Specific Laboratory Test for Diagnosis of Multiple Sclerosis

Lymphocytes from patients with multiple sclerosis are much more susceptible to the inhibitory activity of linoleic acid (0·08 mg/ml) when tested for sensitization to thyroid by the macrophage electrophoretic mobility test (91% inhibition) than are those from normal subjects (57% inhibition). Cells from patients with a variety of other neurological diseases give 47% inhibition with linoleic acid. These differences are specific for multiple sclerosis and can be used as an in-vitro diagnostic test for the disease. Nearly 43% of clinically normal near relatives of patients with multiple sclerosis show an “anomalous” figure of about 77%; in the remainder the figure is the same as in the general population (57%). An anomalous result is compatible with lifelong freedom from M.S. Possibly a congenital anomalous handling of unsaturated fatty acids is a constant feature of the disease.     

Regulation of the activity of p38 mitogen-activated protein kinase by Akt in cancer and adenoviral protein E1A-mediated sensitization to apoptosis

The adenoviral early region 1A (E1A) protein mediates sensitization to different stimulus-induced apoptosis, such as tumor necrosis factor alpha, UV and gamma irradiation, and different categories of anticancer drugs. However, the molecular mechanisms underlying E1A-mediated sensitization to apoptosis are still not completely defined. Here, we show that E1A-mediated sensitization to apoptosis by the inactivation of a key survival factor Akt and the activation of a pro-apoptotic factor p38. Also, inactivation of Akt by either a specific inhibitor or a genetic knockout of Akt1 results in p38 activation, possibly through the release of the activity of p38 upstream kinases, including ASK1 and MEKK3. In addition, we showed that p38 phosphorylation is downregulated and Akt phosphorylation is upregulated in multiple human tumor tissues, and this correlates with tumor stage in human breast cancer. A deletion mutation of a conserved domain of E1A, which is required for E1A-induced downregulation of Akt activity, disrupts E1A-mediated upregulation of p38 activity and also eliminates E1A-mediated chemosensitization. Thus, activation of p38 and inactivation of Akt may have general implications for tumor suppression and sensitization to apoptosis.     

Carcinoembryonic Antigen in Management of Colorectal Carcinoma

Carcinoembryonic antigen (C.E.A.) estimation has been used in the preoperative assessment of colorectal carcinoma patients and has been shown to give a useful guide to the presence of metastatic disease and ultimately to a poor prognosis if the serum concentration is 100 ng/ml or more. C.E.A. has been shown to be a more reliable index of tumour spread than either clinical examination or serum alkaline phosphatase estimation. Raised C.E.A. levels of less than 100 ng/ml do not, however, necessarily imply a poor prognosis. Routine C.E.A. estimation may have a valuable role in the assessment of the colorectal cancer patient by identifying those likely to benefit from postoperative chemotherapy.The test has also been assessed in a group of patients attending cancer follow-up clinics after radical resection of a colorectal tumour. Raised C.E.A. occurred in most of those developing recurrent disease, and in several patients a rising C.E.A. level preceded clinical or biochemical evidence of recurrence. C.E.A. estimation is a superior guide and of clinical importance when applied to the follow-up of the colorectal cancer patient.     

Coronavirus isolates SK and SD from multiple sclerosis patients are serologically related to murine coronaviruses A59 and JHM and human coronavirus OC43, but not to human coronavirus 229E.

