Severe right heart failure in a patient with chronic obstructive lung disease: a diagnostic challenge.

A 55-year-old male was admitted for evaluation of severe dyspnoea and hypoxaemia. Physical examination upon admission showed elevated jugular venous pressure and an accentuated second heart sound. Chest radiograph showed cardiomegaly with increased bibasilar markings. Arterial blood gas analysis while breathing room air showed marked hypoxaemia. High resolution computed tomography angiography of the chest showed modestly enlarged mediastinal lymph nodes with discrete diffuse ground-glass attenuation especially at the lower lung zones. Positron emission tomography using 18F labelled 2-deoxy-D-glucose (FDG) demonstrated the mediastinal lymph nodes were FDG-avid. Transthoracic echocardiography showed dilated hypokinetic right heart chambers with bulging of the interventricular septum to the left, compatible with acute cor-pulmonale. From the tricuspid regurgitation jet measurement a systolic pulmonary artery pressure (PAP) of 48 mmHg was estimated. Patent foramen ovale was suspected on bubble test. Right heart catheterisation confirmed pulmonary arterial hypertension: mPAP 47 mmHg, pulmonary artery occlusion pressure 5 mmHg, cardiac index 1.1 L/min/m2, pulmonary vascular resistance (PVR) 959 dyne.sec.cm(-5). Pulmonary function tests showed a marked diffusing capacity for carbon monoxide (DLCO) decrease of 32% predicted but no obstructive lung deficit. Before an open lung biopsy could be scheduled the patient developed acute cardiogenic shock. At autopsy pulmonary veno-occlusive disease with marked pulmonary hypertension was diagnosed.

Contribution of postnatally formed small beta cell aggregates to functional beta cell mass in adult rat pancreas.

AIMS/HYPOTHESIS: Neogenesis of beta cells and their clustering to small aggregates is a key process in prenatal development of beta cell mass. We investigated the contribution of postnatally formed small aggregates to functional beta cell mass in adult rats. METHODS: Conditions were defined for (1) counting total beta cell number in pancreases with relative error of <10% and (2) determining their distribution over aggregates of different size and over functionally different subpopulations. RESULTS: Pancreases of 10-week-old male Wistar rats contained 2.8 ± 0.2 × 106 beta cells, of which >90% was generated postnatally, involving: (1) neo-formation of 30,000 aggregates with diameter <50 µm including single cells; and (2) growth of 5,500 aggregates to larger sizes, accounting for 90% of the increase in cell number, with number of growing aggregates in the tail 50% greater than elsewhere. At 10 weeks, 86% of aggregates were <50 µm; compared with aggregates >200 µm, their beta cells exhibited a higher basal insulin content that was also resistant to glibenclamide-induced degranulation. The pool of Ki67-positive beta cells was sixfold larger than at birth and distributed over all aggregate sizes. CONCLUSIONS/INTERPRETATION: We describe a method for in situ counting of beta cell numbers and subpopulations with low relative error. In adult rats, >90% of beta cells and beta cell aggregates are formed after birth. Aggregates <50 µm are more than 100-fold more abundant than aggregates >200 µm, which are selected for isolated islet studies. Their topographic and functional properties contribute to the functional heterogeneity of the beta cell population; their growth to larger aggregates with characteristic beta cell functions may serve future metabolic needs.

Human islet cell implants in a nude rat model of diabetes survive better in omentum than in liver with a positive influence of beta cell number and purity.

