Use of serum antibody and lysozyme levels for diagnosis of leprosy and tuberculosis.

Active tuberculosis (TB) and leprosy are difficult to diagnose early because there are few organisms to detect and the specific immune response does not distinguish between active and inactive disease. We developed an immunoassay for lysozyme to see whether serum lysozyme levels could be used to identify individuals with clinical leprosy or TB. The immunoassay for lysozyme proved superior to standard enzyme assays that were less sensitive and reliable. The lysozyme assay was compared with assays for antibodies to Mycobacterium tuberculosis lipoarabinomannan (LAM) and M. leprae phenolic glycolipid-1. The sera tested were from Ethiopian leprosy (paucibacillary and multibacillary) and TB patients and from healthy Ethiopian and U.S. controls. The lysozyme assay was able to detect more of the individuals with TB (sensitivity, 100% for 19 patients) or leprosy (sensitivity, 86% for 36 patients) than either antibody assay. In particular, lysozyme levels were raised in a higher proportion of the paucibacillary leprosy patients (83% of 17), for whom the antibody assays were less sensitive; the LAM IgG and the phenolic glycolipid-1 IgM levels were raised in only 62 and 44% of 16 patients, respectively. The data suggest that lysozyme measurements may be useful in the diagnosis of mycobacterial infections and other chronic infectious granulomatoses.

The value of IgM antibodies to PGL-I in the diagnosis of leprosy.

An ELISA has been used to measure IgM antibodies to phenolic glycolipid-I (PGL-I) in previously undiagnosed patients who were suspected of leprosy on purely clinical grounds. The certainty of clinical diagnosis was classified as either "firm" or "indefinite." Leprosy was confirmed in 133 of 161 patients on the basis of positive slit-skin smears and/or skin and/or nerve histopathology. All 58 patients with multibacillary leprosy (BB, BL, or LL) were correctly diagnosed clinically, as were 50 of 54 patients (93%) with a firm diagnosis of BT or TT leprosy. The firm clinical diagnoses were more accurate than either the slit-skin smear or ELISA data. However, there were 44 patients (27% of total), designated "rule out leprosy" (RO), for whom the clinical diagnosis was indefinite. The clinical suspicion of leprosy (RO) was correct in only 24 (55%) of these patients who had BT leprosy. The slit-skin smears were positive in only 20% of these patients compared to 50% for the ELISA. It was concluded that the PGL-I IgM ELISA may have its greatest diagnostic confirmatory value in paucibacillary disease because paucibacillary leprosy comprises the major source of clinical diagnostic difficulty.

Urinary phenolic glycolipid 1 in the diagnosis and management of leprosy.

A simplified assay to measure the phenolic glycolipid 1 (PGL-1) of Mycobacterium leprae in the urine was applied to the diagnosis of leprosy and the monitoring of antileprosy chemotherapy. One hundred seventy-nine previously untreated patients and 25 normal controls were tested. The specificity of the assay was 100%. There were no false-positive results. The sensitivity of the assay varied with the type of leprosy from 92% for lepromatous leprosy to 56% for borderline lepromatous and 18% for borderline tuberculoid patients. After the onset of chemotherapy in lepromatous leprosy patients, there was often a transient increase of urinary PGL-1, followed by a steady decline. Within 3 months of multiple drug therapy, urinary PGL-1 levels were reduced by 90%-99% and were often undetectable. This assay appears to have considerable potential for monitoring chemotherapy and detecting treatment failure and relapse in patients with Hansen's disease.

Anti-neural antibodies in leprosy sera: further characterization of the antigens.

