Azoximer bromide and hydroxyapatite: promising immune adjuvants in cancer.

Immune adjuvants are immune modulators that have been developed in the context of infectious vaccinations. There is currently a growing interest in immune adjuvants due to the development of immunotherapy against cancers. Immune adjuvant mechanisms of action are focused on the initiation and amplification of the inflammatory response leading to the innate immune response, followed by the adaptive immune response. The main activity lies in the support of antigen presentation and the maturation and functions of dendritic cells. Most immune adjuvants are associated with a vaccine or incorporated into the new generation of mRNA vaccines. Few immune adjuvants are used as drugs. Hydroxyapatite (HA) ceramics and azoximer bromide (AZB) are overlooked molecules that were used in early clinical trials, which demonstrated clinical efficacy and excellent tolerance profiles. HA combined in an autologous vaccine was previously developed in the veterinary field for use in canine spontaneous lymphomas. AZB, an original immune modulator derived from a class of heterochain aliphatic polyamines that is licensed in Russia, the Commonwealth of Independent States, and Slovakia for infectious and inflammatory diseases, is and now being developed for use in cancer with promising results. These two immune adjuvants can be combined in various immunotherapy strategies.

Opportunities and challenges of active immunotherapy in dogs with B-cell lymphoma: a 5-year experience in two veterinary oncology centers.

BACKGROUND: Pet dogs spontaneously develop lymphoma. An anthracycline-based multidrug chemotherapy regimen represents the treatment cornerstone; however, cure is rarely achieved. We have been treating dogs with B-cell lymphoma with an autologous vaccine (APAVAC®) and CHOP-based chemotherapy since 2011. METHODS: To better characterize the safety and efficacy of APAVAC®, and to find the best candidates for immunotherapy, we designed a retrospective study on all dogs treated with chemo-immunotherapy to date and compared them with those dogs treated with chemotherapy only. All dogs were completely staged and re-staged at the end of treatment. The primary endpoint was the effectiveness of chemo-immunotherapy, measured as time to progression (TTP), lymphoma-specific survival (LSS), and 1-, 2-, and 3-year survival rates. The secondary objective was safety. RESULTS: Three hundred dogs were included: 148 (49.3%) received chemotherapy and 152 (50.7%) chemo-immunotherapy. Overall, the latter survived significantly longer (median LSS, 401 vs 220; P <  0.001). Among dogs with diffuse large B-cell lymphoma, the 1-, 2- and 3-year survival rates were 20, 13 and 8% for chemotherapy, and 51, 19 and 10% for chemo-immunotherapy. The benefit of chemo-immunotherapy was particularly relevant in dogs with concurrent high serum LDH, stage V, substage a disease and not previously treated with steroids (median LSS, 480 vs 85 days; P <  0.001). Among dogs with nodal marginal zone lymphoma, those having at least 3 of the aforementioned characteristics significantly benefited from chemo-immunotherapy (median LSS, 680 vs 160 days, P <  0.001). The 1-, 2- and 3-year survival rates were 30, 16 and 10% for chemotherapy, and 55, 28 and 10% for chemo-immunotherapy. Among dogs with follicular lymphoma, lack of immunotherapy administration was the only variable significantly associated with increased risk of tumor-related death. Chemo-immunotherapy was remarkably well tolerated, with no local or systemic adverse events. CONCLUSIONS: Overall, the addition of immunotherapy to a traditional CHOP protocol is associated with improved outcome in dogs with B-cell lymphoma, regardless of histotype and evaluated prognostic factors. Moreover, the identikit of the best candidate for immune-therapy was delineated for the most common histotypes. The study also confirms the excellent tolerability of the vaccine.

Minimal residual disease detection by flow cytometry and PARR in lymph node, peripheral blood and bone marrow, following treatment of dogs with diffuse large B-cell lymphoma.

