Ventricular arrhythmias in aortic valve disease: a further marker of impaired left ventricular function.

One hundred and twenty stable patients with pure and severe aortic valve disease and without coronary lesions (aortic stenosis, 43 patients; aortic regurgitation, 45 patients; combined aortic stenosis and regurgitation, 32 patients) who had been submitted to haemodynamic studies were prospectively studied with standard electrocardiograms, M-mode echocardiograms, and 24-hour ambulatory electrocardiography (Holter recording). The frequency and complexity of ventricular arrhythmias were related to clinical parameters such as functional class, type of lesion and presence of syncope, and to parameters of left ventricular hypertrophy and function. Ventricular arrhythmias were present in 92% of patients. A high number of ventricular premature beats was directly correlated with parameters of complexity of the arrhythmia. A significant relation was found between electrocardiographic left ventricular hypertrophy and Ryan class (P less than 0.05), and an inverse relation between maximal number of ventricular premature beats in any hour and left ventricular ejection fraction (P less than 0.05). The group of patients with aortic regurgitation showed a higher total number of ventricular premature beats per 24 hours (P less than 0.001), a higher maximal number of these in any hour (P less than 0.01), a higher number of patients with pairs (P less than 0.001), and a higher number of patients in Ryan classes 3, 4A, 4B (P less than 0.01). This study shows a high incidence of ventricular arrhythmias in aortic valve disease, and especially in aortic regurgitation, with a significant relation between left ventricular hypertrophy and function, and number and complexity of arrhythmias.


Absent left-sided atrioventricular connexion, with right atrium connected to left ventricle: prospective diagnosis in infancy, and outcome.

Prospective echocardiographic diagnosis of absence of the left atrioventricular connexion, with the right atrium connected to a morphologic left ventricle through a bileaflet morphologically mitral valve, was made in six infants. The rudimentary right ventricle was left-sided in all patients, and separated from the left atrium by sulcus tissue. The ventriculoarterial connexions were discordant. Associated defects included subpulmonary stenosis (2 patients), pulmonary atresia (1 patient), and a patent duct (4 patients). All patients developed early left atrial hypertension due to a restrictive interatrial septum, and required transcatheter septostomy (5 patients), or surgical septectomy (3 patients). One patient who had a severely restrictive ventricular septal defect died following cardiac catheterization. In three others the ventricular septal defect has become progressively restrictive on serial catheterization. Successful intermediate term palliation has been performed in two patients using a bidirectional Glenn anastomosis, together with enlargement of the ventricular septal defect and a Damus-Kay-Stansel procedure in one. It is possible to distinguish this malformation from "mitral atresia" using cross-sectional echocardiography. The long-term outlook is influenced by early relief of left atrial hypertension. Balloon atrial septostomy alone is usually inadequate, and either blade septostomy or surgical septectomy are required. Serial cardiac catheterization is mandatory for planning definitive palliation.


HLA class II variation in the Gila River Indian Community of Arizona: alleles, haplotypes, and a high frequency epitope at the HLA-DR locus.

A genetic distribution for the HLA class II loci is described for 349 "full-blooded" Pima and Tohono O'odham Indians (Pimans) in the Gila River Indian Community. A high frequency epitope in the *DRw52 family was defined by reactions with 31 alloantisera, which we have designated *DR3X6. It segregates as a codominant allele at HLA-DR with alleles *DR2, *DR4, and *DRw8, and has the highest frequency yet reported for an HLA-DR specificity, 0.735. It forms a common haplotype with *DRw52 and *DQw3 that is a valuable marker for genetic admixture and anthropological studies. Phenotype and allele frequencies, and haplotype frequencies for two and three loci, are presented. Variation at these loci is highly restricted, the mean heterozygosity for HLA-DR and HLA-DQ being 0.361. The Pimans represent a contemporary model for the Paleo-Indians who first entered North America 20,000 to 40,000 years ago.


Identification of residues involved in polymorphic antibody binding epitopes on HLA-DR molecules.

Based on previous studies it was predicted that amino acids 4 or 25 of the DR4 beta 1 and DR7 beta 1 chains are involved in polymorphic antibody binding epitopes on DR4 or DR7 molecules. These predictions were tested by analyzing monoclonal antibody (mAb) binding to transfectants expressing mutant DR4 beta 1 or DR7 beta 1 chains with single amino acid substitutions at positions 4 or 25. Antibody binding to transfectants expressing additional DR4/7 beta 1 hybrids was also analyzed to assess further the contributions of four segments of the DR4 beta 1 or DR7 beta 1 chains: amino acids 1-20, 21-40, 41-97, and the beta 2 domain. Single amino acid substitutions at positions 4 and 25 of the DR4 beta 1 chain or DR7 beta 1 chain eliminate binding of several mAb to DR4 or DR7 molecules, documenting that these residues are involved in antibody epitopes. However, the data with the hybrid DR4/7 beta 1 chains indicate that some of these epitopes require contributions from both segments 1-20 and 21-40 of these DR beta chains, whereas other epitopes can be generated by placing the appropriate segment in the context of the other DR beta chain. In addition, the data with other mAb indicate that their epitopes are determined primarily by sequences within the 41-97 segment or in the beta 2 domain.


The dream in terminal illness: a Jungian formulation.

It is a central assumption of Jungian theory that psychical transformation occurring during the critical developmental stages of the life cycle is anticipated, inspired, and orchestrated by the archetypal symbol. In this way, archetypal dreams are afforded particular significance during these transitional stages. The present paper purports to consider the clinical and theoretical implications of this understanding with reference to the dying process. The concepts discussed are illustrated by a series of dreams of a terminally ill cancer patient, which are elucidated by way of the method of amplification. Thematic analysis of the dream series supports Jung's conceptualization of death and dying as being a critical stage of the individuation process, characterized by profound psychical development of a specific and purposeful nature. The value of using dreams in the psychotherapeutic care of dying patients and their families is discussed, with case illustrations. It is suggested that such an approach may foster creative development, assist patients to integrate meaningfully subjective experiences pertaining to dying, and counteract the sense of isolation experienced by the terminally ill. The need for further research and the development of specific treatment modalities is highlighted.


Sperabillins, new antibacterial antibiotics with potent in vivo activity. Taxonomy, fermentation, isolation and biological activity.

A Gram-negative bacterium was found to produce new antibacterial antibiotics, sperabillins A, B, C and D, and the producing bacterium was characterized and identified as Pseudomonas fluorescens YK-437. Sperabillins were isolated by column chromatographies using cation-exchange resins, activated carbon and cation-exchange Sephadex, and preparative reverse-phase HPLC. Sperabillins showed antibacterial activity against Gram-negative and Gram-positive bacteria including antibiotic-resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus. Sperabillin A inhibited DNA, RNA, protein, and cell wall biosynthesis in Escherichia coli. Sperabillins showed good protective effects in experimentally infected mice.


Microbial transformation of immunosuppressive compounds. I. Desmethylation of FK506 and immunomycin (FR 900520) by Actinoplanes sp. ATCC 53771.

The immunosuppressants FK506 and FR 900520 were desmethylated by Actinoplanes sp. ATCC 53771 to yield various O-desmethylated products. The products were isolated and purified by solvent extraction and HPLC chromatography, and identified by NMR and MS spectroscopy.


Protactin, a new antibiotic metabolite and a possible precursor of the actinomycins.

Streptomyces cucumerosporus strain L703-4 (ATCC 53784) produces a new 4-methyl-3-hydroxyanthraniloylpentapeptide lactone for which we have proposed the name protactin, in addition to several actinomycin components. Protactin is rather resistant to air oxidation but it can be converted to a new actinomycin, actinomycin Zp by ferricyanide oxidation. Actinomycin Zp possesses in vitro antibacterial activity and in vivo antitumor activity against P-388 leukemia in mice.


Antibiotics from basidiomycetes. XLI. Clavicoronic acid, a novel inhibitor of reverse transcriptases from Clavicorona pyxidata (Pers. ex Fr.) Doty.

A novel inhibitor of RNA-directed DNA-polymerases was isolated from fermentations of Clavicorona pyxidata. Its structure was elucidated by spectroscopic methods. Clavicoronic acid (1) is a noncompetitive inhibitor of avian myeloblastosis virus (Ki 130 microM) and Moloney murine leukemia virus (Ki 68 microM) reverse transcriptases. In permeabilized cells and isolated nucleic DNA- and RNA-synthesis are not affected. Clavicoronic acid markedly inhibits the multiplication of vesicular stomatitis virus in baby hamster kidney cells by interfering with this virus's RNA-directed RNA-polymerase. 1 exhibits no cytotoxic and very weak antimicrobial activities.


Expression of an early gene in the flagellar regulatory hierarchy is sensitive to an interruption in DNA replication.

Genes involved in the biogenesis of the flagellum in Caulobacter crescentus are expressed in a temporal order and are controlled by a trans-acting regulatory hierarchy. Strains with mutations in one of these genes, flaS, cannot transcribe flagellar structural genes and divide abnormally. This gene was cloned, and it was found that its transcription is initiated early in the cell cycle. Subclones that restored motility to FlaS mutants also restored normal cell division. Although transcription of flaS was not dependent on any other known gene in the flagellar hierarchy, it was autoregulated and subject to mild negative control by other genes at the same level of the hierarchy. An additional level of control was revealed when it was found that an interruption of DNA replication caused the inhibition of flaS transcription. The flaS transcript initiation site was identified, and an apparently unique promoter sequence was found to be highly conserved among the genes at the same level of the hierarchy. The flagellar genes with this conserved 5' region all initiate transcription early in the cell cycle and are all sensitive to a disruption in DNA replication. Mutations in these genes also cause an aberrant cell division phenotype. Therefore, flagellar genes at or near the top of the hierarchy may be controlled, in part, by a unique transcription factor and may be responsive to the same DNA replication cues that mediate other cell cycle events, such as cell division.


Premature termination of in vivo transcription of a gene encoding a branched-chain amino acid transport protein in Escherichia coli.

Previous studies have suggested that control of expression of genes of the LIV-I permease system for the high-affinity transport of branched-chain amino acids in Escherichia coli involves modulation in the frequency of mRNA elongation. Mutation of the Rho transcription termination factor and shortages of charged leucyl-tRNA have been shown to alter LIV-I transport activity. Rho-dependent transcription termination regulated by shortages of charged leucyl-tRNA at sites preceding structural genes has been proposed to account for their role in regulation of LIV-I transport. Transcription of the livJ-binding protein gene, encoding one of the periplasmic components of the LIV-I system, was analyzed in vivo with strains which lack repression of the LIV-I genes and harbor a temperature-sensitive allele for either leucyl-tRNA synthetase or Rho factor. Analysis of mRNA synthesis by DNA-RNA hybridization in the various mutant strains indicated that both shortages of leucyl-tRNA caused by inactivation of the temperature-sensitive leucyl-tRNA synthetase and inactivation of the Rho factor were associated with increased synthesis of livJ mRNA. Nuclease protection and gel electrophoresis studies detected prematurely terminated transcripts corresponding in size to the leader region of livJ mRNA. Accumulations of these short transcripts were suppressed in strains harboring temperature-sensitive alleles for either leucyl-tRNA synthetase or Rho factor. These results provide support for the hypothesis that expression of livJ involves Rho-dependent transcription termination in which antitermination is associated with the intracellular availability of aminoacyl leucyl-tRNA.


Expression of Erwinia amylovora hrp genes in response to environmental stimuli.

Seven hrp loci that are essential for the hypersensitive reaction elicited by Erwinia amylovora were transcriptionally fused with a derivative of transposon Tn5, containing the promoterless Escherichia coli beta-glucuronidase reporter gene. The seven hrp fusions were used to monitor expression of the hrp loci in vitro and in planta. No significant expression was detected in rich medium for any of the fusions. However, five of them were expressed highly in planta and in inducing medium that contains mannitol, salts, and 5 mM (NH4)2SO4. Expression of these five hrp loci is regulated by ammonium, nicotinic acid, complex-nitrogen sources, certain carbon sources, temperature, and pH. Under well-defined conditions, i.e., in inducing medium, no specific plant components were required for transcriptional activation of the hrp loci. The high levels of expression detected in vitro were comparable to those determined during the development of the hypersensitive reaction in tobacco. Differential levels of expression of the hrp loci occurred in host and nonhost plants. In pear, a host plant, expression of the hrp loci was delayed and greatly reduced compared with expression in tobacco leaves, a nonhost.


Cloning, sequencing, and transcriptional regulation of the Vibrio cholerae fur gene.

Many genes involved in the transport of iron by bacteria as well as in pathogenesis are regulated by the environmental concentration of iron, with increased expression under low-iron conditions. In Escherichia coli, transcriptional regulation by iron depends on the fur gene. A virulence gene in Vibrio cholerae (irgA) is also transcriptionally regulated by iron, and the promoter of irgA contains a dyad repeat homologous to Fur binding sites in E. coli. Southern hybridization of V. cholerae chromosomal DNA, using an internal fragment of E. coli fur as a probe, showed a single hybridizing sequence under conditions of low stringency. We derived a restriction map in the vicinity of this hybridizing sequence; overlapping PstI and HindIII fragments were identified at the center of this map. The cloned PstI fragment failed to complement an E. coli fur mutant; sequence analysis revealed an open reading frame that began just downstream of the PstI site, suggesting that the clone was not functional because it lacked its promoter. The overlapping HindIII fragment contained the intact V. cholerae fur gene with its promoter and complemented an E. coli fur mutant. DNA sequencing of the HindIII fragment demonstrated a single open reading frame of 150 amino acids that was 76% homologous to E. coli Fur. Primer extension analysis localized two promoters for the V. cholerae fur gene; no significant homology to an E. coli Fur binding site was identified for either promoter. Northern blot analysis showed that the two fur transcripts were not strongly regulated by iron. These studies identify a gene in V. cholerae homologous in both function and sequence to the fur gene of E. coli, and we have designated this gene the fur gene of V. cholerae.


Immunochemical and structural analysis of the O polysaccharides of Salmonella zuerich [1,9,27,(46)].

Salmonella zuerich [1,9,27,(46)] has been shown to exhibit two levels of structural heterogeneity. The bacterium carries two distinct O-polysaccharide molecules with and without O:factor 1. Both sets of molecules (1+ and 1-) carry the two O:factors 27 and 46 on the same O chain, but they are expressed unevenly; in contrast to factor 27, O:factor 46 is always weakly expressed. Part of this weak expression was thought to be due to strong inhibition of factor 46 by factor 1. In this study, serological analysis gave more detailed information on the sizes of the different O:factor epitopes. Structural analysis of S. zuerich O polysaccharides showed that they are constructed of the expected chemical sequences characteristic of factors 1, 9, 27, and 46. No modification in the sugar sequence could account for the weak expression of O:factor 46. Factors 27 and 46 are present on oligosaccharides carrying either an alpha-Man (factor 27) or a beta-Man (factor 46) residue. In S. zuerich, the alpha-Man configuration is predominant, corroborating the expression of strong factor 27 and weak factor 46 on the bacteria. Questions raised by the existence of such heterogeneous O polysaccharides on the specificity of the O chain polymerase, as well as the place of S. zuerich in Salmonella evolution, are dicussed in this paper.


Construction of Salmonella strains with both antigen O4 (of group B) and antigen O9 (of group D).

A Salmonella live vaccine causing both O4- and O9-specific immune responses would be of use, but no reported Salmonella serotype has both of these O antigens. Constructed Salmonella typhimurium strains with an rfb (O-antigen-specifying) gene cluster of type D in the chromosome and one of type B in an F'-rfb+ factor, and those with the reverse combination reacted strongly with both anti-O4 (and anti-O5) and anti-O9 sera and, if they carried recA, could be maintained in this state by growth conditions selective for retention of the F' factor. One of the two B.rfb+ gene clusters of a (P22-lysogenic) S. typhimurium strain with a tandem duplication of a chromosomal segment including hisD and B.rfb+ was replaced (by transduction) by a D.rfb+ gene cluster; the resulting strain was O1+ O4+ O5+ O9+ and stable as such after being made recA. A stable O4+ O9+ derivative of a virulent S. enteritidis (O-group D) strain was made by transducing into it first the join point of an appropriate tandem duplication strain, together with the adjacent B.rfb+ gene cluster, and then srl::Tn10 recA.


Structural and immunochemical studies of the lipopolysaccharides of Salmonella strains with both antigen O4 and antigen O9.

Two Salmonella hybrid strains, SL5313 (Salmonella typhimurium with a D.rfb+ gene cluster) and SL5396 (S. enteritidis with a B.rfb+ gene cluster), each expressing both O-antigen 4 (of serogroup B) and O-antigen 9 (of serogroup D) were studied by immunofluorescence using a mixture of O4-specific mouse monoclonal and O9-specific rabbit polyclonal antibodies. Bound antibodies, detected by anti-mouse antibody labelled with fluorescein isothiocyanate and anti-rabbit antibody labelled with tetramethylrhodamine isothiocyanate showed that more than 98% of the bacteria expressed both the O4 and O9 epitopes. Phenol-water-extracted lipopolysaccharide from batch-grown cultures subjected to sugar and methylation analyses by gas-liquid chromatography and mass spectrometry were shown to contain abequose (of the O4 epitope) and tyvelose (of the O9 epitope) in ratios of 1:1.5 and 1:2.5 for SL5313 and SL5396, respectively. Isolated polysaccharide chains, obtained by weak-acid hydrolysis of the lipopolysaccharides, were found to contain both O4 and O9 specificities in the same molecule, since polysaccharide bound to O4 antibody attached to a solid-phase-adsorbed O9-specific antibody and vice versa. This demonstrates that in strains SL5313 and SL5396 O chains containing both O4 repeating units (from S. typhimurium) and O9 units (from S. enteritidis) are present.


Effects of analogs of 1,25(OH)2 vitamin D3 on the proliferation and differentiation of the human chronic myelogenous leukemia cell line, RWLeu-4.

We evaluated the proliferative and differentiative effects of analogs of 1,25(OH)2 vitamin D3 [1,25(OH)2D3] on a chronic myelogenous leukemia cell line, RWLeu-4, which is growth-inhibited and differentiates in response to 1,25(OH)2D3 (ED50 = 3-10 nM). Side-chain-fluorinated analogs were more potent (ED50 = 0.7-2 nM) while most of those with altered saturation of the D ring or side-chain carbon-carbon bonds were equally or less effective than 1,25(OH)2D3. However, the two analogs with either two additional double bonds or an extra double and triple bond in the D ring had greater antiproliferative activity [1,25(OH)2-16,23-diene D3 (ED50 = 2.7 nM) and 1,25(OH)2-16-ene-23-yne D3 (ED50 = 0.7 nM)]. Since the latter of these has been reported to be less potent at mobilizing calcium than 1,25(OH)2D3, it (or a similar compound) may be a candidate for clinical use as an antineoplastic agent.


Membrane transport in multidrug resistance, development, and disease. AACR special conference in cancer research.

The main goal of this meeting was to provide the scientists and clinicians active in this field with a comprehensive overview of the progress that has been made. The meeting was a forum in which new advances in membrane transport were discussed in depth and which gave new impulses for clinical applied research. Again, the importance of intensive cooperation between basic research and clinical use became evident during this symposium.


Expression of serum insulin-like growth factors, insulin-like growth factor-binding proteins, and the growth hormone-binding protein in heterozygote relatives of Ecuadorian growth hormone receptor deficient patients.

Recently, an isolated population of apparent GH-receptor deficient (GHRD) patients has been identified in the Loja province of southern Ecuador. These individuals presented many of the physical and biochemical phenotypes characteristic of Laron-Syndrome and are believed to have a defect in the GH-receptor gene. In this study, we have compared the biochemical phenotypes between the affected individuals and their parents, considered to be obligate heterozygotes for the disorder. Serum GH, insulin-like growth factor I and II (IGF-I and IGF-II) levels were measured by RIA Insulin-like growth factor binding proteins. (IGFBPs) were measured by Western ligand blotting (WLB) of serum samples, following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and relative quantitation of serum IGFBPs was performed with a scanning laser densitometer. Serum GH-binding protein (GHBP) levels were measured with a ligand-mediated immunofunctional assay using a monoclonal antibody raised against the GHBP. These values were then compared to values obtained from normal, sex-matched adult Ecuadorian controls, to determine if the above parameters were abnormal in the heterozygotes. The serum IGF-I levels of the GHRD patients were less than 13% of control values for adults and 2% for children. However, the IGF-I levels of both the mothers and fathers were not significantly different from that of the control population. The serum IGF-II levels of the GHRD patients were approximately 20% of control values for adults and 12% for the children. The IGF-II levels of the mothers were reduced, but were not significantly different from that of the control population. However, IGF-II levels of the fathers were significantly lower than those of controls (64% of control male levels). WLB analysis of serum IGFBP levels of the affected subjects demonstrated increased IGFBP-2 and decreased IGFBP-3, suggesting an inverse relationship between these IGFBPs. The GHRD patients who had the lowest serum IGFBP-3 levels (as measured by WLB) demonstrated a serum protease activity that could proteolyze 125I-IGFBP-3. GHRD patients who had higher serum IGFBP-3 levels lacked this serum protease activity. There were no differences in the serum IGFBP profiles of the mothers or the fathers for either IGFBP-2 or IGFBP-3, and serum from both groups lacked the ability to significantly proteolyze 125I-IGFBP-3. While GHRD patients had very low levels of serum GHBP, some patients did have measurable GHBP levels.(ABSTRACT TRUNCATED AT 400 WORDS)


Insulin-like growth factor binding protein profiles in human ovarian follicular fluid correlate with follicular functional status.

The insulin-like growth factors (IGF), IGF-I and IGF-II, influence ovarian follicular development. The IGF binding proteins (IGFBPs) comprise at least six distinct species which may modulate IGF action. We examined IGFBP profiles in follicular fluid (FF) from developing human ovarian follicles with aromatase activity as well as from androgen-dominant, atretic follicles. Western ligand blot analysis of FF from both atretic and active follicles revealed the presence of IGFBP-3 and IGFBP-2, confirmed by immunoprecipitation with specific antiserum, as well as 28 kilodalton (kDa) and 24 kDa IGFBPs. In FF specimens obtained during the follicular phase, IGFBP-2 and the 28 kDa and 24 kDa species were present in 3-, 6-, and 19-fold higher amounts in atretic compared to healthy developing follicles, respectively, and were present in greater amounts in atretic FF than in serum. In atretic FF obtained during the luteal phase, lower levels of IGFBP-2 and the 28 and 24 kDa IGFBPs were seen than in atretic follicles in the follicular phase. IGFBP-3 levels were comparable in all types of follicles and in serum. The 28 kDa IGFBP is glycosylated, and its level varied in parallel with the 24 kDa IGFBP, suggesting that these are variants of the recently identified IGFBP-4. IGFBP-1 was not detectable by immunoprecipitation and ligand blotting of FF obtained during the follicular phase. These data suggest that one or more IGFBPs present in higher levels in FF from atretic compared to healthy, developing follicles may play an important role in folliculogenesis and follicular atresia.


Characterization of urinary insulin-like growth factor binding proteins.

The insulin-like growth factors (IGFs) are important mitogens that are present in many body fluids, where they are commonly bound with high affinity to IGF binding proteins (IGFBPs). We investigated human urine for the presence of IGFBPs. Western ligand blots of concentrated, dialyzed normal urine disclosed the presence of two major bands with IGF binding activity, one at 40-44 kilodaltons and another at 31 kDa. Deglycosylation with endoglycosidase F, and immunoprecipitation with alpha HEC1 antibody revealed these proteins to be hIGFBP-3 and hIGFBP-2, respectively. Comparison of IGFBPs in normal serum and urine showed a reversal of the hIGFBP-2/hIGFBP-3 ratio in urine compared to serum, with hIGFBP-2 being the predominant binding protein in normal urine. The 150 kDa form of hIGFBP-3 was absent in normal urine. In patients with renal disease, the urinary IGFBP (U-IGFBP) pattern was altered. Patients with glomerular disease and proteinuria had elevated U-hIGFBP-3, whereas patients with renal failure who displayed increased urinary beta-2-microglobulin had a dramatic increase in U-hIGFBP-1, in the face of normal serum IGFBP profiles. In conclusion, we have documented the presence of IGFBPs in the urine of normal and diseased individuals. The presence of IGFBPs in urine may complicate the assessment of IGF measurements in urine. U-IGFBPs may be potential clinical markers in renal diseases. Additional studies are required before the origin of urinary IGFBPs in both normal and pathological conditions will be definitively established.


Myasthenia gravis patients with a thymoma have antibodies against a high molecular weight protein in sarcoplasmic reticulum.

Our purpose was to investigate whether components of the sarcoplasmic reticulum (SR) are relevant antigens in myasthenia gravis (MG). Using enzyme-linked immunosorbent assay (ELISA), 75 MG sera and 120 control sera were examined for IgG antibodies against SR prepared from rabbit skeletal muscle. 16/30 thymoma MG patients had IgG antibodies that reacted with SR. 1/30 MG patients with thymic hyperplasia and 3/15 MG patients with thymic atrophy had SR antibodies in low concentrations. Control sera were negative. Using immunoblot, SR antibodies were detected in the thymoma group only. 14/30 sera from thymoma patients reacted with a protein of 320 kDa relative molecular weight. The only reported SR protein with similar electrophoretic mobility is the subunit of the spanning protein which links junctional SR to sarcolemma and functions as a calcium-release channel.


Hausdorff dimension as a quantification of local roughness of protein surfaces.

Based on structural information from the Brookhaven Protein Data Bank, the contact surfaces of the proteins lysozyme, trypsin, and BPTI (bovine pancreatic trypsin inhibitor) with a spherical test particle of the size of a water molecule have been calculated and systematically analyzed. It is our purpose to establish (i) self-similarity as a statistical concept for the characterization of surface roughness and (ii) the Hausdorff dimension as a measure of the local surface complexity. It is found that the proteins statistically show self-similarity within a yardstick range 1.2 A less than R less than 20 A, and that this concept also holds reasonably for parts of the surface which are not too small.


Studies of V beta 8 T cell receptor peptide treatment in experimental autoimmune encephalomyelitis.

Lewis rats immunized with T cell receptor (TCR) variable region peptide V beta 8 in complete Freund's adjuvant (CFA) were protected against experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein in CFA, although variable protection was also observed in rats injected with control peptide in CFA, or CFA alone. However, this adjuvant-mediated protection could be avoided by immunizing with TCR peptide in incomplete adjuvant (IFA). Clinical, but not histologic EAE was suppressed in rats given V beta 8 peptide in IFA, whereas control animals injected with V beta 14 peptide in IFA, or IFA alone developed severe clinical EAE. Anti-V beta 8 antibodies were present in the sera of all V beta 8-treated rats. These findings lend support to the hypothesis that autoimmune disease can be suppressed by inducing an immune response against the TCR-idiotope of autoreactive T cells.


IgG anti-ganglioside antibodies and their subclass distribution in two patients with acute and chronic motor neuropathy.

IgG anti-ganglioside antibodies were found in two patients with motor neuropathy. The first patient had a chronic axonal neuropathy with persistently elevated anti-GM1 antibodies. The second patient had an acute axonal neuropathy with anti-GM1, GD1b and asialoGM1 antibodies. In both, the IgG subclass study showed that the antibodies belonged to the IgG1 subclass. An enzyme-linked immunosorbent assay (ELISA) for light chains revealed anti-ganglioside antibodies of the lambda type.


T cells in the spinal cord in experimental autoimmune encephalomyelitis are matrix adherent and secrete tumor necrosis factor alpha.

We examined T cells isolated from an autoimmune tissue lesion and from lymphoid organs for their ability to secrete tumor necrosis factor-alpha (TNF-alpha) and to adhere to extracellular matrix (ECM) proteins. CD4+ T cells were obtained from spleens, popliteal lymph nodes, and spinal cords of Lewis rats that had been immunized with myelin basic protein (MBP) to induce experimental autoimmune encephalomyelitis (EAE). We now report that, irrespective of whether or not the T cells were activated with MBP or the T cell mitogen concanavalin A (ConA), the T cells isolated from the spinal cord lesions secreted greater amounts of TNF-alpha and adhered better to ECM than did T cells from the draining lymph node. Thus, the lesions of EAE concentrate a subpopulation of CD4+ T cells with enhanced ability to interact with blood vessel wall components and to secrete TNF-alpha.


Microglial and astroglial reactions to inflammatory lesions of experimental autoimmune encephalomyelitis in the rat central nervous system.

Gliosis is a repair process of lesions appearing in the central nervous system (CNS). Although gliosis by astrocytes (astrocytic gliosis) has been well documented, that by microglia (microglial gliosis) remains poorly understood. In the present study we induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats and examined microglial and astroglial reactions to EAE lesions at various stages of the disease by immunohistochemistry. For the demonstration of microglia and astrocytes, antibodies against complement receptor type 3 (OX42) and glial fibrillary acidic protein (GFAP) were used, respectively. It was revealed that the whole course of microglial and astroglial reactions to EAE lesions is divisible into three stages, i.e., initial, peak and recovery stages. Microglial and astroglial reactions to EAE lesions at each stage correspond well with the clinical and histological stages of EAE. At the initial stage, rats showed mild clinical signs and a few inflammatory foci were found in the CNS. Microglia were increased in number in close association with inflammatory cell aggregates, whereas astrocytes showed no significant reaction in spite of the presence of inflammatory cells. At the peak stage, rats showed full-blown EAE and the number of inflammatory cells reached maximum. The most characteristic finding at this stage was 'encasement' of inflammatory lesions by astrocytic fibers. Microglia were increased in number, but association of microglia with lesions was prevented by astrocytes. Interestingly, however, such characteristic distribution of microglia and astrocytes was not observed at the recovery stage. Residual inflammatory cell aggregates were intermingled with dense microglial and astrocytic gliosis, forming 'micro-astroglial scars'. Double immunofluorescence staining with anti-GFAP and anti-bromodeoxyuridine (BrdU), or with OX42 and anti-BrdU revealed that BrdU-incorporated microglia, but not astrocytes, were present mainly at the initial and peak stages, suggesting that microglia would proliferate by cell division to create gliosis, whereas astrocytic gliosis would be a result of migration of astrocytes and/or up-regulation of expression of GFAP molecule. Taken together with previous in vitro findings that microglia, but not astrocytes, stimulate encephalitogenic T cell proliferation, these in vivo findings suggest that microglia augment, whereas astrocytes suppress, inflammatory processes in the CNS.


Increase of peripheral B lymphocytes committed to the production of monoreactive and high affinity antibodies to HTLV-1 in patients with HAM/TSP.

We quantitated the frequency of B lymphocytes capable of producing antibodies to HTLV-1 in the peripheral blood from patients with HAM/TSP, non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. Epstein-Barr virus (EBV) was used as a polyclonal activator of B lymphocytes in a limiting dilution condition. We found that B lymphocytes committed to the production of monoreactive-IgG and -IgA antibodies to recombinant HTLV-1 (gag + env) hybrid protein were significantly increased in a number in patients with HAM/TSP as compared to non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. By transforming these B lymphocytes with EBV and fusing them with human-mouse heteromyeloma (F3B6), a stable hybridoma producing IgG monoclonal antibody (mAb) to HTLV-1 (gag + env) protein was generated from a patient with HAM/TSP. This mAb (IgG1, kappa), designated F31.1, specifically bound to the amino acid residues from 235 to 254 of HTLV-1 envelope glycoproteins (gp46) with high affinity (Kd = 4.0 x 10(-9) mol/l). These data indicate that the antigen-driven process of B lymphocytes maturation by HTLV-1 antigens is markedly increased in patients with HAM/TSP.


Continuous in vivo treatment with catecholamines suppresses in vitro reactivity of rat peripheral blood T-lymphocytes via alpha-mediated mechanisms.

A 20 h continuous treatment of rats with catecholamines, using subcutaneously implantable retard tablets, had either no (adrenaline, isoproterenol, midodrine) or a slight (noradrenaline) suppressive effect on the in vitro responsiveness of peripheral blood T-lymphocytes. A marked suppression of the mitogen response ensued when adrenaline, noradrenaline or midodrine, but not isoproterenol, was applied together with the beta-receptor blocker propranolol, whereas the combination with the alpha-receptor blocker phentolamine had no effect. The mitogen response of splenic lymphocytes was not affected by any of these treatments. This alpha-mediated adrenergic suppression of peripheral blood T-cells was not correlated with general metabolic alterations, shifts in white blood cell counts or CD4+/CD8+ subsets, or with elevated glucocorticoid levels. The data suggest that to consistently influence the reactivity of rat peripheral blood lymphocytes by chronic adrenergic stimuli in vivo requires both high catecholamine levels and a bias towards alpha-adrenergic receptivity.


Immunoregulatory effects of neuropeptides. Stimulation of interleukin-2 production by substance p.

Substance P (SP), a tachykinin neuropeptide, has been previously reported to stimulate T cell proliferation, and SP receptors have been identified on subpopulations of T lymphocytes. The effect of SP on the interleukin-2 (IL-2) production has been investigated by using the murine EL-4.IL-2 and LBRM-T6G T cell lines. SP synergized with phorbol 12-myristate 13-acetate (PMA) in a dose-dependent manner to induce IL-2 production. The generated interleukin was identified as IL-2 by neutralization with a specific anti-murine IL-2 monoclonal antibody. The effect of SP was specific, since spantide and physalaemin which have affinity for SP receptors inhibited the generation of IL-2 by SP. These results provide additional evidence for the immunoregulatory role of neuropeptides, and suggest that the immunostimulatory action of SP could be mediated, at least in part, through the upregulation of IL-2 expression.


Copolymer-1-induced inhibition of antigen-specific T cell activation: interference with antigen presentation.

Copolymer-1 (Cop-1) has been shown to inhibit in vivo development of experimental allergic encephalomyelitis (EAE) in animals and has been reported to have some therapeutic benefit in relapsing/remitting multiple sclerosis (MS). The mechanism by which Cop-1 acts in vivo is not known. The present study demonstrates that Cop-1 inhibits the in vitro response of several antigen-specific murine T cell hybridomas restricted to I-A, and to a lesser extent, I-E. The ability of human myelin basic protein (MBP)-specific T cell lines (TCL) to lyse targets in the context of three HLA-DR types associated with MS was also impaired by Cop-1. The results suggest that the observed inhibition was due to competition between Cop-1 and nominal antigen for the class II major histocompatibility complex (MHC) peptide binding site.


Anti-MAG IgM paraproteins from some patients with polyneuropathy associated with IgM paraproteinemia also react with sulfatide.

Sera from five of 11 patients with neuropathy associated with IgM paraproteinemia (NAIgMPP) and reactivity against myelin-associated glycoprotein (MAG) also had elevated levels of IgM against sulfatide. Although three patients had anti-sulfatide IgM titers of less than or equal to 1:1000, two patients had titers of greater than or equal to 1:50,000. Absorption of patient serum with sulfatide revealed that anti-MAG IgM paraproteins in two patients with high titer anti-sulfatide IgM crossreacted with sulfatide. Our study is the first to show that some anti-MAG IgM paraproteins crossreact with sulfatide, a major acidic glycolipid of myelin. Moreover, some patients with NAIgMPP have polyclonal anti-sulfatide IgM in addition to anti-MAG IgM paraproteins. Therefore, sulfatide may be a target antigen in some patients with NAIgMPP.


Monoclonal antibody binding to peptide epitopes conjugated to synthetic branched chain polypeptide carriers. Influence of the carrier upon antibody recognition.

The peptide C A P D T R P A P G has been linked covalently to defined branched polypeptides with a polylysine backbone and side chains of DL-alanine or D-leucine-DL-alanine oligopeptides. The peptide was coupled via its N terminal cysteine to the side chains of the macromolecular carrier to ensure uniform orientation. The compounds were subjected to compositional analyses to characterise the degrees of substitution and secondary structural studies were performed using circular dichroism spectroscopy. The peptide selected for investigation contains the immunodominant sequence P D T R P A P which is expressed in the protein core of epithelial mucins. It is to this region that many anti-mucin monoclonal antibodies bind (Burchell et al., 1989; Price et al., 1990a,b). With these characterised constructs, it has been possible to evaluate the influence of secondary structure upon the binding of monoclonal antibodies which recognise short linear sequences in the synthetic antigenic peptide. The findings are relevant to the design and construction of synthetic immunogens and vaccines as well as to the production of synthetic analogues of clinically relevant antigens (in this case, epithelial mucins associated with breast and ovarian carcinomas).


Quantitative western blot analysis and spot immunodetection using time-resolved fluorometry.

We describe a new method of staining and quantification of proteins blotted or spotted on nitrocellulose. Blotted or spotted proteins are first reacted with specific antibodies followed by reaction with biotinylated secondary antibodies. The immunocomplex is then reacted with a streptavidin-based macromolecular complex labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl) 1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). The fluorescent spots or bands can then be assessed by visual inspection under UV illumination, by instant photography or quantified by scanning with a time-resolved fluorometer. The method does not involve enzyme detection, is simple, sensitive and gives sharp bands which remain fluorescent for long periods of time (months to years).


A novel solid-phase test to study the binding of IFN-gamma to its receptor.

A novel solid phase assay for interferon-gamma (IFN-gamma) binding to the human IFN-gamma receptor was developed. The receptor binding assay is carried out using a soluble form of the recombinant IFN-gamma receptor protein corresponding to the extracellular portion of the IFN-gamma receptor. Using different IFN molecules and anti-IFN monoclonal antibodies, we show that the specificity of the soluble IFN-gamma receptor coated to the plastic surface is not altered. In consequence, this new generation binding test can be used to characterize the interactions with the specific ligand under controlled conditions. In comparison with ELISA or RIA tests using antibodies specific for IFN-gamma, the solid-phase binding assay has the advantage of detecting only the active molecules. Finally, since the test has a large capacity, it is being applied for the screening of agents that are able to neutralize the IFN-gamma activity either by blocking the active site of the lymphokine or the binding site of the specific receptor.


Amplification of viral RNA for the detection of dengue types 1 and 2 virus.

In vitro DNA amplification by means of the polymerase chain reaction (PCR) was used to amplify dengue types 1 and 2 viral genomes in cultured cells and in the serum of persons infected with dengue virus. Results of the present investigation suggest that the PCR method is type-specific in detecting dengue virus and has a detection sensitivity of less than 100 plaque-forming units (pfu) for both serotypes of the virus. The PCR method may be useful for detecting and typing dengue virus in clinical and epidemiological specimens.


Degradation of fibronectin and vitronectin in chronic wound fluid: analysis by cell blotting, immunoblotting, and cell adhesion assays.

We used a combination of cell blotting, immunoblotting, and cell adhesion assays to analyze fibronectin and vitronectin in wound fluid from acute and chronic wounds. Acute wound fluid (e.g., suction blister fluid, mastectomy fluid) contained intact fibronectin and vitronectin as major cell adhesion proteins. In marked contrast, chronic wound fluid samples from three of 11 patients with venous stasis ulcers showed complete degradation of vitronectin and degradation of fibronectin into small molecular mass polypeptides less than 125 kDa. Three of these polypeptides--54, 93, and 125 kDa--were biologically active in promoting cell attachment and were recognized by monoclonal antibodies that bind fibronectin near the arg-gly-asp (RGD) domain. In wound fluid samples from the other eight of 11 patients, only slight degradation of vitronectin and fibronectin occurred, which resulted in a mixture of mostly intact molecules along with large fragments. Intact fibronectin in chronic wound fluid samples contained the ED-A domain, which showed that fibronectin synthesis occurred locally in the wound bed. Wound fluid containing extensively degraded vitronectin and fibronectin reversibly inhibited cell adhesion, and excess fetal bovine serum, but not purified fibronectin, neutralized the inhibitory effect. We suggest that protease activity in some chronic wounds may cause degradation of adhesion proteins and prevent cell adhesion necessary for normal wound closure.


Substance P is diminished and vasoactive intestinal peptide is augmented in psoriatic lesions and these peptides exert disparate effects on the proliferation of cultured human keratinocytes.

An involvement of neurogenic components in the pathogenesis of psoriatic lesions has been suggested and neuropeptides are thought to play a modulatory role in cutaneous inflammation. In this study, we evaluated the immunoreactivity of the neuropeptides vasoactive intestinal polypeptide (VIP) and substance P (SP) in the skin of patients with chronic plaque psoriasis, by immunohistochemistry and radioimmunoassay. No differences were observed, by immunohistochemistry, in the expression and localization of VIP and SP between psoriatic and normal skin. Using the radioimmunologic technique on whole skin homogenates, VIP levels were significantly increased in psoriatic lesions as compared to normal skin. By contrast, SP levels were significantly lower in lesional and non-lesional psoriatic skin than in normal skin. In addition, we examined the effect of VIP and SP on the proliferation of cultured normal human keratinocytes. VIP (1-28) (1 nM-1 microM) as well as VIP fragments (10-28) (1 nM-1 microM) and (22-28) (1 nM-1 microM) stimulated the proliferation of keratinocytes in a dose-dependent manner, whereas the VIP fragment (1-12) (1 nM-1 microM) was ineffective. The VIP antagonist (N-Ac-Tyr1, D-Phe2)-GRF (1-29)-NH2 (0.1 microM) significantly inhibited the VIP effect on keratinocytes. On the other hand, SP (0.1 microM) not only failed to stimulate keratinocyte growth, but also blocked the VIP-induced stimulation of these cells. The imbalance of cutaneous VIP and SP and their disparate effects on the proliferation of normal human keratinocytes in culture would suggest that these peptides are involved in the pathogenesis of psoriasis and may exert different modulatory activities in the mechanisms underlying the psoriatic lesion.


CHILD syndrome: lack of expression of epidermal differentiation markers in lesional ichthyotic skin.

Congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD) syndrome is a rare genetic disorder. The epidermal abnormalities associated with the unilateral ichthyosis have previously been examined only by morphology. In order to describe these abnormalities more completely we analyzed the expression of markers of epidermal differentiation (keratins and filaggrin), grew keratinocytes in culture, and correlated the results with ultrastructural features. Expression of all differentiation markers was significantly reduced or absent, whereas keratins K5 and K14 and keratins K6 and K16 were strongly expressed in lesional epidermis, suggesting that basal cell keratin expression was not down-regulated as in normal epidermis and that lesional keratinocytes mature via an abnormal pathway. When removed from the tissue and grown in culture, keratinocytes from lesional and non-lesional biopsies had similar phase microscopic morphology as well as keratin and profilaggrin expression, in contrast to the extreme differences in vivo. Lesional keratinocytes also had similar contents of keratin filaments and keratohyalin, but showed abnormal accumulation of intercellular vesicles and debris and altered cell-cell and cell-substratum interaction. Comparison of the results in tissue and in culture suggests that systemic or dermal factors influence the abnormal structural protein expression and ichthyosiform epidermal differentiation seen in CHILD syndrome, but that lesional keratinocytes maintain abnormalities in the secretion and accumulation of extracellular material in vitro similar to the lesional tissue in vivo.


Protoporphyrin and long-wave ultraviolet light modulate metabolic events in rat peritoneal mast cells.

We have previously shown that protoporphyrin (PP) plus long-wave ultraviolet light (UVA) has an inhibitory effect on the release of histamine from rat peritoneal mast cells in response to various stimuli, without compromising cell viability. In the present study, we observed that protoporphyrin at a noncytolytic dose (3 ng/ml) plus UVA irradiation (0.038 J/cm2) is also able to suppress prostaglandin D2 generation by rat peritoneal mast cells in response to calcium ionophore A23187, compound 48/80, or anti-IgE antibody by 64%, 92%, and 100%, respectively. Because of the participation of protein kinase C in stimulus-secretion coupling in mast cells, we also investigated the effect of PP plus UVA on the release of histamine induced by the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). PP plus UVA inhibited histamine release induced by PMA. The release of histamine induced by the synergistic combination of PMA (50 nM) and a low dose of calcium ionophore A23187 (0.1 microM) was also inhibited. PP plus UVA inhibited the release of histamine induced by the non-fluorescent calcium ionophore, 4-Br-A23187, by 47.8%, but had essentially no effect on changes in intracellular calcium induced by this stimulus. In contrast, both the release of histamine and changes in intracellular calcium stimulated by compound 48/80 were inhibited. We conclude from these results that PP plus UVA may affect both early and late biochemical events involved in mast cell mediator release.


Nootropic drugs positively modulate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-sensitive glutamate receptors in neuronal cultures.

Micromolar concentrations of piracetam, aniracetam, and oxiracetam enhanced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-stimulated 45Ca2+ influx in primary cultures of cerebellar granule cells. Nootropic drugs increased the efficacy but not the potency of AMPA and their action persisted in the presence of the voltage-sensitive calcium channel blocker nifedipine. Potentiation by oxiracetam was specific for AMPA receptor-mediated signal transduction, as the drug changed neither the stimulation of 45Ca2+ influx by kainate or N-methyl-D-aspartate nor the activation of inositol phospholipid hydrolysis elicited by quisqualate or (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid. Piracetam, aniracetam, and oxiracetam increased the maximal density of the specific binding sites for [3H]AMPA in synaptic membranes from rat cerebral cortex. Taken collectively, these results support the view that nootropic drugs act as positive modulators of AMPA-sensitive glutamate receptors in neurons.


First characterization of 6-hydroxytryptamine in the rat midbrain by using specific antibodies.

The visualization of serotonin, 5-methoxytryptamine, and tryptamine in the rat midbrain has been made possible by the development of antibodies raised against these conjugated molecules. It has been suggested that 6-hydroxytryptamine (6-HT) might also be a neurotransmitter in this region. To test this hypothesis, 6-HT was synthesized and antibodies were raised in the rabbit. The high avidity (IC50 = 5 x 10(-9) M) and specificity [cross-reactivity ratio between 6-HT-glutaraldehyde (G)-bovine serum albumin (BSA) and 5-HT-G-BSA, the most immunoreactive compound, was 1,500] rendered these antibodies reliable tools for specific molecular detection of 6-HT in the G-fixed tissues. In the dopaminergic region, 6-HT immunoreactivity was noted in the substantia nigra but was particularly intense in the red nuclei, where it seems to be localized in the magnocellular division in the form of large 6-HT neurons. In contrast, there were few 6-HT neurons in the raphe nuclei. Thus, 6-HT may be a new putative neurotransmitter existing in the red nuclei, in addition to the other neurotransmitters already described in this region, in the nigro-rubral pathway, and in the rubral projection from the dorsal raphe nuclei. 6-HT is possibly implicated in motor control and might exert hallucinogenic properties as do other 6-hydroxylated indoleamines.


Drug-induced changes in blood pressure lead to changes in extracellular concentrations of epinephrine, dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid in the rostral ventrolateral medulla of the rat.

Neurochemical changes in the extracellular fluid of the rostral ventrolateral medulla (RVLM) were produced by changes in arterial blood pressure. Blood pressure was raised or lowered with systemic infusions of phenylephrine or nitroprusside and neurochemicals were recovered from RVLM by in vivo microdialysis. A dialysis probe 300 microns in diameter and 500 microns in length was stereotaxically implanted in the RVLM of the urethane-anesthetized rat. Sterile physiological Ringer's solution was perfused at a rate of 1.5 microliter/min. The perfusate was collected under ice-cold conditions every 15 min for the assay of epinephrine, dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), ascorbic acid, and uric acid. After stable baseline neurochemical concentrations were achieved, animals were infused with phenylephrine or nitroprusside intravenously to raise or lower the blood pressure. Increasing blood pressure 50 mm Hg above the baseline value by phenylephrine led to a significant reduction in heart rate and a reduction in extracellular epinephrine and DOPAC concentrations. The 5-HIAA concentration was increased during the hypertensive drug infusion. There were no changes in the concentrations of ascorbic acid or uric acid. Hypotension produced by nitroprusside (-20 mm Hg) led to neurochemical changes which were the reciprocal of those seen during hypertension. During hypotension, heart rate increased as did the extracellular fluid epinephrine concentration. The 5-HIAA concentration fell with hypotension and remained depressed following the nitroprusside infusion. Ascorbic acid and uric acid concentrations did not change during hypotension but ascorbic acid did increase after the nitroprusside infusion stopped. These data provide direct evidence that epinephrine release in RVLM is linked to changes in systemic blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)


Octanoic acid produces accumulation of monoamine acidic metabolites in the brain: interaction with organic anion transport at the choroid plexus.

Effects of octanoic acid on monoamines and their acidic metabolites in the rat brain were analyzed by HPLC. Octanoic acid (1,000 mg/kg i.p.) elevated homovanillic acid levels by 54% in the caudate and 338% in the hypothalamus but increased 5-hydroxyindoleacetic acid (5-HIAA) levels in both the caudate and the hypothalamus by approximately 50% compared with the control. A lower dose of octanoic acid (500 mg/kg) increased 5-HIAA levels by 29% in the caudate and 20% in the hypothalamus. However, it did not produce any changes in the concentration of homovanillic acid in either the caudate or the hypothalamus. Treatment with octanoic acid also failed to change the level of dopamine, serotonin, and 3,4-dihydroxyphenylacetic acid in the caudate and the hypothalamus. The role of carrier-mediated transport in the clearance of 5-HIAA from the rabbit CSF was also evaluated in vivo by ventriculocisternal perfusion. Steady-state clearance of 5-HIAA from CSF exceeded that of inulin and was reduced in the presence of octanoic acid. Because this transport system in the choroid plexus is normally responsible for the excretion of the serotonin metabolite from the brain to the plasma, accumulation of endogenously produced organic acids in the brain, secondary to reduced clearance by the choroid plexus, could be a contributing factor in the development of encephalopathy in children with medium-chain acyl-CoA dehydrogenase deficiency who have elevated levels of octanoic acid systematically.


MK-801 increases extracellular 5-hydroxytryptamine in rat hippocampus and striatum in vivo.

The effect of MK-801 (0.25 or 0.5 mg/kg) on the extracellular concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in rat hippocampus and striatum was studied using intracerebral dialysis. The dialysate 5-HT concentration was dose-dependently increased by MK-801 in both regions. In the hippocampus, at the higher drug dose a slow increase in the 5-HIAA level was observed, and this became significant 3 h after treatment. In contrast to this, the extracellular 5-HIAA content in the striatum was significantly decreased 150 min after administration of both doses of MK-801. The data are discussed in the light of the known behavioural effects of MK-801 and possible N-methyl-D-aspartic acid receptor regulation of 5-HT release.


Chromatographic evidence for the presence of multiple tachykinins in the bovine adrenal medulla.

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.


Endothelial cell activation in vasculitis of peripheral nerve and skeletal muscle.

To clarify the role of endothelial cells in the pathogenesis of vasculitis affecting peripheral nerve and skeletal muscle, the endothelial expression of adhesion molecules and major histocompatibility antigens (MHC) in different vasculitic syndromes were studied, and related to the presence of anti-endothelial cell antibodies (AECA). Increased expression of the intercellular adhesion molecule ICAM-1 in vasculitic lesions in nerve and muscle was shown, and this was associated with increased expression of MHC class I and II antigens. AECA were detected in low titre in only a minority of patients. The findings suggest that endothelial cells have a critical role in mediating the tissue injury in vasculitis affecting nerve and muscle and that the process is triggered by cellular and not antibody-mediated mechanism in the majority of patients.


High-dose intravenous human immunoglobulin in polymyositis resistant to treatment.

Two patients were treated with treatment-resistant polymyositis with intravenous immunoglobulin over four days at a dose of 0.4 g/kg/day. Clinical recovery followed within two months. Serum creatine kinase (CK) activity decreased to normal, and a clear improvement in muscle strength was observed. One patient showed neither clinical relapses nor increase in serum CK activity after 20 months. The other showed a mild increase in serum CK activity after 24 months and was successfully retreated with intravenous immunoglobulin. There were no significant adverse side effects.


Long-term effects of chemotherapy in patients with testicular cancer.

Combination chemotherapy regimens that include cisplatin (CDDP) and bleomycin (BLE) result in the cure of the majority of patients with malignant germ cell tumors of the testis. We investigated the long-term damage of such chemotherapy to renal, pulmonary, and hearing function. Forty-three patients with disseminated testicular carcinoma were studied 1.5 to 9.3 years (median, 4.1 years) after completion of chemotherapy. All 43 patients received CDDP; of these, 39 also received BLE, 27 vinblastine (VLB), and 27 etoposide (VP-16). Mean cumulative doses of individual cytotoxic drugs administered were CDDP 483 mg/m2 (range, 189 to 1,173 mg/m2), BLE 160 mg/m2 (range, 81 to 311 mg/m2), VLB 31 mg/m2 (range, 19 to 158 mg/m2), and VP-16 667 mg/m2 (range, 242 to 1,455 mg/m2). In the majority of cases, values of renal, pulmonary, and hearing function were within the normal range before treatment. An initial decrease in renal, pulmonary, and hearing function was observed, with recovery of pulmonary function at late follow-up. On average, a decrease of 15% in creatinine clearance rates was observed at late follow-up. Long-term effect on audiometric function was considerable, but frequencies affected were outside the range of conversational speech. With multivariate analysis, no overall relation between the cumulative doses of the individual drugs and the loss in organ function was found; the cumulative doses of CDDP and BLE only contributed approximately 30% to the loss in renal function and vital capacity, respectively. Chemotherapy-induced pulmonary toxicity is reversible, whereas nephrotoxicity and ototoxicity are not. However, the long-term effects of chemotherapy in testicular cancer patients were minor and not invalidating.


Activated lymphocyte subsets in adult periodontitis.

The activation state of T and B lymphocytes in the peripheral blood of periodontitis patients may be a reflection of disease activity. We have utilized 2- and 3-color flow cytometric analyses using a new chromophore, peridinin chlorophyll A protein, and conventional dyes, fluorescein isothiocyanate and phycoerythrin, conjugated to monoclonal antibodies against activated lymphocyte surface markers to measure blood lymphocyte subsets from 18 periodontitis patients and 16 periodontally healthy control subjects. Two-color flow cytometric analysis demonstrated that the frequency of CD4+ and CD5+ T cells, CD20+ B cells, and CD16+ NK (natural killer) cells were increased in periodontitis patients. Of particular interest, CD4+ activated "memory" T cells, CD5+ B cells, and CD56+ NK effector cells were increased significantly in periodontitis patients (p less than 0.05). While the relationship of lymphocyte activation to periodontal disease activity remains unclear, there may be potential for using 2- and 3-color flow cytometry to subcategorize periodontitis patients into high- and moderate-risk groups.


Susceptibility of HIV-1 isolates to zidovudine: correlation between widely applicable culture test and PCR analysis.

Thirteen isolates of human immunodeficiency virus type 1 (HIV-1) obtained in coculture with peripheral blood lymphocytes were tested for in vitro susceptibility to zidovudine (ZDV). Seven isolates were obtained from patients who had never been treated with ZDV and six from patients receiving the drug. The seven isolates from untreated patients and four of six from treated patients were susceptible to ZDV. The two isolates from the patients treated for the longest periods were resistant to the drug. The presence of mutations at critical positions of the reverse transcriptase gene was investigated by direct sequencing of polymerase chain reaction (PCR)-amplified DNA and four isolates were found to be mutants. An isolate from an untreated patient showed a change at residue 70 of the reverse transcriptase and an isolate from a patient treated for 4 months showed a change at residue 67. A change at residue 215 was found only for the two drug-resistant isolates, which correlated with the results obtained by Larder et al. using isolates from MT-2 cell cocultures. These results suggest that any HIV isolate provided by conventional coculture could be confidently tested for ZDV susceptibility in order to study the emergence of resistance during long-term therapy.


Specific antibody responses to synthetic peptides of HIV-1 p17 correlate with different stages of HIV-1 infection.

Antibodies were determined against five synthetic peptides (epitopes) of HIV-1 p17 in the sera of an immunologically and clinically well-characterized cohort (N = 292) of HIV-1 seronegative and HIV-1 seropositive high-risk homosexual men, HIV-1 seropositive i.v. drug abusers (IVDA), and AIDS patients. The synthetic peptides, representing the entire HIV-1 p17 protein sequence were: HGP-33 (aa 1-33), HGP-19 (aa 34-52), HGP-35 (aa 51-85), HGP-30 (aa 85-114), and HGP-17 ala (aa 114-131). The presence of one or more peptide-specific antibodies in the sera of all of the HIV-1 p17-positive subjects indicated that all five peptides contain B-cell epitopes. No antibodies were found in the sera of heterosexual controls, HIV-1 seronegative high-risk men, or asymptomatic HIV-1 seropositive but p17 antibody-negative study subjects. Significant differences in antibody recognition profiles to the peptide epitopes were found among the various study groups. A significantly higher proportion of HIV-1 seropositive IVDA had antibodies specific to HGP-17 ala (aa 114-131), HGP-35 (aa 51-85), and HGP-33 (aa 1-33) compared to the HIV-1 p17-positive asymptomatic homosexuals. The epitope-specific antibody responses reflected the clinical status of the HIV-1-infected study subjects, and declined to nondetectable levels as the patient progressed to ARC/AIDS. This decline preceded by several months the reduction in the antibody titer against the intact HIV-1 p17 and p24 proteins.(ABSTRACT TRUNCATED AT 250 WORDS)


T-lymphocyte responses to Pneumocystis carinii in healthy and HIV-positive individuals.

Pneumocystis carinii pneumonia (PCP) is a well-recognized cause of morbidity in patients with impaired T-cell function. In this study of cellular immunity to P. carinii, peripheral blood mononuclear cells from 25 HIV antibody-positive (HIV+) patients and 11 healthy individuals were stimulated in vitro with P. carinii antigen. The responding T-cell blasts were cocultured with autologous P. carinii antigen-pulsed macrophages to measure P. carinii-specific cytolytic T-lymphocyte activity (CTL). T-cell blasts from two healthy donors were used to generate P. carinii-specific clones by limiting dilution. T cells from HIV+ patients proliferated less to P. carinii antigen than T cells from healthy volunteers. In contrast, the level of specific cytotoxicity was identical in all groups when equal numbers of CTLs were used. Within the group of symptomatic patients, CTL activity was higher in those with a history of PCP (p = 0.033). Pneumocystis carinii antigen-specific T-cell clones proved to be CD4+ and MHC class II restricted; six of eight clones tested showed P. carinii-specific cytolytic activity. Cell-mediated immune response to P. carinii in healthy individuals include CD4+, class II MHC-restricted T cells with P. carinii-specific cytotoxicity. There is an increasing loss of P. carinii-specific proliferative responses in HIV+ patients as disease progresses, but a cytotoxic response is still detected in the absence of proliferation.


Common mechanisms govern the expression of p21ras and class II MHC antigens in the murine placenta.

It has been shown that there is an inverse as well as a direct correlation between class II MHC antigen expression and the p21ras protein, depending on the cell type. By using trophoblastic cells derived from the spongiotrophoblast and labyrinthine trophoblast, the two major components of the murine placenta, we have examined the p21ras expression in these populations and how this correlates with the induction of class II MHC antigens. It has been shown that although only a small number of cells express the p21ras and class II proteins, they can be induced to express higher levels of these markers by two different treatments. Thus, gamma-interferon (gamma-IFN) induces p21ras and class II protein expression in spongiotrophoblast-derived cells, whereas 5-Azacytidine (5-AzaC) induces expression of these antigens in labyrinthine trophoblast-derived populations. Furthermore, it has been demonstrated that there is a direct correlation between the two markers, as blocking antibody to p21ras cancels the ability of gamma-IFN and 5-AzaC to induce class II antigens. As previous work from this laboratory has shown that in vivo class II induction in the placenta leads to fetal abortion, the present results suggest that both proteins are involved in a common signal transduction pathway which may lead to disruption of fetal membranes and fetal loss.


Rare causes of elevated maternal serum alpha-fetoprotein. A report of three cases.

Three rare conditions--amniotic band disruption sequence, placental chorioangioma and congenital nephrosis--were diagnosed in midtrimester because of elevated maternal serum alpha-fetoprotein. The diagnosis is important for genetic counseling and obstetric follow-up.


Syntheses and thymidylate synthase inhibitory activity of the poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-thenoyl]-L-glutamic acid (ICI D1694) and other quinazoline antifolates.

Thirteen poly-gamma-glutamates derived from several novel antifolates have been synthesized by a convergent route. The syntheses of poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2- methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theno yl]-L-glutamic acid (8) (ICI D1694), 2-desamino-N10-propargyl-5,8-dideazafolic acid (6), 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (7), 2-desamino-2-methyl-N10-propargyl-2'-fluoro-5,8-dideazafolic acid (9), and 2-desamino-2-methyl-4-chloro-N10-propargyl-2'-fluoro-3,5,8-trideazafo lic acid (11) are described. A key step in the route involves coupling of an alpha-tert-butyl-protected poly-gamma-glutamate of the required chain length to the appropriate 5,8-dideazapteroic acid, obtained by carboxypeptidase G2 cleavage of the parent monoglutamate, if available, or by chemical synthesis. Deprotection with trifluoroacetic acid in the final step gave the desired poly-gamma-glutamyl antifolates as their trifluoroacetate salts. As inhibitors of thymidylate synthase, these polyglutamates were more potent in every case than the corresponding non-polyglutamylated drug.


Novel benzothiophene-, benzofuran-, and naphthalenecarboxamidotetrazoles as potential antiallergy agents.

The synthesis and antiallergic activity of a series of novel benzothiophene-, benzofuran-, and naphthalenecarboxamidotetrazoles are described. A number of the compounds inhibit the release of histamine from anti-IgE stimulated basophils obtained from allergic donors. Optimal inhibition is exhibited in benzothiophenes with a 3-alkoxy substituent in combination with a 5-methoxy, 6-methoxy, or a 5,6-dimethoxy group. Compound 13c (CI-959) also inhibited respiratory burst of human neutrophils and the release of mediators from anti-IgE-stimulated human chopped lung.


Use of recombinant canine granulocyte colony-stimulating factor to decrease myelosuppression associated with the administration of mitoxantrone in the dog.

Ten dogs were given mitoxantrone at a dose of 5 mg/m2 body surface area intravenously. Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously daily for 20 days after an infusion of mitoxantrone in five of these dogs to determine the effect of the hematopoietic growth factor on the duration and severity of myelosuppression. The median neutrophil counts dropped below normal (less than 3,000/uL) for 2 days in the dogs that received rcG-CSF, and for 5 days in the dogs that received only mitoxantrone. Four of five dogs not treated with rcG-CSF and none of those receiving rcG-CSF developed serious neutropenia (less than 1,500/uL). The neutrophil counts were significantly (P less than 0.05) higher in the rcG-CSF treated dogs at all time points except before the administration of the colony-stimulating factor, and the sixth day after the mitoxantrone was administered. These findings demonstrate that rcG-CSF is capable of reducing the duration and severity of mitoxantrone-induced myelosuppression.


Localisation of the mitogenic epitope of staphylococcal enterotoxin B.

Limited digestion of staphylococcal enterotoxin B (SEB) with trypsin resulted in the generation of a 12-Kda amino-terminal fragment and a 17-Kda carboxy-terminal fragment which were isolated by preparative iso-electric focusing. The carboxy-terminal fragment exhibited significant mitogenic activity for murine splenocytes, whereas the isolated amino-terminal fragment possessed little detectable mitogenic activity. Monoclonal antibodies (MAbs) specific for the carboxy-terminal fragment neutralised most of the mitogenic activity of both the intact toxin and the carboxy-terminal fragment. MAbs specific for the amino-terminal fragment had no detectable neutralising activity. These results support the hypothesis that the epitope(s) responsible for mitogenic activity is located in the carboxy-terminal region of SEB.


Isolation of a novel siderophore from Pseudomonas cepacia.

A novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudomonas cepacia cultures. This compound, named azurechelin, was produced by 88% of P. cepacia strains isolated from the respiratory tract. Production of azurechelin was regulated by the iron concentration in the culture medium. Azurechelin enhanced the growth of P. cepacia in a medium containing transferrin 200 mg/L. Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could complete with transferrin for iron. Azurechelin could also stimulate iron uptake by P. cepacia. This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.


Host factors versus virulence-associated bacterial characteristics in neonatal and infantile bacteraemia and meningitis caused by Escherichia coli.

Possession of P fimbriae, virulence-associated O and K antigens, haemolysin and aerobactin production, and susceptibility to 10 antimicrobial agents were studied in 63 Escherichia coli strains isolated from blood or CSF of infants who were grouped according to their clinical characteristics. These isolates were compared with 35 faecal E. coli strains from healthy infants. Individual virulence factors showed a relatively weak association with invasive infection except for P fimbriae in urosepsis and aerobactin production in meningitis. Combinations of factors were generally more predictive for defining virulent clones, particularly in infants defined as being at normal risk of developing septicaemia. Thus, 62% of isolates from such infants had characteristics typical of previously described uropathogenic or meningitis-associated clones of E. coli, compared with 32% of the isolates from high-risk infants (i.e., those defined as being at high risk of developing septicaemia) and only 9% of the faecal isolates (p less than 0.001 and less than 0.05, respectively). Overall, 45% of the episodes of invasive infection were caused by such clones, whereas risk factors (conditions considered to be associated with increased risk of invasive infection) were present in 59% of the infected infants (39% in meningitis and urosepsis, 78% in cryptogenic septicaemia and untreated bacteraemia). The results indicated that bacterial factors played a significant causative role in neonatal meningitis and urosepsis, particularly in normal-risk infants, whereas predisposing host factors contributed greatly to cryptogenic septicaemia and untreated bacteraemia.


Identification of an endoflagellar associated protein in Borrelia burgdorferi.

DNA of Borrelia burgdorferi was cleaved by the endonuclease EcoRI and ligated with the bacteriophage expression vector lambda gt11. After infection of the Escherichia coli strain Y1089, the plaques of recombinant phages were screened with a B. burgdorferi antiserum (human) for fusion proteins containing borrelia antigen.s A positive clone produced a hybrid protein (p200) of c. 200 Kda. The corresponding native borrelia protein (p97) was identified as having an Mr of 97 Kda. To localise protein p97 in the B. burgdorferi cell, immunoelectronmicroscopy and a Western blot of isolated flagella were used. Antibodies directed against proteins p200 and p97 recognised epitopes associated with the flagella.


Variation of the O antigen of Campylobacter jejuni in vivo.

During the course of a clinical study on patients with campylobacteriosis, three consecutive isolates of Campylobacter jejuni from the same patient were sent for O-serotyping. Marked differences in the specificities of the O antigens of the isolates were observed between the first and third isolates when a passive haemagglutination assay system developed for serotyping C. jejuni was used. Differences in specificity were also demonstrated by immunoblots of lipopolysaccharides (LPS) from proteinase K-digested whole cells. The three isolates could not be distinguished either by restriction endonuclease analysis of chromosomal DNA, by gel electrophoresis of whole-cell proteins or by silver-stained LPS gels, thus providing evidence that they were of the same strain and that antigenic variation had occurred in vivo.


Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators.

Escherichia coli RNA polymerase can terminate transcription efficiently at rho-independent terminators in a purified transcription system in the absence of accessory factors. This process of "intrinsic termination" involves direct recognition of the terminator by the core RNA polymerase, and provides an important model system for the study of the molecular interactions involved in the switch between elongation and termination. We have analyzed the intrinsic termination efficiency (%T) of 13 rho-independent terminators, under a variety of in vitro reaction conditions. Although all of these sites share the general sequence features of typical rho-independent terminators, we find a wide range of %T (2% to 90%) for the different sites under our standard transcription conditions. While %T for a particular site is characteristic of that site, the efficiency can be altered considerably by the nature and concentration of salts in the reaction, by alteration of the concentrations of the nucleoside triphosphate substrates, or by transcription from supercoiled rather than linear templates. Surprisingly, different conditions can alter %T to a different extent for different terminators. For neutral salts such as potassium chloride or potassium glutamate, changes in the range from 0.1 to 1 M affect %T for different terminators in a distinct manner, depending on the terminator and the anion involved. At some sites, %T is greatly increased by Cl- concentrations up to 1 M, while at other sites %T is reduced or unaffected by these conditions. At some sites K+ concentrations up to 1 M give a modest increase in %T, while at other sites %T is slightly reduced under the same conditions. Thus the actual values of %T, as well as the order of terminator sites ranked according to %T, can be altered greatly according to the choice of reaction conditions. Reduction of the Mg2+ concentration below 1 mM has a dramatic and quite different effect, enhancing termination to approximately 100% for all terminators tested. Transcription of supercoiled DNA templates gives somewhat reduced %T as compared with linear DNA templates. However, the effect is no greater than twofold. Our results are not consistent with those expected for models in which %T is determined by the differential stability of DNA, RNA and hybrid duplex structures at the melted region in the transcription complex. Thus, the Cl anion does not affect the stability of nucleic acid duplexes even at 1 M concentrations, but can enhance termination tenfold. Also, the alterations of monovalent cation concentration that affect %T are not expected to have a differential effect on Tm for DNA, RNA and hybrid duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)


Parameters affecting transcription termination by Escherichia coli RNA. II. Construction and analysis of hybrid terminators.

Rho-independent terminators are characterized by two major functional regions, one upstream from the termination site having a sequence capable of forming an RNA hairpin in the nascent transcript, the second extending, from the base of this hairpin, seven to nine nucleotides along the transcript to the actual sites of termination (3'-tail region). This latter region of the transcript is often rich in uridine residues. Both regions are postulated to play central roles in the termination process. We have constructed a series of hybrid rho-independent, transcription terminators in which sequences upstream and downstream from the RNA hairpin for the Escherichia coli trp attenuator (trpatt+) are interchanged with sequences from trpatt mutant (1419) or from the phage T7 early terminator (T7Te). Similar hybrids have been constructed for T7Te, replacing flanking sequences with trpatt regions. The effects of such changes on transcription termination have been tested in vitro with purified E. coli RNA polymerase to determine the intrinsic termination efficiency (%T) of each hybrid terminator. Both the trpatt+ terminator and T7Te are highly efficient rho-independent terminators in vitro. However, replacement of trpatt+ sequences upstream and downstream from the RNA-terminator hairpin with the comparable T7Te sequences reduces %T dramatically, suggesting that the RNA-terminator hairpin does not function independently from its flanking regions. Regions downstream from the actual termination/release site are shown to be of considerable importance in determining %T for terminators bearing the T7Te or trpatt1419 3'-tail region, but have little effect on terminators with the trpatt+ 3'-tail region. For terminators bearing the T7Te or trpatt1419 3'-tail region that are inefficient, efficient termination is restored by elevated concentrations of KCl in the reaction. The results do not fit well with models for termination in which %T is determined by a two-step process in which the terminator-RNA hairpin, and a seven to 12 base-pair DNA-RNA hybrid structure rich in uridine residues, act independently to cause the polymerase to pause, and to release the transcript, respectively. DNA sequences both upstream and downstream from these regions, as well as DNA sequences downstream from the transcript termination site, can significantly affect the termination process. Conversely, terminators lacking a 3'-tail region rich in uridine residues can be highly efficient, but only when joined with appropriate sequence immediately downstream from the termination site. This suggests that the 3'-tail region acts in some manner other than the formation of an unstable DNA-RNA hybrid that facilitates termination.


Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol.

The mode of action of gliotoxin against poliovirus has been analyzed in detail. This fungal metabolite inhibits the appearance of poliovirus proteins when present from the beginning of infection but has no effect on viral translation when added at late times. In agreement with previous findings, this toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium. Analysis of the synthesis of poliovirus plus- or minus-stranded RNA in the presence of gliotoxin suggests that this compound effectively hampered both processes. This result contrasts with the mode of action of other inhibitors of poliovirus RNA synthesis, such as guanidine or flavones, that selectively block plus-stranded RNA synthesis and suggests that the target of gliotoxin differs from the target of guanidine, i.e., poliovirus protein 2C. Indeed, gliotoxin was found to be a potent inhibitor of poliovirus RNA synthesis in cell-free systems, using membranous crude replication complexes, a reaction that is not blocked by guanidine or Ro 09-0179. Moreover, in vitro activity of the purified poliovirus polymerase 3Dpol was efficiently inhibited by gliotoxin. These results indicate that this toxin acts on the poliovirus polymerase 3Dpol, providing the first description of an inhibitor of this viral enzyme.


