Triplex metallohelices have enantiomer-dependent mechanisms of action in colon cancer cells.

Self-assembled enantiomers of an asymmetric di-iron metallohelix differ in their antiproliferative activities against HCT116 colon cancer cells such that the compound with Λ-helicity at the metals becomes more potent than the Δ compound with increasing exposure time. From concentration- and temperature-dependent 57Fe isotopic labelling studies of cellular accumulation we postulate that while the more potent Λ enantiomer undergoes carrier-mediated efflux, for Δ the process is principally equilibrative. Cell fractionation studies demonstrate that both enantiomers localise in a similar fashion; compound is observed mostly within the cytoskeleton and/or genomic DNA, with significant amounts also found in the nucleus and membrane, but with negligible concentration in the cytosol. Cell cycle analyses using flow cytometry reveal that the Δ enantiomer induces mild arrest in the G1 phase, while Λ causes a very large dose-dependent increase in the G2/M population at a concentration significantly below the relevant IC50. Correspondingly, G2-M checkpoint failure as a result of Λ-metallohelix binding to DNA is shown to be feasible by linear dichroism studies, which indicate, in contrast to the Δ compound, a quite specific mode of binding, probably in the major groove. Further, spindle assembly checkpoint (SAC) failure, which could also be responsible for the observed G2/M arrest, is established as a feasible mechanism for the Λ helix via drug combination (synergy) studies and the discovery of tubulin and actin inhibition. Here, while the Λ compound stabilizes F-actin and induces a distinct change in tubulin architecture of HCT116 cells, Δ promotes depolymerization and more subtle changes in microtubule and actin networks.

Control of apoptosis in autoimmunity.

Apoptosis and the subsequent removal of apoptotic cells underpin a healthy immune system. They are crucial for both the maintenance of self-tolerance and the contraction of clonally expanded lymphocytes at the conclusion of immune responses. Aberrant apoptosis and the disposal of apoptotic cells is implicated in the development of both systemic and organ-specific autoimmune disease and is a major contributing factor in disease susceptibility. Dissection of the molecular mechanisms involved in dysregulated apoptosis may reveal pathways which can be targeted for more effective therapeutic intervention. This review highlights the molecular events underlying programmed cell death and apoptotic cell uptake, and summarizes recent studies that link impaired apoptotic death to autoimmunity.

Monocytosis in BXSB mice is due to epistasis between Yaa and the telomeric region of chromosome 1 but does not drive the disease process.

The BXSB murine model of systemic lupus erythematosus is differentiated from other murine models of lupus by a severe monocytosis. The recently identified Y-linked autoimmune accelerator locus, Yaa, which is fundamental to accelerated disease in male BXSB mice, is required for the monocytic phenotype in BXSB. It has also recently been shown to induce monocytosis in combination with the Nba2 locus from NZB. To dissect the genetic basis and associated pathogenicity of BXSB-related monocytosis, a panel of existing congenic mice were studied and a novel sub-congenic mouse B10.Y(BXSB).BXSB-Bxs3 was generated. Monocytosis was found to be caused by an epistatic interaction between Yaa and the telomeric region of chromosome 1, an area of approximately 30 cM. Bxs3 and Yaa together were sufficient to generate monocytosis equivalent to that of BXSB. In contrast to the NZB model, however, where monocytosis tightly correlated with autoantibody production and lethal lupus nephritis, this was not the case in BXSB. While Yaa(+) mice bearing the Bxs3 locus drive monocytosis, glomerulonephritis and autoantibody production, both autoantibody production and nephritis are discreet events that occur in the absence of the Bxs3 locus. Yaa is a pre-requisite for monocytosis, demonstrating a novel synergistic interaction between Yaa and Bxs3.

A rapid radiochemical bacterial bioassay to evaluate copper toxicity in freshwaters.

