McCoy cell line as a possible model containing CD4+ receptors for the study of HIV-1 replication

Several studies have recently shown the use of recombinant rabies virus as potential vector-viral vaccine for HIV-1. The sequence homology between gp 120 and rabies virus glycoprotein has been reported. The McCoy cell line has therefore been used to show CD4+ or CD4+ like receptors. Samples of HIV-1 were isolated, when plasma of HIV-1 positive patients was inoculated in the McCoy cell line. The virus infection was then studied during successive virus passages. The proteins released in the extra cellular medium were checked for protein activity, by exposure to SDS Electrophoresis and blotting to nitro-cellulose filter, then reacting with sera of HIV positive and negative patients. Successive passages were performed, and showed viral replication, membrane permeabilization, the syncytium formation, and the cellular lysis (cytopathic effect). Flow cytometry analysis shows clear evidence that CD4+ receptors are present in this cell line, which enhances the likelihood of easy isolation and replication of HIV. The results observed allow the use of this cell line as a possible model for isolating HIV, as well as for carrying out studies of the dynamics of viral infection in several situations, including exposure to drugs in pharmacological studies, and possibly studies and analyses of the immune response in vaccine therapies.

Effect of one-week ethanol treatment on monoamine levels and dopaminergic receptors in rat striatum

We studied the effects of ethanol on the levels of norepinephrine, dopamine, serotonin (5-HT) and their metabolites as well as on D1- and D2-like receptors in the rat striatum. Ethanol (2 or 4 g/kg, po) was administered daily by gavage to male Wistar rats and on the 7th day, 30 min or 48 h after drug administration, the striatum was dissected for biochemical assays. Monoamine and metabolite concentrations were measured by HPLC and D1- and D2-like receptor densities were determined by binding assays. Scatchard analyses showed decreases of 30 and 43%, respectively, in D1- and D2-like receptor densities and no change in dissociation constants (Kd) 48 h after the withdrawal of the dose of 4 g/kg. Ethanol, 2 g/kg, was effective only on the density of D2-like receptors but not on Kd of either receptor. Thirty minutes after the last ethanol injection (4 g/kg), decreases of D2 receptor density (45%) as well as of Kd values (34%) were detected. However, there was no significant effect on D1-like receptor density and a 46% decrease was observed in Kd. An increase in dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), a decrease in norepinephrine, and no alteration in 5-HT levels were demonstrated after 48-h withdrawal of 4 g/kg ethanol. Similar effects were observed in dopamine and DOPAC levels 30 min after drug administration. No alteration in norepinephrine concentration and a decrease in 5-HT levels were seen 30 min after ethanol (4 g/kg) administration. Our findings indicate the involvement of the monoaminergic system in the responses to ethanol.

Typhoid fever as cellular microbiological model

The knowledge about typhoid fever pathogenesis is growing in the last years, mainly about the cellular and molecular phenomena that are responsible by clinical manifestations of this disease. In this article are discussed several recent discoveries, as follows: a) Bacterial type III protein secretion system; b) The five virulence genes of Salmonella spp. that encoding Sips (Salmonella invasion protein) A, B, C, D and E, which are capable of induce apoptosis in macrophages; c) The function of Toll R2 and Toll R4 receptors present in the macrophage surface (discovered in the Drosophila). The Toll family receptors are critical in the signalizing mediated by LPS in macrophages in association with LBP and CD14; d) The lines of immune defense between intestinal lumen and internal organs; e) The fundamental role of the endothelial cells in the inflammatory deviation from bloodstream into infected tissues by bacteria. In addition to above subjects, the authors comment the correlation between the clinical features of typhoid fever and the cellular and molecular phenomena of this disease, as well as the therapeutic consequences of this knowledge.

