Inhalable microparticles containing a large payload of isoniazid (INH) and rifabutin (RFB) in equal proportions show extremely high efficacy against experimental animal tuberculosis (TB). It was investigated whether inhaled microparticles affect the cytokine environment in the lung lumen, and cytokine secretion by airway and lung macrophages recovered from mice infected with Mycobacterium tuberculosis (Mtb). We attempted to determine whether the cytokine environment of the mouse lung receives significant contribution by lung macrophages, and whether these macrophages maintain a profile of cytokine secretion that is consistent with the cytokine environment of the lung. Groups of mice were infected intravenously with Mtb H37Ra and treated with (a) 5 mg/Kg each of INH and RFB administered by oral gavage; or, (b) 2.5 mg/Kg of the same and an additional ∼2.5 mg/Kg in the form of inhaled microparticles; or, (c) ∼2.5 mg/Kg by inhalation alone. Bronchioalveolar lavage (BAL) was carried out and recovered macrophages cultured. BAL Fluid and culture supernatants were assayed for tumor necrosis factor (TNF-α), interferon (IFN-γ), interleukin (IL)-12 and IL-10 by ELISA and amounts compared with both infected and uninfected, untreated controls. Inhaled microparticles enhanced secretion of TNF-α and supported IFN-γ secretion despite upregulated IL-10. Oral chemotherapy with the same drugs enhanced IL-12 and downregulated TNF-α. Differences in cytokine profiles suggest distinct effects of drug delivery modalities on innate immune strategies mobilized during host response. These differences might account, in part, for the extraordinary efficacy of the microparticles.

Phenolic antioxidants of the hydroxychroman class, alpha-tocopherol (alpha-TOC) and 2,2,5,6,7-pentamethyl-6-hydroxychroman (PMHC), and the hindered phenols 2,3-dihydro-5-hydroxy-2,2,4-trimethylnaphtho[1,2-b]furan (NFUR), 2,6-di-tert-butyl-4-methoxyphenol (DBHA), and 2,6-di-tert-butyl-4-methyl phenol (BHT), were delivered into oxidizable (ACCEPTOR) liposomes of dilinoleoylphosphatidylcholine (DLPC) or 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) from saturated DONOR liposomes of dimyristoylphosphatidylcholine (DMPC) by liposomal transfer. The antioxidant activities, k(inh), by the inhibited oxygen uptake method were compared with the k(inh)s determined when the antioxidants were introduced into the liposomes by coevaporation from organic solvents. The peroxidations were initiated using either thermal initiators, water-soluble azo-bis-amidinopropane hydrochloride (ABAP), lipid-soluble azo-bis-2,4-dimethylvaleronitrile (ADVN) and di-tert-butylhyponitrite (DBHN), or the photoinitiator benzophenone. The antioxidants PMHC, NFUR, DBHA, and BHT transferred rapidly between liposomes, but several hours of incubation were needed to transfer alpha-TOC. The average k(inh)s in liposomes, in the relative order NFUR approximately DBHA>> PMHC>> BHT approximately alpha-TOC, were markedly lower than known values in organic solvent. k(inh) values in liposomes appear to be controlled by effects of hydrogen bonding with water and by restricted diffusion of antioxidants, especially in the case of alpha-TOC. Product studies of the hydroperoxides formed during inhibited oxygen consumption were carried out. The cis,trans/trans,trans (c,t/t,t) product ratios of the 9- and 13-hydroperoxides formed from PLPC during inhibited peroxidation by PMHC were similar for both the coevaporated and liposomal transfer procedures. The c,t/t,t ratio for the same concentration of alpha-TOC, 1.52, compares to a value of 1.69 for PMHC at the start of the inhibition period. The higher c,t/t,t ratio observed for NFUR in DLPC, which varied between values of 7.0 at the start of the inhibition to about 1.8 after the break in the induction period, is a reflection of the increased hydrogen atom donating ability of the antioxidant plus the increased concentration of oxidizable lipid provided by DLPC.

