The toxicokinetics of mercury in mice offspring after maternal exposure to methylmercury--effect of selenomethionine.

Human evidence indicates fetotoxicity of methylmercury at exposure levels inducing only slight and reversible maternal toxicity, but experimental animal data demonstrate, that fetotoxicity may occur despite absence of noticeable maternal toxicity. However, in contrast to the long-term exposure in humans, the key point in the experimental design of the majority of experimental studies has been administration of few doses of methylmercury late in gestation. The present study in mice therefore used long-term maternal exposure to methylmercury (1 nmol/ml in drinking water) and a cross-fostering design to investigate separately in different offsprings the toxicokinetics of transplacentally absorbed mercury and mercury retained during lactation. Further, the influence of seleno-L-methionine (3 micrograms/ml in drinking water) on the toxicokinetics of methylmercury in these mice was studied. The present study demonstrated, that independent on seleno-L-methionine supplementation, offspring deposited equal amounts of mercury during lactation and during gestation. Moreover, the organ distribution and rate of excretion of mercury in transplacentally exposed mice were considerably different from those in mice exposed postnatally and from adult mice in studies using comparable dosages. Seleno-L-methionine only slightly affected the toxicokinetics of mercury in offspring.


Proliferative responses of quail aortic smooth muscle cells to benzo[a]pyrene: implications in PAH-induced atherogenesis.

Repeated exposure of avian and rodent species to benzo(a)pyrene (BaP) has been associated with the development of aortic lesions of atherosclerotic etiology. Because the occurrence of these lesions may involve alterations in the regulation of smooth muscle cell (SMC) growth, the present studies were conducted to evaluate the proliferative responses of quail aortic SMCs to BaP treatment in vivo and in vitro. Measurements of [3H]thymidine incorporation and cell growth were conducted in cultured aortic SMCs isolated from male Japanese quail treated with 10 mg/kg BaP or vehicle weekly for 10 weeks or in naive aortic SMCs exposed in vitro to BaP (0.003-30 microM). Inhibition of DNA synthesis was observed in primary and early passage cultures of aortic SMCs isolated from BaP-treated quail relative to controls. Continued propagation of these cultures yielded a population of BaP cells which proliferated at faster rates than controls. The proliferative phenotype induced by BaP was first observed after the tenth passage and preserved in all subsequent passages tested. In vitro growth of SMCs from BaP-treated animals was serum- and anchorage-dependent. A 24-h exposure of cycling SMC cultures to BaP (0.003-30 microM) was associated with a dose-dependent decrease in DNA synthesis and significant delay in the progression of SMCs through the cell cycle. A time-course study revealed that maximal inhibition of DNA synthesis occurred 10 h after addition of 3 microM BaP to cycling cultures of SMCs. As seen in SMCs isolated from BaP-treated quail, serial subculture of SMCs exposed to 0.3 microM BaP in vitro for 24 h yielded a fast-growing population of cells. In these cultures, expression of the proliferative phenotype was observed after the fifth passage. These data suggest that BaP induces the expression of a proliferative phenotype in aortic SMCs characterized by enhanced serum responsiveness. This phenotypic modulation may contribute to the initiation and/or progression of vascular lesions of atherosclerotic etiology induced by BaP.


A review of the literature on potential reproductive and developmental toxicity of electric and magnetic fields.

The potential of electric and magnetic fields to adversely affect the health of the human population is an issue which continues to receive a great deal of attention in both public and scientific forums. One of the critical issues is the possibility that such fields may adversely affect the reproductive process. Numerous studies investigating the potential of electric and/or magnetic fields to alter reproduction in vertebrates have been conducted. These studies have, in many instances, yielded seemingly contradictory results. A number of epidemiological studies have been conducted as well. This review of the literature examines relevant studies and attempts to draw biologically rational conclusions from them. The studies are ordered in broad categories based upon both classification of the species studied (i.e. submammalian, mammalian exclusive of man and human) and the agent used (i.e. extremely low frequency electric, very low frequency electric, and magnetic fields). From our review we conclude that laboratory experimental and epidemiological results to date have not yielded conclusive data to support the contention that such fields induce adverse reproductive effects under the test or environmental conditions studied. Additional studies may, however, be warranted to clarify some of the experimental results obtained.


Pioneers of anti-venomous serotherapy: Dr Vital Brazil (1865-1950).

Dr Vital Brazil was a great humanitarian and pioneer of medical science. His main work arose from his concern with poisonous snakebite accidents to labourers working the land. Vital Brazil estimated that, at the beginning of this century, deaths due to crotaline snakebites in the State of São Paulo, Brazil, were nearly 3000 per year, representing a mortality rate of about 25%, the majority being due to bothropic envenomation. After reading a report of Calmette's anti-Naja serum, Vital Brazil raised monovalent serum against the venom of Bothrops jararaca and the venom of Crotalus durissus terrificus. In 1989 this led to the first demonstration of the specificity of anti-venomous serum and later, the first production of polyvalent serum for therapeutic use. As Director of the newly founded Institute Butantan in São Paulo, Vital Brazil was actively engaged in every aspect of serotherapeutic treatment. This included organizing a unique system of exchanging anti-ophidic serum for snakes as well as a wide-ranging teaching programme. His many outstanding contributions to the fields of immunology, public health, toxinology and herpetology required not only a very high level of observational, deductive and practical ability but also an unswerving vision and sense of duty; this was allied to great administrative skill and exceptional energy.


Inhibition of the proteolytic activity of hemorrhagin-e from Crotalus atrox venom by antihemorrhagins from homologous serum.

Antihemorrhagic proteins from Crotalus atrox serum were tested for their ability to inhibit the proteolytic activity of the hemorrhagic toxin-e from Crotalus atrox venom and of several other proteolytic enzymes: trypsin, collagenase and thermolysin. The antihemorrhagic proteins inhibited the proteolytic activity of hemorrhagin-e when tested on gelatin type I and collagen type IV, the proteolytic activity of trypsin on photofilm gelatin and the proteolytic activity of whole venom when tested on azocollagen and photofilm gelatin. The antihemorrhagins failed to inhibit the proteolytic activity of trypsin when tested on the specific synthetic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester (APNE), the activity of microbial collagenase on N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) or on azocollagen and the activity of thermolysin on N-(3-[2-furyl]acryloyl)-Gly-Leu amide (FAGLA). It is tentatively suggested that the antihemorrhagins from snake blood serum are proteinase inhibitors that underwent specialization towards the neutralization of the proteolytic activity of hemorrhagic toxins.


Envenoming by viper bites in France: clinical gradation and biological quantification by ELISA.

Viper bites are frequent in France but the evaluation of the severity of envenomings and consequently patient treatment has not yet been properly evaluated. The purpose of this study was to measure venom antigens in blood and/or urine of bitten patients and to establish a quantitative relationship with clinical observations. A prospective enquiry was conducted in 1990 in France to collect epidemiological, clinical and biological data from hospitals. Urine and blood samples were tested for their content of Vipera aspis venom antigens by a sandwich enzyme linked immunosorbent assay (ELISA). One hundred and two charts were analysed, from patients presenting documented viper bites. Oedema was the prominent local feature (81 cases). Systemic signs consisted of vomiting and/or diarrhoea (22 cases), slight or severe hypotension (15 cases), shock (2 cases) and bleeding (1 case). A relationship was observed between these systemic signs and the extent of the oedema, which permitted the establishment of a grading scale. Grade 0 (no envenoming) was identified by fang marks and absence of oedema and local reaction; grade 1 (mild envenoming) by local oedema and absence of systemic symptoms; grade 2 (moderate envenoming) by regional oedema and moderate systemic symptoms; and grade 3 (severe envenoming) by extensive oedema and severe systemic symptoms. Quantification of venom antigens in blood or urine of patients by ELISA revealed a significant correlation between clinical signs of envenoming and the level of venom antigens in blood or urine. This indicated that the ELISA test is a useful and predictive tool for clinically grading viper envenomings.


[Pharmacologic and enzymatic effects of snake venoms from Antioquia and Choco (Colombia)].

We compared several pharmacological and enzymatic effects induced by 11 snake venoms from seven species, six of them from different geographic areas of Antioquia and Choco, north-west of Colombia, South America (Bothrops atrox, B. nasutus, B. schlegelii, B. punctatus, Lachesis muta, Micrurus mipartitus), and Crotalus durissus terrificus venom, from specimens captured in other provinces of the country (Tolima, Huila, Meta and Atlantico). Differences were observed in edema-forming, hemorrhage, defibrination, indirect hemolysis, myonecrosis, proteolysis and lethal activity between venoms from different genera or species, as well as according to the geographic area of origin in B. atrox and B. nasutus snake venoms. Bothrops venoms, in particular B. atrox and L. muta, produced major local effects. All of the venoms, including M. mipartitus, had myotoxic effects. The most defibrinating venoms were B. atrox, L. muta, B. punctatus and C. d. terrificus. All of the venoms had indirect hemolytic activity; the venom of M. mipartitus being greatest. The most lethal venoms were those of C. d. terrificus and M. mipartitus. Within Bothrops species, the venom of B. schlegelii was the least active in terms of local and systemic pathologic effects.


Specificity of action of beta-bungarotoxin on acetylcholine release from synaptosomes.

Presynaptically acting phospholipase A2 (PLA2) neurotoxins such as beta-bungarotoxin (beta-BuTX) specifically modify the release of acetylcholine (ACh) in the periphery, whereas in the central nervous system (CNS) the release of other neurotransmitters such as norepinephrine (NE) and serotonin (5-HT) are also modified. In addition, ACh release in the periphery is modified in a triphasic manner (decrease, then increase, then block), while in the CNS only the increase has been demonstrated. To determine the specificity of the central effects of beta-BuTX we compared the effects of beta-BuTX and N. n. atra PLA2 on the release from rat cerebrocortical synaptosomes of ACh, NE, and 5-HT. We also measured the leakage of lactate dehydrogenase (LDH) in order to determine whether membrane permeablization was responsible for neurotransmitter leakage. Both the PLA2 neurotoxin (5.0 nM) and the non-neurotoxic enzyme (0.5 nM) stimulated the loss of NE and 5-HT, but only at concentrations which induced leakage of LDH. Conversely, beta-BuTX stimulated the release of ACh at a concentration (0.5 nM) which caused no leakage of LDH, while N. n. atra PLA2 (0.5 nM) did not stimulate ACh release. beta-Bungarotoxin thus exerts a specific effect on cholinergic nerve terminals, while the leakage of NE and 5-HT induced by beta-BuTX and N. n. atra PLA2 correlates with membrane disruption due to their PLA2 activities. Within 20 min, 0.5 nM beta-BuTX increased the resting release of ACh and decreased the stimulated release induced by depolarization with 4-aminopyridine, while N. n. atra PLA2 (0.5 nM) did not stimulate ACh release and required 45 min to exert an inhibitory effect. beta-BuTX (5.0 nM) also exerted an inhibitory effect on ACh release stimulated by veratridine, but not by high KCl. It is concluded that in low concentrations that do not disrupt membrane permeability, beta-BuTX acts specifically on cholinergic terminals in rat synaptosomes, where it exerts both stimulatory and inhibitory effects.


