RhIL-11 treatment normalized Th1/Th2 and T-bet/GATA-3 imbalance in in human immune thrombocytopenic purpura (ITP).

Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder characterized by reduction in platelet counts. T helper 1 (Th1) cells polarization with an increased shift of Th1/Th2 ratio has been reported in ITP. This shift is associated with transcription factor T-box expressed in T cells (T-bet) upregulation and GATA-binding protein 3 (GATA-3) downregulation, leading to an increased T-bet/GATA-3 ratio. Our previous in vitro study showed that recombinant human interleukin-11 (rhIL-11) could normalize Th1/Th2 imbalance in the peripheral blood mononuclear cells (PBMCs) isolated from adult ITP patients, which co-occurred with T-bet/GATA-3 ratio restoration. In this report, we investigated whether rhIL-11 had therapeutic effect in clinical ITP patients and whether rhIL-11 treatment could normalize Th1/Th2 and T-bet/GATA-3 levels in vivo. We found rhIL-11 treatment had a response rate of 67.7% and significantly decreased Th1 and T-bet levels but increased Th2 and GATA-3 levels in ITP patients who showed good response, normalizing Th1/Th2 and T-bet/GATA-3 ratios similar to that in healthy controls. Thus our study suggested rhIL-11 was effective with tolerable adverse effects in ITP. The treatment strategy warrants further clinical investigation.

Downregulation of T-bet/GATA-3 ratio induced by IL-11 treatment is responsible for Th1/Th2 balance restoration in human immune thrombocytopenic purpura (ITP).

Abnormal cellular immunity induced by deranged Th1/Th2 profile has been revealed to play a critical role in the pathogenesis of immune thrombocytopenic purpura (ITP). Correction of the shifted Th1/Th2 balance represents a potential therapeutic approach to treat ITP. Here, we investigated the effects of IL-11 on the restoration of Th1/Th2 balance in the peripheral blood mononuclear cells (PBMCs) isolated from adult ITP patients. As shown here, we observed a higher ratio of T-bet/GATA-3 gene expression by quantitative real-time PCR in the PBMCs from ITP patients, consistent with the presence of an abnormally high Th1/Th2 ratio. Remarkably, upon IL-11 treatment, a reversal of T-bet/GATA-3 ratio in ITP was achieved and was shown to be responsible for the restoration of Th1/Th2 balance, with IL-11 at 100 ng/ml demonstrating the highest efficiency. T-bet and GATA-3 are the two transcriptional factors that have been indicated to be the master regulators for Th1 and Th2 lineage commitment, respectively. In the presence of 100 ng/ml IL-11, GATA-3 transcript abundance rose up to ~85-fold of that measured in untreated cells, whereas T-bet transcripts were lowered merely to ~41%, suggesting that GATA-3 was the major contributor for the reversal of T-bet/GATA-3 ratio. Thus, our findings may very well encourage the development of novel medicines that specifically target and correct the T-bet/GATA-3 imbalance identified in ITP.

Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells.

To establish a method for efficient and relatively easy isolation of a cell population containing epithelial prostate stem cells, we developed two transgenic mouse models, K5/CFP and K18/RFP. In these models, promoters of the cytokeratin 5 (Krt5) and the cytokeratin 18 (Krt18) genes regulate cyan and red fluorescent proteins (CFP and RFP), respectively. CFP and RFP reporter protein fluorescence allows for visualization of K5(+) and K18(+) epithelial cells within the cellular spatial context of the prostate gland and for their direct isolation by FACS. Using these models, it is possible to test directly the stem cell properties of prostate epithelial cell populations that are positively selected based on expression of cytoplasmic proteins, K5 and K18. After validating appropriate expression of the K5/CFP and K18/RFP transgenes in the developing and adult prostate, we demonstrate that a subset of CFP-expressing prostate cells exhibits stem cell proliferation potential and differentiation capabilities. Then, using prostate cells sorted from double transgenic mice (K5/CFP + K18/RFP), we compare RNA microarrays of sorted K5(+)K18(+) basal and K5(-)K18(+) luminal epithelial cells, and identify genes that are differentially expressed. Several genes that are over-expressed in K5(+) cells have previously been identified as potential stem cell markers. These results suggest that FACS isolation of prostate cells from these mice based on combining reporter gene fluorescence with expression of potential stem cell surface marker proteins will yield populations of cells enriched for stem cells to a degree that has not been attained by using cell surface markers alone.

Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis.

Claudin-3 (CLDN3) is a tight junction protein that is overexpressed in 90% of ovarian tumors. Previous in vitro studies have indicated that CLDN3 overexpression promotes the migration, invasion, and survival of ovarian cancer cells. Here, we investigated the efficacy of lipidoid-formulated CLDN3 siRNA in 3 different ovarian cancer models. Intratumoral injection of lipidoid/CLDN3 siRNA into OVCAR-3 xenografts resulted in dramatic silencing of CLDN3, significant reduction in cell proliferation, reduction in tumor growth, and a significant increase in the number of apoptotic cells. Intraperitoneal injection of lipidoid-formulated CLDN3 siRNA resulted in a substantial reduction in tumor burden in MISIIR/TAg transgenic mice and mice bearing tumors derived from mouse ovarian surface epithelial cells. Ascites development was reduced in CLDN3 siRNA-treated mice, suggesting the treatment effectively suppressed metastasis. Toxicity was not observed after multiple i.p. injections. Importantly, treatment of mice with nonimmunostimulatory 2'-OMe modified CLDN3 siRNA was as effective in suppressing tumor growth as unmodifed siRNA. These results suggest that lipidoid-formulated CLDN3 siRNA has potential as a therapeutic for ovarian cancer.

Nanoparticulate delivery of suicide DNA to murine prostate and prostate tumors.

BACKGROUND: Currently available treatments for benign prostatic hyperplasia (BPH) and localized prostate cancer are generally effective but are often attended by serious side effects that impact on the quality of life. In particular, most current therapies are non-specific, with surgery, radiation, and chemical ablation having the potential to cause damage to surrounding tissue. Here, we demonstrate the effectiveness of a prostate-specific, locally delivered gene therapy for the targeted killing of prostate cells. METHODS: Using a degradable, poly(beta-amino ester) polymer, poly(butane diol diacrylate co amino pentanol) (C32), we developed a nanoparticulate system to deliver a diphtheria toxin suicide gene (DT-A) driven by a prostate specific promoter to cells. These C32/DT-A nanoparticles were directly injected to the normal prostate and to prostate tumors in mice. RESULTS: Nearly 50% of normal prostates showed a significant reduction in size, attributable to cellular apoptosis, whereas injection with naked DT-A-encoding DNA had little effect. Significant apoptosis was also observed in C32/DT-A injected prostate tumors. Importantly, no damage to surrounding tissue was observed. CONCLUSIONS: These results suggest that local delivery of poly(beta-amino ester) polymer/ DT-A nanoparticles may have application in the treatment of BPH and prostate cancer.

[Effect of hypoxic radiosensitizer sodium glycididazole on long-term result of radiotherapy for nasopharyngeal carcinoma].

