Analysis of chemoresistance characteristics and prognostic relevance of postoperative gemcitabine adjuvant chemotherapy in pancreatic cancer.

AIM: To investigate the relationship between chemoresistance in pancreatic cancer patients receiving postoperative gemcitabine adjuvant therapy and specific clinical/pathological characteristics, as well as its impact on patient prognosis. METHODS: From June 2018 to June 2021, clinical and pathological data of 148 pancreatic cancer patients were collected, and 101 patients were followed up for tumor recurrence/metastasis and survival status. The correlation between chemoresistance and specific clinical/pathological characteristics or patient prognosis was retrospectively analyzed. RESULTS: Of the 148 patients, 78 were in the chemoresistance group and 70 in the non-chemoresistance group. Univariate analysis showed that the development of chemoresistance may be related to patient age, combined diabetes, preoperative CA19-9 level, tumor size, AJCC stage, vascular invasion, and positive lymph node ratio. Furthermore, subsequent multivariate analysis incorporating these variables indicated that tumor size may be a key factor influencing chemoresistance (p < 0.001, OR = 1.584). Log-rank test showed patients in the chemoresistance group had worse overall survival (OS) (HR = 2.102, p = 0.018) and progression free survival (PFS) (HR = 3.208, p = 0.002) than patients in the non-chemoresistance group; and patients with smaller size tumors (diameter ≤3 cm) had significantly better OS (HR = 2.923, p < 0.001) and PFS (HR = 2.930, p = 0.003) than those with larger size tumors (diameter >3 cm). CONCLUSIONS: Patients with pancreatic cancer receiving postoperative gemcitabine adjuvant therapy are more likely to develop chemoresistance when their tumor sizes are larger (diameter >3 cm). Development of chemoresistance exacerbates the prognosis of patients with pancreatic cancer, and larger tumor size is also a risk factor for poor prognosis in these patients.

Expanding the reach of commercial cell therapies requires changes at medical centers.

The clinical application of cell therapies is becoming increasingly important for the treatment of cancer, congenital immune deficiencies, and hemoglobinopathies. These therapies have been primarily manufactured and used at academic medical centers. However, cell therapies are now increasingly being produced in centralized manufacturing facilities and shipped to medical centers for administration. Typically, these cell therapies are produced from a patient's own cells, which are the critical starting material. For these therapies to achieve their full potential, more medical centers must develop the infrastructure to collect, label, cryopreserve, test, and ship these cells to the centralized laboratories where these cell therapies are manufactured. Medical centers must also develop systems to receive, store, and infuse the finished cell therapy products. Since most cell therapies are cryopreserved for shipment and storage, medical centers using these therapies will require access to liquid nitrogen product storage tanks and develop procedures to thaw cell therapies. These services could be provided by the hospital pharmacy or transfusion service, but the latter is likely most appropriate. Another barrier to implementing these services is the variability among providers of these cell therapies in the processes related to handling cell therapies. The provision of these services by medical centers would be facilitated by establishing a national coordinating center and a network of apheresis centers to collect and cryopreserve the cells needed to begin the manufacturing process and cell therapy laboratories to store and issue the cells. In addition to organizing cell collections, the coordinating center could establish uniform practices for collecting, labeling, shipping, receiving, thawing, and infusing the cell therapy.

The Potential Use of THP-1, a Monocytic Leukemia Cell Line, to Predict Immune-Suppressive Potency of Human Bone-Marrow Stromal Cells (BMSCs) In Vitro: A Pilot Study.

Adoptive transfer of cultured BMSCs was shown to be immune-suppressive in various inflammatory settings. Many factors play a role in the process, but no master regulator of BMSC-driven immunomodulation was identified. Consequently, an assay that might predict BMSC product efficacy is still unavailable. Below, we show that BMSC donor variability can be monitored by IL-10 production of monocytes/macrophages using THP-1 cells (immortalized monocytic leukemia cells) co-cultured with BMSCs. Using a mixed lymphocyte reaction (MLR) assay, we also compared the ability of the different donor BMSCs to suppress T-cell proliferation, another measure of their immune-suppressive ability. We found that the BMSCs from a donor that induced the most IL-10 production were also the most efficient in suppressing T-cell proliferation. Transcriptome studies showed that the most potent BMSC batch also had higher expression of several known key immunomodulatory molecules such as hepatocyte growth factor (HGF), PDL1, and numerous members of the PGE2 pathway, including PTGS1 and TLR4. Multiplex ELISA experiments revealed higher expression of HGF and IL6 by the most potent BMSC donor. Based on these findings, we propose that THP-1 cells may be used to assess BMSC immunosuppressive activity as a product characterization assay.

