Autologous Leukocytes Research Articles

Calcifying epithelial odontogenic tumour with an atypical clinical presentation in a paediatric patient: A case report and literature review

AbstractAimWe report a rare case of a calcifying epithelial odontogenic tumour with atypical clinical presentation in an adolescent and present a literature review of this tumour in paediatric patients.Materials and MethodsA 12‐year‐old female patient presented with a painless swelling of the left mandible, with a history of 6 months. Clinical examination revealed a sessile mass with a reddish‐pink colour and firm consistency. Tomographic images showed a hypodense, unilocular lesion involving unerupted tooth #34, with a hyperdense focus in contact with the tooth crown disrupting the buccal cortex.ResultsEnucleation, curettage and peripheral osteotomy of the lesion were performed, and the impacted tooth was removed, followed by the application of autologous leucocyte and platelet‐rich fibrin matrices to the region. The patient evolved favourably with good clinical healing and evidence of new bone formation.ConclusionsDespite its aggressive potential, the conservative approach for CEOT in paediatric patients proved to be effective. Our review found reports of recurrence up to 13 years after surgery. Therefore, long‐term clinical and radiographical follow‐up is indicated.

Effectiveness of autologous leucocyte and platelet-rich fibrin (L-PRF) in management of chronic diabetic foot ulcers.

Effectiveness of autologous leucocyte and platelet-rich fibrin (L-PRF) in management of chronic diabetic foot ulcers.

Post-transplant lymphoproliferative disorders after allogeneic hematopoietic stem cell transplantation: a case report, meta-analysis, and systematic review

BackgroundPost-transplant lymphoproliferative disorders (PTLD) are rare but severe complications that occur after solid organ or allogeneic hematopoietic stem cell transplantations (allo-HSCT), with rapid progression and high mortality. Primary central nervous system (CNS)-PTLD are rarely recognized histo-pathologically. In addition, the diagnostic value of the Epstein–Barr virus (EBV) DNA copies in CNS-PTLD remains poorly understood.ObjectivesWe herein report a case of monomorphic EBV-associated CNS-PTLD (diffuse large B-cell lymphoma, DLBCL) after allo-HSCT and perform a meta-analysis to assess the efficacy of PTLD treatment strategies in recent years.MethodsWe present the case report covering clinical manifestations, diagnosis, treatment, and outcomes of a patient with primary CNS-PTLD. Additionally, we include a systematic review and meta-analysis of the clinical characteristics of 431 patients with PTLD after allo-HSCT. We evaluate the main treatment options and outcomes of PTLD management, including rituximab, chemotherapies, and autologous or human leukocyte antigen (HLA)-matched EBV-specific cytotoxic T lymphocyte infusion (EBV-CTLs)/donor lymphocyte infusion (DLI).ResultsThe meta-analysis revealed an overall response rate of 69.0% for rituximab alone (95% CI: 0.47–0.84), 45.0% for rituximab plus chemotherapies (95% CI: 0.15–0.80), and 91.0% for rituximab plus EBV-CTLs/DLI (95% CI: 0.83–0.96). The complete response (CR) rate after treatments for PTLD was 67.0% (95% CI: 0.56–0.77). Moreover, the 6-month and 1-year overall survival (OS) rate was 64.0% (95% CI: 0.31–0.87) and 49.0% (95% CI: 0.31–0.68), respectively.ConclusionsThis case highlighted the urgent need for effective, low-toxic treatment regimens for CNS-PTLD. Our meta-analysis suggested that rituximab combined with EBV-CTLs/DLI could be a favorable strategy for the management of PTLD after allo-HSCT.

Development and evaluation of a radiolabelling agent for white blood cell scans

Radiolabelled autologous leukocytes have been used for the clinical diagnosis of inflammation and infection. To develop a stable and efficient radiopharmaceutical for labelling leukocytes, we prepared a novel radioiodinated cell-penetrating peptide, 125I-TAT, using a bi-functional linker. 125I-TAT was stable for two days under three different temperature conditions of −20 °C, 4 °C, and 40 °C, with its radiochemical purity remaining over 99%. Iodinated TAT was non-toxic to leukocytes with an IC50 value of over 100 μM. The labelling efficiency of 125I-TAT using 1ｘ107 cells ranged from 27% to 53% when the three leukocyte cell lines were pre-treated with DMSO. This is comparable to the labelling efficiency recommended by the guideline for conventional labelling agents using 2ｘ108 cells. Radioiodinated cell-penetrating peptide may be an improved radiopharmaceutical for white blood cell scans by further optimization.

