Effects of morphine during Mycobacterium tuberculosis H37Rv infection in mice

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Life Sciences 82 (2008) 308 – 314 www.elsevier.com/locate/lifescie

Effects of morphine during Mycobacterium tuberculosis H37Rv infection in mice Raman Preet Singh, Sarbjit Singh Jhamb ⁎, Prati Pal Singh National Institute of Pharmaceutical Education and Research, Phase-X, S. A. S Nagar-160 062, India Received 12 October 2007; accepted 30 November 2007

Abstract The effects of opiates in various infections are well known; however, very little is known about tuberculosis infection. Therefore, in the present study, we report for the first time, the effects of morphine during murine tuberculosis. Mice were infected intravenously with Mycobacterium tuberculosis H37Rv, administered morphine (0.1–100 mg/kg subcutaneously on day 0 and day +15) and sacrificed on day +30 for CFU enumeration in lungs and spleen. Morphine exerted maximum suppression of infection at 5 mg/kg, and sometimes completes elimination of infection; naloxone, silica and aminoguanidine blocked the protective effect of morphine. In vitro, morphine lacked direct antimycobacterial activity up to 1 × 10− 4 M concentration, as assessed by radiometric BACTEC method. In macrophage model of infection, morphine showed maximal killing at 1 × 10− 7 M concentration, the activity was blocked by naloxone and aminoguanidine. These observations suggest that morphine exerts a dose-dependent effect in murine tuberculosis, the protective effect being naloxone-reversible and may involve macrophage-mediated protective mechanisms. These results may be helpful in developing new opioid-like chemical entities against tuberculosis infection. © 2007 Elsevier Inc. All rights reserved. Keywords: Morphine; Mycobacterium tuberculosis; Macrophage; Mice

Introduction Opioid addiction is known to exert alterations in the host immune system, thus, resulting in immunosuppression, which, in turn, leads to an increased susceptibility of host towards infections (Risdahl et al., 1998; Wang et al., 2005; Tubaro et al., 1983). These effects of opioids are mediated through the opioid receptors, which are transmembrane G-protein coupled receptors. The binding of morphine on the extracellular domain of the receptors initiates a cascade of intracellular events, which lead to changes in the normal cellular functions of the immune cells, particularly macrophages and lymphocytes (Mellon and Bayer, 1998; Sharp et al., 1998). The opioid-mediated effects on immune cells are mediated via direct and indirect mechanisms. The binding of opioids on the surface receptors of the immunocytes accounts for the direct ⁎ Corresponding author. Tel.: +91 172 2214682-87x2079; fax: +91 172 2214692. E-mail address: [email protected] (S.S. Jhamb). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.11.024

effects, which include suppression of migration, phagocytic activity and burst phenomena in macrophages and the cytokine and antibody elaboration profile in lymphocytes. The indirect effects involve opioid receptors in central nervous system, which play a crucial role in modulating the immune system via autonomic nervous system (Sharp et al., 1998; Stefano et al., 2001; Vallejo et al., 2004). However, morphine can also augment the protective immune responses in the host leading to suppression of infection (Fuggetta et al., 2005; Sheridan and Moynihan, 2005). The effects observed are dose-dependent and biphasic in nature (Singal et al., 2003; Singal and Singh, 2005; Singh et al., 1994); acute treatment with low doses of morphine show protection from infection (Singal et al., 2003; Singal and Singh, 2005; Singh et al., 1994) while high doses (Singal et al., 2003; Singal and Singh, 2005; Singh et al., 1994) or chronic treatment resulted in suppression of immune responses (Wang et al., 2005). Opioid addicts are more susceptible to mycobacterial infections than the non-addicts (Durante et al., 1998) and can also show anergy towards tuberculin test, thus obscuring the

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diagnosis of TB (MacGregor et al., 1994). These observations support the immunosuppressive action of morphine. In vitro, morphine increases the uptake of Mycobacterium tuberculosis by microglial cells (Peterson et al., 1995), indicating immunostimulant activity of morphine. The reactive nitrogen intermediates are an important defense mechanism against TB (Kaufmann, 2002) and morphine can modulate iNOS expression and hence NO release (Pacifici et al., 1995; Stefano et al., 2001). Apparently, no experimental study has been reported to study the effect of morphine in murine tuberculosis (TB). Hence, we studied the effect of morphine in murine TB and the role of macrophage- and nitric oxide (NO)-dependent mechanisms.

after infection) and day +15 of infection, subcutaneously. Control groups (vehicle-treated) were given saline on similar schedule. Naloxone and silica were administered either alone or with morphine. Naloxone (4 mg/kg, i.p.) was administered 30 min before morphine (5 mg/kg) treatment. Silica (3 mg/ mouse, i.v.) was administered 15 min after morphine (5 mg/kg) treatment. AG treatment (1% solution in drinking water) was started 12 h after infection and continued till 4 weeks. Isoniazidtreated group was included as a positive control, which received 25 mg/kg of isoniazid 6 days/week for 4 weeks by oral gavage. The experiments were performed three times and the data are the means of the three independent experiments.

