Tectona grandis leaf extract ameliorates hepatic fibrosis: Modulation of TGF- ß /Smad signaling pathway and upregulating MMP3/TIMP1 ratio.

ETHNOBOTANICAL RELEVANCE: Tectona grandis L.f (or syn: Jatus grandis (L.f.) Kuntze Revis), from family Lamiaceae, also known as Teak, is widely recognized in ayurvedic system of medicine and confer curative potential against inflammation, liver disorders, biliousness, diabetes, bronchitis, leprosy and dysentery. Its leaves are rich source of edible food colorant and reported nontoxic for liver and various organs. AIM OF STUDY: Hepatic injury progression to liver cirrhosis and cancer is a serious health issue across the world. Currently, anti-fibrotic therapeutic options are limited and expensive with no FDA approved direct anti-hepato-fibrotic drug validated in clinic. Thus, the aim of this study was to understand ameliorative effect of Tectona grandis L.f, leaves in early liver fibrosis. METHOD AND RESULTS: C57BL/6 mice suffering from CCl4 induced liver injury, were orally administered at three different doses (50, 100 & 200 mg/kg) of Tectona grandis L.f, leaf extract, thrice a week, up to 4 and 8 weeks. Anti-fibrotic effect was evaluated through animal body/liver weight measurements, serological tests (AST, ALT, GSH, MDA and LDH assays), tissue hydroxyproline content, and histochemical analysis (H&E, Masson trichrome, Sirius red and αSMA localization). Moreover, transcriptional and post-transcriptional expression of fibrosis associated biomarkers and TGF-ß/Smad cascade were analyzed. It was observed that 100 mg/kg dose optimally downregulated TGF-ß1/Smad2 with upregulation of Smad7 and regulated αSMA, Col 1, PDGF, TIMP1 and MMP3 expression, post 8 weeks of treatment. In addition, MMP3/TIMP1 ratio was upregulated to 0.7, 2.5 and 1.7 fold at 50 mg/kg, 100 mg/kg & 200 mg/kg treatments respectively, in comparison to untreated liver fibrosis models. The extract contains gallic acid, caffeic acid, sinapinic acid and myricetin when analyzed through high performance liquid chromatography. CONCLUSION: Tectona grandis L.f, leaves have potential to ameliorate liver fibrosis induced by CCl4 in mice via modulation of TGF-ß1/Smad pathway and upregulated MMP3/TIMP1 ratio.

Clofazimine analogs with efficacy against experimental tuberculosis and reduced potential for accumulation.

The global tuberculosis crisis urgently demands new, efficacious, orally available drugs with the potential to shorten and simplify the long and complex treatments for drug-sensitive and drug-resistant disease. Clofazimine, a riminophenazine used for many years to treat leprosy, demonstrates efficacy in animal models of tuberculosis via a novel mode of action. However, clofazimine's physicochemical and pharmacokinetic properties contribute to side effects that limit its use; in particular, an extremely long half-life and propensity for tissue accumulation together with clofazimine's dye properties leads to unwelcome skin discoloration. We recently conducted a systematic structure-activity study of more than 500 riminophenazine analogs for anti-Mycobacterium tuberculosis activity. We describe here the characteristics of 12 prioritized compounds in more detail. The new riminophenazine analogs demonstrated enhanced in vitro activity compared to clofazimine against replicating M. tuberculosis H37Rv, as well as panels of drug-sensitive and drug-resistant clinical isolates. The new compounds demonstrate at least equivalent activity compared to clofazimine against intracellular M. tuberculosis and, in addition, most of them were active against nonreplicating M. tuberculosis. Eleven of these more water-soluble riminophenazine analogs possess shorter half-lives than clofazimine when dosed orally to mice, suggesting that they may accumulate less. Most importantly, the nine compounds that progressed to efficacy testing demonstrated inhibition of bacterial growth in the lungs that is superior to the activity of an equivalent dose of clofazimine when administered orally for 20 days in a murine model of acute tuberculosis. The efficacy of these compounds, along with their decreased potential for accumulation and therefore perhaps also for tissue discoloration, warrants further study.