A sensitive enzyme-linked immunosorbent assay (ELISA) for testosterone: use of a novel heterologous hapten conjugated to penicillinase.

A microplate enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of testosterone in plasma. The assay uses a heterologous system consisting of a novel hapten 4-(17 beta-hydroxy-3-oxoestra-4,9-dien-11 beta-yl)butanoic acid (1) conjugated to penicillinase (beta-lactamase). The key reaction in the synthesis of the hapten was the cuprate-mediated 1,4-conjugate addition on 3,3,17,17-bis-ethylenedioxy-5 alpha,10 alpha-oxido-estr-9(11)-ene by the Grignard reagent derived from trimethyl 4-bromoorthobutyrate; this regiospecifically introduces the 11 beta-butanoate function. The hapten-penicillinase conjugate was used in the assay in conjunction with the immunoglobulin G (IgG) fraction derived from a previously characterized, highly specific, antitestosterone serum raised against a testosterone-19-O-carboxymethyl ether-bovine serum albumin (T-19-O-CME-BSA) conjugate. This unique system represents one incorporating three elements of structural heterology: bridge, site, and ring heterology between the antigen hapten and enzyme-linked hapten. The limit of detection was 10 pg of testosterone with a sensitivity range between 15 and 1,000 pg. A low level of cross-reactivity with 5 alpha-dihydrotestosterone (6.17%) and 11 beta-hydroxytestosterone (1.03%) was noted. No interference was noted with other common androgens, estradiol, or progesterone. The sensitivity and selectivity observed in the assay may be attributable to the selection of penicillinase as the enzyme marker and the elements of conformational heterology between the antigen-linked and enzyme-conjugated steroid haptens.


Synthesis of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid from 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid: configuration in the bile of Alligator mississippiensis.

Synthesis of 25R- and 25S-diastereoisomers of 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid from 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid is described. The 25S-diastereoisomer of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan- 26-oic acid was obtained by vigorous hydrolysis of the bile of Alligator mississippiensis followed by repeated crystallization of the hydrolysate, and the 25R-diastereoisomer was isolated by hydrolysis of the bile salts in bile of A mississippiensis with rat feces. Acetylation of the 25R- or 25S-diastereoisomer of methyl 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid under controlled conditions yielded the corresponding 3 alpha,7 alpha-diacetate in approximately 70% yield. The diacetate was quantitatively oxidized to methyl 3 alpha,7 alpha-diacetoxy-12-oxo-5 beta-cholestan-26-oate, which was converted into the 12-tosylhydrazone in approximately 58% yield. Reduction of the tosylhydrazone with sodium borohydride in acetic acid yielded the 25R- or the 25S-diastereoisomer of 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid as the major product. Purification via column chromatography yielded the pure diastereoisomers in approximately 25% overall yield. The two diastereoisomers were resolved on thin-layer chromatography and high-performance liquid chromatography. When the bile of A mississippiensis was hydrolyzed with rat fecal bacteria, the 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid isolated via chromatographic purification was shown to be the 25R-diastereoisomer.


Isolation of immunogenic and lethal peptides of alpha-toxin from Clostridium novyi type B.

The lethal alpha-toxin was isolated from the culture filtrate of Clostridium novyi type B using ammonium sulfate precipitation and ion exchange chromatography. The alpha-toxin has a mol. wt of 190,000 and does not contain any disulfide cross-linkages. It consists of a single polypeptide chain. The peptide fragments resulting from the cyanogen-bromide cleavage were isolated using reversed phase and gel filtration HPLC. The immunogenic actions of these peptides and peptide mixtures were studied in Balb/c mice. Three polyclonal antisera recognizing the uncleaved native toxin could be found using an ELISA test (Br3, Bro2, Bro5). One peptide mixture (Tx5), which was proved lethal in shell-less quail eggs (in vitro), was rechromatographed with gel filtration HPLC that resulted in one peptide with mol. wt 3000 (Txleth), which again proved lethal in the shell-less quail egg lethality test. The immunogenic peptides differ from the lethal one, therefore we assumed different locations on the polypeptide chain. The separation of the immunogenic, non-toxic fragment from the lethal one may allow the production of a highly specific non-toxic vaccine. By using synthetically produced immunogenic peptides, time-consuming purification methods and working with the whole toxin will become unnecessary.


The study of properties of immunoreactive estradiol secreted by adrenals and ovaries of immature female rats.

Some properties of immunoreactive estradiol secreted by adrenals and ovaries of immature female rats were studied. It was shown that adrenals and ovaries of 15-day-old animals secrete approximately equal amounts of immunoreactive estradiol, with thin-layer chromatogram of immunoreactive material being identical for both glands. Adrenal- as well as ovarian-derived estradiol can bind to uterine cytosol estradiol receptors with the formation of complexes, which can be activated in the cell-free system. The removal of either adrenals or ovaries causes a decrease of cytosol estradiol receptor levels in the uterus 6 hours after the operation. From these results and previously reported data, nothing suggests that immunoreactive estradiol secreted by the adrenals of immature rats should be regarded as "spurious" hormone.


Comparison of paralytic toxins in aquaculture of purple clam in Taiwan.

Food poisoning incidents due to ingesting the cultured purple clam Soletellina diphos occurred in western Taiwan in February 1991. Clam specimens, sediment and the dinoflagellate Alexandrium tamarensis were collected and assayed for lethality as paralytic shellfish poison (PSP). The lethality of purple clam, wet sediment and phytoplankton was 1700 mouse units per specimen (MU/specimen), 0.05 MU/g and 3.6 x 10(-5) MU/cell, respectively. The toxins obtained from clam, sediment and phytoplankton consisted of gonyautoxins 1-4, along with trace amounts of neosaxitoxin.


Enzyme immunoassay for serum dexamethasone using 4-(carboxymethylthio)dexamethasone as a new hapten.

A sensitive and simple enzyme immunoassay for direct quantitation of serum dexamethasone was established. An antiserum with high specificity was produced by the immunization of rabbits with a newly synthesized 4-(carboxymethylthio)dexamethasone-bovine serum albumin conjugate. Alkaline phosphatase was used as a labeling enzyme. The minimum amount of dexamethasone detected was 2 pg per tube on the basis of B/Bo 100 - 2 SD (%) of standard curve. However, taking into account the cross-reaction with steroids such as cortisol in dexamethasone-free serum, the measurable range was from approximately 0.13 to 10 micrograms/dl. Intra- and interassay coefficients of variation were 1.5 - 5.4% and 0.6 - 6.5%, respectively. Serum levels of dexamethasone and cortisol in four normal subjects after an oral administration of 1 mg of dexamethasone are also reported.


Diarrhoeic shellfish toxins in Adriatic Sea mussels evaluated by an ELISA method.

A competitive enzyme immunoassay (ELISA) was used to determine the presence of okadaic acid (OA) and/or dinophysistoxin-1 (DTX-1) in mussels. The accuracy and sensitivity of the ELISA method has been checked. The sensitivity of the method (100 ng/g of hepatopancreas) makes it possible to determine OA concentrations ten times lower than the tolerance limits established by the Health Authorities of many countries. For the first time, OA and/or DTX-1 were detected in mussels (Mytilus galloprovincialis) collected in different stations along the Adriatic Sea coasts.


Tyrosinase-like polypeptides in the uterus and in the central nervous system of rats.

Nuclear and cytosolic fractions of rat uteri and tissues from the central nervous system contain proteins that are recognized by a polyclonal tyrosinase antibody. This antibody eliminates the cresolase activity of uterine nuclear extract when estradiol is used as substrate. Thus, it appears that tyrosinase-like proteins might be present in tissues not generally considered to chain such an enzyme.


Synthesis of sulfonate analogs of bile acids.

Sulfonate analogs of C23 and C24 bile acids were synthesized from norcholic, norchenodeoxycholic, norursodeoxycholic, nordeoxycholic, norhyodeoxycholic, cholic, deoxycholic, hyodeoxycholic, and lithocholic acids. The principal reactions used were (1) reduction of the bile acids with NaBH4 to the corresponding bile alcohols, (2) selective tosylation of the terminal hydroxyl group, (3) iodination of the tosyl esters with NaI, and (4) treatment of the iodides with Na2SO3 to form the sulfonate analogs of the bile acids. The sulfonate analogs showed polarity similar to that of taurine-conjugated bile acids on thin-layer chromatography. The carbon 13 nuclear magnetic resonance spectral data for the sulfonate analogs were tabulated.


Synthesis and evaluation for uterotrophic and antiimplantation activities of 2-substituted estradiol derivatives.

Two novel series of 2-substituted estradiol derivatives have been synthesized and evaluated for uterotrophic and antiimplantation activities. Among the compounds tested in the rat, 2-acetylestradiol 17 beta-acetate (1), 2-(3'-dimethylamino-1'-propionyl)estradiol 3,17 beta-diacetate (7), 2-(3'-diethylamino-1'-propionyl)estradiol 3,17 beta-diacetate (8), 2-(3'-piperidino-1'-propionyl)estradiol 3,17 beta-diacetate (9), 1'-(2-estradiol 3,17 beta-diacetate-3'-diethylaminopropionyl thiosemicarbazone (12), and 1'-(2-estradiol 3,17 beta-diacetate)-3'-morpholinopropionyl thiosemicarbazone (14) displayed estrogenic activity. At dosages of 4 microliters/rat/day, none of the tested compounds elicited antiimplantation activity. All compounds shared a similar characteristic: nuclear substitution at the C-2 position of the steroid nucleus, a property previously thought to be markedly inhibitory for estrogenic activity.


Synthesis of 6 alpha-tritiated beta-muricholic acid for use as a molecular probe.

Regioselective oxidation of methyl beta-muricholate to give the 6-ketoderivative is described. Stereoselective reduction of this ketone with tritiated NaBH4 furnishes labeled methyl beta-muricholate. The structure of all compounds was confirmed by infrared, 1H, and 13C nuclear magnetic resonance spectroscopy. Data obtained by circular dichroism and mass spectroscopy were in agreement with the structure of the ketone 3.


Early English steroid history.

Following the revelation of the presence of vitamin D in fish liver oils and of estrogenic hormones in pregnancy urine in the 1920s, active interest in the steroids began in England. Most of this interest originated from the studies of Ian Heilbron at Liverpool and of Otto Rosenheim at the National Institute for Medical Research in London.