Extração de DNA a partir de sangue humano coagulado para aplicação nas técnicas de genotipagem de antígenos leucocitários humanos e de receptores semelhantes à imunoglobulina

O objetivo deste estudo foi padronizar uma metodologia de extração de DNA de alta qualidade a partir de amostras de sangue coagulado. Quarenta e oito amostras de sangue humano coagulado foram utilizadas para a extração de DNA pelo kit comercial EZ-DNA® (Biological Industries, Beit Haemek, Israel), pelo kit de coluna Neoscience® (One Lambda Inc., San Diego, CA) e pelo método modificado de salting out. Apenas o método de salting out foi capaz de extrair altas concentrações de DNA (média, 180ng/µL), as quais foram medidas pelo detector de fluorescência Qubit® (Invitrogen, USA). Este método permitiu a amplificação dos genes HLA (human leukocyte antigens) pela tecnologia PCR-SSO (polymerase chain reaction - specific sequence of oligonucleotides) Luminex, a qual exige DNA de boa qualidade, e de genes KIR (killer cell immunoglobulin-like receptors) pela técnica made in house PCR-SSP (polymerase chain reaction-sequence specific of primers), a qual demanda uma concentração específica de DNA (10ng/µL). Concluímos que a técnica de salting out modificada foi muito eficiente, simples e rápida para a extração de DNA de amostras de sangue humano coagulado, com o objetivo de realizar a genotipagem de genes HLA e KIR.

The interaction between Histoplasma capsulatum cell wall carbohydrates and host components: relevance in the immunomodulatory role of histoplasmosis

Histoplasma capsulatum is an intracellular fungal pathogen that causes respiratory and systemic disease by proliferating within phagocytic cells. The binding of H. capsulatum to phagocytes may be mediated by the pathogen's cell wall carbohydrates, glucans, which consist of glucose homo and hetero-polymers and whose glycosydic linkage types differ between the yeast and mycelial phases. The ±-1,3-glucan is considered relevant for H. capsulatum virulence, whereas the ²-1,3-glucan is antigenic and participates in the modulation of the host immune response. H. capsulatum cell wall components with lectin-like activity seem to interact with the host cell surface, while host membrane lectin-like receptors can recognize a particular fungal carbohydrate ligand. This review emphasizes the relevance of the main H. capsulatum and host carbohydrate-driven interactions that allow for binding and internalization of the fungal cell into phagocytes and its subsequent avoidance of intracellular elimination.

Polymorphisms IL10-819 and TLR-2 are potentially associated with sepsis in Brazilian patients

Genetic variation in immune response is probably involved in the progression of sepsis and mortality in septic patients. However, findings in the literature are sometimes conflicting or their significance is uncertain. Thus, we investigated the possible association between 12 polymorphisms located in the interleukin-6 (IL6), IL10, TLR-2, Toll-like receptor-4 (TLR-4), tumor necrosis factor-α and tumor necrosis factor-β (lymphotoxin α - LTA) genes and sepsis. Critically ill patients classified with sepsis, severe sepsis and septic shock and 207 healthy volunteers were analyzed and genotyped. Seven of the nine polymorphisms showed similar distributions in allele frequencies between patients and controls. Interestingly, our data suggest that the IL10-819 and TLR-2 polymorphisms may be potential predictors of sepsis.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Insights from animal models on the immunogenetics of leprosy: a review

A variety of host immunogenetic factors appear to influence both an individual's susceptibility to infection with Mycobacterium leprae and the pathologic course of the disease. Animal models can contribute to a better understanding of the role of immunogenetics in leprosy through comparative studies helping to confirm the significance of various identified traits and in deciphering the underlying mechanisms that may be involved in expression of different disease related phenotypes. Genetically engineered mice, with specific immune or biochemical pathway defects, are particularly useful for investigating granuloma formation and resistance to infection and are shedding new light on borderline areas of the leprosy spectrum which are clinically unstable and have a tendency toward immunological complications. Though armadillos are less developed in this regard, these animals are the only other natural hosts of M. leprae and they present a unique opportunity for comparative study of genetic markers and mechanisms associable with disease susceptibility or resistance, especially the neurological aspects of leprosy. In this paper, we review the recent contributions of genetically engineered mice and armadillos toward our understanding of the immunogenetics of leprosy.

