Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Immunological potential of cytotoxic T lymphocyte antigen 4 immunoglobulin in murine autoimmune cholangitis.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig) is an important regulator of T cell activation and a fusion protein directed at CD80 and CD86; it blocks co-stimulatory signalling and T cell activation. We have taken advantage of a murine model of human primary biliary cirrhosis (PBC), mice expressing a transforming growth factor (TGF)-ß receptor II dominant negative (dnTGF-ßRII) transgene to address the potential therapeutic efficacy of CTLA-4 Ig. To mimic patients with PBC at different stages or duration of disease, we treated mice with either CTLA-4 Ig or control IgG three times weekly from 3 to 12 or 24 weeks of age, or from 12 to 24 weeks of age. CTLA-4 Ig treatment from 3 weeks of age significantly reduced liver inflammation to 12 weeks of age. Treatment initiated at 12 weeks of age also ameliorated the autoimmune cholangitis at 24 weeks of age. However, in mice treated at 3 weeks of age, suppression of liver inflammation was not sustained and colitis was aggravated when treatment was extended to 24 weeks of age. Our data indicate that, in dnTGF-ßRII mice, CTLA-4 Ig treatment has short-term beneficial effects on autoimmune cholangitis, but the effect varies according to duration of treatment and the time in which therapy was initiated. Further dissection of the events that lead to the reduction in therapeutic effectiveness of CTLA-4 Ig will be critical to determining whether such efforts can be applied to human PBC.

An anti-CD154 domain antibody prolongs graft survival and induces Foxp3(+) iTreg in the absence and presence of CTLA-4 Ig.

The use of monoclonal antibodies targeting the CD154 molecule remains one of the most effective means of promoting graft tolerance in animal models, but thromboembolic complications during early clinical trials have precluded their use in humans. Furthermore, the role of Fc-mediated deletion of CD154-expressing cells in the observed efficacy of these reagents remains controversial. Therefore, determining the requirements for anti-CD154-induced tolerance will instruct the development of safer but equally efficacious treatments. To investigate the mechanisms of action of anti-CD154 therapy, two alternative means of targeting the CD40-CD154 pathway were used: a nonagonistic anti-CD40 antibody and an Fc-silent anti-CD154 domain antibody. We compared these therapies to an Fc-intact anti-CD154 antibody in both a fully allogeneic model and a surrogate minor antigen model in which the fate of alloreactive cells could be tracked. Results indicated that anti-CD40 mAbs as well as Fc-silent anti-CD154 domain antibodies were equivalent to Fc-intact anti-CD154 mAbs in their ability to inhibit alloreactive T cell expansion, attenuate cytokine production of antigen-specific T cells and promote the conversion of Foxp3(+) iTreg. Importantly, iTreg conversion observed with Fc-silent anti-CD154 domain antibodies was preserved in the presence of CTLA4-Ig, suggesting that this therapy is a promising candidate for translation to clinical use.

Parametric study of horizontally vibrated grain packings: comparison between Discrete Element Method and experimental results.

Numerical and experimental studies have been undertaken to analyze three parameters controlling the compaction of granular media submitted to sinusoidal horizontal vibrations. We have characterized the influence of the dimensionless acceleration Γ, the geometry of the container and the friction coefficients on the grain velocities and on the packing densities. Above a critical acceleration Γ, the velocities increases with Γ. For low values of Γ, the surface layers are compacted, whereas the bottom layers remain at their initial density. For high values of Γ, the bottom layers get compacted, the surface layers are fluidized so that the bulk dynamic and relaxed densities decreased. In the same way, the effect of the dimensions of the container and of the friction coefficients on the packing properties has been studied for given heights of sand, acceleration and frequency. It has been shown that the influence of the two last parameters is similar to that of acceleration. The numerical results given by the Discrete Element Method appear to be in good agreement with experimental results.

Transcriptional profiling of liver and effect of glucocorticoids in a rat adjuvant-induced arthritis model.

Glucocorticoids (GCs), despite having many undesirable side effects, remain effective for the treatment of many inflammatory diseases and are commonly used as benchmark drugs in animal models of disease. However, the molecular mechanisms underling systemic GC effects in these models are poorly characterized. In this study, prednisolone and dexamethasone were evaluated in the fully established Lewis rat adjuvant-induced arthritis (AIA) model. In AIA, adjuvant administration induced polyarticular and systemic inflammation, which included spleen and liver. In the liver, multifocal hepatic granulomas were observed. To characterize the systemic response and the pathways responsible for GC effects, histology, transcriptional profiling, and immunohistochemistry (IHC) were performed. There was a decrease in the incidence and histologic severity score for granulomas with GC treatment. There was no effect on cellular composition of granulomas as assessed by IHC for CD3+ lymphocytes, macrophages, and B cells, but there was a significant reduction in infiltrating lymphocytes in the hepatic parenchyma. By Affymetrix microarray analysis, 10% of hepatic transcripts were altered (P<.01) in livers from AIA rats, with ~31% of them partially reversed with treatment with dexamethasone and ~13% with prednisolone. Many of these altered hepatic transcripts correspond to human genes that are dysregulated in the synovium in human rheumatoid arthritis (RA), indicating that the rat AIA model shares features with human RA. These data establish molecular changes in the liver and the effect of GCs in rat AIA, which can be used to aid in understanding the mechanism of action of novel anti-inflammatory compounds in this animal model.

