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Biomedical subjects

J N Whitaker

Publications and source records attributed to J N Whitaker.

At least 19 recordsLinked to original sources
PubMed1992

Interstrain cross-reactive idiotypes on monoclonal antibodies to an encephalitogenic myelin basic protein peptide.

In order to assess the role of idiotype (Id) and the anti-Id network in murine experimental autoimmune encephalomyelitis (EAE), Id-bearing monoclonal antibodies (mAb) to human myelin basic protein (MBP) peptide acetyl 1-9, as well as mAb anti-Id, were developed in EAE-susceptible PL/J mice (H-2u). These mice recognize MBP residues acetyl 1-9 as an encephalitogenic determinant. Reactivities of PL/J Id-bearing mAbs to MBP and to MBP peptides were identical to those of mAbs generated against the same MBP peptide in EAE-resistant BALB/c mice (H-2d), even though isotypes of the mAbs differed. By using an inhibitory ELISA and immunoblotting, it was demonstrated that one PL/J mAb anti-Id recognized a public or framework Id, whereas another PL/J mAb-anti Id was directed to a private Id more restricted to the paratopic site. Two Id-bearing PL/J mAbs shared a cross-reactive Id (IdX) on the light chain, and an interstrain IdX was present on both the heavy and light chains of mAbs raised in PL/J and BALB/c mice to the same MBP peptide. The PL/J mAb anti-Id was capable of cross-regulating the production of Id-bearing mAbs by hybridomas across murine strains. These findings suggest that a restrictive family of germ-line genes encode for these Id-bearing antibodies to MBP peptide, irrespective of whether the MBP peptide is encephalitogenic in the murine strain immunized. Manipulation of the Id network may provide a means for modifying autoimmune demyelinating diseases of the central nervous system.

Animals↗
PubMed1992

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8.

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.

Adult↗
PubMed1991

Immunocytochemical localization of cathepsin D in rat ventral prostate: evidence for castration-induced expression of cathepsin D in basal cells.

Cathepsin D (EC3.4.23.5) is an aspartyl endopeptidase involved in lysosomal proteolysis. Its functional role is uncertain. This study was undertaken to determine the cellular and subcellular distribution of cathepsin D in the normal rat ventral prostate and its possible role in the castration-induced atrophy of the gland. Cathepsin D was localized immunohistochemically to perinuclear lysosomes in secretory cells, in capillary endothelial cells, and, occasionally, in stromal cells of the untreated animal. Castration resulted in an increased number of cathepsin D-positive cells in the stroma within 24 hr. By 48 hr after castration autophagolysosomes formed in secretory cells and apoptotic bodies appeared in the epithelium. Although apoptotic bodies generally contained immunoreactive cathepsin D, a subpopulation of larger apoptotic bodies, which commonly rested on the basement membrane and contained multiple inclusions, were more variable in cathepsin D expression. The induction of cathepsin D in dendritic cells basally oriented in the epithelium was noted at 4 days of castration. These cells had a phagocytic phenotype, were distributed periodically along the basement membrane, and were not found in ductal epithelia. Treatment with actinomycin D or hydrocortisone to reduce the rate of regression of the ventral prostate blocked the appearance of these cathepsin D-positive, basally oriented epithelial cells. Our data indicate that this cathepsin D-positive, phagocytic cell differentiates from a cell resident in the prostatic epithelium. We suggest that it differentiates from basal cells in the secretory tubuloalveolar portion of the gland and that it is involved in the destruction of regressed secretory cells.

Animals↗
PubMed1991

The use of protease inhibitors in experimental allergic neuritis.

In experimental allergic neuritis (EAN) break-down of myelin is attributed to macrophages, which among other factors contain and secrete proteases. In vitro studies have shown that cathepsin D, an acidic aspartyl endopeptidase, and plasmin can degrade myelin proteins. In order to elucidate a potential therapeutic effect of protease inhibitors we treated Lewis rats, immunized with bovine peripheral nervous system myelin, with epsilon-amino-caproic acid (EACA) or pepstatin. EACA or pepstatin was administered twice daily by intraperitoneal injection beginning on day 6 postimmunization or from the onset of disease (on day 12) through day 24. Compared to saline-treated controls, animals treated with either of the inhibitors showed delayed development of clinical signs and electrophysiological abnormalities. Maximal severity and the further course of disease, however, were not different in control and treated groups. Immunohistological evaluation of sciatic nerve specimens on day 24 postimmunization showed equal numbers of cells positive for ED1 (macrophages) and cathepsin D in all animal groups. There was also no difference in the spontaneous proteolytic activity of the sciatic nerve homogenates at pH 2.8, 5.0, and 7.4. Incubation of the homogenates with pepstatin, however, significantly reduced proteolytic activity at pH 2.8 and 5.0, while EACA had no effect at any pH tested. These results imply that treatment to limit the infiltration of cathepsin D-positive cells or to reduce the induction or activity of cathepsin D may provide a therapeutic avenue for treating inflammatory demyelination of the peripheral nervous system.

