A distinct human cell type expressing MHCII and RORγt with dual characteristics of dendritic cells and type 3 innate lymphoid cells.

Recent studies have characterized various mouse antigen-presenting cells (APCs) expressing the lymphoid-lineage transcription factor RORγt (Retinoid-related orphan receptor gamma t), which exhibit distinct phenotypic features and are implicated in the induction of peripheral regulatory T cells (Tregs) and immune tolerance to microbiota and self-antigens. These APCs encompass Janus cells and Thetis cell subsets, some of which express the AutoImmune REgulator (AIRE). RORγt+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota-specific Tregs. While RORγt+ APCs have been actively investigated in mice, the identity and function of these cell subsets in humans remain elusive. Herein, we identify a rare subset of RORγt+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term RORγt+ DC-like cells (R-DC-like), exhibit DC morphology, express the MHC class II machinery, and are distinct from all previously reported DC and ILC3 subsets, but share transcriptional and epigenetic similarities with DC2 and ILC3. We have developed procedures to isolate and expand them in vitro, enabling their functional characterization. R-DC-like cells proliferate in vitro, continue to express RORγt, and differentiate into CD1c+ DC2-like cells. They stimulate the proliferation of allogeneic T cells. The identification of human R-DC-like cells with proliferative potential and plasticity toward CD1c+ DC2-like cells will prompt further investigation into their impact on immune homeostasis, inflammation, and autoimmunity.

Immunoglobulin light chain transcript detection by ultrasensitive RNA in situ hybridization for B-cell lymphoma diagnosis.

Evaluation of B-cell clonality can be challenging in the interpretation of lymphoid infiltrates on tissue sections. Clonality testing based on IG gene rearrangements analysis by PCR (IG-PCR) is the gold standard. Alternatively, B-cell clonality can be assessed by the recognition of immunoglobulin light chain (IgLC) restriction, by immunohistochemistry (IHC), chromogenic in situ hybridization (ISH) or flow cytometry (FC). IG-PCR requires molecular facilities, and FC requires cell suspensions, both not widely available in routine pathology units. This study evaluates the performance of B-cell clonality detection by IgLC-RNAscope® (RNAsc) in a group of 216 formalin-fixed, paraffin-embedded samples including 185 non-Hodgkin B-cell lymphomas, 11 Hodgkin lymphomas (HL) and 20 reactive samples. IgLC-RNAsc, performed in parallel with FC in 53 cases, demonstrated better performances (93% vs 83%), particularly in diffuse large B-cell lymphoma (98% vs 71%) and follicular lymphoma (93% vs 83%) diagnosis. IgLC-RNAsc was also superior to IHC and ISH especially in samples with limited tumor cell content, where IG-PCR was not informative. Performed for the first time on mediastinal lymphomas, IgLC-RNAsc identified monotypic IgLC transcripts in 69% of primary mediastinal large B-cell lymphoma (PMBCL) and 67% of mediastinal gray zone lymphomas (MGZL). IGK/L double-negative cells were detected in 1 PMBCL, 2 MGZL, and all classical HL, while monotypic IgLC expression appeared to be a hallmark in nodular lymphocyte-predominant HL. IgLC-RNAsc demonstrates to be a powerful tool in B-cell lymphoma diagnosis, above all in challenging cases with limited tumor cell content, ensuring in situ investigations on mechanisms of Ig regulation across lymphoma entities.

Impaired activation of plasmacytoid dendritic cells via toll-like receptor 7/9 and STING is mediated by melanoma-derived immunosuppressive cytokines and metabolic drift.

Introduction: Plasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production. Methods: The activation of tumor-associated pDCs was evaluated by in silico and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by in vitro stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer. Results: Based on a set of microscopic, functional and in silico analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs via TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape. Discussion: These findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM).

Electromagnetic distal targeting system does not reduce the overall operative time of the intramedullary nailing for humeral shaft fractures.

INTRODUCTION: We aimed to evaluate the efficacy of the use of the electromagnetic distal targeting system in treating humeral shaft fracture. METHODS: Patients were divided in: Group 1) patients that received a distal locking screw placement following the free-hand technique; Group 2) patients in which the distal locking screw was performed using the SURESHOT device. RESULTS: No differences were noted comparing Group 1 (freehand) [71,9 range 40-135 min] to Group 2 (SURESHOT)[70, range 25-125 min]. CONCLUSION: The use of the EM distal targeting system doesn't reduce the overall operative time of the humeral shaft fracture fixation using IMN.

Revision Anterior Cruciate Ligament Reconstruction with Contralateral Hamstring Tendon Grafts: 6 Years Follow-Up.