Materials and methods

CFU enumeration

Animals

Mice were sacrificed on day +30 of infection by cervical dislocation. Lungs and spleen were aseptically removed and homogenized in saline. Viable bacterial counts were determined by plating 10-fold serial dilutions on Middlebrook 7H10 medium with ADC supplement. CFUs were enumerated after 4–6 weeks of incubation at 37 °C in 5% CO2 atmosphere.

Swiss albino mice weighing 20 ± 2 g, of both sexes, were obtained from the Central Animal Facility of the institute and maintained in 12 h light/dark cycle. The animals were provided food and water ad libitum. All studies were carried out as per protocol approved by Institutional Animal Ethics Committee (IAEC) complying with the guidelines of the Care and Use of Animals in Scientific Research, Indian National Science Academy, New Delhi. Bacteria and infection M. tuberculosis H37Rv was obtained from Tuberculosis Research Centre, Chennai, India. The bacteria were grown in Middlebrook 7H9 medium (HiMedia, India) supplemented with 10% ADC (HiMedia). Log phase cultures were centrifuged and washed twice with sterile saline and adjusted to McFarland standard corresponding to 1 × 107 colony forming units (CFU)/ ml. The bacterial suspension was sonicated (20 kHz, 10 s, 10 cycles; Bandelin, Germany) to obtain single cell suspension and mice were infected intravenously with 0.1 ml of the suspension. The viability of bacteria was consistently N 90%, as confirmed by plating 10-fold serial dilutions on Middlebrook 7H10 agar medium with 10% ADC supplement. Drugs

Spleen pathology Lungs and spleen of mice were removed on day +30 and gross organ morphology was observed. Spleen weights were also obtained. Touch smears of lungs and spleen were prepared as follows. The organs were cut into small pieces and spotted on microscope slides. The slides were then heat-fixed and stained with acid-fast stain (HiMedia, India) as per manufacturer's instructions. Direct antimycobacterial activity of drugs Direct antimycobacterial activity of morphine was tested against M. tuberculosis H37Rv in concentration range of 1 × 10− 4 M to 1 × 10− 9 M by radiometric method (BACTEC 460 TB, Becton and Dickinson, USA) (Siddiqi, 1995). Simultaneously, direct antimycobacterial activity of naloxone (1 × 10− 5 M) and AG (1 × 10− 4 M) was also determined. Isoniazid (Becton and Dickinson, USA) and rifampicin (Becton and Dickinson, USA) were used as positive controls.

Morphine was obtained from Government Opium and Alkaloid Factory, Ghazipur, India. Isoniazid was obtained from Sigma, USA. Morphine was dissolved in sterile saline and administered subcutaneously (5 mg/kg). Stock solution of isoniazid was prepared in distilled water. Naloxone (Sigma, USA) and silica (Sigma, USA) were prepared in sterile saline. Aminoguanidine (AG; Sigma, USA) was dissolved in drinking water for in vivo studies or distilled water for in vitro studies.

Elicited macrophages were harvested 96 h after intraperitoneal injection of 2 ml of 1% thioglycollate, as described elsewhere (Singal et al., 2003). The cells were adjusted to 1 × 106 cells/well of a 24-well tissue culture plate and incubated overnight at 37 °C in humid 5% CO2 atmosphere for adherence and formation of monolayer.

Drug treatment

Intramacrophage killing of M. tuberculosis

Mice were divided in groups of 10 each for treatment. Animals were administered indicated doses of morphine (0.1, 0.25, 0.5, 5, 10, 20, 30, 40, 50 and 100 mg/kg), on day 0 (3 h

After overnight incubation in 24-well tissue culture plate, non-adherent cells were removed by washing with warm Hank's balanced salt solution (HBSS; PAA Lab, Austria).

