parasitism and inflammation, heart ultrastructure, recruitment of monocytes/macrophages and nitric oxide, and arginase production by these cells. Although both rats were able to infect, only Holtzman rats developed a marked infection in the cardiac and skeletal muscles, in parallel to a high recruitment of first-line defense cells. A high number of inflammatory macrophages directed parasite clearance. By the end of the acute phase, Holtzman rats showed consistent disease control. Interestingly, parasite killing was not related to nitric oxide production likely inhibited by an arginase-dependent mechanism. Our work demonstrates differential responses of Holtzman and Wistar rats to T. cruzi, and highlights the use of Holtzman rats as useful models for further studies of cardiac/skeletal muscle tropism and innate immune responses that protect the host against parasite replication. This is important for the development of proper therapeutic interventions.