The most promising techniques for detecting minimal residual disease (MRD) in canine lymphoma are flow cytometry (FC) and polymerase chain reaction amplification of antigen receptor genes (PARR). However, the agreement between these methods has not been established. MRD was monitored by FC and PARR following treatment of dogs affected with diffuse large B-cell lymphoma (DLBCL), comparing results in lymph node (LN), peripheral blood (PB) and bone marrow (BM) samples. The prognostic impact of MRD on time to relapse (TTR) and lymphoma-specific survival (LSS) was also assessed. Fourteen dogs with previously untreated DLBCL were enrolled into the study; 10 dogs eventually relapsed, while four dogs with undetectable MRD were still in remission at the end of the study. At diagnosis, the concordance rate between FC and PARR was 100%, 78.6%, and 64.3% for LN, PB and BM, respectively. At the end of treatment, the agreement rates were 35.7%, 50%, and 57.1% for LN, PB and BM, respectively. At least one of the follow-up samples from dogs experiencing relapse was PARR(+); conversely, FC was not able to detect MRD in seven of the dogs that relapsed. PARR was more sensitive than FC in predicting TTR, whereas the combination of PARR and FC was more sensitive than either technique alone in predicting LSS using PB samples. The results suggest that immunological and molecular techniques should be used in combination when monitoring for MRD in canine DLBCL.

Randomized, placebo-controlled, double-blinded chemoimmunotherapy clinical trial in a pet dog model of diffuse large B-cell lymphoma.

PURPOSE: Active immunotherapy is a promising antitumoral strategy; however its use in combination with chemotherapy in dogs with large B-cell lymphoma (DLBCL) remains largely untested. Heat shock proteins (HSP) bind the small peptides they chaperone (HSPPC), allowing for immunization of the host against a large repertoire of tumor-associated antigens. Hydroxylapatite vehicles HSPPCs and acts as an immunologic adjuvant. The aim of this study was to show that an autologous vaccine with hydroxylapatite and tumor-derived HSPPCs is safe and therapeutically effective in dogs with DLBCL. EXPERIMENTAL DESIGN: Nineteen dogs with naturally occurring DLBCL were entered into a prospective randomized placebo-controlled double-blinded trial of HSPPCs-hydroxylapatite plus chemotherapy versus chemotherapy alone. Endpoints included time to progression (TTP), lymphoma-specific survival (LSS), and incidence of toxicoses. RESULTS: Median first TTP after randomization to the vaccine arm was 304 days versus 41 days for the control arm (P = 0.0004). There was also a statistically significant difference in duration of second remission between the two groups (P = 0.02). Median LSS was 505 days for the vaccinated dogs versus 159 days for the unvaccinated dogs (P = 0.0018). Six vaccinated dogs achieved molecular remission, as shown by clonal immunoglobulin H (IgH) rearrangement. Toxicoses were comparable between the two treatment arms. CONCLUSIONS: The results of this trial demonstrate that the autologous vaccine tested here is safe and efficacious in prolonging TTP and LSS in dogs with DLBCL when used in combination with dose-intense chemotherapy. On the basis of these results, additional evaluation of this novel therapeutic strategy is warranted in human DLBCL.

NLRP3 inflammasome plays a critical role in the pathogenesis of hydroxyapatite-associated arthropathy.

The proinflammatory and catabolic cytokine IL-1ß has been implicated in the pathogenesis of osteoarthritis (OA) by mediating synovial inflammation and cartilage degeneration. Although synovial macrophages are suggested to be the source of IL-1ß, the mechanism remains unclear. Ectopic deposition of hydroxyapatite (HA) crystals in joints is closely associated with OA and other arthropathies, but the precise role of HA in arthritis pathogenesis has not been clearly demonstrated. Here we show that HA crystals of a particular size and shape can stimulate robust secretion of proinflammatory cytokines IL-1ß and IL-18 from murine macrophages in a NLRP3 inflammasome-dependent manner. HA-induced inflammasome activation is dependent on potassium efflux, generation of reactive oxygen species (ROS), and lysosomal damage, but independent of cell death. Mice lacking the inflammasome components are protected against HA-induced neutrophilic inflammation in the air-pouch model of synovitis, and they show decreased joint pathology accompanying spontaneous HA deposition in the ank-deficient mouse model of arthritis. Moreover, calcium crystal positive synovial fluids from some OA patients exhibited inflammasome-stimulatory activity in vitro. These results demonstrate that the NLRP3 inflammasome mediates the pathological effect of HA crystals in vitro and in vivo and suggest a critical role for the inflammasome in the pathogenesis of OA.

