Chemical properties of bronchorrhea sputum in bronchial asthma

Bronchorrhea, defined as watery sputum of 100 ml or more per day, was seen in 18 of 207 patients (8.7 percent) with bronchial asthma during attack. Fifteen bronchorrhea sputum samples were chemically examined using ten parameters: dry weight, albumin, IgA, pH, Na+, Cl-, K+, prostaglandins E and F and histamine, and compared with eight saliva samples and 17 mucoid sputum samples. Bronchorrhea sputum differed from saliva in its chemical parameters. Bronchorrhea sputum exhibited parameter values intermediate between those of saliva and mucoid sputum, except for the two following parameters. The pH of bronchorrhea sputum was significantly lower than that of mucoid sputum and histamine concentration, expressed as weight per dry weight of sample, was significantly higher in bronchorrhea than in mucoid sputum. Administration of corticosteroid or an histamine H1-blocker to five to nine asthmatic patients with associated bronchorrhea sputum during asthmatic attacks, significantly reduced the volume of bronchorrhea sputum, whereas anticholinergics and H2-blocker did not alter the sputum volume.     

Identification of four novel mutations in F5 associated with congenital factor V deficiency

Coagulation factor V (FV) deficiency is a rare bleeding disorder characterized by low coagulant and antigen levels of FV with bleeding symptoms ranging from mild to severe. Only a limited number of mutations have been reported because of the large size of the factor V gene (F5) as well as the low prevalence. In this study, we have identified four novel mutations in F5 in five unrelated patients with congenital FV deficiency. All the patients, including two with undetectable FV activity, were asymptomatic and were found to have prolonged prothrombin time and activated partial thromboplastin time during preoperative screening or routine examinations. All four mutations found in this study are either missense or in-frame deletion. This is in contrast with previous reports of a high frequency of mutations introducing premature termination codons in inherited FV deficiency. Missense mutations of F5 might produce a mild phenotype and are not frequently diagnosed. Although FV deficiency is a very rare disorder with a predicted incidence of one in 1 million, this study suggests that the numbers of F5 mutations, especially missense mutations, are higher than estimated.     

Quantitative immunoferritin microscopy of Fya, Fyb, Jka, U, and Dib antigen site numbers on human red cells

The Fya, Fyb, Jka, U, and Dib antigen site numbers and ultrastructural distribution patterns on the human erythrocyte membrane were determined using quantitative immunoferritin microscopy. For homozygous antigen- positive red cells, the average number of determinants per red cell was about 14,000 for Jka, 17,000 for Fya and Fyb, 19,000 for Dib, and 23,000 for the U antigen, assuming that the equilibrium binding observed represented 80% saturation of the accessible antigen sites. The site numbers for this group of antigens were less than that for the Rh antigens, but considerably more than the Kell and Cellano antigens. The technique used was capable of demonstrating a twofold difference in antigen density between heterozygous and homozygous Fy (a+) red cells. More than 85% of the Fya and Fyb antigen sites were lost following pretreatment of the red cells with papain, consistent with the serologic lability of the Fy antigens following proteolysis. The ferritin distribution observed following conjugate staining of antibody- sensitized ghost membranes was similar for all five antigens studied and showed a random, clustered ferritin pattern. Although the quantitative estimates are valid, the remarkable similarity in antigen distribution pattern for this diverse group of antigens, as well as other considerations, suggest that the findings with ghose membranes probably do not reflect faithfully the antigen arrangement on the intact red cell membrane.     

''VA'', a New Type of Erythrocyte Polyagglutination Characterized by Depressed H Receptors and Associated with Hemolytic Anemia

With help of immunoflorescence, best with anti-AHP from Helix pomatia, a stippled structure could be demonstrated on the patient"s red blood cells. Thus an "A-like" receptor could be detected on the erythrocyte membrane of this group O patient. The reactive antigen was proved not to be a crypt antigen exposed by the action of neuraminidase. The same stippled fluorescence with antiAhp was observed on the red blood cells of a patient suffering from hemolytic anemia induced by influnza A2 virus. In this case this virus was shown not to be responsible for polyagglutination. No virus or microorganism could be isolated from the patient"s blood. Also by immunofluorescence the weak expression of the H antigen could be demonstrated with an extract of Evonymus europaeus. Electron microscopy of erythrocytes was normal. The neuraminic acid content and the electrophoretic mobility were found to be decreased to a minor degree. No distinct cell populations could be observed.     