Evaluation and management of symptomatic duodenal diverticula: a single-center retrospective analysis of 647 patients.

Aims: To explore the clinical characteristics of patients with symptomatic duodenal diverticula and to generalize how to make appropriate treatment choices for this group of patients. Materials and methods: From January 2010 to September 2020, a total of 647 patients with duodenal diverticula (DD) were included in this study. 345 of them with relevant symptoms were divided into the symptomatic group and the other 302 patients were in the asymptomatic group. Results: Among all patients, most DD were located in the periampullary area, <1 cm in size, and single in number. The distribution of DD localized in the 2nd portion/periampullary (P = 0.002/P < 0.001) and with a 1 cm size cut-off value (P = 0.003) was significantly different between the symptomatic and asymptomatic groups. Multivariate Logistics analysis further suggests that diverticular size (<1 cm, 1-3 cm) and combined biliary comorbidities (bile duct stones and gallstones, primary bile duct stones, cholangitis without bile duct stones) may be factors influencing the choice of treatment modality. Of all patients undergoing surgical treatment, a total of 7 cases developed various postoperative complications, and no one died. Conclusions: Patients with DD ≥1 cm or located in the periampullary were more likely to be symptomatic. The specific size of the DD and the combination of specific biliary comorbidities may have an impact on the choice of treatment modality.

Prognostic nomogram for primary splenic lymphoma: a SEER database-based study.

This study aimed to establish a nomogram model based on the clinicopathological factors affecting the prognosis of patients with primary splenic lymphoma (PSL) to predict the overall survival (OS) and cancer-specific survival (CSS) of patients. A total of 4074 patients diagnosed with PSL were included in this study. Among them, 4052 cases from the SEER (Surveillance, Epidemiology, and End Results) database were randomized into a training set and an internal validation set in a 7:3 ratio. Another 22 patients from the First Affiliated Hospital of Xi'an Jiaotong University were used as an external validation set. The prognostic factors affecting the OS and CSS of patients were analyzed using univariate and multivariate Cox regression models. Survival analysis was performed using Kaplan-Meier (KM) method and compared by Log-rank test. Then, a nomogram model was established to predict OS and CSS. Finally, the model was validated both internally and externally using the concordance index (C-index), receiver operating characteristic curve (ROC), and calibration curve to evaluate its predictive value, and the decision curve analysis (DCA) was conducted to assess its clinical utility. Our results showed that the model displayed a good prediction ability. In the training set, the OS rates at 1, 3, and 5 years were 85.9%, 75.8% and 70.1%, respectively, while the CSS rates at 1, 3, and 5 years were 91.9%, 86.2% and 82.3%, respectively. Predictors in the prediction model of OS included age, sex, marital status, Ann Arbor stage, histology, surgery, chemotherapy and year at diagnosis. On the other hand, predictors in the model of CSS included age, Ann Arbor stage, histology, chemotherapy, and year at diagnosis. Internal and external validation of the nomogram model showed that the C-index for predicting OS was 0.678 (0.662, 0.694) in the training set, 0.672 (0.648, 0.696) in the internal validation set, and 0.704 (0.565, 0.843) in the external validation set; the C-index for predicting CSS was 0.685 (0.661, 0.709) in the training set, 0.683 (0.650, 0.716) in the internal validation set, and 0.676 (0.488, 0.864) in the external validation set. The calibration curves for several groups showed good consistency, and DCA suggested its clinical usability. In conclusion, the nomogram constructed in this study has a good predictive value for the survival of patients with PSL, and can be a clinically applicable and practical prediction tool, facilitating rapid and accurate individualized predictions of the patient survival.

