Intracellular pathways triggered by galanin to induce contraction of pig ileum smooth muscle cells. 1. In order to determine the intracellular mechanisms by which galanin induces contraction of isolated smooth muscle cells from pig ileum, we examined the effects of external Ca2+, relaxing agents, pertussis toxin and forskolin on the galanin-induced contraction and compared these effects to those observed on the cholecystokinin derivative CCK8-induced contraction. 2. Galanin induced a concentration-dependent cell contraction. The maximal contraction (24.5 +/- 2.1% of the length of resting cells) was observed at 1 nM of galanin. When cells were incubated in the simultaneous presence of concentrations of galanin (10 fM) and CCK8 (1 pM) which were ineffective alone, or galanin (10 fM) and acetylcholine (100 pM), a synergistic action was observed corresponding to a submaximal contraction. 3. Incubation of cells in Ca(2+)-free medium caused a significant decrease in galanin- but not in CCK-induced contraction. Nifedipine, a Ca2+ channel blocker, provoked a concentration-dependent inhibition of galanin-induced contraction while it had no effect on the contraction induced by CCK8. 4. Vasoactive intestinal polypeptide (VIP) and isoprenaline, known to induce cell relaxation through an increase in intracellular cAMP level, inhibited CCK-induced cell contraction at concentrations ranging from 1 pM to 1 microM but failed to inhibit cell contraction induced by galanin. 5. When cells were pre-incubated for 3 h in the presence of 200 ng/ml of pertussis toxin, the contraction induced by galanin was abolished while the CCK-induced contraction remained unchanged. On the contrary, 10 microM forskolin abolished the contraction induced by 10 nM CCK but had no effect on galanin-induced contraction. 6. These results indicate that galanin induces a concentration-dependent contraction of pig ileum smooth muscle by a direct myogenic effect. This effect of galanin involves the activation of a pertussis toxin-sensitive G protein, which results in an influx of Ca2+ into the cell. This intracellular pathway is insensitive to the relaxing effect of cAMP.


Caffeine-induced release and reuptake of Ca2+ by Ca2+ stores in myocytes from guinea-pig urinary bladder. 1. Voltage-clamped isolated smooth muscle cells from guinea-pig urinary bladder were studied with 3.6 mM extracellular Ca2+ at 36 degrees C. The fluorescence of the Ca(2+)-sensitive dye Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transient). Fast application of caffeine (10 mM) to the cell was used to release the intracellular Ca2+ from a 'caffeine-sensitive Ca2+ store'. 2. At the holding potential -60 mV, a short (1 s) caffeine application increased [Ca2+]i within less than 1 s from the resting 118 +/- 22 nM to 1490 +/- 332 nM. Following the caffeine wash-out, [Ca2+]i fell from this peak to a subresting level of 47 +/- 12 nM, i.e. an 'undershoot' of [Ca2+]i occurred. Subsequent caffeine-induced [Ca2+]i transients had attenuated peaks suggesting that the caffeine-sensitive Ca2+ store had lost a part of the releasable Ca2+. 3. In the continuous presence of caffeine, [Ca2+]i decayed from its peak to control resting [Ca2+]i values. The wash-out of caffeine following prolonged (10-30 s) treatment also resulted in [Ca2+]i undershoot. Subsequent caffeine-induced [Ca2+]i transients were largely abolished as if the caffeine-sensitive Ca2+ store had lost a large part of releasable Ca2+. During the undershoot, hyperpolarization to -100 mV did not affect [Ca2+]i. In most cells studied, recovery of [Ca2+]i from the undershoot to the resting level required depolarizations inducing Ca2+ influx through L-type Ca2+ channels. 4. Block of plasmalemmal Ca(2+)-ATPase (PMCa) with extracellular La3+ (3 mM) did not modify the decay of the [Ca2+]i transients induced by depolarization or by a 1 s caffeine application suggesting that decay rate of both is not limited by PMCa rate. La3+ abolished the undershoot of [Ca2+]i. In the continuous presence of caffeine, La3+ largely prevented the decay of [Ca2+]i. 5. When the depolarizing steps from -60 to 0 mV (160 ms duration) were applied during the period of [Ca2+]i undershoot, the half-time of decay of the corresponding [Ca2+]i transients was up to three times faster than in control. Repetitive depolarizations restored the rate of decay and [Ca2+]i recovered to the resting value. Both processes recovered along a similar time course. 6. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 0.1 mM) or of 8-Br-cAMP (0.1 mM) did not mimic the above caffeine effects suggesting that stimulation of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCa) by cAMP-dependent phosphorylation is not the underlying mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)


[Channel-forming activity at planar lipid bilayer of the membrane active polypeptide B form venom of Bungarus fasciatus]. Using planar lipid bilayer formed by lecithin and cholesterol (20 and 5 mg/ml respectively in N-decane) the channel-forming activity of the membrane active polypeptide B(BMAP B) from the venom of Bungarus fasciatus was investigated. Under the existence of a voltage or a salt concentration gradient between two sides of the bilayer, unit conductance fluctuation and a decrease in steady state resistance accompanying BMAP B incorporation and channel formation were observed. By measuring the reversal potential in an asymmetric solution, the selectivity of the BMAP B-channel was estimated having a value of PK/PC1 = 1.4. Divalent cations, such as Ba2+, Ca2+ inhibited the channel activity as they did in biomembranes. These data might provide an explanation for the depolarizing effect of the membrane active polypeptide on the native membranes.


[Interferon theory of schizophrenia].

[Interferon theory of schizophrenia]. The author presents a literature review of the eventual role of interferon in the etiopathogenesis of schizophrenia. Research material is presented of 20 chronic schizophrenic patients treated with Hu-alpha which seems to substantiate the beneficial effects of this substance in some cases which are resistant to current treatment methods.


[The effect of chemical agents on lysosome fusion with phagosomes and on the F-actin content in murine peritoneal macrophages]. The influence of natural and synthetic polyamines, phalloidin, cytochalasin D, vinblastine, colchicine, puromycin, chlorpromazine, urea, glutaraldehyde, and ethanol on the phagosome-lysosome fusion and the content of F-actin in murine peritoneal macrophages has been studied. Fluorescent phallotoxin FITC-phalloidin was used to stain F-actin. Natural polyamines (spermine, spermidine, putrescine), phalloidin, ethanol (0.1 M) stimulated the phagosome-lysosome fusion and increased the mid-content of F-actin in macrophages. Cytochalasin D, vinblastine, colchicine, puromycin, chlorpromazine, urea, glutaraldehyde, ethanol (0.15 and 0.2 M) inhibited this process and decreased the mid-content of F-actin. Possible mechanisms of the interconnection of cytoskeleton and the phagosome-lysosome fusion are discussed.


The fate of "small twins": a four-year follow-up study of low birthweight and prematurely born twins. This follow-up study of twins includes 35 families from the Stockholm area. One section concerns the physical and mental development of the twins. The other sections compare the development of the children with their parents' expectations of them and the parents' feelings about the delivery. Seven twin pairs (20%) in the total group were born before 37 complete gestational weeks. Thirty-seven of the twins weighed less than 2,500 g at birth; of these, 23 (9 boys and 14 girls) weighed less than 2,500g, although they were born after 37 gestational weeks. At four years of age, 22 of the 68 twins still showed some impairment of locomotor, language/speech and emotional development, as assessed by the Griffiths Mental Development Scales. All the prematurely born and small-for-date infants were in this group. There also seems to be a relationship between developmental problems and the mother's negative or ambivalent expectations concerning the twin pregnancy.


[Modulation of 3,4-diaminopyridine-evoked norepinephrine release from rat hippocampal slices by activation of alpha 2-adrenoceptor]. The effects of the alpha 2-adrenoceptor agonist clonidine and antagonist yohimbine were investigated on 3,4-diaminopyridine(DAP)-evoked [3H]norepinephrine ([3H]NE) release from rat hippocampal slices in the presence and absence of extracellular Ca2+. The slices were preincubated with [3H]NE and superfused in the presence of desipramine 1 mumol.L-1.[3H]overflow was evoked by addition of DAP 100 mumol.L-1 for 10 min to the superfusion medium. Clonidine and yohimbine inhibited and enhanced the 3,4-DAP-evoked [3H]NE release in a concentration-dependent manner both in the presence and absence of extracellular Ca2+. The effect of yohimbine was abolished by clonidine and was additive with the effect of ruthenium red. In the presence of extracellular Ca2+ the clonidine effect was not altered by addition of omega-conotoxin GVIA 0.1 mumol.L-1 or by removal of extracellular Ca2+, suggesting that the Ca2+ entry was not involved in the modulatory mechanisms of DAP-evoked [3H]NE release by activation of alpha 2-receptor. In the absence of extracellular Ca2+ the clonidine effect was reduced by the presence of ruthenium red 10 mumol.L-1, supporting the hypothesis that alpha 2-adrenoceptor activation might affect the intracellular mechanism of Ca2+ homeostasis.


[The effect of polymeric modification on the biological activity of pancreatic ribonuclease]. The effect of modification of dextran on pharmacokinetic properties of pancreatic RNAse and on its ability to suppress the proliferation of cells has been studied. It has been shown that the basic contribution to biological activity of polymer form of RNAse is making by azo-bonds which are forming in the process of chemical bonding of the protein with dextran support.


[Effects of prostatic massage on serum levels of prostatic acid phosphatase and specific antigen]. We studied the influence of prostatic massage on the seric levels of acid prostatic phosphatase (PAP) and prostatic specific antigen (PSA) before and 1, 24 and 48 h after the examination. Three groups of patients were studied: 13 patients with prostatic benign hypertrophy (PBH), six patients with metastatic prostate carcinoma and 10 control patients (urinary lithiasis). We observed a significant elevation of both seric markers 1 h after prostatic massage in the PBH and control groups. In both groups, seric marker levels returned to normal values within 24 h with PAP and 48 h with PSA. We recommend to assay serum PSA and PAP with an interval of at least 48 h after rectal digital examination.


[Experimental study on tea in inhibiting mutational specificity of 6 antineoplastic drugs]. According to the principles of SOS response, the authors tested the mutational specificity of tea and its inhibitory effects to the mutational specificity of 6 antineoplastic drugs by using the method of mutational and anti-mutational synchronous test. The results revealed that the tea had no mutational toxicity but anti-mutation effect. It also had the inhibitory effect on mutational toxicity of 6 antineoplastic drugs, including Mitomycin C, Bleomycin, Fluorouracil, Cisdiaminodichoroplatinum, Arabinosylcytosin and Mustargen. These results have provided referential basis for further study on anti-cancer effect and clinical use of tea.


Cloning and mapping of the alpha-adducin gene close to D4S95 and assessment of its relationship to Huntington disease. The genetic defect underlying Huntington's disease (HD) has been mapped to 4p16.3. Refined localization using recombinant HD chromosome analysis and allelic association analyses have identified two distinct candidate regions. Using a cDNA hybrid selection procedure we have cloned the gene for alpha-adducin, a subunit of a cytoskeletal protein crucial for spectrin-actin membrane plasticity. This gene maps to the proximal 2.2 Mb candidate region within 20 kb of D4S95. Alleles of markers at this locus have been shown to exhibit significant linkage disequilibrium with HD. A 4 kb alpha-adducin transcript was identified which is abundantly expressed in the caudate nucleus, the site of major neuronal loss in HD. Sequencing of the brain alpha-adducin cDNA from two HD patients and an age-matched control did not detect any sequence alterations specific to HD. However, we identified in brain cDNA of both patients and control samples, two alternately spliced brain exons, not previously described in the erythrocyte cDNA. A 93 bp exon is inserted in frame between codon 471 and 472 while a 34 bp exon inserted within codon 621 disrupts the frame and introduces a stop codon after 11 novel amino acids. The mapping of the adducin gene adjacent to D4S95 and its pattern of expression, as well as its potential for distinct alternately spliced variants, reinforces the necessity to accurately assess the role of the expression of this gene in the pathogenesis of HD.


Prostate-specific antigen levels and subsequent prostate cancer: potential for screening. Prostate-specific antigen levels are increased in men with prostatic disease, including prostate cancer, and have been used clinically to monitor the response of prostate cancer to therapy. More recently, prostate-specific antigen levels, usually in combination with digital rectal examination or transrectal prostatic ultrasonography, have been suggested to be useful for the detection of prostate cancer. To evaluate the association between a single serum prostate-specific antigen level and the subsequent development of prostate cancer, we measured serum levels in 35 men who donated blood to a community-based serum bank in 1974 and who subsequently developed prostate cancer and in 35 matched controls from the same group of volunteers. Levels of prostate-specific antigen were significantly higher in men who went on to develop prostate cancer, up to 6 years prior to the time of diagnosis in the cases. The level of prostate-specific antigen decreased with increasing time to diagnosis. The mean level for prostate cancer cases diagnosed within the first 3 years of follow-up was 16.2 micrograms/liter compared to 2.4 micrograms/liter for controls (P = 0.002). The mean level for cancer cases diagnosed in years 4 through 6 following blood sampling was 9.6 micrograms/liter compared to 1.3 micrograms/liter for controls (P = 0.0002). The sensitivity and specificity of a prostate-specific antigen level > or = 4 micrograms/liter up to 3 years prior to the time of clinical diagnosis were both 75% and up to 6 years were 67% and 85%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Point-mutational MspI and Ile-Val polymorphisms closely linked in the CYP1A1 gene: lack of association with susceptibility to lung cancer in a Finnish study population. In this study of 87 lung cancer patients, 23 patients with lung disease other than cancer, and 121 healthy controls, no association was found between the MspI restriction fragment length polymorphism (RFLP) of the CYP1A1 gene and lung cancer risk. In the lung cancer population, histological type, smoking, and occupational histories were also examined with respect to increased lung cancer risk. No association was found between the MspI RFLP in the CYP1A1 gene and any of these variables. This is in contrast to the results of an earlier report describing an association between the rare genotype m2m2 and susceptibility to lung cancer in a Japanese population; but another study in Norway found no such association. It is evident that, in the Nordic population, MspI polymorphism in the CYP1A1 gene does not indicate individual susceptibility to lung cancer. We also studied a new point mutation which has recently been closely linked to the MspI restriction site polymorphism in a Japanese study population. This mutation results in an isoleucine-valine amino acid replacement in the heme binding region of human CYP1A1. We obtained a similar linkage in our study, so the discrepancy between the Japanese and the Nordic MspI RFLP findings cannot be based on a different degree of linkage between these two point mutations.


Racial differences in prostate-specific antigen levels in patients with local-regional prostate cancer. Prostate cancer is a significant health problem for blacks. The incidence and mortality rates are higher in blacks than in whites; blacks often present with a higher stage. Prostate-specific antigen (PSA) is a very useful serum marker in prostate cancer. We analyzed data from a cohort of 161 patients to determine whether there were any racial differences in PSA levels prior to treatment in local-regional prostate cancer. The immunoradiometric method was used to determine the PSA values. The mean PSA levels were significantly higher in blacks than in whites (P = 0.022), and the difference remained significant in multivariate analysis after adjusting for stage and grade (P = 0.020). However, when analyzed further, the difference was statistically significant in one hospital (P = 0.001) and not in another (P = 0.493). Thus, our results are not unequivocal, but our data do suggest that racial differences in PSA levels not accounted for by tumor stage or grade may exist. Assuming that the data truly reflect a racial difference, the cause(s) of this difference remains to be determined. It may exist because, within each clinical stage, blacks are presenting with a higher tumor cell burden, or it may be indicative of more aggressive biological behavior. The possibility that racial differences are due to socioeconomic factors was considered by estimating median income level from zip code of residence; although a correlation between socioeconomic status and PSA level was found, racial differences remained borderline significant (P = 0.055) after adjusting for income level (in addition to stage and grade).


Human apurinic endonuclease gene (APE): structure and genomic mapping (chromosome 14q11.2-12). Abasic (AP) sites in DNA are produced spontaneously and by many genotoxic agents. The repair of such damages is initiated by AP endonucleases, which are evidently ubiquitous. We employed the recently cloned cDNA, APE, that encodes the major human AP endonuclease, to isolate large genomic fragments that contain the intact APE gene. The sequence of 3 kb encompassing APE was determined (GenBank Accession No. M99703). The APE gene contains four small introns (ranging 130 to 566 bp) and five exons, the first of which is untranslated. The 0.5 kb of DNA sequence upstream of APE did revealed only a possible CCAAT box, but no other regulatory sites or a TATA box, consistent with the constitutive expression of AP endonuclease activity observed in other studies. The location of APE in the human genome was mapped to chromosome 14, bands q11.2-12, by fluorescence in situ hybridization of metaphase cells with DNA from the genomic clones and subclones. Although this locus has not been associated causally with genetic diseases of DNA repair, some translocations that affect 14q11.2-12 could compromise APE and lead to genetic instability.


Genetic linkage of autosomal dominant neovascular inflammatory vitreoretinopathy to chromosome 11q13. Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is an inherited eye disease characterized by retinal and iris neovascularization, abnormal retinal pigmentation, anterior chamber and vitreous inflammation, cystoid macular edema, vitreous hemorrhage, and traction retinal detachment. Some of these clinical features are shared by more common, potentially blinding, conditions including diabetic retinopathy, uveitis, and retinitis pigmentosa. Elucidation of the molecular pathogenesis of ADNIV has the potential to provide insight into the mechanisms of these common disorders. One hundred and sixteen members of an eight generation family affected with ADNIV were examined. A combination of slit lamp biomicroscopy, ophthalmoscopy, and electroretinography was used to establish the diagnosis and 34 family members were found to be affected. Blood samples were obtained from thirty-three of these individuals and nine spouses and used for chromosome linkage analysis with denaturing gradient gel and short tandem repeat polymorphisms. Two markers that map to chromosome 11q13 were found to be significantly linked to the ADNIV phenotype. There were no recombinants between the disease phenotype and marker D11S527 and multipoint analysis yielded a maximum LOD score of 11.9 centered on this marker.


Molecular isolation and characterization of an expressed gene from the human Y chromosome. Using a positional cloning approach, we have isolated an expressed gene from a flow-sorted Y chromosome cosmid library. The isolation of this gene was based on the identification of the Y-231 cosmid that contains CpG rich sequences (HTF islands) in its human insert. The Y-231 cosmid was capable of detecting a 1.3 kb transcript in poly (A)+ RNA samples from human testis. Several cDNA clones were isolated from a human testis cDNA library constructed in lambda gt10. In addition, DNA-mediated gene transfer and restriction enzyme mapping experiments demonstrated that two functional transcriptional units are present within the Y-231 cosmid. DNA sequencing analysis showed that the largest cDNA clone contains 1075 bp of unique sequence and a poly (A) track at the 3' end of the corresponding mRNA. An open reading frame of 762 bp that encodes a predicted protein of 253 amino acids with a calculated molecular weight of 28.9 kD was identified. The Y-231 structural gene encompasses approximately 2.7 kb of genomic sequence and contains six exons that are interrupted by five introns. The Y-231 gene shares very high (97%) identity at the DNA level to a previously described Y-specific gene, testis specific protein Y-encoded (TSPY) gene, suggesting the possibility that these two genes are related, if not identical. However, the TSPY gene has been postulated to be intronless. Further PCR and RT-PCR analyses of these two genes and their transcripts have provided evidence supporting the hypothesis that they are the same gene and are members of a Y-specific repeated gene family containing intronic sequences. The Y-231 (TSPY) gene is conserved in the male genome and expressed in the testis of the chimpanzee, suggesting that it may play an important role in the physiology of this organ in man and the great ape.


