Metabolic profiling identifies Qrich2 as a novel glutamine sensor that regulates microtubule glutamylation and mitochondrial function in mouse sperm.

In our prior investigation, we discerned loss-of-function variants within the gene encoding glutamine-rich protein 2 (QRICH2) in two consanguineous families, leading to various morphological abnormalities in sperm flagella and male infertility. The Qrich2 knockout (KO) in mice also exhibits multiple morphological abnormalities of the flagella (MMAF) phenotype with a significantly decreased sperm motility. However, how ORICH2 regulates the formation of sperm flagella remains unclear. Abnormal glutamylation levels of tubulin cause dysplastic microtubules and flagella, eventually resulting in the decline of sperm motility and male infertility. In the current study, by further analyzing the Qrich2 KO mouse sperm, we found a reduced glutamylation level and instability of tubulin in Qrich2 KO mouse sperm flagella. In addition, we found that the amino acid metabolism was dysregulated in both testes and sperm, leading to the accumulated glutamine (Gln) and reduced glutamate (Glu) concentrations, and disorderly expressed genes responsible for Gln/Glu metabolism. Interestingly, mice fed with diets devoid of Gln/Glu phenocopied the Qrich2 KO mice. Furthermore, we identified several mitochondrial marker proteins that could not be correctly localized in sperm flagella, which might be responsible for the reduced mitochondrial function contributing to the reduced sperm motility in Qrich2 KO mice. Our study reveals a crucial role of a normal Gln/Glu metabolism in maintaining the structural stability of the microtubules in sperm flagella by regulating the glutamylation levels of the tubulin and identifies Qrich2 as a possible novel Gln sensor that regulates microtubule glutamylation and mitochondrial function in mouse sperm.

Assessing the clinical diagnostic value of anti-Müllerian hormone in polycystic ovarian syndrome and its correlation with clinical and metabolism indicators.

BACKGROUND: This study investigated the association between Anti-Müllerian Hormone (AMH) and relevant metabolic parameters and assessed its predictive value in the clinical diagnosis of polycystic ovarian syndrome (PCOS). METHODS: A total of 421 women aged 20-37 years were allocated to the PCOS (n = 168) and control (n = 253) groups, and their metabolic and hormonal parameters were compared. Spearman correlation analysis was conducted to investigate associations, binary logistic regression was used to determine PCOS risk factors, and receiver operating characteristic (ROC) curves were generated to evaluate the predictive value of AMH in diagnosing PCOS. RESULTS: The PCOS group demonstrated significantly higher blood lipid, luteinizing hormone (LH), and AMH levels than the control group. Glucose and lipid metabolism and hormonal disorders in the PCOS group were more significant than in the control group among individuals with and without obesity. LH, TSTO, and AMH were identified as independent risk factors for PCOS. AMH along with LH, and antral follicle count demonstrated a high predictive value for diagnosing PCOS. CONCLUSION: AMH exhibited robust diagnostic use for identifying PCOS and could be considered a marker for screening PCOS to improve PCOS diagnostic accuracy. Attention should be paid to the effect of glucose and lipid metabolism on the hormonal and related parameters of PCOS populations.

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying a bispecific T cell engager against hepatocellular carcinoma.

We previously established a hepatocellular carcinoma (HCC) targeting system of conditionally replicative adenovirus (CRAd) delivered by human umbilical cord-derived mesenchymal stem cells (HUMSCs). However, this system needed to be developed further to enhance the antitumor effect and overcome the limitations caused by the alpha-fetoprotein (AFP) heterogeneity of HCC. In this study, a bispecific T cell engager (BiTE) targeting programmed death ligand 1 controlled by the human telomerase reverse transcriptase promoter was armed on the CRAd of the old system. It was demonstrated on orthotopic transplantation model mice that the new system had a better anti-tumor effect with no more damage to extrahepatic organs and less liver injury, and the infiltration and activation of T cells were significantly enhanced in the tumor tissues of the model mice treated with the new system. Importantly, we confirmed that the new system eliminated the AFP-negative cells on AFP heterogeneous tumor models efficiently. Conclusion: Compared with the old system, the new system provided a more effective and safer strategy against HCC.

