Showing papers in "Journal of Translational Medicine in 2003"

Translational Medicine: A two-way road.

Abstract: The purpose of translational research is to test, in humans, novel therapeutic strategies developed through experimentation. Translational research should be regarded as a two-way road: Bench to Bedside and Bedside to Bench. However, Bedside to Bench efforts have regrettably been limited because the scientific aspects are poorly understood by full time clinicians and the difficulty of dealing with humans poorly appreciated by basic scientists. Translational research would be most useful to the scientific community at large if journals would foster specific interest for the publication of ex vivo human observation. The review process for such work should be assigned to clinical scientists competent not only in the intricacies of molecular or cell biology but also intimate with the reality of Internal Review Boards, ethics committees, Governmental Regulatory Agencies and most importantly the humane aspects of dealing with sick individuals and their families. This approach may focus both basic and clinical scientists and those struggling to fill the gap between them on the effective treatment of diseases affecting women, men and children making translational research more than an interesting concept.

The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity.

Abstract: The interferon-γ (IFN-γ) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-γ secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-γ secretion. We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-γ ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (αEN-EBV and αJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. When the GrB ELISPOT was directly compared to the IFN-γ ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-γ secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-γ ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses.

Polymorphism in clinical immunology - From HLA typing to immunogenetic profiling.

Abstract: The pathology of humans, in contrast to that of inbred laboratory animals faces the challenge of diversity addressed in genetic terms as polymorphism. Thus, unsurprisingly, treatment modalities that successfully can be applied to carefully-selected pre-clinical models only sporadically succeed in the clinical arena. Indeed, pre-fabricated experimental models purposefully avoid the basic essence of human pathology: the uncontrollable complexity of disease heterogeneity and the intrinsic diversity of human beings. Far from pontificating on this obvious point, this review presents emerging evidence that the study of complex system such as the cytokine network is further complicated by inter-individual differences dictated by increasingly recognized polymorphisms. Polymorphism appears widespread among genes of the immune system possibly resulting from an evolutionary adaptation of the organism facing an ever evolving environment. We will refer to this high variability of immune-related genes as immune polymorphism. In this review we will briefly highlight the possible clinical relevance of immune polymorphism and suggest a change in the approach to the study of human pathology, from the targeted study of individual systems to a broader view of the organism as a whole through immunogenetic profiling.

Integration of gene expression, clinical, and epidemiologic data to characterize Chronic Fatigue Syndrome

Abstract: Background Chronic fatigue syndrome (CFS) has no diagnostic clinical signs or diagnostic laboratory abnormalities and it is unclear if it represents a single illness The CFS research case definition recommends stratifying subjects by co-morbid conditions, fatigue level and duration, or functional impairment But to date, this analysis approach has not yielded any further insight into CFS pathogenesis This study used the integration of peripheral blood gene expression results with epidemiologic and clinical data to determine whether CFS is a single or heterogeneous illness

Cross-reactivity between HLA-A2-restricted FLU-M1:58-66 and HIV p17 GAG:77-85 epitopes in HIV-infected and uninfected individuals.

Abstract: Background: The matrix protein of the influenza A virus and the matrix and capsid proteins of the human immunodeficiency virus (HIV) share striking structural similarities which may have evolutionary and biological significance. These similarities led us to hypothesize the existence of cross-reactivity between HLA-A2-restricted FLU-M1:58–66 and HIV-1 p17 GAG:77–85 epitopes. Methods: The hypothesis that these two epitopes are cross-reactive was tested by determining the presence and extent of FLU/GAG immune cross-reactivity in lymphocytes from HIVseropositive and seronegative HLA-A2+ donors by cytotoxicity assays and tetramer analyses. Moreover, the molecular basis for FLU/GAG cross-reactivity in HIV-seropositive and seronegative donors was studied by comparing lymphocyte-derived cDNA sequences corresponding to the TCR-β variable regions, in order to determine whether stimulation of lymphocytes with either peptide results in the expansion of identical T-cell clonotypes. Results: Here, we report evidence of cross-reactivity between FLU-M1:58–66 and HIV-1 p17 GAG:77–85 epitopes following in vitro stimulation of PBMC derived from either HIV-seropositive or seronegative HLA-A2 + donors as determined by cytotoxicity assays, tetramer analyses, and molecular clonotyping. Conclusion: These results suggest that immunity to the matrix protein of the influenza virus may drive a specific immune response to an HLA-A2-restricted HIV gag epitope in HIV-infected and uninfected donors vaccinated against influenza.

