Dorsal horn neurons in the young rat spinal cord-hindlimb preparation were physiologically classified as wide dynamic range (WDR), nociceptive specific (NS) or low threshold (LT) according to their excitatory responses to low and high intensity mechanical stimuli applied to the hindlimb skin. Two additional types were classified: neurons displaying only sub-threshold excitations (SUB) and neurons displaying inhibitory events (INH), such as inhibitory post-synaptic potentials or interruption of spontaneous spiking following cutaneous stimulation. Direct intracellular current injection revealed four different patterns of spiking behaviour: group A neurons were characterized by tonic firing in response to depolarizing current pulses; group B neurons were strongly phasic, producing only one spike at the beginning of the pulse; group A-B neurons generated an early unsustained (< 300 ms) burst of spikes; and group C neurons exhibited anomalous rectification in response to hyperpolarizing current which was followed by a voltage-dependent rebound excitation. A statistically significant (P < or = 0.01) association existed between a neuron's physiological classification and its electrophysiological profile. The majority of WDR neurons responded with tonic firing and were assigned to group A, while NS neurons were strongly represented in group A-B. All INH neurons were assigned to group C. LT neurons were distributed between groups A and A-B, and SUB neurons were distributed between groups A and B. These data indicate, firstly, that dorsal horn neurons possess heterogeneous membrane properties and, secondly, that a relationship exists between a neuron's biophysical profile and its excitatory or inhibitory response to peripheral cutaneous afferent stimulation. The implications for dorsal horn somatosensory processing are discussed.

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.

The occurrence of eight pharmaceuticals (beta-blockers: acebutolol, atenolol, metoprolol and sotalol; antiepileptic: carbamazepine; fluoroquinolone antibiotics: ciprofloxacin, norfloxacin, ofloxacin) were assessed in the raw and treated sewage of 12 sewage treatment plants (STPs) in Finland. The average concentrations in the raw and treated sewage ranged from 100 to 1060 ng L(-1) and from <24 to 755 ng L(-1), respectively. The average daily loads ranged from 36 to 405 mg/1000 inh and from 2 to 302 mg/1000 inh, respectively. In the treatment plants, fluoroquinolones were eliminated by >80%. Carbamazepine was not eliminated during the treatment and in fact even higher concentrations were frequently found in the treated than in the raw sewages. The increase in concentration was shown to be most likely due to enzymatic cleavage of the glucuronic conjugate of carbamazepine and release of the parent compound in the treatment plant. The beta-blockers were eliminated in average by less than 65% and the elimination varied greatly between the treatment plants. Especially the dilution of raw sewage by rainwater and a consequent decrease in the hydraulic retention time of a treatment plant was found to deteriorate the elimination of the beta-blockers. The work shows that especially carbamazepine and the beta-blockers may reach the recipient waters and there is a need to enhance their elimination in the sewage treatment plants. In this attempt, a denitrifying biofilter as a tertiary treatment could be of minor importance since in this study it did not result in further elimination of the target compounds.

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.

Inhibin and activin are members of the transforming growth factor beta (TGF-beta) family of ligands produced and secreted primarily by the gonads and adrenals. Inhibin-null (INH(-/-)) mice develop gonadal tumors and-when gonadectomized-adrenocortical carcinoma. The mechanisms leading to adrenal tumorigenesis have been proposed to involve the lack of a gonadal factor and/or a compensatory increase in gonadotropins. In order to achieve elevation of gonadotropins without the concomitant loss of a gonadal hormone, we crossed INH(-/-) mice with a transgenic mouse strain that has chronically elevated luteinizing hormone (LH) levels (LH-CTP). Compound INH(-/-)-LH-CTP mice die within 6 weeks of age from severe cancer cachexia induced by large, activin-secreting ovarian tumors. Unexpectedly, INH(-/-)-LH-CTP mice not only fail to develop adrenal tumors but have smaller adrenals, with a regressed x zone, indicating that elevated LH levels are not sufficient to induce adrenal tumor formation. However, following gonadectomy, INH(-/-)-LH-CTP mice develop large, sex steroid-producing adrenal tumors that arise from the x zone, indicating a growth-promoting effect of high levels of LH on the adrenal cortex in the absence of ovarian tumors. In addition, in vivo and in vitro data indicate that activin induces apoptosis specifically in the adrenal x zone. The restricted expression of activin receptor subunits and Smad2 in cells of the adrenal x zone, together with the elevated activin levels in INH(-/-)-LH-CTP mice, supports the conclusion that activin inhibits adrenal tumor growth by inducing x-zone regression.