Investigations of peritoneal and intestinal infections of adult hookworms (Uncinaria spp.) in northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups on San Miguel Island, California (2003).

The peritoneal cavity (PNC) and intestine of northern fur seal (Callorhinus ursinus) pups and California sea lion (Zalophus californianus) pups that died in late July and early August, 2003, on San Miguel Island, California, were examined for hookworms. Prevalence and morphometric studies were done with the hookworms in addition to molecular characterization. Based on this and previous molecular studies, hookworms from fur seals are designated as Uncinaria lucasi and the species from sea lions as Uncinaria species A. Adult hookworms were found in the PNC of 35 of 57 (61.4%) fur seal pups and of 13 of 104 (12.5%) sea lion pups. The number of hookworms located in the PNC ranged from 1 to 33 (median = 3) for the infected fur seal pups and 1 to 16 (median = 2) for the infected sea lion pups. In addition to the PNC, intestines of 43 fur seal and 32 sea lion pups were examined. All of these pups were positive for adult hookworms. The worms were counted from all but one of the sea lion pups. Numbers of these parasites in the intestine varied from 3 to 2,344 (median = 931) for the fur seal pups and 39 to 2,766 (median = 643) for the sea lion pups. Sea lion pups with peritoneal infections had higher intensity infections in the intestines than did pups without peritoneal infections, lending some support for the hypothesis that peritoneal infections result from high-intensity infections of adult worms. There was no difference in intestinal infection intensities between fur seal pups with and without peritoneal infections. Female adult hookworms in the intestines of both host species were significantly larger than males, and sea lion hookworms were larger than those in fur seals. Worms in the intestine also were larger than worms found in the PNC. Gene sequencing and (RFLP) analysis of (PCR) amplified (ITS) ribosomal DNA were used to diagnose the species of 172 hookworms recovered from the PNC and intestine of 18 C. ursinus and seven Z. californianus hosts. These molecular data revealed that U. lucasi (hookworm of C. ursinus) and Uncinaria species A (of Z. californianus) infrequently mature in the intestine of the opposite host species in California rookeries. However, there is no support from molecular data for the hypothesis that cross-infection with "the wrong" Uncinaria species is a contributing factor in these cases of host peritonitis. The major significance of this research is the unusual finding of adult hookworms in the PNC of so many dead pups. No obvious explanation for this occurrence could be determined. Further research, like in the present study, should help understand and monitor the apparent ever changing role of hookworm disease in the health of northern fur seal and California sea lion pups on SMI.

Interaction of the immunosuppressant deoxyspergualin with a member of the Hsp70 family of heat shock proteins.

Deoxyspergualin (DSG) is a potent immunosuppressant whose mechanism of action remains unknown. To elucidate its mechanism of action, an intracellular DSG binding protein was identified. DSG has now been shown to bind specifically to Hsc70, the constitutive or cognate member of the heat shock protein 70 (Hsp70) protein family. The members of the Hsp70 family of heat shock proteins are important for many cellular processes, including immune responses, and this finding suggests that heat shock proteins may represent a class of immunosuppressant binding proteins, or immunophilins, distinct from the previously identified cis-trans proline isomerases. DSG may provide a tool for understanding the function of heat shock proteins in immunological processes.

Disparate rates of molecular evolution in cospeciating hosts and parasites.

DNA sequences for the gene encoding mitochondrial cytochrome oxidase I in a group of rodents (pocket gophers) and their ectoparasites (chewing lice) provide evidence for cospeciation and reveal different rates of molecular evolution in the hosts and their parasites. The overall rate of nucleotide substitution (both silent and replacement changes) is approximately three times higher in lice, and the rate of synonymous substitution (based on analysis of fourfold degenerate sites) is approximately an order of magnitude greater in lice. The difference in synonymous substitution rate between lice and gophers correlates with a difference of similar magnitude in generation times.

The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection.

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.

Endothelin inhibits vasopressin-stimulated water permeability in rat terminal inner medullary collecting duct.