Indomethacin-induced spectral responses of naphthalimide-based dyes to serum albumin: effects of substituent and spacer.

Four indomethacin-naphthalimide binaries with different proton receptors at 4-position of naphthalimide were designed and synthesized. N,N-Dimethylethylenediamine and N-methyl piperazine were served as proton receptors as well as solubility regulators. Indomethacin, an inhibitor for cyclooxygenase-2 overexpressed on cancer cells, was connected at the imine N through different spacers. The attachment of indomethacin significantly quenched the fluorescence of all compounds with obvious red-shift in the absorption maxima due to the strong photo-induced electron transfer process of the folded-state. Human serum albumin (HSA) triggered about 15-fold fluorescence enhancements of DMN-IMC-5 with 30 nm blue-shift. However, it caused much smaller fluorescence increments of other compounds, suggesting that indomethacin, the linker and proton receptor play critical roles in HSA identification. Fluorescence bioimaging results show that indomethacin enables the naphthalimide-based compounds to fluorescent imaging living cells. Molecular docking reveals that the introduction of indomethacin improved the binding affinity of the dyes to HSA.

The small silver nanoparticle-assisted homogeneous sensing of thiocyanate ions with an ultra-wide window based on surface-enhanced Raman-extinction spectroscopy.

For the first time, we present an original sensing strategy with an ultra-wide detection window from 17 nM to 20 mM to detect SCN- ions. Initially, we investigated the clustering and optical properties of noble metal sol nanoparticles (NPs) due to the competitive interaction of thiocyanate ions (SCN-) and cetyltrimethylammonium bromide (CTAB) under weak acidic conditions, and found that different dimensions and scales of nanoclusters containing the alkyne-embedded Au@Ag NPs and relatively small Ag NPs could be achieved by the mediation of CTAB through electrostatic forces and hydrophobic interaction, in which SCN- could be covalently bonded with the silver surface of NPs to form a compact molecular layer (-Ag-S-C[triple bond, length as m-dash]N), and CTAB could only occupy remaining sites. In this process, we found that SCN- always runs counter to CTAB and tends to dissolve nanoclusters, so that they occupy the exposed surface of NPs in nanoclusters rather than the binding sites of one another. Remarkably, when the concentration of SCN- initially increased, two highly recognizable SERS emissions, which were assigned to alkyne reporter molecules (2208 cm-1) and C[triple bond, length as m-dash]N of SCN- (2110 cm-1), respectively, were rapidly detected, and their ratios (I2110/I2208) increased linearly proportional to the concentration of SCN- over a range of 17 nM to 172 µM, with a limit of detection (LOD) of 10 nM. With the further increase of SCN-, small Ag NPs started to desorb from the surface of individual Au@Ag NPs and dissociated in the solution but did not contribute to SERS signals. Instead, the surface plasmon resonance (SPR) peak of pure silver NPs at 385 nm increased gradually in the range from 0.5 to 20 mM with an LOD of 0.2 mM. Of particular significance, this simple sensor in conjunction with surface-enhanced Raman-extinction spectroscopy can be used for the rapid detection of extensive samples with an ultra-wide detection window, such as body fluids (saliva, urine, and serum) and food (milk powder and brassica vegetables), which is far superior to that of ion chromatography (IC).

Group O plasma as a media supplement for CAR-T cells and other adoptive T-cell therapies.

