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.

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.

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.

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.

Down regulation of macrophage activation in Brugia pahangi-infected jirds (Meriones unguiculatus).

The macrophage is a major component of the inflammatory response induced by lymphatic tissue-dwelling filariae. Intraperitoneal (i.p.) infections with Brugia pahangi in Mongolian gerbils, or jirds (Meriones unguiculatus), induce a peritoneal inflammatory response characterized by accumulation of numerous macrophages and fewer eosinophils. This inflammatory response is associated with the release of microfilariae by female worms. The aim of this study was to investigate the activation state of the peritoneal macrophages during the course of i.p. infections with either male or female worms. Activation was determined by a toxoplasmacidal assay and assays which measured the production of tumor necrosis factor (TNF)-like activity and nitric oxide (NO) production. The development of these assays with jirds was initially conducted in parallel with the mouse system, which served as a positive control. Jird macrophages became activated to kill Toxoplasma gondii by in vivo immunization with Mycobacterium bovis BCG in a pattern similar to that of mouse macrophages. However, unlike the mouse system, supernatants from purified protein derivative- or concanavalin A-stimulated jird splenocytes plus lipopolysaccharide failed to activate jird macrophages in vitro or induce NO production. These results indicate that factors involved in jird macrophage activation may differ from those demonstrated in the mouse system and other systems. i.p. infections of 15 days in duration with either male or female worms induced macrophage activation as measured by Toxoplasma killing and TNF production. These responses decreased as the infection progressed to the chronic period on a time course that parallels the down regulation of experimental B. pahangi granulomas. There was no evidence of NO production by activated jird macrophages. These data indicate that macrophage function is down modulated during filarial infection and suggest that mechanisms involved in macrophage deactivation are related to those that induce down modulation of the systemic granulomatous inflammatory response in the jird. This response is not dependent on the microfilarial stage of the parasite and is also independent of mechanisms which induce peritoneal accumulations of macrophages.

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.

Comparison of the roles of reactive oxygen and nitrogen intermediates in the host response to Mycobacterium tuberculosis using transgenic mice.

OBJECTIVE: To study the role of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) in host response to Mycobacterium tuberculosis. DESIGN: M. tuberculosis infection (i.v.) was compared in B6 control and two strains of knockout (KO) mice. X-CGD mice with a nonfunctional allele for the gp91phox subunit of the phagocyte oxidase cytochrome b are unable to produce ROI whereas iNOS KO mice lack a functional inducible nitric oxide synthase (iNOS) gene and fail to make RNI. RESULTS: M. tuberculosis growth was markedly enhanced in the lungs of X-CGD mice compared to B6 mice, but was controlled in the spleen and liver. In iNOS KO mice, M. tuberculosis growth was exacerbated in the spleen, but was unremarkable in the lungs compared to B6 mice until later (Day 60) in the infection. In vitro, X-CGD alveolar and peritoneal macrophages (M phi) produced no ROI, but did produce RNI and inhibited growth of M. tuberculosis when activated with interferon gamma. iNOS KO M phi produced ROI, but failed to produce RNI and could not cope with M. tuberculosis in vitro when activated. The inhibition of M. tuberculosis growth observed in activated B6 and X-CGD M phi) was reversed in the presence of aminoguanidine. CONCLUSION: These KO mouse strains demonstrate the relative potent effects of ROI and RNI in resistance to M. tuberculosis and should prove useful for the study of regulatory and compensatory mechanisms of immunity.

Differences in the kinetics of T cell accumulations in C3H/HeN (Bcg-resistant) and C57BL/6 (Bcg-susceptible) mice infected with Mycobacterium paratuberculosis.

The accumulation of various T cell subsets in Bcg-susceptible (C57BL/6) and- resistant (C3H/HeN) strains of mice were compared following an intraperitoneal infection with Mycobacterium paratuberculosis. Groups of mice from both strains were killed at 3, 5, 10, 15, 30, and 150 days after infection and lymphocytes were harvested from the peritoneal exudate cells (PEC), spleen, intestinal epithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), Peyer's patches, and mesenteric lymph node (MLN) and labelled with monoclonal antibodies to CD3, CD4, CD8, gamma delta TCR, CD25, and CD44 for flow cytometric analysis. Uninfected C3H/HeN mice had higher proportions of CD4+ cells in the spleen, MLN, LPL, IEL, and Peyer's patches, while uninfected C57BL/6 mice had higher proportions of CD8+ and/or gamma delta T cells. Significant increases in accumulation of CD8+ and gamma delta T cells were detected in the peritoneum and other tissues in both strains of mice after infection. Higher CD4/CD8 ratios were observed in most lymphoid tissues of C3H/HeN mice, while increased proportions of CD8+ and/or gamma delta T cells were present in C57BL/6 mice. These results indicate that significant differences in T cell profiles exist between these two strains of mice, both inherently and in response to infection with M. paratuberculosis. Innately lower levels of CD4+ cells and/or higher percentages of CD8+ and gamma delta T cells may play a role in the increased suspectibility of C57BL/6 mice to infection with M. paratuberculosis.

Comparison of the resistance of C57BL/6 and C3H/He mice to infection with Mycobacterium paratuberculosis.

Susceptibility of C57BL/6 (Bcgs) and C3H/HeN (Bcgr) mice to an intraperitoneal infection with Mycobacterium paratuberculosis strain 19698 was compared (by histopathology and the number of mycobacteria isolated from the spleen). Mycobacterial counts from the spleen of Bcgr mice progressively decreased over the course of infection but remained unchanged in Bcgs mice. Granulomatous lesions and acid-fast bacteria were consistently present in the liver and lymph nodes of Bcgs mice, whereas lesions were transient or absent in Bcgr mice. These results indicate that Bcgr mice are inherently resistant to M. paratuberculosis, whereas Bcgs mice are inherently susceptible. These differences may prove useful in elucidating the mechanisms of resistance and susceptibility to paratuberculosis and other mycobacterial infections.

Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis.

The susceptibility of C57BL/6 mice to oral inoculation with Myobacterium paratuberculosis was evaluated histopathologically. Granulomatous lesions containing acid-fast bacteria developed in the mesenteric lymph nodes in over 50% of the mice by 11 months after inoculation. The results suggest that C57BL/6 mice may be useful for studying infection, pathogenesis, and other aspects of paratuberculosis.

Exacerbation of acute and chronic murine tuberculosis by administration of a tumor necrosis factor receptor-expressing adenovirus.

Tumor necrosis factor (TNF) plays a pivotal role in inflammatory phenomena that culminate in either pathogenesis or resistance in mycobacterial disease. The regulatory role of TNF in murine tuberculosis was examined by administering a recombinant adenovirus encoding a fusion protein consisting of the human 55-kDa TNF receptor extracellular domain and the mouse IgG heavy chain domain (AdTNFR). During acute infections with Mycobacterium tuberculosis, AdTNFR pretreatment induced elevated mycobacterial burdens of 1 log10 in the tissues of H37Ra-infected mice and 2 log10 (spleen and liver) and 4 log10 (lungs) in H37Rv-infected mice. In mice infected chronically with H37Rv, AdTNFR treatment induced a 3-log10 increase of M. tuberculosis in the lungs, in which a tuberculous bronchopneumonia developed with numerous acid-fast bacilli visible in alveoli and bronchi. Administration of AdTNFR may serve as a useful model for studying the pathogenesis and chemotherapy of progressive primary tuberculosis.

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.