Induction of granulomas in interferon-gamma gene-disrupted mice by avirulent but not by virulent strains of Mycobacterium tuberculosis

Tumor necrosis factor-alpha (TNF-alpha) levels were measured in the serum (sTNF-alpha) or bone marrow (BM) biopsies of 43 patients with myelodysplastic syndromes (MDS) who subsequently received therapy with a combination of pentoxifylline and ciprofloxacin (PC) with or without dexamethasone (PCD). All 43 patients received only PC therapy for 12 weeks, after which 18 of 36 nonresponders received PCD. A total of 18 of 43 patients showed a hematologic or cytogenetic response or both. BM TNF-alpha levels were semiquantitatively assessed using immunohistochemistry on a scale of 0-8+ and in the serum using enzyme linked immunoassay. The median TNF-alpha for the entire group was 3.0 in BM and 6.9 pg/ml in the serum, and 14 patients had no detectable levels. Responders had higher BM levels (median 3.5 vs. 2.0) than nonresponders, although this was not statistically significant. During PC therapy, a decline in BM TNF-alpha level was seen in the entire group, which was significant at 2 weeks (p = 0.02), 8 weeks (p = 0.001), and 12 weeks (p = 0.0001). Both responders (p = 0.01) and nonresponders (p = 0.03) had a decline at 8 weeks, but at 12 weeks, only the responders continued to show a significant decline (p = 0.03). We conclude that MDS patients with high BM TNF-alpa levels have a better chance of responding to PCD therapy and that the therapy is quite successful in reducing the TNF-alpha levels in a sustained fashion. Future studies need to be directed at identifying agents that would be more potent suppressors of the proapoptotic cytokines in these patients.     

Specific differentiation between Mycobacterium bovis BCG and virulent strains of the Mycobacterium tuberculosis complex

A PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying Mycobacterium bovis BCG. The senX3-regX3 IR contains a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs within the senX3-regX3 IR, whereas all non-BCG M. tuberculosis complex strains contained a 53-bp MIRU, in addition to the 77-bp MIRUs. All 148 strains analyzed so far could be divided into eight different groups according to the copy numbers of the 77-bp MIRU and to the presence or absence of the 53-bp MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The other strains contained one to five 77-bp MIRUs invariably followed by a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains and its presence in virulent strains allowed us to develop an enzyme-linked immunosorbent assay using specific capture oligonucleotide probes to distinguish between BCG and other M. tuberculosis complex strains.     

Diminished adherence and/or ingestion of virulent Mycobacterium tuberculosis by monocyte-derived macrophages from patients with tuberculosis

The interaction between the macrophage and Mycobacterium tuberculosis is mediated by a variety of macrophage membrane-associated proteins. Complement receptors have been implicated in the adherence of M. tuberculosis to macrophages. In the present work, the adherence and/or ingestion of M. tuberculosis H37Rv to human monocyte-derived macrophages (MDM) from patients with tuberculosis (TB) and healthy controls was measured by microscopical examination, [3H]uracil incorporation, and CFU. The adherence and/or ingestion was enhanced by fresh serum and inhibited by heat inactivation, EDTA treatment, and anti-CR1 and anti-CR3 antibodies. Comparison of MDM from TB patients and healthy controls showed that the former exhibited a significantly decreased capacity to adhere and/or ingest M. tuberculosis, as determined by the number of CFU and 3H incorporation. The expression of CR1 (CD35) and CR3 (CD11b/CD18) on MDM from TB patients and healthy controls, as determined by flow cytometry, did not show significant differences. These results suggest that the lower ingestion of M. tuberculosis by MDM from TB patients is not due to defects in complement receptors, and therefore, there might be other molecules involved in the adherence and/or ingestion process that render MDM from TB patients ingest less mycobacteria than those from healthy controls.     

Cytokine induction in murine macrophages infected with virulent and avirulent Rhodococcus equi

To look for a possible correlation between the virulence of Rhodococcus equi and its cytokine-inducing capacity, we evaluated intracellular survival and measured cytokine induction by mouse macrophages infected with a virulent strain containing an 85-kb plasmid and expressing VapA (103+), its avirulent plasmid-cured derivative (103-), and heat-killed 103+ (HK). After incubation with similar numbers of bacteria, macrophages infected with 103- contained significantly more organisms than those infected with 103+ or HK. The number of bacteria in the macrophages infected with 103- and HK decreased progressively, whereas the 103+ numbers remained constant over 48 h. Interleukin 1beta (IL-1beta), IL-6, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-alpha) mRNA induction peaked at 4 h and returned to baseline between 12 and 48 h postinfection. IL-1beta, IL-6, IL-10, and TNF-alpha concentrations assessed by enzyme-linked immunosorbent assay generally agreed well with mRNA expression; IL-12 could, however, not be detected. For all the cytokines detected, mean concentrations in the supernatants were consistently higher in the 103(-)-infected monolayers than in those infected with 103+, although, with the exception of IL-1beta, the differences were not statistically significant. R. equi HK was a poor inducer of cytokine production. In conclusion, virulent and avirulent R. equi strains induced similar levels of cytokine synthesis. The slightly greater induction of most cytokines observed following infection with 103- is likely secondary to greater uptake by macrophages rather than to a direct role of VapA or another plasmid-encoded product in downregulating cytokine induction.     

Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis-specific cytolytic activity is mediated mostly by CD4+CTL in humans. CD4+CTL kill infected target cells by inducing Fas (APO-1/CD95)-mediated apoptosis. We have examined the effect of Fas ligand (FasL)-induced apoptosis of human macrophages infected in vitro with M. tuberculosis on the viability of the intracellular bacilli. Human macrophages expressed Fas and underwent apoptosis after incubation with soluble recombinant FasL. In macrophages infected either with an attenuated (H37Ra) or with a virulent (H37Rv) strain of M. tuberculosis, the apoptotic death of macrophages was associated with a substantial reduction in bacillary viability. TNF-induced apoptosis of infected macrophages was coupled with a similar reduction in mycobacterial viability, while the induction of nonapoptotic complement-induced cell death had no effect on bacterial viable counts. Infected macrophages also showed a reduced susceptibility to FasL-induced apoptosis correlating with a reduced level of Fas expression. These data suggest that apoptosis of infected macrophages induced through receptors of the TNF family could be an immune effector mechanism not only depriving mycobacteria from their growth environment but also reducing viable bacterial counts by an unknown mechanism. On the other hand, interference by M. tuberculosis with the FasL system might represent an escape mechanism of the bacteria attempting to evade the effect of apoptosis.     

A polymorphism in a DNA polymerase alpha gene intron differentiates between murine virulent and avirulent strains of Toxoplasma gondii

The IC intron, found within the DNA polymerase alpha gene of Toxoplasma gondii, was used to evaluate the genetic relationship among 10 strains of T. gondii. Sequence comparison detected polymorphisms within this 652 bp intron which correlated with murine virulence. The results reported here suggest that T. gondii contains two lineages, corresponding with their virulence, evolving independently following their separation. The extensive homology of the IC sequences within the virulent and avirulent groups affirms the close relationship of the strains within the group, as reflected by the identical nucleotide substitutions and dinucleotide insertions/deletions observed. In addition, the presence of the Nde I restriction enzyme site within the IC intron of avirulent strains allows definition of a T. gondii strain as murine virulent or avirulent without needing to test it in vivo.     

Lipoarabinomannan inhibits nonopsonic binding of Mycobacterium tuberculosis to murine macrophages

The initial phagocytic interaction between Mycobacterium tuberculosis and macrophages (M phi) in the lung is probably nonopsonic, which would mean that M phi receptors will bind directly to bacterial ligands without the involvement of serum opsonins. Lipoarabinomannan (LAM) is a major component of the cell wall of mycobacteria. The possibility that LAM is involved in the nonopsonic binding of M. tuberculosis to M phi was investigated by using competitive inhibition assays. LAM inhibited binding of M. tuberculosis to murine peritoneal M phi in a dose-dependent manner. LAM also inhibited the binding of Mycobacterium avium and Mycobacterium bovis BCG to M phi. Phosphatidylinositol mannoside and lipomannan have the same phosphatidylinositol (PI) moiety as LAM, but differ in their glycosylation patterns. Both molecules inhibited binding of M. tuberculosis to M phi. Deacylation of LAM abrogated its capacity to inhibit binding of M. tuberculosis to M phi. These observations indicated that it was the PI moiety of LAM that was important in mediating its inhibitory properties. In support of this hypothesis, commercial PI was shown to inhibit the binding of M. tuberculosis to M phi. Our results suggest that cellfree LAM is able to inhibit the binding of mycobacteria to M phi, but that it does not do so by competing with any interaction between M phi receptors and cell-associated LAM, because the PI end of the molecule is believed to be anchored in the bacterial plasma membrane, and therefore not available as a ligand on the cell surface. However, LAM that is released from M. tuberculosis in the course of its active replication during infection may be able to interfere with the phagocytic clearance of mycobacteria.     

Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis

Intracellular parasites (e.g., Mycobacterium tuberculosis, Toxoplasma gondii, and some Chlamydiae) may promote their survival within the host by acting from within phagosomes to prevent phagolysosome formation, thus avoiding exposure to the lysosomal hydrolases. The present studies demonstrate that when sulfatides of M. tuberculosis (anionic trehalose glycolipids largely responsible for the neutral red reactivity of virulent strains) are administered to cultured mouse peritoneal macrophages, they accumulate in the secondary lysosomes, which are rendered incompetent for fusion with phagosomes containing suitable target particles such as viable yeasts. This antifusion effect is also exhibited when small amounts of sulfatide are introduced directly into phagosomes by attachment to the target yeasts prior to their ingestion. The sulfatides evidently exert a selective inhibitory influence on membrane fusion, analogous to what occurs typically when macrophage cultures are infected with tubercle bacilli. This effect may be due to ionic interaction between the polyanionic micelles of bacterial sulfatide and organelle membranes, modifying the latter and inducing dysfunction.     

Relationship between streptomycin susceptibility and rpsL mutations of Mycobacterium tuberculosis strains

The relationship between streptomycin (SM) susceptibility and rpsL mutations of Mycobacterium tuberculosis strains was studied. Of 18 clinically isolated SM-resistant M.tuberculosis strains, mutation was suspected in 9 strains (50%) with SM MICs of > or = 256 micrograms/ml by PCR-single strand conformation polymorphism targeting rpsL gene. On the other hand, using PCR-direct sequence method, amino acid substitution caused by single nucleotide point mutation in rpsL gene was demonstrated in 11 out of 18 strains (61%). The same amino acid substitution at codon 43 (Lys-->Arg) was observed in all 11 strains with SM MICs of > or = 256 micrograms/ml. In addition, PCR products obtained from these 11 strains could not be cut by a restriction enzyme, Mbo II, while H37Rv strain and the other 32 strains with SM MICs of < 256 micrograms/ml were cut into 2 fragments. In conclusion, our results suggest that highly SM-resistant M.tuberculosis strains with MICs of > or = 256 micrograms/ml could be rapidly and easily detected by the restriction enzymatic method.     

Growth of human cytomegalovirus in primary macrophages

An enzyme with a cholinesterase (ChE) activity, produced by Pseudomonas fluorescens, was purified to homogeneity in a three-step procedure. Analysis by non-denaturing and SDS-PAGE, and by isoelectric focusing, indicated that the enzyme was a monomer of 43 kDa, with a pI of 6.1. The N-terminal sequence, AEPLKAVGAGEGQLDIVAWPGYIEA, showed some similarities with proteins of the ChE family and a strong similarity with a protein from Escherichia coli with unknown structure and function. Cholinesterase activity at pH 7.0 and 25 degreesC was maximum with propionylthiocholine as substrate (kcat,app=670 min-1), followed by acetylthiocholine, and significantly lower with butyrylthiocholine. Catalytic specificity (kcat/Km) was the same for propionylthiocholine and acetylthiocholine, but was two orders of magnitude lower for butyrylthiocholine. Kinetics of thiocholine ester hydrolysis showed inhibition by excess substrate which was ascribed to binding of a second substrate molecule, leading to non-productive ternary complex (Km=35 microM, KSS=0.49 mM with propionylthiocholine). There was low or no reactivity with organophosphates and carbamates. The enzyme inhibited by echothiophate (kII=0.44x102 M-1 min-1) was not reactivated by pralidoxime methiodide. However, the P. fluorescens enzyme had affinity for procainamide and decamethonium, two reversible ChE inhibitors used as affinity chromatography ligand and eluant, respectively. Although similarity of the N-terminal amino acid sequence of the enzyme with an internal sequence of ChEs is weak, its catalytic activity towards thiocholine esters, and its affinity for positively charged ligands supports the contention that this enzyme may belong to the ChE family. However, we cannot rule out that the enzyme belongs to another structural family of proteins having cholinesterase-like properties. The reaction of the enzyme with organophosphates suggests that it is a serine esterase, and currently this enzyme may be termed as having a cholinesterase-like activity.     