Interleukin-10 rs1800896 and CXCR2 rs1126579 polymorphisms modulate the predisposition to septic shock

Despite major improvements in its treatment and diagnosis, sepsis is still a leading cause of death and admittance to the intensive care unit (ICU). Failure to identify patients at high risk of developing septic shock contributes to an increase in the sepsis burden and rapid molecular tests are currently the most promising avenue to aid in patient risk determination and therapeutic anticipation. The primary goal of this study was to evaluate the genetic susceptibility that affects sepsis outcome in 72 sepsis patients admitted to the ICU. Seven polymorphisms were genotyped in key inflammatory response genes in sepsis, including tumour necrosis factor-α,interlelukin (IL)-1β, IL-10,IL-8, Toll-like receptor 4, CXCR1and CXCR2. The primary finding showed that patients who were homozygous for the major A allele in IL-10rs1800896 had almost five times higher chance to develop septic shock compared to heterozygotes. Similarly, selected clinical features and CXCR2rs1126579 single nucleotide polymorphisms modulated septic shock susceptibility without affecting survival. These data support the hypothesis that molecular testing has clinical usefulness to improve sepsis prognostic models. Therefore, enrichment of the ICU portfolio by including these biomarkers will aid in the early identification of sepsis patients who may develop septic shock.

Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40% reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.

Hydrogen sulfide in posthemorrhagic shock mesenteric lymph drainage alleviates kidney injury in rats

Posthemorrhagic shock mesenteric lymph (PHSML) is a key factor in multiple organ injury following hemorrhagic shock. We investigated the role of hydrogen sulfide (H2S) in PHSML drainage in alleviating acute kidney injury (AKI) by administering D,L-propargylglycine (PPG) and sodium hydrosulfide hydrate (NaHS) to 12 specific pathogen-free male Wistar rats with PHSML drainage. A hemorrhagic shock model was established in 4 experimental groups: shock, shock+drainage, shock+drainage+PPG (45 mg/kg, 0.5 h prehemorrhage), and shock+drainage+NaHS (28 µmol/kg, 0.5 h prehemorrhage). Fluid resuscitation was performed after 1 h of hypotension, and PHMSL was drained in the last three groups for 3 h after resuscitation. Renal function and histomorphology were assessed along with levels of H2S, cystathionine-γ-lyase (CSE), Toll-like receptor 4 (TLR4), interleukin (IL)-10, IL-12, and tumor necrosis factor (TNF)-α in renal tissue. Hemorrhagic shock induced AKI with increased urea and creatinine levels in plasma and higher H2S, CSE, TLR4, IL-10, IL-12, and TNF-α levels in renal tissue. PHSML drainage significantly reduced urea, creatinine, H2S, CSE, and TNF-α but not TLR4, IL-10, or IL-12. PPG decreased creatinine, H2S, IL-10, and TNF-α levels, but this effect was reversed by NaHS administration. In conclusion, PHSML drainage alleviated AKI following hemorrhagic shock by preventing increases in H2S and H2S-mediated inflammation.

The imidazoline receptors and the central regulation of the arterial blood pressure: a minireview

Recently, we proposed the hypothesis according to wich the central hypotensive effect of clonidine and related substances could be related to an action upon specific receptors, requiring the imidazoline or imidazoline-like structures, rather than alpha2-adrenoceptors. Since then, direct evidences have been accumulated to confirm the existence of a population of imidazoline specific binding sites in the brainstem of animals and man, more precisely in the Nucleus Reticularis Lateralis (NRL) region of the ventrolateral medulla (VLM), site of the antihypertensive action of clonidine. The purification of the putative endogenous ligand of the imidazoline receptors - named endazoline - is currently being attempted from human brain extracts. This new concept might at last lead to the expected dissociation of the pharmacological mechanisms involved, on the one hand, in the therapeutic antihypertensive effect, and on the other, in their major side-effect, which is sedation. In fact, it has been recently confirmed that hypotension is mediated by the activation of imidazoline preferring receptors (IPR) within the NRL region, while sedation is attributed to the inhibition of alpha2-adrenergic mechanisms in the locus coeruleus, which is involved in the control of the sleep-waking cycle. The IPRmay constitute on interesting target for new drugs in the treatment of arterial hypertension. Finally, dysfunctions of this modulatory system which could be involved in the pathophysiologyof some forms of the hypertensive disease are under investigation.