Cultivation of mouse peritoneal macrophages

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Adherent macrophages were infected with M. tuberculosis at a multiplicity-of-infection (MOI) of 1:10 for 4 h at 37 °C in 5% CO2 atmosphere. After phagocytosis, the unphagocytosed bacteria were removed by washing (5×) with HBSS and replaced with fresh Dulbecco's modified Eagle's medium (DMEM; PAA Lab, Austria) with or without drugs, and incubated at 37 °C in 5% CO2 atmosphere. After incubation for 0, 4 and 7 days, macrophages were lysed with 0.4 ml of 0.25% sodium dodecyl sulphate (SDS) and 1.1 ml of 7H9 medium for 10 min. After incubation at 37 °C for 10 min, 0.5 ml of 20% bovine serum albumin was added and again incubated for 10 min. Ten-fold serial dilutions of macrophage lysates were plated (50 µl each) on 7H10 plates in triplicate and incubated for 3–4 weeks at 37 °C in 5% CO2 atmosphere (Reddy et al., 1994). Statistical analysis CFU counts were converted to logarithmic scale and were evaluated by one way ANOVA followed by Tukey test using Sigma Stat program. All the morphine-treated groups were compared with control group. Results Direct antimycobacterial activity of morphine To determine the direct antimycobacterial activity of morphine, if any, radiometric BACTEC method was employed. M. tuberculosis H37Rv was exposed to varying concentrations of morphine from 1 × 10− 4 M to 1 × 10− 9 M. Morphine showed no direct activity in the given concentration range. These results correspond to earlier studies suggesting that morphine lacks direct antibacterial activity (Simon, 1964). Effect of morphine treatment on lung CFU counts Administration of two very low doses of morphine, 0.1, and 0.25 mg/kg on day 0 and day +15 of infection, had no effect on

infection as compared to vehicle-treated control. The mean log10CFU counts were 6.37, 6.2 and 6.6 in control, 0.1 mg/kg and 0.25 mg/kg groups respectively. However, treatment with higher doses of 0.5 and 5 mg/kg morphine following similar schedule, resulted in significant (P b 0.05) suppression of infection and showed mean log10CFU counts of 4.5 and 3.3 respectively. Treatment with 5 mg/kg morphine showed maximum activity and sometimes resulted in complete suppression of infection. Further, higher doses of morphine at 10, 20, 30, 40, 50 and 100 mg/kg following the similar schedule, significantly (P b 0.05) suppressed infection as compared to the control group and showed mean log10CFU counts of 3.9, 4.1, 4.2, 4.4, 4.3 and 5.5 respectively (Fig. 1). Isoniazid-treated group was included as a positive control and showed complete clearance of infection as determined by CFU enumeration. No CFU were detected even after 8 weeks of incubation. Effect of morphine treatment on spleen CFU counts Administration of morphine at very low doses, 0.1 and 0.25 mg/kg, showed no significant (P b 0.05) change in CFU counts in spleen. The mean log10CFU counts were 5.0, 5.1 and 4.9 in control, 0.1 mg/kg and 0.25 mg/kg groups. However, treatment with 0.5 mg/kg morphine resulted in significant (P b 0.05) reduction in mean log10CFU counts to 4.1. Treatment with 5 mg/kg morphine resulted in maximum suppression of infection and reduced the mean log10CFU counts to 3.7 (P b 0.05). With further increase in dose, the protective effects of morphine were reduced in a dose-dependent manner. Treatment with doses of 10, 20, 30, 40 and 50 mg/kg showed significant (P b 0.05) suppression of infection with mean log10CFU counts of 3.8, 4.1, 4.3, 4.4 and 4.5 respectively. Treatment with 100 mg/kg showed no significant (P b 0.05) reduction in CFU counts (mean log10CFU = 4.8). Isoniazidtreated group was included as a positive control and showed clearance of infection as determined by CFU enumeration. No CFU were detected even after 8 weeks of incubation.

Fig. 1. Effect of morphine on lungs and spleen CFU values. The animals were treated with morphine as described in Materials and methods. CFUs were enumerated after 4–6 weeks of incubation at 37 °C. Data are the mean ± SD of three separate experiments, run in triplicate. ⁎Significant difference (P b 0.05) as compared to control.

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Fig. 2. Effect of naloxone pre-treatment on lungs and spleen CFU values. The animals were treated with morphine as described in Materials and methods. CFUs were enumerated after 4–6 weeks of incubation at 37 °C. Data are the mean ± SD of three separate experiments, run in triplicate. ⁎Significant difference (P b 0.05) as compared to control.

