Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR: a versatile monitoring and discovery tool

BACKGROUND: Monitoring of cellular immune responses is indispensable in a number of clinical research areas, including microbiology, virology, oncology and autoimmunity. Purification and culture of peripheral blood mononuclear cells and rapid access to specialized equipment are usually required. We developed a whole blood (WB) technique monitoring antigen specific cellular immune response in vaccinated or naturally sensitized individuals. METHODS: WB (300 microl) was incubated at 37 degrees C with specific antigens, in the form of peptides or commercial vaccines for 5-16 hours. Following RNAlater addition to stabilize RNA, the mixture could be stored over one week at room temperature or at 4 degrees C. Total RNA was then extracted, reverse transcribed and amplified in quantitative real-time PCR (qRT-PCR) assays with primers and probes specific for cytokine and/or chemokine genes. RESULTS: Spiking experiments demonstrated that this technique could detect antigen specific cytokine gene expression from 50 cytotoxic T lymphocytes (CTL) diluted in 300 microl WB. Furthermore, the high sensitivity of this method could be confirmed ex-vivo by the successful detection of CD8+ T cell responses against HCMV, EBV and influenza virus derived HLA-A0201 restricted epitopes, which was significantly correlated with specific multimer staining. Importantly, a highly significant (p = 0.000009) correlation between hepatitis B surface antigen (HBsAg) stimulated IL-2 gene expression, as detectable in WB, and specific antibody titers was observed in donors vaccinated against hepatitis B virus (HBV) between six months and twenty years before the tests. To identify additional markers of potential clinical relevance, expression of chemokine genes was also evaluated. Indeed, HBsAg stimulated expression of MIP-1beta (CCL4) gene was highly significantly (p = 0.0006) correlated with specific antibody titers. Moreover, a longitudinal study on response to influenza vaccine demonstrated a significant increase of antigen specific IFN-gamma gene expression two weeks after immunization, declining thereafter, whereas increased IL-2 gene expression was still detectable four months after vaccination. CONCLUSION: This method, easily amenable to automation, might qualify as technology of choice for high throughput screening of immune responses to large panels of antigens from cohorts of donors. Although analysis of cytokine gene expression requires adequate laboratory infrastructure, initial antigen stimulation and storage of test probes can be performed with minimal equipment and time requirements. This might prove important in "field" studies with difficult access to laboratory facilities

Economic Burden of Surgical Site Infections at a European University Hospital

Objective. To quantify the economic burden of in-hospital surgical site infections (SSIs) at a European university hospital. Design. Matched case-control study nested in a prospective observational cohort study. Setting. Basel University Hospital in Switzerland, where an average of 28,000 surgical procedures are performed per year. Methods. All in-hospital occurrences of SSI associated with surgeries performed between January 1, 2000, and December 31, 2001, by the visceral, vascular, and traumatology divisions at Basel University Hospital were prospectively recorded. Each case patient was matched to a control patient by age, procedure code, and National Nosocomial Infection Surveillance System risk index. The case-control pairs were analyzed for differences in cost of hospital care and in provision of specialized care. Results. A total of 6,283 procedures were performed:187 SSIs were detected in inpatients, 168 of whom were successfully matched with a control patient. For case patients, the mean additional hospital cost was SwF19,638 (95% confidence interval [CI], SwF8,492-SwF30,784); the mean additional postoperative length of hospital stay was 16.8 days (95% CI, 13-20.6 days); and the mean additional in-hospital duration of antibiotic therapy was 7.4 days (95% CI, 5.1-9.6 days). Differences were primarily attributable to organ space SSIs (n = 76). Conclusions. Ina European university hospital setting, SSIs are costly and constitute a heavy and potentially preventable burden on both patients and healthcare provider

Immunogenic capacities of recombinant vaccinia virus expressing CD154 : effects on CTL priming