G-protein-coupled receptors (GPCRs) such as the vasopressin-2 receptor (V(2)R) are an important class of drug targets. We developed an efficient screen for GPCR-induced cAMP elevation using as read-out cAMP activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. Fischer rat thyroid cells expressing CFTR and a halide-sensing yellow fluorescent protein (H148Q/I152L) were transfected with V(2)R. Increased cell Cl(-) conductance after agonist-induced cAMP elevation was assayed using a plate reader from cell fluorescence after solution I(-) addition. The Z' factor for the assay was approximately 0.7 with the V(2)R agonist [deamino-Cys1, Val4, d-Arg8]-vasopressin (1 nM) as positive control. Primary screening of 50,000 small molecules yielded a novel, 5-aryl-4-benzoyl-3-hydroxy-1-(2-arylethyl)-2H-pyrrol-2-one class of V(2)R antagonists that are unrelated structurally to known V(2)R antagonists. The most potent compound, V(2)R(inh)-02, which was identified by screening 35 structural analogs, competitively inhibited V(2)R-induced cAMP elevation with K(i) value of approximately 70 nM and fully displaced radiolabeled vasopressin in binding experiments. V(2)R(inh)-02 did not inhibit forskolin or beta(2)-adrenergic receptor-induced cAMP production and was more than 50 times more potent for V(2)R than for V(1a)R. The favorable in vitro properties of the pyrrol-2-one antagonists suggests their potential usefulness in aquaretic applications. The CFTR-linked cAMP assay developed here is applicable for efficient, high-throughput identification of modulators of cAMP-coupled GPCRs.

The effect of isonicotinic acid hydrazide (INH), a potent haem inhibitor, on globin chain synthesis was studied in reticulocytes from the following groups of patients: four non-thalassaemic patients (group i); five beta thalassaemia heterozygotes (group ii); three Hb S/beta thalassaemia heterozygotes (group iii); and two additional patients--one with homozygous beta thalassaemia and the other with thalassaemia intermedia (group iv). This was done to determine whether haem inhibitors depress alpha globin chain synthesis. The progressive increase of INH concentration (10-40 mmol l-1) in reticulocytes from a beta thalassaemia heterozygote resulted in a remarkable decrease of the alpha and beta chain synthesis, ranging from 80% to 97% and from 74% to 96% of control values, respectively, and in a gradual drop of alpha:beta ratio from 1.87 to 1.38. Furthermore, in the samples incubated with 40 mmol l-1 INH, a pronounced inhibition of globin chain synthesis 77 (19%) for alpha chain and 67 (27%) for beta or beta S chain) and a substantial drop of the alpha:beta or beta S ratio in samples with INH (median 1.16) compared with that in samples without INH (median 1.70) were observed. The inhibitory effect of INH was significantly or completely corrected by adding exogenous haem. It is suggested that haem inhibition and the resulting preferential diminution of alpha chain synthesis could provide a new approach to the treatment of homozygous beta thalassaemia with an excess of detrimental free alpha chain in erythroid cells.

Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Recently, S-adenosylhomocysteine hydrolase deficiency was confirmed for the first time in an adult. Two missense mutations in codons 89 (A>V) and 143 (Y>C) in the AdoHcyase gene were identified [N.R.M. Buist, B. Glenn, O. Vugrek, C. Wagner, S. Stabler, R.H. Allen, I. Pogribny, A. Schulze, S.H. Zeisel, I. Barić, S.H. Mudd, S-Adenosylhomocysteine hydrolase deficiency in a 26-year-old man, J. Inh. Metab. Dis. 29 (2006) 538-545]. Accordingly, we have proven the Y143C mutation to be highly inactivating [R. Beluzić, M. Cuk, T. Pavkov, K. Fumić, I. Barić, S.H. Mudd, I. Jurak, O. Vugrek, A single mutation at tyrosine 143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound co-factor NAD, Biochem. J. 400 (2006) 245-253]. Now we report that the A89V exchange leads to a 70% loss of enzymatic activity, respectively. Circular dichroism analysis of recombinant p.A89V protein shows a significantly reduced unfolding temperature by 5.5 degrees C compared to wild-type. Gel filtration of mutant protein is almost identical to wild-type indicating assembly of subunits into the tetrameric complex. However, electrophoretic mobility of p.A89V is notably faster as shown by native polyacrylamide gel electrophoresis implicating changes to the overall charge of the mutant complex. 'Bioinformatics' analysis indicates that Val(89) collides with Thr(84) causing sterical incompatibility. Performing site-directed mutagenesis changing Thr(84) to 'smaller' Ser(84) but preserving similar physico-chemical properties restores most of the catalytic capabilities of the mutant p.A89V enzyme. On the other hand, substitution of Thr(84) with Lys(84) or Gln(84), thereby introducing residues with higher volume in proximity to Ala(89) results in inactivation of wild-type protein. In view of our mutational analysis, we consider changes in charge and the sterical incompatibility in mutant p.A89V protein as main reason for enzyme malfunction with AdoHcyase deficiency as consequence.

