Irreversible oxidations of platelet proteins after riboflavin-UVB pathogen inactivation.

Pathogen inactivation technologies are known to alter in vitro phenotype and functional properties of platelets. Because pathogen inactivation generates reactive oxygen species, oxidative stress is considered as one of the plausible cause at the origin of the platelet storage lesion acceleration after treatment. To date proteomics has been used to document the protein variations to picture out the impact. Here, platelet concentrates were prepared from buffy-coats in Intersol additive solution, leukoreduced and pathogen inactivated using a riboflavin/UVB treatment. At day 2 of storage the platelet proteomes of control (untreated) and treated platelet concentrates were investigated against the site specific oxidation by liquid chromatography coupled to tandem mass spectrometry in a shotgun experiment. The shotgun approach detected 9350 peptides (and 2534 proteins) of which 1714 were oxidized. Eighteen peptides were found exclusively oxidized in treated platelets whereas 3 peptides were only found oxidized in control. The present data evidenced an interference with several proteins involved in platelet aggregation and platelet shape change (such as talin and vinculin).

Towards the understanding of the UV light, riboflavin and additive solution contributions to the in vitro lesions observed in Mirasol®-treated platelets.

OBJECTIVES: Pathogen reduction technologies are implemented to increase the safety of blood products. We previously showed that the UVB alone significantly contributes to the storage lesions observed in platelets treated with riboflavin/UVB using a home-made illuminator. The present study aims at confirming these observations using the commercial Mirasol® technology. METHODS: A three-arm study (untreated, UV-, Mirasol®-treated platelets) was conducted to investigate the platelet storage lesions throughout storage (n=4). A two-arm study was then designed to compare Intersol and T-PAS+ additive solutions (n=3). Phenotype and functional platelet characteristics were assessed using flow cytometry, aggregometry, antioxidant assays and metabolic parameters. RESULTS: Mirasol®-treated platelets exhibit enhanced storage lesions compared to controls (increase of activation markers and glycolysis rate, lower hypotonic shock and double-agonist activation responses, and decrease of total antioxidant capacity). Here, we also confirmed that the UV radiation alone is causing platelet lesions. Riboflavin tends to have an intracellular protective role while it decreases the extracellular antioxidant defenses. Furthermore, benefits of platelet additive solutions containing potassium and magnesium were confirmed as it reduces the extent of storage lesions. CONCLUSIONS: The photosensitizer, UV illumination and composition of the platelet additive solutions are key parameters influencing the platelet storage lesion. The clinical relevance of these findings is not fully understood and recent published clinical studies could not show increase in bleeding in patients receiving Mirasol-treated platelets. New developments in storage solutions might help to improve storage conditions of PRT-treated platelets and should be prioritised as research subject in the future.

The storage lesions: From past to future.

Red blood cell (RBC) concentrates are stored in additive solutions at 4oC for up to 42 days, whereas platelets concentrates (PCs) are stored at 22oC with continuous agitation for up to 5 to 7 days, according national regulations, and the use or not of pathogen inactivation procedures. Storage induces cellular lesion and alters either RBC or platelet metabolism, and is associated with protein alterations. Some age-related alterations prove reversible, while other changes are irreversible, notably following protein oxidation. It is likely that any irreversible damage affects the blood component quality and thus the transfusion efficiency. Nevertheless, there still exists a debate surrounding the impact of storage lesions, for both RBCs and PCs. Uncertainty is not completely resolved. Several studies show a tendency for poorer outcomes to occur in patients receiving older blood products; however, no clear significant association has yet been demonstrated. The present short review aims to promote a better understanding of the occurrence of storage lesions, with particular emphasis on biochemical modifications opening discussions of the future advancement of blood transfusion processes. The paper is also an advocacy for the implementation of an independent international organization in charge of planning and controlling clinical studies in transfusion medicine, in order to base transfusion medicine practices both on security principles, but also on clinical evidences.

