Evaluation of monocot and eudicot divergence using the sugarcane transcriptome.

Over 40,000 sugarcane (Saccharum officinarum) consensus sequences assembled from 237,954 expressed sequence tags were compared with the protein and DNA sequences from other angiosperms, including the genomes of Arabidopsis and rice (Oryza sativa). Approximately two-thirds of the sugarcane transcriptome have similar sequences in Arabidopsis. These sequences may represent a core set of proteins or protein domains that are conserved among monocots and eudicots and probably encode for essential angiosperm functions. The remaining sequences represent putative monocot-specific genetic material, one-half of which were found only in sugarcane. These monocot-specific cDNAs represent either novelties or, in many cases, fast-evolving sequences that diverged substantially from their eudicot homologs. The wide comparative genome analysis presented here provides information on the evolutionary changes that underlie the divergence of monocots and eudicots. Our comparative analysis also led to the identification of several not yet annotated putative genes and possible gene loss events in Arabidopsis.

Immunological basis for reactivation of tuberculosis in mice.

In this study different inbred strains of mice appeared to control and contain a low dose aerosol infection with Mycobacterium tuberculosis in a similar manner, giving rise to a chronic state of disease. Thereafter, however, certain strains gradually began to show evidence of regrowth of the infection, whereas others consistently did not. Using C57BL/6 mice as an example of a resistant strain and CBA/J mice as an example of a strain susceptible to bacterial growth, we found that these animals revealed distinct differences in the cellular makeup of lung granulomas. The CBA/J mice exhibited a generally poor lymphocyte response within the lungs and vastly increased degenerative pathology at a time associated with regrowth of the infection. As a possible explanation for these events, it was then observed that the CBA/J mouse strain was also less able to upregulate adhesion molecules, including CD11a and CD54, on circulating lymphocytes. These results therefore suggest that a failure to control a chronic infection with M. tuberculosis may reflect an inability to localize antigen-specific lymphocytes within the lung.

The progression of chronic tuberculosis in the mouse does not require the participation of B lymphocytes or interleukin-4.

The aging process is associated with alterations in the immune system. Some of the changes reported are an increase in the proportion of B lymphocytes, and a shift to a TH2-like cytokine environment. It has been hypothesized that the development of immunopathology within the lung during tuberculosis is linked to increased interleukin-4 (IL-4) production. In addition, a role for B cells in maintaining granuloma integrity has been recently proposed. This study investigated the role of B cells and IL-4 during the long-term course of chronic tuberculosis in mice and showed that the course of Mycobacterium tuberculosis infection in the lungs was not influenced by the absence of B lymphocytes or the TH2 cytokine IL-4.

Boosting vaccine for tuberculosis.

An effective new vaccine for the control of tuberculosis is badly needed. While current research attempts to improve vaccination are concentrating on new prophylactic or immunotherapeutic vaccines, virtually no emphasis has been placed on boosting individuals already inoculated with Mycobacterium bovis BCG. It is shown here that mice vaccinated with BCG gradually lose their capacity to resist an aerosol challenge infection as they age. However, if these mice are inoculated with the 30-kDa mycolyl transferase A protein in midlife, after challenge when aged they express levels of protection in the lungs comparable to those of young mice, associated with minimal pathological damage.

Disruption of mitochondrial respiration by melatonin in MCF-7 cells.

