Expression and characterization of recombinant interferon gamma (IFN-gamma) from the nine-banded armadillo (Dasypus novemcinctus) and its effect on Mycobacterium leprae-infected macrophages.

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.

The continuing challenges of leprosy.

Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases.

The continuing challenges of leprosy

Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases.

Viable M. leprae as a research reagent.

Mycobacterium leprae remain a rare research resource. They cannot be cultivated on artificial media, and the only established means to quantify viability of M. leprae has been by its relative growth in the foot pads of conventional mice (MFP). The MFP method is technically difficult and requires several months to yield results. More effective methods are needed. We examined the association between M. leprae's ability to oxidize 14C-palmitate in axenic culture and the MFP growth results of a large number of suspensions. Oxidative activity was assessed by radiorespirometry (RR) using the Buddemeyer-type biphasic culture vessels containing 7H12 liquid medium and 14C-palmitate, or with commercially prepared BACTEC 12B vessels containing the same medium. The RR results were highly correlated (r = 0.71) with the growth level that each M. leprae suspension achieved by the MFP technique. In using this technique to examine the effects that many common laboratory practices have on M. leprae viability, we found that viability varies markedly between bacillary suspensions derived from different hosts and tissues. The highest viabilities were obtained with bacilli from moderately enlarged nude MFP (< 1 g). Viability tended to be lower among very large nude MFP or long-duration infections and from armadillo tissues. After their harvest from host tissues, leprosy bacilli lost viability quickly. Suspensions stored in 7H12 liquid medium retained < 1% of their viability within 3 weeks of harvest, and freezing bacillary preparations or incubating them at 37 degrees C resulted in nearly an immediate equivalent loss in metabolic activity and viability. M. leprae viability is maintained best when bacilli are stored for only short periods of time at 4 degrees C-33 degrees C. Palmitate oxidation is a rapid, reliable and objective means by which to estimate the viability of M. leprae and can be used effectively as a surrogate for the conventional MFP technique in many studies.

Role of inducible nitric oxide synthase in resistance to Mycobacterium leprae in mice.

The manifestation of leprosy in humans is largely determined by host immunity to Mycobacterium leprae and is a model for immunoregulation in a human disease. However, animal models available for exploration of the leprosy spectrum are inadequate. This study explored M. leprae infection in mice deficient in inducible nitric oxide synthase, and this report describes elements resembling borderline tuberculoid leprosy in humans.

Inhibition of metabolism and growth of Mycobacterium leprae by gamma irradiation.

Mycobacterium leprae is uncultivable on artificial medium, but viability can be maintained without multiplication for a limited time in vitro. In this study, we evaluated gamma-irradiation (gamma-irr) as a means to kill this slowly growing organism. Freshly harvested, viable, athymic, nu/nu mouse-derived M. leprae were exposed to varying doses of gamma-irr from a 60Co source. Two indicators of bacterial viability were determined: metabolism, measured by oxidation of 14C-palmitic acid to 14CO2 in the BACTEC 460 system, and multiplication, measured by titration in the mouse foot pad. gamma-Irr of both M. leprae and M. lufu, a cultivable control mycobacterium, resulted in a dose-dependent inhibition of viability. gamma-Irr of up to 10(3) rad had little effect on the metabolic activity of either organism. For M. leprae, 10(4)-10(5) rad caused an intermediate inhibitory effect; whereas 10(6) rad yielded almost total inhibition. In the mouse foot pad assay, up to 10(4) rad had little effect on M. leprae growth; however, 10(5) rad resulted in at least a 2-log reduction in the number of bacilli recovered and no M. leprae growth was measurable after exposure to 10(6) rad. With M. lufu, 10(5) rad inhibited metabolic activity by 99% and caused > or = 2-log reduction in the number of colony forming units (CFU). No CFU of M. lufu were recovered after exposure to 10(6) rad. Scanning electron microscopy revealed the presence of some aberrant protrusions on the cell surface of lethally irradiated M. leprae; whereas boiling and autoclaving caused obvious morphological denaturation. These data suggest that gamma-irr is an effective way to kill M. leprae without causing extensive damage to the cell architecture. Killing M. leprae by gamma-irr may be preferable when comparing cellular responses to live versus dead bacilli in vitro and in vivo.

Single lesion paucibacillary leprosy: baseline profile of the Brazilian Multicenter Cohort Study.