Whooping cough is a re-emerging respiratory tract infection. It has become clear that there is a need for better understanding of protective immune responses and variation between Bordetella pertussis strains to aid the development of improved vaccines. In order to survive in the host, B. pertussis has evolved mechanisms to evade complement-mediated killing, including the ability to bind complement-regulatory proteins. Here we evaluate the variation in interactions with the complement system among recently isolated strains. Isolates whose genomes appear highly similar and cluster together on a SNP-based dendrogram were found to vary significantly in resistance to complement-mediated killing and in the deposition of C3b/iC3b, C5b-9 and C1 esterase inhibitor (C1-INH). The key role of Vag8 as a receptor for C1-INH was confirmed and its expression was shown to vary in a panel of isolates. A Vag8 knockout mutant showed increased sensitivity to complement-mediated killing. Antibodies in convalescent sera blocked C1-INH binding to B. pertussis and may play an important role in natural immunity.

<p><div>(<em>b</em>)OBJECTIVES</<em>b</em>)</div>Prothionamide, a structural analogue of isoniazid, is used mainly for treating multidrug-resistant tu<em>b</em>erculosis (MDR-TB). Both drugs have a common target InhA, so prothionamide can <em>b</em>e ineffective against isoniazid-resistant (<em>INH</em>^R) Myco<em>b</em>acterium tu<em>b</em>erculosis. We aimed to investigate the prevalence of mutations in katG, ethA, ndh, ethR, mshA, inhA and/or its promoter associated with independent resistance and cross-resistance to <em>INH</em>^R and/or prothionamide-resistant (PTO^R) M. tu<em>b</em>erculosis isolates.</p><A<em>b</em>stractText>We sequenced the a<em>b</em>ove genes in 206 M. tu<em>b</em>erculosis isolates with suscepti<em>b</em>ility testing against ten drugs.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Of the 173 <em>INH</em>^R PTO^R isolates, 170 (98.3%) har<em>b</em>oured mutations in katG, 111 (64.2%) in ethA, 58 (33.5%) in inhA or its promoter, 5 (2.9%) in ndh, 3 (1.7 %) in ethR and 2 (1.2%) in mshA. Among the 18 <em>INH</em>^R PTO^S isolates, mutations in katG were found in all of them; one had a mutation in the inhA promoter and another in ndh. Of the five <em>INH</em>^S PTO^R isolates, four showed mutations in ethA and two in the inhA promoter. Nota<em>b</em>ly, 55 novel non-synonymous mutations were found in them and 20.2% of the PTO^RM. tu<em>b</em>erculosis isolates har<em>b</em>oured no known mutations.</p><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>This is the first report to investigate cross-resistance <em>b</em>etween <em>INH</em>^R and/or PTO^R isolates. Among <em>INH</em>^R (94.4% MDR-TB) M. tu<em>b</em>erculosis isolates, the high diversity of mutations for independent resistance and cross-resistance with prothionamide highlight the importance of <em>b</em>oth phenotypic suscepti<em>b</em>ility and genotypic diagnosis when using it to treat patients with <em>INH</em>^R-TB. The high proportion (one-fifth) of PTO^RM. tu<em>b</em>erculosis isolates showed no known mutation related to PTO^R genes, so uncovered resistance mechanism(s) of prothionamide exist.</p>

Responses of threeHylastes species,Dryocoetes autographus, and twoHylobius species to terpenes and ethanol were studied in field experiments on clear-cut forest sites in Sweden using baited ground traps.α-Pinene alone did not attract any of the six species. A terpene blend (spruce turpentine consisting mainly ofα-pinene,β-pinene, and 3-carene) attractedHylastes cunicularius, H. brunneus, andHylobius abietis in some experiments, but not in others. The attractiveness of ethanol also varied; the only species consistently attracted wasH. abietis. Baits containing both terpenes and ethanol, particularly the combination of spruce turpentine and ethanol, were attractive to all species exceptHylobius pinastri. InH. abietis, the terpene plus ethanol/ ethanol catch ratios increased during early summer. Seasonal differences in catch levels were observed inH. cunicularius andH. abietis. The addition ofα-pinene reduced the attractiveness of the combination of spruce turpentine and ethanol toH. cunicularius, H. opacus, andD. autographus. The differences in response to the volatiles between species are probably related to differences in reproductive behavior and host preferences.

