The Vitamin D Analogue Calcipotriol Reduces the Frequency of CD8+ IL-17+ T Cells in Psoriasis Lesions.

The vitamin D analogue calcipotriol is an immunomodulatory drug widely used to treat psoriasis; however, how calcipotriol affects the immune cells in psoriasis lesions is not fully understood. The aim of this study was to investigate the effect of calcipotriol on the frequency of CD4(+) and CD8(+) T cells and innate lymphoid cells (ILC) and their production of IL-17A, IFN-γ and IL-22 in psoriasis lesions in patients with chronic plaque psoriasis. Eighteen patients with psoriasis were included, and two similar psoriasis lesions were chosen for each patient. One lesion was treated with calcipotriol (50 µg/g) and the other with vehicle twice a day for 14 days. The clinical effect was measured by degree of erythema, scaling and induration in each lesion (SUM score). Skin biopsies were collected for histological and immunohistochemical analyses. Skin-derived cells were isolated and analysed by flow cytometry. After 14 days of treatment with calcipotriol, a significant clinical and histological effect was seen; however, we found no differences in the frequency of CD4(+) and CD8(+) T cells or ILC between calcipotriol- and vehicle-treated skin. The main finding was a significant decrease in CD8(+) IL-17(+) T cells in skin-derived cells from calcipotriol-treated skin, which was further supported by the absence of CD8(+) IL-17(+) T cells in immunohistochemical staining of calcipotriol-treated skin. No changes in the frequency of IL-22(+) or IFN-γ(+) cells were observed. Our findings show that the vitamin D analogue calcipotriol reduces the frequency of CD8(+) IL-17(+) T cells in psoriasis lesions concomitant with clinical improvement.

Correlation between thymidylate synthase gene variants, RNA and protein levels in primary colorectal adenocarcinomas.

This study was designed to compare thymidylate synthase (TS) genotype, mRNA and protein levels in primary colorectal adenocarcinoma, and to examine the correlation between microsatellite instability (MSI) and TS expression. The TS genotype of 68 patients with colorectal cancer was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis in peripheral blood mononuclear cells and tumour tissue. The TS mRNA levels in tumour tissue were measured by reverse-transcription PCR, and TS protein levels and MSI status were assessed using immunohistochemistry. Significantly higher mRNA and protein levels were observed in patients with the TS 3R/3R versus the 2R/2R and 2R/3R genotypes. There was no correlation between TS single nucleotide polymorphism and TS expression. Individuals homozygous for the six base-pair insertion in the 3'-untranslated region had significantly higher TS mRNA levels than heterozygous and homozygous wild type individuals. The TS mRNA and protein levels were significantly higher in microsatellite unstable tumours compared with microsatellite stable tumours. There was a significant association between the number of TS enhancer region repeats (in blood) and intratumoural TS mRNA and protein levels. A larger case series investigating the role of TS gene polymorphisms as predictors of sensitivity to 5-fluorouracil-based chemotherapy is required.

The human placenta from heavy smokers: evaluation of vasoactive peptides by immunohistochemistry.

The study aimed to demonstrate the expression of nitric oxide converting enzyme, nitric oxide synthase (e-NOS), and endothelin-1 (Et-1) in formalin-fixed paraffin-embedded placental tissue, and to demonstrate a difference in staining intensity between heavy smokers and non-smokers. Term placentas from pregnancies from otherwise healthy women smoking 15 or more cigarettes per day (heavy smokers) and term placentas from a matching group of non-smokers were included. The antibodies for Et-1 and e-NOS are recommended for cryostat sections. We evaluated the antibodies on paraffin-embedded tissue combined with the streptavidin-biotin-peroxidase technique. Et-1 and e-NOS were demonstrated in the placental vasculature, the trophoblast, and the amnion. A blinded comparative study showed no reproducible significant differences in the staining intensity of the antigen-antibody reaction to Et-1 and e-NOS between the two groups.

Folate receptor in malignant effusions of ovarian carcinoma.

Binding of 3H-folate in human ovarian adenocarcinoma tissue was of a high-affinity type (K approximately 10(10) M-1) and displayed apparent positive cooperatively. A high-affinity folate receptor was also present in ascitic fluid and pleural effusion. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. The folate analogues methotrexate and in particular 5-formyltetrahydrofolate acted as inhibitors of 3H-folate binding in ascitic fluid. Ovarian adenocarcinoma tissue showed immunostaining with rabbit antibodies against human milk folate-binding protein. The gel filtration diagram contained two peaks of radiolabelled folate (at 25 and 100 kDa). The 25 kDa peak was predominant in ascitic fluid and pleural effusion. A single band of 70 kDa was seen on SDS-PAGE immunoblotting of tissue and malignant effusions. The concentration of folate receptor in tissue and fluid specimens could be determined by an immunochemical method (ELISA) utilizing antibodies against human milk folate-binding protein.

A high-affinity soluble folate receptor in fluids of non-neoplastic ovarian cysts: radioligand binding, molecular size, hydrophobic residue, and immunological properties.

The presence of a soluble folate receptor in fluids of non-neoplastic ovarian cysts was demonstrated. Radioligand binding exhibited characteristics typical of high-affinity folate-binding proteins. These included positive cooperativity, a tendency to increased binding affinity with decreasing receptor concentration, a slow ligand dissociation at pH 7.4 and inhibition by folate analogues. The folate receptor was probably synthesized in the lining epithelial cells of the cysts which showed positive immunostaining with antibodies against human milk folate-binding protein. The gel filtration profile of cystic fluid contained two radioligand-bound peaks, 25 and 100 kDa, whereas a single band of 70 kDa was seen on SDS-PAGE immunoblotting. Treatment with the enzyme phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100 kDa peak to the 25 kDa peak. This suggests that insertion of a hydrophobic glycosylphosphatidylinositol tail into Triton X-100 micelles could give rise to large molecular size forms of the receptor on gel filtration.

The high-affinity folate receptor of normal and malignant human colonic mucosa.

The hypothesis that folate depletion is a risk factor for development of colonic neoplasia prompted us to study the presence of a putative folate receptor in human colon mucosa. Binding of 3H-folate to normal and malignant mucosa studied by equilibrium dialysis was of high-affinity type (K = 10(10) L/mol) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate was a potent inhibitor of binding. Gel filtration revealed a 25 kDa and a 100 kDa peak of folate-binding activity. Immunoreactivity studies were performed with rabbit antibodies against human 25 kDa milk folate-binding protein. Immunoblotting showed a single band at 65 kDa, and tissue sections exhibited immunostaining of mucosal areas. The present folate receptor with characteristics similar to those of other high-affinity folate-binding proteins may serve as a regulator of intracellular folate concentration in colon mucosa.

Folate receptor of human mammary adenocarcinoma.

Binding of 3H-folate to human mammary tumor homogenate was of a high-affinity type (K = 10(10) M-1) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate acted as a strong inhibitor of radioligand binding. Gel chromatography of radioligand-labeled homogenate of tumor tissue revealed three peaks: a small > or = 110 kDa peak and two major peaks of folate-binding activity (M(r) approximately 25 kDa and M(r) approximately 100 kDa). Mammary tumor tissue showed immunostaining with rabbit antibodies against human milk folate binder. A parallel elevation in the concentrations of folate-binding protein and triglyceride in tumor tissue as compared to normal tissue adjacent to the tumor was compatible with the localization of folate-binding protein in the triglyceride-rich fraction of mammary gland homogenate.