A rapid, highly sensitive bacterial bioassay to determine copper toxicity in freshwaters was developed based on the inhibition of cellular assimilation of radiolabeled glucose. The test used a copper-sensitive bacterium isolated from a freshwater stream. Employing sensitive radiochemical techniques enabled environmentally relevant concentrations of the test bacterium (10(5) cells mL(-1)) and a short incubation period (4 hours) to be used, which minimized the potential for changes in copper speciation during the test. The 4-hour median effective concentration (EC(50)) for inorganic copper at pH 7.5 in synthetic freshwater was 0.6 microg L(-1) (95% confidence limits 0.4 to 1.0 microg L(-1)). This compared well with chronic growth inhibition of this bacterium in minimal medium (48-hour EC(50) of 0.9 microg L(-1) [95% confidence limits 0.7 to 1.0 microg L(-1)]). MINEQL + software (Environmental Research Software) was used to calculate copper (II) ion concentrations in synthetic freshwater at pH 7.5, giving an EC(50) value of pCu(2+) 8.8. However, using nitrilotriacetic acid metal-ion buffers (Cu-NTA), 50% inhibition occurred at a pCu(2+) of 9.7, suggesting this bacterium was markedly more inhibited by copper in these Cu(2+)-buffered solutions. This may indicate that the Cu-NTA species was contributing to toxicity. The radiochemical bioassay was evaluated further using freshwater samples from both copper-impacted and pristine environments. Measured EC(50) values ranged from 3.4 to 34.0 microg L(-1)inorganic copper and were strongly correlated with dissolved organic carbon (DOC) concentrations (r = 0.88, p < 0.05).

Hardness corrections for copper are inappropriate for protecting sensitive freshwater biota.

Toxicity testing using a freshwater alga (Chlorella sp.), a bacterium (Erwinnia sp.) and a cladoceran (Ceriodaphnia cf. dubia) exposed to copper in synthetic and natural freshwaters of varying hardness (44-375 mg CaCO3/l), with constant alkalinity, pH and dissolved organic carbon concentration, demonstrated negligible hardness effects in the pH range 6.1-7.8. Therefore, the use of a generic hardness-correction algorithm, developed as part of national water quality guidelines for protecting freshwater biota, is not recommended for assessing the toxicity of copper to these, and other, sensitive freshwater species. Use of the algorithm for these sensitive species will be underprotective because the calculated concentrations of copper in water that cause a toxic effect will be higher.

Allorecognition.

Until recently, the vigorous T cell response via the direct pathway has overshadowed studies involving the indirect pathway. Thus, while the direct pathway has previously been considered to be the main driving force in alloimmune responses, there is an increasing body of data to support a prominent role of the indirect pathway in transplant rejection. Most importantly, while the direct anti-donor alloresponse diminishes with time after transplantation, the indirect alloresponse is likely to be permanently active due to traffic of recipient dendritic cells (DCs) through the graft. Consequently, the future challenge in the induction of allograft tolerance is to design interventions that will target the cells involved in the indirect pathway, especially the T cells with indirect allospecificity.

Alleviation of aluminum toxicity to Rhizobium leguminosarum bv. viciae by the hydroxamate siderophore vicibactin.

Acid rain solubilises aluminum which can exert toxic effects on soil bacteria. The root nodule bacterium Rhizobium leguminosarum biovar viciae synthesises the hydroxamate siderophore vicibactin in response to iron limitation. We report the effect of vicibactin on the toxicity of aluminum(III) to R. leguminosarum and kinetic studies on the reaction of vicibactin with Al(III) and Fe(III). Aluminum (added as the nitrate) completely inhibited bacterial growth at 25 microM final concentration, whereas the preformed Al-vicibactin complex had no effect. When aluminum and vicibactin solutions were added separately to growing cultures, growth was partly inhibited at 25 microM final concentration of each, but fully inhibited at 50 microM final concentration of each. Growth was not inhibited at 50 microM Al and 100 microM vicibactin, probably reflecting the slow reaction between Al and vicibactin; this results in some aluminum remaining uncomplexed long enough to exert toxic effects on growth, partly at 25 microM Al and vicibactin and fully at 50 microM Al and vicibactin. At 100 microM vicibactin and 50 microM Al, Al was complexed more effectively and there was no toxic effect. It was anticipated that vicibactin might enhance the toxicity of Al by transporting it into the cell, but the Al-vicibactin complex was not toxic. Several explanations are possible: the Al-vicibactin complex is not taken up by the cell; the complex is taken up but Al is not released from vicibactin; Al is released in the cell but is precipitated immediately. However, vicibactin reduces the toxicity of Al by complexing it outside the cell.

