Frequencies of an Immunogenic HER-2/neu Epitope of CD8+ T Lymphocytes Predict Favorable Clinical Outcomes in Prostate Cancer.

HER-2/neu is the human epidermal growth factor receptor 2, which is associated with the progression of prostate cancer (PCa). HER-2/neu-specific T cell immunity has been shown to predict immunologic and clinical responses in PCa patients treated with HER-2/neu peptide vaccines. However, its prognostic role in PCa patients receiving conventional treatment is unknown, and this was addressed in this study. The densities of CD8+ T cells specific for the HER-2/neu(780-788) peptide in the peripheral blood of PCa patients under standard treatments were correlated with TGF-ß/IL-8 levels and clinical outcomes. We demonstrated that PCa patients with high frequencies of HER-2/neu(780-788)-specific CD8+ T lymphocytes had better progression-free survival (PFS) as compared with PCa patients with low frequencies. Increased frequencies of HER-2/neu(780-788)-specific CD8+ T lymphocytes were also associated with lower levels of TGF-ß and IL-8. Our data provide the first evidence of the predictive role of HER-2/neu-specific T cell immunity in PCa.

Identification of novel breast cancer-associated transcripts by UniGene database mining and gene expression analysis in normal and malignant cells.

Breast cancer is a heterogeneous and complex disease. Although the use of tumor biomarkers has improved individualized breast cancer care, i.e., assessment of risk, diagnosis, prognosis, and prediction of treatment outcome, new markers are required to further improve patient clinical management. In the present study, a search for novel breast cancer-associated genes was performed by mining the UniGene database for expressed sequence tags (ESTs) originating from human normal breast, breast cancer tissue, or breast cancer cell lines. Two hundred and twenty-eight distinct breast-associated UniGene Clusters (BUC1-228) matched the search criteria. Four BUC ESTs (BUC6, BUC9, BUC10, and BUC11) were subsequently selected for extensive in silico database searches, and in vitro analyses through sequencing and RT-PCR based assays on well-characterized cell lines and tissues of normal and cancerous origin. BUC6, BUC9, BUC10, and BUC11 are clustered on 10p11.21-12.1 and showed no homology to any known RNAs. Overall, expression of the four BUC transcripts was high in normal breast and testis tissue, and in some breast cancers; in contrast, BUC was low in other normal tissues, peripheral blood mononuclear cells (PBMCs), and other cancer cell lines. Results to-date suggest that BUC11 and BUC9 translate to protein and BUC11 cytoplasmic and nuclear protein expression was detected in a large cohort of breast cancer samples using immunohistochemistry. This study demonstrates the discovery and expression analysis of a tissue-restricted novel transcript set which is strongly expressed in breast tissue and their application as clinical cancer biomarkers clearly warrants further investigation.

Long-term culture of patient-derived mammary organoids in non-biogenic electrospun scaffolds for identifying metalloprotein and motor protein activities in aging and senescence.

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotype in tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactone scaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids and patient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.

Chemotherapy ± cetuximab modulates peripheral immune responses in metastatic colorectal cancer.

OBJECTIVE: To identify changes in peripheral immune responses in patients with metastatic colorectal cancer (mCRC) treated with irinotecan/5-fluorouracil/leucovorin (IFL) alone or in combination with cetuximab (C-IFL). METHODS: Peripheral blood mononuclear cells (PBMCs) collected from healthy donors (n = 20) and patients with mCRC receiving treatment with either IFL (n = 30) or C-IFL (n = 30) were tested for cytokine production upon polyclonal stimulation with anti-CD3 monoclonal antibody, T cell proliferation in the autologous mixed lymphocyte reaction (auto-MLR) and T regulatory cell (Treg) frequency. The respective results were evaluated over two treatment cycles and further assessed in relation to response to treatment. RESULTS: PBMCs prior to treatment exhibited significantly lower production of IL-2, IFN-γ, IL-12 and IL-18 cytokines and lower auto-MLR responses, whereas Treg frequency, IL-4, IL-10 cytokines were increased compared to healthy donors. During treatment, IL-2, IFN-γ, IL-12, IL-18 and auto-MLR responses increased, while Treg frequency and IL-10 secretion decreased significantly compared to the baseline. Responders to treatment exhibited a significantly higher increase in IL-2, IFN-γ, IL-12 and IL-18 production and auto-MLR responses, and higher decrease in IL-4, IL-10 secretion and Treg frequency. Among all patient subgroups analysed, responders to C-IFL demonstrated significantly higher increase in auto-MLR responses, IL-12 and IL-18 secretion and higher decrease in Treg frequency. CONCLUSION: The disturbed immune parameters observed in patients with mCRC at presentation can be significantly improved during treatment with IFL and this effect can be potentiated by the addition of cetuximab. Monitoring of the peripheral immune system function could be used as surrogate marker in predicting treatment-related outcome.