Tuberculosis occurs at higher rates in renal transplant recipients than in the general population. It would be desirable to use isoniazid prophylaxis in renal transplant recipients at risk for reactivation of tuberculosis; yet many transplant centers do not routinely employ INH prophylaxis because they perceive transplant recipients to be an enhanced risk of hepatotoxicity from isoniazid. Data on the risk of isoniazid in renal transplant patients receiving cyclosporine-based immunosuppression are limited. We retrospectively studied 83 renal transplant recipients (mean age 39.1 +/- 11.7 yr) who had received INH prophylaxis between 1985 and 1994. Eight patients had laboratory evidence of chronic hepatitis B or chronic hepatitis C infection. The mean duration of INH therapy was 344 +/- 163 d. The mean serum glutamate oxalacetic transferase (SGOT) at the start of INH therapy was 24.1 +/- 10.9 I.U., and 10% of patients had mildly elevated SGOTs. Mean peak SGOT during therapy was 36.4 +/- 15.3 I.U. (p < 0.001 compared to start (SGOT). In follow up, 31% of patients had an abnormal SGOT >> 40 I.U.); however, the elevations were small (the highest SGOT was 88.I.U.) and never necessitated discontinuation of INH. No patient had jaundice or other evidence of clinical hepatotoxicity. The 95% confidence interval for the observed frequency of clinical hepatitis was 0% to 4.3%. At the end of INH therapy the mean SGOT was 22.7 +/- 6.2 I.U. (p>> 0.2, compared with start SGOT) and only one patient had an abnormal SGOT. In conclusion, it appears that the risk of renal transplant recipients developing serious hepatotoxicity with the administration of INH is low and not different from normal individuals.

Examination of the crocin bleaching assay performance and in-house validation were focused on probe and test compound characteristics, conditions for peroxyl radical generation, reaction monitoring, and expression of results. HPLC and spectrometric examination showed that any authentic commercial saffron (origin, grade) can be used for probe preparation given that (a) interferences, such as tocopherols, are removed, (b) working solution concentration is adequately adjusted, and (c) stock probe solution changes during storage are not neglected. As suggested by log P values, calculated for a great number of radical scavengers (AHs), any AH more polar than Trolox (common reference compound) can be tested in the aqueous environment of the assay. AH activities order obeyed principles of structure-activity relationships. The assay was robust toward preheating of the azo-initiator (2,2'-azobis(2-aminopropane) dihydrochloride). Reaction monitoring through periodic UV-vis spectra recording was very informative. An alternative expression of results as "percent inhibition of crocin bleaching value", % Inh = [(DeltaA(0) - DeltaA)/DeltaA(0))] x 100, is proposed for [AH]/[crocin] = 1, instead of the so far used k(rel) values. The above findings also lead to analysis cost and time reduction.

OBJECTIVE

To observe the clinical efficacy of bushen huoxue recipe (BHR) combined estrogen and progesterone in treating premature ovarian failure (POF), and to explore an effective treatment program of POF by integrative medicine.

METHODS

Totally 265 POF patients were randomly assigned to 3 groups, i.e., Group I (86 cases, treated by BHR),Group II (88 cases,treated by conjugated estrogens and medroxyprogesterone acetate), and Group III (91 cases,treated by BHR +conjugated estrogens and medroxyprogesterone acetate). The therapeutic course for each group was 6 months. The main symptoms (including menstrual cycle, hectic fever, night sweat, vaginal dryness, and low libido), laboratory indices [including follicle stimulating hormone (FSH), luteotropic hormone (LH), estradiol (E2), and inhibin B (INH-B)], B-ultrasound indicators (including endometrial thickness, ovarian volume, and antral follicle count), and adverse reactions were observed in the three groups at the end of treatment and 6 months after treatment.

RESULTS

Compared with before treatment, the main symptoms, laboratory indices, and B-ultrasound indicators were statistically improved in the three groups at the end of treatment and 6 months after treatment (P <0.05, P <0.01). Better effects were obtained in Group III in improving symptoms of the menstrual cycle, vaginal dryness, and low libido, lowering levels of FSH and LH, elevating levels of E2and INH-B, and ameliorating the endometrial thickness, the ovarian volume, and the antral follicle count (P <0.05, P <0.01). No obvious adverse reaction occurred in the three groups.

CONCLUSIONS

BHR combined estrogen and progesterone showed better clinical efficacy than use of BHR or estrogen/progesterone alone, indicating it was an effective treatment program for POF.

BACKGROUND

Meconium aspiration syndrome has a complex pathophysiology. Meconium activates the complement system and meconium-induced cytokine formation is differentially mediated by complement and CD14. C1-inhibitor (C1-INH) regulates complement and contact-system activation mainly by protease inhibition, but may reduce inflammation by other mechanisms as well.

OBJECTIVE

The aim of the study was to investigate the initial mechanisms of meconium-induced complement activation and to study the effect of C1-INH on the meconium-induced inflammatory reaction.

