Relation of Infection to Tissue Temperature in Mice Infected with Mycobacterium marinum and Mycobacterium leprae

Intravenous and footpad infections with Mycobacterium marinum and footpad infections with M. leprae were compared in the following mouse strains: A/He, BALB/C, CBA, C3H, C57BL, C57L, DBA, 101, and CFW. The results varied a great deal according to mouse strain used. Intravenous injection of high doses of M. marinum resulted in deaths after 28 days of 100% of strain A/He, and none of strain 101; 27 days after injection, the feet and noses of all strain CBA mice, but few of the C57BL, 101, or CFW mice, were involved. Injection of a small dose of M. marinum into the footpad produced visible disease in 5 days in all of the C57BL and 101 mice, but in not more than 60% of the A/He, DBA, and CFW mice; the average amount of swelling at 17 days varied from 4.40 mm in strain C57L to 0.92 in strain 101. After footpad injection of M. leprae, the average plateau harvests varied from 1.3 x 10(7) acid-fast bacteria in strain CBA to 6.5 x 10(5) in strain C57L. The infections in CBA mice extended from the site of inoculation throughout the foot. The temperature was measured rectally, in the footpad, and in the tail. Analysis of all the results revealed little correlation among the three types of infection. There was a strong negative correlation between the tail temperature and the death rate after intravenous injection of M. marinum, and a strong positive correlation between footpad temperature and plateau harvest of M. leprae.     

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.     

Emergence of an effective adaptive cell mediated immune response to Mycobacterium leprae is not impaired in reactive oxygen intermediate-deficient mice

Cytokine-activated macrophages (MPhi) employ reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to combat pathogens. The requirement for ROI for an effective host response to experimental leprosy using mice which have a disruption in the 91-kD subunit of the NAPDH oxidase cytochrome b (phox91-/-) was examined. Mycobacterium leprae multiplication in phox91-/- foot pads (FP) was elevated early in infection but subsequently arrested similarly to control mice within a noninvasive granuloma. Using a modified lepromin test model, a similar cellular composition in the M. leprae-induced FP granuloma in both strains with lymphocyte infiltration consisting primarily of CD4+CD44(hi)CD62L(lo) effector cells was found. Of great interest was the disparity in the T cell population between the granuloma and the draining lymph node which contained predominantly na?ve CD4+CD44(lo)CD62L(hi) cells and was, therefore, not representative of the infection site. TH1 cytokines, chemokines and inducible nitric oxide synthase were comparably expressed in the FP of both strains. When infected in vitro, normal MPhi from B6 and phox91-/- mice supported bacterial viability, whereas IFNgamma-activated MPhi killed M. leprae in a RNI-dependent manner, emphasizing that ROI was dispensable. These data show that phox91-/- mice generate a strong adaptive immune response and control long-term infection with M. leprae.     

Lethal effect of fresh sea water on Vibrio parahaemolyticus and isolation of Bdellovibrio parasitic against the organism.

Halophilic Bdellovibrio, which is parasitic and lytic to Vibrio pharahaemolyticus, was ioslated from fresh sea water in the winter. It had a lethal effect on V. parahaemolyticus. The optimum temperature ofr multiplication ranged from 25 C to 30 C and growth was not observed at 35 C. Plaque numbers of the isolate reached a maximum in 17 hr under conditions of shaking at 25 C in autoclaved sea water supplemented with V. parahaemolyticus cells, and were as high as ten times the number of host cells. With respect to the host-suspended medium, the isolate multiplied in natural sea water ten times more than in Herbst's artificial sea water but did not grow in saline. V. parahaemolyticus, Vibrio alginolyticus and several species in the Vibrio genus were susceptible to the parasite on the basis of plaque formation but Escherichia coli and Staphylococcus aureus were not.     

An alternative route for infecting armadillos with Mycobacterium leprae

A nine-banded armadillo was inoculated with Mycobacterium leprae in both hind footpads. The animals were usually inoculated intravenously, or intradermally in the abdominal skin. Profuse multiplication of the bacilli occurred at the injection sites after more than two years. Eventually bacteraemia developed, and large numbers of the organisms were found in skin biopsies and in lymph nodes. There was limited dissemination of the bacteria into the spleen and the liver, and peripheral nerve invasion by the bacilli was also detected. M. leprae remained viable in the liver tissue, kept frozen at -80 degrees C for three years. This experimental system would be useful in testing the effects of certain immunological and chemotherapeutic agents against M. leprae by injecting them directly at the infection site.     

In vitro vegetative propagation of Leucaena leucocephala (Lam.) de Wit

A method for in vitro clonal multiplication of Leucaena leucocephala cv K-8 is described. On MS with BAP (3×10^–6M), at the optimum temperature of 30°C, shoots from seedling and adult trees multiplied at a rate of 6–7 fold every three weeks. The addition of adenine or glutamine reduced precocious leaf drop. All shoots rooted on MS with IAA (5×10^–6M). Micropropagated plants have been successfully transferred to soil.     

Mycobacterium leprae inhibits dendritic cell activation and maturation

Leprosy presents with a clinical spectrum of skin lesions that span from strong Th1-mediated cellular immunity and control of bacillary growth at one pole to poor Ag-specific T cell immunity with extensive bacillary load and Th2 cytokine-expressing lesions at the other. To understand how the immune response to Mycobacterium leprae is regulated, human dendritic cells (DC), potent inducers of adaptive immune responses, exposed to M. leprae, Mycobacterium tuberculosis (Mtb), and Mycobacterium bovis bacillus Calmette-Guerin (BCG) were studied for their ability to be activated and to prime T cell proliferation. In contrast with Mtb and BCG, M. leprae did not induce DC activation/maturation as measured by the expression of selected surface markers and proinflammatory cytokine production. In MLR, T cells did not proliferate in response to M. leprae-stimulated DC. Interestingly, M. leprae-exposed MLR cells secreted increased Th2 cytokines as well as similar Th1 cytokine levels as compared with Mtb- and BCG-exposed cells. Gene expression analysis revealed a reduction in levels of mRNA of DC activation and maturation markers following exposure to M. leprae. Our data suggest that M. leprae does not induce and probably suppresses in vitro DC maturation/activation, whereas Mtb and BCG are stimulatory.     

Lethal Effect of Fresh Sea Water on Vibrio parahaemolyticus and Isolation of Bdellovibrio Parasitic against the Organism

Halophilic Bdellovibrio, which is parasitic and lytic to Vibrio parahaemolyticus, was isolated from fresh sea water in the winter. It had a lethal effect on V. parahaemolyticus. The optimum temperature for multiplication ranged from 25 C to 30 C and growth was not observed at 35 C. Plaque numbers of the isolate reached a maximum in 17 hr under conditions of shaking at 25 C in autoclaved sea water supplemented with V. parahaemolyticus cells, and were as high as ten times the number of host cells. With respect to the host-suspended medium, the isolate multiplied in natural sea water ten times more than in Herbst's artificial sea water but did not grow in saline. V. parahaemolyticus, Vibrio alginolyticus and several species in the Vibrio genus were susceptible to the parasite on the basis of plaque formation but Escherichia coli and Staphylococcus aureus were not.     

Use of tuftsin in experimental leprosy]

The effect of tuftsin was studied in vivo using CBA mice infected with M. leprae by Shepard's technique and in vitro using macrophage-like cell line P. 388 (Co-cultivated with M. leprae) and the cultivated leproma tissue. It was found out that tuftsin acted as a stimulator of M. leprae multiplication in foot-pads of mice and as a prolongator of M. leprae survival in the cells of macrophage-like cell line P .388. It is concluded that using tuftsin might be useful in view of studying different aspects of experimental leprosy.     

Enumeration of Mycobacterium leprae using real-time PCR

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.     

Factors influencing the in vitro growth of Mycobacterium leprae: effect of oxygen.

In our efforts to evaluate factors responsible for in vitro growth of Mycobacterium leprae in DH medium and also to improve the system, effects of oxygen tension on in vitro growth of M. leprae were determined. This was achieved by varying the ratio between DH medium and free air space above the medium in the culture tubes. Growth-competent M. phlei (ATCC 11758) could tolerate all the oxygen it can get in the medium. On the other hand, M. leprae seemed to be of microaerophilic nature. In vivo-grown M. leprae cells were more sensitive than their counterparts that were adapted to in vitro environment. In vivo-grown cells grew better when 70% of the space in culture tube was occupied by DH medium. These in vitro-adapted cells gave optimum growth in subcultures when the air spaces in the culture tubes were 40-50%. The role of oxygen tensions in the development of lesions in leprosy patients and armadillos has been discussed.     