In Brazil, there is little information about the clinical and epidemiological characteristics of paucibacillary, single skin lesion leprosy patients (SSL-PB). Only recently has the official notification system distinguished leprosy patients with a single lesion as a clinical entity, for whom the single-dose ROM (rifampin, ofloxacin and minocycline) regimen has been recommended. In this paper, we describe the baseline clinical features and the immunological background of a multicenter cohort of SSL-PB leprosy cases enrolled between December 1997-1998. Patients were recruited at health centers located in the following regions: Southeast = Rio de Janeiro; North = Amazon and Rondônia states and Center-West = Goiás state. Eligible cases were newly detected, untreated single-lesion leprosy patients without thickened nerve involvement, and were assessed by clinical, bacilloscopic and histopathological exams. The Mitsuda skin test and anti-PGL-I serology (ELISA) were also performed. Of the 299 SSL-PB leprosy patients, 259 (86.6%) fulfilled the criteria for single-dose ROM intervention. Our results showed that patients recruited from different sites had similar features, considering the clinical and immunological profiles. There was a predominance of adults (mean age 32.4; S.D. = 16.0), and a BCG scar was detected in 76.7% of the children (< or = 15 years old). Only 7 cases were diagnosed as the multibacillary type, representing less than 3% of the patients being misclassified. Our data indicate that in Brazil SSL-PB case ascertainment based on clinical and bacilloscopic criteria can be accurately defined under a routine control program; 75.0% of SSL-PB cases were Mitsuda positive (> or = 5 mm) and seropositivity for anti-PGL-I was detected in 17.3% of the patients. These data are compatible with effective cell-mediated immunity and low bacillary load, suggesting favorable clinical outcomes for most SSL-PB participants of this cohort.

Causative organism and host response. International Leprosy Congress, Beijing, 7-12 September 1998. Workshop report.

Whether or not the leprosy elimination target is met in all endemic countries by the year 2000, the MDT programme will have greatly reduced worldwide prevalence. However, our workshop chairmen were asked to ignore the prevalence-based leprosy 'elimination' programme and focus on recommendations for a long term, incidence-based eradication target where transmission is blocked. They were asked to be concerned with basic leprosy research goals in the post 2000 era. The members of our workshops are actively productive workers, committed to their special interests. They are fully cognizant of the obstacles faced daily in working with leprosy and M. leprae, the requirement for clever experimental design even with the availability of the powerful tools of molecular biology which can now be brought to bear on some of the research obstacles. They are also aware of our lack of understanding about leprosy and M. leprae. How do you block transmission if you don't know how infection is transmitted? Can infection be detected, diagnosis made earlier? Is there a non-human reservoir host, a carrier state, an environmental source? What is the basis of M. leprae's predilection for nerves, the mechanisms underlying reactions? What needs to be targeted to treat reactions? Can a vaccine play a role? There is nothing startling in the workshops' recommendations. Other individuals and groups of experts have made the same suggestions, with slightly varying priorities. What one can read between the lines of these reports, is a sense of urgency to get as much done as soon as possible. Worldwide interest in leprosy will soon be diminished, not by design but as a consequence of the laudable success of the MDT programme. The experiment is still underway, but chemotherapy alone, killing bacilli in the detectable human host, does not appear to be the answer to blocking transmission. A number of goals must be addressed while there are still intact national and international leprosy programmes, while there are still leprosy treatment and research centres that can co-ordinate and facilitate the necessary trials for early diagnosis, early detection of reactions, evaluation of immunosuppressive regimens for reactions. A key recommendation is concerned with the means of measuring progress. A clear and explicit means of reporting incidence, prevalence and 'case detection' should be implemented to avoid a distorted picture of worldwide leprosy. These recommendations are non-controversial. What should be done is clear. The uncertainty is in determining who will do the work. Who will fund the laboratories engaged in this work? Look around you. There are fewer scientists attending this Congress but browsing the abstracts and attending our sessions and posters clearly revealed to me that fewer of us are doing far better work than in the past. Alternative sources of funding will help. Tuberculosis research is enticing researchers away from leprosy in the developed countries but is visibly sustaining leprosy research in many centres in developing countries. Formation of alliances was a key goal of this Congress. I asked my colleagues from Carville to identify in their own discipline, dedicated people, committed laboratories that will sustain their leprosy research efforts over the next 5, 10 or more years. These are the people with whom we wish to collaborate, form alliances, share resources and expertise, address the future of worldwide leprosy.