Pathomorphological Features of The Cornea in Secondary Posttraumatic Dystrophy (Clinical Case)

Secondary corneal dystrophy is a group of diseases characterized by an inflammatory–dystrophic process in the cornea of the eye. This pathology is accompanied by a pronounced pain syndrome and leads to an irreversible decrease in vision, up to complete blindness. To date, many aspects of pathomorphological changes of the cornea in secondary dystrophies remain insufficiently studied due to the impossibility of conducting an intravital study of the cornea tissue. The article presents a case of morphological analysis of a tissue fragment of a pathologically altered cornea in secondary posttraumatic corneal dystrophy. Destructive changes in corneal tissue revealed during light microscopy indicate the presence of signs of fibrinoid swelling in the shell stroma with gross disorganization of the base substance and neovascularization. The study made it possible to correlate the data of pathomorphological changes and the clinical picture, as well as to evaluate the effectiveness of the chosen method of treatment of this pathology (intrastromal injection of a suspension of autologous mononuclear leukocytes of blood into the stroma of the damaged cornea).

Immunomodulatory effects of a cell processing device to ameliorate dysregulated hyperinflammatory disease states

Cell directed therapy is an evolving therapeutic approach to treat organ dysfunction arising from hyperinflammation and cytokine storm by processing immune cells in an extracorporeal circuit. To investigate the mechanism of action of the Selective Cytopheretic Device (SCD), in vitro blood circuits were utilized to interrogate several aspects of the immunomodulatory therapy. SCD immunomodulatory activity is due to its effects on circulating neutrophils and monocytes in a low ionized calcium (iCa, Ca2+) blood circuit. Activated neutrophils adhere to the SCD fibers and degranulate with release of the constituents of their exocytotic vesicles. Adhered neutrophils in the low iCa environment display characteristics of apoptotic senescence. These neutrophils are subsequently released and returned back to circulation, demonstrating a clear potential for in vivo feedback. For monocytes, SCD treatment results in the selective adhesion of more pro-inflammatory subsets of the circulating monocyte pool, as demonstrated by both cell surface markers and cytokine secretory rates. Once bound, over time a subset of monocytes are released from the membrane with a less inflammatory functional phenotype. Similar methods to interrogate mechanism in vitro have been used to preliminarily confirm comparable findings in vivo. Therefore, the progressive amelioration of circulating leukocyte activation and immunomodulation of excessive inflammation observed in SCD clinical trials to date is likely due to this continuous autologous leukocyte processing.

A randomised Trial of Autologous Blood products, leukocyte and platelet-rich fibrin (L-PRF), to promote ulcer healing in LEprosy: The TABLE trial.

Autologous blood products like Platelet Rich Plasma (PRP) and Leukocyte and Platelets Rich Fibrin (L-PRF) have been used for many years across many types of skin ulcers. However, the effectiveness of autologous blood products on wound healing is not well established. We evaluated the 'second generation' autologous product- Leukocyte and Platelet- Rich Fibrin (L-PRF). Our trial was undertaken on patients suffering from neuropathic leprosy ulcers at the Anandaban hospital which serves the entire country of Nepal. We conducted a 1:1 (n = 130) individually randomised trial of L-PRF (intervention) vs. normal saline dressing (control) to compare rate of healing and time to complete healing. Rate of healing was estimated using blind assessments of ulcer areas based on three different measurement methods. Time to complete healing was measured by the local unblinded clinicians and by blind assessment of ulcer images. The point estimates for both outcomes were favourable to L-PRF but the effect sizes were small. Unadjusted mean differences (intervention vs control) in mean daily healing rates (cm2) were respectively 0.012 (95% confidence interval 0.001 to 0.023, p = 0.027); 0.016 (0.004 to 0.027, p = 0.008) and 0.005 (-0.005 to 0.016, p = 0.313) across the three measurement methods. Time to complete healing at 42 days yielded Hazard Ratios (unadjusted) of 1.3 (0.8 to 2.1, p = 0.300) assessed by unblinded local clinicians and 1.2 (0.7 to 2.0, p = 0.462) on blind assessment. Any benefit from L-PRF appears insufficient to justify routine use in care of neuropathic ulcers in leprosy. ISRCTN14933421. Date of trial registration: 16 June 2020.

