Constitutive IL-1RA production by modified immune cells protects against IL-1-mediated inflammatory disorders.

Dysregulation of the interleukin-1 (IL-1) pathway leads to immune diseases that can result in chronic tissue and organ inflammation. Although IL-1 blockade has shown promise in ameliorating these symptoms and improving patients' quality of life, there is an urgent need for more effective, long-lasting treatments. We developed a lentivirus (LV)-mediated gene transfer strategy using transplanted autologous hematopoietic stem/progenitor cells (HSPCs) as a source of IL-1 receptor antagonist (IL-1RA) for systemic delivery to tissues and organs. Transplantation of mouse and human HSPCs transduced with an IL-1RA-encoding LV ensured stable IL-1RA production while maintaining the clonogenic and differentiation capacities of HSPCs in vivo. We examined the efficacy of cell-mediated IL-1RA delivery in three models of IL-1-dependent inflammation, for which treatment hindered neutrophil recruitment in an inducible model of gout, prevented systemic and multi-tissue inflammation in a genetic model of cryopyrin-associated periodic syndromes, and reduced disease severity in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Our findings demonstrate HSPC-mediated IL-1RA delivery as a potential therapeutic modality that can be exploited to suppress tissue and organ inflammation in diverse immune-related diseases involving IL-1-driven inflammation.

Brillouin-Raman micro-spectroscopy and machine learning techniques to classify osteoarthritic lesions in the human articular cartilage.

In this study, Brillouin and Raman micro-Spectroscopy (BRamS) and Machine Learning were used to set-up a new diagnostic tool for Osteoarthritis (OA), potentially extendible to other musculoskeletal diseases. OA is a degenerative pathology, causing the onset of chronic pain due to cartilage disruption. Despite this, it is often diagnosed late and the radiological assessment during the routine examination may fail to recognize the threshold beyond which pharmacological treatment is no longer sufficient and prosthetic replacement is required. Here, femoral head resections of OA-affected patients were analyzed by BRamS, looking for distinctive mechanical and chemical markers of the progressive degeneration degree, and the result was compared to standard assignment via histological staining. The procedure was optimized for diagnostic prediction by using a machine learning algorithm and reducing the time required for measurements, paving the way for possible future in vivo characterization of the articular surface through endoscopic probes during arthroscopy.

Randomised, double-blind comparison of a fixed co-formulation of intra-articular polynucleotides and hyaluronic acid versus hyaluronic acid alone in the treatment of knee osteoarthritis: two-year follow-up.

BACKGROUND: A first-year interim analysis of this two-year study suggested that intra-articular injections of highly purified, natural-origin polynucleotides and hyaluronic acid (HA) as a fixed combination (PNHA) might improve knee function and joint pain more effectively than HA alone in patients with knee osteoarthritis (OA). The purpose of the second-year analysis herein described was to verify whether the first-year interim outcomes persist over the whole two-year period. METHODS: Randomised, double-blind, HA-controlled clinical trial in 100 knee OA patients (98 randomised, 79 completing the study) in a high-specialisation tertiary care setting. The hypothesised difference of efficacy between PNHA and HA for the original sample size estimate is 20%. Treatment cycle: three intra-articular knee injections of either PNHA or HA, at baseline and weekly for two weeks. EVALUATIONS: Western Ontario and McMaster Universities (WOMAC) score and Knee Society Score (KSS) as, respectively, primary and secondary endpoints, evaluated at baseline and after 2, 6, 12, and 24 months; synovial fluid levels of mediators (at baseline and the end of the treatment cycle). Adverse effects investigated at each control visit. STATISTICAL ANALYSIS: Kruskal-Wallis test for independent samples (nonparametric one-way analysis of variance) after correction of means for age, Body Mass Index and Kellgren-Lawrence grade. If significant, pairwise post-hoc Sidak multiple comparisons. RESULTS: KSS total score and KSS pain item: significant improvement in both groups, with significantly more pain improvement in patients treated with PNHA (2-point reduction) than HA (1-point reduction). Both groups experienced significant long-term reductions in WOMAC total scores: significantly stronger in PNHA-treated patients after 24 months with a steady difference of 16% favouring PNHA in WOMAC pain subscore. No clinically significant adverse events in either group. CONCLUSIONS: The outcomes of the 2-year study confirmed that a short cycle of intra-articular treatment (3 weekly double-blind injections) with polynucleotides (long-acting viscosupplementation properties, chondrocyte activation, pain-relieving properties) in fixed combination with high molecular weight hyaluronic acid is more effective in improving knee function and pain in knee OA patients than HA alone. PNHA may be elective for viscosupplementation in knee OA patients with fastidious and resistant pain and worsening disease. TRIAL REGISTRATION: NCT02417610 . Registration, 15/04/2015. ClinicalTrials.gov database link.