Aminocaproic Acid↗
PubMed1991

Cerebrospinal fluid levels of myelin basic protein-like material and soluble interleukin-2 receptor in multiple sclerosis.

The presence, level and disease activity relationships of soluble interleukin-2 receptor (sIL-2R) in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients are unresolved. We measured CSF immunoreactive myelin basic protein (MBP), a marker of acute myelin damage, and sIL-2R levels in the CSF from 11 patients with active relapsing remitting (RR) MS, five with stable RR MS, eight with chronic progressive (CP) MS, five with other neurologic diseases, and three normal controls. No measurable (less than 100 units/ml) sIL-2R was present in any of the samples. Conversely, MBP levels were elevated in the active RR group compared to the other four groups. These results indicate that, at the sensitivity of assays currently available, levels of CSF sIL-2R do not correlate with the diagnosis or disease activity of MS.

Humans↗
PubMed1991

Changes induced in astrocyte cathepsin D by cytokines and leupeptin.

Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were greater than 95% astrocytes and less than 3% microglia. Under serum-free conditions, leupeptin induced a 1.4- to 2.0-fold increase, maximal by 48 hours, in cathepsin D protein quantified by a radioimmunoassay. Cathepsin D enzymatic activity, inhibitable by pepstatin, also increased. Northern blot analysis demonstrated that leupeptin also increased cathepsin D mRNA expression. Kinetic analysis indicated that maximal cathepsin D mRNA levels are detected 24 h after stimulation with leupeptin. Exposure of astrocytes under the same conditions to rat recombinant interferon-gamma, human recombinant tumor necrosis factor-alpha, human recombinant interleukin-1 beta, lipopolysaccharide, calcium ionophore, or a combination of these reagents did not increase the level of cathepsin D above controls. These results indicate that astrocytic cathepsin D mRNA and protein can be induced by selected materials. Furthermore, the effects attributed to leupeptin as a proteinase inhibitor may be modified by its ability to increase cathepsin D activity.

Analysis of Variance↗
PubMed1991

Proteolysis of microtubule-associated protein 2 and tubulin by cathepsin D.

The in vitro degradation of microtubule-associated protein 2 (MAP-2) and tubulin by the lysosomal aspartyl endopeptidase cathepsin D was studied. MAP-2 was very sensitive to cathepsin D-induced hydrolysis in a relatively broad, acidic pH range (3.0-5.0). However, at a pH value of 5.5, cathepsin D-mediated hydrolysis of MAP-2 was significantly reduced and at pH 6.0 only a small amount of MAP-2 was degraded at 60 min. Interestingly, the two electrophoretic forms of MAP-2 showed different sensitivities to cathepsin D-induced degradation, with MAP-2b being significantly more resistant to hydrolysis than MAP-2a. To our knowledge, this is the first clear demonstration that MAP-2 is a substrate in vitro for cathepsin D. In contrast to MAP-2, tubulin was relatively resistant to cathepsin D-induced hydrolysis. At pH 3.5 and an enzyme-to-substrate ratio of 1: 20, only 35% of the tubulin was degraded by cathepsin D at 60 min. The cathepsin D-mediated hydrolysis of tubulin was optimal only at pH 4.5. These results demonstrate that MAP-2 and tubulin are unequally susceptible to degradation by cathepsin D. These data also imply a potential for rapid degradation of MAP-2 in vivo by cathepsin D either in lysosomes or perhaps autophagic vacuoles of the neuron.

Animals↗
PubMed1991

Neuropathological changes in scrapie and Alzheimer's disease are associated with increased expression of apolipoprotein E and cathepsin D in astrocytes.

With the rationale that the neuropathological similarities between scrapie and Alzheimer's disease reflect convergent pathological mechanisms involving altered gene expression, we set out to identify molecular events involved in both processes, using scrapie as a model to study the time course of these changes. We differentially screened a cDNA library constructed from scrapie-infected mice to identify mRNAs that increase or decrease during disease and discovered in this way two mRNAs that are increased in scrapie and Alzheimer's disease. These mRNAs were subsequently shown by sequence analysis to encode apolipoprotein E and cathepsin D (EC 3.4.23.5). Using in situ hybridization and immunocytochemistry to define the cellular and anatomic pathology of altered gene expression, we found that in both diseases the increase in apolipoprotein E and cathepsin D mRNAs and proteins occurred in activated astrocytes. In scrapie, the increase in gene expression occurred soon after the amyloid-forming abnormal isoform of the prion protein has been shown to accumulate in astrocytes. In Alzheimer's disease, the increased expression of cathepsin D also occurred in association with beta-amyloid. These studies reveal some of the molecular antecedents of neuropathological changes in scrapie and Alzheimer's disease and accord new prominence to the role of astrocytes in neurodegenerative conditions.