Allorecognition.

Until recently, the vigorous T-cell response via the direct pathway has overshadowed studies involving the indirect pathway. Thus, while the direct pathway has previously been considered to be the main driving force in alloimmune responses, there is an increasing body of data to support a prominent role of the indirect pathway in transplant rejection. Most importantly, the direct antidonor alloresponse diminishes with time after transplantation, possibly due to the tolerogenic effects of alloantigen presentation by the parenchymal cells of the transplant. In contrast, the indirect alloresponse is likely to be permanently active, due to traffic of recipient dendritic cells (DCs) through the graft. The challenge that this poses in the pursuit of clinical transplant tolerance is how to induce tolerance in T cells with indirect allospecificity.

Porcine CTLA4-Ig lacks a MYPPPY motif, binds inefficiently to human B7 and specifically suppresses human CD4+ T cell responses costimulated by pig but not human B7.

The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue.

A model for sequestration of the transmission stages of Plasmodium falciparum: adhesion of gametocyte-infected erythrocytes to human bone marrow cells.

With the aim of developing an appropriate in vitro model of the sequestration of developing Plasmodium falciparum sexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays of P. falciparum cytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.

Costimulatory blockade by the induction of an endogenous xenospecific antibody response.

Xenogeneic tissues induce vigorous T cell immunity, reflecting the ability of costimulatory molecules to function across species barriers. We describe a strategy to inhibit costimulation that exploits species differences using the model of porcine pancreatic islet transplantation into mice. Mice were immunized with chimeric peptides that contained a known T cell epitope and selected sequences of the porcine costimulatory molecule CD86. This resulted in anti-peptide antibody responses that recognized intact porcine CD86, blocked costimulation by porcine CD86 but not murine CD86 in vitro, and prolonged the survival of porcine islet grafts in vivo. This strategy of inducing endogenous donor-specific costimulatory blockade has potential clinical applicability.

Xenotransplantation: steps towards a clinical reality.

It was clear that significant research progress has been made in the two years since the last congress, although, as the chairman J.P. Soulillou (Nantes) concluded, there were no singular advances announced during the week; rather, it was a congress for assimilation of information. It was also apparent that there is now an attitude of realism in the xenotransplantation community. The prevailing mood is one of cautious optimism, to contrast with the unguarded optimism of the previous meeting. The future for clinical xenotransplantation now appears more secure than it has been previously.

Loss of cochlear nucleus neurons following aminoglycoside antibiotics or cochlear removal.

This study compared the effects of aminoglycoside ototoxicity and surgical ablation of the cochlea in infancy on the survival of neurons in the rat cochlear nucleus (CN). Ototoxicity was induced by a single, systemic dose of gentamicin sulfate and furosemide on postnatal day 6 (P6), P7, or P10, and assessed by the elevation of auditory brain stem response thresholds, as described in a companion paper. Unilateral cochlear removals were performed under Saffan anesthesia on P6, P9, and P12. Rats were painlessly sacrificed in adulthood, and the formalin-perfused brains and cochleas were embedded in wax, sectioned, and stained. Ototoxic treatment at P6 through P10 did not reduce neuron counts in the CN. Cochlear removal at P6 resulted in a 40% loss of CN neurons, but removal at P12 did not result in CN neuron loss. These data suggest that the critical period for the dependence of CN neurons on afferent input from the cochlea ends at the same time that susceptibility to aminoglycoside ototoxicity begins.

Plasmodium falciparum gametocyte adhesion to C32 cells via CD36 is inhibited by antibodies to modified band 3.