Fig. 4. Effect of AG treatment on lungs and spleen CFU values. The animals were treated with morphine as described in Materials and methods. CFUs were enumerated after 4–6 weeks of incubation at 37 °C. Data are the mean ± SD of three separate experiments, run in triplicate. ⁎Significant difference (P b 0.05) as compared to control.

Effect of naloxone-pretreatment on morphine-induced suppression of infection

morphine-treated group were 6.37, 3.3, 7.4 and 6.7 in lungs and 5.0, 3.7, 6.3 and 5.96 in spleen respectively.

Pretreatment with naloxone, a non-selective antagonist of opioid receptors, abrogated morphine-induced suppression of infection in lungs and spleen (P b 0.05). Naloxone, per se, did not alter the course of infection. The CFU values in the target organs of naloxone-pretreated animals showed significant (P b 0.05) difference from the control group (Fig. 2). The mean log10CFU counts of control, morphine-treated, naloxonetreated and naloxone + morphine-treated group were 6.37, 3.3, 6.14 and 5.43 in lungs and 5.0, 3.7, 5.04 and 4.8 in spleen respectively.

Effect of AG on morphine-induced suppression of infection

Effect of silica on morphine-induced suppression of infection

Spleen and lung pathology

Administration of silica, a selective killer of macrophages, resulted in a significant (P b 0.05) increase in CFU counts in lungs and spleen as compared to the control group. However, treatment with silica significantly (P b 0.05) inhibited morphineinduced suppression of infection (Fig. 3). The mean log10CFU counts of control, morphine-treated, silica-treated and silica +

Spleen weights of mice were taken on the day of sacrifice. There appeared to be a direct correlation between the weight and number of CFUs in the spleen (Table 1). Touch-smear of lungs and spleen showed the presence of acid-fast M.

Fig. 3. Effect of silica treatment on lungs and spleen CFU values. The animals were treated with morphine as described in Materials and methods. CFUs were enumerated after 4–6 weeks of incubation at 37 °C. Data are the mean ± SD of three separate experiments, run in triplicate. ⁎Significant difference (P b 0.05) as compared to control.

Treatment with AG, an inhibitor of inducible nitric oxide synthase (iNOS), resulted in significant (P b 0.05) increase in CFU counts in lungs and spleen with respect to control group. Treatment with AG abrogated the protective effect of morphine in tuberculosis infection (Fig. 4). The mean log10CFU counts of control, morphine-treated, AG-treated and AG + morphinetreated group were 6.37, 3.3, 7.2 and 6.8 and in lungs and 5.0, 3.7, 5.7 and 5.3 in spleen respectively.

Table 1 Effect of morphine on spleen weight Morphine dose (mg/kg)

Spleen weight (mg)

CFU counts (Mean log10CFU)

0 (uninfected) 0 (control) 0 (isoniazid) 0.1 0.25 0.5 5 10 20 30 40 50 100

64∗ 362 76∗ 344 364 258∗ 158∗ 194∗ 240∗ 270∗ 210∗ 312∗ 394

ND 5.0 ND 5.1 4.9 4.1∗∗ 3.7∗∗ 3.8∗∗ 4.1∗∗ 4.3∗∗ 4.4∗∗ 4.5∗∗ 4.8

The animals were treated with morphine as described in Materials and methods. Spleen weights were obtained on day +30. Data are the mean ± SD of three separate experiments, run in triplicate. ⁎Significant difference (P b 0.001) as compared to control. ⁎⁎Significant difference (P b 0.05) as compared to control.

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Fig. 5. Effect of morphine on intramacrophage growth of M. tuberculosis. M. tuberculosis-infected mouse peritoneal macrophages were incubated with morphine and lysed on day 0, day 4 and day 7. CFUs were enumerated after 4–6 weeks of incubation at 37 °C. Data are the mean ± SD of three separate experiments, run in triplicate. ⁎Significant difference (P b 0.05) as compared to control.

tuberculosis and no other type of bacteria was observed indicating that the organ pathology was due to M. tuberculosis only. Effect of morphine on intramacrophage growth of M. tuberculosis M. tuberculosis-infected macrophages were incubated with 2-log serial dilutions of morphine over a wide range of concentrations, between 1 × 10− 5 M and 1 × 10− 11 M. The intramacrophage survival of bacteria appeared to be concentration-dependent with maximum killing at 1 × 10− 7 M followed by 1 × 10− 9 M. It is notable here that very low and high concentrations of morphine failed to induce intramacrophage killing of bacteria. Thus, the macrophage model supports the observed in vivo results (Fig. 5).

