Anti-C1s monoclonal antibody BIVV009 in late antibody-mediated kidney allograft rejection-results from a first-in-patient phase 1 trial.

The classical pathway (CP) of complement may contribute to the pathogenesis of antibody-mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first-in-patient phase 1b trial was to evaluate the safety/tolerability and CP-blocking potential of 4 weekly doses (60 mg/kg) of the anti-C1s antibody BIVV009 in complement-mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years posttransplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement-fixing donor-specific antibodies (DSA). During 7 weeks follow-up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA-triggered CP activation in serum. Five of 8 C4d-positive recipients turned C4d-negative in 5-week follow-up biopsies, while another 2 recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody-triggered CP activation, even though short-course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials.gov NCT#02502903.

Effect of the Anti-C1s Humanized Antibody TNT009 and Its Parental Mouse Variant TNT003 on HLA Antibody-Induced Complement Activation-A Preclinical In Vitro Study.

The classic pathway (CP) of complement is believed to significantly contribute to alloantibody-mediated transplant injury, and targeted complement inhibition is currently considered to be a promising approach for preventing rejection. Here, we investigated the mode of action and efficacy of the humanized anti-C1s monoclonal antibody TNT009 and its parental mouse variant, TNT003, in preclinical in vitro models of HLA antibody-triggered CP activation. In flow cytometric assays, we measured the attachment of C1 subcomponents and C4/C3 split products (C4b/d, C3b/d) to HLA antigen-coated flow beads or HLA-mismatched aortic endothelial cells and splenic lymphocytes. Anti-C1s antibodies profoundly inhibited C3 activation at concentrations >20 µg/mL, in both solid phase and cellular assays. While C4 activation was also prevented, this was not the case for C1 subcomponent attachment. Analysis of serum samples obtained from 68 sensitized transplant candidates revealed that the potency of inhibition was related to the extent of baseline CP activation. This study demonstrates that anti-C1s antibodies TNT009 and TNT003 are highly effective in blocking HLA antibody-triggered complement activation downstream of C1. Our results provide the foundation for clinical studies designed to investigate the potential of TNT009 in the treatment or prevention of complement-mediated tissue injury in sensitized transplant recipients.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro.

Women's experiences with tailored use of a combined oral contraceptive: a qualitative study.

STUDY QUESTION: What are women's experiences with tailored use of combined oral contraceptive pills (COCPs)? SUMMARY ANSWER: Some women reported very positive experiences with tailored use of COCPs, others did not like the unpredictability about when they would bleed and some women reported increased anxiety about possible pregnancy. WHAT IS KNOWN ALREADY: While many studies have investigated views toward extended use of COCPs, little research has examined women's actual experiences with these regimens. STUDY DESIGN, SIZE, DURATION: This was a semi-structured qualitative interview study that was part of a larger randomized trial of a standard (21 daily pills followed by a 7-day pill-free interval) versus a tailored regimen (daily pills until 3-consecutive-day bleeding triggers a 3-day pill-free interval) of Microgynon 30® mcg (Ethinyl estradiol 30 mcg, Levonorgestrel 150 mcg). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Interviews were conducted with 26 women (17 in the tailored group and 9 who switched their assigned treatment group) . Data were analyzed using thematic analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Women discussed positive changes associated with tailored use of COCPs, as well as some negative consequences. The major themes identified in the interview data were: ease of tailored regimen; changes in cycle-related symptoms; adjustment to reduced/absent bleeding and unpredictability about bleeding. LIMITATIONS, REASONS FOR CAUTION: The sample comprised mainly young, nulliparous women. The majority of women were using COCPs at the start of the study. WIDER IMPLICATIONS OF THE FINDINGS: Clinicians discussing extended-use regimes with patients should mention that women may need time to adjust to an extended-use regime. Future research should attempt to identify predictors of response to extended use of COCPs.

Expression of an electrically silent voltage-gated potassium channel in the human placenta.

