Morphometric evaluation of the hypothalamic-ovarian axis of the ketonuric,diabetic Chinese hamster: Relationship to the reproductive cycle

Summary The relationship between diabetes and the morphological alterations which occur in hypothalamic and ovarian tissue was examined in the long-term, ketonuric-diabetic Chinese hamster. Matched diabetic and non-diabetic control hamsters were inspected daily for changes in the reproductive cycle by vaginal lavage. On dioestrus, animals were perfused, the hypothalamus and ovaries collected, prepared for microscopy and morphometrically analyzed. The nuclei in the medial basal hypothalamus of diabetic hamsters exhibited a decreased area (p<0.01) and neuronal population (p<0.05–0.01) compared with controls. The ovaries of the diabetic animals had a reduced follicular population (p0.05) and an increased atresia rate (p0.05) compared with controls. In addition, all diabetic hamsters were acyclic. In diabetic animals, the corpora luteal cells contained a reduced lipid content (p0.001) which was possibly functionally related to a significant decline in serum progesterone levels (p0.01). Based on these results it is suggested that the hypothalamic-ovarian axis is both morphologically and functionally impaired in the diabetic hamster.     

Rapid molecular characterization of mutations leading to unstable hemoglobin β-chain variants

Summary Characterization of unstable hemoglobins by protein analysis is often difficult. However, it is facilitated by DNA analysis, especially in the case of hyperunstable -chain variants, which produce a -thalassemia phenotype. We have applied an efficient strategy to the detection of such variants at the DNA level, based on computer-designed denaturing gradient gel electrophoresis (DGGE) of amplified DNA fragments. This approach makes it possible to detect any anomaly in the -globin gene. We describe the use of the DGGE method for rapid characterization of -chain variants and report a new missense mutation in the -globin gene third exon, 127 CAG-CGG/Gln-Arg, which is responsible for the synthesis of a highly unstable hemoglobin.     

No evidence for linkage between hla and maturity onset type of diabetes in young people

Summary Studies with 20 different genetic marker systems were performed in a large kindred including 18 members affected with maturity onset type of diabetes of young people. Linkage closer than 0.1 could be excluded for ABO and Gm, and linkage closer than 0.05 for HLA GLO, and haptoglobin. No significant positive lod scores were found.     

Incipient nephropathy in Type 1 (insulin-dependent) diabetes

Summary Patients with Type 1 (insulin-dependent) diabetes without proteinuria were studied to define those patients who will later develop persistent proteinuria (more than 0.5 g protein/24 h). Two investigations were performed; 71 patients were studied longitudinally for 6 years and another 227 patients were studied cross-sectionally. All were less than 50 years of age and had developed diabetes before the age of 40 years. At entry into the study they had no proteinuria (Albustix method), had normal blood pressure and urinary albumin excretion rates < 200 g/min (normal 20 g/min). The best predictor of persistent proteinuria or an albumin excretion rate > 200 g/min was the initial urinary albumin excretion rate. During the longitudinal study, seven patients with an urinary albumin excretion rate of more than 70 g/min at the start of the study developed persistent proteinuria or an albumin excretion rate > 200 g/min. In contrast, only three out of the remaining 64 patients with urinary albumin excretion rate 70 g/min developed urinary albumin excretion rate > 200 g/min. Patients with an urinary albumin excretion rate > 70 g/min are thus at risk of developing diabetic nephropathy. We designate this stage of renal involvement incipient nephropathy. Patients with incipient nephropathy were further characterized in the cross-sectional study. Compared with normoalbuminuric patients, patients with incipient nephropathy had increased systolic and diastolic blood pressure, but normal serum creatinine. The glomerular filtration rate was higher than normal in patients with incipient nephropathy though not different from that of normoalbuminuric patients.     

