Comparison of cryopreservation methods on T-cell responses to islet and control antigens from type 1 diabetic patients and controls.

BACKGROUND: Type 1 diabetes (T1D) is a cell-mediated autoimmune disease characterized by destruction of the pancreatic islet cells. The use of cryopreserved cells is preferable to the use of freshly isolated cells to monitor clinical trials to decrease assay and laboratory variability. METHODS: The T-Cell Workshop Committee of the Immunology of Diabetes Society compared two widely accepted T-cell freezing protocols (warm and cold) to freshly isolated peripheral blood mononuclear cells from patients with T1D and controls in terms of recovery, viability, cell subset composition, and performance in functional assays currently in use in T1D-related research. Nine laboratories participated in the study with four different functional assays included. RESULTS: The cold freezing method yielded higher recovery and viability compared with the warm freezing method. Irrespective of freezing protocol, B cells and CD8+ T cells were enriched, monocyte fraction decreased, and islet antigen-reactive responses were lower in frozen versus fresh cells. However, these results need to take in to account that the overall response to islet autoantigens was low in some assays. CONCLUSIONS: In the current study, none of the tested T-cell functional assays performed well using frozen samples. More research is required to identify a freezing method and a T-cell functional assay that will produce responses in patients with T1D comparable to responses using fresh peripheral blood mononuclear cells.

Genetic variation of the platelet- surface integrin GPIIb-IIIa (PIA1/A2-SNP) shows a high association with Type 2 diabetes mellitus.

AIMS/HYPOTHESIS: The gene encoding the beta(3)-subunit (GPIIIa) of the platelet alpha(2)beta(3)-integrin (fibrinogen receptor) shows a polymorphism PlA1/A2 with the A2 allele putatively associated with an increased risk of acute ischaemic events. This study investigated whether Type 2 diabetes as a particular macrovascular risk factor associates with the thrombogenic PIA2 genotype. METHODS: The PlA genotype was determined in 112 consecutive Type 2 diabetic patients additionally classified according to the presence of macrovascular disease. Forty-four non-diabetic patients with angiografically documented cardiovascular disease (CAD/ AMI) and a further 59 non-diabetic subjects with no angiografical signs of CAD were investigated as genomic background control (n=103). PIA-genotyping was carried out by standard restriction fragment length analysis (RFLA) of PCR amplified lymphocyte template DNA. RESULTS: The overall allelic PlA2- prevalence accounted to 34.8% (39/112) in diabetic patients as compared to 14.6% (15/103) in non-diabetic patients [OR 3.1 (1.6-6.1), p<0.01]. This odds ratio increased to 7.0 (2.5-19.7), (p<0.01) in subjects free of criteria of macrovascular disease. In non-diabetic control subjects without CAD there was an allelic PIA2 frequency of 10.2% (6/59) as compared to 20.5% (9/44) in patients with CAD and a history of AMI being less than either diabetes subgroup. The PIA2 prevalence in the subgroup of diabetes patients with macrovascular complications did not differ from the respective value in patients without macrovascular disease. [29.0% (20/69) vs. 44.2% (19/43)]. CONCLUSION/INTERPRETATION: This study confirms a trendwise association of PlA2 with severe coronary artery disease, but rather suggests an even stronger, highly significant association with the metabolic condition of Type 2 diabetes mellitus. This justifies the speculation that pathways dependent on the platelet alpha(2)beta(3) integrin physiology could be implicated in the pathogenesis of Type 2 diabetes which lends further support to the "common soil" hypothesis of diabetes and vascular disease.

Leukocyte apoptosis in whole blood involves platelet-dependent coaggregation.

BACKGROUND: Activated leukocytes and platelet-leukocyte interaction are involved in the pathogenesis of thrombotic and inflammatory events. Because apoptosis is a prerequisite for the successful resolution of an inflammatory response, we investigated the amount of apoptotic peripheral blood leukocytes (PBLs) in whole blood and their possible functional relation with the platelet-leukocyte interaction by a flow cytometric assay using APO 2.7 antibody for the detection of apoptosis METHODS: Thirty healthy subjects volunteered for the study. PBL apoptosis in seven volunteers was induced by phorbol 12-myristate 13-acetate or while standing at rest. RESULTS: Apoptosis was observed in all types of leukocytes (0.7% neutrophils, 1.5% monocytes, and 0.3% lymphocytes). Apoptosis was found predominantly in platelet and leukocyte coaggregates (<1% of nonaggregated leukocytes vs. 9% of platelet and leukocyte coaggregates). This phenomenon was even more pronounced after induction of leukocyte apoptosis in vitro (66% of platelet and leukocyte coaggregates). CONCLUSIONS: Apoptosis and platelet-leukocyte interaction seemed to be closely related phenomena, and apoptotic leukocytes seemed to trigger adhesion and, hence, activation of platelets. Because platelet-leukocyte interaction is involved in the pathogenesis of thrombotic events, apoptotic leukocytes may constitute an additional prothrombotic trigger.