From allergy to schistosomes: role of Fc receptors and adhesion molecules in eosinophil effector function

The dual function of eosinophils has been evidenced in protective immunity against parasites as well as in pathological manifestations during allergic disorders. We have demonstrated that a new class of IgE receptors, FcepsilonRII/CD23, was involved in the functional duality of eosinophils and other proinflammatory cells. More recently, we have shown that FcepsilonRI, the high affinity IgE receptor thought to be only expressed by basophils and mast cells, was involved in eosinophil-mediated cytotoxicity against schistosomes as well as in mediator release. These results favour the view that both IgE and its receptors have been primarily associated to a protective immune response, rather than to pathology. Not only IgE receptors but also members belonging to the family of adhesion molecules can participate as co-receptors in eosinophil effector function. The inhibitory role of monoclonal antibodies to LewisX (LeX, CD15) or to selectins in eosinophil-mediated cytotoxicity towards schistosomes and the detection of LeX and 'selectin-like' molecules on schistosomula surface indicate a double interaction mediated by selectins and their carbohydrate ligands between eosinophils and schistosomula. These results suggest new functions for these adhesion molecules, previously known to be involved mainly in cell infiltration.

Insulin-like growth factor and growth hormone receptor in postpartum lactating beef cows

The objective of this study was to evaluate the plasma concentrations of insulin-like growth factor-I (IGF-I), and the mRNA hepatic expression of IGF-I and of the growth hormone receptors GHR and GHR 1A, in postpartum beef cows. Four Angus and four crossbred (Angus x Nelore) postpartum suckled beef cows were used. Liver and blood samples were collected every 10 days, from calving to 40 days postpartum, for gene expression and for β-hydroxybutyrate and IGF-I assays, respectively. Samples for progesterone assay were collected every other day, from day 10 to 40 postpartum. Three cows ovulated before 40 days postpartum. IGF-I concentration was higher in Angus x Nelore than in Angus cows. There was no difference in the expression of GHR, GHR 1A and IGF-I according to breed or ovulatory status. IGF-I concentrations were higher in crossbred cows, but have not changed according to postpartum ovulatory status. Moreover, changes in postpartum IGF-I concentrations are not associated with changes in liver GHR, GHR 1A and IGF-I mRNA expression in either breed.

Involvement of dopamine receptors in diethylpropion-induced conditioning place preference

Diethylpropion (DEP) is an amphetamine-like agent used as an anorectic drug. Abuse of DEP has been reported and some restrictions of its use have been recently imposed. The conditioning place preference (CPP) paradigm was used to evaluate the reinforcing properties of DEP in adult male Wistar rats. After initial preferences were determined, animals weighing 250-300 g (N = 7 per group) were conditioned with DEP (10, 15 or 20 mg/kg). Only the dose of 15 mg/kg produced a significant place preference (358 ± 39 vs 565 ± 48 s). Pretreatment with the D1 antagonist SCH 23390 (0.05 mg/kg, sc) 10 min before DEP (15 mg/kg, ip) blocked DEP-induced CPP (418 ± 37 vs 389 ± 31 s) while haloperidol (0.5 mg/kg, ip), a D2 antagonist, 15 min before DEP was ineffective in modifying place conditioning produced by DEP (385 ± 36 vs 536 ± 41 s). These results suggest that dopamine D1 receptors mediate the reinforcing effect of DEP