Resting peripheral blood lactate elevation in survivors of prehospital cardiac arrest: Correlation with hemodynamic, electrophysiologic and oxyhemoglobin dissociation indexes

Thirteen patients who were survivors of sudden unexpected cardiac arrest in the community were followed up for up to 3 years. All showed an anomalous relation between erythrocyte levels of oxygen dissociation (P₅₀) and 2,3-diphosphoglyceric acid (2,3-DPG). This could not be explained by hemoglobinopathy, carbon monoxide or methemoglobinemias. Because lactate accumulation in red blood cells may alter oxygen dissociation, whole blood and red blood cell lactate levels were measured. An average of 4.4 measurements per patient were obtained over a mean time of 5.6 months of the post-hospital phase of the follow-up period, which had a total mean duration of 22 months. The patients did not have overt congestive heart failure and were not acidotic (mean venous pH = 7.35). Lactate levels were elevated (mean = 15.1 mg/100 ml ± 0.8 mg/100 [standard error of the mean], compared with normal values of 7.6 mg/100 ml ± 1.4 mg/100 ml; P < 0.01). When lactate was plotted against red blood cell 2,3-diphosphoglycerate, a positive curvilinear relation was found (r² = 0.12, P < 0.05). The production of lactate in chronic ischemia may increase red blood cell 2,3-diphosphoglycerate through glycolysis. The expected effect on oxygen dissociation of this increase in 2,3-diphosphoglycerate is offset by a counterbalancing leftward shift of the oxyhemoglobin dissociation curve by an increase in red blood cell lactic acid. When lactate was compared with left ventricular ejection fraction, there was a significant negative correlation (r = 0.86, P < 0.01). Serial 24 hour ambulatory electrocardiograms (mean 4 per patient) were analyzed for changes in quantity and severity of ventricular arrhythmia at the time of lactate determinations. Six patients had lactate level variation of more than 30 percent, and five of these six patients had an increase in quantity and severity of ventricular ectopic activity when their lactate levels were in the higher range. We conclude that elevated resting lactate levels correlate with impaired ventricular function, and fluctuations in a given patient may identify changes in clinical and electrophysiologic status.     

Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency

We identified 3 mutations in the factor V (FV) gene (F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity, <1%; FV antigen, 9%) and was a compound heterozygote for a novel 5-bp deletion in exon 22 and the V1813M mutation. Patient 2 had moderate bleeding symptoms (plasma FV activity, <1%; FV antigen, 4%) and was homozygous for the V1813M mutation. Patient 3 had very mild symptoms (plasma FV activity, 1%; FV antigen, 5%) and was homozygous for the novel R2174L mutation. A study of recombinant protein expression revealed that the FV coagulant-specific activities in conditioned media for the FV-R2174L and FV-V1813M mutants were reduced to approximately 40% and 28% of wild-type FV, respectively. The amounts of FV-R2174L protein and messenger RNA in the platelets of patient 3 were similar to those of healthy subjects; however, the amount of FV-V1813M protein in patient 2 was decreased. Our data suggest that the severity of the bleeding tendency in patients with FV deficiency is correlated not only with plasma FV activity but also with the amount of FV protein in the platelets.     

鼠疫抗原致敏红血球诊断试剂制备方法的改进

本文对鼠疫抗原致敏红血球诊断试剂的常规制备方法进行了改进。利用鞣酸作为粘附剂同时作用于绵羊红血球和鼠疫F1抗原,而建立了改良法。该法致敏成功率高于常规法,所用致敏红血球的F1抗原比常规法少5倍,制备流程短,PHA反应凝集相清晰。建议在诊断试剂的生产实践中用改良法替代常规法。     

Covalent crosslinking of human coagulation factor V by activated factor XIII from guinea pig megakaryocytes and human plasma