Autologous leucocyte and platelet rich in fibrin (L-PRF) - is it a competitive solution for bone augmentation in maxillary sinus lift? A 6-month radiological comparison between xenografts and L-PRF.

Much of the research published involves using artificial products, like xenografts and resorbable collagen membranes, after a prior Cone Beam Computer Tomography (CBCT) investigation. Nowadays, more accessible access, less financial costs, a biological approach, and faster healing are objectives that surround this procedure. Leucocytes and platelets rich in Fibrin (L-PRF) are a natural component with a high concentration of growth factors. Due to its regenerative properties and lack of complications, it is used in several medical fields, like orthopedics, dermatology, and oral surgery. This retrospective study aims to compare results in bone height and volume obtained through external sinus lift, either by using xenografts or autologous plasma rich in fibrin, by evaluating the quantity of new bone formation from a radiological point of view. Fifty-eight Caucasian patients were included in this retrospective study; 48 were submitted to xenograft procedure, and 10 were selected for L-PRF grafting material with simultaneous implant placement. Lack of clinical and histological studies performed on patients with L-PRF surgeries limited us in choosing a larger group for the radiological analysis. CBCT evaluation was performed before surgery and 6 months after. All patients selected for the study presented good general and oral health, acute oral and sinus infections excluded; smoking and periodontal disease were also criteria of exclusion. Two operators performed the measurements in pre-established landmarks in different time frames. The two independent groups were compared with the Wilcoxon rank-sum test for quantitative data. Qualitative characteristics were described as counts and percentages. All analyses were performed in an R environment for statistical computing and graphics. Mean bone height gain in the xenograft group in the regions was as follows: 7.44 for the anterior landmark, 12.14 for the median and 8.28 for the distal. The mean group height gained for the L-PRF group was 0.1 anteriorly, -0.18 for the median measurement, and 0.23 distally. We obtained excellent overall reliability for all the height measurements between the two operators. Further studies must be conducted to establish new sets of surgical protocols in case L-PRF alone is found to be a reliable, stable, biological alternative to the well-documented xenografts in external sinus lifts. Radiological results, although promising, must be further applied in long term clinical survival of the implants in the grafted sites. Also, studies combining L-PRF in conjunction with xenograft might bring improved clinical results in terms of reduced postoperative complications and accelerated healing.

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations.

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopre-served PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples.

Updated Follow-up of a Phase 1 Trial of Bispecific CART19/20 Cells for Relapsed or Refractory Non-Hodgkin Lymphoma

CART19/20 was developed by engineering autologous naïve/memory T (T N/MEM) cells with a bispecific anti-CD19/CD20 chimeric antigen receptor (CAR) with the goal of mitigating antigen escape and lack of CAR T-cell persistence. We report an update of an ongoing first-in-human phase 1 clinical trial of CART19/20 for patients with relapsed/refractory non-Hodgkin lymphoma (R/R NHL) (NCT04007029). Eligible patients were ≥18 years old with R/R diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B-cell lymphoma (PMBCL) after ≥2 prior lines of therapy, or with mantle-cell lymphoma (MCL), follicular lymphoma (FL), or CLL/SLL after ≥3 prior lines of therapy. Autologous leukocytes were obtained by leukapheresis and sorted for CD14-/CD25-/CD62L+ T N/MEM cells, followed by lentiviral transduction of the bispecific CD19/CD20 CAR. Lymphodepletion chemotherapy with fludarabine 30 mg/m 2/day and cyclophosphamide 500 mg/m 2/day was administered on Days −5 to −3 prior to CAR T-cell infusion. The primary endpoint was assessment of safety. Secondary endpoints were overall response rate (ORR), progression free survival (PFS), overall survival (OS), and CART19/20 transgene persistence. As of data cutoff on July 21, 2023, 11 patients had received CART19/20 cells (6 DLBCL, including 4 transformed FL; 3 primary FL; 1 PMBCL; 1 MCL). Patients were treated at two dose levels, either 50 x 10 6 ± 30% CAR+ cells (8 patients) or 200 x 10 6 ± 30% CAR+ cells (3 patients). The median age was 58 (range: 34 to 70). Patients had received a median of 3 prior lines of therapy (range: 2 - 4). None of the treated patients had received prior CAR-T cell therapy; one patient had received a prior CD19-directed therapy. All patients had stage IV disease and 9 of 11 patients received bridging therapy. The engineered CART19/20 cells were enriched with central-memory T cells (T CM: CD45RA-/CD45RO+/CD62L+) which composed a median of 30.8% (range: 5.3 - 80.1%) of the CAR+ T-cells in the infused products. There were no occurrences of CRS grade ≥ 2 or neurotoxicity of any grade. Six patients had grade 1 cytokine release syndrome (CRS) with a median onset of 8 days from infusion (range: 5 - 11 days). One patient met criteria for dose-limiting toxicity with persistent thrombocytopenia after Day+120. Ten of the 11 patients achieved an objective response (91% ORR), with 8 patients (73%) achieving complete response (CR). One patient with FL, who progressed 18 months after receiving CART19/20, received a second infusion of CART19/20 cells and achieved a second CR for 14.9 months. With a median follow-up of 32.3 months (range: 5.1 - 44.0 months), the median PFS (95% CI 2.6 months - not estimable) and median OS (95% CI 5.7 months - not estimable) have not been reached. Overall, CART19/20 demonstrated a favorable safety profile with no severe CRS or neurotoxicity of any grade. Moreover, CART19/20 showed robust activity in patients with R/R NHL with durable responses seen. By utilizing a bispecific CAR and enriching for T N/MEM cells, this phase 1 trial of CART19/20 demonstrates the possibility of improving outcomes through targeting common mechanisms of CAR-T cell resistance.

