Decision analysis of allogeneic hematopoietic stem cell transplantation for patients with myelodysplastic syndrome stratified according to the revised International Prognostic Scoring System.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) represents the only curative treatment for patients with myelodysplastic syndrome (MDS), but involves non-negligible morbidity and mortality. Crucial questions in clinical decision-making include the definition of optimal timing of the procedure and the benefit of cytoreduction before transplant in high-risk patients. We carried out a decision analysis on 1728 MDS who received supportive care, transplantation or hypomethylating agents (HMAs). Risk assessment was based on the revised International Prognostic Scoring System (IPSS-R). We used a continuous-time multistate Markov model to describe the natural history of disease and evaluate the effect of different treatment policies on survival. Life expectancy increased when transplantation was delayed from the initial stages to intermediate IPSS-R risk (gain-of-life expectancy 5.3, 4.7 and 2.8 years for patients aged ⩽55, 60 and 65 years, respectively), and then decreased for higher risks. Modeling decision analysis on IPSS-R versus original IPSS changed transplantation policy in 29% of patients, resulting in a 2-year gain in life expectancy. In advanced stages, HMAs given before transplant is associated with a 2-year gain-of-life expectancy, especially in older patients. These results provide a preliminary evidence to maximize the effectiveness of allo-SCT in MDS.

Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.

A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype.

Photokinetic and ultrastructural studies on porphyrin photosensitization of HeLa cells.

Liposome-bound haematoporphyrin or haematoporphyrin dimethylester, as well as haematoporphyrin dissolved in phosphate-buffered saline, were added to HeLa cell monolayers at a dose of 1 microgram of porphyrin per 10(5) cells. After 2 min or 20 min incubation liposome-bound porphyrins were accumulated by cells in an about two-fold larger amount than the water-dissolved haematoporphyrin. This caused a more efficient photosensitization of HeLa cells by liposome-delivered porphyrins upon illumination with 366 nm light. Ultrastructural studies of HeLa cells, which had been incubated in a physiological medium for 24h after the end of irradiation, showed that liposomal porphyrins induce an early and extensive endocytoplasmic damage, leading to swelling of the mitochondria and vesiculation; changes of the permeability of the cytoplasmic membrane are also evident, especially in the case of haematoporphyrin dimethylester. On the other hand, water-dissolved haematoporphyrin predominantly photosensitizes damage of the plasma membrane. The different pattern of cell photodamage probably reflects a different subcellular distribution of the photosensitizing drugs.

Efficient photosensitization of malignant human cells in vitro by liposome-bound porphyrins.

Comparative kinetics of porphyrin uptake and release by HeLa cells, incubated with equivalent concentrations of either hematoporphyrin (Hp) in aqueous solution or Hp and its dimethylester (HpDME) bound to unilamellar liposomes, show that liposomal porphyrins are bound at a higher rate and in considerably larger amounts. Moreover, the release of cell-bound porphyrins into the medium is remarkably reduced and slowered after cell loading with liposome-bound porphyrins. The presence of 1% bovine or human serum albumin (but not serum globulins) in the medium has no effect on uptake and release of liposome-bound porphyrins by HeLa cells, whereas it remarkably decreases the uptake of aqueous Hp. Parallel studies of cell photodamages under known concentrations of cell-bound porphyrin unequivocally demonstrate that the photodynamic effect is strictly related to the porphyrin load. As a consequence a dramatic increase of cell-photosensitizing efficiency is obtained by binding Hp (and even more HpDME) with liposomes. Electron microscopy investigations on cell damages caused by loading with liposome-bound porphyrins and subsequent illumination show that the plasmatic membrane is one important cell site of porphyrin interaction and photodynamic effect.

Familial hypoplastic glomerulocystic kidney. A new entity?

Two pairs of female siblings of French and Italian origin presented with the histological picture of glomerulocystic kidneys. The cases differ from the patients previously described with glomerulocystic kidneys by the absence of major extrarenal malformations, the reduction of kidney size with absence of renal papillae and by the presence of stable chronic renal failure, starting during the first months of life. Both mothers of the patients also had chronic renal failure with similar urographic abnormalities.

Disruption of cytoskeleton by methylmercury in cultured CHO cells.

