Expression of monovalent fragments derived from a human IgM autoantibody in E. coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM. We discuss the expression of recombinant scFv fragments in E. coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.     

Severe bradyarrythmia induced by tricyclic antidepressants in an elderly patient

The fluoroquinolones have an established role in treatment of infection with aerobic gram negative rods. The increased importance of gram positive nosocomial infection and of acquired fluoroquinolone resistance has stimulated a search for new compounds with enhanced potency and spectrum. CP-99,219 is a novel compound in this class with enhanced activity against gram positive organisms. We have studied the activity of CP-99,219 relative to ciprofloxacin, fleroxacin, ofloxacin, and sparfloxacin using test panels of organisms with a high proportion of ciprofloxacin resistance. CP-99,219 is more potent than any of the other four compounds against both gram positive and gram negative bacteria. The activity of CP-99,219 against many bacteria resistant to the established agents, warrants further in vitro and clinical studies.     

A contact-free method for computerized functional assessment of mandibular movements

A specialized computer system for real-time study of some dynamic characteristics of the maxillodental system has been designed. The system consists of a TV unit compatible to IBM PC/AT and software. The method of non cintact recording of dynamic characteristics of the mandible permits, among other things, measurements and comparison of mandibular movements before and after orthodontic treatment and, hence, may be used for assessing the results of occlusion correction.     

Uptake and metabolism of L-serine by Pneumocystis carinii carinii

Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid-precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin-layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 11% in long chain bases and other compounds. Four long chain bases were detected by thin-layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were tentatively identified by their migrations on thin-layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidylethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L-Cycloserine and beta-Cl-D-alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids.     

STZ-induced diabetes in mice and heme pathway enzymes. Effect of allylisopropylacetamide and alpha-tocopherol

We compared the systemic and regional hemodynamic effects of nifedipine and lisinopril in 26 elderly hypertensive patients with the use of the pulsed Doppler ultrasound technique. Nifedipine is a dihydropyridine calcium antagonist, and lisinopril is an angiotensin-converting enzyme inhibitor. The study had a single-blind crossover design: nifedipine and lisinopril were given for 8 weeks each after washout periods of 4 weeks. Both nifedipine and lisinopril significantly reduced mean arterial pressure to the same extent (P < .01); cardiac output remained unchanged in both nifedipine- and lisinopril-treated groups. Lisinopril increased renal flow significantly (P < .01), but nifedipine did not. Common carotid, vertebral, celiac, and superior mesenteric arterial and diaphragmatic and terminal aortic flows did not show a significant change with either nifedipine or lisinopril. The specific action of lisinopril on the thoracic aorta was a marked improvement of aortic compliance compared with nifedipine, which might be partly responsible for an increase in renal flow. Lisinopril may provide more desirable regional hemodynamic effects and additional benefits for elderly hypertensive patients.     

Gender and the transition into practice

Since 1990, over half the enrollees in advanced education pediatric dentistry programs have been women. The higher proportion of women in pediatric dentistry should permit examination of the practice patterns of groups of men and women at similar stages in their careers. In 1991 the American Academy of Pediatric Dentistry surveyed 4,950 dentists about a variety of issues related to practice patterns and demographics, obtaining 2,362 responses. This study conducted a secondary analysis of the survey data by developing three age-matched graduation cohorts based on gender and years since graduation: 1 to 5 years, 6 to 10 years, and over 10 years. Four areas were investigated: practice patterns, practice arrangements, distribution of time, and income. The overall differences in practice patterns between males and females were statistically significant for the Early Career Group (1 to 5 years). More males were in private practice and a higher proportion of them were practice owners. More women were dental school faculty or in private practice as an employee or contractor. The differences in practice patterns for males and females were not statistically significant for the Intermediate Career Group (6 to 10 years). In the Established Career group (over 10 years), the differences were again statistically significant, with more males as practice owners and shareholders and more women in solo practices. Analysis of time distribution showed that, in the two earlier career groups, women spend about twice as much time as men in child care. These findings may help to explain why many women in the early stages of their careers might prefer the flexibility of working for someone else. When the effects of gender and employment status on income were tested, significant differences were found for the Intermediate and Established groups, with males and practice owners having higher income levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase I trial of an anti-CD19 deglycosylated ricin A chain immunotoxin in non-Hodgkin's lymphoma: effect of an intensive schedule of administration

