The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) promote HIV type 1 viral replication in vitro. In the present studies, HIV production was increased in the macrophagic U1 cell line expressing the HIV genome after exposure to IL-1beta, osmotic stress, or surface adhesion, suggesting a confluence of signaling pathways for proinflammatory cytokines and cell stressors. The p38 mitogen-activated protein kinase (MAPK) mediates both cytokine and stress responses; thus the role of this kinase in HIV production was investigated. HIV production as measured by p24 antigen correlated with changes in the expression of a specific (non-alpha) isoform of p38 MAPK. In the presence of a specific p38 MAPK inhibitor (p38 inh), IL-1beta-induced HIV production was suppressed by more than 90% and IL-1beta-induced IL-8 production was suppressed completely, both with IC50 of 0.01 microM. p38 inhibition blocked cell-associated p24 antigen and secreted virus to a similar extent. The p38 inh also decreased constitutive HIV production in freshly infected peripheral blood mononuclear cells by up to 50% (P < 0.05). Interruption of p38 MAPK activity represents a viable target for inhibition of HIV.

METHODS

A major concern surrounding the use of tumor necrosis factor-alpha (TNF-alpha) inhibitors is their potential to increase the risk of opportunistic infections, particularly tuberculosis (TB).

OBJECTIVE

To estimate the incidence of active TB in patients with rheumatic diseases receiving anti-TNF drug therapy and to evaluate the effectiveness of an antituberculosis chemoprophylaxis regimen.

METHODS

Retrospective study of the files of 613 patients with rheumatic diseases who had received anti-TNF agent (etanercept, infliximab and adalimumab) therapy from July 2000 to June 2004 at the Aristotle University of Thessaloniki, Greece. All patients had a tuberculin skin test (TST) and a postero-anterior chest radiograph (CXR) prior to anti-TNF therapy. When indicated (TST>> or =10 mm and/or fibrotic lesions on CXR), treatment for latent TB was established (6 months isoniazid [INH] or 3 months INH and rifampicin [RMP]). Anti-TNF agent therapy was started again 2 months later.

RESULTS

Of 45 patients who fulfilled the criteria for chemoprophylaxis, only 36 were treated correctly. Eleven patients developed active TB 2-35 months after the beginning of anti-TNF therapy. Six patients developed pulmonary and five extra-pulmonary TB. Eight of these had received infliximab and three adalimumab.

CONCLUSIONS

The incidence of active TB in this study population was estimated at 449 cases per 100,00 population annually. Anti-tuberculosis chemoprophylaxis was only of partial preventive success in these patients.

Airway submucosal gland serous cells express the cystic fibrosis transmembrane conductance regulator (CFTR) and secrete antimicrobial, anti-inflammatory, and antioxidant molecules. In cystic fibrosis, diminished gland secretion may impair innate airway host defenses. We used Calu-3 cells as a serous cell model to study the types of proteins released, the pathways that release them, and the possible involvement of CFTR activity in protein release. Many proteins were secreted constitutively into the apical fluid and showed increased release to agonists. We identified some of them by high pressure liquid chromatography-mass spectrometry and reverse transcriptase PCR, including lysozyme, siderocalin (the protein NGAL), which inhibits bacterial growth by binding iron-containing siderophores, HSC-71, which is thought to have anti-inflammatory properties, and the serine protease inhibitors alpha-1-antitrypsin and alpha-1-antichymotrypsin, which may function as antimicrobials as well as play a potential role in diminishing the activation of epithelial Na(+) channels by serine proteases. We used an enzyme-linked immunosorbent assay to quantify lysozyme secretion by Calu-3 cells in response to various agonists and inhibitors. Forskolin increased the lysozyme secretion rate (J(lyz)) from 32 to 77 ng/hr/cm(2) (n = 36, p < 0.005). Thapsigargin increased J(lyz) from 40 to 63 ng/h/cm(2) (n = 16, p < 0.005), and forskolin plus thapsigargin further increased the forskolin-stimulated J(lyz) by 48% (n = 9, p < 0.05). 1-Ethyl-benzimidazolinone and carbachol were less effective. Glibenclamide inhibited basal and stimulated J(lyz), but clotrimazole was without effect. CFTR(inh)172 caused a small (15%) but significant inhibition of forskolin-stimulated J(lyz) without affecting basal J(lyz). Thus, Calu-3 cells secrete diverse proteins that in aggregate would be expected to suppress microbial growth, protect the airways from damage, and limit the activation of epithelial Na(+) channels via serine proteases.

