Gender effects on intramuscular interferon beta-1a in relapsing-remitting multiple sclerosis: analysis of 1406 patients.

BACKGROUND: We aimed to evaluate effects of gender on efficacy and safety of intramuscular (IM) interferon beta (IFNß)-1a in patients with relapsing-remitting MS (RRMS) or clinically isolated syndromes (CIS) characteristic of early MS. METHODS: Pooled data from 1406 (1027 women; 379 men) patients enrolled in five clinical studies of IM IFNß-1a were analyzed. One analysis examined data for all patients treated with IM IFNß-1a from all studies. Separate analyses were conducted of pooled IM IFNß-1a-treated groups from all studies and pooled IFNß-1a-treated and placebo-treated patients from the placebo-controlled studies. Outcome measures included time to first relapse, annualized relapse rate, time to disability progression, number of gadolinium-enhanced lesions, adverse events, laboratory evaluations, and neutralizing antibodies. RESULTS: All efficacy assessments indicated similar treatment effects of IM IFNß-1a in men and women with no significant treatment-by-gender interactions. Women reported more headaches, urinary tract infections, and depression in the analysis; however, these were also common in women who received placebo. Men reported more frequent flu-like symptoms in the placebo-controlled studies only. There were no other differences in the safety profile of IM IFNß-1a between men and women. CONCLUSIONS: We conclude that no significant gender-related differences were found in the efficacy and safety of IM IFNß-1a in patients with RRMS or CIS.

Cerebellar granule cells in vitro. A light and electron microscope study.

The behavior of granule cells in mature cerebellar cultures derived from newborn mice was studied by light and electron microscopy. Many granule cells remained in the explants as an external granular layer. These cells were differentiated, as evidenced by formation of bundles of parallel fibers and by development of synapses between granule cell axons and Purkinje cell branchlet spines, and between Golgi cell axons and granule cell dendrites. Although the over-all architecture of the cerebellar explants after 18-33 days in vitro was similar to that of the newborn mouse, the evident differentiation of the granule cells suggested that interneuronal relationships resemble those of the mature cerebellum in vivo.

Selective destruction of neurons by a transmitter agonist.

Microinjection of nanomole amounts of kainic acid, a heterocyclic analog of glutamate, into the cerebellums of adult hamsters and rats causes rapid degeneration of Purkinje, basket, stellate, and Golgi II cells, neurons that receive synaptic input from granule cells, whereas the granule cells themselves are spared. This selectivity is consistent with the evidence that glutamate is the granule cell transmitter and supports the hypothesis that kainic acid exerts its neurotoxic effects through glutamate receptors.

Regeneration of oligodendroglia during recovery from demyelinating disease.

Infection of mice with the JHM strain of mouse hepatitis virus causes demyelination as a result of a cytolytic infection of oligodendroglia. In recovery, animals show remyelination, which could result either from surviving oligodendrocytes extending their territory or by generation of new oligodendroglia. Electron microscopic autoradiographic studies with 3H-labeled thymidine demonstrate that the cells associated with remyelination are newly generated oligodendroglia.

Virions from progressive multifocal leukoencephalopathy: rapid serological identification by electron microscopy.

Virions were extracted directly from the brain of a patient with progressive multifocal leukoencephalopathy (PML). They were treated with antiserum to SV40, with rabbit antiserum to previous PML isolates, or with serum from another patient with the same disease and observed directly by electron microscopy. This procedure could be used for the rapid identification of the antigenic nature of virions in cases of PML.

The fine structure of macrophages in lysolecithin-induced demyelination: a freeze-fracture study.

Focal injection of lysolecithin into the thoracic spinal cord was used to induce experimental demyelination in rats. The macrophages which invaded the demyelinating area to phagocytose the myelin breakdown products were examined by electron microscopy using conventional thin-section and freeze-fracture techniques. The cells had the typical foamy cytoplasm characteristic of lipid macrophages. In freeze-fracture preparations, the P-face of the plasmalemma had scattered 10-18 nm round to oval or elongate particles. The cytoplasm was filled with phagocytic vacuoles containing myelin debris. Occasionally, membranous connections could be demonstrated between lysosomes and phagocytic vacuoles. The debris in the vacuoles varied in structure from material with a normal myelin periodicity to partially digested lamellated structures and pseudocrystalline structures to homogeneous-like, lipid-filled vacuoles. These patterns appeared to represent successive stages in the digestion of the ingested myelin.

Immunoperoxidase labelling of rat brain sections with sera from patients with paraneoplastic cerebellar degeneration and systemic neoplasia.