A Blood-Based Immune Gene Signature with Prognostic Significance in Localized Prostate Cancer.

Prostate cancer (PCa) is one of the most common male cancers worldwide and one of the deadliest if unsuccessfully treated. Τhe need for reliable, easily accessible immune-related molecular biomarkers that could be combined with clinically defined criteria, including PSA and Gleason score, to accurately predict PCa patients' clinical outcomes is emerging. Herein, we describe for the first time a blood-identified immune-related gene signature comprising eight upregulated multi-functional genes associated with poor prognosis. Next-generation sequencing (NGS) analysis of PCa patients' peripheral blood samples revealed a more than three-fold upregulation of each of the eight genes as compared to samples originating from healthy donors. The construction of gene and protein interaction networks revealed different extents of the functional implications of these genes in the regulation of cell proliferation and immune responses. Analysis of the available data from The Cancer Genome Atlas (TCGA) regarding gene expression and survival of prostate adenocarcinoma (PRAD) and pan-cancer (PANCAN) patients revealed that intra-tumoral upregulation of this eight-gene signature (8-GS) was associated with poor 5-year progression-free intervals in PCa patients, even in those with high Gleason scores, and also with an unfavorable prognosis for cancer patients irrespective of the cancer type and even in the early stages. These observations suggest that further investigation of the 8-GS prospectively in randomized clinical trials, in which clinical benefit in terms of evaluating time to disease progression can be assessed, is warranted.

Correction: Kogionou et al. Radiotherapy-Related Gene Signature in Prostate Cancer. Cancers 2022, 14, 5032.

The authors wish to make the following corrections to this paper [...].

Ontak reduces the immunosuppressive tumor environment and enhances successful therapeutic vaccination in HER-2/neu-tolerant mice.

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT+ (neuT+) triple transgenic mice represent an improvement over neuT+ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85-94) (p85) CTL and HER-2(776-790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT+ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8+ CTLs and CD4+ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4+ and CD8+ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4+ and CD8+ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.

Combined treatment with bevacizumab and standard chemotherapy restores abnormal immune parameters in advanced colorectal cancer patients.

BACKGROUND: Bevacizumab, a monoclonal antibody (mAb) targeting vascular endothelial growth factor (VEGF), has produced promising results when combined with chemotherapy in the treatment of advanced colorectal cancer (CRC). The aim of the present study was to define the immunological profile of metastatic CRC patients at baseline and following chemotherapy with either irinotecan/5-fluorouracil/leucovorin (IFL) alone or IFL in combination with.bevacizumab (B-IFL). METHODS: Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (HD) (n = 20) and patients (n = 40) were tested for T-cell proliferation in the autologous mixed lymphocyte reaction (auto-MLR), and cytokine production following stimulation with anti-CD3 mAb. RESULTS: PBMCs obtained from CRC patients prior to treatment exhibited lower auto-MLR responses and low production of IL-2, IFN-γ, IL-12 and IL-18 cytokines, whereas IL-4 and IL-10 cytokines were increased as compared to HD (p < 0.001, for all parameters) following in vitro stimulation with anti-CD3 mAb. During treatment, and in particular in week 12 of evaluation, IL-2 (p < 0.001 for both IFL and B-IFL groups), IFN-γ (p < 0.001 for IFL and p = 0.001 for B-IFL), IL-12 (p < 0.001 for both IFL and B-IFL) and IL-18 (p < 0.001 for both IFL and B-IFL) production, as well as auto-MLR responses increased (p < 0.001 for both IFL and B-IFL), whereas IL-4 (p < 0.001 for IFL and p = 0.001 for B-IFL) and IL-10 [p < 0.001 for IFL and p = 0.067 (non-significant) for B-IFL] production decreased over baseline in the two treatment groups, yet their respective values never reached those of HD. Moreover, IL-2, IFN-γ production, and auto-MLR were higher in the B-IFL over the IFL treatment group (p < 0.001, p < 0.04, p < 0.001, respectively). CONCLUSION: Our study demonstrates that the abnormal immune parameters observed in metastatic CRC patients at presentation can substantially improve during treatment with either IFL or B-IFL. The immune parameters examined can provide a sensitive and valuable tool for monitoring immune function in CRC patients, and could be applied as surrogate markers predicting treatment-related outcome.

