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D J McCormick

Publications and source records attributed to D J McCormick.

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

In vitro binding and CNS effects of novel neurotensin agonists that cross the blood-brain barrier.

Animal studies with neurotensin (NT) directly injected into brain suggest that it has pharmacological properties similar to those of antipsychotic drugs. Here, we present radioligand binding data for some novel hexapeptide analogs of NT(8-13) at the molecularly cloned rat and human neurotensin receptors (NTR-1), along with behavioral and physiological effects of several of these peptides after intraperitoneal (i.p.) administration in rats. One unique analog, NT66L, which had high affinity (0.85 nM) for the molecularly cloned rat neurotensin receptor (NTR-1), caused a drop in body temperature and antinociception at doses as low as 0.1 mg/kg after i.p. injection. At 30 min post-injection, the ED50 for NT66L-induced hypothermia (rectal temperature) and antinociception (hot plate test) was 0.5 and 0.07 mg/kg, respectively. At a dose of 1 mg/kg i.p., NT66L caused 100% of the maximum possible effect for antinociception for up to 2 h after administration. At this dose body temperature lowering was greater than -2.5 degrees C from 20 to 120 min after i.p. administration. These results in animals suggest that NT66L has agonist properties at NTR-1 in vivo after extracranial administration and provide support for its further study in behavioral tests predictive of neuroleptic activity.

Animals↗
PubMed1999

Hb Chile [beta28(B10)Leu-->Met]: an unstable hemoglobin associated with chronic methemoglobinemia and sulfonamide or methylene blue-induced hemolytic anemia.

Among the causes of life-long cyanosis are congenital methemoglobinemia due to M hemoglobins, congenital methemoglobinemia due to methemoglobin reductase deficiency, a small number of low oxygen affinity hemoglobins, and a small number of unstable hemoglobins that spontaneously form methemoglobin in vivo at an accelerated rate. We report an unstable hemoglobin with these characteristics that was observed in a family of indigenous (native American) origin living near Santiago, Chile. This variant has the substitution beta28(B10)Leu-->Met, unambiguously corresponding to the DNA mutation of CTG-->ATG in beta-globin gene codon 28.

Anemia, Hemolytic↗
PubMed1998

Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo.

Peptide nucleic acids (PNAs) are DNA analogs that can hybridize to complementary sequences with high affinity and stability. Here, we report the first evidence of intracellular delivery of PNAs in vivo. Two CNS receptors, an opioid (mu) and a neurotensin (NTR-1), were targeted independently by repeated microinjection of PNAs into the periaqueductal gray. Behavioral responses to neurotensin (antinociception and hypothermia) and morphine (antinociception) were lost in a specific manner. Binding studies confirmed a large reduction in receptor sites. The loss of behavioral responses was long lasting but did fully recover. The implications of specifically and readily turning off gene expression in vivo are profound.

Animals↗
PubMed1998

Thyroglobulin peptides of specific primary hormonogenic sites can generate cytotoxic T cells and serve as target autoantigens in experimental autoimmune thyroiditis.

Previously we demonstrated that thyroxine (T4)-containing, 12-mer peptides from positions 5 (1-12) and 2553 (2549-2560), as well as thyronine (T0)-substituted 2553 peptide, derived from human (H) thyroglobulin (Tg) are capable of activating T cells that infiltrate the thyroid (thyroiditogenic). In contrast, peptides T4(2567) and T0(2567) (2559-2570) are not. To determine if these thyroiditogenic peptides, T4(5), T4(2553), and T0(2553), activated cytotoxic T cells (Tc) and served as target autoantigens when loaded onto indicator cells (BW5147 lymphoma, H2k), lymph node cells from CBA mice immunized with mouse (M) Tg were cultured in vitro with MTg, HTg, or Tg peptide. After MTg or HTg activation, Tc were detected for both MTg- and HTg-loaded target cells in an 18-h, 51Cr-release assay at an effector:target cell ratio of 50:1. These Tc also killed target cells labeled with T4(5), T4(2553), or T0(2553), but not the control peptide T4(2567). When MTg-primed lymphocytes were cultured with T4(5), T4(2553), or T0(2553), specific Tc were also generated against target cells labeled with the respective peptide. The data suggest that one of the thyroiditogenic properties of these peptides previously shown by adoptive transfer of thyroiditis is related to the generation of Tc. In addition, these conserved autoepitopes of Tg also serve as target antigens for Tc.

Animals↗
PubMed1998

Interlaboratory comparison of the assessment of P450 activities in human hepatic microsomal samples.

1. Although the importance of in vitro technology in supporting drug development is widely accepted, there is no real consensus about which approaches should be taken, which substrates should be used, or on the reliability and application of in vitro data. Consequently, as part of a collaborative project to characterize human liver with respect to the major forms of cytochrome P450, an interlaboratory comparison of the analysis of samples for form-specific activities was undertaken. 2. Microsomal fractions were isolated from five different human liver samples taken from the liver bank maintained at the Royal Postgraduate Medical School (RPMS). Aliquots from the five samples were sent to the 11 collaborating laboratories for characterization using their in-house, form-specific assays for cytochrome P450 activities. Although each laboratory assayed protein concentration, total cytochrome P450 content and enzyme activities were calculated using the protein estimation generated by RPMS to eliminate this possible source of variability. 3. With the exception of one laboratory, all estimates of protein concentration were similar (coefficient of variation, CoV, 9-13%) and the rank-order of the five samples was consistent across the laboratories. There was greater variability in the estimates of total cytochrome P450 content (CoV 28-43%), although again rank order of the samples across laboratories was fairly consistent. 4. The various laboratories used a number of different probe substrates, together with a range of conditions (substrate concentration, time of incubation, amount of protein), to assay for activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. However, apart from the occasional outlier, the five samples were ranked for activity of all these forms of cytochrome P450 with a high degree of consistency by the various laboratories and the choice of substrate had no appreciable effect on the ranking of the samples. 5. While this interlaboratory comparison has shown that greater consistency in the approach to in vivo determination of drug-metabolizing activity is desirable, there was little indication that any particular approach or substrate was superior to the others.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Certain activating mutations within helix 6 of the human luteinizing hormone receptor may be explained by alterations that allow transmembrane regions to activate Gs.

Male-limited gonadotropin-independent precocious puberty (MPP) is frequently associated with mutations of the human LH/CG receptor (hLHR) that result in constitutively active hLHRs. Many such activating mutations have been identified in transmembrane 6 of the hLHR, with the substitution of Asp-578 being the most frequently observed mutation. Mutagenesis of a transmembrane helix of a G protein-coupled receptor can cause local alterations in the conformation near the mutated residue, allosteric changes elsewhere in the protein, and/or changes in the interhelical packing of the receptor. Therefore, while it has been hypothesized that activation of the receptor by mutations of Asp-578 may arise via alterations in the interactions of helix 6 with other transmembrane helices and/or by allosterically altering the conformation of the third intracellular loop, it has not been possible to ascertain the role of the sixth transmembrane helix per se in activating Gs in the mutated full-length receptor. Recently, however, we have shown that a peptide KMAILIFT, corresponding to the juxtacytoplasmic portion of helix 6 of the hLHR, is capable of activating Gs. These results suggest that helix 6 itself can directly interact with Gs. Importantly, the KMAILIFT peptide did not include Asp-578, which lies just C-terminal to this sequence. We show herein that a peptide extended to include Asp-578 (KMAILIFTDFT) is a poor activator of Gs. However, if the peptide is synthesized with the aspartate replaced with either a glycine or tyrosine, substitutions that are found in some patients with MPP, these peptides have Gs-stimulating activity. Additionally, a transmembrane 6 peptide with the substitution of Ile-575 with leucine, another mutation found in MPP, mimicked the activating effects of this mutation in the full-length receptor. The ability of peptides in which Asp-578 or Ile-575 is substituted to mimic the activating effects of these mutations in the full-length receptor suggests that the sixth transmembrane helix represents a site for direct interaction with Gs. In addition to the stimulatory effects of transmembrane 6 peptides, peptides corresponding to the juxtacytoplasmic portions of the fourth, fifth, and seventh helices were also able to stimulate Gs. These results are consistent with the hypothesis that the transmembrane helices may form a pocket for interaction with Gs and that constitutive activation of the hLHR may involve the opening of the pocket formed by these helices, thus exposing Gs-binding sites on these helices.

Adenylyl Cyclases↗
PubMed1998

Flexibility of the thyroiditogenic T cell repertoire for murine autoimmune thyroiditis in CD8-deficient (beta2m -/-) and T cell receptor Vbeta(c) congenic mice.

In murine experimental autoimmune thyroiditis (EAT), previous studies have revealed a highly adaptable thyroiditogenic T cell repertoire which involves both CD4+ and CD8+ T cells in the susceptible H2k strain. To further test this flexibility, congenic B10.K mice lacking CD8+ T cells (B2m -/-) or harboring 70% T cell receptor (TCR) Vbeta gene deletions (Vbeta(c)) were immunized with mouse thyroglobulin (MTg) and evaluated for EAT 28 days later. All B2m -/- mice developed moderate antibodies to MTg, and thyroidal inflammation was comparable to B10.K mice, averaging 35-40%. Spleen cells (SC) from MTg-immunized mice were then injected into syngeneic recipients after stimulation in vitro with MTg or with conserved, thyroxine (T4)- or thyronine (T0)- containing 12mer peptides, hT4(5), hT0(2553), or hT4(2553), derived from the primary hormonogenic sites at position 5 or 2553 of human Tg. As previously shown in another H2k strain (CBA/J), all three peptides activated MTg-primed SC to transfer EAT in B10.K mice. hT4(5) and hT4(2553) were further tested in B10.K-Vbeta(c) and beta2m- B10.K mice. Both peptides expanded thyroiditogenic T cells in either strain, resulting in severe thyroiditis in syngeneic recipients. That EAT can develop in the absence of CD8+ T cells or in the presence of a severely restricted TCR repertoire underscores the remarkable flexibility of the thyroiditogenic T cell profile in the susceptible k haplotype.

Adoptive Transfer↗
PubMed1998

Hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, codon 143 CAC-->TAC--a variant with altered oxygen affinity that compromises measurement of glycated hemoglobin in diabetes mellitus: structure, function, and DNA sequence.

OBJECTIVE: To determine the nature and characteristics of a unique hemoglobin variant that causes a spurious increase in glycated hemoglobin (HbA1c). MATERIAL AND METHODS: Blood specimens from four unrelated persons with this hemoglobin variant were examined by conventional laboratory methods, including electrophoresis, high-performance ion-exchange chromatography, and isoelectric focusing; by amino acid sequence analysis, polymerase chain reaction-based DNA sequence analysis, and electrospray ionization mass spectrometry, to establish the molecular structure; and by studies of oxygen affinity under varied conditions, to define the functional characteristics of the hemoglobin variant. RESULTS: The unique hemoglobin variant observed in these four cases is due to the mutation CAC-->TAC, at beta-globin gene codon 143, corresponding to beta 143 (H21) His-->Tyr. This amino acid substitution affects an important 2,3-diphosphoglycerate binding site and slightly increases the oxygen affinity of the hemoglobin variant. CONCLUSION: A hitherto unrecognized hemoglobin variant, encountered in four unrelated persons of Irish or Scots-Irish ancestry, hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, has now been characterized at the molecular, structural, and functional levels. Although it is associated with a slight increase in oxygen affinity, it is without hematologic effect, and its only clinical significance is that it coelutes with HbA1c on ion-exchange chromatography and thereby causes a spurious increase in HbA1c and compromises the use of this analyte to monitor the treatment of diabetes mellitus.

Adult↗
PubMed1997

Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis.

Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 mu mol/L etoposide. After treatment with 17 mu mol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 mu mol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.

Antineoplastic Agents, Phytogenic↗
PubMed1997

A 17-Mer self-peptide of acetylcholine receptor binds to B cell MHC class II, activates helper T cells, and stimulates autoantibody production and electrophysiologic signs of myasthenia gravis.

We have identified in an extracellular segment of the alpha1 subunit of nicotinic acetylcholine receptor (AChR) of homologous muscle a 17-residue autoantigen that, without conjugation to a carrier, activates Th lymphocytes and induces production of autoantibodies that cause electrophysiologic signs of experimental autoimmune myasthenia gravis. A panel of overlapping synthetic peptides revealed two T cell epitopes, one encompassed by residues 121-136 and the other by 129-145. Residues 129 (glutamic acid) and 130 (isoleucine) were implicated in a pathogenic B cell epitope. Peptide 129-145 (Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu-Gln-Asn-Cys-Ser-Met-Lys, a conserved sequence in rat and human AChR) induced autoantibody production in 76% of rats. All seropositive rats had evidence of experimental autoimmune myasthenia gravis; five of five tested had electrophysiologic signs, and all had loss of immunochemically measured autologous muscle AChR. Analogues of 129-145, with single residues substituted by alanine, revealed phenylalanine 135, phenylalanine 137, and glutamic acid 139 as most important determinants of Ag/MHC-II/TCR interactions; phenylalanine 137 is critical for T cell activation. B cells were the major MHC-II-positive cell type to which the self-peptide 129-145 bound in a population of nonimmune splenic cells. More efficient processing and presentation of the Th cell epitope by an expanded population of immune B cells selected by specifically binding another epitope of the same peptide would greatly amplify the production of autoantibodies. Peptide autoantigens of this type could plausibly perpetuate the autoantibody response in myasthenia gravis, and are a rational target for strategies aimed at Ag-specific therapeutic intervention.

Amino Acid Sequence↗
PubMed1997

Activation of multiple interleukin-1beta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis.

Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1. 5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Primary hormonogenic sites as conserved autoepitopes on thyroglobulin in murine autoimmune thyroiditis: role of MHC class II.

A few synthetic peptides corresponding to amino acid sequences on human thyroglobulin (Tg) have been reported to induce moderate thyroiditis or activate mouse Tg (MTg)-primed T cells to transfer thyroiditis in mice susceptible to experimental autoimmune thyroiditis. Using three pairs of 12-mer peptides (1-12, 2549-2560, 2559-2570), with thyroxine (T4) or noniodinated thyronine (T0) at the conserved, hormonogenic site 5, 2553, or 2567 respectively, we reported that iodination was not required for a Tg hormonogenic site to be a thyroiditogenic autoepitope. To determine the relative importance of MHC class II and T cell receptor (TCR) repertoire, we compared two EAT-susceptible k and s (CBA and A.SW) haplotypes and their respective MHC-identical strain (C57BR and SJL) with approximately 50% genomic deletion of TCR Vbeta genes. Whereas k and s strains develop MTg-induced EAT, vigorous immunization with peptides containing T4 or T0 at either 5 or 2553, but not at 2567, led to mild (10-20%) thyroiditis only in some mice of either k strain. TCR Vbeta gene differences played a minor role. T cell responses to all peptide pairs were quite similar in CBA and C57BR mice, and both hT0(2553) and hT4(2553) reciprocally primed and stimulated their T cells. In adoptive transfer, SJL mice were somewhat more responsive to peptide activation than A.SW but much weaker than k strains. By comparing T4- and T0-containing peptides in different haplotypes, we show further that antigenicity of conserved hormonogenic sites is intrinsic, dependent more on amino acid sequence and binding to appropriate class II molecules and less on TCR repertoire or iodination of T0.

Adoptive Transfer↗
PubMed1997

TCR vbeta usage of TSH receptor-specific CD4+ T cells in Graves' disease patients and healthy humans.

Healthy humans have CD4+ T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+ cells present in healthy persons. We studied CD4+ cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves' disease (GD) patients. We used short-term CD4+ cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject. We determined their TCR Vbeta usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vbeta region family, in conjunction with a constant region primer. Six lines preferentially used one Vbeta family (42-94%), different for each line. In all lines, three or less Vbeta families accounted for approximately 60% or more of the Vbeta usage. Different Vbeta regions were used by each subject. There was no obvious difference between the Vbeta usage of the lines from GD patients and healthy controls. These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4+ cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4+ cells specific for the same autoantigen in healthy subjects.

Adult↗
PubMed1997

Monoclonal antibodies specific for peptide epitopes of the epidermal growth factor receptor's extracellular domain.

The ErbB tyrosine kinase receptor family plays an important role in normal cellular growth and differentiation. In addition, ErbB receptor family members are commonly amplified and overexpressed in various human neoplasms and tumor-derived cell lines, where it is believed that increased signalling as a result of receptor overexpression may play an important role in oncogenesis. Consequently, ErbB receptor family members are being investigated rigorously as potential biomarkers of cancer and as therapeutic targets in malignant tissues. Numerous studies now demonstrate the existence of "soluble" ErbB (sErbB) analogs in normal and cancerous tissues. These sErbB proteins embody the extracellular domain (ECD) of the receptor only; they are generated by either proteolytic cleavage or from truncated, alternatively spliced mRNA transcripts. Recently, we have identified an alternate transcript of the human c-erbB1 (Epidermal Growth Factor Receptor) proto-oncogene from placenta that encodes a sErbB1 protein of 60-kDa. This protein, p60 sErbB1, is glycosylated and secreted when expressed in transfected tissue culture cells in vitro. Although "soluble" receptor analogs may play important physiological roles in intercellular communication, tissue morphogenesis, tissue regeneration and repair, and embryogenesis by inhibiting or stimulating specific mitogenic and pattern forming signals, their mechanism of action has not been thoroughly elucidated. To further characterize sErbB1 expression in human tissues and cell lines and to better understand their role in carcinogenesis and normal development, we have generated monoclonal antibodies (MAbs) toward specific peptide epitopes of ErbB1 extracellular subdomains III and IV. These antibody reagents are described here and should be useful experimental, preparative, analytical, diagnostic, and therapeutic reagents for the study of sErbB1 molecules in normal development and cancer.

Amino Acid Sequence↗
PubMed1997

Hb Watts [alpha 74(EF3) or alpha 75(EF4)Asp-->0]: a shortened alpha chain variant due to the deletion of three nucleotides in exon 2 of the alpha 2-globin gene.

We have identified a new, slightly unstable alpha chain hemoglobin variant, present in a Mexican-American family. Amino acid sequencing and mass spectral analysis of the aberrant peptide (alpha T-9) of the variant revealed that the aspartic acid is deleted either at position 74 or 75 of one of the alpha-globin chains. Sequencing of the amplified alpha 2- or alpha 1-globin genes revealed a trinucleotide deletion (GAC) at codon 74 or 74 of the alpha 2 gene. Although the aspartic acid residues of 74 and 75 of the alpha chain are neither a heme nor an inter chain contact, the slight instability of Hb Watts may be due to disturbance of the central cavity of hemoglobin by the deletion of an aspartic acid residue in the EF helix. Hb Watts is the first example of a trinucleotide deletion in the alpha 2-globin gene.

Adult↗
PubMed1996

Induction of blister-causing antibodies by a recombinant full-length, but not the extracellular, domain of the pemphigus vulgaris antigen (desmoglein 3).

Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.

Amino Acid Sequence↗