Retrospective Data Analysis of the Use of an Autologous Multilayered Leukocyte, Platelet, and Fibrin Patch for Diabetic Foot Ulcers Treatment in Daily Clinical Practice.

To describe the healing outcome of chronic, hard-to-heal diabetic foot ulcers (DFUs) treated with an autologous multilayered leukocyte, platelet, and fibrin (MLPF) patch in addition to the best standard of care, in a real-world clinical setting of two US amputation preventive centers. In this retrospective study of patients treated between September 2021 and October 2022, the authors analyzed DFU healing outcomes based on Wound, Ischemia, and foot Infection-derived amputation risk. All 36 patients had a diagnosis of type 2 diabetes and 29 (81%) were male. Their average age was 61.4 years, body mass index was 29.2 kg/m2, and glycated hemoglobin was 7.9. Twenty-seven patients (78%) were diagnosed with peripheral vascular disease, 20 (56%) underwent a peripheral vascular procedure, 15 (42%) had a prior amputation, and 6 (17%) were on hemodialysis. Average wound size was 4.9 cm2, and wound age was 9.5 months. Twelve patients (32%) were classified as low risk, 15 (39%) as moderate risk, and 11 (29%) as high risk for amputation. Within 12 weeks of the first MLPF patch application, nine wounds (24%) healed. After 20 weeks, 23 wounds (61%) were closed, and by follow-up, 30 wounds (79%) healed. No amputations were noted. Compared with published data, 40% fewer patients underwent readmission within 30 days, with 72% shorter admission duration. Real-world clinical experiences using the MLPF patch to treat hard-to-heal DFUs resulted in the majority of wounds healing. Few patients experienced a readmission within 30 days, and the average admission duration was short.

Leukocyte- and platelet-rich fibrin in endoscopic endonasal skull base reconstruction: study protocol for a multicenter prospective, parallel-group, single-blinded randomized controlled non-inferiority trial

BackgroundRecent advances in endoscopic endonasal transsphenoidal approaches (EETA) for skull base lesions have resulted in a significant increase in extent and complexity of skull base defects, demanding more elaborate and novel reconstruction techniques to prevent cerebrospinal fluid (CSF) leakage and to improve healing. Currently, commercially available fibrin sealants are often used to reinforce the skull base reconstruction. However, problems have been reported regarding hypersensitivity reactions, efficacy, and costs. This trial aims to investigate autologous leukocyte- and platelet-rich fibrin (L-PRF) membranes as an alternative for commercially available fibrin glues in EETA-related skull base reconstruction reinforcement.Methods/designThis multicenter, prospective randomized controlled trial aims to demonstrate non-inferiority of L-PRF membranes compared to commercially available fibrin sealants in EETA cases (1) without intra-operative CSF-leak as dural or sellar floor closure reinforcement and (2) in EETA cases with intra-operative CSF-leak (or very large defects) in which a classic multilayer reconstruction has been made, as an additional sealing. The trial includes patients undergoing EETA in three different centers in Belgium. Patients are randomized in a 1:1 fashion comparing L-PRF with commercially available fibrin sealants. The primary endpoint is postoperative CSF leakage. Secondary endpoints are identification of risk factors for reconstruction failure, assessment of rhinological symptoms, and interference with postoperative imaging. Additionally, a cost-effectiveness analysis is performed.DiscussionWith this trial, we will evaluate the safety and efficacy of L-PRF compared to commercially available fibrin sealants.Trial registrationClinicalTrials.gov NCT03910374. Registered on 10 April 2019.

An autologous blood-derived patch as a hemostatic agent: evidence from thromboelastography experiments and a porcine liver punch biopsy model

Perioperative bleeding is a common complication in surgeries that increases morbidity, risk of mortality, and leads to increased socioeconomic costs. In this study we investigated a blood-derived autologous combined leukocyte, platelet, and fibrin patch as a new means of activating coagulation and maintaining hemostasis in a surgical setting. We evaluated the effects of an extract derived from the patch on the clotting of human blood in vitro, using thromboelastography (TEG). The autologous blood-derived patch activated hemostasis, seen as a reduced mean activation time compared to both non-activated controls, kaolin-activated samples, and fibrinogen/thrombin-patch-activated samples. The accelerated clotting was reproducible and did not compromise the quality or stability of the resulting blood clot. We also evaluated the patch in vivo in a porcine liver punch biopsy model. In this surgical model we saw 100% effective hemostasis and a significant reduction of the time-to-hemostasis, when compared to controls. These results were comparable to the hemostatic properties of a commercially available, xenogeneic fibrinogen/thrombin patch. Our findings suggest clinical potential for the autologous blood-derived patch as a hemostatic agent.Graphical

Abstract CT023: Phase 1 trial of CD19/CD20 bispecific chimeric antigen receptor-engineered naïve/memory T cells for relapsed or refractory non-Hodgkin lymphoma

Abstract Despite excellent responses to anti-CD19 chimeric antigen receptor (CAR)-T cell therapy in patients with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL), more than half of patients will relapse due to poor CAR-T persistence or CD19 antigen escape. Engineering naïve/memory T (TN/MEM) cells with a bispecific anti-CD19/CD20 CAR could improve outcomes by mitigating these limitations. Here we report an update of the first-in-human phase 1 clinical trial with TN/MEM cells expressing a bispecific anti-CD19/CD20 CAR (CART19/20) for patients with R/R NHL (NCT04007029). Eligible patients were ≥18 years old with R/R diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B-cell lymphoma (PMBCL) after ≥2 prior lines of therapy, or with mantle-cell lymphoma (MCL), follicular lymphoma (FL), or CLL/SLL after ≥3 prior lines of therapy. Prior CAR-T cell therapy was excluded, but other forms of CD19- or CD20-targeted therapies were allowed. Autologous leukocytes were obtained by leukapheresis and sorted for CD14-/CD25-/CD62L+ TN/MEM cells, followed by lentiviral transduction of the bispecific CD19/CD20 CAR. Bridging therapy after leukapheresis was allowed. Lymphodepletion chemotherapy with fludarabine 30 mg/m2/day and cyclophosphamide 500 mg/m2/day was administered for 3 days from Day −5 to Day −3 prior to CAR-T cell infusion. The primary endpoint was safety and the secondary endpoints were ORR, progression free survival (PFS), overall survival (OS), and CART19/20 transgene persistence. As of December 19, 2022, 11 patients have been treated with CART19/20 cells (7 DLBCL, including 4 transformed FL and 1 PMBCL; 3 FL; and 1 MCL). Patients were enrolled at two dose levels, including 50 × 106 CAR+ cells (8 patients) and 200 × 106 CAR+ cells (3 patients). The median age was 58 (range: 34 to 70). All patients had stage IV disease and 9 of 11 patients received bridging therapy. Six patients had grade-1 cytokine release syndrome (CRS), without any occurrence of higher-grade CRS. Neurotoxicity was not observed. Ten of the 11 patients achieved an objective response (91% ORR), with 8 patients (73%) achieving complete response (CR). One patient with FL relapsed at 18 months, received a second CART19/20 cell infusion (112 × 106 CAR+ cells), and re-achieved a CR that has persisted for &amp;gt;6 months at last follow-up. With a median follow-up of 20.9 months (range: 3.4 - 37 months), 7 patients remain in CR with a median PFS of 18.2 months (95% CI 3.4 months - not estimable) and the median OS was not reached. This phase 1 study demonstrates robust safety and tolerability of CART19/20 T-cells in patients with R/R NHL, without occurrence of severe CRS or neurotoxicity. CART19/20 cells, utilizing a bispecific CAR and TN/MEM cells, may be an effective strategy to overcome the challenges of poor CAR T-cell persistence and antigen escape. Citation Format: Benjamin R. Puliafito, Christopher Walthers, Brenda Ji, Sanaz N. Ghafouri, Jacob Naparstek, Jacqueline Trent, Jia M. Chen, Mobina Roshandell, Caitlin Harris, Mobina Khericha, Thomas Schweppe, Beata Berent-Maoz, Stanley B. Gosliner, Amr Almaktari, Melanie Ayala Ceja, Martin S. Allen-Auerbach, Jonathan Said, Karla Nawaly, Monica Mead, Sven de Vos, Patricia A. Young, Caspian Oliai, Gary J. Schiller, John M. Timmerman, Antoni Ribas, Yvonne Y. Chen, Sarah M. Larson. Phase 1 trial of CD19/CD20 bispecific chimeric antigen receptor-engineered naïve/memory T cells for relapsed or refractory non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT023.