Bone marrow aspirate concentrate/platelet-rich fibrin augmentation accelerates healing of aseptic upper limb nonunions.

INTRODUCTION: Nonunions remain a significant burden in orthopedics, often afflicting young males of working age. Positive findings have been published using bone marrow aspirate concentrate (BMAC) and platelet-rich fibrin (PRF) for the treatment augmentation of lower limb nonunions. The aim of this study was to investigate if the treatment augmentation with BMAC and PRF can also accelerate the healing of nonunions of the upper limb. MATERIALS AND METHODS: Sixty-eight patients (45 men, 23 women) affected by 75 nonunions of long bones of the upper limb were treated and divided into two groups. The first series was treated with standard surgery alone (group A); afterwards, the second series benefited from standard surgery with the addition of BMAC and PRF applied on lyophilized bone chips. Nonunions were classified radiographically according to the Weber-Cech method and prognostically using the Calori and Moghaddam scores. All patients were radiographically assessed at 1.5, 3, 6, 12, and 24 months of follow-up. RESULTS: Baseline demographic characteristics did not present differences between groups. No differences were documented in terms of complications (two in group A and three in group B). Significant differences were instead documented in terms of healing time. The first healing signs were observed 1.5 months after surgery in 90.7% of patients in group B and 34.4% of group A (p < 0.0005). At 1.5, 3, 6, and 12 months, a higher radiographic score was found for group B (all p < 0.0005), while no difference was found at final follow-up of 24 months (90.6% of group A and 97.7% of group B achieved radiological healing). Faster healing with BMAC/PRF augmentation was confirmed for all bones, as well as for the subgroup of patients affected by atrophic nonunions (p = 0.001). CONCLUSION: This study showed the benefits of restoring both mechanical and biological aspects when addressing nonunions of the long bones of the upper limb. In particular, the association of BMAC and PRF to lyophilized bone chips was safe and able to accelerate healing time. These good results were confirmed for humerus, radius, and ulna sites, as well as for challenging atrophic nonunions of the upper limb.

Giant hemorrhagic trochanteric bursitis mimicking a high-grade soft tissue sarcoma: report of two cases.

BACKGROUND: The giant haemorrhagic bursitis of the hip joint is a rare clinical condition that requires evidence-based guidelines for adequate diagnosis and management. Usually, this pathology requires conservative treatment; however, when abnormal size or clinical symptoms of compression of the surrounding noble structures are reported, an accurate differential diagnosis is required, in order to exclude other malignant conditions that can be included into differential diagnosis, and a surgical approach should be considered. The purpose of this work is to provide an appropriate description of the diagnostic and therapeutic path, providing an accurate analysis of the possible differential diagnoses. METHODS: We report 2 cases of symptomatic haemorrhagic bursitis of the hip joint, confirmed by histological investigation. In both cases, the patients complained a peripheral nerve deficit of a single limb: one patient presented paresthesia of lateral femoral cutaneous nerve while the second peripheral edema due to compression of the proximal venous and lymphatic circulation. RESULTS: Both cases were successfully managed by complete surgical excision of the mass, with no  recurrence. There were no major complications, but in first case the nerve deficit was permanent. CONCLUSIONS: Giant hemorrhagic trochanteric bursitis is a rare condition, but it should be included in the differential diagnosis of soft tissue masses arising from the hip joint. Due to the rarity of this entity, a cautious exclusion process of all plausible differential diagnosis must be undertaken, in order to not miss the possibility of soft-tissue tumors, primarily malignant high-grade sarcomas.

Comparison of a fast track protocol and standard care after hip arthroplasty in the reduction of the length of stay and the early weight-bearing resumption: study protocol for a randomized controlled trial.