Sera or plasmas from 129 leprosy patients were tested by immunoblotting for antibodies that bound to proteins in a Triton-insoluble fraction enriched in neural intermediate filaments (IF fraction) from human or bovine spinal cord. Sixty samples (47%) showed positive staining of proteins at 35 kDa, 42 kDa or both. The presence of these antibodies appeared to be evenly distributed across the spectrum of disease. The frequency of these antibodies in samples from 12 healthy Ethiopians was similar to that in the leprosy group. Similar antibodies were found in only three of 28 samples from U.S. patients with neurologic diseases and in seven of 35 normal U.S. sera. Sera from U.S. tuberculosis patients stained multiple bands in the 50-30 kDa region of the blots; 11 of 16 stained bands corresponding to the 35 kDa or 42 kDa bands along with a number of other bands in this region. The 35 kDa and 42 kDa antigens do not appear to be breakdown products of neural filaments or glial fibrillary acidic protein, since antibodies to these proteins do not react with the 35 kDa or 42 kDa antigen. Further, the staining pattern with the leprosy sera is unchanged following Ca2+-mediated proteolysis of the IF-enriched fraction. The two antigens differ from one another in isoelectric point: the pI of the 35 kDa antigen is 5.9, and the pI of the 42 kDa antigen is 4.8. Staining of the immunoblots with antibodies against a number of known neural antigens failed to identify the 35 kDa and 42 kDa antigens. The 42 kDa antigen appears to be a component of axolemma, since 42 kDa-positive leprosy sera stained a protein with identical migration in preparations of bovine peripheral nervous system and human central nervous system axolemma. In some sera, antibodies reacting with the 35 kDa antigen were adsorbed by D-O bovine serum albumin, a synthetic analogue of the terminal disaccharide portion of the phenolic glycolipid 1 of Mycobacterium leprae. Antibodies to the 42 kDa antigen were not removed by this treatment.

Methods for the detection of a specific Mycobacterium leprae antigen in the urine of leprosy patients.

Two methods for detecting the phenolic glycolipid, PGL-1, a Mycobacterium leprae-specific molecule, in the urine of leprosy patients are described. Both methods rely on the 100-fold preconcentration of the urine, which can be accomplished by a single-step ultrafiltration procedure. The equivalent of approximately 2.5 micrograms of PGL-1/ml was detected in the urine of LL patients with an inhibition ELISA. The second method, a direct dot-blot assay on nitrocellulose paper, was much simpler and more sensitive. As little as 3 ng of antigen was detected by the dot-blot technique. PGL-1 was detected in the urine of LL patients.

Characterization of the anti-neural antibodies in the sera of leprosy patients.

Sera from 43 leprosy patients were tested for antibodies that bound to normal human nerve. Thirty-eight percent showed positive staining as demonstrated by indirect immunofluorescence. Only 1 out of 30 control sera tested displayed similar staining. Western blots of myelin and neural intermediate filament (IF) proteins were tested with patient sera. Two of the anti-neural antibody (ANeAb)-positive leprosy sera bound to the P0 protein of PNS myelin. All 17 ANeAb-positive leprosy sera displayed 2 or more bands in the molecular weight range of Mr 45 000-55 000, when tested against IF proteins. One explanation for these findings is that leprosy patients produce antibodies to intermediate filament (IF) proteins released subsequent to the bacterial invasion of the peripheral nerves. The importance of these autoantibodies in the pathogenesis of leprosy is discussed.

Mycobacterium lepraemurium infection of nude athymic (nu/nu) mice.

Nude athymic (nu/nu) mice on a BALB/c background and their heterozygous euthymic litter mates (nu/+) were infected with either 10(8) or 10(6) Mycobacterium lepraemurium organisms intravenously or in the left hind footpad (LHF). After LHF infection with 10(8) M. lepraemurium organisms, nu/+ mice slowly developed a response that consisted of LHF swelling and local resistance to Listeria monocytogenes. The lower inoculum induced a proportionately lower response in nu/+ mice, but the nu/nu mice developed neither LHF swelling nor resistance to L. monocytogenes in response to either dose of M. lepraemurium. Counts of M. lepraemurium in the LHF revealed no difference between the nu/+ mice and nu/nu mice. After intravenous infection the nu/+ mice developed splenomegaly, but did not otherwise differ from nu/nu mice with respect to resistance to intravenous challenge with L. monocytogenes or growth of M. lepraemurium in the spleen. In light of the poor responsiveness of nu/+ mice in this experiment, they were then compared with CB6 and B6D2 mice, which are genetically susceptible and resistant to M. lepraemurium, respectively. These mice were infected with either 10(8) or 10(6) M. lepraemurium cells or 10(6) Mycobacterium bovis BCG cells in the LHF. Once again the nu/+ mice responded poorly to M. lepraemurium, the CB6 mice responded very strongly, and the B6D2 mice gave an intermediate response with respect to LHF swelling and resistance to L. monocytogenes. However, M. lepraemurium grew to higher numbers in the LHF of nu/+ and CB6 mice than in B6D2 mice, revealing, in CB6 mice, a dissociation between resistance to L. monocytogenes and M. lepraemurium. All three mouse strains responded strongly to M. bovis BCG, but there was a suggestion that nu/+ mice might be more susceptible to this agent than the other two strains. I concluded that the failure of nu/+ mice to restrict the growth of M. lepraemurium more than nu/nu mice was due to the intrinsic genetic susceptibility of both types of mice. In effect, the nu/+ mice behaved like nu/nu mice, as if they too were deficient in T lymphocytes that were responsive to M. lepraemurium.