Recombinant poxviruses expressing immuno-modulatory molecules together with specific antigens might represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in clinical trials, that co-expression of costimulatory molecules (CD80 and CD86) could increase the immunogenic capacity of a recombinant Vaccinia virus (rVV) also encoding different tumor associated antigens. In order to further investigate the capacity of these vectors to provide ligands for different co-stimulatory pathways relevant in the generation of CD8+ T cell responses, we designed a recombinant virus expressing CD40 ligand (CD154rVV). This co-receptor, expressed on activated CD4+ T cells, upon binding CD40 expressed on antigen presenting cells (APC) has been reported to increase their antigen presentation and immunomodulatory capacities. To investigate the potency of CD154rVV in CTL generation, different types of infection were performed in cultures containing APC and CD8+ T cells. Phenotypic characterization of infected iDC showed that CD154rVV enhances their activation and maturation, measured by increased expression of CD83 and CD86, as compared to wild type Vaccinia virus as control. Cytokine gene expression was evaluated by quantitative real time PCR. As expected, control virus infection triggered cytokine gene expression in APC and T-cells. However, typical APC cytokines such as GM-CSF, TNF-α and IL-15 and, on the other hand, typical T cells cytokines such as IL-2 and IFN-γ seemed to be expressed to a higher extent in CD154rVV infected cultures. Furthermore, as a landmark of the CD40/CD154 pathway, IL-12p40 gene transcription in APC was exclusively induced by CD154rVV infection. The latter factor is also known to play a major role in CTL priming. Complementary, as expected, control virus infection triggered IL-6 gene expression, which is known to render tumor specific T cells refractory to T regulatory cell activity. Nevertheless, this expression could not be further enhanced by CD154rVV. Activation of specific CD8+ T cells was investigated by using the MART-1/Melan-A27-35 epitope as model antigen and monitored by tetramer staining and cytotoxicity assays. We found that increased numbers of specific cytotoxic CD8+ T cells were induced by the specific peptide in the presence of the CD154rVV activated APCs, as compared to control virus. Finally, CD154rVV was demonstrated to enhance T cell proliferation, mainly CD4+ T cell, in culture of infected APCs with autologous T cells. Taken together, these data indicate that functional CD154 expression from recombinant Vaccinia virus infected cells induces APC activation and maturation, thereby enhancing antigen specific CD8+ T cell generation. Such recombinant vector might help bypassing the requirement for activated helper cells, thus qualifying as a potentially relevant reagent in the generation of CD8+ T cell responses in cancer immunotherapy

New dimensions in tumor immunology: what does 3D culture reveal?

Experimental models indicate that tumor cells in suspension, unlike solid tumor fragments, might be unable to produce life-threatening cancer outgrowth when transferred to animal models, irrespective of the number of cells transferred, although they induce specific immune responses. Human tumor cells cultured in three dimensions display increased pro-angiogenic capacities and resistance to interferons, chemotherapeutic agents or irradiation, as compared with cells cultured in two-dimensional (2D) monolayers. Tumor cells cultured in three dimensions were also shown to be characterized by defective immune recognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens (TAAs) and by a capacity to inhibit CTL proliferation and dendritic cell (DC) functions. Downregulation of human leukocyte antigen (HLA) or TAA expression and high production of lactic acid might play a role in the elicitation of these effects. Here, we propose that growth in 3D architectures might provide new insights into tumor immunology and could represent an integral missing component in pathophysiological tumor immune escape mechanisms

Economic burden of surgical site infections at a European university hospital

OBJECTIVE: To quantify the economic burden of in-hospital surgical site infections (SSIs) at a European university hospital. DESIGN: Matched case-control study nested in a prospective observational cohort study. SETTING: Basel University Hospital in Switzerland, where an average of 28,000 surgical procedures are performed per year. METHODS: All in-hospital occurrences of SSI associated with surgeries performed between January 1, 2000, and December 31, 2001, by the visceral, vascular, and traumatology divisions at Basel University Hospital were prospectively recorded. Each case patient was matched to a control patient by age, procedure code, and National Nosocomial Infection Surveillance System risk index. The case-control pairs were analyzed for differences in cost of hospital care and in provision of specialized care. RESULTS: A total of 6,283 procedures were performed: 187 SSIs were detected in inpatients, 168 of whom were successfully matched with a control patient. For case patients, the mean additional hospital cost was SwF-19,638 (95% confidence interval [CI], SwF-8,492-SwF-30,784); the mean additional postoperative length of hospital stay was 16.8 days (95% CI, 13-20.6 days); and the mean additional in-hospital duration of antibiotic therapy was 7.4 days (95% CI, 5.1-9.6 days). Differences were primarily attributable to organ space SSIs (n = 76). CONCLUSIONS: In a European university hospital setting, SSIs are costly and constitute a heavy and potentially preventable burden on both patients and healthcare providers

