Absence of autoantibodies to human chorionic gonadotropin in women with a history of habitual abortion. To determine if immunization to human chorionic gonadotropin (hCG) has occurred in women with habitual abortion. Comparisons between nonpregnant patients with a history of at least three consecutive miscarriages (n = 48) and normal controls (n = 38). In addition, 28 habitual aborters were compared, while pregnant, with 37 pregnant control women. Antibodies to hCG were assessed by a solid-phase immunometric assay using europium-labeled antihuman immunoglobulin (Ig)G as tracer; this method is capable of detecting antibodies toward hCG in serum of patients immunized with beta-hCG-tetanus toxoid conjugate. Departments I and II of Obstetrics and Gynecology, University Central Hospital of Helsinki, Helsinki, Finland. Three patients (1 primary and 2 secondary aborters, 1 both while pregnant and not pregnant) showed evidence of Ig binding to hCG, but the binding was not inhibited by an excess of hCG. Antibodies against endogenous hCG may not be responsible for habitual abortion.


Insulin-like growth factor binding protein-1 in female reproductive functions. Multiple lines of evidence suggest that growth factors and related proteins are involved in the regulation of reproductive functions. It appears that hormones and local regulators control each other's production and action. Thus, the same regulatory factor may have different effects depending on the context in which it acts. Insulin-like growth factor-binding protein-1 (IGEBP-1) is a member of the family of soluble proteins that bind insulin-like growth factors (IGF-I and IGF-II) and modulate their biological actions at the cellular level. In the reproductive tract, endometrium and ovarian granulosa-luteal cells express IGFBP-1 mRNA and secrete the protein at a certain stage of differentiation. During pregnancy, IGFBP-1 is a major secretory product of decidualized endometrium. This report summarizes the current views on IGFBP-1 with special regard to its synthesis, regulation and potential role in female reproductive tissues.


Medial edge epithelium fate traced by cell lineage analysis during epithelial-mesenchymal transformation in vivo. Vital cell labeling techniques were used to trace the fate of the medial edge epithelial (MEE) cells during palatal fusion in vivo. Mouse palatal tissues were labeled in utero with DiI. The fetuses continued to develop in utero and tissues of the secondary palate were examined at several later stages of palatal ontogeny. The presence and distribution of DiI was correlated with the presence of cell phenotype-specific markers. During the initial stages of palatal fusion the DiI-labeled MEE were present in the midline position. These cells were attached to an intact laminin-containing basement membrane and contained keratin intermediate filaments. At later stages of palatogenesis the DiI-labeled MEE were not separated from the mesenchyme by an intact basement membrane and did not contain keratin. In late fetal development, DiI-labeled cells without an epithelial morphology were present in the mesenchyme. The transition of the DiI-labeled cells from an epithelial phenotype to a mesenchymal phenotype is consistent with a fate of epithelial-mesenchymal transformation rather than programmed cell death.


Ribosomes as carriers for antigenic determinants of the surface of micro-organisms. Over the past twenty-five years, many authors have reported evidence of the immunoprotective capacity of ribosomes isolated from bacteria, fungi and parasites. Since 1971 we have explored the protective capacity of ribosomes isolated from a large variety of micro-organisms responsible for human and animal diseases. Accurate biochemical characterization of ribosomes always reveals trace amounts of non-ribosomal components such as short polysaccharides strongly linked to ribosomal RNA after phenol extraction even under denaturing conditions. rRNA-antigen complexes have been purified from Klebsiella pneumoniae ribosomes inducing high level of protection against homologous experimental infection in mice. Monoclonal antibodies raised against ribosomes and then selected for their ability to confer passive immunity to mice have been used to study the mechanism of the protection induced by ribosomes and to characterize their "immunogenic principle". These investigations have clearly shown the presence on ribosomes of epitopes corresponding to antigens normally exposed on the membrane of the bacteria. In the original concept of "ribosomal immunotherapy" that we have developed, ribosomes can be considered as natural carriers for cell surface epitopes, presenting them to the immune system in a highly immunogenic configuration.


Immunomodulatory properties of soluble recombinant human CD58 (LFA-3) molecules. The interaction between the E-rosette receptor (CD2) expressed on the cell surface of T lymphocytes and its natural ligand CD58 (LFA-3), a broadly distributed cell surface molecule, represents a potential target for immune modulation. To explore this possibility, we have expressed a soluble recombinant form of human CD58 which exerts potent biological effects in that it acts as a competitive inhibitor for cell surface CD58 in its binding to CD2 positive lymphocytes. Thus, recombinant CD58 blocks NK-mediated cytotoxicity, the mixed lymphocyte reaction, and the T lymphocyte adhesion to CD58 positive cells. In contrast, recombinant CD58 synergized with mitogenic CD2R monoclonal antibodies (anti-T11(2/3) or 9.1) in T cell triggering.


Monoclonal antibody MT2 identifies an extracellular matrix glycoprotein that is co-localized with tenascin during adult newt limb regeneration. Using immunohistochemical techniques and mAb MT2, we describe here a novel extracellular matrix (ECM) molecule that is developmentally regulated during limb regeneration in adult newts. The MT2 antigen appears during preblastema stages, is most abundant during blastema stages, and persists, near undifferentiated cells, until digit stages. The MT2 antigen is located in an acellular layer under the wound epithelium and throughout the ECM of the undifferentiated mesenchyme as a thick, cord-like component. In unamputated limbs mAb MT2 reactivity is restricted to tendons, myotendinous junctions, periosteum and to a layer of material beneath the epidermis. In both unamputated limbs and regenerating limbs, the reactivity to mAb MT2 colocalizes closely with urodele tenascin. Immunoblot analysis of blastema extracts showed that the unreduced form of the MT2 antigen is a large, polydispersed protein of approximately the same size as tenascin. However, based upon (a) molecular weights of reduced subunits, (b) competition experiments on tissue sections, and (c) analysis of molecules immunoprecipitated by mAb MT2, we conclude that the MT2 substance is unrelated biochemically to tenascin. The results from immunoblots, enzyme digestions and DEAE-Sephacell binding studies suggest that the unreduced MT2 antigen is a large protein composed of subunits which are connected by disulfide bonds. Reduction of the MT2 antigen results in three components recognized by mAb MT2. The largest of these reduced components is a chondroitin sulfate-like glycoprotein with a molecular weight (Mr) of 310-325 x 10(3). A second component (Mr, 285-300 x 10(3)) is the core protein of the 310-325 x 10(3) glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)


Structural features and sites of expression of a new murine 65 kD and 48 kD hair-related keratin pair, associated with a special type of parakeratotic epithelial differentiation. In the course of studies on local keratin phenotypes in the epidermis of the adult mouse, we have identified a new 65 kD and 48 kD keratin pair. In mouse skin, this keratin pair is only expressed in suprabasal cells of adult mouse tail scale epidermis which is characterized by the complete absence of a granular layer and the formation of a remarkably compact stratum corneum. A second site in which the 65 kD and 48 kD keratin pair is suprabasally expressed and whose morphology corresponds to that of tail scale epidermis is found in the posterior unit of the complex filiform papillae of mouse tongue. The causal relationship of the expression of the 65 kD and 48 kD keratins with this particular type of a non-pathological epithelial parakeratosis is emphasized by the suppression of the mRNA synthesis of the two keratins during retinoic acid mediated orthokeratotic conversion of tail scale epidermis. Apart from tail scale epidermis and the posterior unit of the filiform papillae, the 65 kD and 48 kD keratin pair is, however, also coexpressed with "hard" alpha keratins in suprabulbar cells of hair follicles and in suprabasal cells of the central core unit of the lingual filiform papillae. The non alpha-helical domains of the two new keratins are rich in cysteine and proline residues and lack the typical subdomains into which epithelial keratins of both types can be divided. This structural resemblance of the 65 kD and 48 kD keratins to "hard" alpha keratins is supported by comparative flexibility predictions for their non alpha-helical domains. Phylogenetic investigations then show that the 65 kD and 48 kD keratin pair has evolved together with hair keratins, but has diverged from these during evolution to constitute an independent branch of a pair of hair-related keratins. In view of this exceptional position of the 65 kD and 48 kD keratins within the keratin multigene family, their expression has apparently been adopted by rare anatomical sites in which an orthokeratinized stratum corneum would be too soft and a hard keratinized structure would be too rigid to meet the functional requirement of the respective epithelia.


Separation of the toxic and glutathione-enhancing effects of the naturally occurring nitrile, cyanohydroxybutene. Cyanohydroxybutene (CHB) is reported to be hepatotoxic in male Fischer 344 rats at an oral dose of 300 mg/kg and, while no longer hepatotoxic, pancreatotoxic at 200 mg/kg. In addition, the 200 mg/kg dose causes a persistent elevation in hepatic and pancreatic glutathione (GSH). This study was conducted to determine if smaller doses of CHB could cause GSH elevation in the absence of toxicity. A single oral dose of 100 mg/kg or multiple lower doses (50 mg/kg daily for 3 days or 30 mg/kg for 6 days) caused a significant and persistent increase in pancreatic GSH, although hepatic levels were unchanged. Ten milligrams per kilogram, even daily for 24 days, was without effect on hepatic or pancreatic GSH. Neither a single oral dose of 100 mg/kg nor multiple lower doses were associated with toxicity. However, when either 100 or 50 mg/kg were administered intravenously, pancreatic apoptosis was observed. In animals dosed with 100 mg/kg iv, mixed histiocytic and suppurative inflammation and frank pancreatic necrosis also developed and were associated with elevated plasma lipase and amylase. The animals receiving CHB intravenously also exhibited elevated GSH levels in both pancreas and liver. This study shows that oral doses between 30 and 100 mg CHB/kg can be used to elevate GSH levels without any pancreatotoxicity. However, a single 50 mg CHB/kg dose given intravenously causes apoptosis, while 100 mg/kg causes severe pancreatotoxicity with necrosis.


A two-signal model for regulation of immunoglobulin isotype switching. A characteristic feature of the humoral immune response is a switch from IgM to other Ig isotypes that typically occurs subsequent to a first exposure to antigen. Ultimately, isotype switching involves a DNA rearrangement that recombines a variable region gene, initially juxtaposed to the mu constant region gene (C mu), with a constant region gene located downstream of C mu. Isotype switching is controlled by T lymphocyte-derived cytokines, such as interleukin-4 (IL-4), gamma-interferon (gamma-IFN), and TGF beta, which direct B lymphocytes to switch to specific Ig classes. For example, IL-4, directs murine B cells to produce IgG1 and IgE, and human B cells to produce IgE and IgG4. IL-4 appears to direct switching to IgE and IgG1 by inducing transcription of the epsilon and gamma 1 constant region genes before switch recombination. However, IL-4 is not a sufficient stimulus for isotype switching, and additional signals are required to complete this process. This second signal can be provided by physical contact with activated T cells, which may involve, at least in part, ligation of the CD40 molecule. For murine B cells the second signal may also be provided by IL-5. Isotype switching in B lymphocytes may provide a useful model for directed DNA recombination in higher eukaryotes.


