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.

[Propositions of interpretative comments for thyroid function tests]. / Propositions de commentaires interprétatifs pour les bilans biologiques thyroïdiens.

In the field of thyroid disease, a number of governmental organisms or professional associations have published practice guidelines containing laboratory-related recommendations, eg the Haute autorité de la santé (HAS), or the American Thyroid Association (ATA). Among the physicians who prescribe thyroid function tests, all have not read and memorized all these recommendations. In order to help them to better integrate these recommendations in their practice, we have composed a thesaurus of ready-made interpretative comments, trying to adapt our proposed comments to each possible combination of results of TSH and/or free T4 and/or free T3. The laboratorians who would prefer to use only the comments based strictly on the recommendations of HAS and/or ATA, will be able to select among our comments what is really validated by these two organizations. In addition, our work aims at enabling the patients who want it, to benefit from written information, which may be complementary to the more often spoken information provided by the clinicians.

[Disseminated tuberculosis and profound thrombopenia]. / Tuberculose disséminée et thrombopénie profonde.

Thrombocytopenia within the context of disseminated tuberculosis can lead to complications requiring rapid treatment. Although the origin is generally central, thrombocytopenia can arise from an immune disorder. We hereby report a case of disseminated tuberculosis associated with thrombocytopenia, which required, in addition to antituberculosis therapy initiated before bacteriological proof, corticosteroid treatment and multiple platelet transfusions. The discovery of anti-platelet antibodies along with the success of immunomodulator therapy confirmed the auto-immune origin of this thrombocytopenia.

Binding sites for a peripheral type benzodiazepine antagonist ([3H]PK 11195) in human iris.

Peripheral-type benzodiazepine binding sites have been characterized on sections of 8 normal human iris/ciliary-body preparations. Saturability was determined at 25 degrees C with [3H] PK 11195 (1 nM) a specific ligand of peripheral type sites. The studies revealed a single class of binding sites for PK 11195 with a nanomolar range affinity (KD = 1.45 nM) and a maximal capacity (Bmax) of 35.5 fmol/mg protein. The displacement potency order of the benzodiazepines tested suggest that these sites belong to the peripheral type: PK 11211 (IC50 = 12 nM) greater than Ro 5-4864 (IC50 = 770 nM) greater than clonazepam (IC50 = 20,000 nM). The present data demonstrate that high affinity binding sites for peripheral type benzodiazepines are present in human iris/ciliary-body. This tissue is therefore a suitable tool for evaluation of the putative functional role of these binding sites.

Cell transplantation of genetically altered cells on biodegradable polymer scaffolds in syngeneic rats.

Many severe metabolic deficiencies in children are caused by a single gene defect with a resultant single gene product deficiency. These diseases may be amenable to permanent cure using new techniques of gene transfer and cell transplantation. In many in vivo models of retroviral mediated gene therapy, a significant limiting factor is the ability to transplant a sufficient number of modified cells. To potentially circumvent this problem, we have developed a biodegradable polymer implant system capable of supporting large numbers of genetically modified cells. In this study, we inserted a reporter gene into syngeneic cultured normal fibroblasts and then transplanted these genetically modified cells into animals using synthetic biodegradable polymer fibers as temporary cell delivery scaffolds. To begin to develop a system capable of delivering desirable proteins secreted by genetically modified cells, Fischer 344 adult rat fibroblasts were transduced in tissue culture with a retrovirus containing the reporter gene Lac Z. These genetically modified cells (1.1 x 10(7) cells/graft) were then attached to the biodegradable polymer fibers and the polymer-cell graft was transplanted subdermally into syngeneic recipients (n = 9). There was persistence of the modified cells with expression of the reporter gene for at least 30 days. The estimated number of genetically modified cells per implanted graft decreased from a pretransplant value of 1.1 +/- 0.6 x 10(7) to 3.2 +/- 0.7 x 10(6) by 15 days after transplantation (P < 0.01). Thereafter, the cell number did not vary significantly to the conclusion of the study at day 30 (3.6 +/- 1.0 x 10(6) cells/graft). Evidence of ingrowth and incorporation of other stromal elements was present in the graft by 1 week post-transplantation, as judged by counterstained hematoxylin and eosin micrograph sections. Migration of modified cells to areas outside of the polymer-cell graft was not detected. Over the course of the study, there was little degradation of the polymer implant, although by day 30, evidence of early dissolution was evident. The number of polymer fibers per high power field increased slightly from 62.5 +/- 5.8 on day 1 to 77.3 +/- 26.6 on day 30 (P > 0.2). These data suggest that the use of biodegradable polymer fibers may permit the transplantation of genetically modified cells in sufficient numbers to deliver a therapeutically useful product. Polymer matrices allow for the attachment and site-specific transplantation of genetically modified cells.