Immunity to leprosy. II. Genetic control of murine T cell proliferative responses to Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured after immunization of mice at the base of the tail with antigen and challenging lymphocytes from draining lymph nodes in culture with M. leprae. This T cell response to M. leprae has been compared in 18 inbred strains of mice. C57BL/10J mice were identified as low responder mice. The congenic strains B10.M and B10.Q were found to be high responders, whereas B10.BR and B10.P were low responders. F1 (B10.M X C57BL/10J) and F1 (B10.Q X C57BL/10J) hybrid mice were found to be low responders, similar to the C57BL/10J parent, indicating that the low responsive trait is dominant. Whereas B10.BR mice were shown to be low responders to M. leprae, B10.AKM and B10.A(2R) were clearly high responders, indicating that the H-2D region influences the magnitude of the T cell proliferative response. Gene complementation within the H-2 region was evident. Genes outside the H-2 region were also shown to influence the response to M. leprae. C3H/HeN were shown to be high responder mice, whereas other H-2k strains, BALB.K, CBA/N, and B10.BR, were low responders. Gene loci that influence the T cell proliferation assay have been discussed and were compared to known background genes which may be important for the growth of intracellular parasites. Because mycobacteria are intracellular parasites for antigen-presenting cells, genes that affect bacterial growth in these cells will also influence subsequent immune responses of the host.     

Growth and Development in the Young Tomato: III. THE EFFECT OF NIGHT AND DAY TEMPERATURES ON VEGETATIVE GROWTH

Tomato seedlings were grown in a 12-hour day at constant andalternating day and night temperatures ranging from 10°to 30° C. The pattern of results was similar at light intensitiesof 400 and 800 f.c. The maximum rate of dryweight accumulationoccurred at a constant temperature close to 25° C. The effectsof day and night temperatures on total dry weight showed a considerabledegree of independence. The optimum day temperature was 25°C irrespective of the night temperature; the optimum night temperatureincreased from 18° to 25° C over the whole range ofday temperature. On average, day temperature affected totaldry weight twice as much as night temperature. High night temperaturesto some extent compensated for low day temperatures. The optimumday and night temperatures for leaf growth were both 25°C. On average day temperature affected leaf growth one and ahalf times as much as night temperature. By 12-hourly sampling it was shown that the cotyledons and leavesgrow throughout both day and night and that high night temperatureaccelerates nocturnal growth (cotyledons by cell expansion,young leaves by cell multiplication). Plants having receivedonly one night at 25° C, as compared with 15° C, showa slightly greater assimilation during the following light period,apparently as a consequence of increased photosynthetic surface.The respiratory loss in dry weight during darkness was not significantlyaffected by temperature over the range 15–25° C.     

Genetic control of murine T cell proliferative responses to Mycobacterium leprae. V. Evidence for cross-reactivity between host antigens and Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured by immunization of mice at the base of the tail with Ag and challenging lymphocytes from draining lymph nodes in culture with M. leprae. C57BL/10J and B10.BR mice were identified as low responder mice and the congenic strains B10.M, B10.Q, and B10.AKM as high responders whereas F1 (high x low) hybrid mice were found to be low responders. The cellular basis of low responsiveness did not appear to result from a defect in Ag-presenting cells or the activation of suppressor T cells by M. leprae. The influence of the environment in which T cells developed on responsiveness to M. leprae was analyzed in chimeric mice prepared by irradiating F1(C57BL/10J x B10.M) mice and reconstituting with bone marrow from C57BL/10J, B10.M, or F1 donors. Six weeks later, chimeric mice were immunized with M. leprae, lymph node cells were subsequently prepared, and H-2 phenotyped and challenged in culture with M. leprae Ag. T cell proliferative responses were found to be low in all cases, similar to those observed using lymph node cells from F1 hybrid mice. These results suggested that high responder B10.M lymphocytes developing in the irradiated F1 mice became tolerized to antigenic determinants found on M. leprae. This implied cross-reactive epitopes existed between some mouse strains and M. leprae. Low responsiveness to M. leprae in low responder and F1 hybrid mice may result from tolerance to H-2-encoded Ag that show cross-reactivity with M. leprae.     