BACKGROUND

Hereditary angioedema (HAE) caused by functional deficiency of C1-inhibitor (C1-INH) is a rare disease that manifests with recurrent spontaneous nonallergic edema of the subcutaneous tissues and mucous membranes. In cases of laryngeal edema that are not treated immediately, HAE is associated with high mortality rates. Attenuated androgens (e.g., danazol) are usually administered for prophylaxis, but associated side effects may limit their use. This study investigated the efficacy, safety, and quality of life (QoL) associated with a pasteurized plasma-derived C1-inhibitor (pC1-INH) concentrate for individual replacement therapy (IRT) in patients with severe HAE suffering from frequent attacks who were intolerant or not responding to danazol.

METHODS

Twenty-two patients with severe HAE and danazol incompatibility or insufficient efficacy of danazol were recruited. Intraindividual comparisons of efficacy, safety, and QoL with pC1-INH concentrate IRT versus danazol treatment were made using retrospective and prospective patient data. Pharmacokinetic data were collected for 15 of the 22 patients.

RESULTS

In patients receiving pC1-INH regularly, the median number of attacks per year decreased significantly compared to danazol prophylaxis (p < 0.001), and the 24 laryngeal edema episodes per year ceased. Superior efficacy of pC1-INH was found for all QoL variables (e.g., general condition, social activities). No transmission of human immunodeficiency virus or hepatitis A, B, or C was observed.

CONCLUSIONS

In patients with severe HAE who experience severe side effects and/or lack of efficacy of danazol prophylaxis, very early substitution with pC1-INH can completely abolish the incidence of potentially fatal laryngeal edema and can reduce the incidence of acute attacks.

The spirochete Borrelia recurrentis is the causal agent of louse-borne relapsing fever and is transmitted to humans by the infected body louse Pediculus humanus. We have recently demonstrated that the B. recurrentis surface receptor, HcpA, specifically binds factor H, the regulator of the alternative pathway of complement activation, thereby inhibiting complement mediated bacteriolysis. Here, we show that B. recurrentis spirochetes express another potential outer membrane lipoprotein, termed CihC, and acquire C4b-binding protein (C4bp) and human C1 esterase inhibitor (C1-Inh), the major inhibitors of the classical and lectin pathway of complement activation. A highly homologous receptor for C4bp was also found in the African tick-borne relapsing fever spirochete B. duttonii. Upon its binding to B. recurrentis or recombinant CihC, C4bp retains its functional potential, i.e. facilitating the factor I-mediated degradation of C4b. The additional finding that ectopic expression of CihC in serum sensitive B. burgdorferi significantly increased spirochetal resistance against human complement suggests this receptor to substantially contribute, together with other known strategies, to immune evasion of B. recurrentis.

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

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.

In this paper we demonstrate the synthesis of the components of the classical complement pathway, namely C1q, C1r, C1s, C1-Inh, C2, C4, and C5, by human glioma cell lines (U118MG, T193, and T98G). All these components were structurally, antigenically, and functionally similar to their serum counterparts as determined by biosynthetic labeling experiments, Western blot analysis, and hemolytic assays. Northern blot analysis of mRNA demonstrated that, for each of these components, their specific mRNA had the same size as the equivalent mRNA from hepatic tissue. We could not detect the synthesis of C4bp by these cell lines, and the secretion of C1q was only detected after stimulation by interferon-gamma. All these syntheses were up-regulated by interferon-gamma and tumor necrosis factor. Interleukin-1 beta only increased C2 expression and reproducibly down-regulated C5 secretion when used at high doses. Glioma cell lines appear to be an efficient and convenient model for the analysis of complement expression in human astrocytic cells.