Structural basis of antigenic specificity and design of new vaccines. This manuscript describes the design of new vaccines based on synthetic peptides. To this end, we first analyze the structural basis of antigenic reactivity and specificity and the various types of epitopes that form the mosaics of macromolecular antigens, as well as the regulatory mechanisms involved in immune recognition. A distinction is made between sequential or continuous epitopes, and discontinuous or conformational ones, which are the majority of epitopes in globular proteins. In this context it is of particular interest to identify epitopes reacting with B cells and T cells, respectively, or with cytotoxic T cells, in association with the major histocompatibility cell-surface antigens, and the role of these interactions in protective immunity. Identification of such epitopes in proteins of viral, bacterial, or parasitic organisms led to the synthesis of peptides, which when used in conjunction with appropriate carriers and/or adjuvants induced neutralizing antibodies. Particular examples are described, including: bacterial epitopes and mainly those of toxins of diphtheria, cholera, and shigella, leading not only to neutralizing antibodies but also to protective immunity against the deleterious effects of the respective toxins; parasite epitopes, such as those leading to anti-malaria vaccine, based on either the sporozoite or the merozoite stage antigens; viral epitopes leading to protective immunity, with special emphasis on influenza virus where induction of CTL is crucial; and finally, synthetic peptide vaccines against HIV, which should lead to broad specificity protective immunity while avoiding the risks of a vaccine based on the infectious agent. The rapid recent progress in this field, as described in this review, increases the prospect of constructing successful synthetic peptide vaccines in the not too distant future.


Receptor tyrosine kinase substrates: src homology domains and signal transduction. Among the intracellular milieu of proteins are molecules with defined biochemical functions that serve as substrates for ligand-activated tyrosine kinase receptors. It seems likely that some of these substrate molecules are elements of a critical signaling pathway used by growth factors to control cell proliferation and subverted by oncogenes to deregulate this process. Although the process of cell growth and division is relatively slow compared with other hormonally regulated responses, homeostasis in a human being requires approximately 20 x 10(6) cell divisions per second for the renewal of various cell populations. This review summarizes the present understanding of tyrosine kinase substrates that seem likely to have key roles in the signal transduction pathway that regulates cell proliferation. This includes structural features of these molecules, the influence of tyrosine phosphorylation on their functions, the biological roles of these proteins, and the capacity of these substrates to associate with activated receptor tyrosine kinases.


Mechanism of bradycardia-dependent appearance of manifest extrasystoles in concealed bigeminy. A theoretical model derived from the concepts of longitudinal dissociation and multilevel block in the reentrant pathway of extrasystoles. A case of bradycardia-dependent appearance of manifest extrasystoles in concealed bigeminy is presented. To explain the mechanism of such bradycardia-dependent appearance, a theoretical model is derived from the concepts of "longitudinal dissociation" and "multilevel block" in the reentrant pathway of extrasystoles. In the theoretical model, functional longitudinal dissociation divides the reentrant pathway into dual pathways F and S. When manifest extrasystoles are not found for a long time, alternate sinus impulses pass through both pathways F and S, but become concealed extrasystoles because of insufficient conduction delay in the pathways. The other alternate sinus impulses are blocked in the pathways; in pathway F, the impulses are blocked at the entrance, while in pathway S, the impulses are blocked at a more distal level. When sinus cycles gradually lengthen, one of such alternate sinus impulses passes through the entrance of pathway F and, traveling very slowly, is blocked at a more distal level. The next sinus impulse is blocked at the entrance of pathway F; namely, 3:2 Wenckebach block occurs at the entrance of pathway F. Thus this sinus impulse enters only pathway S and passes through pathway S with enough conduction delay to become a manifest reentrant extrasystole.


[Transfection of pancreatic acinar cells (AR4-2J) by bFGF modifies cell morphology and biosynthesis of pancreatic secretory enzymes]. Basic fibroblast growth factor (bFGF or FGF-2) is present in the basal membrane of pancreatic cells during the pancreatic embryonic development. The expression of bFGF receptors has been described in normal pancreatic cells. By contrast, pancreatic cancer cells express not only the bFGF receptors but also the bFGF itself. With the aim of understanding the effects induced by the production of bFGF by pancreatic cancer cells, the pancreatic acinar cell line (AR4-2J) was used. AR4-2J cells do not produce bFGF but express bFGF receptors. These cells were transfected with a vector containing the bFGF cDNA encoding the three different forms of bFGF characterized in tumor cells. Results showed that the bFGF expression induced important phenotypic and enzymatic modifications. The transfected cells lost some morphological features of the acinar cells and expressed amylase and lipase at low levels (a 90% decrease for amylase activity, whereas lipase activity was barely detectable). These results suggest that bFGF could be involved in maintaining pancreatic cells in a slightly differentiated state.


Effects of tetragastrin on mucus glycoprotein in rat gastric mucosal protection. The effects of tetragastrin on mucus glycoprotein (mucin) metabolism and mucosal protection in rat gastric mucosa were investigated. Rats were administered with various doses of tetragastrin (12, 120, or 400 micrograms/kg body weight; s.c.), followed by 50% ethanol-induced gastric injury. Tetragastrin caused a significant increase in mucin content in the corpus mucosa and prevented 50% ethanol-induced gastric mucosal damage in a dose-dependent manner. For assessment of the effects of tetragastrin on the metabolism of gastric mucin in detail, changes in mucin distribution in the three different layers of rat gastric mucosa were examined one hour after single administration of tetragastrin. A significant increase in the mucin content was noted in the mucus gel and surface mucosal layer. Mucin content in the deep mucosa corresponding mainly to the mucus neck cell mucin underwent virtually no change by this treatment. An increase in mucin in the mucus gel and surface mucosa would thus appear due to the administration of tetragastrin and may possibly be related to the protective action of the gastric mucosa against injury. The data demonstrate a possibility that gastrin may have potential for enhancing gastric mucosal protection associated with mucus secretion and/or mucus synthesis on the surface mucosa of rat gastric mucosa.


Acute reflux pancreatitis in rats: a comparison between two experimental models. This study was undertaken in order to compare the reliability of two acute reflux pancreatitis models in rats, one performed by positioning a silicon tube in the duodenum and the other by creating a gastro-jejunal anastomosis. In two groups (A = 10 and B = 10 rats) a silicon tube was positioned in the duodenum; in the remaining two groups (C = 12 and D = 6 rats) a latero-lateral antecolic anisoperistaltic gastro-jejunal anastomosis was performed 30 days before surgery. A closed duodenal loop was created for 12 hours in groups A and C but not in B and D. Rats in both groups A and C developed acute pancreatic inflammation of a mild degree. Sham operated rats with silicon tube placement had higher histological damage scores than those with gastro-jejunal anastomosis. The difference found between the two groups of rats which underwent gastro-jejunal anastomosis was more marked than that between the two groups which had silicon tube placement. It was concluded that the creation of a gastro-jejunal anastomosis is probably the safer procedure to allow gastro-intestinal flow in acute reflux pancreatitis in rats.


Hepatitis C virus detection is facilitated by the combined use of c100 protein and GOR epitope. Assay for the antibody to the c100 protein (anti-c100) lacks sensitivity in terms of detection of hepatitis C virus (HCV) in all samples. The author used anti-c100 and antibody to the GOR epitope (anti-GOR) by the enzyme-linked immunosorbent assay to examine 524 patients with chronic liver disease and 682 volunteer blood donors in Fukuoka, Japan. The prevalence of HCV infection, as revealed by the presence of anti-c100 and/or anti-GOR, was 3.9% in 540 volunteer blood donors, 12.7% in 142 volunteers with abnormal liver function, 7.4% in 135 patients with HBsAg-positive liver disease and 89.5% in 389 patients with non-A, non-B (NANB) liver disease. These results show a higher prevalence than demonstrated only by the anti-c100 in NANB liver disease patients (82.5%, P < 0.01). The concurrence of anti-c100 and anti-GOR in subjects with HCV infection was 23.8% in 21 volunteer blood donors, 44.4% in 18 volunteers with abnormal liver function and 61.2% in 348 NANB liver disease patients. The concurrence seems to increase with deterioration of liver function. We concluded that combination assay for anti-c100 and anti-GOR demonstrated a more accurate prevalence of HCV infection than single assay for anti-c100 among NANB liver disease patients, and that the presence of anti-GOR plays a role in liver disease in anti-HCV-positive subjects.


Alpha-fetoprotein producing carcinoma of the gallbladder associated with anomalous arrangement of the pancreaticobiliary ductal system--early detection through an attack of acute pancreatitis. A 56-year-old female was admitted to our hospital with a diagnosis of acute pancreatitis. Ultrasonography revealed a hypoechoic tumor in the gallbladder. The serum alpha-fetoprotein (AFP) level was 971 ng/ml. After healing of the acute pancreatitis, the anomalous arrangement of the pancreaticobiliary ductal system (APDS) was demonstrated by ERCP with mild dilatation of the common bile duct. Within one month after admininon, AFP level reached 4390 ng/ml. On operation, a pedunculated tumor, 6.5 x 3 cm in size, was found in the gallbladder. Histological examination revealed a moderately differentiated adenocarcinoma and positive immunohistochemical staining of cancer cells for AFP. After absolutely curative (stage II) resection, normal serum AFP levels were recognized. This is the first report of an AFP-producing cancer of the gallbladder associated with APDS and it was detected as a result of an attack of acute pancreatitis.


Thyroid hormone differentially regulates rat intestinal brush border enzyme gene expression. Thyroid hormone [triiodothyronine (T3)] has been shown to play a critical role in the growth and maturation of the mammalian small intestine, but its mechanism of action has not been well studied. In the current study, an animal model of hypothyroidism and hyperthyroidism was used to study the effects of T3 on the small intestine. Adult rats were treated with propylthiouracil for a 6-week period and then given injections of either saline (hypothyroid) or 30 micrograms/100 g body wt of T3 (hyperthyroid). Northern blot analyses showed marked differential regulation of brush border enzyme gene expression. Lactase messenger RNA (mRNA) levels decreased approximately 75% along the length of the small intestine, whereas sucrase levels were unchanged. The intestinal alkaline phosphatase mRNA species were upregulated by T3, especially the 3-kilobase band, which increased most dramatically in jejunum. Further experiments showed significant levels of both the alpha-1 and beta-1 T3 receptor mRNAs within the small intestinal mucosa. Histological examination showed that T3 treatment causes marked villus hyperplasia throughout the length of the small intestine. These results provide insight into the mechanism by which T3 exerts its influence on the growth and differentiation of the intestinal epithelium.


