Enhanced therapeutic effect of APAVAC immunotherapy in combination with dose-intense chemotherapy in dogs with advanced indolent B-cell lymphoma.

The aim of this non-randomized controlled trial was to compare time to progression (TTP), lymphoma-specific survival (LSS), and safety of an autologous vaccine (consisting of hydroxyapatite ceramic powder and Heat Shock Proteins purified from the dogs' tumors, HSPPCs-HA) plus chemotherapy versus chemotherapy alone in dogs with newly diagnosed, clinically advanced, histologically confirmed, multicentric indolent B-cell lymphoma. The vaccine was prepared from dogs' resected lymph nodes and administered as an intradermal injection. Forty-five client-owned dogs were enrolled: 20 dogs were treated with dose-intense chemotherapy, and 25 received concurrent immunotherapy. Both treatment arms were well tolerated, with no exacerbated toxicity in dogs also receiving the vaccine. TTP was significantly longer for dogs treated with chemo-immunotherapy versus those receiving chemotherapy only (median, 209 versus 85 days, respectively, P=0.015). LSS was not significantly different between groups: dogs treated with chemo-immunotherapy had a median survival of 349 days, and those treated with chemotherapy only had a median survival of 200 days (P=0.173). Among vaccinated dogs, those mounting an immune response had a significantly longer TTP and LSS than those with no detectable response (P=0.012 and P=0.003, respectively). Collectively these results demonstrate that vaccination with HSPPCs-HA may produce clinical benefits with no increased toxicity, thereby providing a strategy for enhancing chemotherapy in dogs with advanced indolent lymphoma.

[Engineering of osseous cells and bioartificial tissues]. / Ingénierie des cellules osseuses et tissus bioartificiels.

The association of osteogenic stem cells to a synthetic carrier makes possible the elaboration of bioartificial tissue. Numerous phosphocalcic ceramics does not trigger a foreign body reaction when implanted in bone tissue and thus, a number of materials are available osteogenic stem cell carriers to replace the bone tissue. Several methods can be used to harvest these cells. Their multiplication in vitro can lead to the appearance of anomalies of their metabolism or their karyotype. The culture method also seems to have a major influence on their appearance. The presence of these anomalies could explain the variability of results in terms of bone extracellular matrix synthesis after cell reimplantation. The surgical technique used for the implantation is also of influence. A method suppressing the in vitro period has been developed to avoid any cell metabolism modification. This method allows for a very reproducible bone synthesis in ectopic site. The availability of human embryonic stem cells could help to develop cell graft techniques for bone reconstruction.

Osseointegration of composite calcium phosphate bioceramics.

The resistance of macroporous calcium phosphate ceramics to compressive strength generally is low and depends on, among other factors, porosity percentage and pore size. A compromise always is adopted between high porosity, required for a good integration, and mechanical strength, which increases with material density. We improved the strength of macroporous calcium phosphate ceramics of interconnected porosity by filling the pores with a highly soluble, self-setting calcium phosphate cement made of TCP and DCPD. Cylinders of the resulting material were implanted in sheep condyles and subjected to histological analysis after 20, 60, and 120 days. Microradiographs were made of the histological sections. The control material consisted of ceramic that had not been loaded with cement. Progressive ingrowth of bone into the ceramic pores occurred as the cement was degraded during the first implantation period. Marked degradation of the cement was apparent after 2 months, with fragmentation of the cement in most of the pores and the presence of bone tissue between the fragments. All the cement had been replaced by bone after 4 months. Some fragments of cement still were embedded in the newly formed bone. There was no significant difference between the integration of loaded and nonloaded ceramics. Filling the macroporous ceramic pores with a calcium phosphate cement significantly improved the mechanical strength of these ceramics without modifying their integration in the healing bone.

Tissue reaction against a self-setting calcium phosphate cement set in bone or outside the organism.

Calcium phosphate cements are able to set in situ when injected into bone tissue. We evaluated the tissue reaction occurring when a DCPD-based calcium phosphate cement was either set within the bone or implanted when already set. The samples were implanted in rabbit condyles and examined histologically after 8 and 16 weeks. The relative bone surface, the fibrous capsule around the implants and the implant section surface were measured. Solid material seemed to be better tolerated than paste implants. More bone was found at the solid implant contact whatever the implantation time and the solid material degraded much less rapidly. In conclusion, the physico-chemical modification of the biological environment occurring during setting increases the foreign body reaction against the material.