Abstract 1031: Specific gene alterations of HER2 positive single circulating tumor cell (CTC) compared to autologous leukocytes in metastatic breast cancer (MBC)

Abstract Introduction: Monitoring of CTC in MBC showed ability to predict treatment resistance and outcome. HER2 is one of the most important tumor markers which is associated with rapid cell division and the metastasis and prognosis of MBC in the clinic. More recently, we reported single CTC gene mutation is a key point to distinguish metastasis capability in MBC (2022 AACR and 2022 ASCO). Herein, we report specific gene alterations in HER2+ single CTC compared to autologous leukocytes in MBC. Methods: Whole blood sample (7.5ml/each) was collected from MBC patient before therapy. CTC enumeration was performed in FDA approved CellSearch™ System and then the single HER2+ CTC and leukocytes were isolated by using DEPArrayTM System (Menarini). The single cell DNA was isolated and then the initial library was prepared by using Ampli1™ Whole Genome Amplification, Ampli1™ QC Kit (Menarini) and AMPure XP kit (Beckman Coulter). The exome capture was performed by TruSeq DNA Exome kit (illumine). The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). Results: We identified 107 CTCs including 55 HER2+ CTCs and 14 CTC-clusters. Autologous single HER2+ CTC (HER2+CK+CD45−, Group 1), HER2+CK−CD45+ leukocyte (Group 2) and HER2−CK−CD45+ leukocyte (Group 3) were isolated and sequenced respectively. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 486,119 counts (56.69%) for intron variants, 175,819 counts (20.51%) for intergenic variants, 70,334 counts (8.20%) for exon variants, 50,370 counts (5.87%) for downstream genes, 45,915 counts (5.36%) for upstream genes and others (3.37%) in HER2+ CTC. Meanwhile, there were 71,848 (8.76%) and 0 counts for exon variants found in Group 2 and Group 3 respectively. There were 79 gene variants ((SNP and Ins-Del) identified to have the highest impact effect (≥20) on HER2+ CTC chromosome, when there were 85 and 0 highest impact gene variants were identified in Group 2 and Group 3 respectively. The specific gene alterations in Group 1 compared to Group 2 includes CCNA2, FOLH1, BRD4, SAMHD1, CYP17A1, IDE, HPGDS and CTNNB1 Among the top 50 high impact gene variants, there were 26 genes alteration sites are same in both Group 1 and Group 2, including FECH, HDAC8, CYP11B2, TTN(6 sites), RAN, CD207, HK1, CASP1(2 sites), BRCA1, PIK3CG (3 sites), APEX1(4 sites), KIF11, SIRT5 (2 sites), XYLB and CHKA. Conclusion: Genomic characterization of HER2 positive single CTCs elucidated specific gene alterations associated with disease metastasis compared to autologous leukocytes in MBC. The newfound HER2+CK−CD45+ cells with gene alterations maybe a new kind of cancer transformed leukocytes which need further validation. Specific gene alterations will help to develop novel drugs aimed at the eradication of CTCs using molecularly driven therapies for disease metastasis. Citation Format: Qiang Zhang, Lorenzo Gerratana, Zhe Ji, Xinkun Wang, Paolo D’Amico, Seema Singhal, Youbin Zhang, Andrew A. Davis, Ami N. Shah, William Gradishar. Specific gene alterations of HER2 positive single circulating tumor cell (CTC) compared to autologous leukocytes in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1031.

