Conservative management of a lymphocele that formed shortly after creation of an arteriovenous fistula for hemodialysis.

Patients with end-stage renal disease require to establish vascular access for regular hemodialysis. The creation of arteriovenous fistula (AVF) is usually a safe procedure; however, there could be complications such as bleeding, hematoma, pseudoaneurysm, thrombosis, infection, and steal syndrome. A rare complication of such vascular manipulation could be formation of lymphocele. We present a case of a 67-year-old man who presented with a progressively enlarging mass 12 days after the surgery for AVF creation at the site of surgery in the right upper arm. Ultrasonographic examination revealed a fluid-filled cystic structure measuring about 4.2 × 3.6 × 1.9 cm under the skin just above the anastomosis. The fluid was aspirated using ultrasound-guided fluoroscopy that relieved the swelling. The analysis of aspirate suggested the cyst to be a lymphocele. The mass re-enlarged to its previous size in the next 3 days. While under observation for signs of complication, regular intermittent compression and a low-fat diet completely resolved the lymphocele over the subsequent 3 months. The less common occurrence of such lymphocele post AVF creation needs to be evaluated for its potential for complication, in the absence of which the lymphocele is amenable to conservative management using regular intermittent compression and low-fat oral diet.

Chimeric antigen receptors containing the OX40 signalling domain enhance the persistence of T cells even under repeated stimulation with multiple myeloma target cells.

Persistence of CAR-T cell function is associated with relapse rate after CAR-T therapy, while co-stimulatory agents are highly concerned with the persistence of CAR-T cells. In this study, we designed and constructed a series of BCMA-targeting second-generation CAR constructs containing CD28, 41BB, and OX40 molecules, respectively, to identify the costimulatory domains most favorable for persistence. The results of routine in vitro studies showed that OX40-CAR-T and 41BB-CAR-T had similar antitumor effects and were superior to CD28-CAR-T in terms of proliferation and cytotoxicity. Although difficult to distinguish by conventional functional assays, OX40-CAR-T cells exhibited greater proliferation and enhanced immune memory than 41BB-CAR-T cells with the repeated stimulation assay by BCMA-expressing target cells. In vivo studies further demonstrated that OX40-CAR-T cells had stronger proliferative activity than 41BB-CAR-T cells, which was highly consistent with the in vitro antitumor activity and proliferation results. Our study provides for the first time a scientific basis for designing OX40-CAR-T cell therapy to improve relapse in patients with MM after CAR-T treatment.

Feasibility study of a novel preparation strategy for anti-CD7 CAR-T cells with a recombinant anti-CD7 blocking antibody.

Although chimeric antigen receptor (CAR) T cell immunotherapy has shown promising significance in B cell malignancies, success against T cell malignancies remains unsatisfactory because of shared antigenicity between normal and malignant T cells, resulting in fratricide and hindering CAR production for clinical treatment. Here, we report a new strategy of blocking the CD7 antigen on the T cell surface with a recombinant anti-CD7 antibody to obtain a sufficient amount of CD7-targeting CAR-T cells for T cell acute lymphoblastic leukemia (T-ALL) treatment. Feasibility was evaluated systematically, revealing that blocking the CD7 antigen with an antibody effectively blocked CD7-derived fratricide, increased the expansion rate, reduced the proportion of regulatory T (Treg) cells, maintained the stem cell-like characteristics of T cells, and restored the proportion of the CD8+ T cell population. Ultimately, we obtained anti-CD7 CAR-T cells that were specifically and effectively able to kill CD7 antigen-positive target cells, obviating the need for complex T cell modifications. This approach is safer than previous methods and provides a new, simple, and feasible strategy for clinical immunotherapies targeting CD7-positive malignant tumors.

Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase-mediated cassette exchange system.

Cell line development (CLD) by random integration (RI) can be labor intensive, inconsistent, and unpredictable due to uncontrolled gene integration after transfection. Unlike RI, targeted integration (TI) based CLD introduces the antibody-expressing cassette to a predetermined site by recombinase-mediated cassette exchange (RMCE). The key to success for the development of a TI host for therapeutic antibody production is to identify a transcriptionally active hotspot that enables highly efficient RMCE and antibody expression with good stability. In this study, a genome wide search for hotspots in the Chinese hamster ovary (CHO)-K1-M genome by either RI or PiggyBac (PB) transposase-based integration has been described. Two CHO-K1-M derived TI host cells were established with the Cre/Lox RMCE system and are described here. Both TI hosts contain a GFP-expressing landing pad flanked by two incompatible LoxP recombination sites (L3 and 2L). In addition, a third incompatible LoxP site (LoxFAS) is inserted in the GFP landing pad to enable an innovative two-plasmid based RMCE strategy, in which two separate vectors can be targeted to a single locus simultaneously. Cell lines generated by the TI system exhibit comparable or higher productivity, better stability and fewer sequence variant (SV) occurrences than the RI cell lines.

Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3.

Combined effects of fluoride and cadmium on liver and kidney function in male rats.