Human placental expression of K(V)9.3, a voltage-gated K channel linked to tissue oxygenation responses, has been suggested at the messenger RNA level but tissue localisation has not been described. We aimed to: (1) produce an antibody to human K(V)9.3 and (2) assess channel expression and distribution in human placental tissue. We determined human placental protein expression and localisation using an antibody to K(V)9.3. Antibody specificity was confirmed by Western blotting. Staining was observed in syncytiotrophoblast microvillous membrane, endothelial cells (in intermediate, stem villi and chorionic plate blood vessels) and vascular smooth muscle cells (large diameter vessels only) by immunohistochemistry. Expression was unchanged in tissue from women with small-for-gestational age babies. It was concluded that K(V)9.3 is localised to human placental vascular tissues and syncytiotrophoblast.

Molecular epidemiology of HBV among HIV infected individuals in Chennai, south India.

Hepatitis B is a major co-infection among people with HIV (PWHIV) worldwide. There is a paucity of data on HBV genetic diversity in India, which would be useful for targeted preventive and management interventions. To characterize the distribution of HBV genotypes and sub-genotypes, samples of 180 HIV-HBV co-infected individuals from a study previously conducted to estimate the prevalence of HBV co-infection were analyzed. Nested PCR using type-specific primers was used to identify the various HBV genotypes. Partial HBV S sequences were generated for a subset of samples using Sanger sequencing. Mutation analysis was done using the online HBVseq program. PCR based genotyping documented D (69.4 %) and A (5.6 %) to be the major genotypes in the study population. Infection with multiple genotypes was observed in 25 % co-infected individuals. D2, D5, A2, and A1 were the sub-genotypes detected. Mutations 184K and 173L were identified. HBV genotypes/ sub-genotypes play a pivotal role in the clinical outcome of chronic hepatitis B (CHB). Therefore, monitoring of CHB cases is needed to track disease progression, including early detection of hepatocellular carcinoma.

Oral glucosamine modulates the response of the liver and lymphocytes of the mesenteric lymph nodes in a papain-induced model of joint damage and repair.

OBJECTIVES: To assess whether glucosamine (GlcN), an oral supplement commonly taken to relieve the symptoms of osteoarthritis, modulates the immune and inflammatory responses to joint injury in organs proximal to GlcN absorption; namely, the liver and the gut-draining lymph nodes. METHOD: Using a papain-injected knee mouse model, standard histological methods were used to validate our model and document the impact of GlcN (100mg/kg/day) on groups of C57BL/6 mice (n=5). Circulating inflammatory cytokines were assessed by Luminex-based immunoassays and the relevance of this cytokine profile on proteoglycan biosynthesis evaluated using a patellar-cartilage assay. Real-time PCR was used to document the role of the liver in cytokine production. Finally, we appraised the activation of mesenteric lymph nodes (MLNs) lymphocytes by flow cytometry. RESULTS: Papain significantly degraded the proteoglycans in the injected knees by 2 days. Cartilage proteoglycan content was significantly higher in GlcN-treated, papain-injected knees at Day 14. The peak concentration of serum pro-inflammatory cytokines occurred earlier and decreased sooner in the injected, GlcN-supplemented mice; this trend was in agreement with the expression of these factors by the liver. GlcN did not alter the percentage of MLN populations but accelerated their activation. CONCLUSIONS: Oral GlcN alters the physiology of the liver and MLNs, which in turn, could indirectly alter the biology of the injured joint.

Characterisation of 3' end formation of the yeast HIS3 mRNA.

The nucleotide (nt) sequence of the 3' end of the yeast HIS3 mRNA was determined by PCR amplification of the 3' end. Analysis of 28 individual clones revealed that at least 13 distinct polyadenylation sites are present. The sites of polyadenylation are extremely heterogeneous and do not show any obvious similarity other than that they occur after pyrimidine residues in most cases. Most mutants carrying internal deletions of the 3' untranslated region (3' UTR) did not abolish 3' end formation and showed polyadenylation at normal sites. Deletion of a 90-nt region that contains an A+T-rich sequence close to the 3' end of the HIS3 coding sequence and a subset of processing sites resulted in a drastic reduction in the levels of full-length HIS3 mRNA and concomitant transcription past the normal HIS3 3' end. The 90-nt region appears to be sufficient to direct the formation of at least a subset of the HIS3 3' ends since mutants that carry deletions of flanking regions of this sequence show detectable levels of HIS3 mRNA. Spacing between the upstream A-T sequence and the site of processing is variable. In the light of the extreme heterogeneity of the sites, a possible mechanism for 3' processing is discussed.

Evidence that high-density lipoprotein cholesterol is an independent predictor of acute platelet-dependent thrombus formation.

