Blocking FcRn in humans reduces circulating IgG levels and inhibits IgG immune complex-mediated immune responses.

The neonatal crystallizable fragment receptor (FcRn) functions as an intracellular protection receptor for immunoglobulin G (IgG). Recently, several clinical studies have reported the lowering of circulating monomeric IgG levels through FcRn blockade for the potential treatment of autoimmune diseases. Many autoimmune diseases, however, are derived from the effects of IgG immune complexes (ICs). We generated, characterized, and assessed the effects of SYNT001, a FcRn-blocking monoclonal antibody, in mice, nonhuman primates (NHPs), and humans. SYNT001 decreased all IgG subtypes and IgG ICs in the circulation of humans, as we show in a first-in-human phase 1, single ascending dose study. In addition, IgG IC induction of inflammatory pathways was dependent on FcRn and inhibited by SYNT001. These studies expand the role of FcRn in humans by showing that it controls not only IgG protection from catabolism but also inflammatory pathways associated with IgG ICs involved in a variety of autoimmune diseases.

Controlled release recombinant human interferon-α2b for treating patients with chronic hepatitis C genotype 1: a phase 2a clinical trial.

Better convenience and tolerability and sustained therapeutic concentrations might improve interferon (IFN) treatment for chronic hepatitis C virus (HCV) infection. In an open-label, randomized study, controlled release free (chemically unmodified) recombinant human IFN-α(2b) in poly(ether-ester) microspheres (CR-rhIFN-α(2b)), was injected at doses of 160, 320, 480 or 640 µg every 2 weeks for 12 weeks with concomitant weight-based oral ribavirin in 32 treatment-naïve patients with chronic HCV genotype 1. Treatment was well tolerated, with 31 patients (97%) successfully completing the study. Full doses of CR-rhIFN-α(2b) were administered on 96% of scheduled occasions. Flu-like symptoms were generally mild and brief. Injection site reactions developed in 13 patients (41%), and neutropenia occurred in six of eight patients receiving 640 µg. In the 320, 480 and 640 µg groups, 62-75% of patients achieved a ≥2 log(10) HCV RNA reduction by 4 weeks and 88-100% by 12 weeks. For those groups, the pooled median time to ≥2 log(10) reduction was 11 days (95% confidence interval, 7-35 days). In those groups, viral reduction below the limit of detection was accomplished in 25% of patients by 4 weeks and in 62% by 12 weeks. The 160-µg dose was less potent. After CR-rhIFN-α(2b) injection, stable plateau levels of serum IFN-α(2b) were generally reached within 72 h. Treatment-emergent neutralizing antibodies to IFN-α(2b) were observed in one patient. No antibodies to host plant proteins were detected. CR-rhIFN-α(2b) with ribavirin cotherapy was well tolerated and displayed potent early antiviral activity in patients with chronic HCV genotype 1.

Signal transduction in larval trematodes: putative systems associated with regulating larval motility and behaviour.

The multi-host lifestyle of parasitic trematodes necessitates their ability to communicate with their external environment in order to invade and navigate within their hosts' internal environment. Through recent EST and genome sequencing efforts, it has become clear that members of the Trematoda possess many of the elaborate signal transduction systems that have been delineated in other invertebrate model systems like Drosophila melanogaster and Caenorhabditis elegans. Gene homologues representing several well-described signal receptor families including receptor tyrosine kinases, receptor serine tyrosine kinases, G protein-coupled receptors and elements of their downstream signalling systems have been identified in larval trematodes. A majority of this work has focused on the blood flukes, Schistosoma spp. and therefore represents a narrow sampling of the diverse digenean helminth taxon. Despite this fact and given the substantial evidence supporting the existence of such signalling systems, the question then becomes, how are these systems employed by larval trematodes to aid them in interpreting signals received from their immediate environment to initiate appropriate responses in cells and tissues comprising the developing parasite stages? High-throughput, genome-wide analysis tools now allow us to begin to functionally characterize genes differentially expressed throughout the development of trematode larvae. Investigation of the systems used by these parasites to receive and transduce external signals may facilitate the creation of technologies for achieving control of intramolluscan schistosome infections and also continue to yield valuable insights into the basic mechanisms regulating motility and behaviour in this important group of helminths.

Protein kinase C regulation of cell spreading in the molluscan Biomphalaria glabrata embryonic (Bge) cell line.

Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMA-induced expression of activated MAPK, suggesting that PKC-, Ras- and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek.

Receptor-ligand interactions and cellular signalling at the host-parasite interface.

Although the effects of trematode infection on snail host physiology or host responses on parasite development have been well described in the literature, very little is known regarding the underlying mechanisms and specific molecules responsible for mediating those effects. It is presumed that many host-parasite interactions are communicated through receptor-mediated events, in particular those involving haemocytic immune responses to invading parasites, larval motility and migration through host tissues, and larval acquisition of host molecules either as nutrients or critical developmental factors. The intent of this chapter is to review current knowledge of molecules (both receptors and their ligands or counter-receptors) involved in molecular communication at the interface between larval trematodes, especially the mother or primary sporocyst stage, and host cells/tissues in intimate proximity to developing larvae. Information to date suggests that the molecular exchange at this interface is a highly complex and dynamic process, and appears to be regulated in specific cases. Topics discussed will focus on snail cell receptor interactions with the sporocyst tegument and its secretions, host cell-cell and cell-substrate adhesion receptors and their related signal transduction pathways, and sporocyst tegumental surface receptors and ligands involved in the binding of soluble host molecules.

Local adenovirus-mediated delivery of hirudin in a rabbit arterial injury model.

UNLABELLED: Intravascular delivery of an E1/E3 deleted adenovirus encoding the hirudin protein reduces neointimal formation in the rat arterial injury model. Given the interspecies variability in response to adenoviral vectors, we tested this same construct in the hirudin-sensitive cholesterol-fed rabbit arterial balloon injury model. We hypothesized that local delivery of an E1/E3-deleted adenovirus encoding hirudin (Ad-Hir) in addition to early hirudin infusion would limit neointimal formation compared to early hirudin alone. METHODS AND RESULTS: Local delivery of Ad-Hir, 2.5 x 10(10) PFU/ml, using a double balloon catheter [n = 6 vessels (v)] produced a 79% reduction in vessel wall thrombin activity at 48 h after balloon angioplasty (BA) compared with vehicle (Veh, n = 6v; p = 0. 05). In chronic experiments, hypercholesterolemic rabbits underwent femoral BA, and received either early hirudin alone (n = 9v) or early hirudin plus locally delivered Ad-Hir (early hirudin + Ad-Hir; n = 9v), an E1/E3-deleted adenovirus encoding beta-galactosidase (early hirudin + AdGal; n = 7v), or Veh (early hirudin + Veh; n = 10v). Early hirudin + Ad-Hir did not limit the arterial response to injury versus the other groups at 4 weeks after BA. Plaque area, cross-sectional luminal area narrowing by plaque, and T cell infiltration were significantly increased in the adenovirus- versus non-adenovirus-treated arteries. Plaque area correlated with T cell density. CONCLUSION: Following BA in cholesterol-fed rabbits, local transduction with A-Hir produced a marked reduction in vessel wall-associated thrombin activity. However, this strategy increased rather than decreased the arterial response to BA injury. Our results suggest that the lack of therapeutic effect resulted from adenovirus-stimulated plaque formation, possibly resulting from a T cell-mediated inflammatory response.

Transcatheter regional urokinase therapy in the management of inferior vena cava thrombosis.

PURPOSE: To study the efficacy of local infusion of urokinase (UK) in the treatment of symptomatic inferior vena cava (IVC) thrombosis. MATERIALS AND METHODS: Eight patients (five men and three women) who ranged in age from 19 years to 75 years (mean, 56 years) with symptomatic IVC thrombosis underwent local catheter-directed infusion of UK with use of up to three access sites. Infrarenal IVC thrombus and iliac vein thrombus was identified in all patients. Four patients had extension of thrombus proximal to the renal veins. Seven of eight patients had at least one risk factor for IVC thrombosis: hypercoagulable state (n = 3), IVC filter (n = 3), malignancy (n = 2), recent surgery (n = 2), and oral contraceptive use (n = 1). No serious procedure-related complications were encountered, although one patient died 5 days after UK therapy of pulmonary failure due to advanced lung cancer. UK was infused for an average of 79 hours (range, 24-140 hours) and a mean total dose of 7.4 million U of UK (range, 2.9-14.4 million U). Adjunctive balloon angioplasty was performed in three patients. No vascular stents were placed. Clinical and/or radiographic follow-up was obtained in all eight patients. RESULTS: Thrombolysis was successful in seven of eight (88%) IVCs with no or minimal residual thrombus. The remaining seven patients had no lower extremity swelling 2-24 months (mean, 11 months) after the procedure. Three of seven patients had computed tomographic or venographic follow-up (mean, 9 months; range, 1.5-15 months), demonstrating unchanged or improved IVC patency. CONCLUSIONS: Transcatheter regional infusion of UK for re-establishing venous patency in acute IVC thrombosis appears to be effective with good short-term and mid-term clinical benefit.

Early plus delayed hirudin reduces restenosis in the atherosclerotic rabbit more than early administration alone: potential implications for dosing of antithrombin agents.

BACKGROUND: A 2-hour infusion of r-hirudin at the time of balloon angioplasty limits restenosis in atherosclerotic rabbits. Because thrombin activity in the vessel wall after angioplasty remains high for 48 to 72 hours, we hypothesized that a second infusion of hirudin at 24 hours would reduce restenosis more than early treatment alone. METHODS AND RESULTS: Femoral atherosclerosis was induced in 35 rabbits by air desiccation injury and a high-cholesterol diet. At the time of angioplasty, rabbits were randomly assigned to 1 of 4 groups: controls: heparin bolus, saline infusion at 24 hours; early hirudin: hirudin bolus+2 hours' infusion, saline infusion at 24 hours; delayed hirudin: heparin bolus, hirudin infusion+/-bolus at 24 hours; and early+delayed hirudin: hirudin bolus+2 hours' infusion, hirudin infusion+/-bolus at 24 hours. Rabbits were euthanized after 28 days. The early+delayed hirudin treatment group had less loss of minimal lumen diameter by angiography at 28 days. By histomorphometry, cross-sectional area narrowing by plaque was least in the early+delayed treatment group compared with controls (P=0.0001), early hirudin (P=0.01), or delayed hirudin (P=0.001). The early+delayed hirudin group also had a significant reduction in absolute plaque area and an improvement in lumen area compared with the other groups. No differences were observed between treatment groups with respect to the cross-sectional area encompassed by the internal or external elastic laminae. CONCLUSIONS: Combined early+delayed administration of hirudin significantly reduces angiographic restenosis and cross-sectional area narrowing by plaque compared with early or late treatment alone. These results suggest that restenosis after balloon angioplasty is markedly influenced by thrombin-mediated events not only occurring early but also extending beyond the first 24 hours in this model.

Establishing an institution-specific therapeutic range for heparin.

The relationship between heparin concentration and activated partial thromboplastin time (aPTT) in pooled plasma was compared with that in patient samples to assess the feasibility of using heparin-spiked pooled plasma in determining a therapeutic range for aPTT. Blood samples were taken from 32 patients who had been receiving intravenous unfractionated heparin sodium for more than 24 hours. The samples were stored at -70 degrees C until anti-Xa assay within three months of collection. Pooled normal plasma was spiked with unfractionated heparin sodium to produce nominal anti-Xa concentrations of 0, 0.05, 0.1, 0.2, and 0.5 unit/mL. Heparin concentrations and a aPTT values were measured, and the relationship between the two was determined by linear regression. For the ex vivo samples, the range of aPTT values corresponding to therapeutic heparin concentrations of 0.3-0.7 anti-Xa unit/mL was 64-106 seconds, which corresponds to an aPTT range of 2.3-3.9 times the mean of the normal range (compared with the traditionally defined therapeutic range of 1.5-2.5 times the control value). For the in vitro samples, the aPTT range corresponding to heparin concentrations of 0.3-0.7 unit/mL was 121-256 seconds, which corresponds to an aPTT range of 4.4-9.4 times the mean of the normal range. Each institution should establish a therapeutic aPTT range by calibrating aPTT values against heparin concentrations from blood samples of patients receiving intravenous heparin.

The inhibitor antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with factor VIII autoantibodies. Recombinate and Kogenate Study Groups.

Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as > or = 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

Cholinergic, serotoninergic and peptidergic components of the nervous system of Haematoloechus medioplexus (Trematoda, Digenea), characterised by cytochemistry.

Cholinergic, serotoninergic and peptidergic neuronal pathways have been demonstrated in whole-mount preparations of the frog-lung digenean trematode, Haematoloechus medioplexus, using enzyme cytochemical methodologies and indirect immunocytochemical techniques in conjunction with confocal scanning laser microscopy. All 3 classes of neuroactive substance were found throughout both central and peripheral elements of a well-developed orthogonal nervous system. Peptidergic immunoreactivity was particularly strong, using antisera directed to native flatworm neuropeptides, neuropeptide F, and FMRF amide-related peptides (FaRPs), and there was significant overlap in the staining with that for cholinergic components. The serotoninergic system appeared quite separate, with the staining localised to a different set of neurons.

Infectivity and growth of Echinostoma revolutum (Froelich, 1802) in the domestic chick.

Infectivity and growth studies in domestic chicks were carried out on a strain of Echinostoma revolutum isolated from Lymnaea elodes snails in Indiana, U.S.A. Of 21 chicks, each fed 40 +/- 10 cysts of Echinostoma revolutum, 16 (64%) were infected with a total of 269 (32%) worms from approximately 840 cysts. Worms were found only in the ceca and rectum at 2-14 days p.i. In vivo excysted metacercariae were obtained in the lower ileum and ceca at 4 h p.i. Excysted metacercariae averaged 0.2 mm in length and 0.02 mm2 in body area. Worm length averaged 1.3 mm on day 6, 2.3 mm on day 8 and 3.6 mm on day 14. Mean body area averaged 0.29 mm2 on day 6, 0.62 mm2 on day 8 and 1.93 mm2 on day 14. Worms first became ovigerous on day 12. Growth of E. revolutum in the chick was delayed compared to previous findings on E. trivolvis, a closely related species of 37-collar-spined echinostome in the E. revolutum complex.

Vaccination with fixed feline immunodeficiency virus (FIV) infected cells: protection, breakthrough and specificity of response.

Infection of cats with feline immunodeficiency virus (FIV), a naturally occurring lentivirus infection of cats which causes an AIDS-like disease, has generated considerable interest as an animal model for HIV vaccination. This paper reports on experiments performed to examine the potential of a fixed infected cell vaccine to confer protection against intraperitoneal challenge with cell-free FIV. The cell vaccine was highly immunogenic and elicited antibody responses to virus core antigen, p24, high virus neutralizing (VN) antibody titres, and antibodies which recognized cellular components of the vaccine. Whilst protection, assessed by the inability to detect infectious virus by virus isolation or polymerase chain reaction, against homologous but not heterologous FIV isolates was apparent up to week 12 post-challenge, when cats were monitored longer up to week 50 post-challenge a breakthrough in vaccine protection against homologous virus was observed. Protection could not be correlated with levels of antibody to p24 or VN antibody titres. In contrast with simian immunodeficiency virus vaccine studies in macaques there was no clear evidence that antibodies recognizing cellular components of the vaccine, including MHC class I and II antigens, conferred any protective effect following challenge. These results indicate that long-term post-challenge monitoring for infection is essential in lentivirus vaccine trials.

Arterial thrombin activity after angioplasty in an atherosclerotic rabbit model: time course and effect of hirudin.