Effective treatment of acute and chronic murine tuberculosis with liposome-encapsulated clofazimine.

The therapeutic efficacy of liposomal clofazimine (L-CLF) was studied in mice infected with Mycobacterium tuberculosis Erdman. Groups of mice were treated with either free clofazimine (F-CLF), L-CLF, or empty liposomes twice a week for five treatments beginning on day 1 (acute), day 21 (established), or day 90 (chronic) postinfection. One day after the last treatment, the numbers of CFU of M. tuberculosis in the spleen, liver, and lungs were determined. F-CLF at the maximum tolerated dose of 5 mg/kg of body weight was ineffective; however, 10-fold-higher doses of L-CLF demonstrated a dose response with significant CFU reduction in all tissues without any toxic effects. In acutely infected mice, 50 mg of L-CLF/kg reduced CFU 2 to 3 log units in all three organs. In established or chronic infection, treated mice showed no detectable CFU in the spleen or liver and 1- to 2-log-unit reduction in the lungs. A second series of L-CLF treatments cleared M. tuberculosis in all three tissues. L-CLF appears to be bactericidal in the liver and spleen, which remained negative for M. tuberculosis growth for 2 months. Thus, L-CLF could be useful in the treatment of tuberculosis.

Leprosy.

Leprosy is an ancient disease which is still poorly understood and often feared by the general public and even by some healthcare professionals. Fortunately, improvements in the management of leprosy over the past three decades have diminished the stigma and greatly altered the outlook for patients. Public understanding of the disease has benefited from WHO's goal of eliminating leprosy as a public health problem by the year 2000. Unfortunately that goal has also led many to believe that leprosy has been or will soon be eradicated. This will not happen in the near future because, despite a fall in registered cases, the incidence of the disease has changed very little, and eradication of a bacterial infectious disease such as this is unlikely with chemotherapy alone. Nevertheless, as a result of the WHO's efforts, patients nearly everywhere should have access to care, and the incidence may begin to diminish if adequate control efforts are maintained beyond the year 2000. Given the mobility of patients today a physician anywhere may occasionally see a case or be asked about the disease so a basic understanding of leprosy and its management should prove useful.

Effects of essential fatty acid deficiency on prostaglandin E2 production and cell-mediated immunity in a mouse model of leprosy.

Results from animal and in vitro studies suggest that essential fatty acid (EFA) deficiency enhances cell-mediated immunity by reducing production of prostaglandins with immunosuppressive actions. However, direct experimental evidence that EFA deficiency enhances T-lymphocyte function in vivo has not been obtained. In this study, athymic (nu/nu) mice were infected in the footpads with Mycobacterium leprae and fed a linoleic acid-free diet. These mice, and infected nu/nu mice on control diets, were given an adoptive transfer of M. leprae-primed, T-cell-enriched lymphocytes. After 2 weeks, M. leprae bacilli were harvested from the recipient mice and bacterial viability was determined by the BACTEC system. M. leprae recovered from recipient mice fed control diets displayed little reduction in metabolic activity. In contrast, M. leprae from recipient mice fed the EFA-deficient (EFAD) diet exhibited markedly reduced viability. In vitro, donor cells from M. leprae-primed mice secreted elevated levels of gamma interferon upon exposure to the bacilli. These cells also exhibited an enhanced proliferative response, which was reduced by exogenous prostaglandin E2 (PGE2). In addition, M. leprae-infected granuloma macrophages (Mphi) from EFAD recipient nu/nu mice secreted significantly less PGE2 than granuloma Mphi from mice on control diets. These data suggest that enhanced levels of Mphi-generated PGE2, induced by M. leprae or its constituents, could act as an endogenous negative modulator of the immune response occurring in the microenvironment of the lepromatous granuloma.

Hydrolysis of thalidomide abrogates its ability to enhance mononuclear cell synthesis of IL-2 as well as its ability to suppress the synthesis of TNF-alpha.