BACKGROUND

The current study was designed to compare antral follicle count (AFC) and basal ovarian volume (BOV), the exogenous FSH ovarian reserve test (EFORT) and the clomiphene citrate challenge test (CCCT), with respect to their ability to predict poor and hyper responders.

METHODS

One hundred and ten regularly menstruating patients, aged 18-39 years, participated in this prospective study, randomized, by a computer designed 4-blocks system study into two groups. Fifty six patients underwent a CCCT, and 54 patients underwent an EFORT. All patients underwent a transvaginal sonography to measure the basal ovarian volume and count of basal antral follicle. In all patients, the test was followed by a standard IVF treatment. The result of ovarian hyperstimulation during IVF treatment, expressed by the total number of follicles, was used as gold standard.

RESULTS

The best prediction of ovarian reserve (Y) was seen in a multiple regression prediction model that included, AFC, Inhibin B-increment in the EFORT and BOV simultaneously (Y = -3.161 + 0.805 x AFC (0.258-1.352) + 0.034 x Inh. B-incr. (0.007-0.601) + 0.511 BOV (0.480-0.974) (r = 0.848, p < 0.001). Univariate logistic regression showed that the best predictors for poor response were the CCCT (ROC-AUC = 0.87), the bFSH (ROC-AUC = 0.83) and the AFC (ROC-AUC = 0.83). Multiple logistic regression analysis did not produce a better model in terms of improving the prediction of poor response. For hyper response, univariate logistic regression showed that the best predictors were AFC (ROC-AUC = 0.92) and the inhibin B-increment in the EFORT (ROC-AUC = 0.92), but AFC had better test characteristics, namely a sensitivity of 82% and a specificity 89%. Multiple logistic regression analysis did not produce a better model in terms of predicting hyper response.

CONCLUSIONS

In conclusion AFC performs well as a test for ovarian response being superior or at least similar to complex expensive and time consuming endocrine tests. It is therefore likely to be the test for general practise.

BACKGROUND

There is little information on nasopharyngeal (NP) flora or bacteremia in HIV-infected children. Our aim was to describe the organisms and antimicrobial resistance patterns in children enrolled in a prospective study comparing daily and three times weekly trimethoprim-sulfamethoxazole (TMP-SMX) and isoniazid (INH) or placebo prophylaxis.

METHODS

NP swabs were taken at baseline from HIV-infected children enrolled in the study. Standard microbiological techniques were used. Children were grouped according to previous or current exposure to TMP-SMX and whether enrolled to the study during a period of hospitalization. Blood culture results were also recorded within 12 months of baseline.