Clinical and laboratory studies have provided evidence of oncostatic activity by the pineal neurohormone melatonin. However, these studies have not elucidated its mechanism of action. The following series of MCF-7 breast tumor cell studies conducted in the absence of exogenous steroid hormones provide evidence for a novel mechanism of oncostatic activity by this endogenous hormone. We observed a 40--60% loss of MCF-7 cells after 20-h treatment with 100 nM melatonin, which confirmed and extended previous reports of its oncostatic potency. Interestingly, there were no observed changes in tritiated thymidine uptake, suggesting a lack of effect on cell cycle/nascent DNA synthesis. Further evidence of a cytocidal effect came from morphologic observations of acute cell death and autophagocytosis accompanied by degenerative changes in mitochondria. Studies of mitochondrial function via standard polarography revealed a significant increase in oxygen consumption in melatonin-treated MCF-7 cells. Enzyme-substrate studies of electron transport chain (complex IV) activity in detergent permeabilized cells demonstrated a concomitant 53% increase (p < 0.01) in cytochrome c oxidase activity. Additional studies of succinate dehydrogenase activity (complex II) as determined by reduction of (3-4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide demonstrated a significant increase (p < 0.05) in melatonin-treated cells and further confirmed the accelerated ET activity. Finally, there was a 64% decrease (p < 0.05) in cellular ATP levels in melatonin-treated cells. The G-protein-coupled melatonin receptor antagonist luzindole abrogated the cytotoxic and mitochondrial effects. These studies suggest a receptor-modulated pathway of cytotoxicity in melatonin-treated MCF-7 tumor cells with apparent uncoupling of oxidative phosphorylation.

Pentoxifylline treatment of mice with chronic pulmonary tuberculosis accelerates the development of destructive pathology.

It is well established in animal models that production of the cytokine tumour necrosis factor-alpha (TNF-alpha) is essential to the proper expression of acquired specific resistance following infection with Mycobacterium tuberculosis. This gives rise to an apparent state of chronic disease which over the next 100-200 days is characterized by slowly worsening pathological changes in the lung. To determine whether continued TNF-alpha production was harmful during this phase mice were treated with a TNF-alpha inhibitor, pentoxifylline. It was observed that although this therapy did not alter the numbers of bacteria recovered from the lungs of the infected mice, tissue damage within the lung was accelerated. These data thus demonstrate that production of TNF-alpha, already known to be important during the early expression of resistance to tuberculosis, remains important and beneficial during the chronic stage of the disease.

Tuberculosis in aged gammadelta T cell gene disrupted mice.

In young mice exposed to aerosol infection with Mycobacterium tuberculosis removal of the gammadelta T cell population by targeted gene disruption does not affect the expression of host resistance, but does influence the integrity of the early granulomatous response. The current study demonstrates that in aged gammadelta T cell gene disrupted mice similar immunopathologic changes ensued in both gene knockout and wild type control mice. Changes in cell surface marker expression, evident in other gene knockout models, was not observed in the aged gammadelta T cell knockout mice. These data imply that gammadelta T cell functions previously observed in young mice become much less important as the animal ages.

CD8- and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis.

The role of CD8 T lymphocytes in the immune response to Mycobacterium tuberculosis infection remains enigmatic, with persuasive reports of both cytolytic and noncytolytic (cytokine-mediated) responses to infection. To address the importance of the cytolytic mechanisms, mice with targeted disruptions for CD8 and perforin or with gene mutations in the CD95/ CD95L signaling pathway were exposed to pulmonary infection. All mice tested showed no differences in their ability to contain the growth of infection during the early phase of disease. As the chronic phase of the disease ensued, however, both CD8- and CD95/CD95L-deficient mice gradually lost their ability to limit bacterial growth. This was associated with a tendency toward pyogenic inflammation in the lung. This tendency was not seen in the perforin gene-disrupted mice. In CD8 gene-disrupted mice, the ability to generate interferon-gamma secreting T cells was unimpaired. Although these cells were capable of entering the lung they were unable to influence the increasing bacterial load in this organ.

A novel nonclassic beta2-microglobulin-restricted mechanism influencing early lymphocyte accumulation and subsequent resistance to tuberculosis in the lung.

