A post-genomic perspective.

The complete genome sequence of Mycobacterium tuberculosis, along with novel genetic tools, provides the foundation for a new era of post-genomic research. The challenge is now to translate these opportunities into an improved understanding of the complex biology of tuberculosis infection.

Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection.

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.

Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine.

The current vaccine against tuberculosis, Mycobacterium bovis strain bacille Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for the expression and delivery of protective recombinant antigens (Stover, C.K., V.F. de la Cruz, T.R. Fuerst, J.E. Burlein, L.A. Benson, L.T. Bennett, G.P. Bansal, J.F. Young, M.H. Lee, G.F. Hatfull et al. 1991. Nature [Lond]. 351:456; Jacobs, W.R., Jr., S.B. Snapper, L. Lugosi and B.R. Bloom. 1990. Curr. Top. Microbiol. Immunol. 155:153; Jacobs, W.R., M. Tuckman, and B.R. Bloom. 1987. Nature [Lond.]. 327:532); but as an attenuated intracellular bacterium residing in macrophages, BCG would seem to be best suited for eliciting cellular responses and not humoral responses. Since bacterial lipoproteins are often among the most immunogenic of bacterial antigens, we tested whether BCG expression of a target antigen as a membrane-associated lipoprotein could enhance the potential for a recombinant BCG vaccine to elicit high-titered protective antibody responses to target antigens. Immunization of mice with recombinant BCG vaccines expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi as a membrane-associated lipoprotein resulted in protective antibody responses that were 100-1,000-fold higher than responses elicited by immunization with recombinant BCG expressing OspA cytoplasmically or as a secreted fusion protein. Furthermore, these improved antibody responses were observed in heterogeneous mouse strains that vary in their immune responsiveness to OspA and sensitivity to BCG growth. Thus, expression of protective antigens as chimeric membrane-associated lipoproteins on recombinant BCG may result in the generation of new candidate vaccines against Lyme borreliosis and other human or veterinary diseases where humoral immunity is the protective response.

A serological test for leprosy with a glycolipid specific for Mycobacterium leprae.

A phenolic glycolipid from Mycobacterium leprae was purified and used as antigen in an enzyme-linked immunosorbent assay. Antibodies directed against the lipid were seen in serums from leprosy patients but not in serums from uninfected controls or patients infected with other mycobacteria, including Mycobacterium tuberculosis. The antibody response distinguished between the Mycobacterium leprae lipid and the structurally related phenolic glycolipid from Mycobacterium kansasii. This assay has considerable potential as a specific serodiagnostic test for leprosy infection.

IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Analysis of long-term potassium regulation.

Potassium regulation is maintained by a system which affects the rate of renal excretion of the ion and its distribution between the intra- and extracellular spaces. Long-term regulation is accomplished by the interactions of several components of the control system. The direct effect of changes in plasma potassium concentration on potassium secretion by the cells of the distal nephron is the most powerful regulator of K excretion. Changes in aldosterone concentration interact multiplicatively with the effect of plasma K on K excretion, and changes in aldosterone also affect the distribution of the ion so that elevations in aldosterone shift a greater proportion of K into the cells of the body. Sodium intake affects K excretion, increases in intake resulting in a higher rate of K excretion. Aldosterone secretion is regulated by a multiplicative interaction between angiotensin II and potassium concentration. An hypothesis that states in essence that long-term K regulation is determined by the interaction of several component systems is developed. A mathematical model constructed from the hypothesis employing data from experiments designed to quantitatively analyze the functions of the individual components is presented. The model is used to test the hypothesis by comparing the operation of the model with the results of experiments in which the potassium control system is subjected to a wide range of challenges and perturbations. General agreement between model predictions and experimental results was obtained, thereby providing support for the validity of the hypothesis. Building the model drew attention to several areas in which experimental investigation is required to obtain understanding of several important physiological processes. Finally, several predictions obtained from the model may have clinical relevance in the areas of renal medicine and hypertension.

Tuberculosis. Beating the bacillus.

The identification of the target of isoniazid in the mycobacterial tuberculosis pathogen opens the way for the developoment of new drugs and diagnostic tests to combat drug-resistant pathogen strains.

Heat-shock proteins: immunity and autoimmunity.

Antigens from a wide variety of pathogens have been identified as members of conserved heat-shock protein families, sharing upwards of 50% amino acid identity with corresponding host-cell proteins. Analysis of the responses to these conserved antigens may provide insights into regulation of the immune system during infection and autoimmunity.

IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.

Effect of skin testing and segregation on the prevalence of bovine tuberculosis, and molecular typing of Mycobacterium bovis, in Ethiopia.

In 2002, the prevalence of bovine tuberculosis (tb) among 500 cattle on Holeta Farm, near Addis Ababa, Ethiopia, was 48 per cent, and the farm was divided into positive and negative herds. After three consecutive rounds of skin testing and segregation of skin test-positive and -negative animals, the prevalence of bovine tb was reduced from 14 per cent to 1 per cent in the negative herd in a year. Spoligotyping of 41 isolates from 17 cows gave an identical and unique spoligotype pattern, which can be represented as the binary number 1100000101111110111111100010000000000100000, where 1 indicates the presence of a spacer and 0 represents a loss. This spoligotype pattern had not previously been reported on the Mycobacterium bovis spoligotype database, and it was therefore designated SB1176, Ethiopian M bovis strain 1 (EMbs1). The variable number tandem repeat (VNTR) profile of the strain was 5254(*)33.1, which differed from the VNTR profile of strains reported in Great Britain.

Frequency and implications of pyrazinamide resistance in managing previously treated tuberculosis patients.

OBJECTIVE: To determine the extent of pyrazinamide (PZA) resistance in isolates from previously treated patients from the Western Cape, South Africa. DESIGN: Drug-resistant isolates, isolates resistant to one or more drugs other than PZA (PZA resistance is not routinely determined) (n = 127), and drug-susceptible (n = 47) clinical isolates of Mycobacterium tuberculosis from previously treated patients from the Western Cape were phenotypically (BACTEC MGIT 960) and genotypically (pncA gene sequencing) analysed for PZA resistance. RESULTS: MGIT analysis found that 68 of the 127 drug-resistant isolates were PZA-resistant. Nearly all (63/68) PZA-resistant isolates had diverse nucleotide changes scattered throughout the pncA gene, and five PZA-resistant isolates had no pncA mutations. Of the 47 phenotypically susceptible isolates, 46 were susceptible to PZA, while one isolate was PZA-monoresistant (OR = 53.0, 95% CI = 7.1-396.5). A pncA polymorphism (Thr114Met) that did not confer PZA resistance was also identified. PZA resistance was strongly associated with multidrug-resistant tuberculosis (MDR-TB). CONCLUSION: An alarmingly high proportion of South African drug-resistant M. tuberculosis isolates are PZA-resistant, indicating that PZA should not be relied upon in managing patients with MDR-TB in the Western Cape. A method for the rapid detection of PZA resistance would be beneficial in managing patients with suspected drug resistance.

Leprosy lipid provides the key to Schwann cell entry.

A recent study has demonstrated that the species-specific phenolic glycolipid of Mycobacterium leprae triggers uptake into Schwann cells by interaction with laminin-2 and the alpha-dystroglycan receptor. This finding emphasizes the importance of lipids in the biology of mycobacterial infection and suggests possible strategies to combat nerve damage in leprosy.

Molecular mechanisms of isoniazid: a drug at the front line of tuberculosis control.

The resurgence of tuberculosis and emergence of multidrug-resistant isolates has focused attention on the need for an improved understanding of molecular aspects of the disease, and for elucidation of the factors responsible for drug action and resistance. Recent research has probed the mechanism of action of isoniazid (INH), a key drug in the chemotherapy of tuberculosis.

Chronic hypokalemia and the left ventricular responses to epinephrine and preload.

The effects of moderate, chronic (5 days) potassium depletion on cardiac function were assessed in 14 normokalemic and 13 hypokalemic open chest, anesthetized dogs. Cardiac responses to intravenous bolus injection of 2.5 micrograms/kg body weight epinephrine (10 normokalemic and 11 hypokalemic dogs) and to rapidly increased preload (8 dogs in each group) were evaluated. Hypokalemic dogs received a low potassium diet plus chlorthalidone. Plasma potassium levels were lower (p less than 0.001) in the hypokalemic dogs (3.2 +/- 0.1 mEq/liter [mean +/- SEM]) than in the normokalemic dogs (4.1 +/- 0.1). The inotropic response to epinephrine was lower in hypokalemic than in normokalemic dogs, the response of the maximal rate of rise of left ventricular pressure was 20% greater (p less than 0.03) and the response of the peak rate of change of ejection power was 60% greater in the normokalemic dogs. The relaxation response to epinephrine (the maximal rate of fall of left ventricular pressure) was 33% lower (p less than 0.02) in hypokalemic dogs. Responses to rapid volume expansion were impaired by hypokalemia; maximal stroke volume index was 31% lower (p less than 0.01), maximal cardiac index was 26% lower (p less than 0.01) and the peak response to the maximal rate of filling was 51% lower (p less than 0.01). There were no differences in basal cardiac function. Therefore, modest potassium depletion within the clinical range impaired the contractile and relaxation responses to epinephrine and preload and impaired rapid filling.