Degradation of hydroxylapatite, fluorapatite, and fluorhydroxyapatite coatings of dental implants in dogs.

Calcium phosphate coatings on dental implants enhance integration of the material. Resorption of the ceramic coatings has raised some concern about the behavior of the bone-implant interfaces after the coating disappearance. Substitution of the OH- ions by fluoride in the hydroxylapatite (HA) lattice makes the calcium phosphate more stable. We investigated the degradation rate of dental implants with 50- and 100-microm coatings of HA, fluorapatite (FA), or fluorhydroxylapatite (FHA). The implants were inserted in dog jaws and retrieved for histological analysis after 3, 6, and 12 months. The thickness of the calcium phosphate coatings was evaluated using an image analysis device. A relative resorption index and its standard deviation were studied. HA and FA coatings (even at 100-microm thickness) were almost totally degraded within the implantation period. In contrast, the FHA coatings did not show significant degradation during the same period. The standard deviation showed that the resorption process for FHA with thicknesses of 50 or 100 microm was the same. Such a difference was not observed between the 50- and 100-microm thick coatings of FA and HA. In conclusion, the FHA coatings showed good integration in the bone tissue and lasted much longer than classic calcium phosphate coatings.

LPS challenge in D-galactosamine-sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock.

Experimental models of sepsis using endotoxin challenges, including studies with sensitized animals with D-galactosamine, have largely contributed to the basic rationale for innovative clinical trials in human septic shock, which have, to date, failed. The ability of these models to reproduce human disease has been highly discussed. We report here that the widely used D-galactosamine/LPS model does not account for septic shock. Treatment with YVAD-CMK, a potent tetrapeptide inhibitor of caspases of the interleukin (IL)-1beta converting enzyme (ICE) family, protects from LPS-induced liver apoptosis and mortality in D-galactosamine-sensitized mice when administered either before or up to 2 h after the lethal challenge. This curative effect is related to complete inhibition of caspase-3 activity in the liver. However, YVAD-CMK does not affect LPS-induced release of IL-1beta and does not protect from a lethal dose of LPS in unsensitized mice. These experiments demonstrate the difference between these two widely recognized experimental models of sepsis. LPS toxicity in D-galactosamine-treated mice, leading to blocked gene transcription, results from tumor necrosis factor (TNF)-alpha-induced caspase-3-dependent liver injury, not from the systemic inflammatory response. These results provide evidence that inhibitors of the ICE caspase family can prevent or even overcome the ongoing hepatic injury induced by TNF-alpha during sepsis, ischemia-reperfusion, or severe hepatitis.

Silver methenamine staining for scanning electron microscopy of bone sections containing biomaterials.

Sections of tissue containing orthopedic materials are currently used to study the compatibility of those materials and to perform electron probe microanalysis at the material-tissue interface. Identification of the cells in contact with the material by Scanning electron microscopy (SEM) is of interest. We have developed a method for staining cells and tissue structures embedded in polymethyl methacrylate with silver methenamine once the sections have been obtained. Sections were prepared by grinding, and the silver methenamine was applied after oxidation with periodic acid. The procedure was carried out in a microwave oven. Backscatter SEM showed staining of the cell nucleus membrane, chromatin, the nuclear organizers, and the chromosomes of dividing cells. The cytoplasm and the cytoplasmic membrane were also stained. Collagen fibers of the extracellular matrix and the mineralized matrix of bone were labeled. Material particles in the macrophages were easily recognizable and Energy-Dispersive Spectrometer were not impaired by the presence of silver in the preparation.

Short-term implantation effects of a DCPD-based calcium phosphate cement.