In a phase I trial, eight patients with non-Hodgkin's B-cell lymphoma received mouse IgG1k monoclonal antibody HD37 specific for CD19 conjugated to deglycosylated ricin A chain (dgA) administered in four doses at 4-h intervals with total doses ranging from 4-12 mg/m2. This schedule generated serum levels of immunotoxin which were sustained over 36 h. The plasma half-life of HD37-dgA was 17 +/- 4 (SD) h. The HD37-dgA conjugate was stable in vivo as demonstrated by serum levels of HD37-dgA conjugate comparable to those of total HD37 antibody. Peak serum levels attained after the fourth dose ranged from 0.36 to 5.63 micrograms/ml. Two of seven evaluable patients developed modest human anti-immunotoxin antibody responses. Toxicity in patients 1-7 consisted of dose-dependent capillary leak syndrome with hypoalbuminemia, orthostatic hypotension, and weight gain. Patient 8 died on day 8 with severe capillary leak, bronchopneumonia, and rhabdomyolysis. All patients had progressive disease at 4 weeks except patient 8, who exhibited a near-complete remission before his death. This intensive schedule appears to produce inordinate toxicity with a maximal tolerated total dose of 8 mg/m2.     

Isolation and study of the properties of the catalytic subunit of cAMP-dependent protein kinase of the small intestinal mucosa of the rabbit

cAMP-dependent and casein proteinkinase were found in cytosol of the rabbit small intestine mucosa. cAMP-dependent proteinkinase of cytosol is represented by two forms of types I and II. The activity of enzymes of types I and II constitutes 10 and 90%, respectively. Casein proteinkinase is represented by a single form. The catalytic subunit of cAMP-dependent proteinkinase of type II was isolated in a homogenous state. The catalytic subunit phosphorylates histones H1, H2a, H2b and protamine and to a far less degree histones H3, H4 and casein (H2b greater than H1 greater than H2a greater than protamine much greater than H3 greater than casein). The Km value for histone H1 is equal to 65 mkM, and that for Mg-ATP 12 mkM. Chloromethylpyrophosphonate and adenosine p-fluorosulfobenzoate were studied as affine modifiers of the active center of the catalytic subunit from the small intestine mucosa. It was shown that only adenosine p-fluorosulfonate is an irreversible inhibitor of the catalytic subunit.     

Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate

PURPOSE: The purpose of the study is to determine the effect of exogenous vascular endothelial growth factor (VEGF) on the primate retina and its vasculature. METHODS: Ten eyes of five animals were studied. Physiologically relevant amounts of the 165 amino acid isoform of human recombinant VEGF were injected into the vitreous of six healthy cynomolgus monkey eyes. Inactivated human recombinant VEGF or vehicle was injected into four contralateral control subject eyes. Eyes were assessed by slit-lamp biomicroscopy, tonometry, fundus color photography, fundus fluorescein angiography, light microscopy, and immunostaining with antibodies against proliferating cell nuclear antigen and factor VIII antigen. RESULTS: All six bioactive VEGF-injected eyes developed dilated, tortuous retinal vessels that leaked fluorescein. Eyes receiving multiple injections of VEGF developed progressively dilated and tortuous vessels, venous beading, edema, microaneurysms, intraretinal hemorrhages and capillary closure with ischemia. The severity of the retinopathy correlated with the number of VEGF injections. None of the four control eyes exhibited any abnormal retinal vascular changes. The endothelial cells of retinal blood vessels were proliferating cell nuclear antigen positive only in the bioactive VEGF-injected eyes. CONCLUSION: Vascular endothelial growth factor is sufficient to produce many of the vascular abnormalities common to diabetic retinopathy and other ischemic retinopathies, such as hemorrhage, edema, venous beading, capillary occlusion with ischemia, microaneurysm formation, and intraretinal vascular proliferation.