Biomedical subjects
Publications and source records attributed to J N Whitaker.
Bovine brain cathepsin D cleaved bovine P2 protein to produce three major and several minor peptides. The major P2 peptides formed were shown by amino acid analysis and partial sequencing to be peptides 17-54, 20-58 and 65-131 with the latter predominating. In preliminary experiments, P2 peptide 65-131 did not induce experimental allergic neuritis in Lewis rats in equimolar amounts to the neuritogenic P2.
Defining the immunochemical basis for the detection of BP peptides in body fluids has led to an appreciation of the complexity of the number and location of epitopes in BP peptide 43-88. There appear to be roles for epitopes based on both sequence and conformation of these small peptides. Antisera to some of the fragments of BP peptide 43-88 may have restricted reactivity with BP peptide 43-88 and BP. The amount of BP-like material that enters CSF and cross-reacts with BP peptide 43-88 is related to the recognition of an epitope or epitopes in the C-terminal portion of this peptide. The immunochemical detection of BP peptides in body fluids is dependent on the precise identification of the BP peptides present.
The central nervous system myelin unit (the myelin sheath and the supporting oligodendrocyte) may be damaged by a number of immune-mediated mechanisms involving cellular or humoral factors. In this review the following areas will be considered: the composition and properties of central nervous system myelin, the chemical and biological characteristics of oligodendrocytes, the participation of the central nervous system myelin unit in experimentally induced diseases, and a description and analysis of immunological abnormalities in humans with multiple sclerosis.
The distribution of cathepsin D, an aspartyl endopeptidase, was measured in selected, discrete nuclei of the forebrain of the Brattleboro rat by means of microdissection and radioimmunoassay. The results indicate that cathepsin D is widely distributed, but in varying amounts among nuclear groups in this region of the brain. High concentrations were detected in the supraoptic and paraventricular nuclei. In studies of the vasopressin-deficient Brattleboro rat, an increased content of cathepsin D in the supraoptic nucleus was observed compared to the heterozygous control. No differences were detected between homozygous and heterozygous Brattleboro rats in the caudate, medial preoptic, suprachiasmatic or paraventricular nuclei or globus pallidus. These results raise the possibility that brain cathepsin D may be involved in the physiological events related to fluid homeostasis.
Reversed-phase (RP) high-performance liquid chromatography (HPLC) using a solvent mixture of triethylammonium formate buffer and methanol permitted the rapid separation of myelin basic protein peptide 43-88 and a mixture of synthetic fragments from this peptide. The elution times for some of the peptides were markedly affected by minimal changes in the solvent mixture. Attempts to separate the same peptides by gel permeation HPLC resulted in poor resolution and an aberrant elution pattern unrelated to molecular size. With the use of the volatile triethylammonium formate buffer in the RP-HPLC, material could be more readily separated and easily recovered by freeze-drying. Analysis of the components separated by this system of RP-HPLC demonstrated that the preparation of normal human BP peptide 43-88 results in an admixture of peptides 43-87 and 43-88. This procedure of RP-HPLC should make it possible to analyze the degradation of myelin basic protein peptide 43-88 and to isolate the degradation products for characterization.
The distribution of cathepsin D (CD) was surveyed in rat tissues by light microscopic immunocytochemistry. Although immunoreactive CD was detected in all tissues examined, there was a marked difference in the amount in the cytoplasm of different cell types of the same organ. In the retina large amounts of CD were present in the pigment epithelium, ganglion cells, and Müller cells. Moderate to large amounts of CD were also found in neuronal perikarya of the gastrointestinal tract and adrenal medulla; in macrophages in the lung, liver, and spleen; in some secretory cells of the submandibular and lacrimal glands; in parts of renal distal convoluted and collecting tubules; and in the surface transitional epithelium of the calyx, ureter, and urinary bladder. Other cells adjacent to cells containing large amounts of the enzyme had little or no detectable CD themselves. These included hepatocytes, the proximal tubular cells of the kidney, selected cells of the submandibular gland, cells of the zona glomerulosa of the adrenal cortex, and lymphocytes in lymphoid organs. The localization of CD indicates that its degradative effect is exerted preferentially in certain cell types and suggests that physiological influences on CD may have a variety of effects in different organs.
