Nerve cell differentiation in hydra requires two signals

Endogenous signals controlling nerve cell commitment in hydra were investigated using an assay for committed nerve precursors. Extracts of hydra tissue were prepared and tested for their ability to induce nerve cell commitment. The active component in such extracts was identified as a neuropeptide, the head activator [H. C. Schaller and H. Bodenmüller (1981) Proc. Natl. Acad. Sci. USA 78, 7000–7004], based on its chromatographic properties and reaction with anti-head activator antibody. In addition, synthetic head activator (10−13–10−11 M) was shown to cause nerve cell commitment. Additional experiments demonstrated that committed nerve precursors require a second signal to differentiate nerve cells. Committed precursors induced by treatment of hydra with head activator do not differentiate in whole hydra; but do differentiate when pieces of treated tissue are explanted or when whole animals are simply injured with transverse cuts. The injury stimulus is long-lived. It cannot be replaced with head activator (10−12–10−10 M) but is contained in a methanol extract of hydra tissue

Synthesis and biodistribution of immunoconjugates of a human IgM and polymeric drug carriers

The synthesis and purification of radiolabelled immunoconjugates, composed of a human IgM monoclonal antibody directed against an intracellular tumour-associated antigen and either poly (alpha-L-glutamic acid) (PGA) or poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG) is described. Coupling of polymers to the antibody was performed through disulfide bond formation involving a single thiol group at the C-terminus of the polymer chain and 2-pyridyldisulfide groups introduced onto the antibody. The antibody was iodinated with 131I before conjugation. The polymers contained tyrosinamide in a low degree of substitution and were radiolabelled with 125I. 125I-labelled PGA and PHEG were found to be stable for at least 3 days in murine and human plasma. The biodistribution in mice of the doubly labelled immunoconjugates was studied and was compared with the pharmacokinetics of the individual components.\ud \ud PHEG showed a relatively slow blood clearance, the half-life being approximately 10 h with low uptake in liver, kidneys and spleen. PGA was rapidly cleared from the circulation and was significantly taken up in liver, kidneys and spleen. The biodistribution of both immunoconjugates was indistinguishable from that of the IgM proper, with plasma half-lives of approximately 6 h, indicating that the pharmacokinetic properties of the immunoconjugates are largely determined by the antibody part

Further evidence of the circulation of PMV-4 and influenza viruses with N2 - 1957 enzyme in migratory waterfowls

I n the years 1980—1984, one paramyxovirus type 4 and 11 influenza viruses were isolated from cloacal swabs collected from migratory waterfowls in Fed. Rep. Germany. One influenza virus of H4N8 subtype was isolated from swabs of commercial ducks collected at an abbatoir. Seven of 10 influenza strains, isolated from mallard clucks and coot were identified as a mixture of 2 —3 strains of H l , H4, and Ho subtype; 3 virus strains from the same locality relate antigenically to subtype H4 w i t h enzyme serologically identical with N2 — Singapore/57 as demonstrated by means of polyclonal and monoclonal antibody

Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

BACKGROUND Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample. RESULTS EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10² to 1.3 × 10⁸ copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³ to 2.0 × 10⁵ copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴ to 1.1 × 10⁵ copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10² to 5.6 × 10³ copies/ml in HIV-infected patients (n = 12), and 2.0 × 10² to 9.1 × 10⁴ copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11). CONCLUSION Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.The Ausimmune Study is funded by the National Multiple Sclerosis Society of the USA, the National Health & Medical Research Council (Project Grant 316901) and Multiple Sclerosis Research Australia

Single-cell western blotting.

To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution

Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

© 2015 Elsevier Inc.Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies

Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus.

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0-2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human antibody response to foreign Ags and for generating human mAbs of predetermined specificity and high affinity