RESULTS

Two hundred and three children, median age 1.8 (Interquartile [IQ]: 0.7-4) years had NP swabs submitted for culture. One hundred and eighty-four (90.7%) had either stage B or C HIV disease. One hundred and forty-one (69.8%) were receiving TMP-SMX and 19 (9.4%) were on antiretroviral therapy. The majority, 168 (82%) had a history of hospitalization and 91 (44.8%) were enrolled during a period of hospitalization. Thirty-two subjects (16.2%) died within 12 months of study entry. One hundred and eighty-one potential pathogens were found in 167 children. The most commonly isolated organisms were Streptococcus pneumoniae (48: 22.2%), Gram-negative respiratory organisms (Haemophilus influenzae and Moraxella catarrhalis) (47: 21.8%), Staphylococcus aureus (44: 20.4%), Enterobacteriaceae 32 (14.8%) and Pseudomonas 5 (2.3%). Resistance to TMP-SMX occurred in>> 80% of pathogens except for M. catarrhalis (2: 18.2% of tested organisms). TMP-SMX resistance tended to be higher in those receiving it at baseline (p = 0.065). Carriage of Methicillin resistant S. aureus (MRSA) was significantly associated with being on TMP-SMX at baseline (p = 0.002). Minimal inhibitory concentrations (MIC) to penicillin were determined for 18 S. pneumoniae isolates: 7 (38.9%) were fully sensitive (MIC <or= 0.06 microg/ml), 9 (50%) had intermediate resistance (MIC 0.12 - 1 microg/ml) and 2 (11.1%) had high level resistance (MIC>>or=2 microg/ml). Fifty percent of Enterobacteriaceae produced extended spectrum beta-lactamases (ESBL) (resistant to third generation cephalosporins) and 56% were resistant to gentamicin. Seventy-seven percent of S. aureus were MRSA. Carriage of resistant organisms was not associated with hospitalization.On multivariate logistic regression, risk factors for colonization with Enterobacteriaceae were age <or= one year (Odds ratio 4.4; 95% Confidence Interval 1.9-10.9; p = 0.0008) and CDC stage C disease (Odds ratio 3.6; 95% Confidence Interval 1.5-8.6; p = 0.005)Nineteen (9.4%) subjects had 23 episodes of bacteremia. Enterobacteriaceae were most commonly isolated (13 of 25 isolates), of which 6 (46%) produced ESBL and were resistant to gentamicin.

CONCLUSIONS

HIV-infected children are colonized with potential pathogens, most of which are resistant to commonly used antibiotics. TMP-SMX resistance is extremely common. Antibiotic resistance is widespread in colonizing organisms and those causing invasive disease. Antibiotic recommendations should take cognizance of resistance patterns. Antibiotics appropriate for ESBL-producing Enterobacteriaceae and MRSA should be used for severely ill HIV-infected children in our region. Further study of antibiotic resistance patterns in HIV-infected children from other areas is needed.

Using cytostatic factor metaphase II-arrested extracts as a model system, we show that protein phosphatase 1 is regulated during early embryonic cell cycles in Xenopus. Phosphatase 1 activity peaks during interphase and decreases shortly before the onset of mitosis. A second peak of activity appears in mitosis at about the same time that cdc2 becomes active. If extracts are inhibited in S-phase with aphidicolin, then phosphatase 1 activity remains high. The activity of phosphatase 1 appears to determine the timing of exit from S-phase and entry into M-phase; inhibition of phosphatase 1 by the specific inhibitor, inhibitor 2 (Inh-2), causes premature entry into mitosis, whereas exogenously added phosphatase 1 lengthens the interphase period. Analysis of DNA synthesis in extracts treated with Inh-2, but lacking the A- and B-type cyclins, shows that phosphatase 1 is also required for the process of DNA replication. These data indicate that phosphatase 1 is a component of the signaling pathway that ensures that M-phase is not initiated until DNA synthesis is complete.