Thalidomide is effective in the treatment of inflammatory conditions like erythema nodosum leprosum in leprosy patients, and aphthous ulcers in AIDS patients. Its mechanism of action is uncertain and reports of its effect on the synthesis of inflammatory cytokines such as IL-2 and TNF-alpha are contradictory. As thalidomide is labile to spontaneous hydrolysis at pH 7.4, studies were carried out to explore the effects of deliberate hydrolysis or the ability of thalidomide to modulated cytokine production by human mononuclear cells stimulated in vitro with Staphylococcal enterotoxin A (SEA)(IL-2) or lipopolysaccharide from Salmonella minnesota (LPS)(TNF-alpha). Unhydrolyzed thalidomide at 4.0 micrograms/ml consistently enhanced the synthesis of IL-2 in SEA-stimulated cells, and suppressed the synthesis of TNF-alpha in LPS-stimulated cells; whereas, hydrolyzed thalidomide had no enhancing effect on SEA stimulated-cell synthesis of IL-2 or suppressive effect on LPS stimulated-cell synthesis of TNF-alpha. These findings demonstrate that thalidomide's ability in vitro to enhance IL-2 and to suppress TNF-alpha in stimulated cells is dependent on the intact molecule and underscore the necessity to employ thalidomide under appropriate physicochemical conditions.

Public health applications of Hansen's disease research and treatment.

Because of the similarities in causative agents of Hansen's disease and tuberculosis, Hansen's disease research is now being used in the identification, treatment, and prevention of tuberculosis. Numerous studies are under way to screen and develop new drugs to combat the threat of multiple drug-resistant tuberculosis. Additional studies focus on factors to reduce the transmission of tuberculosis and on the development of techniques for early diagnosis and identification of drug resistance. Advances in Hansen's disease research and treatment also are being applied to the prevention of ulcers and amputations in diabetics and others without protective sensation in their feet. The Lower Extremity Amputation Prevention Program, developed at the Gillis W. Long Hansen's Disease Center in Carville, LA, is a multidisciplinary approach that includes screening, risk assessment, and the development of a treatment plan with an emphasis on patient involvement. Expected to prevent up to 90 percent of diabetes-related amputations, the program is being implemented in Jackson, MS, in a community-based diabetic foot program and will be replicated throughout the United States.

Regulation of murine macrophage effector functions by lipoarabinomannan from mycobacterial strains with different degrees of virulence.

Lipoarabinomannan (LAM) is the major arabinose- and mannose-containing phosphorylated lipopolysaccharide (LPS) in mycobacterial cell walls. LAM preparations from a virulent strain (Erdman) (LAM(Erdman)) and an attenuated strain (H37Ra) (LAMH37Ra) of Mycobacterium tuberculosis, as well as from M. leprae (a virulent mycobacterium), were analyzed for their effects on various macrophage (M phi) effector functions. LAMH37Ra, like gram-negative LPS, exhibited a dose-dependent ability to induce tumor necrosis factor alpha (TNF-alpha) production in normal M phi, and gamma interferon (IFN-gamma) priming of the M phi greatly augmented the levels of TNF-alpha. However, the effects of LAMH37Ra were unaffected by polymyxin B, which totally abrogated the effects of LPS. LAM(Erdman) and LAM from M. leprae, on the other hand, induced virtually no TNF-alpha production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of TNF-alpha mRNA induced by the various preparations correlated with the levels of TNF-alpha protein detected. Interestingly, both LAMH37Ra and LAM(Erdman) could block subsequent IFN-gamma- and LPS-induced M phi activation, a previously reported measure of the potent ability of LAM to down-regulate M phi effector functions. Two lines of evidence suggested, however, that M phi cyclooxygenase products did not play a role in this down-regulation. LAMH37Ra and LPS could induce the production of NO2- in both normal and IFN-gamma-primed M phi, whereas LAM(Erdman) could stimulate NO2- production only in primed M phi. Both LAMH37Ra and LAM(Erdman) could substitute for LPS as a triggering signal for IFN-gamma-primed M phi in a toxoplasma killing assay. The triggering ability of LAM(Erdman), however, was abrogated by an anti-TNF-alpha antibody, suggesting that sufficient TNF-alpha production was stimulated by LAM(Erdman) to drive a M phi function relevant in host resistance. Thus, mycobacterial LAM is a potent regulator of M phi functions, a fact that may have important consequences in mycobacterial disease.

L-arginine-dependent macrophage effector functions inhibit metabolic activity of Mycobacterium leprae.