In vitro host range, multiplication and virion forms of recombinant viruses obtained from co-infection in vitro with a vaccinia-vectored influenza vaccine and a naturally occurring cowpox virus isolate.

BACKGROUND: Poxvirus-vectored vaccines against infectious diseases and cancer are currently under development. We hypothesized that the extensive use of poxvirus-vectored vaccine in future might result in co-infection and recombination between the vaccine virus and naturally occurring poxviruses, resulting in hybrid viruses with unpredictable characteristics. Previously, we confirmed that co-infecting in vitro a Modified vaccinia virus Ankara (MVA) strain engineered to express influenza virus haemagglutinin (HA) and nucleoprotein (NP) genes with a naturally occurring cowpox virus (CPXV-NOH1) resulted in recombinant progeny viruses (H Hansen, MI Okeke, Ø Nilssen, T Traavik, Vaccine 23: 499-506, 2004). In this study we analyzed the biological properties of parental and progeny hybrid viruses. RESULTS: Five CPXV/MVA progeny viruses were isolated based on plaque phenotype and the expression of influenza virus HA protein. Progeny hybrid viruses displayed in vitro cell line tropism of CPXV-NOH1, but not that of MVA. The HA transgene or its expression was lost on serial passage of transgenic viruses and the speed at which HA expression was lost varied with cell lines. The HA transgene in the progeny viruses or its expression was stable in African Green Monkey derived Vero cells but became unstable in rat derived IEC-6 cells. Hybrid viruses lacking the HA transgene have higher levels of virus multiplication in mammalian cell lines and produced more enveloped virions than the transgene positive progenitor virus strain. Analysis of the subcellular localization of the transgenic HA protein showed that neither virus strain nor cell line have effect on the subcellular targets of the HA protein. The influenza virus HA protein was targeted to enveloped virions, plasma membrane, Golgi apparatus and cytoplasmic vesicles. CONCLUSION: Our results suggest that homologous recombination between poxvirus-vectored vaccine and naturally circulating poxviruses, genetic instability of the transgene, accumulation of non-transgene expressing vectors or hybrid virus progenies, as well as cell line/type specific selection against the transgene are potential complications that may result if poxvirus vectored vaccines are extensively used in animals and man.

ErbB2 receptor tyrosine kinase signaling mediates early demyelination induced by leprosy bacilli.

Demyelination is a common pathologic feature in many neurodegenerative diseases including infection with leprosy-causing Mycobacterium leprae. Because of the long incubation time and highly complex disease pathogenesis, the management of nerve damage in leprosy, as in other demyelinating diseases, is extremely difficult. Therefore, an important challenge in therapeutic interventions is to identify the molecular events that occur in the early phase before the progression of the disease. Here we provide evidence that M. leprae-induced demyelination is a result of direct bacterial ligation to and activation of ErbB2 receptor tyrosine kinase (RTK) signaling without ErbB2-ErbB3 heterodimerization, a previously unknown mechanism that bypasses the neuregulin-ErbB3-mediated ErbB2 phosphorylation. MEK-dependent Erk1 and Erk2 (hereafter referred to as Erk1/2) signaling is identified as a downstream target of M. leprae-induced ErbB2 activation that mediates demyelination. Herceptin (trastuzumab), a therapeutic humanized ErbB2-specific antibody, inhibits M. leprae binding to and activation of ErbB2 and Erk1/2 in human primary Schwann cells, and the blockade of ErbB2 activity by the small molecule dual ErbB1-ErbB2 kinase inhibitor PKI-166 (ref. 11) effectively abrogates M. leprae-induced myelin damage in in vitro and in vivo models. These results may have implications for the design of ErbB2 RTK-based therapies for both leprosy nerve damage and other demyelinating neurodegenerative diseases.