We report characterization of the soluble form of the low density lipoprotein receptor-related protein (sLRP) which circulates in human plasma. Amino acid sequence analysis confirmed that sLRP isolated from human plasma contains the alpha-chain of LRP1. In addition, Western blot analysis identified a truncated beta-chain noncovalently associated with the purified alpha-chain. The molecular size (M(r) 55K) of the peptide portion of the truncated beta-chain indicates that the subunit comprises the extracellular portion of the beta-chain and terminates in a membrane-proximal region. We investigated the mechanism by which sLRP may be generated using the trophoblast cell line, BeWo, which releases sLRP in culture. Cell surface labeling experiments indicate that LRP is released from BeWo cells following expression at the cell surface. Incubation of BeWo cells in the presence of a metalloproteinase inhibitor, INH-3855-PI, results in a dose-dependent inhibition of LRP shedding. The metalloproteinase responsible for the shedding of LRP by BeWo cells is not up-regulated by phorbol ester and is not dependent on serine proteases, such as plasmin, for activity. The BeWo cell line is derived from a human gestational choriocarcinoma and preliminary studies suggest that LRP may be shed within the placenta during gestation. Increased levels of sLRP were detected in cord blood. In term placenta, LRP is expressed in the syncytium, which comprises the maternal-fetal interface. Increased levels of sLRP in cord blood may reflect cellular dysfunction and increased metalloproteinase activity at this important interface.

Antibodies are considered to play a specific pathogenic role in certain disease states such as myasthenia gravis, Graves' disease and autoimmune haemolytic anaemia. Autoantibodies which interfere with the function of enzyme cascade systems have also been described in diseases such as acquired haemophilia (anti-factor VIII antibodies) and glomerulonephritis (C3 nephritic factor). The identification of these autoantibodies is crucial to an understanding of the aetiology of such diseases and is also of importance in revealing the inter-relationships of the immune system with other biological pathways. This is the first report of an immunoglobulin G (IgG) autoantibody reactive with C1-inhibitor (C1-Inh), a pivotal inhibitor of the inflammatory response which is known to inactivate proteins of the complement, kinin, fibrinolytic and 'contact phase' systems. This autoantibody was isolated from a patient with a novel variant of acquired angioedema and C1-Inh dysfunction. This finding highlights the involvement of the immune system in the pathogenesis of disorders characterized by the presence of dysfunctional inflammatory response proteins.

Inhibition of the enzyme Mycobacterium tuberculosis InhA (enoyl-acyl carrier protein reductase) due to formation of an isonicotinoyl-NAD adduct (IN-NAD) from isoniazid (INH) and nicotinamide adenine dinucleotide cofactor is considered central to the mode of action of INH, a first-line treatment for tuberculosis infection. INH action against mycobacteria requires catalase-peroxidase (KatG) function, and IN-NAD adduct formation is catalyzed in vitro by M. tuberculosis KatG under a variety of conditions, yet a physiologically relevant approach to the process has not emerged that allows scrutiny of the mechanism and the origins of INH resistance in the most prevalent drug-resistant strain bearing KatG[S315T]. In this report, we describe how hydrogen peroxide, delivered at very low concentrations to ferric KatG, leads to efficient inhibition of InhA due to formation of the IN-NAD adduct. The rate of adduct formation mediated by wild-type KatG was about 20-fold greater than by the isoniazid-resistant KatG[S315T] mutant under optimal conditions (H2O2 supplied along with NAD+ and INH). Slow adduct formation also occurs starting with NADH and INH, in the presence of KatG even in the absence of added peroxide, due to endogenous peroxide. The poor efficiency of the KatG[S315T] mutant can be enhanced merely by increasing the concentration of INH, consistent with this enzyme's reduced affinity for INH binding to the resting enzyme and the catalytically competent enzyme intermediate (Compound I). Origins of drug resistance in the KatG[S315T] mutant enzyme are analyzed at the structural level through examination of the three-dimensional X-ray crystal structure of the mutant enzyme.

