Proliferative response of lymphocytes from ovarian cancer patients to autologous tumor cells

Summary Peripheral blood lymphocytes (PBL) from 43 patients with histologically confirmed ovarian carcinoma were stimulated in mixed lymphocyte-tumor culture (MLTC) with purified autologous tumor cells. Positive results, assessed as lymphocyte proliferation, were observed in 21 cases (48.8%). Lymphoid cells associated with ascitic fluid or infiltrating solid masses were in general less reactive than PBL as only 3/11 cases had positive MLTC. Tumor cells isolated from peritoneal effusions showed no significant difference in stimulatory potential as compared to the primary tumor. These results suggest that in an appreciable proportion of ovarian carcinoma patients (approximately 50%), lymphocytes have the potential to react to autologous tumor cells. Comprehension of the immunological mechanisms of antitumor resistance may have direct practical relevance for more effective treatment of neoplasms.     

卵巢癌患者术后阴道微生态分析

目的 对卵巢癌患者术后阴道微生态特征进行回顾性分析,为临床预防及诊疗提供参考.方法 选取158例卵巢癌术后患者为卵巢癌组,150例同期健康体检者为对照组,对留取样本进行革兰染色镜检及阴道五项联检检测,并对阴道微生态结果进行统计分析.结果 卵巢癌组阴道五联检各指标异常率高于对照组(均P＜0.05).革兰染色油镜镜检联合五...     

Bone marrow-derived mesenchymal stromal cells from patients with end-stage renal disease are suitable for autologous therapy

Background aimsMesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive and reparative properties in vitro and in vivo. Although autologous bone marrow (BM)-derived MSCs are already clinically tested in transplant recipients, it is unclear whether these BM cells are affected by renal disease. We assessed whether renal failure affected the function and therapeutic potential of BM-MSCs.MethodsMSCs from 10 adults with end-stage renal disease (ESRD) and 10 age-matched healthy controls were expanded from BM aspirates and tested for phenotype and functionality in vitro.ResultsMSCs from ESRD patients were >90% positive for CD73, CD90 and CD105 and negative for CD34 and CD45 and showed a similar morphology and differentiation capacity as MSCs from healthy controls. Of importance for their clinical utility, growth characteristics were similar in both groups, and sufficient numbers of MSCs were obtained within 4 weeks. Messenger RNA expression levels of self-renewal genes and factors involved in repair and inflammation were also comparable between both groups. Likewise, microRNA expression profiling showed a broad overlap between ESRD and healthy donor MSCs. ESRD MSCs displayed the same immunosuppressive capacities as healthy control MSCs, demonstrated by a similar dose-dependent inhibition of peripheral blood mononuclear cell proliferation, similar inhibition of proinflammatory cytokines tumor necrosis factor-α and interferon-γ production and a concomitant increase in the production of interleukin-10.ConclusionsExpanded BM-MSCs procured from ESRD patients and healthy controls are both phenotypically and functionally similar. These findings are important for the potential autologous clinical application of BM-MSCs in transplant recipients.     

Pre-clinical assessment of total knee replacement anterior-posterior constraint

Pre-clinical, bench-top assessment of Total Knee Replacements (TKR) can provide information about the inherent constraint provided by a TKR, which does not depend on the condition of the patient undergoing the arthroplasty. However little guidance is given by the ASTM standard on test configurations such as medial-lateral (M:L) loading distribution, flexion angle or restriction of secondary motions. Using a purpose built rig for a materials testing machine, four TKRs currently in widespread clinical use, including medial-pivot and symmetrical condyle types, were tested for anterior-posterior translational constraint. Compressive joint loads from 710 to 2000 N, and a range of medial-lateral (M:L) load distributions, from 70:30% to 30:70% M:L, were applied at different flexion angles with secondary motions unconstrained. It was found that TKA constraint was significantly less at 60 and 90° flexion than at 0°, whilst increasing the compressive joint load increased the force required to translate the tibia to limits of AP constraint at all flexion angles tested. Additionally when M:L load distribution was shifted medially, a coupled internal rotation was observed with anterior translation and external rotation with posterior translation. This paper includes some recommendations for future development of pre-clinical testing methods.     

Proteomics advances for precision therapy in ovarian cancer

ABSTRACT

Introduction: Due to the relatively low mutation rate and high frequency of copy number variation, finding actionable genetic drivers of high-grade serous carcinoma (HGSC) is a challenging task. Furthermore, emerging studies show that genetic alterations are frequently poorly represented at the protein level adding a layer of complexity. With improvements in large-scale proteomic technologies, proteomics studies have the potential to provide robust analysis of the pathways driving high HGSC behavior.