Impact of ADMA, endothelial progenitor cells and traditional cardiovascular risk factors on pulse wave velocity among prediabetic individuals.

BACKGROUND: Central arterial stiffness represents a well-established predictor of cardiovascular disease. Decreased circulating endothelial progenitor cells (EPCs), increased asymmetric dimethyl-arginine (ADMA) levels, traditional cardiovascular risk factors and insulin resistance have all been associated with increased arterial stiffness. The correlations of novel and traditional cardiovascular risk factors with central arterial stiffness in prediabetic individuals were investigated in the present study. METHODS: The study population consisted of 53 prediabetic individuals. Individuals were divided into groups of isolated impaired fasting glucose (IFG), isolated impaired glucose tolerance (IGT) and combined IGT-IFG. Age, sex, family history of diabetes, smoking history, body mass index (BMI), waist to hip ratio (WHR), waist circumference (WC), blood pressure, lipid profile, levels of high sensitive C-reactive protein (hsCRP), glomerular filtration rate (GFR), and history of antihypertensive or statin therapy were obtained from all participants. Insulin resistance was evaluated using the Homeostatic Model Assessment (HOMA-IR). Carotid -femoral pulse wave velocity was used as an index of arterial stiffness. Circulating EPC count and ADMA serum levels were also determined. RESULTS: Among studied individuals 30 (56.6%) subjects were diagnosed with isolated IFG, 9 (17%) with isolated IGT (17%) and 14 with combined IFG-IGT (26.4%). In univariate analysis age, mean blood pressure, fasting glucose, total cholesterol, LDL cholesterol, and ADMA levels positively correlated with pulse-wave velocity while exercise and GFR correlated negatively. EPC count did not correlate with PWV. In multivariate stepwise regression analysis PWV correlated independently and positively with LDL-Cholesterol (low density lipoprotein) and ADMA levels and negatively with exercise. CONCLUSIONS: Elevated ADMA and LDL-C levels are strongly associated with increased arterial stiffness among pre-diabetic subjects. In contrast exercise inversely correlated with arterial stiffness.

Combination Immunotherapy in Prostate Cancer.

During the last decade, there has been significant progress in the field of prostate cancer therapeutic treatments based on androgen receptor-axis-targeted therapies, which resulted in improved clinical outcomes [...].

T-Cell Repertoire in Tumor Radiation: The Emerging Frontier as a Radiotherapy Biomarker.

Radiotherapy (RT) is a therapeutic modality that aims to eliminate malignant cells through the induction of DNA damage in the irradiated tumor site. In addition to its cytotoxic properties, RT also induces mechanisms that result in the promotion of antitumor immunity both locally within the irradiation field but also at distant tumor lesions, a phenomenon that is known as the "abscopal" effect. Because the immune system is capable of sensing the effects of RT, several treatment protocols have been assessing the synergistic role of radiotherapy combined with immunotherapy, collectively referred to as radioimmunotherapy. Herein, we discuss mechanistic insights underlying RT-based immunomodulation, which also enhance our understanding of how RT regulates antitumor T-cell-mediated immunity. Such knowledge is essential for the discovery of predictive biomarkers and for the improvement of clinical trials investigating the efficacy of radio-immunotherapeutic modalities in cancer patients.

Radiotherapy-Related Gene Signature in Prostate Cancer.

Radiotherapy for localized prostate cancer has increased the cure and survival rates of patients. Besides its local tumoricidal effects, ionizing radiation has been linked to mechanisms leading to systemic immune activation, a phenomenon called the abscopal effect. In this study, we performed gene expression analysis on peripheral blood from prostate cancer patients obtained post- radiotherapy and showed that 6 genes, including CCR7, FCGR2B, BTLA, CD6, CD3D, and CD3E, were down-regulated by a range of 1.5-2.5-fold as compared to pre-radiotherapy samples. The expression of the signature consisting of these six genes was also significantly lower post- vs. pre-radiotherapy. These genes are involved in various tumor-promoting immune pathways and their down-regulation post-radiotherapy could be considered beneficial for patients. This is supported by the fact that low mRNA expression levels for the 6-gene signature in the prostate tumor tissue was linked to better survival. Importantly, we report that this 6-gene signature strongly correlated with a favorable prognosis regardless of poor standard clinicopathological parameters (i.e., Gleason score ≥ 8 and T3 (including T3a and T3b). Our pioneering data open the possibility that the 6-gene signature identified herein may have a predictive value, but this requires further long-term studies.

Association between Intratumoral CD8+ T Cells with FoxP3+ and CD163+ Cells: A Potential Immune Intrinsic Negative Feedback Mechanism for Acquired Immune Resistance.