Systematic thin layer chromatographic (TLC) analysis of apolar lipids in Mycobacterium kansasii revealed the presence of a previously uncharacterized novel component. The product was ubiquitously found in a panel of M. kansasii clinical isolates, as well as other pathogenic and non-pathogenic mycobacterial species. TLC analysis of [(14)C]-acetate- or [(14)C]-glycerol-labelled M. kansasii cultures tentatively assigned the novel product as an unusual triacylglycerol-related lipid. Subsequent purification, followed by structural determination using (1)H-nuclear magnetic resonance (NMR) and electrospray mass spectrometry (ES/MS), led to the identification of this product as a monomeromycolyl-diacylglycerol (MMDAG). Treatment of M. kansasii with either isoniazid (INH), a well-known type II fatty acid synthase (FAS-II) and mycolic acid biosynthesis inhibitor, or tetrahydrolipstatin (THL), a drug approved for treating obesity, correlated with a reduced incorporation of [(14)C]-acetate into both mycolic acids and MMDAG. Addition of INH or THL to the cultures induced major morphological changes and, surprisingly, resulted in an increased number of lipid storage bodies, as determined by electron microscopy. The potent antimycobacterial activity of THL was confirmed against a variety of mycobacterial species, including INH-susceptible and -resistant Mycobacterium tuberculosis strains. Therefore, THL and other beta-lactones may be promising drugs for the development of new antitubercular therapy.

Isoxyl (ISO), a thiourea (thiocarlide; 4, 4'-diisoamyloxythiocarbanilide), demonstrated potent activity against Mycobacterium tuberculosis H37Rv (MIC, 2.5 micrograms/ml), Mycobacterium bovis BCG (MIC, 0.5 microgram/ml), Mycobacterium avium (MIC, 2.0 microgram/ml), and Mycobacterium aurum A+ (MIC, 2.0 microgram/ml), resulting in complete inhibition of mycobacteria grown on solid media. Importantly, a panel of clinical isolates of M. tuberculosis from different geographical areas with various drug resistance patterns were all sensitive to ISO in the range of 1 to 10 microgram/ml. In a murine macrophage model, ISO exhibited bactericidal killing of viable intracellular M. tuberculosis in a dose-dependent manner (0.05 to 2.50 microgram/ml). The selective action of ISO on mycolic acid synthesis was studied through the use of [1, 2-14C]acetate labeling of M. tuberculosis H37Rv, M. bovis BCG, and M. aurum A+. At its MIC for M. tuberculosis, ISO inhibited the synthesis of both fatty acids and mycolic acids (alpha-mycolates by 91.6%, methoxymycolates by 94.3%, and ketomycolates by 91.1%); at its MIC in M. bovis BCG, ISO inhibited the synthesis of alpha-mycolates by 87.2% and that of ketomycolates by 88.5%; and the corresponding inhibitions for M. aurum A+ were 87.1% for alpha-mycolates, 87.2% for ketomycolates, and 86.5% for the wax-ester mycolates. A comparison with isoniazid (INH) and ethionamide (ETH) demonstrated marked similarity in action, i.e., inhibition of the synthesis of all kinds of mycolic acids. However, unlike INH and ETH, ISO also inhibited the synthesis of shorter-chain fatty acids. ISO showed no acute toxicity against primary macrophage cell cultures as demonstrated by diminution of redox activity. A homologous series of ISO derivatives were synthesized. Most derivatives were as effective or more effective than the parent compound in the agar proportion assay. Thus, these thioureas, like INH and ETH, specifically inhibit mycolic acid synthesis and show promise in counteracting a wide variety of drug-sensitive and -resistant strains of M. tuberculosis.

Previous studies demonstrated that C1-inhibitor (C1-INH), a complement and contact-kinin systems inhibitor, is neuroprotective in cerebral ischemia. To investigate the mechanism of this action, we evaluated the expression of neurodegeneration and inflammation-related factors in mice subjected to 2-h ischemia and 2 or 46 h reperfusion. C1-INH significantly dampened the mRNA expression of the adhesion molecules P-selectin and ICAM-1 induced by the ischemic insult. It significantly decreased the pro-inflammatory cytokine (TNF alpha, IL-18) and increased the protective cytokine (IL-6, IL-10) gene expression. C1-INH treatment prevented the decrease of NFH gene, a marker of cellular integrity and counteracted the increase of pro-caspase 3, an apoptosis index. Furthermore, C1-INH markedly inhibited the activation and/or recruitment of microglia/macrophage, as shown by immunohistochemistry. In conclusion, C1-INH exerts an anti-inflammatory and anti-apoptotic action on ischemia-reperfusion injury. Our present and past data support a major effect of C1-INH on cell recruitment from the vasculature to the ischemic site.

