A single crystal row-column-array for 3D ultrasound imaging.

In vivo 3D ultrasound imaging with 2D-array transducers is of great importance for both clinical application and biomedical research, but it is complicated in fabrication and also very expensive in hardware due to thousands of electronic channels. In this work, we demonstrate a new fabrication process of 7-MHz 128 + 128 elements row-column-array (RCA) transducer with relaxor ferroelectric PMN-0.28PT single crystal. With piezoelectric single crystal and improved acoustic matching, the optimized performance of -6 dB bandwidth of ∼82 % and insertion loss of -44.6 dB is achieved. The axial and lateral imaging resolutions at different depth of the RCA transducer are quantified by the point spread function (PSF), and the results are respectively 0.20 mm and 0.41 mm at the depth of 7.7 mm, and 0.22 mm and 0.47 mm at the depth of 16.7 mm. The transducer is validated experimentally on a hyperechoic phantom, and 3D view and slices of B-mode images are obtained. The experimental results indicate that our developed RCA transducer can obtain high-quality 3D ultrasound images, demonstrating great potential on ultrafast 3D and functional imaging.

[Application of super-resolution and ultrafast ultrasound to reveal the characteristics of vascular blood flow changes after rat spinal cord injury at different segments].

Objective: To investigate the changes of spinal vascular blood flow in SD rats after cervical, thoracic and lumbar spinal cord injury (SCI) using super-resolution ultrafast ultrasound technology. Methods: A total of 9 SD rats were used to construct SCI models at different segments using a 50 g aneurysm clip. Super-resolution ultrafast ultrasound technology was used to perform vascular blood flow imaging on the spinal cord of rats before and after injury at 6 hours, obtaining quantitative information such as spinal cord vascular density and blood flow velocity. Results: Ultrasound imaging showed that after SCI, the vascular density in the thoracic segment decreased (18.16%±1.04%) more than in the cervical segment (11.42%±1.39%) and lumbar segment (13.88%±1.43%, both P<0.05). The length of the spinal cord with decreased vascular density in the thoracic segment [(4.80±0.34)mm] was longer than that in the cervical segment [(2.80±0.57)mm] and lumbar segment [(3.10±0.36)mm, both P<0.05]. After injury, the decrease of blood flow in the thoracic segment [(8.87±0.85)ml/min] was higher than that in the cervical segment [(4.88±0.56)ml/min] and lumbar segment [(6.19±0.71)ml/min, both P<0.05]. HE staining and Nissl staining showed that the proportion of cavity area after thoracic SCI (11.53%±0.93%) was higher than that in the cervical segment (4.90%±1.72%) and lumbar segment (7.64%±0.84%, both P<0.05). The number of Nissl bodies in the thoracic segment (18.0±5.3) was also lower than that in the cervical segment (32.3±5.1) and lumbar segment (37.0±5.6) (both P<0.05). Conclusions: There are different changes in vascular blood flow after SCI in different segments of rats. The same injury causes the most severe damage to blood vessels in the thoracic spinal cord, followed by the lumbar spinal cord, and the cervical spinal cord has the least damage.

[Observation of liver indexes in patients with relapsed/refractory multiple myeloma treated with CAR-T-cells based on BCMA].

Objective: To observe the characteristics of the evolution of liver indexes in patients with relapsed/refractory multiple myeloma (RRMM) treated with CAR-T-cells based on BCMA. Methods: Retrospective analysis was performed of patients with RRMM who received an infusion of anti-BCMA CAR-T-cells and anti-BCMA combined with anti-CD19 CAR-T-cells at our center between June 1, 2019, and February 28, 2023. Clinical data were collected to observe the characteristics of changes in liver indexes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and direct bilirubin (DBIL) in patients, and its relationship with cytokine-release syndrome (CRS) . Results: Ninety-two patients were included in the analysis, including 41 patients (44.6%) in the group receiving a single infusion of anti-BCMA CAR-T-cells, and 51 patients (55.4%) in the group receiving an infusion of anti-BCMA combined with anti-CD19 CAR-T-cells. After infusing CAR-T-cells, 31 patients (33.7%) experienced changes in liver indexes at or above grade 2, which included 20 patients (21.7%) with changes in one index, five patients (5.4%) with changes in two indexes, and six patients (6.5%) with changes in three or more indexes. The median time of peak values of ALT and AST were d17 and d14, respectively, and the median duration of exceeding grade 2 was 5.0 and 3.5 days, respectively. The median time of peak values of TBIL and DBIL was on d19 and d21, respectively, and the median duration of exceeding grade 2 was 4.0 days, respectively. The median time of onset of CRS was d8, and the peak time of fever was d9. The ALT, AST, and TBIL of patients with CRS were higher than those of patients without CRS (P=0.011, 0.002, and 0.015, respectively). CRS is an independent factor that affects ALT and TBIL levels (OR=19.668, 95% CI 18.959-20.173, P=0.001). The evolution of liver indexes can be reversed through anti-CRS and liver-protection treatments, and no patient died of liver injury. Conclusions: In BCMA-based CAR-T-cell therapy for RRMM, CRS is an important factor causing the evolution of liver indexes. The evolution of liver indexes after CAR-T-cell infusion is transient and reversible after treatment.