Effect of intravenous glucose infusion on renal function in normal man and in insulin-dependent diabetics

Summary The effect of intravenous glucose infusion on glomerular filtration rate and renal plasma flow (constant infusion technique using ¹²⁵I-iothalamate and ¹³¹I-hippuran) and on urinary excretion of albumin and -2-microglobulin were studied in ten normal subjects and seven metabolically well-controlled insulin-dependent diabetics. Following glucose infusion in normal subjects (n = 10) blood glucose increased from 4.7±0.1 to 10.9±0.4 mmol/1 (SEM) (p 0.01). Glomerular filtration rate increased from 116±2 to 123±3ml/min x 1.73 m² (p 0,01), while no change in renal plasma flow was seen 552±11 versus 553±18 ml/min × 1.73 m². Volume expansion with intravenous saline infusion in six of the normal subjects induced no changes in blood glucose or kidney function. In seven strictly controlled insulin-dependent diabetics, blood glucose values were raised from 4.6±0.4 to 16.0±0.6 mmol/1 and clamped by means of an artificial beta cell. Glomerular filtration rate increased in all patients, from 133 ±5 to 140±6 ml/min × 1.73 m² (p 0.02), as did renal plasma flow from 576±26 to 623±38 ml/ min × 1.73 m² (p 0.02). Urinary albumin excretion remained unchanged in both normal subjects and diabetics. -2-microglobulin excretion rate increased significantly in the diabetics following glucose infusion, while no significant change was seen in the normal subjects. Our results show that hyperglycaemia per se contributes to the increased glomerular filtration rate and renal plasma flow in insulin-dependent diabetes.     

Primary hyperoxaluria type 1: Genotypic and phenotypic heterogeneity

Summary Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT). The disease is notable for its extensive heterogeneity at the clinical, biochemical, enzymic and molecular genetic levels. A study of 116 PH1 patients over the past 8 years has revealed four main enzymic phenotypes: (1) absence of both AGT catalytic activity and immunoreactive AGT protein (40% of patients); (2) absence of AGT catalytic activity but presence of immunoreactive protein (16% of patients); (3) presence of both AGT catalytic activity and immunoreactive protein (41% of patients), in most of which cases the AGT is mistargeted to the mitochondria instead of the peroxisomes; and (4) a variation of the mistargeting phenotype in which AGT is equally distributed between peroxisomes and mitochondria, but in which that in the peroxisomes is aggregated into matrical core-like structures (3% of patients). Various point mutations, all occurring at conserved positions in the coding regions of the AGT gene, have been identified in these patients. The five mutations discussed in the present study, which have been found in individuals manifesting all of the four major enzymic phenotypes, account for the expressed alleles in about half of all Caucasian PH1 patients. The most common mutation found so far leads to a Gly170 Arg amino acid substitution. This mutation, in combination with a normally occurring Pro11 Leu polymorphism, appears to be responsible for the unprecedented peroxisome-to-mitochondrion mistargeting phenotype.     

Intracoronary hyperosmotic mannitol during reperfusion does not affect infarct size in ischemic,reperfused porcine hearts

Summary We investigated the effect of reperfusion with hyperosmotic mannitol on the infarct size in porcine hearts. The distal half of the left anterior descending coronary artery was occluded in each of 21 anesthetized pigs for 75 min and was reperfused for 2 h. During reperfusion mannitol (1075 mosmol/kg) was intracoronarily infused at a dose of 0.5 ml/min in 6 pigs (low mannitol group), at a dose of 1.5 ml/min in another 6 pigs (high mannitol group), and at a dose of 5 ml/min in 3 pigs for the first 8 min of reperfusion (very high mannitol group). 6 pigs served as controls. Although mannitol infusion increased plasma osmolality in the ischemic, reperfused myocardium in all experiments, the infarct size expressed as the ratio of the infarcted tissue over the area at risk of necrosis was not significantly influenced. Infarct size amounted to 72±25% in the control group, to 75±14% in the low mannitol group, to 78±18% in the high mannitol group, and to 93±8% in the very high mannitol group. These results clearly indicate that reperfusion with hyperosmotic mannitol after 75 min of ischemia does not exert any beneficial effect on the infarct size.     

Impaired cytokine production in whole blood cell cultures of patients with urological carcinomas

The production of the cytokines interferon (IFN ), interleukin-1 (IL-1 ) and tumor necrosis factor (TNF ) was investigated in the mitogen-stimulated whole blood cell culture media from 51 patients with urinary bladder carcinomas, 52 patients with renal carcinomas, 31 patients with prostatic carcinomas and 360 healthy controls. The cytokines were measured 4 days after induction by a sensitive enzymo-immunological assay. In the blood cell culture supernatants of the patients with urinary bladder carcinomas significancy lower levels of IFN (P0.001), IL-2 (P0.001) and TNF (P0.05) were found as compared to the controls. Blood cells of patients with renal carcinomas had lower production of IFN (P0.01), IL-2 (P0.001) and IL-1 (P0.01), whereas the values of the total group of patients with prostatic carcinomas were not significantly different from those of the controls. Lymphocyte and monocyte counts were almost identical in the control and all tumor patient groups. When the patients with renal carcinomas and prostatic carcinomas were analyzed according to their different clinical stages we could show a gradual depression of the IFN- levels, which was related to tumor burden.Abbreviations PHA phytohemagglutinin - PWM pokeweed mitogen - ELISA enzyme-linked immunoassay - IL interleukin - IFN interferon - TNF tumor necrosis factor, mAb, monoclonal antibody     

Early Outcomes Following Alternative Treatment Strategies in the Management of the Acutely Ischemic Limb

The modem emergency management of the acutely ischemic limb has evolved considerably with the introduction of catheter-directed thrombolysis. Following preservation of life, the next goal of intervention in these emergency cases, whether with surgery or thrombolysis, is limb salvage. We report our own experience with both approaches in the early outcome of the emergency management of the acutely ischemic limb. This is a retrospective review of a tertiary-level vascular units experience. The inclusion criteria consisted of acute limb ischemia as defined by the guidelines of the Vascular Surgical Society of Great Britain and Ireland. Data acquisition used the hospitals inpatient enquiry system, and its operative and radiology databases. Analysis used the ² test and the Yates correction factor (p < 0.05). Patients: N=84. Events: 103. Median age: females, 82 yrs; males, 71 yrs. 17–91 yrs. Females vs. males > 70 years: ²=5.4, d.f.=1, 0.01 < p, 0.02) (²: p= 0.05). Seventy-one patients underwent preoperative angiography. Successful limb salvage was achieved in 75% of cases. Overall, the amputation and mortality rates were 16.5% and 13.1%, respectively. We initially treated 62% of events with surgery and 38% with thrombolysis. In those patients who underwent thrombolysis there, 31% went on to have reconstructive surgery. Thrombolytic treatment resulted in a limb salvage rate of 69%. Thrombolysis was discontinued in six cases due to complications. Significant differences in outcome were not demonstrable between the two treatment groups (²=1.1, d.f.=1, 0.5 < p < 0.1). Acute limb ischemia has significant morbidity and mortality rates. The use of catheter-directed thrombolysis offers the surgeon an alternative to emergency surgery. This approach does not demonstrate any increase in the rate of limb loss.     

Effect of insulin on glucagon enhanced lipolysis in vitro

Summary Glucagon in concentrations similar to those found in human plasma markedly stimulates lipolysis in rat adipose tissuein vitro. The effects of these physiological concentrations of glucagon are reduced or abolished by insulin at concentrations of 25 and 100U/ml. Considering the marked insulinogenic effect of glucagon these observations may provide an explanation for the delayed increase of blood FFA observed after glucagon injectionin vivo.This work was supported by the Fonds National de la Recherche Scientifique and the Fonds de la Recherche Scientifique Médicale, Belgium.     

A Three-dimensional Analysis of Blood Vessels in Bronchioloalveolar Carcinoma

The three-dimensional architecture of blood vessels within lung adenocarcinomas has not been well studied. In 19 cases with bronchioloalveolar carcinoma with central fibrosis, we three-dimensionally examined blood vessel architecture in 150 m thick sections stained with elastin staining and anti-CD34 antibody. We examined four regions: normal alveoli and three regions within the tumor including an area adjacent to the normal alveoli (external area), an area in which tumor cells were replacing epithelial cells (replacement area), and a central fibrotic area (fibrotic area). Elastin staining showed that elastic fibers formed the framework of the alveoli, and the alveolar structure shrank more strongly to the center of the tumor due to folding of alveolar walls invaded by adenocarcinoma cells. We also measured three vessel parameters in these four regions. The vessel diameters were 4.08±1.10 m, 3.95±1.02 m, 5.04±1.56 m, and 6.11±2.23 m, the circumferences of those vessels seen as complete circles were 43.11±12.78 m, 43.71±12.87 m, 95.21±39.32 m, and 126.77±54.65 m; the lengths between vessel bifurcations were 13.28±3.08 m, 13.47±4.58 m, 24.91±9.66 m, and 41.82±28.08 m in the normal alveoli, and the external, replacement, and fibrotic areas, respectively. Blood vessel architecture changed such that the vessels became larger and coarser towards the center of the tumor. Our three-dimensional analysis suggests continuous remodeling of alveolar capillaries rather than angiogenesis within bronchioloalveolar carcinoma.     

Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease

Canavan disease, an inherited leukodystrophy, is caused by mutationsin the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups.Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. Of these, 14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAGTGG, X314W), and one splice acceptor site mutation (IVS1–2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1–2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frameshift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.     

Serum concentrations of resistin-like molecules β and γ are elevated in high-fat-fed and obese db/db mice, with increased production in the intestinal tract and bone marrow

Aims/hypothesis Resistin and the resistin-like molecules (RELMs) comprise a novel class of cysteine-rich proteins. Among the RELMs, RELM and RELM are produced in non-adipocyte tissues, but the regulation of their expression and their physiological roles are largely unknown. We investigated in mice the tissue distribution and dimer formation of RELM and RELM and then examined whether their serum concentrations and tissue expression levels are related to insulin resistance.Methods Specific antibodies against RELM and RELM were generated. Dimer formation was examined using COS cells and the colon. RELM and RELM tissue localisation and expression levels were analysed by an RNase protection assay, immunoblotting and immunohistochemical study. Serum concentrations in high-fat-fed and db/db mice were also measured using the specific antibodies.Results The intestinal tract produces RELM and RELM, and colonic epithelial cells in particular express both RELM and RELM. In addition, RELM and RELM were shown to form a homodimer and a heterodimer with each other, in an overexpression system using cultured cells, and in mouse colon and serum. Serum RELM and RELM levels in high-fat-fed mice were markedly higher than those in mice fed normal chow. Serum RELM and RELM concentrations were also clearly higher in db/db mice than in lean littermates. Tissue expression levels revealed that elevated serum concentrations of RELM and RELM are attributable to increased production in the colon and bone marrow.Conclusions/interpretation RELM and RELM form homo/heterodimers, which are secreted into the circulation. Serum concentrations of RELM and RELM may be a novel intestinal-tract-mediating regulator of insulin sensitivity, possibly involved in insulin resistance induced by obesity and a high-fat diet.     

Effects of ischemia, preconditioning, and adenosine deaminase inhibition on interstitial adenosine levels and infarct size

In order to examine the relationship between local adenosine concentrations before, during, and after ischemia and the extent of ischemic myocardial damage, measurements of interstitial fluid (ISF) nucleosides were made using microdialysis probes implanted in the ischemic region of isoflurane anesthetized Micropigs undergoing 60 coronary artery occlusion (CAO) and 3h of reperfusion (REP). Nucleoside concentrations in the dialysate collected from the microdialysis probes were used as an index of ISF levels. Dialysate nucleoside concentrations (ADO, inosine and hypoxanthine), myocardial infarct size, and myocardial blood flow (MBF) were determined in control animals (n=6), animals preconditioned with a single 10 cycle of CAO and REP (PC, n=6), and those treated with the adenosine deaminase inhibitor pentostatin (n=6, 0.2 mg/Kg IV 30 prior to CAO). The brief PC occlusion resulted in a transient but significant increase in dialysate ADO (6.7±1.8 M vs. 0.67±0.1 M at baseline). Pentostatin administration had no significant effect on either dialysate nucleosides or MBF at baseline. During the 60 CAO, dialystate ADO increased in control animals. In PC animals, however, dialysate ADO during CAO was lower than control. Pretreatment with pentostatin resulted in a six-fold augmentation in dialysate ADO during the 60 min CAO when compared to the control values (110.62±30.2 M vs. 16.31±2.1 M at 60 min of ischemia). Pentostatin also resulted in a significant reduction in the accumulation of inosine and hypoxanthine, indicating inhibition of adenosine deaminase activity. There were no significant differences in MBF between groups at any time point. Following 3 h REP, infarct size was 35.4±5.5%, 8.1±1.5% and 8.3±1.8% of the region at control, PC, and pentostatin groups, respectively. These data suggest that marked increase in ISF ADO during CAO, may be as effective in reducing INF as a modest increase in ISF ADO prior to prolonged CAO.Supported in part by NIH grant R01 HL32043     

Reperfusion Injury: Does It Exist and Does It Have Clinical Relevance?

Although reperfusion is an absolute prerequisite for the survival of ischemic tissue, it is not necessarily without hazard. Many (but not all) cardiologists are of the opinion that some components of reperfusion may be detrimental and able to inflict injury over and above that attributable to the ischemia. In this article we define four sequelae of reperfusion that might be designated as reperfusion injury. We identify possible underlying mechanisms and consider whether any of these forms of reperfusion injury are of clinical relevance.     

Mechanical properties and imaging characteristics of remanufactured intravascular ultrasound catheters

Intravascular ultrasound (IVUS) as a routine device in interventional cardiology is handicapped by its high price. 19 factory-made, remanufactured IVUS catheters which consist of sterilized, used phased-array IVUS transducers inserted into a new catheter shaft were compared with 23 new IVUS catheters. 3 mechanical and 4 imaging characteristics were assessed on a 5 point scale (1 = unacceptable, 5 = excellent). Mechanical as well as imaging properties of remanufactured IVUS catheter were comparable to new catheters with excellent ratings for each of the evaluated characteristics in 38 to 94% of remanufactured catheters and 50 to 96% of new catheters. The initial failure rate for remanufactured IVUS catheters was 31.6% vs. 4.3% for new catheters (P < 0.05). Overall failure rate was 47.3% for remanufactured catheters vs. 8.7% for new catheters (P < 0.05). The failure was due to an electronic connecting problem occurring during mechanical stress to the IVUS catheter. In conclusion, remanufactured IVUS catheters offer mechanical and imaging characteristics which are comparable to new catheters. Improvements in the remanufacturing process to resolve the high rate of electronic connecting problems may make this a promising approach to substantially lower the price of IVUS catheters.     

Oligosaccharides accumulated in the bovineβ-mannosidosis kidney

Summary The phenotype of bovine-mannosidosis (-mannosidase deficiency), recently identified in Salers cattle, is similar to the caprine form of the disease (Abbittet al., 1991). This investigation was designed to characterize accumulated kidney oligosaccharides in bovine-mannosidosis. Oligosaccharides were extracted from the kidney of an affected Salers calf and purified by chromatographic techniques. The amount of accumulating oligosaccharides in 1 g of wet tissue was about 21µmol. Structures of derivatized oligosaccharides were characterized by high-performance liquid chromatography, mass spectrometry, methylation analysis and sequential exoglycosidase digestions. The major accumulating oligosaccharides were Man1-4GlcNAc and Man1-4GlcNAc1-4GlcNAc. Oligosaccharides accumulating in minor amounts were Man1-4GlcNAc1-4Man1-4GlcNAc, Man1-6Man1-4GlcNAc1-4GlcNAc and Man1-4GlcNAc1-4Man1-4GlcNAc1-4GlcNAc. As in caprine-mannosidosis, oligosaccharides with terminal-mannose residues and cleaved as well as uncleaved chitobiose linkages were identified in bovine-mannosidosis kidney. The accumulating oligosaccharides in tissue were thus identical in bovine and caprine-mannosidosis; however, the source of the novel oligosaccharides remains to be determined.     

Treatment of clinical stage I testicular cancer and a possible role for new biological prognostic parameters

Three different treatment strategies for patients with stage I non-seminomatous testicular cancer are available that will all result in long-term survival in more than 98% of the patients: a wait and see strategy with follow-up and chemotherapy in cases of tumour progression, retroperitoneal lymphadenectomy, with or without application of systemic chemotherapy, in cases of retroperitoneal metastases (pathological stage II disease) or primary adjuvant chemotherapy following inguinal orchiectomy. Each treatment strategy is associated with specific side-effects. In several studies histological characteristics of the primary tumour, particularly the presence of vascular invasion and of embryonal carcinoma cells, have been demonstrated to be significant prognostic factors for the risk of occult retroperitoneal metastases in patients with stage I disease. In addition, new biological prognostic factors determined by flow cytometry, cytogenetic analysis or molecular-biological DNA or RNA analysis have been investigated, among which alterations of thep53 tumour-suppressor gene may represent a promising new prognostic factor. Although alterations ofp53 gene expression seem to be associated with advanced tumour stage and may predict retroperitoneal metastatic disease, the independent role of these molecular genetic alterations needs to be prospectively studied. Currently a risk-adapted treatment strategy based on the histological criteria of vascular invasion and the presence of embryonal carcinoma can be used to stratify patients into a high- and low-risk group with respect to tumour progression. While primary-nervesparing retroperitoneal lymphadenectomy or adjuvant chemotherapy with two cycles of platinum, etoposide and bleomycin may be appropriate for patients with a high risk (above 40%) for tumour progression, a wait-and-see strategy can be used for low-risk (less than 15% risk of progression) patients. Molecular investigations of prognostic factors may be able to improve further the stratification of patients into these different risk categories.Abbreviations RLA retroperitoneal lymphadenectomy - SCF stem-cell factor     

Die Beziehungen zwischen Mastopathia chronica cystica und Fibroadenom der Mamma zum Mammacarcinom

Zusammenfassung Sowohl Mastopathia fibrocystica als auch Mamma-Fibroadenom sind morphologisch präcanceröse Gewebsveränderungen, aus denen sich besondere Krebse bilden.Die Brustkrebse können in folgender Weise unterteilt werden: Carcinoma ex fibroadenoma (16,09%), Carcinoma ex mastopathia (37,56%), von Fibroadenom und Mastopathie unabhängige Krebse (46,35%).Es wird aus gutem Grund angenommen, daß die Induktionszeit des Carcinoma ex fibroadenoma ungefähr 20 Jahre ausmacht, die des Carcinoma ex mastopathia ungefähr 8 Jahre beträgt.Zentrum für die Diagnose und Behandlung bösartiger Tumoren, Histopathologische Abteilung     

A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C)

Summary Both the - and -anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA: glucosaminideN-acetyltransferase. Using the -anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed <1% of mean normalN-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl--d-glucosaminide requires the sequential action of theN-acetyltransferase and -hexosaminidase. Normal -hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl--d-N-acetylglucosaminide formed by theN-acetyltransferase. In cell extracts with a -hexosaminidase deficiency, however, a second incubation in the presence of excess -hexosaminidase is needed to avoid underestimation of theN-acetyltransferase activity.