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Background: Paroxysmal nocturnal hemoglobinuria (PNH), which is characterized by intravascular hemolysis and venous thrombosis, is an acquired clonal disorder associated with a somatic mutation in a totipotent hematopoietic stem cell. An understanding of the natural history of PNH is essential to improve therapy., Methods: We have followed a group of 80 consecutive patients with PNH who were referred to Hammersmith Hospital, London, between 1940 and 1970. They were treated with supportive measures, such as oral anticoagulant therapy after established thromboses, and transfusions., Results: The median age of the patients at the time of diagnosis was 42 years (range, 16 to 75), and the median survival after diagnosis was 10 years, with 22 patients (28 percent) surviving for 25 years. Sixty patients have died; 28 of the 48 patients for whom the cause of death is known died from either venous thrombosis or hemorrhage. Thirty-one patients (39 percent) had one or more episodes of venous thrombosis during their illness. Of the 35 patients who survived for 10 years or more, 12 had a spontaneous clinical recovery. No PNH-affected cells were found among the erythrocytes or neutrophils of the patients in prolonged remission, but a few PNH-affected lymphocytes were detectable in three of the four patients tested. Leukemia did not develop in any of the patients., Conclusions: PNH is a chronic disorder that curtails life. A spontaneous long-term remission can occur, which must be taken into account when considering potentially dangerous treatments, such as bone marrow transplantation. Platelet transfusions should be given, as appropriate, and long-term anticoagulation therapy should be considered for all patients.

Implantation of tumor during minimal access procedures such as laparoscopic colectomy is a disturbing phenomenon. Although the actual incidence of this problem is unknown, its existence is a threat to the further clinical development of minimally invasive oncological procedures. We have adopted the hypothesis that these recurrences are the result of suboptimal technique or instrumentation and therefore that the problem can be avoided by identifying and correcting these technical factors. Our purpose is to analyze the possible mechanisms by which tumor cells become implanted in the trocar sites and to propose rational solutions to the problem.

Objective: To test our hypothesis that tumors would be more easily established and grow more aggressively after laparotomy than after laparoscopy. This hypothesis was based on studies that have demonstrated that surgery can suppress immune function and facilitate tumor growth and that have shown preservation of immune function after laparoscopic procedures., Design: Double-blinded, randomized, control trial., Setting: Research laboratory and animal care facility., Animals: One hundred forty 5- to 6-week-old C3H/He female mice., Interventions: Three experiments with three groups each: laparotomy, insufflation, and anesthesia controls. All animals received an intradermal inoculation of tumor cells in the dorsal skin. The anesthesia control cohort underwent no procedure. The laparotomy cohort underwent a midline laparotomy from the xiphoid process to the pubis, which was closed after 30 minutes. The insufflation cohort underwent peritoneal insufflation with carbon dioxide for 30 minutes., Main Outcome Measures: Tumor volume, tumor mass, and incidence of tumor establishment., Results: In the first experiment, the tumor volumes of the anesthesia control and insufflation groups followed a similar pattern of plateau and regression. The tumor volumes of the laparotomy group followed a different pattern and were significantly larger than those of the control and insufflation groups on postoperative days 6 and 12 (P < .05 for all comparisons). In the second experiment, tumors in the laparotomy group were approximately three times larger than those of the control group (P < .01) and almost twice as large as insufflation group tumors (P < .01) by mass. In the third experiment, there was a significantly higher incidence of tumor establishment in the laparotomy group than in the insufflation (P < .04) or control (P < .01) groups. The incidence was not different between the control and insufflation groups., Conclusions: Tumors were more easily established and grew more aggressively after laparotomy than after insufflation. These results, coupled with those that demonstrate an immune advantage to laparoscopy over laparotomy, suggest that the difference in observed tumor growth may be related to immune function. While much work remains to be done, we believe these data provide evidence of a previously undemonstrated benefit of laparoscopic intervention.