Calcium phosphate cements can be handled in paste form and set in a wet medium after precipitation of calcium phosphate crystals in the implantation site. Depending on the products entering into the chemical reaction leading to the precipitation of calcium phosphates, different phases can be obtained with different mechanical properties, setting times and injectability. We tested a cement composed of a powder, containing beta-tricalcium phosphate (beta-TCP) and sodium pyrophosphate mixed with a solution of phosphoric and sulphuric acids. The cement set under a dicalcium phosphate dihydrate (DCPD)-based matrix containing beta-TCP particles. This was injected with a syringe into a defect drilled in rabbit condyles, the control being an identical defect left empty in the opposite condyle. The condyles were analysed histologically 2, 6 and 18 weeks after implantation. After injection into the bone defect the cement set and formed a porous calcium phosphate structure. Two different calcium phosphate phases with different solubility rates could be identified by scanning electron microscopy (SEM) observation. The less-soluble fragments could be degraded by cell phagocytosis in cell compartments of low pH or integrated in the newly formed bone matrix. The degradation rate of the material was relatively high but compatible with the ingrowth of bone trabeculae within the resorbing material. The ossification process was different from the creeping substitution occurring at the ceramic contact. Bone did not form directly at the cement surface following the differentiation of osteoblasts at the material surface. The trabeculae came to the material surface from the edges of the implantation site. Bone formation in the implantation site was significantly higher than in the control region during the first week of implantation. In conclusion, this material set in situ was well tolerated, inducing a mild foreign-body reaction, which did not impair its replacement by newly formed bone within a few weeks.

Histological integration of allogeneic cancellous bone tissue treated by supercritical CO2 implanted in sheep bones.

UNLABELLED: Different chemical or physical methods of bone processing have been developed to decrease the antigenicity of allogeneic bone which may delay or prevent graft integration. We have developed a method based on delipidation and deproteination of the bone with a supercritical fluid and hydrogen peroxide. Cylinders of cancellous allogeneic bone treated in this way were implanted for four weeks, four months or eight months in holes drilled in sheep condyles or tibial plateau. Histological sections were then processed and analysed qualitatively and quantitatively using an image analysis software coupled to a light microscope. Measurements were made of the trabecular bone surface (BS/TS), the relative osteoid surface (OS/BS), the active osteoid surface (OS/BS), active resorption surface (Oc.S/BS) and the relative surface of newly formed bone. After four weeks, the control cylinders (non-treated allogeneic bone) had been invaded by cellular tissue composed of lymphocytes and plasmocytes surrounding remnants of the donor bone marrow tissue. The processed cylinders showed osteoid apposition at the surface of their external trabeculae. The trabecular bone and osteoid surfaces were significantly higher in the processed bone sections than in the control bone sections. After four months, most of the control material had been osteolysed and replaced by connective tissue containing lymphocyte islets, while the processed materials showed a large amount of bone synthesized at the surface of implant trabeculae which appeared fragmented and disseminated within the newly formed bone. All the histomorphometric parameters measured were significantly different from those of the control. By eight months, most of the control material had been totally osteolysed with very little bone ingrown in the implantation site. Only one control implant had been integrated. The processed cylinders were difficult to discern from the bone in which they were implanted. The parameters measured on the processed cylinders were significantly higher than those measured on the control sections. IN CONCLUSION: the treatment applied to the bone enhanced allogeneic bone integration and could provide a new kind of tissue treatment for bone banking.

[Osseointegration of 2 different types of calcium phosphate materials: ceramics and ionic cements]. / Ostéointégration de deux matériaux phosphocalciques de natures différentes: céramiques et ciments ioniques.

UNLABELLED: Different kinds of calcium phosphate biomaterials can be used as bone substitutes. Ceramics are constituted by HA or TCP grains linked by grain boundaries. Their porosity depends on the powder characteristics and the sintering temperature. It can be very low with a pore size inferior to one micron. The setting of calcium phosphate hydraulic cements results from the precipitation of a calcium phosphate phase different from the one in suspension in the paste. The strength of the cement is given by the entanglement of the growing mineral crystals. Calcium phosphate hydraulic cements and ceramics have very different physico-chemical characteristics. We have studied the histological integration of both kinds of material. The first material was constituted by macroporous ceramics composed of 75% HA and 25% beta-TCP, the cement was made of beta-TCP grains dispersed in a DCPD matrix. The sequence of events which leads to the ceramic integration is always the same: a/ ingrowth of a loose connective tissue; b/ osteoblast differentiation from fibroblast-like cells of the connective tissue in close proximity to the implant surface; c/ osteoid synthesis at the ceramic surface toward the pore center; d/ remodeling of the immature bone and the ceramic itself. The cement is differently integrated. The osteoblasts differentiate at some distance from the implant and there is a trabeculae ingrowth toward the material. CONCLUSIONS: The early stages of both materials osteointegration are different. The integration is centrifugal for ceramics and centripetal for the cement.