Suppression of adoptive antituberculosis immunity by normal recipient animals.

Adoptive immunity is poorly expressed in normal syngeneic mice. This phenomenon was studied by using experimental antituberculosis immunity as a model system representing pure cell-mediated immunity. Expression of adoptive immunity was facilitated by pretreating recipients with sublethal ionizing radiation (500 rads) or high doses (200 mg/kg) of cyclophosphamide or by using adult thymectomized, lethally irradiated, bone-marrow-reconstituted (TXB) mice. Adult thymectomy was less effective, and a low dose of cyclophosphamide (20 mg/kg) was completely ineffective. The beneficial effect of sublethal irradiation was reduced over time; it persisted for 4 weeks and was absent after 8 weeks. Attempts to restore the suppressed state of normal mice to sublethally irradiated mice by using normal spleen or thymus cells did not succeed. Even in rats, which express adoptive antituberculosis immunity without immunosuppressive treatment, the use of sublethally irradiated or TXB recipients potentiated adoptive immunity. It was concluded that suppression of adoptive immunization in normal recipient mice is mediated predominantly, if not exclusively, by T lymphocytes that are sensitive to a number of immunosuppressive agents. The suppressor cells are long-lived and can be regenerated from precursors that are resistant to 500 but not to 900 rads of ionizing radiation.

Induction and suppression of cross-reactive antituberculosis immunity after Mycobacterium lepraemurium infection of mice.

Mice immunized with 10(8) live Mycobacterium lepraemurium in the footpad showed increased resistance to infection with BCG or M. tuberculosis R1Rv. This resistance could be transferred adoptively with lymphoid cells, signifying that the immunity was cross-reactive rather than nonspecific. Adoptive cross-reactive immunity to M. tuberculosis was also conferred by spleen cells from mice immunized with large doses of living or dead M. lepraemurium intravenously, a route of immunization that suppresses the induction of cell-mediated immunity to that organism. The presence of specific suppressor activity was sought in mice immunized intravenously with M. lepraemurium. It was found that mice preimmunized intravenously with living or dead M. lepraemurium and then infected with BCG did not confer levels of adoptive antituberculosis immunity as high as those conferred by mice immunized with BCG alone. Similarly, a mixture of BCG-sensitized and M. lepraemurium-sensitized cells did not convey as much immunity as BCG-sensitized cells alone, signifying suppression of the effector lymphocytes.

Properties of peritoneal exudate lymphocytes that mediate tuberculin delayed-type hypersensitivity and anti-tuberculosis immunity. I. The effect of cytotoxic agents.

Acute peritoneal exudates were raised in rats that had been immunized by BCG, in order to draw into the peritoneal cavity sensitized immunoblasts. The peritoneal exudate cells (PEC) were harvested 24 h after exudate induction. The ability of these cells to confer tuberculin delayed-type hypersensitivity (DTH) and anti-tuberculosis immunity upon syngeneic recipients was examined after treatment with various cytotoxic agents. The transfer of DTH and immunity was entirely unaffected by in vitro exposure of PEC to high specific activity [3H]-thymidine, 5-bromodeoxyuridine or a low dose of mitomycin C. agents that adversely affect DNA-synthetic cells. Treatment of immunized rats with vinblastine reduced the number of PEC, but the cells were able to transfer DTH and immunity. In vitro exposure of PEC to vinblastine ablated the transfer of DTH but spared immunity. A moderate dose of mitomycin C suppressed immunity but not DTH; but a high dose abolished both expressions of cell-mediated immunity. The transfer of immunity was highly susceptible to ionizing radiation whereas DTH was resistant. These results suggest that the lymphocytes mediating DTH and anti-tuberculosis immunity are not actively replicating cells.

Properties of peritoneal exudate lymphocytes that mediate tuberculin delayed-type hypersensitivity and anti-tuberculosis immunity.