Binding of negatively charged macromolecules to factor XII induces a conformational change such that it becomes a substrate for trace amounts of activated factor present in plasma (less than 0.01%). As activated factor XII (factor XIIa or factor XIIf) forms, it converts prekallikrein (PK) to kallikrein and kallikrein cleaves high molecular weight kininogen (HK) to release bradykinin. A far more rapid activation of the remaining unactivated factor XII occurs by enzymatic cleavage by kallikrein (kallikrein-feedback) and sequential cleavage yields two forms of activated factor XII; namely, factor XIIa followed by factor XII fragment (factor XIIf). PK circulates bound to HK and binding induces a conformational change in PK so that it acquires enzymatic activity and can stoichiometrically cleave HK to produce bradykinin. This reaction is prevented from occurring in plasma by the presence of C1 inhibitor (C1 INH). The same active site leads to autoactivation of the PK-HK complex to generate kallikrein if a phosphate containing buffer is used. Theoretically, formation of kallikrein by this factor XII-independent route can activate surface-bound factor XII to generate factor XIIa resulting in a marked increase in the rate of bradykinin formation as stoichiometric reactions are replaced by Michaelis-Menton, enzyme-substrate, kinetics. Zinc-dependent binding of the constituents of the bradykinin-forming cascade to the surface of endothelial cells is mediated by gC1qR and bimolecular complexes of gC1qR-cytokeratin 1 and cytokeratin 1-u-PAR (urokinase plasminogen activator receptor). Factor XII and HK compete for binding to free gC1qR (present in excess) while cytokeratin 1-u-PAR preferentially binds factor XII and gC1qR-cytokeratin 1 preferentially binds HK. Autoactivation of factor XII can be initiated as a result of binding to gC1qR but is prevented by C1 INH. Yet stoichiometric activation of PK-HK to yield kallikrein in the absence of factor XII can be initiated by heat shock protein 90 (HSP-90) which forms a zinc-dependent trimolecular complex by binding to HK. Thus, endothelial cell-dependent activation can be initiated by activation of factor XII or by activation of PK-HK. Hereditary angioedema (HAE), types I and II, are due to autosomal dominant mutations of the C1 INH gene. In type I disease, the level of C1 INH protein and function is proportionately low, while type II disease has a normal protein level but diminished function. There is trans-inhibition of the one normal gene so that functional levels are 30% or less and severe angioedema affecting peripheral structures, the gastrointestinal tract, and the larynx results. Prolonged incubation of plasma of HAE patients (but not normal controls) leads to bradykinin formation and conversion of PK to kallikrein which is reversed by reconstitution with C1 INH. The disorder can be treated by C1 INH replacement, inhibition of plasma kallikrein, or blockade at the bradykinin B-2 receptor. A recently described HAE with normal C1 INH (based on inhibition of activated C1s) presents similarly; the defect is not yet clear, however one-third of patients have a mutant factor XII gene. We have shown that this HAE has a defect in bradykinin overproduction whether the factor XII mutation is present or not, that patients' C1 INH is capable of inhibiting factor XIIa and kallikrein (and not just activated C1) but the functional level is approximately 40-60% of normal, and that α2 macroglobulin protein levels are normal. In vitro abnormalities can be suppressed by raising C1 INH to twice normal levels. Finally, aggregated proteins have been shown to activate the bradykinin-forming pathway by catalyzing factor XII autoactivation. Those include the amyloid β protein of Alzheimer's disease and cryoglobulins. This may represent a new avenue for kinin-dependent research in human disease. In allergy (anaphylaxis; perhaps other mast cell-dependent reactions), the oversulfated proteoglycan of mast cells, liberated along with histamine, also catalyze factor XII autoactivation.

C1 inhibitor (C1-INH) is a serine protease inhibitor (serpins) that inactivates several different proteases in the complement, contact, coagulation, and fibrinolytic systems. By its C-terminal part (serpin domain), characterized by three beta-sheets and an exposed mobile reactive loop, C1-INH binds, and blocks the activity of its target proteases. The N-terminal end (nonserpin domain) confers to C1-INH the capacity to bind lipopolysaccharides and E-selectin. Owing to this moiety, C1-INH intervenes in regulation of the inflammatory reaction. The heterozygous deficiency of C1-INH results in hereditary angioedema (HAE). The clinical picture of HAE is characterized by bouts of local increase in vascular permeability. Depending on the affected site, patients suffer from disfiguring subcutaneous edema, abdominal pain, vomiting and/or diarrhoea for edema of the gastrointestinal mucosa, dysphagia, and dysphonia up to asphyxia for edema of the pharynx and larynx. Apart from its genetic deficiency, there are several pathological conditions such as ischemia-reperfusion, septic shock, capillary leak syndrome, and pancreatitis, in which C1-INH has been reported to either play a pathogenic role or be a potential therapeutic tool. These potential applications were identified long ago, but controlled studies have not been performed to confirm pilot experiences. Recombinant C1-INH, produced in transgenic animals, has recently been produced for treatment of HAE, and clinical trials are in progress. We can expect that the introduction of this new product, along with the existing plasma derivative, will renew interest in exploiting C1-INH as a therapeutic agent.

