Serodiagnosis of viral hepatitis A: rise in antibody titre and evaluation of three methods for detecting early and late antibodies

A serological investigation was made on patients with viral hepatitis A and individuals with a past history of this disease. Titration of antibody in sequential samples was found to be of no help in diagnosis. Separation of early (IgM) from late (IgG) antibodies by protein A or by 2-mercaptoethanol did not prove to be convenient for the serodiagnosis. A chromatographic separation of late and early antibody was found to be satisfactory, and equivalent to a radioimmunoassay for IgM-antibodies.     

Novel approaches to the treatment of small-cell lung cancer

Small-cell lung cancer (SCLC) is characterized by its initial responsiveness to chemotherapy and the appearance of early metastases. Although combination chemotherapy, in some instances together with radiation, has improved the prognosis of this disease, in most patients SCLC ultimately recurs in a drug-resistant form. Several new strategies for the eradication of SCLC are being explored at the preclinical level. The identification of selective target molecules on the surface of SCLC cells, together with the progress made in antibody engineering, have provided new generations of antibodies and immunoconjugates as well as growth factor antagonists and inhibitors. In addition, recent advances in understanding the biology of SCLC have stimulated new investigations searching to counter the molecular basis underlying the increased proliferation and the apoptosis deficiency of SCLC cells. This can be achieved using antisense oligodeoxynucleotides that repress the expression of growth factor receptors and anti-apoptosis genes, or by gene replacement to compensate for the loss or inactivation of tumor suppressor genes.     

Studies on the modification of Escherichia coli ribosomal protein L7/L12 by succinic anhydride

Lysine modification by increasing quantities of succinic anhydride in the Escherichia coli ribosomal protein L7/L12 produces loss of its ability in reconstitution of elongation-factor-G-dependent GTP hydrolysis and polyphenylalanine synthesis activities, showing lower antigenicity and loss of antigenic determinants.     

Loss of antibody production accompanied by chromosome loss in a cloned hybrid line secreting antibodies to sheep red blood cells

Somatic cell hybrids between Sp2/O-Ag14 mouse myeloma cells and lymphocytes derived from BALB/c mice hyperimmunized with sheep red blood cells (SRBC) were produced. One hybrid producing IgG1 antibody to SRBC was selected, cloned twice and subsequently transferred to BALB/c mice. After a number of transfers it was found that the antibody titer in ascites fluid gradually decreased. Cytogenetic analysis revealed gradual chromosome loss in the hybrid clone, which produced progressively less antibody.     

Loss of antibody production accompanied by chromosome loss in a cloned hybrid line secreting antibodies to sheep red blood cells

Summary Somatic cell hybrids between Sp2/O-Ag14 mouse myeloma cells and lymphocytes derived from BALB/c mice hyperimmunized with sheep red blood cells (SRBC) were produced. One hybrid producing IgG₁ antibody to SRBC was selected, cloned twice and subsequently transferred to BALB/c mice. After a number of transfers it was found that the antibody titer in ascitec fluid gradually decreased. Cytogenetic analysis revealed gradual chromosome loss in the hybrid clone, which produced progressively less antibody.     

Induction of autotomy in the American bird grasshopperSchistocerca americana (Drury) by the ecdysone agonist RH-5849 and investigation of its mode of action

The non-steroidal ecdysone agonist RH-5849 (1,2-dibenzoyl-l-tert-butylhydrazine) was found to be an effective neurotoxicant on injection into the American bird grasshopper,Schistocerca americana (Drury). Treated grasshoppers became immediately hyperactive, followed by loss of coordination, paralysis and eventually death. We also discovered that this compound induced bilateral autotomy of the metathoracic legs during the early stages of intoxication. However, no evidence of ecdysonergic or morphogenetic activities was observed. Synergism studies with neurotoxins of known mode of action suggested that RH-5849 has a mechanism of action similar to that of 4-amino pyridine, which blocks potassium channels.     