Recently, L-arginine has been shown to be a necessary substrate for murine-activated macrophage-mediated tumor cytostasis and microbiostasis of certain fungi, bacteria, and intracellular protozoa. We report here the effects of the L-arginine-dependent pathway of activated mouse macrophages (MO) on the obligate intracellular prokaryote, Mycobacterium leprae. Due to the inability to culture M. leprae in vitro, a simple, quantitative assay was employed to measure the metabolism/viability of M. leprae released from MO: the metabolic capacity of M. leprae to oxidize 14C-palmitic acid to 14CO2. Murine normal MO or MO activated in vitro with IFN-gamma or in vivo by injection with Corynebacterium parvum were infected with viable M. leprae freshly harvested from the footpads of nu/nu mice. Activated MO strikingly inhibited the metabolism of M. leprae; however, in L-arginine-free medium or in medium containing L-arginase, the inhibitory effects of activated MO on M. leprae metabolism were abolished. The competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, also blocked the inhibitory effects of activated MO for M. leprae, but the addition of supplemental L-arginine overcame the NG-monomethyl-L-arginine-induced block. Furthermore, in the culture supernatants, the levels of NO2-, an end product of L-arginine degradation, were directly proportional to the ability of the activated MO to inhibit M. leprae metabolism. These data present five lines of evidence that suggest that activated MO utilize the L-arginine-dependent pathway to cope with M. leprae.

Effects of activated macrophages on Mycobacterium leprae.

Five alternative methods were used to explore in vitro the effects of normal and activated murine macrophages on the metabolic well-being of intracellular Mycobacterium leprae: fluorescein diacetate-ethidium bromide staining, ATP content, synthesis of phenolic glycolipid 1, and two techniques to quantitate oxidation of palmitic acid. In relatively short-term experiments (7 to 10 days), each of these procedures provided strong evidence that activated macrophages exerted a deleterious effect on the leprosy bacillus. These findings appear to confirm the contention that activated macrophages underlie host resistance to clinical leprosy and limitation of M. leprae growth in paucibacillary leprosy.

Gamma interferon in experimental leprosy.

We have shown that preactivated MACs are able to cope with newly acquired leprosy bacilli, and a high intracellular burden of live M. leprae induces a refractory response of the host MAC to activation by IFN-gamma. Our studies underscore the fact that MAC function in LL is dependent on localized conditions, influenced by the high intracellular burden of leprosy bacilli and, in part, involving the production of prostanoids. These findings preclude inferences about the function of granuloma MAC s in leprosy based on the responses of MACs from other easily accessible anatomical compartments such as the peritoneal cavity or peripheral blood. We feel that the clearance of bacilli from the LL lesion as a consequence of local immunotherapeutic measures or chemotherapy likely depends on the influx of new competent MAC rather than the activation of resident lepromatous MACs.

Inhibition of interferon-gamma-mediated activation in mouse macrophages treated with lipoarabinomannan.

Lipoarabinomannan (LAM), purified from the cell walls of Mycobacterium leprae and M. tuberculosis, is a potent inhibitor of interferon-gamma (IFN-gamma) mediated activation of macrophages. The capability of LAM to inhibit IFN-gamma activation of macrophages in vitro was dose dependent and required a 24-h pre-exposure. Defective activation was evident as a block in IFN-gamma-induced cytocidal activity for tumour cell targets and microbicidal capacity for intracellular Toxoplasma gondii. Additionally, LAM treatment blocked the induction of surface Ia antigens on peritoneal macrophages by IFN-gamma. The requirement for pretreatment with LAM was further substantiated by the finding that peritoneal macrophages that were activated in vivo were not affected by LAM treatments and retained full microbicidal function. However, once inhibited by LAM treatment in vitro, macrophages remained fully refractory to IFN-gamma activation for up to 5 days in culture. Inhibition of IFN-gamma activation in macrophages treated with LAM was not overcome by 100-fold increases in the dose of IFN-gamma used or by a constant dose of IFN-gamma in combination with 100-fold increases in the level of endotoxin used to trigger cytotoxic activity. The defect in IFN-gamma unresponsiveness was not due to altered receptor function, as control and LAM-treated macrophages showed similar capacity to bind, internalize, and digest radiolabelled IFN-gamma. Based on the in vitro findings reported here, the inhibition of IFN-gamma-mediated macrophage activation by exposure to LAM may contribute to defective macrophage function observed in lepromatous granulomas and thus constitutes an important aspect of pathogenesis in mycobacterioses.