Two coronaviruses (SK and SD), isolated from fresh autopsy brain tissue from two multiple sclerosis patients, were compared with known human and murine coronaviruses. In plaque neutralization assays, antisera prepared against multiple sclerosis isolates SK and SD demonstrated significant cross-reactivity to each other and to murine coronavirus A59, weak cross-reactivity to murine coronavirus JHM, but no cross-reactivity to the human coronavirus 229E. Antiserum to SK or SD failed to inhibit hemagglutination of chicken erythrocytes by the human coronavirus OC43. However, OC43 antiserum neutralized both SD and SK. Specific coronavirus polypeptides were identified and compared by immunoprecipitation and polyacrylamide gel electrophoresis. Infected and mock-infected 17Cl-1 cells were pretreated with actinomycin D and labeled with [35S]methionine. Polypeptides in Nonidet P-40 cytoplasmic extracts were immunoprecipitated with homologous and heterologous antisera. Identical polypeptides were precipitated from A59-, SD-, or SK-infected cell extracts by SD, SK, OC43, or A59 antisera. The polypeptides of human virus 229E were antigenically distinct, with the exception of weak recognition of a polypeptide of 50,000 molecular weight. We conclude that the two multiple sclerosis virus isolates SK and SD are closely related serologically to the murine coronavirus A59 and the human coronavirus OC43.     

Measles Virus Antibody in Cerebrospinal Fluids from Patients with Multiple Sclerosis

A strong measles-specific gel precipitation reaction was found in the cerebrospinal fluid (C.S.F.) of two patients with multiple sclerosis (M.S.) (total of 15 tested). The serum and C.S.F. specimens from these two patients were tested for measles antibody by six assay methods. The results were compared with those obtained from serum and C.S.F. specimens of a patient with subacute sclerosing panencephalitis (S.S.P.E.). The gel precipitation line produced by the C.S.F. from the M.S. patients was identical with one of the three lines produced by the C.S.F. from the S.S.P.E. patient. The main antigenic component responsible for measles antibody appearing in the C.S.F. of the S.S.P.E. patient and the M.S. patients was also electrophoretically similar, and the corresponding antibody was associated with IgG. The serum/C.S.F. antibody titre ratios with the various assay methods used suggest that the C.S.F. antibodies are mainly to other than envelope components of measles virus. No complement-fixing antibody against 27 other viruses or Mycoplasma pneumoniae was found in the C.S.F. of the two M.S. patients.     

Radioimmunoassay of myelin basic protein in cerebrospinal fluid and its clinical application to patients with neurological diseases

A double antibody radioimmunoassay for myelin basic protein (BP) was developed that detects BP concentration greater than 0.5 ng/ml in cerebrospinal fluid ( CSF ). By this method, the amount of BP in CSF of 62 patients with various neurological disorders including 9 cases of multiple sclerosis was measured. The amount of BP in CSF obtained from 2 cases of multiple sclerosis in the exacerbation had significantly higher values than that in remission or during the gradual recovering stage. Also two of the 6 patients with myelopathy ( etiology unknown ) and one of the 11 patients with cerebrovascular disease, having acute attack, had significantly high BP values in CSF. The amount of BP in CSF from the other neurological patients showed normal level, compared with that from control patients. The presence of cross-reacting materials with bovine BP in CSF appears to be characteristic of acute myelin sheath destruction on not only patients with multiple sclerosis but also those with myelopathy and cerebrovascular disease.     

Mitochondrial protein nitration primes neurodegeneration in experimental autoimmune encephalomyelitis

The mechanisms of axonal and neuronal degeneration causing visual and neurologic disability in multiple sclerosis are poorly understood. Here we explored the contribution of mitochondria to neurodegeneration in the experimental autoimmune encephalomyelitis animal model of multiple sclerosis. Oxidative injury to the murine mitochondrion preceded the infiltration of inflammatory cells, classically heralded as the mediators of demyelination and axonal injury by transection. Nitration of mitochondrial proteins affected key subunits of complexes I and IV of the respiratory chain and a chaperone critical to the stabilization and translocation of proteins into the organelle. Oxidative products were associated with loss of mitochondrial membrane potential and apoptotic cell death. Reductions in the rate of synthesis of adenosine triphosphate were severe and even greater than those associated with disorders caused by mutated mitochondrial DNA. Mitochondrial vacuolization, swelling, and dissolution of cristae occurred in axons as early as 3 days after sensitization for experimental autoimmune encephalomyelitis. Our findings implicate mitochondrial dysfunction induced by protein inactivation and mediated by oxidative stress initiates a cascade of molecular events leading to apoptosis and neurodegeneration in experimental autoimmune encephalomyelitis that is not mediated by inflammatory cells.     