Modulation of macrophage heat shock proteins (HSPs) expression in response to intracellular infection by virulent and avirulent strains of Leishmania donovani

We have examined the differential expression of heat shock proteins of murine macrophage-like cell line J774 G8 following infection with Leishmania donovani. In response to infection with virulent promastigotes, the up-regulation of HSP70 and 90 and a selective down-regulation of HSP60 was observed using monoclonal antibodies specific for host HSPs. However, infection with avirulent strain failed to alter the expression of host HSPs. The maximum alterations in HSPs expression were noted at 18 h post infection, a time period which coincided with the transformation of parasite from promastigote to the amastigote form. Data indicates that host HSPs may play a role in parasite differentiation/survival during infection with L. donovani.     

Mycobacterium avium complex and Mycobacterium tuberculosis in patients infected with the human immunodeficiency virus

Primary care physicians play an important role in identifying and treating bacterial infections in adults infected with the human immunodeficiency virus (HIV). Mycobacterium avium complex and Mycobacterium tuberculosis are pathogens that can cause systemic or local infection in these patients. We review the epidemiology, pathogenesis, clinical presentation, and principles of treatment for these two mycobacterial pathogens. Because M tuberculosis disease is preventable and curable and yet communicable, physicians should maintain a high degree of suspicion for tuberculosis in HIV-infected adults. In comparison, the goal of treating M avium complex in patients with advanced HIV disease is to reduce constitutional symptoms and improve survival.     

Similar responses by macrophages from young and old mice infected with Mycobacterium tuberculosis

Repeated ethanol withdrawal experience has been shown to result in exacerbated seizures associated with future withdrawal episodes. This sensitization of the withdrawal response has been postulated to represent a "kindling" phenomenon. The present study employed an established model of repeated ethanol withdrawals to examine the potential role of GABA(A), and NMDA and non-NMDA glutamate receptor systems in mediating enhanced seizure activity, as assessed by sensitivity to seizures induced by pentylenetetrazol (PTZ), NMDA, and kainic acid (KA) i.v. infusions, respectively. Adult C3H mice were chronically exposed to ethanol vapor in inhalation chambers. A multiple withdrawal (MW) group received four cycles of 16-h ethanol vapor exposure interrupted by 8-h periods of abstinence; a single withdrawal (SW) group was tested after a single 16-h bout of ethanol intoxication; and the third group was ethanol-naive, serving as controls (C). Results indicated that the MW group evidenced significantly lower PTZ and NMDA seizure thresholds compared to SW and C groups at 8 and 24 h post-withdrawal. In contrast, MW and SW groups exhibited reduced sensitivity (higher seizure threshold) to KA in comparison to controls, and this effect only emerged at 24 h post-withdrawal. Further, MW mice required significantly less additional PTZ or NMDA to induce more severe convulsions once initial signs of seizures were elicited. Conversely, latency and amount of KA required to transition from initial seizure signs to more severe end-stage convulsions was significantly greater for MW and SW groups compared to controls. Taken together, these results suggest that repeated ethanol withdrawal experience does not result in a global non-specific lowering of threshold to convulsive stimuli, but rather, selective changes in CNS mechanisms associated with neural excitability may underlie potentiated withdrawal responses. Thus, reduced GABA(A) receptor function and increased NMDA receptor activity may become exaggerated as a consequence of repeated withdrawal experience, while reduced sensitivity to KA induced seizures may represent a compensatory response to withdrawal-related CNS hyperexcitability.     

Drug-resistant strains of Mycobacterium tuberculosis exhibit a range of virulence for mice

A panel of clinical isolates of Mycobacterium tuberculosis, several of which were resistant to one or more antimycobacterial drugs, were tested for their capacity to give rise to active disease following aerogenic infection of normal immunocompetent mice. The panel exhibited a range of virulence in this model, which followed no clear trend in terms of geographical source, degree of drug resistance, or rate of growth in vitro. Several isolates grew very quickly over the first 20 days in mouse lungs before being contained by emerging immunity. In view of this latter observation, we hypothesize that it is possible that such so-called fast growers may be responsible for the rapid fatality sometimes seen in immunocompromised patients with tuberculosis. Moreover, the results of the study do not support the belief that increased drug resistance usually associates with loss of virulence of the isolate.