Strategic regulatory approaches for the qualification of a biomarker assay for safety use.

Biomarkers can be defined as key molecular or cellular events that link a specific biological event to a health outcome. As such, biomarkers play an important role in understanding the relationships between exposure to a xenobiotic, the development of chronic human diseases, and the identification of subgroups that are at increased risk of disease. Much progress has been made in identifying and validating new biomarkers to be used in population-based studies. The increasing availability and use of biomarkers to aid informed decision-making in risk-benefit decisions highlights the need for careful assessment of the validity of such models. In particular, models involving new biomarkers require careful validation and regulatory acceptance.

Herpesvirus saimiri transformation may help disclose inherent functional defects of mucosal T lymphocytes in patients with gastric adenocarcinoma.

To dissect the phenotypic and functional features of mucosal T lymphocytes in patients with gastric adenocarcinoma, we have used the Herpesvirus saimiri transformation procedure to achieve T-cell lines from gastric origin. Once achieved, cell function was assessed by in vitro stimulation with mitogens. CD2-specific monoclonal antibodies (alpha-CD2), alone or in combination with interleukin (IL)-2, rendered fewer counts in patients (34 408+/-3965 and 52 157+/-6473 c.p.m., respectively) than in controls (67 471+/-11 755 c.p.m., P<0.01 and 77 864+/-12 545 c.p.m., P<0.05, respectively). Likewise, CD3-based responses were defective in cancer cell lines: alpha-CD3 (54 794+/-9269 vs 86 104+/-10 341 c.p.m., P<0.01), alpha-CD3+IL-2 (57 789+/-8590 vs 88855+/-8516 c.p.m., P<0.01) and alpha-CD3+alpha-CD2 (52 130+/-7559 vs 120 852+/-16 552 c.p.m., P<0.01). Finally, IL-2 failed to adequately stimulate patient cell lines (39 310+/-4023 vs 60 945+/-9463 c.p.m., P<0.05). These results suggest that mucosal T lymphocytes in cancer patients are inherently impaired in their proliferative ability. This may be crucial in the control of tumour growth.

FcRgamma chain does not replace CD3zeta chain in CD3zeta-deficient T lymphocytes of patients with gastric adenocarcinoma.

Defective CD3zeta chain expression has been reported in T lymphocytes of patients with inflammatory diseases, such as systemic lupus erythematosus or osteoarthritis, and with cancer. In lupus, the absent CD3zeta chain is replaced by the FcRgamma chain, rendering the T cells hyper responsive. However, there are no data on T lymphocytes from patients with cancer. In this study, the presence of the FcRgamma chain and its associated kinase, Syk, was analysed in patients with gastric adenocarcinoma and healthy subjects. Western blot and immunoprecipitation experiments were carried out with total cell or lipid raft extracts from fresh peripheral blood mononuclear cells or T lymphocytes, and Herpesvirus saimiri-derived T-cell lines (of blood or tissue origin). Our results revealed that the absent CD3zeta chain in cancer T lymphocytes was not replaced by FcRgamma either in fresh T cells or T-cell lines, in contrast to lupus T cells. This altered expression of signalling molecules in T lymphocytes of cancer patients, would explain their low proliferative capacity. Our T-cell lines represent tools to unveil the signalling abnormalities of cancer T lymphocytes.

Expression of CD45 and proliferative response to CD3 as suitable classification markers of patients with gastric adenocarcinoma.

25 patients with resectable gastric adenocarcinoma, subdivided according to the absence or presence of residual neoplasic disease (RND- or RND+, respectively), were studied. Cytofluorometric analysis and proliferative responses to mitogens was performed in peripheral blood mononuclear cells of patients. When compared to healthy subjects, the percentage of CD3-expressing cells was significantly reduced in both groups of patients studied (p < 0.0001 in all instances). However, when CD45 is considered instead of (CD3, its expression is found to be significantly reduced only in the RND+ patients (72% +/- 11), when compared with the control group (96 +/- 1%, p < 0.0001). Likewise, cells from these patients significantly less proliferated when stimulated with monoclonal antibodies to CD3 than control cells (18,920 +/- 6,019 cpm vs. 42,697 +/- 1,798 cpm, p = 0.0036); a difference not found if RND- patients (33,619 +/- 11,733 cpm) were considered. We propose that the low expression of CD45 and the poor response to CD3 are markers that are able to identify the subgroup of patients in whom the disease will tend to progress more rapidly. We also suggest the use of such markers as additional criteria for the classification of patients with gastric adenocarcinoma or to identify patients who require more aggressive therapeutic strategies.

Defective CD3zeta chain expression in Herpesvirus saimiri (HVS)-derived T-cell lines in gastric adenocarcinoma.

Low expression of the CD3zeta chain has been reported in patients with cancer and it has been suggested that tumor-derived factors are involved in its downregulation. The expression of CD3zeta chain was measured in T-cell lines from patients with gastric adenocarcinoma and healthy volunteers and grown in vitro for several months and, hence, in the absence of any tumor-derived factors. T-cell lines of mucosal origin were obtained by Herpesvirus saimiri transformation from gastric cancer patients. The expression of CD3zeta and CD3epsilon was measured by flow cytometry and Western-blot analysis. Calcium mobilization and apoptosis rate were also measured. The levels of CD3zeta, but not CD3epsilon, chain on the cell surface were significantly reduced in T-cell lines derived from patients with gastric cancer when cultured in the absence of IL-2. Western-blot analysis of total cell extracts or lipid raft fractions confirmed this finding. Calcium mobilization, a measure of signal transduction, was reduced in T cell lines from patients with gastric cancer. We conclude that T cells from patients with cancer express lower levels of CD3zeta. This downregulation is not caused by a direct effect of tumor-derived factors but, rather, it appears to be inherent to the patient cells. The low CD3zeta expression would render T lymphocytes unable to control the growth of tumor cells.

Increased levels of bovine serum albumin antibodies in patients with type 1 diabetes and celiac disease-related antibodies.

OBJECTIVES: To detect the presence of antibodies against bovine serum albumin in a cohort of Spanish patients with type 1 insulin-dependent diabetes. METHODS: Antibodies were measured using an in-house enzyme-linked immunosorbent assay test in 80 patients with type 1 diabetes, subdivided according to the presence or absence in their serum of celiac disease-related antibodies. For comparison, 30 patients with celiac disease (nondiabetic), 13 patients with autoimmune thyroiditis, and 45 healthy volunteers were used. RESULTS: Thirty-one percent of patients with diabetes yielded a positive result, with a mean value of 26.1 +/- 21.8 arbitrary units (AU). If the group was split into those with celiac disease-related antibodies and those lacking them, the percentages were 53% and 25%, respectively, with a mean value of 39.6 +/- 28.4 AU and 22.4 +/- 18.3 AU (P = 0.003), respectively. Seventy-three percent of celiac patients showed bovine serum albumin antibodies with a mean level of 38.8 +/- 27.7 AU, comparable to that of patients with diabetes with celiac antibodies, but higher than the group lacking them (P = 0.001). Although 46% of patients with autoimmune thyroiditis had positive results, the level detected (22.1 +/- 8.7 AU) was significantly lower than that recorded in patients with type 1 diabetes who had celiac disease antibodies (P = 0.04) and celiac patients (P = 0.04). Healthy volunteers showed no antibodies against bovine serum albumin. CONCLUSIONS: These data suggest that bovine serum albumin antibodies appears in patients with a compromised epithelial permeability, and they reflect a general defect in the process of immunologic tolerance associated with a predisposition to autoimmunity, rather than immunity specific to beta cells.