BACKGROUND: To date, hip arthroplasty is one of the most commonly performed surgical procedures, with growing worldwide demand. In recent decades, major progress made in terms of surgical technique, biomechanics, and tribology knowledge has contributed to improve the medical and functional management of the patient. This study aims to assess if the application of a fast track protocol, consisting of a preoperative educational intervention, adequate postoperative pain control, and intensive rehabilitation intervention, reduces the length of stay (LOS) and allows the early functional recovery compared to standard clinical practice for patients undergoing hip arthroplasty. METHODS: The study population consists of 90 patients with primary arthrosis of the hip with an anterior indication of hip arthroplasty. The exclusion criteria are older than 70 years, a contraindication to performing spinal anesthesia, and bone mass index (BMI) greater than 32. Participants, 45 for each group, are randomly allocated to one of two arms: fast track clinical pathway or standard care protocol. During allocation, baseline parameters such as Harris Hip Score (HHS) and Western Ontario and McMaster Universities (WOMAC) index are collected. On the third postoperative day, the functional autonomy for each patient is assessed by the Iowa Level of Assistance (ILOA) scale, and it is expected the discharge for patients in the fast track group (primary outcome). On the other hand, standard care patient discharge is expected after 5-7 days after surgery. During follow-up fixed at 6 weeks and 3, 6, and 12 months, HHS and WOMAC scores are collected for each patient (secondary outcomes). DISCUSSION: Although total hip replacement has become a widespread standardized procedure, to the authors' knowledge, only few randomized controlled trials were performed to evaluate the effectiveness of fast track pathway vs. standard care procedure in the reduction of the LOS after hip arthroplasty. It is expected that our results collected by the application of minimally invasive surgical interventions with concomitant management of perioperative pain and bleeding and early functional rehabilitation will contribute to enriching the understanding of clinical and organizational aspects linked to fast track arthroplasty. TRIAL REGISTRATION: ClinicalTrials.gov NCT03875976 . Registered on 15 March 2019-"retrospectively registered".

Core decompression with bone chips allograft in combination with fibrin platelet-rich plasma and concentrated autologous mesenchymal stromal cells, isolated from bone marrow: results for the treatment of avascular necrosis of the femoral head after 2 years minimum follow-up.

INTRODUCTION: Avascular necrosis of femoral head (AVN) is 1 of the main factors causing disability in young adults. Hip prosthesis can be considered an effective treatment of the painful symptoms but it is a major surgical intervention for this type of population. Thus, a large space should be left to therapeutic alternatives such as regenerative medicine.This retrospective study evaluates 52 AVN treated by core decompression, bone chips allograft, fibrin platelet-rich plasma (PRF) and concentrated autologous mesenchymal stromal cells (MSCs). METHODS: The AVN was diagnosed using magnetic resonance imaging (MRI) and graded according to ARCO classification: a patient was classified stage 1 (21 patients), stage 3 (26 patients), and 4 patients were classified as stage 4. We evaluated patients with functional scores (Harris Hip Score) and radiological analysis at 3, 6, 12 and 24 months after the procedure. Patients requiring prosthetic replacement of the joint were included; in these cases, follow-up was interrupted at the time of the joint replacement procedure. RESULTS: Our statistical analysis showed differences between survived and failed treatments, in terms of patient profile and ARCO radiological classification.The best result occurred in patients with ARCO grades 1 and 2, while the more advanced grades showed a high failure rate. It is interesting to note that ARCO quantification, conceived as the joint surface involved in the necrosis, has a negative influence on the outcome of the procedure. Indeed, patients affected by ARCO 3a, where necrosis involved a small portion of the femoral epiphysis and the collapse of the articular surface was limited to 2 mm, showed results similar to those obtained in patients with ARCO 1 and 2. CONCLUSIONS: In conclusion, compared with the alternative technique of decompression, our data suggest that post-collapse cases with a small area of necrosis and the use of bone grafts may show better results compared to those of the literature.

Loss of gut barrier integrity triggers activation of islet-reactive T cells and autoimmune diabetes.

Low-grade intestinal inflammation and alterations of gut barrier integrity are found in patients affected by extraintestinal autoimmune diseases such as type 1 diabetes (T1D), but a direct causal link between enteropathy and triggering of autoimmunity is yet to be established. Here, we found that onset of autoimmunity in preclinical models of T1D is associated with alterations of the mucus layer structure and loss of gut barrier integrity. Importantly, we showed that breakage of the gut barrier integrity in BDC2.5XNOD mice carrying a transgenic T cell receptor (TCR) specific for a beta cell autoantigen leads to activation of islet-reactive T cells within the gut mucosa and onset of T1D. The intestinal activation of islet-reactive T cells requires the presence of gut microbiota and is abolished when mice are depleted of endogenous commensal microbiota by antibiotic treatment. Our results indicate that loss of gut barrier continuity can lead to activation of islet-specific T cells within the intestinal mucosa and to autoimmune diabetes and provide a strong rationale to design innovative therapeutic interventions in "at-risk" individuals aimed at restoring gut barrier integrity to prevent T1D occurrence.

Comparison between absorbable pins and mini-screw fixations for the treatment of radial head fractures Mason type II-III.

