Analysis of ammonia-oxidizing bacteria populations in acid forest soil during conditions of moisture limitation.

Ammonia-oxidizer numbers decreased under conditions of moisture limitation in litter, fermentation and humus layers of forest soil in the field, but the extent of regrowth after rehydration varied between layers. Nitrosospira 16S rRNA genes were amplified from all layers, regardless of moisture content or soil pH which varied between 4.1 and 5.2. Nitrosomonas spp. were detected less often, but appeared to exhibit more rapid recovery than the Nitrosospira spp. when drought conditions were relieved by rainfall.

Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12. Preferential expansion of interferon-gamma-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms.

In an adoptive transfer model of experimental allergic encephalomyelitis, stimulation of lymph node cells with proteolipid protein and recombinant murine interleukin (rmIL)-12 before cell transfer accelerated the onset and exacerbates clinical disease. In vitro stimulation with proteolipid protein in the presence of rmIL-12 was associated with an increase in interferon-gamma-producing cells and a decrease in IL-4-producing cells, indicating a preferential expansion of Th1 effector cells. This was supported by the finding that severe disease with rapid onset could be transferred with as few as 10 x 10(6) rmIL-12-stimulated lymph node cells. Immunohistochemical analysis confirmed that the accelerated onset of disease after in vitro stimulation with rmIL-12 coincided with an acute inflammatory response in the central nervous system. At peak disease, both control and rmIL-12 treatment groups exhibited extensive cellular infiltration with characteristic perivascular cuffing. No notable differences in either the cellular composition or cytokine expression within the lesions were seen between groups. However, the frequency of macrophages that stained positively for inducible nitric oxide synthase was increased in animals challenged with rmIL-12-treated lymph node cells. The results suggest that, in addition to promoting the preferential expansion of interferon-gamma-producing cells by rmIL-12 in vitro, secondary in vivo effects leading to macrophage activation and inducible nitric oxide synthase expression may contribute to the severe and protracted course of central nervous system inflammation in this model.

Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment.

Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed.

beta-Lactamase activity in mycobacteria including Mycobacterium avium and suppression of their growth by a beta-lactamase-stable antibiotic.

beta-Lactamase has been reported from only a few mycobacteria. It is widely assumed that Mycobacterium avium strains do not contain the enzyme, but earlier assays were done using insensitive methods. Thus the beta-lactamase activity in cell-free extracts of ten selected strains of mycobacteria, including four strains of M. avium, was determined using a highly sensitive spectrophotometric method. The results showed that all the mycobacteria tested possess the enzyme, which explains their resistance to beta-lactam antibiotics. However, some of the bacteria differed from others in the action of the inhibitors, clavulanate, sulbactam and tazobactam against their beta-lactamases. Growth of the mycobacteria was suppressed by novel combinations of the beta-lactam/beta-lactamase-inhibitors, and by a new beta-lactamase-stable cephalosporin, Cefepime (aminothiazolyl methoxyimino cephalosporin). The results presented, as well as reports of previous studies in vivo, suggest that the intracellular growth of the bacilli or the high partition coefficient of a beta-lactamase inhibitor such as sulbactam does not impede the antimycobacterial action of these compounds.

Multiple effects on peripheral hematology following administration of recombinant human interleukin 12 to nonhuman primates.

Interleukin 12 has demonstrated a wide spectrum of bioactivity on T and NK cells both in vitro and in vivo. Therapeutic potential of these activities has been suggested by studies in murine tumor, viral and microbial models of disease. We have investigated the in vivo effect of rhlL-12 in cynomolgus monkeys treated with 1 micrograms/kg/day by bolus i.v. or s.c. injection for 5 days. Treated group transient decreases in total WBC counts as compared to controls, with reversable decreases seen mainly in the lymphocyte and monocyte subsets. Phenotypic analysis showed a decrease in the number of CD4+ and CD8+ cells/microL on Days 2 and 4. Reversible thrombocytopenia and anemia were noted in both treated groups as compared to controls. Plasma neopterin concentrations were increased in both treated groups as compared to controls. These data show that rhlL-12 has multiple effects on peripheral hematology and suggests that this model may be useful to investigate in vivo bioactivity of rhlL-12.

Thalidomide does not perturb CD2, CD4, CD5, CD8, HLA-DR, or HLA-A, B, C molecules in vitro on the membranes of cells with immune potential.

Thalidomide dramatically relieves the signs and symptoms of erythema nodosum leprosum (ENL). ENL is an acute inflammatory complication of lepromatous leprosy. The cause(s) of ENL as well as the mechanism of action of thalidomide in arresting ENL are unknowns. It has been suggested that ENL is the consequence of a transient activation of a cell-mediated-immune (CMI) response to Mycobacterium leprae. To initiate a CMI response, an interaction between adhesion and/or signal transducing molecules on T-cells and molecules on antigen presenting cells would occur. An alteration, induced by thalidomide, of one or more of the molecules on T-cells or antigen presenting cells that are essential to maintaining the reactive state of ENL, could explain Thalidomide's ability to attenuate ENL. Thalidomide did not modify: (a) adhesion and/or signal transducing molecules such as CD2, CD4, CD5 and CD8, or (b) molecules that facilitate antigen presentation such as HLA-DR, HLA-A, HLA-B, or HLA-C.