OBJECTIVE: To evaluate the long-term effect of sodium glycididazole (CMNa) as a hypoxic radiosensitizer on the radiotherapy for nasopharyngeal carcinoma. METHODS: Between May 1999 and May 2002, 211 patients with pathologically confirmed nasopharyngeal carcinoma were randomized into group-A treated by radiotherapy plus CMNa or group-B by radiotherapy alone. The staging was determined according to 92' Fuzhou staging systerm. The type, procession and dosage of radiotherapy were identical in both groups. The early adverse effect grade was assessed based on the CTC2.0 criteria and the late adverse effects were evaluated according to the RTOG/EORTC criteria. The median follow-up time was 52 months. All the data was analyzed by the SPSS 13.0 software. Characteristics and adverse events of these patients were compared between the two groups using t-test and the Wilcoxin rank sum test. Time-to-event curves were estimated using the Kaplan-Meier method. The prognostic parameters were analyzed using univariate analysis and the Cox multivariate regression analysis. RESULTS: The clinical data of the two groups were comparable. The 3-year survival was 88.4% in group-A, while 75.2% in group-B, with a statistically significant difference between two groups (P = 0.010). Univariate analysis showed that the 3-year survival was statistically correlated with N-staging ((N0-1, 86.9%, N2-3 73.8%, P < 0.001), T-staging (T1-2 85.6%, T3-4 79.3%, P = 0.014), TNM staging (P = 0.039), and whether using CMNa or not during rediotherapy (Group-A 88.4%, Group-B 75.2%, P = 0.010). The 5-year recurrence-free survival, 5-year metastasis-free survival and 5-year overall survival were 75.8%, 74.9% and 77.7% in Group-A, while 63.0%, 63.0% and 62.4% in Group-B with a statistically significant difference between two groups (0.013, 0.022 and 0.010, respectively). If stratified in the subgroups, the overall survival of stage III - IV patients was statistically different between group A and B (P = 0.009), however, not of stage I - II patients (P = 0.502). Cox multivariate regression analysis showed that the independent prognostic parameters for survival were N-stage (RR = 3.288) , T-stage (RR = 2.147) and use of CMNa during rediotherapy (RR = 0.407). However, there was no statistically significant difference between two groups in acute or late adverse effects on nervous system or heart, which suggested that use of CMNa during radiotherapy would not aggravate the toxicity caused by radiotherapy. CONCLUSION: Sodium glycididazole is well tolerable effective as a hypoxic radiosensitizer, which can improve the efficacy of radiotherapy and the long-term result of nasopharyngeal carcinom a patients, especially for the stage III - IV patients.

Human coxsackie adenovirus receptor (CAR) expression in transgenic mouse prostate tumors enhances adenoviral delivery of genes.

BACKGROUND: Transgenic mice that recapitulate the progression of human diseases are potentially useful models for testing the effectiveness of new therapeutic strategies. Their use in pre-clinical testing of adenovirally-delivered gene therapies, however, is limited because of restricted cell surface expression of Coxsackie adenovirus receptor (CAR) in mice. METHODS: To develop a more suitable transgenic mouse model for testing adenoviral-based gene therapies for prostate cancer, we generated prostate specific antigen/human CAR (PSA/hCAR) transgenic mice in which a chimeric enhancer/promoter sequence of the human PSA gene drives expression of a functional hCAR coding sequence. RESULTS: Expression of an adenovirally-delivered luciferase reporter gene in prostate tumor cells in bigenic mice (PSA/hCAR + TRAMP) was enhanced compared to the level in tumor cells lacking the PSA/hCAR transgene. CONCLUSIONS: Breeding PSA/hCAR mice to existing transgenic mouse models for prostate cancer (e.g., TRAMP) results in improved mouse models for testing adenovirally-delivered therapeutic genes.

[Selected CD34+ cell autologous transplantation for advanced malignant tumors].

OBJECTIVE: To determine the clinical results of selected CD34(+) cell autologous transplantation in advanced malignant tumors. METHODS: After pretreatment, fifteen patients aged 12 - 70 (49.5) years with various Stage III or IV malignant tumors were given the sorted CD34(+) cells collected by magnetic-activated cell sorting (Clini MACS, Milteny Biotech, Germany). RESULTS: Peripheral blood progenitor cells (PBPC) from the patients were mobilized by chemotherapy and G-CSF 5 micro g/kg per day. CD34(+) cells gave 2.0 - 5 log depletion after cell sorting, with a median yield of CD34(+) selected cells of 2.4 (0.15 - 12.03) x 10(6)/kg. It gave a median recovery of 64 (52 - 81.4)% and median purity of 98.2 (83.2 - 99.7)%. The median time of neutrophil recovery > 1.0 x 10(9)/L and platelet recovery > 20 x 10(9)/L post-transplantation were 14 (8 - 26) days and 13 (11 - 35) days, respectively. On follow-up of 2 - 33 (11) months, the event-free survival rate was 53.3% (8/15) and the overall survival rate was 66.7% (10/15). CONCLUSION: Transplantation of autologous selected PBPC CD34(+) cells gives prompt and stable engraftment. Selected CD34(+) cell transplantation, being a safe approach, may improve the clinical outcome even in patients with advanced malignant tumors.