Leukocyte- and platelet-rich fibrin in cranial surgery: study protocol for a prospective, parallel-group, single-blinded randomized controlled non-inferiority trial {1}

BackgroundCSF leakage is a major complication after cranial surgery, thus, adequate dural closure must be performed. Commercially available fibrin sealants are currently considered the gold standard for dural closure, but problems have been reported regarding safety, efficacy, and costs. This trial aims to investigate autologous leukocyte- and platelet-rich fibrin (L-PRF) as an alternative to commercially available fibrin sealants.Methods/designThis single-blinded, prospective randomized controlled interventional trial aims to demonstrate the non-inferiority of L-PRF compared to commercially available fibrin sealants for dural closure. This trial will include patients undergoing cranial neurosurgery (supratentorial and infratentorial) with intentional opening of the dura. Patients are randomized in a 1:1 fashion comparing L-PRF to commercially available fibrin sealants. The primary endpoint is postoperative CSF leakage within 12 weeks after surgery. Secondary endpoints are complications such as bleeding or wound infections. Additionally, a cost-effectiveness analysis is performed.DiscussionWith this trial, we will evaluate the safety and efficiency of L-PRF compared to commercially available fibrin sealants.Trial registrationClinicalTrials.gov NCT03812120. Registered on 22 January 2019.

Features of the local cytokine profile of patients with bullous keratopathy by using personalized therapy with cellular technologies

Background. Today we have active development of ophthalmic surgery, but the role of cytokines in the pathogenesis of bullous keratopathy of the cornea remains insufficiently studied at present.The aim. To study the dynamics of the local cytokine profile in bullous keratopathy by using personalized treatment with suspension of autologous blood mononuclears.Material and methods. Two groups of patients with bullous keratopathy were formed: the main group (30 people, 30 eyes), who received an intrastromal injection of a suspension of autologous blood mononuclears and comparison (28 people, 28 eyes), who received a course of pharmacotherapy. The level of interleukins (IL) – IL-10, IL-4, IL-6, IL-1ß, transforming growth factor beta-2 (TGF-β2), tumor necrosis factor alpha (TNF-α) was determined in the lacrimal fluid of the patient’s eye by enzyme immunoassay.Results. All patients before treatment showed an increase of IL-1ß in the lacrimal fluid by 3.3 times, IL-6 – by 4.2 times, TNF-α – by 2.0 times (p &lt; 0.05); an increase in the level of IL-4, IL-10 and TGF-β2 by 1.1 times (p &gt; ˂ 0.05). In the main group, after treatment, there was a decrease in IL-1ß by 2.0 times, IL-6 – by 2.1 times and TNF-α – by 1.8 times, and an increase in IL-10 by 1.5 times, IL-4 – by 1.9 times, TGF-β2 – by 1.4 times (p &lt; 0.05), persisting for 12 months. There was a short-term decrease of IL-1ß by 1.7 times, IL-6 and TNF-α – by 1.2 times and an increase of IL-10, IL-4, TGF-β2 by 1.2 times (p &gt;&lt; 0.05) in the comparison group which then reached the initial values. Conclusion. There is an imbalance in the system of pro- and anti-inflammatory cytokines in bullous keratopathy in the lacrimal fluid. Injection of autologous mononuclear cells in the stroma of the cornea reduces the severity of the imbalance of the local cytokine system compared to the course of pharmacotherapy. Key words: bullous keratopathy, cellular technologies, autologous mononuclear leukocytes, cytokines&gt;˂ 0.05), persisting for 12 months. There was a short-term decrease of IL-1ß by 1.7 times, IL-6 and TNF-α – by 1.2 times and an increase of IL-10, IL-4, TGF-β2 by 1.2 times (p ˂ 0.05) in the comparison group which then reached the initial values.Conclusion. There is an imbalance in the system of pro- and anti-inflammatory cytokines in bullous keratopathy in the lacrimal fluid. Injection of autologous mononuclear cells in the stroma of the cornea reduces the severity of the imbalance of the local cytokine system compared to the course of pharmacotherapy.