Sera from patients with systemic cancer found by immunofluorescence staining to have antibodies to human cerebellar cell populations were reacted with vibratome sections of rat cerebellum and examined by peroxidase-antiperoxidase (PAP) methods. Seven patients with clinically or pathologically confirmed paraneoplastic cerebellar degeneration and two neurologically normal patients with high titers of anticerebellar antibodies were studied. Sera from all antibody-positive patients, but not from controls, produced intense staining of brain sections. Sera from patients with ovarian adenocarcinoma reacted predominantly with Purkinje cells and neurons within brainstem nuclei. Sera from patients with oat cell carcinoma and one patient with ductal carcinoma of the breast produced nuclear and cytoplasmic staining of neurons throughout the central nervous system. Serum from a patient with Hodgkin's disease labeled the peripheries of Purkinje cells and Golgi II cells. Serum from a patient with mixed mesodermal sarcoma of the ovary labeled Purkinje cells, basket cells, and scattered astrocytes. Staining of extraneural tissues was not observed. This study confirms the presence of antineural antibodies in patients with systemic neoplasia with and without paraneoplastic cerebellar degeneration and suggests that the antigens recognized by this antibody response may vary with the associated neoplasm.

The development of the cerebellar cortex of the Syrian hamster, Mesocricetus auratus. Foliation, cytoarchitectonic, Golgi, and electron microscopic studies.

Although a number of investigations of abnormalities of cerebellar development have been carried out in the hamster, no detailed Golgi or ultrastructural studies of cerebellar development in this species have been reported. This report describes the development of the hamster cerebellar cortex from birth (day 0) through postnatal day 78, as studied by light, Golgi, and electron microscopic methods. Foliation patterns correlate with the expansion of the cerebellar layers and of total cerebellar area. Cytologic and morphologic development of the major cerebellar cell types--Purkinje, Golgi, basket, stellate, granule, and Bergmann glial cells--correlate with those of other species, such as the rat and mouse. Electron microscopic observations at selected developmental ages allow identification and classification of synapses in the early postnatal hamster. Parallel fiber and climbing fiber synapses are already present at birth. Although synaptogenesis probably continues through the first two postnatal months, all major cell types have developed initial synapses by postnatal day 6, at a time when little cellular maturation has occurred. By using gestational rather than natal age, close developmental correlations between hamsters and rat and mouse are possible. Since the gestational period of the hamster is only 16 days, the hamster cerebellum is less mature at birth than that of either the rat or mouse. Thus, the hamster is a convenient animal in which to investigate the effects of various procedures on early cerebellar development.

The pathogenesis of parvovirus-induced cerebellar hypoplasia in the Syrian hamster, Mesocricetus auratus. Fluorescent antibody, foliation, cytoarchitectonic, Golgi and electron microscopic studies.

Cerebellar histogenesis was studied in hamsters infected at birth with a parvovirus, rat virus strain PRE 308. Cerebellar granule cell precursors in these animals were selectively infected and lysed in the external germinal layer before their migration to form the internal granular layer. The effects of the absence of granule cells on cerebellar development and especially on the development of the Purkinje cells and their dendrites was analyzed using fluorescent antibody. Golgi, conventional paraffin, and electron microscopic methods. This study represents the first Golgi and ultrastructural study of the pathogenesis of rat virus infections in the cerebellum. The destruction of the granule cell precursors resulted in a dysplastic cerebellar hypoplasia with total disruption of normal cerebellar stratification and cytoarchitectonics. The Purkinje cells developed misshapen, progressively disoriented dendritic stems lacking tertiary dendrites and studded with numerous spines, devoid of afferent synaptic contacts (naked spines) and encased by glial processes. These developmental studies, together with the mouse mutant studies, demonstrated that the spines of the Purkinje cells were elaborated in the absence of both tertiary dendrites and afferent parallel fiber contacts. Such data suggested that spine formation, once triggered, was intrinsically programmed rather than being dependent on the development of parallel fiber contacts. Despite the loss of a major interneuronal component and disintegration of normal cytoarchitectonic relationships, synapses in the cerebellar cortex developed normally as long as both the pre- and post-synaptic elements were present. Thus synaptic specificity is maintained in the face of gross disruption of cytoarchitectonic relationships. If either the pre- or post-synaptic portion of a contact was absent, then glial processes isolated the persisting element or aberrant contacts formed. In addition to glial encasement of naked spines, there were dendrodendritic articulations between Purkinje cell dendrites, some of which were joined by septate, plaque-like junctions. Aberrant synaptic contacts between mossy and climbing fiber glomeruli and the smooth surface of the Purkinje cell somata were found rarely. In addition to these contacts which also occur in the hypoplastic cerebella produced by other methods, previously undescribed non-synaptic spine-articulations between Purkinje cell dendrites were seen. The role played by granule cells and their axons in Purkinje cell development appeared to be two-fold. First, the development of the orderly array of parallel fibers in the normal animal played a role in orienting and flattening the dendritic trees of Purkinje cells. Second, the formation of tertiary dendritic branches appeared to depend primarily upon the presence of an external germinal layer throughout this stage of Purkinje cell development. By contrast, dendritic spines developed and persisted in the absence of granule cells.

