Safety and efficacy of plasmid DNA expressing two isoforms of hepatocyte growth factor in patients with critical limb ischemia.

VM202, a plasmid DNA that expresses two isoforms of hepatocyte growth factor, may elicit angiogenic effects that could benefit patients with critical limb ischemia (CLI). In a phase 2, double-blind trial in 52 CLI patients, we examined the safety and potential efficacy of intramuscular injections of low-dose (n=21) or high-dose (n=20) VM202 or placebo (n=11) in the affected limb (days 0, 14, 28 and 42). Adverse events and serious adverse events were similar among the groups; no malignancy or proliferative retinopathy was seen. In exploratory efficacy analyses, we found no differences in ankle or toe-brachial index, VAS, VascuQuol or amputation rate among the groups. Complete ulcer healing was significantly better in high-dose (8/13 ulcers; P<0.01) versus placebo (1/9) patients. Clinically meaningful reductions (>50%) in ulcer area occurred in high-dose (9/13 ulcers) and low-dose (19/27) groups versus placebo (1/9; P<0.05 and P<0.005, respectively). At 12 months, significant differences were seen in TcPO2 between the high-dose and placebo groups (47.5 ± 17.8 versus 36.6 ± 24.0 mm Hg, respectively; P<0.05) and in the change from baseline among the groups (P<0.05). These data suggest that VM202 is safe and may provide therapeutic bioactivity in CLI patients.

Lymphocytes expressing Fc gamma receptors suppress antigen-induced proliferation in cells from guinea pigs infected with virulent Mycobacterium tuberculosis.

The immunomodulatory role of T lymphocytes expressing receptors for the Fc portion of IgG (T gamma cells) in BCG-vaccinated guinea pigs following pulmonary infection with a low dose of virulent Mycobacterium tuberculosis H37Rv was studied. Compared to uninfected animals, guinea pigs infected 2 or 4 weeks previously harbored significantly increased percentages of T gamma cells in the peripheral blood (twofold increase) and the spleen (50% increase), and at 4 weeks had nearly fourfold increases in T gamma cells in bronchotracheal lymph nodes draining the infected lungs. Removal of T gamma cells by panning on plastic dishes coated with a monoclonal antibody specific for guinea pig Fc gamma R resulted in significant increases in proliferative responses of splenocytes to Con A and PPD in vitro. Removal of T gamma cells from peripheral blood lymphocytes resulted in significantly increased responses to PPD and to recombinant mycobacterial hsp 65 and hsp 70 antigens. The isolated T gamma cells themselves did not proliferate when stimulated with Con A, PPD, or either of the specific mycobacterial antigens, even in the presence of syngeneic accessory cells. These results suggest that FcR gamma-bearing T cells may play an important immunomodulatory role in pulmonary tuberculosis, principally by suppressing antigen-induced proliferation in the rest of the lymphocyte population.

Formoterol. An update of its pharmacological properties and therapeutic efficacy in the management of asthma.

Formoterol, a selective beta 2-adrenoceptor agonist, produces effective dose-proportional bronchodilation, which persists for up to 12 hours, in patients with reversible obstructive respiratory disease. Bronchodilation is significant within minutes of inhalation, maximal within 2 hours, and at therapeutic doses is equivalent to that produced by standard doses of traditional beta 2-agonists. In single-dose studies comparing the two long-acting beta 2-agonists formoterol and salmeterol, significant bronchodilation is achieved more rapidly with formoterol than salmeterol. Duration of bronchodilation is similar with both drugs. The therapeutic efficacy of inhaled formoterol has been equal to or greater than that of salbutamol (albuterol), fenoterol and terbutaline in both short and long term clinical trials. Formoterol reduces symptoms of nocturnal asthma and reduces the need for rescue medication compared with salbutamol. Recent studies have shown that the addition of inhaled formoterol 12 or 24 micrograms twice daily to existing inhaled corticosteroid regimens improves lung function and reduces asthma symptoms compared with placebo. In one well designed study, the frequency of severe exacerbations of asthma over 12 months was decreased by adding formoterol to existing regimens of inhaled corticosteroids. Tolerance to the bronchodilator response of formoterol has not been observed in long term clinical trials. Because of its long duration of action, formoterol offers significant therapeutic advantages over shorter-acting beta 2-agonists in the treatment of nocturnal and exercise-induced asthma. Formoterol is effective in preventing exercise-induced asthma in adults and children and confers significantly more protection than salbutamol when administered 3 and 12 hours before exercise. In general, inhaled formoterol is well tolerated. The most commonly reported adverse effects, tremor and palpitations, are those traditionally associated with the use of beta 2-agonists. Oral formoterol and high doses of inhaled formoterol are associated with more adverse events than are the recommended doses of 6 to 24 micrograms. Formoterol is currently recommended for use as an alternative to increasing inhaled steroid dosage in patients whose symptoms are inadequately controlled despite therapy with low to moderate doses of inhaled steroids and intermittent short-acting beta 2-agonists, and results of recent studies support therapeutic guidelines. Long term clinical studies comparing formoterol and salmeterol have not yet been published. Further studies to evaluate the earlier use of formoterol in patients with mild to moderate asthma are needed to determine the role and long term safety of formoterol in the management of asthma.