We studied the safety and efficacy of laparoscopic jejunal free flap harvesting with total intracorporeal small-bowel anastomosis in an animal model. Eight dogs underwent laparoscopic resection of 8 to 15 cm of jejunum with endoscopic GIA anastomoses and jejunal segment harvesting through the periumbilical laparoscopic port. In six animals, the harvested jejunum was implanted subcutaneously and revascularized by anastomosis of the mesenteric artery and vein to the femoral vessels. Both the microvascular and intracorporeal jejunal anastomoses were studied at 10 days. Mean laparoscopic operative time was 2.9 hours, with the last five procedures all completed in fewer than 2 hours. Mean ischemic time was 1.9 hours. The laparoscopically performed small-bowel anastomoses were all successful. All dogs took regular diets within 1 day, with normal bowel function returning by the second day. Both clinically and histologically, the bowel wall and mesenteric vessels of all the segments harvested demonstrated no injury despite their laparoscopic harvest. Five of the free flaps remained fully viable at 10 days. One flap failed after prolapse of the flap resulting from inadequate fixation. Laparoscopic harvesting of the jejunal free flap is safe and efficacious and offers all of the potential advantages of laparoscopic abdominal surgery.

We investigated the effects of laparotomy and insufflation on tumor establishment and growth in a murine model. Twenty female mice received intradermal inoculation of a low dose of tumor cells (2 x 10(3)) derived from the MC2 mouse mammary carcinoma cell line. Ten of these mice underwent laparotomy and ten received intraperitoneal insufflation with carbon dioxide gas at a pressure of 5 mmHg for 30 min. Tumor growth was followed postoperatively. By postoperative day 14, tumors had grown in zero of the ten insufflated mice and in seven of the ten laparotomy-group mice (P < 0.005). By postoperative day 30, tumors had grown in one of the ten insufflated mice and in eight of the ten laparotomy-group mice (P < 0.007). Ten additional mice received a high-dose inoculum of cells (1 x 10(6)) followed by either laparotomy or intraperitoneal insufflation. Upon sacrifice 12 days later, all mice had developed tumors, but the laparotomy group's tumors were almost three times as large, by mass, as tumors in the insufflated group (70.5 +/- 23.5 mg vs 25.8 +/- 9.5 mg; P < 0.02). These results suggest that laparotomy confers a permissive effect on tumor establishment and growth in a murine model not seen after peritoneal insufflation. We hypothesize that this may be a function of relative immunosuppression following laparotomy which is not present following peritoneal insufflation. These data may be important when choosing a route of access to the peritoneal cavity for cancer resection.

We evaluated the immunologic responses following laparoscopic and open surgery by comparing delayed type hypersensitivity induration size before and after each method of accessing the abdominal cavity. One hundred and thirty-two male Sprague-Dawley rats were sensitized with keyhole limpet hemocyanine (KLH). Animals were challenged with KLH and phytohemaglutanin (PHA) 10 days after sensitization. On day 14 after initial sensitization animals were randomly divided into three groups. Group one served as controls and had no procedure performed, group two underwent peritoneal insufflation with carbon dioxide gas to a pressure of 6-8 mm Hg for one half hour, and rats in group three had a midline laparotomy which was closed after one half hour. Each rat was challenged with KLH immediately and at three days postoperatively. The area of induration in response to each of the challenges was measured with calipers 24 and 48 hours after the challenge. Results of this skin testing showed that the group of animals that underwent laparotomy, despite having normal responses preoperatively, had significantly diminished responses to both KLH and PHA when challenged postoperatively. The insufflated group showed no differences from control animals at any time point examined. We conclude that DTH response in this model is better preserved after laparoscopy than laparotomy. We further conclude that the defect in DTH response is in the effector arm. The question of the clinical significance of these findings is addressed.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic disorder characterized by complement-mediated hemolytic anemia, pancytopenia, and venous thrombosis. These clinical manifestations arise from an underlying molecular defect of bone marrow stem cells. Specifically, somatic mutations in the phosphatidylinositol glycan class A gene result in the ability of blood cells to anchor complement-regulatory proteins (CD59 and DAF) to the cell surface via glycosyl phosphatidylinositol (GPI). In an attempt to circumvent the functional defect in PNH cells, a recombinant transmembrane form of CD59 (CD59-TM) was analyzed for the ability to regulate complement activity. Balb/3T3 stable transfectants expressing similar levels of either CD59-TM or native CD59 (CD59-GPI) were equally protected against human complement-mediated membrane damage. Treatment of these cells with phosphatidylinositol-specific phospholipase C failed to release CD59-TM from the cell surface. Retroviral transduction of GPI-anchoring deficient mouse L cells with CD59-TM resulted in surface expression of the protein and rendered these cells resistant to human complement-mediated membrane damage. Conversely, L cells transduced with CD59-GPI failed to express this protein on the cell surface. A GPI-anchoring deficient complement-sensitive B-cell line derived from a PNH patient was successfully transduced with CD59-TM, resulting in surface expression of the protein. The PNH B cells expressing CD59-TM were protected against classical complement-mediated membrane damage by human serum. Taken together, these data establish that a functional recombinant transmembrane form of CD59 can be expressed on the surface of GPI-anchoring deficient PNH cells and suggest that retroviral gene therapy with this molecule could provide a treatment for PNH patients.