Effect of cholylsarcosine on hepatic cholesterol and bile acid synthesis and bile secretion in rats. The regulatory and secretory properties of cholylsarcosine (C-sar), a synthetic conjugated bile acid analogue that resists deconjugation and dehydroxylation, were compared with those of the natural conjugates of cholic acid. After continuous intraduodenal infusion of cholylsarcosine (C-sar), cholyltaurine (C-tau), or cholylglycine (C-gly) at 36 mumol/100 g.h, the infused bile acid in each case became the predominant biliary bile acid. After 48 hours, infusion of C-sar, C-tau, and C-gly suppressed the activity of cholesterol 7 alpha-hydroxylase (C7 alpha H; rate-limiting for bile acid synthesis) by 65%, 78%, and 92%, respectively, compared with biliary fistula controls. After C-sar infusion, levels of C7 alpha H protein, messenger RNA, and transcriptional activity were depressed to the same extent as specific activity, indicating that C-sar, like C-tau, down-regulates C7 alpha H principally at the level of gene transcription. All three bile acids also suppressed activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (rate-limiting for cholesterol synthesis). Both short- and long-term, the three cholyl conjugates caused similar increases in bile flow and in biliary secretion of cholesterol and phospholipid. It is concluded that in the rat, cholyl conjugates per se can suppress cholesterol and bile acid biosynthesis without prior conversion to deoxycholate. The effects of C-sar on hepatic cholesterol and bile acid synthesis as well as on induced bile flow and biliary lipid secretion are essentially identical to those of the naturally occurring cholyl conjugates.


Serum and liver hepatitis B virus DNA in chronic hepatitis B after sustained loss of surface antigen. Polymerase chain reaction (PCR) was used to detect hepatitis B virus DNA in the sera and livers of nine patients with chronic hepatitis B after treatment-induced or spontaneous loss of serum hepatitis B surface antigen. Patients were evaluated at intervals ranging from 3 to 67 months after disappearance of hepatitis B surface antigen. PCR was performed using primer pairs from the surface and core gene regions, and surface gene products were quantitated. Liver tissue was also evaluated by in situ hybridization to assess viral transcription. Five of the nine patients had viral DNA detectable in serum by PCR. Quantitation of polymerase chain reaction products in serum and liver showed that the DNA levels tended to decline progressively after antiviral therapy. Six of seven surface antigen-negative patients tested had detectable viral DNA in the liver, and four of the six DNA-positive patients were negative for DNA in serum by PCR. None had surface gene messenger RNA. Thus, it is concluded that hepatitis B virus DNA may be detectable by PCR in liver tissue years after the disappearance of hepatitis B surface antigen, even in the absence of detectable hepatitis B virus DNA in serum.


The origin of and subcellular mechanisms causing pancreatic bicarbonate secretion. In recent years, there has been a rapid growth in knowledge about the subcellular mechanisms involved in pancreatic ductal secretion of bicarbonate. The mechanisms governing anion transport across the luminal membrane of duct cells have been well characterized. Evidence suggests that the cystic fibrosis transmembrane conductance regulator is a cyclic adenosine monophosphate-regulated Cl- conductance in the luminal membrane that plays a pivotal role in ductal bicarbonate secretion by recirculating the Cl- imported into duct cells through Cl(-)-HCO3- exchange. The mechanisms governing ion transfer across the basolateral plasma membrane of duct cells are less well defined. There is some evidence suggesting that secretin may cause exocytotic insertion of proton pumps into the basolateral plasma membrane. Once inserted into the plasma membrane, proton pumps could engage in primary active electrogenic H+ ion transport to interstitial tissue while secondary active HCO3- secretion occurs over the luminal membrane through Cl(-)-HCO3- exchangers coupled in parallel with the cystic fibrosis transmembrane conductance regulator. These and other subcellular phenomena related to ductal secretory function are reviewed.


[Antigenic composition of serum proteins of the Inv system in normal conditions and in patients with hematologic diseases among the Armenian population]. Inv (1) antigen distribution was studied in 568 normal subjects and in 354 hematological patients in the Armenian population. Inv (1) antigen was detected in 16.7% of the normal Armenians studied. The incidence rate of Inv (1) factor does not depend on the distribution of phenotypes of ABO system, rhesus factor (D), and the sex of the subjects investigated. Inv (1) antigen incidence rate in patients with acute leukemia, chronic lymphocytic leukemia, iron deficiency anemia, lymphogranulomatosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia was similar to that in the control, and only patients with chronic myeloid leukemia had significantly decreased levels of Inv (1) antigen: 6.8% as compared to 16.7% in the population.


GM1-ganglioside regulation of EGF-induced gastric mucosal calcium channel activation. 1. Calcium channels, isolated from gastric epithelial cell membranes when reconstituted into phosphatidylcholine vesicles exhibited active 45Ca2+ uptake as evidenced by a dose dependent response to calcium channel activator, BAY K8644, and antagonist, PN200-110. 2. The channels on epidermal growth factor (EGF) binding in the presence of ATP showed an increase in tyrosine phosphorylation of 55 and 170 kDa calcium channel proteins. Such phosphorylated channels following reconstitution into the vesicles displayed a 48% greater 45Ca2+ uptake than that of the controls. 3. The binding of EGF to calcium channel protein was inhibited by GM1-ganglioside reaching maximum inhibition of 65% at 40 nM GM1. In contrast, calcium channel antagonist, PN200-110, had no effect on EGF binding. 4. The EGF-stimulated calcium channel protein phosphorylation was inhibited by GM1. This inhibitory effect was mainly reflected in the decrease of tyrosine phosphorylation of 55 and 170 kDa proteins. 5. The results suggest the participation of GM1-ganglioside in the regulation of EGF-stimulated gastric mucosal calcium channel activation.


Effects of calcium channel antagonists and Bay K 8644 on the analgesic response to pentazocine and U 50488H. 1. The effects of diltiazem, nifedipine and verapamil and the calcium channel agonist Bay K 8644 on the analgesic responses to the subcutaneous (s.c.) or intracerebroventricular (i.c.v.) administration of pentazocine and U 50488H were investigated in mice. 2. The three calcium channel antagonists and Bay K 8644 reduced the number of writhes induced by the intraperitoneal administration of acetic acid. 3. The analgesic responses to the low doses of pentazocine (s.c.) were additive with the effects of diltiazem, nifedipine or Bay K 8644; while, in contrast, the higher doses produced underadditive responses. Only verapamil increased the effects of the i.c.v. administration of the opioid. 4. The effects of U 50488H (s.c.) were additive with those of diltiazem and Bay K 8644; verapamil only increased the response to the lower dose of the opioid. Nifedipine plus pentazocine always induced underadditive responses. The i.c.v. effects of U 50488H were only increased by verapamil. 5. These findings are discussed in relation with a possible interaction of kappa agonists with calcium channels in the central nervous system.


Hormones regulating hepatic glycogenolysis in two chelonians use cyclic AMP, and not Ca2+, as intracellular messenger. In teleosts, lungfish, amphibians, and a reptile, Amphibolurus nuchalis, hormonal stimulation of hepatic glycogenolysis is mediated by a rise in intracellular cyclic AMP concentration. In mammals, by contrast, the inositol trisphosphate/Ca2+/diacylglycerol signal transduction pathways are also involved. The present study describes the hormonal regulation of hepatic glycogenolysis in adult long-necked turtles, Chelodina longicollis, and hatchlings of the loggerhead turtle, Caretta caretta. Adrenaline and glucagon, but not neurohypophysial peptides, stimulated glycogenolysis, glycogen phosphorylase activity, and accumulation of cAMP in cultured liver pieces from either C. longicollis or C. caretta. The actions of adrenaline were blocked by a beta-adrenergic antagonist, propranolol, but were unaffected by an alpha-adrenergic antagonist, phentolamine. The effects of adrenaline were maintained in Ca(2+)-free medium containing EGTA, and were not mimicked by the Ca2+ ionophore, A23187. The beta-adrenergic ligand, [125I]iodocyanopindolol (ICP), specifically bound to membranes prepared from C. longicollis liver, with a calculated KD of 59 pM and a Bmax of 171 fmol/mg protein. The adrenergic ligands, propranolol, isoprenaline, adrenaline, phenylephrine, phenoxybenzamine, noradrenaline, and phentolamine displaced ICP with KD's of 50 nM, 5 microM, 22 microM, 140 microM, 180 microM, 250 microM, and 1 mM, respectively. The alpha-adrenergic ligands, prazosin and yohimbine, did not bind specifically to the membranes, although prazosin did bind to membranes prepared similarly from rat liver. Thus the glycogenolytic actions of adrenaline are mediated via beta-adrenergic receptors in liver from C. longicollis and C. caretta and alpha-adrenergic receptors may play no role in the control of hepatic metabolism in these chelonians.


Effects of estradiol valerate on the uterus of the musk shrew (Suncus murinus L.). The effects of exogenous estradiol valerate on some uterine characteristics of the musk shrew were investigated. Treatment with the steroid for either 1 day or 7 days did not noticeably alter luminal epithelial cell height, endometrial gland epithelial cell height or diameter, or number of endometrial glands. The reciprocal values of cell density of circular muscle and deep endometrial layers and endometrium-to-myometrium ratio of the uterus increased significantly in response to 7 days of steroid administration. After 1 day of steroid treatment the numbers of mast cells in different layers of the uterus (i.e., meso-, myo-, and endometrium) were unchanged, but after 7 days there were significant increases in the number of mast cells in meso- and myometria. The number of eosinophils in all three layers of the uterus increased significantly in response to the treatment of estradiol for 1 day or for 7 days. The increase was greater in the 7-day group. Neither uterine DNA nor RNA contents changed following administration of the steroid for either group, although protein content was elevated significantly in the 7-day group. Estradiol administration thus evokes small but subtle changes in the uterus of the musk shrew.


Semi-automatic analysis of unit channel conductance from voltage ramp records. An IBM PC-compatible computer program, RAMP, for evaluation of single-channel recordings acquired using voltage ramp protocols is presented. The program uses semi-automatic procedures to make necessary corrections to a record (e.g. subtraction of baseline shift) and to measure all channel slope conductances as well as reversal potentials. The output is either a hardcopy of graphic display, which includes the calculated parameters, or data in ASCII format for further use (e.g. plots using various graphic software). Originally, the software was developed for the evaluation of voltage ramp records of single channel data from maxi chloride channels in myoblasts of a muscle cell line (Hurnák and Zachar 1992). Records from these membrane patches were also used in this work to demonstrate basic principles of the software and its practical use in evaluating single channel records obtained in response to the application of voltage ramps. The channel conductances calculated from ramp records were compared with those obtained by classical evaluation procedures from voltage step records.


RNA sequence analysis shows that the symbionts in the ciliate Metopus contortus are polymorphs of a single methanogen species. The polymerase chain reaction was used to amplify and partially sequence the 16S ribosomal RNA genes of symbiotic bacteria within the anaerobic ciliate Metopus contortus. In situ probing with fluorescent oligonucleotides showed that the amplified sequences originated from a single species of archaebacterium which is closely related to Methanocorpusculum parvum. The probed symbionts exhibited a variety of shapes and sizes. These data support the hypothesis, first proposed on the basis of electron microscopy, that the symbionts undergo a morphological transformation as part of the symbiotic process.


Phylogenetic and chemotaxonomic characterization of Acidaminococcus fermentans. The phylogenetic position of Acidaminococcus fermentans was determined by comparative sequence analysis of the 16S rRNA. This Gram-negative bacterium is a member of the Sporomusa cluster that is defined by other Gram-negative bacteria, i.e. Sporomusa, Megasphaera, Selenomonas, Butyrivibrio, Pectinatus, and Zymophilus. The branching point of this group within the radiation of Gram-positive bacteria of the Clostridium/Bacillus subphylum and adjacent to Peptococcus niger could be confirmed. Chemotaxonomic data were provided for a more detailed characterization of A. fermentans.


Cell wall-less, free-living spirochetes in Antarctica. The phylogeny of an Antarctic, cell wall-less, bacterial strain was determined by sequencing PCR amplified 16S rDNA, and comparison of the sequence with other bacterial 16S rRNA sequences available in databanks. Although the strain was phenotypically very similar to members of the genus Anaeroplasma, phylogenetic analyses showed it was a member of the order Spirochaetales. Until now, the order was one of the few bacterial orders in which phylogeny was reflected in a uniform morphology of its members. The viability of wall-less cells in cultures of spirochetes and spirochetal infective material warrants reinvestigation.


A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. We previously developed a cladistic approach to identify subsets of haplotypes defined by restriction endonuclease mapping or DNA sequencing that are associated with significant phenotypic deviations. Our approach was limited to segments of DNA in which little recombination occurs. In such cases, a cladogram can be constructed from the restriction site or sequence data that represents the evolutionary steps that interrelate the observed haplotypes. The cladogram is used to define a nested statistical design to identify mutational steps associated with significant phenotypic deviations. The central assumption behind this strategy is that any undetected mutation causing a phenotypic effect is embedded within the same evolutionary history that is represented by the cladogram. The power of this approach depends upon the confidence one has in the particular cladogram used to draw inferences. In this paper, we present a strategy for estimating the set of cladograms that are consistent with a particular sample of either restriction site or nucleotide sequence data and that includes the possibility of recombination. We first evaluate the limits of parsimony in constructing cladograms. Once these limits have been determined, we construct the set of parsimonious and nonparsimonious cladograms that is consistent with these limits. Our estimation procedure also identifies haplotypes that are candidates for being products of recombination. If recombination is extensive, our algorithm subdivides the DNA region into two or more subsections, each having little or no internal recombination. We apply this estimation procedure to three data sets to illustrate varying degrees of cladogram ambiguity and recombination.


Mutagenic analysis of the promoter of the Streptomyces fradiae beta-lactamase-encoding gene. The Streptomyces fradiae beta-lactamase promoter (PblaF) was sequenced and characterized by promoter probing, primer extension, and exonuclease III-mediated deletions. The transcription start point (tsp) was the same in both S. lividans and S. fradiae. Oligodeoxyribonucleotide-directed random mutations and site-specific mutations were introduced in the promoter region. The effects of these mutations on transcription were assayed by an RNA colony hybridization method. This analysis identified cis-acting sequence determinants located similarly to the -10 and -35 regions of a typical Escherichia coli promoter. Also, a change in the distance between these regions from 19 to 17 bp drastically reduced promoter activity. PblaF was shown not to be recognized by sigma-whiG or by sigma-hrdA, hrdC, or hrdD. Sequence alignment of PblaF to sigma factor-classified Streptomyces promoters revealed little homology. Thus, PblaF is probably recognized by an as yet unidentified sigma factor.


Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors. We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.


[Experimental research on the efficacy of terrilytin in silicosis]. Experimental silicosis was induced by quartz-containing dust administered intratracheally to Wistar male rats. Proteoclastic enzymes terrilytine was found to arrest pulmonary fibrosis, which was proved by inhibited development of silicotic granulomas and their lowered fibrosis. Terrilytine was most effective when inhaled in a dose of 0.08 PU per rat. Injected intraperitoneally, terrilytine in the dose elevated from 0.1-0.2 to 0.3 PU inhibited fibrosis developing in the presence of marked serous desquamative alveolitis. Incorporation of the enzyme in the cholesterol-lecithin liposomes prevents this side effect in the lungs. Liposomes injected intraperitoneally do not influence the development of pulmonary fibrosis in silicosis.


[The evaluation of alternative fuels for automobile transport and the resolution of hygienic and ecological tasks]. Automobile transport ranks first among the pollutants of big cities by nitrogen and carbogen oxides nowadays. Other kinds of fuel are to reduce the level of pollution. Evaluation of the liquid and gaseous fuel showed their positive hygienic and ecological prospects.


Role of platelet activating factor in pathogenesis of acute pancreatitis in rats. The importance of platelet activating factor in acute pancreatitis was examined by determining the tissue content of endogenous platelet activating factor and the protective effects of TCV-309, a highly selective platelet activating factor blocker, against caerulein induced pancreatitis in rats. Infusion of caerulein (10 micrograms/kg/h) for five hours resulted in about 70% increase in pancreatic weight, 22% rise in protein content, 50% reduction in tissue blood flow, nine fold increase in tissue level of platelet activating factor and 165% rise in plasma amylase as well as histological evidence of acute pancreatitis. Such infusion of caerulein in chronic pancreatic fistula rats caused a marked increase in protein output from basal secretion of 10 mg/30 minutes to 40 mg/30 minutes in the first hour of infusion followed by a decline in protein output to 15-20 mg/30 minutes in the following hours of the experiment. Exogenous platelet activating factor (50 micrograms/kg) injected ip produced similar alterations in weight, protein content, blood flow, and histology of the pancreas but the increment in serum amylase was significantly smaller and pancreatic secretion was reduced below the basal level. TCV-309 (50 micrograms/kg) given ip before caerulein or platelet activating factor administration significantly reduced the biochemical and morphological alterations caused by caerulein and abolished those induced by exogenous platelet activating factor. These results indicate that platelet activating factor plays an important role in the pathogenesis of acute pancreatitis probably by reducing the blood flow and increasing vascular permeability in the pancreas.


Acute pancreatitis complicating Crohn's disease: mere coincidence or causality? An example of acute pancreatitis developing five weeks after initial treatment with 5-aminosalicylic acid (5-ASA) and methylprednisolone for severe Crohn's disease is reported in a 37 year old female patient. She had undergone cholecystectomy for gall stones some years earlier. There was no evidence of acute or chronic pancreatitis. No morphological changes of the upper gastrointestinal tract were found except for some irregularity of the main pancreatic duct and the secondary ducts on endoscopic retrograde pancreatography. Rechallenge with 5-ASA did not induce recurrent pancreatitis or changes in pancreatic enzymes. This case report supports the concept of an association between acute pancreatitis and Crohn's disease.


The CD34 hemopoietic progenitor cell associated antigen: biology and clinical applications. CD34, which was first detected in hemopoietic and lymphopoietic progenitors, is a heavily glycosylated Type I transmembrane protein that does not share any significant similarity with other transmembrane proteins. Its functions are still unknown. Several monoclonal antibodies were raised against CD34, and at least 4 different epitopes could be recognized. CD34 expression is confined to a few cell lines, to 1-4% of adult bone marrow mononuclear cells (including marrow-repopulating cells, all multipotent and committed myeloid progenitors, B and T lymphoid precursors, osteoclast precursors, and most likely the precursors for stromal cells), and to less than 1% of peripheral blood mononuclear cells. In non-lymphohemopoietic tissues its expression is confined to endothelial cells and to some cells of the skin. In malignancies, CD34 expression is not fully elucidated. Immature hemolymphopoietic malignancies (namely acute leukemias) and the blast cells of chronic myeloid leukemia are frequently positive. Chronic lymphoproliferative disorders and lymphomas are negative. Among other tumors, only vascular derived tumors are positive. Clinical applications of CD34+ cells include autologous transplantation of putative CD34+ stem cells isolated by positive selection from the bone marrow, and transplantation of autologous peripheral blood stem cells, using the proportion and number of CD34+ cells as a guideline for the harvesting procedure.


[Experimental study using porous, vascularizable polytetrafluoroethylene in preformed free flaps]. Following radical tumor resections or severe trauma, particularly in the head and neck region, cartilage or bone grafts serve as frame work in defect reconstruction. As an alternative, porous plastic material was used for preformed free flaps in an experimental study with 28 New Zealand rabbits. Due to ingrowth of fibrous tissue and capillaries, it was possible to obtain prefabricated flaps consisting of vascularized porous plastic, a thin layer of fatty gliding tissue and full thickness skin graft. The blood supply of all three layers of the transplant was confirmed by microangiography, Tc 99 m labeled red blood cells and histological examination.


[Effect of Z-103 on wound healing by dermal incision in guinea pigs]. We investigated the effects of Z-103, ZnSO4, L-carnosine and solcoseryl on wound healing by dermal incision in guinea pigs. The tensile strength, hydroxyproline contents and the value of angiogenesis (carmine contents) at the wounded site of dorsal skin were used as indices of wound healing. Z-103, given daily s.c., increased the tensile strength and hydroxyproline contents on day 4 after operation in a dose-dependent manner; in particular, the effect of 10 mg/kg of Z-103 was nearly equal to that of solcoseryl at 0.5 ml/animal. Moreover, Z-103 10 mg/kg increased the value of angiogenesis on day 3 after the operation. On the other hand, ZnSO4 and L-carnosine, components of Z-103, also similarly increased the tensile strength and hydroxyproline contents. These results suggest that Z-103 possessed an accelerative action on wound healing, and these effects may be due to the activity of its components, ZnSO4 and L-carnosine.


CD34-positive cell selection by immunomagnetic beads and chymopapain. Pluripotent hemopoietic stem cells, progenitors of all hemolymphopoietic lineages, and clonogenic cells from many patients with acute nonlymphocytic leukemia (ANLL) and chronic myeloid leukemia (CML) express the CD34 antigen on their surface. Isolation of these cell populations is of primary experimental and clinical importance. Six bone marrow (BM) and 10 peripheral blood (PB) samples were obtained from 2 normal individuals, 3 patients with CML and 9 with ANLL. The CD34+ cell fraction was isolated using MY10 antibody, sheep anti-mouse immunomagnetic beads and the enzyme chymopapain. Indirect immunofluorescence and semisolid culture were employed to evaluate the percentage of CD34+ cells and that of clonogenic cells in each cell fraction. The frequency of CD34+ cells in the original unseparated populations was (mean +/- SE) 24.3 +/- 7.3%, and reached 85.0 +/- 2.7% in the isolated CD34-positive fractions; in the negative fractions it was only 2.7 +/- 1.7%. According to these results, the great majority of clonogenic cells was separated in the CD34-positive fractions and depleted in those CD34-negative. Moreover, chymopapain was shown to be non-toxic to the clonogenic cells. Positive immunoselection using My10 Ab, immunomagnetic beads and chymopapain is a method for isolating almost pure progenitors from the BM and PB of normal individuals and patients with myeloid leukemias.


Effect of pesticides on oestradiol-receptor complex formation in rat uterus cytosol. The effect of gamma-1,2,3,4,5,6-hexachlorocyclohexane (lindane), 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) and 2-methylthio-4,6-bis-isopropylamino-s-triazine (prometryne) on the formation of a specific oestradiol-receptor complex in the rat uterus cytosol has been examined in vitro and in vivo. Both in vitro and in vivo, the pesticides significantly (P < 0.001) inhibited the formation of the complex in the rat uterus cytosol. The decrease in the number of free specific binding sites on the receptors was determined. The affinity of binding was not modified under the influence of pesticides, and the Kd value was of the same order of magnitude (10(-9) M). The inhibition was found to be fully non-competitive.


Genotoxicity studies in vitro and in vivo on carminic acid (natural red 4). The potential genotoxic activity of carminic acid (CAS no. 1260-17-9; EINECS no. 215-023-3; C.I. no. 75410), a component of natural red colouring products (cochineal: CAS no. 1343-78-8; EINECS no. 215-680-6; C.I. no. 75470), used in food, cosmetics and drugs, has been evaluated by means of a series of short-term tests in vitro and in vivo, namely Salmonella reverse mutation, chromosome aberrations and sister chromatid exchanges in vitro on Chinese hamster ovary cells, and the mouse micronucleus test. All studies have produced negative results. The data obtained strongly support the non-mutagenic/non-carcinogenic activity of this compound. Genotoxicity data previously obtained for carminic acid, concerning the induction of a series of other genetic endpoints in different test systems, have also been considered, as have recent findings that indicate lack of carcinogenic activity in the cochineal preparation containing 29.8% carminic acid.


CEOP/PEB alternating chemotherapy in advanced intermediate and high-grade non-Hodgkin's lymphomas. From February, 1987 to July, 1990, 28 patients (M/F = 16/12; median age = 60.5 yrs) affected by intermediate (22) or high-grade (6) advanced stage (III = 8; IV = 20) NHL were given a median (range 4-10) of 8 cycles of CEOP (Cyclophosphamide, Epirubicin, Vincristine and Prednisone), alternated every 21 days with PEB (Cisplatin, Etoposide, Bleomycin). Nineteen (68%) pts. achieved a CR, 5 (18%) a PR, and 4 (14%) experienced progressive disease (PD); 11/19 CRs subsequently relapsed within a median (range 3-15) time of 8 mos.. After a follow-up ranging from 6 to 42+ (median 18) mos., the 3-year actuarial overall survival (OS) was 51% and shifted to 18% at 42 mos. After 7-42+ (median 19) mos., 14/19 (73%) Crs were still alive with 63% of them predicted to survive at 3 years. The projected 3-year disease-free survival (DFS) for these pts. after 1-36+ (median 8) mos. was 28%. CEOP/PEB alternating chemotherapy failed to improve the therapeutic results we obtained in a previous study with CEOP alone. Toxicity was moderate, but higher than expected.


A case of spinal cord compression by extramedullary haemopoiesis in a thalassaemic patient: a putative role for hydroxyurea? We report a case of homozygous beta thalassaemia who developed chronic paraparesis due to spinal cord compression by paravertebral extramedullary masses. Our patient was successfully treated with hypertransfusion and hydroxyurea. This drug in addition to its well-known cytostatic effects, may be a good alternative in conditions analogous to our case. This action of hydroxyurea can also be attributed to its favourable effect on foetal haemoglobin production.


Effects of riluzole (2-amino-6-trifluoromethoxy benzothiazole) on striatal neurochemical markers in the rat, with special reference to the dopamine, choline, GABA and glutamate synaptosomal high affinity uptake systems. Riluzole, a new compound with anticonvulsant properties, was found to induce a dose-dependent decrease in the uptake of 3H-dopamine, 3H-GABA and 3H-glutamate into striatal synaptosomes when added to the incubation medium or after in vivo administration, whereas an inhibition of 3H-choline uptake was detected only in the in vitro experiments. Interestingly, riluzole affected 3H-dopamine and 3H-glutamate uptake differentially since 3H-dopamine uptake was found to be more sensitive to the compound. Moreover, riluzole inhibited 3H-dopamine uptake competitively and 3H-glutamate uptake non-competitively, which further suggests that the action of the compound is selective. After in vivo injection, riluzole did not affect the striatal dopamine, DOPAC, serotonin, 5HIAA, glutamate, aspartate or GABA contents. Since this compound was previously reported to induce a decrease in the spontaneous release of glutamate, serotonin, dopamine and possibly acetylcholine, the hypothesis is put forward that riluzole may, at least at high concentrations, have general effects on the striatal nerve terminals affecting both the uptake and release processes. This action may be correlated with the recently identified blocking properties of the compound on the sodium channels, as previously shown for local anaesthetics.


Prevalence of antibodies to hepatitis C virus among Saudi patients with chronic liver diseases. The prevalence of antibodies against hepatitis C virus (anti-HCV) was determined in 55 patients with chronic liver diseases including liver cirrhosis (42 patients), liver cirrhosis and hepatocellular carcinoma (8 patients), and chronic active hepatitis (4 patients). A total of 63.6% of these patients were positive for anti-HCV, a significantly higher prevalence than the rate of 3.9% observed in 488 asymptomatic volunteers. Of the 42 patients with liver cirrhosis 16 (38.1%) had positive anti-HCV without any markers of hepatitis B virus (HBV), while 12 (28.6%) had markers of neither HCV nor HBV infection. Our findings suggest that HCV infection may play a significant role in the pathogenesis of chronic liver disease in Saudi Arabia, which is an area of endemic HBV infection. Screening for anti HCV should be considered mandatory in patients with chronic liver disease (CLD) especially where the etiology appears obscure.


Intrahepatic lymphocyte subpopulations and HLA class I antigen expression by hepatocytes in chronic hepatitis C. The lymphocyte subpopulations in the peripheral blood and liver were studied in 17 patients with chronic active hepatitis type C (CAH C) by immunoenzymatic and immunofluorescence techniques, using mono-specific T cell antibodies and other reagents. The peripheral blood subsets showed no significant differences between the patients with CAH C and normal controls. In the liver, CD8-positive cells were predominant over CD4- and Leu 7-positive cells. Leu 7-, CD4-, and CD11-positive lymphocytes were all few in number. HLA class I antigen was expressed diffusely on the cell surfaces of the hepatocytes. Serum levels of soluble interleukin 2 receptor (sIL2R) in the CAH C patients were significantly higher than in normal controls (p less than 0.01). In CAH C, sIL2R levels were higher during exacerbations than during remissions (p less than 0.01). These results suggest that cytotoxic T cells (CD8- positive, CD4-negative, and CD11-negative) may play an important role in the pathogenesis of hepatocyte injury in patients with CAH C.


[Experimental allergic encephalomyelitis: immunopathological analysis of antigenic reactivity and loss of encephalitogenicity]. Experimental allergic encephalomyelitis (EAE) is an autoimmune demyelinating disease of the central nervous system (CNS). EAE can be induced by immunization with myelin basic protein (MBP) or passive transfer of MBP-reactive T cell lines and clones. We established several T-cell clones from SJL/J mice by immunization with whole rat MBP or a synthetic peptide encompassing guinea pig MBP 89-101 which contains the encephalitogenic determinant for SJL/J mice. One clone was found to have lost its encephalitogenicity during long-term passages in vitro, although this clone maintains its specific reactivity to the encephalitogenic determinant. To clarify the difference between the encephalitogenic T cell clone (4b. 14a) and the non-encephalitogenic T-cell clone (4b. 14a/n), we examined the suppressive activity of 4b. 14a/n on the reactivity to antigen of 4b. 14a, various lymphokine production and adhesion molecules expression of 4b. 14a and 4b. 14a/n. The culture fluid of the both 4b. 14a/n and 4b. 14a revealed a suppressive effect on the proliferation of 4b. 14a stimulated by MBP 89-101, and the effect was not different between these clones. In lymphokine production, the activities of lymphotoxin, interferon or interleukin-2 were not different between encephalitogenic clones (4b.14a and TNT-1) and 4b. 14a/n, whereas the activity of tumor necrosis factor-alpha, passively secreted by antigen presenting cell, was higher in culture media of 4b. 14a/n. Examination of adhesion molecule expression of 4b.14a/n failed to show any differences in expression of lymphocyte function-associated antigen-1 (LFA-1) alpha and CD2 in the comparison with 4b. 14a. However, LFA-1 beta expression of 4b. 14a/n was always less than of 4b. 14a. The present studies indicated that the lack of encephalitogenicity of T-cell clones which were responsive to an encephalitogenic determinant depends not on the difference in major lymphokines production but partially on adhesion molecules expression which was decreased in non-encephalitogenic T-cell clone.


Synergistic effects of murine stem cell factor in combination with a variety of cytokines on the expansion of murine hematopoietic progenitor cells in short-term suspension cultures. In the present study, it is represented that the ability of murine stem cell factor (SCF) to expand hematopoietic progenitor cells in short-term suspension culture when used alone or with IL-1 beta, IL-3, IL-6, M-CSF and IL1 beta Plus IL-3. SCF alone had a limited effect on the expansion of early primitive hematopoietic progenitor cells (CFU-HPP: high proliferative potential colony forming unit, and CFU-S: colony forming unit in spleen) even at a high concentration, but expanded mature hematopoietic progenitor cells (CFU-GM: colony forming unit-granulocyte/macrophage, and BFU-E: burst forming unit-erythroid) markedly at low concentrations. When SCF was used in combination with other cytokines, the expansion of primitive hematopoietic progenitor cells was significantly increased; namely, CFU-HPP were expanded approximately 2 to 5-fold compared with SCF alone. A marked expansion of hematopoietic progenitor cells was observed in a combination of SCF plus IL-1 beta plus IL-3. In this setting, CFU-S was increased 2.2-fold compared with the number of CFU-S in fresh bone marrow and CFU-HPP were increased 8.5-fold compared with the number of primary CFU-HPP. These results suggest that these factors may be utilized in experiments of murine bone marrow transplantation (BMT) and also in human BMT. Namely, the adequate number of hematopoiesic progenitor cells and stem cells required for the successful engraftment can be obtained from small volume of peripheral or bone marrow blood by this procedure, thus obtiating the donor's burden.


Aflatoxin B1 DNA adducts in smeared tumor tissue from patients with hepatocellular carcinoma. Aflatoxins are well-known animal hepatocarcinogens, but the association between aflatoxins and human hepatocellular carcinoma remains to be elucidated. A study method consisting of indirect immunofluorescence assay combined with densitometry was developed to quantitate aflatoxin B1 DNA adducts in smeared liver tissue obtained at the time of biopsy for diagnosis in 50 hepatocellular carcinoma patients in Taiwan. Monoclonal antibody 6A10, generated against the persistent form of the major N7 guanine adduct of aflatoxin B1, was used for detection of adduct. Thirty-five (70%) of the hepatocellular carcinoma samples had detectable levels of aflatoxin B1 DNA adducts (> or = 1/10(6) nucleotides). The detection rate was slightly lower in men (69%) than in women (75%), and younger patients had a significantly higher rate of adducts (83%) than did older ones (58%). Carriers of both HBsAg and HBeAg, carriers of HBsAg only and noncarriers had different rates of detection: 29%, 74% and 82%, respectively. Patients with family histories of hepatocellular carcinoma had a higher detection rate (100%) than did those patients without such histories (67%). No association was found between aflatoxin B1 DNA adducts in liver tissue and Child's score for severity of liver disease. The results suggest that aflatoxin B1 may be involved in the pathogenesis of hepatocellular carcinoma in Taiwan. Our immunohistochemical method for analysis of adducts in small numbers of cells from the target organ should improve results of monitoring for the biologically effective dose of aflatoxin.


Appearance of hepatocytelike cells in the interlobular bile ducts of human liver in various liver disease states. Among 1,098 liver biopsy specimens obtained from patients with various liver diseases characterized by liver injury, 58 epithelial cells whose cytoplasms stained positively by the periodic acid-Schiff stain (digested with diastase) were recognized in the interlobular bile ducts of 37 specimens from 36 patients. Light microscopic study revealed that the cytoplasms of these cells were clear or stained weakly eosinophilic on hematoxylin and eosin staining and that the cell limits were distinct. From their reaction with periodic acid-Schiff stain and from electron microscopic observation it was clear that these cells contained an abundance of glycogen and were located among the normal bile duct cells surrounded by basement membrane. On electron microscopy, these cells had microvilli of equal sizes on their luminal surfaces and many irregularly sized microvilluslike cell membrane projections on their basal surfaces. They rested on basement membrane with basal spaces. These cells varied in size from 25.0 to 452.2 microns 2 (mean = 212.2 microns 2). In contrast, the sizes of normal bile duct cells and hepatocytes ranged from 20.0 to 69.3 microns 2 (mean = 34.2 microns 2) and from 113.0 to 860.3 microns 2 (mean = 447.0 microns 2), respectively. Immunohistochemical study with antiserum to cytokeratin 19, albumin and alpha 1-antitrypsin on serially cut frozen sections showed that some of these cells expressed markers of bile duct cells and hepatocytes. Some cells expressed only the markers of hepatocytes. Computer graphic three-dimensional reconstruction clearly demonstrated that these cells were located sparsely (but sometimes in groups) among normal interlobular bile duct cells, without any connection to the surrounding parenchymal hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)


Acyclovir vs isoprinosine (immunovir) for suppression of recurrent genital herpes simplex infection. To compare the efficacy and safety of oral acyclovir (400 mg twice daily) with oral isoprinosine (500 mg twice daily) in the suppression of recurrent genital herpes. Double-blind, double-dummy, randomised, controlled, parallel group trial. 13 centres in UK, Belgium and Germany. 127 immunocompetent patients with frequently recurring genital herpes. Proportions of patients reporting recurrences, recurrence frequency, and mean duration of lesions during breakthrough recurrences in each treatment group during a 6 month treatment period; time to first recurrence during treatment and follow-up after treatment cessation. During treatment, acyclovir recipients showed significant differences (p < 0.05) when compared with isoprinosine recipients in terms of a lower proportion reporting recurrences (31% vs 96%), a reduced mean number of reported recurrences per patient (0.6 vs 3.6), a shorter mean duration of breakthrough lesions (6.4 days vs 8.2 days), and a longer mean time (standard error) to first recurrence (143.7 (9.1) days vs 40.5 (5.4) days. The mean time to first recurrence after treatment cessation did not differ between the two groups. As compared with placebo recipients, isoprinosine treated patients had an increased recurrence frequency (3.6 vs 2.5) during treatment, and a shorter time to first recurrence after treatment cessation. All treatments were well tolerated without serious adverse events or toxicity. Acyclovir is very effective in suppressing recurrent genital herpes and is clearly superior to isoprinosine which is not clinically useful in the dosage studied.


Genetic and physical map of the interferon region on chromosome 9p. A region of chromosome 9, surrounding the interferon-beta (IFNB1) locus and the interferon-alpha (IFNA) gene cluster on 9p13-p22, has been shown to be frequently deleted or rearranged in a number of human cancers, including leukemia, glioma, non-small-cell lung carcinoma, and melanoma. To assist in better defining the precise region(s) of 9p implicated in each of these malignancies, a combined genetic and physical map of this region was generated using the available 9p markers IFNB1, IFNA, D9S3, and D9S19, along with a newly described locus, D9S126. The relative order and distances between these loci were determined by multipoint linkage analysis of CEPH (Centre d'Etude du Polymorphisme Humain) pedigree DNAs, pulsed-field gel electrophoresis, and fluorescence in situ hybridization. All three mapping approaches gave concordant results and, in the case of multipoint linkage analysis, the following gene order was supported for these and other closely linked chromosome 9 markers present in the CEPH database: pter-D9S33-IFNB1/IFNA-D9S126-D9S3-D9S19 -D9S9/D9S15-ASSP3-qter. This map serves to extend preexisting chromosome 9 maps (which focus primarily on 9q) and also reassigns D9S3 and D9S19 to more proximal locations on 9p.


Synteny mapping in the bovine: genes from human chromosome 5. In an effort to generate a more complete bovine syntenic map of Type I comparative anchor loci, seven homologs to genes found on HSA5 were mapped using a panel of bovine x rodent hybrid somatic cells. Five HSA5 genes, CSF2, RPS14, PDGFRB, FGFA, and CSF1R, were assigned to bovine syntenic group U22 (chromosome 7), while two others, C9 and HGMCR, mapped to U10 and U5, respectively. Previous studies had assigned the HSA5 marker SPARC to bovine syntenic group U22. The mapping of genes spanning the length of HSA5 in cattle and also in mouse permits syntenic comparisons between prototypic genomes of three mammalian orders, providing insight into the evolutionary history of this region of the ancestral mammalian genome.


The human APO-1 (APT) antigen maps to 10q23, a region that is syntenic with mouse chromosome 19. The APO-1 (APT) antigen is a cell surface antigen expressed on a variety of normal and malignant cells. Binding of anti-APO-1 antibody to the APO-1 antigen induces programmed cell death (apoptosis). The APO-1 antigen shows homology to the members of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. Using cosmid DNA containing the APO-1 gene as a probe for fluorescence in situ hybridization, we have mapped the gene to a subregion of chromosomal band 10q23. The human APO-1 locus lies within a conserved synteny segment present on mouse chromosome 19 consistent with the previous chromosomal assignment of the corresponding mouse antigen.


Chromosome aberrations in 450 sperm complements from eight controls and lack of increase after chemotherapy in two patients. Four hundred fifty sperm complements from eight controls were analyzed. A conservative estimate of aneuploidy was 1.8% with a hyperhaploid rate of 0.9% (4/450). The overall frequency of structural aberrations was 8.9% (40/450). The proportion of X-bearing (47.5%) and Y-bearing (52.5%) sperm did not differ significantly. Sperm complements were analyzed from a cancer patient 9 months after polychemotherapy (n = 63) and from a patient being treated with Imurek (azathioprine) (n = 30). There was no significant increase in the incidence of numerical and structural chromosome aberrations in the sperm of either patient. The percentages of X-bearing and Y-bearing sperm were not significantly different from the expected 50%.


Linkage relations between A2M, HOX3, INT1, KRAS2, and PAH on bovine chromosome 5. There is a high level of conservation between human chromosomes and bovine syntenic groups. One such comparison is between human chromosome 12 and bovine chromosome 5, where at least 16 loci have been shown to be conserved in an homologous segment. However, the degree of conservation of order of the loci on bovine chromosome 5 is unknown, and in general the conservation of order in comparisons between humans and cattle can only be speculated. We have estimated the recombination fractions between five of the loci that were previously published as mapping to bovine chromosome 5 by a combination of in situ hybridization and analysis of bovine-rodent somatic cell hybrid lines to determine whether order has been conserved in the homologous segment of bovine chromosome 5 and human chromosome 12. Recombination fractions were estimated in reference pedigrees of cattle. The loci were A2M, GSNL, HOX3, INT1, KRAS2, and PAH. Restriction fragment length polymorphisms for all loci were defined by screening a panel of eight restriction endonucleases. The linkage between loci was estimated using the lod score method, and all possible pairwise comparisons were made. A preliminary map was created by joining together loci that showed the smallest recombination fractions and the largest lod scores. A multipoint analysis was performed to estimate support for the most likely order. This order shows the relative inversion of some of the loci. Moreover, the distance spanned in cattle is less than a quarter the distance spanned in humans. Together, these data indicate that several chromosomal evolutionary events have occurred in the homologous segment shared by humans and cattle.


Characterization of the human kallikrein locus. The human kallikrein gene family is composed of three members: tissue kallikrein (KLK1), prostate-specific antigen (PA or APS), and human glandular kallikrein-1 (hGK-1 or KLK2). The three genes have previously been isolated and mapped to chromosome 19q13.2-q13.4. Further analysis of an area of 110 kb surrounding the kallikrein genes by CHEF electrophoresis and chromosome walking showed clustering of the three genes. The KLK1 gene is positioned in the opposite orientation of the APS and KLK2 genes in the order KLK1-APS-KLK2. The APS and KLK2 gene are separated by 12 kb; the distance between KLK1 and APS is 31 kb. A CpG island was detected in the region between KLK1 and APS. Preliminary data indicate that this CpG island is located directly adjacent to a gene that is unrelated to the kallikreins and seems to be ubiquitously expressed.