A central role for calcineurin in protein misfolding neurodegenerative diseases

Accumulation of misfolded/unfolded aggregated proteins in the brain is a hallmark of many neurodegenerative diseases affecting humans and animals. Dysregulation of calcium (Ca²⁺) and disruption of fast axonal transport (FAT) are early pathological events that lead to loss of synaptic integrity and axonal degeneration in early stages of neurodegenerative diseases. Dysregulated Ca²⁺ in the brain is triggered by accumulation of misfolded/unfolded aggregated proteins in the endoplasmic reticulum (ER), a major Ca²⁺ storing organelle, ultimately leading to neuronal dysfunction and apoptosis. Calcineurin (CaN), a Ca²⁺/calmodulin-dependent serine/threonine phosphatase, has been implicated in T cells activation through the induction of nuclear factor of activated T cells (NFAT). In addition to the involvement of several other signaling cascades, CaN has been shown to play a role in early synaptic dysfunction and neuronal death. Therefore, inhibiting hyperactivated CaN in early stages of disease might be a promising therapeutic strategy for treating patients with protein misfolding diseases. In this review, we briefly summarize the structure of CaN, inhibition mechanisms by which immunosuppressants inhibit CaN, role of CaN in maintaining neuronal and synaptic integrity and homeostasis and the role played by CaN in protein unfolding/misfolding neurodegenerative diseases.     

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 – 30 detectable by the 3F4 antibody against human PrP109 – 112. We recently identified a new PK-resistant PrP species, designated PrP^*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 – 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP^*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP^*20 but also a small amount of 3F4-detected PK-resistant PrP27 – 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP^*20 to the occurrence of the 3F4-detected PrP27 – 30 was observed. Our study suggests that an increase in the level of PrP^*20 characterizes the early stages of prion diseases. Received 17 October 2007; received after revision 5 December 2007; accepted 14 December 2007     

Distribution of repetitious DNA in human chromosomes

Zusammenfassung Studien von in situ DNS/RNS-Hybriden (oder Mischflüssigkeiten) zwischen RNS und verschiedenen Fraktionen von DNS und Metaphase-Chromosomen des Menschen ergaben, dass hauptsächlich die wiederholt vorkommende DNS-Fraktion C _o t = 0 0.005 sich in der centromeren und telomeren Region befindet.

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Supported in part by grants No. G-267 and No. G-373 from the Robert A. Welch Foundation, and grants No. DRG-269 and No. 1061 from the Damon Runyon Memorial Fund for Cancer Research     

Chymotrypsin gene expression during the intermolt cycle in the shrimpPenaeus vannamei (Crustacea; Decapoda)

InPenaeus vannamei, chymotrypsin is present as two isoenzymes in the hepatopancreas. The enzyme has been localized in F-cells by immunocytochemistry using a specific antibody. By in situ hybridization, with a 510 pb cDNA probe encoding for the first 170 amino acids of the shrimp chymotrypsin, mRNA was localized in the same cells. Gene expression was followed during the intermolt cycle by measuring changes in specific activity in crude extracts, and by the estimation of mRNA levels by Northern blots using the same probe. The increase in specific activity in premolt is preceded in early premolt by an increase in the amount of chymotrypsin mRNA. A second increase is observed in postmolt, suggesting a different mode of regulation of gene expression.     

Increased activity of dipeptidyl peptidase IV in serum of hepatoma-bearing rats coincides with the loss of the enzyme from the hepatoma plasma membrane

The specific activity of dipeptidyl peptidase IV (DPP IV E.C. 3.4.14.-) in the plasma membrane of Morris hepatoma 9121 or hepatoma 7777 was 3.5% and 2.9%, respectively, of that in the plasma membrane of rat liver. The enzyme activity in the serum of hepatoma-bearing rats was 141% (hepatoma 91219) and 162% (hepatoma 7777) of the normal value. Cytochemical investigation showed that the DPP IV activity was almost completely absent from the hepatoma cell plasma membrane and was not sequestered within these cells. Indirect immunofluorescence staining with a polyclonal antibody directed against DPP IV indicated that the loss of activity was due to the absence of DPP IV molecules in the plasma membrane. The possibility that the enzyme is transferred from the membrane into the serum as a result of structural alterations is discussed.     