BACKGROUND: The treatment of comminuted radial head fractures can include prosthetic replacement or open reduction and internal fixation. The purpose of this study is to evaluate the results of two different internal fixation systems for Mason type II-III radial head fractures. METHODS: Between 2005 and 2015, 82 patients were treated using pins and 65 patients by mini-screws. The follow-up protocol included: a clinical evaluation 15 days after surgery, and clinical and radiographic evaluations performed at 30 and 60 day intervals, unless any complications were reported by the patient. Over a period of at least 12-months of follow-up, patients were checked and interviewed. Clinical examinations included elbow range of motion (ROM), arm, shoulder and hand Disabilities, (DASH), and the Mayo Elbow Performance Score (MEPS). RESULTS: Sixty-one subjects who had been treated with mini-screws were clinically reviewed at a mean 47.3 ± 35.8 month of follow-up; all patients who had been treated using absorbable pins were evaluated at a mean 82.5 ± 20.6 month of follow-up. No significant statistically differences were observed between the two groups in the mean ROM, DASH, and MEPS scores. Residual pain was reported in 15.8%of the patients treated by pins and 9.2% patients treated by mini-screws. Secondary displacement of fracture fragments was observed in 8.5% patients treated by pins and 1.6% using mini-screws. CONCLUSIONS: Both absorbable pins and mini-screws provided adequate strength and rigidity, allowing good clinical and functional scores at a mid-term follow-up. However, a higher rate of secondary displacement of the fracture fragments was reported among subjects who had been treated using absorbable pins.

NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease.

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and ß genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.

Infiltrative therapy as conservative treatment in hip osteoarthritis: a literature review.

Osteoarthritis (OA) is a very common disease, its prevalence increases with age and is a frequent cause of disability.Osteoarthritis is characterised by joint pain, stiffness and loss of range of motion. Overall, as many as 40% of those aged over 65 years in the community may have symptomatic OA of the knee or hip (1). OA results from a complex interaction of biomechanical and biochemical factors and is characterised by cartilage disruption and hypertrophy of bone. Intraarticular proinflammatory cytokines and proteinases in OA interfere with the synthesis of hyaluronic acid (HA), a complex glycosaminoglycan composed of repeated disaccharide units to form a linear polymer, resulting in an HA with a significantly reduced molecular weight and a reduction in synovial fluid viscoelasticity (2-3). Loss of normal characteristics of HA leads to the degradation of the articular cartilage and the disruption of the mechanical and homeostasis of the joint.Several pharmaceutical approaches, such as analgesics, non steroidal antiinflammatory drugs, COX-2 inhibitors and steroids, have been proposed (4), with the aim of reducing pain and maintaining and/or improving joint function. However, none of these options has shown to delay the progression of osteoarthritis or reverse joint damage.Infiltrative hip therapy involves injecting into the joint the drugs or medicinal substances that are used primarily to control the symptoms of the disease, such as pain and functional limitation.The aim of this review is to analyse existing infiltrative alternatives for hip osteoarthritis, and describe our experience.

Targeting Macrophages Sensitizes Chronic Lymphocytic Leukemia to Apoptosis and Inhibits Disease Progression.

The role of monocytes/macrophages in the development and progression of chronic lymphocytic leukemia (CLL) is poorly understood. Transcriptomic analyses show that monocytes/macrophages and leukemic cells cross talk during CLL progression. Macrophage depletion impairs CLL engraftment, drastically reduces leukemic growth, and favorably impacts mouse survival. Targeting of macrophages by either CSF1R signaling blockade or clodrolip-mediated cell killing has marked inhibitory effects on established leukemia also. Macrophage killing induces leukemic cell death mainly via the TNF pathway and reprograms the tumor microenvironment toward an antitumoral phenotype. CSF1R inhibition reduces leukemic cell load, especially in the bone marrow, and increases circulating CD20(+) leukemic cells. Accordingly, co-targeting TAMs and CD20-expressing leukemic cells provides a survival benefit in the mice. These results establish the important role of macrophages in CLL and suggest therapeutic strategies based on interfering with leukemia-macrophage interactions.

SIGLEC-G deficiency increases susceptibility to develop B-cell lymphoproliferative disorders.