Cellular and humoral immune responses to mycobacterial stress proteins in experimental pulmonary tuberculosis.

OBJECTIVE: Immunity to mycobacterial stress protein antigens was studied in response to vaccination and/or virulent infection. DESIGN: Guinea pigs, either vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), infected by the pulmonary route with virulent M. tuberculosis, or vaccinated then infected, were studied for the development of cellular and humoral immunity to two recombinant mycobacterial stress proteins, hsp 65 and hsp 70. RESULTS: Recombinant hsp 70 stimulated good proliferation in blood lymphocytes and, to a lesser extent, spleen and bronchotracheal lymph node lymphocytes from BCG-vaccinated guinea pigs. The proliferative responses to hsp 70 were diminished in both the spleen and lymph node cells following subsequent pulmonary challenge alone, but were boosted significantly by prior vaccination. Recombinant hsp 65 was much less active at inducing the proliferation of spleen and lymph node cells, with lowest responses observed in blood lymphocytes occurring in the cells from BCG-vaccinated, aerosol-challenged guinea pigs. Using a semi-quantitative dot blot procedure, serum antibodies to both hsp 65 and hsp 70 developed gradually following BCG vaccination, with all guinea pigs studied exhibiting significant seroreactivity after 15 weeks post-vaccination. In guinea pigs exposed to virulent M. tuberculosis by aerosol, serologic reactivity to hsp 70 was consistently stronger 6 weeks post-challenge in both vaccinated and non-vaccinated guinea pigs. In fact, 6 weeks following pulmonary exposure to M. tuberculosis in previously naive guinea pigs, 3 out of 6 animals had no detectable serum antibodies to hsp 65. Somewhat surprisingly, antibody levels to both hsp 65 and hsp 70 were only slightly increased by prior BCG vaccination in guinea pigs exposed to virulent M. tuberculosis by the respiratory route. CONCLUSION: These results demonstrate that both hsp 65 and hsp 70 stimulate detectable humoral and cell-mediated immunity in guinea pigs vaccinated and/or infected under highly relevant conditions. There is little evidence that vaccination with BCG primes the guinea pig to make an anamnestic response to hsp 65 following virulent pulmonary challenge. The precise contribution of immunity to mycobacterial stress proteins to the pathogenesis of tuberculosis in this model remains to be elucidated.

Immunosuppression and alteration of resistance to pulmonary tuberculosis in guinea pigs by protein undernutrition.

The impact of chronic moderate protein deficiency on resistance to pulmonary tuberculosis was studied in a guinea pig model. Inbred and outbred guinea pigs were maintained on isocaloric diets containing 30% or 10% ovalbumin, vaccinated with Mycobacterium bovis BCG vaccine and infected by the respiratory route with virulent Mycobacterium tuberculosis. Protein deficiency was associated with significant loss of dermal tuberculin hypersensitivity, reduced purified protein derivative (PPD)-driven lymphoproliferation in vitro and diminished interleukin-2 production. The proportion of E rosette receptor (CD2) positive lymphocytes was significantly lower in the blood and thymus of low-protein guinea pigs. Increased levels of circulating anti-PPD antibodies were associated with loss of delayed hypersensitivity in protein-deprived animals. Immune complexes containing these antibodies may act on T cells bearing Fc receptors for immunoglobulin G (T gamma cells), which appear to exert a suppressive effect on antigen-induced lymphoproliferation of Tnon-gamma cells in vitro. These results imply an important immunoregulatory role for T gamma cells in tuberculosis and suggest one mechanism whereby resistance to tuberculosis is altered in protein malnutrition.

Erythrocyte receptor (CD2)-bearing T lymphocytes are affected by diet in experimental pulmonary tuberculosis.

Outbred (Hartley) and inbred (strain 2 and 13) guinea pigs were vaccinated with Mycobacterium bovis BCG and placed on isocaloric, purified diets containing either 10 or 30% ovalbumin or on commercial guinea pig chow. Six weeks later, the animals were challenged by the respiratory route with virulent M. tuberculosis H37Rv. At intervals postchallenge, groups were tuberculin tested and sacrificed. Thymus-dependent (T) lymphocytes were enumerated either by rosette formation with rabbit erythrocytes or by the indirect immunofluorescence assay (IFA) with a guinea pig pan-T-cell monoclonal antibody, 8BE6. Protein-deficient guinea pigs of all three strains had significantly fewer erythrocyte rosette-forming (CD2+) T cells in the peripheral blood, and malnourished strain 2 and Hartley guinea pigs exhibited reduced levels of CD2+ T cells in the thymus. In contrast, animals of all three strains fed the low-protein diet harbored more CD2+ T cells in the bronchotracheal lymph nodes than did their control-fed counterparts. A larger proportion of lymphocytes from the blood and lymph nodes of all three strains were IFA positive than formed erythrocyte rosettes regardless of diet treatment. Diet had no effect on IFA-positive lymphocytes in those organs. Protein deficiency is associated with significant alterations in the number and/or distribution of T lymphocytes expressing functional CD2-receptors in BCG-vaccinated animals exposed to virulent mycobacteria by the pulmonary route. These alterations may contribute to the reduction in BCG vaccine efficacy observed in this model.

Protein malnutrition alters the distribution of Fc gamma R+ (T gamma) and Fc mu R+ (T mu) T lymphocytes in experimental pulmonary tuberculosis.

Inbred, strain 2 guinea pigs were given isocaloric diets containing either 30% (control diet) or 10% (low-protein diet) ovalbumin and infected 4 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. By using an Fc receptor rosette assay, the proportions of T lymphocytes bearing Fc receptors for immunoglobulin G (T gamma cells) or immunoglobulin M (T mu cells) were quantified in blood and lymphoid tissues taken postinfection. A significant elevation in the proportion of the putative suppressor T subset (T gamma) in the blood of protein-deprived guinea pigs was observed at all intervals postinfection. Conversely, the levels of the putative helper T subset (T mu) in the bronchotracheal lymph nodes draining the site of virulent infection in malnourished animals were significantly reduced. Diet did not influence T gamma or T mu cells in the spleens. Diet-induced loss of purified protein derivative-specific T-cell functions in tuberculosis may be associated with alterations in the proportions of or the balances between T gamma and T mu subsets.

Micronutrient status and immune function in tuberculosis.

Both macro- and micronutrients have been shown to affect resistance to tuberculosis, which is mediated by macrophages activated by T lymphocytes. Others have demonstrated inhibition of mycobacterial replication in macrophage cultures treated with vitamin D or retinoic acid. We examined the influence of dietary zinc and vitamin D on resistance to tuberculosis. Guinea pigs were fed diets containing varying levels of zinc or vitamin D, and infected 6 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. Zinc-deficient guinea pigs had fewer circulating T cells and reduced tuberculin (PPD) hypersensitivity. The response of peritoneal exudate macrophages to the lymphokine MIF was impaired. Zinc deprivation did not influence disease resistance in BCG-vaccinated or nonvaccinated animals. Vitamin D deficiency adversely affected the tuberculin reaction and ability to control the infection. Lymphocytes from vitamin D-deprived animals did not proliferate normally when cultured with PPD. A diet supplemented with vitamin D enhanced T cell responses to PPD in vivo. These results suggest that zinc and vitamin D status affect immunity to tuberculosis.

Dietary protein deficiency and Mycobacterium bovis BCG affect interleukin-2 activity in experimental pulmonary tuberculosis.

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated. They were maintained for 6 weeks on defined, isocaloric diets containing either 30% (control animals) or 10% (animals receiving low protein) ovalbumin as the sole protein source. Animals were challenged by the respiratory route with a low dose of virulent M. tuberculosis H37Rv and killed 4 weeks later. Protein-malnourished animals were not protected by previous vaccination with BCG. Lymphocytes isolated from various tissues were tested in vitro for proliferative responses to mitogen (concanavalin A) and antigen (purified protein derivative [PPD]), production of interleukin-2 (IL-2), and response to exogenous recombinant IL-2 (rIL-2). Protein-malnourished guinea pigs responded only weakly to PPD skin tests, and their blood and lymph node lymphocytes exhibited impaired proliferation when cultured with PPD in vitro. IL-2 levels were consistently low in cultures of stimulated blood and spleen lymphocytes from protein-deprived animals. BCG vaccination of nutritionally normal guinea pigs, on the other hand, induced significantly more IL-2 production by PPD- and concanavalin A-stimulated lymphocytes. The addition of exogenous mouse rIL-2 (40 and 80 U/ml) in vitro to PPD-stimulated blood and lymph node cells from nonvaccinated, protein-deprived guinea pigs resulted in no improvement of the proliferative response. Previous vaccination of malnourished guinea pigs did not consistently enhance the response of PPD-stimulated lymphocytes to added rIL-2. Dietary protein deficiency and BCG vaccination appear to modulate antigen-driven cellular immunity in animals with tuberculosis by altering the production of, and the response to, IL-2 by PPD-stimulated lymphocytes.