Acquired immune resistance (AIR) describes a situation in which cancer patients who initially responded clinically to immunotherapies, after a certain period of time, progress with their disease. Considering that AIR represents a feedback response of the tumor against the immune attack generated during the course of immunotherapies, it is conceivable that AIR may also occur before treatment initiation as a mechanism to escape endogenous adaptive antitumor immunity (EAAI). In the present study, we assessed the EAAI in paraffin-embedded breast primary tumor tissue samples and drew correlations with the clinical outcomes. In particular, we analyzed densities of CD8+ cells as elements mediating antitumor cytotoxicity, and of CD163+ and FoxP3+ cells as suppressor elements. We found a direct correlation between the densities of CD8+ cells and of CD163+ and/or FoxP3+ cells in the vast majority of patients' tumors. Importantly, the vast majority of patients whose tumors were overpopulated by CD8+ cells developed AIR, which was characterized by high intratumoral CD163+ and/or FoxP3+ cell densities and reduced overall survival (OS). We also showed that AIR depends on the levels of CD8+ cell-ratios in the tumor center to the invasive margin. Our data suggest that tumors develop AIR only when under a robust endogenous immune pressure.

The impact of radiation therapy on the TCR Vß chain repertoire in patients with prostate cancer.

Radiation therapy (RT) is an essential component in the therapeutic treatment of patients with localized prostate cancer (LPCa). Besides its local effects, ionizing radiation has been linked to mechanisms leading to systemic immune activation. The present study explored the effect of RT on the T­cell receptor variable ß (TCR Vß) chain repertoire of peripheral blood T cells in patients with LPCa. High­throughput TCR Vß sequencing was performed on 20 blood samples collected from patients with LPCa at baseline and 3 months post­RT. The diversity index was altered, as were TCR Vß clonal evenness and convergence before and post­RT; however, these findings were not significant. Notably, marked changes in the frequencies among the top 10 TCR Vß clonotypes were detected and some patients developed new clonotypes of high abundance. These data provided initial evidence that RT in patients with LPCa may induce systemic immune changes, which could be exploited by future therapies for improved clinical results.

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy.

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828-836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu (+) tumor cell lines. HER-2/neu(828-836), [HER-2(9(828))], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9(828)) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8(+) T-lymphocytes specifically recognizing HER-2(9(828)) in 8 out of 20 HLA-A*0201(+) HER-2/neu (+) breast cancer patients. Moreover, HER-2(9(828))-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9(828)) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9(828)) as a promising candidate for peptide-based cancer vaccines.

Integrating Microstructured Electrospun Scaffolds in an Open Microfluidic System for in Vitro Studies of Human Patient-Derived Primary Cells.

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

Amifostine enhances recovery and expansion of peripheral FAS/CD95+ T- and NK-cell subpopulations during radiotherapy of patients with head-neck cancer.

PURPOSE: Experimental studies suggest that the FAS/APO-1/CD95 (cytokine receptor protein TNF-receptor superfamily, member 6) cell surface molecule is involved in the apoptotic effect of radiotherapy. In this study we investigated the role of amifostine in protecting the CD95+ (CD: cluster of differentiation) lymphocytic subpopulation in patients with head and neck cancer undergoing radiotherapy. MATERIALS AND METHODS: Using flow-cytometry we examined the expression of FAS/CD95 antigen on CD4+ (helper/inducer T cells), CD8+ (suppressor/cytotoxic T cells) and CD56+ (NK, natural killer) T-lymphocytes of 28 patients with head and neck cancer undergoing radiotherapy (with and without amifostine). RESULTS: The numbers of peripheral blood lymphocytes were significantly reduced after treatment from (mean value +/- STD error) 1477 +/- 129 to 1015 +/- 77 for T lymphocytes, 700 +/- 70 to 454 +/- 38 for CD4, 449 +/- 46 to 296 +/- 34 for CD8 and, 140 +/- 18 to 118 +/- 13 for NK, before and after treatment, respectively. CD95 expressing lymphocytes showed a faster recovery rate in patients receiving amifostine. CD95 expressing CD56 lymphocytes increased during radiotherapy in patients receiving daily cytoprotection with amifostine to values higher than the pre-treatment levels (p = 0.004). CONCLUSION: It is suggested that amifostine enhances recovery of T- and NK-lymphocyte subpopulations expressing the CD95 antigen in head-neck cancer patients undergoing RT and may enhance the efficacy of the later by interfering FAS-related immunological pathways.

Improvement of Sperm Quality in Hyperviscous Semen following DNase I Treatment.