[The effect of platelet infusion on the repair of bone marrow hematopoietic niche damage in mice induced by (60)Co radiation and hematopoietic reconstruction after bone marrow transplantation].

Objective: To observe the effect of platelets on hematopoietic stem cell (HSCs) implantation in mice with radiation-induced bone marrow injury and bone marrow transplantation models. Methods: ①Male C57BL/6 mice were divided into a single irradiation group and a radiation infusion group after receiving (60)Co semimyeloablative irradiation for 18-10 weeks. The irradiation infusion group received 1×10(8) platelets expressing GFP fluorescent protein. ② The allogeneic bone marrow transplantation model was established. The experimental groups included the simple transplantation group (BMT) and the transplantation infusion group (BMT+PLT). The BMT group was infused through the tail vein only 5 × 10(6) bone marrow cells, the BMT+PLT group needs to be infused with bone marrow cells at the same time 1× 10(8) platelets. ③ Test indicators included peripheral blood cell and bone marrow cell counts, flow cytometry to detect the proportion of hematopoietic stem cell (HSC) and hematopoietic progenitor cells, bone marrow cell proliferation and apoptosis, and pathological observation of vascular niche damage and repair. Results: ①On the 3rd, 7th, 14(th), and 21st days after irradiation, the bone marrow cell count of the infusion group was higher than that in the single irradiation group (P<0.05), and the peripheral blood cell count was also higher. A statistically significant difference was found between the white blood cell count on the 21st day and the platelet count on the 7th day (P<0.05). In the observation cycle, the percentage of bone marrow cell proliferation in the infusion group was higher, while the percentage of apoptosis was lower. ② The results of bone tissue immunofluorescence after irradiation showed that the continuity of hematopoietic niche with red fluorescence was better in the irradiation infusion group. ③The chimerism percentage in the BMT+PLT group was always higher than that in the BMT group after transplantation.④ The BMT+PLT group had higher bone marrow cell count and percentage of bone marrow cell proliferation on the 7th and 28th day after transplantation than that in the BMT group, and the percentage of bone marrow cell apoptosis on the 14th day was lower than that in the BMT group (P<0.05). After the 14th day, the percentage of stem progenitor cells in the bone marrow cells of mice was higher than that in the BMT group (P<0.05). ⑤The immunohistochemical results of bone marrow tissue showed that the continuity of vascular endothelium in the BMT+PLT group was better than that in the BMT group. Conclusion: Platelet transfusion can alleviate the injury of vascular niche, promotes HSC homing, and is beneficial to hematopoietic reconstruction.

[Analysis of prognostic factors of extranodal NK/T-cell lymphoma treated with pegaspargase/L-asparaginase: a multicenter retrospective study].

Objective: To explore the prognostic factors of extracellular NK/T cell lymphoma (ENKTL) treated with pegaspargase/L-asparaginase. Methods: The clinical data of 656 ENKTL patients diagnosed at 11 medical centers in the Huaihai Lymphoma Working Group from March 2014 to April 2021 were retrospectively analyzed. The patients were randomly divided into two groups: a training set (460 cases) and a validation set (196 cases) at 7∶3, and the prognostic factors of the patients were analyzed. A prognostic scoring system was established, and the predictive performance of different models was compared. Results: Patients' median age was 46 (34, 57) years, with 456 males (69.5% ) and 561 nasal involvement (85.5% ). 203 patients (30.9% ) received a chemotherapy regimen based on L-asparaginase combined with anthracyclines, and the 5-year overall survival rate of patients treated with P-GEMOX regimen (pegaspargase+gemcitabine+oxaliplatin) was better than those treated with SMILE regimen (methotrexate+dexamethasone+cyclophosphamide+L-asparaginase+etoposide) (85.9% vs 63.8% ; P=0.004). The results of multivariate analysis showed that gender, CA stage, the Eastern Cooperative Oncology Group performance status (ECOG PS) score, HGB, and EB virus DNA were independent influencing factors for the prognosis of ENKTL patients (P<0.05). In this study, the predictive performance of the prognostic factors is superior to the international prognostic index, Korean prognostic index, and prognostic index of natural killer lymphoma. Conclusion: Gender, CA stage, ECOG PS score, HGB, and EB virus DNA are prognostic factors for ENKTL patients treated with pegaspargase/L-asparaginase.