BACKGROUND: Most chimeric antigen receptor T (CAR-T) cells and other adoptive T-cell therapies (ACTs) are currently manufactured by ex vivo expansion of patient lymphocytes in culture media supplemented with human plasma from group AB donors. As lymphocytes do not express A or B antigens, the isoagglutinins of non-AB plasmas are unlikely to cause deleterious effects on lymphocytes in culture. STUDY DESIGN AND METHODS: Seeding cultures with peripheral blood mononuclear cell (PBMNC) concentrates from group A1 donors and using a CAR-T culture protocol, parallel cultures were performed, each with unique donor plasmas as media supplements (including group O plasmas with high-titer anti-A and group AB plasmas as control). An additional variable, a 3% group A1 red blood cell (RBC) spike, was added to simulate a RBC-contaminated PBMNC collection. Cultures were monitored by cell count, viability, flow cytometric phenotype, gene expression analysis, and supernatant chemokine analysis. RESULTS: There was no difference in lymphocyte expansion or phenotype when cultured with AB plasma or O plasma with high-titer anti-A. Compared to controls, the presence of contaminating RBCs in lymphocyte culture led to poor lymphocyte expansion and a less desirable phenotype-irrespective of the isoagglutinin titer of the plasma supplement used. CONCLUSIONS: This study suggests that ABO incompatible plasma may be used as a media supplement when culturing cell types that do not express ABO antigens-such as lymphocytes for CAR-T or other ACT. The presence of contaminating RBCs in culture was disadvantageous independent of isoagglutinin titer.

Sustainable remediation of diesel-contaminated soil by low temperature thermal treatment: Improved energy efficiency and soil reusability.

Thermal treatment can effectively remediate diesel-contaminated soil, but is considered unsustainable because of its energy-intensive nature and potential to damage soil properties. Here, we used low temperature thermal treatment (LTTT) as an energy-efficient technique to remediate diesel-contaminated soil. The impacts of LTTT on the physiochemical and ecological properties of soils were investigated to evaluate the reusability of heated soil. Heating at 250 °C for 10 min reduced the concentration of the total petroleum hydrocarbons from 6271 mg/kg to 359  mg/kg, which is lower than the Chinese risk screening level of 826 mg/kg. After LTTT, most soil physiochemical properties were nearly unchanged, and the NO3--N and NH4+-N contents increased. Moreover, LTTT-remediated soil was favorable for the germination and early growth of wheat. The microbial community changed substantially, but recovered after being mixed with uncontaminated soil. Finally, exploration of the mechanisms of LTTT revealed that pyrolysis was the dominant mechanism of diesel removal. A biochar-like pyrolytic carbon was formed, which improved the soil reusability.

Production of a cellular product consisting of monocytes stimulated with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for human use.

BACKGROUND: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. METHODS: Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. RESULTS: Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. CONCLUSION: Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.

Accurate Clinical Diagnosis of Liver Cancer Based on Simultaneous Detection of Ternary Specific Antigens by Magnetic Induced Mixing Surface-Enhanced Raman Scattering Emissions.

Establishing an accurate, simple, and rapid serodiagnosis method aiming for specific cancer antigens is critically important for the clinical diagnosis, therapy, and prognostication of cancer. Currently, surface-enhanced Raman scattering (SERS) readout techniques challenge fluorescent-based detection methods in terms of both optical stability and more importantly multiple detection capability, which become more desirable for clinical diagnostics. We thus started using an interference-free mixing SERS emission (m-SERS) readout to simultaneously indicate, for the first time, three specific liver cancer antigens, including α-fetoprotein (AFP), carcinoembryonic antigen (CEA), and ferritin (FER), even in one clinical serum sample. Here, three triple bonds (C≡N and C≡C) coded SERS tags contribute separate SERS emissions located at 2105, 2159, and 2227 cm-1, respectively; must have one-to-one correspondence from AFP, to FER, to CEA, In the process of detection, the mature double antibody sandwich allows the formation of microscale core-satellite assembly structure between a magnetic bead (MB) and single SERS tags, and therefore a pure and single SERS emission can be observed under the routine excitation laser spot. Because of the action of magnetic force, the uniform 3D packing of SERS tags absorbed MBs will in contrast generate a so-called m-SERS signals. With the help of enrichment and separation by MBs, the proposed m-SERS immunoassay provides an extremely rapid, sensitive, and accurate solution for multiplex detection of antigens or other biomarkers. Herein, the limit of detection (LOD) for simultaneous m-SERS detection of AFP, CEA, and FER was 0.15, 20, and 4 pg/mL, respectively. As expected for 39 clinical serum samples, simultaneous detection of ternary specific antigens can significantly improve the accuracy of liver cancer diagnosis.

Single cell sequencing reveals gene expression signatures associated with bone marrow stromal cell subpopulations and time in culture.