Steroid hormones and the Luftwaffe. A venture into fundamental strategic research and some of its consequences: the Birch reduction becomes a birth reduction.

I recount my involvement in the development of biologically active, totally synthetic steroid hormones since 1941. The reasons for my approach to the first total synthesis of a potent androgen are given. I touch on the even more important general synthetic outcomes from ideas generated by the search for useful amounts of a cortically active hormone, such as novel angular methylations, partial hydrogenations of aromatic systems (Birch reductions), and novel usages in synthesis based on the unique structures so generated. The Birch process was critical for synthesis of the first oral contraceptives. A need to explain the structures of Birch reduction products and the experimental requirements resulted in further elucidations of the mechanism of reduction processes, notably for aromatic compounds. The first theoretical and practical distinction between structures of products as determined by a reaction rate or an equilibrium position was first exploited experimentally in the deconjugation of cholest-4-en-3-one in the final step of the first facile total synthesis of cholesterol. A knowledge gained of the biosynthesis of steroids and its specific enzymes helped to initiate my general polyketide theory of biosynthesis and also my idea of outdoing enzyme achievements with organometallic complexes ("inorganic enzyme chemistry"). I assert the high historic importance of steroids in promoting the advance of general organic chemistry within many fields.


Sterol molecule: structure, biosynthesis, and function.

This review briefly summarizes key researches on the structure of the sterol molecule from its very beginnings to the definitive elucidation in 1932. Cholesterol biosynthesis treated in somewhat greater detail covers the period from the 1930s to the 1960s. As a historic contribution, it presents researches previously published in numerous books, reviews, and original papers. The selection of topics, dictated by limits of time and space, is necessarily arbitrary and a personal choice. Readers of this journal will be familiar with the relevant chemical structures. Structural formulas are therefore omitted.


Historic reflection on steroids: Lederle and personal aspects.

Steroid research at Lederle was initiated in 1946 and continued until about 1971. The areas of research interest involved primarily a study of delta 5,7-steroids, provitamin D3 variants, dehydroepiandrosterone variants, steroidal ketals, corticoids, and steroid conjugates. One of the principal goals was to develop novel antiinflammatory steroids for the treatment of, e.g., rheumatoid arthritis. A rational design led to the development of 16 alpha-hydroxycorticoids, culminating in the synthesis and therapeutic use of triamcinolone and related compounds.


Silent stroke in patients with transient ischemic attack or minor ischemic stroke. The Dutch TIA Trial Study Group.

We studied silent stroke (i.e., infarcts on computed tomographic scan not related to later symptoms) in patients after transient ischemic attack or minor ischemic stroke. Ours is a cross-sectional study of 2,329 patients who were randomized in a secondary prevention trial after transient ischemic attack or minor ischemic stroke and had no residual deficit after the qualifying event. Silent stroke was observed in 13% of the 2,329 patients. Lacunes formed 79%, cortical lesions 14%, and border zone lesions 7% of all silent strokes. Silent lacunes were most often located in the basal ganglia and symptomatic lacunes most often in the corona radiata. Age, hypertension, and current cigarette smoking were related to the presence of silent stroke. Silent stroke was equally common in different types of transient ischemic attack, including transient monocular blindness. Residual symptoms of any kind were more common in patients with silent stroke than in those without. Because only the sites of silent stroke infarcts differed slightly from those of symptomatic infarcts and the frequency of vascular risk factors was similar to that of symptomatic infarcts, silent stroke may have the same bearing on future risk as known prior stroke.


Dementia associated with lacunar infarction.

The purpose of this study was to assess the number of patients with lacunar lesions who develop dementia and to evaluate in patients with and without dementia the relevance of risk factors for cerebrovascular disease, the occurrence of leukoaraiosis, the volume and location of vascular lesions, the size of ventricular and subarachnoid spaces, and stroke recurrence. One hundred eight patients in whom computed tomograms revealed lacunar lesions that could account for their clinical neurological pictures were followed up for an average of 4 years after their first lacunar stroke. Twenty-five patients (23.1%) developed dementia. The prognosis regarding occurrence of dementia during the follow-up period, evaluated by the Kaplan-Meier method, was significantly worse in subjects with the greatest evidence of cerebral atrophy (p less than 0.009) and in subjects who underwent new focal cerebrovascular episodes (p less than 0.000001). No differences were seen in the frequency of vascular risk factors or the site or volume of lesions between the demented and nondemented groups. Patients with lacunar infarcts suffer from dementia 4-12 times more frequently than the normal population. Cerebral atrophy and recurrent stroke, as well as other as-yet unclarified factors, are involved in producing dementia.


Some epidemiological aspects of stroke: mortality/morbidity trends, age, sex, race, socioeconomic status.

The reasons for decreasing stroke mortality could be related to either decreasing incidence and/or improved prognosis. Thus far, secular trends of stroke have been analyzed either through mortality or morbidity data. This report examines both aspects simultaneously, on a nationwide basis, for the period 1968-1988. Mortality statistics were based on the Compressed Mortality File. Estimates of morbidity were based on the National Hospital Discharge Survey. The Area Resource File was used to obtain county-specific socioeconomic statistics. The decline in stroke mortality continued through the 1970s and 1980s, whereas morbidity remained constant and possibly even increased. Mortality and morbidity rates were similar in both sexes, higher in blacks, and lower in other (primarily Asian) Americans. There was an inverse correlation between death rates and socioeconomic status, which was particularly marked in blacks. Temporal decline occurred in all strata. The observed decrease in stroke mortality rates results most probably from an improved survival rather than from a decline in incidence. The abundance of new drugs and screening programs may not have affected the overall morbidity of stroke, possibly because of inefficient treatment regimens.


Incidence of stroke in Taiwan.

Few epidemiological studies concerning the incidence of stroke have been conducted in Taiwan. In an attempt to investigate this common disease, we studied the incidence of stroke in Taiwan in a prospective incidence study. A cohort of 8,562 stroke-free people were followed up for 4 years to observe new stroke occurrence. The methods of sampling the study population have been reported elsewhere. In addition to the help of local doctors, who reported the new stroke cases, we also sent public nurses to visit the study population annually to screen the new cases of stroke. There were 104 (61 men and 43 women) first-ever stroke cases identified by a neurologist in a period between October 1, 1986 and December 31, 1990. The average annual incidence rate of first-ever stroke for people aged 36 years or older in this study was 330 per 100,000. Incidence rate was higher in eastern Taiwan and in rural communities. Percentages of the major types of stroke were as follows: cerebral infarction, 71%; cerebral hemorrhage, 22%; subarachnoid hemorrhage, 1%; and unclassified, 6%. The significant risk factors for stroke were hypertension and intake of food with a high sodium content. The age-specific incidence rates in this study are higher than those reported from the United Kingdom and the United States. The rates are close to those in a report from Japan and a report from a city in mainland China at the same latitude. Cerebral hemorrhages are more common among people in Taiwan than among Occidental people.


Cigarette smoking, alcohol use, and subarachnoid hemorrhage.

Subarachnoid hemorrhage remains a devastating disease. Identification of etiologic risk factors would allow the possibility of prevention. We conducted a population-based case-control study in King County, Washington. Patients whose bleeds originated from a source other than an aneurysm were excluded. Two age- and gender-matched control subjects were identified for each case through random digit telephone dialing. A standardized in-person interview was conducted with the patient whenever possible, a proxy respondent for the case in all instances, the two control subjects, and their proxies. Analyses involved conditional logistic regression taking into account matching on age, gender and respondent type. Over 2 years, 169 cases were identified, and 149 participated in the case-control study. Compared with those who never smoked, the odds ratio for current heavy smokers (greater than 20 cigarettes/day) was 11.1 (95% confidence interval [CI], 5.0-24.9); for current light smokers (less than or equal to 20 cigarettes/day), 4.1 (95% CI, 2.3-7.3); and for former smokers, 1.8 (95% CI, 1.0-3.2). The risk associated with smoking was greatest in the 3 hours after a cigarette (odds ratio [OR] = 7.0; 95% CI, 3.7-13.1) and then fell, not reaching the risk in those who had never smoked until more than 10 years had passed since the last cigarette. Heavy alcohol use (greater than 2 drinks/day) was also associated with bleeds (OR = 2.2; 95% CI, 0.9-5.1, after adjusting for smoking status). These associations were not substantially altered after adjusting for several possible confounding factors, including a history of hypertension. Cigarette smoking and heavy alcohol use are associated with the occurrence of subarachnoid hemorrhage.


Relationship of cardiac disease to stroke occurrence, recurrence, and mortality.

This is a study of cardiac diseases associated with the 1,382 cases of first cerebral infarction in residents of Rochester, Minn., during 1960-1984. This is a population-based study in Rochester, Minn., for which the medical record-linkage system was used to identify cardiac disease among patients with first cerebral infarction. There were 318 patients (23%) who had at least one major potential cardiac source of emboli. The proportion of patients with a cardiac source of emboli was significantly higher among patients older than age 75 years (29%) than among younger patients (17%). The 30-day stroke recurrence rate among patients with a cardiac source of emboli (2%) was not significantly different than that among those without a cardiac source of emboli (2%). Among patients with a cardiac source of emboli, there was no difference in the probability of stroke at 30 days and at 90 days for those treated or not treated with anticoagulants. During the first 30 days after cerebral infarction, the risk of death in patients with a cardiac source of emboli (23%) was 14 times that of recurrent stroke. The risk of death at 30 days in patients without a cardiac source of emboli was 8%. Significant independent predictors of death (Cox proportional-hazards analysis) were age, prior myocardial infarction, atrial fibrillation present at onset of stroke, congestive heart failure before the stroke, and an age x congestive heart failure interaction. The only significant independent predictors of recurrent stroke were cardiac valve disease and congestive heart failure.


Low and high density lipoprotein metabolism in atherothrombotic brain infarction.

Elevated low density lipoprotein and reduced high density lipoprotein cholesterol may increase the risk of atherothrombotic brain infarction, but the metabolic mechanisms accounting for this relation are poorly understood. The kinetic parameters of low density and high density lipoprotein were studied in nine subjects with atherothrombotic brain infarction or identifiable (by noninvasive testing) extracranial occlusive disease and in 12 control subjects. Autologous iodine-125-labeled lipoproteins were injected intravenously. Blood samples were drawn 10 minutes after injection and periodically thereafter for 10 days. Kinetic parameters were calculated from the decay curves. The stroke-risk group showed significantly higher triglyceride (p less than 0.05), total cholesterol (p less than 0.02), and low density lipoprotein cholesterol (p less than 0.01). The fractional catabolic rate of low density lipoprotein was significantly lower (p less than 0.001) and the high density lipoprotein rate higher (p less than 0.02) in the stroke-risk group than in the control group. Regression analysis (using all subjects) of serum lipoproteins and their respective fractional catabolic rates correlated significantly (for low density lipoprotein, r = 0.684, p less than 0.001; for high density lipoprotein, r = 0.595, p less than 0.002). Mean percent stenosis showed a significant relation with triglyceride level (r = 0.678, p less than 0.01) and low density lipoprotein cholesterol (r = 0.535, p less than 0.02) but not with high density lipoprotein cholesterol. Mean percent stenosis also showed correlation with both fractional catabolic rate of low density lipoprotein (r = 0.667, p less than 0.002) and with serum high density lipoprotein levels (r = 0.504, p less than 0.02). Our study provides insights into the role of altered low and high density lipoprotein metabolism in the pathogenesis of carotid stenosis. The statistically significant association of serum lipoprotein metabolic rates with carotid stenosis, rather than their respective serum concentrations, implies that metabolic parameters may be more important in predicting stroke risk.


