Aims The excretion of low molecular weight heparin (LMWH) in breast milk was investigated in 15 lactating mothers after Caesarean section. Methods Blood and milk samples were collected before and 3±4 h after once daily routine subcutaneous injection of 2500 IU dalteparin. Anti-Xa activity was measured by an assay utilizing prolonged clotting times in plasma or breast milk as an index of LMWH activity. Results Plasma anti-Xa activities ranged from 0.074 to 0.308 IU ml x1 of plasma. Anti-Xa activities in breast milk ranged from <0.005±0.037 IU ml x1 of milk. This is equivalent to a milk/plasma ratio of <0.025±0.224. Conclusions Therefore, it appears highly unlikely that puerperal thromboprophylaxis with LMWH has any clinically relevant effect on the nursing infant.
In order to confirm and further explore the significance of the overexpression of the CRABP II (cellular retinoic acid binding protein type II) and psoriasin genes in psoriatic versus normal skin, we examined the mRNA expression levels of these two genes by in situ hybridization in skin samples from psoriatic plaques and in one case from the border between a psoriatic plaque and uninvolved skin. Both genes were markedly upregulated in lesional skin, with a shift from low to high expression in the transitional zone of the plaque. Expression of the cytokeratin 1 (K1) gene was, in contrast, high in normal skin and decreased in the transition from uninvolved skin to psoriatic plaque, Examination of mRNA levels of CRABP II and psoriasin in other hyperproliferative and inflammatory skin diseases showed high expression of psoriasin, and in some cases also of CRABP II, in atopic dermatitis, mycosis fungoides, Darier's disease and inflammatory lichen sclerosus et atrophicus. In atrophic lesions of lichen sclerosus et atrophicus that lacked an inflammatory infiltrate, these changes were only weakly expressed. These findings demonstrate that altered epidermal gene expression of K1, psoriasin and CRABP II is not disease-specific and may reflect instead an altered state of epidermal differentiation and/or may be linked to the inflammation and cellular infiltration common to all the conditions studied.
Thrombin has recently been shown not only to exert procoagulant activities, but also to induce mitogenic responses of different cell types involved in wound healing via binding to and cleavage of the thrombin receptor. In order to further explore these aspects of thrombin function, human keratinocytes (HaCaT cell line) were examined for their potential mitogenic responsiveness to thrombin and for the dependency of this process on the expression of the high-affinity thrombin receptor. Quiescent keratinocytes were stimulated in the mitogenic assay with alpha-thrombin and the thrombin receptor activating peptides TRAP42-55 (SFLLRNPNDKYEPY) and TRAP42-46 (SFLLR). A strong induction of cell proliferation was noted with alpha-thrombin, TRAP42-55 and TRAP42-46, but not with the "scrambled" peptide (FSLLR). These findings confirm that keratinocytes express the thrombin receptor and that the sequence of the first two amino acids of the generated neo-N-terminus are important for the activation of the receptor. Using cDNA fragments of the 5' coding sequence of the receptor, Northern blot analysis confirmed that HaCaT keratinocytes express the thrombin receptor. Expression of the receptor was also detected on normal human keratinocytes by immunohistochemistry and in situ hybridization. These data demonstrate the expression and biologic function of the human thrombin receptor on human keratinocytes, suggesting that thrombin, among other mediators, plays an important part in the orchestration of epidermal growth and repair processes.
The aim of this study was to investigate the effects of 13-cis retinoic acid treatment on cellular retinoic acid binding protein II (CRABP II) mRNA expression in sebaceous follicles from acne patients, using in situ hybridization. Biopsies were taken from uninvolved skin areas in close juxtaposition to inflamed comedos before therapy, and at 2-4 or 14-16 weeks of treatment. Paraffin sections were used for in situ hybridization study with riboprobes transcribed from human CRABP II cDNA. After oral treatment with 13-cis retinoic acid, sebaceous glands were reduced in size and atrophic, and the ratio of sebum-free to fully differentiated (sebum-producing) sebocytes was dramatically increased. The CRABP II expression in the sebaceous gland, and to some extent in infundibular structures, was strongly increased compared with the level of expression in the epidermis. The maximum signal was always found in layers of suprabasal sebocytes lacking lipid droplets, but never in the basal layers. These findings indicate a selective activity of 13-cis retinoic acid on CRABP II mRNA expression in the sebaceous glands of acne patients.
In situ hybridization histochemistry (rsH) using cRNA probes (riboprobes) has become a powerful technique for the examination of gene expression in tissue sections. The construction of plasmid templates for the synthesis of riboprobes with phage RNA polymerases is often a difficult and timeconsuming step. We have therefore developed a rapid, Hicient, and flexible method to generate totally artificial riboprobe templates by the polymerase chain reaction (PCR). We have made riboprobe templates using self-priming oligonucleotide primers spanning 146 BP of the 3 end of the human cytokeratin 1 (Kl) gene coding region flanked by T7 and T3 promoters. These PCR-derived riboprobe templates were used to synthesize 35S-labeled anti-sense riboprobes as well as sense riboprobes as negative controls. The riboprobes
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