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.

We have previously shown that despite the presence of mRNA encoding CFTR, renal proximal cells do not exhibit cAMP-sensitive Cl(-) conductance (Rubera I, Tauc M, Bidet M, Poujeol C, Cuiller B, Watrin A, Touret N, Poujeol P. Am J Physiol Renal Physiol 275: F651-F663, 1998). Nevertheless, in these cells, CFTR plays a crucial role in the control of the volume-sensitive outwardly rectifying (VSOR) activated Cl(-) currents during hypotonic shock. The aim of this study was to determine the role of CFTR in the regulation of apoptosis volume decrease (AVD) and the apoptosis phenomenon. For this purpose, renal cells were immortalized from primary cultures of proximal convoluted tubules from cftr(+/+) and cftr(-/-) mice. Apoptosis was induced by staurosporine (STS; 1 microM). Cell volume, Cl(-) conductance, caspase-3 activity, intracellular level of reactive oxygen species (ROS), and glutathione content (GSH/GSSG) were monitored during AVD. In cftr(+/+) cells, AVD and caspase-3 activation were strongly impaired by conventional Cl(-) channel blockers and by a specific CFTR inhibitor (CFTR(inh)-172; 5 microM). STS induced activation of CFTR conductance within 15 min, which was progressively replaced by VSOR Cl(-) currents after 60 min of exposure. In parallel, STS induced an increase in ROS content in the time course of VSOR Cl(-) current activation. This increase was impaired by CFTR(inh)-172 and was not observed in cftr(-/-) cells. Furthermore, the intracellular GSH/GSSG content decreased during STS exposure in cftr(+/+) cells only. In conclusion, CFTR could play a key role in the cascade of events leading to apoptosis. This role probably involves control of the intracellular ROS balance by some CFTR-dependent modulation of GSH concentration.

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900s(-1) and 8-25s(-1), respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC50 values of 0.2-20muM and 50-170muM, respectively. The K(M)s of the enzymes for H2O2 in the catalase reaction were relatively invariant between 3 and 5mM at pH 7.0, but increased to values ranging from 20 to 225mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H2O2 binding to Cpd I. The catalatic k(cat) was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002s(-1), respectively. Only the NADH oxidase reaction varied more widely between 10(-4) and 10(-2)s(-1) with the fastest rate being exhibited by the enzyme from B. pseudomallei.

This review focuses on the role of Toll-like receptors (TLRs) in lupus and on possibilities to treat lupus using TLR modulating inhibitory oligodeoxynucleotides (INH-ODNs). TLRs bridge innate and adaptive immune responses and may play an important role in the pathogenesis of systemic lupus erythematosus. Of particular interest are TLR3, -7, -8, and -9, which are localized intracellularly. These TLRs recognize single-stranded or double-stranded RNA or hypomethylated CpG-DNA. Exposure to higher order CpG-DNA ligands or to immune complexed self-RNA triggers activation of autoreactive B cells and plasmacytoid dendritic cells. INH-ODNs were recently developed that block all downstream signaling events in TLR9-responsive cells. Some of these INH-ODNs can also target TLR7 signaling pathways. Based on their preferential cell reactivity, we classify INH-ODNs into class B and class R. Class B ('broadly reactive') INH-ODNs target a broad range of TLR-expressing cells. Class R ('restricted') INH-ODNs easily form DNA duplexes or higher order structures, and are preferentially recognized by autoreactive B cells and plasmacytoid dendritic cells, rather than by non-DNA specific follicular B cells. Both classes of INH-ODNs can block animal lupus. Hence, therapeutic application of these novel INH-ODNs in human lupus, particularly class R INH-ODNs, may result in more selective and disease-specific immunosuppression.