Hypoxia in human gliomas: demonstration by PET with fluorine-18-fluoromisonidazole.

Positron emission tomography (PET) with the hypoxic-cell tracer [18F]fluoromisonidazole presents a possible means of noninvasively demonstrating tumor hypoxia. PET studies using this tracer were performed in three patients with malignant glioma, and in all patients the tumor was clearly seen at 5 min postinjection and initial tumor activity exceeded cortical activity. In one patient, there was no tumor retention of [18F] fluoromisonidazole and tumor activity fell while cortical activity increased, with the two tissues reaching equality at 40-50 min. The tumor-to-plasma ratio was 0.71 at 3 hr. The other two patients showed variable tumor retention of [18F]fluoromisonidazole, with tumor-to-plasma ratios of 1.10 and 1.49 at 2 and 3 hr. These results demonstrate the feasibility of using [18F]fluoromisonidazole PET to detect hypoxia in human gliomas in vivo. Clinical trials are needed to determine whether a relationship exists between [18F]fluoromisonidazole uptake and tumor radiation response.

The relationship between nerve terminal adenosine triphosphatases and neurotransmitter release: as determined by the use of antidepressant and other CNS-active drugs.

1 The role of adenosine triphosphatases (ATPases) in neurotransmitter release was studied using nerve terminals (synaptosomes) prepared from rat cerebral cortex as a model. 2 Amitriptyline, nortriptyline, protriptyline, desipramine and imipramine were found to inhibit ATPases at concentrations of 10(-5) M and above. The drugs inhibited both the basal and electrically evoked release of acetylcholine (ACh) and noradrenaline (NA) at concentrations of 10(-4) M and above. 3 At low concentrations of antidepressants (10(-8) and 10(-7) M) release of NA was enhanced but there was no effect on ACh release. 4 Other drugs which inhibit Na+, K+-ATPase increase basal NA release as did drugs which inhibited vesicular MG2+-ATPase. 5 A model is proposed suggesting that transmitter release/re-uptake depends on (1) active Na+, K+-ATPase at the presynaptic membrane and (2) an active synaptic vesicular MG2+-ATPase.

Effects of anticonvulsant and convulsant drugs on the ATPase activities of synaptosomes and their components.

1. The effects of anticonvulsants, and other drugs on the Na+, K+-adenosine triphosphatase (ATPase) (ouabain-sensitive) and Mg++-ATPase activities of synaptosomes and their components have been determined. 2. The Mg++-ATPase activity of synaptosomes was not affected by the drugs but the Na+, K+-ATPase activity was inhibited by phenytoin (diphenylhydantoin), ethosuximide and diazepam. 3. Fractions containing mainly membranes, mitochondria or synaptic vesicles, were prepared from synaptosomes by osmotic shock and subsequent density gradient centrifugation. Inhibition of Na+, K+-ATPase activity by phenytoin, ethosuximide and diazepam was apparent only in the membrane fraction. 4. The fraction containing synaptic vesicles exhibited pronounced Md++-ATPase but no Na+, K+-ATPase activity. In contrast to the enzymes of the membranes and mitochondria, the Mg++-ATPase of the vesicles was inhibited by diazepam and all of the anticonvulsants tested.

Effects of delta9-tetrahydrocannabinol and cannabidiol on a Mg2+-ATPase of synaptic vesicles prepared from rat cerebral cortex.

1. delta9-Tetrahydrocannabinol and cannabidiol both exhibited a concentration-related inhibition of Mg2+-ATPase of vesicles prepared from synaptosomes isolated from rat cerebral cortex. Cannabidiol was about 3 times more potent than tetrahydrocannabinol. 2. These results were similar to those obtained previously using drugs with well established anticonvulsant activity. 3. Tetrahydrocannabinol at a sub-inhibitory concentration (1 micronM) increased the activity of the Mg2+-ATPase relative to values obtained with vehicle controls.

The effects of dopamine agonists and antagonists on Na+,K+-ATPase and Mg2+-ATPase activities of synaptosomes.

The effects of dopamine agonists and antagonists on the Na+,K+-ATPase and Mg2+-ATPase activities of rat cerebral cortex synaptosomes have been determined. Dopamine, ADTN, apomorphine and S-584, but not piribedil, stimulated the activities of the enzymes. The stimulatory effect of dopamine was not antagonised by dopamine antagonists and apparently the catechol group is responsible for the enzyme stimulation.

Effects of physostigmine and electrical stimulation on the acetylcholine content of the guinea-pig ileum.