Current cases in which epitope mimicry is considered a component cause of autoimmune disease: Guillain-Barré syndrome

Some patients develop Guillain-Barré syndrome (GBS) after the administration of bovine gangliosides. Patients with GBS subsequent to Campylobacter jejuni enteritis frequently have IgG antibody to GM1 ganglioside. Miller Fisher syndrome (MFS), a variant of GBS, is associated with IgG antibody to GQ1b ganglioside. Molecular mimicry between GM1 and lipopolysaccharide of C. jejuni isolated from patients with GBS, and between GQ1b and C. jejuni lipopolysaccharides from patients with MFS have been demonstrated. The molecular mimicry between infectious agents and gangliosides may function in the production of anti-ganglioside antibodies. This sugar mimicry is one possible cause of the Guillain-Barré and Miller Fisher syndromes; however, unidentified host factors may contribute to the development of these syndromes.     

Regulatory peptide immunocytochemistry at light- and electron microscopical levels

Immunocytochemical techniques applied at both light- and electron microscopical levels are valuable in the study of regulatory peptide distribution in normal and diseased tissue, whether in the form of sections or whole cell preparations. Successful immunolocalization depends on adequate preservation of the peptide antigen and the tissue structure in which it resides; a suitably specific and sensitive labelled antibody detecting system. In general, peptides are stable molecules, most of which retain their antigenicity after conventional cross-linking fixation and tissue processing, allowing standard immunocytochemical methods to be used for light- and electron microscopy. Regulatory peptides are derived from precursor molecules and several 'families' of structurally similar peptides are now generally recognised. Region-specific antibodies may be needed to overcome problems of cross-reactivity or to identify a bioactive form in the presence of its precursor. Multiple co-localisation of different related and unrelated peptides in the same cell or even storage granule is now recognised and can be identified by immunocytochemistry.     

Increased activity of dipeptidyl peptidase IV in serum of hepatoma-bearing rats coincides with the loss of the enzyme from the hepatoma plasma membrane

Summary The specific activity of dipeptidyl peptidase IV (DPPIV E.C. 3.4.14.-) in the plasma membrane of Morris hepatoma 9121 or hepatoma 7777 was 3.5% and 2.9%, respectively, of that in the plasma membrane of rat liver. The enzyme activity in the serum of hepatoma-bearing rats was 141% (hepatoma 91219) and 162% (hepatoma 7777) of the normal value. cytochemical investigation showed that the DPP IV activity was almost completely absent from the hepatoma cell plasma membrane and was not sequestered within these cells. Indirect immunofluorescence staining with a polyclonal antibody directed against DPP IV indicated that the loss of activity was due to the absence of DPP IV molecules in the plasma membrane. The possibility that the enzyme is transferred from the membrane into the serum as a result of structural alterations is discussed.     

Nitric oxide stimulates human neural progenitor cell migration via cGMP-mediated signal transduction

Neuronal migration is one of the most critical processes during early brain development. The gaseous messenger nitric oxide (NO) has been shown to modulate neuronal and glial migration in various experimental models. Here, we analyze a potential role for NO signaling in the migration of fetal human neural progenitor cells. Cells migrate out of cultured neurospheres and differentiate into both neuronal and glial cells. The neurosphere cultures express neuronal nitric oxide synthase and soluble guanylyl cyclase that produces cGMP upon activation with NO. By employing small bioactive enzyme activators and inhibitors in both gain and loss of function experiments, we show NO/cGMP signaling as a positive regulator of migration in neurosphere cultures of early developing human brain cells. Since NO signaling regulates cell movements from developing insects to mammalian nervous systems, this transduction pathway may have evolutionary conserved functions.     

Profile of sequential determinants in tissue polypeptide antigen BrCN:B fragment

A synthetic approach has been applied to determine the profile of sequential determinants of one immunodominant region of Tissue Polypeptide Antigen (TPA). Five overlapping peptides, covering 30 of the 32 amino acid residues of this fragment, were chemically synthesized, and their antibody-binding activities for rabbit anti-TPA antibodies determined by enzyme-linked immunoadsorbant assays. Anti-TPA reacted with two overlapping fragments at the COOH-terminal end of the fragment, but not with peptides that include Arg 15 considered as essential for the antigenicity of the whole fragment. This might suggest that this critical residue is involved in the formation of a complex conformational determinant.     