Pyrosequencing™ : A one-step method for high resolution HLA typing

Abstract: While the use of high-resolution molecular typing in routine matching of human leukocyte antigens (HLA) is expected to improve unrelated donor selection and transplant outcome, the genetic complexity of HLA still makes the current methodology limited and laborious. Pyrosequencing™ is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given nucleotide dispensation order (NDO) that is programmed into a pyrosequencer. Here we describe the design of a NDO that generates a pyrogram unique for any given allele or combination of alleles. We present examples of unique pyrograms generated from each of two heterozygous HLA templates, which would otherwise remain cis/trans ambiguous using standard sequencing based typing (SBT) method. In addition, we display representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion Pyrosequencing is a one-step method for high resolution DNA typing.

Integration of Translational Research in the European Organization for Research and Treatment of Cancer Research (EORTC) Clinical Trial Cooperative Group Mechanisms

Abstract: The landscape for cancer research is profoundly different today from that only one decade ago. Basic science is moving rapidly and biotechnological revolutions in molecular targeting and immunology have completely modified the opportunities and concepts for cancer treatment. In contrast to the recent past where cytotoxic molecules were screened in the laboratory and then tested in early clinical studies with toxicity as endpoint instead of the often poorly defined mechanism for its potential anti-tumor effect, we now have entered the age of molecular therapeutics, rationally designed to target "strategic" checkpoints that underlie the malignant phenotype. Translational research in early clinical trials (Phase I and II) is an integral aspect of the development of the new generation of cancer drugs as it is necessary to implement radically different early phase clinical trial design and to validate new biological end-points if the full potential of these new agents is to be realized. The "proof of principle with mechanistic analysis" strategy will allow optimisation of therapy from the beginning, and provide important feedback to pre-clinical drug developers. Translational research is also essential in late (phase III) clinical trials in defining different patient populations that may benefit to differing degrees from new treatments, and thus provide further insight and refine clinical practice in a more and more patient-tailored approach. In this editorial we will discuss the integration of Translational Research in the Organization for Research and Treatment of Cancer (EORTC).

Peripheral monocytes from diabetic patients with coronary artery disease display increased bFGF and VEGF mRNA expression

Abstract: Macrophages can produce vascular endothelial growth factor (VEGF) in response to hypoxia, transforming growth factor β1 (TGF-β1), angiotensin II, basic fibroblast growth factor (bFGF), and interleukin-1. These factors have been found in the serum of coronary artery disease (CAD) patients as well as in atherosclerotic lesions. The aim of the present study was to test the hypothesis that the expression of VEGF, TGF-β1 and bFGF in peripheral monocytes and lymphocytes is related to CAD. Human Mononuclear cells and lymphocytes from peripheral blood were isolated from 53 donors undergoing angiography. Seventeen were found to be healthy and 36 were diagnosed with CAD. The respective mRNAs were extracted and quantified. The statistical analysis revealed a significant increase of the basal level expression for macrophage VEGF and bFGF in the CAD SA (stable angina) patient group compared to the noCAD (control) (p = 0.041 and p = 0.022 respectively) and CAD UA (unstable angina) (p = 0.024 and p = 0.005 respectively) groups, which was highly dependent on the diabetic status of the population. Furthermore, we demonstrated with an in vitro cell culture model that the levels of VEGF and bFGF in monocytes of healthy donors are not affected by short term exposure to increased glucose levels (usually observed in the diabetic patients) and/or statin. Our findings display a statistically significant association of the increased VEGF and bFGF levels in peripheral monocytes, with stable angina and diabetes in coronary artery disease. The results give new insight to CAD and the impaired collateral vessel formation in diabetics.

A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings.

Abstract: Detection of unknown single nucleotide polymorphism (SNP) relies on large scale sequencing expeditions of genomic fragments or complex high-throughput chip technology. We describe a simplified strategy for fluorimetric detection of known and unknown SNP by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes. The array consists of two sets of 18-mer oligonucleotide probes. One set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected "consensus" sequence (consensus-oligos), the other includes oligos specific for known SNP within the same genomic region (variant-oligos). Fluorescence-labeled DNA amplified from a homozygous source identical to the consensus represents the reference target and is co-hybridized with a differentially-labeled test sample. Lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant-oligos designates a known allele. Lack of hybridization to consensus- and variant-oligos indicates a new allele. Detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal. This method can identify unknown SNP in heterozygous conditions with a sensitivity of 82% and specificity of 90%. This strategy should dramatically increase the efficiency of SNP detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials.

Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

Abstract: The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization.

rIFN-γ-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition

Abstract: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-γ (rIFN-γ), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-γ on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-γ reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-γ-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-γ. Suppression of arginase activity by rIFN-γ in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-γ EOC cells. Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-γ inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-γ. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-γ or rIFN-γ-inducers, it would be helpful to examine these effects in vivo.

Interferon-γ Added During Bacillus Calmette-Guerin Induced Dendritic Cell Maturation Stimulates Potent Th1 Immune Responses

Abstract: Dendritic cells (DC) are increasingly prepared in vitro for use in immunotherapy trials. Mature DC express high levels of surface molecules needed for T cell activation and are superior at antigen-presentation than immature DC. Bacillus Calmette-Guerin (BCG) is one of several products known to induce DC maturation, and interferon (IFN)-γ has been shown to enhance the activity of DC stimulated with certain maturation factors. In this study, we investigated the use of IFN-γ in combination with the powerful maturation agent, BCG. The treatment of immature DC with IFN-γ plus BCG led to the upregulation of CD54, CD80, and CD86 in comparison with BCG treatment alone. In MLR or recall immune responses, the addition of IFN-γ at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-γ-producing Th1 cells. In primary immune responses, on the other hand, BCG and IFN-γ co-treated DC stimulated higher proportions of specific T cells as well as IFN-γ secretion by these T cells. Thus the use of IFN-γ during BCG-induced DC maturation differentially affects the nature of recall versus naive antigen-specific T-cell responses. IFN-γ co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC. These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.

Fibrinogen: a novel predictor of responsiveness in metastatic melanoma patients treated with bio-chemotherapy: IMI (italian melanoma inter-group) trial

Abstract: To evaluate a panel of pretreatment clinical and laboratory parameters in metastatic melanoma (MM) in order to verify their impact on response and survival in a single prospective multi-institutional phase III study comparing out-patient chemotherapy (CT) vs bioCT. A total of 176 patients were randomised to receive CT (cisplatin, dacarbazine, optional carmustine) or bioCT (the same CT followed by subcutaneous IL-2 plus intramuscular α-IFN-2b). Pretreatment total leucocytes, lymphocytes, eosinophyls, C-reactive protein (CRP), lactate dehydrogenase (LDH), erytrosedimentation rate (ESR), and fibrinogen were analyzed. Some clinical parameters (performance status, age, sex, and disease site) were also considered. As we found a positive trend for bio-CT with no statistical significance in OR (25.3% vs 20.2%) and OS (11 Mo vs 9.5 Mo), all analyses are stratified by treatment arm. In univariate analysis, higher value of lymphocytes percentage (P < .0001), lower value of total leucocytes (P=.005), CRP (P=.003), LHD (P < .0001), ESR (P < .027), fibrinogen (P < .0001), and no liver disease were strongly related to a better survival. In a multivariate analysis, using the Cox proportional hazards model, only fibrinogen (P=.004), LDH (P=.009) and liver disease (P=.04) were found to have an independent role on clinical outcome in metastatic melanoma patients. Liver disease and higher LDH and fibrinogen levels had an important impact on survival in MM patients. In particular, fibrinogen has been recently reconsidered both for its determinant role in the host hemostatic system, and for its capability to provide protection against NK and LAK-cell-induced lysis. These observations could have some important implications for therapeutic approaches, in particular when immunological strategies are used.

Introduction of article-processing charges (APCs) for articles accepted for publication in the Journal of Translational Medicine.

Abstract: The Journal of Translational Medicine's Open Access policy changes the way in which articles are published. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [1]. Third, a copy of the full text of each Open Access article is archived in an online repository separate from the journal. The Journal of Translational Medicine's articles are archived in PubMed Central [3], the US National Library of Medicine's full-text repository of life science literature; e-Depot [4], the National Library of the Netherlands' digital archive of all electronic publications; and Potsdam [5] and INIST [6], sites archiving the research articles published by BioMed Central in Germany and France, respectively.