AIMS/HYPOTHESIS: Intraportal human islet cell grafts do not consistently and sustainably induce insulin-independency in type 1 diabetic patients. The reasons for losses in donor cells are difficult to assess in patients. This study in streptozotocin-diabetic nude rats examines whether outcome is better in an extra-hepatic site such as omentum. METHODS: Intraportal and omental implants of human islet cell grafts with the same beta cell number were followed for function and cellular composition over 5 weeks. Their outcome was also compared with that of rat islet cell grafts with similar beta cell numbers but higher purity. RESULTS: While all intraportal recipients of rat islet cell grafts were normoglycaemic until post-transplant (PT) week 5, none was with human islet cell grafts; loss of human implants was associated with early infiltration of natural killer and CD45R-positive cells. Human islet cell implants in omentum achieved plasma human C-peptide positivity and normoglycaemia in, respectively, nine of 13 and five of 13 recipients until PT week 5; failures were not associated with inflammatory infiltrates but with lower beta cell numbers and purity of the grafts. Observations in human and rat islet cell implants in the omentum suggest that a delayed revascularisation can interfere with their metabolic outcome. Irrespective of normalisation, human omental implants presented beta cell aggregates adjacent to alpha cells and duct cells. CONCLUSIONS/INTERPRETATION: In nude rats, human islet cell implants survive better in omentum than in liver, with positive influences of the number and purity of implanted beta cells. These observations can guide studies in patients.

The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation.

AIMS/HYPOTHESIS: Defects in pancreatic beta cell turnover are implicated in the pathogenesis of type 2 diabetes by genetic markers for diabetes. Decreased beta cell neogenesis could contribute to diabetes. The longevity and turnover of human beta cells is unknown; in rodents <1 year old, a half-life of 30 days is estimated. Intracellular lipofuscin body (LB) accumulation is a hallmark of ageing in neurons. To estimate the lifespan of human beta cells, we measured beta cell LB accumulation in individuals aged 1-81 years. METHODS: LB content was determined by electron microscopical morphometry in sections of beta cells from human (non-diabetic, n = 45; type 2 diabetic, n = 10) and non-human primates (n = 10; 5-30 years) and from 15 mice aged 10-99 weeks. Total cellular LB content was estimated by three-dimensional (3D) mathematical modelling. RESULTS: LB area proportion was significantly correlated with age in human and non-human primates. The proportion of human LB-positive beta cells was significantly related to age, with no apparent differences in type 2 diabetes or obesity. LB content was low in human insulinomas (n = 5) and alpha cells and in mouse beta cells (LB content in mouse <10% human). Using 3D electron microscopy and 3D mathematical modelling, the LB-positive human beta cells (representing aged cells) increased from >or=90% (<10 years) to >or=97% (>20 years) and remained constant thereafter. CONCLUSIONS/INTERPRETATION: Human beta cells, unlike those of young rodents, are long-lived. LB proportions in type 2 diabetes and obesity suggest that little adaptive change occurs in the adult human beta cell population, which is largely established by age 20 years.

Physiological regulation of total tubulin and polymerized tubulin in tissues.

Polymerized and depolymerized forms of tubulin were measured in rat and mouse liver, rat islets, human lymphocytes, and platelets. The percent of the total tubulin present in the polymerized form varied from 30.3 +/- 1.5% in the liver of the fed rat to 89.2 +/- 0.2% in human platelets. Fasting decreased the total tubulin and to a greater extent the polymerized form of tubulin in both rat and mouse liver. Glucose feeding increased the polymerized tubulin without affecting the total tubulin content in rat liver. Phytohemagglutinin-stimulated lymphocytes exhibited at least a three-fold increase in total tubulin (expressed in terms of DNA content), which during the initial 48 h of incubation was accounted for in toto by an increase in polymerized tubulin. It is suggested that the lectin not only accelerates tubulin synthesis but also stimulated the polymerization process. Storage of platelets at 4 degrees C for 6 days resulted in a marked decrease in total tubulin and an even greater reduction in the polymerized form. It is concluded that both the total tubulin content and its degree of polymerization can be modulated independently by a wide variety of physiological factors.

A sensitive method for measuring polymerized and depolymerized forms of tubulin in tissues.

A rapid method for measuring polymerized and depolymerized forms of tubulin in tissues has been developed. The procedure consists of homogenization and centrifugation of the tissue in a microtubule-stabilizing solution and depolymerization of the precipitated microtubules; polymerized and depolymerized forms of tubulin are quantitated by a colchicine-binding assay. The validity of the technique was assessed by electron microscopy and recovery studies with labeled and unlabeled preparations of polymerized and depolymerized forms of rat brain tubulin. The sensitivity of this technique allows quantitation of tubulin in 150 micrograms of tissue, wet weight. The method demonstrated that both the polymerized and depolymerized forms of tubulin were present in rat liver cells, and that in the fed state 31.3 +/-0.7% of the total tubulin pool was in the polymerized form.

Microtubule assembly and the intracellular transport of secretory granules in pancreatic islets.

Polymerized and depolymerized tubulin were measured in pancreatic islets under various physiological and pharmacological conditions. Variations in insulin release from islets of fed or fasted rats were accompanied by concomitant changes in tubulin polymerization. Glucose induced the formation of microtubules in vitro independent of extracellular calcium. Total and polymerized tubulin content were decreased by fasting and restored by glucose feeding.

Transplantation of purified islet cells in diabetic rats. III. Immunosuppressive effect of cyclosporin.

This study examined the effect of cyclosporin on the survival of islet beta-cell allografts in streptozocin (STZ)-induced diabetic rats. At a daily oral dose of 5 mg/kg, the agent prevented the rejection of isolated islets, provided they were little contaminated by other pancreatic tissue. The immunosuppressive effect rapidly disappeared after discontinuation of the drug, except when the donor tissue had been pretreated to reduce its nonendocrine content. All recipients of cultured and selected islets maintained a normalized state for greater than 15 wk beyond the 5-wk drug course; this was not the case for shorter periods of treatment. A long-term beneficial effect was also observed in all recipients of purified islet beta-cell grafts, which reversed without treatment in half of the cases. Cyclosporin markedly reduced the mononuclear cell infiltration in each type of islet beta-cell allograft; aggregates of mixed endocrine islet cells were kept virtually infiltration free. Conditions with minimal initial infiltration were associated with long-term graft survival without the need for continuous pharmacological immunosuppression. We conclude that a short-term cyclosporin treatment can induce long-term survival of allografted islet beta-cells, provided the grafts are only slightly contaminated by nonendocrine elements. In rodents, sufficient immunosuppression was achieved by circulating cyclosporin levels of 100-400 ng/ml. Higher concentrations were cytotoxic for cultured islet beta-cells and islet non-beta-cells. A 5-wk treatment with the immunomodulator ciamexone also resulted in long-term survival of purified beta-cell allografts but not of cultured islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of purified islet cells in diabetic rats. II. Immunogenicity of allografted islet beta-cells.

This study examines whether the survival of allografted rat islet beta-cells is influenced by the presence of other pancreatic donor cells. Grafts (RT1u/l) of different cellular composition were intraportally transplanted in streptozocin-induced diabetic rats (RT1n/n). All grafts corrected the diabetic state within 3 days. Implants of freshly isolated islets contained various endocrine and nonendocrine cell types; they became diffusely infiltrated within 1 wk and were completely destroyed within 2 wk. A 4-day culture period did not lead to major changes in the cellular composition of the islets or in their survival as allograft. Islet cell aggregates prepared after islet dissociation and cell purification were less acutely infiltrated and less rapidly rejected. Aggregates composed of sorted MHC class II-negative cells maintained basal normoglycemia in 3 of 5 recipients for 5 wk but only in 1 of 5 for 20 wk. Aggregates of purified islet beta-cells remained relatively free of diffuse infiltrations during the 1st wk and preserved the normalized state in 7 of 13 recipients for 5 wk; after 20 wk, 6 of 13 were still aglucosuric, but 40% of the implants were diffusely infiltrated and depleted of insulin. Reaggregation of purified islet beta-cells with purified islet endocrine non-beta-cells promoted their long-term survival as allograft: 11 of 13 recipients of mixed islet endocrine cells maintained normal basal glycemia over 20 wk; their implants contained relatively constant insulin reserves and remained virtually devoid of diffuse infiltrations. These results demonstrate that techniques aiming at the elimination of surface MHC class II-positive cells are less successful in preparing rat islet allografts of low immunogenicity than methods of positive cell selection. Pure islet beta-cells are immunogenic as an allograft but illicit a milder and less-acute immune attack than undissociated islet tissue. Nonendocrine and damaged islet cells are suspected of enhancing the rapidity and intensity of the cytotoxic reaction. Survival of allografted beta-cells is markedly prolonged by the presence of islet endocrine non-beta-cells within the graft. The mechanisms underlying this effect have not yet been elucidated; they may involve immune and metabolic interactions of the endocrine non-beta-cells. We conclude that purification of islet endocrine cells represents a new and powerful method for preparing insulin-producing allografts that can survive in hosts without pharmacological immunosuppression.

