High Specificity of C4d/CD68 Staining for the Diagnosis of Late Antibody-Mediated Rejection in Heart Transplantation.

In heart transplantation (HTx), acute antibody-mediated rejection (AMR) is infrequent but carries high mortality and increased risk of graft vasculopathy. The diagnosis requires evidence of acute graft dysfunction, capillary lesions on endomyocardial biopsy (EMB), and immunopathological criteria of antibodymediated injury. Multiple markers of antibody-mediated injuries have been proposed, but there is ample debate on their usefulness. In kidney transplantation, C4d deposition in peritubular capillaries is a reliable marker of alloantibody-dependant graft injury. In this study, we prospectively screened all EMBs for C4d and CD68 in new HTx recipients, and correlated pathological fi ndings with immunological evidence of donor-specifi c antibodies (DSA) and graft dysfunction. Methods Between Nov 05 and Aug 08, we had 22 HTx, and 17 cases were analysed. All recipients received polyclonal rabbit anti-thymocytes globulin, calcineurin inhibitors, mycophenolate mofetil, and corticosteroids (weaning in 6 -12 months). They had EMB every 1-2 weeks in the fi rst 3 months, and then monthly for 9 months. C4d and CD 68 were assessed by immunochemistry. Echocardiography and DSA assessment or crossmatch (early phase) were realised if C4d or CD68 staining was positive. Results There was 1 early and 1 late AMR. Table 1 C4d and CD68 positive, at least 1 EMB 6 / 17; 35% 1 treated C4d and CD68 positive, at least 2 consecutive EMBs 3 / 17; 17.5% 1 treated C4d and CD68 positive, and graft dysfunction 1 / 17; 6% 1 treated C4d and CD68 positive, with DSA and crossmatch + 1 / 17; 6% 1 treated Table 2 C4d and CD68 positive, at least 1 EMB 1 / 17; 6% 1 treated C4d and CD68 positive, at least 2 consecutive EMBs 1 /17; 6% 1 treated C4d and CD68 positive and graft dysfunction 1 / 17; 6% 1 treated C4d and CD68 positive, and + DSA 1 / 17; 6% 1 treated Conclusion In this single-center experience, C4d / CD68 positive staining was frequent in the early phase and raised the question of false positive cases of AMR. However, these markers showed high specifi city for the diagnosis of AMR in the late phase. Of course these data need to be confi rmed in larger multi-center studies.

An anti-CD19 antibody coupled to a tetanus toxin peptide induces efficient Fas ligand (FasL)-mediated cytotoxicity of a transformed human B cell line by specific CD4+ T cells.

Treatment of B cell lymphoma patients with MoAbs specific for the common B cell marker (CD20) has shown a good overall response rate, but the number of complete remissions is still very low. The use of MoAbs coupled to radioisotopes can improve the results, but induces undesirable myelodepression. As an alternative, we proposed to combine the specificity of MoAbs with the immunogenicity of T cell epitopes. We have previously shown that an anti-Ig lambda MoAb coupled to an MHC class II-restricted universal T cell epitope peptide P2 derived from tetanus toxin induces efficient lysis of a human B cell lymphoma by a specific CD4+ T cell line. Here we demonstrate that the antigen presentation properties of the MoAb peptide conjugate are maintained using a MoAb directed against a common B cell marker, CD19, which is known to be co-internalized with the B cell immunoglobulin receptor. In addition, we provide evidence that B cell lysis is mediated by the Fas apoptosis pathway, since Fas (CD95), but not tumour necrosis factor receptor (TNFr) or TNF-related receptors, is expressed by the target B cells, and FasL, but not perforin, is expressed by the effector T cells. These results show that B cell lymphomas can be 'foreignized' by MoAb-peptide P2 conjugates directed against the common B cell marker CD19 and eliminated by peptide P2-specific CD4+ T cells, via the ubiquitous Fas receptor. This approach, which bridges the specificity of passive antibody therapy with an active T cell immune response, may be complementary to and more efficient than the present therapy results with unconjugated chimeric anti-CD20 MoAbs.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.

Role of mTOR, Bad, and Survivin in RasGAP Fragment N-Mediated Cell Protection.