A pilot study with a therapeutic vaccine based on hydroxyapatite ceramic particles and self-antigens in cancer patients.

We describe an approach to produce an autologous therapeutic antitumor vaccine using hydroxyapatite (HA) for vaccinating cancer patients. The novel approach involved (1) the purification of part of the self-tumor antigens/ adjuvants using column chromatography with HA, (2) the employ of HA as a medium to attract antigen-presenting cells (APCs) to the vaccination site, and (3) the use of HA as a vector to present in vivo the tumor antigens and adjuvants to the patient's APCs. The vaccine was prepared using and combining HA particles, with at least 3 heat shock proteins (gp96 was one of them possibly with chaperoned proteins/peptides as shown in the slot blots) and with proteins from the cell membrane system (including Hsp70, Hsp27, and membrane proteins). The timing of HA degradation was tested in rats; the HA particles administered under the skin attracted macrophages and were degraded into smaller particles, and they were totally phagocytized within 1 week. In patients (n = 20), the vaccine was then administered weekly and showed very low toxicity, causing minor and tolerable local inflammation (erythema, papule, or local pain); only 1 patient who received a larger dose presented hot flashes, and there were no systemic manifestations of toxicity or autoimmune diseases attributed to the vaccine. Our study suggests that this therapeutic vaccine has shown some efficacy producing a positive response in certain patients. Stable disease was noted in 25% of the patients (renal carcinoma, breast carcinoma, and astrocytoma), and a partial response was noted in 15% of the patients (breast carcinoma and astrocytoma). The most encouraging results were seen in patients with recurrent disease; 4 patients in these conditions (20%) are disease free following the vaccine administration. However, we do not want to overstate the clinical efficacy in this small number of patients. The therapeutic vaccine tested in our study is working by activating the T-cell response as was shown in the comparative histological and immunohistochemical study performed in the pre- and postvaccine biopsy taken from a patient with inflammatory breast carcinoma. However, we cannot ruled out that the vaccine could also be producing an antibody(ies)-mediated response. In conclusion, this therapeutic vaccine based on HA ceramic particles and self-antigens can be safely administered and is showing some encouraging clinical results in cancer patients.

Involvement of toll-like receptor 4 in the inflammatory reaction induced by hydroxyapatite particles.

Hydroxyapatite (HA) is widely used to coat metal parts in order to improve their biocompatibility. Analysis of retrieved tissues associated with failed implants, suggest that phagocytosis of HA wear debris by monocytes/macrophages might provide a potent stimulus for the release of a variety of cytokines. Phagocytosis involved a large variety of cellular receptors like toll-like receptors that results in activation of the transcriptional nuclear factor-kappaB (NF-kappaB) via a cell-signalling pathway. In the present paper, we aimed to evaluate the role of the toll-like receptor 4 (TLR4) in the production of inflammatory cytokines induced by HA particles using TLR4(+) and TLR4(-) peritoneal macrophages. We investigated the production of TNF-alpha and the activation of the nuclear transcription factor NF-kappaB. Our data clearly show for the first time that the production of TNF-alpha by macrophages exposed to HA particles was TLR4 dependent but not the activation of NF-kappaB. All these results open future therapies to reduce the inflammatory response induced by HA biomaterials.

Bone cell transfection in tissue culture using hydroxyapatite microparticles.