Rats were immunized with living BCG and acute peritoneal exudates were induced on the ninth day of infection. The peritoneal exudate cells (PEC), that confer adoptive anti-tuberculosis immunity and tuberculin delayed-type hypersensitivity (DTH), were subject to velocity sedimentation analysis. It was found that the ability to confer immunity of DTH was limited to a population of cells that sedimented at a rate of 3-4 mm/h. This sedimentation rate corresponds to that of small lymphocytes. No significant immunological activity was detected in large lymphocytes that incorporate [3H]-thymidine in vitro, regardless of whether the exudates were obtained 14 to 24 h after induction of peritoneal inflammation. The failure of large lymphocytes to confer immunity and DTH was not due to adherent cells with suppressor activity, because removal of adherent cells failed to amplify the transfer of immunological activity by non-adherent cells. The persistence of the ability to express immunity and DTH in adoptively immunized rats was studied. There was no decay of adoptive immunity during a 4 week period following cell transfer, but there was a rapid reduction in the expression of DTH.

Effect of Mycobacterium bovis BCG vaccination upon Mycobacterium lepraemurium infection.

Mice were infected with 10(8) Mycobacterium lepraemurium in the footpad (unsuppressed mice), and some of these animals were concurrently given 10(9) heat-killed M. lepraemurium intravenously (suppressed mice). These groups of mice were preimmunized with 10(7) viable organisms of Mycobacterium bovis BCG by several routes. BCG inhibited the proliferation of M. lepraemurium in the unsuppressed mice, but not in the suppressed mice. In effect, the intravenous administration of heat-killed M. lepraemurium suppressed the immunity to M. lepraemurium that BCG vaccination had engendered. BCG did not protect normal mice against intravenous infection with M. lepraemurium. It appears that normal mice against intravenous infection with M. lepraemurium. It appears that the inhibitory effect of BCG vaccination upon M. lepraemurium infection is due to cross-reactive immunity rather than to nonspecific immunity or immunopotentiation. Thus, the route of BCG vaccination was immaterial, and vaccination 12 weeks before M. lepraemurium infection was as beneficial as vaccination 4 weeks before infection. Moreover, spleen cells from M. lepraemurium-immunized mice conferred adoptive immunity to BCG. The implications of this study for the use of BCG as a prophylactic and therapeutic agent in human leprosy are discussed.

Macrophage activation and resistance to pulmonary tuberculosis.

Mice were vaccinated with 300 micrograms of BCG cell walls (BCG-CW) in oil-in-water emulsion intravenously or with a high or low dose of living BCG by inhalation (BCG-HD or BCG-LD, respectively). The consequences of vaccination were evaluated in terms of the growth of BCG in the lungs and spleen, lung and spleen weight, resistance to intravenous and airborne challenge with Listeria monocytogenes, airborne challenge with virulent Mycobacterium tuberculosis H37Rv, and transfer of adoptive immunity. BCG-CW and BCG-HD mice developed increased lung weight, which was associated with transiet, low-level resistance to airborne L. monocytogenes and initial resistance to airborne H37Rv. Only BCG-CW mice developed splenomegaly, which was accompanied by high resistance to intravenous challenge with L. monocytogenes. The initial resistance of BCG-CW mice to H37Rv was not sustained, whereas that of BCG-HD mice persisted. There was no initial resistance to H37Rv in BCG-LD mice, but immunity was generated later. Overall, BCG-HD mice were most resistant to H37Rv, and BCG-CW and BCG-LD mice were less but equally resistant.

Listeria pneumonitis: influence of route of immunization on resistance to airborne infection.

Mice that are immunized with an airborne inoculum of BCG are more highly resistant to airborne challenge with Mycobacterium tuberculosis than are mice that are immunized by the subcutaneous or intravenous route. To discover whether this phenomenon is peculiar to tuberculosis, we studied the influence of the route of immunization upon pulmonary resistance in Listeria monocytogenes infection. Mice were immunized by the airborne, intravenous, or footpad route and were subsequently challenged by the same route at 1 to 4 weeks after immunization. Mice were highly and uniformly resistant to intravenous challenge, regardless of the route of immunization. The route of immunization bore no influence upon resistance to footpad infection, but resistance was appreciably better in mice challenged within 2 weeks of immunization than it was at later time points. In mice immunized by the footpad and intravenous routes, the pattern of resistance to airborne and footpad challenges was similar, in that there was substantially less immunity at 4 weeks than at 2 weeks after immunization. However, mice immunized by the airborne route were highly resistant to airborne challenge, regardless of the interval between immunization and reinfection. In this last respect, resistance of the lungs to reinfection was similar after Listeria and tuberculosis pneumonitis. It is suggested that a similar pattern of resistance may prevail in pneumonitis caused by other facultative intracellular parasites.