The purpose of this preliminary study was to evaluate immunologic responses to laparoscopic vs standard open colon resection and to evaluate possible mediators of any differences found. Specifically, we compared cortisol levels and delayed-type hypersensitivity response after each method of colon resection in a group of 20 pigs. Two groups of 10 animals each were treated in identical fashion including bowel preparation, anesthesia, and postoperative management. The only difference between groups was that one underwent laparoscopic and the other an open colon resection. Blood specimens for cortisol were drawn before, during, and immediately postoperatively as well as at 11 A.M. on postoperative days 1 and 2. All animals had been previously immunized as piglets with Sow Bac-E (Oxford Veterinary, Worthington, MN), an antigen preparation of common pig pathogens. At the conclusion of the operative procedure 0.5 cc of the antigen was injected intradermally on the right forelimb of the animals. At 48 and 72 h postoperatively the largest diameters of induration surrounding the injection site were measured and averaged. Cortisol levels were measured in serum samples by radioimmunoassay (Met-Path, Rockville, MD). Statistical significance was determined by t-test. Results of skin antigen testing showed that the group of pigs that underwent laparoscopic resection had a 20% greater response, 1.54 cm +/- 0.28 cm at 48 h and 1.53 cm +/- 0.18 cm at 72 h. For the open-surgery group results were 1.24 cm +/- 0.26 cm at 48 h and 1.32 cm +/- 0.21 cm at 72 h, P < 0.05 for the difference between groups at both 48 and 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Paroxysmal nocturnal haemoglobinuria (PNH) is due to the absence or marked reduction of glycan phosphatidylinositol (GPI)-anchored proteins on the surface of blood cells. Affected patients may have a population of red blood cells that are completely deficient (PNH III) or partially deficient (PNH II) in these proteins, or they may have both. PNH III has recently been shown to be due, in all cases examined, to a somatic mutation in the PIG-A gene, whose product is required for an early step in GPI anchor synthesis. We now show that two patients with PNH II cells also have somatic mutations of the same gene: these produce a partial rather than a total loss of PIG-A function.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic anaemia, clonal in nature, due to somatic mutation. PNH may evolve to aplastic anaemia; more rarely to a myelodysplastic syndrome (MDS) or to acute myeloid leukaemia (AML). We have studied a patient who suffered from PNH and later developed refractory anaemia with ringed sideroblasts (RARS) associated with trisomy 8. By testing peripheral blood cells with appropriate antibodies we have shown that all of the red cells, neutrophils and monocytes, as well as 20% of the lymphocytes, belonged to the PNH clone; in contrast, only 43% of neutrophils and 22% of monocytes belonged to the MDS clone. We infer that the MDS must have arisen from within the PNS clone.

The PIG-A gene, whose product is involved in one of the early steps in the synthesis of glycan phosphatidylinositol (GPI) anchors, has been recently found to be defective in all cases of paroxysmal nocturnal haemoglobinuria (PNH). By isolating genomic clones from a human phage library we now show that the PIG-A gene consists of six exons (the first of which is non-coding) spanning 17 kb of DNA, and we have mapped the gene to chromosomal position Xp22.1. The PIG-A promoter has features of a housekeeping gene. We have also isolated additional clones which cross-hybridize to PIG-A cDNA, and we have thus identified an intronless PIG-A pseudogene (psi PIG-A), which we have mapped to chromosomal position 12q21. psi PIG-A cannot be functional because it contains several stop codons and a frameshift. These data make it possible to design primers for amplification of the entire PIG-A coding region, with exclusion of psi PIG-A sequences, which will facilitate characterization of PIG-A mutations in patients with PNH. Database searches revealed that PIG-A contains homologies with a number of glycosyl transferases and is highly homologous (45%) to the protein encoded by the yeast SPT14 gene.

Patients with paroxysmal nocturnal haemoglobinuria (PNH) have in their blood two red-cell populations, one normal and one deficient in proteins anchored to the membrane through a glycan phosphatidylinositol (GPI) structure. The PNH abnormality is due to a somatic mutation in the PIG-A gene, whose product is required for an early step in GPI anchor biosynthesis. We show that in two patients, two PNH clones with different mutations co-exist, and must therefore have arisen independently. This finding supports the concept that PNH develops under the pressures of a positive selection mechanism whereby GPI-anchor-deficient haemopoietic cells have a survival advantage.