Use of multicellular tumor spheroids to dissect endothelial cell-tumor cell interactions: A role for T-cadherin in tumor angiogenesis

This study addresses establishment of an ``in vitro`` melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRTPCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A] and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor micro-environment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis

Differential responsiveness to IL-2, IL-7, and IL-15 common receptor gamma chain cytokines by antigen-specific peripheral blood naive or memory cytotoxic CD8+ T cells from healthy donors and melanoma patients

Common receptor gamma chain (c-gamma) cytokines (CKs) support proliferation of CD8+ T cells in presence or absence of antigen triggering and help maintaining the immunologic memory. We addressed the effects of low (> or = 5 ng/mL)-dose interleukin (IL)-2, IL-7, or IL-15 on human naive and memory antigen-specific CD8+ T cells. Peripheral blood CD8+ lymphocytes proliferated with decreasing efficiency in response to IL-15, IL-7, and IL-2. Of note, IL-15 preferentially promoted expansion of CD45RA/CD8+ T-cell memory subset. Accordingly, cytotoxic T lymphocytes specific for cytomegalovirus-derived antigens from seropositive donors proliferated in response to IL-15 and, to lesser extent to IL-7, but poorly to IL-2. CD8+ T cells were then pretreated with CK before antigen stimulation using, as read out, specific cytotoxic activity. After the pretreatment with IL-15, but not IL-2, previously experienced viral antigens induced vigorous cytotoxic responses. Minor effects of IL-7 were also detectable. In contrast, IL-2 best supported the cytotoxic T lymphocyte generation from prevailingly naive CD8 T cells from HLA-A*0201 healthy donors, specific for L27Melan-A/MART-126-35 melanoma-associated antigen. Cells from melanoma patients were tested before and after Melan-A/MART-1-targeted antigen-specific immunotherapy. Untreated patients showed heterogeneous patterns of responsiveness to c-gamma CK. However, when naive patients whose CD8+ T cells best responded to IL-2 were vaccinated, a modified responsiveness pattern was detectable. After immunization, cells displayed a significantly higher response to IL-15 than to IL-2 pretreatment. Thus, responsiveness to c-gamma CK is critically influenced by naive or memory status of peripheral blood CD8+ T cells

Differential patterns of Large Tumor Antigen specific immune responsiveness in patients with BK polyomavirus positive prostate cancer or benign prostatic hyperplasia

The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as target of immune response in patients with prostate cancer (PCa) has not been investigated so far. In this study we have comparatively analyzed humoral and cellular L-Tag specific responsiveness in age matched patients bearing PCa or benign prostatic hyperplasia (BPH), expressing or not expressing BKV L-Tag specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) following surgery (PSA≥0.2ng/ml).In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag positive tumors, stimulation with peptides derived from BKV L-Tag, but not those derived from Epstein Barr virus, influenza virus or Cytomegalovirus, induced a peculiar cytokine gene expression profile, characterized by high expression of IL-10 and TGFβ-1 and a low expression of IFN-γ genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti BKV L-Tag IgG. Furthermore, in PBMC from these PCa bearing patients, L-Tag derived peptides significantly expanded an IL-10-secreting CD4(+)CD25(+(high))CD127(-(dim))FoxP3(+) T cell population with an effector memory phenotype (CD103(+)) capable of inhibiting proliferation of autologous anti-CD3/CD28 triggered CD4(+)CD25(-) T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag specific immune response are significantly associated with tumor progression in patients with BKV+ PCa