Plasma total and low-density lipoprotein (LDL) cholesterol are established risk factors for atherosclerotic vascular disease and may also contribute to a prothrombotic risk via enhanced platelet reactivity. This study examines whether high-density lipoprotein (HDL) cholesterol, which is inversely correlated with coronary artery disease, is associated with a reduced thrombogenic potential. Platelet thrombus formation was evaluated by exposing porcine aortic media placed in Badimon perfusion chambers to flowing nonanticoagulated venous blood for 5 minutes at a shear rate of 1,000 s(-1). Forty-five subjects, 23 normal (LDL 104 +/- 31, HDL 50 +/- 15 mg/dl) and 22 hypercholesterolemic (LDL 181 +/- 45, HDL 41 +/- 10 mg/dl) patients without coronary artery disease were studied. Platelet aggregation and CD62 antigen expression, and assay for circulating prothrombotic factors were also performed. In univariate analysis platelet thrombus formation correlated with weight (r = 0.33, p = 0.03), diastolic blood pressure (r = 0.39, p = 0.01), HDL cholesterol (r = -0.45, p = 0.003), total/HDL cholesterol (r = 0.43, p = 0.004) and LDL/HDL (r = 0.38, p = 0.01) ratios, and platelet CD62 expression (r = 0.41, p = 0.02). In multiple regression analysis only HDL cholesterol showed significant correlation with platelet thrombus formation (p = 0.03). Platelet aggregation and circulating prothrombotic factors did not correlate with platelet thrombus formation. A comparison between normal and hypercholesterolemic subjects revealed enhanced thrombus area (0.026 +/- 0.20 vs 0.045 +/- 0.039 mm2/mm; p = 0.04), resting CD62 expression (6 +/- 7% vs 15 +/- 10% positive platelets, p = 0.02), and platelet aggregation (16.7 +/- 5.2 vs 21.7 +/- 6.7 ohms, p = 0.04) in hypercholesterolemic subjects. Our results demonstrate that HDL cholesterol is a significant independent predictor of ex vivo platelet thrombus formation.

Oxidative cyclisation of cinnamyl ethers mediated by CAN: a stereoselective synthesis of 3,4-trans disubstituted tetrahydrofuran derivatives.

The oxidative cyclisation of cinnamyl ethers mediated by cerium(IV) ammonium nitrate results in the stereospecific formation of 3,4-trans disubstituted tetrahydrofuran derivatives in moderate to good yields.

Cocaine euphoria, dysphoria, and tolerance assessed using drug-induced changes in brain-stimulation reward.

The time course of cocaine-induced changes in self-stimulation thresholds were used to evaluate cocaine euphoria and dysphoria as a function of the chronicity of drug treatment, dosage level, and the spacing of injections. It was assumed that cocaine-induced decreases in thresholds were indicative of cocaine euphoria, while increases in thresholds reflected rebound dysphoric responses to cocaine administration. Three experiments were performed using self-stimulating rats implanted with ventral tegmental area electrodes. Cocaine's threshold-lowering effects were evident 15 min postinjection (IP) with thresholds returning to baseline by approximately 3.0 h after treatment. Little evidence for cocaine-induced increases in thresholds was observed during periods of chronic cocaine treatment. However, thresholds were slightly elevated upon withdrawal from chronic cocaine treatment in Experiments 2 and 3. No evidence of tolerance or sensitization to cocaine-induced shifts in thresholds was noted with single daily injections, while multiple daily injections produced tolerance to cocaine's threshold-lowering effects. It is concluded that cocaine's ability to enhance brain-stimulation reward is highly reliable and robust, while decreases in brain-stimulation reward associated with chronic cocaine treatment are less reliable and difficult to demonstrate. The possible influence of drug dosage on the induction of cocaine dysphoria and the ability of various self-stimulation procedures to measure dysphoric effects are discussed.

Banded krait minor satellite (Bkm) contains sex and species-specific repetitive DNA.

A novel class of repetitive DNA was isolated from a Bkm DNA library by exclusion hybridization. This sequence was mapped to the short arm of the W chromosome of banded krait, Bungarus fasciatus. Southern blot hybridization showed that these sequences are sex and species specific. Sequence analysis of a 206 bp long clone, BR87, revealed the presence of a tandem array of two internal repeat units of 18-19 bp alternating with each other with a gap of 1, 2 of 3 nucleotides. To our knowledge, this is the first report of an exclusively W chromosome- and species-specific repeat isolated from any reptile. The functional significance of this sequence based on its organisation is discussed.