Partial cleavage of p120 RasGAP by caspase-3 in stressed cells generates an N-terminal fragment, called fragment N, which activates an anti-apoptotic Akt-dependent survival response. Akt regulates several effectors but which of these mediate fragment N-dependent cell protection has not been defined yet. Here we have investigated the role of mTORC1, Bad, and survivin in the capacity of fragment N to protect cells from apoptosis. Neither rapamycin, an inhibitor of mTORC1, nor silencing of raptor, a subunit of the mTORC1 complex, altered the ability of fragment N from inhibiting cisplatin- and Fas ligand-induced death. Cells lacking Bad, despite displaying a stronger resistance to apoptosis, were still protected by fragment N against cisplatin-induced death. Fragment N was also able to protect cells from Fas ligand-induced death in conditions where Bad plays no role in apoptosis regulation. Fragment N expression in cells did neither modulate survivin mRNA nor its protein expression. Moreover, the expression of cytoplasmic survivin, known to exert anti-apoptotic actions in cells, still occurred in UV-B-irradiated epidermis of mouse expressing a caspase-3-resistant RasGAP mutant that cannot produce fragment N. Additionally, survivin function in cell cycle progression was not affected by fragment N. These results indicate that, taken individually, mTOR, Bad, or Survivin are not required for fragment N to protect cells from cell death. We conclude that downstream targets of Akt other than mTORC1, Bad, or survivin mediate fragment N-induced protection or that several Akt effectors can compensate for each other to induce the pro-survival fragment N-dependent response.

Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death in mice.

Tissue transglutaminase (TG2) is a protein cross-linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha-1b-adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti-Fas antibodies, which is sublethal for wild-type mice, kills all the TG2 knock-out mice within 20 hours. Although TG2-/- thymocytes exposed to anti-Fas antibodies die at the same rate as wild-type mice, TG2-/- hepatocytes show increased sensitivity toward anti-Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIP(L) (FLICE-inhibitory protein), or the rate of I-kappaBalpha degradation, but a decrease in the Bcl-xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl-xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand-induced hepatocyte proliferation with a simultaneous inhibition of the Fas-death pathway plays a determinant role.

Selective accumulation of mature DC-Lamp+ dendritic cells in tumor sites is associated with efficient T-cell-mediated antitumor response and control of metastatic dissemination in melanoma.

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

RecA-mediated strand exchange traverses substitutional heterologies more easily than deletions or insertions.

RecA protein in bacteria and its eukaryotic homolog Rad51 protein are responsible for initiation of strand exchange between homologous DNA molecules. This process is crucial for homologous recombination, the repair of certain types of DNA damage and for the reinitiation of DNA replication on collapsed replication forks. We show here, using two different types of in vitro assays, that in the absence of ATP hydrolysis RecA-mediated strand exchange traverses small substitutional heterologies between the interacting DNAs, whereas small deletions or insertions block the ongoing strand exchange. We discuss evolutionary implications of RecA selectivity against insertions and deletions and propose a molecular mechanism by which RecA can exert this selectivity.

Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1β stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

Hypotensive effect of the active fragment derived from factor XII is mediated by an activation of the plasma kallikrein-kinin system.

Plasma protein fraction (PPF) contaminated by factor XII active fragment (XIIf) may cause hypotensive reactions when infused to patients. This study was planned to assess in conscious normotensive rats whether the blood pressure response to the factor XIIf is mediated by an activation of the plasma kallikrein-kinin system or by stimulation of prostaglandin synthesis. To test whether the factor XIIf-induced blood pressure fall is due partially to an enhanced generation of vasodilating prostaglandins, the blood pressure effect of XIIf (1 microgram i.v.) was investigated 15 min after treatment with indomethacin (5 mg i.v.), an inhibitor of cyclo-oxygenase. Factor XIIf reduced mean blood pressure similarly in indomethacin- and vehicle-treated rats (-23 +/- 4 mmHg, n = 5, and -23 +/- 5 mmHg, n = 4, respectively). Other rats received factor XIIf 15 min after depletion of circulating prekallikrein by the administration of dextran sulfate. Thirty minutes after a 0.25 mg i.v. dose of this agent, plasma prekallikrein activity averaged 0.12 +/- 0.015 mumol/min/ml (n = 6) as compared to 2.48 +/- 0.31 mumol/min/ml in control rats (n = 4, P less than .001). Factor XIIf decreased mean blood pressure by only 4 +/- 2 mm Hg in rats pretreated with dextran sulfate. Thus, it was possible to blunt the acute hypotensive effect of factor XIIf by depleting circulating prekallikrein, but not by inhibiting prostaglandin production. This strongly suggests that the blood pressure effects of factor XIIf is mediated by a stimulation of the plasma kallikrein-kinin system.

Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein-disaggregation under stress.

Active protein-disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress-induced protein aggregates in vitro and in Escherichia coli cells. K-glutamate and glycine-betaine (betaine) naturally accumulate in salt-stressed cells. In addition to providing thermo-protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone-mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K-glutamate or betaine. The combined protective, counter-negative and net activatory effects of K-glutamate and betaine, allowed protein disaggregation and refolding under heat-shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte-activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat-shock conditions.

Biosphere/lithosphere interface : microbially-mediated CaCO3 precipitation in hypersaline and freshwater environments

Abstract: Microbial mats very efficiently cycle elements, such as C, 0, N, S and H, which makes them key players of redox processes at the biosphere-lithosphere interface. They are characterized by high metabolic activities and high turnover rates (production and consumption) of biomass, which mainly consists of cell material and of extracellular organic matter (EOM). The EOM forms a matrix, embedding the microbial cells and fulfilling various functions within the microbial mat, including: mat attachment to surfaces; creation of micro-domains within the mat; physical stabilization under hy- drodynamic stress and the protection of the cells in multiple other stress conditions. EOM mainly consists of polysaccharides, amino acids, and a variety of chemical func-tional groups {e.g., -C00H, - SH -OH). These groups strongly bind cations such as Ca2+ and Mg2+ and thus exert a strong control on carbonate mineral formation within the microbial mat. A feedback mechanism between community metabolisms, their prod¬ucts, and the surrounding physicochemical microenvironment thus influences the de¬gree of carbonate saturation favoring either carbonate precipitation or dissolution. We investigated the driving forces and mechanisms of microbialite formation in the Sari ne River, FR, Switzerland, the hypersaline lake, Big Pond, Bahamas and in labo¬ratory experiments. The two fundamentally different natural systems allowed us to compare the geochemical conditions and microbial metabolisms, necessary for car¬bonate formation in microbial mats. Although carbonates are oversaturated in both environments, precipitation does not occur on physicochemical substrates (i.e. out¬side the microbial mats). In the Sarine a high crystal nucleation threshold exceeds the carbonate saturation, despite the high carbonate alkalinity in the water column. Cyanobacterial photosynthesis strongly locally enhances the carbonate alkalinity, whereas the EOM attract and immobilize calcium, which increases the saturation state and finally leads to carbonate precipitation within the EOM (in this case the cyanobacterial sheath) as nucleation template. In Big Pond, the presence of calcium- chelating anions (i.e. sulfate) and EOM, as well as the presence of magnesium, lowers the calcium activity in the water column and mat, and thus inhibits carbonate pre¬cipitation. Coupled with other heterotrophic metabolisms, sulfate reduction uses the EOM as carbon source, degrading it. The resulting EOM consumption creates alkalin¬ity, releases calcium and consumes sulfate in mat-micro domains, which leads to the formation of carbonate layers at the top of the microbial mat. Résumé: Interface biosphère/lithosphère: médiation microbienne de la précipitation de CaC03 dans des environnements en eaux douces et hypersalines Les tapis microbiens engendrent une circulation très efficace des éléments, tels que C, 0, N, S et H, ce qui en fait des acteurs clé pour les processus d'oxydoréduction à l'inter¬face biosphère-lithosphère. Ils sont caractérisés par des taux élevés d'activité méta¬bolique, ainsi que par la production et la consommation de biomasse, principalement constituée de cellules microbiennes et de matière organique extracellulaire (MOE). Dans un tapis microbien, les cellules microbiennes sont enveloppées par une matrice de MOE qui a différentes fonctions dont l'attachement du tapis aux surfaces, la créa¬tion de micro-domaines dans le tapis, la stabilisation physique en situation de stress hydrodynamique, et la protection des cellules dans de multiples autres conditions de stress. La MOE se compose principalement de polysaccharides, d'acides aminés, et d'une variété de groupes fonctionnels chimiques (par exemple, COOH, -SH et -OH). Ces groupes se lient fortement aux cations, tels que Ca2+ et Mg2+, et exercent ainsi un contrôle fort sur la formation de CaC03 dans le tapis microbien. Un mécanisme de rétroaction, entre les métabolismes de la communauté microbienne, leurs produits, et le microenvironnement physico-chimique, influence le degré de saturation de car¬bonate, favorisant soit leur précipitation, soit leur dissolution. Nous avons étudié le moteur et les mécanismes de minéralisation dans des tapis de la Sarine, FR, Suisse et du lac hypersalin, Big Pond, aux Bahamas, ainsi que durant des expériences en laboratoire. Les deux systèmes naturels, fondamentalement dif¬férents, nous ont permis de comparer les conditions géochimiques et les métabolis¬mes nécessaires à la formation des carbonates dans des tapis microbiens. Bien que les carbonates soient sursaturés dans les deux environnements, la précipitation ne se produit pas sur des substrats physico-chimiques (en dehors du tapis microbien). Dans la Sarine, malgré un taux d'alcalinité élevé, les valeurs de seuil pour la nucléa- tion de carbonates sont plus hautes que la saturation du carbonate. La photosynthèse cyanobactérienne augmente localement l'alcalinité, alors que la MOE attire et immo¬bilise le calcium, ce qui augmente l'état de saturation et conduit finalement à la pré¬cipitation des carbonates, en utilisant la MOE comme substrat de nucléation. À Big Pond, la présence de chélateurs de calcium, notamment les anions (p.ex. le sulfate) et la MOE, ainsi que la présence de magnésium, réduit l'activité du calcium et inhibe en conséquence la précipitation des carbonates. Couplée avec d'autres métabolismes hétérotrophes, la réduction des sulfates utilise la MOE comme source de carbone, en la dégradant. Cette consommation de MOE crée l'alcalinité, consomme des sulfates et libère du calcium dans des micro-domaines, conduisant à la formation de couches de carbonates dans le haut du tapis microbien.

