Plasminogen activation in vivo upon intravenous infusion of DDAVP. Quantitative assessment of plasmin-alpha 2-antiplasmin complex with a novel monoclonal antibody based radioimmunoassay.

Infusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established. A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor alpha 2-antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated alpha 2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system. Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex. We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.


[Conjugate vaccines against bacterial infections: typhoid fever].

Capsular polysaccharides have been studied as possible vaccines against infectious diseases. However, they are capable to induce only short-run protection because of their T-independent properties and they would not be protective against infection in high-risk populations. The alternative to face this problem is to develop methods to join covalently the polysaccharide and proteins to both increase the immunogenicity of and to confer the property of T-dependence to this antigen. In order to obtain a conjugate vaccine against typhoid fever, in our laboratory we have tried to synthesize a conjugate immunogen between the Vi antigen and porins from Salmonella typhi.


[Development of oral vaccines based on recombinant proteins derived from cholera toxin].

In this paper a new approach to create antigens through genetic engineering is discussed. In this particular case the subunits of V. cholerae toxin are used as heterologous epitope carries. In this paper the manipulation of A and B subunits is described. This manipulation allows both the insertion of epitopes to the B subunit and the use of subunit A in the construction of recombinant antigens similar to the ones derived from subunit B.


Neutralization of the antiheparin activity of platelet factor 4 by a monoclonal antibody.

We have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4's ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.


Fine mapping of monoclonal antibody epitopes on human von Willebrand factor using a recombinant peptide library.

A recombinant human von Willebrand factor (vWF) cDNA fragment library was constructed in lambda gt11 for the localization of anti-vWF monoclonal antibody epitopes. Twelve of 21 monoclonal antibodies screened identified epitopes expressed in lambda gt11 as beta-galactosidase fusion proteins. By sequence analysis, these antigenic determinants were localized to segments ranging from 17 to 105 amino acids in length. Four epitopes apparently shared by more than one antibody were identified, suggesting the presence of immuno-dominant epitopes within vWF. Monoclonal antibody C3, which blocks factor VIII (FVIII) binding to vWF, bound to the same epitope previously identified by a second monoclonal antibody which also blocks this function, suggesting that this region may be at or near the vWF/FVIII binding domain. Three antibodies recognize the same region within the vWF A2 repeat. Mutations near this region appear to be responsible for Type IIA von Willebrand's disease. The co-localization of these antibodies suggests that this domain might be exposed on the surface of vWF, consistent with its apparent increased sensitivity to plasma proteases.


Aprotinin does not inhibit the release of PGI2 or vWF from cultured human endothelial cells.

The release of prostacyclin (PGI2) and von Willebrand factor (vWF) from human umbilical vein endothelial cells (HUVEC) was examined to determine if aprotinin had any effects on these endothelial cell reactions. These end-points were chosen to indicate if this serine protease inhibitor caused alterations in the control of haemostatic function by endothelium, in the light of the improvement in haemostasis seen in patients given aprotinin therapy at the time of open heart surgery. Stimuli used to promote secretion of prostacyclin and vWF were human alpha-thrombin, histamine, protamine sulphate, poly-L-lysine and phorbol myristate acetate. Aprotinin (30 microMs) had no significant effect on the basal or stimulated release of PGI2 or vWF from HUVEC.


Characterization of human tissue-type plasminogen activator with monoclonal antibodies: mapping of epitopes and binding sites for fibrin and lysine.

The study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding. Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D'). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA. The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F.(ABSTRACT TRUNCATED AT 250 WORDS)


An enzyme-linked immunosorbent assay for urokinase-type plasminogen activator (u-PA) and mutants and chimeras containing the serine protease domain of u-PA.

An enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 +/- 0.6 ng/ml (mean +/- SD, n = 27). The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising AA 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with alpha 2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)


Alteration of adenine nucleotide metabolism in coronary smooth muscle cells by activated platelets.

Cultured porcine coronary smooth muscle cells were preloaded with [3H]adenine and the inside and outside radioactive metabolites of the cells were analyzed following exposure to activated platelets. Incubation of the cells with human platelets activated by collagen enhanced intracellular conversion of ATP to ADP and caused dose- and time-dependent increase in radioisotopic release, mainly adenosine. Isolation of cyclic AMP revealed decreased cyclic AMP levels in the treated cells, both intra- and extracellularly. Of the substances released by the activated platelets, thromboxane A2 and serotonin enhanced radioisotopic release. The modulation of adenine metabolism by the activated platelets was preceded by increase in accumulation of inositol phosphates in the cells and was prevented by Iloprost (1 microM), a prostacyclin analog, cilostamide (10 microM), a cyclic AMP-specific phosphodiesterase inhibitor, or dibutyryl cyclic AMP (1 mM). Nifedipine showed only minor preventive effect. The agents which elevate cyclic AMP accumulation also attenuated phosphoinositide hydrolysis, whereas nifedipine had no effect. These results suggest that activated platelets may stimulate adenine metabolism in coronary smooth muscle cells, presumably due to activation of phosphoinositide turnover resulting in increased intracellular calcium. Enhanced adenosine release from the cells exposed to activated platelets may be a compensatory mechanism to prevent further platelet aggregation and contraction of coronary smooth muscle.


Effect on primary haemostasis of prophylactic regimens of low molecular weight heparin, unfractionated heparin, dextran and their combinations. An animal experimental study.

The aim of the study was to determine whether an impairment of the haemostasis could be observed experimentally when thromboprophylactic substances, which act differently on the haemostatic mechanism, were given single or in combination in prophylactic doses. In 36 rabbits we measured the primary haemostatic plug formation time (PHT), rebleedings and total haemostatic plug formation time (THT) after transection of venules and arterioles using an intravital microscope. We combined unfractionated heparin (UH) and low molecular weight heparin (LMWH) in low dose with either dextran 70 or polygeline (placebo volume expander) in a randomized double-dummy set up. In the placebo group (NaCl and polygeline) the median PHT was 55 and 101 seconds for arterioles and venules respectively, which are well-comparable to earlier results from our group. Most prolonged PHT and THT for arterioles were seen for dextran+NaCl, actually less prolongation was seen for UH+dextran. We did not observe any differences, except for a prolongation of THT for venules in rabbits given dextran+NaCl (p less than 0.05). Thus, in thromboprophylactic doses used, there does not seem to be an impaired or additive effect between heparins and dextran 70 in primary haemostasis in rabbits.


An essential 'set' of K+ channels conserved in flies, mice and humans.

The molecular genetic approach to studying K+ channels has revealed that at least four subfamilies of voltage-gated K+ channels originally discovered in Drosophila are conserved in mice and humans. This conservation of the K+ channel subfamilies Shaker, Shal, Shab, and Shaw suggests that not only the broad outlines of membrane electrical properties but also many molecular details as well evolved in the parent species ancestral to both invertebrate and vertebrate life. Shaker, Shal, Shab, and Shaw K+ channels have similar structures, but appear to be independent channel systems: when co-expressed in Xenopus oocytes, all four function independently. These four K+ channel subfamilies may be part of an essential 'set' of excitable channels required by most nervous systems. The task now remaining is to understand the functions of each member of the set.


Target influences on the development of leech neurons.

A pair of Retzius neurons is found in each segmental midbody ganglion of the CNS of the leech Hirudo medicinalis. Although all Retzius neurons appear to have the same cell lineage and are indistinguishable from one another through the initial phases of axonogenesis, later in development two pairs of Retzius neurons--those in the segments containing the male and female reproductive ducts--acquire distinctive morphological and physiological characteristics. Experimental manipulations of the reproductive ducts in early embryos have indicated that the outgrowing Retzius axons receive a signal from these peripheral targets that triggers major changes in their developmental program. Such 'end-organ specification' has been shown to contribute to the differentiation of neurons in other nervous systems as well, and the mechanisms underlying such control can be investigated in great detail in the relatively simple, segmented nervous system of the leech.


Control of eye-head coordination during orienting gaze shifts.

Combined eye and head displacements are routinely used to orient the visual axis rapidly (gaze). Humans can use a wide variety of head movement strategies. However, in the cat, comparatively limited eye motility forces a more routine and stereotyped use of head motion. Nevertheless, the same general principles of gaze control may be applicable to humans, rhesus monkeys and cats. The gaze control system can be modeled using a feedback system in which an internally created, instantaneous, gaze motor error signal--equivalent to the distance between the target and the gaze position at that time--is used to drive both eye and head motor circuits. The visual axis is moved until this error equals zero. Recent studies suggest that the superior colliculus of the cat provides brainstem eye and head motor circuits with the gaze motor error signal; such studies have led to speculation that information on ongoing gaze motion is fed back to the superior colliculus. It is still uncertain whether comparable collicular and brainstem neuronal mechanisms control gaze in the monkey.


Getting the message from the gene to the synapse: sorting and intracellular transport of RNA in neurons.

A key question in cellular neurobiology is how neurons target molecules to cellular microdomains at a distance from the nucleus. Of special importance are the thousands of postsynaptic sites that form the basis for synaptic communication. Recent evidence suggests that an important aspect of molecular trafficking involves differential sorting, selective intracellular transport, and docking of particular mRNA molecules and associated protein synthetic machinery at postsynaptic sites. This offers the potential for local regulation of the production of key proteins in response to conditions at individual synapses. This article reviews what is known about the mechanisms of mRNA trafficking in neurons and in other cells ranging from oocytes to oligodendrocytes, and considers the possible role that mRNA trafficking and the resulting local synthesis of particular proteins may play in cellular function.


Neural substrates of opiate withdrawal.

Drug withdrawal is an integral part of most types of dependence and, to a large extent, opiate withdrawal has been considered the prototypic, classic measure of opiate dependence. The opiate withdrawal syndrome is characterized by multiple behavioral and physiological signs such as behavioral activation, ptosis, diarrhea, 'wet dog' shakes and motivational dysfunction, which may be represented in the CNS at multiple sites. It seems that the activating effects associated with the opiate withdrawal syndrome may be mediated by the nucleus locus coeruleus. Other signs such as wet dog shakes may involve sites in the hypothalamus important for temperature regulation. Certain other signs such as diarrhea and lacrimation may be dependent on peripheral opiate receptors. The motivational aspects of opiate withdrawal as demonstrated by the aversive stimulus effects or negative reinforcing effects (e.g. disrupted lever-pressing for food and place aversions) may involve those elements of the nucleus accumbens that are known to be important for the acute reinforcing effects of opiates in nondependent rats. Evidence exists at the cellular and molecular level for both 'within-system' and 'between-system' adaptations to dependence. Elucidation of the neural networks, cellular mechanisms and molecular elements involved in opiate withdrawal may provide not only a model for our understanding of the adaptive processes associated with drug dependence but also of those associated with other chronic insults to CNS function.