E1A sensitizes cancer cells to TRAIL-induced apoptosis through enhancement of caspase activation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis of cancer cells. Sensitization of cancer cells to TRAIL, particularly TRAIL-resistant cancer cells, could improve the effectiveness of TRAIL as an anticancer agent. The adenovirus type 5 E1A that associates with anticancer activities including sensitization to apoptosis induced by tumor necrosis factor is currently being tested in clinical trials. In this study, we investigated the sensitivity to TRAIL in the E1A transfectants ip1-E1A2 and 231-E1A cells and the parental TRAIL-resistant human ovarian cancer SKOV3.ip1 and TRAIL-sensitive human breast cancer MDA-MB-231 cells. The results indicated that the percentage of TRAIL-induced apoptotic cells was significantly higher in the E1A transfectants of both cell lines than it was in the parental cell lines. To further investigate the cellular mechanism of this effect, we found that E1A enhances TRAIL-induced activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity by a specific inhibitor, Z-DEVD-fmk, abolished TRAIL-induced apoptosis. In addition, E1A enhanced TRAIL expression in ip1-E1A2 cells, but not in 231-E1A cells, and the anti-TRAIL neutralizing antibody N2B2 blocked the E1A-mediated bystander effect in vitro. Taken together, these results suggest that E1A sensitizes both TRAIL-sensitive and TRAIL-resistant cancer cells to TRAIL-induced apoptosis, which occurs through the enhancement of caspase activation; activation of caspase-3 is required for TRAIL-induced apoptosis; and E1A-induced TRAIL expression is involved in the E1A-mediated bystander effect. Combination of E1A and TRAIL could be an effective treatment for cancer.     

Human papillomavirus (HPV) 16 E6 sensitizes cells to atractyloside-induced apoptosis: Role of p53, ICE-like proteases and the mitochondrial permeability transition

Infection of cervical epithelial cells with certain high risk HPV genotypes is thought to play an etiologic role in the development of cervical cancer. In particular, HPV type 16 and 18 early protein 6 (E6) is thought to contribute to epithelial transformation by binding to the tumor suppressor protein p53, targeting it for rapid proteolysis, resulting in loss of its cell cycle arrest and apoptosis-inducing activities. Recent data indicate that factors responsible for triggering apoptosis reside in the cytoplasm of cells, and not in the nucleus. In particular, the findings that mitochondria are required in certain cell-free models for induction of apoptosis and that bcl-2 is localized to mitochondria have focused attention on the role of the mitochondrial membrane permeability transition (MPT) in apoptosis. Here we present data to indicate that HPV 16 E6 expression sensitizes cells to MPT-induced apoptosis. We also report that HPV 16 E6 sensitization of cells to MPT-induced apoptosis occurs only in the presence of wildtype (wt) p53 expression. The extent of apoptosis induced by atractyloside (an inducer of the MPT) in normal, temperature-sensitive (ts) p53, and HPV-16 E6 transfected J2-3T3 cells, and the HPV expressing cervical carcinoma cell lines SiHa, Hela and CaSki was determined. C33A cells, which express mutant p53 but not HPV, were also exposed to atractyloside in the presence or absence of HPV 16 E6 expression. Dose-dependent apoptosis induced by atractyloside in normal J2-3T3 cells and cervical carcinoma cells was measured by loss of cell viability, nuclear fragmentation and DNA laddering. The sensitivity of cells to atractyloside-induced apoptosis was found to be: HPV 16 E6-J2-3T3 > CaSki > normal-J2-3T3 cells ≈ ts p53-J2-3T3 ≈ vector-J2-3T3 cells > Hela > SiHa > C33A ≈ C33A 16 E6. Cyclosporin A (CsA), an inhibitor of the MPT, and ICE-I, a protease inhibitor, provided protection against atractyloside-induced apoptosis. These findings indicate that: 1) high risk HPV 16 E6 protein is capable of sensitizing cells to apoptosis; 2) HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis occurs in a p53-dependent fashion; 3) the target of HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis is the mitochondria; and 4) HPV 16 E6 sensitization of cells to atroctycoside-induced apoptosis involves an ICE-like protease-sensitive mechanism, regulating the onset of the MPT. These findings constitute the first evidence that mitochondria play a role in HPV 16 E6 modulation of apoptosis. J. Cell. Biochem. 66:245-255. © 1997 Wiley-Liss, Inc.     