BACKGROUND: A 2-hour infusion of the direct thrombin inhibitor hirudin at the time of balloon angioplasty limits restenosis in the focally atherosclerotic rabbit. Although short-term administration of hirudin may have a prolonged biological effect, the effect of hirudin on vessel thrombin activity has not been previously studied in an animal model of angioplasty. We hypothesized that a short intravenous infusion of hirudin would result in prolonged inhibition of arterial wall-associated thrombin activity (ATA) after angioplasty. METHODS AND RESULTS: Sixty-one rabbits received recombinant hirudin (r-hirudin)(1 mg/kg bolus plus 1 mg x kg(-1) x h(-1)x2hours) or bolus heparin (controls, 150 U/kg) intravenously at the time of femoral balloon angioplasty. ATA was measured through exposure of arterial segments ex vivo to fibrinogen and conducting an assay for fibrinopeptide A (FPA). ATA was low in nonballooned, atherosclerotic vessels (FPA=0.5+/-0.3 ng x mL(-1) x mg(-1)) but increased significantly at 24 hours after angioplasty in the heparin group (3.7+/-0.9 ng x mL(-1) x mg(-1), P<.01 versus baseline, n=9) but not in the hirudin group (FPA = 1.4+/-0.3; P=NS versus baseline, P<.02 versus heparin controls, n=8). The time course of ATA after angioplasty was assessed in 44 rabbits. Thrombin activity peaked at 48 hours and declined to baseline at 72 hours and 7 days. FPA values between the heparin and r-hirudin groups were similar at these later time points. CONCLUSIONS: A 2-hour intravenous infusion of r-hirudin suppressed ATA measured 24 hours after angioplasty in the focally atherosclerotic rabbit. This prolonged biological effect may account, in part, for the reduction in restenosis seen in this model.

D-dimer testing and acute venous thromboembolism. A shortcut to accurate diagnosis?

D-dimer fragments can be measured easily in plasma and whole blood, and the presence or absence of D-dimer could be useful in the diagnostic evaluation of venous thromboembolism. We systematically reviewed the English literature for articles that compared D-dimer results with those of other tests for deep venous thrombosis or pulmonary embolism. Twenty-nine studies were selected for detailed review, and we noted wide variability in assay performance, heterogeneity among subjects, and failure to define absence or presence of venous thromboembolism by a comprehensive criterion standard for diagnosis. These methodologic problems limit the generalizability of the published estimates of D-dimer accuracy for deep venous thrombosis or pulmonary embolism, and the clinical utility of this potentially important test remains unproved.

Accuracy of laboratory and portable monitor international normalized ratio determinations. Comparison with a criterion standard.

BACKGROUND: Portable instruments that measure the prothrombin time and automatically calculate the international normalized ratio (INR) with the use of a drop of whole blood have simplified the treatment of patients who are receiving warfarin therapy. The accuracy of these portable monitors has never been determined by comparing INR results with a criterion (gold) standard INR determination. METHODS: Duplicate whole-blood INR determinations were made with two commercially available portable INR monitors. Duplicate frozen-plasma samples were measured with four different thromboplastin reagents, each with a different international sensitivity index. The criterion standard INR was determined by using an international reference thromboplastin and the manual tilt-tube technique. Agreement was evaluated by determining how accurately laboratory and monitor INR determinations matched criterion standard values in designating a sample to be within or outside of currently recommended INR target ranges. RESULTS: Two of the laboratory methods, which used relatively sensitive thromboplastins, showed close agreement with the criterion standard, whereas two laboratory methods that used less sensitive thromboplastin reagents showed poor agreement. Both of the portable monitors fell between these extremes. The two best laboratory methods ere significantly better (P < .003) than both monitors, which in turn were better (P < .003) than the remaining two laboratories. CONCLUSIONS: There is large interlaboratory variation in the accuracy of INR determinations. Laboratory methods that used insensitive (high international sensitivity index) thromboplastins performed poorly. Accuracy of monitor measurements appears satisfactory.

Failure of the synthetic androgen, danazol, to increase protein S levels and prevent thrombosis in a patient with severe protein S deficiency.

A 34-year-old man with recurrent deep and superficial thromboses was found to have severe protein S deficiency. Treatment with both warfarin and adjusted-dose subcutaneous heparin failed to completely prevent thrombosis. Based on reports of increases in the endogenous anticoagulants (protein C, protein S, antithrombin III and plasminogen) with synthetic androgen therapy, the patient was treated with danazol for 8 weeks. Although the levels of antithrombin III, protein C and plasminogen increased, no change in the levels of total or free protein S or C4b binding protein was observed. Treatment was discontinued at 8 weeks when the patient developed a recurrence of superficial thrombophlebitis. The role of synthetic androgens in the treatment of patients with inherited thrombotic disorders is reviewed and potential reasons for treatment failure in this patient are discussed.