In this study, we compared the course of a low-dose aerosol Mycobacterium tuberculosis infection in mice bearing gene disruptions for the beta2-microglobulin molecule, the CD8 molecule, and the CD1 molecule. Over the first 50 d of infection, the CD8- and CD1-disrupted mice were no more susceptible to infection than were the control mice. In contrast, the bacterial load in beta2-microglobulin gene-disrupted mice increased rapidly and attained much higher levels than that observed in the other gene-disrupted mice and in control mice. A second major difference between the beta2-microglobulin gene-disrupted mice and the other animals was the development of lung granulomas; both the CD8- and CD1-disrupted mice developed essentially normal granulomas except for an apparent increased lymphocyte influx in the CD8-disrupted mice. The beta2-microglobulin gene-disrupted mice, on the other hand, developed granulomas virtually devoid of lymphocytes, with these cells instead localized within prominent perivascular cuffing adjacent to the lesions. These data support the hypothesis that a beta2-microglobulin-dependent, non-CD8- and non-CD1-dependent mechanism controls the early and efficient influx of protective lymphocytes into infected lesions, and that the absence of this mechanism decreases the capacity of the animal to initially deal with pulmonary tuberculosis.

Interleukin-6 induces early gamma interferon production in the infected lung but is not required for generation of specific immunity to Mycobacterium tuberculosis infection.

Immunity to Mycobacterium tuberculosis is dependent upon the generation of a protective gamma interferon (IFN-gamma)-producing T-cell response. Recent studies have suggested that interleukin-6 (IL-6) is required for the induction of a protective T-cell response and that IL-4 may suppress the induction of IFN-gamma. To evaluate the role of the cytokines IL-6 and IL-4 in the generation of pulmonary immunity to M. tuberculosis, IL-6 and IL-4 knockout mice were infected with M. tuberculosis via aerosol. The absence of IL-6 led to an early increase in bacterial load with a concurrent delay in the induction of IFN-gamma. However, mice were able to contain and control bacterial growth and developed a protective memory response to secondary infection. This demonstrates that while IL-6 is involved in stimulating early IFN-gamma production, it is not essential for the development of protective immunity against M. tuberculosis. In contrast, while the absence of IL-4 resulted in increased IFN-gamma production, this had no significant effect upon bacterial growth.

Lack of protection in mice and necrotizing bronchointerstitial pneumonia with bronchiolitis in guinea pigs immunized with vaccines directed against the hsp60 molecule of Mycobacterium tuberculosis.

In this study, the hsp60 and hsp70 heat shock protein antigens of Mycobacterium tuberculosis were tested as potential vaccine candidates, using purified recombinant protein antigens or antigens encoded in the form of a DNA plasmid vaccine. Guinea pigs vaccinated with a mixture of the two proteins showed no evidence of resistance to low-dose aerosol challenge infection and quickly developed severe lung damage characterized by necrotizing bronchointerstitial pneumonia and bronchiolitis. As a result, we turned instead to a DNA vaccination approach using a plasmid encoding the hsp60 antigen of M. tuberculosis. Although immunogenic in mice, vaccination with plasmid DNA encoding hsp60 was not protective in that model or in the guinea pig model and again gave rise to similar severe lung damage. This study seriously questions the safety of vaccines against tuberculosis that target highly conserved heat shock proteins.

Transient loss of resistance to pulmonary tuberculosis in p47(phox-/-) mice.

Mycobacterium tuberculosis is an important respiratory pathogen the growth of which is controlled primarily by cytokine-activated macrophages. One of the principal mediators of this control is nitric oxide; however, superoxide has recently been shown to be protective in systemic mycobacterial infections. To determine whether superoxide is important in controlling M. tuberculosis during primary pulmonary infection, mice lacking the cytosolic p47(phox) gene (which is essential for effective superoxide production by the NADPH oxidase) were infected aerogenically. The lack of superoxide during an aerosol infection with M. tuberculosis resulted in a significant increase in bacterial growth over the early period of infection. Once antigen-specific gamma interferon-producing lymphocytes were detected in the draining lymph nodes, however, bacterial growth in the lung stopped. One interesting consequence of the lack of superoxide was an increase in neutrophilic infiltrates within the granuloma. This may be a consequence of increased tissue damage due to more rapid bacterial growth or may reflect a role for superoxide in controlling inflammation.

Effective preexposure tuberculosis vaccines fail to protect when they are given in an immunotherapeutic mode.