Activation products of the early complement components C1, C4 and C3 can be found colocalized with diffuse and fibrillar beta-amyloid (beta/A4) deposits in Alzheimer's disease (AD) brains. Immunohistochemically, C1-esterase inhibitor (C1-Inh) and the C1 subcomponents C1s and C1r can not, or only occasionally, be detected in plaques or in astrocytes. The present finding that C1q, C1s and C1-Inh mRNA are present in both AD and control brains suggests that the variable immunohistochemical staining results for C1r, C1s and C1-Inh are due to a rapid consumption, and that the inability to detect C1s, C1r or C1-Inh is probably due to the dissociation of C1s-C1-Inh and C1r-C1-Inh complexes from the activator-bound C1q into the fluid phase. Employing monoclonal antibodies specific for different forms of C1-Inh, no complexed C1-Inh could be found, whereas inactivated C1-Inh seems to be present in astrocytes surrounding beta/A4 plaques in AD brains. These findings, together with our finding (using reverse transcriptase-polymerase chain reaction) that C1-Inh is locally produced in the brain, suggest that in the brain complement activation at the C1 level is regulated by C1-Inh. Immunohistochemically, no evidence for the presence of the late complement components C5, C7 and C9, or of the membrane attack complex (MAC), was found in beta/A4 plaques. In contrast to the mRNA encoding the early components, that of the late complement components appears to be hardly detectable (C7) or absent (C9). Thus, without blood-brain-barrier impairment, the late complement components are probably present at too low a concentration to allow the formation of the MAC, which is generally believed to be responsible for at least some of the neurodegenerative effects observed in AD. Therefore, the present findings support the idea that in AD, complement does not function as an inflammatory mediator through MAC formation, but through the action of early component activation products.

Angioedema due to acquired C1-inhibitor (C1-INH) deficiency (also referred to as "acquired angioedema") is a rare, life-threatening disease with poorly defined etiology, therapy, and prognosis. To define the profile of acquired C1-INH deficiency and to facilitate the clinical approach to these patients, we report on 23 patients with acquired C1-INH deficiency followed for up to 24 years (median, 8 yr), and review the literature. We measured C1-INH activity with chromogenic assay and detected autoantibodies to C1-INH by enzyme-linked immunosorbent assay (ELISA). Median age at onset of angioedema was 57 years (range, 39-75 yr). All patients had C1-INH function and C4 antigen below 50% of normal. C1q was reduced in 17 patients. Autoantibodies to C1-INH were present in 17 patients. Long-term prophylaxis of attacks with danazol was effective in 2 of 6 patients, and with tranexamic acid, in 12 of 13 patients. Therapy with C1-INH plasma concentrate was necessary in 12 patients: 9 had rapid positive response and 3 became progressively resistant. Associated diseases at the last follow-up were non-Hodgkin lymphomas (3 patients), chronic lymphocytic leukemia (1 patient), breast cancer (1 patient), monoclonal gammopathies of uncertain significance (13 patients). In 4 patients no pathologic condition could be demonstrated. Compared with the general population, patients with acquired C1-INH deficiency present higher risk for B-cell malignancies, but not for progression of monoclonal gammopathies of uncertain significance to malignancy. Antifibrinolytic agents are more effective than attenuated androgens in long-term prophylaxis. Patients with acquired C1-INH deficiency may be resistant to replacement therapy with C1-INH plasma concentrate.

In the present study, the biochemical manifestations of liver toxicity caused by co-administration of anti-TB drugs, rifampicin (RIF), isoniazid (INH) and pyrazinamide (PZA), in a sub-chronic mode (12 weeks), were investigated. Significant alterations were revealed in (a) increased levels of alanine aminotrasferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) and a high bilirubin content in serum; (b) elevated lipid peroxidation (LPO), intracellular calcium [Ca(2+)](i) and CYP4502EI activity in liver; and (c) decreased glutathione (GSH) content, glutathione peroxidase (GPx) and catalase activities in liver. Silymarin reversed these abnormal alterations. The biochemical changes were supported by histological observations.

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.