Hyperhidrosis as a reflection of autonomic failure in patients with acute hemispheral brain infarction. An evaporimetric study.

Sweating dysfunction is one of the most frequently encountered symptoms of autonomic failure but has received scant attention in patients with cerebrovascular diseases. Our purpose was to evaluate the prevalence, pathogenesis, and clinical correlates of sweating dysfunction in stroke. We studied sweating at baseline and after a heating stimulus in 53 patients with acute hemispheral brain infarction and in 40 healthy control subjects by using a quantitative evaporimetric method. Significant hyperhidrosis on the paretic side of the body was verified in 55% of the patients at baseline, in 74% after 5 minutes of heating, and in 77% after 10 minutes of heating. Hyperhidrosis was established throughout the body and correlated with the severity of paresis, the presence of reduced muscle tone, and the extensor plantar response. The phenomenon of hyperhidrosis in hemiparetic patients reflecting autonomic dysfunction seems to be a common manifestation that should be listed among the expected consequences of brain infarction. This sweating disturbance might be attributed to a lesion of a putative sympathoinhibitory pathway controlling sweating. The failure of this pathway could also be related to other manifestations of sympathetic hyperfunction, e.g., cardiac complications. Therefore, assessment of sweating may provide a new, important aspect in the evaluation of stroke patients.


Low blood-to-cerebrospinal fluid passage of sorbitol after intravenous infusion.

Compared with mannitol, the osmotherapeutic agent sorbitol is less prone to accumulate in the blood and the same quantity may be infused in a smaller volume. Because of these advantageous characteristics, we studied the pharmacokinetics of sorbitol in serum and cerebrospinal fluid. Six patients (five women and one man; age range, 46-70 years) with an external ventriculostomy and suffering from brain edema due to cerebrovascular disease received sorbitol as part of their therapy. Before and after the first dose of 50 g infused over 20 minutes, sorbitol concentrations in serum and cerebrospinal fluid were determined repeatedly using an enzymatic procedure. Maximal sorbitol concentrations ranged from 2,705 to 5,821 (median, 3,227) mg/l in serum compared with 6.7-130.7 (median, 19.5) mg/l in cerebrospinal fluid. Cerebrospinal fluid maxima were observed 0.17-3 hours after the end of the infusion. Sorbitol elimination in serum was adequately described by a two-compartment pharmacokinetic model (distribution half-life, 0.05-0.14 hour; elimination half-life, 0.23-0.61 hour). Elimination in cerebrospinal fluid followed a single-exponential decay and was considerably slower than that in serum (half-life, 1.3-7.7 hours). The maximal cerebrospinal fluid concentration/maximal serum concentration ratio was low for sorbitol, thus suggesting a small potential risk of inducing an increase of intracranial pressure after osmotherapy (rebound effect).


Evaluation of the vertebrobasilar-posterior system by transcranial color duplex sonography in adults.

The transcranial color duplex sonography technique was applied to the vertebrobasilar-posterior system to provide normal data for clinical application. The intracranial posterior circulation was studied in 49 healthy volunteers (mean +/- SD age, 35 +/- 12 years) by a transcranial and suboccipital approach with a 2.0-MHz sector transducer of a computed sonography system. The posterior cerebral artery and the vertebrobasilar system were depicted clearly in the color Doppler mode. Pulsed Doppler signals could be recorded in the posterior cerebral (100%), basilar (92%), and vertebral arteries (89%). The following normal values were provided for all vessels: systolic peak, end-diastolic maximum, time-averaged, and time-averaged maximum velocities; resistance and pulsatility indexes; and a spectral broadening index. Mean +/- SD values were 45.9 +/- 9.6, 45.5 +/- 10.8, and 39.2 +/- 10.6 cm/sec for time-averaged maximum velocity, and 28.3 +/- 6.5, 30.6 +/- 7.2, and 24.7 +/- 8.4 cm/sec for time-averaged velocity in the posterior cerebral, basilar, and vertebral arteries, respectively. In a reproducibility study, duplex measurements of the posterior cerebral arteries were repeated in 27 subjects. The correlation between the two examiners was high (r = 0.56, p less than or equal to 0.0001 for time-averaged maximum velocity). Color duplex sonography of the vertebrobasilar-posterior system is a new, noninvasive, bedside investigative technique. It permits visualization of artery flow in real time, relating these to adjacent brain and cranial structures, as well as angle-corrected duplex measurement of "true" flow velocities at defined sites of the vessels. Thus, it will open new diagnostic possibilities in disorders of the posterior circulation.


A phenothiazine derivative reduces rat brain damage after global or focal ischemia.

We previously reported that 2-(10H-phenothiazin-2-yloxy)-N,N-dimethylethanamine hydrochloride is a potent inhibitor of iron-dependent lipid peroxidation in vitro and can protect primary cultures of rat hippocampal neurons from hydrogen peroxide-induced toxicity. Because oxidants may play an important role in mediating postischemic tissue injury, we evaluated this agent in two rat models of transient cerebral ischemia. In a model of global forebrain ischemia, 23 male Wistar rats were subjected to 10 minutes of four-vessel occlusion followed by 72 hours of reperfusion. The rats received three intraperitoneal injections of either vehicle (2% aqueous acacia) or test agent (40 mg/kg). In a model of focal stroke, 19 spontaneously hypertensive rats were subjected to 2 hours of tandem middle cerebral and ipsilateral common carotid artery occlusion followed by 24 hours of reperfusion. The rats received three intraperitoneal injections of either vehicle (2% aqueous acacia) or test agent (40 mg/kg). In the global model, the phenothiazine significantly protected the CA1 layer of the hippocampus, with a reduction in mean damage score from 2.1 +/- 0.3 for control rats to 1.0 +/- 0.4 for treated rats (p less than 0.05). In the transient focal stroke model, the compound reduced cortical infarct volume from 130.1 +/- 10.3 mm3 for control rats to 95.2 +/- 24.5 mm3 for treated rats (p less than 0.02). Although the primary mechanism responsible for the protective effect is unclear at the present time, our study is consistent with the hypothesis that oxidant-mediated lipid peroxidation may be involved in the pathophysiology of postischemic brain injury.


Distribution of the 72-kd heat-shock protein as a function of transient focal cerebral ischemia in rats.

The significance and physiological implications of the expression of the 72-kd heat-shock protein in ischemic tissue are unknown. To enhance our understanding of the relation between ischemic cell damage and 72-kd heat-shock protein expression, we evaluated the cellular expression and the anatomic distribution of 72-kd heat-shock protein in conjunction with the morphological analysis of rat brain, as a function of the duration of a single arterial occlusion. Adult Wistar rats were subjected to graded transient middle cerebral artery occlusion (for a duration of 10, 20, 30, 60, 90, and 120 minutes and sham; n = 4 per group). Forty-eight hours after reopening the artery, brain tissue sections were analyzed to determine the extent of neuronal damage (hematoxylin and eosin staining), the extent of astrocytic reactivity (immunohistochemistry, using anti-glial fibrillary acidic protein), and the distribution of 72-kd heat-shock protein (immunohistochemistry, using a monoclonal antibody to 72-kd heat-shock protein). We found that 72-kd heat-shock protein was sequentially expressed in morphologically intact neurons, microglia, and endothelial cells with increasing duration of ischemia; 72-kd heat-shock protein immunoreactivity was not detected in astrocytes. The duration of ischemia required to evoke a 72-kd heat-shock protein response in neurons was dependent on the anatomic site and followed a pattern of increasing neuronal sensitivity to ischemic cell damage with duration of ischemia: 72-kd heat-shock protein and neuronal damage were sequentially detected in the caudate putamen, globus pallidus, cerebral cortex, amygdala, and hippocampus with increasing duration of ischemia. With ischemia of long duration (greater than or equal to 90 minutes), neurons expressing 72-kd heat-shock protein were localized to a zone peripheral to the severely damaged ischemic core. These studies suggest that 1) the expression of 72-kd heat-shock protein in neurons precedes the development of ischemic cellular alterations detectable by conventional hematoxylin and eosin light microscopy methods; 2) there is a hierarchy of cell types and anatomic sites that express 72-kd heat-shock protein, and this hierarchy reflects cellular and anatomic vulnerability to ischemic cell damage; and 3) 72-kd heat-shock protein induction in neurons bordering a necrotic ischemic core may be the morphological equivalent of the ischemic penumbra.


Magnetoencephalography of focal cerebral ischemia in rats.

The purpose of this study was to use magnetoencephalography to record magnetic field changes in the brain during middle cerebral artery occlusion. A direct-current electrocorticogram (two channels) and a direct-current magnetoencephalogram (seven channels) were simultaneously recorded from five rats subjected to middle cerebral artery occlusion for 1-2 hours. Direct-current electrocorticographic and direct-current magnetoencephalographic signal deflections were observed after the onset of middle cerebral artery occlusion and occurred repeatedly throughout the ischemic period, with a mean +/- SD time interval of 12 +/- 5 minutes. A one-to-one correspondence of the electrocorticographic and magnetoencephalographic signal deflections was demonstrated. Direct-current magnetoencephalography can provide a new noninvasive technique for studying depolarization and/or spreading depression in focal cerebral ischemia.


Reversible focal ischemic injury demonstrated by diffusion-weighted magnetic resonance imaging in rats.

Diffusion-weighted magnetic resonance imaging (DWI) can quantitatively display focal brain abnormalities within minutes after the onset of ischemia. We performed the present study to determine the effects of 1 and 2 hours of temporary ischemia on DWI. We examined DWI and T2-weighted magnetic resonance images (T2WI) during and after 1 and 2 hours of temporary middle cerebral artery occlusion in rats (n = 10 for each group). In a subgroup of four animals from each group, we employed perfusion magnetic resonance imaging to monitor cerebral perfusion. Neurological outcome and infarct size after survival for 24 hours were compared between the groups and correlated with DWI and T2WI studies. Perfusion studies qualitatively documented hypoperfusion and reperfusion during and after temporary occlusion. Lesion size on DWI during reperfusion was significantly less than that during ischemia for 1 (55% decline, p less than 0.02) but not 2 hours of occlusion. The DWI signal intensity ratio (intensity compared with that in the contralateral homologous area) just before withdrawal of the occluder was significantly less in regions where the hyperintensity disappeared after withdrawal than in regions with persistent hyperintensity (p less than 0.002). The T2WI studies revealed few or no abnormalities, except after 2 hours of occlusion. The neurological outcome was significantly better in the 1-hour than in the 2-hour group (p less than 0.05). Postmortem infarct volume was significantly smaller in the 1-hour group than in the 2-hour group (p less than 0.05). The postwithdrawal DWI accurately predicted infarct size (R = 0.96, p less than 0.0001). The present study indicates that DWI can rapidly display not only irreversible but also reversible ischemic brain damage and enhances the importance of DWI as a diagnostic modality for stroke.


Prevention of postischemic canine neurological injury through potentiation of brain energy metabolism by acetyl-L-carnitine.

Mechanisms of ischemia/reperfusion brain injury include altered patterns of energy metabolism that may be amenable to pharmacological manipulation. The purpose of this study was to test the effectiveness of postischemic acetyl-L-carnitine administration on potentiation of metabolic recovery and prevention of neurological morbidity in a clinically relevant model of complete, global cerebral ischemia and reperfusion. Neurological deficit scoring as well as spectrophotometric and fluorescent assays of frontal cortex lactate and pyruvate levels were used in a canine model employing 10 minutes of cardiac arrest followed by restoration of spontaneous circulation for 2 or 24 hours. Dogs treated with acetyl-L-carnitine exhibited significantly lower neurological deficit scores (p = 0.0037) and more normal cerebral cortex lactate/pyruvate ratios than did vehicle-treated control animals. Postischemic administration of acetyl-L-carnitine potentiates normalization of brain energy metabolites and substantially improves neurological outcome in a clinically relevant model of global cerebral ischemia and reperfusion.


Neuroprotective mechanism of (+)SKF 10,047 in vitro and in gerbil global brain ischemia.

The N-methyl-D-aspartate receptor is believed to mediate part of the ischemic neuronal damage caused by the excitatory amino acid glutamate. (+)SKF 10,047, the prototypic sigma-agonist, interacts with the N-methyl-D-aspartate receptor. Therefore, we studied the neuroprotective effect of (+)SKF 10,047 on cultured rat cerebellar neurons and on CA1 hippocampal neurons of gerbils exposed to brain ischemia. Mechanisms of neuroprotection were studied in vitro by measuring calcium influx into cultured rat cerebellar granule cells loaded with fura 2-AM. In vivo neuroprotection of gerbil CA1 hippocampal neurons was studied in a posttreatment regimen following 5 minutes of bilateral carotid artery occlusion and 7 days of reperfusion. In primary cultured rat cerebellar granule cell neurons, (+)SKF 10,047 in a dose-dependent manner diminished intracellular calcium levels of N-methyl-D-aspartate-stimulated neurons by a maximum of 87% (n = 8), with a 50% inhibitory concentration of 0.8 microM. (+)SKF 10,047 did not prevent subsequent calcium influx stimulated by kainic acid or KCl, nor did it interfere with modulation of the kainate response by quisqualic acid. Neuroprotection of 64% (p = 0.006, n = 15) of gerbil CA1 hippocampal neurons was achieved by posttreatment injection followed by minipump infusion. Neuroprotection by (+)SKF 10,047 most likely involves interaction at the N-methyl-D-aspartate receptor. These results suggest that the benzomorphan class of sigma-agonists may provide neuroprotection in cerebral ischemia and stroke.


Increases in cerebral blood flow in rat hippocampus after medial septal injection of naloxone.

In a previous study, we occasionally found that the rat given naloxone in the preoptic region develops behavioral seizures. In view of knowledge that the forebrain including the medial septal nucleus provides cholinergic projections to the hippocampal formation, the present study examined the effects of naloxone injected into the medial septal nucleus on the local blood flow in the hippocampus. A polyurethane-coated platinum electrode with a 1-mm bare tip for measurement of blood flow and a guide cannula made of stainless steel tube for naloxone injection were implanted chronically into the brain. The cerebral blood flow was measured by the hydrogen clearance method in freely moving rats. The injection of 50 micrograms naloxone caused a significant increase in hippocampal blood flow, with its peak at 20 minutes. Twenty micrograms naloxone caused a similar increase, but 10 micrograms caused only a slight increase that peaked at 30 minutes, suggesting a dose-response of naloxone effect. Hippocampal blood flow was not changed after the injection of saline into the medial septal nucleus and after the injection of naloxone into the caudate nucleus. Taken together with previous findings, the results suggest that endogenous opioids exert a decreasing effect on the local blood flow in the hippocampus, probably mediated by the magnocellular cholinergic neurons projecting to the hippocampus.


Laser doppler flowmetry in CA1 sector of hippocampus and cortex after transient forebrain ischemia in gerbils.

Local differences in the hemodynamic response to transient ischemia could be involved in the development of selective vulnerability. These differences were studied in vulnerable and nonvulnerable regions of the brain. Five gerbils were subjected to 10 minutes of bilateral forebrain ischemia, and cerebral blood flow was measured continuously in the frontal cortex and CA1 sector of the hippocampus using laser Doppler flowmetry. Carotid artery pressure was recorded simultaneously with a pressure transducer. After induction of ischemia, blood flow in the cortex and CA1 sector decreased to 11.8% and 18.0% of the baseline value, respectively. After release of the vascular occlusion, blood flow in the cortex returned to the preischemic level at 7.5 minutes (recovery time), reached the hyperemic peak (123.8%) at 12.4 minutes (peak latency), and again decreased to the preischemic level at 27.2 minutes. In the CA1 sector, blood flow returned to the preischemic level at 2.1 minutes, reached the hyperemic peak (122.2%) at 5.7 minutes, and decreased again to the preischemic level at 21.3 minutes. In both the cortex and CA1 sector, recovery time and peak latency correlated inversely with the amount of residual blood flow during ischemia. Histologically, cortical neurons were not injured but only 14.6% of CA1 neurons survived 1 week after ischemia. CA1 neurons were selectively injured despite the milder percentage decrease of blood flow during ischemia and the more prompt recovery of flow after ischemia. These findings stress the importance of intrinsic rather than hemodynamic factors in the pathogenesis of selective vulnerability of CA1 neurons after transient bilateral forebrain ischemia.


Level of function predicts first stroke in the elderly.

Our aim in this study was to assess physical function as a predictor of stroke incidence in a probability sample of noninstitutionalized elderly subjects with no previous history of stroke. The data were obtained from a prospective longitudinal study of 2,812 individuals aged 65 years of age and older living in New Haven, Connecticut. Incidence of stroke was monitored from the baseline interview in 1982 until December 1988 (n = 167). Physical function was measured by the Katz scale of activities of daily living and a three-item scale measuring gross mobility function (Rosow scale). Both measures of impairment of function were independently associated with stroke incidence controlling for age, sex, diabetes, hypertension, and angina (p less than 0.001). Our findings suggest that in elderly persons, physical disability is a newly identified risk factor for stroke.


The preponderance of posterior circulatory events is independent of the route of cardiac catheterization.

Central nervous system complications of cardiac catheterization are most often attributed to embolic events that occur at the time of catheter manipulation. Nevertheless, the reason that over 50% of these events are localized to the posterior circulation remains unexplained. One potential explanation offered for this preponderance is the use of the brachial artery approach. In this report, we examined the relation between the route of catheterization and central nervous system complications. We retrospectively analyzed all central nervous system complications that occurred after cardiac catheterization through a femoral route at our institution over a 3 1/2-year period. Thirteen patients were identified as having central nervous system complications. Using defined criteria, posterior circulatory events still accounted for at least 54% of central nervous system complications. The preponderance of posterior circulatory events is apparently independent of the route of catheterization. Furthermore, given the array of neurological symptoms and their often complete resolution, we feel it is unlikely that embolism is the sole pathophysiological mechanism involved in these events.


Caudate hemorrhage with moyamoya-like vasculopathy from atherosclerotic disease.

Caudate hemorrhage usually results from hypertension, rupture of arteriovenous malformation or aneurysm, or rarely, moyamoya disease. Moyamoya-like changes related to severe atherosclerotic occlusive disease, usually causing ischemic stroke, have been reported. A 51-year-old normotensive patient was admitted with headache due to a left caudate hematoma with ventricular extension. There was a history of smoking, leg claudication, elevated cholesterol, and coronary artery disease. Angiography demonstrated complete extracranial carotid occlusion on the left and atherosclerotic stenosis at the bifurcation on the right, with supraophthalmic occlusion distally. At the base of the brain, bilateral moyamoya-like vessels, presumed to be secondary to atherosclerotic occlusion, were evident, but neither aneurysm nor arteriovenous malformation was present. Cerebral blood flow and transcranial Doppler studies indicated severely impaired cerebral perfusion that improved after bilateral extracranial-to-intracranial bypass surgery. Atherosclerotic occlusive carotid disease with moyamoya-like changes may be a rare cause of caudate hemorrhage. A decrease in moyamoya vessels with bypass surgery may reduce the risk of recurrent hemorrhage.


Postpartum cerebral angiopathy with cerebral infarction due to ergonovine use.

Earlier cases of stroke due to postpartum cerebral angiopathy have been reported. The mechanism of this angiopathy has not been explained. We present a case of cerebral infarction with evidence of occlusive change in the vertebrobasilar system as a complication of intravenous ergonovine use after cesarean section delivery. Ergonovine therapy is a likely cause of so-called postpartum cerebral angiopathy and stroke.


Polymorphonuclear leukocytes and monocytes/macrophages in the pathogenesis of cerebral ischemia and stroke.

The extent to which polymorphonuclear leukocytes and monocytes/macrophages contribute to the pathobiology of cerebral ischemia and stroke is an issue of long-standing contradiction and controversy. Recent developments in the ability to selectively modify leukocyte adhesion with antiadhesion antibodies and the potential clinical application of this therapeutic approach have spurred a resurgence of experimental studies examining the role of leukocytes in cerebral ischemia and stroke. We review studies examining leukocyte accumulation, initiation of thrombosis, and exacerbation of ischemic brain injury in stroke, and we examine other proposed contributions of leukocytes to cerebrovascular pathophysiology. The importance of specific characteristics of a given ischemia model and of underlying stroke risk factors in determining the degree of leukocyte involvement and effectiveness of therapies directed against these cells is discussed.


Interactions between clobazam and standard antiepileptic drugs in patients with epilepsy.

We retrospectively collected plasma level assessments performed in 96 adult patients with epilepsy on stable monotherapy, including 9 patients on clobazam (CLB), 34 on carbamazepine (CBZ), 24 on phenobarbital (PB), 9 on phenytoin (PHT), and 20 on valproate (VPA); these results were compared to those obtained in 54 adult patients on stable bitherapy with the association of CLB with either CBZ (n = 17), PB (n = 17), PHT (n = 5), or VPA (n = 15). Our results show that CLB has no significant effect on the level to dose ratio (LDR) of CBZ, PB, PHT, or VPA. Conversely, CBZ, PB, and PHT significantly decrease the LDR of CLB. CBZ and PHT significantly increase the LDR of N-desmethylclobazam (NCLB), the major metabolite of CLB. A significant increase in the NCLB/CLB ratio was found in CBZ + CLB, PB + CLB, and PHT + CLB bitherapies. These findings are of clinical significance: clobazam is useful as adjunctive treatment in human epilepsy and is often chosen as the benzodiazepine adjunctive drug in chronic resistant epilepsy. Sedative side effects may occur, especially in patients treated by a CBZ + CLB or PHT + CLB bitherapy, and both CLB and NCLB plasma levels should be monitored in such patients.


Blood cyclosporin concentrations but not doses correlate with acute changes in renal function following heart and heart-lung transplantation.

The relationship between changes in cyclosporin (CyA) dose or CyA blood concentration and the reciprocal creatinine concentration was investigated by cross-correlation analysis over the first 3 postoperative months in 32 consecutive heart and heart-lung recipients. Exploratory analysis suggested that early changes in renal function, probably attributable to recovery from preoperative cardiac failure, obscured later underlying correlations. Therefore, all data up to the first nadir in plasma creatinine following transplantation were excluded from the analysis. Five-day mean CyA doses or blood concentrations were cross-correlated with 5-day mean reciprocal creatinine concentrations measured either in the same 5-day period or with the creatinine measured up to two 5-day periods later. Although a significant correlation was found between CyA dose and blood concentration (the 95% confidence interval of the population correlation coefficient did not overlap zero), there was no relationship between dose and changes in renal function. The blood CyA concentration, however, correlated significantly with the reciprocal creatinine concentration measured in the same 5-day period and was also predictive of changes in creatinine measured in the subsequent 5-day period. Thus, a major criterion for therapeutic drug monitoring had been fulfilled: CyA dosage adjustment based on blood CyA concentrations, as the intermediate therapeutic end point, is helpful in the management of acute nephrotoxicity in heart and heart-lung transplant recipients because of the lack of a dose-effect relationship. Regular CyA monitoring and appropriate dosage adjustment is essential for the management of acute nephrotoxicity in the first 3 months following heart or heart-lung transplantation.


Pharmacokinetics of zidovudine phosphorylation in patients infected with the human immunodeficiency virus.

The relationship between zidovudine phosphorylation inside mononuclear cells and plasma zidovudine pharmacokinetic was assessed in six subjects. Plasma and intracellular concentrations were measured by radioimmunoassay over an 8-h period after administration of 100 or 200 mg of zidovudine. Plasma pharmacokinetics followed expected patterns, with considerable interpatient variability in area under the concentration-versus-time curve (AUC), and a terminal half-life of 1.5 h. Intracellular AUC was even more variable than plasma AUC, but the data suggested a crude linear relationship between these parameters. The intracellular half-life of 3.5 h was consistently longer than the plasma half-life, and varied little between patients. The prolonged intracellular half-life suggested that total phosphorylated zidovudine, as measured by the method described, is not greatly dominated by the 5'-monophosphate as predicted from the in vitro studies reported in the literature. Plasma concentrations of zidovudine have shown little correlation with clinical effect. Study of the relationship between phosphorylated zidovudine and clinical outcome could lead to a more effective management of therapy.


Predictive performance of Bayesian and nonlinear least-squares regression programs for lidocaine.

The predictive performance of two computer programs for lidocaine dosing were evaluated. Two-compartment Bayesian and nonlinear least-squares regression programs were used in two groups of patients (15 acute arrhythmia patients and 14 chronic arrhythmia patients). Lidocaine was given as a 1.5 mg/kg bolus and a 2.8 mg/min infusion for 48 h. A second bolus (0.5 mg/kg) was given 10 min after the first bolus over 2 min. Serum samples of the patients receiving lidocaine were drawn at 2, 15, 30 min and 1, 2, and 4 h and were used in forecasting the serum concentrations at 6, 8, 12, and 48 h. Predictive performance was assessed by mean error and mean-squared error. The results (mean +/- 95% confidence intervals) demonstrated the Bayesian program predicted a significant (p less than 0.05) difference at 12 h between the two arrhythmia groups (acute 0.52 [-0.95; -0.09] and chronic 0.28 [0.12; 0.44]). The results also demonstrated the Bayesian method was significantly more precise compared to the nonlinear least-squares regression program at 8, 12, and 48 h for the acute group. While caution is warranted, this study demonstrated that the predictive performance by a two-compartment Bayesian model is more accurate in predicting future lidocaine serum concentrations than that by nonlinear least-squares regression.


Specific monoclonal radioimmunoassay and fluorescence polarization immunoassay for trough concentration and area-under-the-curve monitoring of cyclosporine in renal transplantation.

In order to study the performance of a specific fluorescence polarization immunoassay (FPIA) for monitoring of the area under the concentration time curve (AUC) of cyclosporine (CsA), a total of 170 24-h CsA AUC studies were prospectively collected from 40 consecutive adult renal transplant recipients during the first 6 months after transplantation. Each AUC study included whole blood samples that had been collected at 0, 2, 4, 6, 10, 14, and 24 h after CsA administration. Each sample was subjected to CsA analysis in whole blood using a 3H-tracer specific monoclonal radioimmunoassay (wb-sRIA), specific FPIA (wb-sFPIA), and polyclonal FPIA (wb-pcFPIA). Furthermore, the performance of the specific assays was assessed against high-performance liquid chromatography (HPLC). Correlations between HPLC and specific assays were good, namely correlation coefficients of 0.94. Contrariwise, correlations between specific and nonspecific assay were poorer. As compared to HPLC, wb-sRIA overestimated CsA concentrations by 7.0%, and wb-sFPIA overestimated concentrations by 26.8%. The correlation between the AUC of CsA, as determined by wb-sRIA and wb-sFPIA, was excellent (r = 0.96). The ratio between wb-sFPIA and wb-sRIA was higher at low concentrations and small AUCs of CsA. On the average, CsA AUCs were 14% larger when assayed by wb-sFPIA. However, the appearance of the CsA concentration versus time curve was almost identical for the two assays. The apparent oral clearance (clearance/bioavailability) of CsA decreased with time after transplantation. Furthermore, the oral clearance of CsA was significantly higher in patients experiencing acute rejection than in those who were quiescent (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Performance of a fluorescence polarization immunoassay system evaluated by therapeutic monitoring of four drugs.

Fluorescence polarization immunoassays (FPIA) for amikacin, gentamicin, quinidine, and theophylline (supplied by Roche Diagnostic Systems, made using a Cobas Fara centrifugal analyzer) were evaluated and compared with widely used monitoring analysis methods. For each drug, the between-assay imprecision was ascertained by calibration on the day of assay and by a stored calibration curve made at the beginning of the study. The precision of the amikacin and theophylline assays was acceptable [total coefficient of variation (CV) less than 7.5%] at all concentrations tested for each calibration mode. Imprecision of quinidine and gentamicin assays was significant at low concentrations (1.9 mg/L): total CV = 9.0% for quinidine assessed with stored calibration curve and total CV greater than 8.5% for gentamicin measured with the two calibration modes. The calibration curves for all four assays had a good stability (greater than 30 days). Linear regression analysis demonstrated close agreement between the FPIA (y) and the following comparative techniques (x): Abbott TDx assay for amikacin and gentamicin (r = 0.988, r = 0.974, respectively); Stratus fluorometric enzyme immunoassay for quinidine (r = 0.979); and EMIT Syva assay for theophylline (r = 0.993). It is concluded that fluorescence polarization immunoassay is a rapid and reliable method for the therapeutic monitoring of the four drugs tested. Moreover, the use of reagents on an instrument that can be implemented for a wide range of chemistries has significant advantages and cost benefits over dedicated instruments.


Determination of fluconazole in human serum by solid-phase extraction and reversed-phase high-performance liquid chromatography.

A simple, rapid, and accurate reversed-phase octadecylsilyl high-performance liquid chromatographic method using solid-phase column extraction is described for measuring fluconazole in human serum. The column eluent was monitored by ultraviolet absorption at 210 nm. Fluconazole was extracted from diluted serum by adsorption on a small Bond-Elut C18 cartridge after the addition of UK48,134 as the internal standard and recovered by elution with methanol. The methanol was then evaporated to dryness and the residue reconstituted in 200 microliters of mobile phase and filtered prior to injecting an aliquot (50 microliters) onto an Adsorbosphere C18 column (4.6 x 250 mm, 5 microns particle size), using a mobile phase of 25 mM tris(hydroxymethyl)aminomethane-phosphate buffer (pH 7.0):acetonitrile (75:25, vol/vol). The retention times were 6.6 min for fluconazole and 9.0 min for the internal standard. The assay was precise, with inter- and intraassay coefficients of variation of less than or equal to 2.9% and less than or equal to 2.1%, respectively, and with good linearity (r = 1.000) in the range of 0.1 to 25 micrograms/ml. The duration of each analysis was 15 min and the minimum detectable serum concentration was 0.1 microgram/ml.


A quick, sensitive high-performance liquid chromatography assay for monoethylglycinexylidide and lignocaine in serum/plasma using solid-phase extraction.

Recent advances in the investigation of liver disease and transplantation have seen the introduction of lignocaine as a probe of liver function. For this purpose, an assay that is sensitive and rapid is required for the major metabolite of lignocaine, monoethylglycinexylidide (MEGX). We have developed an accurate, low-cost high-performance liquid chromatography (HPLC) method using Bond-Elut phenyl (1 cc) cartridges for sample preparation. The total preparation time for five samples is less than 10 min and the run time is approximately 10 min/sample. Each cartridge can be used at least four times. Simultaneous measurement of another metabolite of lignocaine, glycinexylidide (GX), can be achieved by adjustment of the mobile phase and flow rate. The chromatogram is monitored with an UV detector at 210 nm. The inter- and intra-assay coefficients of variation for MEGX (10-250 micrograms/L) and lignocaine (100-2,000 micrograms/L) are less than 9.5 and less than 2%, respectively, with recoveries for MEGX, trimethoprim (internal standard), and lignocaine all greater than 85%. This method offers a rapid, sensitive assay that is clinically useful in the new role for lignocaine/MEGX in dynamic liver function testing.


Rapid and sensitive liquid chromatographic assay of mefenamic acid in plasma.