[Optimization of CD19 chimeric antigen receptor T cell establishment and observation of the killing effect in vitro and in vivo].

Objective: To optimize the stimulation and activation system of mouse CD3(+) T cells in vitro and explore the optimal infection time of CD3(+) T cells to establish mouse CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and to also verify its killing effect in vivo and in vitro. Method: Splenic CD3(+)T cells were isolated and purified using magnetic beads, and the cells were cultured in Soluble anti-CD3/CD28, PMA+Ionomycin, and Plated anti-CD3/CD28. Cell activation and apoptosis were assessed by flow cytometry after 8, 24, 48, and 72 hours. ScFv plasmid of mouse CD19 antibody was transfected to plat-E cells to package retrovirus. Activated CD3(+) T cells were infected to construct mouse-specific CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and mCD19 CAR-T cells were co-cultured with B-cell lymphoma cell line A20 in vitro. The specific toxicity of A20 was detected by flow cytometry, and mCD19 CAR-T cells were infused into the lymphoma mouse model to detect its killing effect and distribution. Results: The activation effect of Plated anti-CD3/CD28 on CD3(+) T cells was superior, with the cells exhibiting good viability 24-48 hours after stimulation. Established mCD19 CAR-T cells with stable efficiency[ (32.27±7.56) % ] were specifically able to kill A20 tumor cells (The apoptosis rate was 24.3% at 48 h) . In vivo detection showed a non-significant decrease in the percentage[ (1.83±0.58) % ] of splenic CD19(+) cells 6 days after mCD19 CAR-T cell infusion. A marked clearance in bone marrow and spleen appeared on day 12 compared with the A20 group, and this difference was statistically significant[spleen: (0.36±0.04) % vs (47.00±13.46) % , P<0.001; bone marrow: (1.82±0.29) % vs (37.30±1.44) % , P<0.0001]. Moreover, mCD19 CAR-T cells were distributed in high proportions in the peripheral blood, spleen, and bone marrow[ (2.90±1.12) % , (4.96±0.80) % , (13.55±1.56) % ]. Conclusion: This study demonstrated an optimized activation system and the optimal infection time of CD3(+) T cells. Furthermore, stable constructed mCD19 CAR-T cells showed a remarkable killing ability in vitro and in vivo.

[Long-term safety and activity of humanized CD19 chimeric antigen receptor T cells for children and young adults with relapsed/refractory acute lymphoblastic leukemia].

Objective: To investigate the efficacy and safety of humanized CD19-specific chimeric antigen receptor T cells (hCART19s) in treating children and young adults with relapsed/refractory acute lymphoblastic leukemia (R/R ALL) and to analyze relevant factors affecting its curative effect and prognosis. Methods: We conducted a single-center clinical trial involving 31 children and young adult patients with R/R B-ALL who were treated with humanized CD19-specific CAR-T cells (hCART19s) from May 2016 to September 2021. Results: Results showed that 27 (87.1%) patients achieved complete remission (CR) or CR with incomplete count recovery (CRi) one month after CAR-T cell infusion. During treatment, 20 (64.5%) patients developed grade 1-2 cytokine release syndrome (CRS) , and 4 (12.9%) developed grade 3 CRS. Additionally, two patients had grade 1 neurological events. During the follow-up with a median time of 19.3 months, the median event-free survival (EFS) was 15.7 months (95% CI 8.7-22.5) , and the median overall survival (OS) was 32.2 months (95% CI 10.6-53.9) . EFS and OS rates were higher in patients who have undergone hemopoietic stem cell transplantation (HSCT) than in those without [EFS: (75.0 ± 12.5) % vs (21.1 ± 9.4) %, P=0.012; OS: (75.0 ± 12.5) % vs (24.6 ± 10.2) %, P=0.035]. The EFS and OS rates were significantly lower in patients with >3 treatment lines than in those with <3 treatment lines [EFS: 0 vs (49.5±10.4) %, P<0.001; OS: 0 vs (52.0±10.8) %, P<0.001]. To the cutoff date, 12 patients presented with CD19(+) relapse, and 1 had CD19(-) relapse. Conclusion: hCART19s are effective in treating pediatric and young adult R/R ALL patients, with a low incidence of severe adverse events and reversible symptoms. Following HSCT, the number of treatment lines can affect the long-term efficacy and prognosis of pediatric and young adult R/R ALL patients.