Coagulation factor V (FV) has been shown to be synthesized in both the liver and megakaryocytes. We now present evidence that FV can be covalently crosslinked by an enzyme originating from megakaryocytes to form polymeric multimers of factor V. The guinea pig megakaryocyte enzyme appears to be factor XIIIa since the FV-crosslinking activity (1) had an absolute requirement for Ca++, (2) was completely inhibited by iodoacetamide, 5,5'-dithiobis- (2-nitrobenzoic acid), p- chloromercuribenzene sulfonic acid, and N-ethylmaleimide, all known alkylators of the thiol group at the active site of the factor XIIIa, (3) was blocked by known pseudoamine donor substrates of factor XIIIa including dansylcadaverine and putrescine, and (4) could be directly demonstrated in the guinea pig megakaryocyte lysate by a specific activity staining procedure. No tranglutaminase was detected in guinea pig megakaryocytes in contrast to red cells and liver. A similar pattern of covalent crosslinking of human FV by purified activated human plasma factor XIII was also demonstrated. Analysis of the crosslinked products of FV formed by the guinea pig enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicates the formation of intermediate as well as higher molecular weight polymers, suggesting that the crosslinking is a stepwise polymerization process.     

Regression of atherosclerosis in cholesterol-fed rabbits: effects of fish oil and verapamil

Previous studies have shown that either fish oil or verapamil can attenuate the development of atherosclerosis in the lipid-fed rabbit. The present study was designed to evaluate the individual and combined effects of these two interventions on regression. Seventy New Zealand rabbits in seven groups (10 each) were fed a 0.3% cholesterol diet for 10 weeks. Control group C10 was then killed. Control group C20 was fed a 0.3% cholesterol diet and the other five groups were fed a normal diet for an additional 10 weeks. Group F in three treated groups received 2 ml/day of fish oil (Proto-Chol, eicosapentaenoic acid, 180 mg/ml and docosahexaenoic acid, 120 mg/ml) by gavage. Group V received verapamil, 2 g/1,000 ml drinking water, and group FV received both fish oil and verapamil for an additional 10 weeks. Group CF (control for fish oil) received 2 ml/day of water by gavage and group CV (control for verapamil) received water without gavage for an additional 10 weeks. The percent of aortic and pulmonary atherosclerosis was measured by planimetry of sudanophilic lesions. The percent of aortic lesions in the four control groups (C20, C10, CF and CV) was 57 +/- 22, 40 +/- 15, 40 +/- 14 and 33 +/- 25%, respectively. The fish oil or verapamil groups (F, V, FV) showed a significant reduction in aortic lesions: 15 +/- 17%, p less than 0.05; 16 +/- 12%, p less than 0.05; and 26 +/- 24%, p = NS, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood group D antigen content of nucleated red cell precursors.

The D antigen content of nucleated red cell precursors in human bone marrow was estimated using autoradiography and 125I-anti-D. D antigen first appeared in the pronormoblast, and the quantity of antigen progressively increased during red cell maturation. Maximal anti-D binding occurred on mature red blood cells. Pronormoblasts, basophilic normoblasts, polychromatophilic normoblasts, and orthochromatic normoblasts, respectively, had approximately 1/4, 1/2, 2/3, and 3/4 the quantity of antigen found on mature red cells. None of the other cell types were found in bone marrow labeled with anti-D.     

Mutagenic effects of nitrofurylacrylic acid on V79 cells under hypoxic conditions

Induction of 6-thioguanine-resistant mutations was studied in Chinese hamster V79 cells treated with nitrofurylacrylic acid under aerobic and hypoxic conditions. The results obtained demonstrated that the mutagenic activity of this substance, less significant in aerobic conditions, was very high under hypoxic conditions. A gradual increase in mutation frequencies according to the applied concentrations of nitrofurylacrylic acid was observed, the maximal values being at 0.8 mg of the substance per 1 ml of growth medium. The mutagenic effects of nitrofurylacrylic acid were independent of its cytostatic effect that was higher in aerobic than hypoxic conditions. Mutagenic capacity of this substance found in vitro in mammalian V79 cells suggests its possible carcinogenic potential in vivo under conditions favorable for metabolic activation of this nitrofuran-derived compound.     