METHODS

Human serum from five donors was preincubated with an anti-MBL monoclonal antibody and then incubated with meconium for 30 min at 37 degrees C. Human cord whole blood, anticoagulated with lepirudin, from six donors was preincubated with C1-INH and then incubated with meconium for 30 min and 4h at 37 degrees C. Complement activation products specific for the different pathways were measured by ELISAs: classical pathway C1rs/C1-INH complexes, classical and lectin pathway C4d, alternative pathway C3bBbP, and terminal pathway sC5b-9 complex (TCC). A Bio-Plex Array Reader was used to measure 27 inflammatory mediators.

RESULTS

The anti-MBL monoclonal antibody significantly reduced meconium-induced formation of C4d by 63% (p=0.0159) and TCC by 27% (p=0.0079). C1-INH dose-dependently inhibited meconium-induced formation of C1rs/C1-INH complexes, C4d, C3bBbP, and TCC compared to albumin (p<0.002 for all). C1-INH induced a dose-dependent and substantial inhibition of meconium-induced formation of the proinflammatory cytokines TNFalpha, IL-1 beta, IL-6 and IFN-gamma (p<0.01 for all), the chemokines IL-8, MCP-1, MIP-1 alpha, MIP-1 beta, and eotaxin (p<0.02 for all), the growth factors G-CSF, GM-CSF, basic FGF, and PDGFbb (p<0.05 for all), and the anti-inflammatory cytokine IL-1ra (p<0.001).

CONCLUSIONS

Meconium activated the lectin complement pathway as well as the alternative pathway. C1-INH efficiently reduced a broad spectrum of inflammatory mediators even at the lowest concentration. Administration of C1-INH may thus reduce the inflammatory response in MAS.

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.

OBJECTIVE

Inhibin A and B (Inh A and B), activin A (Act A) as well as FSH may play an important role in bone turnover in perimenopausal women. Data in men are lacking. The aim was to investigate the relationship between circulating concentrations of Inh B and Act A and FSH/LH/testosterone (T) and their contribution to bone mineral density (BMD) in a male population.

METHODS

Cross-sectional case-control study of 156 men, 63 with osteoporosis and 93 controls, aged (mean [SD]) 57.7 [13.7] years.

METHODS

Areal (aBMD) was measured at the femoral neck, total hip and lumbar spine. Volumetric BMD (vBMD) was calculated at the femoral neck and lumbar spine. Risk factors were assessed including the measurement of LH/FSH/T, Inh B and Act A.

RESULTS

After correction for age and body mass index (BMI), associations were found between Inh B and FSH (beta regression coefficient beta = -0.326; P < 0.0001), T (beta = -0.36; P = 0.019) and Act A (beta = -0.4; P = 0.007) and between Inh B and LH (beta = 0.23; P < 0.0001) in all patients. The controls had higher Inh B concentrations compared to the cases (Inh B: controls: 139 [86] pg/ml vs. cases 88 [51] pg/ml; P = 0.005). Act A tended to be lower in the controls (Act A: controls 0.63 [0.24] ng/ml vs. cases 0.75 [0.4] ng/ml; P = 0.056). Univariate regression analyses showed a positive association between Inh B and BMD (P < 0.01) at the lumbar spine and total hip. In contrast a negative association was seen between FSH and BMD at the lumbar spine and femoral neck (P < 0.01). In a partial multivariate regression model that included the gonadal factors only, a positive association was seen between Inh B and BMD at the hip (beta = 0.088; P = 0.04). When all hormones including the gonadotrophins were entered in a full multivariate model, FSH and LH were found to be better predictors of BMD than Inh B or Act A in the controls and cases.

CONCLUSIONS

These data suggest that the gonadal peptides and gonadotrophins may play a role in the maintenance of bone mass in men. Future confirmatory longitudinal studies are needed.