ErbB2 receptor tyrosine kinase signaling mediates early demyelination induced by leprosy bacilli

Demyelination is a common pathologic feature in many neurodegenerative diseases including infection with leprosy-causing Mycobacterium leprae. Because of the long incubation time and highly complex disease pathogenesis, the management of nerve damage in leprosy, as in other demyelinating diseases, is extremely difficult. Therefore, an important challenge in therapeutic interventions is to identify the molecular events that occur in the early phase before the progression of the disease. Here we provide evidence that M. leprae-induced demyelination is a result of direct bacterial ligation to and activation of ErbB2 receptor tyrosine kinase (RTK) signaling without ErbB2-ErbB3 heterodimerization, a previously unknown mechanism that bypasses the neuregulin-ErbB3-mediated ErbB2 phosphorylation. MEK-dependent Erk1 and Erk2 (hereafter referred to as Erk1/2) signaling is identified as a downstream target of M. leprae-induced ErbB2 activation that mediates demyelination. Herceptin (trastuzumab), a therapeutic humanized ErbB2-specific antibody, inhibits M. leprae binding to and activation of ErbB2 and Erk1/2 in human primary Schwann cells, and the blockade of ErbB2 activity by the small molecule dual ErbB1-ErbB2 kinase inhibitor PKI-166 (ref. 11) effectively abrogates M. leprae-induced myelin damage in in vitro and in vivo models. These results may have implications for the design of ErbB2 RTK-based therapies for both leprosy nerve damage and other demyelinating neurodegenerative diseases.

MAP kinase protects G protein-coupled receptor kinase 2 from proteasomal degradation.

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195-199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenomenon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3.

Naturally acquired and experimental leprosy in nonhuman primates.

Naturally-acquired leprosy has been observed in chimpanzees and sooty mangabey monkeys. Experimental multibacillary leprosy was established in 24 of 36 mangabey monkeys, 7 of 34 rhesus monkeys, and 15 of 19 African green monkeys following intravenous and intradermal inoculation of Mycobacterium leprae. The experimental disease strongly resembles leprosy in humans clinically, histopathologically, and immunologically. Thus, in addition to nine-banded armadillos in Louisiana and Texas, chimpanzees and sooty mangabeys in Africa, in the wild or in captivity, may serve as a zoonotic source of M. leprae. Investigators using chimpanzees and monkeys should be alerted to the possibility of naturally-acquired leprosy.

Intraocular pressure changes and postural changes of intraocular pressure in experimentally induced Hansen's disease of rhesus, mangabey, and African green monkeys.

In our long term evaluation of patients with Hansen's disease we have frequently found reduction of their intraocular pressure. Furthermore, we noted changes in their intraocular pressure on change of posture. To determine if these changes have any significance we measured the intraocular pressures of 24 experimentally infected and 39 control monkeys in both sitting and reclining positions. We found significant reduction of intraocular pressure in 66.7% compared with controls in the sitting position, and a significant increase in intraocular pressure in 79% when checked first in the sitting then in the reclining position. We offer a possible pathophysiological explanation as to why the changes occur.

[Peripheral nerve lesions of experimental leprosy in monkeys. VII. Intrafascicular-edema and small blood vessels].

Histopathological findings of intrafascicular-edema found in n. ulnaris running at forearm, palmside of hand and fingers of rhesus monkey 8664 and african green monkey 8175 were studied by semithin section method. These two monkeys were inoculated with leprosy bacilli from lepromata of mangabey monkey A022. The mangabey monkey A022 had been experimental leprosy after inoculum of the leprosy bacilli from lepromata of the mangabey monkey A015-natural infection. Period between date of inoculum and sacrifice of the rhesus monkey 8664 was about 2 years, and the african green monkey 8175 was about 5 years. Results found were: 1. Intrafascicular-edema found in n. ulnaris running at forearm, palmside of hand and fingers was remarkable at some of peculiar anatomical areas where were put on mechanical pressure. 2. The intrafascicular-edema inside many fascicles was remarkable around the small blood vessels. 3. Obsruction of the small blood vessels and congestion of blood stream at surrounded of the intrafascicular-edema with fibrous elements and cellular elements were observed inside some of fascicles. 4. Bundles of peripheral nerve fibers inside some of fascicles were pressed by the intrafascicular-edema, and the fibrous elements, especially collagen fibers, were increased around the pressed bundles of nerve fibers and each nerve fiber inside the fascicles. 5. Vacuolar degeneration and swelling of axon were observed at some of myelinated nerve fibers. 6. Not only the intrafascicular-edema but also a large amount of intracytoplasmic foamy structures with solid leprosy bacilli were observed inside many fascicles.(ABSTRACT TRUNCATED AT 250 WORDS)

[Peripheral nerve lesions of experimental leprosy in monkeys. VI. Tuberculoid leprous granuloma in an African green monkey].