Incubation with physostigmine (7.7 muM) caused an approximately 2 fold increase in the acetylcholine content of the myenteric plexus--longitudinal muscle preparation of the guinea-pig ileum. This effect was due mainly to an increase in 'free' acetylcholine, which was directly assayable in either the homogenate after removal of cell debris or the supernatant fraction (100,000 g for 60 min) after subcellular fractionation. Acetylcholine output during stimulation at 0.017, 0.1 or 1 Hz was maintained for 60 min at a rate 2--4 times greater than the non-stimulated output; there was no change in content. At 10 HZ, output was high at the start of stimulation and then decreased continuously; there was a proportionate loss of mainly 'free' acetylcholine from the tissue. Mn2+, hexamethonium, morphine and noradrenaline, which depressed acetylcholine output during stimulation at 0.1 HZ, had no effect on the acetylcholine content nor did they affect the increase in acetylcholine content during incubation with physostigmine.

Quantitative autoradiographic determination of binding sites for a peripheral benzodiazepine ligand ([3H]PK 11195) in human iris.

Specific binding sites of peripheral-type benzodiazepines were investigated in human iris/ciliary body (8 eyes). Examination of color-coded prints and densitometric quantification of autoradiograms were performed on slides (20 micron) labelled with [3H]PK 11195 (1 nM) at 25 degrees C. Nonspecific binding was determined with PK 11211 (5 microM) or Ro 5-4864 (5 microM). Binding sites were present on all the slides, with equivalent density in the 3 regions of the preparation (ciliary body, iris, and pupil margin). The numbers of binding sites in ciliary body, iris, and pupil margin, respectively, were: 42.7 +/- 0.2, 30.1 +/- 0.5, and 37.4 +/- 0.4 femtomol/mg protein. Labelling on the pupil margin seemed to coincide with the iris sphincter muscle. The presence of peripheral benzodiazepine binding sites in iris muscular tissue, and particularly in the pupil margin, suggests that the iris preparation may be a valuable tool to detect putative physiological effects of peripheral benzodiazepines on muscular motility.

MRI-monitored cryosurgery in the rabbit brain.

The inability to observe the transient, irregular shape of the frozen region that develops during cryosurgery has inhibited the application of this surgical technique to the treatment of tumors in the brain and deep in visceral organs. We used proton NMR spin-echo and spoiled gradient-echo imaging to monitor the development of frozen lesions during cryosurgery in the rabbit brain and the resulting postcryosurgical changes up to 4 hr after freezing. Spoiled gradient-echo images (TE = 14 ms; TR = 50 ms) were acquired during freezing and thawing at a rate of 15 s/slice. Although the frozen region itself is invisible in MR images, its presence is distinguished easily from the surrounding unfrozen soft tissue because of the large contrast difference between frozen and unfrozen regions. T2-weighted spin-echo images (TE = 100 ms, TR = 2 s) obtained after thawing suggest that edema forms first at the margin of the region that was frozen (cryolesion) and then moves into the region's core. Histological examination showed complete necrosis in the cryolesion and a sharp transition to undamaged tissue at the margin of the lesion and its image. Blood-brain barrier (BBB) damage was investigated using gadolinium-DTPA. The region of edema in the T2-weighted spin-echo images was coincident with the area of BBB damage in the Gd-DTPA-enhanced T1-weighted spin-echo images (TE = 33 ms, TR = 400 ms) and both were distinguishable as areas of high signal relative to the surrounding normal tissue. The results of these experiments indicate that MR can both effectively monitor the cryosurgical freezing and thawing cycle and characterize the postcryosurgical changes in tissue during follow-up.

Temperature determination in the frozen region during cryosurgery of rabbit liver using MR image analysis.

Cryosurgery currently is being used clinically to treat tumors in internal organs such as the liver and prostate. Although performed at present under ultrasound monitoring, magnetic resonance imaging (MRI)-guidance of these procedures not only permits monitoring of the frozen region during cryosurgery but also makes it possible to determine the temperature distribution in the frozen region, which is not possible using ultrasound monitoring. A good estimate of the region of destruction in the tissue can be obtained from correlating the temperature distribution and the time course of the freezing with the image of the frozen region. Unfortunately, MR pulse sequence-based temperature determination techniques such as diffusion, relaxation time, and chemical shift cannot be used for measuring the temperature in the frozen region because the T2 of the frozen regions is so short that there is effectively no RF signal from the frozen region. This paper describes a numerical technique for determining the two dimensional temperature distribution in the frozen region during MR image-guided cryosurgery of normal liver in rabbits. The technique involves solving the energy equation numerically in the frozen region to determine the temperature distribution there. The boundary conditions needed to solve the equation are determined from MR images of the frozen tissue during cryosurgery and from the measured temperature of the cryoprobe. The calculated temperature in the frozen region is then correlated with the damaged region (cryolesion) determined from post mortem histologic evaluation.

In vivo cytogenetic activity of sulphonylurea drugs in man.