Compound heterozygosity for a recessive glycine substitution and a splice site mutation in the COL7A1 gene causes an unusually mild form of localized recessive dystrophic epidermolysis bullosa.

Type VII collagen is the major component of anchoring fibrils, adhesion structures of stratified epithelia that span the basement membrane region and papillary dermis. Mutations in the gene COL7A1 encoding type VII collagen cause dystrophic epidermolysis bullosa, a clinically heterogeneous autosomal dominant or recessive blistering disorder of the skin and mucous membranes. In this report, we investigate three siblings affected by an unusually mild form of localized recessive dystrophic epidermolysis bullosa who were shown to be compound heterozygotes for novel mutations affecting COL7A1. The maternally inherited mutation is a G-->C transversion that converts a codon for glycine to a codon for arginine (G1347R). The paternal mutation is a neutral G-->A transition at the last base of exon 70(5820G-->A) that alters the correct splicing of COL7A1 pre-mRNA, giving rise to an aberrant mRNA carrying the in-frame skipping of exon 70 in addition to the full-length RNA transcript carrying the G-->A substitution. Consistent with the normal levels of COL7A1 mRNA transcripts detected by northern analysis, immunoblotting and immunofluorescence studies evidenced that the patient keratinocytes synthesize and secrete normal amounts of stable type VII collagen, which is correctly deposited at the dermal-epidermal junction. In addition, mutated type VII collagen molecules assemble to form numerous, normally shaped anchoring fibrils, as shown by electron microscopic examination. The combination of a recessive glycine substitution with a splice site mutation that permits partially correct splicing therefore leads to a normal expression of mutated type VII collagen molecules with marginally altered biologic activity, and to the extremely mild phenotype observed in our patients.

Survival and prognostic factors in patients with localised cutaneous melanoma observed between 1980 and 1991 at the Istituto Dermopatico dell'Immacolata in Rome, Italy.

530 patients with localised cutaneous melanoma consecutively observed between 1980 and 1991 at a hospital for skin diseases in Rome, Italy, were studied. Crude survival proportions were calculated with the method of Kaplan and Meier. Cox proportional hazards regression analysis was used to estimate the effect of prognostic factors on death rates. Females and younger patients had better 5- and 10-year survival rates, while increasing tumour thickness was associated with a decrease in survival time. In the multivariate analysis, an independent association with survival was found for tumour thickness, presence of ulceration, age, sex and cross-sectional profile of neoplasia. Our study confirms that females and young patients with thin melanomas have a better prognosis, while the importance of cross-sectional profile needs further study.

Time trends in features of cutaneous melanoma at diagnosis: central-south Italy, 1962-1991.

A total of 1360 cases of cutaneous malignant melanoma (CMM) consecutively diagnosed at the Istituto Dermopatico dell'Immacolata (IDI) in Rome, during the period 1962-1991, were reviewed. There was a positive trend of thin (Breslow thickness < 1.49 mm) lesions at diagnosis in comparison to thick lesions, with more severe prognosis (p < 0.05). CMM on the trunk and upper legs increased more than CMM on the face (p < 0.05). There are suggestions that the incidence of CMM in the hospital referral population, resident in Central-South Italy, has been steadily rising. This trend could be due not only to a referral bias related to a growing public concern about "bad moles", but also to a real increase in the incidence of CMM.

HBV infection in Italian family groups.

Intrafamilial spread of HBV infection was evaluated in 85 Italian family groups. Secondary infected cases were observed in 43% of these families, of which 73% showed clusters of HBs positive members, with a prevalence among the family members (children-siblings) of the mother/wife index cases, and no strict relation to their "e" system. The presence of the delta agent in the index case was correlated to a higher percentage of secondary HBs-positive cases, but the intrafamilial spread of delta agent was a rare event. A similar higher incidence of HBV markers was observed among the relatives of the index cases positive for auto-antibodies. The evidence presented suggests the importance of genetic factors in the acquisition and clearance of HBV and delta infections.