Studies on tumour inhibitory activity of indigenous drugs: Part I. Tumour inhibitory activity of Hippophae salicifolia, D.DON

The extracts of the bark of Hippophae salicifolia D.DON have been found to possess significant inhibitory activity on mouse fibrosarcoma. The tumour tissues showed that there was a degeneration and even necrosis of tumour calls. The alcoholic extract (B) of the bark also possesses an inhibitory activity against Yoshida sarcoma (ascites), as evidenced by the increase in survival period of the experimental animals. The chemistry of the bark is under investigation

Cytokine and Protein Markers of Leprosy Reactions in Skin and Nerves: Baseline Results for the North Indian INFIR Cohort

Leprosy affects skin and peripheral nerves. Although we have effective antibiotics to treat the mycobacterial infection, a key part of the disease process is the accompanying inflammation. This can worsen after starting antibacterial treatment with episodes of immune mediated inflammation, so called ‘reactions’. These reactions are associated with worsening of the nerve damage. We recruited a cohort of 303 newly diagnosed leprosy patients in North India with the aim of understanding and defining the pathological processes better. We took skin and nerve biopsies from patients and examined them to define which molecules and mediators of inflammation were present. We found high levels of the cytokines Tumour Necrosis Factor alpha, Transforming Growth Factor beta and inducible Nitric Oxide Synthase in biopsies from patients with reactions. We also found high levels of bacteria and inflammation in the nerves. These experiments tell us that we need to determine which other molecules are present and to explore ways of switching off the production of these pro-inflammatory molecules

Analysis of Antibody and Cytokine Markers for Leprosy Nerve Damage and Reactions in the INFIR Cohort in India

Leprosy is one of the oldest known diseases. In spite of the established fact that it is least infectious and a completely curable disease, the social stigma associated with it still lingers in many countries and remains a major obstacle to self reporting and early treatment. The nerve damage that occurs in leprosy is the most serious aspect of this disease as nerve damage leads to progressive impairment and disability. It is important to identify markers of nerve damage so that preventive measures can be taken. This prospective cohort study was designed to look at the potential association of some serological markers with reactions and nerve function impairment. Three hundred and three newly diagnosed patients from north India were recruited for this study. The study attempts to reflect a model of nerve damage initiated by mycobacterial antigens and maintained by ongoing inflammation through cytokines such as Tumour Necrosis Factor alpha and perhaps extended by antibodies against nerve components

A DNA Vaccine Encoding Multiple HIV CD4 Epitopes Elicits Vigorous Polyfunctional, Long-Lived CD4+ and CD8+ T Cell Responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4+ T cells are important for the generation and maintenance of functional CD8+ cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4+ T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4+/CD8+ T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4+ and CD8+ T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4+ T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4+ T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4+ T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8+ T cells and antibody responses- elicited by other HIV immunogens

A Synthetic Uric Acid Analog Accelerates Cutaneous Wound Healing in Mice

Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice. Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions

TRPV1 in Brain Is Involved in Acetaminophen-Induced Antinociception

Background: Acetaminophen, the major active metabolite of acetanilide in man, has become one of the most popular overthe- counter analgesic and antipyretic agents, consumed by millions of people daily. However, its mechanism of action is still a matter of debate. We have previously shown that acetaminophen is further metabolized to N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) by fatty acid amide hydrolase (FAAH) in the rat and mouse brain and that this metabolite is a potent activator of transient receptor potential vanilloid 1 (TRPV1) in vitro. Pharmacological activation of TRPV1 in the midbrain periaqueductal gray elicits antinociception in rats. It is therefore possible that activation of TRPV1 in the brain contributes to the analgesic effect of acetaminophen. Methodology/Principal Findings: Here we show that the antinociceptive effect of acetaminophen at an oral dose lacking hypolocomotor activity is absent in FAAH and TRPV1 knockout mice in the formalin, tail immersion and von Frey tests. This dose of acetaminophen did not affect the global brain contents of prostaglandin E-2 (PGE(2)) and endocannabinoids. Intracerebroventricular injection of AM404 produced a TRPV1-mediated antinociceptive effect in the mouse formalin test. Pharmacological inhibition of TRPV1 in the brain by intracerebroventricular capsazepine injection abolished the antinociceptive effect of oral acetaminophen in the same test. Conclusions: This study shows that TRPV1 in brain is involved in the antinociceptive action of acetaminophen and provides a strategy for developing central nervous system active oral analgesics based on the coexpression of FAAH and TRPV1 in the brain

Clinical oxidative stress during leprosy multidrug therapy:impact of dapsone oxidation

This study aims to assess the oxidative stress in leprosy patients under multidrug therapy (MDT; dapsone, clofazimine and rifampicin), evaluating the nitric oxide (NO) concentration, catalase (CAT) and superoxide dismutase (SOD) activities, glutathione (GSH) levels, total antioxidant capacity, lipid peroxidation, and methemoglobin formation. For this, we analyzed 23 leprosy patients and 20 healthy individuals from the Amazon region, Brazil, aged between 20 and 45 years. Blood sampling enabled the evaluation of leprosy patients prior to starting multidrug therapy (called MDT 0) and until the third month of multidrug therapy (MDT 3). With regard to dapsone (DDS) plasma levels, we showed that there was no statistical difference in drug plasma levels between multibacillary (0.518±0.029 μg/mL) and paucibacillary (0.662±0.123 μg/mL) patients. The methemoglobin levels and numbers of Heinz bodies were significantly enhanced after the third MDTsupervised dose, but this treatment did not significantly change the lipid peroxidation and NO levels in these leprosy patients. In addition, CAT activity was significantly reduced in MDT-treated leprosy patients, while GSH content was increased in these patients. However, SOD and Trolox equivalent antioxidant capacity levels were similar in patients with and without treatment. These data suggest that MDT can reduce the activity of some antioxidant enzyme and influence ROS accumulation, which may induce hematological changes, such as methemoglobinemia in patients with leprosy. We also explored some redox mechanisms associated with DDS and its main oxidative metabolite DDS-NHOH and we explored the possible binding of DDS to the active site of CYP2C19 with the aid of molecular modeling software

TGF-β-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates

Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth

Partial activity of the kidney and the "all or nothing" principle

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