The distribution of cathepsin D, an acidic endopeptidase, was localized by immunocytochemistry in human skeletal muscle obtained from 34 persons with a variety of neuromuscular disorders. Normal human skeletal muscle contained small amounts of cathepsin D, all of which was found close to the sarcolemmal membrane. Immunoreactive cathepsin D was present in the cytoplasm of many infiltrating phagocytic cells and was increased in skeletal muscle fibers from patients with muscular dystrophies, inflammatory myopathies, rhabdomyolysis, acid maltase deficiency, and neurogenic atrophy. In cases of Duchenne type muscular dystrophy, the increase in cathepsin D was especially prominent in small regenerating fibers, in which it was visualized at the ultrastructural level in lysosome-like organelles and extralysosomal locations. The function of cathepsin D in skeletal muscle is unclear, but the present findings suggest a possible role in muscle regeneration and repair. Such a role would necessitate careful selection of drugs which interfere with proteolytic activity if they are to be used as therapeutic agents in treating neuromuscular diseases.
Myelin basic protein (BP) has the capacity to induce experimental allergic encephalomyelitis (EAE) in animals as well as to prevent and suppress EAE. Immunoreactive BP or BP fragments appear in cerebrospinal fluid (CSF) in persons with multiple sclerosis in acute disease periods and in individuals with acute damage to central nervous system myelin. Humoral and cellular immune responses, especially in the CSF, may occur but are of uncertain effect in the etiology and pathogenesis of MS. Attempts to use BP to suppress MS have disclosed no therapeutic benefit.
Previous investigations have demonstrated that the detection and measurement of peptides of myelin basic protein (BP) by immunochemical procedures are heavily dependent on the conformation of BP peptides and the specificities of antisera to these peptides. In the present study a double antibody-radioimmunoassay was used to assess the species specificity of antisera from 4 rabbits and 2 sheep against myelin basic protein (BP) peptide 43-88. Although some antisera showed broad reactivity, others were selective for species of BP used in the immunogen. By optimizing the reaction of one of the sheep anti-human peptide 43-88 for detection of rabbit BP peptide 43-88, it was possible to detect immunoreactive BP peptide 43-88 in the plasma of rabbits previously injected with bovine BP peptide 43-88 and complete Freund's adjuvant (CFA). Peptide could be detected in plasma for 1-2 days immediately after subcutaneous injection and again between days 20 and 50. Plasma antibody to rabbit BP peptide 43-88 also appeared 15-20 days after immunization and persisted often in an oscillatory pattern reciprocal for the peptide level. Neither peptide nor antibody was detected in the plasma of normal rabbits or of rabbits injected with bovine spinal cord homogenate-CFA, bovine renal homogenate-CFA or CFA alone. There was no relationship of the level of immunoreactive BP peptide 43-88, antibody to this peptide or their patterns to clinical or neuropathological changes in the animals. Quantitation and analysis of BP peptides in body fluids of animals with diseases affecting central nervous system myelin may provide information relevant to human demyelinating diseases.
1. A species comparison was conducted on the proteolytic activity in human, dog, rabbit, guinea-pig and rat kidney which can degrade human myelin basic protein peptide 43-88. 2. In rat kidney the degrading activity occurred over a pH range of 4-11.5 with the greatest activities at pH 5 and 9. The peptide degrading activity in human, dog, rabbit and guinea-pig kidney was considerably less than in the rat and occurred predominantly at pH 7 with lesser activity at pH 9. 3. The effects of inhibitors of proteolytic enzymes indicated that the peptide degrading activities at the same two pH's of dog, rabbit and guinea-pig were similar to one another but differed from that of human. 4. These results indicate that the activity for degrading a potential autoantigenic material is widespread in renal tissue among different species and that different enzymes are involved. More generally, these findings suggest that renal proteinases differ among commonly used laboratory animals and also differ from the human enzymes.