The effect of methylmercury (MM) on three main cytoskeletal components [i.e. microtubules (MT), microfilaments (MF) and intermediate filaments (IF)] and on specific biochemical parameters (i.e. glutathione transferase (GST), glutathione reductase (RED), glutathione peroxidase (GSH-Px), glyoxalase 1 (GLY 1) and total -SH groups (TSH) of the cytosolic fraction) was studied in cultured Chinese hamster ovary (CHO) cells. The experiments were conducted with increasing doses of MM (i.e. 1, 4 and 8 mum), using an exposure time of 16 hr; and with a fixed dose of MM (2 mum), using increasing exposure periods (i.e. 0-24 hr). The morphological changes observed by immunofluorescence seemed to indicate that MF were damaged as much as (if not more than) MT after 16 hr of exposure to 4 mum-MM. At a concentration of 1 mum, MM only affected MF. The time-course experiments revealed that IF as well as MF and MT were severely disorganized after 3 and 6 hr of incubation in the presence of 2 mum-MM. However, an obvious reorganization was observed after 24 hr of exposure. In experiments using increasing MM doses, changes in the enzymatic activities were less noticeable than those observed in the morphology; only a modest decrease in TSH and RED activities (<30%) was recorded at the highest dose of MM used (i.e. 8 mum). In contrast, increasing the time of exposure to MM induced changes in both the cytoskeletal structures and the biochemical parameters: the lowest RED activity and TSH were observed after 3-6 hr exposure; control values were obtained after an exposure period of 24 hr. Ultrastructural observations on cells treated with increasing doses of MM showed changes in plasmamembrane profile, cytoskeleton organization and mitochondrion structure. The results confirm that MM causes non-specific damage to CHO cells and suggest that a functional interaction may exist between GSH-dependent enzymes and cytoskeletal structures.

Morphological aspects of an experimental tumour photosensitized with a meso-substituted cationic porphyrin.

The meso-substituted cationic porphyrin, meso-tetra(4N-methyl-pyridyl)porphine (T4MPyP) appears to be a selective tumour localizer on the basis of pharmacokinetic studies. Irradiation (at 600-680 nm) of an intramuscularly implanted MS-2 fibrosarcoma in Balb/c mice at 24 h after injection of T4MPyP causes tumour necrosis: histological and ultrastructural analyses of tumour specimens taken at different times after phototherapy indicate slowly-appearing tissue damage which involves both malignant cells and the vascular endothelium. At the subcellular level, the membranous systems and nuclei are the main targets of the photoprocess. The tumour necrosis is particularly extensive upon injection of 4.1 mg kg-1 T4MPyP.

Apoptosis of mouse MS-2 fibrosarcoma cells induced by photodynamic therapy with Zn (II)-phthalocyanine.

The destructive process of mouse MS-2 fibrosarcoma induced by photodynamic therapy (PDT) with liposome-administered Zn(II)-phthalocyanine (ZnPc) was studied by electron microscopy. Pronounced ultrastructural changes characteristic of apoptosis were observed for several tumour cells, including early occurrence of condensation and margination of chromatin, disappearance of nuclear pores, karyopyknosis, karyorrhexis, protuberance formation at the cell surface and cell fragmentation. The findings indicate that apoptosis was involved in the process of tumour cell death induced by ZnPc-PDT. The detailed mechanism and pathways controlling this phenomenon need to be elucidated further.

Tumour-localizing and tumour-photosensitizing properties of zinc(II)-octapentyl-phthalocyanine.

Zn(II)-octapentyl-phthalocyanine (ZnOPPc), incorporated into a Cremophor oil emulsion, was found to be a highly selective tumour-targeting agent (50-fold larger concentration in the tumour than in the peritumoral tissue) when injected at a dose of 1.46 mumol (kg body weight)-1 in Balb/c mice bearing an intramuscularly transplanted MS-2 fibrosarcoma. The pharmacokinetic properties of this phthalocyanine were closely similar to those found for the analogous octadecyl derivative, whereas the unsubstituted Zn(II)-phthalocyanine showed a lower efficiency and selectivity of tumour targeting than the octaalkyl-substituted phthalocyanines. Irradiation of the ZnOPPc-loaded tumour with 620-700 nm light 24 h after injection caused a significant delay of tumour growth with a gradual shrinkage of the neoplastic mass; the damage involved important contributions from both random necrosis and apoptosis of malignant cells.

[Membranous nephropathy associated with chronic hepatitis caused by B virus]. / Nefropatia membranosa associata ad epatite cronica da virus B.

We report 6 cases with membranous glomerulonephritis associated with chronic hepatitis B virus infection, who came under our observation for microscopic haematuria in 2 cases, haematuria and proteinuria in 1 case, and nephrotic syndrome in the others. At the examination all 6 children were found to have hepatomegaly with raised serum transaminase activity. All the patients were positive in the serum for HBsAg and anti-HBc, 3 were HBeAg and anti-HBe negative. Liver biopsy showed features of chronic hepatitis with moderate signs of activity. Renal biopsy was consistent with membranous glomerulonephritis in all patients. With a fluorescent antibody technic HBeAg was found to be deposited in diffuse granular fashion, along glomerular capillary walls, together with IgG, in 2 out of 3 cases stained, but no deposition of HBsAg was detected in all the patients. Steroid therapy was started from 18 to 33 months. Urine analysis became negative in 4 cases and persisted normal in the follow-up. One patient with haematuria developed nephrotic syndrome and one died from miliary tuberculosis. Our findings suggest that the clinical outcome is favourable in children with membranous glomerulonephritis and chronic active hepatitis and that the pathogenesis probably is not unique.

Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Mucous, endocrine and plicated cells.

The following five cell types have been recognized and defined on the basis of their fine structure in the gastric epithelium of B. schlosseri: vacuolated and zymogenic cells (described in a previous paper); ciliated mucous, endocrine and plicated cells. The ciliated mucous cells are distributed at the apex and the bottom of the gastric folds and along the dorsal groove. The mucus droplets appear to form from the Golgi complex as secretory granules of variable density and texture, which are released from the cell after fusion of their membranes with the apical plasma membrane. Holocrine or apocrine secretion has not been observed. The endocrine cells are scattered and are characterized by electron dense granules, especially numerous in the basal region of the cell. Finally, the plicated cells, present in the pyloric caecum, show rod-like microvilli, a well developed Golgi complex and abundant, deep infoldings of the basal plasma membrane, which are associated with numerous mitochondria. The possible role of the gastric cell types is discussed taking into account information concerning morphologically similar cells in other animals, as well as previously reported data on the biochemistry and physiology of digestion and excretion in ascidians.

Fine structure of the intestinal epithelium of the colonial ascidian Botryllus schlosseri.

In the intestine of the filtering zooids of B. schlosseri three segments can be distinguished. In the intermediate segment the epithelium, which is encrusted by the ampullae of the pyloric gland, shows marked aspects of alteration. In the proximal and distal segments, ciliated mucous, vacuolated and endocrine-like cells are recognizable. Ciliated mucous cells, widely distributed along the intestine, possess the apical region filled with numerous mucous granules, which are extruded with merocrine modality. Variations in morphology of the granules are visible especially between cells of different regions. Vacuolated cells appear involved in absorptive function. They are characterized by developed microvilli, numerous apical small vesicles and great supranuclear vacuoles containing heterogeneous material. The vacuolated cells of the proximal segment resemble the gastric vacuolated cells of B. schlosseri. The vacuolated cells of the distal segment show many morphological similarities with protein absorbing cells of various animals for the presence of a giant vacuole and an apical network of vesicles and tubules with fuzzy coating on the luminal face. The intestinal endocrine-like cells are rare and characterized by strongly electron dense granules distributed in all the cytoplasm, but predominantly in the basal region.

[Ultrastructure of the gills of Carcinus maenas]. / Ultrastruttura delle branchie di Carcinus maenas.

In the present paper it is reported the ultrastructure of the gills from Carcinus maenas, showing the different morphology of the epithelium of the anterior, respect to the posterior gills which can justify, according to some biochemical data, a different function mechanism.

Electron microscopy for measurement of glomerular basement membrane width in children with benign familial hematuria.

Glomerular basement membrane (GBM) width was measured in 13 children with benign familial hematuria (BFH) and in 13 controls (6 children and 7 adults), using a standardized procedure and examining 3 glomeruli in each case. GBM widths were evaluated by comparing arithmetic means and considering the frequency of segments with a thickness less than 150 and 200 nm. With the former method, only 6 children with BFH fell below the normal values, while with the latter, 9 out of 13 were below the normal range. When BFH is suspected, it appears that GBM width is best evaluated by considering only the thinner segments, because the arithmetic mean can be influenced by the presence of thick segments and therefore has less diagnostic value.

Zn(II)-phthalocyanine as a photodynamic agent for tumours. II. Studies on the mechanism of photosensitised tumour necrosis.

The mechanism of tumour necrosis photosensitised by liposome-delivered Zn(II) phthalocyanine (Zn-Pc) has been studied in mice bearing a transplanted MS-2 fibrosarcoma. Ultrastructural analyses of tumour specimens obtained at different times after red light-irradiation (300 J cm-2, dose-rate 180 mW cm-2) indicate an early (3 h) photodamage of malignant cells especially at the level of the mitochondria and rough endoplasmic reticulum. The cellular damage becomes more evident between 6 h and 15 h after photodynamic therapy. On the other hand, the capillaries supplying the tumour tissue are modified at a much slower rate and appear to be severely damaged only after 15 h from irradiation, when the whole tissue becomes necrotic. Occasionally, mildly damaged capillaries are observed even at 72 h after irradiation. These findings support the hypothesis that low density lipoproteins (LDL) play a major role in the delivery of Zn-Pc to the tumour tissue; the photosensitiser is released specifically to malignant cells as a consequence of a receptor-mediated endocytosis of LDL.

Ultrastructural mucosal appearance in the ileal neobladder.