Mefenamic acid (MA) is a nonsteroidal antiinflammatory analgesic agent widely used clinically. A simple and sensitive liquid-chromatographic assay has been developed for the quantitative determination of MA in human plasma. A reverse phase, 10-microns cyano column (25 x 0.4 cm), a mobile phase of water-acetonitrile-methanol-17 M acetic acid (69:15:15:1 by volume), and an ultraviolet detection (290 nm) are used for the separation of MA and internal standard (methyl-clonazepam). MA and internal standard are extracted from acidified plasma into diethyl ether and after evaporation of the organic phase, the residue is redissolved in methanol. Calibration curves are linear in the range 0.05-3.20 micrograms/ml, and at the plasma level corresponding to the quantification limit (0.05 microgram/ml) coefficients of variation are 7.5% and 10.2% for repeatability and reproducibility studies, respectively. This assay was successfully used in the study of pharmacokinetics of MA in human plasma after a single oral administration of a low MA dose.


Comparison of cyclosporine measurement in whole blood by high-performance liquid chromatography, monoclonal fluorescence polarization immunoassay, and monoclonal enzyme-multiplied immunoassay.

Monitoring of cyclosporine concentrations in whole blood is used routinely as a guide to adjusting dose so as to achieve optimal therapeutic benefit with minimal adverse effects. In the present study, we have compared a specific high-performance liquid chromatography (HPLC) assay with a fluorescence polarization immunoassay (TDx) and an enzyme-multiplied immunoassay (Emit). Both Emit and TDx assays employ a monoclonal antibody to cyclosporin A and therefore have the potential for a high degree of specificity. Blood specimens (EDTA as anticoagulant) were obtained from 113 patients (71 renal transplants, 17 liver transplants, and 25 other categories) taking cyclosporine and analysed by all three methods. There were significant correlations between results for HPLC and Emit (Emit = 10.54 + 1.07 x HPLC; r2 = 0.82, p less than 0.001) and between results for HPLC and TDx (TDx = 9.16 + 1.42 x HPLC; r2 = 0.82, p less than 0.001). Compared to HPLC analysis, 74% and 96%, respectively, of Emit and TDx results were to the left of the line of identity. The TDx monoclonal antibody appears to have a lesser degree of specificity than that used in the Emit assay. Mean concentrations of cyclosporine measured by Emit and TDx were 17% and 51% higher, respectively, than those measured by HPLC. Because of this overestimation, we suggest that both Emit and TDx methods may find their most appropriate use in routine therapeutic monitoring of renal transplant patients in whom metabolite concentrations are less variable over time.


Is the monoclonal fluorescence polarization immunoassay for cyclosporine specific? Comparison with specific radioimmunoassay.

Trough concentrations of cyclosporine (CsA) in whole blood were measured by specific monoclonal 3H-radioimmunoassay (S-RIA) and compared with those obtained by monoclonal fluorescence polarization immunoassay (S-FPIA) in 89 transplant recipients. Differences in the S-RIA:S-FPIA relationship between kidney transplant (KT, n = 59) and heart transplant (HT, n = 24) recipients were investigated. Mean concentrations of CsA were significantly higher by S-FPIA than by S-RIA [191 +/- 127 vs. 166 +/- 124 ng/ml (mean +/- SD), p less than 0.001], with a S-FPIA:S-RIA ratio of 1.25 +/- 0.33. Higher ratios were observed either in patients with low CsA concentrations or in those with a high proportion of metabolites. HT recipients had higher S-FPIA:S-RIA ratios than did KT recipients (1.37 vs. 1.22, t = 1.87, p = 0.065). Within-assay coefficients of variation were lower for S-FPIA than for S-RIA (2.4% vs. 12.3%, p less than 0.001). Monoclonal FPIA is a precise and fast method, more suitable than RIA for therapeutic CsA monitoring in clinical practice. However, our results indicate a 25% higher mean CsA concentration by S-FPIA than by S-RIA (37% in HT recipients), which should be borne in mind until therapeutic and toxic ranges are established or a more specific FPIA method is developed.


Interaction of tricyclic antidepressants with cholestyramine in vitro.

The adsorption of amitriptyline, desipramine, doxepin, imipramine, and nortriptyline onto cholestyramine was demonstrated in vitro with use of 1.2 mol/L HCl at 37 degrees C to simulate gastric fluid. Binding to cholestyramine was approximately 80% for each of the tricyclic antidepressants, and this was about the same degree of binding noted with a nonpharmaceutical, non-ionic resin widely used in the diagnostic toxicology laboratory (Amberlite XAD-2). In contrast, five other non-antidepressants (acetaminophen, chlordiazepoxide, procainamide, quinidine, and theophylline) showed only minimal binding to cholestyramine under these conditions. Activated charcoal completely bound all drugs studied. These findings suggest that cholestyramine should be used with caution in patients receiving tricyclic antidepressants. They also suggest that cholestyramine may be a potentially useful adjunctive therapy in treatment of overdose with the tricyclic antidepressants.


Effect of pH changes and ethanol on the binding of tricyclic antidepressants to cholestyramine in simulated gastric fluid.

Tricyclic antidepressants have previously been shown by this laboratory to bind significantly (greater than 80%) to cholestyramine in 1.2 mol/L HCl, used to simulate gastric fluid. This finding has important implications for individuals using tricyclic and cholestyramine concurrently, and it may also have potential therapeutic implications in the treatment of tricyclic overdose. In the present study the effects of pH changes and ethanol on this binding were evaluated. Percent binding of amitriptyline, desipramine, doxepin, imipramine, and nortriptyline decreased from 75-90% (pH 1.0) to 35-50% (pH 4.0). Beyond pH 4.0, however, the binding of each antidepressant increased up to 60-75% at pH 6.5. In contrast, ethanol consistently reduced the percent binding of each antidepressant to cholestyramine in 1.2 mol/L HCl until, in the presence of pure ethanol, the binding ranged from 0% (nortriptyline) to only 32% (doxepin). These findings are important since pH varies widely within the gastrointestinal tract and since ethanol may be co-ingested with cholestyramine and antidepressants.


Effects of 2-acetyl-4-tetrahydroxybutyl imidazole (THI) on the thymus of rats.

2-Acetyl-4-tetrahydroxybutyl imidazole (THI), a component of the food colouring ammonia caramel, has been shown to produce suppression of cell-mediated immunity and a reduction in circulating TH and TC/S lymphocytes in rats. Accordingly in this study the effects of THI on the thymus has been investigated. THI (1 mg/kg/day) was given for up to 7 days in the drinking water to Fischer 344 rats on a vitamin B6 deficient diet. No marked change in thymus weight was found but the cellularity was marginally decreased and flow cytometric analysis of the lymphocyte subsets revealed an increase in the number of CD4+CD8- and CD4-CD8+ single positive (SP) cells and a reduction in the number of CD4+CD8+ double positive (DP) thymocytes. This reduction was in agreement with histological findings of increased numbers of pyknotic cells in the cortex, mainly engulfed by macrophages. Mitogen-induced proliferation of thymocytes prepared from THI-treated animals was increased, concordant with the gradual increase in the percentage of mature SP cells. No change in normal proliferation of thymocytes cultured in vitro, or, in proliferation in vivo, detected as 5-bromo-2'-deoxyuridine (BrdU) incorporation, was found. It is concluded that THI produced an increase in death of immature DP cells. However, THI did not affect thymocyte proliferation or their differentiation into mature SP cells in the thymus, but rather impairs their migration into the circulation. The mechanism of action of THI appears to be indirect, but THI does not act through increasing the release of adrenal corticosteroids to supra-physiological levels as the same histopathological changes in the thymus were found in adrenalectomized rats.


The influence of thymus extracts on the chemotaxis of polymorphonuclear neutrophils (PMN) from patients with insulin-dependent diabetes mellitus (IDD).

The main goal of this study was to evaluate the possible influence of thymus extracts on the chemotaxis of PMN isolated from diabetic patients. The analyzed group consisted of fifty patients with insulin-dependent diabetes mellitus (IDD), and twenty healthy adults were taken as controls. The results showed, that PMN isolated from patients with IDD manifested the impaired chemotaxis toward zymosan-activated plasma (ZAP), plasma incubated with cellophane and supernate from E. coli culture. Both thymus extracts did not change the directed migration toward control plasma and toward ZAP. THYMEX-L did improve the migration of diabetic PMN toward plasma incubated with cellophane, whereas TFX-THYMOMODULIN ameliorated the impaired chemotaxis toward supernate from E. coli culture. Both thymus extracts can attract PMN obtained from healthy donors, but not the cells isolated from diabetic patients.


5-Lipoxygenase gene expression in the thymus.

Eicosanoids are arachidonic acid metabolites issued both the cyclooxygenase and the lipoxygenase pathways. Many of these products were reported to modulate the immune response. Since most of eicosanoids have a short half life they are considered as local immunomodulators. Interactions between eicosanoids and thymocytes appear to be complex within the thymus. It was reported that cyclooxegenase derivatives of arachidonic acid are produced in this primary lymphoid organ mostly by cells of the thymic microenvironment. On the other hand it is not yet clearly established (1) what is the location of the lipoxygenase-positive cells within the gland and (2) what is the ratio of cells producing lipoxygenase metabolites of arachidonic acid when compared to the whole thymocyte population. Using two oligonucleotides complementary to the rat 5-lipoxygenase mRNA we demonstrated (by both hybridization on Northern blots and in situ hybridization) the expression of the 5-lipoxygenase gene in the thymus. 5-lipoxygenase positive cells appear to be associated in "clusters" and are mostly located in the thymic cortex. It is likely that they belong to the thymic microenvironment.


Adenine nucleotide content of thymoma-derived lymphocytes.

Purine and pyrimidine metabolites are essential substances for cells. We have measured the adenine nucleotide (AN) contents of thymocytes from 15 human thymomas, 11 adjacent non-neoplastic thymuses and 3 children's thymuses. There was no significant difference in AN content of thymocytes between thymoma and children's thymus. But, AN content of adjacent non-neoplastic thymuses was significantly lower than that of thymoma or children's thymus. In mixed type thymoma, ATP content in thymocytes was significantly higher than that in lymphocytic type thymoma. These data indicate that thymocytes in thymoma may show further T cell maturation probably associated with the functional microenvironment of the neoplastic epithelial cells just like the children's thymus.


Localization of estrogen receptors and estrogen receptor-mRNA in female mouse thymus.

The present study was performed to demonstrate estrogen receptor (ER) and ER-mRNA in female mouse thymus. The results are as follows: (i) thymic tissue contains ER in both reticuloepithelial(RE)- and T-cell fractions, the ER level being three-fold higher in the former fraction than in the latter; and (ii) thymic tissue contains ER-mRNA at 6.2 kb, a large amount of which was localized in the RE cells and less in the T cells. From these results it is suggested that estrogen (E) mediates some immune function of the mouse thymus through its receptor within RE cells and/or T cells.


Metoclopramide increases the number of solid epithelial islands in the adult rat thymuses.

The effect metoclopramide on thymocyte proliferation and the histological picture of the rat thymuses was investigated. A significant increase in the epithelial component of the thymus was found. On the other hand, metoclopramide administration did not influence the thymocyte proliferation.


The ontogeny of bovine thymostimulin production in fetal and postnatal age.

The development of thymostimulin production in the bovine fetal thymus was determined, starting at month 2 of gestation until birth. Production of fetal thymostimulin, identified electrophoretically as a peptide with 4-5000 Da, started at month 4 of gestation and achieved its maximum expression three months after birth, followed by a rapid decrease until month 18. Thymus of fetuses from the early gestational phase (2-3 months) yielded no electrophoretically detectable thymostimulin band. Biological activity of the fractions, determined by increased E-rosetting, fairly correspond to the content of the 4-5000 Da peptide moiety.


Immunodeficiency, growth hormone deficiency and central nervous system involvement in a girl.

We describe a mentally retarded 12-year-old girl with ataxia in whom diagnostic evaluation for short stature revealed isolated growth hormone (GH) deficiency and multiple central nervous system (CNS) lesions. Assessment of immunologic status, performed because of the persistence of recurrent respiratory tract infections, showed associated deficiencies of IgG2-IgG4 and specific antibody response; in addition, in vitro lymphocyte response to mitogens was low, in vitro production of interleukin-2 and of IgM was absent, and natural killer activity was decreased. The possibility that association of the CNS lesions, GH deficiency and immune defects could be due to alterations of the neuro-immuno-endocrine network secondary to a disturbance of neurotransmitters induced by precocious CNS damage of a viral or ischemic nature is discussed.


Testosterone binding sites in the rat thymus during late embryonal and postnatal period.

Using immunoperoxidase staining with anti-testosterone Ab, has been shown that the cells binding testosterone were present in the rat thymus and that these cells are localized in the outer thymic cortex as well as in cortico-medullary region and medulla. Immunoperoxidase staining with that Ab at electron microscopy level showed that thymocytes as well as thymic epithelial cells bind this hormone. Combined immunoperoxidase staining with anti-testosterone Ab and immunofluorescence method with mAbs specific for thymocytes or thymic epithelial cells, revealed that thymocytes binding this hormone are localized mainly in the outer cortex, while thymic epithelial cells binding testosterone were found in cortico-medullary region and medulla. These testosterone binding cells were found, for the first time, in the thymus of 18-day-old fetus. It has also been shown that their density increased markedly by the day 3 of postnatal life and continued to increase up to the adult stage of organ development. These results indicate that testosterone can influence upon function of specific thymic epithelial cells, localized in the corticomedullary region and medulla. Thus, the results also suggest that this hormone can modulate T cell proliferation and/or differentiation, not only directly acting on the T cells localized in the outer thymic cortex, but also indirectly modulating function of the thymic epithelial cells that bind this hormone.


Dexamethasone and hydrocortisone enhance the in vitro migration of prethymic stem cells to thymus supernatant.

The migration of prethymic stem cells from the bone marrow to thymus supernatant was examined in a blind well migration assay following treatment with glucocorticoids (GCS). To study the in vivo effects of GCS, time release pellets containing dexamethasone (DEX) were implanted subcutaneously in young adult CBA/J mice for 7, 14 and 21 days. After seven and 21 days of DEX treatment, enhanced migration of bone marrow lymphoid cells to thymus supernatant occurred. To study the in vitro effects of GCS on prethymic stem cell migration, bone marrow cells were incubated for one or six hours in media containing dexamethasone (DEX), hydrocortisone (HCS), or medium alone. After a six-hour incubation with GCS, significantly more bone marrow cells migrated toward thymus supernatant in vitro than bone marrow cells incubated in medium alone. The enhanced migration of cells to thymus supernatant seen in this assay may reflect a feedback mechanism whereby bone marrow cell migration is enhanced to restore the thymic lymphocyte reserves depleted following treatment with GCS.


A report of 104 transfusion errors in New York State.

In New York State, significant incidents involving the collection, processing, or transfusion of blood must be reported. Incident reports received over a 22-month period involving transfusion of blood to other than the intended recipient or release of blood of an incorrect group were analyzed. Among 1,784,600 transfusions of red cell components; there were 92 cases of erroneous transfusion that met study criteria (1/19,000). There were 54 ABO-incompatible transfusions (1/33,000); three of these (1/600,000) were fatal. Correction for underreporting of ABO-compatible errors resulted in an estimate of 1 per 12,000 as the true risk of transfusion error. National application of New York State data results in an estimate of 800 to 900 projected red cell-associated errors in the United States annually. The majority of reported errors occurred outside of the blood bank (43% resulted solely from failure to identify the patient and/or unit prior to transfusion and 11% resulted from phlebotomist error), while the blood bank was responsible for 25 percent of errors and contributed, with another hospital service, to 17 percent. The risk of transfusion of ABO-incompatible blood remains significant, and additional precautions to minimize the likelihood of such events should be considered.


In vitro storage and in vivo survival studies of red cells from persons with the In(Lu) gene.

Red cells (RBCs) of individuals with the In(Lu) gene are characterized by suppression of the Lutheran, P1, i, and other blood group antigens, acanthocytosis, and abnormal electrolyte metabolism. To determine the clinical significance of these abnormalities, the survival of autologous RBCs was determined by 51Cr in two siblings with the dominant Lu(a-b-) [In(Lu)] phenotype. Both subjects studied had normal hemoglobin, hematocrit, reticulocyte count, haptoglobin, and ferritin values. RBC indices were mildly hypochromic. Examination of the peripheral smear showed mild acanthocytosis in one individual. Analysis of RBC distribution on discontinuous density gradients showed a shift to lighter fractions than normal control RBCs. Storage of these Lu (a-b-) RBCs at 4 degrees C showed significant hemolysis within a few days; this was confirmed by increased autohemolysis, which was reduced by glucose and ATP. RBC cation content (sodium and potassium) was higher than that in control cells, which indicated increased cell hydration, which explains the lighter density and mild hypochromia of the Lu(a-b-) RBCs. 51Cr survival of autologous Lu(a-b-) RBCs was normal in both subjects studied. The data indicate that the morphologic and cation abnormalities of RBCs of persons with the In(Lu) gene are clinically insignificant, as these cells have normal in vivo survival. Such RBCs, however, are susceptible to increased hemolysis in vitro under standard blood banking storage conditions. Individuals of the Lu(a-b-) phenotype, associated with In(Lu), may not be suitable candidates for routine blood donation.


The binding of human alloantibodies to recombinant glycophorin A.

Chinese hamster ovary (CHO) cells were transfected with the wild-type, M allele of glycophorin A cDNA. The binding of human alloantibodies to recombinant glycophorin A was assessed with a modified hemagglutination-inhibition assay. Patient sera were incubated with acetone powders derived from CHO cells, and the adsorbed supernatants were tested in standard hemagglutination assays. Five M antibodies and one sample containing anti-En(a) bound to transfected CHO cells expressing glycophorin A but did not bind to untransfected CHO cells. Three N antibodies as well as 21 other alloantibodies (representing other major red cell blood group specificities) bound to neither CHO cell line. The M allele specificity of recombinant glycophorin A was further verified by the demonstration that a high-titer D alloantibody maintained the same titer of agglutination after incubation with recombinant glycophorin A. Transfected CHO cells thus express an M blood group antigen that appears to be serologically equivalent to that found on human red cells. A panel of cell lines expressing mutant glycophorin A molecules with defined variations in amino acid sequence and carbohydrate composition will be useful in studies of the fine specificity of human glycophorin alloantibodies. This approach may also provide an abundant source of artificial antigens for clinical use in blood group serology.


Selection of platelets for refractory patients by HLA matching and prospective crossmatching.

A multi-site clinical study compared platelets chosen for refractory patients by prospective platelet crossmatching using stored donor platelets and HLA-based selection. Seventy-three patients who were refractory to random-donor platelets received two plateletpheresis components, one chosen by HLA-based criteria and the other by crossmatching. Patients were carefully evaluated to exclude nonimmune factors that could adversely affect transfusion results. Each of the five study sites used a crossmatch procedure with which it had experience. Results from this study indicate the following: 1) The overall rate of successful transfusion was similar when an HLA-based method of donor selection that includes all grades of matching and mismatching was compared to a crossmatch-based method of donor selection. 2) HLA-based selection that restricts recipients to grade A and BU matches was superior to a selection method based upon crossmatching alone. Donor selection based on HLA matching (grades A or BU) was also superior to selection based on any degree of HLA mismatching (grades BX, C, or D). 3) Selection of donors based on HLA-cross-reactive groups (defined by in vitro serologic crossreactivity) was no more successful than that based on grade C and D mismatches and was no more successful than selection by crossmatching alone. 4) Lymphocytotoxic and platelet antibodies were not detected in many of the enrolled patients, even though patients demonstrating nonimmune factors were eliminated from the study. It can be concluded that HLA-compatible (grades A and BU) platelets provide optimal support for refractory patients, but that crossmatch-selected platelets are acceptable as an alternative component.


Identifying elective orthopedic surgical patients transfused with amounts of blood in excess of need: the transfusion trigger revisited.

The discharge hematocrit has been analyzed as a clinical indicator of the transfusion trigger by which to identify patients undergoing elective orthopedic surgery who were transfused with blood in excess of need. The volume of red cells lost by each patient during surgical hospitalization was compared to the volume of red cells transfused. Three clinical indicator levels were considered. Red cell losses of 10, 20, and 30 percent of each patient's baseline red cell volume at admission were considered to be appropriate before subsequent blood transfusion replacement, representing generous, intermediate, or strict clinical indicator levels, respectively. With Level I as a generous clinical indicator, 110 (25%) of 525 patients were transfused in excess of blood needs; by Level II (intermediate) and Level III (strict) criteria, 221 (42%) and 314 (60%) of 525 patients, respectively, were transfused in excess of blood needs. Significant differences were found for transfused patients analyzed by gender (26% of women vs. 13% of men; Level I, p less than 0.001) and preoperative autologous blood donation (25% of autologous blood donors vs. 11% of those who did not donate autologous blood; Level I, p less than 0.001). It can be concluded that the discharge hematocrit and amount of blood lost during hospitalization can be used as clinical indicators with which to identify patients receiving transfusions in excess of needs in the elective surgical setting. With this method, it was found that the transfusion trigger is different for women and for men as well as for autologous blood donors and those who did not donate autologous blood undergoing elective orthopedic surgery [corrected].