Semen hyperviscosity impairs sperm motility and can lead to male infertility. This prospective study aimed at assessing the ability of exogenous DNase in improving sperm quality, taking into consideration that DNase has been found in the seminal plasma of several species and that neutrophils release chromatin in order to trap bacteria. A total of seventy-seven semen samples with high seminal viscosity (HSV) as the study group and sixty-two semen samples with normal seminal viscosity (NSV) as the control group were compared in this analysis. These semen samples were divided into three groups of receiving treatment (a) with DNase I at 37°C for 15 min, (b) by density gradient centrifugation, and (c) with a combination of the above two methods. Following a fifteen-minute treatment of hyperviscous semen, the motility of spermatozoa in 83% of semen samples increased to a statistically significant degree. On the contrary, DNase treatment of semen with normal viscosity had no such effects. The above treatment was also accompanied by a significant increase in the percentage of normal spermatozoa, resulting in a major decrease of the teratozoospermia index. Comparison between semen samples that underwent density gradient centrifugation following DNase I treatment, to those collected after density gradient treatment alone, showed that in the first case the results were more spectacular. The evaluation of each preparation in terms of yield (% total progressively motile sperm count after treatment in relation to the initial total sperm count) revealed that the combined approach resulted in 29.8% vs. 18.5% with density treatment alone (p=0.0121). DNase I treatment results in an improvement of sperm motility and morphology and could be beneficial to men with hyperviscous semen in assisted reproduction protocols.

Vaccination with human HER-2/neu (435-443) CTL peptide induces effective antitumor immunity against HER-2/neu-expressing tumor cells in vivo.

HER-2/neu is a self-antigen expressed by tumors and nonmalignant epithelial tissues. The possibility of self-tolerance to HER-2/neu-derived epitopes has raised questions concerning their utility in antitumor immunotherapy. Altered HER-2/neu peptide ligands capable of eliciting enhanced immunity to tumor-associated HER-2/neu epitopes may circumvent this problem. The human CTL peptide HER-2/neu (435-443) [hHER-2(9(435))] represents a xenogeneic altered peptide ligand of its mouse homologue, differing by one amino acid residue at position 4. In contrast to mHER-2(9(435)), vaccination of HLA-A*0201 transgenic (HHD) mice with hHER-2(9(435)) significantly increased the frequency of mHER-2(9(435))-specific CTL and also induced strong protective and therapeutic immunity against the transplantable ALC tumor cell line transfected to coexpress HLA-A*0201 and hHER-2/neu or rHER-2/neu. Similar results were also obtained with wild-type C57BL/6 mice inoculated with HER-2/neu transfectants of ALC. Adoptive transfer of CD8(+) CTL from mice immunized with hHER-2(9(435)) efficiently protected naive syngeneic mice inoculated with ALC tumors. In conclusion, our results show that HER-2(9(435)) serves as a tumor rejection molecule. They also propose a novel approach for generating enhanced immunity against a self-HER-2/neu CTL epitope by vaccinating with xenogeneic altered peptide ligands and provide useful insights for the design of improved peptide-based vaccines for the treatment of patients with HER-2/neu-overexpressing tumors.

A novel quantitative flow cytometric method for measuring glucocorticoid receptor (GR) in cell lines: correlation with the biochemical determination of GR.

Currently, a time consuming biochemical method is used for GR quantification. Here we compare the biochemical approach with a newly developed flow cytometric method of measuring GR in cell lines, which is less time consuming and does not requires the use of radioactive materials. The biochemical assay is easy to apply but the cells need to be grown in media free of endogenous glucocorticoids, in order to prevent them from interfering with radiolabelled hormone binding to the receptor. The presence of endogenous GR ligands is known to reduce receptor levels and to often produce false negative results. The immunofluorescent method is free of such limitations, as it depends entirely on detecting the receptor using a highly specific monoclonal antibody. Additionally, the biochemical assay cannot measure heterogeneity in individual cells, in contrast the flow cytometric one enables the enumeration of the receptor on a per cell basis, allowing exact description of differences in receptor levels amongst intact cells. Our results demonstrate that the flow cytometric method is of similar accuracy but of higher precision compared to the biochemical one. Also, the data we obtained using the immunofluorescent method correlated well with the biochemical one (R(2)=0.9712). In conclusion, flow cytometric method requires small cell numbers, is more accurate and lesser time consuming than the biochemical one. Thus, it could be useful for the quantification of GR in lymphocyte subpopulations, in lymphoproliferative disorders and in tumor cells from cancer patients, in order to design more efficient clinical treatment protocols.