A cytogenetic investigation of diabetic patients undergoing treatment with sulphonylurea drugs, particularly chlorpropamide, shows that significantly more chromatid aberrations and chromosome exchange aberrations are present in the lymphocytes of these patients compared with controls. This is taken as evidence of possible mutagenic activity by these drugs, although the possibility cannot be ruled out that the diabetic state itself is a contributory factor.

Minor pediatric injuries.

Injuries are a common source of childhood morbidity and mortality. The initial evaluation should follow in a sequential fashion to determine the extent of injuries. Most minor injuries can be treated safely and cost-effectively in an office setting. The principles of wound care include adequate hemostasis, tissue debridement, removal of imbedded foreign bodies, and appropriate closure or coverage of the wound to optimize healing. Appropriate use of antibodies, tetanus prophylaxis, and rabies immunization will minimize complications. With proper selection and treatment, the outcome of children with minor injuries should be excellent.

Dopamine-sensitive adenylate cyclase in canine renal artery.

To characterize further a putative dopamine receptor in the renal artery, the effects of dopamine on canine renal artery adenylate cyclase activity were studied. Since the femoral artery is thought to be devoid of a similar dopamine receptor, the effects of dopamine on the adenylate cyclase activity of the canine femoral artery were also studied. In tissues from dogs with or without phenoxybenzamine pretreatment, renal artery adenylate cyclase was maximally stimulated by 4 muM dopamine, compared to 20 muM required for the femoral artery enzyme. The concentrations of isoprenaline required to maximally stimulate renal and femoral artery adenylate cyclase were 0-04 and 0-2 muM, respectively. In tissue from the phenoxybenzamine-pretreated dog, the sitmulatroy effect of dopamine on the renal artery enzyme was selectively blocked by 0-01 muM haloperidol, but not by 0-2 muM propranolol. In the femoral artery, however, the dopamine stimulation was blocked by both antagonists. Stimulation by isoprenaline of renal and femoral artery adenylate cyclase was blocked by propranolol. These data suggest the concept that dopamine interacts with a specific artery receptor apparently different from alpha-and beta-adrenoceptors.

Quantitation of transplanted hepatic mass necessary to cure the Gunn rat model of hyperbilirubinemia.

The minimal hepatic mass necessary to reverse the metabolic defect of unconjugated hyperbilirubinemia in the rat model of Crigler-Najjar type I deficiency was determined using heterotopic (auxiliary) partial liver transplantation (HLT) and orthotopic liver transplantation (OLT). In HLT, the donor graft consisted of the right upper and/or right lower hepatic lobe(s) depending on the final mass of liver tissue desired for transplantation. The mass of the donor graft ranged from 12% to 23% of the whole organ (n = 12). The serum unconjugated bilirubin levels decreased quickly after HLT from a preoperative value of 8.98 +/- 0.34 mg/dL to 0.63 +/- 0.11 mg/dL in 24 hours, which was similar to OLT in which the levels decreased from a preoperative value of 8.20 +/- 0.44 mg/dL to 0.24 +/- 0.07 mg/dL in 24 hours. Conjugated bilirubin was excreted from the graft liver shortly after OLT and also from both the host and graft livers after HLT. This study demonstrates that using as little as 12% of the whole liver mass in HLT reduces serum bilirubin significantly in 24 hours in a fashion similar to whole-organ OLT. The clinical application of alternative therapies to whole-organ OLT such as HLT or hepatocyte transplantation may provide sufficient replacement therapy in metabolic disease.

Does early ultrasonography affect management of pediatric appendicitis? A prospective analysis.

BACKGROUND: Appendicitis remains a difficult diagnosis in children. Ultrasonography is increasingly used for the diagnosis of appendicitis, although the proper clinical role for this test remains unclear. METHODS: To evaluate the clinical utility of ultrasonography in appendicitis, the authors analyzed prospectively all children evaluated for possible appendicitis from January 1 through December 31, 1997. Children with a high clinical suspicion of appendicitis were referred for surgery (n = 122). Children with equivocal findings of appendicitis were referred for early ultrasonography (EUS) and formed the study cohort (n = 103). An initial management plan was made to operate or observe each patient, and a risk of appendicitis (doubtful, possible, probable) was assigned by a pediatric surgery fellow. EUS was then performed, and its effect on management was assessed. RESULTS: Using clinical judgment to operate at initial presentation, the sensitivity was 38% and specificity was 95%. Using EUS alone, the sensitivity was 87% and specificity was 88%. The management of 30 of 103 patients (30%) was changed after EUS, including a decision to operate in 28 patients and a decision not to operate in two patients. CONCLUSIONS: EUS appears to have substantial clinical utility in children with equivocal findings of appendicitis, and its use complements the clinical management. The use of EUS can improve patient care and reduce hospital resource utilization.