Altered nuclear localization of bax protein in BCNU-resistant glioma cells.

To investigate the role of apoptosis suppression in glioma chemotherapy resistance, protein levels and subcellular localization of bcl-2 family members were investigated in 2 pairs of sensitive cell lines and their in vitro generated resistant derivatives. The alkylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), induced apoptosis in both sensitive cell strains and apoptosis was suppressed in both resistant derivatives. Both resistant cell lines contained altered regulation of a bcl-2 related protein consistent with the suppression of apoptosis. Independent of which bcl-2 family member was dysregulated, resistance was associated with altered regulation in the subcellular localization of bax protein. Following BCNU treatment, bax accumulated in nucleoli and a nuclei containing fraction of sensitive cells but not their resistant derivatives. Nuclear accumulation was an early event in apoptosis induction. These data indicates altered subcellular localization of bax may play a role in resistance. In addition, the association between an early, nucleolar localization of bax and the induction of apoptosis suggests that localization of bax to nucleoli may play a role in apoptosis-induction of glioma cells.

Banded krait minor-satellite (Bkm)-associated Y chromosome-specific repetitive DNA in mouse.

The mouse Y chromosome remains highly condensed in all somatic tissues but decondenses extensively in testis. We have isolated a mouse Y chromosome-specific repeat M34 (11.5 kb) and shown that this is distributed along the Y chromosome except the sex-determining region (the Y short arm) in which GATA repeats are predominantly concentrated. It has 32 copies of GATA repeats in a 2.7 kb fragment. About 200-300 copies of M34 on the Y chromosome are interspersed among other sequences. A 1.2 kb fragment (p3) of M34, containing GATA repeats, also has scaffold attachment region (SAR) motifs which bind to nuclear matrices. A strong affinity of histone H1 to SAR motifs is implicated in maintaining the condensed state of the Y chromosome in somatic tissues. The probable significance of molecular organization of the Y chromosome is discussed.

A new technique - semicontinuous rapid flow, high volume exchange - for effective peritoneal dialysis in shorter periods.

During the period of study, 85 dialysis clearance periods were studied in a total of 42 dialyses. Peritoneal clearance of urea and creatinine were observed at varying flow rates of dialysing fluid ranging from 2 litres/hour to 8.5 litres/hour. Flow rates were adjusted to various speeds with indigineously designed diaphragm pump. The diaphragm pump is cheap and simple and has an electronically controlled timer. A linear correlation was observed between dialysate flow rate and peritoneal clearances. With this semicontinuous rapid flow, high volume exchange peritoneal dialysis technique described in this study, correction of serum potassium disturbance is found to be excellent even in shorter periods.

Novel compound heterozygous laminina2-chain gene (LAMA2) mutations in congenital muscular dystrophy. Mutations in brief no. 159. Online.

The laminina2-chain gene (LAMA2) encodes a basal lamina protein, laminina2, known to be deficient in one form of congenital muscular dystrophy (CMD). In a laminina2 deficient-CMD patient, we screened the entire LAMA2 cDNA (953bp) by reverse transcriptase polymerase chain reaction combined with single strand conformational polymorphism analysis. Direct sequencing of aberrant conformers in this patient revealed two loss-of-function mutations, consistent with autosomal recessive inheritance. The patient had two novel heterozygous mutations: 1) an exon 4 nonsense mutation caused by a G-->A substitution at cDNA position 547, changing the TGG codon for tryptophan into a TGA stop codon (W166X) in the N-terminus domain VI;ii) an exon 54 frameshift mutation due to a deletion of nucleotide 'C' at cDNA position 7707 (S2553Y), resulting in a premature stop codon (V2587X) in exon 55 in the globular G domain of laminina2 at the C-terminus. These mutations cause a disruption of the open reading frame of LAMA2. The absence of laminina2 observed in the patient's muscle biopsy could result from diminished levels of the LAMA2 transcript. Alternatively, the mutations might lead to translation of a truncated laminina2. By either mechanism the phenotype of congenital muscular dystrophy is believed to be the result of disruption of linkage between the extracellular matrix and the dystrophin glycoprotein complex.