study indicate that morphine can modulate the mycobacterial growth and survival in the target organs, as is evident from the bacterial load in these organs. The bacterial load is modulated in a dose-dependent manner. Higher doses of morphine (up to 50 mg/kg) have less suppression, whereas 100 mg/kg dose showed no effect. The analgesic dose of morphine in mice is 10 mg/kg, therefore 100 mg/kg was chosen as the highest dose in this study. Low doses of morphine (5 mg/kg) were found to be more effective in the containment of infection in target organs as compared to higher doses, thus supporting earlier reports of dose-dependent immunomodulatory effects of morphine (Singal et al., 2003; Singh et al., 1994). Further, bacterial load in control group was more in lungs as compared to spleen. This is a characteristic feature of the virulent strains, which preferentially infect lungs. Interestingly, morphine showed preferential suppression of infection in lungs as compared to spleen; an observation of direct clinical relevance. In macrophage model of infection, the results obtained also show a dose-dependent effect on intramacrophage killing of M. tuberculosis. Thus, the in vivo and in vitro results are complementary and further support the involvement of immunomodulatory mechanisms responsible for M. tuberculosis killing. Opioid-mediated immunomodulation comprises a highly complex intertwining network of common receptors and ligands shared by the immune system and the central nervous system, and may involve the endocrine system (Sharp, 2004; Vallejo et al., 2004). The mechanisms of neuroimmunomodulation still remain elusive and may involve classical opioid receptors present in the central nervous system and on the immune cells (Vallejo et al., 2004), the former accounting for the indirect actions of the opioids and involve communication via the autonomic nervous system and the hypothalamus–pituitary– adrenal (HPA) axis. The corticosteroids released due to HPA axis activation may account for the long-term immunosuppressive effects of opioids (Mellon and Bayer, 1998; Vallejo et al.,

Effect of drugs on morphine-induced intracellular killing of M. tuberculosis M. tuberculosis-infected macrophages were incubated with 1 × 10− 7 M morphine in the presence or absence of naloxone (1 × 10− 5 M) or AG (1 × 10− 4 M). In the presence of these drugs, morphine-induced intramacrophage killing of M. tuberculosis was significantly (P b 0.05) inhibited (Fig. 6). Discussion Our laboratory has been involved in studying immunomodulatory effects of opioids on various parasitic diseases. Earlier, we have reported that morphine can modulate Leishmania donovani and Plasmodium berghei infection biphasically, at least in part, via macrophage-mediated host defense mechanism (s) (Singal et al., 2003; Singh et al., 1994). The results of this

Fig. 6. Effect of drugs on morphine-induced intracellular killing of M. tuberculosis. M. tuberculosis-infected mouse peritoneal macrophages were incubated with morphine in presence or absence of naloxone (1 × 10− 5 M) or AG (1 × 10− 4 M) and lysed on day 0, day 4 and day 7. CFUs were enumerated after 4–6 weeks of incubation at 37 °C. Data are the mean ± SD of three separate experiments, run in triplicate. ⁎Significant difference (P b 0.05) as compared to control.