A Novel CD3/BCMA Bispecific T-cell Redirecting Antibody for the Treatment of Multiple Myeloma.

Multiple myeloma (MM) is a B-cell malignancy for which new treatments are urgently needed. Redirecting the activity of T cells by bispecific antibodies against tumor cells is a potent approach. The B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein and therefore is an ideal therapeutic target for T-cell redirecting therapies. The main objective of this work is to target the BCMA by generating BCMA-specific murine monoclonal antibody and construct a cluster of differentiation 3 (CD3)/BCMA-directed tandem diabodies (Tandab). In brief, using standard hybridoma technology, we developed a novel BCMA-specific monoclonal antibody (clone 69G8), that specifically bind with BCMA+ cell lines and MM patient sample; whereas BCMA- cells were not recognized. For T cells by bispecific antibodies application, we constructed a Tandab (CD3/BCMA) simultaneously targeting both CD3 and BCMA and our studies demonstrated that Tandab (CD3/BCMA) was functional with specific binding capability both for CD3+ cells and BCMA+ cells. It induced selective, dose-dependent lysis of BCMA+ cell lines, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA- cells were not affected. Furthermore, we demonstrated that Tandab activity correlates with BCMA expression, with higher potency observed in highly BCMA expressing tumor cells. In vivo, the purified Tandab (CD3/BCMA) significantly inhibited the tumor growth in a subcutaneous NCI-H929 xenograft model. Taken together, these results show that the Tandab (CD3/BCMA) displays potent and selective anti-MM activities and represents a promising immunotherapeutic for the treatment of MM.

A Novel Blockade CD47 Antibody With Therapeutic Potential for Cancer.

Macrophages as components of the innate immune system play a critical role in antitumor responses. Strategies for targeting CD47 are becoming a hot spot for cancer therapy. The expression of CD47 is exercised by macrophages to make a distinction between "self" or "nonself." Anti-CD47 antibodies block the interaction between macrophage signal regulatory protein-α (SIRPα) and tumor surface CD47. In this study, we report and assess a novel anti-CD47 blocking antibody named 2C8, which exhibits high affinity and tremendous anticancer effects. More concretely, 2C8 significantly induces macrophages, including protumorigenic subtype M2 macrophages killing tumor cells in vitro, and is revealed to be more effective than commercially available anti-CD47 mAb B6H12.2. In vivo, 2C8 controls tumor growth and extends survival of xenograft mice. The antitumor ability of 2C8 might be applicable to many other cancers. The generation of a novel CD47 antibody contributes to consolidating clinical interest in targeting macrophages for the treatment of malignancy and, moreover, as a supplement therapy when patients are resistant or refractory to other checkpoint therapies or relapse after such treatments.

An Engineered Fusion Protein Anti-CD19(Fab)-LDM Effectively Inhibits ADR-Resistant B Cell Lymphoma.

The 5-year survival rate of patients with B cell lymphoma is about 50% after initial diagnosis, mainly because of resistance to chemotherapy. Hence, it is necessary to understand the mechanism of chemo-resistance and to explore novel methods to circumvent multidrug resistance. Previously, we showed that an engineered cytotoxic fusion protein anti-CD19(Fab)-LDM (lidamycin), can induce apoptosis of B-lymphoma cells. Herein, we successfully established an adriamycin (ADR)-resistant B cell lymphoma cell line BJAB/ADR. The mRNA and protein level of ATP-binding cassette subfamily B member 1 (ABCB1) were significantly overexpressed in BJAB/ADR cells. Increased efflux function of ABCB1 was observed by analyzing intracellular accumulation and efflux of Rhodamine 123. The efflux of Rhodamine 123 could be significantly ameliorated by verapamil. Treatment with anti-CD19(Fab)-LDM at different concentrations induced cytotoxic response of BJAB/ADR cells similar to that of the sensitive cells. In vivo studies showed that anti-CD19(Fab)-LDM had better antitumor effect in BJAB and BJAB/ADR cell lymphoma xenografts compared with ADR or LDM treatment alone. Taken together, anti-CD19(Fab)-LDM can effectively inhibit the growth of BJAB/ADR cells both in vitro and in vivo. Anti-CD19(Fab)-LDM could be a promising molecule for the treatment of drug resistant cancers.