The genomic organization of a novel regulatory myosin light chain gene (MYL5) that maps to chromosome 4p16.3 and shows different patterns of expression between primates. Myosin participates in a varying repertoire of cellular functions ranging from cytokinesis, receptor capping and secretion to sarcomere contraction. In vertebrates this functional complexity is achieved through the regulated expression of gene families encoding isoproteins for each of the myosin subunits. We report here the identification and characterization of a gene (MYL5) that encodes a novel regulatory myosin light chain isoprotein and maps 700 kb from the human chromosome 4p telomere. Identical cDNAs have been isolated from human adult retina and fetal muscle cDNA libraries. A full length 519 bp open reading frame was identified in the cDNA sequence encoding a predicted protein of 173 residues. Sequence analysis of a 5.6 kb genomic region that encodes these cDNAs revealed the presence of 7 exons which span 4 kb. Expression of this gene has been detected in human adult retina, cerebellum, basal ganglia and fetal skeletal muscle. Whereas Northern analysis fails to detect transcription of this gene in human adult skeletal muscle it reveals an abundant transcript in monkey skeletal muscle. Phylogenetic comparison of the predicted proteins primary structure to those of related myosin light chains from Drosophila, rat and human reveal evolutionarily conserved structural motifs important for both calcium binding and phosphorylation.


[The coupling of phosphoinositide metabolism sensitive to ACTH with the adenylate cyclase system in the synaptosomes]. The effect of cAMP on the phosphoinositide metabolism increased by the ACTH, was studied in the rat brain synaptosomes. The data obtained suggest that a coupling of the phosphoinositide metabolism and the adenylate cyclase system occurs under these conditions.


Effect of reducing agents on proteolytic and keratinolytic activity of enzymes of Microsporum gypseum. The effect of sodium sulphite, cysteine, glutathione, mercaptoethanol and dithioerythritol (0.1-10 mmol l-1) on the activity of proteases of Microsporum gypseum was studied using azocasein, cross-linked bovine serum albumin and keratin as substrates. With the substrate without disulphide bonds (casein) no stimulation was found, and reducing agents inhibited proteolysis in most cases. With the remaining two substrates, all substances enhanced the activity of proteases probably through the cleavage of the substrate disulphide bonds. Sulphite was more effective than the four used thiols and enhanced the activity against serum albumin up to 3.2 times and against keratin up to 2.9 times. Using sulphitolysed sheep wool, keratinolytic activity increased after sulphitolysis of more than 20% of disulphide bonds. With the fully sulphitolysed wool the activity increased 43 times. The obtained results support the author's hypothesis on keratin degradation by sulphite excretion prior to attack by fungal proteases. Stimulation of proteolysis and keratinolysis by cleavage of disulphide bonds is not specific for dermatophytic proteases because trypsin and pronase behaved similarly in the experiments.


[Changes of luminol-dependent chemiluminescence of rabbit peripheral blood leukocytes during incubation with a specific allergen during the development of Arthus phenomenon]. The authors studied luminol-dependent chemiluminescence of rabbit peripheral blood leukocytes during the development of Arthus' phenomenon. Samples with a specific allergen were previously incubated. It was established that in this type of allergic reactions the intensity of leukocyte chemiluminescence was determined by the concentration of the allergen with which they were incubated. Small doses of the allergen stimulate and large doses inhibit it. The inhibition is linked with the direct effect of the allergen on the cells and is not attended with diminution of their viability.


[Pain and stress: biological protection]. The biological methods of animal organism protection, the role of stress and the nociceptive signals, the hierarchy of defence mechanism--from the autonomic reflexes, neuroendocrine regulations, emotional reactions and expressions up to peculiar to the human being intellectual safeguard have been considered. The latest data on the role of tachykinins in transmission have been examined. The discovery of mediator function of "substance P" (SP), other related peptides, which coexists and realizes from the primary afferents and other terminals of SP-containing, capsaicin-sensitive neurons--opened a new chapter in the neurobiology and medicine. The necessity to support the medico-biological (fundamental) experiments by the modern society is grounded.


[An electrophoretic analysis of human glycoprotein hormones and their subunits]. Stability of heterodimers of human glycoprotein hormones with gonadotropic and thyrotropic activities in sodium dodecylsulfate (SDS) under non-reducing conditions at low temperature permits to resolve the native molecules of these hormones in SDS-PAG and to distinguish from their dissociated subunits by electrophoretical mobility. The analysis of dimers and alpha-, beta-subunits in one polyacrylamide gel allows to detect certain human glycoprotein hormones and to study some of their physico-chemical properties. Using two polyclonal antisera against human LH and FSH by the Western blot immunoassay it was shown that heterodimers as well as alpha and beta subunits after SDS-PAGE retain antigenic activity of native hormones. The method gave possibility to characterize the specificity of the given sera to different glycoprotein hormones.


Study on the formation of neural network in the tongue of Gallus domesticus (White Leghorn) as revealed by cholinesterase technique. The neural network in the tongue of Gallus domesticus was visualized using a cholinesterase technique. The network of acetylcholinesterase positive nerve fibres was formed by the tortuous myelinated as well as non-myelinated, preganglionic and postganglionic nerve fibres. The network was closely associated with a chain of acetylcholinesterase positive ganglia, the nerve fibres of the fungiform, filiform, circumvallate and foliate papillae and with the fibres of the neural terminal network.


Inhibition of S-antigen-induced experimental autoimmune uveoretinitis by active immunization against anti-idiotypic antibody to an S-antigen epitope. Experimental autoimmune uveoretinitis (EAU) was induced in rnu/+ rats by one injection of retinal S-antigen (S-Ag) in complete Freund's adjuvant. Immunization of rats, before the S-Ag challenge, with a polyclonal antibody directed at the monoclonal antibody (mAb) S2D2, prevented the development of EAU in rats at clinical and histological levels. Lymph node cells from rats hyperimmunized with this anti-idiotype (anti-Id) S2D2 antibody were stimulated in vitro by anti-Id S2D2, S-Ag, and mAb S2D2. These findings suggest that anti-S2D2 antibody acts as an internal image at the T-cell level for the epitope of S-Ag recognized by mAb S2D2 (epitope S2). From these results, it appears that cells bearing receptors for the internal image of the epitope S2, which is not pathogenic when injected emulsified in adjuvants, are able to inhibit S-Ag-induced EAU.


Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP. The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.


Production and secretion of nerve growth factor by clonal striated muscle cell line, G8-1. Two specific methods, Northern blot analysis using a 50 nucleotides probe to the conserved region of the nerve growth factor (NGF) gene, and enzyme immunoassay using a monoclonal biotinylated rat anti-NGF IgG-avidin conjugated peroxidase system, were used to demonstrate the production and secretion of NGF by mouse striated muscle cell line G8-1. Calcium ionophore, A23187 (0.1-1 microM), forskolin (0.1-100 microM) and dibutyryl cyclic AMP (0.1-10 mM) strongly decreased the secretion of ir-NGF. The level of NGF mRNA was decreased by veratridine, A23187, forskolin and cyclic AMP but not by cyclic GMP. Consequently, we conclude that the secretion of NGF molecules paralleled the changes of NGF mRNA levels in the cells induced by all agents tested. Carmamylcholine also decreased the level of NGF mRNA. Immunoblot analysis suggested that denatured ir-NGF molecules exist in a higher molecular weight form (22 KDa) than those of mouse submaxillary gland (13 KDa). Both Ca(2+)- and cAMP mediated mechanisms contribute to the decreased production of NGF mRNA in the cells and the consequent inhibition of secretion of NGF molecules. Finally, molecular cloning of NGF of G8-1 cells was conducted and confirmed the structure of the gene that consists of 1, 3, and 4 exons deleting exon 2. Thus, G8-1 NGF is derived from transcript B.


Acidic fibroblast growth factor delays in vitro ischemia-induced intracellular calcium elevation in gerbil hippocampal slices: a sign of neuroprotection. Using microfluorometry, effects of acidic fibroblast growth factor (aFGF) on the in vitro ischemia-induced intracellular calcium elevation were investigated in gerbil hippocampal slices at 35 degrees C. When slices were superfused with hypoxic and glucose-free medium, the mean latency of the in vitro ischemia-induced calcium elevation was 209 +/- 51 s. The addition of aFGF in medium (25 micrograms/l) delayed the calcium elevation throughout the experiments: the mean latency was 541 +/- 94 s. This retardation in calcium elevation may be indicative of neuroprotective nature of aFGF.


Behavioural, biochemical and histological effects of trimethyltin (TMT) induced brain damage in the rat. To assess the nature and extent of behavioural, biochemical and histological changes induced by trimethyltin (TMT), rats were treated with a single injection of TMT over a dose range of 6, 7 and 8 mg/kg i.p. Behavioural observations were performed at a minimum of 21 days after the administration of TMT. The behavioural consequences of TMT were hyperactivity in the open-field test, increased locomotor activity and deficits in passive and active avoidance behaviour, T-maze alternation and Morris Water Maze behaviour. The behavioural changes were dose dependent and were accompanied by a degree of pathological damage to the hippocampal pyramidal cells which was particularly apparent at the highest dose. The main biochemical effects of TMT involved deficits in the serotonergic and GABA-ergic systems and a decrease in M1 and M2 binding sites in the hippocampus. These results suggest that the toxic interaction of TMT with the hippocampus and other limbic brain regions may be responsible for its effect on learning and memory.


Acidic fibroblast growth factor prevents death of hippocampal CA1 pyramidal cells following ischemia. Ischemic insult induces neuronal death in the CA1 subfields of the hippocampus which are designated generally as the most vulnerable brain region. Recent studies have shown that acidic and basic fibroblast growth factors are potent trophic factors that support the survival of neurons in many brain regions including the hippocampus. Here we demonstrate that continuous infusion of acidic fibroblast growth factor into the lateral cerebral ventricles beginning 2 days before ischemia prevents the death of the CA1 pyramidal cells in the hippocampus of gerbils. Furthermore, delayed continuous administration of acidic fibroblast growth factor starting 5 min after ischemia is equally protective. The results suggest a possible physiological function for acidic fibroblast growth factor in the normal support of hippocampal CA1 pyramidal cells and neurons in some other brain regions in considering the broad spectrum of responsive neurons.


Canine systemic lupus erythematosus. II: Antinuclear antibodies. The frequency and the specificities of antinuclear antibodies (ANAb) were studied in dogs with systemic lupus erythematosus (SLE) and compared to those found in normal dogs and in dogs with various infectious diseases. Whole ANAb were detected by immunofluorescence. Anti-double-stranded DNA Ab were found in only 2% of SLE dogs, whereas anti-single-stranded DNA Ab were present in 21.4% of SLE dogs and in 26.8% of dogs with infectious disease. Antihistone Ab were frequently observed in SLE dogs (71%) and are essentially directed against trypsin-resistant epitopes of H3, H4 and H2A. The Western blots of nuclear extracts of HeLa cells were recognized mainly by type 1 Ab (30%, reacting with bands of 43, 36, 35, 34, 30 and 27 kDa) and by anti-Sm Ab (12%) associated with anti-RNP Ab. Anti-SSA and anti-SSB Ab were rare.


Detection of over 98% cystic fibrosis mutations in a Celtic population. We have conducted a large systematic study of 365 cystic fibrosis (CF) chromosomes in a Celtic population from Brittany, France, in which we have been able to identify more than 98% of the cystic fibrosis gene mutations. We detected 19 different CFTR mutations located in 9 exons. Eleven of these mutations have not been described previously and nine of them are presented in this study. The denaturing gradient gel electrophoresis strategy we have used, can be applied to other populations suggesting that population screening for CF on a large scale might be possible.


Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice. Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.


Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. An important issue for in vivo gene therapy for cystic fibrosis (CF) is the percentage of cells within the CF airway that will require correction. In this study, we mixed populations of a CF airway cell line expressing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA (corrected cells) or a reporter gene in defined percentages. As few as 6-10% corrected cells within an epithelial sheet generated C1-transport properties similar to sheets comprised of 100% corrected cells. Cell-cell coupling may serve as the mechanism for amplification of the functional effects of corrected cells. These data suggest that in vivo correction of all CF airway cells may not be mandatory.


The murine pallid mutation is a platelet storage pool disease associated with the protein 4.2 (pallidin) gene. Pallid is one of 12 independent murine mutations with a prolonged bleeding time that are models for human platelet storage pool deficiencies in which several intracellular organelles are abnormal. We have mapped the murine gene for protein 4.2 (Epb4.2) to chromosome 2 where it co-localizes with pallid. Southern blot analyses suggest that pallid is a mutation in the Epb4.2 gene. Northern blot analyses demonstrate a smaller than normal Epb4.2 transcript in affected pallid tissues, such as kidney and skin. This is the first gene defect to be associated with a platelet storage pool deficiency, and may allow the identification of a novel structure or biological pathway that influences granulogenesis.


[Bacterial flora of two sources of natural mineral waters; characterization by restriction patterns of the genes coding for ribosomal RNA; specificity and biological stability]. The bacterial flora of natural mineral waters was analysed using ribosomal ribonucleic acid gene restriction patterns in addition to customary identification techniques. Two springs were studied between 1989 and 1990 and six water samples per source were examined. Among the 206 bacterial strains collected at the spring, 91 (49%) were identified at species level using the API system and 99 representative strains from the two sampling points were selected for ribosomal ribonucleic acid gene restriction analysis. Forty six patterns were detected without common patterns to two springs. The bacterial association of each mineral water has a specific character (100% of specific patterns) shown by a series of patterns, not common to the other spring. This bacteriological specificity is preserved over a marked period of time, as, for each source, a great number of characteristics patterns was repeatedly found.


[Is it possible to predict the anti-arrhythmic effect of beta blockers based on various relations between premature ventricular beat and the preceding cardiac cycle?]. The aim of our study was to correlate betablocker effects on premature ventricular beats (PVBs) with different patterns between PVB frequency and cardiac cycle. The computer program we used correlates each PVB to the preceding cycle length in a 24-hour electrocardiogram recording. The arrhythmic patterns obtained were defined as tachycardia-dependent, indifferent and bradycardia-dependent. We selected 51 patients (39 males, 12 females, mean age 52 years) with different cardiac diseases, all in sinus rhythm, with > 25 PVBs/h, characterized by PVB stability and reproducible pattern in 2 Holter monitoring recorded at a 3-5 day interval. A tachycardia-dependent pattern (TDP) was demonstrated in 20 patients; 22 patients showed an indifferent pattern (IP) and 9 a bradycardia-dependent pattern (BDP). A third Holter monitoring was performed 4-5 days after nadolol administration (80 mg/day). Nadolol caused a 87.7% PVB suppression in TDP patients (p < 0.001), a 34.8% PVB reduction in IP patients (p < 0.01) and a 36.3% increase in BDP patients (NS). The different effect of nadolol on these groups was highly significant (chi 2 30.9; p < 0.0001). These results indicate that pattern definitions is useful in identifying PVB subsets which are likely to be improved, or not, by betablockers.


The angiogenesis inhibitor beta-cyclodextrin tetradecasulfate inhibits ecto-protein kinase activity. The growth of new blood vessels plays an important role in the pathogenesis of several diseases including cancer, diabetes, and arthritis. Beta-cyclodextrin tetradecasulfate, when administered with an appropriate steroid inhibits angiogenesis, and can stimulate angiogenesis when given alone. The regulation of angiogenesis is not well understood, and the mechanism of action of beta-cyclodextrin tetradecasulfate is similarly not well defined. Ecto-protein kinase activity that utilizes extracellular ATP has recently been reported on several types of cells. Human neutrophils appear to possess two distinct ecto-protein kinase activities; one that phosphorylates exogenous substrates including vitronectin and basic fibroblast growth factor, and one that phosphorylates endogenous cell-surface proteins. This report shows that beta-cyclodextrin tetradecasulfate inhibits the phosphorylation of the exogenous substrates casein, vitronectin (the major ecto-protein kinase substrate in serum), and basic fibroblast growth factor by human neutrophil ecto-protein kinase activity. In contrast, beta-cyclodextrin tetradecasulfate had no effect on the phosphorylation of endogenous cell-surface proteins by the neutrophil ecto-protein kinase activity. Ecto-protein kinase activity that was inhibited by beta-cyclodextrin tetradecasulfate was also detected on porcine aortic and human umbilical vein endothelial cells. The effects of beta-cyclodextrin tetradecasulfate on ecto-protein kinase activities may play a role in its effects on angiogenesis.


Human CD8+ T cell clone regulates autologous CD4+ myelin basic protein specific T cells. Normal human CD8+ T cell clones were co-isolated from the same culture wells as CD4+ T effector cell clones specific for myelin basic protein (MBP). Microcultures from which the CD8+ clones were isolated initially proliferated weakly to whole MBP and to an MBP peptide spanning residues 90-170. This pattern of response was similar to strongly proliferating wells that yielded CD4+ T cell clones specific for the 90-170 peptide. After repeated stimulation, however, no response to MBP or MBP 90-170 was detected, even though the number of cells increased after stimulation. Phenotyping and TCR analyses revealed the presence of two CD8+, CD4-, IL-2R+ T cell isolates that expressed a single V beta gene (V beta 17) that differed from the CD4+ isolates that uniformly expressed V beta 14. One of these CD8+ clones (C9) inhibited the antigen-driven proliferation of an autologous MBP 90-170 reactive clone but not an autologous clone specific for Herpes simplex virus (HSV), without affecting MHC non-restricted mitogen responses of the same clones. Moreover, C9 did not inhibit heterologous CD4+ T cell clones specific for MBP 1-38 or 90-170. A culture supernatant of the CD8+ clone showed the same pattern but lower levels of inhibition. C9 had mild cytolytic activity when incubated at high ratios with an autologous MBP-specific CD4+ clone. Lysis was blocked completely by anti-MHC class I antibodies, but not by anti-MHC II antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)


A study of mast cells in autoimmune NZB/W F1 mice: possible relationship between mast cells and increased vascular permeability in the thymus of NZB/W F1 mice. We examined the possible relationship between thymic mast cells and increased vascular permeability in the thymus of autoimmune NZB/W F1 mice. Light-microscopic observation of tissue sections showed that non-autoimmune BDF1 mast cells increased with age. In contrast, autoimmune NZB/W F1 mast cells did not increase in the thymic parenchyma at the age of 9 weeks. However, NZB/W F1 mast cells resumed the age-associated increase from the age of 12 weeks and exceeded the number of BDF1 mast cells at the age of 30 weeks. Blood histamine levels of 9-week-old NZB/W F1 mice were higher than those of BDF1 mice of comparable age. Furthermore, peritoneal mast cells of NZB/W F1 mice were more sensitive to compound 48/80 than those of BDF1 mice. Increased blood histamine levels of NZB/W F1 mice seem to be due to the enhanced histamine release from mast cells. These results suggest a possible correlation between the high histamine levels by degranulation of mast cells and increased vascular permeability in the thymus of NZB/W F1 mice.


Pathophysiological significance of brain acidic fibroblast growth factor. The acidic fibroblast growth factor (aFGF) in rat cerebrospinal fluid (CSF) increased 1000 times in the 2 hr period after food intake, or intraperitoneal (IP) or intracerebroventricular (ICV) glucose infusion. It diffused into the brain parenchyma and was taken up into neurons in the hypothalamus, hippocampus, etc.... aFGF is produced in the ependymal cells and released into CSF in response to increased glucose. ICV application of aFGF dose dependently inhibits, and anti-aFGF antibody facilitates food intake. IP injection of glucose 2 hr before a task that combined acquisition with passive avoidance significantly increased retention of avoidance by mice tested 24 hr later. In a Morris water maze task, IP glucose injection 2 hr before a first trial block reduced time to find and climb onto a platform hidden just below the water surface. These facilitation by glucose of affective and spatial memory were abolished by pretreatment with anti-aFGF antibody applied ICV. Continuous ICV infusion of aFGF into rats also significantly increased the reliability of passive avoidance for several days. The memory facilitation by aFGF was significantly attenuated by CA1 neuron death in the hippocampus caused by 5 min ischemia of the brain, in gerbils. After food intake, centrally-released aFGF reaches the hippocampus and facilitates memory, while peripherally released cholecystokinin reaches the endings of the afferent vagal nerves in the portal vein and changes the vagal nerve activity, which modulates hippocampal activity, to lead to memory facilitation. This, however, is blocked by vagotomy below the diaphragm.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of a lipopolysaccharide encoded by a recombinant Shigella sonnei plasmid in Escherichia coli K-12. The genetic information to synthesize the S-specific region of Shigella sonnei phase I lipopolysaccharide (LPS) is localized on a 180 kb plasmid which is lost quite readily. A recombinant plasmid derivative remaining stable in the bacteria was shown to determine the S-specific region of the LPS which is completely identical with that of a S. sonnei phase I strain following transfer in Escherichia coli K-12. However, the length control in polysaccharide biosynthesis is lost at least partially.


Antigen-antibody interactions: elucidation of the epitope and strain-specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K. The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain-specific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-Phe-Ile-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain-specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.


A single nucleotide change within a plant virus satellite RNA alters the host specificity of disease induction. Some RNA plant viruses contain satellite RNAs which are dependent upon their associated virus for replication and encapsidation. Some cucumber mosaic virus satellite RNAs induce chlorosis on any of several host plants, including either tobacco or tomato. The exchange of sequence domains between cDNA clones of chlorosis-inducing and non-pathogenic satellite RNAs delimited the chlorosis domain for both tobacco and tomato plants to the same region. Site-directed mutagenesis of one nucleotide (149) within this domain changed the host plant specificity for a chlorotic response to satellite RNA infection from tomato to tobacco. Within the chlorosis domain, three conserved nucleotides are either deleted or altered in all satellite RNAs that do not induce chlorosis. Deletion of one of these nucleotides (153) did not affect satellite RNA replication but rendered it non-pathogenic. Thus, two single nucleotides have been identified which play central roles in those interactions between the virus, its satellite RNA and the host plant, and together result in a specific disease state.


Differential response of maize catalases and superoxide dismutases to the photoactivated fungal toxin cercosporin. Many fungi of the genus Cercospora produce a light-induced, photoactivated polyketide toxin called cercosporin. In the presence of light an excited form (triplet state) of the toxin molecule is produced which, depending on the reducing potential of the environment, reacts with molecular oxygen to produce singlet oxygen and/or superoxide radicals. In this paper a system is presented for analysis of antioxidant defense gene response using purified cercosporin under conditions demonstrated to favor superoxide formation. Under the assay conditions employed, changes in total catalase activity, as well as individual isozyme protein levels generally mirrored the changes observed in corresponding steady-state RNA levels in response to applied cercosporin. In contrast, while transcript accumulation for most maize superoxide dismutases increased dramatically, both total superoxide dismutase activity and individual isozyme protein levels remained constant in all toxin treatments. In one case, the analyses indicated that there are two distinct transcripts that hybridize with a gene-specific probe for Sod3. These two transcripts responded differentially to applied toxin (levels of the larger transcript increased while the smaller decreased), whereas corresponding steady-state levels for the SOD-3 isozyme proteins remained constant. This suggests that protein turnover might play a role in the response of these SODs to activated oxygen species.


[The role of substance P in the modulation of myotension in paralyzed limbs after spinal transsection on rats]. It is a common clinical observation that stretch reflex excitability increases progressively after spinal transsection in man, and causes many severe complications. The purpose of the present study was to investigate the role and mechanism of substance P (SP) on the spinal reflex following spinal cord transsection. We first observed the changes of spontaneous electromyography (EMG) recorded in the paralyzed limbs of spinally transsected rats, and found that the EMG amplitude increased progressively within 40 days. The results were as follows: 1) Spontaneous EMG increased significantly following intrathecal injection of 1 microgram capsaicin, and the sensitivity of EMG to capsaicin increased progressively with time. But this effect could be partly blocked by pretreatment with SP antiserum. 2) Spontaneous EMG increased significantly following intrathecal injection of SP. Yet the time course was relatively short and the amplitude of EMG was lower. These results indicate that SP in the spinal dorsal horn participates in the regulation of myotension, and it is suggested that to decrease SP content in the spinal dorsal horn or block the afferent fibers of dorsal roots might be an effective therapy to reduce the hypermyotension caused by spinal cord injury, multiple sclerosis or other reasons.


Communication effects of prenatal alcohol exposure. This paper surveys the literature on prenatal alcohol exposure. The focus is on studies of speech, language, and communication skills evidenced by children diagnosed with fetal alcohol syndrome and fetal alcohol effects. Concomitant physical, behavioral, intellectual, and learning patterns are reviewed. Symptoms presented by alcohol-exposed children are compared to those seen in other developmentally delayed children. Future needs in areas of identification, assessment, and treatment are discussed.


Effects of trigeminal neurotransmitters on piglet pial arterioles. We investigated effects of calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) on pial arterioles in newborn pigs. Pial arteriolar diameter was determined using a closed cranial window and intravital microscopy. Initial diameters were approximately 100 microns. Calcitonin-gene related peptide dilated pial arterioles by 22 +/- 8% at 10(-9)M and by 34 +/- 6% at 10(-8)M (n = 8), and this response was not significantly altered by prior administration of indomethacin (5mg/kg, iv) (n = 6) or administration of NG-methyl-L-arginine (5mg/kg, iv, and 10(-3)M in CSF) (n = 10). Substance P dilated arterioles at 10(-10)M through 10(-5)M (maximal response = 23 +/- 3%) (n = 6), and this response was unaffected by indomethacin administration (n = 6). In contrast, NG-methyl-L-arginine blocked much of the pial arteriolar dilation to SP. Unlike the other two peptides, NKA did not change pial arteriolar diameter. Radioimmunoassay determinations indicated that cerebrospinal fluid levels of 6-keto-prostaglandin F1 and prostaglandin E2 did not change appreciably during application of CGRP or SP. We conclude that CGRP and SP but not NKA are dilator stimuli in the piglet pial circulation. Dilation by CGRP probably involves direct activation of receptors on vascular smooth muscle, while SP probably partially dilates pial arterioles via release of an endothelium-dependent relaxing factor.


Determination of hemoglobin Bart's in alpha thalassemia traits by two-site immunoradiometric assay. I. Purification of hemoglobin Bart's and preparation of specific anti-Hb Bart's. This research report describes methods for the preparation of hemolysate and the isolation and purification of hemoglobins Bart's, A, A2, E, F and H. Procedures for the preparation of anti-Hb Bart's by injecting purified Hb Bart's into rabbits is indicated in the time schedule. The rabbit antisera were evaluated by antigen-antibody reaction in agar gel. Although the antiserum reacted with Hb Bart's but not with Hb A, A2,E and H, it also cross-reacted with Hb F. After the rabbit antisera were absorbed with Hb F, the antisera were highly specific because it only reacted with Hb Bart's. The purified specific anti-Hb Bart's was labelled with radioactive 125I by chloramine-T method. After passing through Sephadex G-100 column, the 125I labelled specific anti-Hb Bart's was obtained in the first peak. This radioactive labelled anti-Hb Bart's was ready to use in the two-site immunoradiometric assay.


Cerebral abscesses due to Pseudomonas pseudomallei. Two cases of cerebral abscesses caused by Pseudomonas pseudomallei are reported. The first case, a 51-year-old women had a sudden onset of progressive right hemiparesis and right facial palsy and died within 7 days. Postmortem examination disclosed brain abscess in association with disseminated infection outside the central nervous system. The second case, a 9-year-old boy displayed cerebral abscesses as an isolated manifestation. Recovery occurred after treatment with ceftazidime. Review of the ten case reports of cerebral melioidosis revealed that the lesion occurred in patients of all ages and was more common in men than in women. The frontoparietal lobe was the most common location. Fever, headache, and hemiparesis were frequent clinical manifestations while seizures, ataxia were uncommon. CT scanning, serum antibody titer along with hemoculture were useful investigate tools. The importance of early diagnosis and prompt treatment is emphasized for this fatal but treatable disease.


[Renal adenocarcinoma with secretion of renin, simulating a oncocytoma. Apropos of a case in a pregnant woman with histological and immunohistochemical study]. The authors report a new case of primary renin-secreting renal carcinoma in a pregnant woman. The clinical, radiological and pathological findings mimicked an oncocytoma and the diagnosis was confirmed by immunohistochemical study.


Early ontogeny of the vagus nerve: an analysis of the medulla oblongata and cervical spinal cord of the postnatal rat. Retrograde transport of cholera toxin conjugated with horseradish peroxidase in the postnatal rat has revealed remarkable features of dendritic fields of vagal motor neurons in the medulla oblongata and cervical spinal cord during the period of early development (0-10 days). At birth, vagal motor neurons in the dorsal motor nucleus of the vagus, nucleus ambiguus, nucleus retroambigualis, nucleus dorsomedials and the spinal nucleus of the accessory nerve are small with relatively few, unbranched processes. The span of the dendritic tree is much smaller than that found in adult animals. By the postnatal Day 2 there are marked changes in the soma as well as in the dendritic tree of these neurons. There is dispersion of the cell bodies within the neuropil as well as an expansion of the total area of the brain stem occupied by these motor neurons and their dendritic processes which show extensive growth and branching. By postnatal Day 3 the most extensive proliferation of these neurons is seen and appears to represent the peak of dendritic growth of vagal motor neurons such that the area occupied by the dendritic tree of a single neuron is three times that seen in an adult rat. This proliferation gradually decreased during the subsequent seven days of early development (i.e. Days 4-10) so that by Day 10 the dendritic span of vagal motor neurons was reduced to about twice the adult size. This growth progressively decreased from Days 10 to 30 at which time adult levels were reached. Ultrastructural examination of these horseradish peroxidase labeled dendrites showed a positive correlation between the number of dendritic processes and the number of axo-dendritic synapses. This was accompanied by an increase in the number of identifiable synaptic junctions. These morphological complexities observed during the period of early development of vagal motor neurons indicate that the vagus nerve undergoes dramatic changes during the period of early development including the establishment of numerous synaptic contacts between vagal afferents and efferents in the brainstem. A number of these changes occur in developing dendritic fields of vagal motor neurons during the first three days of neonatal life. It is reasonable to assume that developmental abnormalities during this "critical period" could produce significant functional changes in the pattern of respiration as well as in the control of airway smooth muscle.


c-fos mRNA in mouse brain after MPTP treatment. The neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces a transient increase of mRNA for the immediate-early gene c-fos in the mouse brain. The c-fos mRNA level is MPTP dose-dependent and is evident in all brain regions tested including striatum, hypothalamus, cortex, hippocampus, cerebellum and midbrain. There are regional differences in the time-course for the rise of c-fos mRNA. Pretreatment with deprenyl, a selective monoamine oxidase B inhibitor, pargyline, a nonselective monoamine oxidase inhibitor, or mazindol, a dopamine uptake transport inhibitor, does not prevent the c-fos mRNA increase, suggesting that the elevation is due to the action of MPTP and not its neurotoxic metabolite MPP+.


Calcium-ganglioside interactions and synaptic plasticity: effect of calcium on specific ganglioside/peptide (valinomycin, gramicidin A)-complexes in mixed mono- and bilayers. A controlled exchange of calcium between the extracellular space (mM Ca2+) and the neuroplasm (microM Ca2+) is considered to be an essential prerequisite for almost every stage of neuronal activity. Our research interest is focused on those compounds, which due to their physico-chemical properties and localization within the synaptic membrane might fulfill the task as neuromodulators for functional synaptic proteins. Because of this specific binding properties towards calcium and their peculiar interactions with calcium in model systems gangliosides (amphiphilic sialic acid containing glycosphingolipids) are favorite candidates for a functional involvement in synaptic transmission of information. In this study we used monolayers to investigate the molecular packing and surface potential at the air/water interface, the interaction of gangliosides with the depsipeptide valinomycin (= monovalent ion carrier), and its influenceability by calcium. Furthermore we looked at calcium effects on the single channel conductance and mean channel life-time of the monovalent ion channel gramicidin A in mixed PC/ganglioside bilayers. In pure ganglioside monolayers the addition of 0.01 mM Ca2+ induces monolayer condensation, a rise in collapse pressure (= higher film stability), a shift of phase transition (= change of conformation), and a more negative head group potential (change of electric properties). In mixed ganglioside-valinomycin monolayers the addition of Ca2+ causes phase separation and/or aggregate formation between the ganglioside and the peptide. Single channel conductance fluctuations as well as mean channel life-time were analyzed for gramicidin A incorporated into binary mixed black lipid membranes of negatively charged gangliosides (GM1, GD1a, GT1b, GMix) and neutral lecithin (DOPC) in different molar ratios. At monovalent electrolyte concentrations up to < 250 mM CsCl the single channel conductance was significantly larger in the negatively charged mixed DOPC/ganglioside membranes than in the neutral DOPC membrane. Additionally, in the presence of gangliosides the mean channel life-time is increased. The addition of calcium (0.05 mM) induced a reduction of single channel conductance of gramicidin A in DOPC- and mixed DOPC/ganglioside membranes. These physico-chemical data in connection with new electromicroscopical evidences for a precise localization of calcium, a calcium pump (Ca(2+)-ATPase), a clustered arrangement of gangliosides in synaptic terminals, and biochemical results with regard to activatory nature of exogenous gangliosides for neuronal protein phosphorylation and ATPases, support the hypothesis of a modulatory function of gangliosides in synaptic transmission.


Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA. The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC]n where n > or = 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds approximately 70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo (rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho. Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.


Crystallization of proton channel peptides. Crystals have been grown of two similar peptides that form ion-conducting channels in diphytanoyl phosphatidylcholine bilayers. These crystals were grown by slow evaporation of the organic solvent, 2,2,2-trifluoroethanol. Crystals of one of the peptides have been characterized by X-ray diffraction, and X-ray data have been measured to 2.3 A resolution. Earlier it was proposed that the ion-conducting channels formed by these peptides consist of four peptides associated as a parallel alpha-helical tetramer. On the basis of the space group and unit cell dimensions of the crystals, a packing scheme for the peptide is proposed that is consistent with a tetrameric channel.


Biotechnology in transfusion medicine.

Biotechnology in transfusion medicine. In an overview, this paper summarizes the new possibilities based on biotechnology, such as monoclonal antibodies and genetic engineering. Examples are given where these new techniques are already used in transfusion work. In addition, some perspectives for the future in relation to these techniques are given.


Automated blood component preparation with the Opti system: three years' experience. Removal of buffy coat from the red cell preparations reduces the content of microaggregates and leukocytes and decreases transfusion side effects. However, buffy coat removal is difficult to standardize when performed manually. An automated method using a new type of plastic container system, the Optipac, and a new type of device, the Optipress, greatly improves blood component production. An initial hard centrifugation of the original whole blood provides the best results, leaving approximately 20 ml of plasma and 0.5 x 10(9) leukocytes per unit of red cells suspended in 100 ml of an additive solution (SAG-M). Improved quality and standardization of the products, reduced technician time and better working conditions for the technician are the major advantages as compared to traditional manual technique.


[Reduction of leukocytes and loss of erythrocytes in production of erythrocyte concentrates using the "top and bottom" principle, a comparison between the equipment combinations Biopack-U/Biotrans Separator and Optipac/Optipress]. Using four different centrifugation methods, we compared the combinations of top and bottom bags and blood component separators made by Biotrans and Baxter. Compared with the cell counts of the donated blood, there remained 25% of the leukocytes and 88% of the red blood cells in the red cell preparations following application of the Biotrans system; the respective values for the Baxter system were 20% leukocytes and 85% red cells. The results were not influenced by the centrifugation method used. Thus, febrile nonhemolytic transfusion reactions and immunization against leukocyte antigens can be avoided by neither system.


Improvement of the separation of blood by modification of separators Optipress and Biotrans Separator. By use of the new top-and-bottom bag system and automatic separators the quality of red cell concentrates (RCC) and fresh frozen plasma (FFP) could be markedly improved. Simultaneous preparation of storable single-donor platelet concentrates (PC) resulted, however, in too high losses of red cells and plasma. Through modification of the pressure plate of the separators and variation of the residual buffy coat (BC) volumes we tried to optimize the separation in this respect. In a second study we tried to establish the preparation of platelet-rich plasma (PRP) using top-and-bottom bag systems and automatic separators. 1st study: 149 whole-blood units (Biopack U, quadruple systems) were separated into components either with Optipress (Opt, n = 58) or with Biotrans Separator (Bio, n = 91). To Optipress additional plates, 3-6 mm thick, were attached and the residual BC volume was varied by different adjustments of the distance screw. The Biotrans Separator was used with a plane pressure plate as well as with a modified plate having a transverse groove; the residual BC volume was adjusted to 50-120 ml. 2nd study: 15 whole-blood units were separated into PRP and RCC either using the Optipress (standard version, n = 5) or the Biotrans Separator (alternating opening of clamps, standard plate, n = 10). 1st study: Both separations could be improved. The loss of red cells was significantly lower in case of the Biotrans Separator: Bio 15.6%, Opt 25.9%, p < 0.001. Other separation parameters showed no relevant difference between both separators. 2nd study: By use of the Biotrans Separator significantly less platelets could be separated (Bio 75.1 x 10(9), Opt 87.0 x 10(9), p < 0.05), but the PRP clearly was contaminated with less leukocytes (Bio 51.0 x 10(6), Opt 458.9 x 10(6), p < 0.01). The contamination of the RCC with leukocytes (about 20%) was markedly improved in both methods when compared with conventional preparations. The mean loss of red cells was 12.8% in both methods.


[Procedure for production of leukocyte depleted erythrocyte and thrombocyte concentrates (PRP method) using top and bottom bags, Optipress automated separators and a computer controlled Cryofuge 6000]. PRP methods for production of WBC-poor red blood cell (RBCC) and platelet concentrates (PC) are investigated using top-and-bottom bags and Optipress separators. A standard centrifugation method (profile A) and 2 methods (profiles B and C) for a computer run centrifugation using 21 (profile B) or 19 (profile C) varying time segments and g numbers. An MS-DOS-compatible personal computer runs a Cryofuge 6000. 500 ml fresh whole blood are collected in triple and/or quadruple top-and-bottom blood bags. Samples of 163 blood donors, 163 RBCCs and 161 PCs are analyzed by a Coulter Counter T 540. Blood smears of 5 RBCCs made by profile B are evaluated. Mean WBC contamination of RBCCs produced by profiles A, B and C is found to be lower than 5 x 10(8)/RBCC. None of the 5 blood smears can be counted out completely. The full number of 100 WBCs is not detected. All WBCs found are polymorphous nuclear cells. Mononuclear cells (MNC) are not evident. PCs produced by profile B contain a mean platelet yield of (76.4 +/- 21.2) x 10(9)/PC and a WBC contamination of (1.2 +/- 0.7) x 10(7)/PC. The PCs of profile B differ significantly (p < 0.001) from those of profile A and profile C. The results show a high quality of RBCC and PC produced by PRP methods using top-and-bottom blood bags and Optipress separators. Employing a computer run centrifuge, PC and RBCC contain a similar WBC contamination compared with concentrates produced by buffy coat methods.(ABSTRACT TRUNCATED AT 250 WORDS)


[Differentiation of leukocytes in thrombocyte concentrates by APAAP staining]. The preparation of platelet concentrates (PC) from pooled buffy coats has raised concern as to whether activation of lymphocytes might take place during the period of storage. Therefore it appears to be essential to investigate the degree of leukocyte contamination and identify the subtypes of these cells. Especially lymphocytes are of relevance as they may induce either a mixed lymphocyte reaction (MLR), the production of cytokines or a graft-versus-host reaction. As the number of leukocytes is very low we adopted the alkaline phosphatase-antialkaline phosphatase (APAAP) technique to determine the composition of leukocytes. We used mouse monoclonal antibodies to specifically stain T lymphocytes, B lymphocytes, monocytes, HLA-DR-positive cells and NK cells. All subtypes could easily be identified and their relative amounts were determined. In samples from 15 PCs the percentages of T lymphocytes, B lymphocytes, monocytes, NK cells and HLA-DR-positive cells was 40.3, 6.4, 18.9, 7.2 and 17.1% of total leukocytes, respectively. The standard deviation ranged between 2 and 5%. We highly recommend this technique, which is a very sensitive method to determine leukocyte contamination in PC.


[Hepatitis C virus antibodies in patients and the normal population]. With regard to the controversial issue of a reduction of transfusion-associated infections by non-remunerated donations, epidemiological data on the prevalence of HIV-1, HIV-2 and hepatitis C virus (HCV) are of particular interest in our country. We investigated four sample categories: (1) healthy employees and workers from Hamburg; (2) hemodialysis patients; (3) hemato-oncological patients, and (4) blood donors, and tried to differentiate between the three disputed vectors of community-acquired (sexually or pregnancy-transmitted), nosocomial and transfusion/transplantation-associated HCV infections. We conclude from our results that--prior to the implementation of blood screening--our carefully selected 'paid blood donors' conferred no higher HCV risks than the general (working) population (0.66 vs. 0.82% HCV antibody prevalence). Besides transfusions/transplantations, significant nosocomial risks apparently exist in hemodialysis units (21.0 vs. 9.5% HCV seroprevalence in polytransfused patients). Preventive measures, e.g. separate dialysis machines for HCV-positive patients, seem to be advisable.


[Comparison of erythrocyte concentrations of ATP, ADP, AMP and 2,3-BPG in storage of five different additive solutions and in serum]. In this study, we compared the ability of serum and of 5 additive solutions for the liquid storage of red blood cells to preserve the cellular contents of 2,3-BPG and the adenylate nucleotides ATP, ADP and AMP. As to the adenylates, PAGGS-M showed the best results. However, the difference to SAG-M was significant only after 7 weeks. On the other hand, serum appears to be the best medium for the conservation of 2,3-BPG. Between the 1st and 3rd week of storage, the decrease of 2,3-BPG was somewhat slower in PAGGS-M and SAG-M compared with the other 3 additive solutions.


[Oxygen affinity and regeneration capacity of SAG-M and PAGGS-M stored erythrocytes]. In order to estimate the leftward shift of the oxygen dissociation curve of hemoglobin following storage of red blood cells (rbc) in the additive solutions SAG-M and PAGGS-M, respectively, we performed blood gas analyses after equilibrating the cells with a gas mixture containing 4% of O2 and 5% of CO2 at pH 7.4 and 37 degrees C. Additionally, we took advantage of these nearly physiological conditions to measure the recovery of the hemoglobin function in vitro. We observed a good correlation between 2,3-BPG and p50 (i.e. the oxygen tension, at which hemoglobin is half-saturated with oxygen). Within the first 3 weeks of storage, the 2,3-BPG content fell to one fifth of its original value, whereas the p50 declined from 26.6 to about 20 mm Hg. Compared to fresh cells, rbc stored for longer than 3 weeks will thus deliver 30% less oxygen to the myocardium. The rbc's ability to restore these parameters remained unchanged throughout 7 weeks of storage.


[Lymphocytograms in relation to storage time of CPDA1 whole blood concentrates]. Immunocompetent cells have been shown to be effective in immunosuppression after blood transfusion. The analysis of leukocytes by electronic cell counter and of lymphocyte subsets by flow cytometry in stored whole blood revealed a decay of granulocytes within the first day of storage. T-lymphocyte subpopulations and natural killer cells continuously decrease over the time of storage, whereas B lymphocytes are very stable during the first 2 weeks. After 4 weeks lymphocyte subsets had decreased by the following values: total lymphocytes 46.0%, CD3+ 78.3%, CD4+ 80.1%, CD8+ 81.3%, CD19+ 65.9%, CD16/56+ 74.8% and CD3/DR+ 89.5%. Therefore leukocyte removal should be achieved as soon as possible after collection.


Hemotherapy in neonatology.

Hemotherapy in neonatology. The intensive care of very immature preterm infants is of special significance in the treatment of neonates with blood and blood components. A new, practicable transfusion concept has been developed for this group with its high and often acute need of hemotherapeutic measures: 30-ml packs containing packed red cells of blood group 0 Rh neg, free of 'immune' anti-A/B and anti-CMV-free, or deep-frozen fresh plasma of blood group AB. This concept has the advantages of simplified pretransfusional serology, reduced blood requirement with simplified storage, fresher stored blood due to more rapid turnover, reduced risk of infection through reduction in number of donors per patient, implying a reduced strain on patients and personnel, and the opportunity provided by the reduced thawing time and the eliminated risk of error to commence therapy immediately after the decision.


[Determining clinical effectiveness of plasmapheresis thrombocyte preparations]. Automated plasmapheresis with Haemonetics-Ultralite plasma collecting system was tested to prepare platelet-rich plasma. 19 patients with bone marrow aplasia/hypoplasia received 112 platelet-rich plasma. The efficiency of platelet transfusions was determined by corrected count increment (CCI) and resonance thrombography in correlation to the clinical situation of patients. Fever and chemotherapy did not impair the values 1 h after transfusion, but the 24-hour CCI showed a reduced survival of the transfused platelets in this group. The CCI was 11.3 1 h after transfusion and 9.5 1 day after transfusion in the patient group without any factor reducing the efficiency of transfusion. In this group, the resonance thrombography significantly improved 1 and 24 h posttransfusion compared to pretransfusion. The results demonstrate an acceptable clinical efficiency of platelet-rich plasma.


[Leukocyte depletion, a comparison of the "Leukotrap in-line" filter system with a sterile added 4-bag Speacell-R-500-A filter system]. Leukocytes in RBC preparations have a variety of immunologic effects in transfused patients. Not only their elimination at time of transfusion but before storage seems to be advantageous. Filtration with the Leukotrap system at the day of preparation led to an overall reduction of leukocytes of 82.2%, i.e. an average of 5.4 x 10(8) leukocytes/RBC concentrate. Filtration with a Sepacell R 500 A eliminated 99.0% of leukocytes, 3.4 x 10(7) cells remained per blood unit. The determination of lymphocyte subpopulations by flow cytometry after filtration with the Leukotrap system showed a more effective adsorption of B cells than of T and natural killer cells.


[Permselect--a new substance for leukocyte elimination]. Permselect particles consisting of purified plant cell wall ghosts obtained from the biomass of a suspension culture of Chenopodium album L. show a very high efficiency for leukocyte binding. A package containing 1 g dry Permselect eliminates 99-100% of leukocytes from 80 ml of fresh blood, preserved whole blood, or red blood cell concentrates. At comparable conditions Permselect shows a higher efficiency of leukocyte binding than commercial filter material (cotton, cellulose acetate). The vitality parameter of filtered erythrocytes and their stability during storage were not altered. The data are discussed with respect to causality of leukocyte binding to the pectin-containing surface of the cell wall particles.


[Filtration of buffy coat-free erythrocyte suspensions in additive solution]. Buffy coat-free red cell concentrates in SAG-M (RCC) were produced by BAT system (leukocyte content 132 x 10(6)/RCC). They were stored for 4 weeks and filtered by Erypur Optima (E), Sepacell R 500 B (S) und PALL RC 50 TM (P). Leukocyte depletion was very effective (E: 0.56 x 10(6)/RCC; S: 0.36 x 10(6)/RCC; P: 0.55 x 10(6)/RCC) but hemolysis was remarkable (E: 301 +/- 195; S: 127 +/- 123; P: 368 +/- 256 mg/RCC). Therefore preparation of two RCC per filter was only acceptable with S. In S and P loss of red cells was tolerable (E: 90.5; S. 41.8; P: 38.2). In contrast, E should be rinsed with sodium chloride at the end of the preparation. In E and S filtration times were short without additional pressure (E: 6.6 min; S: 4.7 min; P: 20.3 min). We conclude from our results that the use of buffy coat-free red cell concentrates in additive solution considerably reduces the problems of filtration, e.g. storage interval, leukocyte reduction, hemolysis, filtration flow. Despite this, bedside filtration is not recommended because quality assurance is necessary.


[Production of rejuvenated and stable, leukocyte depleted erythrocyte concentrates using the heated centrifugation method]. We report on an alternative to filtration for the preparation of leukocyte-poor red cell concentrates (LP-RCC). It is based on the method of Schneider. Using RCC with buffy coat it is comparably effective in leukocyte reduction [98.3 +/- 1.0%, (3.7 +/- 2.6) x 10(6) leukocytes] and more effective in platelet reduction (96.9 +/- 2.5%). Addition of PAGGS-M before heating (30 min, 37 degrees C) as well as after preparation significantly reduces hemolysis (free hemoglobin, LDH, HBDH) and improves the quality of the LP-RCC (ATP, 2,3-DPG) during storage for 24 h after preparation. LP-RCCs prepared with PAGGS-M after 6-day storage show still better quality than before preparation and about the same quality as LP-RCCs 24 h after conventional preparation with saline solution. In conclusion, by use of PAGGS-M and sterile docking LP-RCCs of adequate quality for 6-day storage can be prepared, improving the supply of the patients concerned.


New-generation RIBA hepatitis C strip immunoblot assays. Second-generation hepatitis C virus (HCV) ELISAs are currently in use in Europe and have been submitted for approval in the United States. These new assays contain additional antigens from the putative nucleocapsid and NS-3 regions of the HCV genome in addition to the c100-3 antigen present in first-generation ELISAs. A supplementary test, the second-generation RIBA HCV strip immunoblot assay (2-RIBA HCV SIA) has also been developed. The strip immunoblot assay uses four recombinant HCV antigens [5-1-1 (NS-4), c100-3 (NS-4), c33c (NS-3), and c22-3 (NS-3) (nucleocapsid)] slot blotted on nitrocellulose. Screening of random volunteer blood donors with the Ortho second-generation HCV ELISA (ORTHO HCV 2.0 ELISA) indicates that a substantial change in the repeat reactive donor population is observed with the new test. Two notable features of this change are: (1) A large number of samples reactive in the 2-RIBA HCV SIA for the second-generation antigens, c33c and c22-3, are detected by the ORTHO HCV 2.0 ELISA; (2) the percentage of ORTHO HCV 2.0 ELISA reactive specimens found indeterminate (reactive for only one HCV antigen) by the 2-RIBA HCV SIA is higher than in first-generation HCV ELISAs (approximately 25 vs. 5%). In addition, ORTHO HCV 2.0 ELISA repeat reactive, 2-RIBA HCV SIA indeterminate samples are dominated by reactivity to c22-3 instead of c100-3, which is the case for first-generation HCV ELISA repeat reactive samples. Resolution of 2-RIBA HCV SIA indeterminate samples as either containing anti-HCV antibodies or not, is important in both diagnostic and blood screening environments, especially where donor notification is required. Our approach to resolution of these troublesome samples evolved from initial work with HCV peptides. Early studies with an experimental strip immunoblot assay containing 5 peptides from the nucleocapsid, E2 (NS-1), NS-4, and NS-5 regions of the viral genome indicated that peptides from the nucleocapsid and NS-4 regions of the genome could provide additional evidence for the presence of anti-HCV antibodies with good specificity, but other peptides suffered from poor specificity. In addition, no immunoreactive peptide from the NS-3 (c33c) region of the virus is available, presumably because the major epitope(s) of this key second-generation antigen is a conformational determinant.(ABSTRACT TRUNCATED AT 400 WORDS)


[Effect of leukocyte depleting polyester filters on thrombocytapheresis concentrates]. We studied platelet loss, leukocyte depletion, T/B cell ratio, platelet morphology and platelet function (aggregation, hypotonic shock reaction, retention and retraction) prior to and after filtration through two different polyester filters (Sepacell PL-5A, Pall PL100) as well as the posttransfusional platelet increment 1 h after transfusion in 8 pairs of single-donor platelet concentrates (Cobe Spectra, Fresenius AS 104). Leukocyte depletion was effective (>99.9%) and platelet loss acceptable in both filters, all tests showing no indication of a diminished platelet function by filtration.


[Effect of storage duration and irradiation dose on in vitro aggregation of thrombocytes]. We studied the influence of storage (1-5 days) and gamma irradiation (0, 25, 50, 100 Gy) on the in vitro aggregability of stored platelets. Platelet aggregation was measured using the method of Born and Breddin and the aggregating agents ADP, collagen, ristocetin and arachidonic acid. With increasing time of storage the ADP-, collagen- and ristocetin-induced platelet aggregation was significantly diminished. On the other hand, the irradiation even with 100 Gy had no additional effect.


[Graft versus host disease with fatal outcome after administration of filtered erythrocyte concentrates]. Transfusion-associated graft-versus-host disease (TA-GVHD) resulting from the engraftment of competent lymphocytes contained in blood products has been well described in immunocompromised patients and more recently in immunocompetent patients. Prophylactic irradiation of blood products prior to transfusion is the most efficient way to prevent TA-GVHD. Standard blood bank measures to reduce mononuclear cell contamination in red blood cell units, such as freezing, washing and filtration, may reduce the number of viable lymphocytes to prevent immunizations. However, it is unknown whether the depletion of leukocytes with these techniques would decrease the risk of TA-GVHD. In this report we describe the first case of TA-GVHD following transfusion of filtrated red blood cells given to a patient receiving cytotoxic therapy for Hodgkin's disease.


Decreased formation of the complement component C4A in leukocyte-depleted stored whole blood. As reported earlier, factors of the complement cascade get activated in CPD-A1-stabilized whole blood. While leukocyte depletion inhibited partially the activation of C4, it had no effect on C3a concentrations. Therefore cleavage of C4 during storage of whole blood seems to be partially leukocyte-dependent, whereas the activation of C3 is possibly due to the activation of the alternate pathway of the complement system by contact of blood to plastic surfaces. Even though the radioimmunologically measured C3a might be inactive as an anaphylatoxin, these observations are of clinical importance since the inactivated C3a-desArg still possesses biological activities, like activation of platelets which may lead to hypercoagulability and thrombosis.


Age-dependent regeneration of plasma proteins after donor plasmapheresis. To analyze age-dependent effects of a plasma donation on the regeneration of plasma proteins, two groups of first-time plasma donors were selected (< 25 and > 50 years). The regeneration kinetics of different plasma proteins until 3 weeks after donation was followed up. It can be concluded that a single plasma donation has an age-dependent effect on regeneration, especially on the prolonged regeneration of cholinesterase and immunoglobulins.


[Effect of plasma and plasma components on lymphocyte transformation]. Blood transfusions have an immunosuppressive effect on the recipient and induce changes of several immunological parameters. We therefore studied the effect of homologous and autologous fresh plasma, fresh-frozen plasma, heparinized plasma, as well as the influence of i.v. immunoglobulins on the MLC- and mitogen-induced lymphocyte transformation. We conclude from our study that, besides unspecific mechanisms, an unknown plasma factor enriched in the immunoglobulin fraction suppresses the MLC- and mitogen-induced T-cell immune response.


The role of lipoproteins in inactivation of endotoxin by serum. Lipopolysaccharide (LPS)-induced release of cytokines from human monocytes can be prevented by previous interference of serum with LPS. This inactivation is a function of lipoproteins. Here we show that low-density lipoproteins as well as high-density lipoproteins can inactivate LPS. The effects of heparin and EDTA on LPS inactivation by serum are also described.


Prevention of cytomegalovirus infection after organ transplantation with passive immunization. An analysis of 6 randomized clinical trials. We performed a meta-analysis of 6 randomized clinical trials to study the efficacy of passive immunization against CMV in solid organ transplantation. The results indicate that passive immunization against CMV prevents (fatal) CMV disease in CMV seronegative recipients of a CMV seropositive allograft, even when treated with additional immunosuppression in case of rejections. Passive immunization with anti-CMV immunoglobulin preparations did not reduce the incidence of CMV infection or disease in CMV seropositive recipients.


[Evaluating the safety with reference to intravenous tolerance of immunoglobulins]. The section 'Plasmatic Blood Constituents' of DGTI (German Society for Transfusion Medicine and Immunohematology) performed four ACA (anticomplementary activity) ring tests with human immunoglobulins for intravenous application (IVIG). The laboratories carrying out the analyses presented relatively consistent ACA results for IVIG commercial preparations that were obtained by means of the ACA method elaborated based on these tests. The tolerance trials evaluating the IVIGs solely by means of in vitro tests are not sufficient to serve as safety controls since there is no clear correlation existing between the ACA values and their compatibility in the animal model (rat).


The 'critical hematocrit': a figure differing from patient to patient. In my opinion, the problem of a 'critical hematocrit' can be summarized in five contentions: First, it is inadmissible to label any single hemoglobin or hematocrit value as being generally acceptable, the reason being, second, that the adequate values differ between patients and sometimes also between various stages of their individual course--for instance during the intra- and the postoperative period. Third, a hemoglobin or hematocrit within the normal range constitutes a natural buffer against encroachments upon the oxygen supply from non-Hb causes. Intentional manipulation of this buffer requires a careful assessment of potential benefits vs. risks. Fourth, a patient in otherwise perfect condition tolerates a hemoglobin or hematocrit below 10 g/dl or 30%, respectively, down to approximately 8 g/dl or 25%- but tolerance is not necessarily equivalent to an optimum. And fifth, the patient most dependent on his 'hemoglobin buffer' is the individual who has to overcome troubles without the monitoring facilities of an intensive care unit, for instance in the peripheral hospital equipped only for primary care.


[Critical hematocrit from the viewpoint of the clinician]. The hazards of homologous blood transfusions have been controlled but not eliminated by modern blood banking methods. In addition to the possibility of transmission of infectious diseases, blood transfusion has been reported to induce immunosuppression and thereby impair the host resistance of surgical patients. Experimental and clinical research, particularly in cardiac surgery, have encouraged physicians to accept postoperative anemia. The influence of hemodilution on oxygen supply to the tissues was found to be an increase of local oxygen tension. When hematocrit is progressively lowered under normovolemic conditions, blood cell flux per time and tissue unit is maintained at a hematocrit level as low as 20 vol% as a result of an increase of red blood cell velocity. An increasing number of cardiac operations have been performed upon patients belonging to Jehova's witnesses, inducing an average hematocrit of 11 vol% during extracorporeal circulation. The outcome of these patients was similar to that of patients having received blood transfusion in order to elevate oxygen supply. Some recent studies in critically ill patients demonstrate the importance of oxygen consumption as an important criterion in estimating the effect of oxygen transport. It seems to be proven that, in presence of cardiovascular stability, elevation of the hemoglobin level above 9 g/dl does not result in an increase of oxygen utilization. In accordance with the topical literature we suggest that during the perioperative period a hemoglobin level of 8.5 g/dl will become an accepted transfusion trigger. Particular patients will tolerate even much lower levels.


[The critical hemoglobin value in the therapy of chronic anemia]. Adequate oxygen delivery to the tissues can usually be assured until the hemoglobin concentration falls below 7-8 g/dl in patients with chronic anemia provided their compensatory mechanisms are not impaired. Such a reduction of hemoglobin concentration is usually tolerated without symptoms at rest. Under increased physical activity or by an underlying impaired function of the lung or the cardiovascular system, adaptation to anemia may not be sufficient. For this reason the impact of anemia and the resulting hypoxemia must be assessed by determining oxygen needs at various levels of physical activity. The clinical assessment for adequate oxygenation of tissues can be sufficiently estimated by simple parameters as tachycardia, dyspnea etc. Benefits from replenishing O2 carrying capacity by transfusion must be always balanced against transfusion-associated risks. Below a hemoglobin concentration of 7 g/dl oxygen transport is impeded and transfusions are necessary to avoid jeopardizing the patient by additional stressful illnesses such as fever.


[The critical hemoglobin/hematocrit value in obstetrics]. During pregnancy, there are characteristics changes in the hemoglobin and hematocrit values. Compared with the norm for nonpregnant women, there is an increase in the total number of erythrocytes and in the plasma volume. An overproportional increase of the latter results in hydremia. The normal physiologic range for hemoglobin during pregnancy is 11.5-13.0 (13.5) g/dl; anemia is, by definition, present when the values are under 11 g/dl and is quite common in pregnancy. Since it is caused almost exclusively (95%) by iron deficiency, iron therapy or routine iron supplementation can influence its incidence. Values outside the norm range are associated with complications during pregnancy and with growth retardation of the fetus.