Effect of periodate oxidation of glycoconjugates of lymphocyte membranes on the immune response in vitro]

Mouse spleen cells treated with sodium periodate for 10 min. at 4 degrees C are stimulated to undergo blastogenesis and to incorporate thymidine. The effect of such treatment on the antibody response in vitro induced by Sheep red blood cells has been evaluated. Periodate-induced proliferation is accompanied by a marked inhibition of the immune response to this antigen. At concentrations leading to mitogenesis, no cytotoxic effect of periodate was observed and treated cells survived well on tissue culture. Cell recoveries from samples treated with periodate at the optimal mitogenic dose, were markedly enhanced when harvested at different days after culturing wheras lower antibody forming cells numbers wereconsistently observed during the culture period.     

Long-term production and culture of clones of human T-lymphocytes

Culture of blood T lymphocytes collected from normal individuals and cancer patients were carried out in presence of T cell growth factor (TCGF); these cultures presented cytotoxic activity directed against different targets (lectin activated cells, autologous cancer cells, antibody coated cells and K 562). In order to study separately the different effector subpopulations, isolation of single cultured cells were performed with the help of a micropipette under microscope and monoclonal cultures were carried out in presence of TCGF. In the preliminary cytotoxic assays performed in the clones: (1) a marked activity directed against lectin targets was observed in many clones and (2) an important N K activity was exhibited by the clone 45 B9 (65% of the tested cells lysed human lymphoma K 562 cells).     

Drug susceptibility of PCA in WBB6F1-W/Wv mice

Our previous study revealed that passive cutaneous anaphylaxis (PCA) can be produced in congenitally mast cell-deficient WBB6F1-W/W^v (abbreviated as W/W^v) mice on sensitization with undiluted or slightly diluted allogeneic and xenogeneic antisera but not on sensitization with allogeneic monoclonal immunoglobulin (Ig)E and IgG1 antibodies regardless of the antibody concentration [1]. In view of these findings, the present study was conducted to characterize PCA in this strain from its drug susceptibilities using mast cell-bearing WBB6F1-+/+ (abbreviated as +/+) and B6D2F1 mice as references. PCA in W/W^v mice mediated by a low dilution (1  4) of hyperimmune serum to bovine serum albumin of the B6D2F1 mouse origin was markedly suppressed by CV-6209, an antagonist of platelet-activating factor (PAF), but not by antihistamines such as cyproheptadine and oxatomide. In contrast, PCA in +/+ and B6D2F1 mice mediated by a high dilution (1  128) of the anti-serum (virtually by IgG1 antibody) was nearly completely suppressed by antihistamines but not by CV-6209. A remarkable difference between PCA in W/W^v and reference mice was also observed in the susceptibility to monoclonal anti mouse granulocyte (Gr-1) antibody PCA in W/W^v mice was potently suppressed by the 1- to 3-day pretreatment with this antibody but that in references was not at all. Putting these present results together with the previous finding that anti-granulocyte antibody greatly reduces circulatory Gr-1⁺ leukocytes, 1 to 3 days after the treatment [2], it is highly probable that PCA in W/W^v mice mediated by some antibody isotypes other than IgE and IgG1 is produced by PAF mainly released from Gr-1⁺ cells, while IgG1 antibody-mediated PCA in mast cell-bearing reference mice is evoked by histamine derived from mast cells. PCA homologous to that in W/W^v mice could also be produced in the reference mice on sensitization with undiluted or slightly diluted antiserum, when generalized blueing due to excess IgG1 antibody was removed by the oxatomide treatment be fore the antigen challenge. Received 10 December 1997; received after revision 2 February 1998; accepted 23 February 1998     

Studies on the modification ofEscherichia coli ribosomal protein L7/L12 by succinic anhydride

Summary Lysine modification by increasing quantities of succinic anhydride in theEscherichia coli ribosomal protein L7/L12 produces loss of its ability in reconstitution of elongation-factor-G-dependent GTP hydrolysis and polyphenylalanine synthesis activities, showing lower antigenicity and loss of antigenic determinants.This study was supported in part by Fondo de investigaciones sanitarias del INSALUD.