Patterns of disease activity in multiple sclerosis: clinical and magnetic resonance imaging study.

OBJECTIVE--To compare the abnormalities shown by magnetic resonance imaging of the brain in three clinically distinct groups of patients with multiple sclerosis, and to correlate the extent of abnormality with the degree of clinical disability in the three groups. DESIGN--All patients underwent magnetic resonance imaging and full neurological examination, and their disability was scored according to the expanded Kurtzke disability state scale. SETTING--National Hospital for Nervous Diseases (Multiple Sclerosis NMR Research Group). PATIENTS--Three groups of patients with confirmed multiple sclerosis were studied: 12 patients with minimal disability despite a long (greater than 10 years) duration of illness (benign multiple sclerosis), 16 who had developed progressive disability after a relapsing and remitting course (secondary progressive multiple sclerosis), and 13 who had had progressive disability from the onset of the disease (primary progressive multiple sclerosis). MAIN OUTCOME MEASURES--Number and size of lesions in 17 anatomically defined sites; total lesion load, estimated with an arbitrary scoring system weighted for the size of lesions; and disability score. RESULTS--Magnetic resonance imaging showed that all 41 patients had abnormalities. These were extensive in the groups with secondary progressive and benign disease compared with the group with primary progressive disease. The lesions in the patients with secondary progressive disease were larger and more confluent than those in the two other groups (p = 0.007). Most lesions (85%) in the patients with primary progressive disease were under 5 mm in diameter; this percentage was higher than that in the two other groups (p = 0.032). Consequently the patients with primary progressive disease had the lowest mean lesion load (36.7); that in the patients with benign disease was 52.7 and that in the patients with secondary progressive disease 64.6 (p = 0.05). No correlation existed between disability and total lesion load. The distribution of brain lesions and of detectable lesions of the spinal cord, and the frequency of cortical atrophy, were similar in all groups. CONCLUSIONS--No relation was found between the degree of clinical disability and the extent of abnormality shown by magnetic resonance imaging: patients with clinically benign disease often had extensive abnormalities and those with primary progressive disease had surprisingly few lesions. Though magnetic resonance imaging increases knowledge of the disease process in multiple sclerosis and is invaluable in diagnosis, it is not helpful in predicting disability in individual patients.     

Activation of Cdc2 contributes to apoptosis in HPV E6 expressing human keratinocytes in response to therapeutic agents

Infection with human papillomaviruses (HPV) is strongly associated with the development of cervical cancer. The HPV E6 oncogene induces apoptosis in cervical cancer precursor lesions but the mechanism is poorly understood. While it is expected that inactivation of p53 by E6 should lead to a reduction in apoptosis, E6 also sensitizes cells to apoptosis under some experimental conditions. Here, we demonstrate that expression of E6 in human keratinocytes rendered sensitization to chemotherapeutic agents. The cell death was shown to be by apoptosis involving caspase activation and the mitochondria pathway. To explore mechanisms involved in sensitization of E6 expressing cells to apoptosis, we used a proteomic approach to identify proteins differentially expressed in E6 expressing and control keratinocytes. Among nearly a thousand proteins examined, Cdc2 was demonstrated to be the most dramatically up-regulated protein in E6 expressing cells. p53 degradation appears to be important for the up-regulation of Cdc2 by E6. Using genetic, pharmacologic, and siRNA strategies, a role for Cdc2 in E6 expression-conferred apoptosis was demonstrated. Thus, these results have important therapeutic implications in enhancing the efficacy of chemotherapy.     