Two vaccine formulations previously shown to induce protective immunity in mice and prevention of long-term necrosis in guinea pigs were tested as potential immunotherapeutic vaccines in mice earlier infected by aerosol with Mycobacterium tuberculosis. Neither vaccine had any effect on the course of the infection in the lungs, but both reduced the bacterial load in the spleen. Similarly, inoculation with Mycobacterium bovis BCG had no effect whatsoever and, if given more than once, appeared to induce an increasingly severe pyogranulomatous response in the lungs of these mice.

Granuloma formation is required to contain bacillus growth and delay mortality in mice chronically infected with Mycobacterium tuberculosis.

Previous studies in this laboratory have shown that mice with a gene disruption to the intracellular adhesion molecule-1 (ICAM-K/O) express normal cell-mediated immunity but cannot mount delayed-type hypersensitivity reactions following Mycobacterium tuberculosis infection. However, even in the absence of any appreciable granuloma formation, these mice control bacterial growth for at least 90 days. While not required to control the infection initially, we hypothesized that granuloma formation was required to control chronic infection, acting by surrounding infected cells to prevent bacterial dissemination. To test this, ICAM-1 knockout mice were infected with a low dose aerosol of M. tuberculosis Erdman and were found to succumb to infection 136+/-30 days later, displaying highly elevated bacterial loads compared to wild-type mice. Lung tissue from ICAM-K/O mice displayed extensive cellular infiltration and widespread tissue necrosis, but no organized granulomatous lesions were evident, whereas the control mice displayed organized compact granulomas. These data demonstrate that while a granulomatous response is not required initially to control M. tuberculosis infection, absence of granulomas during chronic infection leads to increased bacterial growth and host death. Thus these data support the hypothesis that granuloma formation is required to control chronic infection, acting by surrounding and walling off sites of infection to prevent bacterial dissemination and maintain a state of chronic infection.

Role of gamma delta T cells in immunopathology of pulmonary Mycobacterium avium infection in mice.

Several studies have shown that gamma delta T cells influence granuloma development after infection with intracellular pathogens. The role of gamma delta T cells in controlling the influx of inflammatory cells into the lung after Mycobacterium avium infection was therefore examined with gene-disrupted mice (K/O). The mice were infected with either M. avium 724, a progressively replicating highly virulent strain of M. avium, or with M. avium 2-151 SmT, a virulent strain that induces a chronic infection. gamma delta-K/O mice infected with M. avium 2-151 SmT showed early enhanced bacterial growth within the lung compared to the wild-type mice, although granuloma formation was similar in both strains. gamma delta-K/O mice infected with M. avium 724 showed identical bacterial growth within the lung compared to the wild-type mice, but they developed more-compact lymphocytic granulomas and did not show the extensive neutrophil influx and widespread tissue necrosis seen in wild-type mice. These data support the hypothesis that isolates of M. avium that induce protective T-cell-specific immunity are largely unaffected by the absence of gammadelta T cells. Whereas with bacterial strains that induce poor protective immunity, the absence of gamma delta T cells led to significant reductions in both the influx of neutrophils and tissue damage within the lungs of infected mice.

Occult osseous metastasis of a colonic adenocarcinoma visualized with technetium tc 99m hydroxymethylene diphosphate scintigraphy in a horse.

A 5-year-old Arabian horse was admitted with a 5-day history of undulant pyrexia of unknown origin, inappetence, obtundation, and acute collapse. Physical examination results were unremarkable except for a grade II/VI left-sided systolic cardiac murmur and abdominal splinting. Mild chronic inflammatory changes were evident on clinicopathologic evaluation. Echocardiography revealed moderate aortic insufficiency. A solitary soft tissue opacity was found on thoracic radiography but not on ultrasonography. Palliative treatment was ineffective. Nuclear scintigraphy with WBC labeled with technetium Tc 99m hexamethylpropyleneamine oxime did not identify abnormalities, but a second nuclear scan with technetium Tc 99m hydroxymethylene diphosphate identified polyostotic disease. Examination of a biopsy specimen from an affected rib revealed disseminated adenocarcinoma. The horse was euthanatized. Necropsy and histologic examination revealed a colonic adenocarcinoma with osseous metaplasia that had disseminated to multiple parenchymal organs, muscle, and bone.