Bispecific CD3-HAC carried by E1A-engineered mesenchymal stromal cells against metastatic breast cancer by blocking PD-L1 and activating T cells.

BACKGROUND: PD-1/PD-L1 blockade can confer durable benefits in the treatment of metastatic cancers, but the response rate remains modest and potential adverse effects occur sometimes. Concentrating immunotherapeutic agents at the site of disease was believed to break local immune tolerance and reduce systemic toxicity. E1A-engineered mesenchymal stromal cell (MSC.E1A) was an attractive transfer system that preferentially homing and treating cancer metastasis, through which the tumor cells were modified by locally replicated adenoviruses to release CD3-HAC, a bifunctional fusion protein that anti-CD3 scfv linked with high-affinity consensus (HAC) PD-1. Subsequently, CD3-HAC, wbich was bound on PD-L1-positive breast cancer cells, recruited T cells to exhibit a potent antitumor immunity incombination with immune checkpoint blockade. METHODS: We constructed the CD3-HAC gene driven by human telomerase reverse transcriptase (hTERT) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. The homing property of MSCs in vivo was analyzed with firefly luciferase-labeled MSCs (MSC.Luc) by bioluminescent imaging (BLI). The cytotoxicity of T cells induced by CD3-HAC towards PD-L1-positive cells was detected in vitro and in vivo in combination with 5-FU. RESULTS: Our data suggest that CD3-HAC could specifically bind to PD-L1-positive tumor cells and induce lymphocyte-mediated lysis effectively both in vitro and in vivo. The intervention with HAC diminished the effects of PD-1/PD-L1 axis on T cells exposed to MDA-MB-231 cells and increased lymphocytes activation. MSCs infected by AdCD3-HAC followed by LentiR.E1A could specially migrate to metastasis of breast cancer and produce adenoviruses in the tumor sites. Furthermore, treatment with MSC.CD3-HAC.E1A in combination with 5-FU significantly inhibited the tumor growth in mice. CONCLUSIONS: This adenovirus-loaded MSC.E1A system provides a promising strategy for the identification and elimination of metastasis with locally released immuno-modulator.

Up-regulated lncRNA XIST contributes to progression of cervical cancer via regulating miR-140-5p and ORC1.

BACKGROUND: The study purpose was to make investigation into the influence of XIST on cervical cancer progression and what's more its potential mechanism. METHODS: The cervical cancer data sets (lncRNA, miRNA, and mRNA) obtained from TCGA were analyzed with the "mixOmics" R package. Then, the expression of XIST, miR-140-5p, and ORC1 were detected using qRT-PCR and western blot in both tissues and cervical cancer cell lines (Hela and C33A) to verify the bioinformatics analyses results. CCK-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) assays, cell cycle assay and cell apoptosis assay were practiced. Besides, immunohistochemistry staining was operated for the detection of the Ki-67, E-cadherin and vimentin expression in cervical cancer tissues and the apoptosis-related proteins expression (c-caspase3, Bcl-2, total PARP and cleaved PARP) was verified through western blot. And in vivo experiments were implemented. RESULTS: MiR-140-5p was down-regulated but XIST and ORC1 were up-regulated in cervical cancer tissues and cell lines. Knocking down of the XIST or ORC1 memorably suppressed cell proliferation, blocked cell cycle, decreased the expression of Bcl-2 while increased the apoptosis rate and the expression of c-caspase3 and cleaved PARP in HeLa and C33A cells. Besides, the results of immunohistochemistry staining showed knocking down the expression of XIST improved the expression levels of E-cadherin and decreased Ki-67 and vimentin expression. And overexpression of miR-140-5p also could inhibit the progression and reverse the influence of XIST and ORC1 in HeLa and C33A cells. CONCLUSION: Our study indicated the effects of XIST/miR-140-5p/ORC1 axis on the progression of cervical cancer which will shed new light on epigenetic diagnostics and therapeutics in cervical cancer.