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2004). The surface opioid receptors are postulated to be involved in direct effects of opioids and account for their acute effects. Multiple lines of evidence have confirmed the presence of µ-, κ- and δ-opioid receptors, as well as nonclassical opioid-like receptors on the surface of macrophages (Navolotskaya et al., 2003a,b; Sharp, 2004). Naloxonepretreatment abrogated the in vivo morphine-mediated suppression of infection, thus indicating the involvement of naloxone-sensitive opioid receptors. Further, naloxone blocked morphine-mediated intramacrophage killing of M. tuberculosis. These observations are in consonance with previous reports from our laboratory suggesting the involvement of naloxonesensitive opioid receptors in protective responses against P. berghei infection in mice (Singh et al., 1994) and L. donovani infection in hamsters (Singal et al., 2003). Macrophages are the key cells involved in protection against mycobacterial infections. The protection is afforded by a complex network involving many biomolecules affecting various cellular processes (Kaufmann, 2002). Macrophages are a key cellular target for opioids and morphine can alter normal functions like phagocytic activity and burst phenomena, the primary phenomena accounting for host defense against infection and may account for the immunosuppressive effects of morphine. However, morphine also exhibits immunostimulant effects. The observed dose-dependent effects of morphine can be attributed to its ability to inhibit at all the concentrations, but the duration of inhibition is concentration-dependent. Following inhibition, the cells may enter into a hyperactive phase and may lead to immunostimulation. Hence, following exposure to low concentrations, cells rebound faster into an excitatory state leading to augmented immune functions like phagocytosis (Pacifici et al., 1994). This indicates the possibility of involvement of a macrophage-mediated mechanism in the protective effect of morphine, at least in part. This is supported by the abrogation of protective effect by silica, a selective killer of macrophages (Allison et al., 1966). In macrophage model of M. tuberculosis infection, intramacrophage killing of bacteria followed the same pattern as observed in in vivo study. M. tuberculosis and other related species reside in the very cells that are responsible for killing of the bacteria, i.e. macrophages. The intracellular killing of these bacteria is mediated via generation of reactive nitrogen intermediates generated from NO by the activity of NOS; which, in turn, is controlled by cytokines like interferon-γ and tumor necrosis factor-α (Kaufmann, 2002). Blockade of NOS activity leads to reduced production of NO thus leading to an increased susceptibility to TB (Chan et al., 1995). Morphine can alter the generation of NO through modulation of NOS activity (Pacifici et al., 1995; Stefano et al., 2001) or cytokine production by immune cells (Singal and Singh, 2005), leading to alteration in host susceptibility to infections. Treatment with AG abrogated the protective effect of morphine in in vivo and in vitro models of TB indicating a role of NO in morphine-induced suppression of infection. The reduction in NO production, due to AG, leads to an increased susceptibility to infection. Morphine lacked direct activity against M. tuberculosis H37Rv in a concentration range of 1 × 10− 4 to 1 × 10− 9 M, thus

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providing evidence that morphine exerted its effects via the immune system and not due to direct activity against the bacteria. Also, the dose-dependent effect substantiates the role of immune system in host defense against the infection. On the basis of findings of this study, we conclude that low doses of morphine can modulate the immune system in such a way that bacterial load in target organs is lowered. Also, the preferential elimination of bacteria in the lungs of infected mice is of direct clinical relevance. Albeit preliminary, these findings open new vistas in the treatment of TB. We believe that in light of this preliminary report, newer opioid compounds may be developed that may be used alone or in combination with the existing antitubercular drugs, with the aim of treating multidrug resistant cases, shortening the duration of therapy and reducing the dose of presently available antitubercular drugs. Met-enkephalin, administered in combination with azidothymidine, provided enhanced protection from Friend leukemia virus in mice (Specter et al., 1994) and biphalin (a bivalent opioid analogue containing two tyrosine residues) has been reported to act synergistically with azidothymidine in inhibiting in vitro replication of Friend leukemia virus in Mus dunni cells (Tang et al., 1998). The results reported herein must be confirmed in higher animals before applying to humans. Further, the dose-dependent biphasic effect of morphine is not known in humans. This is because the data available on effects of opioids in humans is available from either drug addicts or patients receiving opioids (Vallejo et al., 2004). It is notable here that the dose(s) showing immunostimulation in animals (2.5–5 mg/kg in mouse (Singh et al., 1994) and hamster (Singal et al., 2003)) is lower than addictive dose (Mamiya et al., 2004) and also administered acutely; such studies have not been reported in humans. Hence, due skepticism and caution should be exercised in extrapolating these results to humans. Acknowledgements We are grateful to Prof. P. Ramarao, Director, National Institute of Pharmaceutical Education and Research (NIPER), for his help and encouragement. We thank Mr. Vijay Kumar Misra for excellent technical assistance. This is NIPER communication No. 411. References Allison, A.C., Harington, J.S., Birbeck, M., 1966. An examination of the cytotoxic effects of silica on macrophages. The Journal of Experimental Medicine 124 (2), 141–154. Chan, J., Tanaka, K., Carroll, D., Flynn, J., Bloom, B.R., 1995. Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis. Infection and Immunity 63 (2), 736–740. Durante, A.J., Selwyn, P.A., O'Connor, P.G., 1998. Risk factors for and knowledge of Mycobacterium tuberculosis infection among drug users in substance abuse treatment. Addiction 93 (9), 1393–1401. Fuggetta, M.P., Di Francesco, P., Falchetti, R., Cottarelli, A., Rossi, L., Tricarico, M., Lanzilli, G., 2005. Effect of morphine on cell-mediated immune responses of human lymphocytes against allogeneic malignant cells. Journal of Experimental & Clinical Cancer Research 24 (2), 255–263.

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