Immunohistochemical detection of acidic fibroblast growth factor in bladder transitional cell carcinoma.

Acidic fibroblast growth factor (aFGF) is a regulatory peptide which, on account of its structural homologies with the products of oncogenes, is involved in cell proliferation, differentiation, and motility. We previously reported the presence of aFGF in the urine of patients with transitional cell carcinoma (TCC). aFGF can also induce the motility of a rat-derived bladder carcinoma cell line (NBTII). This immunohistochemical study used polyclonal rabbit antibodies against acidic and basic FGF and peroxidase detection. Native NBTII nude mice xenografts and aFGF transfected NBTII (NFS14) nude mice xenografts were used as tissue controls for antibody specificity. The samples included 4 normal urothelia and 12 TCC. In addition, cytospins of 4 different tumoral cell lines of human bladder and normal bladder cells were stained. The results showed strong immunostaining in all tumoral urothelium samples using anti-aFGF and a very low amount of staining or none at all in healthy tissues. A primary analysis suggested that the strongest reaction was obtained in high-grade tumors (3 + vs + for lower-grade tumors). Using bFGF antibody, strong immunohistochemical staining was detected on basal membranes and stromal vessels and none in urothelium. These data confirm aFGF expression in the epithelial cell compartment of bladder cancer and the likely involvement of this regulatory peptide in the biology of TCC.


[Epidemiology and natural course of benign prostatic hyperplasia].

Benign prostatic hyperplasia (BPH) is a frequent condition in men over 50 years of age. At autopsy the histological characteristics of BPH are seen in 80% of all men older than 80 years. Although BPH is so frequent, its precise etiology is still unknown. Each of the various sources from which epidemiological data on BPH can be obtained has its own specific problems in terms of potential errors and biases, which have to be taken into account in interpretation of the data from these sources. There are clear differences in the incidence of BPH in various races. In negroids BPH is more frequent than in Caucasians, while in Asiatic races BPH is less frequent. However, in Asiatics BPH occurs more frequently in men who have immigrated into western countries. Age and hormonal status are well known to be involved in the development of BPH. Other factors, such as marital status, socioeconomic class, smoking, diet, diabetes, hypertension, and liver cir-rhosis have been implicated, but their relation with the risk of developing BPH has never been proven. The natural history of BPH is characterized by an age-dependent increase in histological changes and an increase in prostate size. The histological changes compatible with BPH are found in men below the age of 30 with an incidence of less than 10%. During the 6th decade, 42% of prostatic specimens have histological signs of BPH and during the 8th decade of life more than 80% of prostatic specimens. The histological changes of BPH precede prostatic enlargement by roughly 1 decade. There is an initial spurt of prostate growth during the period of maturation and puberty.(ABSTRACT TRUNCATED AT 250 WORDS)


[Clinical manifestations and indications for therapy of benign prostatic hyperplasia].

The high incidence of benign prostatic hyperplasia (BPH), together with the wide variability in its clinical manifestations and in the natural course of disease, requires a careful evaluation of the patient. Technical progress and continuing development of established methods mean that a wide range of diagnostic procedures is available and an "objective" correlate of infravesical obstruction can be obtained. In addition to these objective criteria, the patient's subjective perception of the impact of the symptoms on his quality of life also affects the decision as to whether therapeutic intervention should be attempted and the degree of success that can be attained, as is becoming increasingly evident. The diagnostic methods available for the routine assessment of patients with BPH is reviewed, and their relative value as a basis for deciding on therapeutic intervention is analysed.


[Value of functional examination techniques for the assessment of the clinical aspects of benign prostatic hyperplasia].

The urodynamic relevance of benign prostatic hyperplasia (BPH) is determined by evaluation of the symptoms of prostatism, the degree of infravesical obstruction and the size of adenoma. The combination of all three findings, but also the presence of at least of two of these findings suggest a diagnosis of clinical BPH. Standard diagnostic procedures consist in elicitation of the history, evaluation of symptoms, physical examination, urinalysis and laboratory examination of serum creatinine; with evaluation of residual urine and uroflowmetry in addition, surgical therapy can be expected to be successful in 93%. Urinary flow rates exceeding 15 ml/s and/or discrepancies between symptoms and findings need further assessment by synchronous pressure-flow studies for differential diagnosis between unobstructed flow and high-flow outflow obstruction. Complete videourodynamic investigation is indicated both in patients with combined BPH and urinary incontinence without residual urine and in patients with BPH and suspected or known neurological disorder. Surgical treatment of BPH involves the risk of postoperative incontinence in patients with detrusor hyperreflexia combined with a functional or morphological lesion of the external urinary sphincter.


[Evaluation of new technical alternative procedures for therapy of symptomatic benign prostatic hyperplasia].

Transurethral resection of the prostate is still the gold standard in the treatment of symptomatic benign prostatic hyperplasia. It has proved possible to reduce the mortality of this treatment almost to zero, while the morbidity has remained unchanged at 18% for decades. Therefore, understandably, procedures are being looked for that will be similarly effective but have lower morbidity. Current developments in this field are transurethral implants (spiral, intraurethral catheter, Wall-Stent), the balloon dilatation, transurethral incision (TUIP), laser therapy (TULIP, ITK), ultrasound-induced aspiration of tissue and heat treatment (hyperthermia, thermotherapy). Chances and risks inherent in these different technical procedures and the amount of investment they involve are reviewed. Potential for the future can at best be attributed to the Wall-Stent, TUIP, the application of laser and the thermotherapy.


[Benign prostatic hyperplasia and growth factors: mechanisms and hypotheses].

The aetiology and pathogenesis of benign prostate hyperplasia (BPH) are still unresolved questions, although a number of hypotheses have been developed, most of which have still not been confirmed by experimentation. BPH has been regarded as a kind of adenoma, as a stromal disease, as the result of either hormonal imbalance (altered oestrogen/testosterone ratio) or testosterone or dihydrotestosterone stimulation, and finally as the result of oestrogen stimulation, perinatally or involutionally. More recently, scientific interest has focused on the presence and possible function of growth factors and their receptors in the human prostate and their autocrine or paracrine stimulatory effects in BPH development. Hypotheses on their hormonal regulation as well as their interplay during epithelial-stromal interaction have been developed. The intact human prostate produces epithelial (EGF) and basic fibroblast (bFGF) growth factors. Normally, they do not appear to have autocrine or paracrine effects. In androgen deficiency, however, as shown experimentally in castrated rats, the stromal cells express increased amounts of TGF beta, of TGF beta receptor, and of bFGF. Platelet-derived growth factor (PDGF), but not the corresponding receptor, has been shown in prostate. The growth factor receptor-associated tyrosine protein kinase is present in the human prostate in two different forms, but its functional significance in BPH development has not yet been elucidated. A more significant role may be attributed to the recently described growth factors in cultured human stromal cells, which exert multifarious mitogenic and non-mitogenic effects on prostatic epithelium as well as neuronal and non-neuronal cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Nondetectable prostate-specific antigen in moderately differentiated adenocarcinoma of prostate.

Prostate-specific antigen (PSA) is a glycoprotein derived from prostatic ductal and acinar epithelial cells. The main clinical use of PSA is as a marker of prostate tumor progression/recurrence. We present a case of a sixty-nine-year-old patient with recurrent endometrioid carcinoma of the prostate (status post-radical prostatectomy, hormonal therapy, and external beam radiation therapy) with normal serum PSA.


Prostate-specific antigen and digital rectal examination in long-term follow-up of stage A1 prostatic carcinoma.

Twenty-six individuals with Stage A1 carcinoma of the prostate (less than or equal to 5%, Gleason score less than or equal to 4) diagnosed from 1969 to 1980 were evaluated with digital rectal examination (DRE) and prostate-specific antigen (PSA). This unique cohort, sixty-one to eight-two years of age (median 72 years), had a mean interval from diagnosis of thirteen years (median 12.5 years). Abnormal findings on DRE were found in 6 individuals, whereas only one elevated PSA was detected. Ninety-six percent of the PSA levels were less than 3.0 ng/mL and nearly 60 percent of the group had 1.0 ng/mL or less. These levels compare favorably with healthy control subjects under forty years of age and with the limited data available for PSA in healthy men over seventy years of age (87% and 26%, respectively). While biopsy showed persistent or recurrent carcinoma in 2 of 5 individuals, further evaluation disclosed only localized disease. Though the PSA provided little additional information to DRE in the individual patient, it appears from an overview of this group that a low level of PSA in Stage A1 prostatic carcinoma may be associated with long-term survival.


Enhanced chemiluminescent enzyme immunoassay for the detection of trichinellosis antibodies in pigs.

A modified enhanced chemiluminescent enzyme assay (ECIA) was developed for mass screening of pigs for trichinellosis antibodies in abattoirs. Using Bionectics beads as solid support, the assay time could be reduced to 45 min. Optimal conditions for washing, blocking, incubation, concentration of serum, antigens and conjugates as well as timing of film exposure were determined. The sensitivity and specificity of the assay were found to be comparable to those of the triple antibody-IgG ELISA. The assay was tested in an abattoir and its efficacy was found to be satisfactory. However, the major disadvantage of the assay is the high cost of magnetic beads.


An improved passive hemagglutination test for the serological diagnosis of bovine fascioliasis using the specific antigen f2.

Optimum conditions for coupling the specific antigen f2 of Fasciola hepatica to sheep red cells and for the preparation of control cells coated with an unrelated protein are described. With a careful selection of donor sheep for erythrocytes, the problem of anti-species antibodies in bovine sera has been overcome and results may be obtained within 1 h as only one step is required in the assay system. Incorporation of a concentration of 25 mM CaCl2 in the diluent achieved increased stability of the agglutinates and enabled a more precise estimation of the end-point to be made. Analyses performed on bovine sera obtained at slaughter provided results in good agreement with the presence of flukes or fascioliasis lesions in livers at routine slaughter inspection. The developed assay is simpler, faster, more sensitive (P less than 0.01) and has a cut-off between populations of negative sera more easy to define than a recently marketed enzyme-linked immunosorbent assay.


In vitro properties and experimental pathogenic effect of three strains of feline immunodeficiency viruses (FIV) isolated from cats with terminal disease.

Three strains of virus isolated from peripheral blood mononuclear cells (PBMC) of sick cats were identified as feline immunodeficiency virus (FIV) on the basis of in vitro cytopathic effect, T-lymphotropism, ultrastructural morphology and magnesium-dependent reverse-transcriptase activity. The pathogenic properties of two isolates were studied in 13 experimentally infected cats. The primary phase of infection was characterised by a range of haematological (neutropenia, lymphopenia, presence of atypical lymphocytes) and clinical alterations (fever, various signs lasting several weeks, generalised lymphadenopathy persisting for several months) and specific seroconversion. A correlation between the inoculated dose of virus and the intensity and duration of clinical signs was observed. The primary phase was followed in the 10 surviving cats by a stage of asymptomatic seropositivity of undetermined duration but which has persisted for over 35 months for the earliest infections. Viruses reisolated several weeks or months after experimental infection retained the same in vitro properties as the initial isolates.


Metabolism by rat liver cytosol of illudin S, a toxic substance of Lampteromyces japonicus. II. Characterization of illudin S-metabolizing enzyme.

1. Enzyme systems responsible for formation of cyclopropane ring-cleavage metabolites (M1 and M2) of illudin S in rat liver were characterized. 2. The enzymes were localized in the cytosol fraction and utilized NADPH alone as electron donor; they were not affected by oxygen and had low pH optima. 3. Formation of metabolites M1 and M2 was inhibited completely by dicumarol (10(-4) M), an inhibitor of DT-diaphorase. 4. Menadione (10(-4) M) and quercetin (10(-4) M) both inhibited formation of M1 and M2 by 35% and 15%, respectively, but quinacrine, barbital, pyrazole and p-chloromercuribenzoic acid had no significant effect. 5. Results show that the enzyme systems may differ from DT-diaphorase, aldehyde oxidase, xanthine oxidase, ketone reductase, aldose reductase, aldehyde reductase and alcohol dehydrogenase, known cytosolic enzymes responsible for xenobiotic metabolism.


Stimulation of the summit of the right ventricular aspect of the ventricular septum during orthodromic atrioventricular reentrant tachycardia.

Application of ventricular premature complexes (VPCs) from the right ventricular (RV) apex during orthodromic atrioventricular (AV) reentrant tachycardia has limitations both in the ability to shorten the succeeding atrial cycle length and in helping to identify accessory pathway location. Stimulation from the summit of the RV aspect of the septum during AV reentrant tachycardia was investigated as a new technique to improve the diagnostic utility of applying VPCs during AV reentrant tachycardia. VPCs were induced during AV reentrant tachycardia at 10 ms decrements in patients with left free wall (n = 15), posteroseptal (n = 5), and right free wall (n = 3) accessory pathways from the RV apex and then from the summit of the RV septum. When the His was refractory, shortening of the atrial cycle length was noted in 13% of patients with left free wall pathways, in 60% of patients with posteroseptal pathways, and in 100% of patients with right free wall pathways with VPCs from the RV apex, and in 47, 100 and 100%, respectively, with VPCs from the summit of the septum. When all VPCs were considered, there was a significant shortening of the atrial cycle length in 67% of patients with left free wall pathways when stimulated from the RV apex, which increased to 93% with summit stimulation. An extrastimulus applied on or after the His effected a significant shortening of the atrial cycle length in no patients with left free wall pathways.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis C testing. Comparison of Ortho's EIA and RIBA II tests in 1,182 patients undergoing primary liver transplantation.

Plasma samples from 1,182 patients undergoing primary liver transplantation were tested for anti-hepatitis C (HCV) virus by two methods: Ortho HCV ELISA Test System (EIA) and Chiron RIBA HCV Test System (RIBA II). The EIA results, 0 or +, were recorded first, followed by RIBA results, N = negative, P = positive, or I = indeterminate. Concordant results--0N, + P, + I--were found in 1,076 (91%), and discordant results were found in 106 (9%). The EIA optical density did not relate to concordant or discordant results. Band patterns were described by stating the band position (1, 2, 3, or 4) and inserting a dash (-) if no band was visualized. Most + P samples fell into two patterns: 47% showed all four bands, pattern 1234, and 15% showed the two-band pattern, 34. When the EIA was negative, 0P, the opposite was seen: 8% showed the 1234 pattern and 81% showed the 34 pattern. There were 226 samples that formed bands (+ P, 149; 0P, 31; + I, 15; 0I, 31). The frequency of bands was as follows: 4, 32%; 3, 31%; 2, 19%; and 1, 18%. Band 2 and the EIA test detected antibodies to the same c100-3 fragment and showed 74% concordance. No explanation is apparent for the lower concordance rate here than that between the EIA test and bands 3 = 96% or 4 = 88%. The EIA and RIBA II tests, together with positive liver function tests and abnormal tissue pathologic findings, provide a basis for the diagnosis of HCV.


Ca(2+)-activated nonselective cation channel in apical membrane of vestibular dark cells.

Patch-clamp recordings were made on cell-attached and excised apical membrane from dark cells of the semicircular canal of the gerbil. These cells are thought to secrete K+ and absorb Na+ from the luminal fluid (endolymph). Single-channel events were identified as being equally conductive (27.6 +/- 0.4 pS; n = 48) for K+, Na+, Rb+, Li+, and Cs+ and 1.4 times more permeable to NH4+ but not permeable to Cl-, Ca2+, Ba2+, nor to N-methyl-D-glucamine. The channels displayed linear current-voltage relations that passed nearly through the origin (intercept: -2.6 +/- 0.5 mV; n = 48) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face; there was no activity at less than or equal to 10(-7) M Ca2+ and full activity at greater than or equal to 10(-5) M Ca2+. Cell-attached recordings had a mean reversal voltage of -36.4 +/- 7.9 mV (n = 7), which was interpreted to reflect the intracellular potential of dark cells under the present conditions. We have identified a nonselective cation channel in the apical membrane of vestibular dark cells that might participate in K+ secretion or Na+ absorption under stimulated conditions, but the density appears to be insufficient to fully account for the transepithelial K+ flux.


InsP3 and Ins(1,3,4,5)P4 act in synergy to stimulate influx of extracellular Ca2+ in Xenopus oocytes.

To investigate the role of D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in the regulation of Ca2+ influx, we injected inositol phosphates into Xenopus oocytes and measured Ca(2+)-gated Cl- current to assay intracellular free Ca2+ concentration ([Ca2+]i). To assess Ca2+ influx, we removed extracellular Ca2+ or added the inorganic Ca2+ channel blocker Mn2+ to the extracellular bath and measured the resulting change in Cl- current. Ins(1,3,4,5)P4 did not cause Ca2+ influx when injected alone or when preceded by an injection of Ca2+. In contrast, Ins(1,3,4,5)P4 stimulated Ca2+ influx when injected after the poorly metabolized inositol trisphosphate (InsP3) analogues D-myo-inositol 1,4,5-trisphosphorothioate [Ins(1,4,5)P3S3] or D-myo-inositol 2,4,5-trisphosphate [Ins(2,4,5)P3]. These results indicate that Ins(1,3,4,5)P4 is not sufficient to stimulate Ca2+ influx but acts in synergy with InsP3s to cause Ca2+ influx. We also studied the effect of Ca2+ influx on the immediate metabolism of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in single oocytes. Ca2+ influx shunted the metabolism of Ins(1,4,5)P3 toward the formation of Ins(1,3,4,5)P4 and away from D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2]. These results suggest that there is a positive feedback regulatory mechanism in which Ca2+ influx stimulates Ins(1,3,4,5)P4 production and Ins(1,3,4,5)P4 stimulates further Ca2+ influx.


Blockers of platelet-derived growth factor-activated nonselective cation channel inhibit cell proliferation.

In serum-deprived G(o)-arrested cells, the addition of serum or growth factors initiates a cascade of events that culminates in DNA synthesis and mitosis. Recently, we showed that in mouse L-M(TK-) fibroblasts a 28-pS nonselective cation channel (NS channel) becomes quiescent at G(o) arrest and rapidly active within seconds of platelet-derived growth factor (PDGF) or serum addition, placing this response very early in the postreceptor signaling cascade. However, lack of specific channel blockers hindered determination of whether channel activation was necessary for mitogenesis. Derivatives of N-phenylanthranilic acid (DCA) have been reported to block a pancreatic nonselective channel. Therefore, using single-channel analysis, we examined the effect of these agents on the L-M(TK-) NS channel. Flufenamic acid and mefenamic acid rapidly produced reversible channel block with an inhibitory constant (Ki) approximately 10 microM. Furthermore, the component of the macroscopic K+ efflux shown to be mediated by the NS channel was blocked with a similar Ki value. DCA effects on cell proliferation were tested by measuring cloning efficiency and growth rate. Both were inhibited over the range of concentration that affected channel activity, and a 50% inhibitory dose of 50-100 microM was determined. This observation further substantiates the hypothesis that NS channel activation forms a necessary component in the transduction of the mitogenic signal from the PDGF receptor.


Gs and Gi protein subunits during cell differentiation in intestinal crypt-villus axis: regulation at the mRNA level.

The expression of subunits of the guanine nucleotide regulatory protein that mediates hormonal stimulation of adenylyl cyclase (Gs) and of the guanine nucleotide regulatory protein that mediates hormonal inhibition of adenylyl cyclase (Gi) was studied during cell migration and differentiation in the rat small intestine crypt-villus axis. Proliferative crypt cells were separated from nonproliferative mature villus cells and the following data were obtained: 1) alpha s subunits were more abundant in crypt cells than in villus cells as evidenced by cholera toxin-catalyzed [32P]NAD ribosylation and Western blotting of this relative molecular weight (M(r)) 42,000 protein; 2) alpha i2- and alpha i3-subunits (M(r) 40,000 and M(r) 41,000, respectively) were preferentially expressed in villus cells as evidenced by pertussis toxin-catalyzed [32P]NAD ribosylation and Western blotting (alpha i1-subunit was not detectable in intestinal epithelium by using these techniques); 3) Western blotting showed a higher expression of the common beta- (M(r) 36,000) subunit of G proteins in villus cells than in crypt cells; and 4) Northern blot analysis using an alpha s-subunit oligonucleotide probe showed a 1.9-kb mRNA that was more abundant in crypt cells than in villus cells. In contrast, alpha i2- and alpha i3-mRNA species (2.3 and 3.5 kb, respectively), analyzed by using specific cDNA probes, were much more abundant in villus cells than in crypt cells. Finally, two beta-subunit mRNA species of 3.3 and 1.8 kb were detectable in intestinal epithelial cells and were more abundant in villus cells than in crypt cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Sepsis-induced changes in protein synthesis: differential effects on fast- and slow-twitch muscles.

Sepsis is associated with severe muscle wasting. Mechanisms responsible for sepsis-induced alterations in muscle protein metabolism were investigated in vivo and compared with changes induced by nonseptic inflammation. The rate of protein synthesis in mixed hindlimb muscles was not altered in inflammation but was inhibited 50% in sepsis. This inhibition did not result from a decreased RNA content. Instead, the translational efficiency was significantly reduced by 50% in skeletal muscle of septic animals compared with control. The effect of sepsis to lower the rate of protein synthesis was further examined using individual muscles containing different fiber types. Both the protein concentration and protein synthetic rate in fast-twitch muscles were reduced by sepsis, whereas neither of these parameters was affected in slow-twitch muscles or heart. The decreased translational efficiency did not result from a change in the rate of peptide-chain elongation. Instead, the sepsis-induced inhibition of protein synthesis resulted from a restraint in peptide-chain initiation because sepsis caused a 1.6-fold increase in free ribosomal subunits. Overall, sepsis, but not inflammation, caused an inhibition of protein synthesis primarily in muscles composed of fast-twitch fibers. The mechanism involved in the reduced rates of protein synthesis in muscles resulted from an inhibition of peptide-chain initiation, with no change in peptide-chain elongation.


Regulation of alpha 1-beta 3-NA(+)-K(+)-ATPase isozyme during meiotic maturation of Xenopus laevis oocytes.

During progesterone-induced maturation of Xenopus oocytes, the transport and ouabain binding capacity of Na(+)-K(+)-ATPase at the plasma membrane is completely downregulated. To elucidate the mechanism and the physiological significance of this process, we have followed the fate of oocyte alpha-beta 3-Na(+)-K(+)-ATPase complexes during meiotic maturation and early embryonic development. An immunocytochemical follow-up of the catalytic alpha-subunit, ouabain binding studies, cell surface iodination, and oocyte cell fractionation combined with immunochemical subunit detection provides evidence that following progesterone treatment Na(+)-K(+)-ATPase molecules are retrieved from the oocyte plasma membrane. The enzyme complexes are recovered in an active form in an intracellular compartment in both in vitro and in vivo matured eggs. Exogenous Xenopus alpha 1- and beta 1-complexes expressed in the oocyte from injected cRNAs are regulated by progesterone similar to endogenous Na(+)-K(+)-ATPase complexes. Finally, active Na(+)-K+ pumps internalized during oocyte maturation appear to be redistributed to plasma membrane fractions during blastula formation in Xenopus embryos. In conclusion, our data suggest that endocytosis of alpha 1- and beta 3-complexes during meiotic maturation of Xenopus oocytes is responsible for downregulation of Na(+)-K(+)-ATPase activity and results in an intracellular pool of functional enzymes, which might be reexpressed during early development in response to physiological needs.


Effect of osmotic swelling on K+ conductance in jejunal crypt epithelial cells.

To further elucidate differences in ion transport properties between jejunal crypt and villus cells, we compared the responses of purified cell suspensions to hypotonic stress using electronic cell sizing to evaluate volume changes and 86Rb and 36Cl efflux. After hypotonic swelling, villus enterocytes undergo a regulatory volume decrease (RVD) due to the loss of K+ and Cl- through volume-activated conductances. After 0.6x isotonic challenge in Na(+)-free medium, crypt cells exhibited only partial RVD, with t1/2 congruent to 15 min. The addition of a cation ionophore, gramicidin (0.25 microM), to hypotonically swollen crypt cells caused an accelerated RVD, which was complete with t1/2 congruent to 5 min. Crypt epithelial cells showed no volume-activated 86Rb efflux, but villus enterocytes had an increased rate of 86Rb efflux after hypotonic dilution (P less than 0.001). Gramicidin added to hypotonically diluted crypt cells greatly increased the rate of 86Rb efflux compared with controls. Both villus (30 s; P less than 0.005) and crypt (2 min; P less than 0.001) cells exhibited volume-activated 36Cl efflux in absence of gramicidin. Cl- channel blockers anthracene-9-carboxylate (9-AC, 300 microM) and indanyloxyacetic acid (IAA-94, 100 microM) prevented crypt RVD (P less than 0.001) in the presence of gramicidin. Ouabain (P less than 0.001) or K(+)-free Na(+)-containing medium, but not Ba2+ (5 mM) or quinine (100 microM), prevented crypt partial RVD. We conclude that crypt cells lack volume-activated K+ conductance. The RVD exhibited by crypt cells, although partial, was due to Cl- loss through a volume-activated Cl- conductance and Na+ loss via Na(+)-K(+)-ATPase.


Anion channels in rat liver canalicular plasma membranes reconstituted into planar lipid bilayers.

Previous studies from this laboratory have demonstrated a Cl(-)-HCO3- exchanger and have provided evidence for a Cl- conductance in rat liver canalicular plasma membrane vesicles. To further investigate the apical Cl- conductance, we performed single-channel analysis after incorporation of canalicular liver plasma membrane vesicles into planar lipid bilayers. This was necessary, because the canalicular membrane is not accessible for the patch-clamp technique. Two types of anion channels could be identified (30- and 90-pS conductance) corresponding to the class of small and intermediate channels, respectively. The kinetics of the small channel were found to be voltage dependent with a maximum for the open probability at -20 mV. In contrast, intermediate channel kinetics were voltage independent. The anion channels described above could allow electrogenic Cl- efflux, to compensate Cl- influx via the electroneutral Cl(-)-HCO3- exchanger. Further studies will be required to prove their functional importance in bile formation.


Dietary regulation of pancreatic amylase in transgenic mice mediated by a 126-base pair DNA fragment.

Expression of the mouse pancreatic amylase gene Amy-2.2 is increased approximately 10-fold in response to increasing the carbohydrate content of the diet from 9.6 to 74%. The DNA sequence mediating this response has been localized to the 5' flanking region of the amylase gene by analysis of hybrid constructs in transgenic mice. The results define a 127-base pair dietary response unit that includes two previously described regulatory elements, an insulin-responsive element and a pancreatic enhancer. Fragments containing these two elements alone fail to respond to diet, demonstrating a requirement for additional regulatory sequences. Another mouse amylase gene Amy-2.1 is only minimally responsive to insulin and to diet. The data are consistent with the hypothesis that the insulin-response element is necessary but not sufficient for regulation of amylase by dietary carbohydrate.


Growth and developmental regulation of wnt-2 (irp) gene in mesenchymal cells of fetal lung.

The wnt gene family encodes a group of proteins implicated as intercellular signaling molecules in vertebrate development. Because many wnt genes are also expressed in the lung, we have examined whether the wnt family member wnt-2 (irp) plays a role in lung development. We have cloned rat wnt-2 and found that this cDNA detects multiple mRNAs expressed at high levels in fetal rat lung. Much lower levels were found in adult rat lung and other tissues, including, surprisingly, the mammary gland. The wnt-2 mRNA was also detected in human fetal lung fibroblast cell lines, where the mRNA levels were dramatically regulated by growth state as well as growth factor stimulation. In situ hybridization showed that, in fetal rat lung, wnt-2 mRNA expression is restricted to the mesenchyme; levels in the developing epithelium were indistinguishable from background. Based on the known properties of other wnt proteins, our data lead us to propose that wnt-2 may play a role in lung development by mediating intercellular interaction(s) between mesenchyme and epithelium.


Monocrotaline pyrrole alters DNA, RNA and protein synthesis in pulmonary artery endothelial cells.

Administration of monocrotaline pyrrole (MCTP) to animals results in pulmonary vascular injury. Pulmonary vascular endothelium is a likely target for this pneumotoxicant. Cultured porcine pulmonary artery endothelial cells (PECs) treated with MCTP remain viable but are unable to divide and exhibit an altered morphology. Such responses raise a question about the extent to which affected cells carry out normal functions such as RNA and protein synthesis. Accordingly, the cellular activity of MCTP-treated PECs was examined in this study. PECs were treated with a single administration of MCTP or vehicle, and determinations of cell number, protein, and DNA content were made at times up to 7 days posttreatment. DNA, RNA, and protein synthesis were quantified by incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine, respectively. Increases in cell number that occurred with time in the control cells were reduced in MCTP-treated cells. At 7 days posttreatment, both protein and DNA content increased above control levels. Synthesis of DNA, RNA, and protein continued in all treatment groups throughout the posttreatment period, but cells treated with high concentrations of MCTP showed less synthetic activity than controls during the initial 48 h posttreatment. By 7 days, MCTP-treated cells were producing significantly more DNA, RNA, and protein. These results indicate that cells treated with MCTP continue to synthesize DNA, resulting in an increased DNA content. In addition, treated cells continue to synthesize RNA and translate RNA into protein. Thus, cellular activity is maintained but altered substantially by MCTP exposure.


Cloning and sequence analysis of rat cystic fibrosis transmembrane conductance regulator.