SPP1 inhibition improves the cisplatin chemo-sensitivity of cervical cancer cell lines.

PURPOSE: Cisplatin (DDP)-based chemotherapy is a standard strategy for cervical cancer, while chemoresistance remains a huge challenge. In the present study, we aimed to explore the effects of SPP1 on the proliferation and apoptosis rate of the HeLa cervical cancer cell line with cisplatin (DDP) resistance. METHODS: Microarray analysis was employed to select differentially expressed genes in cervical cancer tissues and adjacent tissues. Then, we established a DDP-resistant HeLa cell line (res-HeLa). Western blotting was used to detect SPP1 expression in both tissue and cells. After the transfection with si-SPP1 and pcDNA3.1-SPP1, colony formation and MTT assays were applied to detect cell proliferation changes. Flow cytometry was employed to detect the cell apoptosis rate. Western blotting was performed to verify the activation of PI3K/Akt signal pathway proteins related to DDP resistance. RESULTS: SPP1 was overexpressed in cervical cancer tissues and cell lines. Compared to normal HeLa cells, expression of SPP1 was significantly enhanced in res-HeLa cells. SPP1 knockdown resulted in repressed proliferation and enhanced apoptosis of res-HeLa cells, which could be reversed by SPP1 overexpression in HeLa cells. Additionally, downregulation of SPP1 improved the DDP sensitivity of HeLa by inhibiting the PI3K/Akt signaling pathway. CONCLUSION: SPP1 inhibition could suppress proliferation, induce apoptosis and increase the DDP chemo-sensitivity of HeLa cells.

Long noncoding RNA OPA-interacting protein 5 antisense transcript 1 upregulated SMAD3 expression to contribute to metastasis of cervical cancer by sponging miR-143-3p.

OBJECTIVES: SMAD3 is pivotal in the biology functions of various tumors. This study is aiming to study the relationship among SMAD3, long noncoding RNAs (lncRNAs) OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1), and miR-143-3p, and their effects on cervical cancer. METHODS: In our research, real-time polymerase chain reaction and western blot assay were conducted to detect the expression level of messenger RNA and protein in tumor tissues and cells. Transfection of lncRNA OIP5-AS1, miR-143-3p, or SMAD3 was performed to investigate their potential effects on the function of cell as well as the relationship among them in cervical cell lines via 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) together with transwell assays or dual-luciferase reporter assay respectively. RESULTS: SMAD3, lncRNA OIP5-AS1 expression is significantly enhanced in cervical cancer tissues and cell lines, but miR-143-3p was inhibited. LncRNA OIP5-AS1 is demonstrated to mediate the physiological process of cervical cancer cells. Moreover, silencing SMAD3 via siRNA suppressed cell number, viability, migration and invasion, whereas overexpression of OIP5-AS1 promoted these abilities. Furthermore, lncRNA OIP5-AS1 exert its function via sponging miR-143-3p to regulate SMAD3 expression. CONCLUSIONS: LncRNA OIP5-AS1 promoted SMAD3 expression via mediating miR-143-3p to promote migration and invasion of cervical cancer cells.

Induced CD20 Expression on B-Cell Malignant Cells Heightened the Cytotoxic Activity of Chimeric Antigen Receptor Engineered T Cells.

CD20 is an effective immunotherapy target for CD20+ B-cell malignant cells. Monoclonal antibody, especially rituximab, has been a conventional strategy in the treatment of B-cell malignancies such as non-Hodgkin's lymphoma. However, treatment with monoclonal antibodies has not been enough to overcome the refractory/relapse problems. Chimeric antigen receptor engineered T (CAR-T) cells have exhibited excellent therapeutic effect on lymphocytic leukemia in recent years. In this study, a CD20-specific CAR was constructed and the cytotoxic efficacy of CD20 CAR-T cells on B-cell malignant cells was evaluated by CD107a degranulation, pro-inflammation cytokine production, and true lytic ability in vitro and in vivo. It was found that CD20 CAR-T cells possessed stronger cytotoxic ability against CD20 highly expressed cells. Furthermore, when histone deacetylase inhibitor was used to enhance the expression of CD20 antigen on the surface of B-cell malignant cells via inducing acetylation of H3K9 on CD20 promoter site, it revealed that the cytotoxicity of CD20 CAR-T cells against histone deacetylase inhibitor-treated B-cell malignant cells was significantly enhanced.

