Generation and expansion of multipotent mesenchymal progenitor cells from cultured human pancreatic islets.

Cellular models and culture conditions for in vitro expansion of insulin-producing cells represent a key element to develop cell therapy for diabetes. Initial evidence that human beta-cells could be expanded after undergoing a reversible epithelial-mesenchymal transition has been recently negated by genetic lineage tracing studies in mice. Here, we report that culturing human pancreatic islets in the presence of serum resulted in the emergence of a population of nestin-positive cells. These proliferating cells were mainly C-peptide negative, although in the first week in culture, proliferating cells, insulin promoter factor-1 (Ipf-1) positive, were observed. Later passages of islet-derived cells were Ipf-1 negative and displayed a mesenchymal phenotype. These human pancreatic islet-derived mesenchymal (hPIDM) cells were expanded up to 10(14) cells and were able to differentiate toward adipocytes, osteocytes and chondrocytes, similarly to mesenchymal stem/precursor cells. Interestingly, however, under serum-free conditions, hPIDM cells lost the mesenchymal phenotype, formed islet-like clusters (ILCs) and were able to produce and secrete insulin. These data suggest that, although these cells are likely to result from preexisting mesenchymal cells rather than beta-cells, hPIDM cells represent a valuable model for further developments toward future replacement therapy in diabetes.

Effects of cigarette smoke in mice with different levels of alpha(1)-proteinase inhibitor and sensitivity to oxidants.

The role of strain difference in the response to cigarette smoke was investigated in mice. Mice of the strains DBA/2 and C57BL/6J responded to acute cigarette smoke with a decrease of the antioxidant defenses of their bronchoalveolar lavage (BAL) fluids. On the other hand, under these conditions ICR mice increased their BAL antioxidant defenses. Mice of these three strains were then exposed to cigarette smoke (three cigarettes/d, 5 d/wk) for 7 mo. Lung elastin content was significantly decreased in C57BL/6J and DBA/2 but not in ICR mice. Also, emphysema, assessed morphometrically using three methods, was present in C57BL/6J and DBA/2 but not in ICR mice. In an additional study pallid mice, with a severe serum alpha(1)-proteinase inhibitor (alpha(1)-PI) deficiency and that develop spontaneous emphysema, were exposed to cigarette smoke for 4 mo. This resulted in an acceleration of the development of the spontaneous emphysema assessed with morphometrical and biochemical (lung elastin content) methods. All these results indicate that sensitivity to the effects of cigarette smoke is strain-dependent and cigarette smoke accelerates the effects of alpha(1)-PI deficiency.

Human SLPI inactivation after cigarette smoke exposure in a new in vivo model of pulmonary oxidative stress.

The role of oxidative stress in inactivating antiproteases is the object of debate. To address this question, we developed an in vivo model of pulmonary oxidative stress induced by cigarette smoke (CS) in mice. The major mouse trypsin inhibitor contrapsin is not sensitive to oxidation, and the mouse secretory leukoprotease inhibitor (SLPI) does not inhibit trypsin. Instead, human recombinant (hr) SLPI inhibits trypsin and is sensitive to oxidation. Thus we determined the effect of CS in vivo on hrSLPI antiproteolytic function in the airways of mice. CS caused a significant decrease in total antioxidant capacity in bronchoalveolar lavage fluid (BALF) and significant changes in oxidized glutathione, ascorbic acid, protein thiols, and 8-epi-PGF(2alpha). Intratracheal hrSLPI significantly increased BALF antitryptic activity. CS induced a 50% drop in the inhibitory activity of hrSLPI. Pretreatment with N-acetylcysteine prevented the CS-induced loss of hrSLPI activity, the decrease in antioxidant defenses, and the elevation of 8-epi-PGF-(2alpha). Thus an inactivation of hrSLPI was demonstrated in this model. This is a novel model for studying in vivo the effects of CS oxidative stress on human protease inhibitors with antitrypsin activity.

Genetic deficiency of alpha1-PI in mice influences lung responses to bleomycin.

It has recently been suggested that proteinase inhibitors modulate the fibrotic response in the lung. This study investigated the development of bleomycin-induced pulmonary changes in pallid mice, deficient in serum alpha1-proteinase inhibitor, and with a lower elastase inhibitory capacity, and in congenic C57Bl/6J mice. Male pallid and C57Bl/6J mice received a single intratracheal instillation of either saline or bleomycin. The investigation was carried out by means of biochemical, morphological and morphometrical methods. In both strains, 21 and 72 h after bleomycin, the lungs showed foci of inflammatory cell infiltration associated with emphysema. Fibrosis developed with time after bleomycin. At 14 days fibrosis affected 23.46+/-9.48% (mean +/- SD) and 40.62+/-13.34% (p < 0.01) of the lungs of C57Bl/6J and pallid mice, respectively. Emphysema affected 3.68+/-3.11% and 12.57+/-4.13% (p<0.01) of lung in C57Bl/6J and pallid mice, respectively. In C57Bl/6J mice bleomycin increased lung hydroxyproline content by 34% and desmosine content by 44% (p < 0.01 for both). In pallid mice these increases were only 21% (p < 0.01) and 6% which may reflect parenchymal loss. Thus, the lung destructive response (emphysema) and the subsequent proliferative reaction (fibrosis) to bleomycin are potentiated in alpha1-proteinase inhibitor deficiency.

