Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct.

Vasopressin increases both the urea permeability and osmotic water permeability in the terminal part of the renal inner medullary collecting duct (terminal IMCD). To identify the second messengers that mediate these responses, we measured urea permeability, osmotic water permeability, intracellular calcium concentration, and cyclic AMP accumulation in isolated terminal IMCDs. After addition of vasopressin, a transient rise in intracellular calcium occurred that was coincident with increases in cyclic AMP accumulation and urea permeability. Half-maximal increases in urea permeability and osmotic water permeability occurred with 0.01 nM vasopressin. The threshold concentration for a measurable increase in cyclic AMP accumulation was approximately 0.01 nM, while measurable increases in intracellular calcium required much higher vasopressin concentrations (greater than 0.1 nM). Exogenous cyclic AMP (1 mM 8-Br-cAMP) mimicked the effect of vasopressin on urea permeability but did not produce a measurable change in intracellular calcium concentration. Conclusions: (a) Cyclic AMP is the second messenger that mediates the urea permeability response to vasopressin in the rat terminal IMCD. (b) Vasopressin increases the intracellular calcium concentration in the rat terminal IMCD, but the physiological role of this response is not yet known.     

Comparison of two different methods for CD34+ selection and T cell depletion in peripheral blood stem cell grafts--our experiences with CellPro, E rosetting and CliniMACS technique

The aim of this study was to establish a suitable method for in vitro T cell depletion in peripheral blood stem cell grafts for mismatched/haploidentical transplantation in children and adults with severe hematological disorders and for autologous transplantation in patients with autoimmune diseases refractory to conventional immunosuppressive treatment. Two different selection techniques have been used: CD34+ selection using immunoaffinity columns (CellPro Ceprate) followed by T cell depletion by E-rosetting or CD34+ selection using submicroscopic paramagnetic beads (CliniMACS device) with T cell depletion in a one step procedure. The mean purity and recovery of CD34+ cells and efficiency of T cell removal in the final product were compared. From March 1995 to December 1998 we prepared twelve allografts using Cell Pro system for eight children with high-risk hematological malignancies and six autografts for six patients with severe autoimmune diseases. From January 1999 to October 2000 we prepared fifteen allografts using CliniMACS system for ten children with high-risk hematological diseases and inborn metabolic disorders or primary immunodeficiences, five allografts for three adult patients with high-risk hematological malignancies and two autografts for two patients with autoimmune diseases. In allogeneic transplantation the median purity of CD34+ cells in the final products after CellPro and E-rosetting was 85.6% (55.3%-95.7%); median recovery was 24.8% (17%-35%), median transplanted doses of T cells per kilogram of body weight were 0.66x10(4) (0-2.8); in autologous transplantation the median purity of CD34+ was 92.6% (55.6%-96%), median recovery was 28% (22%-46.2%), median transplanted doses of T cells per kilogram of body weight were 0.39x10(4) (0.0-3.6). After CliniMACS technique the median purity of CD34+ cells was 94.87% (69.15%-99%),medianrecoverywas 58% (30%-79.6%), median transplanted doses of T cells per kg of body weight were 0.254x10(4) (0-14.15); in autologous transplantation the median purity of CD34+ was 94% (94%-94%, median recovery was 97.4% (95%-99.8%), median transplanted doses of T cells per kilogram of body weight were 0.87x10(4) (0.49-1.24). We consider both methods of CD34+ selection and T cell depletion suitable for peripheral blood stem cell processing before mismatched hemopoietic stem cell transplantation in patients without identical donor or before autologous transplantation for severe autoimmune diseases. However, magnetic separation using CliniMACS system results in higher levels of purity and recovery with efficient T cell depletion.     

Sclerotic osseous metastases from renal cell carcinoma

This case series describes and illustrates three cases of sclerotic osseous metastases from untreated renal cell carcinoma (RCC). RCC is commonly metastatic to the skeleton but almost always produces lytic metastases, with only three prior reports of sclerotic metastases identified in the literature. Sclerotic metastasis causing low back pain was the initial disease presentation in two of the three patients in this case series and the first manifestation of metastatic disease in one. The most common metastatic sites of RCC, i.e., retroperitoneal lymph nodes, lung, and liver, were not identified in any of the cases, and skeletal involvement with epidural extension was the only site of metastasis in two. Pathologic specimens from all three cases revealed RCC of high nuclear grade.     

The Number of Nucleoli and Main Nucleolar Types in Lymphoblasts of Children Suffering from Acute Lymphoid Leukemia

Nucleoli were studied in lymphoblasts of children (untreated with cytostatic therapy) suffering from acute lymphoblastic leukemias (ALL) by means of a simple cytochemical procedure for the demonstration of RNA to provide information on the incidence of the main nucleolar types and the number of nucleoli in these cells. The values of the nucleolar coefficient reflecting the number of nucleoli per lymphoblast ranged between 1.66 and 2.03. The slightly larger values of the nucleolar coefficient in T-ALL were not statistically significant in comparison with those in nonT-ALL. Lymphoblasts in the bone marrow as well as the peripheral blood of both nonT and T-ALL patients mostly contained, “active” (RNA transcribing) large nucleoli with a relatively uniform distribution of RNA. “Inactive” micronucleoli or particularly “resting” ring shaped nucleoli were noted less frequently in these cells. On the other hand, the larger percentage of lymphoblasts determined in specimens stained with the panoptic staining (May-Grünwald-Giemsa) in comparison with that of lymphoblasts with “active nucleoli” in specimens stained for RNA apparently indicates the absence of such nucleoli in some of these cells. These cells might represent ageing, not proliferating, cells the existence of which has been already suggested by previous studies based on a different methodical approach.     

Quantitative light dosimetry in vitro and in vivo

Knowledge of the radiant energy fluence rate in tissues during laser irradiation is important for the understanding and improvement of the results of preclinical as well as clinical treatments. Quantitative data are extremely rare, however. In this paper, quantitative measurements of energy fluence rates are reported, in vitro as well as in vivo, with emphasis on light dosimetry for photodynamic therapy. Examples are given of fluence rate distributions that may occur during surface irradiation, intracavity irradiation of hollow organs (bladder) and interstitial irradiation. For the same incident irradiance, the energy fluence rate in tissue may vary considerably, depending on type of tissue, wavelength and geometry. During experimental and clinical interstitial PDT-treatments a considerable decrease in light penetration into tumours was observed, apparently indicating changes in optical properties as a result of treatment. This demonstrates the importance of in vivo light dosimetry.     

Photodynamic therapy of oral cancer A review of basic mechanisms and clinical applications

Photodynamic therapy (PDT) is an experimental cancer treatment modality. PDT is based on the accumulation of a photosensitive dye in premalignant and malignant lesions. A certain period of time after the dye has been administered, tumor tissue may contain more of the sensitizer then the surrounding normal tissues. When tissue containing the sensitizer is exposed to light of a proper wavelength and dose, a photochemical reaction between sensitizer and light will occur. The activated photosensitizer reacts with available oxygen which subsequently damages cells and eventually may cause necrosis of the tumor. Photosensitizers can also be used for fluorescence detection. If a tumor contains more of the photosensitizer than the surrounding normal tissue, its fluorescence can potentially be utilized to detect tumors. Analogous to PDT, this can therefore be referred to as photodynamic detection (PDD). This paper reviews the basic mechanisms and clinical applications of PDT and PDD. Emphasis is placed on PDD and PDT with the photosensitizer Photofrin for detection and treatment of premalignant epithelial lesions and squamous cell carcinomas of the oral mucosa.     

In vivo fluorescence and photodynamic activity of zinc phthalocyanine administered in liposomes.

Zinc(II) phthalocyanine, a hydrophobic photosensitiser, was incorporated in unilamellar liposomes and studied in vivo for fluorescence kinetics and photodynamic activity. An observation chamber mounted in a dorsal skinfold of female WAG/Rij rats was used as a model system. In the chamber, an isogeneic mammary carcinoma was transplanted in the subcutaneous tissue. Phthalocyanine fluorescence was excited at 610 nm with a power density of 0.25 mW cm-2 and was detected above 665 nm through a high-pass filter using a two-stage image intensifier coupled to a charge-coupled device (CCD) camera. Following i.v. administration of 0.14 mg kg-1 of the drug, the fluorescence pharmacokinetics of the dye in vasculature, normal tissue and tumour tissue was determined as a function of time. Tumour fluorescence increased slowly to a maximum about 3 h post injection (p.i.), and remained well above the normal tissue fluorescence till 24 h p.i. Fluorescence in the circulation was always stronger than in the tissues. A treatment light dose at a wavelength of 675 nm was delivered 24 h p.i. One group of six animals received a total light dose of 150 J cm-2 (100 mW cm-2). A second group of six animals received a total light dose of 450 J cm-2 at the same dose rate. Vascular damage resulting from treatment was observed only at the final stages of the irradiation, despite the relatively high levels of fluorescence in the circulation. Immediate post-treatment (re)transplantation of the content of the chamber into the flank always resulted in tumour regrowth, confirming the presence of viable tumour cells following photodynamic therapy (PDT). When the chamber was left intact, the light dose of 450 J cm-2 yielded complete tissue necrosis. The role of the dye-carrier complex in shielding the vascular surrounding from photoproducts was studied in a third group of animals. The presence of peroxides was demonstrated in the serum of these animals after PDT with zinc phthalocyanine in liposomes (ZnPc-lip) using a total light dose of 450 J cm-2. This ex vivo observation supports the previously reported observations in vitro that the carrier complex is able to quench the photoproducts resulting from photoactivation of the photosensitiser which is present in the circulation.