Surface expression of anti-CD3scfv stimulates locoregional immunotherapy against hepatocellular carcinoma depending on the E1A-engineered human umbilical cord mesenchymal stem cells.

Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy.

The development of bispecific antibodies and their applications in tumor immune escape.

During the past two decades, a great evolution of bispecific antibodies (BsAbs) for therapeutic applications has been made. BsAbs can bind simultaneously two different antigens or epitopes, which leads to a wide range of applications including redirecting T cells or NK cells to tumor cells, blocking two different signaling pathways, dual targeting of different disease mediators, and delivering payloads to targeted sites. Aside from approved catumaxomab (anti-CD3 and anti-EpCAM) and blinatumomab (anti-CD3 and anti-CD19), many more BsAbs are now in various phases of clinical development. Here, this review focus on the development of bispecific antibodies and their applications in tumor immune escape.

Mesenchymal stromal cells as vehicles of tetravalent bispecific Tandab (CD3/CD19) for the treatment of B cell lymphoma combined with IDO pathway inhibitor D-1-methyl-tryptophan.

BACKGROUND: Although blinatumomab, a bispecific T cell engaging antibody, exhibits high clinical response rates in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia (B-ALL) and B cell non-Hodgkin's lymphoma (B-NHL), it still has some limitations because of its short half-life. Mesenchymal stromal cells (MSCs) represent an attractive approach for delivery of therapeutic agents to cancer sites owing to their tropism towards tumors, but their immunosuppression capabilities, especially induced by indoleamine 2,3-dioxygenase (IDO), should also be taken into consideration. METHODS: Human umbilical cord-derived MSCs (UC-MSCs) were genetically modified to secrete Tandab (CD3/CD19), a tetravalent bispecific tandem diabody with two binding sites for CD3 and two for CD19. The tropism of MSCs towards Raji cells in vitro was determined by migration assays, and the homing property of MSCs in vivo was analyzed with firefly luciferase-labeled MSCs (MSC-Luc) by bioluminescent imaging (BLI). The cytotoxicity of T cells induced by MSC-secreting Tandab (CD3/CD19) was detected in vitro and in vivo in combination with D-1-methyl-tryptophan (D-1MT), an IDO pathway inhibitor. RESULTS: The purified Tandab (CD3/CD19) was functional with high-binding capability both for CD3-positive cells and CD19-positive cells and was able to induce specific lysis of CD19-positive cell lines (Raji, Daudi, and BJAB) in the presence of T cells. Additionally, results from co-culture killing experiments demonstrated that Tandab (CD3/CD19) secreted from MSCs was also effective. Then, we confirmed that D-1MT could enhance the cytotoxicity of T cells triggered by MSC-Tandab through reversing T cell anergy with down-regulation of CD98 and Jumonji and restoring the proliferation capacity of T cells. Furthermore, MSC-Luc could selectively migrate to tumor site in a BALB/c nude mouse model with Raji cells. And mice injected with MSC-Tandab in combination with D-1MT significantly inhibited the tumor growth. CONCLUSIONS: These results suggest that UC-MSCs releasing Tandab (CD3/CD19) is an efficient therapeutic tool for the treatment of B cell lymphoma when combined with D-1MT.

Mesenchymal stem cells deliver and release conditionally replicative adenovirus depending on hepatic differentiation to eliminate hepatocellular carcinoma cells specifically.