Cross-reactive and serotype-specific antibodies against foot-and-mouth disease virus generated by different regions of the same synthetic peptide.

Synthetic peptides based on the VP1 proteins of two serotypes of foot-and-mouth disease virus (FMDV) and having the general formula C-C-(200-213)-P-P-S-(141-158)-P-C-G induce heterologous as well as homologous protection against challenge. Substitution of the sequence consisting of residues 200 to 213 (200-213 sequence) with a second copy of the homologous 141-158 sequence (i.e., homodimers) resulted in failure of either serotype peptide to protect heterologously. The antiviral and antipeptide titers of sera from guinea pigs immunized with the homodimeric 141-158 peptides showed serotype specificity and, with the data from the heterodimeric peptide vaccines, suggested that the C-terminal 141-158 sequence was more effectively recognized by the immune system than the N-terminal sequence. Whereas heterologous antiviral titers as measured by enzyme-linked immunosorbent assay and virus neutralization tests have not been observed with sera from cross-protected animals, epitope-mapping studies established that there was heterologous recognition of an octapeptide within the 200-213 sequence. That the 200-213 sequence was required for the induction of heterologous protection was also confirmed with a number of peptides, including hybrids based on the 200-213 sequence of one virus and the 141-158 sequence of a second virus. Thus, peptides of the general formula given above induce serotype-specific and serotype-cross-reactive protective antibodies and are unique in their induction of significant levels of heterologous protection, a property which has never been reported for whole FMDV vaccines.


Homologous recombination of copackaged retrovirus RNAs during reverse transcription.

According to prevailing models, the high frequency of recombination in retroviruses occurs during reverse transcription of two genetically different genomes copackaged into virion particles. This view has been tested in our studies of the mechanism of recombination within homologous sequences of two retroviral genomes during a single round of virus replication and in the absence of helper virus. The recombination substrates were Moloney murine leukemia virus-based vectors, each of which contains an altered defective neomycin gene (neo) under the transcriptional control of the 5' long terminal repeat; the 3' sequences of each construct contain either the Moloney murine leukemia virus or simian virus 40 large-T polyadenylation sequence. One neo gene contained a linker insertion mutation at the 5' end (neo minus), and the other contained a deletion and linker insertion at the 3' end (neo delta 3). Each of the mutant neo constructs was introduced into the packaging helper cell line psi 2 by sequential cotransfection, and individual psi 2 double transformants were selected. Supernatant fluids from the cloned psi 2 double transformants were used to infect NIH 3T3 cells, and recombinant neo+ proviruses were detected by their ability to confer G418 resistance during infection of NIH 3T3 cells. Our results show that (i) recombination between a homologous sequence of about 560 bp occurred with a frequency of about 10(-4) per virus replication cycle; (ii) recombination occurred only after the viral RNAs had been packaged into particles, i.e., recombination between the two vector DNAs or between viral RNAs prior to packaging was not detected; and (iii) copackaging of two different genomic RNAs as a heterodimer is a prerequisite for recombination. Furthermore, our results indicate that recombination can occur during the DNA negative-strand synthesis of reverse transcription.


Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.


Generation of "natural killer cell-escape" variants of Pichinde virus during acute and persistent infections.

Pichinde virus (PV) strain AN 3739 was determined to be sensitive to natural killer (NK) cells in vivo by enhanced replication in NK-cell-depleted mice. An NK-sensitive subclone (PV-NKs1) was serially passed in mice whose NK cells had previously been activated by an interferon inducer, and two plaque isolates were shown to be resistant to NK cells but not to interferon. Inoculation of severe-combined-immunodeficient mice with PV-NKs1 led to a persistent infection resulting in an NK-resistant viral population. This is the first demonstration of the isolation of viral "NK-escape" variants, as defined by the ability of the virus to replicate in vivo.


Pneumocystis carinii in corticosteroid-treated voles: a comparison of three different staining methods.

Pneumocystis carinii (PC) is an opportunistic pathogen which causes clinical disease in immunocompromised hosts. Three different staining protocols were employed to detect this organism in lung samples of corticosteroid treated voles in order to discover a suitable method for large-scale screening. The procedures employed were: Grocotts methenamine silver (GMS)-stained paraffin sections, toluidine blue O-stained impression smears, and methenamine-silver-stained frozen sections. GMS-stained paraffin sections were relatively easy to interpret and gave more positive results than the other methods. It seemed to be the satisfactory method for large-scale population analyses. An unexpected result was that methylprednisolone treatment did not induce in voles a similarly fatal pneumocystosis infection as occurred in rats. All infections found in voles were mild. This might be due to species-dependent differences in metabolizing methylprednisolone.


Endothelial function in thrombosis and thrombolysis.

The localization of tissue factor (TF) in atherosclerotic plaques of human aortas was immunohistochemically examined using rabbit anti-IgG against recombinant TF, which was expressed in E. coli. TF, the initiator of the extrinsic coagulation pathway, was ubiquitously present in atherosclerotic intima, and was expressed mainly by macrophages, but not by endothelial cells. It has been suggested that some macrophages in atherosclerotic intima co-express both molecules of TF and platelet-derived growth factor-B chain. We have developed a morphometrically quantitative in vitro assay for angiogenesis, using endothelial cultures on collagen gel incorporating plasminogen. With this method, we have obtained findings suggesting that plasminogen and plasminogen activators (PAs), especially urokinase-type PA (uPA) derived from endothelial cells, enhance angiogenic activity, probably by increasing endothelial migration. uPA was immunohistochemically observed to be primarily cell-associated on the focal contract areas, probably via its receptors on endothelial cells. These findings may support the hypothesis that the activation and regulation of the pericellular fibrinolysis system is closely related to angiogenesis.


Immunohistochemical characteristics of adenosquamous carcinoma of the pancreas.

Six patients with adenosquamous carcinoma (ASqC) of the pancreas were studied clinicopathologically and immunohistochemically. In five of six ASqC tumors, both malignant squamous and glandular elements were reactive with CA 19-9, ST 439, and keratin antibodies. In contrast, a portion of the glandular element in the remaining one ASqC was reactive with CA 19-9 and ST 439 antibodies, but that of the squamous cell carcinoma (SqCC) was not reactive. However, SqCC of this tumor was intensely reactive with keratin antibody. These immunohistochemical results suggest that the histogenesis in one ASqC tumor was different from that of the other 5 ASqCs, and that this tumor may be a collision tumor rather than transformation to SqCC from adenocarcinoma, which is a very rare pattern of histogenesis in ASqC. The patients with ASqC of the pancreas showed shorter survivals following operations because of systemic metastasis including liver metastasis.


Alpha-fetoprotein production by hepatocellular carcinoma is prognostic of poor patient survival.

Immunohistochemistry using the avidin-biotin-peroxidase complex method was performed to study the production of alpha-fetoprotein (AFP) in hepatocellular carcinoma (HCC) tissue specimens which were obtained surgically. The relationship between staining for AFP and serum AFP levels or pathological findings was examined. The prognosis of the patients with HCC who underwent curative hepatic resections was studied with respect to the staining for AFP in their tumors. The mean serum AFP level in patients with positive AFP staining was significantly higher than in those with negative AFP staining. No significant relationship was found between AFP positivity and tumor size, tumor encapsulation, degree of vascular invasion, or the histological differentiation grade of the tumor. The patients with AFP-positive carcinomas had a poorer prognosis than did those with AFP-negative carcinomas (5-year survival rate of AFP-positive and negative groups were 26.7% and 56.5%, respectively.


Prostate-specific antigen, digital rectal examination, and transrectal ultrasound in predicting the probability of cancer.

Over a 4 1/2 year period, 1,940 asymptomatic men were entered in a prostate cancer detection program consisting of digital rectal examination (DRE), prostate-specific antigen (PSA), and transrectal prostate ultrasound (TRUS). Four hundred and sixteen biopsies were performed resulting in the diagnosis of 79 cancers; 82% had clinically organ confined tumors. A recommendation for biopsy was made in 260 (62%) based on the TRUS alone, 55 (13%) by DRE alone, 92 (22%) when the DRE and TRUS were both abnormal, and in 9 (2.2%) cases when only PSA levels were elevated. The DRE, PSA, and TRUS were abnormal in 1,261 (65%), 989 (51%), and 1,552 (80%) of the patients with cancer, respectively. Prostate cancer detection increased as the serum PSA level increased above 4 ng/ml. The positive predictive value of both DRE and TRUS were significantly influenced by an elevated PSA, (P = .042 and P less than .00005, respectively). The results of this study support the idea that, although the prostate cancer detection rate is influenced by these three modalities and the detection rate of localized disease can be improved by early detection programs, its effect on mortality rates remains undefined at this time.


Influence of preoperative treatment and surgical operation on immune function of patients with esophageal carcinoma.

Multiple immunological parameters, including total lymphocyte count, lymphocyte subpopulations (CD2+, CD19+, CD3+, CD4+ and CD8+), phytohemagglutinin (PHA) response, and natural killer (NK) activity, were measured in 66 patients with previously untreated esophageal carcinoma. The influence of preoperative treatment and/or surgical operation on the immune function were evaluated in 40 patients. The PHA response and NK activity of the patients with esophageal carcinoma were 229 +/- 103 S.I.% and 18.5 +/- 11.9% lysis, respectively, and were significantly depressed as compared with the control. The CD4+/CD8+ ratio, PHA response, and NK activity in stage IV were also significantly depressed compared to that in stages I-III. Preoperative treatment induced significant reductions in the total lymphocyte count (1,994 +/- 644 to 670 +/- 274/mm3), PHA response (219 +/- 77 to 159 +/- 59 S.I.%), and NK activity (19.7 +/- 13.2 to 11.1 +/- 10.3% lysis) as well as a significant gradual decrease in the CD4+/CD8+ ratio (2.09 +/- 1.42 to 0.69 +/- 0.48), while the surgical operation significantly influenced only the total lymphocyte count. This study demonstrates that preoperative treatment induces a more pronounced influence on the immune function than surgical operation alone, in patients with esophageal carcinoma in which the immune function is disturbed prior to these treatments.