The sialic-acid-binding immunoglobulin-like lectin SIGLEC-G is a negative regulator of B-cell receptor-mediated calcium signaling. Its deficiency leads to reduced turnover and increased proliferation and survival of murine B-1a cells. Siglecg(-/-) mice show a premature expansion of polyclonal CD5(+) B cells in the spleen and the peritoneal cavity. Here we studied the fate of B lymphocytes in Siglecg(-/-) mice over time. We demonstrate that in aging animals SIGLEC-G deficiency promotes progressive accumulation of monoclonal B lymphocytes and increases the susceptibility to develop B-cell lymphoproliferative disorders. Lymphoid tumors arising in aged Siglecg(-/-) mice are monoclonal and histologically heterogeneous as they include diffuse large B-cell lymphoma, follicular lymphoma, and medium-to-large B-cell monomorphic lymphoma but surprisingly not chronic lymphocytic leukemia. The tumors express high levels of BCL-2 and are transplantable. In keeping with these findings we have also observed a remarkable down-regulation of the human ortholog SIGLEC10 in human B-cell lymphoma and leukemia cell lines. Taken together, these observations indicate that the down-regulation of negative B-cell receptor regulators such as SIGLEC-G/SIGLEC10 may represent another mechanism relevant to the pathogenesis of B-cell lymphomas.

Vaccine-instructed intratumoral IFN-γ enables regression of autochthonous mouse prostate cancer in allogeneic T-cell transplantation.

Vaccination can synergize with transplantation of allogeneic hematopoietic stem cells to cure hematologic malignancies, but the basis for this synergy is not understood to the degree where such approaches could be effective for treating solid tumors. We investigated this issue in a transgenic mouse model of prostate cancer treated by transplantation of a nonmyeloablative MHC-matched, single Y chromosome-encoded, or multiple minor histocompatibility antigen-mismatched hematopoietic cell preparation. Here, we report that tumor-directed vaccination after allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion is essential for acute graft versus tumor responses, tumor regression, and prolonged survival. Vaccination proved essential for generation of CD8(+) IFN-γ(+) tumor-directed effector cells in secondary lymphoid organs and also for IFN-γ(+) upregulation at the tumor site, which in turn instructed local expression of proinflammatory chemokines and intratumoral recruitment of donor-derived T cells for disease regression. Omitting vaccination, transplanting IFN-γ-deficient donor T cells, or depleting alloreactive T cells all compromised intratumoral IFN-γ-driven inflammation and lymphocyte infiltration, abolishing antitumor responses and therapeutic efficacy of the combined approach. Our findings argue that posttransplant tumor-directed vaccination is critical to effectively direct donor T cells to the tumor site in cooperation with allogeneic hematopoietic cell transplantation.

Lack of TIR8/SIGIRR triggers progression of chronic lymphocytic leukemia in mouse models.

Inflammation is involved in the initiation and progression of several chronic lymphoid malignancies of B-cell type. Toll-like receptors (TLR) are transmembrane inflammatory receptors that on recognition of pathogen-associated molecular patterns trigger an innate immune response and bridge the innate and adaptive immune response by acting as costimulatory signals for B cells. Fine tuning of TLR and IL-1R-like (ILR) activity is regulated by TIR8 (SIGIRR), a transmembrane receptor of the TLR/ILR family which inhibits other family members. To test the hypothesis that TLR and/or ILR may play a role in the natural history of chronic B-cell tumors, we crossed Eµ-TCL1 transgenic mice, a well established model of chronic lymphocytic leukemia (CLL), with mice lacking the inhibitory receptor TIR8 that allow an unabated TLR-mediated stimulation. We here report that in the absence of TIR8 the appearance of monoclonal B-cell expansions is accelerated and mouse life span is shortened. The morphology and phenotype of the mouse leukemic expansions reproduce the progression of human CLL into an aggressive and frequently terminal phase characterized by the appearance of prolymphocytes. This study reveals an important pathogenetic implication of TLR in CLL development and progression.

HS1 has a central role in the trafficking and homing of leukemic B cells.

The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1(-/-) mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2(-/-)γ(c)(-/-) mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eµ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eµ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.

A novel Rag2-/-gammac-/--xenograft model of human CLL.

Easily reproducible animal models that allow for study of the biology of chronic lymphocytic leukemia (CLL) and to test new therapeutic agents have been very difficult to establish. We have developed a novel transplantable xenograft murine model of CLL by engrafting the CLL cell line MEC1 into Rag2(-/-)gamma(c)(-/-) mice. These mice lack B, T, and natural killer (NK) cells, and, in contrast to nude mice that retain NK cells, appear to be optimal recipient for MEC1 cells, which were successfully transplanted through either subcutaneous or intravenous routes. The result is a novel in vivo model that has systemic involvement, develops very rapidly, allows the measurement of tumor burden, and has 100% engraftment efficiency. This model closely resembles aggressive human CLL and could be very useful for evaluating both the biologic basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents.