Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

Toxic Immunoglobulin Light Chain Autoantibodies are Associated with a Cluster of Severe Complications in Older Adult Type 2 Diabetes.

AIMS: To assess neuronal depolarization evoked by autoantibodies in diabetic depression compared to depolarization evoked by autoantibodies in control patients. To determine whether a subset of severe (late-onset) diabetic complications may be mediated in part by toxic immunoglobulin light chains that may increase in diabetic nephropathy. METHODS: Protein-A eluates from plasma of 21 diabetic depression patients and 37 age-matched controls were tested for depolarization in hippocampal or immature neurons. Subsets of depolarizing or non-depolarizing autoantibodies were tested for neurite outgrowth inhibition in N2A neuroblastoma cells or the ability to modulate Ca2+ release in HL-1 atrial cardiomyocytes or in endothelial cells. The stability of depolarizing autoantibodies was investigated by heat treatment (56°C × 30 minutes) or following prolonged exposure to the pro-protein convertase, furin. Gel filtration of active depolarizing autoantibodies was performed to determine the apparent molecular mass of peak neurotoxicity associated with the autoantibodies. RESULTS: Diabetic depression (n = 21) autoantibodies caused significantly greater mean depolarization in neuroblastoma cells (P < 0.01) compared to autoantibodies in diabetic (n = 15) or non-diabetic (n = 11) patients without depression. Depolarizing autoantibodies caused significantly more (P=0.011) inhibition of neurite outgrowth in neuroblastoma cells than non-depolarizing autoantibodies (n = 10) and they evoked sustained, global intracellular Ca2+ release in atrial cardiomyocytes or in endothelial cells. A subset of older diabetic patients suffering with a cluster of nephropathy, non-ischemic cardiomyopathy and/or depression demonstrated the presence of stable light chain dimers having apparent MW of 46 kD and associated with peak neurotoxicity in neuroblastoma cells. CONCLUSION: These data suggest that autoantibodies in older adult diabetic depression cause long-lasting depolarization in hippocampal neurons including adult dentate gyrus neural progenitor cells. The autoantibodies may impair adult dentate gyrus neurogenesis associated with treatment-refractory depression via several mechanisms including suppression of neurite outgrowth, and alteration of membrane excitability. Stable, toxic light chain autoantibody components may contribute to a cluster of severe (late-onset) complications characterized by dysfunction in highly vascularized tissues.

Loss of heterozygosity at 9p23 defines a novel locus in non-small cell lung cancer.

Genetic studies have previously demonstrated cytogenetic deletions and allelic imbalance or loss of heterozygosity (LOH) on the p arm of chromosome 9, in a number of tumour types. We have analysed 45 Non-Small Cell Lung Cancers (NSCLC) with a panel of highly polymorphic microsatellite markers on chromosome 9. Our results indicate that loss on 9p is concentrated within the D9S156-D9S161 region with 44% (20/45) LOH, however the area with minimal loss in this set of lung tumours was found at D9S157 (9p23), with 30% LOH (10/33), whereas loss at the IFNA locus was only found in 6% (2/34) tumours. Five of the lung tumours in this study which demonstrated LOH at D9S157 retained heterozygosity at the adjacent informative markers lying centromeric and telomeric to D9S157. No correlations were found between any of the clinico-pathological parameters and LOH on 9p or at the D9S157 locus. The results of this study indicates the presence of a further putative tumour suppressor gene on 9p at the D9S157 locus (9p23) to be most likely involved in the pathogenesis of non-small cell lung cancer.

Multiple forms of beta-amyloid peptide precursor RNAs in a single cell type.

The longest open reading frames (ORFs) of three different cDNAs ([10, 12, 18, 26], and this report) contain the exact 42 amino acid (aa) sequence of the beta-amyloid peptide (BAP) which is selectively deposited in Alzheimer's diseased (AD) brains. Each of the three cDNAs for the putative amyloid peptide precursor (APP) has been cloned from a different cell type. Using an HL 60 library, we have cloned two of these three APP cDNAs from a single cell type. The sequences of the HL 60 cDNAs are identical to the APP 751 and to the APP 770 forms of APP cDNAs. Northern blots show that oligonucleotide probes drawn from unique regions of the APP 751 and APP 770 cDNAs both hybridize to 4.0 Kilobase (Kb) and to 1.6 Kb APP RNAs from HL 60 cells. In human adult brain, an oligonucleotide probe drawn from the unique region of the APP 751 cDNA hybridizes to a 3.5 Kb APP RNA. However, a DNA probe drawn from the BAP region, which is common to the 695, 751, and 770 forms of APP cDNAs, hybridizes to 3.5, 3.2 and 1.6 Kb APP RNAs. Taken together, these results show that at least two forms of APP RNAs can exist within a single cell type and that the diversity of possible APP RNAs and complexity of their expression may have been underestimated. Thus, in addition to identifying the cells which produce BAP, a new challenge consists of determining which form of forms of APP RNAs and hence APP proteins are associated with BAP deposition in AD and Down syndrome (DS).

The intracellular domain of the second chain of the interferon-gamma receptor is interchangeable between species.

In this report we show that the mouse interferon (IFN)-gamma R1 and IFN-gamma R2 subunits expressed in hamster cells are capable of rendering the cells sensitive to mouse IFN-gamma as measured by induction of class I MHC antigens and the activation of the transcription factor Stat1 alpha. However, these cells showed no antiviral protection in response to IFN-gamma when challenged with vesicular stomatitis virus (VSV) but limited protection when challenged with encephalomyocarditis virus (EMCV). Furthermore, the cytoplasmic domains of the IFN-gamma R2 subunits, like the cytoplasmic domains of the IFN-gamma R1 chains, can be interchanged between species with no loss of biologic activity, demonstrating that the species-specific interaction of the IFN-gamma R1 and IFN-gamma R2 chains involves only the extracellular domains of the two proteins.

The dynamics of bone marrow stromal cells in the proliferation of multipotent hematopoietic progenitors by substance P: an understanding of the effects of a neurotransmitter on the differentiating hematopoietic stem cell.

Communication within the hematopoietic-neuroendocrine-immune axis is partly mediated by neurotransmitters (e.g. substance P, SP) and cytokines. SP mediates neuromodulation partly through the stimulation of bone marrow (BM) progenitors. This study shows that SP, through the neurokinin-1 receptor, stimulates the proliferation of primitive hematopoietic progenitors: cobblestone-forming cells (CAFC, CD34+). This effect is optimal when macrophage is included within the fibroblast support. Indirect induction of IL-1 could be important in the proliferation of CAFC colonies by SP. Phenotypic and functional studies suggest that SP might directly interact with the CD34+/CD45(dim) population. These studies indicate that SP can initiate a cascade of biological responses in the BM stroma and stem cells to stimulate hematopoiesis.

Stapler design and strictures at the esophagogastric anastomosis.

An apparent reduction in the rate of benign anastomotic stricture after stapled esophagogastrectomy prompted us to review the results obtained with different stapling devices since 1988. We present a retrospective review of 125 consecutive patients undergoing esophageal resection for malignancy with stapled intrathoracic anastomoses. Benign anastomotic stricture was deemed present when a patient required endoscopic dilatation to treat postoperative dysphagia. We found no difference in risk factors not related to stapler size (tumor histologic characteristics, adjuvant therapy) between patients with stricture and patients without stricture. Event-free survival was compared for different stapler diameters as well as for different stapler designs. We found that staplers of smaller diameter were associated with significantly more strictures (p < 0.005). In a comparison of different designs of 25 mm stapler, the newer CDH device (Ethicon Ltd., Edinburgh, United Kingdom) was associated with a similar stricture rate to that associated with other designs (ILP [Ethicon] and EEA [Autosuture Company Division, United States Surgical Corp., Norwalk, Conn.]). For a given stapler diameter, it appears that different stapler designs have no effect on stricture rate.

Neurokinin-1 expression and co-localization with glutamate and GABA in the hypothalamus of the cat.

Recent behavioral studies using pharmacological techniques have demonstrated that the high affinity substance P (SP) receptor, neurokinin-1 receptor (NK-1), in the medial hypothalamus could be important in mediating defensive rage behavior in the cat. These observations prompted us to use molecular techniques to determine the distribution of NK-1 in the hypothalamus and in other regions of the forebrain relevant to the control of rage behavior. We cloned a 650 bp fragment of the cat NK-1 cDNA. Partial DNA sequence analyses of this fragment indicate 90% homology with the human cDNA. By in situ hybridization (ISH), we showed that NK-1 mRNA was localized in the cytoplasm but not nuclei of cat forebrain neurons. Furthermore, NK-1 mRNA was co-localized in neurons that displayed positive immunolabeling for glutamate or GABA. Moderate labeling was visualized in the anterior medial hypothalamus which receives significant SP input via the stria terminalis from the medial amygdala. Strong labeling was also observed in the basal amygdaloid complex. The functional significance of this labeling pattern is suggested from the observation that both the medial and basal complex of amygdala serve as powerful modulators of defensive rage behavior. Weaker labeling was seen over the posterior medial and lateral hypothalamus. The distribution of NK-1 in the hypothalamus was matched by that of SP-immunoreactive axons and pre-terminals that were observed in the hypothalamus. The overall findings provide anatomical evidence to show that the high affinity SP receptor, NK-1, is linked to glutamate and GABA neurons in the anterior medial hypothalamus and further suggests its likely role in the regulation of feline aggression.

Anastomotic narrowing after esophagogastrectomy with the EEA stapling device.

We studied a series of 176 patients undergoing esophageal resection with the aid of the EEA surgical stapling device (Auto Suture U.K. Limited, Great Britain) during a period of 7 1/2 years. A total of 160 patients (91%) were operated on for malignant disease. Operative death occurred in 15 patients (8.5%), and there were three anastomotic leaks (1.7%). The prevalence of dysphagia caused by both benign and malignant strictures after esophageal resection in which the EEA stapler was used was 17.4%. The rate of benign anastomotic narrowing in discharged patients was 12.5%. Anastomotic stricture resulting from recurrent tumor caused dysphagia in 6.2% of the patients undergoing resection for malignant disease. The highest rate of benign anastomotic narrowing occurred in patients who had undergone esophageal resection for benign, nondilatable strictures. In these patients, the prevalence of benign anastomotic narrowing was 37.5%, compared with 9.6% in the patients undergoing resection for malignant disease (p less than 0.001). An additional trend was noted: The smaller the stapling head used to construct the anastomosis, the higher the prevalence of benign anastomotic narrowing; however, a statistically significant difference could not be documented. Ninety-five percent of patients with benign anastomotic narrowings complained of dysphagia within the first 6 months after the operation; 79% of these patients required two or fewer dilatations to relieve the dysphagia. Dysphagia after esophageal resection with the aid of EEA stapler occurred in just over one of six patients. The usual cause of the dysphagia was benign anastomotic narrowing, which responds well to dilatation.(ABSTRACT TRUNCATED AT 250 WORDS)

Early and late results of aortoplasty with a left subclavian flap for coarctation of the aorta in infancy.

Between February, 1969, and December, 1976, 45 consecutive infants younger than 6 months old underwent aortoplasty with a subclavian flap for the relief of coarctation of the aorta. All infants had persistence of the ductus arteriosus, and 58 percent had associated intracardiac anomalies. The over-all hospital mortality rate was 24 percent, nad no deaths occurred in infants with coarctation associated with patent ductus arteriosus only. All the deaths were in the group of patients under 2 months of age who had associated intracardiac defects. Follow-up over a 7 year period shows no clinical, hemodynamic, or angiographic evidence of recoarctation in any of the survivors.

Surgical correction of a case of common atrium with anomalous systemic and pulmonary venous drainage.

A case of common atrium with anomalous systemic and pulmonary venous connections and pulmonary stenosis is described. Surgical correction was performed by pulmonary valvotomy and the insertion of an intra-atrial pericardial baffle. To our knowledge this is the first reported case of surgical correction of total anomalous connections of both systemic and pulmonary veins. The embryologic development of this condition is discussed.

Results of diaphragmatic plication for unilateral diaphragmatic paralysis.

Seven adult patients with dyspnea resulting from nonmalignant unilateral diaphragmatic paralysis underwent plication of the affected hemidiaphragm. Preoperatively, the patients complained of exertional dyspnea and orthopnea and had a reduced arterial oxygen tension, total lung capacity, vital capacity, expiratory reserve volume, and functional residual capacity. Plication was performed by imbricating the diaphragm in layers through a thoracotomy. After plication there was a significant increase in arterial oxygen tension and all lung volumes except residual volume. The patients' symptoms were improved with plication and a significant decrease was recorded in breathlessness on a visual analogue scale. There were no postoperative complications and mean hospital stay was 12 days.

Generation of random internal deletion derivatives of YACs by homologous targeting to Alu sequences.

To facilitate the manipulation of human genomic DNA in yeast artificial chromosome (YAC) clones, a plasmid to integrate the selective marker for antibiotic G418 resistance into YACs and to delete some of the human DNA fragments from YACs was constructed. The linearized integration/deletion plasmid, which contains Alu family sequences at both ends, can recombine with YACs containing human repetitive sequences via homologous recombination. The homologous recombination results in a random integration of the antibiotic G418-resistant gene into a human genomic Alu sequence, and in most cases, an internal deletion within the YAC. The YACs with internal deletions can be useful to identify the location of the genes if they produce functional knockouts. In those cases when the integration/deletion event disrupts the integrity of the gene so it no longer can produce a viable and functional mRNA in fused eukaryotic cells, the site of integration in the YAC thus serves as a marker for the inactivated gene. In this report we describe a model system to locate specific genes in YACs.

Complications after esophagogastrectomy using stapling instruments.