BACKGROUND: Bone marrow stromal cells (BMSCs) are a heterogeneous population that participates in wound healing, immune modulation and tissue regeneration. Next generation sequencing was used to analyze transcripts from single BMSCs in order to better characterize BMSC subpopulations. METHODS: Cryopreserved passage 2 BMSCs from one healthy subject were cultured through passage 10. The transcriptomes of bulk BMSCs from designated passages were analyzed with microarrays and RNA sequencing (RNA-Seq). For some passages, single BMSCs were separated using microfluidics and their transcriptomes were analyzed by RNA-Seq. RESULTS: Transcriptome analysis by microarray and RNA-Seq of unseparated BMSCs from passages 2, 4, 6, 8, 9 and 10 yielded similar results; both data sets grouped passages 4 and 6 and passages 9 and 10 together and genes differentially expressed among these early and late passage BMSCs were similar. 3D Diffusion map visualization of single BMSCs from passages 3, 4, 6, 8 and 9 clustered passages 3 and 9 into two distinct groups, but there was considerable overlap for passages 4, 6 and 8 cells. Markers for early passage, FGFR2, and late passage BMSCs, PLAT, were able to identify three subpopulations within passage 3 BMSCs; one that expressed high levels of FGFR2 and low levels of PLAT; one that expressed low levels of FGFR2 and high levels of PLAT and one that expressed intermediate levels of FGFR2 and low levels of PLAT. CONCLUSIONS: Single BMSCs can be separated by microfluidics and their transcriptome analyzed by next generation sequencing. Single cell analysis of early passage BMSCs identified a subpopulation of cells expressing high levels of FGFR2 that might include skeletal stem cells.

Splicing Nanoparticles-Based "Click" SERS Could Aid Multiplex Liquid Biopsy and Accurate Cellular Imaging.

Here, a completely new readout technique, so-called "Click" SERS, has been developed based on Raman scattered light splice derived from nanoparticle (NP) assemblies. The single and narrow (1-2 nm) emission originating from triple bond-containing reporters undergoes dynamic combinatorial output, by means of controllable splice of SERS-active NPs analogous to small molecule units in click chemistry. Entirely different to conventional "sole code related to sole target" readout protocol, the intuitional, predictable and uniquely identifiable "Click" SERS is relies on the number rather than the intensity of combinatorial emissions. By this technique, 10-plex synchronous biomarkers detection under a single scan, and accurate cellular imaging under double exposure have been achieved. "Click" SERS demonstrated multiple single band Raman scattering could be an authentic optical analysis method in biomedicine.

Plasma from some cancer patients inhibits adenoviral Ad5f35 vector transduction of dendritic cells.

BACKGROUND: Pooled AB serum is often used as a media supplement for cell culture but it has the potential to transmit infectious diseases. To avoid this risk, we used autologous plasma as a media supplement for manufacturing dendritic cells (DCs) for cancer immunotherapy. We noticed inconsistencies in the DCs and investigated their nature and cause. METHODS: Adenovirus human epidural growth factor receptor 2 (adHER2/neu) DCs for 21 patients were manufactured from autologous peripheral blood monocytes that were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 3 days, transduced with Ad5f35HER2ECTM and then treated with lipopolysaccharide and interferon (IFN)-γ for 1 day. The cells were cultured in RPMI-1640 supplemented with either 10% heat inactivated autologous or AB plasma. RESULTS: Twenty-eight adHER2/neu DCs were manufactured for 21 patients using autologous plasma and 68 were manufactured for 20 of those patients using AB plasma. The expression of human epidural growth factor receptor 2 (HER2/neu) was less for DCs manufactured with autologous plasma (70.3 ± 33.3% versus 86.1 ± 22.8%; P <0.01). Manufacturing adHER2/neu DCs using monocytes from three healthy subjects and plasma from one patient with low HER2/neu expression (18%) resulted in low HER2/neu expression by all three DCs (13%, 16% and 23%). Analysis of the levels of 1322 proteins in eight plasma samples associated with low HER2/neu expression and in 12 associated with high HER2/neu expression revealed that the levels of 14 predicted HER2/neu transduction efficiency. CONCLUSION: The manufacture of adHER2/neu DC using autologous plasma as a media supplement resulted in inconsistent HER2/neu expression. It is likely that variability in the levels of multiple proteins in autologous plasma contributed to low HER2/neu expression.

Comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum.

BACKGROUND: Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture. METHODS: A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared. RESULTS: Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF. CONCLUSIONS: Traditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

BACKGROUND: Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. METHODS: TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. RESULTS: The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. CONCLUSION: Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

Rapid and Reliable Detection of Alkaline Phosphatase by a Hot Spots Amplification Strategy Based on Well-Controlled Assembly on Single Nanoparticle.

The first appeal of clinical assay is always accurate and rapid. For alkaline phosphatase (ALP) monitoring in medical treatment, a rapid, reliable surface-enhanced Raman scattering (SERS) test kit is designed based on a "hot spots" amplification strategy. Consisting of alkyne-tagged Au nanoparticles (NPs), Ag+, and enzyme substrate, the packaged test kit can achieve one-step clinical assay of ALP in human serum within several minutes, while the operation is simple as it directly inputs the sample into the test kit. Here, Ag+ ions are adsorbed onto the surface of Au core due to electrostatic interaction between Ag+ and the negatively charged donor surface, then enzymatic biocatalysis of ALP triggers the reduction of Ag+ and subsequently silver growth occurs on every Au core surface in a controllable manner, forming "hot spots" between the Au core and Ag shell, in which the SERS signal of alkyne Raman reporters would be highly amplified. Meanwhile, ALP mediates a redox reaction of Ag+ as well as the dynamic silver coating process so the increase of SERS intensity is well-controlled and can be recognized with increasing amounts of the targets. Instead of conventional NP aggregation, this leads to a more reproducible result. In particular, the distinct Raman emission from our self-synthesized alkyne reporter is narrow and stable with zero background in the Raman silent region, suffering no optical fluctuation from biosystem inputs and the detection results are therefore reliable with a limit of detection of 0.01 U/L (2.3 pg/mL). Along with ultrahigh stability, this SERS test kit therefore is an important point-of-care candidate for a reliable, efficacious, and highly sensitive detection method for ALP, which potentially decreases the need for time-consuming clinical trials.

Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers.

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells.

BACKGROUND: Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. METHODS: Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products. RESULTS: When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells. CONCLUSIONS: Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

Autologous lymphapheresis for the production of chimeric antigen receptor T cells.

BACKGROUND: The first step in manufacturing chimeric antigen receptor (CAR) T cells is to collect autologous CD3+ lymphocytes by apheresis. Patients, however, often have leukopenia or have other disease-related complications. We evaluated the feasibility of collecting adequate numbers of CD3+ cells, risk factors for inadequate collections, and the rate of adverse events. STUDY DESIGN AND METHODS: Apheresis lymphocyte collections from patients participating in three CAR T-cell clinical trials were reviewed. Collections were performed on the COBE Spectra by experienced nurses, with the goal of obtaining a minimum of 0.6 × 109 and a target of 2 × 109 CD3+ cells. Preapheresis peripheral blood counts, apheresis parameters, and product cell counts were analyzed. RESULTS: Of the 71 collections, 69 (97%) achieved the minimum and 55 (77%) achieved the target. Before apheresis, the 16 patients with yields below the target had significantly lower proportions and absolute numbers of circulating lymphocytes and CD3+ lymphocytes and higher proportions of circulating blasts and NK cells than those who achieved the target (470 × 106 lymphocytes/L vs. 1340 × 106 lymphocytes/L, p = 0.008; 349 × 106 CD3+ cells/L vs. 914 × 106 CD3+ cells/L, p = 0.001; 17.6% blasts vs. 4.55% blasts, p = 0.029). Enrichment of blasts in the product compared to the peripheral blood occurred in four patients, including the two patients whose collections did not yield the minimum number of CD3+ cells. Apheresis complications occurred in 11 patients (15%) and, with one exception, were easily managed in the apheresis clinic. CONCLUSIONS: In most patients undergoing CAR T-cell therapy, leukapheresis is well tolerated, and adequate numbers of CD3+ lymphocytes are collected.