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Biomedical subjects

A Henry

Publications and source records attributed to A Henry.

At least 37 records · Page 2Linked to original sources
PubMed1999

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus.

HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka approximately 1x10(9) M-1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection.

Algorithms↗
PubMed1999

Enhancing compliance not a prerequisite for effective eradication of Helicobacter pylori: the HelP Study.

OBJECTIVE: The aim of this study was to compare cure rates of Helicobacter pylori (H. pylori) infection, compliance, and side effects in patients given 10 days of omeprazole 20 mg b.d., amoxycillin 500 mg t.d.s., and metronidazole 400 mg t.d.s. (OAM) or 10 days OAM plus compliance enhancing measures. METHODS: A total of 119 H. pylori-positive patients were prospectively randomized to receive either 10 days OAM or 10 days OAM plus compliance enhancing measures (medication in a dose dispensing unit, medication chart, an information sheet about H. pylori treatment, and phone call 2 days after starting therapy). H. pylori eradication was assessed by 13C-UBT at least 4 wk after cessation of therapy, compliance by phone interview on the last day of therapy and returned pill count, and side effects by phone interview and returned side effects form. RESULTS: In 113 patients attending 13C-UBT H. pylori was eradicated in 51 of 57 patients (89.5%) after 10 days OAM and in 48 of 56 (85.7%) after 10 days OAM plus compliance enhancing measures (p = 0.54). In both groups 97% of medications were taken. Side effects were common (82% of patients). Both side effects (p = 0.001) and ulcer versus nonulcer at endoscopy (p = 0.016) were independent predictors of treatment failure; side effects also predicted noncompliance (p = 0.02). CONCLUSIONS: Ten days of OAM was effective for H. pylori eradication in our clinical population. Patient compliance was excellent and attempts to increase compliance had no impact on outcome or compliance. Side effects were very common and were significantly associated with treatment failure and decreased compliance.

Adult↗
PubMed1999

Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the V(L) and V(H) regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.

Amino Acid Sequence↗
PubMed1998

Processing of the Alzheimer's disease amyloid precursor protein in Pichia pastoris: immunodetection of alpha-, beta-, and gamma-secretase products.

betaA4 (Abeta) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP). Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage within the betaA4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP695 cDNA. In addition to the full-length integral transmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culture medium. Most sAPP comprised the N-terminal moiety of betaA4 and corresponds to sAPPalpha, the product of alpha-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPbeta (the ectodomain released by beta-secretase cleavage). Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of alpha and beta cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the betaA4 domain yielded a 4 kDa product with the same electrophoretic mobility as betaA4 synthetic peptide. These results suggest that the alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P. pastoris may offer an alternative to mammalian cells to identify the proteases involved in the generation of AD betaA4 amyloid.

Alzheimer Disease↗
PubMed1998

[Teaching ethics in French medical schools: evolution or revolution ?].

Ethics was introduced as subject matter in French medical schools only recently despite a rich historical context where scientific legitimacy, humanistic exigencies and anglo-saxon influence have all played a role. Ten years after the thesis presented by Bastian in 1986, a survey of French medical schools shows that ethics has become an integral part of the curriculum. Ethics has tended to become a discipline on its own, separate from law and deontology. However, the lack of specific courses on concentration in the first years of the curriculum show that there is much room for growth in the discipline of medical ethics.

Curriculum↗
PubMed1998

Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

Alternative Splicing↗
PubMed1998

Genetic and developmental influences on infant mouse ultrasonic calling. II. Developmental patterns in the calls of mice 2-12 days of age.

Infant house mice, Mus musculus, produce ultrasonic calls that reliably lead to retrieval by adult mice. While individual differences in calls have been demonstrated both among and within species, the influences of age and sex on call characteristics have not been systematically investigated in mice. This study examined the influences of age, sex, and genotype (inbred versus hybrid) on the rate, length, and frequency characteristics of the calls of 486 male and female mice from 2 to 12 days of age. Rate of calling followed a shallow inverted U-shaped function across days. Call lengths decreased and call frequency characteristics increased, in a linear manner, with age. Females emitted fewer calls, with a smaller bandwidth, at some ages than males. Hybrid pups produced more calls of greater length and a lower frequency than inbred pups. These results indicate the presence of cues that could allow adult mice to behave differentially toward pups as a function of their age and sex.

Age Factors↗
PubMed1998

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein.

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.

Adenosine Diphosphate↗
PubMed1998

Subcellular localization of the Alzheimer's disease amyloid precursor protein and derived polypeptides expressed in a recombinant yeast system.

Different isoforms and derived polypeptides of the Alzheimer's disease amyloid protein precursor (A beta PP) have been expressed in the yeast Pichia pastoris. The expression characteristics of the different A beta PP polypeptides were studied by post-embedding immunogold electron microscopy with various A beta PP antibodies. The site of intracellular expression could be readily identified with specific antibodies. Full length A beta PP was expressed in association with the nuclear membrane and the endoplasmic reticulum. Secretory derivatives of A beta PP were localized in membrane-bound secretory vesicles. A construct encoding two copies of beta A4[1-42] linked head-to-tail (beta A4duplex) accumulated as irregular dense cytoplasmic and intranuclear inclusions which reacted with all beta A4 antibodies tested. A beta A4-C-terminal construct accumulated into membranous structures in the cytoplasm and nucleus and reacted with most antibodies to beta A4 and the cytoplasmic domain of A beta PP. The two shorter constructs containing the beta A4 sequence formed similar intranuclear aggregates to those reported for intranuclear inclusions of polyglutamine peptides from huntingtin (in Huntington's disease) and ataxin protein fragments (in spinocerebellar ataxia). This is of interest because intracellular aggregation of the polyglutamine and beta A4 peptides may affect cells by similar toxic mechanisms. These studies demonstrate clear differences in the expression properties of different A beta PP polypeptides.

Alzheimer Disease↗
PubMed1998

Processing load and the extended optional infinitive stage.

Spontaneous language data from fifteen language-impaired children in the Extended Optional Infinitive stage of morphosyntactic development are examined with respect to processing load. Clauses with a target finite verb form are categorised according to finiteness and their preverbal constituents. Patterns of occurrence are discussed with regard to the Optional Infinitive framework itself and the implications for remediation.

Child↗
PubMed1997

Expression of human amyloid precursor protein ectodomains in Pichia pastoris: analysis of culture conditions, purification, and characterization.

We have examined the use of the yeast Pichia pastoris for expression of the human amyloid precursor protein (APP). The ectodomains of the isoforms APP695, APP751, and APP770 were expressed in both P. pastoris protease-deficient strain SMD1163 and wild-type strain GS115, using the secretion vector pHIL-S1. Expression of recombinant APP in each of these strains produced intact recombinant protein, together with a small number of breakdown products. The levels of these breakdown products were not significantly altered by expression in the protease-deficient strain compared with wild-type GS115. The effects of induction time and medium composition on recombinant APP stability were also examined. After optimization of expression and culture conditions, baffled shaker flask cultures of clones selected for high expression routinely yielded 13-24 mg/liter recombinant protein following a two-step purification procedure. The recombinant isoforms possessed the heparin binding, metal binding, and Kunitz-type protease inhibitor properties of human brain-derived APP. These data indicate that P. pastoris is an appropriate laboratory-scale expression system for production of sufficient quantities of recombinant APP for use in biological studies.

Amyloid beta-Protein Precursor↗
PubMed1997

Tethered thoracic cord resulting from spinal cord herniation.

Tethered cord syndrome (TCS) usually involves tethering of the lower cord at the conus medullaris from dural abnormalities, but may occur after spinal cord herniation. A tethered thoracic spinal cord is rare. We present an unusual case of a 30-year-old woman with a history of myelopathy presumed to be secondary to T6 cord compression resulting from T6-T8 arachnoid cyst. She continued to deteriorate after partial excision of the cyst. Repeat magnetic resonance imaging suggested recurrence of the presumed arachnoid cyst with cord compression and showed tethering at T6-T8. Surgical exploration revealed myelocele with cord herniation through the anterior thoracic dura. Pathologic diagnosis showed neural tissue with gliosis. After physical therapy treatments, the patient had increased lower extremity strength, ambulated with a cane, and regained some bladder control. Progressive myelopathy may represent tethering of the cord resulting from cord herniation. Early recognition of TCS, even in patients with minimal neurologic deficits, could prevent progressive disability.

Adult↗
PubMed1997

Genetic and developmental influences on infant mouse ultrasonic calling. I. A diallel analysis of the calls of 3-day olds.

Ultrasonic calls produced by young mice reliably elicit investigation and retrieval by adults. While there are large individual differences in the characteristics of these calls, little work has been done to partition that variation. We completed a 4 x 4 diallel cross and Hayman analyses on several characteristics of these cries. The major result was the detection of directional dominance toward a higher rate of calling, longer calls, and calls of lower overall frequency with a greater bandwidth. Within the context of biometrical genetic theory, we conclude that calls with such characteristics may have important fitness value. Extending this idea, we propose that within the population sampled for this study (the animals of the four inbred strains and 12 F1 hybrid groups), the calls most effectively eliciting investigation and retrieval would be calls with the average hybrid values of the diallel cross.

Animals↗
PubMed1996

Secreted glypican binds to the amyloid precursor protein of Alzheimer's disease (APP) and inhibits APP-induced neurite outgrowth.

The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican bound strongly to the APP affinity column. Purified glypican bound to APP with an equilibrium dissociation constant of 2.8 nM and inhibited APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican was specific for APP, as glypican did not inhibit laminin-induced neurite outgrowth. Furthermore, treatment of cultures with 4-methylumbelliferyl-beta-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibited APP-induced neurite outgrowth but did not inhibit laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP.

Alzheimer Disease↗