Reorganization of organotypic cultures of mouse cerebellum exposed to cytosine arabinoside: a timed ultrastructural study.

This study was designed to examine the sequential changes in the developing granuloprival cerebellar culture. In this model of anomalous cerebellar development, organotypic cultures derived from newborn Swiss-Webster mice were exposed to the DNA synthesis inhibitor, cytosine arabinoside, at explantation and were fixed for electron microscopic examination on successive days in vitro. Similar developmental stages were compared in control explants. Granule cell destruction began early, and was widespread by 2 days in vitro, when oligodendrocyte destruction also began in treated cultures. A few granule cells survived, but no recognizable oligodendrocytes remained by 7 days in vitro, at a time when myelin was initially evident in control explants. Purkinje cell recurrent axon collateral sprouting began at 3 days in vitro in cultures exposed to cytosine arabinoside, and the sprouted terminals initially synapsed with Purkinje cell somata, somatic spines and dendritic shafts. Synapses with Purkinje cell dendritic spines developed later, at approximately the same time as parallel fiber-Purkinje cell dendritic spine synapses formed in control cultures. Astrocytic ensheathment of control Purkinje cells was well underway by 6 days in vitro and Purkinje cell somata were relatively rounded and almost completely ensheathed by 9 days in vitro. Glial ensheathment did not occur in cytosine arabinoside treated cultures, and Purkinje cell somata were scalloped at 7 days in vitro by excess impinging recurrent axon collateral terminals, and never developed the smooth contours characteristic of control Purkinje cells. Purkinje cell somatic spines persisted in treated explants, and reduction of excess extracellular space was delayed until 12 days in vitro, when most of the developmental changes had been completed. The earlier development of synapses by excess recurrent axon collateral terminals with Purkinje cell somata, somatic spines and dendritic shafts, followed by the later development of heterotypical synapses with dendritic spines, in parallel with synapse formation by normal presynaptic elements, suggests that the sequence of development of synapses is a function of the maturational state of the postsynaptic components.

Evasion of immunologic defenses and emerging viral threats.

New viruses and virus strains appear at irregular but frequent intervals in the human population. Those that represent a widespread serious threat to human populations have evolved mechanisms for either short-term or longer-term evasion of immunologic recognition and neutralization. These mechanisms include (1) antigenic shift, (2) intracellular transport, (3) intracellular latency, (4) direct infection and destruction of cells of the immune system, (5) infection in utero or early infancy so the developing immune system regards it as part of the host repertoire of antigens, and probably (6) nonantigenicity. In this article, I discuss these mechanisms and their relationship to the threat constituted by emerging viral agents.

The effect of dexamethasone on L-alpha-lysophosphatidyl choline (lysolecithin)-induced demyelination of the rat spinal cord.

Adult male Sprague-Dawley rats were injected intraspinally with 2 microL of 1% lysolecithin to induce demyelination. This was followed by systemic treatment with dexamethasone acetate for two weeks. Control groups were injected with lysolecithin but not treated. The animals were perfused at 8, 12, 28, and 60 days postoperatively, and the spinal cords were processed and examined histopathologically. The intraspinal injection of lysolecithin produced a focal area of primary demyelination followed by remyelination. In the steroid-treated groups, there was a suppression of the mononuclear phagocytic response, accompanied by slow clearance of the disrupted myelin sheaths and an apparent reduction in lesion size. Remyelination was substantially retarded in comparison with the controls. The findings suggest that steroids interfere with the process of remyelination, as they are known to do with myelination during development. Thus, dexamethasone appears to have a dual effect, decreasing demyelination but delaying remyelination.

Mature oligodendrocytes. Division following experimental demyelination in adult animals.

Primary demyelination can be caused by injury to oligodendrocytes or to the myelin sheaths that these cells maintain. Although remyelination does take place in multiple sclerosis (MS), its possible role in the recovery from MS attacks has been inadequately considered, partly because of the belief that oligodendrocytes, once destroyed, cannot be replaced in the adult. The injection of lysolecithin into the mouse spinal cord causes primary demyelination, followed by the generation of new oligodendrocytes and remyelination. By using a pulse label of tritiated thymidine, this electron-microscopic autoradiographic study demonstrated a source of these regenerated oligodendrocytes. The replacement of oligodendrocytes can occur through the division of preexisting oligodendrocytes. This is the first demonstration that mature oligodendrocytes are capable of dividing in older animals. These results lend support to recent observations of an apparent proliferation of these cells in an active MS lesion. We believe that the ability of mature oligodendrocytes to divide and to remyelinate axons in the adult may play an important role in the recovery from MS attacks.

Mouse hepatitis virus-induced recurrent demyelination. A preliminary report.

Four-week-old BALB/c mice inoculated intracerebrally with the JHM strain of mouse hepatitis virus developed an acute demyelinating disease followed by apparent recovery with remyelination. When surviving mice were examined 16 months later, small areas of active demyelination were still present. This is the first reported example, to our knowledge, of an experimental viral infection in which acute demyelination with recovery is followed by persisting or recurring demyelination.

Multiple sclerosis. The problem of incorrect diagnosis.

Various neurologic disorders may be diagnosed incorrectly as multiple sclerosis (MS) since there is no test that is entirely specific for the disease. We report ten patients who met clinical criteria for probable or definite MS and who were given incorrect diagnoses. All of the patients were subsequently shown to have alternative diagnoses, three of which were directly treatable. From these illustrative cases, five characteristics were identified that alerted us to the possibility of an alternative diagnosis. We have called these characteristics "red flags," and suggest that they may be useful as features casting doubt on the diagnosis of MS if used judiciously in conjunction with clinical diagnostic criteria.

Pulmonary function and dysfunction in multiple sclerosis.

Pulmonary function was studied in 25 patients with clinically definite multiple sclerosis with a range of motor impairment. Forced vital capacity (FVC), maximal voluntary ventilation (MVV), and maximal expiratory pressure (MEP) were normal in the ambulatory patients (mean greater than or equal to 80% predicted) but reduced in bedridden patients (mean, 38.5%, 31.6%, and 36.3% predicted; FCV, MVV, and MEP, respectively) and wheelchair-bound patients with upper extremity involvement (mean, 69.4%, 50.4%, and 62.6% predicted; FVC, MVV, and MEP, respectively). Forced vital capacity, MVV, and MEP correlated with Kurtzke Expanded Disability Status scores (tau = -0.72, -0.70, and -0.65) and expiratory muscle weakness occurred most frequently. These findings demonstrate that marked expiratory weakness develops in severely paraparetic patients with multiple sclerosis and the weakness increases as the upper extremities become increasingly involved.

Metachromatic leukodystrophy: comparison of early-and late-onset forms.

Comparative study of peripheral nerve biopsies and cultured skin fibroblasts from patients with late infantile and early adult-onset forms of metachromatic leukodystrophy revealed similar pathologic changes in the early stages of nerve degeneration. However, the peripheral nerves in the adult-onset cases eventually reached a more chronically demyelinated fibrotic state than did nerves in the infantile cases. Cultured skin fibroblasts from the adult-onset patients, although clearly abnormal, were able to catabolize sulfatide significantly more effectively than the cultured skin fibroblasts from late infantile patients.

A longitudinal study of T1 hypointense lesions in relapsing MS: MSCRG trial of interferon beta-1a. Multiple Sclerosis Collaborative Research Group.

BACKGROUND: T1 hypointense lesions (T1 black holes) are focal areas of relatively severe CNS tissue damage detected by MRI in patients with MS. OBJECTIVE: To determine the natural history of T1 hypointense lesions in relapsing MS and the utility of T1 hypointense lesions as outcome measures in MS clinical trials. METHODS: MR studies were from the Multiple Sclerosis Collaborative Research Group trial. Longitudinal results are reported in 80 placebo- and 80 interferon beta-1a (IFNbeta-1a)-treated patients with mild to moderate disability relapsing-remitting MS. RESULTS: There was a small but significant correlation between T1 hypointense lesion volume and disability at baseline and on trial (r = 0.22, r = 0.28). In placebo patients there was a 29.2% increase in the mean volume of T1 hypointense lesions (median 124.5 mm3) over 2 years (p < 0.001 for change from baseline), as compared to an 11.8% increase (median 40 mm3) in the IFNbeta-1a-treated patients (change from baseline not significant). These treatment group comparisons did not quite reach significance. The most significant contributor to change in T1 hypointense lesions was the baseline number of enhancing lesions (model r2 = 0.554). Placebo patients with more active disease, defined by enhancing lesions at baseline, were the only group to show a significant increase in T1 hypointense lesion volume from baseline. CONCLUSION: The development of T1 hypointense lesions is strongly influenced by prior inflammatory disease activity, as indicated by enhancing lesions. These results suggest that treatment with once weekly IM IFNbeta-1a (30 mcg) slows the 2-year accumulation of these lesions in the brain.