GFAP is expressed as a major soluble pool associated with glucagon secretory granules in A-cells of mouse pancreas.

To elucidate the role of intermediate filament proteins in endocrine cells, we investigated the expression and subcellular distribution of GFAP in mouse islets of Langerhans. For this purpose, combined immunocytochemical and biochemical analysis with a panel of antibodies was carried out to identify GFAP-immunoreactive cells in mouse endocrine pancreas. Cell fractionation into NP-40-soluble and detergent/high salt-insoluble components was performed to assess whether GFAP was located in the cytosolic and/or cytoskeletal compartments of immunoreactive cells. Immunoelectron microscopic analysis was carried out to determine the subcellular distribution of the protein. Peripheral islet cells were stained with anti-GFAP antiserum. These cells were identified as glucagon-secreting cells by immunocytochemical staining of consecutive sections with anti-somatostatin, anti-GFAP, and anti-glucagon antisera. Western blotting analysis of both NP-40-soluble and detergent/high-salt insoluble fractions of isolated islets of Langerhans allowed detection of GFAP in both cytosolic and cytoskeletal compartments. Interestingly, however, the former location was highly predominant. In addition, immunoelectron microscopy localized GFAP associated with the periphery of secretory granules. On the basis of these results, an intriguing role for GFAP in secretory events should be strongly suspected.(J Histochem Cytochem 48:1233-1242, 2000)

Neutrophil influx into the lungs of beige mice is followed by elastolytic damage and emphysema.

The beige mouse is currently used as a model of elastase and cathepsin G deficiency to demonstrate or exclude the role of these proteases in a variety of pathologic conditions. We recently demonstrated that beige cathepsin G is tightly bound to neutrophil lysosomal membranes but is released in near normal quantities during exocytosis. Also, beige neutrophils contain a latent form of elastase that undergoes spontaneous activation when released under in vitro or in vivo conditions. However, the pathogenic potential of this enzyme in matrix degradation has not been ascertained previously. The possibility that in beige mice elastolytic proteases from neutrophils recruited into the lung have the capability to damage alveolar septa was investigated following an intratracheal instillation of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (200 microg). Neutrophil influx was followed by a decrease in lung elastin content (-18%) and by a significant increase of the mean linear intercept (+30%) and of morphologic emphysema. The onset of pulmonary lesion was preceded by a marked increase of neutrophil elastase burden on the alveolar interstitium. The appearance of emphysema was prevented by administration of the serine protease inhibitor 4-(2-aminoetyl)-benzenesulfonyl fluoride hydrochloride (2. 4 microg/ml saline). These results demonstrate that the lung elastin degradation and emphysema can occur in beige lungs. The fact that the beige mouse does develop lung elastolytic changes after neutrophil recruitment indicates that this mutant cannot be considered a model of neutrophil function deficiency and used as a model of elastase deficiency.

Neutrophils in beige mice secrete normal amounts of cathepsin G and a 46 kDa latent form of elastase that can be activated extracellularly by proteolytic activity.

Among other phenotypic defects, the beige mouse is susceptible to infection and has large neutrophil granules that apparently secrete a decreased amount of elastolytic activity. We have shown using in vitro methods that cytosolic inhibitors in beige neutrophils are normal. Although cathepsin G is tightly bound to lysosomal membranes, normal amounts of activity are released in response to degranulating agents. Decreased elastolytic activity is secreted by beige neutrophils because elastase is present in the granules as a 46 kDa proenzyme, which can be activated extracellularly by a protease-dependent mechanism. The current experiments were undertaken to explore the in vivo functions of neutrophils from C57 BI/6J (bg/bg) beige mice using the model of casein-induced acute peritonitis; normal C57 BI/6J (+/+) mice served as controls. The kinetics of neutrophil accumulation in the peritoneum were normal, suggesting normal neutrophil migration. Cathepsin G activity in the cell-free supernatant of peritoneal lavage fluid was normal; elastolytic activity was initially very low but increased to about twice baseline level after 4 h at 25 degrees C and to about 20-fold at 36 h. The appearance of this activity was inhibited to varying degree (54 to 83%) by different protease inhibitors (pepstatin, antipain, aprotinin, leupeptin and chymostatin). We conclude that the decreased amount of elastolytic activity secreted by beige neutrophils into an inflammatory exudate is due to a genetic defect that results in production of a 46 kDa proelastase rather than the normal 29 kDa active elastase; the proelastase can be spontaneously activated by a protease-dependent mechanism. In light of these data, the use of the beige mouse as a model for the Chediak-Higashi syndrome, and as a model in which neutrophils do not produce elastase, must be reconsidered.

Neutrophil recruitment into the lungs is associated with increased lung elastase burden, decreased lung elastin, and emphysema in alpha 1 proteinase inhibitor-deficient mice.

The possibility that polymorphonuclear leukocytes (PMN) recruited into the lung have the capability to damage alveolar septa was investigated in several strains of mice with different serum alpha 1 proteinase inhibitor levels and PMN lysosomal functions. After an intratracheal instillation of FMLP (200 micrograms), all strains of mice showed a similar PMN influx in alveolar spaces with an increase (approximately 4- to 5-fold) in bronchoalveolar lavage total cell count, which peaked at 24 to 48 hours. At this time, differential cell count in all strains revealed an approximately 40-fold increase in neutrophils. In C57BL/6J and pallid mice but not in NMRI mice, PMN influx was followed by a decrease in lung elastin content (-17% and -37%, respectively) and by the development of significant emphysema (mean linear intercept, +28% and +56%, respectively). The onset of the pulmonary lesion was preceded by a marked increase of neutrophil elastase burden in alveolar interstitium. Compared with NMRI mice, C57BL/6J and pallid mice have lower serum elastase inhibitory capacity levels. The degree of lung destruction was inversely correlated with elastase inhibitory capacity levels. Lung elastin degradation and emphysema may be induced by eliciting PMN into the lungs only in animals with a deficient anti-elastase screen. Compared with C57BL/6J mice, pallid mice showed a significantly greater lung elastin loss and a higher degree of emphysema after FMLP treatment. These differences may be accounted for by the higher baseline levels of interstitial elastase burden. It may be assumed that an enzymatically active elastase was already working on the lung interstitium before FMLP instillation in pallid mice.

[A case of thrombotic thrombocytopenic purpura]. / Considerazioni a proposito di un caso di porpora trombotica trombocitopenica.

Thrombocytopenic thrombotic purpura is a comparatively rare disease and little is known about its aetiopathogenesis. Once almost invariably negative, the prognosis has been changed by the introduction of plasmapheresis. A particularly complex clinical case is described and a number of conclusions are drawn from it: 1) there is no criterion for opting for any particular therapeutic approach; 2) evaluation of the clinical response is the only criterion for monitoring the effectiveness of ongoing treatment; 3) plasmapheresis is proposed as the treatment of choice; 4) vincristine is a useful alternative in the treatment of recalcitrant cases.

[2 cases of metabolic acidosis during therapy of portosystemic encephalopathy with branched-chain amino acids]. / Due casi di acidosi metabolica in corso di terapia con soluzioni di aminoacidi a catena ramificata per encefalopatia porto-sistemica.

Two patients known to be suffering from liver cirrhosis were admitted to hospital in a state of coma. The apparent ineffectiveness of treatment with branched chain amino acids was found in reality to be due to the onset of metabolic acidosis. The suspension of treatment and balancing of the metabolic imbalance with an infusion of bicarbonate solutions led to lessening of the neurological symptomatology.

[Plasmodium falciparum malaria transmitted through human contact in a group of drug addicts]. / Malaria da Plasmodium falciparum trasmessa per contagio interumano in un gruppo di tossicomani.

The Authors describe a case of malaria from P. falciparum occurred in a group of young drug-addicts. Noteworthy is the fact that such a way of transmission did never occur before in Italy. Current concepts in treatment of malaria are briefly reviewed.

[Fiberoptic endoscopic examination in the diagnosis and therapy of digestive hemorrhages of the supra-mesocolic tract. Retrospective analysis of 46 cases]. / L'indagine fibroendoscopica nella diagnosi e nella terapia delle emorragie digestive del tratto sporamesocolico. Analisi retrospettiva di 46 casi.

Forty-six cases of upper gastrointestinal haemorrhage examined during three years have been revised. The patients were 39 men and 7 women, from 22 to 73 years old: twenty-six of them were first examined by endoscopic technique, while in the other twenty-ones the first examination was a bariummeal X-ray. All the twenty-six patients of the first group were examined within 48 hours from admission to hospital while the twenty patients of the second group could be examined not before the third day from admission. The evaluation of our results has confirmed an overt superiority of endoscopic versus radiologic technique, mainly in detecting non-ulcerative mucosal alterations responsible of bleeding.