Numerous recent findings indicate the involvement of a neuroinflammatory reaction in the neurodegeneration in idiopathic Parkinson's disease (PD). We examined 29 consecutive patients with PD, ages 54-84 years, most of whom were moderately impaired (median UPDRS 19; Hoehn-Yahr 3; MMSE 28). A series of serum biomarkers were investigated, and their levels were correlated with the degree of the motor and cognitive impairment. There were no abnormalities of IL-6, acute phase proteins (C-reactive protein, serum amyloid A, alpha 1-antitrypsin, orosomucoid, ceruloplasmin, alpha 2-macroglobulin, transferrin, prealbumin) and factors of the complement system (C1q, C1-INH, C3, C4). A decrease in Mannan-binding lectin (MBL) levels was observed in six patients; an elevation of tumor necrosis factor-alpha (TNF-alpha) was found in 12 patients. No statistically significant correlation was found between the patient's clinical state (neuropsychologic and motor, as expressed by UPDRS III, Hoehn-Yahr, and MMSE) and the immunomarker changes. Our results indicate that the inflammatory process may be reflected in the serum; nevertheless, further research is needed to elucidate the possible clinical implications.

BACKGROUND

B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion.

METHODS

We have developed a new generation of DNA-like compounds named class R inhibitory oligonucleotides (INH-ODNs). We tested their effectiveness in autoimmune B cells and interferon-alpha-producing dendritic cells in vitro and in lupus-prone MRL-Faslpr/lpr mice in vivo.

RESULTS

Class R INH-ODNs have 10- to 30-fold higher inhibitory potency when autoreactive B cells are synergistically activated through the BCR and associated TLR7 or 9 than when stimulation occurs via non-BCR-engaged TLR7/9. Inhibition of TLR9 requires the presence of both CCT and GGG triplets in an INH-ODN, whereas the inhibition of the TLR7 pathway appears to be sequence-independent but dependent on the phosphorothioate backbone. This difference was also observed in the MRL-Faslpr/lpr mice in vivo, where the prototypic class R INH-ODN was more effective in curtailing abnormal autoantibody secretion and prolonging survival.

CONCLUSIONS

The increased potency of class R INH-ODNs for autoreactive B cells and dendritic cells may be beneficial for lupus patients by providing pathway-specific inhibition yet allowing them to generate protective immune response when needed.

BACKGROUND

Isoniazid (INH), a key drug of antituberculosis therapy, is metabolized by arylamine N-acetyltransferase2 (NAT2), cytochrome P450 2E1 (CYP2E1) and glutathione S-transferase (GST). We studied the possible influence of genetic polymorphisms of INH-metabolizing enzymes on serum concentrations of INH and its metabolites, as well as on the incidence of hepatotoxicity.

METHODS

A total of 144 tuberculosis patients who received antituberculosis treatment were followed prospectively. Their NAT, CYP2E1 and GST genotypes were determined using a polymerase chain reaction with restriction fragment length polymorphism method. Blood samples were collected from the patients and serum concentrations were determined by HPLC. The severity of hepatotoxicity was judged by the increases in either aspartate aminotransferase or alanine aminotransferase levels from the upper limit of the corresponding normal range.

RESULTS

Incidence of hepatotoxicity was highest in pulmonary tuberculosis patients with the slow acetylator (SA) phenotype and lowest in those with the rapid acetylator (RA) phenotype, although no clear relationship of genetic polymorphism of INH-metabolizing enzymes on the severity of hepatotoxicity were confirmed.

CONCLUSIONS

The risk of side-effects, such as hepatic disorder, may rise in these patients with an SA phenotype, because of an increase in serum INH concentration. The evidence presented in this study, albeit based on the examination of a low number of patients, suggests that a safe INH dosage for tuberculosis patients with SA phenotype should be less than the dosage which is usually recommended for tuberculosis patients.