Purpose The purpose of this study was to retrospectively evaluate the clinical outcome of revision anterior cruciate ligament (ACL) reconstruction with contralateral hamstring tendon autografts, specifically with regard to patient satisfaction, return to preinjury activity level, and postoperative functional outcomes. Methods Between 2004 and 2011, 23 patients underwent revision ACL reconstruction with contralateral autogenous hamstring tendon grafts and were retrospectively reviewed at an average follow-up of 6.3 years. Subjective and functional evaluations were performed. The Tegner score, Knee Injury and Osteoarthritis Outcome Score (KOOS), and International Knee Documentation Committee (IKDC) Subjective Knee Form were used. Objective evaluation included range of motion, Lachman test, pivot-shift test, and KT-1000 instrumented laxity testing. Wilcoxon test was used to compare the preoperative and follow-up status. Differences with a p -value of <0.05 were considered statistically significant. Results No major complications were reported. The mean KOOS significantly increased from a preoperative mean of 62.8 ± 8.3 to 85.8 ± 6.9 ( p < 0.001). IKDC subjective score significantly improved from 29.2 ± 10.4 to 72.8 ± 5.2 ( p < 0.001). The median Tegner activity score significantly improved from a preoperative mean of 6.5 (range: 4-10) to 7.5 (range: 7-10) ( p < 0.001). Most of the patients increased or returned to the same activity level, with 61% of the patients returning to cutting and pivoting sports. Conclusion The use of contralateral hamstring tendon autografts for ACL revision surgery represents a valid option following a failed primary ACL reconstruction and confirms subjective and objective clinical improvement 6 years after surgery. Level of Evidence Level IV, therapeutic case series.

Revision anterior cruciate ligament reconstruction with ipsi- or contralateral hamstring tendon grafts.

OBJECTIVES: With the increasing number of primary anterior cruciate ligament (ACL) reconstructions, the need for revision ACL surgery has risen over the past few years. The purpose of the present study is to retrospectively compare the clinical outcome of ipsilateral versus contralateral hamstring tendon autografts for ACL revision surgery, specifically with regard to patient satisfaction, post-operative functional outcomes, and return to sports. METHODS: Between 2004 and 2011, 64 patients underwent ACL revision surgery. Forty-five were successfully recontacted and retrospectively reviewed at an average follow-up of 6.3 years. Twenty-two subjects underwent revision ACL reconstruction with ipsilateral autogenous hamstring tendon grafts; in 23 subjects contralateral hamstring were used for reconstruction. Clinical, arthrometric, and functional evaluations were performed. The Tegner activity level, Knee Injury and Osteoarthritis Outcome Score (KOOS), and International Knee Documentation Committee (IKDC) Subjective Knee Form were used. Objective evaluation included range of motion, Lachman test, pivot shift test and KT-1000 instrumented laxity testing. RESULTS: No major complications were reported. Follow-up examination showed that there were no significant differences in the IKDC and KOOS scores between the groups. No differences in anterior tibial translation as measured with KT-1000 arthrometer were reported between the groups, although there was a trend for more of the patients undergoing ipsilateral DGST reconstruction to have a glide on the pivot shift test. The percentage of patients returning to pre-injury level was high in both groups. CONCLUSIONS: The use of contralateral hamstring tendon autografts for ACL revision surgery produced similar subjective and objective outcomes at 6-years follow-up compared to revision with ipsilateral hamstring tendon autografts. Patients undergoing revision surgery with contralateral autografts experienced a quicker return to sports compared to patients who underwent ipsilateral DGST revision surgery.

Can graft choice affect return to sport following revision anterior cruciate ligament reconstruction surgery?

INTRODUCTION: With the increasing number of primary anterior cruciate ligament (ACL) reconstructions, revision surgery has become more frequent. The purpose of the present study is to retrospectively compare the clinical outcome of contralateral hamstring tendon autografts vs. allografts for ACL revision surgery, specifically with regard to patient satisfaction, return to preinjury activity level, and postoperative functional outcomes. MATERIALS AND METHODS: Between 2004 and 2011, 59 patients underwent ACL revision surgery. 44 were successfully recontacted and retrospectively reviewed at an average follow-up of 5.2 years. 23 subjects underwent revision ACL reconstruction with contralateral autogenous hamstring tendon grafts; 21 underwent allograft revision ACL surgery. Clinical, arthrometric, and functional evaluations were performed. The Tegner, Knee Injury and Osteoarthritis Outcome Score (KOOS), International Knee Documentation Committee (IKDC) Subjective Knee Form were used. Objective evaluation included range of motion, Lachman test, pivot-shift test and KT-1000 instrumented laxity testing. RESULTS: No major complications were reported. Follow-up examination showed that there were no significant differences IKDC and KOOS scores between the groups. The percentage of patients returning to pre-injury level was high in both groups. Anterior tibial translation according to manual laxity testing and as measured with KT-1000 arthrometer was not statistically different in the two groups. With regard to return to sports, patients undergoing revision surgery with autografts experienced a quicker return to sports compared to patients who underwent allograft revision surgery. CONCLUSIONS: The use of contralateral hamstring tendon autografts for ACL revision surgery produced similar subjective and objective outcomes at 5.2 years follow-up compared to revision with allograft patellar or Achilles tendon. Patients undergoing revision surgery with autografts experienced a quicker return to sports compared to patients who underwent allograft revision surgery.