Comparison of the breadth and complexity of bovine viral diarrhea (BVDV) populations circulating in 34 persistently infected cattle generated in one outbreak.

Exposure to bovine viral diarrhea viruses (BVDV) results in acute and persistent infections. Persistent infections result from in utero exposure during the first trimester of gestation. Clinical presentation, in persistently infected cattle (PI), is highly variable. The reasons for this variation is largely unknown. The BVDV circulating in PI exist as quasispecies (swarms of individual viruses). An outbreak resulting in 34 PI cattle presented an opportunity to compare a large number of PI׳s. Methods were developed to compare the circulating viral populations within PI animals. It was found that PI animals generated in the same outbreak carry circulating viral populations that differ widely in size and diversity. Further, it was demonstrated that variation in PI viral populations could be used as a quantifiable phenotype. This observation makes it possible to test the correlation of this phenotype to other phenotypes such as growth rate, congenital defects, viral shed and cytokine expression.

The antihypertensive mechanism of verapamil. Alteration of glomerular filtration rate regulation.

The renal hemodynamic and renin release responses to verapamil were analyzed to determine if the antihypertensive action of the calcium entry blocker could be due to its renal effects. Hemodynamic and renin release measurements were compared in a control group of nine anesthetized rabbits and in a group of 10 rabbits given verapamil (200 micrograms/kg i.v. initially, 4 micrograms/kg/min thereafter), starting 30 minutes before data collection. Measurements were made over a range of controlled renal perfusion pressure from 100 to 40 mm Hg. The renal blood flow at 100 mm Hg of the verapamil-treated group was 18% greater (p less than 0.02) than that of the control group, while the glomerular filtration rate was 51% greater (p less than 0.001) than that of the control group. Renal blood flow and glomerular filtration rate autoregulation were highly effective in the control group down to 80 mm Hg, but both variables were poorly regulated in the verapamil-treated group. The filtration fraction of the treated group was 36.9 +/- 1.5% versus 28.5 +/- 1.6% in the control group (p less than 0.003) at 100 mm Hg, and the filtration fraction of the treated group remained significantly greater down to 40 mm Hg. Renin release rates of the two groups were similar at the 100 mm Hg pressure level, but the increase in release due to the progressive reduction in perfusion pressure was significantly greater in the treated group than in the control group. At the 80 mm Hg pressure level, the mean release rate for the treated group was more than three times greater (p less than 0.05) than that of the control group. These findings demonstrate that verapamil is an effective renal vasodilator and that the effect is proportionally greater on the preglomerular than on the postglomerular resistance. This action could be the basis for its antihypertensive efficacy.

Experimental aldosterone hypertension in the dog.

Sequential changes in arterial pressure, renal function, body fluid, and electrolyte balance, and several hemodynamic variables were examined during chronic intravenous infusion of aldosterone (14 micrograms/kg/day) in eight conscious dogs maintained on 250 mEq/day sodium and 140 mEq/day potassium intake. Arterial pressure gradually increased and stabilized at 132% +/- 3% (p less than 0.05) of the control value on the 16th day of aldosterone infusion, and cardiac output remained within the normal range. Coinciding with the rise in arterial pressure on the first 2 days of infusion was a marked retention of water and sodium and a rise in extracellular fluid volume and blood volume. Blood volume increased from a baseline value of 64.0 +/- 0.3 ml/kg to 70.7 +/- 1.9 ml/kg (p less than 0.05) on Day 4 and extracellular fluid volume increased from 318 +/- 5 ml/kg to 352 +/- 11 ml/kg (p less than 0.05) on Day 3 of infusion. Both blood volume and extracellular fluid volume remained elevated during infusion. Mean circulatory filling pressure increased from the baseline volume of 9.7 +/- 0.4 mm Hg to an average of 11.7 +/- 0.3 mm Hg (p less than 0.05) during the experimental period. The elevation of mean circulatory filling pressure suggested that this increase may be an essential component in the onset and maintenance of hypertension.