The content and distribution of cathepsin D, a lysosomal acidic endopeptidase, were determined by immunochemical methods in rat sciatic nerve near the site of a ligature or after exposure of animals to neurotoxins. In normal sciatic nerve, cathepsin D was localized predominantly in the perinuclear regions of Schwann cells. In ligated nerve, cathepsin D increased equally in both the proximal and distal nerve segments adjacent to the ligature. Although orthograde and retrograde axonal transport of cathepsin D may have contributed to this increase, immunocytochemical methods indicated that Schwann cells or other phagocytic cells accounted for the bulk of the increased cathepsin D content of nerve. Axonal function was nontraumatically altered by the administration of 2,5-hexanedione, acrylamide, B,B'-iminodipropionitrile or zinc pyridinethione. Exposure to any of these neurotoxins raised cathepsin D content throughout the sciatic nerve twofold or more, and greater amounts of immunoreactive cathepsin D in the cytoplasm of Schwann cells could be demonstrated immunocytochemically. These results indicate that changes in cathepsin D content of Schwann cells may be a reflection of their catabolic activity. The increased Schwann cell cathepsin D content in toxic axonopathies is further proof for an enhanced Schwann cell role as a phagocyte resulting from axonal injury.
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Cranial computed tomography (CT) was used to estimate the frequency and permanence of brain abnormalities in 108 consecutive children with acute lymphoblastic leukemia (ALL). Fifty-five patients received cranial irradiation (1,800 rad) with intrathecal methotrexate (RT group) and 53 patients received intravenous and intrathecal methotrexate without irradiation (IVIT group). Continuation treatment included sequential drug pairs for the RT group and periodic IVIT methotrexate for the other group. After 12 to 24 months of serial evaluation, five (9%) of the 55 patients in the RT group have had CT scan abnormalities, compared to 10 (19%) of 52 in the IVIT group (p = 0.171). Fourteen of the 15 patients with CT scan abnormalities had focal or diffuse white-matter hypodensity; these have reverted to normal in most cases, reflecting a dynamic process. While such CT findings are of concern and may be an early indicator of central nervous system toxicity, this remains to be proven. Therapy should not be altered on the basis of abnormal CT scans alone but in the context of the entire clinical situation.
Normal human kidney contains a neutral endopeptidase that can degrade human myelin basic protein peptide 43-88. In the present study, renal homogenates prepared from postmortem tissue obtained from four persons with multiple sclerosis, two with amyotrophic lateral sclerosis, one each with olivopontocerebellar atrophy, subacute sclerosing panencephalitis, and Guillain-Barré syndrome, and four controls were analyzed for the enzymes present that degrade human myelin BP peptide 43-88. There was no evidence that the activity in renal tissue for degrading human BP peptide 43-88 is qualitatively different in persons with MS, other neurologic diseases, or controls. Gel filtration of digested peptide demonstrated the action of an endopeptidase capable of hydrolyzing BP peptide 43-88 into large fragments.
Twenty patients with malignant gliomas were selected for active immunization within 4 weeks following surgery. Each patient had a Karnofsky Functional Rating equal to or greater than 70, a peripheral blood lymphocyte count equal to or greater than 1000 cells/cu mm, skin test responses to one or more of four recall antigens, peripheral blood T-cells equal to or greater than half that of control, and was not receiving steroid therapy at the time of entry into the study. Each patient received subcutaneous inoculations with one of two human glioma tissue culture cell lines (D-54MG or U-251MG) monthly, with 500 micrograms of bacillus Calmette-Guérin cell wall (BCG-CW) being included with the first inoculation. Each patient also received levamisole, 2.5 mg/kg 3 days per week every other week. Radiotherapy and chemotherapy with BCNU were begun after the first month of immunization. Follow-up evaluations included computerized tomography brain scans, neurological examinations, Karnofsky Functional Ratings, and studies of general immune competence. No evidence of allergic encephalomyelitis was noted clinically, nor was any gross or microscopic evidence of such pathology obtained upon autopsy of three of these patients. Serial studies of general immune competence showed no alterations from those previously described with non-immunized patients. Patients who were inoculated with the U-251MG cell line have had a longer survival time compared to those inoculated with the D-54MG cell line (p less than 0.0590) or compared to 58 historical cases of glioma patients treated with levamisole, radiation therapy, and chemotherapy alone (p less than 0.02).