Coprecipitates of calcium phosphate and DNA have been used in vitro for several decades for cell transfection. We evaluated the efficiency of calcium phosphate ceramics associated to plasmid DNA in the transfection of bone cells in vitro when they are grown in tissue culture. Newborn rat calvariae and tibia epiphyses were grown on an agar surface for a period of 48 h to 30 days. The hydroxyapatite (HA)-particles were loaded with a plasmid bearing a galactosidase reporter gene by incubation of the plasmid solution in PBS with the particles. One milligram of HA-particles was then placed in contact with the bone explants for 8 and 30 days. Histological sections were then performed and the galactosidase activity was revealed using an X-gal solution. At eight days, very few cells expressing the galactosidase activity were detected. By 30 days, however, the explants appeared uniformly stained blue. The staining of sections showed that the osteoblasts, chondroblasts, perichondroblasts, and perisoteal cells all expressed the lacZ gene while the number of cells stained in the control was negligible. The time dependence of the transfection suggests that transfection using ceramics is linked to the degradation of the ceramic by the cells. Furthermore, the cells are stained remote from the particles suggesting that the particles induce a coprecipitate of DNA in the explant.

Biological performance of a new beta-TCP/PLLA composite material for applications in spine surgery: in vitro and in vivo studies.

The objective of this research was to carry out an in vitro and in vivo study of the biological performance of PLLA/beta-TCP composite materials, to estimate the scope of their potential applications in bone surgery. Samples with increasing beta-TCP (0-60% w/w) contents were processed by injection molding. The in vitro study consisted of an evaluation of inflammatory potential by assaying the IL-1alpha secreted by monocytes, and then cell proliferation (counting) and phenotype expression (PAL and I collagen) in human osteogenous cells. The in vivo study was carried out using cylindrical implants of composite materials composed of composite materials containing 0 or 60% beta-TCP and pure beta-TCP, respectively. The implants were inserted in femoral sites in rabbits, using the Kathagen protocol. Each animal received a 60% implant, with either a 0 or a 100% implant in the contralateral femur, so that the materials could be compared with one another. Five animals were examined for each material and implantation period, giving a total of 30 animals. This study showed that adding increasing percentages of beta-TCP to a lactic acid polymer matrix stimulated the proliferation of human osteogenous cells and synthesis of the extracellular bone matrix in a dose-dependent manner. In vivo results indicate that, in comparison with pure PLA, tricalcium phosphate-containing composite materials had faster degradation kinetics, caused less inflammatory reaction, and promoted contact osteogenesis. The composite material containing 60% beta-TCP demonstrated a similar performance to pure tricalcium phosphate bone grafts in terms of osteogenesis, and is apparently compatible with the production of intra-osseous implants for situations representing high levels of mechanical strain.

Poly-L-lactide surfaces subjected to long-term cell cultures: cell proliferation and polymer degradation.

Short term cell cultures are usually grown in contact with biomaterials to assess cytocompatibility. Depending on the rate of material degradation or corrosion, the time of culture can be a key-point in the method which, if too short, may not show any effect of the released material on the cells. A long term culture was therefore carried out with L929 fibroblast cells in contact with PLLA/PDLA samples for up to eight months. The degradation was measured in terms of shear-strength properties, intrinsic viscosity of the material and its cristallinity. The effect of the material on the cells was evaluated by measuring the growth rate of the cells. A significant decrease in the shear strength of the material was measured after three months. The rate of modification of the intrinsic viscosity was regular and decreased progressively throughout the culture period. Differential scanning calorimetry showed that the samples were initially essentially amorphous and that contact with the cell culture and its medium did not change its crystallinity level. The growth rate of the cells was not modified by the presence of the material when compared to the control. This study showed this material to be cytocompatible for a long period of time, even after detection of modifications of its physico-chemical properties.

Histomorphometric comparison of three bioabsorbable GTR barrier membranes in the canine model.