The influence of sintering temperature on the proliferation of fibroblastic cells in contact with HA-bioceramics.

HA-ceramics used in human surgery as osteoconductive surfaces show a great variety of characteristics. Certain characteristics such as grain size, porosity, and surface area, are controlled by the sintering temperature of the slurry. We grew L-929 fibroblast cells on HA-ceramic disks that had been sintered at different temperatures ranging from 850 degrees-1350 degrees C. The cell line growth rate was lower on ceramic disks than on the culture-grade polystyrene used as a negative control. Cell growth correlated with the ceramic sintering temperature although no significant difference in the cell adhesion to the different ceramics was shown. Growth rate on ceramics sintered at low temperatures (850 degrees and 950 degrees C) was negative whereas it was positive on disks sintered at higher temperatures. When the cells were separated from the disks by a polycarbonate membrane, the growth rate was negative on those membranes in contact with low-temperature sintered disks and positive on the high-temperature sintered disks. The calcium and phosphorus concentration in the culture medium in contact with ceramics sintered below 1050 degrees C decreased during the culture period. Ceramics sintered between 1100 degrees and 1250 degrees C brought about an increase in Ca and P concentrations while ceramics sintered at higher temperatures did not induce any changes. SEM examination of the 850 degrees and 1200 degrees C sintered ceramics showed that the 850 degrees C sintered ceramics consisted of small grains with pores between them and the 1200 degrees C sintered ceramics were made of larger grains without any visible pores, thereby decreasing the surface of material in contact with the culture medium. This difference in surface area was confirmed by the fact that the amount of albumin absorbed onto the ceramic was dependent on the sintering temperature. In conclusion, the modification of the culture medium brought about by high-surfaced ceramics could influence the growth of cells with which such ceramics come in contact.

Multiple pathways of Fas-induced apoptosis in primary culture of hepatocytes.

Fas (Apo1/CD95) is a member of the tumour necrosis factor/nerve growth factor receptor superfamily and mediates apoptosis in various cell types (for review sec [1]). Although this apoptotic activity has been clearly related to homeostasis in the immune system and pathological situations in non-lymphoid organs, the Fas signaling pathway remains mostly elusive. We and others previously showed that Fas-induced apoptosis of primary culture hepatocytes requires either an inhibitor of translation or a protein kinase inhibitor, suggesting that two distinct pathways of Fas signaling exist in hepatocytes. We report here that activation of ICE-like and CPP32-like cysteine proteases are required for Fas-mediated apoptosis, but that these pathways involve different subclasses of serine proteases and are selectively modulated by inhibitors of protein tyrosine kinases. These results confirm that distinct pathways can lead to Fas-induced apoptosis in hepatocytes. Further understanding of these pathways could facilitate the rational design of anti-apoptotic drugs in liver diseases associated with massive Fas-mediated hepatocyte apoptosis, including fulminant hepatitis.

ICE inhibitor YVADcmk is a potent therapeutic agent against in vivo liver apoptosis.

In liver, apoptosis is a physiological process involved in the clearance of injured cells and in homeostatic control [1]. However, in patients with viral fulminant hepatitis or with nonacute liver diseases [2], dramatic liver failure or secondary cirrhosis results from the death of hepatocytes, which express the cell-surface receptor Fas, by apoptosis. To date, treatment of fulminant hepatitis relies mainly on orthotopic liver transplantation, which is limited by immunological complications and graft availability. Unravelling the molecular mechanisms that underlie acute liver failure could allow the design of an appropriate therapy. Ligand-bound Fas and tumour necrosis factor alpha (TNF-alpha) induce hepatic apoptosis in mice [3-6]. In various cell types, Fas- or TNF-alpha-induced apoptosis is blocked by viral proteins (such as p35 and CrmA) as well as by a decoy peptide (YVADcmk) [7-11], suggesting that these mechanisms of apoptosis involve ICE (interleukin-1 beta converting enzyme)-like proteases. Here, we report that, in vivo, pre-treatment of mice with YVADcmk protects them from the lethal effect of anti-Fas antibody and from liver failure induced by injection of TNF-alpha. Remarkably, YVADcmk administration is also highly effective in rescuing mice that have been pretreated with anti-Fas antibody from rapid death, despite extensive hepatic apoptosis. This dramatic curative effect could be of clinical benefit for the treatment of viral and inflammatory liver diseases.