Renal tubule solute and water transport is subject to regulation by numerous factors. To characterize direct effects of the recently discovered peptide endothelin (ET) on renal tubule transport, we determined signaling mechanisms for ET effects on vasopressin (AVP)-stimulated water permeability (PF) in rat terminal inner medullary collecting duct (IMCD) perfused in vitro. ET caused a rapid, dose-dependent, and reversible fall in AVP- but not cyclic AMP-stimulated PF, suggesting that its effect on PF is by inhibition of cyclic AMP accumulation. Indomethacin did not block ET actions, ruling out a role for prostaglandins in its effect. The protein kinase C (PKC) inhibitor calphostin, or pretreatment of perfused tubules with pertussis toxin, blocked ET-mediated inhibition of AVP-stimulated PF. ET caused a transient increase in intracellular calcium ([Ca2+]i) in perfused tubules, an effect unchanged in zero calcium bath or by PT pretreatment. ET effects on PF and [Ca2+]i desensitized rapidly. Inhibition of PF was transient and largely abolished by 20 min ET preexposure, and repeat exposure to ET did not alter [Ca2+]i. In contrast, PGE2-mediated inhibition of AVP-stimulated PF and increase of [Ca2+]i were sustained and unaltered by prior exposure of IMCD to ET. Thus desensitization to ET is homologous. We conclude that ET is a potent inhibitor of AVP-stimulated water permeability in rat terminal IMCD. Signaling pathways for its effects involve both an inhibitory guanine nucleotide-binding protein and phospholipase-mediated activation of PKC. Since ET is synthesized by IMCD cells, this peptide may be an important autocrine modulator of renal epithelial transport.

Evolution of plant parasitism among nematodes.

Despite extraordinary diversity of free-living species, a comparatively small fraction of nematodes are parasites of plants. These parasites represent at least three disparate clades in the nematode tree of life, as inferred from rRNA sequences. Plant parasites share functional similarities regarding feeding, but many similarities in feeding structures result from convergent evolution and have fundamentally different developmental origins. Although Tylenchida rRNA phylogenies are not fully resolved, they strongly support convergent evolution of sedentary endoparasitism and plant nurse cells in cyst and root-knot nematodes. This result has critical implications for using model systems and genomics to identify and characterize parasitism genes for representatives of this clade. Phylogenetic studies reveal that plant parasites have rich and complex evolutionary histories that involve multiple transitions to plant parasitism and the possible use of genes obtained by horizontal transfer from prokaryotes. Developing a fuller understanding of plant parasitism will require integrating more comprehensive and resolved phylogenies with appropriate choices of model organisms and comparative evolutionary methods.

Putrescine transport in human platelets.

Putrescine transport has been studied in human platelets. The uptake of putrescine is saturable and appears to be an energy-dependent process, since it is inhibited by the uncoupler 2,4-dinitrophenol and low temperature. The evidence presented suggests that the uptake process is complex and may be dependent upon pH gradient, membrane potential, and other unidentified factors. Putrescine transport is not inhibited by amino acids and is only slightly inhibited by spermidine and spermine. A membrane protein involved in putrescine transport has been identified and partially purified. Differential labeling with N-ethylmaleimide identified proteins with apparent molecular weights of 65000 and 23000 as determined by SDS-polyacrylamide gel electrophoresis. Column chromatographic purification on a putrescine affinity column revealed a Mr 55000 protein which copurified with the Mr 65000 protein. Additional evidence supporting the involvement of these proteins in putrescine transport was seen in putrescine protection against N-ethylmaleimide inhibition of putrescine uptake. Putrescine uptake may occur via the serotonin transport system, since imipramine inhibits transport and because of the similarities in the molecular weights of the proteins implicated in transport.

Genetic obesity unmasks nonlinear interactions between murine type 2 diabetes susceptibility loci.

Nonlinear interactions between obesity and genetic risk factors are thought to determine susceptibility to type 2 diabetes. We used genetic obesity as a tool to uncover latent differences in diabetes susceptibility between two mouse strains, C57BL/6J (B6) and BTBR. Although both BTBR and B6 lean mice are euglycemic and glucose tolerant, lean BTBR x B6 F1 male mice are profoundly insulin resistant. We hypothesized that the genetic determinants of the insulin resistance syndrome might also predispose genetically obese mice to severe diabetes. Introgressing the ob allele into BTBR revealed large differences in diabetes susceptibility between the strain backgrounds. In a population of F2-ob/ob mice segregating for BTBR and B6 alleles, we observed large variation in pancreatic compensation for the underlying insulin resistance. We also detected two loci that substantially modify diabetes severity, and a third locus that strongly links to fasting plasma insulin levels. Amplification of the genetic signal from these latent diabetes susceptibility alleles in F2-ob/ob mice permitted discovery of an interaction between the two loci that substantially increased the risk of severe type 2 diabetes.

Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex.

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.

Agonistic activity of a CD40-specific single-chain Fv constructed from the variable regions of mAb G28-5.

A single-chain Fv (sFv) was expressed from the variable regions of the CD40-specific mAb G28-5. The molecule bound CD40 with a high affinity (2.2 nM) and was a monomer in solution. Surprisingly, G28-5 sFv was a potent CD40 agonist that rapidly crosslinked CD40 on the cell surface but did not crosslink CD40-Ig in solution. G28-5 sFv was a more potent agonist than G28-5 IgG and was able to stimulate CD40 responses by B cells and monocytes. G28-5 IgG partially blocked, whereas G28-5 sFv augmented CD40 responses during stimulation with natural ligand (gp39-CD8 fusion protein). These results indicate that the functional activity of ligands built from the binding site of G28-5 is highly dependent upon the size and physical properties of the molecule both in solution and on the cell surfaces.

Revising the standard wisdom of C. elegans natural history: ecology of longevity.

Here, we consider that most of the research concerning Caenorhabditis elegans has been laboratory focused and that only limited research has directly considered the worm's biology relative to its natural history in the wild. We describe that, although the worm has traditionally been considered a soil nematode, we could not find it in soil but frequently recovered it from snails. Finally, we discuss how a better understanding of the natural history of C. elegans may enhance its usefulness as a model organism for studying aging and other phenomena.

Biochemical polymorphism in Parascaris equorum, Toxocara canis and Toxocara cati.

Vertical starch gel electrophoresis was used to resolve proteins encoded by 18 gene loci in ascaridoid nematodes. Estimates of genetic variability were made from population samples of the dog ascarid (Toxocara canis), cat ascarid (Toxocara cati), and the horse ascarid (Parascaris equorum). Levels of polymorphism and mean heterozygosity were high, which is not consistent with the hypothesis that the intestinal environment selects for monomorphism among endoparasites. Most observed allele frequencies conformed to Hardy-Weinberg equilibrium expectations as tested by chi2 goodness-of-fit, suggesting that the proteins evaluated are inherited in a Mendelian fashion and that these nematodes are mating at random. Subunit structures of the following enzymes, deduced from electrophoretic phenotypes of heterozygotes, corresponded to those of vertebrates: lactate dehydrogenase; malate dehydrogenase; 6-phosphogluconate dehydrogenase; phosphoglucomutase; esterase D; peptidase B; peptidase D; and mannose-6-phosphate isomerase. This observation substantiates the conservative nature of polypeptide subunit number across phylogenetically diverse groups of organisms.

Peritransplant tolerance induction with anti-CD3-immunotoxin: a matter of proinflammatory cytokine control.

BACKGROUND: Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance. METHODS: Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B. CONCLUSIONS: In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

Effect of an intravenous iron dextran regimen on iron stores, hemoglobin, and erythropoietin requirements in hemodialysis patients.

Iron deficiency is a common cause of delayed or diminished response to erythropoietin (EPO) in hemodialysis patients. Although oral iron is often prescribed to replete iron stores, this approach to iron supplementation may not be adequate with chronic EPO therapy. Intravenous (IV) iron dextran may be an effective alternative approach to replete iron stores and may facilitate more cost-effective use of EPO. The purpose of this study was to evaluate an IV iron dextran regimen that consisted of a loading dose phase followed by monthly maintenance doses of iron dextran. The effect of this regimen on iron stores, hemoglobin, and EPO doses was evaluated. This was an open prospective study in adult hemodialysis patients who were iron deficient as defined by a serum ferritin less than 100 ng/mL or transferrin saturation (TSAT) of less than 20%. Patients were loaded with 1 g iron dextran in five divided doses and then received monthly maintenance doses of 100 mg for the 4-month study period. Values of serum ferritin, TSAT, hemoglobin, and EPO dose were followed for the 4-month study period. Thirty hemodialysis patients receiving EPO were identified as being iron deficient and were enrolled in the study. The mean serum ferritin increased significantly from 49 ng/mL at baseline to 225 ng/mL at the end of the study period (P < 0.0001). Mean TSAT also increased significantly from 27% to 33% (P = 0.002). Values for hemoglobin did not change significantly during the study period; however, there was a significant reduction in EPO dose from a mean baseline dose of 112 U/kg/wk to 88 U/kg/wk at the end of the study period (P = 0.009). Seventeen patients experienced an increase in hemoglobin or a decrease in EPO dose. Economic analysis showed that approximately $580 (Cdn) per patient per year could be saved by use of IV iron dextran. The administration of the IV iron dextran regimen in the iron-deficient hemodialysis population was effective at repleting and maintaining iron stores and reducing EPO use.