Bone Marrow Fractionation by the Haemonetics System: Reduction of Red Cell Mass before Marrow Freezing, with Special Reference to Pediatric Marrow Volumes

For purposes of freezing autologous marrow or transplants of allogeneic marrow with major ABO blood group incompatibility, 54 freshly harvested bone marrows from children of 7-65 kg of weight were depleted of their red cells with the Haemonetics V50 system. The marrow volumes ranged from 230 to 1,145 ml, with 17 small (200-399 ml), 18 intermediate (400-799 ml) and 19 large (800-1,200 ml) volumes. After processing, the median recoveries were: volume 24%, red cell mass 18%, and nucleated cells 75%. In the small marrow volume group, a good nucleated cell recovery was achieved at the expense of red cell depletion. The colony-forming units, granulocytes-macrophages (CFU-GM) were normal after thawing of processed, cryopreserved marrows, and good engraftment of both allogeneic and autologous marrows were achieved. We conclude that marrow processing with the Haemonetics V50 system results in adequate red cell depletion and good nucleated cell recovery without open-air contact of marrow or excessive time consumption. For small marrow volumes, however, the red cell depletion was suboptimal, and a bowl size smaller than 125 ml is desirable for pediatric use.     

Comparison of clinical features of rotavirus infection with rotavirus antigen titers in feces obtained during the acute phase

When fecal specimens obtained from infants with acute gastroenteritis during the acute phase between January 1986 and March 1987 were examined by enzyme immunoassay (EIA), 72 cases were found to have the rotavirus antigen. Such 3 clinical signs as diarrhea (D), fever (F) and vomiting (V) were all observed in 38 cases. These cases showing DFV syndrome demonstrated the highest geometric mean titer of rotavirus antigen among the rotavirus antigen-positive cases. Geometric mean titers (GMTs) were calculated to be as high as more than 10(3.1) in 26 of the 38 DFV cases (68%) and in 11 of 34 cases with DV, DF, FV or D syndromes (32%), showing a significant difference between the 2 groups (p less than 0.005). GTMs were slightly higher in cases having fever of 38 degrees C or more, than in those having fever of less than 38 degrees C.     

Serum vitamin B 12 and folic acid levels in patients with fasciolopsiasis.

Serum vitamin B12, serum and red cell folate concentrations and vitamin B12 absorption were studied in 100 patients with fasciolopsiasis. A mean value of serum vitamin B12 level in the patient group was found to be significantly lower than that of normal subjects and 14% of these patients had serum vitamin B12 level less than 100 pg/ml. Serum UBBC and TBBC levels in the patients were significantly higher than those of the normal subjects. Serum TCI and TCIII increased significantly while TCII decreased. Vitamin B12 absorption was found to be impaired in 3 of 9 patients studied. There was no relationship seen between serum vitamin B12 level and vitamin B12 absorption. The mean values of serum folate and red cell folate levels in the patient group were significantly lower than those of normal subjects. Fifteen of 100 patients (15%) had serum folate level less than 3 ng/ml, while all of them had red cell folate higher than 100 ng/ml. Serum folic acid binding protein levels (FABP) in these patients, were not significantly different from those of normal subjects.     

Biology of human megakaryocyte factor V

To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S-methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.     

Increased potassium transport and ouabain binding in human Rhnull red blood cells.

Potassium (K+) influx and 3H-ouabain binding were studied in human red cells completely lacking the rhesus (Rh) antigens (Rhnull cells) and compared with normal Rh(D) red cells. The Rhnull cells, originally described by Seidl, Spielmann, and Martin (Vox Sang. 23:182, 1972) were normal in size, cation, and water content, indicating no significant increase in cell volume as occurs in young human red cells. However, the ouabain-insensitive K+ permeability, as well as the ouabain sensitive active K+ transport, were increased 1.6 1.8-and 1.4-1.5-fold, respectively, above the values found in Rh(D) control cells. The Na+K+ ATPase activity of membranes from Rhnull cells was also higher than from Rh(D) cells. Binding studies with 3H-ouabain revealed that at 100% K+ pump inhibition Rhnull cells bound 670 and Rh(D) cells 450-500 ouabain molecules per cell. Since the rate of ouabain binding was identical in Rhnull and Rh(D) control cells, we concluded that the Rhnull cell had about 35%-45% more cation pumps than the Rh(D) cell. These additional pumps in Rhnull cells appeared to be indistinguishable from those in control cells. Anti-D or the serum from the Rhnull individual did not alter cation permeability in Rh(D) red cells. The data suggested that the Rhnull cell, known for its hematologic malfunction, was not a young or prematurely released red cell, but had a pleiotropic membrane defect which also affected the passive and active cation transport system on the molecular level. Our finding precludes a structural identity of the rhesus antigen with the molecules composing the Na+K+ pump system.     

Identification of three F5 gene mutations associated with inherited coagulation factor V deficiency in two Chinese pedigrees

To investigate the molecular defects in two Chinese pedigrees with inherited factor V (FV) deficiency. A 37-year-old male (proband 1) and an 18-month-old boy (proband 2) were diagnosed as inherited coagulation FV deficiency by severely reduced plasma levels of FV activity and antigen. All 25 exons and their flanking sequence of F5 gene were amplified by polymerase chain reaction (PCR) for both probands and the PCR products were directly sequenced. Total RNA was extracted from the peripheral lymphocytes of proband 1 for detecting the changes at mRNA level. The homozygous deletion IVS8 -2A>G was identified in the F5 gene of proband 1 and complementary DNA (cDNA) analysis revealed the abolishment of the canonical splicing site by the mutation and the activation of the cryptic acceptor site 24 bp upstream instead. The insertion introduced eight additional amino acids (AA) into the FV protein. Two heterozygous mutations of F5 gene were discovered in proband 2. The 2238-9del AG in exon 13 introduced a premature termination code at 689 AA and the substitution of G6410 by T in exon 23 lead to the missense mutation Gly2079Val. Three F5 gene mutations, IVS8 -2A>G, 2238-9del AG and G6410T, have been identified in two Chinese pedigree with congenital FV deficiency, respectively.     

High-dose intravenous immunoglobulin treatment in two patients with acquired factor V inhibitors

We report two patients who developed acquired factor V (FV) inhibitors not related to exposure to bovine thrombin. Associated conditions were found in one patient (surgery, antibiotic administration) but none in the other one. Bleeding complications occurred only in the patient with idiopathic FV inhibitor, leading to packed red cell infusion. Laboratory findings showed the presence of specific FV inhibitors with titers of 5.5 and 5 Bethesda units, respectively. These two patients received high-dose intravenous immunoglobulin and FV levels normalized within a few days with a concomitant disappearance of FV inhibitors.     

Demonstration of ferritin-labelled antibodies bound to human erythrocytes fixed with glutaraldehyde

In this immuno-electron microscopic study, the capability of the combining antibody and the agglutinability was examined in glutaraldehyde-fixed erythrocytes. Glutaraldehyde-fixed human red cells preserved a strong ability to bind specific antibodies as unfixed cells, even though they decrease in agglutinability. As the fixed cells were separated from one another, even when were agglutinating, the localization of antibodies on a cell in the fixed cells was very clear. The number of A antigen sites per red ceil of different phenotypes was estimated individually as each values of single cell but not as mean value of many cells. It was confirmed that the ability of the combining antibody varied greatly from cell to cell in the same blood of umbilical cord.     

Adenosine 3',5-cyclic monophosphate phosphodiesterase activity in granulosa cells from Booroola x Romney ewes with and without the F gene

Granulosa cells from ovarian follicles (greater than or equal to 1 mm diameter) in Booroola ewes which are homozygous (FF) or heterozygous (F+) for the F gene have previously been shown to produce significantly more cAMP in response to FSH or LH than those from similar sized follicles in ewes without the F gene (++). The aim of these studies was to test whether these F gene-specific differences arose because of differences in cAMP-phosphodiesterase (cAMP-PDE) activity. In the first study using 1 mumol cAMP/l as substrate, no F gene-specific effects were noted in cAMP-PDE activity in granulosa cells from small (1-2.5 mm diameter, n = 4 per genotype) or large (greater than or equal to 3 mm diameter, n = 4 per genotype) follicles from FF, F+ or ++ ewes, despite F gene-specific effects in FSH (1 microgram/ml)- and LH (0.1 microgram/ml)-induced cAMP accumulation in these same cell preparations. The overall mean levels of cAMP-PDE across all genotypes in cells from small and large follicles were 0.47 +/- 0.04 (S.E.M., n = 12) and 0.28 +/- 0.03 pmol cAMP/10(6) cells per min respectively; the mean PDE activity in cells from small follicles was significantly (P less than 0.05) higher compared with that in cells from large follicles. In a second study, granulosa cells from each genotype were pooled over all follicle sizes (greater than or equal to 1 mm diameter, one pool per genotype) and the rates of cAMP hydrolysis tested over a range of substrate concentrations (0-16 mumol/l) but no gene-specific differences with respect to the Michaelis constant and maximum velocity were noted.(ABSTRACT TRUNCATED AT 250 WORDS)