A complementary DNA (cDNA) encoding the rat cystic fibrosis transmembrane conductance regulator (CFTR) has been isolated and the tissue distribution of the rat CFTR mRNA has been determined. Northern blot analysis revealed that the highest levels of the 6.3 kilobase (kb) CFTR mRNA were expressed in the colon, with expression also noted in uterus, lung, stomach, and small intestine. A 7.5-kb mRNA was expressed in skeletal muscle, and in testes both the 7.5-kb mRNA and a 6.0-kb mRNA were expressed. Five cDNAs were isolated from a rat colon library, the longest corresponding to codons 684 through the poly A tail. Three other clones, corresponding to codons 213 through 245, 372 through 574, and 656 through 886 were also isolated. Polymerase chain reaction amplification of cDNA prepared from rat colon mRNA was utilized to clone the remainder of the cDNA. The predicted amino acid sequence of the rat CFTR is 79% identical to the human CFTR, with 73% identity noted in the R domain, and 81 and 83% identities noted in nucleotide binding folds 1 and 2, respectively. Thirty-two of the 38 potential phosphorylation sites identified in the human CFTR were also present in the rat CFTR.


[Histamine release during induction of combination anesthesia using nalbuphine or fentanyl. Modulation of the reaction by premedication with promethazine/pethidine].

In a controlled clinical trial in patients admitted for general surgery (mainly abdominal and thyroid), histamine release following nalbuphine 1 mg/kg i.v. versus fentanyl 5 micrograms/kg i.v. was studied in the course of an otherwise routine induction with promethazine/pethidine as premedication 30 min before the opioids and alcuronium-flunitrazepam-thiopental 5 min later. Succinylcholine was given before intubation and further analgesia was obtained by repeated administration of either nalbuphine or fentanyl. Plasma histamine levels were measured by a specific fluorometric assay, heart rate and blood pressure were measured for assessing hemodynamics, and clinical signs of anaphylactoid reactions such as skin eruptions and arrhythmias were registered. RESULTS. Nalbuphine and fentanyl both released histamine with an incidence of more than 40%. In addition, nalbuphine potentiated the histamine release evoked by the sequential administration of alcuronium-flunitrazepam-thiopental in one complex of application. The incidence of histamine release in the nalbuphine group was 6/13 = 46%, in the fentanyl group only 1/11 = 9% (chi2 test, P less than 0.05). Furthermore, this study showed high histamine levels after succinylcholine and intubation in a relation to time of administration that suggested histamine release as a stress response to intubation. Finally, the incidence of histamine release after a second injection of the opioids was still 30%. A direct correlation between plasma histamine levels, hemodynamic changes, and skin reactions could not be shown. A detailed causality analysis with histamine release as a contributory determinant showed histamine release less detrimental to hemodynamic stability than the opposite, which had been expected. However, the promethazine administered 30 min before induction of anaesthesia had strong H1- and H2-receptor antagonistic activity and was given with optimum timing for H1- and H2-prophylaxis. CONCLUSION. The study demonstrated that histamine release during anaesthesia and surgery depends strongly on the time sequence of drugs and measures used. Histamine release is not predictable from studies in human volunteers alone; studies in patients have to be added. Histamine release is not always detrimental. H3-receptor-mediated effects after H1- and H2-prophylaxis may help patients to counteract the effects of a series of vasoactive drugs given during induction of anaesthesia.


Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates.

We report an ultrasensitive, enzymatically amplified, time-resolved fluorescence immunoassay with a terbium chelate as the detectable moiety. In this immunoassay, the primary label is the enzyme alkaline phosphatase (ALP). ALP cleaves phosphate out of a fluorogenic substrate, 5-fluorosalicyl phosphate, to produce 5-fluorosalicylic acid (FSA). 5-Fluorosalicylic acid can then form a highly fluorescent ternary complex of the form FSA-Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. In this assay, exceptional sensitivity is achieved because of the enzymatic amplification introduced by ALP and the quantification by laser-induced microsecond time-resolved fluorometry. Time-resolved fluorometry is applicable because of the long fluorescence lifetime of the Tb3+ complexes. It is shown that in a model AFP assay 10(6) or 1.5 x 10(5) molecules can be detected (final assay volume, 100 microL) by using monoclonal or polyclonal detection antibodies, respectively. The assay demonstrates excellent precision (approximately 4%), and it seems to be highly suited for automated, sensitive, and rapid immunoassays.


Influence of hydroxyethyl starch on coagulation in patients during the perioperative period.

The perioperative use of hydroxyethyl starch (HES) has been implicated as a possible cause of intracranial bleeding. The purpose of this study was to compare the influence on blood coagulation of the isovolemic replacement of 1-L blood loss with either 6% HES (molecular weight [MW] average: 450,000) or 5% human albumin during neurosurgery or lower abdominal surgery. Twenty patients scheduled for brain tumor surgery and 20 patients undergoing transabdominal hysterectomy were studied. The activated partial thromboplastin time, prothrombin time, fibrinogen concentration, factor VIII coagulant, von Willebrand factor antigen, platelet count, and the activated clotting time were compared after induction of anesthesia, after administration of 500 and 1000 mL of colloid solution, and 24 and 48 h postoperatively. All measured coagulation variables remained within physiologic range. Changes in coagulation indices were identical in neurosurgical and hysterectomy patients, except for a larger increase in fibrinogen concentration 24 and 48 h after hysterectomy. The acute phase reaction of factor VIII coagulant and von Willebrand factor, which plays a role in postoperative hypercoagulability, was attenuated by the use of HES. We conclude that isovolemic replacement of 1-L blood loss with either 6% HES (MW average: 450,000) or 5% human albumin does not interfere with normal hemostasis during and after neurosurgery or lower abdominal surgery.


Halothane relaxes preconstricted small and medium isolated porcine coronary artery segments more than isoflurane.

To compare the putative vasodilatory effects of isoflurane versus halothane on porcine coronary arteries, we studied the capacity of isoflurane and halothane to relax K(+)-constricted (30 mM) small (0.5-1.0 mm outside diameter [OD]) and medium (1.0-1.5 mm OD) porcine coronary arteries with use of in vitro tension recording. We also examined the effect of the dihydropyridine calcium channel agonist BAY K8644 on previously constricted epicardial porcine coronary artery segments in the presence of halothane or isoflurane. Our purpose was to determine (a) whether anesthetic effect on coronary arteries varied with arterial diameter, and (b) whether halothane and isoflurane inhibited BAY K8644-induced contraction of coronary vessels. Small and medium porcine coronary artery segments were constricted with K+ (30 mM) and the resulting contraction was allowed to stabilize. This was followed by exposure to 0.5%, 1.0%, 2.0%, and 3.0% isoflurane or halothane and the resultant tension was again measured. Potassium-induced contractions were significantly relaxed by halothane in small coronary artery segments at 0.5%, 1.0%, 2.0%, and 3.0% and in medium coronary artery segments at 1.0%, 2.0%, and 3.0%. Potassium-induced contractions were significantly reduced by isoflurane only at 3.0% in both small and medium coronary artery segments. Halothane caused significantly more relaxation of both small and medium porcine coronary arteries previously constricted with K+ (30 mM) than did isoflurane. There were no significant differences in coronary artery response to isoflurane or halothane with respect to coronary artery diameter. These experiments indicate that in porcine coronary arteries greater than 0.5 mm OD, studied in vitro after K(+)-induced contraction, isoflurane was not a potent coronary vasodilator.(ABSTRACT TRUNCATED AT 250 WORDS)


Treatment of gastric lymphoma.

A review of 52 patients with gastric lymphoma at the Texas A&M University College of Medicine Affiliated Scott and White Memorial Hospital (Temple, TX). was performed to determine the influence of different treatment modalities. Thirty-one patients had a potentially curative resection, while 21 underwent a palliative procedure or biopsy alone. Overall 5-year survival was 73.4 per cent after curative resection and 38.3 per cent for lesser operative procedures (P less than .005). Adjuvant radiation was given to 14 patients after curative resection with a 5-year survival rate of 71.5 per cent compared to 82.4 per cent in the 17 patients treated by curative resection alone (nonsignificant). Patients who underwent palliative surgery or biopsy who received postoperative radiation therapy had a 38.0 per cent 5-year survival rate compared to a 0.0 per cent 5-year survival rate in patients who received no therapy (P = .18). The authors conclude that curative resection is the treatment of choice for gastric lymphoma, but radiation therapy may offer some benefit when complete resection is not feasible.


Tracheo-esophageal fistula complicating carcinoma of the esophagus.

In a 35-year review of 557 patients with esophageal carcinoma, 26 tracheal esophageal fistulas were encountered. Seventeen arose among patients with advanced disease or poor performance status and these patients were given comfort measures only. Treatment was attempted in 9 patients, 4 of whom died peri-operatively. The remaining 5 avoided early pulmonary deaths. In principle, esophageal exclusion and bypass should provide the best palliation in patients whose performance status permits it, but extensive surgical procedures are futile in patients with established pulmonary sepsis. For most patients, not candidates for exclusion procedures, endoprostheses are more appropriate.


[Staging evaluation and surveillance in tumors of the testicle].

In 1992, the staging and follow-up of testicular germ cell tumours is based on a combination of computed tomography and tumour markers. Due to the development of medical imaging over the last decade, abdominal and thoracic CT has now replaced the combination of lymphography and pulmonary tomographies. Testicular ultrasonography is valuable for the diagnosis and contributes to staging and follow-up. The chest x-ray is still performed and MRI has very exceptional indications. Tumour markers, essentially alpha-foetoprotein and the free beta fraction of human chorionic gonadotrophin, are useful in more than 80% of NSGCTs and about 15% of seminomas (beta-HCG alone). A very high initial level often indicates a poor prognosis. Monitoring of markers is essential after exclusive orchidectomy and to assess the efficacy of chemotherapy.


Antikeratin antibodies: diagnostic and prognostic markers for early rheumatoid arthritis.

Antibodies to the stratum corneum of rat oesophagus (antikeratin antibodies) were assayed by indirect immunofluorescence in a prospective study of patients with early rheumatoid arthritis (RA). At the beginning of the study, antikeratin antibodies of IgG class were detected in serum samples from 27/71 (38%) patients compared with 1/20 (5%) control patients with reactive arthritis, and 1/38 (3%) healthy blood donors. At the end of the two year follow up, 27/67 (40%) patients with RA were positive for antikeratin antibodies. The patients with RA who were initially positive for antikeratin antibodies had a more active disease course than the patients negative for antikeratin antibodies as measured by clinical, laboratory, and radiological variables. The prevalence of positivity for antikeratin antibodies fluctuated during the follow up, the variation paralleling the disease activity. The occurrence of HLA-DR4 was similar in patients with RA who were positive and negative for antikeratin antibodies. Antikeratin antibodies were also found in seronegative patients with RA, confirming that antikeratin antibodies do not have rheumatoid factor activity. These results show that antikeratin antibodies are detectable at the time of the initial diagnosis of RA and that the positivity for antikeratin antibodies may have prognostic significance in early RA.


Neurotensin augments intestinal regeneration after small bowel resection in rats.

Massive small bowel resection (SBR) is characterized by increased proliferation of residual gut mucosa and pancreas. Neurotensin (NT), a gut tridecapeptide, stimulates growth of normal gut mucosa and pancreas. This study examined whether NT affected growth of the small intestine and the pancreas after either distal or proximal SBR. Male Fischer 344 rats were divided into four groups. Group 1 underwent ileal transection with reanastomosis (SHAM) and group 2 underwent 70% distal SBR. Group 3 underwent SHAM operation (jejunal transection), and group 4 underwent 70% proximal SBR. After operation, each group was further subdivided to receive either saline (control) or NT (300 micrograms/kg) subcutaneously in gelatin every 8 hours for 7 days. At death, the pancreas and proximal jejunum (from groups 1 and 2) or distal ileum (from groups 3 and 4) were removed, weighed, and analyzed for DNA, RNA, and protein content. Both proximal and distal SBR significantly increased mucosal growth in the remnant intestine; a more pronounced effect was noted with proximal SBR. Administration of NT significantly augmented the adaptive changes in both groups of rats by mechanisms involving increases in both cell size (hypertrophy) and cell number (hyperplasia). Pancreatic growth was stimulated by distal (but not proximal) SBR; NT did not augment this response. The authors conclude that NT augments intestinal growth after SBR by mechanisms involving an increase in overall mucosal cellularity. Administration of NT may be therapeutically useful to enhance mucosal regeneration during the early period of adaptive hyperplasia after SBR.


Extracellular collagenous spherules in salivary gland tumors. Immunohistochemical analysis of laminin and various types of collagen.

Collagenous spherulosis is a benign breast lesion involving lobular acini and ductules and containing eosinophilic spherules measuring up to 100 microns in diameter. We present an immunohistochemical analysis of similar collagen-rich spherules that are also found in salivary gland tumors. These collagenous spherules contain varying amounts of acidic mucins, elastin, basement membrane proteins including type IV collagen and laminin, and considerable amounts of interstitial collagen types I and III. Types II and VI collagen were not detected in collagenous spherules of salivary gland tumors. The cells surrounding these collagenous spherules expressed muscle actin, S100 protein, vimentin, and cytokeratins 8, 18, and 19, indicating that these cells have myoepithelial characteristics.


Developments in the control of testicular function.

Clinicians and clinical investigators have developed improved means for controlling testicular function in men. New and refined approaches for stimulation and inhibition of the hypothalamic-pituitary-testicular axis are now available. This chapter reviewed the most successful ways to inhibit the reproductive axis in men and its current application to the treatment of precocious puberty, metastatic prostate cancer, benign prostate hyperplasia and as prospective male contraceptives. Safe, effective and reversible medical approaches to male contraception are now approaching reality. Azoospermia and severe oligozoo/azoospermia can now be accomplished in the majority of men with combined GnRH antagonists and replacement doses of testosterone. Androgens and androgen-progestogen concentrations will induce azoospermia in over 90% of Asian men and azoospermia or severe oligospermia in Caucasian ethnic groups. Field trials are ongoing to determine whether testosterone administration will be more effective than condoms as contraceptives. True precocious puberty can now be managed more effectively than in the past by suppression of gonadotropin secretion with GnRH analogues. Precocious puberty due to other causes can be treated more effectively with inhibitors of steroidogenesis and blockers of androgen action. Metastatic prostate cancer, previously treatable with either castration or oestrogens, is now amenable to suppression of androgen secretion. GnRH analogues are given either alone or combined with blockers of androgen action. While significant palliative effects are observed with endocrine ablative therapy in most men with Stage C or D prostate cancer, modest increases in duration of survival may be seen. Benign prostate hyperplasia was previously approachable only with surgical intervention. Recent data have suggested that medical treatment with 5 alpha-reductase inhibitors and/or selective alpha-adrenergic blockers may offer non-surgical alternatives in some patients. More data are needed to determine the role of medical management of this common disorder.


Apparent specificity of bovine seminal ribonucleases can depend on the conditions used for the isolation of substrate.

RNAase SPL, a ribonuclease isolated earlier from bovine seminal plasma, was shown to possess the ability to produce large acid-insoluble fragments of Mg(2+)-containing RNA in a limit digest. The factor which could be responsible for this apparent specificity has been identified as polyvinyl sulphate; it has been shown that polyvinyl sulphate inhibits RNAase SPL at much lower concentrations than required for RNAase A. The earlier results are now reinterpreted based on this effect of polyvinyl sulphate, thus providing a plausible explanation for RNAase SPL's apparent specificity. RNAase SPL has been shown to be a mixture of two ribonucleases, RNAase SPL I and RNAase SPL II. RNAase SPL I is like RNAase A in its activity while RNAase SPL II, the major ribonuclease in seminal plasma, appears to be identical to RNAase BS1.


Mammalian fetuin-binding proteins sarcolectin, aprotinin and calcyclin display differences in their apparent carbohydrate specificity.

The interferon antagonist sarcolectin, the protease inhibitor aprotinin and calcyclin whose expression is regulated by growth stimulation in quiescent fibroblasts display sialic acid-dependent binding to fetuin, visualized by solid-phase assays using biotinylated fetuin. The potential functional importance of this property prompted its comparative characterization with an array of neutral or negatively charged sugars, sulfated polysaccharides and sialoglycoproteins as inhibitors of binding of biotinylated fetuin to the immobilized proteins. The results revealed that this activity of sarcolectin and calcyclin is nearly unaffected by charge-free carbohydrates in contrast to aprotinin, calcyclin exhibits a notable affinity for Neu5Gc and together with aprotinin for phosphorylated sugars, and that sarcolectin's binding is affected to the highest extent by sulfated sugars relative to aprotinin and calcyclin.


Characterization of "modified-self"-induced specific antibody hyporesponsiveness to herpes simplex virus.

Pretreatment of mice iv with syngeneic spleen cells modified with soluble HSV envelope antigens induced an anti-HSV antibody hyporesponsiveness following challenge with infectious virus. The epitope density on the HSV-modified spleen cells was quantitated using a photon-counting spectrofluorimeter so that observed immunological results could be correlated with the HSV antigen dose on the splenocytes. The degree of anti-HSV antibody hyporesponsiveness was found to be related to the epitope density on the HSV-modified spleen cells, but not the number of modified cells used in the pretreatment over the 16-fold range tested. Anti-HSV antibody hyporesponsiveness was induced if 7, but not 3, days had elapsed between pretreatment and challenge. This antibody hyporesponsiveness could be adoptively transferred with T cells. Only mice that had induced an anti-HSV antibody hyporesponsiveness following pretreatment with HSV-modified splenocytes were able to survive an LD50 challenge with infectious virus.


[Meningiomas: immunohistochemical analysis of 26 cases].

We have analyzed, with immunohistochemical staining techniques 26 meningiomas of CNS. Vimentin intermediate filaments was present in all cases in the cytoplasm of tumoral cells. Single epithelium cytokeratin was positive in only 19% cases, same that was obtained with S-100 protein. Whereas 69% of cases expressed epithelial membrane antigen. No case was stained positively with Actin, Desmin and GFAP. The results obtained for others authors are analyzed.


Conformational variations of the cis-syn cyclobutane-type photodimer in DNA and RNA.

The recent NMR study of a cis-syn photodimer B-DNA 10mer-duplex (Taylor et al., Biochemistry 29, 8858 (1990)) showed the cyclobutane (CB) ring with a puckered-twist in a right-handed sense (CB+). This is opposite to that of the crystal structure of cis-syn d-TpT(cyano-ethyl)(d-T[p]T-CE) which has a left-handed puckered-twist (CB-)(Hruska et al., Biopolymers 25, 1399 (1986)). 2D-NOESY experiments were performed on cis-syn d-T[p]T and cis-syn U[p]U at 25 and 35 degrees C, respectively, to investigate the puckering mode of the cyclobutane ring of isolated cis-syn photodimers of the DNA and RNA types. The DNA photodimers showed interconversion of the puckered-twist of the cyclobutane ring between CB- and CB+ and interconversion of the glycosidic angle between syn and anti in both nucleoside residues. Interestingly, in the RNA photodimer only the CB- puckering mode with syn conformation of the glycosidic angle of the U[p]- was observed. These different dynamical behaviors of the photodimer in DNA and RNA might portend differential conformational effects on their corresponding normal nucleic acid regions. In addition these results indicate differences in the cyclobutane ring conformation of the cis-syn d-T[p]T, not only in solution and crystalline states, but also when the dimer is isolated and in duplex forms.


Analysis of an RNA pseudoknot structure by CD spectroscopy.

The RNA PK5 (GCGAUUUCUGACCGCUUUUUUGUCAG) forms a pseudoknotted structure at low temperatures and a hairpin containing an A.C opposition at higher temperatures (J. Mol. Biol. 214, 455-470 (1990)). CD and absorption spectra of PK5 were measured at several temperatures. A basis set of spectra were fit to the spectra of PK5 using a method that can provide estimates of the numbers of A.U, G.C, and G.U base pairs as well as the number of each of 11 nearest-neighbor base pairs in an RNA (Biopolymers 31, 373-384 (1991)). The fits were close, indicating that PK5 retained the A conformation in the pseudoknot structure and that the fitting technique is not hindered by pseudoknots or A.C oppositions. The results from the analysis were consistent with the pseudoknotted structure at low temperatures and with the hairpin structure at higher temperatures. We concluded that the method of spectral analysis should be useful for determining the secondary structures of other RNAs containing pseudoknots and A.C oppositions.


Decreased expression of the membrane inhibitor of complement-mediated cytolysis CD59 on T-lymphocytes of HIV-infected patients.

To study the expression of the membrane inhibitor of complement-mediated cytolysis, CD59, on peripheral blood mononuclear cells (PBMC) from HIV-infected individuals. CD59 membrane expression was investigated by double immunofluorescent staining on purified PBMC from HIV-infected patients and seronegative controls. CD59 expression was decreased on peripheral blood T-lymphocytes from HIV-infected patients. Decreased expression of CD59 antigen did not correlate with clinical stage of disease. Low CD59 expression on T-lymphocytes was mediated by several mechanisms, including: (1) decrease in number of CD4+ cells which normally express high amounts of the antigen; (2) relative increase in CD8+ cells, more specifically in CD8+ CD57+ cells expressing a low density of the antigen; (3) loss of or decreased CD59 expression on a subpopulation of CD3+ belonging either to the CD4+ or to the CD8+ subset. Decreased CD59 expression was consistently observed on CD8+ lymphocytes. CD59 expression was normal on patient red blood cells, monocytes and B-lymphocytes. HIV infection is associated with a T-cell-specific defect in CD59 membrane expression. Decreased CD59 expression may result in increased susceptibility of T-lymphocytes from HIV-infected patients to complement-mediated lysis.


Cytolytic T-cell activity against mycobacterial antigens in HIV.

The declining incidence of tuberculosis (TB) in developed countries has recently been reversed with the advent of HIV disease. This study proposes to document in vitro T-cell responses to mycobacterial antigens in HIV-infected individuals. T-cell-mediated immunity is recognized as one of the mechanisms of defence against TB. The cellular immunodeficiency and the importance of TB in the context of HIV disease has prompted use of in vitro assays of lymphocyte proliferation and cytolytic activity. Peripheral blood mononuclear cells isolated from 29 HIV-infected patients (four with recent TB) and 11 healthy volunteers were stimulated with purified protein derivative (PPD). The responding blasts were presented to autologous antigen-primed macrophages to measure specific cytolytic T-lymphocyte (CTL) activity in vitro. T-cell proliferative responses were significantly lower in late stages of HIV disease. The degree of specific CTL activity was higher in healthy individuals than in Centers for Disease Control (CDC) stage II-III (P = 0.037), and CDC stage IV patients (P = 0.029). The clinical presentation of TB tends to be typical in early stages of HIV disease and atypical in late stages. The manifestations reflect the degree of immunodepression. This study documents the declining proliferative and cytolytic T-cell-mediated responses in HIV patients with progression of immunodeficiency.


Single-step purification on DEAE-sephacel of recombinant polypeptides produced in Escherichia coli.

We describe a method for the purification of recombinant proteins based upon the selective interaction of the choline-binding domain of the pneumococcal murein hydrolase and tertiary amines. Proteins of interest, fused to the binding domain by a peptide linker, containing the cleaving sequence recognized by blood coagulation factor Xa, can either be assayed for biological activities in vitro and in vivo or have the binding moiety removed to yield a totally unmodified form, suitable for clinical and functional studies. The method can also be applied to the production of low molecular mass peptides. The principle of the technique is illustrated with acidic fibroblast growth factor and with a neuropeptide-like fragment of ten amino acids contained within its sequence.


Determination of coexisting nuclear transcription rates and cytoplasmic mRNA levels for gonadotropin subunit genes in rat anterior pituitary.

Nuclear run-on transcription assays allow the study of those factors that regulate the expression of a given gene. Because previously published protocols may have been presented in abbreviated form or not developed for nuclei derived from specific tissues, we have established a comprehensive protocol for those who wish to measure transcription of the gonadotropin subunit genes (alpha, LH-beta, FSH-beta) in the anterior pituitary of the rat. The protocol also allows the determination of simultaneously existing cytoplasmic subunit mRNA levels within the same cells. An especially critical step in nuclear run-on transcription assays is the isolation of newly formed labeled mRNA transcripts from the DNA template. In previously published protocols such isolation has been accomplished through rather tedious, multistep methods. To simplify this isolation process, we have evaluated the use of the commercially available product RNAzol. Nascent mRNA transcripts are attached to chromatin and the DNA template; proteinase K and DNase I are typically utilized to disrupt this attachment, thereby facilitating subsequent extraction. We have found that strict adherence to proteinase K and DNase I digestion prior to RNA extraction with RNAzol, followed by two ethanol/NH4OAc precipitations, will enable one to successfully isolate nascent mRNA transcripts free of unincorporated nucleotides. In summary, we present a comprehensive protocol for determining transcription rates and cytoplasmic mRNA levels for the gonadotropin subunit genes in the anterior pituitary of the rat. RNAzol was found to simplify the extraction of newly formed transcripts and should prove applicable with transcription assays utilizing nuclei from a variety of tissue.


[The palliative treatment of pancreatic cancer: percutaneous drainage versus surgical diversion].

In a retrospective study, the outcome of 56 patients with unresectable carcinoma of the exocrine pancreas undergoing two palliative operations (bypass surgery vs percutaneous transhepatic biliary drainage with endoprosthesis) for the management of biliary obstruction was evaluated. Morbidity and mortality were similar in the two groups. Postop hospital stay was 8 days for the intubated, and 15 days for operated patients (p less than 0.01). Although the symptom-free period was similar in both groups, intubated patients had a shorter period of poor-quality life.


The effect of sodium butyrate on the growth characteristics of human cervix tumour cells.

Sodium butyrate has been shown to affect cell proliferation, and, at concentrations above approximately 0.5 mM, to cause cell death in some tumour cell lines. When combined with cytotoxic drugs increase in chemosensitivity has been observed. We are presently carrying out a study of the combined effects of sodium butyrate and cytotoxic drugs on cultured cervix tumour cells. To provide a baseline for this study we have carried out a systematic investigation of the effects of sodium butyrate alone on the growth characteristics of cervix tumour cells cultured as multicell spheroids. This has shown that concentrations of n-butyrate of 0.005 mM to 0.50 mM decrease cell proliferation without inducing cell death, the effect increasing with increasing concentration. Butyrate concentrations greater than 0.50 mM cause cell death after a period of 5 to 15 days exposure, dependent on concentration. Concentrations of 0.010 mM and above cause fragmentation of, and increased cell shedding from, multicell spheroids, suggesting an effect on the cell surface. Concentrations of butyrate greater than 0.10 mM cause a considerable increase in the synthesis of cytokeratin, as shown by reaction with cytokeratin antibody. Correlated with this is a marked increase in cell size, concentrations of butyrate of 2.0 or 3.0 mM leading to an approximate doubling of cell diameter, followed by cell disintegration. The effects of butyrate less than 0.25 mM are readily reversible. At concentrations greater than 0.25 mM the effects are reversible up to a limit of about 7 to 20 days depending on concentration, even when cytokeratin synthesis has been induced.


Selective enhancement of the tumour necrotic activity of TNF alpha with monoclonal antibody.

The binding and biological activity of human TNF alpha on endothelial and tumour cells has been studied in the presence of monoclonal antibodies (MAbs). In particular, one monoclonal antibody to TNF alpha (MAb 32) has been identified which failed to inhibit binding and cytotoxicity of TNF alpha on WEHI-164 tumour cells but which was a potent inhibitor of TNF alpha-induced endothelial cell procoagulant activity on bovine aortic endothelial cells. The ability of MAb 32 to inhibit selectively the actions of TNF alpha on endothelial cells but not on tumour cells suggests a mechanism for enhancement of the anti-tumour action of TNF alpha in vivo when in complex with this antibody. Treatment of tumour bearing mice (WEHI-164 and Meth A fibrosarcoma) with TNF alpha-MAb 32 complex resulted in a 5- to 10-fold enhancement in the potency of the cytokine in comparison to free TNF alpha. Complexes between this cytokine and other MAbs generally resulted in either no effect or inhibition of TNF alpha activity in vivo and in vitro. Neither intact MAb 32 nor FAb' fragments of MAb 32 showed any tumour regressive activity in the absence of TNF alpha. The FAb' fragments were equipotent to the bivalent form of the antibody in enhancing TNF alpha activity. These data provide evidence that it is possible to segregate the individual biological activities of TNF alpha with concomitant enhancement of the tumour regressive activity of the cytokine in vivo.


A Medical Research Council (MRC) randomised trial of palliative radiotherapy with two fractions or a single fraction in patients with inoperable non-small-cell lung cancer (NSCLC) and poor performance status. Medical Research Council Lung Cancer Working Party.

Two policies of palliative thoracic radiotherapy for NSCLC have been compared in a randomised multicentre controlled trial aimed at simplifying the palliative treatment of patients with poor performance status. A total of 235 patients were entered. They had inoperable, microscopically confirmed disease, too advanced for 'curative' radiotherapy. Their main symptoms were related to the primary intrathoracic tumour even if metastases were present, and they had a poor performance status. Patients were allocated at random to regimens of either 17 Gy given in two fractions of 8.5 Gy 1 week apart (F2 regimen, 117 patients), or a single fraction of 10 Gy (F1 regimen, 118 patients). Two patients (one in each group) were excluded from all analyses because they were found to have had previously treated malignant disease and had been admitted in error. On admission, 95% of the 233 eligible patients had cough, 47% haemoptysis, 59% chest pain, 64% anorexia, and 16% dysphagia. As assessed by the clinicians, these symptoms were palliated in high proportions of patients, ranging in the F2 group from 48% for cough to 75% for haemoptysis, and in the F1 group from 55% for anorexia to 72% for haemoptysis and chest pain. For all five symptoms the median duration of palliation was 50% or more of survival. All these results were similar in the two treatment groups. In contrast, on daily assessment by the patients using a diary card, those treated with the F2 regimen experienced substantially more dysphagia, which was recorded in 56% of the patients compared with 23% in the F1 group (difference 33%: 95% confidence interval 17-48%). The median survival from randomisation was 100 days in the F2 group and 122 days in the F1 group. The F1 regimen, as it requires only a single attendance for treatment, is recommended as a palliative regimen for patients with inoperable NSCLC and a poor performance status.


Advanced breast cancer and its prevention by screening.

In discussions on breast cancer screening, much attention has been focussed on the possible morbidity generated by screening. Favourable effects like the prevention of advanced disease seem underestimated, probably because quantification is that difficult. To analyse the amount of care and treatment given to women with advanced breast cancer, we report on patients followed from first recurrence until death using patient files and national sources. A random sample of 60 female cases from computerised registries of two cancer centres and a sample of 20 cases from a non-computerised hospital registry was taken. A total of 68 patient files were sufficiently documented. A woman with advanced breast cancer is estimated to have a 39% loss in utility compared to a healthy woman (range 27-45%). Hormonal treatment is the main modality during 14 and chemotherapy during 4 months. Total medical cost from diagnosis of advanced disease until death amounts to 17,100 US dollars, or 21,000 when including extramural cost. The effect of breast cancer screening by preventing the occurrence of advanced disease is quantified. The resulting gain in quality of life contributes 70% of the total gain in quality of life. In the long run, almost half of the annual cost of screening will be offset by savings in the cost for advanced disease. Only the changes in palliative surgery and/or radiotherapy will be small in contrast to primary treatment changes. Besides the mortality reduction, screening is justified by the improvements in quality of life and cost savings for women prevented from reaching advanced disease.


Monoclonal antipeptide antibodies to the glucocorticoid receptor.

The generation of monoclonal antibodies to synthetic peptides of the glucocorticoid receptor is described. Two antibodies to sequences from the DNA binding region are IgMs. Two other antibodies to sequences in the steroid binding region and the C-terminus belong to the IgG class. The specificity of the IgG binding to the receptor in an ELISA assay is demonstrated by competition with the relevant peptides. Both IgGs are able to recognize the receptor in Western blots, but do not form stable complexes in sucrose gradients. Steroid binding to the receptor is not influenced by preincubation with antibodies. This indicates that denaturation or distortion of the receptor is necessary for the accessibility of these antibodies to their epitopes. Both antibodies can be used to stain the glucocorticoid receptor in neoplastic cells of patients suffering from chronic lymphatic leukemia.


Management of stage II Hodgkin's disease: 15 years experience at St. Bartholomew's Hospital.

One hundred seventy-seven consecutive patients with newly diagnosed stage II Hodgkin's disease (HD) (supradragmatic 157; infra diaphragmatic 20) were treated at St. Bartholomew's Hospital on the basis of pathologic stage (PS) in 84 (IIA 69; IIB 15) and clinical stage (CS) in 93 (IIA 33, IIB 60) between January 1968 and December 1984. The median follow up is 13 years. Overall, complete remission (CR) was achieved in 143 patients (75%) of whom 53 have had a recurrence. One hundred twenty-seven patients remain alive, the cumulative predicted survival at 15 yrs being 70%. Mantle radiotherapy was prescribed to 88 patients with supradiaphragmatic HD, of whom 75 entered CR and 9 achieved good partial remission (GPR) (95%). The duration of remission correlated strongly with ESR (greater than 50 mm/h) and mediastinal thoracic ratio (less than 33% vs. greater than 33%) in a multivariate analysis (p = 0.05 and 0.02, respectively). 46/88 patients remain in continuous first remission, the median duration of remission having not reached at 15 years. Combined modality therapy or chemotherapy alone was prescribed to 69 patients with supradiaphragmatic HD, CR being achieved in 51 patients and GPR in 8 at the completion of all therapy. 48/59 patients continue in first remission. The duration of remission of patients receiving combined modality therapy or CT alone was significantly longer (p = 0.002) than that of patients receiving RT alone, in spite of the fact that the former group comprised predominantly of patients with unfavourable features.(ABSTRACT TRUNCATED AT 250 WORDS)


Kinetic immunodominance: functionally competing antibodies against exposed and cryptic epitopes of Escherichia coli beta-galactosidase are produced in time sequence.

The murine antibody response to Escherichia coli beta-galactosidase (GZ) was analyzed in vivo and in vitro by focusing on two families of antibodies that exert distinct conformational/functional activity on the antigen. Activating antibodies--defined by their capacity to increase the enzymatic activity of defective GZ produced by mutant strains of E. coli--are detected early after secondary challenge. Inhibiting antibodies, which interfere with antibody-mediated enzyme activation, appear later and cause the abrupt fall of activating titer, a scenario suggesting either idiotype/anti-idiotype interaction or opposite pulsions exerted on the antigen molecule. Supporting the latter mechanism, the confrontation of mAbs of the two families produced classical competitive inhibition curves when the readout was enzyme activation, although they recognize two different epitopes of the same molecule: the activating mAb a quaternary conformation-dependent site of wild-type GZ, the inhibiting mAb a sequential determinant exposed only in denatured or in defective enzyme. The different timing of generation of these antibodies during the response may depend on a processing step necessary for unfolding of native antigen and consequent display of certain cryptic epitopes before they can trigger specific B cells. A picture emerges where the response to the various epitopes of a complex antigen is sequentially connected and where the uptake by antigen-presenting cells of antigen complexed with antibodies specific for the exposed epitopes may favor revelation of the cryptic ones.


Transfection of HLA-DR-expressing DAP.3 cells with a cDNA clone encoding the glycosyl phosphatidylinositol-linked form of lymphocyte function associated antigen-3: biochemical features and functional consequences.

Structural and functional aspects of the accessory molecule lymphocyte function associated antigen (LFA)-3 (CD58) have been examined following the transfection of DAP.3 and P815 cells with a cDNA clone encoding the glycosyl phosphatidylinositol (GPI)-linked form of human LFA-3. Despite earlier observations that DAP.3 cells are deficient in GPI anchoring LFA-3 was expressed efficiently on DAP.3, as well as on P815 cells. Immunoprecipitation of LFA-3 from 35S-labelled cells revealed that the molecule expressed on the DAP.3 cells had a molecular weight intermediate between the transmembrane and GPI-linked forms expressed by human B cells. This suggests that the DAP.3 cells have a default pathway whereby the RNA transcript which encodes the GPI-linked form of the molecule can also encode an integral membrane protein. Functionally, expression of LFA-3 by DAP.3 which had previously been transfected with the genes encoding HLA-DR1 led to a marked augmentation of the proliferative response of five out of eight anti-DR1 human T cell clones. This effect was not reproduced when DR1 and LFA-3 were expressed by separate populations of DAP.3 cells, suggesting that the ligands for CD2 and for the T cell's receptor must be expressed on the same cell membrane. Expression of human LFA-3 also led to a substantial increase in the proliferative response of human peripheral blood T cells to a DR alloantigen. Separation of T cells into CD45RO+ and CD45RO- populations revealed that the augmentation was more marked for the memory than the virgin population. The mechanisms responsible for these differences are discussed.


Binding of substance P to monolayers and vesicles made of phosphatidylcholine and/or phosphatidylserine.

Analyses of interactions between substance P (SP) and phospholipids were performed by combined surface pressure and surface potential measurements in monolayers and by 13C-NMR experiments on liposomes. This study was carried out using synthetic SP molecules: [1-13C-Gly9]SP and [1-13C-Gly2]SP. Injection of SP into the aqueous subphase led to an expansion of phosphatidylcholine (PtdCho) or phosphatidylserine (PtdSer) monolayer surface area. An apparent association constant of SP for PtdSer was estimated to be around 10(6)-10(-7) M-1. The surface potential delta V/n varied linearly with the molecular area whereas the variation of surface pressure was biphasic, suggesting that at least two binding states contributed to the monolayer expansion. These two states Si (SP is inserted into the bilayer) and Ss (SP is stuck on the surface) were observed on vesicular membranes by 13C-NMR. The kinetic of interconversion between these two states can be estimated by NMR, the Ss state being the stablest one. No perpendicular insertion of SP into these vesicular preparations seemed to occur, as previously postulated. However, SP might form aggregates in contact with these model systems, leading to a loss of permeability of the lipid vesicles.


Ion channel formation by duramycin.

The formation of ion channels by the nonadecapeptide antibiotic duramycin was examined using black lipid membranes and using the patch-clamp technique. In black lipid membranes made from glyceryl monooleate or a phosphatidylcholine/phosphatidylethanolamine mixture, duramycin induced complex fluctuations in membrane conductance, some step-like and some which were incapable of being resolved into discrete conductance states. Both conductance and largest step size increased with time. A similar time-dependent increase in conductance was seen in patch-clamp experiments with HCA-7 Colony 29 human colonic epithelial cell. The channels displayed weak anion selectivity and the smaller channels formed in patches from epithelial cells showed weak inward-rectification. Channel formation by duramycin was achieved at lower concentrations when the black lipid membrane was made with phospholipid rather than with glyceryl monooleate. Lower concentrations were effective in generating conductances in epithelial cells than in bilayers. It is concluded that duramycin forms ion channels in both artificial and biological membranes. Accumulation of duramycin and coalescence of initially small channels into larger ones is considered to be responsible for the recorded behaviour and to final disruption of membranes.


Inhibition of gramicidin channel activity by local anesthetics.

Ondrias et al. ((1986) Stud. Biophys. 115, 17-22) found that dibucaine, butacaine, and tetracaine reduce the conductance of membranes containing multiple (greater than 10(6)) gramicidin channels. Similar experiments with local anesthetics (LA's) added to the bath while gently stirring showed that the inhibition developed slowly over a time course of 5-10 min. We developed a many (10-20) channel membrane technique which demonstrated that when LA's were added to the bath and the membrane was repeatedly broken and reformed, the channel occurrence frequency declined promptly. In standard single-channel membrane experiments at lower gramicidin densities, the mean single channel conductance and lifetime distributions with LA's present in the bath did not differ from the controls. The predominant channel conductance amplitude was lower by 9.1% than those of controls, but channel amplitude distributions were also modified so that the net reduction in overall population channel conductance was only about 2.0%. Channel currents showed no evidence of flicker blocks. The lifetime histograms of control and LA-exposed channel populations were both satisfactorily fit by a single-exponential function with the same mean. Thus, inhibition is due primarily to a reduction in the frequency of occurrence of conducting channels, implying a reduced concentration of active monomers in the membrane.