<A<em>b</em>stractText>The treatment of latent tu<em>b</em>erculosis infection (LTBI) in individuals at risk of reactivation is essential for tu<em>b</em>erculosis control. However, <em>b</em>lood <em>b</em>iomarkers associated with LTBI treatment have not <em>b</em>een identified.</A<em>b</em>stractText><p><div>(<em>b</em>)Methods</<em>b</em>)</div>Blood samples from tu<em>b</em>erculin skin test (TST) reactive individuals were collected <em>b</em>efore and after one and six months of isoniazid (<em>INH</em>) therapy. Peripheral mononuclear cells (PBMC) were isolated, and an in-house interferon-<i>γ</i> release assay (IGRA) was performed. Expression of chemokine ligand 4 (CCL4), chemokine ligand 10 (CXCL10), chemokine ligand 11 (CXCL11), interferon alpha (IFNA), radical S-adenosyl methionine domain-containing 2 (RSAD2), u<em>b</em>iquitin-specific peptidase 18 (USP18), interferon-induced protein 44 (IFI44), interferon-induced protein 44 like (IFI44L), interferon-induced protein tetratricopeptide repeats 1(IFIT1), and interleukin 2 receptor su<em>b</em>unit alpha (IL2RA) mRNA levels were assessed <em>b</em>y qPCR <em>b</em>efore, during, and after <em>INH</em> treatment.</p><A<em>b</em>stractText>We o<em>b</em>served significantly lower relative a<em>b</em>undances of USP18, IFI44L, IFNA, and IL2RA transcripts in PBMC from IGRA-positive individuals compared to levels in IGRA-negative individuals <em>b</em>efore <em>INH</em> therapy. Also, relative a<em>b</em>undance of CXCL11 was significantly lower in IGRA-positive than in IGRA-negative individuals <em>b</em>efore and after one month of <em>INH</em> therapy. However, the relative a<em>b</em>undance of CCL4, CXCL10, and CXCL11 mRNA was significantly decreased and that of IL2RA and USP18 significantly increased after <em>INH</em> therapy, regardless of the IGRA result. Our results show that USP18, IFI44L, IFIT1, and IL2RA relative a<em>b</em>undances increased significantly, meanwhile the relative a<em>b</em>undance of CCL4, CXCL11, and IFNA decreased significantly after six months of <em>INH</em> therapy in TST-positive individuals.</A<em>b</em>stractText><A<em>b</em>stractText>Changes in the profiles of USP18, IL2RA, IFNA, CCL4, and CXCL11 expressions during <em>INH</em> treatment in TST-positive individuals, regardless of IGRA status, are potential tools for monitoring latent tu<em>b</em>erculosis treatment.</A<em>b</em>stractText>

(<em>b</em>)Background and Aims:</<em>b</em>) Rifampicin (RFP) and isoniazid (<em>INH</em>) are widely used as anti-tu<em>b</em>erculosis agents. However, the mechanisms underlying the involvement of reactive oxygen species and mitochondria in RFP- and <em>INH</em>-related hepatotoxicity have not <em>b</em>een esta<em>b</em>lished yet. This study aimed to o<em>b</em>serve the intracellular mechanisms leading to mitochondrial dysfunction and morphological changes in RFP- and <em>INH</em>-induced hepatocyte injury. (<em>b</em>)Methods:</<em>b</em>) Cell injury, changes in mitochondrial function, and expression and activation of dynamin related protein 1 (Drp1), known as the main protein for mitochondrial fission, were analyzed in cultured QSG7701 cells exposed to RFP and <em>INH</em>. (<em>b</em>)Results:</<em>b</em>) <em>INH</em> and RFP treatment induced pronounced hepatocyte injury and increased cell death. In the similar context of aspartate aminotransferase elevation and adenosine triphosphate synthesis decrease, changes in mitochondrial mem<em>b</em>rane permea<em>b</em>ility and reactive oxygen species in hepatocytes induced <em>b</em>y RFP were significantly different from those induced <em>b</em>y <em>INH</em> (<i>p</i> < 0.05). Particularly, we o<em>b</em>served the overactivation and mitochondrial translocation of Drp1 in RFP-induced cell injury, which was not occurred with exposure to <em>INH</em>. (<em>b</em>)Conclusions:</<em>b</em>) RFP-induced hepatotoxicity may <em>b</em>e closely related to mitochondrial dysfunction and Drp1-mediated mitochondrial fission.

Herein, we propose novel quinolones incorporating an <em>INH</em> moiety as potential drug templates against TB. The quinolone-<em>b</em>ased compounds <em>b</em>earing an <em>INH</em> moiety attached <i>via</i> a hydrazide-hydrazone <em>b</em>ond were synthesised and evaluated against <i>Myco<em>b</em>acterium tu<em>b</em>erculosis</i> H37Rv (MTB). The compounds were also evaluated for cytotoxicity against HeLa cell lines. These compounds showed significant activity (MIC<su<em>b</em>)90</su<em>b</em>)) against MTB in the range of 0.2-8 μM without any cytotoxic effects. Compounds (<em>b</em>)10</<em>b</em>) (MIC<su<em>b</em>)90</su<em>b</em>); 0.9 μM), (<em>b</em>)11</<em>b</em>) (MIC<su<em>b</em>)90</su<em>b</em>); 0.2 μM), (<em>b</em>)12</<em>b</em>) (MIC<su<em>b</em>)90</su<em>b</em>); 0.8 μM) and compound (<em>b</em>)15</<em>b</em>) (MIC<su<em>b</em>)90</su<em>b</em>); 0.8 μM), the most active compounds in this series, demonstrate activities on par with <em>INH</em> and superior to those reported for the fluoroquinolones. The SAR analysis suggests that the nature of su<em>b</em>stituents at positions -1 and -3 of the quinolone nucleus influences anti-MTB activity. Aqueous solu<em>b</em>ility evaluation and <i>in vitro</i> meta<em>b</em>olic sta<em>b</em>ility of compound (<em>b</em>)12</<em>b</em>) highlights favoura<em>b</em>le drug-like properties for this compound class.