Paroxysmal nocturnal haemoglobinuria (PNH), an acquired clonal blood disorder, is caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins due to a defect in a specific step of GPI-anchor synthesis. The cDNA of the X-linked gene, PIG-A, which encodes a protein required for this step has recently been isolated. We have carried out a molecular and functional analysis of the PIG-A gene in four cell lines deficient in GPI-linked proteins, obtained by Epstein-Barr virus (EBV) transformation of affected B-lymphocytes from PNH patients. In all four cell lines transfection with PIG-A cDNA restored normal expression of GPI-linked proteins. In three of the four cell lines the primary lesion is a frameshift mutation. In two of these there is a reduction in the amount of full-length mRNA. The fourth cell line contains a missense mutation in PIG-A. In each case the mutation was present in the affected granulocytes from peripheral blood of the patients, but not in normal sister cell lines from the same patient. These data prove that PNH is caused in most patients by a single mutation in the PIG-A gene. The nature of the mutation can vary and most likely occurs on the active X-chromosome in an early haematopoietic stem cell.

Background: Fluorescein angiography is often beneficial in the evaluation and management of many retinal vascular diseases. However, vitreous blood or opacities may prevent fluorescein studies before pars plana vitrectomy. After vitrectomy, fluorescein angiography may be delayed, creating uncertainty over differentiation of preoperative, intraoperative, and postoperative findings., Methods: The authors have modified an intraocular light source and operating microscope to perform intraoperative fluorescein angiography easily. Twenty-five patients underwent fluorescein angiography during pars plana vitrectomy. All studies were recorded using a microscope-mounted video camera for intraoperative and postoperative evaluation., Results: In all cases, intraoperative angiography was performed without complication. Indications for intraoperative studies included confirmation of macular edema, delineation of avascular retina, and localization of retinal and choroidal neovascularization. All videotape recordings of procedures were of high quality, allowing easy review and interpretation., Conclusion: Intraoperative fluorescein angiography is an easily performed adjunctive diagnostic procedure that can aid intraoperative evaluation and treatment of selected retinal vascular disorders.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder arising in a multipotent hemopoietic stem cell. PNH manifests clinically with intravascular hemolysis resulting from an increased sensitivity of the red cells belonging to the PNH clone to complement-mediated lysis. Numerous studies have shown that surface proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor (including proteins protecting the cell from complement) are deficient on the cells of the PNH clone, leading to the notion that GPI-anchor biosynthesis may be abnormal in these cells. To investigate the biochemical defect underlying PNH we have used lymphoblastoid cell lines (LCLs) with the PNH phenotype obtained by Epstein-Barr virus immortalization of lymphocytes from nine patients with PNH. By labeling cells with myo-[3H]inositol we have found that PNH LCLs produce phosphatidylinositol normally. By contrast, PNH LCLs fail to incorporate [3H]mannose into GPI anchor precursors. When cell-free extracts of PNH LCLs and normal LCLs obtained from the same patients (and expected therefore to be isogeneic except for the PNH mutation) were incubated with uridine diphospho-N-acetyl[3H]glucosamine (UDP-[3H]GlcNAc), we observed complete failure or marked reduction in the production of N-acetylglucosaminyl(alpha-1,6)phosphatidylinositol and glucosaminyl(alpha-1,6)phosphatidylinositol by the PNH LCLs in all cases. These findings pinpoint the block in PNH at an early stage in the biosynthesis of the GPI anchor, suggesting that the defective enzyme is UDP-GlcNAc:phosphatidylinositol-alpha-1,6-N- acetylglucosaminyltransferase. The existence of PNH type III cells and type II cells is probably explained by the transferase deficiency being total or partial, respectively.