To clarify the possible link between radicals and the cytotoxicity of eugenol-related compounds, 2-allyl-4-X-phenols (2-allyl-4-chlorophenol (1), 2-allyl-4-phenylphenol (2), 2-allyl-4-methoxyphenol (3), 2-allyl-4-acetylphenol (4), 2-allyl-4-nitrophenol (5), 2-allyl-4-t-butylphenol (6), 2-allyl-4-methyphenol (7), 2-allyl-4-bromophenol (8), 2,4-dimethoxyphenol (9)), and dimeric compounds from eugenol (4-allyl-2-methoxyphenol), BHA (2-t-butyl-4-methoxyphenol) or MMP (2-methoxy-4-methylphenol); bis-EUG (3,3'-dimethoxy-5,5'-di-2-propenyl-1, 1'-biphenyl-2,2'-diol) (10), bis-MMP (3,3'-dimethoxy-5,5'-dimethyl-1,1'-biphenyl-2,2'-diol) (11) bis-BHA (3,3'-di-t-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol) (12) were synthesized. The radical production, radical-scavenging activity and the cytotoxicity of these synthetic compounds and conventional antioxidants (i.e. butylhydroxytoluine, BHT; butylhydroxyanisole, BHA; alpha-tocopherol (alpha-Toc); eugenol, phenol) were studied. Erectron spin resonance (ESR) spectroscopy suggested that compounds of 3, 6, 9, eugenol and BHA, but not compounds of 10, 11, and 12 produced radicals in alkaline solutions (pH>9.5) and compounds, 3, eugenol and 9 most efficiently scavenged reactive oxygen species (ROS, O(2)(-)). The cytotoxic activity of 6 toward human submandibular gland carcinoma (HSG) cells was the highest and was 1000-fold greater than that of eugenol and 100-fold greater than that of BHA, possibly due to the high hydrophobicity and stable phenoxy radicals of this compound. The kinetic polymerization method in the presence of methyl methacrylate (MMA), an antioxidant, and 2,2'-azobisisobutyronitrile (AIBN) was developed for the measurements of the number of moles of peroxy radicals trapped by moles of the relative phenols (stoichiometric factors, n), the inhibition rate of polymerization (R(inh)), and the inhibition rate constants (k(inh), the rate constants for scavenging of radicals by an antioxidant). The n values of conventional phenolic antioxidants decreased in the order: alpha-Toc>BHT>eugenol>phenol. Those for eugenol and phenol, less hindered phenols, were much less than two, whereas those for alpha-Toc and BHT, hindered phenols, were approximately two. The R(inh) of alpha-Toc significantly increased tcompared with that of BHT, eugenol and phenol. The k(inh) of the polymer radicals of the MMA reaction with conventional phenolic antioxidants was a low value of 1-2x10(2) M(-1) s(-1), suggesting that the antioxidants trapped radicals quickly. The comparative cytotoxicity of methoxyphenols against HSG cells was investigated. The cytotoxic activity of dimers of 10 and 12 was markedly lower than that of their corresponding monomers, whereas that of the dimer of MMP, 11 was not reduced even after the dimerization. In particular, visible-light (VL) exposure enhanced the cytotoxicity of 11 similar to the monomers of eugenol, BHA and MMP. Changes in BDE (ph(O-H)) (homolytic bond dissociation energy) for phenols is well known to be associated with the n and k(inh) values, and consequently the cytotoxic activity. Thus, the BDE was calculated using a PM3 semiempirical method. The n and k(inh) values for monophenols, but not for dimers were correlated to the BDE, possibly due to the steric hindrance of orthosubstituents of dimers. The quantitative structure-activity relationship (QSAR) of eugenol-related compounds was investigated, indicating that logP (octanol-water partition coefficients), the redox potential measured in culture medium, was effective as a term for QSAR. A parabolic relation between the cytotoxic activity and the logP or the redox potential, but not the BDE was observed with an optimum value. In conclusion, the cytotocity of eugenol-related compounds was significantly associated with the activity of the production of phenoxyl radicals, their stability of the subsequent quinonemethide (QM) and the hydrophobicity.

The complement system is an essential component of host defense against pathogens. Previous research in our laboratory identified StcE, a metalloprotease secreted by Escherichia coli O157:H7 that cleaves the serpin C1 esterase inhibitor (C1-INH), a major regulator of the classical complement cascade. Analyses of StcE-treated C1-INH activity revealed that surprisingly, StcE enhanced the ability of C1-INH to inhibit the classical complement-mediated lysis of sheep erythrocytes. StcE directly interacts with both cells and C1-INH, thereby binding C1-INH to the cell surface. This suggests that the augmented activity of StcE-treated C1-INH is due to the increased concentration of C1-INH at the sites of potential lytic complex formation. Indeed, removal of StcE abolishes the ability of C1-INH to bind erythrocyte surfaces, whereas the proteolysis of C1-INH is unnecessary to potentiate its inhibitory activity. Physical analyses showed that StcE interacts with C1-INH within its aminoterminal domain, allowing the unaffected serpin domain to interact with its targets. In addition, StcE-treated C1-INH provides significantly increased serum resistance to E. coli K-12 over native C1-INH. These data suggest that by recruiting C1-INH to cell surfaces, StcE may protect both E. coli O157:H7 and the host cells to which the bacterium adheres from complement-mediated lysis and potentially damaging inflammatory events.

Inhibin (INH) is a gonadal glycoprotein hormone that regulates pituitary FSH secretion and may also play a role in the regulation of androgen biosynthesis. There are two forms of inhibin that strongly inhibit pituitary FSH secretion. These share the same alpha subunit that is covalently linked to one of two distinct beta subunits (beta A or beta B). However, dimers of two beta subunits are potent stimulators of FSH synthesis and release in vitro. The beta subunits share extensive sequence similarity with transforming growth factor beta. Recently isolated cDNAs for all three inhibin subunits have been used to map their cognate loci on human and mouse chromosomes by Southern blot analysis of somatic cell hybrid DNAs and by in situ hybridization. INH alpha and INH beta B genes were assigned to human chromosome 2, regions q33----qter and cen----q13, respectively, and to mouse chromosome 1. The INH beta A locus was mapped to human chromosome 7p15----p14 and mouse chromosome 13. The region of mouse chromosome 1 that carries other genes known to have homologs on human chromosome 2q includes the jsd locus (for juvenile spermatogonial depletion). Adult jsd/jsd mice have elevated levels of serum FSH and their testes are devoid of spermatogonial cells. The possibility that the mutation in jsd involves the INH alpha or INH beta B gene was investigated by Southern blotting of DNA from jsd/jsd mice, and no major deletions or rearrangements were detected.

The currently recommended daily dose of ethambutol (EMB) for the treatment of tuberculosis (TB) in children varies from a maximum daily dose of 15 mg/kg body weight daily (without a range) to 15-20 mg/kg and 20 mg/ kg (range 15-25 mg/kg). Published evidence relating to the dosage, toxicity and pharmacokinetics of EMB in children and adults is reviewed and a dose of EMB for use in childhood is recommended. Using key words 'ethambutol', 'childhood', 'TB', 'pharmacokinetics', 'bioavailability' and 'toxicity', Medline searches were conducted; cross-references were sought from original papers, books and conference proceedings dating from 1961. When English summaries were available, data were extracted from papers in languages other than English. EMB has a dose-related efficacy best seen when given to adults alone or with a single other drug. Together with isoniazid (INH), a dose of 15 mg/kg EMB gave better results than 6 mg/kg, and 25 mg/kg better than 15 mg/kg. The occurrence of ocular toxicity was also dose-related; >40% of adults developed toxicity at doses of >50 mg/ kg, and 0-3% at a dose of 15 mg/kg/daily. Peak serum EMB concentrations increase in relation to dose, but are significantly lower in children receiving the same dosage. In only 2 of 3811 children (0.05%) receiving EMB doses of 15-30 mg/kg was EMB stopped due to possible ocular toxicity; children of all ages can be given EMB in daily doses of 20 mg/kg (range 15-25 mg/kg) and three times weekly intermittent doses of 30 mg/kg body weight without undue concern.

Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD(+)/NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-Tyr-Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t-BuOOH and H(2)O(2)), superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct.