Inhibition of vascular smooth muscle cell migration by elevation of extracellular potassium concentration.

The effect of potassium on the migration of vascular smooth muscle cells was analyzed in media made with extracellular potassium concentrations of 3, 4, 5, and 6 mmol/L. The migration of cultured porcine coronary artery cells was stimulated with platelet-derived growth factor (PDGF)-BB. In the first study, cells were exposed to PDGF-BB at concentrations of 0, 10, or 20 ng/mL for 5 hours with the use of a Boyden chamber. Cells were quiescent overnight in 0.5% fetal bovine serum in Dulbecco's modified Eagle's medium with an extracellular potassium concentration of 4 mmol/L. With increasing potassium concentration, migration was significantly inhibited (P<0. 02, 2-way ANOVA). In the cells exposed to 10 ng/mL PDGF-BB, migration ranged from 500+/-86% to 294+/-44% (value in wells with 0 ng/mL PDGF-BB and 4 mmol/L potassium concentration=100%) in medium containing 3 to 6 mmol/L extracellular potassium concentration (P<0. 03). Long-term potassium exposure was investigated in cells grown in 5% serum in Dulbecco's modified Eagle's medium with an extracellular potassium concentration of 3, 4, 5, or 6 mmol/L for 3 to 4 weeks. Migration was assessed with 0 or 20 ng/mL PDGF-BB. Migration was significantly inhibited by the elevation of extracellular potassium concentration (P<0.01, 2-way ANOVA). With 20 ng/mL PDGF-BB, the migration rates ranged from 152+/-11% in medium with 3 mmol/L potassium to 69+/-5% in 6 mmol/L potassium (P<0.01). Increases in extracellular potassium concentration within the physiological range significantly and directly inhibit vascular smooth muscle cell migration.

Roles of prostaglandins and nitric oxide in the effect of endothelin-1 on renal hemodynamics.

It is known that endothelin-1 stimulates the release of nitric oxide and prostaglandins in various vascular beds. We designed the present study to analyze the roles of prostaglandins and nitric oxide in the effect of endothelin-1 on the regulation of renal hemodynamics and renin release. We used N omega-nitro-L-arginine methyl ester (L-NAME) and meclofenamic acid to inhibit the production of nitric oxide and prostaglandins, respectively. With a nonfiltering kidney model, renal blood flow was reduced 21% in dogs treated with L-NAME and 18% in dogs treated with meclofenamic acid. Inhibition of nitric oxide and prostaglandins, however, produced opposite effects on estimated glomerular hydraulic pressure: L-NAME increased glomerular hydraulic pressure from 63.1 +/- 0.9 to 64.6 +/- 1.3 mm Hg (P < .01), and meclofenamic acid reduced glomerular hydraulic pressure from 63.3 +/- 1.4 to 59.8 +/- 1.6 mm Hg (P < .01). Endothelin-1 infusion produced a dose-dependent reduction in renal blood flow after blockade of nitric oxide and prostaglandins. The responses of glomerular hydraulic pressure were different in the two groups during endothelin-1 infusion. Endothelin-1 progressively reduced glomerular hydraulic pressure in a dose-dependent fashion in the meclofenamic acid group. However, endothelin-1 slightly increased glomerular hydraulic pressure until the infusion rate reached 5.0 ng/kg per minute. At that rate, endothelin-1 reduced glomerular hydraulic pressure from 63.3 +/- 1.4 to 47.0 +/- 1.4 mm Hg in the meclofenamic acid group (P < .01), a more than 25% reduction, whereas at the same dose, endothelin-1 reduced glomerular hydraulic pressure only less than 2% in the L-NAME group. In addition, blockade of nitric oxide and prostaglandins did not alter the inhibitory effect of endothelin-1 on renin release in the non-filtering kidney. Therefore, the present study demonstrates that the release of nitric oxide and prostaglandins might modulate the effects of endothelin-1 on the renal circulation. The present findings suggest that the differential vasoconstrictive effects of endothelin-1 on preglomerular and postglomerular vessels are associated with its stimulation of nitric oxide and prostaglandin production.