Currently, it is a key challenge to remove the postsurgical residuals and metastasis of hepatocellular carcinoma (HCC). Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer; however, the difficulty remains regarding its intravenous administration. The aim of this study was to develop a targeted therapeutic system which has great potential to overcome the postsurgical residuals and metastasis of HCC. In this system, we developed a conditionally replicative adenovirus (CRAd) loaded on human umbilical cord-derived mesenchymal stem cells (HUMSCs), in which the CRAd contained an adenovirus E1A gene dual regulated by α-fetoprotein promoter and microRNA-122 target sequence. When HUMSCs homed to the tumor sites and differentiated into hepatocyte-like cells within tumor microenvironment, the CRAds were packaged and released strictly to the local tumor. Subsequently, the CRAd lysed tumor cells selectively with the post-infection regulation. The study showed the specific oncolytic effect of the CRAd to HCC cells and the production of the CRAd by differentiated HUMSCs in vitro. Furthermore, we proved the hepatocyte-like transformation of HUMSC in the microenvironment of orthotopic or heterotopic hepatoma. Finally, this therapeutic system exhibited dramatic tumor inhibition on both orthotopic and subcutaneous hepatic xenograft tumor model mice with less toxicity on normal organs. The study results have demonstrated that this targeted therapeutic strategy is a promising method to resolve the problem of postsurgical residuals and metastasis of HCC.

E1A-engineered human umbilical cord mesenchymal stem cells as carriers and amplifiers for adenovirus suppress hepatocarcinoma in mice.

Gene therapy is an attractive approach for hepatocellular carcinoma (HCC) patients. Nevertheless, efficient transgene delivery remains a challenge. In this study, we explored a new targeted system based on human umbilical cord-derived mesenchymal stem cells (HUMSCs), which were engineered to deliver adenovirus to tumor sites, and to replicate and assemble into new adenovirus against HCC. Our results showed that HUMSCs infected by Ad-hTERTp-IL24 followed by LentiR.E1A infection could specifically migrate to HepG2 tumor cells and support adenoviral replication in vitro and in vivo 36 h after LentiR.E1A infection. Ad-hTERTp-IL24 specifically inhibited HepG2 cells growth, and this inhibitory effect was enhanced by low doses of 5-fluorouracil (5-Fu), because the expression levels of coxsackie adenovirus receptor (CAR) and integrin ανß3 on tumor cells were significantly increased, causing higher viral uptake. Compared with the no treatment groups, Ad-hTERTp-IL24 and LentiR.E1A co-loaded HUMSCs exhibited significant anti-tumor activity in vivo, particularly in combination with low doses of 5-Fu. In summary, this study provides a promising targeted gene therapeutic strategy dependent on the tumor tropism of HUMSCs, to improve the outcome of virotherapy for tumor patients especially those with metastatic diseases.

The protective role of neuregulin-1: A potential therapy for sepsis-induced cardiomyopathy.

The extremely high mortality of sepsis in intensive care units, caused primarily by sepsis-induced cardiomyopathy, is a pressing issue. Current studies have revealed the importance of the neuregulin-1 (NRG-1)/ErbB signaling axis at the cardiovascular level and the positive effect of NRG-1 on cardiac function in patients with heart failure. To investigate the protective mechanism of NRG-1 against myocardial injury in septic rats, a cecal ligation and puncture (CLP) model was applied. Animals were administered either a vehicle or recombinant human NRG-1 (rhNRG-1, 10µg/kg). Their survival rates were noted 24h after CLP. The hemodynamic method was used to evaluate their cardiac function. The myocardial morphology was observed. An enzyme-linked immunosorbent assay was used to detect the level of cardiac troponin-T (cTn-T), cytokines, and angiotensin II (Ang II) in the serum and myocardium. Compared with the vehicle, rhNRG-1 improved survival of rats and prevented hemodynamic derangement, as reflected in the increased mean arterial pressure, left ventricular systolic pressure, ±dp/dt max, and decreased left ventricular end-diastolic pressure (P<0.05). Furthermore, the serum levels of cTn-T and pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and IL-6) were significantly increased in vehicle-treated rats but reduced in rhNRG-1-treated rats. The latter also showed decreased concentration of macrophage inhibitory factor and Ang II in the myocardium (P<0.05). These results suggest that NRG-1 improved cardiac function and protected cardiomyocytes of rats from CLP-induced sepsis by suppressing the immune inflammatory response and excessive activation of the renin-angiotensin-aldosterone system. Ultimately, NRG-1 increased the survival rate of rats.