Although both serum inhibin B (Inh-B) and anti-Müllerian hormone (AMH) concentrations have been proposed as markers of spermatogenesis in men with subfertility, there are wide overlaps with fertile controls. The main aim of this study was to evaluate stimulated serum Inh-B and AMH concentrations in men with non-obstructive azoospermia (NOA). Thirty-seven men with NOA confirmed by testicular fine-needle aspiration and 17 fertile controls participated at this prospective, case-control study. All subjects underwent the Exogenous FSH SErtoli Reserve Test (EFSERT): estimation of serum Inh-B and AMH concentrations before, 24 and 48 hours after administration of 300 IU human recombinant FSH (hrFSH). Basal serum Inh-B and AMH concentrations, as well as Inh-B concentrations at 24 and 48 h were lower in men with NOA as compared to controls. No changes in Inh-B or AMH concentrations were recorded throughout the EFSERT in either men with NOA or controls nor when men with NOA were classified according to their clinical, hormonal and cytological diagnosis. Thus, stimulated serum concentrations of Inh-B and AMH, as obtained by an EFSERT, do not contribute to the diagnostic evaluation of the men with NOA, as the same information can be acquired by the basal serum concentrations of these hormones.

Drug-induced liver injury (DILI) is a growing problem. Diagnostic methods to differentiate DILI caused by an adaptive immune response from liver injury of other causes or to identify the responsible drug in patients receiving multiple drugs, herbals and/or dietary supplements (polypharmacy) have not yet been established. The lymphocyte transformation test (LTT) has been proposed as a diagnostic method to determine if a subject with an apparent hypersensitivity reaction has become sensitized to a specific drug. In this test, peripheral blood mononuclear cells (PBMC) collected from a subject are incubated with drug(s) suspected of causing the reaction. Cell proliferation, measured by the incorporation of [3H]-thymidine into new DNA, is considered evidence of a drug-specific immune response. The objectives of the current studies were to: (1) develop and optimize a modified version of the LTT (mLTT) and (2) investigate the feasibility of using the mLTT for diagnosing DILI associated with an adaptive immune response and identifying the responsible drug. PBMC collected from donors with a history of drug hypersensitivity reactions to specific drugs (manifested as skin rash) were used as positive controls for assay optimization. Following optimization, samples collected from 24 subjects enrolled in the U.S. Drug-Induced Liver Injury Network (DILIN) were tested in the mLTT. Using cytokine and granzyme B production as the primary endpoints to demonstrate lymphocyte sensitization to a specific drug, most samples from the DILIN subjects failed to respond. However, robust positive mLTT responses were observed for two of four samples from three DILIN subjects with hepatitis due to isoniazid (INH). We conclude that the mLTT, as performed here on frozen and thawed PBMC, is not a reliable test for diagnosing DILI caused by all drugs, but that it may be useful for confirming the role of the adaptive immune response in DILI ascribed to INH.

BACKGROUND

Inhibins (INH) are dimeric glycoproteins, composed of an alpha subunit (INH-alpha) and one of two possible beta subunits (INH-betaA or INH-betaB). They have substantial roles in human reproduction and in endocrine-responsive tumours. Therefore, the aims of this study were to determine the frequency and tissue distribution of INH-alpha, INH-betaA and INH-betaB in normal human endometrium and glandular-cystic endometrial polyps, and polyps caused by tamoxifen use.

METHODS

Tissue samples were obtained from women in the proliferative, early secretory and late secretory phase as well as glandular-cystic polyps and endometrial polyps associated with tamoxifen use (n = 5 each). Immunohistochemistry with specific monoclonal antibodies, a semi-quantitative analysis and statistical evaluation was performed.