Effect of cocaine, 95% oxygen and ellagic acid on the development and antioxidant status of cultured rat embryos.

Prenatal exposure to cocaine has been associated with adverse developmental effects and current data suggest cocaine induced malformations are caused by ischemic-reperfusion injury. This study was undertaken to assess a new in vitro model which uses a routine rat whole embryo culture system that incorporates a change in oxygen status, and to examine the effects of altered oxygen status and pretreatment with ellagic acid (EA), an anti-oxidant, after cocaine exposure. Embryos were evaluated by determining a developmental score and by measuring tissue reduced glutathione (GSH) levels. Following re-oxygenation with 95% O2 for the last 6 h of culture, embryos treated with cocaine had reduced developmental scores and GSH levels. Embryos treated with cocaine and not re-oxygenated with 95% O2 did not have reduced developmental scores. EA blocked the effects of cocaine on developmental score and GSH level. These data support ischemia-reperfusion injury as the mechanism of cocaine developmental toxicity.

Disruption of the cellular inflammatory response to Listeria monocytogenes infection in mice with disruptions in targeted genes.

The results of this study to dissect the nature of the acquired immune response to infection with Listeria monocytogenes in mice with targetted gene disruptions show that successful resolution of disease requires the essential presence of alphabeta T cells and the capacity to elaborate gamma interferon. In the absence of either of these entities, mice experience increasingly severe hepatitis and tissue necrosis and die within a few days. The data from this study support the hypothesis that the protective process is the efficient replacement of neutrophils in lesions by longer-lived mononuclear phagocytes; alphabeta-T-cell-knockout mice died from progressive infection before neutrophil replacement could occur, whereas in gammadelta-T-cell-knockout mice this replacement process in the liver has previously been shown to be much slower. In the present study we attribute this delay to reduced production of the macrophage-attracting chemokine MCP-1 in the gammadelta-T-cell-knockout animals. These data further support the hypothesis that gammadelta T cells are important in controlling the inflammatory process rather than being essential to the expression of protection.

Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis.

The results of this study provide the first evidence that two completely separate vaccine approaches, one based on a subunit vaccine consisting of a mild adjuvant admixed with purified culture filtrate proteins and enhanced by the cytokine interleukin-2 and the second based on immunization with DNA encoding the Ag85A protein secreted by Mycobacterium tuberculosis, could both prevent the onset of caseating disease, which is the hallmark of the guinea pig aerogenic infection model. In both cases, however, the survival of vaccinated guinea pigs was shorter than that conferred by Mycobacterium bovis BCG, with observed mortality of these animals probably due to consolidation of lung tissues by lymphocytic granulomas. An additional characteristic of these approaches was that neither induced skin test reactivity to commercial tuberculin. These data thus provide optimism that development of nonliving vaccines which can generate long-lived immunity approaching that conferred by the BCG vaccine is a feasible goal.

Adequate expression of protective immunity in the absence of granuloma formation in Mycobacterium tuberculosis-infected mice with a disruption in the intracellular adhesion molecule 1 gene.

It remains unknown whether the expression of cell-mediated protective immunity and the capacity to mount a delayed-type hypersensitivity (DTH) reaction in tuberculosis infection represent two manifestations of a basic response or are dissociable events. In this study, we present data in favor of the latter hypothesis, by showing that tuberculosis infection in the lungs of mice possessing only a truncated form of intracellular adhesion molecule 1 due to gene disruption was still adequately controlled by the expression of protective immunity in the absence of any sustained influx of macrophages and the lack of formation of appreciable granulomas. These animals also had no detectable DTH response to mycobacterial proteins in the footpad assay, indicating that the accumulation of blood-borne macrophages at sites of mycobacterial infection or antigen deposition is not essential to control of the infection. These data support the hypothesis that the DTH component of the cellular response is not protective but contributes by walling off the sites of infection to prevent dissemination and reactivation disease.