We studied 195 patients undergoing esophageal resection using stapling instruments during a period of over 8.5 years. Of these, 178 (91.2%) underwent operation for malignant disease. Operative death occurred in 19 patients (9.7%). Nine (4.6%) postoperative complications, excluding stricture formation, were related to the use of stapling instruments, including two operative deaths (1.0%). Anastomotic leaks and gastrotomy staple line leaks were the most common complications (four each). In 7 patients (3.6%) the circular stapler tore the esophagus; anastomotic leaks subsequently developed in 43% of these patients. After we began oversewing gastrotomy staple lines in 1984, we experienced only one gastrotomy leak due to a technical error. Postoperative stricture formation occurred soon after resection and responded well to one or two dilations. It is better to err on the side of choosing too small a stapling head when performing an esophageal anastomosis because this may reduce the incidence of leaks, and further strictures are easily dealt with.

Thoracoscopy: a review of 121 consecutive surgical procedures.

The records of 121 patients who underwent surgical thoracoscopy between 1976 and 1987 were reviewed. The indications for thoracoscopy were pleural effusion (88%), pleural thickening (7.4%), and a mass on the chest roentgenogram (15%). All procedures were performed under general anesthesia with a rigid thoracoscope and 91 patients (75%) were diagnosed as having a malignant process. Although thoracotomy was undertaken in 20 patients (16.5%), no patient with benign disease underwent diagnostic thoracotomy. Thoracoscopy had a 98.9% sensitivity and a 93.3% specificity in this series. One patient died of a myocardial infarction after the procedure, and 11 patients (9.1%) had other, predominantly respiratory, complications. We confirm that surgical thoracoscopy is a useful procedure in the diagnosis of pleural processes.

Simultaneous tracheal and esophageal pH monitoring: investigating reflux-associated asthma.

Aspiration of gastric acid into the trachea may cause asthma in some patients who have gastroesophageal reflux. Antireflux surgery has been advocated for such patients, but lack of an objective test for acid aspiration makes patient selection difficult. We report a new technique for demonstrating acid aspiration, simultaneous tracheal and esophageal pH monitoring. Tracheal pH was measured with a 1.0-mm pH electrode introduced through the cricothyroid membrane under bronchoscopic vision. A standard esophageal pH electrode was placed in the usual position. Tracheal and esophageal pH were monitored over a 24-hour period. Peak expiratory flow rate was measured hourly while the patient was awake. We present data obtained in 3 patients with severe asthma and symptomatic gastroesophageal reflux. All 3 patients demonstrated a decrease in tracheal pH to less than 5.5, coinciding with a decrease in esophageal pH to less than 4.0. The test was repeated after antireflux operation and showed that significant decreases in esophageal pH no longer lowered tracheal pH. Asthmatic symptoms were improved, and medication was reduced in 2 of the 3 patients.

Diaphragmatic plication for unilateral diaphragmatic paralysis: a 10-year experience.

Unilateral paralysis of the diaphragm due to nonmalignant disease is an uncommon disorder previously thought to have benign implications. Some patients, however, experience dyspnea and orthopnea with impairment of pulmonary function. Unilateral diaphragmatic plication was performed on 17 patients (16 men and 1 woman with a mean age of 53.7 years [range, 28 to 74 years]) during the last 10 years. Preoperatively each patient was shown to have paradoxical movement of the paralyzed diaphragm on sniffing and to have a reduction in forced vital capacity and lung volumes. These reductions were greater when the patient was in the supine position. All patients had moderate hypoxemia (mean arterial oxygen tension, 73.1 +/- 10.9 mm Hg). Plication was performed by imbricating the diaphragm in layers through a thoracotomy incision. After plication, all patients showed both subjective and objective improvement. Six patients were reassessed 5 or more years after plication (range, 5 to 7 years), and the improvement was maintained. Diaphragmatic plication is a safe and effective procedure for adult patients with dyspnea due to unilateral diaphragmatic paralysis; furthermore, the initial improvement is maintained.

Esophagogastrectomy via left thoracophrenotomy.

Esophagogastrectomy is generally considered to be the treatment of choice for resectable tumors of the esophagus. Although many approaches and techniques have been advocated, since April 1983 we have used a left thoracophrenotomy approach for most lesions of the lower two thirds of the esophagus and gastric cardia. Stapling instruments have been used for mobilization of the stomach and fashioning of the esophagogastric anastomosis. One-hundred fifteen patients undergoing resection of malignant tumors with this technique were retrospectively reviewed. Perioperative mortality was 8.7% (10/115). The rate of anastomotic leakage was 1.7% (2/115), and benign narrowing of the anastomosis requiring dilation developed in 16 patients. The rate of recurrent anastomotic tumor was 4.3%. Eighteen patients had complications, and the mean postoperative hospital stay was 13 days. Survival at 3 years was 22.1%. During the period of study, 22 patients underwent esophageal resection by some other approach; the reasons for this are described. The advantages of the left thoracophrenotomy approach are discussed.