A longitudinal study of brain atrophy in relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group (MSCRG).

OBJECTIVE: To determine if progressive brain atrophy could be detected over 1- and 2-year intervals in relapsing MS, based on annual MR studies from the Multiple Sclerosis Collaborative Research Group (MSCRG) trial of interferon beta-1a (Avonex). METHODS: All subjects had mild to moderate disability, with baseline expanded disability status scores ranging from 1.0 to 3.5, and at least two relapses in the 3 years before study entry. Atrophy measures included third and lateral ventricle width, brain width, and corpus callosum area. RESULTS: Significant increases were detected in third ventricle width at year 2 and lateral ventricle width at 1 and 2 years. Significant decreases in corpus callosum area and brain width were also observed at 1 and 2 years. Multiple regression analyses suggested that the number of gadolinium-enhancing lesions at baseline was the single significant contributor to change in third ventricle width. Atrophy over 1 and 2 years as indicated by enlargement of the third and lateral ventricle and shrinkage of the corpus callosum was greater for patients entering the trial with enhancing lesions. Greater disability increments over 1 and 2 years were associated with more severe third ventricle enlargement. CONCLUSION: In patients with relapsing MS and only mild to moderate disability, significant cerebral atrophy is already developing that can be measured over periods of only 1 to 2 years. The course of cerebral atrophy in MS appears to be influenced by prior inflammatory disease activity as indicated by the presence of enhancing lesions. Brain atrophy measures are important markers of MS disease progression because they likely reflect destructive and irreversible pathologic processes.

Incidence and significance of neutralizing antibodies to interferon beta-1a in multiple sclerosis. Multiple Sclerosis Collaborative Research Group (MSCRG)

BACKGROUND: Interferon beta is an effective treatment for relapsing multiple sclerosis (MS). As with other protein drugs, neutralizing antibodies (NAB) can develop that reduce the effectiveness of treatment. OBJECTIVES: To determine the incidence and biological significance of NAB to interferon beta-la (IFN-beta-1a; Avonex; Biogen, Cambridge, MA) in MS patients. METHODS: A two-step assay for NAB to IFN-beta-1a was developed and used to assay serum samples from participants in the phase III clinical trial of IFN-beta-1a, and from patients in an ongoing open-label study of IFN-beta-1a. The biological significance of NAB to IFN-beta-1a was determined by relating the NAB assay result to in vivo induction of the IFN-inducible molecules neopterin and beta-2 microglobulin, and the clinical significance was determined by comparing clinical and MRI measures of disease activity after 2 years of IFN-beta-1a therapy in patients who were NAB+ and NAB-. The incidence of NAB was compared in MS patients who had used only IFN-beta-1a with the incidence in MS patients who had used only IFN-beta-1b. RESULTS: In patients in the open-label study, development of NAB to IFN-beta-1a resulted in a titer-dependent reduction in neopterin induction after interferon injections. In patients in the phase III study, development of NAB was associated with a reduction in beta-2 microglobulin induction. In the phase III study, a trend toward reduced benefit of IFN-beta-1a on MRI activity in NAB+ versus NAB- patients was observed. The incidence of NAB to IFN-beta-1a in the open-label study was approximately 5% over 24 months of treatment of IFN-beta-1a therapy, but was four- to sixfold higher using the same assay for patients exposed only to IFN-beta-1b for a similar duration. There were no clinical, MRI, or CSF characteristics that were predictive of which patients would develop NAB. CONCLUSIONS: NAB directed against IFN-beta have in vivo biological consequences in patients with MS. The frequency with which MS patients develop NAB against IFN-beta is significantly greater with IFN-beta-1b therapy compared with IFN-beta-1a therapy. Treatment decisions in MS patients treated with IFN-beta should take into account development of NAB.