Metabolic and morphologic studies in intraportal-islet-transplanted rats.

The intraportal injection of 350 to 1,000 isolated islets into streptozotocin-diabetic rats immediately normalized (approximately 24 hours) fasting plasma glucose and insulin levels. Polyuria, polydipsia, and hyperglucagonemia disappeared more gradually over a 2-to-12-week period--the time required for normalization varying with the severity of the diabetes and the number of islets transplanted. In long-term islet-transplanted rats (greater than five months), the hepatic insulin and glucagon reserves averaged 50 per cent and 25 per cent, respectively, of the corresponding normal pancreatic hormone content. Glucagon was increased slightly in the pancreas of streptozotocin-diabetic rats and decreased considerably in transplanted animals. However, total pancreatic glucagon (i.e. pancreatic and hepatic reserves) in transplanted animals was the same as the pancreatic content of normal control rats, indicating the presence of feedback control mechanism(s) in the regulation of pancreatic glucagon reserves. Long-term transplanted islets demonstrated well-granulated A-, B-, and D-cell movement out of the vascular space and the formation of narrow intercellular spaces and junctional complexes with surrounding hepatocytes.

Effects of chronically elevated glucose levels on the functional properties of rat pancreatic beta-cells.

This study examines the effects of chronically elevated glucose levels on the survival and function of purified rat beta-cells. Prolonged exposure (9 days) of beta-cell aggregates to 20 mmol/l glucose did not lead to cell losses, but reduced the amount of insulin secreted in response to glucose. This decrease was not caused by cellular desensitization but resulted from the lower cellular insulin content after a prolonged imbalance between stimulated rates of insulin synthesis and release. Virtually all beta-cells exhibited a state of metabolic and biosynthetic activation, which was maintained for at least 2 h in glucose-depleted media. Their rates of protein and insulin synthesis were amplified by glucose, reaching (half-) maximal stimulation at lower glucose concentrations (2 and 5 mmol/l, respectively) than control cells cultured at 10 mmol/l glucose (5 and 10 mmol/l, respectively). As for insulin release, the net glucose effect on insulin synthesis was markedly reduced as compared with that in control cells. This was also the case after culture at 6 mmol/l glucose. In the latter condition, the lower glucose-inducible activities were caused by cellular desensitization, with 50% of the beta-cells unresponsive to glucose and the other 50% responding with a lower sensitivity (half-maximal stimulation at 7 mmol/l glucose). Comparison of beta-cells cultured at the three glucose concentrations indicated that prolonged exposure to elevated glucose levels increases the number of degranulated cells, of cells with a high proportion of immature insulin granules, and of cells with glycogen deposition-morphologic features previously described in conditions of hyperglycemia. It is concluded that chronic exposure (9 days) of rat beta-cells to elevated glucose levels induces a prolonged state of beta-cell activation and glucose hypersensitivity rather than a glucotoxicity or glucose desensitization. This shift in the functional state of the beta-cell population is responsible for a reduced insulin release in response to glucose, as observed in other conditions of prolonged exposure to high glucose levels.

Transplantation of purified islet cells in diabetic rats. I. Standardization of islet cell grafts.

A standardized procedure was developed for the preparation of rat islet cell grafts with selected cell number and composition. After collagenase digestion of pancreases and elutriation of tissue fragments, islets were isolated and dissociated, and cells were purified by autofluorescence-activated cell sorting. Approximately 30% of the initial beta-cell mass was lost during digestion and elimination of small mostly exocrine particles. Fifty percent was recovered in isolated islet preparations and 30% in the purified beta-cell suspensions of greater than 95% purity and viability. Sorting according to cellular flavin adenine dinucleotide content discriminated islet beta-cells from islet endocrine non-beta-cells, fibroblasts, leukocytes, and exocrine cells. Purified endocrine islet cell grafts were prepared by aggregating 10(6) pure beta-cells with or without 8 x 10(5) pure endocrine non-beta-cells. In contrast to intact islets, the purified aggregates were devoid of nonendocrine and damaged cells. Intraportal implantation of a pure beta-cell graft rapidly and permanently normalized the diabetic state of streptozocin-administered animals. The standardized preparation of purified beta-cell grafts allows us to address several metabolic and immunological questions concerning islet cell transplantation in diabetes.

Presence of pancreatic hormones in islet cells with MHC-class II antigen expression.

In the normal rat pancreas, only few islet cells express MHC-class II antigens. Their nature and function has not yet been elucidated. We report a method for the purification of Ia-positive islet cells by fluorescence-activated cell sorting. The isolated mononuclear cells appear of nonendocrine origin but contain vacuoles with partially digested secretory vesicles. Both insulin- and glucagon-immunoreactive granules were identified in these vacuoles of varying size and composition. It is suggested that at least part of the rat islet cells with class II antigen expression can exhibit phagocytotic properties leading to the uptake of fragments from damaged endocrine cells. This functional characteristic may implicate this particular islet cell type in the pathology of the endocrine pancreas in type I diabetes.

Contribution of the 13C-urea breath test to the detection of Helicobacter pylori gastritis in children.

Serology, 13C-urea breath test, histology, Campylobacter-like organism testing, and culture were performed in 95 consecutive children to evaluate the contribution of these tests to the detection of Helicobacter pylori infection. In analyses considering any combination of three positive tests as "gold standard" for diagnosing H pylori infection, 26 children were Helicobacter positive (27%), which is only one patient more than the number of children with only a positive culture. The accuracy of culture was excellent when "any combination of three positive tests" was used as the gold standard (sensitivity 96%, specificity 100%, positive predictive value 100% [false positivity 0%], negative predictive value 99% [false-negative results 1%]). The results of invasive and noninvasive tests were comparable. When culture was considered as "gold standard," the sensitivity of serology and 13C-urea breath test was 96%; the specificity was 96% and 93%, respectively; the positive predictive value was 89% and 83% (false-positive results in 11% and 17%); and the negative predictive value for both was 99% (false-negative results in 1%). It is concluded that culture can be used as gold standard, but that non-invasive tests such as serology and/or 13C-urea breath test can be used to diagnose H pylori infection in children, since each has at least 95% sensitivity and 92% specificity.

Mixed ductal-endocrine carcinomas of the pancreas and ductal adenocarcinomas with scattered endocrine cells: characterization of the endocrine cells.

We compared the histological and immunohistochemical features of mixed ductal-endocrine carcinomas of the pancreas with those of ductal adenocarcinomas (DACs) containing scattered tumor-associated endocrine cells (SECs). Three pancreatic neoplasms fulfilled the WHO criteria for mixed ductal-endocrine carcinomas. Two of them showed moderately to poorly differentiated glandular structures composed of both mucin producing and neuroendocrine cells. The third mixed ductal-endocrine carcinoma was of the composite type showing DAC structures and a solid component with small epithelial cells, most of them of neuroendocrine nature. In 32 of 34 cases of DAC located in the head (30 cases) and body to tail (4 cases) of the pancreas and showing lymph-node metastases, SECs were found, but they were few in number and irregularly distributed in the tumors. In three DACs a few SECs were also detected in lymph-node metastases. Double staining for chromogranin A and the proliferation marker Ki-S5 revealed that all SECs that were not intimately integrated into the neoplastic glandular epithelium failed to show proliferative activity and changes of the expression of tumor suppressor genes (p53 and DPC 4). These findings suggest that only those SECs that belong to the proliferative cell fraction may be of neoplastic origin, while the majority of SECs probably constitute a tumor-associated but non-neoplastic cell population. These features contrast with those of mixed ductal-endocrine carcinomas, in which all endocrine cells are a component of the neoplasm.

Loss of nuclear BRCA1 localization in breast carcinoma is age dependent.

Abstract. Patients carrying a germ line mutation in the BRCA1 gene are predisposed to breast cancer. Somatic BRCA1 mutations were almost never reported in sporadic breast tumors, but several authors have described a decrease in BRCA1 mRNA and protein. In order to further investigate the possible role of BRCA1 in sporadic breast cancer, an improved immunohistochemical protocol was developed and applied on tissue sections obtained from 102 cancer patients belonging to two nonoverlapping age groups (less than 40 and more than 60 years). Based on the obtained BRCA1 specific nuclear staining we could distinguish two categories of breast cancer. The staining was present in almost all the nuclei of the normal and the cancer cells in about 50% of young (less than 40 years old) as well as older patients (more than 60 years old). Thus, BRCA1 does not seem to be involved in the genesis of these cancers. In the second category of patients, either a fraction of, or all tumor cells showed no nuclear staining. In this category, no nuclear BRCA1 staining could be observed in the tumor cells of 14 (27%) young and 3 (6%) older patients. Among six young patients bearing a breast tumor showing no BRCA1 nuclear staining at all, one was found to carry a BRCA1 germline mutation. Taken together, our results suggest that the molecular pathway in which the BRCA1 protein participates is disturbed in about 50% of sporadic breast cancer, this effect being more pronounced in tumors of young patients.

Increasing serum levels of alpha-fetoprotein in a patient with relapsing gallbladder carcinoma.

Alpha-fetoprotein (AFP) is a useful serum tumor marker especially for hepatocellular carcinoma and yolk-sac tumor. Increased serum levels of AFP have also been found in adenocarcinoma of the stomach, the pancreas, the colon and the lung, and in some squamous cell carcinomas of the lung. We describe a patient with a recurrent gallbladder carcinoma, presenting with increasing serum levels of AFP as the tumor progresses. Remarkable are the histologic changes in the metastases of the tumor, which showed more dedifferentiation as the number of cells containing AFP increased.

Imaging of renal cell cancer with radiolabelled octreotide.

Recently the presence of somatostatin receptors on human renal cell carcinomas has been demonstrated by autoradiographic techniques on surgically removed kidneys. In a prospective study we evaluated, by means of 111In-labelled octreotide scintigraphy, the in vivo tumour imaging in a group of patients with biopsy proven renal cell carcinomas at different tumour stages. Seven patients were studied. In three of them (43%) pathological tracer accumulation was demonstrated. In these patients 20 out of 23 known tumour localizations were clearly visualized. Tracer uptake could be inhibited by prior administration of cold octreotide. We conclude that 111In-octreotide scintigraphy can be used to demonstrate, in vivo, metastatic renal cell carcinoma.

Solitary exulceratio simplex (ulcer of Dieulafoy).

Solitary exulceratio simplex (ulcer of Dieulafoy--Dieulafoy vascular malformation) is a rare and frequently overlooked cause of massive gastric hemorrhage. The source of the bleeding, a large, submucosal artery penetrating the centre of a small mucosal defect, is usually located in the upper portion of the stomach. The pathogenesis of this entity is not known. We report on two patients who were successfully treated by surgery. Clinical, morphological and pathogenetic aspects are discussed.