RESULTS

INH-alpha, INH-betaA and INH-betaB were primarily observed in glandular and luminal epithelial cells, with a variant staining intensity in stromal cells. INH-alpha in glands was significantly higher during the early secretory phase (p < 0.05) and the late secretory phase (p < 0.01) than in the proliferative phase with a significant difference between the early secretory and the late secretory phases (p < 0.01). INH-betaA expression was significantly higher during the late secretory than the proliferative phase (p < 0.05) and the late secretory than the early secretory phase (p < 0.05), with no significant differences for INH-betaB. Glandular-cystic polyps showed significantly lower expression of INH-alpha and INH-betaA than the late secretory endometria (p < 0.05 and p < 0.01 respectively). Additionally, tamoxifen-associated polyps also demonstrated a significantly lower expression of INH-alpha and INH-betaA than late secretory endometria (p < 0.01 and p < 0.01 respectively). No statistical differences were observed between tamoxifen-associated and glandular-cystic polyps.

CONCLUSIONS

INH-alpha, INH-betaA and INH-betaB were expressed in normal endometrium and endometrial polyps. A cyclical expression of INH-alpha and INH-betaA in normal glands may reflect a functional and hormone-dependent role in human endometrium. Significant differences in staining reaction between the late secretory endometria and polyps suggest that this tissue remains in the proliferating state rather than the secretory state. Therefore, endometrial polyps may be tumours of dysregulation with mainly proliferating characteristics, being unable to synchronise with normal endometrium.

A quantitative study of the circulating immune complexes (IC) was carried out on women during normal pregnancy (286) and the post-partum period (20) and women with pre-eclampsia (30). Furthermore, the behaviour of the complement (C) system was followed. Results showed that IC were low in the first trimester of normal pregnancy (25.3%) and decreased in the following trimesters, whereas they were always present in pre-eclampsia. A very significant difference (p less than 0.0001) was seen when we compared the incidence of IC in normal pregnancy at the third trimester and the pre-eclamptic patients. The follow-up study of the IC, carried out on 4 pre-eclamptic women, showed an increase in the IC levels associated with the exacerbation of the pre-eclamptic picture and a decrease after delivery. The study of complement in normal pregnancy showed a decrease in C1-INH, C1s and C1q, whereas C3, C5, C9 and the properdin factor B increased during the following weeks of gestation; CH50 did not vary excepting during the 1st trimester. In the puerperium all values increased. There was no significant difference between the serum levels of the C components in the 3rd trimester of normal pregnancy and pre-eclampsia. High levels of C3d were observed in normal pregnancy at the 3rd trimester and in pre-eclampsia. The study of this split product of C3 showed that there is activation of the C system, but, since the synthesis of the C components is increased, activation could be masked. Alloantibodies and circulating IC could be the factors responsible for this activation in normal pregnancy and in pre-eclampsia, respectively.

<p><div>(<em>b</em>)BACKGROUND AND PURPOSE</<em>b</em>)</div>Coronary artery disease leads to ischaemic heart disease and ultimately myocardial infarction. Thus, it is important to determine the factors that regulate coronary <em>b</em>lood flow. Ca²⁺ -activated chloride channels contri<em>b</em>ute to the regulation of arterial tone; however, their role in coronary arteries is unknown. The aim of this study was to investigate the expression and function of the main molecular correlate of Ca²⁺ -activated chloride channels, TMEM16A, in rat coronary arteries.</p><A<em>b</em>stractText>We performed mRNA and protein analysis, electrophysiological studies of coronary artery myocytes, and functional studies of coronary artery contractility and coronary perfusion, using novel <em>inh</em>i<em>b</em>itors of TMEM16A. Furthermore, we assessed whether any changes in expression and function occurred in coronary arteries from spontaneously hypertensive rats (SHRs).</A<em>b</em>stractText><p><div>(<em>b</em>)KEY RESULTS</<em>b</em>)</div>TMEM16A was expressed in rat coronary arteries. The TMEM16A-specific <em>inh</em>i<em>b</em>itor, MONNA, hyperpolarised the mem<em>b</em>rane potential in U46619. MONNA, T16A<su<em>b</em>)<em>inh</em></su<em>b</em>) -A01, and Ani9 attenuated 5-HT/U46619-induced contractions. MONNA and T16A<su<em>b</em>)<em>inh</em></su<em>b</em>) -A01 also increased coronary flow in Langendorff perfused rat heart preparations. TMEM16A mRNA was increased in coronary artery smooth muscle cells from SHRs, and U46619 and 5-HT were more potent in arteries from SHRs than in those from normal Wistar rats. MONNA diminished this increased sensitivity to U46619 and 5-HT.</p><A<em>b</em>stractText>In conclusion, TMEM16A is a key regulator of coronary <em>b</em>lood flow and is implicated in the altered contractility of coronary arteries from SHRs.</A<em>b</em>stractText>