Distribution of some mycobacterial waxes based on the phthiocerol family

Characteristic waxes, based on methoxy and keto long-chain diols, members of the phthiocerol family, have been isolated from representatives of Mycobacterium bovis, M. kansasii, M. marinum, M. microti and M. tuberculosis. M. kansasii produced essentially di-esters of the ketodiol phthiodiolone A, but the remaining species also had waxes based on the methoxy-diols phthiocerol A and phthiocerol B. Gas chromatography of derivatives of the components of the waxes showed that the phthiocerol A components from M. bovis, M. microti and M. microti and M. tuberculosis were qualitatively similar, being mainly C34 and C36, but potentially significant differences were seen in the proportions of the components from M. bovis. The phthiocerols A from M. marinum were C28 and C30 and the phthiodiolones A from M. kansasii were C25 and C27. The multimethyl-branched acids from the waxes of M. bovis were quantitatively different from those of M. microti and M. tuberculosis but all these mycocerosic acids ranged in size from C23 or C24 to C32, with C29 or C30 being the major component in most cases. M. marinum and M. kansasii strains had mainly C26 or C27 and C29 or C30 multimethyl-branched acids, respectively.     

Preadaptation of protein synthesis in wheat seedlings to high temperature

The optimum temperature of protein synthesis in wheat seedlings (Triticum aestivum L.), measured as ¹⁴C-leucine incorporation, depends on the growing temperature. Plants grown at reduced temperature (4 C) reach their optimum at 27.5 C, whereas plants kept at 36 C have the highest rate of protein synthesis at 35 C. The transition is gradual. The activation energy of protein synthesis for seedlings grown at medium or reduced temperature is lower (about 11 kcal/mole), than for plants grown at higher temperatures (15 keal/mole). The decline of the rate of protein synthesis beyond the temperature optimum is also affected by the growth temperature; only plants kept at 30 or 36 C show a sharp decrease with increasing slope; plants kept at 4, 10, and 20 C exhibit a linear and comparatively moderate decline.     

Comparative studies on the interaction of benzpyrene with cells derived from poikilothermic and homeothermic vertebrates. II. Effect of temperature on benzpyrene metabolism and cell multiplication

The effect of incubation temperature on cell multiplication and on the efficiency of benzpyrene (BP) metabolism to water-soluble derivatives was compared in cell cultures derived from three poikilothermic and three homeo-thermic vertebrate species. The fish cells grew optimally at about 20°C and the amphibian and reptilian cells at about 30°C, and in general, these cells multiplied over broader ranges of temperature than the mouse, hamster or chick cells. In each cell system, the maximum temperature supporting efficient BP metabolism exceeded the maximum temperature supporting cell growth by 4 to 8°, but the range of temperatures supporting near-maximal BP metabolism was also considerably broader in the poikilothermic than in the homeothermic vertebrate cultures.     

Morphological Changes of Mycobacterium lepraemurium Grown in Cultures of Mouse Peritoneal Macrophages

Studies were made on morphological changes of Mycobacterium lepraemurium grown in cultures of mouse peritoneal macrophages. Two types of nonsolid or irregularly stained M. lepraemurium were observed. One type occurred in the growth phase of the organisms during the stage of preparation for bacillary multiplication. The nonsolid bacilli appeared as elongated organisms having pointed ends, isolated acid-fast dots, or faintly stained areas at the ends of the bacilli. It is possible that this irregularity in staining is due to a very gradual, versus an instantaneous, acquisition of acid-fast material during bacillary multiplication and maturation. Solid forms were again observed upon maturation. Nonsolid bacilli were also observed in macrophage cultures infected with autoclave-killed M. lepraemurium. Under these conditions there was an emergence of organisms which showed irregularly stained areas and various forms of deformity unaccompanied by elongation or multiplication. These irregularities were most probably due to the destructive process of digestion of bacillary protoplasm. The present study does not support the current hypothesis that all nonsolid acid-fast organisms are nonviable.     

Effect of ultrasonic waves on the growth intensity of Mycobacterium leprae in liquid nutrient media

Ultrasound-treated M. leprae grow in liquid culture media at a greater rate than untreated ones. Ultrasound-treated M. leprae retain their capacity for intensive growth after their passage in mice.