METHODS: 15 patients with ileal neobladder underwent endoscopic biopsy at different postoperative intervals. The specimens were analyzed by electron microscopy in order to evaluate the evolution of the mucosal changes ultrastructurally. RESULTS: No significant change was observed 3 months after the operation. After 6 months, the number and height of the microvilli were reduced, the cell borders crooked and the terminal web upset. After 12 months disappearance of the glycocalyx, increased lysosomal features, increased activity of the muciparous cells, rounded mitochondria and loss of the polarized disposition of the cytoplasmic organelles were detected in the enterocytes. We observed no other substantial change after 24 months and more. CONCLUSIONS: Progressive modifications occur in the cytoplasmic structures involved in the absorptive process. They do not seem to begin before 3 months and are almost totally completed after 1 year.

Effects of cyclosporin A on paracellular and transcellular transport of horseradish peroxidase in perfused rat livers.

The immunosuppressive agent cyclosporin A (CsA) is known to cause cholestasis. Transcellular and paracellular transport of macromolecules contribute, albeit to a minor extent, to bile formation, but little is known about the effects of CsA on these pathways. The aims of this study were to investigate the influence of CsA on tight junction (paracellular) permeability and on transcytotic vesicular pathways labeled with horseradish peroxidase (HRP) in perfused rat liver. Livers from male Sprague-Dawley rats were perfused with Krebs-Henseleit buffer (albumin 1%, RBC 20%, and amino acid mixture). Taurodehydrocholate (1 microM/min) was coinfused into the portal vein; 1 microg/ml of CsA, dissolved in Cremophor-EL (CsA livers), or the vehicle alone (CEL livers), was added to the medium. Tight junction permeability was assessed by administering HRP (25 mg) as a short pulse to perfused rat livers, operating under single-pass conditions. Under such conditions, HRP output into the bile shows two components: an initial peak at approximately 3-5 min, corresponding to paracellular transfer across tight junctions, and a second peak at approximately 15 min, corresponding to vesicular transport. Furthermore, we assessed the vesicular transport pathway by examining HRP-labeled vesicles in the perisinusoidal (PS) and pericanalicular (PC) areas using ultrastructural morphometric analysis. To analyze HRP in hepatocytes and to study rapid and late transcytotic vesicular pathways, a 1-min pulse of a high dose of HRP (500 and 200 mg, respectively) was given. Two and 18 min after single-pass perfusion the livers were fixed with 2.5% glutaraldehyde-0.8% paraformaldehyde in 0.1 mM cacodylate buffer, pH 7.8. The total pericanalicular area, the HRP-containing structures, were quantified morphometrically in liver samples. At concentrations of 1.2 microg/ml, CsA produced a twofold increase in the paracellular transfer of HRP to bile. The areas under the second peak (transcellular vesicular pathway) of the biliary HRP secretion curve were similar in CEL- and CsA-treated livers. Morphometric analysis confirmed that CsA treatment did not affect the percentage area of HRP-labeled vesicles in either the pericanalicular or in the perisinusoidal area at 2 min (rapid pathway) and 18 min (late pathway). These results indicate that CsA increases tight junctional permeability whereas it does not inhibit rapid or late transcytotic vesicle pathways.

Tumour-localising and -photosensitising properties of a novel zinc(II) octadecylphthalocyanine.

1,4,8,11,15,18,22,25-Octadecylphthalocyaninato zinc(II), ZnODPc, incorporated into a Cremophor emulsion, was assayed for its pharmacokinetic and phototherapeutic properties in Balb/c mice bearing an intramuscularly transplanted MS-2 fibrosarcoma. The phthalocyanine was injected intravenously (i.v.) in three doses, i.e. 1.46, 0.73 and 0.37 mumol kg-1 body weight. In all cases, the octadecyl-substituted phthalocyanine showed an unusually high affinity for serum low-density lipoproteins (LDLs) and a high efficiency and selectivity of tumour targeting: the maximum accumulation in the tumour occurred at 24 h after injection, whereas no detectable amount of phthalocyanine was recovered from the muscle, i.e. the peritumoral tissue, between 1 h and 1 week after injection. At the same time, low amounts of phthalocyanine were recovered from skin and then only at short times after injection, with skin photosensitivity rapidly disappearing and the phthalocyanine present in the serum only. Tumour photosensitisation studies were carried out at 24 h after administration of 1.46 mumol kg-1 ZnODPc and showed that this phthalocyanine has a very high phototherapeutic efficiency; this is probably a consequence of the multiple mechanisms by which the phthalocyanine induces tumour damage, involving both direct modification of malignant cells and impairment of blood flow, as well as the alteration of a variety of subcellular components, such as mitochondria, the rough endoplasmic reticulum, the perinuclear membrane and, occasionally, cell nuclei. Tumour necrosis appears to be the consequence of both random cell death and apoptosis.