Transmission of leprosy in nude mice through thorn pricks.

The dorsum of the feet of 10 nude mice was smeared with 10(7) Mycobacterium leprae and then pricked with cactus thorns contaminated with M. leprae. In 15 months five of them developed lepromatous nodules at the infected site and disseminated lesions in the ears, nose, tail and the organs of the reticuloendothelial system. Penetrating injuries through unprotected skin contaminated with M. leprae from the environment may play a role in the transmission of leprosy in humans.

Armadillos (Dasypus novemcinctus) as a model to test antileprosy vaccines; a preliminary report.

The efficacy of two candidate leprosy vaccines, BCG and a mixture of BCG and killed Mycobacterium leprae, was tested in 62 armadillos caught in the wild. The abilities of the vaccines to convert lepromin-negative armadillos to a positive reaction were compared with a group of control animals. Both vaccines upgraded subsequent lepromin skin-test histopathology. The conversion results parallel the protection values obtained in some BCG vaccine trials against leprosy in humans. Before conducting expensive human trials with new antileprosy vaccines, it would be worthwhile first to evaluate them in the armadillo model.

Lepromin responses in recipients of a candidate antileprosy bacterin vaccine (WHO-IMMLEP Mycobacterium leprae killed preparation) in the USA.

BACKGROUND: Identification of the nine-banded armadillo as a potential source of massive numbers of Mycobacterium leprae led to the development of a candidate bacterin vaccine for possible immunoprophylaxis. METHODS: Volunteers were from a leprosy-hypoendemic, nonBCG-using area (USA). They had been vaccinated intradermally 3 years earlier with a candidate antileprosy bacterin vaccine of irradiated and autoclaved Mycobacterium leprae obtained from experimental nine-banded armadillos. They were tested for dermal responsiveness to standard lepromin A. RESULTS: Values for induration and erythema appeared slightly greater for the vaccinated group; however, the differences were not statistically significant, indicating no appreciable 'anamnestic' effect on either Fernandez (early) or Mitsuda (late) reactions after 3 years. CONCLUSIONS: Because previous studies had demonstrated that administration of this bacterin produced no humoral changes, it now appears less probable that laboratory methods will be of much help in assessing even possible effectiveness of such vaccination.

Use of beta-lactam/beta-lactamase-inhibitor combinations as antimycobacterial agents.

Mycobacterium tuberculosis and Mycobacterium leprae develop resistance against the drugs used to treat tuberculosis and leprosy, respectively. Now multidrug-resistant tuberculosis is spreading in many countries, especially with the emergence of AIDS. Multidrug treatment is being promoted at present to eradicate leprosy. Since M. leprae may also become multidrug-resistant, new approaches have to be adopted for controlling mycobacterial diseases. Mycobacteria usually synthesize beta-lactamase and are insensitive to beta-lactam antibiotics. M. tuberculosis contains a constitutive beta-lactamase; de-repression of beta-lactamase has been reported in M. leprae. Three different beta-lactam/beta-lactamase-inhibitor combinations (ampicillin/sulbactam, amoxicillin/clavulanate and piperacillin/tazobactam) were used to suppress the growth of several strains of mycobacteria (including M. tuberculosis H37Rv) in vitro. Ampicillin/sulbactam is a potent bactericidal agent against M. leprae multiplying in mouse foot pads. In the present work, ampicillin/sulbactam showed higher activity than the other drug combinations. The beta-lactam/beta-lactamase inhibitors are likely to be effective as rational therapeutic agents against mycobacterial infections.

Lepromin skin testing in the classification of Hansen's disease in the United States.

Hansen's disease, or leprosy, although a relatively uncommon disease in the United States, continues to be important because of its implications--physical, psychological, and social--for the patient. Prognosis and treatment of the disease are based largely on clinical classification, which ranges from the multibacillary "lepromatous" to the paucibacillary "tuberculoid" forms, depending on the patient's specific immune capabilities. Traditionally, skin testing with lepromins--suspensions of the etiologic agent of Hansen's disease, Mycobacterium leprae--have been used as adjuncts to clinical parameters for classification in endemic areas. However, these have not been systematically studied in the United States. This report describes the results obtained from skin testing 38 volunteers (22 patients and 16 uninfected persons) with standard lepromin preparations. These results support the adjunctive value of lepromins for clinically classifying Hansen's disease in our "hypoendemic" population.

Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent.

Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, can be used to direct selective killing of human immunodeficiency virus (HIV)-infected cells. This approach takes advantage of the ability of sCD4 to bind with high affinity to gp120, the envelope protein of HIV-1, which is expressed on actively infected cells. However, conjugation of sCD4 via reagents that target amino groups may reduce its affinity for gp120, since at least one such group is important for gp120 binding. Here, we describe a novel cross-linking reagent which enables the conjugation of sCD4 via its carbohydrate moieties rather than its free amino groups. This heterobifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), combines a nucleophilic hydrazide with an electrophilic maleimide, thereby allowing coupling of carbohydrate-derived aldehydes to free thiols. We describe conditions by which MPBH is coupled selectively to the sialic acid residues of sCD4, and exemplify the use of MPBH by conjugating sCD4 to hemoglobin and to beta-galactosidase. We show that, whereas conjugation of sCD4 via amino groups markedly reduces its gp120 binding affinity, conjugation via the carbohydrate chains using MPBH does not affect binding. Moreover, we demonstrate the ability of a sCD4-MPBH-fluorescein conjugate to label HIV-infected human CEM cells selectively. These results indicate that, by targeting its carbohydrate moieties, sCD4 can be cross-linked to other molecules without compromising its function. The approach described here can be useful for glycoproteins in which amino groups, but not carbohydrates, are important for function. More generally, this approach can be considered for use in cross-linking glycoconjugates to compounds which either contain thiols, or to which thiols can be added.

The pathogenesis of leprosy in the nine-banded armadillo and the significance of IgM antibodies to PGL-1.

Twenty-seven nine-banded armadillos captured from the wild and tested free of wild M. leprae infection were distributed into four groups. They were injected at the right hind footpad with saline suspensions of M. Leprae at doses of 10(3), 10(4), 10(5) and 10(6). PGL-1 antibody levels were estimated using an ELISA test, twice during six months before the infection and every two months after the infection. One animal from each group was sacrificed at 6, 12, 18, 24, and 30-month intervals and another eight at unspecified intervals. A thorough autopsy and histopathological examination were conducted on all of them. Of the 27 animals, 18 developed the infection. In 10, there were granulomas at the site of inoculation and in 17 the regional lymph nodes were infected. The disease spread extensively to other lymph nodes and to the liver and spleen and then to the other organs. Peripheral nerves were invaded by M. leprae in only five animals. PGL-1 antibody levels registered a positive reading in only six of the 18 animals with the infection. In armadillo leprosy, the lesions did not persist at the site of entry in all animals M. leprae multiplied in the macrophages at the site of inoculation and the reticuloendothelial cells of the lymph nodes before they spread to other organs. There was evidence of invasion of endothelial cells of capillaries and possible bacteraemia even at an early phase of the infection. Peripheral nerves were not the preferred sites of entry or multiplication of M. leprae. Progressive increase in PGL-1 antibodies was recorded in five lepromatous armadillos with disseminated infection and high bacterial load. However, PGL-1 antibodies response was not sensitive enough to detect early disease.

Reversal of drug resistance in Mycobacterium leprae by ampicillin/sulbactam.

The multiplication of Mycobacterium leprae in foot pads of experimentally-infected mice was suppressed by intramuscular administration of ampicillin combined with sulbactam or YTR-830H, two potent inhibitors of beta-lactamase in the bacteria. The antibiotic or the inhibitors by themselves were inactive. Ampicillin/sulbactam also inhibited the growth of drug-resistant M. leprae which grew in the presence of rifampin or dapsone. The finding provides a new approach to treat leprosy and to overcome drug resistance of the mycobacteria.

Comparison of polymerase chain reaction technique with other methods for detection of Mycobacterium leprae in tissues of wild nine-banded armadillos.

Thirty, nine-banded armadillos weighing between 3 and 5 kilograms trapped from an area endemic for armadillo leprosy were collected at random; killed, autopsied and examined histopathologically. Also, one of the right inguinal lymph nodes was removed under sterile precautions and examined using PCR, direct smear examination, mouse footpad study, culture in laboratory media and histopathology with a view to detecting Mycobacterium leprae. Blood was collected at death and tested for IgM antibodies to PGL-1. According to the PCR study of the inguinal lymph nodes 16 of 30 armadillos (53.3%) had evidence of M. leprae. Significant levels of IgM antibodies to PGL-1 and identifiable lepromatous granuloma in inguinal lymph nodes were found in 2 animals (6.7%) with advanced disseminated disease. The prevalence of generalized leprosy according to autopsy study was 13.3% and according to histopathological examination of ear tissue 3.3%. The presence of M. leprae in the tissues evoked no special tissue reaction in the early stages. The pattern of spread of the disease in 2 animals closely resembled that found in experimental animals infected intracutaneously. Initiation of infection by inoculation of M. leprae through thorn pricks remains a distinct possibility.

The activity of rifabutin against Mycobacterium leprae.

Minimal effective doses of rifabutin and rifampicin were determined in Mycobacterium leprae isolated from skin biopsies of newly diagnosed, previously untreated lepromatous leprosy patients. Rifabutin was more potent than rifampicin. Our previous report that rifabutin was fully active against rifampicin-resistant M. leprae could not be confirmed. Examination of two strains of rifampicin-resistant M. leprae from elsewhere, and a repeat experiment on our original strain of rifampicin-resistant bacilli, showed full cross-resistance between rifampicin and rifabutin. A clinical trial in three newly diagnosed, previously untreated lepromatous patients showed that rifabutin has rapid bactericidal activity.