It has been shown that cadmium and fluoride may both have adverse effects on liver and kidney functions, but most studies focus on a single agent. In this study, we observed the effects of cadmium and fluoride on liver and kidney functions using a rat model. Total of 24 Sprague-Dawley male rats were divided into four groups, one control group and three exposure groups that were given cadmium (50 mg/L) and fluoride (100 mg/L) alone or in combination via drinking water. At the 12th week, urine, blood, and kidney tissues were collected. Aspartate transaminase, alanine transaminase (ALT), urinary ß2-microglobulin, and albumin were determined. Contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in liver and kidney homogenates were measured to evaluate oxidative stress. There was a significant increase in serum ALT and urinary ß2-microglobulin of rats in exposure groups compared with control. Serum ALT and urinary ß2-microglobulin of rats exposed to cadmium and fluoride in combination was significantly higher than those treated with cadmium alone and fluoride alone. SOD declined significantly and MDA increased in combination group compared with control and those treated with cadmium and fluoride alone. Cadmium and fluoride co-exposure increase the liver and kidney damage compared with that exposed to cadmium or fluoride alone.

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases.

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

TIM-4, a receptor for phosphatidylserine, controls adaptive immunity by regulating the removal of antigen-specific T cells.

Adaptive immunity is characterized by the expansion of an Ag-specific T cell population following Ag exposure. The precise mechanisms, however, that control the expansion and subsequent contraction in the number of Ag-specific T cells are not fully understood. We show that T cell/transmembrane, Ig, and mucin (TIM)-4, a receptor for phosphatidylserine, a marker of apoptotic cells, regulates adaptive immunity in part by mediating the removal of Ag-specific T cells during the contraction phase of the response. During Ag immunization or during infection with influenza A virus, blockade of TIM-4 on APCs increased the expansion of Ag-specific T cells, resulting in an increase in secondary immune responses. Conversely, overexpression of TIM-4 on APCs in transgenic mice reduced the number of Ag-specific T cells that remained after immunization, resulting in reduced secondary T cell responses. There was no change in the total number of cell divisions that T cells completed, no change in the per cell proliferative capacity of the remaining Ag-specific T cells, and no increase in the development of Ag-specific regulatory T cells in TIM-4 transgenic mice. Thus, TIM-4-expressing cells regulate adaptive immunity by mediating the removal of phosphatidylserine-expressing apoptotic, Ag-specific T cells, thereby controlling the number of Ag-specific T cells that remain after the clearance of Ag or infection.

Kruppel-like transcription factor 13 regulates T lymphocyte survival in vivo.

Krüppel-like transcription factor (KLF)13, previously shown to regulate RANTES expression in vitro, is a member of the Krüppel- like family of transcription factors that controls many growth and developmental processes. To ascertain the function of KLF13 in vivo, Klf13-deficient mice were generated by gene targeting. As expected, activated T lymphocytes from Klf13(-/-) mice show decreased RANTES expression. However, these mice also exhibit enlarged thymi and spleens. TUNEL, as well as spontaneous and activation-induced death assays, demonstrated that prolonged survival of Klf13(-/-) thymocytes was due to decreased apoptosis. Microarray analysis suggests that protection from apoptosis-inducing stimuli in Klf13(-/-) thymocytes is due in part to increased expression of BCL-X(L), a potent antiapoptotic factor. This finding was confirmed in splenocytes and total thymocytes by real-time quantitative PCR and Western blot as well as in CD4+CD8- single-positive thymocytes by real-time quantitative PCR. Furthermore, EMSA and luciferase reporter assays demonstrated that KLF13 binds to multiple sites within the Bcl-X(L) promoter and results in decreased Bcl-X(L) promoter activity, making KLF13 a negative regulator of BCL-X(L).

Determination of theophylline concentration in serum by chemiluminescent immunoassay.

OBJECTIVE: This study aimed to establish chemiluminescent immunoassay (CLIA) for quantitative determination of theophylline levels in human serum. METHODS: To measure the concentration of theophylline (n=122) and evaluate the assay. RESULTS: The linear range of the CLIA method was 0.51-40 mg/L (Y=1.02X+0.44, r=0.995). The intra and inter CV (coefficient variance) of CLIA were 3.20% and 3.57%, respectively. The average recovery rate was 102.3%. This method was free from interference by brilirubin (<200 micromol/L), hemoglobin (<10 g/L), and triglycerides (<15 mmol/L). CONCLUSION: This method is simple, convenient and precise for clinical pharmacokinetics study of theophylline.

The function role of GATA-3 in Th1 and Th2 differentiation.

GATA-3 plays a central role in regulating Th1 and Th2 cell differentiation. Upon interleukin (IL)-4 binding to its receptor, GATA-3 is induced through the action of Stat6. GATA-3 regulates Th2 cytokine expression not only at the transcription level, such as directly binding to the promoters of the IL-5 and IL-13 gene, but also by the involvement in the remodeling of the chromatin structure and opening the IL-4 locus. As a master control, GATA-3 stabilizes the Th2 phenotype by two methods. First, GATA-3 shuts down Th1 development through the repression the IL-12 receptor beta2-chain expression. Second, GATA-3 augments its own expression by a positive feedback autoregulation. In this article, we review the recent study of the function of GATA-3 in Th1 and Th2 differentiation.