Plasmodium falciparum gametocyte-infected erythrocytes are characterized by their ability to sequester in the microvasculature of various organs, primarily the spleen and bone marrow. This phenomenon is thought to play a critical role in the development and survival of the sexual stages. Little is known, however, about ligands on the gametocyte-infected erythrocyte. Infection of erythrocytes with mature asexual stages of P. falciparum (trophozoites and schizonts) has been shown to induce modification of the erythrocyte anion transporter, band 3, and this has been linked to the acquisition of an adherent phenotype. Here, we demonstrate for the first time that immature gametocyte-infected erythrocytes also express modified band 3. In vitro binding assays demonstrate that gametocyte-infected erythrocytes of the 3D7 strain utilize this surface receptor for adhesion to C32 amelanotic melanoma cells via the host cell receptor CD36 (platelet glycoprotein IIIb). Adhesion of gametocyte-infected erythrocytes to CD36-transfected CHO cells is also dependent on modified band 3. However, modified band 3 does not mediate adhesion of gametocyte-infected erythrocytes to intercellular adhesion molecule 1, a second host receptor for gametocytes expressed on C32 cells.

CD36 and intercellular adhesion molecule 1 mediate adhesion of developing Plasmodium falciparum gametocytes.

Plasmodium falciparum trophozoite-infected erythrocytes adhere to the amelanotic melanoma C32 cell line in vitro. Here we demonstrate for the first time that immature gametocyte-infected erythrocytes also adhere to C32 cells, albeit at lower levels than trophozoites. However, anti-CD36 and anti-intercellular adhesion molecule 1 antibodies inhibit asexual and gametocyte adhesion by comparable percentages, suggesting a common dependency for binding to these cellular receptors.

Prodrugs to improve the oral bioavailability of a diacidic nonpeptide angiotensin II antagonist.

DMP 811 is a diacidic angiotensin II antagonist. It has relatively low oral bioavailability in rats. A prodrug approach to improving oral bioavailability was tested. Five esters were synthesized and their stability in rat plasma in vitro was determined. The hydrolysis rates of these five esters ranged from almost immediate to negligible. A simple n-propyl ester was hydrolyzed very slowly (< 10% in 24 hr) in rat plasma in vitro, and after oral dosing in rats plasma prodrug concentrations were much greater than DMP 811 concentrations. A pivaloyloxymethyl ester (1) was hydrolyzed relatively rapidly in rat plasma in vitro. Prodrug 1 was rapidly hydrolyzed by the intestine in vitro, and the intestinal permeation of DMP 811 was increased. DMP 811 oral bioavailability was 47% in rats dosed with 10 mg/kg 1, compared to 11% for rats dosed with 10 mg/kg DMP 811. However, DMP 811 bioavailability was only 27% after a 2 mg/kg dose of 1. In vitro plasma hydrolysis of 1 was highly species-dependent, with a half-life of 13 hr in human plasma but only 1 min in rat plasma. The prodrug approach has potential for improving the oral bioavailability of DMP 811, but selection of the optimal prodrug must be done in humans or in a species, such as dogs, with hydrolysis characteristics closer to humans.

Deficient expression of co-stimulatory molecules on Leishmania-infected macrophages.

Co-stimulatory signals are necessary for the full activation of T cells for growth and effector function. As co-stimulatory molecules are normally regulated in their expression, it has been suggested that microorganisms enhance their expression on host antigen-presenting cells (APC), thus allowing efficient generation of anti-microbial immunity. We here describe experiments which demonstrate that infection of macrophages, both in vitro and in vivo, by the protozoan parasite Leishmania donovani fails to trigger expression of co-stimulatory molecules B7-1 and heat-stable antigen on these APC. Furthermore, infection with this parasite inhibits the macrophage response to normal regulatory signals, such as bacterial lipopolysaccharide. These changes in the cell surface are mirrored in functional studies of co-stimulation in vitro. Together, these data suggest a further facet of parasite interference in host immunity, but also indicate a potential new target for immunotherapy.