Protection of hepatocytes from Fas-mediated apoptosis by a non-transforming SV40 T-antigen mutant.

Apoptosis is crucial for the normal development of multicellular organisms and is also important for clearing injured cells, such as virus-infected cells or cancer cells. Defective regulation of apoptosis may contribute to viral pathogenesis and aetiology of cancer. Apoptosis of injured cells is principally triggered by the immune system through cytokines such as Fas-ligand and TNF-alpha. Thus, one of the functions of a viral oncogene, such as SV40T-antigen, may be to inhibit cytokine-mediated apoptosis. We previously demonstrated that Fas-mediated apoptosis of hepatocytes is blocked by the wild-type SV40T-antigen during hepatocarcinogenesis. We determined whether this inhibition was directly related to the T-antigen or whether it is a secondary event of cell transformation, by generating transgenic mice expressing a non-transforming T-antigen mutant able to bind endogenous p53 in the liver. This T-antigen mutant cannot induce hepatocarcinoma, unlike the wild-type T-antigen. However, like the wild-type T-antigen, the mutant was a potent inhibitor of apoptosis induced by the Fas-receptor, but not by the TNF-receptor. Therefore, SV40T-antigen has a new property; the inhibition of Fas-mediated apoptosis, which could facilitate the emergence of transformed hepatocytes, but is not sufficient to induce it.

Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice.

Fas is an apoptosis-signalling cell surface antigen that has been shown to trigger cell death upon specific ligand or antibody binding. Treatment of mice with an anti-Fas antibody causes fulminant hepatic failure due to massive apoptosis. To test a putative protective effect of the anti-apoptotic Bcl-2 protein, transgenic mice were generated to express the human bcl-2 gene product in hepatocytes. Early onset of massive hepatic apoptosis leading to death was observed in all nontransgenic mice treated with an anti-Fas antibody. By contrast, hepatic apoptosis was delayed and dramatically reduced in transgenic animals, yielding a 93% survival rate. These results demonstrate that Bcl-2 is able to protect from in vivo Fas-mediated cytotoxicity, and could be of significance for preventing fulminant hepatic failure due to viral hepatitis in humans.

Fas-dependent apoptosis is impaired by SV40 T-antigen in transgenic liver.

A transgenic mouse model for hepatocarcinoma has been previously produced by targeting SV40 T-antigen expression to the liver. To evaluate the perturbation of cell death occurring during hepatocarcinogenesis, we examined the Fas-induced apoptosis on hepatocytes expressing T-antigen. Whereas anti-Fas antibody induced apoptosis in primary cultured normal hepatocytes, they imparted a weak cytotoxicity on primary cultured hepatocytes expressing T-antigen. This resistance of hepatic Fas-mediated apoptosis appears to result in an enhancement of a protective mechanism involving the protein kinase C signaling pathway rather than in a down-regulation of Fas-antigen expression. We further demonstrated that anti-Fas antibody does not have as efficient a lethal effect in T-antigen transgenic mice as in wild-type mice. The livers of transgenic mice injected with anti-Fas mAbs showed large intact regions with a few scattered apoptotic bodies: these regions strictly corresponded with carcinoma nodules, expressing high level of T-antigen. Our results describe a novel function for SV40 T-antigen which could contribute to viral pathogenesis by protecting infected cells against the host apoptotic defense mechanism.

Osseointegration of macroporous calcium phosphate ceramics having a different chemical composition.

Calcium phosphate macroporous ceramics are biocompatible for bone surgery. Their osseointegration is, however, sometimes very poor. To measure the effect of the calcium phosphate content of the ceramic on its osseointegration, macroporous ceramics, differing in their chemical composition, were implanted into sheep femurs. The ceramics were composed of different percentages of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP). All other characteristics were the same. Results were assessed histologically with image analysis and showed significant differences in the amount of bone formed at the contact of the different ceramics. Ceramics containing beta-TCP induced better osseointegration than pure HA ceramics.

New observations on middle term hydroxyapatite-coated titanium alloy hip prostheses.

HA-coated hip prostheses were retrieved from elderly patients after death. Histological analysis, scanning electron microscopy and microanalysis by energy-dispersive X-ray spectrometry were performed on the same sections. These revealed good osseointegration of the implant material and evolution of bone and material.