Mapping of a gene for epidermolytic palmoplantar keratoderma to the region of the acidic keratin gene cluster at 17q12-q21. Epidermolytic palmoplantar keratoderma (EPPK) (Vörner-Unna-Thost) is an autosomal dominantly inherited skin disease of unknown etiology characterized by diffuse severe hyperkeratosis of the palms and soles and, histologically, by cellular degeneration. We have mapped a gene for EPPK to chromosome 17q11-q23, with linkage analysis using microsatellite DNA-polymorphisms, in a single large family of 7 generations. A maximum lod score of z = 6.66 was obtained with the probe D17S579 at a recombination fraction of theta = 0.00. This locus maps to the same region as the type I (acidic) keratin gene cluster. Keratins, members of the intermediate filament family, the major proteins of the cytoskeleton in epidermis, are differentially expressed in a tissue-specific manner. One acidic keratin, keratin 9 (KRT9), is expressed only in the terminally differentiated epidermis of palms and soles. The KRT9 gene has not yet been cloned; however, since the genes for most acidic keratins are clustered, it is highly probable that it too will map to this region. We therefore propose KRT9 as the candidate gene for EPPK.


Homologous chromosomal locations of the four genes for inter-alpha-inhibitor and pre-alpha-inhibitor family in human and mouse: assignment of the ancestral gene for the lipocalin superfamily. The inter-alpha-inhibitor (I alpha I) and pre-alpha-inhibitor (P alpha I) family is composed of three plasma protease inhibitors, I alpha I, P alpha I, and bikunin, whose chains are encoded by a set of three evolutionarily related heavy (H) chain genes designated H1, H2, and H3 and a fourth gene, the so-called alpha 1-microglobulin/bikunin precursor (AMBP) gene. The latter codes for a precursor that splits into: (i) alpha 1-microglobulin, which belongs to the lipocalin superfamily; and (ii) bikunin, which is made up of two tandemly arranged protease inhibitor domains and belongs to the superfamily of Kunitz-type protease inhibitors. The bikunin chain is found in I alpha I and P alpha I molecules and it is also present as a free molecule in plasma. In human, the AMBP and H2 genes have been mapped to 9q32-q34 and 10p14-p15, respectively, while the H1 and H3 genes are tandemly located at 3p21.1-p21.2. In situ hybridization mappings indicate that the mouse AMBP gene (Intin-4) is located at 4C1----C4, and the H1 (Intin-1) and H3 (Intin-3) genes are colocated at 14A2----C1. In interspecific backcrosses (C57BL/6Pas x Mus spretus) a TaqI restriction variant in (and/or near) the H2 (Intin-2) gene identified a linkage of this gene with other polymorphic loci, which assigns Intin-2 to the centromeric area of chromosome 2. All such assignments are in conserved chromosomal regions between human and mouse. Therefore the genetic events that gave rise to the four I alpha I family genes took place prior to the divergence between human and mouse.(ABSTRACT TRUNCATED AT 250 WORDS)


Significantly higher frequency of the MspI 2.2 kb allele of the Duchenne muscular dystrophy intragenic probe P-20 in the Chinese population. The P-20 intragenic marker was used to test for restriction fragment length polymorphisms in unrelated Chinese patients with Duchenne or Becker muscular dystrophy or X-linked mental retardation. In addition to polymorphism at the 6.0/3.5 kb MspI allelic site, we found an independent and high frequency of polymorphism at the 2.2/1.8 kb site. This differs from results found with other populations.


The gene for human transition protein 2: nucleotide sequence, assignment to the protamine gene cluster, and evidence for its low expression. We have isolated the gene for transition protein 2 (TNP2) from a human cosmid clone that contains the genes for protamines 1 and 2. A nucleotide sequence of 1776 bp that comprises 268 bp of the 5'-noncoding region, 400 bp of exon 1, 849 bp of an intron, 17 bp of exon 2, and 242 bp of the 3'-noncoding region was determined. A modified CAT box, a TATAA box, and two possible polyadenylation sites were identified. Transcripts in testicular RNA could be detected only by RT-PCR and RNase protection assays. By direct sequencing of the PCR products, a cDNA sequence was established. It can be deduced from these results that, in contrast to other mammalian genes, the human TNP2 gene is expressed at a very low level. The human gene differs from that of other mammalian species by the absence of a conserved GCCATCAC nucleotide sequence in the 3'-untranslated region. Since both protamine genes are known to be localized on chromosome 16p13.3, this chromosomal localization holds true for the human TNP2 gene as well. The genes for both protamines and TNP2 are arranged in a DNA stretch of 13 kb.


Analysis of the human cysteine-rich protein gene (CSRP), assignment to chromosome 1q24-1q32, and identification of an associated MspI polymorphism. The human cysteine-rich protein (hCRP) is encoded by a highly conserved and widely expressed serum-inducible immediate early response gene. hCRP contains two copies of the "LIM/double zinc-finger" motif. Using a characterized hCRP cDNA probe, we demonstrate that the human CRP gene (CSRP) is present in a single copy and that both mouse and human genomes contain one or more CRP-related genes detected by hybridization at low stringency. Using a panel of human x rodent somatic cell hybrids, the hCRP locus is assigned to chromosome 1. In situ hybridization of 3H-labeled CRP cDNA to human metaphase chromosomes confirms this assignment and permits regional localization to bands 1q24-1q32. A common MspI polymorphism is identified and mapped to intron 4 of the hCRP gene. The chromosomal localization and restriction site polymorphism should prove useful in future studies of the function of this gene.


Mapping of the shortest region of overlap of deletions of the short arm of chromosome 9 associated with human neoplasia. Deletions of the short arm of chromosome 9 with a minimum region of overlap at band 9p22 are frequently observed in acute lymphoblastic leukemia and in gliomas. They also occur at a lower frequency in lymphomas, melanomas, lung cancers, and other solid tumors. These deletions often include the entire interferon (IFN) gene cluster, which comprises about 26 interferon-alpha (IFNA), -omega (IFNW), and-beta-1 (IFNB1) interferon genes, as well as the gene for the enzyme methylthioadenosine phosphorylase (MTAP). By comparing microscopic deletions with the genes lost at the molecular level, we have determined the order of these genes on 9p to be telomere-IFNB1-IFNA/IFNW cluster-MTAP-centromere. In a few cell lines and in primary leukemia cells, we have observed deletions that have breakpoints within the IFN gene cluster and result in partial loss of the IFN genes. These partial deletions allowed us to determine the order of some genes or groups of genes within the IFNA/IFNW gene cluster. Our current results map the shortest region of overlap of these deletions in the various tumors to the region between the centromeric end of the IFNA/IFNW gene cluster and the MTAP gene locus.


Chromosomal mapping of human cytokeratin 13 gene (KRT13). In the present study the human cytokeratin 13 gene (KRT13), encoding a polypeptide characteristic of internal stratified epithelia, has been mapped with the help of the polymerase chain reaction and somatic cell hybrids to chromosome 17. In situ hybridization of a KRT13 cDNA probe to metaphase chromosomes allowed the assignment of the KRT13 gene within the q12-q21.2 region of chromosome 17.


The hypervariable DXS255 locus contains a LINE-1 repetitive element with a CpG island that is extensively methylated only on the active X chromosome. The DXS255 locus at Xp11.22 is highly polymorphic due to a 26-bp variable number of tandem repeats (VNTR) motif. In previous studies, one of the MspI sites flanking the VNTR manifested a correlation between methylation and X chromosome inactivation. Here we show, by DNA sequence analysis, that this MspI site is located within the CpG island at the 5' end of a LINE-1 element, which is 2.5 kb from the VNTR. The methylation status of the CpG island was assessed in Southern blotting experiments using the methylation-sensitive enzymes HpaII, HhaI, and BssHII. All these sites were completely methylated on active X chromosomes, consistent with previously reported findings of full methylation of LINE-1 elements throughout the genome. However, on inactive X chromosomes these sites were predominantly unmethylated, although patterns were found to be heterogeneous. The results suggest that LINE-1 elements on the inactive X chromosome are not suppressed by full methylation of their CpG islands. The differential methylation of the DXS255 CpG island provides the basis for a highly informative X inactivation analysis system.


Localization of brain nitric oxide synthase (NOS) to human chromosome 12. Recent research has shown that nitric oxide is a novel neuronal second messenger and transmitter that may be involved in neuronal cell death and damage in neurological illness. To map the chromosomal localization of this important brain enzyme, a rat cDNA probe was prepared by RNA PCR from rat cerebellum RNA. This rat cDNA was used to isolate a human nitric oxide synthase (NOS) cDNA from a human cerebellum cDNA library. The human cDNA clone containing 1.2 kb of brain NOS cDNA was hybridized to Southern blots containing DNAs obtained from human-rodent hybrid cell line panels using EcoRI and HindIII digestion to ascertain the location of the human NOS gene. These data showed that the human brain nitric oxide synthase mapped within 12q14-qter on human chromosome 12.


Over-expression and characterization of recombinant beta subunit of the human chorionic gonadotropin hormone synthesized in insect cells infected with a genetically engineered baculovirus. A recombinant baculovirus, vAc beta hCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the beta subunit of hCG was used to express beta hCG, an extensively glycosylated hormone, in insect cells. Virus-infected cells, 72 hr pi, secreted approximately 8.02 micrograms beta hCG/2 x 10(6) cells/ml. The recombinant beta hCG purified from insect cells exhibited increased mobility on SDS-PAGE as compared to authentic urinary beta hCG, a reflection on differences in glycosylation between insect and mammalian systems. The insect derived beta hCG, however, was identical to the native hormonal peptide in terms of immunoreactivity and bioactivity on association with alpha-subunit, as evident by its binding to rat testicular receptors and induction of steroidogenesis in a mouse Leydig cell bioassay system. The implications of using the baculovirus system to study the importance of carbohydrates for biological activity are also discussed.


Functional relationship between high-affinity E receptor and CD2-11.3 epitope. The expression of both high-affinity E receptor (EhR) to sheep erythrocytes and CD2-11.3 epitope on activated human T lymphocytes suggests that these two structures may be functionally identical or closely associated. Therefore the aim of the present work was to determine if the CD2-11.3 epitope is involved in the early E rosette formation. The ability of normal and phytohaemagglutin (PHA)-activated human T lymphocytes to express the CD2-11.3 epitope and to form early E rosettes (T cells with EhR) was studied simultaneously. The partial divergence of CD2-11.3 expression on T lymphocytes from the ability of these cells to form early E (Ee) rosettes was found. The results indicated that the expression of CD2-11.3 epitope alone is insufficient to form the Ee rosettes by activated T lymphocytes, yet it may facilitate this phenomenon in the presence of EhR. The above data clearly show that the CD2-11.3 epitope is functionally closely associated although not identical to EhR. Accordingly, it seems that these two structures may co-adhere to the appropriate ligand. Thus it is possible that the CD2-11.3 epitope, as well as its established role in activation signalling, may also act as a co-adhesion molecule.


Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. IV. Cytokines secreted by human thymic epithelial cells in culture and their activities on murine thymocytes and bone marrow cells. In previous reports we described our approach to the cultivation of murine and human thymic epithelial cells in primary cultures, using defined, serum-free growth factor-supplemented medium and extracellular matrix-coated culture plates. The cells in these cultures displayed high metabolic activity and their supernatant was highly active on thymocytes. In the study reported here we analysed cytokine activities in the supernatant of human thymic epithelial cell cultures (HTES), by using the respective cytokine-dependent cell lines and by neutralization with specific monoclonal antibodies. Three cytokine activities were detected--interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) and macrophage (M)-CSF. Other cytokine activities tested for [IL-1, IL-2, IL-7, interferon (IFN) and tumour necrosis factor (TNF)] were negative. The effect of HTES on concanavalin A (Con A)-induced proliferation of murine thymocytes could be completely abolished by anti-IL-6 antibodies, but not by antibodies to CSF, whereas enhancement of bone marrow cell proliferation by HTES was partially inhibited by either anti-G-CSF or anti-M-CSF antibodies and completely inhibited by both antibodies, but not at all by anti-IL-6. We can thus distinguish between thymocyte-related cytokines (IL-6) and bone marrow (myeloid/monocyte) related ones (G-CSF, M-CSF) in HTES.


L-selectin expression differentiates T cells isolated from different lymphoid tissues in cattle but does not correlate with memory. L-selectin (LECAM-1, LAM-1) was expressed by a high proportion of CD4+ and CD8+ T cells, as well as almost all of the gamma delta T-cell receptor (TcR)+ (WC1+) T cells, isolated from blood, lymph nodes or tonsils. CD4+ T cells in the lamina propria of the gut villi and CD8+ T cells in the villous epithelium as well as the majority of WC1+ T cells in the gut mucosa were L-selectin-. The proportion of T cells from Peyer's patches that synthesized the molecule was intermediate between the value for blood and gut mucosa. Expression of L-selectin therefore marks T cells in cattle with a distinct tissue distribution that correlates with its function as the peripheral node homing receptor. The proportion of CD4+ and CD8+ T cells in the circulation that were L-selectin+ decreased with age. Unlike CD45R, expression of L-selectin was not related to CD4 T-cell memory as judged by proliferation in transformation assays to soluble antigen. Three-colour immunofluorescent staining demonstrated four subpopulations of CD4 and CD8 T lymphocytes in peripheral blood mononuclear cells (PBMC) that were CD45R+, L-selectin+; CD45R+, L-selectin-; CD45R-, L-selectin+; CD45R-, L-selectin-. CD4(4) memory cells were CD45R- and L-selectin+ or L-selectin-. Taken with earlier studies the reported observations demonstrated that only one of the four phenotypes of the CD4+ T cells in blood is present in the lamina propria of the gut villi and these are CD45R-, L-selectin-. Two of the four phenotypes of CD8+ T cells were present in the gut epithelium; these were CD45R+, L-selectin- or CD45R-, L-selectin-. Expression of the bovine molecule was not rapidly down-regulated on T cells following activation by exposure to phorbol myristate acetate.


Feeding neonatal rats with IgG antibodies leads to humoral hyporesponsiveness in the adult. Feeding monoclonal IgG2a or IgG1 anti-horseradish peroxidase (HRP) antibodies to 12-16-day-old neonatal rats caused a profound suppression of the humoral anti-HRP response in these rats as adults. The hyporesponsiveness to HRP was specific and long-lasting (up to 5 months). It was shown to be dose dependent, requiring relatively large doses of IgG (100-600 micrograms) for maximum effect. Secondary IgG (IgG1, IgG2a and IgG2b) responses were most depressed. The effect could be reproduced by i.p. injection of antibody. Hyporesponsiveness was not attributable to circulating antiidiotype antibodies directed against the monoclonal IgG, nor to the continued presence of the monoclonal anti-HRP since rats receiving antibody at or some weeks after the time of weaning and gut 'closure' responded well to subsequent HRP challenge. The effect was thus dependent on IgG administered over the identical period during which the neonatal circulation is rich in maternal IgG supplied via the milk. A direct function for maternal IgG in moulding the immune repertoire of the offspring, as well as providing passive protection, is suggested by these results.


Antibody responses to non-immunogenic synthetic peptides induced by co-immunization with immunogenic peptides. Chimeric peptides comprising B- and T-helper cell epitopes from the proteins of infectious agents represent immunogens with potential for use as new vaccines. However, it has become clear that the orientation of the epitopes, the presence of spacer residues and the number of copies of the epitopes influence the specificity, levels and affinity of the antibody produced following immunization with such constructs. Furthermore, the response to peptides is under genetic control leading to major histocompatibility complex (MHC)-linked non-responsiveness. In this study, we have investigated the potential of co-immunization of immunogenic peptides (to provide T-cell help) with non-immunogenic peptides (representing B-cell epitopes) to overcome the non-response to the latter. For this purpose, we have employed peptides representing T- and B-cell epitopes derived from the sequences of the fusion and haemagglutinin glycoproteins of measles virus. The results obtained show that simple co-immunization of a B-cell epitope with a T-cell epitope results in the production of antibody to the B-cell epitope without the requirement for covalent linkage of the two peptides. This approach could thus be used to overcome the problem of poor immunogenicity of peptides and will be of potential value in the design of immunization strategies using synthetic immunogens.


Mycobacterial 65,000 MW heat-shock protein shares a carboxy-terminal epitope with human epidermal cytokeratin 1/2. Molecular mimicry between mycobacterial heat-shock protein (hsp) 65 and host tissue antigens have been implicated in the autoimmune pathogenesis of certain idiopathic diseases. Here, we demonstrated that two of our previously characterized monoclonal antibodies (mAb), Ne5 and Nd4 that were directed to a carboxy-terminal epitope on the mycobacterial hsp 65, specifically cross-reacted with suprabasal cytokeratin of the normal human skin. These mAb also showed similar keratin staining of hair follicle epithelia and produced no reaction with other dermal components. Both mAb strongly stained the cytoplasm of the majority of freshly isolated epidermal keratinocytes from the normal human skin. None of these mAb showed staining with human HeLa cells and with human skin fibroblasts. Immunoblotting using total keratin extract prepared from isolated epidermal keratinocytes revealed that mAb Ne5 and Nd4 specifically reacted with a molecular size of 65,000-67,000 MW keratin protein(s) and such reactivity was not observed from cytoskeletal proteins extracted from HeLa cells and skin fibroblasts. Comparison of immunoblotting reactivity with conventional anti-cytokeratin mAb further revealed that mAb Ne5/Nd4 recognized a 65,000-67,000 MW molecular-sized protein corresponding to cytokeratin 1/2 from the same keratinocyte extract as anti-cytokeratin mAb. Preincubation of mAb Ne5/Nd4 with the purified mycobacterial hsp 65 abolished this keratin cross-reactivity in both immunohistochemistry and immunoblotting. Moreover, these mAb showed no keratin staining in lesional psoriatic skin and also reacted weakly with cultured epidermal keratinocytes. Since mAb Ne5/Nd4 specifically recognized a 67,000-65,000 MW molecular-sized protein(s) derived from epidermal keratinocytes and the known characteristics of epidermal cytokeratin 1/2 appeared to be consistent with present results, we concluded that Ne5/Nd4 cross-reactive protein(s) in the human epidermis is suprabasal cytokeratin 1/2. Comparison of the previously mapped Ne5/Nd4 epitope region of amino acid residues 525-540 of the mycobacterial hsp 65 with the entire sequence of human 65,000 MW keratin revealed that a stretch of nine amino acids of the Ne5/Nd4 epitope sequence resembled certain regions of the carboxy-terminus of the human 65,000 MW keratin. This similarity of the mycobacterial hsp 65 probably contributes to the cytokeratin cross-reactive epitope. Our results presented here demonstrate direct evidence of immunological cross-reactivity between mycobacterial hsp 65 and human epidermal cytokeratin 1/2. We speculate that Ne5/Nd4 cross-reactive epitope of epidermal cytokeratins might be an important target for skin diseases.


Characterization of the C-terminal region of a Trypanosoma cruzi 38-kDa ribosomal P0 protein that does not react with lupus anti-P autoantibodies. A Trypanosoma cruzi cDNA lambda gt11 recombinant, C-P0, encoding the carboxyterminus of a highly antigenic ribosomal P protein, was isolated. Sequence comparisons and immunological evidence allowed its identification as the C-terminal region of the T. cruzi 38-kDa ribosomal P0 protein. This recombinant failed to react with systemic lupus erythematosus (SLE) anti-P antibodies. The T. cruzi ribosomal P0 protein is the first reported eukaryotic ribosomal P protein presenting this immunological property, probably due to its peculiar C-terminal-amino-acid sequence, FGMGALF, which differs from the conserved eukaryotic ribosomal P protein C-terminal consensus, MGFGLFD.


Immune response to a single peptide containing an immunodominant region of hepatitis C virus core protein: the isotypes and the recognition site. We have used one single peptide covering the 17 N-terminal amino acids of the hepatitis C virus (HCV) core protein (c) to analyse the human immune response against B-cell epitope(s) within this region. The sequence MSTNPKPQRKTKRNTNR was obtained from two sequenced HCV genomes, and the peptide was synthesized by a newly developed method. The peptide was assayed with 144 human sera which had all been assayed for antibodies to HCV (anti-HCV) using commercial assays. Forty-nine sera were found to be positive for anti-HCV using these assays; 40 of these were found to be positive with our anti-HCV IgG peptide assay. The class (IgM, IgG) and subclass (IgA1, IgG1-4) specific reactions were determined using the polyclonal and monoclonal anti-HCV peptide enzyme immunoassays. Isotypes of mainly IgG1 and IgG3, but also IgG4, IgM and IgG2, gave specific reactions with this region. Using omission peptide analogues of the region 1-18, the sequence RKTKRNTN within residues 9-16 was common to 34 out of 37 sera of which the IgG antibody binding site could be mapped. It is unusual for a single peptide assay to have such high sensitivity since B cell epitopes within a protein are often discontinuous. It seems that at least 80% of HCV infected individuals develop antibodies of various isotypes to the antigenic site RKTKRNTN, located in the N-terminal portion of the HCV core. Thus, the immune response to this peptide should be further investigated with regard to the reactive Ig isotypes developing during HCV infection.


Intracellular expression of CD1 molecules on PHA-activated normal T lymphocytes. In this study we have analyzed, using immunoperoxidase (IPx) and indirect immunofluorescence (IIF), the intracellular and cell surface expression of CD1 antigens on PHA activated human T cells. By IIF, CD1 isotypes were not detected on the surface of 3 days PHA activated cells. Conversely, CD1a, CD1b and CD1c molecules were found by IPx in the cytoplasm of normal activated cells from 13 different donors. Kinetic studies showed that, while CD25 was already observed 24 h after activation, all 3 isotypes of CD1 molecules started to be detected 48 h after PHA activation, with a peak expression at 72 h.