Bovine myelin basic protein (BP) induced a shape change and endogenous phosphorylation of a 45,000 (45K) molecular weight protein of intact human platelets. This effect occurred rapidly over an effective concentration range of 5-100 microM BP. BP peptides encompassing residues 1-42, 43-88, and 89-169 from the BP molecule of 169 residues, neither induced phosphorylation of platelets nor blocked the effect of intact BP. Subfractionation of disrupted platelets demonstrated the phosphorylated 45K protein in the 100,000 xg supernate. When isolated platelet membranes were used, no BP induced phosphorylation of a 45K protein could be detected. The amino acid composition of the purified, phosphorylated 45K protein differed from those of other known platelet proteins. BP itself was also phosphorylated by an endogenous platelet protein kinase(s) present both in the 100,000 xg supernatant and in the isolated membrane fraction of platelets. These results indicate that the normal or pathological release of BP from myelin may lead to phosphorylation of an internal protein of platelets and possibly other tissue elements with resultant metabolic and functional changes.
Previous studies have demonstrated that the kidney is the major site for clearance and catabolism of a peptide (residues 43-88) of encephalitogenic or basic protein (BP) derived from central-nervous-system myelin. In the present investigation rat renal tissue was shown to be capable of degrading human BP peptide 43-88 over the pH range 4-11.5 with peaks of activity at pH5 and pH9. The enzymic activity at pH5 was localized mainly to the 5900 g pellet (crude mitochondrial fraction) and, on the basis of its sulphydryl features, was inferred to be cathepsin B. The enzyme activity at pH9 was greatly enriched in the 100 000 g pellet (microsomal brush-border fraction), and its sensitivity to inhibitors suggested that it was a metalloproteinase. The activity at alkaline pH in the 100 000 g pellet was stimulated 3-fold by non-ionic detergents and 20-fold by ATP and polyphosphates. Through a series of experiments the ATP stimulation of the alkaline proteinase activity was concluded to be the result of a reversal of inhibition imposed by the presence of another cationic protein, methylated bovine serum albumin. Inhibition by certain bivalent cations, the irregular effects of chelators and the effects of poly-L-lysine supported this conclusion. These studies indicate the availability of renal enzymes of different types and in different cellular compartments that are capable of degrading BP peptide 43-88. In particular, the relative amounts of bivalent cations, anions and charged proteins and peptides are likely to be major influences on the activity of the alkaline proteinase in vivo. The control of this degradation as well as the features of the smaller fragments of the peptide formed may determine biological and immune events subsequent to the release of this potentially autoantigenic material.
Five familial cases (in two families) and one sporadic case of a new congenital myasthenic syndrome were investigated. Symptoms arise in infancy or later life. Typically, one finds selective involvement of cervical, scapular, and finger extensor muscles, ophthalmoparesis, and variable involvement of other muscles. There is a repetitive muscle action potential to single nerve stimulus in all muscles and a decremental response at 2 to 3 Hz stimulation in clinical affected muscles. Microelectrode studies reveal markedly prolonged end-plate potential (epp), miniature end-plate potential (mepp), and miniature end-plate current; normal quantum content of the epp; and a smaller than normal or low-normal mepp amplitude. Light microscopy demonstrates predominance of type I fibers, small groups of atrophic fibers, tubular aggregates and vacuoles near end-plates, abnormal end-plate configuration, and nonspecific myopathic changes. Abundant acetylcholinesterase activity is present at all end-plates, and the activity and kinetic properties of this enzyme in muscle are normal. Calcium accumulated at the end-plate in one patient. Quantitative electron microscopy shows decrease in the size of nerve terminals, increase in the density of synaptic vesicles, and reduction in the length of postsynaptic membranes. There is focal degeneration of junctional folds with corresponding loss of acetylcholine receptor, most marked in cases with the lowest mepp amplitude. There are no immune complexes at the end-plate. Fiber regions near end-plates display dilation, proliferation, and degeneration of the sarcoplasmic reticulum; nuclear, mitochondrial, and myofibrillar degeneration; and vacuoles resembling those found in periodic paralysis. A prolonged open time of the acetylcholine-induced ion channel is considered to be the basic abnormality and may account for the physiological, morphological, and clinical alterations.