Effect of delayed hypersensitivity due to Staphylococcus or BCG vaccine on the sensitivity of animals to infection with heterologous bacteria or influenza virus

Experiments on mice of different strains have demonstrated that sensitization with BCG vaccine slightly increases resistance to infection with Francisella tularensis, Escherichia coli 819 and influenza A2 virus in mice of those strains which are capable of developing a high level of delayed hypersensitivity (DH). On the contrary, sensitization with Staphylococcus aureus b-243 decreases this resistance. A sharp increase in resistance to infection has been achieved in sensitized animals receiving DH-inducing specific antigen (old tuberculin or staphylococcal phagolysate) 24 hours before inoculation. This increased resistance to infection is due mainly to the eliminating capacity of the reticuloendothelial system and not to the bactericidal factors of the serum. The level of sensitization and the manifestation of DH reaction have been found to be genetically determined and to govern the degree of activation of nonspecific immunity.     

Identification of a locus on mouse chromosome 3 involved in differential susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's virus-induced demyelinating disease results from a chronic infection in the white matter of the central nervous system and provides an excellent model for human multiple sclerosis. Like multiple sclerosis, there are genetic risk factors in disease development, including genes associated with the major histocompatibility complex and with those encoding the beta chain of the T-cell receptor. Comparisons of the susceptible DBA/2 and resistant C57BL/6 strains have indicated an important role for the H-2D locus and for a non-H-2 gene (not involving the beta chain of the T-cell receptor) in differential susceptibility. In the present report, analysis of recombinant-inbred strains (BXD) between the DBA/2 and C57BL/6 strains indicated that this non-H-2 locus is located at the centromeric end of chromosome 3 near (4 +/- 4 centimorgans) the carbonic anhydrase-2 (Car-2) enzyme locus.     

Activated suppressor cell dysfunction in progressive multiple sclerosis

Concanavalin A (Con A)-induced suppressor activity has previously been shown to be reduced in multiple sclerosis (MS) patients with active clinical disease. In this study, we demonstrate that OKT3, as well as Con A induced suppressor activity mediated by unfractionated peripheral blood mononuclear cells is reduced in patients with the progressive form of MS. By performing reconstitution experiments involving E+, T4+, or T8+ cells derived from either MS patients or controls, and normal allogeneic macrophages or E- cells, we sought to define the cellular basis for this suppressor defect. In both MS and control groups, E+ cells were required to obtain measurable levels of suppression. Suppressor levels induced by Con A-activated cultures containing E+ cells from MS patients were lower than those induced by those containing control donor E+ cells. Suppression mediated by T8+ cells from MS patients was also lower than for controls. In the control group, suppression mediated by T8+ cells exceeded that mediated by T4+ cells; such differences were not apparent in the MS group. These results suggest that although Con A-induced suppression can be mediated by a number of T and non-T cell subsets, the functional suppressor defect measured in the MS population does involve the T8+ cell subset.     

The role of serum 'adhesive factor' and leukocyte sensitization in pathogenesis of multiple sclerosis

The reactivity to the myelin basic protein, brain gangliosides purified derivative and also the influence of the serum on the cells adherence were determined by micromodification of the leukocyte adherence inhibition (LAI) test. 46 multiple sclerosis patients, 21 patients with hereditary disease and 41 donors were examined. The cellular sensitization to the myelin basic protein and brain gangliosides was revealed in 57% of the multiple sclerosis patients. The leukocyte adherence was increase in 53.5% patients when autologous serum was added. The adherence factor was most often found (90% case) in patients with severe neurological deficit. It is suggested that this factor is a connected t the increased levels of circulating adhesion molecules in multiple sclerosis patients.     