Histopathological finding of left n. peroneus superficialis, left rami calcanei medialis (n. tibialis), left n. medianus, left ramus superficialis, n. radialis and left rami muscularis, m. palmaris brevis of african green monkey 8182 (Delta Regional Primate Research Center, USA) were studied by semithin section method. African green monkey 8182 was inoculated with leprosy bacilli isolated from lepromata of mangabey monkey A 015-natural infection. The length of period between date of inoculum and date of death of the monkey was about 5 years. 1. A typical tuberculoid leprous granuloma was observed in a fascicle of n. peroneus superficialis. 2. In the other fascicles of n. peroneus superficialis, proliferation of collagen fibers was remarkable not only at surrounding of myelinated nerve fibers but also at perineurium. Some of myelinated nerve fibers inside these fascicles were kept in good condition. 3. Infiltration of small amount of lymphocytes, proliferation of collagen fibers, and a few leprosy bacilli were observed inside some of fascicles of rami calcanei medialis. 4. In the fascicles of n. medianus, ramus superficialis of n. radialis and rami muscularis of n. ulnaris at m. palmaris brevis, proliferation of collagen fibres was observed not only at surrounding of myelinated nerve fibers but also at perineurium.

Experimental leprosy in African green monkeys (Cercopithecus aethiops): a model for polyneuritic leprosy.

Three African green monkeys (Cercopithecus aethiops) were inoculated intravenously and intracutaneously with Mycobacterium leprae derived from a naturally infected mangabey monkey. All developed cutaneous lesions at inoculation sites. One developed disseminated cutaneous lesions, while the cutaneous lesions in the other two regressed and eventually disappeared. The animals were examined at necropsy five years after inoculation. All three had active leprosy infection in peripheral nerves with extensive inflammation and fibrosis. The disease histologically resembled borderline-lepromatous leprosy. These findings add a new dimension to animal models of leprosy.

Experimental leprosy in three species of monkeys.

Eleven mangabey monkeys inoculated with Mycobacterium leprae developed lepromatous-type leprosy. Nine of the mangabeys were inoculated with M. leprae isolated from a mangabey with naturally acquired lepromatous leprosy. Immune function was depressed in some of these animals after dissemination of the disease. Two mangabeys developed lepromatous leprosy after inoculation with human M. leprae passaged in an armadillo. Three rhesus and three African green monkeys inoculated with mangabey-derived M. leprae also developed lepromatous leprosy. Mangabeys may be the first reported nonhuman primate model for the study of leprosy. Rhesus and African green monkeys may also prove to be reproducibly susceptible to the disease.

Electron microscopic study of leprosy in a mangabey monkey (natural infection).

Ultrastructural features of the leproma of a) a naturally infected mangabey monkey, and lepromas and liver of b) a passage mangabey monkey, c) a rhesus monkey, d) an African green monkey, and e) a nine-banded armadillo inoculated with leprosy bacilli isolated from the leproma of a naturally infected mangabey monkey were studied by the freeze-etching technique. The size, shape, and ultrastructural features of leprosy bacilli in the phagolysosomes of macrophages in all of these samples were essentially the same as those in humans, nude mice, and armadillos inoculated with human Mycobacterium leprae. Distinct accumulations of small spherical droplets were observed around leprosy bacilli inside lepra cells of all the samples but were scarce in the specimen from the green monkey. The bacilli in all samples were long and slender, and had band structures on the smooth cell wall surfaces. The bacilli were indistinguishable from M. leprae.