[Clinical prognostic analysis of 124 adult patients with hemophagocytic lymphohistiocytosis: a multicenter retrospective study of the Huaihai Lymphoma Working Group].

Objective: Factors influencing the prognosis of hemophagocytic lymphohistiocytosis (HLH) in adults were analyzed based on multicentric data. Methods: Clinical data of 124 adult patients with HLH diagnosed in eight medical centers in the Huaihai Lymphoma Working Group from March 2014 to July 2020 were collected. The optimal truncation value of continuous variables was obtained based on the Maxstat algorithm, X-Tile software, and restricted cubic spline. Cox proportional risk regression model was used to construct the adult HLH risk prediction model, and the visualization of the model was realized through the histogram. The bootstrap resampling method was used to verify the model, C-index and calibration curve was used to verify the histogram, and the prediction accuracy was checked. Kaplan-Meier analysis was used to calculate the survival rate and draw the survival curve. Furthermore, the differences between groups were tested by log-rank. Results: The median age of the 124 patients was 55 (18-84) years, including 61 (49.19%) males. The most common etiology was infection. Serum ferritin increased in 110 cases (88.71%) , hepatosplenomegaly in 57 cases (45.97%) . Of the 124 patients, 77 (62.10%) died, and the median survival time of the patients was 7.07 months. Univariate results showed that the prognosis of adult HLH was influenced by sex, age, fibrinogen, serum creatinine, alanine aminotransferase, and albumin (P<0.05) . The results of multivariate analysis showed that gender, platelet, albumin, alanine aminotransferase, and treatment regimens were independent influencing factors for prognosis. Based on the above five risk factors, the prediction model of the histogram was established, and the C-index of the model was 0.739. Finally, the calibration chart showed good consistency between the observed and predicted values of HLH. Conclusion: The prognosis of the adult hemophagocytic syndrome is influenced by many factors. Gender, platelet, albumin, alanine aminotransferase, and treatment regimens are independent risk factors. Therefore, the established histogram provides a visual tool for clinicians to evaluate the prognosis of adult HLH.

[Comparison of single infusion of anti-BCMA versus combined infusion of anti-CD19 chimeric antigen receptor T cells for immune reconstruction in relapsed/refractory multiple myeloma].

Objective: We observed and compared the differences in immune reconstruction between single-infusion anti-B-cell maturation antigen (BCMA) , chimeric antigen receptor T cells (CAR-T) , and combined infusion of anti-CD19 CAR-T cells in the treatment of recurrent/refractory multiple myeloma (RRMM) . Methods: Sixty-one patients with RRMM who underwent CAR-T cell therapy in our hospital from June 2017 to December 2020 were selected. Among them, 26 patients received anti-BCMA target, and 35 patients received anti-BCMA combined with anti-CD19 target. Using flow cytometry, we determined T cell subsets (CD3(+), CD4(+), CD8(+), CD4(+)/CD8(+)) , B cells (CD19(+)) , and NK cells (CD16(+) CD56(+)) at different time points before and after CAR-T treatment, and detected immunoglobulin IgG, IgA and IgM levels by immunoturbidimetry. We compared the reconstruction rules of lymphocyte subsets and immunoglobulins in the two groups. Results: CD8(+) T lymphocytes recovered most rapidly after the infusion of CAR-T cells, returning to pre-infusion levels at 3 months and 1 month after infusion, respectively[BCMA: 695 (357, 1264) /µl vs 424 (280, 646) /µl; BCMA+CD19: 546 (279, 1672) /µl vs 314 (214, 466) /µl]. NK cells returned to normal levels at 3 months after infusion in both groups[BCMA: 171 (120, 244) /µl, BCMA+CD19: 153 (101, 218) /µl (Normal reference range 150-1100/µl) ]; however, the NK cells were not maintained at stable levels in the BCMA CAR-T cells group. The recovery of CD4(+) T lymphocytes in both groups was slow and remained persistently low within 12 months after infusion, and no recovery was observed in most patients. The reversal of the ratio of CD4(+)/CD8(+) lasted for more than a year. The levels of CD19(+) B cells in both groups returned to baseline 3 months after infusion[BCMA: 62 (10, 72) /µl vs 57 (24, 78) /µl; BCMA+CD19: 40 (4, 94) /µl vs 29 (14, 46) /µl]. IgG returned to the pre-infusion level 12 months after infusion in the group with anti-BCMA cells alone, but not in the group with combined infusion of CD19 CAR T cells[7.82 (6.03, 9.64) g/L vs 6.92 (4.62, 12.76) g/L]. IgA returned to pre-infusion levels at 9 and 12 months after infusion, respectively[BCMA: 0.46 (0.07, 0.51) g/L vs 0.22 (0.12, 4.01) g/L; BCMA+CD19: 0.46 (0.22, 0.98) g/L vs 0.27 (0.10, 0.53) g/L]. IgM in both groups returned to pre-infusion levels 6 months after infusion[BCMA: 0.43 (0.06, 0.60) g/L vs 0.20 (0.13, 0.37) g/L; BCMA+CD19: 0.53 (0.10, 0.80) g/L vs 0.16 (0.11, 0.28) g/L]. There was no significant difference in the indexes of lymphocyte subpopulation reconstruction and immunoglobulin recovery between the two groups at each time point. Conclusion: This study showed that in patients with RRMM treated with CAR-T cells, the appropriate target antigen can be selected without considering the difference of immune reconstruction between anti-BCMA CAR-T and combined anti-CD19 CAR-T therapy.