Guided tissue regeneration (GTR) using bioabsorbable barrier membranes has shown promising and somewhat predictable clinical results. However, limited information is available on the stages of histologic healing when these types of barriers are used. Therefore, the aim of this study was to compare three currently available bioabsorbable GTR barriers (type 1 collagen, polylactide/polyglycolide copolymer, and citric acid copolymer) for the treatment of surgically created periodontal dehiscences in dogs. Eight three-walled periodontal dehiscences were surgically created in the mandibles of six beagle dogs (four on each side of the jaw). The periodontal dehiscences were randomly assigned to receive one of the three barriers. One defect was left untreated to serve as a control. Histomorphometric analyses were conducted at 4 and 12 weeks. Data were analyzed using the Student-Fisher test. The membrane-treated group had a statistically significant increase in new bone formation and connective tissue attachment compared to the nontreated control sites. However, no statistical difference was noted among the various membranes. In addition, no difference was found between test and control sites when new cementum was examined. Sites treated with barrier membranes achieved greater bone regeneration than nontreated control sites in the canine model.

MMP-2, MMP-9 and their inhibitors TIMP-2 and TIMP-1 production by human monocytes in vitro in the presence of different forms of hydroxyapatite particles.

After calcium-phosphates biomaterials based implantation like hydroxyapatite (HA) coating, particles are released in the periprosthetic tissues. Wear-debris induced fibrous membranes contain macrophage subsets that can produce metalloproteinases (MMPs), which are considered to be key enzymes in extra-cellular matrix turnover. Tissue inhibitors of metalloproteinases (TIMPs) are important regulator of MMPs activity. Interleukin-1 mainly produced by monocytes can also regulate MMPs production. In the present work, we have evaluated the effect of HA particles characteristics (size, shape and sintering temperature) on the MMP-2, -9 and their respective inhibitors TIMP-2, -1 production. Our results demonstrate that sintering temperature (that modify crystal size and surface area) have little effect on MMPs and TIMPs production. Non-phagocytable particles induced more MMP-9, although phagocytable particles induced more IL-1beta release. The shape of the particles was the most important factor since needle-shaped particles induced the most significant up-regulated expression of MMPs and IL-1beta.

Growth plate behavior after desepiphysiodesis: experimental study in rabbits.

The aim of this work was to study the potential healing of the growth plate in the case of a central desepiphysiodesis. A central defect was made in the distal femoral growth plate of thirty 3-week-old rabbits. In group A the growth plate defect was left empty as control. The defects of group B were implanted with a polymeric cylinder fixed in the metaphysis with a pin. In group C the cylinder was fixed in the epiphysis. Two months after implantation, clinical, radiologic, and histologic analyses were carried out. In group A, the mean shortening was 12.63%; it was 4.9% in group B and 1.54% in group C. Histologic analysis showed constant appearance of an epiphysiodesis after migration of the implant in the metaphysis. No regeneration of the growth plate was observed. Prevention of migration of the interpositional material is recommended to avoid recurrence of an epiphysiodesis.

Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro.

Calcium phosphate bioceramics have been applied as bone substitutes for several decades. Aseptic loosening after total joint arthroplasty is a major problem in orthopaedic surgery. Hydroxyapatite particles from materials wear have been reported as the main cause of implant failure. For this reason, an investigation into possible wear particles from materials used in the implant may lead to longevity after arthroplasty. Monocytes are among the first cells to colonize the inflammatory site. In the present study, we have evaluated the inflammatory response after exposition to particles with different characteristics (size, sintering temperature and shape). Our data demonstrate that the most important characteristic was the shape and the size of the particles. The needle shaped particles induced the larger production of TNF-alpha, IL-6 and IL-10 by cells. To a less manner, the smallest particles induced an increase of the expression and production of the cytokines studied (TNF-alpha, IL-6 and IL-10). The sintering temperature appeared to be a less important characteristic even though it was involved in the dissolution/precipitation process.