Intermittent azithromycin for treatment of Mycobacterium avium infection in beige mice

The activity of azithromycin (AZI) was evaluated in the beige mouse model of disseminated Mycobacterium avium infection. Mice were infected intravenously with approximately 10(7) viable avium ATCC 49601. AZI at 50, 100, or 200 mg/kg of body weight or clarithromycin (CLA) at 200 mg/kg was given by gavage 5 days per week for 4 weeks. Groups of treated mice were compared with untreated control animals. A dose-related reduction in cell counts in organs was observed with AZI treatment. AZI at 200 mg/kg was more active than CLA at 200 mg/kg against organisms in spleens. The activities of these two agents at 200 mg/kg were comparable against organisms in lungs. In a second study, AZI at 200 mg/kg was given daily for 5 days; this was followed by intermittent AZI treatment for the next 3 weeks. The activities of AZI given on a three-times- and five-times-per-week basis in the continuation phase were comparable. AZI given on a once-weekly basis was less active. The regimen of AZI given in combination with rifapentine on a once-weekly basis for 8 weeks showed promising activity. Clinical evaluation of AZI and rifapentine will help to define the roles of these agents in the treatment of disseminated M. avium complex infection.     

Dietary calcium modulates Mycobacterium paratuberculosis infection in beige mice

A 6-month study was conducted to evaluate the effects of feeding different levels of dietary calcium (Ca) on the persistence of Mycobacterium paratuberculosis infection using a mouse model. Beige mice, averaging 8 weeks of age, were randomly assigned to one of the following dietary treatments: 1) 0.02% Ca, 2) 0.15% Ca, 3) 0.45% Ca, and 4) 1.0% Ca. Mice were infected intraperitoneally with 10(8) CFU viable M. paratuberculosis for 1, 3, and 6 month periods. Plasma Ca levels was unaffected by dietary Ca (x = 7.3 mg/dl). Plasma levels of 1,25(OH)2D3 was elevated significantly in 0.02% and 0.15% Ca groups compared to other treatments at the end of each period, with the highest levels observed for 0.02% Ca mice and intermediate values for 0.15% Ca mice. One month after infection, numbers of viable M. paratuberculosis cultured from the spleen were significantly reduced for 0.15% Ca mice, whereas the number of bacteria isolated from the liver and mesenteric lymph node (MLN) were higher for the 0.02% Ca group. There were no differences in bacterial numbers in the ileum although they tended to be higher for the 0.02% Ca group. Three months after infection, bacterial numbers in the spleen, ileum, and MLN did not differ across treatments, however, significantly lower numbers were found in the liver of 1.0% Ca mice. Reduced bacterial counts were also observed in the liver of 0.15%. 0.45%, and 1.0% Ca mice after a 6-month infection period compared to the 0.02% Ca group, with the lowest numbers isolated from the 1.0% Ca mice. Numbers of viable bacteria cultured from the ileum and MLN after 6 months of infection were also significantly reduced in 1.0% Ca mice. These results suggest that Ca metabolism is an important modulator of M. paratuberculosis infection.     

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

Therapeutic effects of benzoxazinorifamycin KRM-1648 administered alone or in combination with a half-sized secretory leukocyte protease inhibitor or the nonsteroidal anti-inflammatory drug diclofenac sodium against Mycobacterium avium complex infection in mice

The effects of half-sized secretory leukocyte protease inhibitor or diclofenac sodium administered alone or in combination with the benzoxazinorifamycin KRM-1648 on the therapeutic efficacy of KRM-1648 against Mycobacterium avium complex (MAC) in mice were studied. Neither of the two anti-inflammatory drugs affected the efficacy of KRM-1648, while they exerted significant modulating effects on tumor necrosis factor alpha production by MAC-infected macrophages.     

Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium

Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium. This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M. avium. There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M. tuberculosis was found to be four times greater. Examination of in vitro-generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M. avium, however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M. avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.     

Role of iron in the pathogenesis of Mycobacterium avium infection in mice

Mycobacterial infections are of serious concern to HIV-infected patients, and take a heavy toll of such patients. Mycobacterium avium is the most common opportunistic bacterial infection in patients with AIDS. The overload of iron in serum has been implicated in the pathogenicity of a number of bacterial infections. Since iron storage in cells such as macrophages is increased in AIDS, the role of iron as a possible factor in the pathogenesis of M. avium infection was examined. Supplementing iron to normal laboratory chow resulted in accelerated M. avium infection in mice inoculated earlier with the same organism. The bacterial loads in liver, spleen and lungs were approximately 12-fold higher in mice receiving iron supplementation compared with control groups. This is attributed to an increased percentage saturation of iron in the sera of the mice, thus making more iron available for the replication of bacteria. The addition of beef fat to the diet, together with high iron supplementation, further enhanced the infection. Using smaller inocula, mice receiving chow supplemented with high iron and fat developed disseminated M. avium infection faster than control mice. The results provide strong evidence that iron may play a major role in the pathogenesis of M. avium infection.     

Activity of rifabutin, clarithromycin, ethambutol, sparfloxacin and amikacin, alone and in combination, against Mycobacterium avium complex in human macrophages

Disseminated infection with Microbacterium avium complex (MAC) in patients with AIDS is currently treated with a combination of antimycobacterial agents in order to prevent the selection of resistant mutant strains. Although clinical and microbiological responses can generally be achieved within a few weeks, relapses are common and require modification of the combination regimen or identification of effective alternate therapies. In this study we investigated the activities of rifabutin 0.5 mg/L, sparfloxacin 1 mg/L, clarithromycin 4 mg/L, amikacin 16 mg/L and ethambutol 2 mg/L, alone and in combination, against nine strains of M. avium isolated from the blood of patients with AIDS in order to identify regimens with the greatest therapeutic potential. Macrophages derived from human monocytes were infected with M. avium and inoculated with a single drug or a combination of drugs; cfu counts were performed at 0, 4 and 7 days after infection. At day 4 and at day 7, the combination of rifabutin, clarithromycin, amikacin and sparfloxacin displayed the highest degree of activity. However, the activity did not differ significantly from that of the combination of rifabutin, clarithromycin and ethambutol. The results of this study confirm the activity of combinations including rifabutin and clarithromycin (+/- ethambutol) in human monocyte-derived macrophages and suggest potentially useful associations in incorporating sparfloxacin and amikacin.     

Mycobacterium avium complex infection in mice: lack of exacerbation after LP-BM5 murine leukemia virus infection

The murine leukemia virus LP-BM5 has been used to reproduce the model of murine AIDS in order to evaluate the course of infection with the MO-1 strain of Mycobacterium avium complex (MAC). LP-BM5 was inoculated in C57BL/6 mice by intravenous (i.v.) injection either 8 weeks before an i.v. challenge with 10(3) or 10(6) CFU of MAC (coinfection 1) or 10 days after an i.v. challenge with 10(3) CFU of MAC (coinfection 2). During coinfection 2 experiments, the phenotypic alterations in blood lymphocyte subsets were analyzed. During coinfection 1, LP-BM5 infection tended to decrease the mycobacterial growth, with the difference reaching statistical significance for the lower inoculum (10(3) CFU of MAC) (P<0.001). During coinfection 2, LP-BM5 did not exacerbate MAC infection except in the spleen, at day 90 after LP-BM5 challenge (P<0.001). LP-BM5 infection and the LP-BM5-MAC coinfection increased the numbers of activated CD4+ lymphocytes (CD4+ Ly6AE+) (P<0.001), activated CD8+ lymphocytes (CD8+ Ly6AE+) (P<0.001), and activated B lymphocytes (Ly5+ Ly6AE+) (P<0.001). This activation of T lymphocytes could explain the lack of exacerbation of MAC infection and even the trend to a lower level of MAC infection. Thus, this model of retroviral infection of mice does not seem to be a reliable model of immunodepression for the study of MAC infection and its treatments.     

Inhibition of tumor necrosis factor alpha alters resistance to Mycobacterium avium complex infection in mice

Increased production of tumor necrosis factor alpha (TNF-alpha) appears to play an important role in the progression of human immunodeficiency virus disease. One treatment strategy being explored is the use of TNF-alpha inhibitors. TNF-alpha also appears to be important in conferring resistance to infections, and the inhibition of this cytokine may exacerbate the emergence of opportunistic pathogens, such as Mycobacterium avium complex (MAC). The present study examines the possibility that inhibition of TNF-alpha will increase the progression of disease in mice infected with MAC. C57BL/6 beige (bg/bg) mice have been shown to be highly susceptible to infection with MAC and are routinely used for testing of antimycobacterial drugs. However, bg/bg mice are known to exhibit impaired phagocyte and natural killer cell function. Since these cell types are important sources of TNF-alpha, the susceptibility of the bg/bg strain to infection with MAC was compared with those of the heterozygous (bg/+) and wild-type (+/+) strains of C57BL/6 mice. The susceptibilities of the bg/bg and bg/+ strains of mice infected with MAC were found to be comparable. The +/+ strain was the least susceptible. Mycobacterial burden and serum TNF-alpha levels increased over time in all the strains of mice tested. The bg/+ strain of C57BL/6 mice was then chosen to measure the activity of TNF-alpha antagonists. Treatment with dexamethasone decreased serum TNF-alpha levels and increased mycobacterial burden. Treatment with anti-TNF-alpha antibody or pentoxifylline did not significantly alter serum TNF-alpha levels but increased mycobacterial burden. Treatment with thalidomide neither consistently altered mycobacterial burden in the spleens or livers of infected mice nor affected serum TNF-alpha levels.     

Low calcium diet and 1,25-dihydroxyvitamin D(3) infusion modulate immune responses during Mycobacterium paratuberculosis infection in beige mice

A 12-month study was conducted to evaluate the effects of feeding a low calcium (Ca) diet or 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3) infusion on the persistence of Mycobacterium paratuberculosis infection using a mouse model. Male beige mice 6-8 weeks of age were assigned to one of the following treatments: (1) non-infected, (2) infected,(3) non-infected/1,25(OH)(2)D(3), (4) infected/1,25(OH)(2)D(3), and (5) infected/low Ca (0.15 percent) diet. Infected mice were inoculated intravenously with live M. paratuberculosis. At 1, 6 and 12 months postinfection, mice in Treatments 3 and 4 were implanted subcutaneously with mini-osmotic pumps to deliver 1,25(OH)(2)D(3). Infusion with 1,25(OH)(2)D(3) exacerbated M. paratuberculosis infection in most tissues at all time points. Mice infused with 1,25(OH)(2)D(3) had higher bacterial counts in spleen, liver, and ileum compared with control infected mice after 1 month of infection. In contrast, feeding a low Ca diet reduced the number of viable organisms cultured from the liver and ileum of infected mice. Plasma Ca and 1,25(OH)(2)D(3) were increased in mice infused with 1,25(OH)(2)D(3) at all time points but values for low Ca mice were not different than for non-infused mice. Splenocyte production of TNF, IL-1 and IL-6 was higher for mice fed the low Ca diet compared with control infected mice after 1 month of infection. Inducible IL-6 activity remained higher for this treatment at 6 months postinfection. These results suggest that feeding a low Ca diet to mice chronically infected with M. paratuberculosis appears to enhance their ability to clear the infection in a manner distinct from any effect of 1,25(OH)2D3.     

Arthritis caused by use of rifabutine++ in Mycobacterium avium infection

A 60-year-old HIV-seronegative man with a Mycobacterium avium pulmonary infection was treated with rifabutin, ethambutol and clarithromycin. He developed a serious arthritis which disappeared after interruption of the medication and recurred after resumption. The arthritis was attributed to the use of rifabutin. This adverse effect is more frequent with higher doses and in combination with the use of macrolide antibiotics.     

In vitro activities of several diaminomethylpyridopyrimidines against Mycobacterium avium complex

Three recently synthesized dihydrofolate reductase (DHFR) inhibitors designated SoRI 8890, 8895, and 8897 were evaluated for their in vitro activities against 25 isolates of Mycobacterium avium complex. The MICs at which 50 and 90% of isolates were inhibited were 1 and 2, 4 and 8, and 4 and 8 microgram/ml for SoRI 8890, 8895, and 8897, respectively. Although the addition of dapsone at 0.5 microgram/ml did not significantly enhance the in vitro activities of these compounds, their activities alone were comparable to, if not better than, results seen with other DHFR inhibitors, such as pyrimethamine or WR99210.     

Clinical and bacteriologic impact of rifabutin prophylaxis for Mycobacterium avium complex infection in patients with human immunodeficiency virus infection

Carcinoma of the oesophagus is the seventh most common malignancy worldwide. It is a disease with a poor prognosis; more than half of the patients present with surgically irresectable tumours. For such patients, palliative therapy is directed towards the relief of dysphagia. Expandable metallic stents have recently been developed for use in the oesophagus. These have the advantage of being introduced through small diameter delivery catheters. Once released, they can expand to as much as 25 mm in diameter, potentially allowing patients to consume a normal diet. The current designs of metallic stents include the Strecker stent, the Wallstent endoprosthesis, and the Gianturco-Rosch stent. The Strecker is an uncovered stent while the other two are covered on the outside of the stent with plastic to prevent tumour ingrowth. A review of the literature indicates that deployment of these stents is associated with a high technical success rate. Improvement in swallowing function is seen in 83% to 100% of these patients. The overall complication rates are low. However, covered stents are prone to migration while uncovered stents are vulnerable to tumour ingrowth. Further improvements in design promise to expand the role of these endoprostheses in the management of oesophageal carcinoma.