[Influence of prognostic nutritional index and controlling nutritional status on the prognosis of patients with multiple myeloma].

Objective: To explore the influence of prognostic nutritional index (PNI) and controlling nutritional status (CONUT) on the prognosis of patients with multiple myeloma. Methods: Data of 157 patients with multiple myeloma (MM) at the affiliated hospital of Xuzhou medical university from January 2014 to December 2018 were retrospectively evaluated. The operating characteristic (ROC) curve analysis was adopted as the optimal cut-off point. PNI and CONUT were grouped based on the cut-off points of 44.45 and 3.5, respectively, and the differences between age, gender, serum calcium, ß(2)-microglobulin, serum creatinine, lactate dehydrogenase, and hemoglobin were analyzed. The prognostic factors were analyzed via univariate and Cox multivariate regression analyses. Results: The level of PNI and CONUT is the influencing factor of OS time. The univariate analysis revealed that age, LDH, plasma cell ratio, ß(2)-microglobulin, ISS stage, PNI, and CONUT were the risk factors for the prognosis of patients with MM. The multivariate analysis revealed that age (HR=1.636, 95%CI 1.014-2.640) , plasma cell ratio (HR=1.953, 95%CI 1.232-3.096) , and PNI (HR=0.513, 95%CI 0.287-0.917) were the independent prognostic risk factors of patients with MM. Conclusion: Low PNI in patients with MM indicates a poor prognosis, which is an independent prognosis risk factor.

[Effects of L-asparaginase on proliferation, cell cycle and apoptosis of Burkitt lymphoma cell lines].

Objective: To investigate the effect of L-asparaginase on the proliferation, cell cycle, and apoptosis of Burkitt lymphoma cell lines and explore the molecular mechanism. Methods: The effect of L-asparaginase on the cell proliferation of Burkitt lymphoma cell lines was detected using the CCK-8 method. The apoptosis rate and cell cycle were detected using flow cytometry. The expression of related molecules in cell cycle, apoptosis, autophagy, and PI3K/Akt/mTOR signaling pathway was detected and analyzed using qPCR and Western blot assay. Results: L-asparaginase significantly inhibited the proliferation of Burkitt lymphoma cell lines and caused cell cycle arrest at G(0)/G(1) phage. L-asparaginase induced cell apoptosis and autophagy in Burkitt lymphoma cell lines. Further results showed that L-asparaginase inhibited the expression of c-Myc and also inhibited the expression of p-PI3K, p-Akt-S473, p-mTOR, p-70S6K, and p-4E-BP1. Combining PI3K inhibitor LY294002 with L-asparaginase further induced apoptosis. Additionally, L-Asp inhibited STAT and ERK signaling pathways. Conclusion: L-asparaginase inhibited Burkitt lymphoma cell proliferation, arrested cell cycle, activated autophagy, and induced apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway.