The ontogeny and functional characteristics of human B-1 (CD5+ B) cells. We demonstrate that, on average, greater than 90% of B lymphocytes in fetal spleen express CD5 at gestational ages of 17-23 weeks. Similarly, CD5+ B cells (B-1 cells) are the major B cell subset in umbilical cord blood. These findings depend on the optimization of fluorochrome conjugated anti-CD5 reagents for multiparameter fluorescent-activated cell sorter (FACS) analysis. From infancy through childhood the percentage of B-1 cells gradually diminishes in both spleen and peripheral blood. Stable adult levels, 25-35% of the total B cell population, are reached in late adolescence. The decrease in the percentage of B-1 cells in spleen is accompanied by an increase in conventional (CD5-) B cells, keeping the percentage of total B cells per mononuclear cells relatively constant. In contrast, in peripheral blood, the concentration of both B-1 cells and total B cells decreases, while T cells increase. At the functional level, we show that polyreactive IgM autoantibodies are produced by FACS-sorted CD5high B cells, but not by CD5- B cells from adolescent spleen. In contrast, fetal splenic CD5high and CD5- B cells appear functionally uniform, both producing IgM autoantibodies that are typical of B-1 cells. The apparent level of CD5- B cells in fetal spleen, on average 10% of total B cells, may still result from limitations of our reagent. The prominence of B-1 cells in fetal spleen and cord blood, the gradual reduction of B-1 cells with increasing age, and its characteristic repertoire, all suggest a role for this cell type in immunologically immature hosts.


Antibodies of HIV-1 positive subjects and experimentally immunized primates and rodents bind to sequence divergent regions of the third variable domain (V3) of gp120. Several motifs have been found to be the target of the neutralizing antibody response to HIV, the human immunodeficiency virus. One of the well characterized motifs maps to a loop within the third hypervariable region (V3) of the exterior envelope glycoprotein gp120 at amino acid positions 308-331 and is referred to as the principal neutralizing determinant (PND). The sequence of this V3 loop raises the question of the immunogenicity and the degree of diversity of the antibody response to the PND. We show here that this neutralization-related motif is highly immunogenic in HIV-positive subjects and in experimentally immunized primates and rodents submitted to various anti-HIV immunization regimens. In probing the diversity of the antibody response to PNDs corresponding to 11 HIV sequence-divergent isolates in serum samples of 101 HIV-positive individuals we found that human antibodies exhibit binding affinity to up to nine PND synthetic peptides. This antibody binding was in all cases tested inhibitable by the homologous PND synthetic peptide. We additionally demonstrate that this antibody cross-reactivity towards sequence-divergent PNDs is detectable in the sera of mice and chimpanzees experimentally immunized against a single HIV-1 isolate. Finally, we noticed that there is a hierarchy of reactivity among the various PNDs wherein the synthetic peptide corresponding to the MN isolate was generally the most prominently recognized by antibodies of human, non-human primate, and rodent origins. Based on these findings and on features of the sequences analyzed we suggest that, despite its overall sequence variability, the PND encompasses conserved amino acid positions or epitopes that are the targets of antibodies recognizing sequence-divergent isolates. We also propose that the high positive charge density of the most frequently recognized PNDs and the high antigenicity value of some of their residues are critical to the broad immunoreactivity of this neutralization-related motif.


Contribution of the digestive tract microflora to amylomaize starch degradation in the rat. To study in vivo the contribution of the bacterial flora to amylomaize starch degradation in the rat, germ-free and conventional rats were fed on a diet containing either a normal maize starch or an amylomaize starch. In germ-free rats maize starch was almost totally digested in the small intestine, whereas 40% of the ingested amylomaize starch reached the caecum and 30% was excreted, despite the very high endogenous amylase activity. Study by transmission electron microscopy of germ-free caecal contents showed an endocorrosion of the starch granule. In conventional rats, as in germ-free rats, digestibility of maize starch reached 98% in the small intestine, whereas that of amylomaize starch was only 60%. In the caecum of these rats amylomaize starch was fermented, and this led to a decrease in caecal pH and to formation of short-chain fatty acids (SCFA), especially propionate. Comparison between conventional rats fed on maize starch or amylomaize starch showed that caecal SCFA concentrations during a circadian cycle varied in the same way whereas total SCFA and lactic acid concentrations were much higher in rats fed on amylomaize starch. Amylase (EC 3.2.1.1) activity was similar in the caecal contents of conventional rats whatever the ingested starch. It was lower in conventional than in germ-free rats, but no starch granule remained in the caecum of conventional rats. These results showed that bacterial amylase was more efficient at degrading resistant amylomaize starch than endogenous amylase.


The effect of sphingosine and phorbol ester on the signal transduction enzymes and fibronectin release in cell culture. In testing the hypothesis that the stimulation of the release of fibronectin (FN) by 12-O-tetradecanoylphorbol 13-acetate (TPA) from human lung fibroblasts in culture is the result of activation of protein kinase C (PKC), we found that the PKC inhibitor sphingosine strongly inhibited FN release in presence and even in absence of TPA. However, a different PKC inhibitor, calphostin C, despite almost complete inhibition of PKC, had no effect on FN release. We concluded that sphingosine is a potent inhibitor of FN release from the cell surface, independent of its inhibition of PKC; and that TPA stimulates release of FN by a pathway other than activation of PKC. We found that the activation of PKC by TPA was accompanied by inhibition of the cAMP-dependent protein kinase (PKA). When PKA was inhibited by an antagonist (H8, a cAMP analogue) at a concentration specific for PKA inhibition, the release of FN was stimulated similar to the stimulation with TPA. Activation of PKA with forskolin resulted in decreased FN release. In conclusion, we have shown that: (1) sphingosine had a robust effect inhibiting the release of FN from fibroblasts, independent of its action on PKC; (2) TPA treatment of these cells resulted in inhibition of PKA; (3) inhibition of PKA stimulated FN release whereas its activation decreased this release. It is possible that PKA, by phosphorylating a protein, may function, directly or indirectly, in keeping FN attached to the cell surface of fibroblasts.


Pseudo-obstruction in children.

Pseudo-obstruction in children. Pseudo-obstruction of the gastrointestinal tract is a rare disorder of impaired gastrointestinal motility. The more common symptoms of pseudo-obstruction in the infant or child include dysphagia, nausea, vomiting, abdominal distention, abdominal pain, and constipation. The majority of children have symptoms within the first year of life. Chronic cases of pseudo-obstruction are associated with neuropathic or myopathic changes in other parts of the body. Bladder dysfunction and neurological problems have been reported. The diagnosis of pseudo-obstruction is based on history, physical examination, radiographic studies and motility studies. Advances in medical technology have facilitated the identification of abnormal motility patterns in children. Therapy for pseudo-obstruction is primarily supportive. The use of motility agents has been unsuccessful in treating pseudo-obstruction. Nutritional and antibiotic therapy are the mainstays of treatment. Nursing interventions, patient/family education and advances in home care technology have improved the quality of life for children with pseudo-obstruction. Small bowel transplantation offers hope for the future.


Combination of recombinant cytokines fails to produce ex vivo expansion of human blood hematopoietic progenitor cells. The concept of ex vivo expansion of the human progenitor cell population was tested in this study. In the presence of interleukin-3 and -6 we compared the abilities of various liquid culture conditions of human blood-derived hematopoietic progenitor cells to yield a suitable condition for the expansion of blood progenitor cells. Although the best result was obtained in the gastpermeable bag culture system, the enhancement effect (maximum of 3.8-fold for granulocyte/macrophage colony-forming units and 3.0-fold for erythroid burst-forming units) lasted for only a short period (less than 6 d). The results of this study are disappointing for clinical use, and attempts with currently available technology appear to be limited in their potential.


Endothelin modulation of tissue plasminogen activator release from human vascular endothelial cells in culture. To clarify a possible involvement of the vasoconstrictive peptide endothelin in the regulation of endothelial cell-mediated fibrinolytic system, confluent cultures of vascular endothelial cells from human umbilical vein were incubated in serum-free medium in the presence of endothelin-1 at 100 nM and below, and tissue plasminogen activator antigen (t-PA:Ag) in the medium was determined by enzyme immunoassay. Endothelin-1 at 1 nM and above significantly decreased the release of t-PA:Ag from the endothelial cells after a 24 h incubation. The t-PA:Ag release was also decreased by either endothelin-2 or endothelin-3 at 10 nM. The activity of lactate dehydrogenase in the medium was not changed by endothelin-1 at 100 nM and below, suggesting that the peptide did not cause nonspecific cell damage. The decrease in the t-PA:Ag release induced by endothelin-1 occurred in the presence or absence of 8-bromo cyclic AMP, which is an active congener of cyclic AMP; 3-isobutyl-1-methylxanthine, which is an inhibitor of phosphodiesterase; and forskolin, which is a stimulator of adenylate cyclase. These results strongly indicated that cyclic AMP which is known to down-regulate t-PA:Ag release was not involved in the endothelin-1 effect. However, endothelin-1 failed to decrease the t-PA:Ag release in the presence of either calcium ionophore A23187 or EGTA; the ionophore itself markedly decreased the release. The cytosolic calcium accumulation was significantly increased by endothelin-1. These results suggest that endothelin-1 decreases the release of t-PA:Ag from human endothelial cells through an excess accumulation of intracellular, especially cytosolic which would be mediated by an extracellular, calcium-dependent mechanism.


Studies on the mechanism of binding of serpins and serine proteases. The serpin family of inhibitors have an important role in the control of coagulation and fibrinolysis. For a full understanding of how these pathways operate in vivo and correct measurement of enzyme and inhibitor activity, in vitro knowledge of the mechanism of action of serpins is essential. Using alpha 2-antiplasmin as a model inhibitor we find, in contrast to most previous reports, a reversible mechanism: E + I in equilibrium with EI in equilibrium with EI', where complex formation is two stepped, but both steps are reversible. Our work with plasmin in the presence of 50 mM aminohexanoic acid shows that binding of alpha 2-antiplasmin is very tight (but reversible) with an overall Ki (Ki final) = 4.0 pM. With chymotrypsin (a model serine protease) Ki final = 100 pM, so as expected binding of alpha 2-antiplasmin is weaker with chymotrypsin. However, analysis of the individual rate constants shows that the difference in strength of binding is accounted for by the dissociation rate constant for the second step (k-2) = 1.9 x 10(-6) s-1 for plasmin and 1.1 x 10(-4) s-1 for chymotrypsin. Thus k-2, the rate constant previously ignored, explains the different affinities of alpha 2-antiplasmin for these two enzymes. Furthermore, this model of two (or more) step, reversible binding is accepted for protease inhibitors of other families. With one of these, aprotinin (a Kunitz inhibitor) with plasmin we also obtain a two-stage reversible mechanism with a Ki final = 200 pM and the strength of inhibition is also largely determined by k-2 = 3.5 x 10(-5) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)


Aprotinin in cardiopulmonary bypass--effects on the Hageman factor (FXII)--Kallikrein system and blood loss. Aprotinin has been used in our hospital in open heart surgery for almost 20 years and recently published studies have revealed a reduction in postoperative blood loss under this therapy. In the present study patients undergoing aorto-coronary bypass operations received either 20,000 KIU aprotinin/kg body weight (group 2) or 60,000 KIU aprotinin/kg body weight (group 3). Another group of patients without aprotinin served as a control (group 1) and postoperative bleeding was more pronounced in these patients compared with the other groups. In parallel, slight reductions in kallikrein-like activity were observed in patients treated with aprotinin. Furthermore, we have shown that the main inhibitor of the contact phase, C1-esterase-inhibitor, loses some of its activity against beta-FXIIa in the presence of heparin. Aprotinin was able to partly antagonize this phenomenon. All studies dealing with the effect of aprotinin in extracorporeal circulation demonstrate hyperfibrinolysis in untreated patients. Aprotinin is known to inhibit plasmin at low concentrations and thus reduced the postoperative bleeding tendency (group 2). In addition, plasma kallikrein is inhibited by high aprotinin concentrations and is responsible for a reduced activation of the contact phase system. This effect led to a further reduction in blood loss (group 3).


Serotonergic, noradrenergic and galaninergic projections to the nucleus parafascicularis. Immunocytochemical staining for serotonin (5-HT), tyrosine hydroxylase (TH) and galanin (GAL) was combined with horseradish peroxidase (HRP) retrograde tract-tracing technique to analyze the localizations of 5-HT-, catecholamine (CA)- and GAL-containing neurons in the brainstem which project to the nucleus parafascicularis (PF) in rats. It is demonstrated that most of the retrogradely HRP-labeled neurons (70%) in bilateral periaqueductal gray (PAG) and raphe nuclei are positively immunostained by antiserum to 5-HT, and that most of the retrogradely HRP-labeled neurons (over 80%) in bilateral locus coeruleus (LC) are positively immunostained by antisera to both TH and GAL. The possible functions of these PF-petal serotonergic, catecholaminergic (actually noradrenergic) and galaninergic projections are discussed.


Modulation of the behavioural effects of interleukin-1 in mice by nitric oxide. Interleukin-1 is a cytokine which mediates the host response to infection and inflammation and is responsible for sickness behaviour. Inhibition of nitric oxide synthase activity by N omega nitro-L-Arginine-Methyl-ester (30 mg kg-1, i.p.) potentiated the depressive effects of interleukin-1 (375 ng, i.p.) on social investigation in mice. This effect was attenuated by L-arginine (180 mg kg-1, i.p.) but not by D-arginine. The same treatment did not alter the body weight loss induced by interleukin-1. These results suggest that nitric oxide plays a protective role in the neural effects of interleukin-1.


The identification of leaf thionin as one of the main jasmonate-induced proteins of barley (Hordeum vulgare). Jasmonic acid (JA) and its methyl ester (JA-Me) are able to introduce the accumulation of several specific polypeptides in cut leaf segments of barley. Two of the most prominent JA-induced proteins of M(r) 15,000 and 23,000 have been characterized by isolating and sequencing complete cDNA sequences. While the sequence of the M(r) 23,000 polypeptide shows no similarity to published sequences, the sequence of the M(r) 15,000 polypeptide corresponds to the higher-molecular-weight precursor of a leaf thionin previously characterized. Transcripts for the M(r) 23,000 and M(r) 15,000 polypeptides accumulate in leaf segments shortly after the beginning of JA treatment. JA and JA-Me induce the appearance of the two proteins not only in leaf segments but also in intact barley seedlings. However, in seedlings the accumulation of JA-induced proteins occurs much more slowly and requires high concentrations of volatile JA-Me. Thus, in barley it seems unlikely that volatile JA-Me is involved in the interaction between different members of this species, as has been proposed recently for tomato seedlings.


Molecular details of tomato extensin and glycine-rich protein gene expression. In a recent publication (Plant Molecular Biology 16: 547-565 (1991)), Showalter et al. described the isolation and initial characterization of fifteen extensin and extensin-like tomato cDNAs. These cDNAs were determined to fall into five distinct classes; class I and II clones encoded extensins, class III and V clones encoded glycine-rich proteins (GRPs), and class IV clones encoded a portion of a GRP sequence on one DNA strand and a portion of an extensin sequence on the other DNA strand. In this publication, a more detailed analysis of the expression of these cDNA classes was performed with respect to wounding in various tomato organs, development, kinetics and systemic extent of the wound response, ethylene treatment, abscisic acid (ABA) treatment, and drought stress by using RNA gel blot hybridizations. In general, extensin gene expression was readily detected in stems and roots, but not in leaves. With both class I and II extensin cDNA probes, wound-induced accumulation of mRNA in stems was first detected between 4 and 8 h after wounding with maximal accumulation occurring after 12 h. Moreover, these extensin wound responses were detected locally at the wound site but not systemically. Expression of the class III GRP was largely limited to wounded stem tissue. Initial detection and maximal accumulation of the class III GRP mRNA was similar to the extensins mRNAs; however, this GRP wound response occurred both locally and systemically. Additionally, abscisic acid treatment and drought stress resulted in the marked accumulation of the class III GRP mRNA in tomato stems, but did not alter the expression of the other cDNA classes. In contrast, expression of the class V GRP occurred in stems and roots and to a lesser extent in leaves and decreased in response to wounding over a 24 h time period. The class V GRP wound response was further characterized by an early, transient accumulation of mRNA occurring 2-4 h after wounding in stems and by its local nature.


An ethylene-related cDNA from ripening apples. We report the isolation of a ripening-related apple cDNA which is complementary to a mRNA which may be involved in ethylene production. Poly(A)+ RNA was extracted from cortical tissue of ripe apple fruit (Malus domestica Borkh cv. Golden Delicious) and a cDNA library constructed in the plasmid vector pSPORT. The library was screened with pTOM13, a tomato cDNA clone thought to code for ACC oxidase in that fruit. An apple cDNA clone (pAP4) was isolated and sequenced. The 1182 bp cDNA insert includes an open reading frame of 942 bp, and shows strong homology with reported tomato and avocado sequences, both at the nucleic acid and amino acid levels. The polypeptide has a calculated molecular mass of 35.4 kDa and a calculated pI of 5.15. In apple cortical tissue, expression of pAP4-complementary RNA increased with ethylene production by the fruit during ripening. Expression was also enhanced in both ethylene-treated and wounded fruit.


Nucleotide sequence of a tobacco cDNA encoding plastidic glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato. A full-length cDNA encoding glutamine synthetase (GS) was cloned from a lambda gt10 library of tobacco leaf RNA, and the nucleotide sequence was determined. An open reading frame accounting for a primary translation product consisting of 432 amino acids has been localized on the cDNA. The calculated molecular mass of the encoded protein is 47.2 kDa. The predicted amino acid sequence of this precursor shows higher homology to GS-2 protein sequences from other species than to a leaf GS-1 polypeptide sequence, indicating that the cDNA isolated encodes the chloroplastic isoform (GS-2) of tobacco GS. The presence of C- and N-terminal extensions which are characteristic of GS-2 proteins supports this conclusion. Genomic Southern blot analysis indicated that GS-2 is encoded by a single gene in the diploid genomes of both tomato and Nicotiana sylvestris, while two GS-2 genes are very likely present in the amphidiploid tobacco genome. Western blot analysis indicated that in etiolated and in green tomato cotyledons GS-2 subunits are represented by polypeptides of similar size, while in green tomato leaves an additional GS-2 polypeptide of higher apparent molecular weight is detectable. In contrast, tobacco GS-2 is composed of subunits of identical size in all organs examined. GS-2 transcripts and GS-2 proteins could be detected at high levels in the leaves of both tobacco or tomato. Lower amounts of GS-2 mRNA were detected in stems, corolla, and roots of tomato, but not in non-green organs of tobacco. The GS-2 transcript abundance exhibited a diurnal fluctuation in tomato leaves but not in tobacco leaves. White or red light stimulated the accumulation of GS-2 transcripts and GS-2 protein in etiolated tomato cotyledons. Far-red light cancelled this stimulation. The red light response of the GS-2 gene was reduced in etiolated seedlings of the phytochrome-deficient aurea mutant of tomato. These results indicate a phytochrome-mediated light stimulation of GS-2 gene expression during greening in tomato.


The isolation and characterisation of a cDNA clone encoding L-asparaginase from developing seeds of lupin (Lupinus arboreus). An L-asparaginase cDNA clone, BR4, was isolated from a Lupinus arboreus Sims developing seed expression library by screening with polyclonal antibodies to the seed asparaginase. The cDNA hybridised with an oligonucleotide probe designed from amino acid sequence data and was found on sequencing to be 947 bp in length. Six polypeptide sequences obtained previously could be placed along the longest open reading frame. Computer-aided codon use analysis revealed that the cDNA sequence was consistent with other plant genes in terms of codon use. The cDNA insert was used to analyse asparaginase transcription in various tissues by northern blot analysis. A transcript size of approximately 1.2 kb was detected in L. arboreus seed total and poly(A)+ RNA. The level of this transcript declined from 30 days after anthesis to an undetectable level by day 55. Furthermore, under the high stringency conditions used, the seed asparaginase cDNA did not hybridise with total or poly(A)+ RNA isolated from root tips, suggesting that the asparaginase known to be present in this tissue may be the product of a different gene. Southern analysis suggested the seed asparaginase is a single-copy gene. The plant asparaginase amino acid sequence did not have any significant homology with microbial asparaginases but was 23% identical and 66% similar (allowing for conservative substitutions) to a human glycosylasparaginase.


Activation of flavonoid biosynthesis in roots of Vicia sativa subsp. nigra plants by inoculation with Rhizobium leguminosarum biovar viciae. Infective (nodulating) Rhizobium leguminosarum biovar viciae (R.l. viciae) bacteria release Nod factors which stimulate the release of nodulation gene-inducing flavanones and chalcones from roots of the host plant Vicia sativa subsp. nigra (K. Recourt et al., Plant Mol Biol 16: 841-852; H.P. Spaink et al., Nature 354: 125-130). The hypothesis that this release results from increased synthesis of flavonoids was tested by studying the effect of inoculation of V. sativa with infective and uninfective R.l. viciae bacteria on (i) activity of L-phenylalanine ammonia-lyase, (ii) level of chalcone synthase mRNA, and (iii) activity of (eriodictyol) methyltransferase in roots. Consistent with the hypothesis, each of these parameters was found to increase 1.5 to 2-fold upon inoculation with infective R.l. viciae bacteria relative to the situation for uninoculated roots and for roots inoculated with uninfective rhizobia.


Cloning and expression of an embryo-specific mRNA up-regulated in hydrated dormant seeds. Dormant seeds do not germinate when imbibed in water even when conditions are favorable for germination. These hydrated seeds remain viable, but growth-arrested for weeks due to unknown restrictions within the embryo. As a model system for the study of the molecular processes occurring in dormant seeds, we have chosen to examine gene expression in Bromus secalinas, a grass species that produces seeds with high levels of embryonic dormancy. Using differential screening for mRNAs present in hydrated dormant embryos, we have identified a cDNA clone, pBS128, that encodes a mRNA transcript found in the embryos of hydrated seeds of B. secalinus as well as in embryos from mature dry seeds. Striking differences in pBS128 transcript levels appear upon hydration of dormant and nondormant seeds. Upon imbibition pBS128 transcript levels increase over four-fold in dormant seeds, but rapidly decline and disappear in nondormant seeds, which subsequently germinate. The pBS128 transcript appears to be embryo-specific since the transcript is not detectable in either non-stressed or dehydrated seedling tissue. Application of 50 microM ABA to nondormant seeds arrests germination and enhances pBS128 transcript levels. The nucleotide sequence of the nearly full-length pBS128 cDNA shows no homology to other reported genes, and the putative protein sequence does not exhibit the hydrophilic characteristics of the ABA-responsive LEA (late embryogenesis abundant) proteins.


Cloning and characterisation of an oleosin gene from Brassica napus. The sequence of an oleosin gene from Brassica napus has been determined. This gene contains a single intron of 437 bp and encodes a polypeptide of 195 amino acids. The oleosin gene product has an estimated molecular mass of 21.5 kDa and consists of a highly hydrophobic central domain flanked by relatively polar N- and C-terminal domains. The central domain is highly conserved between all oleosins sequenced to date and contains a run of periodically spaced leucine residues similar to that of a leucine-zipper motif. The gene has been shown to be expressed specifically in the embryo, maximally between 9 and 11 weeks after flowering, i.e. during the seed desiccation stage. Two transcriptional start sites have been mapped to -70 and -21 of the ATG and a putative ABA-responsive element and three repeated motifs have been identified in the promoter. These short promoter sequences could correspond to regulatory elements responsible for embryo-specific gene expression. Up to six genes exist in the oleosin gene family.


DNA-binding properties of cloned TATA-binding protein from potato tubers. A full-length cDNA clone encoding the TATA-binding protein (TBP), the DNA-binding component of the general transcription factor TFIID was cloned from potato tubers. The DNA sequence of this cDNA indicated that the predicted potato protein was very similar to cloned TBP from other species. Genomic southern analysis showed that TBP is encoded in the potato genome as a low-copy-number sequence. The potato TBP cDNA clone was shown to encode a functional protein that interacts in a sequence-specific way with the promoter region of a class-1 potato patatin gene. Functional analysis of carboxy-terminal truncated derivatives of potato TBP showed that important components of DNA binding were located within the carboxy-terminal 54 amino acids. Kinetic and thermodynamic properties of in vitro synthesised potato TBP were investigated, and demonstrated strict salt and temperature preferences for maximum DNA binding activity. In addition on and off-rate measurements showed that both association and dissociation of TBP from DNA is slow. The specific and the non-specific equilibrium constants Ks and Kn were calculated as 5 x 10(9) M-1 and 3.65 x 10(4) M-1 respectively. These results indicate that the interaction of potato TBP with the patatin promoter is highly specific.


Three genes encode 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hevea brasiliensis: hmg1 and hmg3 are differentially expressed. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyses an important step in isoprenoid biosynthesis in plants. In Hevea brasiliensis, HMGR is encoded by a small gene family comprised of three members, hmg1, hmg2 and hmg3. We have previously described hmg1 and hmg2 (Plant Mol Biol 16: 567-577, 1991). Here we report the isolation and characterization of hmg3 genomic and cDNA clones. In comparison to hmg1 which is more highly expressed in laticifers than in leaves, the level of hmg3 mRNA level is equally abundant in laticifers and leaves. In situ hybridization experiments showed that the expression of hmg3 is not cell-type specific while hmg1 is expressed predominantly in the laticifers. Primer-extension experiments using laticifer RNA showed that hmg1 is induced by ethylene while hmg3 expression remains constitutive. The hmg3 promoter, like the promoters of most housekeeping genes, lacks a TATA box. Our results suggest that hmg1 is likely to encode the enzyme involved in rubber biosynthesis while hmg3 is possibly involved in isoprenoid biosynthesis of a housekeeping nature.


Structure of gap junction channels.

Structure of gap junction channels. Gap junctions are regions of contact between adjacent cells, consisting of arrays of channels linking the cell interiors. The channels are formed by polypeptides called connexins; the amino acid sequences of many different connexins are known, and they are thought to resemble each other closely in tertiary and quarternary structure. Single channels have recently been isolated and purified, and earlier evidence has been confirmed showing that they consist of six identical subunits arranged around the central pore. Gap junction channels are known to open and close in response to changes in ligand concentrations and electrical potential; in this respect they are very similar to ligand-gated ion channels which act as receptors in the membranes of excitable cells. The similarity is shown to extend to structural features such as the amino acid residues lining the pore, and perhaps the location of the actual gate.


Neurology of language.

Neurology of language. A host of recent investigations and new theoretical models have advanced our understanding of the neural underpinnings of speech and language in humans. The application of positron emission tomography (PET) techniques to the investigation of language has provided corroborating evidence regarding the role of left hemisphere structures previously associated with language, together with some intriguing new findings. Innovative ideas regarding the ontogeny of language have come from studies of deaf infants who appear to babble with signs in much the same way that hearing infants babble vocally. A number of investigators have focused on the controversial syndrome known as progressive aphasia, and new evidence has supported the importance of this syndrome from both diagnostic (e.g. providing clues regarding neuropathology) and scientific (e.g. yielding information about the organization of lexical access structures in left temporal lobe) perspectives.


Susceptibility to experimental allergic encephalomyelitis in animal models of autoimmunity. Experimental allergic encephalomyelitis (EAE) continues to serve as a useful model of organ-specific autoimmunity and as a model for the human demyelinating disease, multiple sclerosis. Current studies focus on the role of the various cellular components involved in mediating disease, the nature and molecular characterization of the immune process, and the target site within the central nervous system (CNS). The results of these studies are providing new therapeutic rationales.


Natural history and treatment of multiple sclerosis. The course of multiple sclerosis (MS) evolves over decades. Recent advances in our understanding of the spectrum and variability of the course of MS over the long term in large geographically based populations, and over the short term in selected subgroups randomized to the placebo limbs of controlled clinical trials are discussed. Trials of several toxic immunosuppressive drugs and plasma exchange (PE) show that these methods of treatment are unlikely to help MS patients, although azathioprine has some rationale in rapidly advancing cases. The triggering of attacks by viral infections makes one await the results of current trials of interferon-beta with special interest.


Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A. The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no alpha-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.


Molecular cloning of feline interferon cDNA by direct expression. A cDNA sequence coding for feline interferon has been cloned for the first time by screening a cDNA library constructed using Okayama-Berg vector and mRNA derived from the feline cells (LSA-I) induced by TPA (12-o-tetradecanoylphorbor 13-acetate) for the ability of transient expression to produce feline interferon in COS1 monkey cells. The amino acid sequence, deduced from the nucleotide sequence by comparing it with the sequences of other mammalian IFNs, consists of 171 amino acids with 6 cysteins and an N-glycosylation site at the amino acid position 79, and has about 60% homology to human IFN alpha 1. The interferon was partially purified through Blue Sepharose, and its molecular weight was estimated to be 2.4 x 10(4). The antiviral activity was acid stable, and glycosylation was suggested.


Prenatal ontogeny of substance P-like immunoreactivity in the trigeminal spinal caudal subnucleus of the human fetus--an immunocytochemical study. Using an immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the trigeminal spinal caudal subnucleus (TSCS) of human fetus, fetal age (menstruation age) 11.5-40 weeks. The time of initial appearance of SP-LI in the human brainstem TSCS was between the fetal age 11.5 weeks and 16 weeks. While the fetus grew, the density of SPLI fibers and terminals in the human fetus brainstem TSCS increased constantly from fetal age 16 weeks to 40 weeks. These findings indicated that substance P (SP) might play an important role in the human trigeminal nerve system development and in its functional establishment during the prenatal period.


A re-examination of the spino-reticulo-diencephalic pathway in the cat. One commonly accepted idea is that affective aspects of pain sensation are derived from a flow of information from the spinal cord through the reticular formation to the intralaminar thalamus and subthalamus. Little is known, however, about the extent to which spinoreticular terminations and reticulodiencephalic neuronal cell bodies overlap. This study used a combination of anterograde and retrograde tracing techniques to compare these distributions in the cat. Whereas spinoreticular terminations were concentrated caudally and laterally, neurons projecting to intralaminar thalamus and subthalamus were concentrated rostrally and medially. Thus, information conveyed from the spinal cord to the reticular formation appears to have direct access to intralaminar thalamus and subthalamus only by way of a few widely scattered neurons. When considered with the results of others, these results encourage less emphasis on a putative spino-reticulo-diencephalic pathway for pain. Rather, the reticular formation's role in pain is more likely to involve its full complement of interconnected descending and ascending connections.


MR imaging characteristics of noncancerous lesions of the prostate. Radical prostatectomy specimens from 53 men with clinical stage A or B prostate cancer were retrospectively reviewed and compared with correlative axial T2-weighted magnetic resonance (MR) images obtained just before surgery. Non-cancerous lesions were evaluated for signal intensity and location. Focal high-signal-intensity areas (n = 72) were present in 81% of patients. The 26% of lesions seen in the central gland all correlated with cystic atrophy. Of the 53 lesions seen in the peripheral prostate, 47 (89%) were cystic atrophy without associated cancer, four (7.5%) cystic atrophy with cancer, and two (3.8%) focal inflammation. Focal low-signal-intensity areas (n = 42) were present in 60% of patients. Of the 31% of lesions in the central prostate, one-fifth correlated with benign prostatic hyperplasia (BPH) and four-fifths with fibrous tissue. Of the 69% of peripheral lesions, 83% corresponded to fibrous tissue, 10% to BPH, and 7% to normal tissue. Mixed lesions (n = 42) were present in 64% of patients; 86% of these were located centrally and 14% peripherally. All mixed central lesions were BPH; the peripheral lesions were areas of combined cystic atrophy and fibrosis. BPH of low or mixed signal intensity can extend into the peripheral prostate and mimic cancer. High-intensity cystic atrophy associated with cancer can mimic normal tissue.


An intracellular calcium store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store. There is considerable evidence, reviewed by Brostrom and Brostrom [1], that Ca2+ stores are involved in the regulation of protein synthesis. We provide evidence in HeLa cells that is consistent with their findings that depletion of Ca2+ stores and not changes in cytosolic free Ca2+ ([Ca2+]i) inhibit protein synthesis, but we also show that the mechanism leading to depletion is critical. Specifically, depletion of stores by the Ca(2+)-mobilizing hormone histamine does not inhibit protein synthesis. In assessing the role of Ca2+ stores in protein synthesis, experiments in certain cell types have been complicated by the use of Ca2+ ionophores, which simultaneously elevate [Ca2+]i and deplete Ca2+ stores. We have measured total cell Ca2+, [Ca2+]i and protein synthesis in HeLa cells under conditions that allowed evaluation of the separate contributions of stores and [Ca2+]i. Using 1,2-bis(2-aminophenoxyethane)-N,N,N'N'-tetraacetic acid (BAPTA) as an intracellular Ca2+, chelator and thapsigargin, which inhibits the membrane Ca(2+)-ATPase of storage vesicles, total cell Ca2+ can be depleted and this depletion is enhanced by extracellular EGTA which blocks Ca2+ influx; [Ca2+]i is actually lowered by BAPTA under these conditions. Protein synthesis is inhibited by BAPTA in the presence of EGTA and by thapsigargin with or without EGTA. However, histamine which with EGTA, affects an equal degree of Ca2+ depletion does not inhibit protein synthesis. Thus, it is suggested that Ca2+ stores are not homogeneous, and that the hormone-sensitive store specifically does not play a role in the regulation of protein synthesis. In this respect, the hormone-sensitive and insensitive stores do not functionally communicate and may be separately regulated.


Prostate cancer screening: current trends and future implications. Screening for prostate cancer represents a clinical dilemma with no clear evidence to suggest decreased mortality from any diagnostic test. We now possess new knowledge regarding optimal combinations of DRE, TRUS, and PSA. While DRE and TRUS may be too subjective and PSA too nonspecific, their combined predictive values identify not only men at high risk but also those for whom continued frequent screening may not be cost effective. A monoclonal PSA decision level of no more than 4.0 ng/ml should be used, since 40 percent of cancers detected from 4.0 to 10.0 ng/ml already have extracapsular extension. Assuming that DRE is performed by experienced examiners, the combination of PSA and DRE should produce cost-effective early detection and minimize missed cancers below 4.0 ng/ml. TRUS should be reserved for those patients with either PSA elevations and/or DRE abnormalities. The use of TRUS gland volume data to further modify PSA decision levels, such as the "predicted" PSA concept, may also improve TRUS biopsy criteria and predictive values. Prostate cancer detection can then be objectively limited to a small percentage of the population and better selected for earlier, more localized disease. The ultimate decrease in mortality from screening remains to be demonstrated in randomized trials or observed only after decades of increased public awareness about prompt early detection combined with effective, definitive therapy.


The human thymic dendritic cell phenotype and its modification in culture. In order to extend our study of human thymic dendritic cells (DC) we have purified DC by density gradient separation followed by treatment with CD1 and CD2 mAb and antibody-coated immunobeads. The resulting population contains 60 to 75% brightly HLA-DR+ cells. Morphological and functional studies demonstrate that these cells share the common characteristics of dendritic cells. Extensive phenotypic analysis of the purified DC has been made using a panel of mAb. Cytofluorometric assays with mAb reactive with common leucocyte antigen confirm that the brightly HLA-DR+ cells are of mesenchymal origin. Thymic DC express HLA-DQ and HLA-class I antigens. They are also positive for the expression of CD45RA molecules and some express the ICAM-1 and the LFA-1 molecules. DC do not stain with a wide variety of anti-T, -B, and -monocyte or -M phi mAb and lack Fc gamma RIII, CR2, and CR3. Freshly isolated DC failed to stain with OKT6 mAb; however, they progressively acquire the CD1 molecule after a few days culture. The acquisition of CD1 molecule is selective since CD4, CD2, and HLA-ABC molecules are not upregulated under the same conditions. From phenotypic results, it was therefore possible to sort brightly HLA-DR+ or -DQ+ cells and so obtain greater than 90 to 95% purified human thymic DC. Such homogeneous DC populations are obviously of great interest for the study of thymic DC functions.


An inducer of NKCF (NK cytotoxic factor) release: localization on target-cell membrane and initial characterization. Natural killer (NK) cells are probably involved in the elimination of virus-infected cells and of certain tumor cells. NK cell-mediated cytotoxicity (NK-CMC) was extensively studied and was found to consist of several steps. Following recognition and conjugation between the effector and the target cell, the latter one induces release of NK cytotoxic factor (NKCF) from the effector cells. The NKCF binds to the target cell which is subsequently killed. None of the molecules involved in these steps was completely characterized. In the present study it is demonstrated that isolated membranes of target cells can effectively induce the release of NKCF. Furthermore, the activity of such isolated membranes was found to be modulated by interferon (IFN) treatment of the cells prior to membrane isolation. It was therefore concluded that an NKCF-inducing structure (NKIS) is present on plasma membranes and is distinct from the NK-recognition structure. Similarly, the sensitivity to NK-CMC could be transferred from sensitive cells to IFN-gamma-treated (NK-resistant) cells by membrane fusion with the aid of Sendai virus envelope glycoproteins. It is proposed that transfer of NKIS is responsible for the acquired sensitivity to NK-CMC. In addition, it is shown that NKIS activity was recovered following membrane solubilization and reconstitution. Its level on cell surface was modulated by treatment of cells with tunicamycin, thus indicating that NKIS was probably a cell surface glycoprotein.


The p21ras protein as an intermediate signaling molecule in the IL-4-induced HLA-DR expression on normal and leukemic human myeloid cells. We have previously demonstrated that the low number of interleukin-4 receptors (IL-4Rc) on HL-60 leukemia cells render this population susceptible to differentiation by IL-4. As it occurs with normal human monocytes, IL-4 induces the expression of HLA-DR surface antigens on HL-60 cells as well. The second messenger pathway(s) involved after the IL-4 stimulation leading to class II up-regulation has not been fully examined. Here we show that IL-4-induced class II antigen expression on the HL-60 cell line or normal human monocytes is calcium/calmodulin-independent since theophylline (TPH, a calmodulin inhibitor) does not block the IL-4 effect. In addition, the pyruvate kinase C (PKC) pathway does not seem to participate in the process either because in our system activation of PKC by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) is insufficient by itself to induce HLA-DR. We found, however, that a second messenger pathway can be mediated by a G protein system since IL-4 concomitantly induces class II and p21ras expression which can be successfully blocked by a highly specific anti-p21ras monoclonal antibody. In addition, using another p21ras inducer, the 5-azacytidine C (5-AzaC), we showed that this agent can also induce the expression of p21ras and class II, both of which can be inhibited by the same antibody. Thus, it appears that IL-4 selects the G protein system as a signaling pathway in order to exert its action for the induction of HLA-DR on human normal monocytes or M2 leukemia target cells. Since monocytes and macrophages participate in virtually all immune reactions, the regulation of class II induction is of obvious importance.


The VLA-4/VCAM-1 molecules participate in gamma delta cell interaction with endothelial cells. The accumulation of T lymphocytes at the site of chronic inflammation depends on a number of factors including adherence of T cells to vascular endothelial cells (EC) and endothelial permeability. We examined the effects of human gamma delta + T lymphocytes on the permeability of EC to macromolecules and characterized the cell surface molecules that are involved in these interactions. In this model, the flux of [125I]albumin was measured across the EC monolayer after a short-term culture with cloned gamma delta cells. Our results show that coculture of activated, but not resting, gamma delta cells with EC enhances endothelial permeability by a cytolytic process. Pretreating gamma delta cells with monoclonal antibodies directed at either LFA-1 or VLA-4 molecules or pretreating EC with monoclonal antibodies directed against either ICAM-1 or VCAM-1 molecules significantly inhibited gamma delta cell-mediated enhancement in endothelial permeability. This indicated that VLA-4/VCAM-1 and LFA-1/ICAM-1 adhesion pathways participate in gamma delta cell-EC interaction.


Stimulation or tolerization of an anti-myelin basic protein T lymphocyte line with membrane fragments from antigen presenting cells. We have investigated the abilities of a cell-free supernatant of splenocytes or thymocytes, which have been incubated with myelin basic protein (MBP), and of membranes prepared by lysing these cells, to stimulate proliferation of a Lewis rat anti-MBP T lymphocyte line in vitro. The supernatant fraction, obtained by low-speed centrifugation, is thought to contain shed membrane fragments bearing class II MHC protein (Ia) and processed antigen. Almost all of 67 preparations of supernatant fraction and about a third (26/70) of the membrane preparations stimulate proliferation of the line cells in the absence of other antigen-presenting cells and antigen. Some membrane preparations bearing the synthetic peptide S69 (residues 69-89 of MBP), containing the immunodominant encephalitogenic determinant for the Lewis rat, instead of processed MBP could also stimulate proliferation. Those membrane preparations bearing either processed MBP or synthetic S69, which do not stimulate proliferation, induce a state of unresponsiveness in which the cells do not proliferate but produce inositol phosphate. Stimulation of proliferation and induction of unresponsiveness were both inhibited by anti-Ia antibody. Addition of cyclosporin A prevents induction of unresponsiveness. Addition of allogeneic splenocytes or the cell-free supernatant fraction of syngeneic or allogeneic splenocytes or thymocytes, prevents induction of unresponsiveness by providing a necessary costimulatory signal. Further fractionation of the cell-free supernatant by high-speed ultracentrifugation showed that the costimulatory signal resided in a particulate fraction which sedimented and not in the supernatant. These results indicate that the encephalitogenic peptide can induce anergy in T cells when presented on class II MHC in the absence of the costimulatory signal. Tolerizing forms of the membrane preparations which lack the costimulatory signal may be useful for in vivo treatment of autoimmune response.


Human anti-pneumococcal polysaccharide antibodies are secreted by the CD5- B cell lineage. To determine whether human antibody responses to T cell-independent pneumococcal polysaccharide antigens are derived from CD5+ or CD5- B cells, we utilized an ELISPOT assay to detect individual anti-polysaccharide antibody-secreting cells. Human anti-type IV pneumococcal polysaccharide antibody-secreting cells were found in the CD5- B cell subpopulation. An EBV transformed anti-pneumococcal antibody-secreting B cell line was also CD5-. The ontogeny of CD5 expressing B cells correlated with the age at which polysaccharide responsiveness is acquired (generally around age 2 years in humans). The CD5- B cell subset represents only 25-30% of the B cells in young children, but this fraction increases throughout childhood to a plateau of 70-80% of the B cells in adults. These results support the hypothesis that the developmental change in responsiveness to T cell-independent polysaccharide antigens in humans is associated with maturation of the CD5- B cell subset.


CD16 on human gamma delta T lymphocytes: expression, function, and specificity for mouse IgG isotypes. We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human gamma delta T cells. CD16 is expressed by the majority of gamma delta T cells in peripheral blood and by part of the gamma delta T cell clones. The amount of CD16 expressed on gamma delta T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated gamma delta T cells. Like CD16 on CD3- CD16+ natural killer (NK) cells, CD16 on gamma delta T cells can act as an activation site triggering cytotoxic activity. CD16+ gamma delta T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of gamma delta T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of Fc gamma R+ target cells by CD16+ gamma delta T cell clones. The mouse IgG-isotype specificity of CD16 on gamma delta T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3- CD16+ NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both gamma delta T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.


Splanchnic and total body oxygen consumption in experimental fecal peritonitis in pigs: effects of dextran and iloprost. Tissue oxygenation in the gastrointestinal tract and in the liver was studied in a porcine model where septic shock was induced by fecal peritonitis. The effects of different fluid regimes were compared. In one group (n = 8) a moderate amount of crystalloid fluids was given, in another (n = 7) crystalloids and colloids, and in a third group (n = 6) iloprost, a prostacyclin analogue, was administered intra-arterially (10 ng x kg-1 b.w. x min-1) in combination with the crystalline and colloid fluid regime. Septic shock induced by fecal peritonitis reduced cardiac index and oxygen supply to splanchnic organs. Iloprost improved the hepatic arterial blood flow, and tended to attenuate the reduction in liver oxygen delivery. Oxygen consumption (VO2) in the gastrointestinal tract and the liver was significantly increased in the group given crystalloids. These animals developed a hypovolemic/hypodynamic septic shock. Liver VO2 in these animals became flow dependent reflected by increasing hepatic venous lactate values and inversion of lactate turnover by the liver. In the two other groups gastrointestinal and liver VO2 remained constant during the observation period. Oxygen extraction over the liver increased when oxygen delivery decreased. The increased liver VO2 is suggested to be secondary to impaired microcirculation and accumulation of macrophages and leukocytes in the septic liver.


A rapid separation of alpha- and beta-lipoproteins by affinity chromatography. A rapid and inexpensive separation of alpha- and beta-lipoproteins has been achieved using sulfated dextran beads as an affinity chromatography medium. The separation is completed in less than 15 min and the cholesterol content of the lipoprotein fractions can then be determined in a separate procedure.


Laboratory monitoring of androgenic activity in benign prostate hypertrophy treated with a 5 alpha-reductase inhibitor. Testosterone and androstenedione are metabolized by 5 alpha- and 5 beta-reductases to androsterone (A) and etiocholanolone (E), respectively. These are excreted in the urine as conjugates, and the A/E ratio in normal men is usually greater than or equal to 1.5 (as opposed to 1 in women) because of the high 5 alpha-reductase activity in the prostate. The A/E ratio can be determined simply by gas chromatography after acid hydrolysis of a urine sample, extraction of steroids, and formation of trimethylsilyl derivatives. A timed collection of urine is unnecessary because the ratio of A/E is used rather than absolute values. In men suffering from benign prostate hypertrophy who are treated with Finasteride (a 5 alpha-reductase inhibitor), the A/E ratio decreases to less than 0.5. The A/E ratio decrease can be detected long before there is clinical improvement.


Radioreceptor assay for quantifying FK-506 immunosuppressant in whole blood. We describe a quantitative radioreceptor assay (RRA) for quantifying FK-506 in whole blood. FK-506 extracted from whole blood with a cyclohexyl-sorbent column competes with [3H]dihydro-FK-506 for binding to a partially purified preparation of FK-506 binding protein (FK-BP). Free and protein-bound FK-506 are separated on LH 20 Sephadex chromatographic columns. We compared the results of this method with those of an enzyme immunoassay that uses a monoclonal antibody: r = 0.97, Sy/x = 0.039. Between-day precisions (CV) at FK-506 concentrations of 8 and 17 micrograms/L were 9.2% and 8.2%, respectively. Within-run precisions were 5.9%, 8.1%, and 9.4%, respectively, at 4, 8, and 15 micrograms/L. Analytical recovery, evaluated at 5, 10, 15, 20, and 25 micrograms/L for FK-506 added to whole blood, ranged from 98% to 103%. The assay can reliably quantify FK-506 blood concentrations between 1.0 and 25 micrograms/L.


Sensory nerves in the airways as a target for drug development. 1. Electrical stimulation (2.5-10 Hz, 80 V, 1 ms for 15 s) within the spinal canal of the pithed guinea-pig pretreated with atropine, D-tubocurarine and pentolinium caused a bronchoconstrictor response, indicated by a rise of insufflation pressure. 2. The magnitude of these non-cholinergic neuronal bronchoconstrictor responses were frequency-dependent, capsaicin-sensitive and temperature-dependent. 3. Responses could be inhibited by the alpha 2-adrenoceptor agonist B-HT920 (3 mg/kg, i.v.) and the mu-opioid agonist H-Tyr-D-Arg-Phe-Lys-NH2 (DALDA; 1 mg/kg, i.v.) and the attenuation observed could be overcome by use of the respective antagonists idazoxan (3 mg/kg, i.v.) and naloxone (3 mg/kg, i.v.). 4. Substance P-induced bronchoconstriction (0.3 micrograms/kg, i.v.), but not that due to electrical stimulation, was attenuated by indomethacin (3 mg/kg, i.v.), indicating an indirect action of substance P via a product of arachidonic acid metabolism. 5. The prostanoid product of the substance P response was probably thromboxane A2. 6. Hence, novel drug development could be directed towards tachykinin receptors or to the synthesis, release and degradation of neuropeptides.


Allotype-dependent stimulation of peripheral blood and synovial lymphocytes by IgG3 in rheumatoid arthritis. The immunopathologic process of rheumatoid arthritis (RA) is primarily expressed in the synovium where rheumatoid factor (RF) synthesis is concentrated. We hypothesized that RF synthesized by rheumatoid synovial cells (RSC) may be driven via a T cell-mediated immune response developed against IgG3 epitopes. To identify and characterize specific RSC RF epitopes and T cell antigens, two 28 amino acid peptides homologous with the C-terminus of IgG1 (P1) and IgG3 [G3m(5)] (P3) were synthesized and used in RF-binding studies and lymphocyte proliferation assays. Our results indicate that (i) the C-terminus of the CH3 domain contains epitopes for IgG3-reactive RSC RF; (ii) IgG3-reactive RSC RF binds primarily to IgG3 [G3m(5)]; (iii) P3 stimulated proliferation of T lymphocytes from both RA peripheral blood and RSC; and (iv) RF production was enhanced by P3 in selected RA cell cultures. These observations suggest that the C-terminus of IgG3 allotype G3m(5) may be important in T cell activation and RF production in RA.


Hepatic glutamine metabolism in the septic rat. 1. The hepatic metabolism of glutamine, alanine, ammonia, urea, glutathione and glucose was studied in rats made septic by caecal ligation and puncture and was compared with that in rats that had undergone sham operation (laparotomy). 2. Sepsis resulted in increases in the plasma activities of gamma-glutamyltransferase (P less than 0.001), alanine aminotransferase (P less than 0.001) and aspartate aminotransferase (P less than 0.001), the serum total and direct bilirubin concentrations (P less than 0.001), and the blood lactate (P less than 0.01), glutamine (P less than 0.05), alanine (P less than 0.001) and urea (P less than 0.05) concentrations, but produced decreases in the blood ketone body (P less than 0.001) and glutathione (P less than 0.05) concentrations and in the plasma cholesterol concentration (P less than 0.05). These changes were associated with marked negative nitrogen balance in septic rats. 3. Sepsis increased total hepatic blood flow (by 22.7%) together with hepatic arterial flow (by 25.8%) and portal venous flow (by 18.7%). Sepsis resulted in marked increases in the net rates of hepatic extraction of glutamine (by 164%), alanine (by 138%) and ammonia (by 259%) with concomitant increases in the net rates of hepatic release of glutamate (by 105%), glutathione (by 87.5%), glucose (by 70.1%) and urea (by 100.4%). 4. Sepsis increased the activities of liver carbamoylphosphate synthase (by 16.4%), ornithine transcarbamylase (by 29.8%), argininosuccinate synthase (by 28.1%) and arginase (by 33.8%). 5. Septic rats exhibited marked increases in hepatic protein (by 46.0%), RNA (by 43.4%) and DNA (by 37.7%) contents. These changes were accompanied by marked increases in the activity of thymidine kinase (by 35.9%).(ABSTRACT TRUNCATED AT 250 WORDS)


The relationship between human long-latency somatosensory evoked potentials recorded from the cortical surface and from the scalp. In scalp recordings, stimulation of the median nerve evokes a number of long-latency (40-300 msec) somatosensory evoked potentials (SEPs) whose neural origins are unknown. We attempted to infer the generators of these potentials by comparing them with SEPs recorded from the cortical surface or from within the brain. SEPs recorded from contralateral sensorimotor cortex can be characterized as "precentral," "postcentral," or "pericentral." The scalp-recorded P45, N60 and P100 potentials appear to correspond to the pericentral P50, N90 and P190 potentials and are probably generated mainly in contralateral area 1 of somatosensory cortex. The scalp-recorded N70-P70 appear to correspond to the precentral and postcentral N80-P80 and are generated mainly in contralateral area 3b of somatosensory cortex. The scalp-recorded N120-P120 appear to correspond to the intracranial N100-P100 and are probably generated bilaterally in the second somatosensory areas. N140 and P190 (the "vertex potentials") are probably generated bilaterally in the frontal lobes, including orbito-frontal, lateral and mesial (supplementary motor area) cortex. The supplementary sensory area probably generates long-latency SEPs, but preliminary recordings have yet to confirm this assumption. Most of the proposed correspondences are speculative because the different conditions under which scalp and intracranial recordings are obtained make comparison difficult. Human recordings using chronically implanted cortical surface electrodes, and monkey studies of SEPs which appear to be analogs of the human potentials, should provide better answers regarding the precise generators of human long-latency SEPs.