A novel antibody-drug conjugate anti-CD19(Fab)-LDM in the treatment of B-cell non-Hodgkin lymphoma xenografts with enhanced anticancer activity.

BACKGROUND: Rituximab is widely used in clinical setting for the treatment of B malignant lymphoma and has achieved remarkable success. However, in most patients, the disease ultimately relapses and become resistant to rituximab. To overcome the limitation, there is still a need to find novel strategy for improving therapeutic efficacy. OBJECTIVE: To construct genetically engineered antibody anti-CD19(Fab)-LDM, and verify the anticancer activity targeted toward B-lymphoma. METHODS: The anticancer activity of anti-CD19(Fab)-LDM in vitro and in vivo was examined. In vitro, the binding activity and internalization of anti-CD19(Fab)-LDP were measured. Using comet assay and apoptosis, the cytotoxicity of energized fusion proteins was observed. From in vivo experiments, targeting of therapeutic effect and anticancer efficacy bythe fusion protein was verified. RESULTS: Data showed that anti-CD19(Fab)-LDM does not only binding the cell surface but is also internalized into the cell. The energized fusion proteins anti-CD19(Fab)-LDM can induce DNA damage. Furthermore, significant in vivo therapeutic efficacy was observed. CONCLUSION: The present study demonstrated that the genetically engineered antibody anti-CD19(Fab)-LDM exhibited enhanced cytotoxicity compared to LDM alone. One of the most powerful advantages of anti-CD19(Fab)-LDM, however, is that it can be internalized within the cells and carry out cytotoxic effects. Therefore, anti-CD19(Fab)-LDM may be as a useful targeted therapy for B-cell lymphoma.

Redirection of CD4+ and CD8+ T lymphocytes via an anti-CD3 × anti-CD19 bi-specific antibody combined with cytosine arabinoside and the efficient lysis of patient-derived B-ALL cells.

BACKGROUND: B-acute lymphoblastic leukemia (B-ALL) is derived from B cell progenitors. Recently, the development of appropriate combinations of chemotherapy and immunotherapy represents a promising approach for eliminating cancer. We previously constructed an anti-CD3 × anti-CD19 bi-specific antibody in a diabody configuration and its disulfide-stabilized format (ds-diabody). The combination of the diabody or ds-diabody and Ara-C was highly effective in enhancing the cytotoxicity of T cells against the CD19+ human leukemia cell-line, Nalm-6, both in vitro and in vivo. This study verified whether B-ALL patient-derived cells were sensitive to the diabody or ds-diabody and low-dosage Ara-C combination. METHODS: This study aimed to detect the B7 family members B7.1 (CD80) and B7.2 (CD86) that were expressed in B-ALL patient-derived cells pre-treated by Ara-C (0.25 µM) and to determine the targeted killing ability of T cell subtypes induced by the diabody or ds-diabody combination with Ara-C both in vitro and in vivo. We also determined the levels of the cytokines that were released by activated CD4+ or CD8+ T cells during therapy. RESULT: Low-dose Ara-C enhanced CD80 and CD86 expression in nearly 50 % of specimens of B-ALL patient-derived cells. A combination of diabody or ds-diabody and Ara-C enhanced T cell against B-ALL cells in vitro and in vivo. Both CD8+ and CD4+ T cells were potently activated. Expression of CD25 and CD69 was augmented equally by CD4+ or CD8+ T cells. However, CD8+ T cells made the major contribution by redirecting target cell lysis in a granzyme B and perforin-dependent mechanism. CD4+ T cells played an important immunomodulatory role by secreting IL2. Consequently, IL3, IL6, TNFα, and IFNγ were also released by CD4+ or CD8+ T cells following diabody-mediated T cell activation. CONCLUSION: T cell therapy induced by diabody or ds-diabody combined with low dose of Ara-C was effective against cancer cell-lines and in clinical trials. In vivo, the ds-diabody was more efficient than its parent diabody due to its enhanced stability.