Regulated expression of diphtheria toxin in prostate cancer cells.

Despite their known potential for effectively killing cells, the therapeutic use of plant and bacterial toxins for the treatment of cancer has been slow to enter the clinical setting. This has been due in large part to the lack of gene regulatory elements that control expression of highly toxic genes in a sufficiently tight manner, such that the toxins are only expressed in specific target cells. "Leaky" promoters result in unwanted and harmful cell death. In this study, we tested a novel gene therapy strategy aimed at expressing diphtheria toxin (DT-A) in androgen-independent prostate cancer cells that express the protein BCL2. This strategy relies on both transcriptional regulation and inducibly regulated DNA recombination mediated by the site-directed Flp recombinase to control expression of the toxin. Adenoviruses are used to introduce the genetic elements required for this approach into cultured cells and xenografts. Administration of 4-hydroxytamoxifen, resulting in recombination and expression of the toxin, effectively kills the cancer cells. Our results suggest that following androgen ablation therapy for the treatment of prostate cancer, use of a regulated recombination system to target expression of DT-A to androgen-independent cancer cells would be an effective way to arrest the development of recurrent tumors.

[Clinical combination therapy for adrenal metastasis from lung cancer].

BACKGROUND: To study the results of combination therapy for adrenal metastasis from lung cancer. METHODS: Thirty patients with adrenal metastasis were treated in our hospital from Feb. 1995 to Apr. 2001. Forteen patients were small cell lung cancer (SCLC) and 16 patients were non-small cell lung cancer (NSCLC). All patients were treated with chemotherapy and/or radiotherapy. Eighteen patients were treated with combination therapy of chemotherapy and radiotherapy, while twelve patients with chemotherapy alone. RESULTS: The median survival time was 8 months. The response rate of chemotherapy alone was 25.0% (partial relief 3 cases), while the response rate of combination therapy was 44.4% (complete relief 1 case and partial relief 7 cases). Pain was relieved quickly after radiotherapy for the patients with pain symptom. CONCLUSIONS: Combination of radiotherapy and chemotherapy is better than chemotherapy alone for the treatment of adrenal metastasis from lung cancer.

[Clinical study on the bone marrow micrometastases in patients with non-small cell lung cancer by immunohistochemical techniques].

BACKGROUND: To investigate the immunohistochemical detected method and the clinical incidence of the bone marrow micrometastases (BMM) in patients with non-small cell lung cancer (NSCLC) and to analyze the sensitivity and specificity and clinical application value. METHODS: Bone marrow samples were collected from the anterior superior iliac spines or posterior superior iliac spines of 53 patients with NSCLC in clinical stage I to III and 15 patients in stage IV, and the BMM was detected by immunohistochemical techniques (IHC) using monoclonal antibodies AE1/AE3 against cytokeratin. Chi-square test was used statistically. RESULTS: The IHC sensitivity could be 10⁻5. The BMM positive rate was 22.6% (12/53) in stage I to III and 53.3% (8/15) in stage IV, and there was a significant difference in the BMM positive rate between stage I to III and stage IV (P < 0.05). No correlation was observed between BMM and sex, age, KPS, pathology classification and cancer cell differentiation. CONCLUSIONS: The detection of BMM by IHC is convenient, sensitive, and specific. It might be helpful to diagnose bone marrow micrometastasis in patients with NSCLC.