A review of recent information on the abnormalities of the blood cell membrane in paroxysmal nocturnal haemoglobinuria (PNH) is presented, with a detailed analysis of biochemical and flow cytometry findings. The complex patterns observed in the various cell lineages of which the PNH clone consists are described, and a simplified monoclonal antibody panel is defined for diagnostic purposes. Available data on in vitro culture of progenitor cells and on the recent establishment of PNH cell lines are summarized. Finally, we discuss speculative hypotheses on the growth advantage of the PNH clone.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disorder thought to result from a somatic mutation in a hemopoietic stem cell. PNH may evolve to aplastic anemia or to acute leukemia. PNH cells are deficient in proteins attached to the cell membrane via a glycosylphosphatidylinositol structure, called the GPI anchor, and the primary lesion in PNH is thought to be a defect in the biosynthesis of the GPI anchor. We have recently established permanent lymphoblastoid cell lines that have the PNH phenotype and we report now the isolation of human-human somatic cell hybrid clones obtained by fusing them with normal lymphoblastoid cells. In all of 21 hybrid clones, obtained from five different patients, the expression of three different GPI-linked proteins on the hybrid cells was normal. These findings indicate that the PNH mutant gene is recessive with respect to the normal allele and that a recessive mutation can cause a clonal preneoplastic disorder.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic disorder caused by a somatic mutation in a hematopoietic stem cell. The fact that, in some cases, not only myeloid but also lymphoid cells are affected suggests that the mutation has occurred in a multipotent stem cell. By studying the expression of CD59 antigen (membrane inhibitor of reactive lysis) and of decay accelerating factor (DAF) on the lymphocytes of 16 patients with PNH, we found an abnormal population of lymphocytes (with absent CD59 and DAF) in 10 cases. From 4 of these patients we were able to produce Epstein-Barr virus-immortalized lymphoblastoid cell lines (LCLs) that have a PNH phenotype (absent CD59, DAF, and CD48). PNH LCL cells have apparently normal DAF messenger RNA despite not having DAF on their surface. These cell lines will be a valuable resource for further investigation of the defect or defects underlying PNH.

The FcRIII on human granulocytes is a glycosyl-phosphatidylinositol-anchored membrane protein. In PNH, proteins with this type of membrane linkage are known to be deficient on blood cells. The purpose of this study was to assess the diagnostic value of flow cytometric FcRIII quantification on granulocytes in PNH. Immunofluorescence measurements were performed in 105 patients, including 7 patients with PNH, 16 patients with aplastic anemia, and 12 with myelodysplastic syndrome, by a whole blood immunofluorescent staining procedure using monoclonal antibodies to FcRIII. In all 7 PNH patients and in 3 patients with aplastic anaemia, a distinct FcRIII-deficient granulocyte population of variable size was found. None of the remaining patients showed a similar population of FcRIII-deficient granulocytes. Quantification of FcRIII-deficient neutrophils is shown to be a highly specific and sensitive diagnostic test for PNH. It is easy, fast and highly reproducible and, therefore, suitable for routine diagnostic and follow-up studies in PNH patients and patients with aplastic anemia.

Studies with human myeloma-derived IgD have demonstrated the existence of IgD-R on peripheral blood T cells. These receptors, which are detected by rosetting with IgD-coated ox E (IgD-rosette-forming cells), are competitively inhibited by IgD, but not by IgM or IgG. Similar results were obtained with human T cell clones and T hybridomas derived from such clones either by rosetting assays or by staining with biotinylated-IgD. In agreement with studies of murine IgD-R+ cells, human IgD-R can be up-regulated by exposure of peripheral blood T cells, T cell clones, and hybridomas derived from such clones, to oligomeric IgD, but not monomeric IgD. Human IgD-R can also be induced by IL-2, IL-4, and IFN-gamma. In contrast with studies of murine IgD-R, which are expressed primarily by CD4+ cells, phenotyping studies show that both the CD4+ and CD8+ human T cell subsets are capable of expressing IgD-R.

We gave one eye of each 21 rabbits an intravitreal injection of 1 mg of triamcinolone acetonide; the other eye received an injection of an equal volume of saline solution as a control. Results of slit-lamp examinations, ophthalmoscopy, intraocular pressure, electroretinography, and light electron microscopy all remained normal throughout the three-month course of the experiment, demonstrating the lack of ocular toxicity of triamcinolone acetonide in the rabbit.

Argon laser intraocular photocoagulation is now available during vitreous surgery, to treat retinal breaks and to give panretinal photocoagulation. Twenty patients, including phakic patients with gas-filled eyes, were treated using a new operative contact lens without complication. This intraoperative technique allows controlled rapid application of laser lesions at a safe distance from the retina, and eliminated the need for attempting treatment through the often hazy media of an irritated postoperative eye. A group of five rhesus monkeys were treated with various power settings to assess the histopathologic features of these laser lesions.

A portable system for external photography combines a focusing lamp and movable flash unit with a wide focusing range. The system is arranged to operate from a single two-position trigger and may be hand-held or mounted on a slitlamp stand. It is designed for use in general ophthalmology in a multilaned office practice or bedside use in a hospital situation.