iNOS in Tuberculosis

Inducible Nitric Oxide Synthase in the Tuberculous Human Lung
The NOS2 isoform, often called iNOS, is a leading candidate to account for the incompletely successful bactericidal activity of macrophages toward M. tuberculosis. The designation "i" denotes that production of nitric oxide by iNOS is independent of elevated intracellular Ca2+ ,a biochemically distinctive and biologically important feature that explains how the enzyme can produce nitric oxide for days after its transcription is induced by immunologic and inflammatory stimuli.
Nitric oxide is the only molecule known to be produced by mammalian cells that can kill tubercle bacilli in vitro with a molar potency comparable to that of chemotherapy. That the primary product of iNOS is mycobactericidal provides one type of evidence consistent with a role for iNOS in controlling tuberculosis. There are four more lines of evidence: immunologically activated, iNOS-expressing mouse macrophages can kill M. tuberculosis in vitro, but not if the macrophages are treated with iNOS inhibitors or bear disrupted NOS2 alleles, iNOS is expressed in infected mouse tissues in which the growth of M. tuberculosis is restrained, but iNOS is scant or absent when immunosuppressive drugs or genetic interventions impair host resistance healthy mice that harbor tubercle bacilli succumb abruptly to tuberculosis after ingestion of specific iNOS inhibitors and mice with disrupted NOS2 alleles die with fulminant tuberculosis in a few weeks, whereas wild-type mice survive the infection for about 9 months.
It is difficult to determine how much of this is relevant to human tuberculosis. No one has knowingly administered iNOS inhibitors to people infected with tubercle bacilli, and no primary genetic deficiency of iNOS has been identified in humans. Thus, only two experimental avenues have been open: the impact of iNOS inhibitors on the mycobactericidal activity of human macrophages in vitro, and a search for iNOS at sites of tuberculosis. Studies of human macrophages in vitro have been more frustrating than informative, because the macrophages tested to date have rarely exerted a bactericidal effect against M. tuberculosis, precluding a determination as to the contribution of iNOS to killing. Both mouse and human macrophages that lack iNOS can exert a bacteriostatic effect against M. tuberculosis by an unknown mechanism, but with respect to iNOS these cells do not model human macrophages at sites of infection or inflammation, which are often intensely iNOS-positive. Thus, one of the major unfulfilled goals for research in the immunology of tuberculosis is to learn how to obtain or culture human macrophages that are as iNOS-positive in vitro as they are in vivo and to test whether they kill M. tuberculosis and use iNOS to do so.

Natural Herbs in Tuberculosis.

Herbal Tuberculosis Care

Azadirachta Indica as a herbal medicine

NEEM EXTRACT MODULATE THE CYTOKINE NETWORKS OF THE IMMUNE AND REPRODUCTIVE SYSTEMS

Extracts of Neem leaves, bark and seeds have a long history as folk remedies for a number of diseases. Neem oil extracted from seeds could prevent pregnancy in rodents and monkeys. Because of the cytokine interactions between the immune system and pregnancy, this led to the suggestion that Neem influences immune cytokine networks. This was strengthened when it was shown that in vivo treatment with Neem oil resulted in higher IFNγ levels when spleen cells were subsequently stimulated with Con A in culture. IFN γ is a central component of the Th1 cytokine pattern, secreted by the TH1 subset of CD4 T cells that carry out cell-mediated immune reactions. In contrast, TH2 cells secrete 1L4, 1L5 and IL1O, and provide help for antibody production. TH1 and TH2 responses cross-inhibit each other, mainly via their characteristic cytokines.
TH1 cytokines are harmful for pregnancy, but enhance the immune response against a number of intracellular pathogens. Neem may act by directly or indirectly enhancing the production of TH1 cytokines, modifying both pregnancy and disease resistance.

1. Neem extract Effects on Cytokine Synthesis, B Cell Proliferation and Parasite Infection:
a). Neem extract preferentially enhances synthesis of the Th1 cytokine IFNγ by spleen cells.
b) Neem extract enhances GM-CSF synthesis.
c) Neem extract stimulate B cell proliferation and antibody production.
d) Neem extract stimulate production of cytokines and other mediators by macrophages.
e) Neem extract effects on Leishmania infection in vitro and in vivo.

2. Analysis of the mode of action of the active component in Neem on immune cells:
a) Mechanism of action of Neem on cytokine synthesis and differentiation of T cells.
b) Neem effects on macrophage pro-and anti-inflammatory mediator synthesis.
c) Neem stimulation of B cell proliferation and antibody production and switching.
3. Neem effects on infections and fertility:
a) Neem enhancement/inhibition of Leishmania infection.
b) Neem effects on placental cytokine production in vitro and in vivo.
c) Neem effects Pre implantation regulated by immune cytokines.

Neem effects on placental cytokine production in vitro and in vivo. The effects of Neem on placental cytokine production will also be investigated. Previous experiments demonstrated substantial levels of TH2 cytokines in placental tissue. The cytokine synthesis ceases immediately after tissue disruption and in vitro culture, and so two approaches will be taken to evaluate placental responses to Neem. In situ hybridization and/or antibody staining will be used to determine the location of TH2 (and possibly Th1)
cytokine synthesis. The possibility of establishing culture conditions for placental cells or tissue that allow continued cytokine synthesis. Possible modifications to the basic culture system include the use of explants instead of single-cell cultures, culture of defined cell types (e.g. trophoblast or uterine epithelial cells), and the addition of cytokines and hormones to mimic
the in vivo environment. If culture conditions can be found that allow the same pattern of cytokine expression as in vivo, this will provide a very powerful system for future characterization of the influences of Neem and other compounds on the placental cytokine regulatory networks.
Neem effects in Pre-implantation: Krishnan has recently found that Leishmania infection significantly compromises concurrent pregnancy. The C57B116 TH1 response can increase the resorption rate, consistent with previous studies showing that IFN γ and NK cells were deleterious to the maintainance of pregnancy. A more unexpected finding was that the early response of C57B1/6 mice against Leishmania could also cause a block in
pregnancy at some stage before implantation. It is tempting to speculate that this effect could also be due to cytokine regulatory changes. Interestingly, others have shown Neem oil prevented pregnancy at a stage before implantation, provided the Neem was administered at an early time. Although it is difficult to compare the effects of Neem seed oil with the water-soluble Neem extract. The
hypothesis that fertilization or implantation is susceptible to the same cytokine regulatory pathways as the maintenance of pregnancy after implantation. This will be tested by first comparing the water soluble extract with seed oil for their effects on fertilization/implantation. In vivo treatments with anti-IFNγ and anti-TNF will be used to determine whether these TH1 cytokines mediate the Neem-induced inhibition of the phase of reproduction. In vivo (the work of two students, Li Li and Yew Hon Lai) the effect of exogenous IL1O for potential blocking of Neem-induced reduction of implantations.