Aged↗
PubMed1991

Steroid-dependent postvaricella encephalomyelitis.

Postinfectious encephalomyelitis is an acute demyelinating illness that usually has its onset 3 to 7 days after the onset of a viral exanthem and has a monophasic course over 2 to 4 weeks. Recurrent bouts of postinfectious encephalomyelitis have been described that have resolved spontaneously or with short courses of steroid therapy. We report a patient who developed a chronic, steroid-dependent encephalomyelitis secondary to a varicella infection at 5 months of age.

Adolescent↗
PubMed1990

An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein.

A mAb of the IgG1/kappa isotype was raised against human myelin basic protein (MBP) peptide acetyl 1-9. This mAb, termed F23, reacted with human MBP and human MBP peptides acetyl 1-9, 1-14, and 1-44, but not with MBP peptides 10-19, 80-89, or 45-89. According to the guidelines of the molecular recognition theory, a complementary peptide to human MBP peptide 1-9 was synthesized and used to raise murine mAb with anti-Id activity. Two mAb anti-Id, F25F7 and F25C8, both of the IgM/kappa isotype, were selected for further study. These anti-Id reacted with F23, the mAb for which they were selected, and also reacted with another mAb, which was of the IgG1/kappa isotype and was raised to human MBP peptide 80-89. There was no reaction with another control mAb of the IgG1/kappa isotype or murine myeloma IgG1. By immunoblotting techniques, it was demonstrated that the Id on each of the mAbs to MBP peptides was located on the kappa L chain but also could be recognized in nonreduced IgG. The cross-reactive anti-Id suppressed antibody secretion of Id-producing hybridoma cells in an Id-specific manner, and kinetic studies suggest an intracellular mechanism for the suppression. These cross-reactive Id among antibodies to different MBP peptides imply that the same V region genes of kappa L chains are involved in the selection of antibodies to an autoantigen, like MBP, and may play a role in the modulation of immune responses against MBP in certain inflammatory demyelinating diseases.

Amino Acid Sequence↗
PubMed1990

Immunocytochemical evidence for the retrograde transport of intraaxonal cathepsin D: possible relevance to the dying-back process.

Immunocytochemical localization of cathepsin D was studied in rat nerves using an antibody to cathepsin D (CD). In normal or control nerves, immunoreactive CD was present in the cytoplasm of Schwann cells but not in axons. In ligated or transected nerves, intraaxonal CD appeared as granular or elongated particles increasing with time at the cut end of the distal stump. In the proximal stump of transected nerves, CD was detected in the nascent axon tips, as well as in a length of axon extending up to the first node of Ranvier. In swollen axons induced by 2,5-hexanedione (HD), CD was associated with granular particles in areas where bidirectional organelle movement was impaired. This study represents the first documentation of intraaxonal CD and provides evidence that immunoreactive CD originates at the nerve terminal in conjunction with the anterograde-to-retrograde (A-R) conversion process of axoplasmic transport. When the A-R conversion process is induced at more proximal sites in the nerve by transection, CD can be observed. In circumstances where axoplasmic transport is impaired, such as nerves exposed to 2,5-HD or ligated and transected nerves, CD-positive granular particles were seen in association with retrogradely moving organelles and their accumulation was related to sites of preferential axonal degeneration.

Animals↗
PubMed1990

Antibody to peptides of human myelin basic protein in post-rabies vaccine encephalomyelitis sera.

Development of neurologic complications after Semple rabies vaccine is closely linked to development of antibody to myelin basic protein (MBP). The portions of MBP against which the antibodies are directed were analyzed by enzyme immunoassay in sera and cerebrospinal fluid from 27 patients with vaccine complications. Most of the antibody was directed to regions of MBP peptides 45-89 and 90-170. There was no apparent correlation between antibody specificity for MBP peptides 1-44, 45-89 and 90-170 and the type of post-vaccinal neurologic complication. We conclude that the immunoglobulin repertoire in human B lymphocytes for responding to human MBP favors the portion of the MBP molecule containing residues 45-170.

Antibodies↗
PubMed1990

Conformational correlates of the epitopes of human myelin basic protein peptide 80-89.

Different epitopes residing within the decapeptide of residues 80-89 of human myelin basic protein (MBP) exist in the MBP-like material detected in human CSF and urine. In the present study, the structure of human MBP peptide 80-89 was examined by a combination of physical measurements and correlated with its varying immunochemical reaction with three polyclonal antisera. At least two epitopes are present in the decapeptide. Progressive shortening and reduction in net negative charge of MBP peptide 80-89 to form peptides 81-89, 82-89, 83-89, and 84-89 revealed an epitope not present in intact MBP. Circular dichroism and Fourier-transform infrared of these MBP peptides in water demonstrated random structure that was partially changed to beta-structure in the shorter peptides. In methanol, used as a model for a lipid environment, the random structure was diminished and was replaced by alpha-helix and beta-structure, especially in the shorter peptides. The findings indicate that the range of epitopes present in this decapeptide is influenced by conformation, which, unexpectedly, becomes progressively less random as the peptide becomes smaller, especially in a hydrophobic environment. This behavior has implications for the immunochemical detection of small antigens or antibodies to them in tissue extracts or body fluids.

Circular Dichroism↗
PubMed1990

Myelin basic protein-specific T lymphocyte lines from MS patients and healthy individuals.

We derived a total of 146 T lymphocyte lines specific for human myelin basic protein (MBP) from the peripheral blood of 20 MS patients and from a control group of 12 healthy donors, and determined the reactivities of T cell lines by [3H]thymidine incorporation on exposure to MBP and MBP peptides 1-44, 45-89, and 90-170. We defined HLA restriction of the T lines by using monoclonal antibodies against monomorphic determinants on human HLA-DR, HLA-DQ, and HLA-DP molecules. MBP-specific T cell lines could be isolated with a comparable efficiency from MS patients and healthy individuals. In both groups, MBP-specific T lymphocytes recognized at least 4 different epitopes in the MBP molecule, and specificities showed comparable patterns for different MBP peptides. MBP-specific T cell lines derived from MS patients and controls were restricted by DR products of the human major histocompatibility class II locus. Notable phenotypic differences of T cell lines existed between the 2 groups. Lines isolated from MS patients expressed predominantly the CD3+ CD4+ CD8- phenotype, while some control lines were composed of up to 87% CD3+CD4+CD8+ T lymphocytes. These findings illustrate the presence of MBP-specific T cells in MS patients and controls that are similarly sensitized to MBP and restricted by HLA-DR products.

Adult↗
PubMed1989

Cathepsin D activity in human peripheral blood mononuclear leukocytes.

To investigate the cellular origins of cathepsin D (CD) in inflammatory lesions, the CD content of lymphocyte subsets, monocytes, and macrophages were compared. Human monocytes, B lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes were separated from peripheral blood of normal donors. CD content was 0.13 +/- .01 micrograms equivalents of CD per million cells and significant differences between different cell types were not found. To determine the CD content of macrophages, differentiation of peripheral blood monocytes was induced by either in vitro culture or treatment with 4 beta-phorbol-12-myristate-13-acetate (PMA). Macrophages induced by five-day culture contained four times more CD than unstimulated monocytes, and macrophages induced by 18-h treatment with 20 mg/ml 4 beta-PMA contained nine times more CD than monocytes treated with 4 alpha-PMA, an inactive stereoisomer of 4 beta-PMA. These results suggest that macrophages are one of the enriched sources of CD in inflammatory lesions.

B-Lymphocytes↗
PubMed1989

Activation of astrocytic lysosomal proteinases by factors released by mononuclear leukocytes.

Lysosomal proteinases are increased in the tissue lesions of experimental allergic encephalomyelitis and have been implicated in the degradation of myelin proteins. The cellular origins of the increased proteinases are not known but reactive astrocytes found in areas of increased activity are candidate cells. To evaluate the potential of astrocytes as the source of these proteinases, cathepsin B (CB) and cathepsin D (CD) levels were measured in lysates of cultured astrocytes from neonatal rats. Because astrocytes are activated by inflammatory mediators in demyelinating lesions the effect of activation on proteinase levels was examined. Culture supernatants from mononuclear leukocytes stimulated with either concanavalin A or phytohemagglutinin (PHA) induced significant increases in the astrocytic proteinases. Neither PHA alone, interleukin-1, interleukin-2, nor gamma-interferon induced significant increases. Fractions of the supernatant from PHA stimulated mononuclear leukocytes were tested and activity was found in fractions corresponding to a molecular weight of 45-50,000. These studies demonstrate that astrocytes contain significant amounts of CB and CD activity which can be increased by a factor or factors released by activated mononuclear leukocytes.

Animals↗