Intralesional adenovirus-mediated interleukin-2 gene transfer for advanced solid cancers and melanoma.

Numerous preclinical and clinical studies have shown that interleukin-2 (IL-2) induces regression of metastatic tumors. We have conducted a phase I/II, multicenter, open-label, dose-escalating study to evaluate the safety, efficacy, and biological effects of repeated intratumoral injections of adenovirus-IL-2 (TG1024) in patients with advanced solid tumors and melanoma. Thirty five patients (twenty-five with metastatic melanoma and ten with other solid tumors) were treated in eight successive cohorts at dose levels ranging from 3 x 10(8) to 3 x 10(11) viral particles (vp). Intratumoral TG1024 injections in combination with dacarbazine (DTIC) were tested in metastatic melanoma in one cohort. No clinical responses were observed at doses below 3 x 10(11) vp. Six local objective responses were recorded in patients receiving 3 x 10(11) vp per treatment [five in metastatic melanoma and one in metastatic squamous cell carcinoma (SCC) of the skin], of which two were complete responses (CRs). Most of the common side effects were injection site reactions and flu-like syndrome. TG1024 dose intensification across cohorts resulted in increased serum IL-2 levels after the injection. Intratumoral TG1024 injection induced pronounced inflammation of the treated lesion, with predominant CD8(+), TIA+ lymphocytic infiltrate. Our results show that intratumoral injections of TG1024 are safe and well tolerated. The clinical activity of TG1024 observed in this study warrants further investigations.

Transepithelial transport of HIV-1 by M cells is receptor-mediated.

Human colon carcinoma Caco-2 cell monolayers undergo conversion into cells that share morphological and functional features of M cells when allowed to interact with B lymphocytes. A lymphotropic (X4) HIV-1 strain crosses M cell monolayers and infects underlying CD4(+) target cells. Transport requires both lactosyl cerebroside and CXCR4 receptors, which are expressed on the apical surface of Caco-2 and M cells. Antibodies specific for each receptor block transport. In contrast, a monotropic (R5) HIV-1 strain is unable to cross M cell monolayers and infect underlying monocytes, despite efficient transport of latex beads. Caco-2 and M cells do not express CCR5, but transfection of these cells with CCR5 cDNA restores transport of R5 virus, which demonstrates that HIV-1 transport across M cells is receptor-mediated. The follicle-associated epithelium covering human gut lymphoid follicles expresses CCR5, but not CXCR4, and lactosyl cerebroside, suggesting that HIV-1 infection may occur through M cells and enterocytes at these sites.