High expression in cancer 1 (Hec1) is an oncogene overly expressed in many human cancers. Small molecule inhibitor of Nek2/Hec1 (INH) targeting the Hec1 and its regulator, Nek2, in the mitotic pathway, was identified to inactivate Hec1/Nek2 function mediated by protein degradation that subsequently leads to chromosome mis-segregation and cell death. To further improve the efficacy of INH, a series of INH analogues were designed, synthesized, and evaluated. Among these 33 newly synthesized analogues, three of them, 6, 13, and 21, have 6-8 fold more potent cell killing activity than the previous lead compound INHand 21 were chosen for analyzing the underlying action mechanism. They target directly the Hec1/Nek2 pathway and cause chromosome mis-alignment as well as cell death, a mechanism similar to that of INHal exploration of structural/functional relationship of INH may advance the progress for developing clinically applicable INH analogue.

Transforming growth factor beta 1 (TGF-beta 1) is the prototype of an increasingly complex superfamily of growth and differentiation factors. To date, a total of 74 TGF-beta-like sequences have been published, probably representing 23 distinct genes. These sequences were obtained from mammalian, avian, amphibian and insect species, thus emphasising the ancient nature of the TGF-beta superfamily peptides. This article summarises current hypotheses concerning the evolutionary history of this protein superfamily, based on the molecular phylogeny of the published sequences. Comparison of the deduced amino acid sequences leads to the definition of five main groups within the superfamily (TGF-beta, Bone Morphogenetic Proteins [BMP], Anti-Müllerian Hormone [AMH], Inhibin alpha [INH alpha] and GDF-9) and six subgroups within the BMPs (60A, Decapentaplegic [dpp], Vg1, BMP-3, Inhibin beta [INH beta A/B] and nodal). This classification predicts possible phylogenetic and functional relationships among these proteins.

Endotoxin shock is a life-threatening syndrome associated with a Gram-negative infection and mediated by a systemic inflammatory response. As a major effector of inflammation, the complement system has been implicated in both the pathogenesis and the protection from endotoxin shock. To clarify the role of complement in endotoxin shock, we have used mice totally deficient in either complement component C3 or C4. We found that both the C3- and C4-deficient mice were significantly more sensitive to endotoxin than wild-type controls. The endotoxin-challenged complement-deficient mice failed to clear endotoxin efficiently from the circulation and this led to excess consumption of C1 inhibitor protein (C1 INH), a major regulator of both complement and the contact system of blood coagulation. Replacement of C1 INH rescued the endotoxin-challenged complement-deficient mice from shock and death. These findings suggest a novel therapy for treatment of endotoxemia with C1 INH protein.

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8-9.4 (peak 9.0-9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt alpha-globulin which was isolated free of alpha(1)-chymotrypsin inhibitor, inter alpha-trypsin inhibitor, alpha(2)-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to alpha(1)-antitrypsin, and it was absent in alpha(1)-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as alpha(1)-antitrypsin.

The use of OCAs has been associated with multiple hemostatic abnormalities and an increased risk of thromboembolic disease. These changes have been attributed to increased synthesis of various clotting factors and decreased titers of antithrombin III. Paradoxically, enhanced in vitro fibrinolytic activity is also found in plasmas of women using OCAs. The present study demonstrates marked elevation of both procoagulant and antigenic HF titers in plasmas of women using OCAs, accompanied by a simultaneous decrease in plasma C-1-INH concentration. Titers of plasma prekallikrein, HMW kininogen, PTA, and alpha 2-Pl were unchanged. The rate of kaolin-assisted fibrinolysis was related directly to the titer of HF and inversely to C-1-INH concentration. Further, the addition of human HF to normal plasma enhanced fibrinolytic activity to a degree similar to that observed in plasmas of women taking OCAs. These data suggest that the increase in plasma HF concentration may participate in the phenomenon of enhanced in vitro fibrinolysis associated with OCA use, possibly augmented by diminished inhibitory control by C-1-INH. The relationship of these phenomena to the increased incidence of thrombosis is not known.

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.

INH-resistant mutants of Mycobacterium aurum and M. smegmatis were isolated and characterized in an attempt to provide fresh insight into the activity of isoniazid (INH), a key antibiotic in the treatment of tuberculosis. In both cases, high levels of resistance were accompanied by slower growth rate, by loss of peroxidase and reduced catalase activities, although mycolic acid production was unaffected. A gene homologous to the katG gene of M. tuberculosis, encoding peroxidase-catalase, was detected in wild-type and INH-resistant strains and it appears that INH resistance may stem from the loss of its product.