Thermal sensitivity and radiosensitization in Chinese hamster V79 cells exposed to 2-aminopurine or 6-thioguanine

Chinese hamster V79 cells were exposed to 10(-6) mol liter-1 2-aminopurine (2-AP) or 6-thioguanine (6-TG) for 18 or 40 h, and then tested for sensitivity to X rays, heat, or a combined treatment of heat and radiation. Cells exposed to 6-TG were sensitive to X rays, while those treated with 2-AP showed little or no sensitivity. At 42 degrees and 45 degrees C moderate sensitization resulted from 2-AP treatment, with greater sensitization resulting from treatment with 6-TG. Combined heat and X-ray treatment of cells exposed to 2-AP yielded sensitization similar to heat treatment alone, while cells exposed to 6-TG before receiving the combined treatment showed a degree of sensitization greater than that due to either treatment by itself but less than that of the two treatments added together. Uptake of the purine analogues into cellular DNA was measured by high-pressure liquid chromatography. At the 1% detection level, after either an 18- or a 40-h exposure of cells to 10(-6) mol liter-1 2-AP or 6-TG, no base substitution was found. Analysis of cell cycle distributions by flow cytometry revealed only very small changes following exposure of cells to the purine analogues.     

Past exposure to sun,skin phenotype,and risk of multiple sclerosis: case-control study

Objective To examine whether past high sun exposure is associated with a reduced risk of multiple sclerosis.Design Population based case-control study.Setting Tasmania, latitudes 41-3°S.Participants 136 cases with multiple sclerosis and 272 controls randomly drawn from the community and matched on sex and year of birth.Main outcome measure Multiple sclerosis defined by both clinical and magnetic resonance imaging criteria.Results Higher sun exposure when aged 6-15 years (average 2-3 hours or more a day in summer during weekends and holidays) was associated with a decreased risk of multiple sclerosis (adjusted odds ratio 0.31, 95% confidence interval 0.16 to 0.59). Higher exposure in winter seemed more important than higher exposure in summer. Greater actinic damage was also independently associated with a decreased risk of multiple sclerosis (0.32, 0.11 to 0.88 for grades 4-6 disease). A dose-response relation was observed between multiple sclerosis and decreasing sun exposure when aged 6-15 years and with actinic damage.Conclusion Higher sun exposure during childhood and early adolescence is associated with a reduced risk of multiple sclerosis. Insufficient ultraviolet radiation may therefore influence the development of multiple sclerosis.     

Studies on the neutralization of herpes simplex virus. VIII. Significance of viral sensitization for inactivation by complement.

Early and late IgG of rabbits immunized with herpes virus showed, respectively, 8-fold and 2-fold enhancement of neutralization endpoint in the presence of complement (C). Kinetic curve experiments employing an appropriate amount of virus revealed that both neutralization and sensitization followed first-order reaction, and each IgG possessed a certain range of concentration where neutralization was negligible while sensitization was marked. Dose responses of neutralization and sensitization velocities demonstrated that the C enhancement of late IgG was about 7-fold and that of early IgG more than 20-fold. These facts suggested that the IgGs contained two different entities of complement-requiring (CRN) and non-requiring neutralizing (N) antibodies at different proportions, only the former being responsible for sensitization. The different CRN: N ratios obtained by the endpoint and kinetic methods may mean either that the two antibodies differ in avidity for the virus or that the number of critical sites per virion for CRN antibody is greater than that for N antibody. In this interpretation, sensitization by CRN antibody as well as neutralization by N antibody is thought to result from attachment of a single antibody molecule to the viral critical site. Alternative explanations, ascribing the mechanism of neutralization to steric hindrance of critical sites or to multiple hit of those sites by antibody, were denied by analyses of the present data.