Intraoperative spinal cord monitoring during surgery for aortic aneurysm: application of spinal cord evoked potential. Spinal cord evoked potentials elicited by direct stimulation of the spinal cord were monitored in 21 patients during thoracic or thoraco-abdominal aortic aneurysm surgery. Flexible catheter-type electrodes were used for both stimulating and recording. The basic pattern of the spinal cord evoked potential consisted of an initial spike and a subsequent polyphasic component. The earliest and most frequent alterations after cross-clamping of the aorta were changes in the configuration or amplitude of the polyphasic component. In 13 patients who exhibited no change except minor alterations of the polyphasic component during the initial test clamping for 15 or 20 min, subsequent graft replacements were safely performed without reimplantation of intercostal vessels. In 2 patients who had sudden cardiac arrests, the evoked potential completely disappeared. The polyphasic component disappeared first, followed by the initial spike. Another patient developed acute loss of the potential after the aneurysm was incised, presumably due to distal aortic hypoperfusion. In this case, prolonged distal hypotension resulted in flaccid paraplegia. Intraoperative monitoring of the spinal cord evoked potential is a useful method for the early detection of spinal cord ischemia during surgery requiring aortic occlusion.


Pyramidal tract function during onset of brain death. In 51 patients with primary brain lesions, who fulfilled the criteria of brain death, sequential recording of transcranial magnetic evoked potentials (TMEPs) and somatosensory evoked potentials (SEPs) were performed. In all comatose patients with apnoeic cranial nerve areflexia the TMEP could not be elicited, while the response after cervical magnetic stimulation was always preserved. Similarly, no cortical SEPs were preserved in apnoeic cranial nerve areflexia; however, the cervical somatosensory response was preserved in 44%. In deteriorating patients with coma grade III TMEPs were preserved in 3 instances, while cortical SEPs were already absent. Current brain death criteria, however, were not challenged, as TMEPs were absent in all 51 patients, at the latest when apnoea was noted.


Serial visual evoked potential recordings in geriatric psychiatry. Serial visual evoked potentials to flash and pattern reversal stimuli were recorded in elderly patients with senile dementia of the Alzheimer type (SDAT), multi-infarct dementia (MID) and functional psychiatric illness, and in a group of elderly control subjects. Recordings were made at 6 monthly intervals over a 2 year period. Latency and amplitude of the main components were measured and the flash P2-pattern reversal P100 latency difference value was calculated. In all groups significant changes over time did not occur for any parameters but in the SDAT group the regression coefficient for the latency of the flash P2 component and the flash P2-pattern reversal P100 latency difference was significant, reflecting a trend towards increasing flash P2 latency as time progressed. The flash P2-pattern reversal P100 latency difference was longer in the SDAT and MID groups than in the functional patients, confirming the findings of previous reports. The latency difference in the SDAT group only was significantly greater than that in the control group.


Event-related EEG potentials in mild dementia of the Alzheimer type. Most studies on event-related EEG potentials in dementia have focussed on the P3 component and used auditory stimuli only. In the present study, N2b was analysed in the usual auditory oddball task in addition to P3, and a visual task was employed ("Push"/"Wait"), with recordings including an occipital scalp site. Seven Alzheimer-type patients, with their mean IQ still in the normal range, were compared to age-matched normal controls. In the oddball task, P3 did not differ between groups but the patients' N2b was delayed. The main difference in the Push/Wait task was in an occipital P270, which component was distinctly larger in the patients. It is suggested that both differences reflect the disintegration of patients' cognition: stimuli are perceived in a normal way but then a gap arises due to uncertainty what to do with the perceived event.


Ventricular premature beats in young subjects without evidence of cardiac disease: histological findings. This report describes the results of right ventricle endomyocardial biopsies from 26 subjects (mean age 27 +/- 10 years) with premature ventricular beats and normal cardiac anatomy and mechanical function. Light microscopy examination revealed normal myocardium in 10 subjects (38%), acute myocarditis in two (7%), borderline myocarditis in one (3.5%), non-specific histological abnormalities including cellular hypertrophy, fibrosis and degenerative changes in 11 (42%), vasculitis in one (3.5%) and findings compatible with right ventricular dysplasia in the final subject (3.5%). The frequency of ventricular premature beats, as assessed by Holter monitoring, and the results of electrophysiological testing did not correlate with histopathological findings and their severity. These data indicate that some young subjects with premature ventricular beats of unknown origin have abnormal right ventricular biopsy findings. Adequate follow-up will probably demonstrate the clinical utility of these observations.


Wave form variations in auditory event-related potentials evoked by a memory-scanning task and their relationship with tests of intellectual function. The inter-subject wave form variability of auditory event-related potentials (ERPs) evoked by digit probe identification in a memory-scanning task (Sternberg paradigm) and the effects of reaction time (RT) and task difficulty were studied in 26 healthy subjects. The response wave forms were compared with the performance of psychological tests of intelligence and memory. ERPs to 1-digit sets consisted of a sequence of waves identified as P100, N170, P250, N290, P400, P560 and N640. The major inter-subject difference in the response wave form was either the presence or absence of the late parietal positive wave P560. This wave occurred significantly more often in responses associated with larger memory sets and slow RT, suggesting that its presence reflects subjective difficulty in performing a task. With increasing set size, the P400 showed variable effects in different subjects, ranging from relative preservation of amplitude, through attenuation, to replacement or overlap by a broad surface-negative wave. This predominantly 'negative-going' effect of increasing task difficulty on the P400 was significantly correlated with scores of psychological tests; the greater the amplitude difference between the responses to easy and more difficult tasks, the better the scores, suggesting that these wave form changes reflect a more effective cognitive processing mechanism.


Area-specific self-regulation of slow cortical potentials on the sagittal midline and its effects on behavior. Exteroceptive feedback was given for negative and positive shifts in slow potentials (SPs) recorded from Fz, Cz, or Pz (between groups design). Slow potentials at the feedback site were referred to adjacent scalp and non-cephalic electrodes, so as to confine SP shifts to the feedback location. Area-specific regulation of SPs was obtained at each midsagittal site after 3 days of feedback training. Subjects reported sensorimotor and emotional arousal when negative SP shifts were trained frontally, but not when negative shifts were trained parietally (cognitive/attentional strategies reported after parietal feedback). Area-specific regulation of SPs was subsequently abolished when behavioral tasks were added to further probe frontal/parietal differences (dual-task procedure). These findings indicate that area-specific self-regulation of SPs is possible on the sagittal midline, and that self-regulated parietal SPs (in contrast to frontal ones) arise from non-motoric generators. The source of SP self-regulation was more readily probed by verbal reports of feedback strategy than by study of dual-task relations, because feedback control was disrupted by the dual-task requirement.


Binaural stimulation reveals functional differences between midline and temporal components of the middle latency response in guinea pigs. Two morphologically distinct auditory middle latency response (MLR) wave forms can be recorded from the surface of the guinea pig brain. The temporal response is recorded from the temporal lobe contralateral to the stimulus ear, and the midline response is recorded over the posterior midline. Experimental evidence suggests that different neural generators contribute to the two responses. Furthermore, it appears that the temporal response principally reflects activity of the primary auditory pathway while the midline response reflects non-primary pathways. Although it is known that neurons throughout the auditory pathway exhibit distinct binaural interaction (BI) properties, thus far there have been no systematic attempts to differentiate the MLR wave forms in response to binaural stimulation. The purpose of this study was to determine if binaural click stimulation could functionally differentiate the midline and temporal MLR responses in the guinea pig. Binaural click stimulation caused a significant decrease in temporal MLR peak amplitudes, and a significant increase in midline MLR amplitudes. The fact that different BI patterns were observed suggests that the two MLR components are functionally distinct. The data further support the hypothesis that the midline and temporal MLR in guinea pigs reflect different neural generators and pathways.


Determinants of left ventricular filling dynamics: alteration in the Doppler-derived transmitral filling profile with progressive impairment of cardiac function in a dog preparation. To clarify the factors determining transmitral filling, left ventricular and atrial pressures (LVP and LAP) and Doppler-derived diastolic indices were analysed in six anaesthetized dogs at various right atrial pacing rates during dextran infusion. The relationship of the late to early diastolic peak velocity ratio (A/E ratio) to end-diastolic LVP (LVEDP) showed a quadratic curve concave to the LVEDP axis in five animals (r2 = 0.320-0.588). An elevation in LVEDP up to 25 mmHg accompanied an increase in A/E ratio (ascending limb), and further LVEDP elevation caused its inverse decline (descending limb). Multiple regression analysis indicated that A/E ratio correlated positively with maximal LVP, a-wave LAP and heart rate, and negatively with v-wave LAP in both limbs. The time constant of isovolumic LVP decline, which was prolonged as LVEDP was elevated, was a positive correlate of A/E ratio in the ascending limb, but lost its influence on A/E ratio in the descending limb. An elevation in v-wave LAP must have masked the expected effect of left ventricular relaxation abnormality on A/E ratio in this limb. Thus, the transmitral filling profile did not alter unidirectionally, but returned to that seen before volume loading, with a simultaneous progressive impairment of cardiac function.


Anergy induced by thymic medullary epithelium. Thymocytes can be rendered tolerant by non-deletional mechanisms upon interaction with major histocompatibility complex (MHC) antigens on thymic epithelium. Whether the epithelial cells in the cortex or medulla could mediate this effect was not clear so far. To address this question, a transgenic mouse was generated in which the bovine keratin IV promoter was used to control expression of the alloantigen Kb. In the periphery the Kb transgene was expressed on a subset of keratinocytes. In the thymus expression was restricted to a subpopulation of medullary epithelial cells. No expression was found in the cortex. Such a tissue distribution has been reported for the keratin IV molecule demonstrating the faithfulness of the promoter used here. To follow the fate of the Kb-reactive thymocytes, this mouse was mated with another transgenic mouse expressing an anti-Kb T cell receptor (TcR). In the double-transgenic mice the CD8+CD4- thymocytes were not deleted but they were found to be anergic as assayed by their failure to be activated in vitro by either Kb-positive spleen cells or by cross-linked anti-TcR antibodies. These observations establish that expression of an MHC class I antigen in the thymic medullary epithelium is sufficient to induce anergy in the mature CD8+CD4- thymocyte population.


Event-related neuronal responses in the human strio-pallido-thalamic system. II. Cognitive functions. Multiunit activity was recorded from the strio-pallido-thalamic system in the same parkinsonian patients (as described in the previous paper) who bore gold electrodes for diagnosis and therapy. The patients voluntarily participated in various tasks designed to study neuronal correlates of cognitive functions: "odd-omissions," "short-term memory," "cued bimodal," "assessment," and "dual-stage delayed response" tasks. Preparatory-related activities were found in multiunits reacting to sensory cues. In a few multiunits these activities depended upon the specific features of the stimuli that were anticipated for evaluation. The most striking characteristic of stimulus-related activity was the suppression of the multiunit responses when the stimuli become behaviourally meaningless. The hypothesis of action programming is discussed: the loop, including the cortex, neostriatum, pallidum and appropriate parts of the thalamus, is involved in the selection of actions, thus providing the organization of sequential behaviour.


Characterization and measurement of CD5+ B cells in normal and Trypanosoma congolense-infected cattle. CD5+ B cells in cattle are present in peripheral blood and spleen, but not in lymph nodes, tonsils or Peyer's patches. Compared to classical B cells, they express similar levels of B cell surface markers, but have higher levels of surface IgM. We failed to find evidence for IgD on bovine B lymphocytes. The CD5+ B cells expressed CD11b (Mac-1). Another small subpopulation of B cells carried CD11b but not CD5. In cattle infected with Trypanosoma congolense, a dramatic increase in the percentage of CD5+ B cells in blood and spleen was observed. This increase occurred 7-10 days after parasites were first detected in the blood and correlated with the increase in serum IgM and the increase in the absolute number of B cells that is typical to trypanosome-infected animals. The increase in B cells was found to be due mainly to the expansion of the CD5+ B cell subpopulation. The cause of the amplification of the CD5+ B cells and their possible involvement in the production of autoantibodies and non-parasite-specific antibodies which have been described in trypanosome-infected animals are discussed.


Restoration of the LFA-3 adhesion pathway in Burkitt's lymphoma cells using an LFA-3 recombinant vaccinia virus: consequences for T cell recognition. Conjugate formation between cytotoxic T lymphocytes (CTL) and target B cells, as observed in vitro, is mediated by interactions between adhesion molecules on the two cell surfaces rather than involving immune recognition through the T cell receptor. It is still not clear to what extent such adhesive contacts facilitate the process of immune recognition and target cell lysis. However, work on the Epstein-Barr virus (EBV)-associated malignancy Burkitt's lymphoma (BL) has suggested that down-regulation of one particular adhesion molecule, the lymphocyte function-associated antigen LFA-3, on the tumor cell surface is a key factor in allowing these target cells to escape EBV-specific T cell surveillance. To examine this directly, we used a cDNA for the full-length transmembrane form of LFA-3 to construct a recombinant vaccinia virus (Vacc-LFA 3), which is capable of restoring surface LFA-3 in adhesion molecule-negative BL cell lines to levels as high as seen in EBV-transformed lymphoblastoid cell lines (LCL); biochemical studies confirmed expression of the authentic N-glycosylated protein. The recombinant vaccinia-encoded LFA-3 was functional as an adhesion molecule since BL cells acutely infected with Vacc-LFA-3 then acquired the ability to form conjugates with activated T cells in vitro. However, there was no clear dependence upon LFA-3 when such BL cell lines were tested as targets for cytotoxic T lymphocytes (CTL). Firstly, LFA-3- BL cells could be killed by allospecific CTL recognizing HLA class I alloantigens, in some cases as efficiently as the corresponding LCL. In other cases where lysis was slightly below that of the LCL, Vacc-LFA-3 infection of the BL cells increased lysis up to, but never beyond, LCL values. Secondly, we studied the sensitivity of BL to EBV-specific HLA class I-restricted CTL using a BL target line which was LFA-3- but which expressed the same spectrum of EBV target proteins as an LCL. This line was not recognized by appropriately HLA-matched effectors, even after restoration of LFA-3 expression. We conclude that the LFA-3 status of BL cells influences their conjugate forming ability in in vitro assays but not necessarily their sensitivity to immune T cell-mediated cytolysis.


Event-related neuronal responses in the human strio-pallido-thalamic system. I. Sensory and motor functions. Multiunit activity was recorded from strio-pallido-thalamic sites in parkinsonian patients bearing gold electrodes for diagnosis and therapy. The patients voluntarily participated in tasks designed to study neuronal correlates of both physical and semantic characteristics of stimuli as well as motor responses. Six modifications of the stimulus-response paradigm were used: visual odd-ball, visual and acoustic odd-ball tasks; tasks in which either the stimulus intensity or the meaning of non-target stimuli varied; single-stage delayed response and dual-stage delayed response tasks, respectively. In each task the patients had to evaluate some of the stimulus characteristics and to respond in a particular way according to the preliminary instructions. Peristimulus time histograms for each multiunit separately as well as profiles of reactions and profiles of reaction differences for the whole set of multiunits were calculated and subjected to statistical analysis. Two functional groups of subcortical neuronal reactions, stimulus-related and response-related activities, were separated. The stimulus-related activities of most multiunits were modality-unspecific. Their most striking feature was dependence on stimulus relevance and also its probability, the strongest reactions observed in response to task relevant stimuli occurring with low probability. The response-related activities occurred prior to initiation of movements, dependent upon the particular action and its probability. The data suggest at least two different and spatially overlapping subcortical channels responsible for goal-directed behaviour: the one related to stimulus assessment and the other to preparation for motor action.


Human monoclonal and polyclonal anti-human immunodeficiency virus-1 antibodies share a common clonotypic specificity. Human monoclonal and purified polyclonal anti-human immunodeficiency virus (HIV)-1 antibodies were tested for binding to a murine monoclonal anti-idiotypic antibody (1F7, IgM, kappa). Four human monoclonal anti-p24 and three human monoclonal anti-gp120 antibodies express the 1F7 clonotype, while one human monoclonal anti-gp41 antibody does not bind to 1F7. Affinity-purified anti-p24 and anti-gp120 antibodies from HIV-1-infected individuals also react with 1F7. Western blot analysis and enzyme-linked immunosorbent assay confirmed that 1F7 reacts with human antibodies of different HIV-1 antigen specificities. A survey of sera from 329 HIV-1-infected individuals showed binding to 1F7 in 239 sera (72.6%) while 1F7 was not reacting with 109 HIV-1-negative sera. These results show that 1F7 idiotype is an HIV-1 infection-associated clonotypic marker shared by anti-HIV-1 antibodies with different epitope specificites.


Improving the reliability of pattern electroretinogram recording. The pattern electroretinogram (PERG) is a small electrical response of the retina to a reversing checkerboard pattern, usually less than 6 microV in amplitude. Unfortunately, the PERG can be obscured by artifacts such as blinks, eye movements, poor fixation, and amplifier saturation. Amplitude criterion artifact rejection systems found on commercial signal averagers eliminate large amplitude artifacts but are insensitive to small amplitude artifacts associated with amplifier saturation. Such saturation often occurs for several recording sweeps after large amplitude signals such as eye blinks are rejected. The presence of post-saturation artifacts complicates clinical PERG analysis. In this paper we describe procedures to remove these small amplitude artifacts from the PERG. These include computer selection of inputs for averaging and use of tracings with small input numbers to approximate PERG amplitudes. These procedures greatly reduce the variability of PERG amplitudes in the normal population, making PERG amplitude a more reliable clinical measure.


CD4+ suppressor cells of autoimmune encephalomyelitis respond to T cell receptor-associated determinants on effector cells by interleukin-4 secretion. We have previously demonstrated that CD4+ suppressor T cells (Ts) inhibit the secretion of interferon (IFN)-gamma, but not interleukin (IL)-2, by effector cells of experimental autoimmune encephalomyelitis (EAE). Moreover, CD4+ Ts appear to regulate IFN-gamma by secretion of transforming growth factor-beta. We now show that CD4+ Ts produce a lymphokine with IL-4 activity in response to a determinant associated with EAE effector cells. CD4+ Ts do not proliferate or secrete IFN-gamma, IL-2, or IL-4 in response to myelin basic protein, nor do CD4+ Ts proliferate or secrete IL-2 when co-cultured with irradiated EAE effector cells. Rather, CD4+ Ts secrete IL-4 when co-cultured with either irradiated effector spleen cells or irradiated encephalitogenic line cells. CD4+ Ts do not secrete IL-4 in response to OVA-primed spleen cells, suggesting that the suppressor cells recognize a determinant specific to encephalitogenic T cells. Furthermore, CD4+ Ts secrete IL-4 when cultured with synthetic T cell receptor (TcR) V beta 8, but not TcR V beta 14 peptide, in the presence of antigen-presenting cells. This response is major histocompatibility complex class II restricted as demonstrated by inhibition of the response with anti-class II monoclonal antibody. These results suggest that CD4+ Ts recognize a determinant associated with TcR on the surface of EAE effector cells and respond by secreting IL-4, in a manner analogous to the Th2 lymphocyte subtype.


GMP-140 (P-selectin/CD62) binds to chronically stimulated but not resting CD4+ T lymphocytes and regulates their production of proinflammatory cytokines. GMP-140 (P-selectin), a 140-kDa granular membrane glycoprotein localized to the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, is thought to play an important role in adhesive interactions predominantly between granulocytes, platelets and vascular endothelial cells during inflammation. Although GMP-140 binds to granulocytes, its binding to lymphocytes has not been demonstrated. Using genetically engineered IgG C gamma 1 fusion protein of the extracellular domains of GMP-140, we demonstrate that GMP-140 binds to chronically antigen (Ag)-stimulated CD4+ T cells. Freshly isolated CD4+ T cells did not bind GMP-140, but priming and subsequent stimulation with alloantigen induced and gradually increased expression of GMP-140-reactive structures on their surface. T cells isolated from rheumatoid synovial fluids also exhibited strong binding to GMP-140. The binding of GMP-140 to primed T cells is not influenced by preactivation with phorbol 12-myristate 13-acetate, is almost completely abolished by pretreatment of T cells with neuraminidase or trypsin, and is also strongly inhibited by EDTA, the soluble sulfated glycans dextran sulfate, fucoidan, and heparin, but not by chondroitin sulfates. In spite of its strong binding to Ag-primed T cells, GMP-140 did not modulate the proliferative responses of these cells to various stimuli. However, GMP-140 in conjunction with anti-T cell receptor alpha beta monoclonal antibodies augmented the production of granulocyte-macrophage colony-stimulating factor GM-CSF and inhibited the production of interleukin-8 by Ag-primed T cells without influencing their tumor necrosis factor-alpha production. These results suggest that GMP-140 binds to chronically stimulated CD4+ T cells and differentially modulates their production of proinflammatory cytokines. The ability of Ag-primed T cells to bind GMP-140 may facilitate interactions with activated platelets and endothelial cells affecting the course of inflammation.


Cell adhesion molecules of the immunoglobulin supergene family as tissue-specific autoantigens: induction of experimental allergic neuritis (EAN) by P0 protein-specific T cell lines. The P0 glycoprotein is a homophilic cell adhesion molecule of the immunoglobulin supergene family which is responsible for maintaining the structure of compact internodal myelin in the peripheral nervous system (PNS). Utilizing a panel of synthetic P0 peptides two distinct T cell epitopes have been identified that can induce T cell-mediated experimental autoimmune neuritis (EAN) in the Lewis rat. One T cell epitope (amino acid residues 56-71), is located within the extracellular, immunoglobulin-like domain of P0, while the other disease-inducing T cell epitope (residues 180-199) is located within the proteins cytoplasmic carboxyterminal domain. The adoptive transfer of 10(6) CD4+ T line cells specific for either of these peptide antigens induced EAN in syngeneic recipients. However, while the pathogenic response induced by both peptide-specific T cell lines was identical, their epitopes differ markedly in their immunologic properties in vivo. In particular while the response to peptide p180-199 was immunodominant in animals immunized with either purified P0 protein or the native membrane-bound P0 protein in autologous rat peripheral nerve myelin, no response to peptide p56-71 was detected, indicating that this epitope is cryptic. This study provides the first experimental evidence that the immunoglobulin-like domains of members of the immunoglobulin supergene family can function as target autoantigens in T cell-mediated autoimmune disease.


Identification of an immunodominant type-II collagen peptide recognized by T cells in H-2q mice: self tolerance at the level of determinant selection. The T cell recognition of type-II collagen (CII) in H-2q mice, susceptible to CII-induced arthritis, was analyzed. With the use of T cell hybridomas derived from rat CII-immunized mice, a peptide corresponding to amino acids 245-270 on chick CII was found to harbor a T cell epitope which is present on heterologous CII (chick, rat, human, and bovine CII) but not on autologous CII. It was shown that this epitope was located within amino acids 260-270, although flanking regions in either direction were necessary for proper recognition. A peptide corresponding to human CII (256-270) was used for further studies. A single amino acid difference at position 266 between mouse CII (aspartic acid) and heterologous CII (glutamic acid) strongly influenced recognition of this peptide. No response towards the mouse peptide was seen with any of the T cell hybridomas. Inhibition studies revealed that the mouse peptide did not bind as well to major histocompatibility complex as the corresponding heterologous peptide. Both peptides gave rise to a T cell response after immunization. However, immunization with the heterologous peptide resulted in a response strictly directed to rat CII and the immunogen while immunization with the autologous peptide elicited T cells which reacted in a heteroclitic fashion, with a stronger response to the heterologous peptide than to the autologous peptide, and did respond to rat CII but not to mouse CII. We suggest that aspartic acid in position 266 results in a cryptic determinant in mouse CII which is neither recognized after CII immunization nor capable of tolerance induction. A glutamic acid at position 266, however, gives rise to an immunodominant epitope which is recognized by a large proportion of the T cells activated after immunization with heterologous CII.


T cell clones with normal or defective O-galactosylation from a patient with permanent mixed-field polyagglutinability. To delineate the extent of O-galactosyltransferase deficiency within the lymphoid lineage, monoclonal antibody specific for the Thomsen-Friedenreich (TF) antigen (Gal beta 1----3GalNAc alpha 1-O-Ser/Thr) and its precursor the Tn antigen (GalNAc alpha 1-O-Ser/Thr) were applied to the flow cytometric analysis of peripheral blood lymphocytes from a patient with permanent mixed-field polyagglutinability (PMFP). We show that only a minor population of 4% expressed the Tn antigen which is in contrast to 93% of the patient's erythrocytes carrying the defect. Tn+ lymphocytes mainly belonged to the CD3+ subset, but were also CD19+ or CD16+. Both Tn+ and TF+ T cell clones from patient R. R. were established and shown to belong to the CD4+ or CD8+ antigenic subset. Three glycosyltransferase activities were determined in lysates from these clones: all Tn+ clones were deficient in UDP-Gal: GalNAc alpha 1-O-Ser/Thr beta 1----3 galactosyltransferase (beta 3Gal-T) activity; by contrast this activity was present in all lysates from TF-expressing clones. UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (GalNAc-T) and UDP-Gal: GlcNAc-R beta 1----4 galactosyl-transferase (beta 4Gal-T) exhibited similar activities in both Tn+ and TF+ T cell clones. As a consequence of defective O-galactosylation in Tn+ T cells, cell surface sialic acid of Tn+ clones was reduced by greater than 50% when compared to TF+ clones as demonstrated by sialic acid-specific labeling using fluoresceinated Limax flavus agglutinin(LA) and flow cytometry. The Tn phenotype of T cell clones was stable for more than 1 year of continuous expansion in vitro. These data demonstrate that in PMFP, T cells may also be affected by the O-galactosyltransferase deficiency which is accompanied by a substantial loss of cell surface sialic acid. However, the frequency of Tn+ lymphocytes in peripheral blood from patient R.R. was strikingly low. These T cell clones should be useful to study the defect at a genetic level and the importance of O-linked carbohydrates for proper T cell function.


CD45 isoform expression during T cell development in the thymus. Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.


Expression of the complement regulatory proteins CD21, CD55 and CD59 on Burkitt lymphoma lines: their role in sensitivity to human serum-mediated lysis. On a panel of nine human B cell lines we showed that the expression of the complement regulatory factors complement receptor type 2 (CR2; CD21), decay-accelerating factor, (DAF; CD55) and homologous restriction factor (HRF20, CD59) is not correlated. All lines expressed DAF, six lines carried detectable amounts of CR2 and three carried HRF20. Upon incubation in human serum, under conditions which allowed the activation of complement through the alternative pathway, the CR2-carrying lines bound C3 fragments and two of them (Ramos and one of its two sublines) were damaged. These two lines had the lowest DAF expression, less than 50% of the cells reacted with the IA10 monoclonal antibody. By modulating the expression of the complement regulatory molecules, the lytic sensitivity of the B cell lines could be altered. Blockade of DAF on the HRF20-, CR2+ lines with the specific monoclonal antibodies increased their sensitivity to lysis by human serum. With the DAF- and HRF20+ cells significant lytic effect was obtained only when they were pretreated with both of the specific antibodies. Interferon-gamma or tumor necrosis factor-alpha treatment elevated the amount of CR2 on the low-CR2 expressor line (Ramos/HR1K) which thereafter bound higher amounts of C3 fragments and was lysed when incubated in human serum. This line had relatively low DAF level and lacked HRF20. The cytokine treatment did not alter the expression of these molecules. The CR2+ Ramos and the CR2- Rael cells were treated with 5-azacytidine which induced HRF20 and increased DAF expression. In parallel with this change Ramos cells became resistant to C-mediated lysis. The experiments with the panel of human B cell lines showed thus that cytolysis through activation of complement in homologous serum can be regulated at several steps by cell surface molecules. While expression of CR2 was required for C3 fixation, DAF and HRF20 inhibited lysis. By independent modulation of the quantities of these molecules, cells acquired or lost their sensitivity.


The calcium-binding proteins MRP8 and MRP14 form a membrane-associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic inflammatory lesions. Monocytes/macrophages expressing an epitope recognized by a monoclonal antibody 27E10 are present in acute but are absent in chronic inflammatory disorders. This report shows that the 27E10 antigen is formed by noncovalent association of the two Ca(2+)-binding proteins MRP8 and MRP14 which belong to the S100 protein family. Identification has been confirmed immunochemically, by matrix-assisted UV-laser desorption/ionization spectrometry and by partial amino acid sequencing. Surface expression of the MRP8/MRP14 complex on a subset of monocytes is reported for the first time and shown to be up-regulated in a Ca(2+)-dependent manner. The 27E10 surface-positive monocytes isolated by cell separation techniques release high amounts of tumor necrosis factor-alpha and interleukin-1 beta in contrast to their 27E10 surface-negative counterparts thus emphasizing their role in inflammation.


Species-specific restriction of complement by HRF20 (CD59) generated by cDNA transfection. The 20-kDa homologous restriction factor (HRF20, CD59) is a phosphatidyl inositol-anchored membrane glycoprotein that inhibits the formation of human complement membrane attack complexes. The cDNA of HRF20 was transfected into Chinese hamster ovary (CHO) cells resulting in expression of human HRF20 protein on the cell surface anchored via glycosylphosphatidyl inositol. The transfected CHO cells were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rat and guinea pig complement. Therefore, species specificity between HRF20 and complement is maintained in HRF20 generated on the CHO cells following transfection with HRF20 cDNA.