Impact of protein malnutrition on exogenous reinfection with Mycobacterium tuberculosis.

Malnutrition may be a predisposing host factor in the development of exogenous-reinfection tuberculosis. Outbred Hartley guinea pigs were given isocaloric diets containing either 30% ovalbumin (control animals) or 10% ovalbumin (low-protein-fed [LP] animals). Equal numbers of control and LP animals were assigned to one of three infection groups: (i) primary pulmonary infection with a low-virulence, streptomycin-resistant (LVsr) isolate of Mycobacterium tuberculosis and then reinfection 6 weeks later by the same route with a high-virulence (HV) isolate; (ii) only the primary infection (LVsr isolate); and (iii) only the secondary infection (HV isolate). Each infection resulted in the development of 4 to 12 pulmonary tubercles. Guinea pigs were skin tested with purified protein derivative and killed 6 weeks after the second infection. Protein deprivation suppressed the dermal responses to purified protein derivative in all infection groups. Primary infection of well-nourished animals with the LVsr isolate induced significant protection against infection with the HV isolate in the reinfected group, based upon the numbers of viable mycobacteria in the lung and spleen. Protein malnutrition did not exacerbate disease in the reinfected group beyond that observed in malnourished animals infected with the HV isolate only, but neither did the infection with the LVsr isolate protect the LP animals against reinfection with the HV isolate. We conclude that malnutrition interferes with the protection normally afforded by primary infection but does not result in more severe disease in reinfected individuals than would be observed in singly infected subjects.

Vaccination with Mycobacterium bovis BCG affects the distribution of Fc receptor-bearing T lymphocytes in experimental pulmonary tuberculosis.

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated and challenged 6 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. By using a double rosette assay with isotype-specific antibody-coated ox and uncoated rabbit erythrocytes, the proportions of T lymphocytes bearing Fc receptors for immunoglobulin G (IgG) (T gamma cells) or IgM (T mu cells) were quantified in tissues taken from animals that were killed within 4 weeks postchallenge. Tuberculin reactivity in vivo and in vitro and antimycobacterial resistance were also measured. BCG vaccination protected the guinea pigs and resulted in significantly enhanced proportions of T mu cells in the blood during the first 3 weeks and in the spleen during weeks 2 and 3 postchallenge. Levels of T gamma cells declined in all tissues during the first 3 weeks of infection and were unaffected by prior vaccination with BCG. Increased proportions of T mu cells in the blood were accompanied by dramatic tuberculin skin reactions and purified protein derivative-induced lymphoproliferation in BCG-vaccinated guinea pigs during the first 2 weeks following virulent pulmonary challenge. Peak levels of T mu cells in the spleens of vaccinated animals at 2 weeks coincided with the first appearance of virulent mycobacteria in that organ. BCG vaccination appears to influence immunoregulatory events in pulmonary tuberculosis through effects on the distribution of IgM Fc receptor-bearing (T mu cell) T lymphocytes.

Effects of diet and genetics on Mycobacterium bovis BCG vaccine efficacy in inbred guinea pigs.

Strain 2 and strain 13 guinea pigs were vaccinated with Mycobacterium bovis BCG and placed on low-protein or protein-adequate diets. Five weeks later all animals were infected by the respiratory route with virulent Mycobacterium tuberculosis H37Rv organisms. Four weeks postchallenge, guinea pigs were skin tested with purified protein derivative and sacrificed. Protein deficiency resulted in significant reductions in body weight and thymus weight and in an impairment in the ability to control the M. bovis BCG vaccine organisms and to mount delayed hypersensitivity reactions. Protein deficiency also adversely affected the efficacy of the BCG vaccine as demonstrated by the numbers of virulent organisms recovered in spleens and lungs. Strain differences were observed in the number of leukocytes, thymus weight, and the responsiveness of blood lymphocytes to purified protein derivative stimulation. In general, strain 13 guinea pigs responded more dramatically to dietary insult than did their strain 2 counterparts. Protein deprivation completely abolished BCG vaccine protection in the lungs and spleens of strain 13 animals and significantly reduced the protection afforded to strain 2 animals. In both strains, the BCG vaccine protected normally nourished guinea pigs. There was no significant difference between strains with respect to susceptibility to pulmonary infection with virulent mycobacteria. Thus, diet and genetic pedigree each had a significant influence on BCG vaccine efficacy.