Advances in Molecular Imaging in Infective Endocarditis.

Infective endocarditis (IE) is a growing epidemiological challenge. Appropriate diagnosis remains difficult due to heterogenous etiopathogenesis and clinical presentation. The disease may be followed by increased mortality and numerous diverse complications. Developing molecular imaging modalities may provide additional insights into ongoing infection and support an accurate diagnosis. We present the current evidence for the diagnostic performance and indications for utilization in current guidelines of the hybrid modalities: single photon emission tomography with technetium99m-hexamethylpropyleneamine oxime-labeled autologous leukocytes (99mTc-HMPAO-SPECT/CT) along with positron emission tomography with fluorodeoxyglucose (18F-FDG PET/CT). The role of molecular imaging in IE diagnostic work-up has been constantly growing due to technical improvements and the increasing evidence supporting its added diagnostic and prognostic value. The various underlying molecular processes of 99mTc-HMPAO-SPECT/CT as well as 18F-FDG PET/CT translate to different imaging properties, which should be considered in clinical practice. Both techniques provide additional diagnostic value in the assessment of patients at risk of IE. Nuclear imaging should be considered in the IE diagnostic algorithm, not only for the insights gained into ongoing infection at a molecular level, but also for the determination of the optimal clinical therapeutic strategies.

Tumor cells fail to present MHC-II restricted epitopes derived from oncogenes to CD4+ T cells.

CD4+ T cells play a critical role in anti-tumor immunity via recognition of peptide antigens presented on MHC class II (MHC-II). Although some solid cancers can be induced to express MHC-II, the extent to which this enables direct recognition by tumor-specific CD4+ T cells is unclear. We isolated and characterized T cell antigen receptors (TCRs) from naturally primed CD4+ T cells specific for two oncoproteins, HPV16 E6 and the activating KRASG12V mutation, from head and neck squamous cell carcinoma (HNSCC) and pancreatic ductal adenocarcinoma (PDAC) patients, respectively, and determined their ability to recognize autologous or human leukocyte antigen (HLA)-matched antigen-expressing tumor cells. We find in both cases that the TCRs are capable of recognizing peptide-loaded target cells expressing the relevant MHC-II or B cell antigen-presenting cells (APC) when the antigens are endogenously expressed and directed to the endosomal pathway but fail to recognize tumor cells expressing the source protein even after induction of surface MHC-II expression by IFN- or transduction with CIITA. These results suggest that priming and functional recognition of both a nuclear (E6) and a membrane-associated (KRAS) oncoprotein is predominantly confined to cross-presenting APC rather than via direct recognition of tumor cells induced to express MHC-II.

Leukocyte and Platelet-Rich Plasma (L-PRP) in Tendon Models: A Systematic Review and Meta-Analysis of in vivo/in vitro Studies.

To perform a systematic review on the application of leukocyte- and platelet-rich plasma (L-PRP) in tendon models by reviewing in vivo/in vitro studies. The searches were performed via electronic databases including PubMed, Embase, and Cochrane Library up to September 2022 using the following keywords: ((tenocytes OR tendon OR tendinitis OR tendinosis OR tendinopathy OR tendon injury) AND (platelet-rich plasma OR PRP OR autologous conditioned plasma OR leukocyte- and platelet-rich plasma OR L-PRP OR leukocyte-richplatelet-rich plasma Lr-PRP)). Only in vitro and in vivo studies that assessed the potential effects of L-PRP on tendons and/or tenocytes are included in this study. Description of PRP, study design and methods, outcomes measured, and results are extracted from the data. A total of 17 studies (8 in vitro studies and 9 in vivo studies) are included. Thirteen studies (76%) reported leukocyte concentrations of L-PRP. Four studies (24%) reported the commercial kits. In in vitro studies, L-PRP demonstrated increased cell proliferation, cell migration, collagen synthesis, accelerated inflammation, and catabolic response in the short term. In addition, most in vivo studies indicated increased collagen type I content. According to in vivo studies reporting data, L-PRP reduced inflammation response in 71.0% of studies, while it enhanced the histological quality of tendons in 67.0% of studies. All 3 studies reporting data found increased biomechanical properties with L-PRP treatment. Most evidence indicates that L-PRP has some potential effects on tendon healing compared to control. However, it appears that L-PRP works depending on the biological status of the damaged tendon. At an early stage, L-PRP may accelerate tendon healing, but at a later stage, it could be detrimental.