Areas covered: This review summarizes recent large-scale proteomics findings across adequately sized ovarian cancer sample sets. Key words combined with ‘ovarian cancer’ including ‘proteomics’, ‘proteogenomic’, ‘reverse-phase protein array’, ‘mass spectrometry’, and ‘adaptive response’, were used to search PubMed.

Expert opinion: Proteomics analysis of HGSC as well as their adaptive responses to therapy can uncover new therapeutic liabilities, which can reduce the emergence of drug resistance and potentially improve patient outcomes. There is a pressing need to better understand how the genomic and epigenomic heterogeneity intrinsic to ovarian cancer is reflected at the protein level and how this information could be used to improve patient outcomes.     

MicroRNAs in ovarian cancer biology and therapy resistance

Epithelial ovarian cancer is the most common cause of death from gynecological malignancies in the Western world. The overall 5-year survival is only 30% due to late diagnosis and development of resistance to chemotherapy. There is, therefore, a strong need for prognostic and predictive markers to help optimize and personalize treatment hence ameliorating the prognosis of ovarian cancer patients.Since 2006, an increasing number of studies have indicated an essential role for microRNAs in ovarian cancer tumorigenesis. In this review, we provide an overview of the microRNAs that have been associated with different aspects of ovarian cancer, such as tumor subtype, stage, histological grade, germline mutations in BRCA genes, prognosis and therapy resistance. We highlight the role of the let-7 and miR-200 families, two major microRNA families that are frequently dysregulated in ovarian cancer and have been associated with poor prognosis. Interestingly, both have been implicated in the regulation of the epithelial-to-mesenchymal transition, a cellular transition associated with tumor aggressiveness, tumor invasion and chemoresistance. Furthermore, we discuss several other microRNAs that have been associated with chemotherapy resistance, such as miR-214, miR-130a, miR-27a and miR-451. In the final section, we speculate on the possibilities of microRNA-based therapies and the use of microRNAs as diagnostic tools.     

Generation of DC-based vaccine for therapy of B-CLL patients. Comparison of two methods for enriching monocytic precursors

BACKGROUND: The generation of Ag-loaded DC under good manufacturing practice (GMP) conditions is logistically challenging and further compounded when the starting precursors need to be purified from B-CLL patients who have overwhelming numbers of circulating B-CLL cells and decreased numbers of monocytes. METHODS: We have previously demonstrated that DC with endocytosed B-CLL apoptotic bodies are powerful stimulators of anti-leukemic T cells. In this study we compared counterflow elutriation and immunomagnetic separation for enriching monocyte precursors, and evaluated the feasibility of generating DC from B-CLL patients and the effects of cryopreservation. RESULTS: Monocyte yield from a single leukapheresis product of a B-CLL patient varied from 1 x 108 to 10 x 108 total cells, from which 40-200 x 106 mature DC could be produced. Adequate numbers of monocytes could not be enriched from one patient with 0.2% monocytes in the leukapheresis product, and the target of 50 x 106 DC was barely achieved in another patient with 0.9% monocytes in the pheresed cells. These results suggested that successful production of DC is dependent on a minimum frequency of 1% CD14(+) monocytes in the leukapheresis product. Cryopreservation of tumor cell-loaded DC yielded a recovery rate of 86+/-4.4% upon thawing, with a total viability of 90+/-2.8%. Most importantly, cryopreserved Ag-loaded DC retained their morphology, phenotype and function. DISCUSSION: The results demonstrate that adequate numbers of functional DC required for clinical therapy can be generated from patients who have >1% of CD14(+) monocytes in the leukapheresis product. Moreover, Ag-loaded DC can be cryopreserved and recovered without significant change in phenotype or function.     

Skin tests with autologous cholesteryl hemisuccinate treated tumour cells in cancer patients

Summary Skin tests with autologous irradiated tumour cells were performed in 20 malignant melanoma, 7 breast and 6 ovarian cancer patients. In the majority of cases evident reaction was noted with cholesteryl hemisuccinate (CHS)-treated cells while the reaction with untreated cells was mostly negative.No correlation was found between this reactivity and the ability of patients to be sensitized to DNCB and to their reactivity to PPD. No correlation was found between reactivity to CHS-treated tumour cells and the stage and course of the disease.     

Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients

Early detection of resistance to platinum-based therapy is critical for improving the treatment of ovarian cancers. We have previously found that increased expression of annexin A3 is a mechanism for platinum resistance in ovarian cancer cells. Here we demonstrate that annexin A3 can be detected in the culture medium of ovarian cancer cells, particularly these cells that express high levels of annexin A3. Levels of annexin A3 were then determined in sera from ovarian cancer patients using an enzyme-linked immunosorbent assay. Compared with those from normal donors, sera from ovarian cancer patients contain significantly higher levels of annexin A3. Furthermore, serum levels of annexin A3 were significantly higher in platinum-resistant patients than in platinum-sensitive patients. To gain insight into the mechanism of secretion, the ovarian cancer cell lines were examined using both transmission electron microscopy and immunoelectron microscopy. Compared with parent cells, there are significantly more vesicles in the cytoplasm of ovarian cancer cells that express high levels of annexin A3, and at least some vesicles are annexin A3-positive. Moreover, some vesicles appear to be fused with the cell membrane, suggesting that annexin A3 secretion may be associated with exocytosis and the release of exosomes. This is supported by our observation that ovarian cancer cells expressing higher levels of annexin A3 released increased numbers of exosomes. Furthermore, annexin A3 can be detected in exosomes released from cisplatin-resistant cells (SKOV3/Cis) by immunoblotting and immunoelectron microscopy.     

Pre-clinical assessment of novel multivalent MSP3 malaria vaccine constructs

Background

MSP3 has been shown to induce protection against malaria in African children. The characterization of a family of Plasmodium falciparum merozoite surface protein 3 (MSP3) antigens sharing a similar structural organization, simultaneously expressed on the merozoite surface and targeted by a cross-reactive network of protective antibodies, is intriguing and offers new perspectives for the development of subunit vaccines against malaria.

Methods

Eight recombinant polyproteins containing carefully selected regions of this family covalently linked in different combinations were all efficiently produced in Escherichia coli. The polyproteins consisted of one monovalent, one bivalent, one trivalent, two tetravalents, one hexavalent construct, and two tetravalents incorporating coiled-coil repeats regions from LSA3 and p27 vaccine candidates.

Results

All eight polyproteins induced a strong and homogeneous antibody response in mice of three distinct genotypes, with a dominance of cytophilic IgG subclasses, lasting up to six months after the last immunization. Vaccine-induced antibodies exerted a strong monocyte-mediated in vitro inhibition of P. falciparum growth. Naturally acquired antibodies from individuals living in an endemic area of Senegal recognized the polyproteins with a reactivity mainly constituted of cytophilic IgG subclasses.

Conclusions

Combination of genetically conserved and antigenically related MSP3 proteins provides promising subunit vaccine constructs, with improved features as compared to the first generation construct employed in clinical trials (MSP3-LSP). These multivalent MSP3 vaccine constructs expand the epitope display of MSP3 family proteins, and lead to the efficient induction of a wider range of antibody subclasses, even in genetically different mice. These findings are promising for future immunization of genetically diverse human populations.     

Insulin and C-peptide secretion in non-obese patients with polycystic ovarian disease

Plasma glucose, immunoreactive insulin (IRI) and C-peptide responses during an oral glucose tolerance test (oGTT) were assessed in 11 non-obese patients with polycystic ovarian disease (PCOD) and 11 reference subjects matched for age, height and weight. Also, 6 patients with PCOD and 6 normal women were subjected to intravenous glucose tolerance testing (ivGTT) On oGTT, all subjects exhibited normal glucose tolerance; however, PCOD patients had significantly higher mean plasma glucose levels at 30, 60, 90 and 120 min and higher mean incremental glucose areas. In addition the patients with polycystic ovaries showed higher mean basal IRI and C-peptide levels, higher mean glucose stimulated IRI and C-peptide levels and higher mean incremental IRI and C-peptide values. The molar ratios of C-peptide/IRI were significantly lower in the PCOD group at all time intervals after glucose stimulation when compared to the normal women. During ivGTT, there were significantly higher mean glucose levels at 5, 40, 50 and 60 min in the PCOD group when compared to the reference group. The IRI response to intravenous glucose in the PCOD women was similar to the reference group. The findings on oGTT suggest that non-obese patients with PCOD have increased pancreatic IRI secretion as well as impaired hepatic extraction of the hormone.     

Noninvasive assessment of cancer response to therapy

Rapid assessment of cancer response to a therapeutic regimen can determine efficacy early in the course of treatment. Although biopsies of cancer can be used to rapidly assess pharmacodynamic response, certain disease sites are less accessible to repeated biopsies. Here, we simultaneously assess response in all sites of disease within days of starting therapy by use of peptide ligands selected for their ability to discern responding from nonresponding cancers. When conjugated to near-infrared imaging agents, the HVGGSSV peptide differentiates between these two types of cancer. Rapid, noninvasive assessment of the pharmacodynamic response within cancer promises to accelerate drug development and minimize the duration of treatment with ineffective regimens in cancer patients.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.