Host defenses in murine malaria: evaluation of the mechanisms of immunity to Plasmodium yoelii infection.

The immune response of random-bred mice to infection with a relatively avirulent strain of Plasmodium yoelii was measured in terms of parasitemia, splenomegaly, immediate and delayed hypersensitivity to a P. yoelii antigen preparation, resistance to challenge with a virulent variant of P. yoelii, and nonspecific resistance to L. monocytogenes. Avirulent P. yoelii produced a self limiting infection which resolved in 21 days. Peak parasitemia and splenomegaly were observed at 14 days, and infected mice were resistant to challenge with virulent P. yoelii from 7 days through at least 126 days. Mice infected with avirulent P. yoelii developed humoral immunity as judged by immediate hypersensitivity reactions and the capacity of their serum to passively protect normal mice against virulent P. yoelii. At no time did mice infected with the avirulent P. yoelii display evidence of cell-mediated immunity, as expressed by delayed-type hypersensitivity and increased resistance to L. monocytogenes. In fact, at the height of avirulent P. yoelii infection there was decreased resistance to L. monocytogenes in both liver and spleen, and the macrophages of the undisturbed peritoneal cavity were similarly defective. It was concluded that the defense mechanism of mice against P. yoelii is mediated by humoral factors in the absence of demonstrable cell-mediated immunity.

Host defenses in murine malaria: successful vaccination of mice against Plasmodium berghei by using formolized blood parasites.

Infections of normal ICR mice with the NYU-2 strain of Plasmodium berghei (Pb) are uniformly fatal. However, a proportion of mice that have been vaccinated with a formalin-killed antigen prepared from the blood stages of Pb survive an otherwise lethal challenge. Such immunity is not induced by immunization with normal mouse erythrocytes. The level of acquired anti-malarial immunity is related to the size and number of doses of antigen, and intravenous injection is superior to the subcutaneous route of vaccination. The addition of the adjuvants BCG and Corynebacterium parvum to the immunizing regimen improved the level of protection to a variable extent, depending on the batch of plasmodial antigen with which they were used. The adjuvants were most efficacious when used with batches of antigen which were poorly protective when used alone. These adjuvants were found never to protect ICR mice against Pb unless used in combination with specific antigen.

Listeria pneumonitis: induction of immunity after airborne infection with Listeria monocytogenes.

After implantation of approximately 10(3) Listeria monocytogenes organisms into the lungs, mice develop an acute pneumonitis with dissemination of infection to a mediastinal lymph node (MedLN), liver, and spleen. The infections in a MedLN and spleen resolve in approximately 7 days, but the lung infection persists for a few days longer. Pneumonitis is accompanied by a lymphoproliferative response in a MedLN and spleen, and immunity to Listeria is conferred adoptively with MedLN and spleen cells but not with mesenteric lymph node cells. Although the spleen appears to be the major repository of sensitized lymphocytes, splenectomized mice combat Listeria pneumonitis as effectively as normal mice. It is concluded that the induction of immunity to lung infection with L. monocytogenes is efficient and that the cause for the rather protracted pneumonitis is due to a defect in the expression of the cell-mediated immunity effector mechanism.

Host defenses in murine malaria: induction of a protracted state of immunity with a formalin-killed Plasmodium berghei blood parasite vaccine.

Random-bred mice were immunized with a nonliving antigen prepared from mixed-blood forms of Plasmodium berghei, strain NYU-2, in combination with Corynebacterium parvum and/or living BCG. A high proportion of intravenously immunized mice survived virulent challenge, but subcutaneous vaccination was less effective. Vaccinated mice developed a patent infection after challenge similar to that observed in normal controls. However, between days 12 to 20 postchallenge, infections in some vaccinated mice became subpatent, whereas infections in all normal controls progressed until death. The incidence of recrudescent infection was low and, eventually, a state of sterile immunity was established. The capacity of vaccinated mice to withstand P. berghei challenge was sustained at a fairly stable level for the 6-month period of observation. Mice that had survived a primary infection with P. berghei almost completely suppressed a second and larger challenge with the same organism.