Masaru Yoshioka

Role of immune activation and cytokine expression in HIV-1-associated neurologic diseases

Central nervous system (CNS) involvement is common during human immunodeficiency virus type-1 (HIV-1) infection. The neurologic disease of the CNS most frequently observed during acquired immunodeficiency syndrome (AIDS) is HIV-1-associated cognitive/motor complex or AIDS dementia complex (ADC), which is most likely a direct consequence of HIV-1 infection of the CNS. The peripheral nervous system (PNS) is also affected in HIV-1-infected individuals and there are several features of immune- and cytokine-related pathogenesis in both the CNS and PNS that are reviewed. Several lines of evidence demonstrate aspects of immune activation in the CNS and peripheral nervous system (PNS) of HIV-1-infected individuals. The relative paucity of HIV-1 expression in contrast to widespread functional and pathologic changes in the CNS and PNS of AIDS patients, and the lack of evidence of productive infection of HIV-1 in neuronal cells in vivo lead to the possibility of indirect or immunopathogenic mechanisms for HIV-1-related neurologic diseases. Proposed mechanisms of neuronal and glial cell damage are injury of oligodendrocytes by tumor necrosis factor-alpha (TNF-alpha) released from activated macrophage/microglia, calcium-dependent excitoneurotoxicity induced by gp120 HIV-1 envelope protein, N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity by quinolinic acid (a product of activated macrophages), cell injury by HIV-1-specific cytotoxic T cells, and apoptosis of oligodendrocytes or neurons triggered by interaction between cell surface receptors and HIV-1 gp120 protein. Common to those mechanisms is the dependence on cellular activation with expression of proinflammatory cytokines (TNF-alpha, interleukin-1). Amplification of activation signals through the cytokine network by macrophage/astrocyte/endothelial cell interactions, and cell-to-cell contact between activated macrophages and neural cells by upregulation of adhesion molecules dramatically enhances the toxic effect of macrophage products. Expression of immunosuppressive cytokines such as interleukin-4, interleukin-6, and transforming growth factor-beta is also increased in the CNS and PNS of HIV-1-infected patients. This may serve as neuroprotective and regenerative mechanism against insults to nervous system tissue.

Expression of cytokines in brain lesions in subacute sclerosing panencephalitis

We analyzed frozen brain specimens from three patients with subacute sclerosing panencephalitis (SSPE) for the presence of interleukin (IL)-1β, IL-2, IL-6, tumor necrosis factor (TNF), lymphotoxin (LT), and interferon (IFN)-γ using immunocytochemical techniques. We detected these cytokines in SSPE brain lesions demonstrating extensive cellular infiltrates, demyelination, and gliosis. Double-label immunocytochemistry, using cell-specific markers, showed that positive immunoreactivity for these cytokines was present on both infiltrating cells and resident brain cells. We also found IL-6, TNF, and IFN-γ at lower levels in brain tissue from a patient with progressive multifocal leukoencephalopathy. In contrast, normal control brain sections showed no reactivity for any of the cytokines. These findings indicate that IL-1β, IL-2, IL-6, TNT, LT, and IFN-γ may be produced in SSPE lesions and may be involved in the lesion pathogenesis of SSPE.

Expression of tumor necrosis factor-α in muscles of polymyositis

Abstract We immunohistochemically examined biopsied muscles from nine untreated patients with polymyositis (PM) and five patients with other neuromuscular diseases (ONMD), using monoclonal antibodies to tumor necrosis factor-α (TNF-α) and lymphoid surface markers. In muscles of three patients with PM, we observed many TNF-α positive macrophages and lymphocytes in endomysium and around vessels in the muscles. By contrast, there were few, weakly TNF-α stained cells in muscles of three patients with ONMD. The ratio of TNF-α-positive cells to the muscle fibers and the ratio of TNF-α-positive cells to the mononuclear cells were significantly higher in PM compared with ONMD. In addition, we observed atrophic muscle fibers more frequently in TNF-α-positive muscles than TNF-α-negative ones. We conclude that, at least, in a part of PM patients, TNF-α produced locally may contribute to the pathogenesis of PM.

Detection of tumor necrosis factor-α-positive cells in cerebrospinal fluid of patients with HTLV-I-associated myelopathy

Tumor necrosis factor (TNF)-alpha-positive cells constituted 1.6-18% and 8.2-23.5% of the total number of cerebrospinal fluid cells from six of 12 patients with HTLV-I-associated myelopathy and in all samples obtained from inflammatory cases, respectively. However, in non-inflammatory cases no TNF-alpha-positive cells were detected. These results suggest that some of the infiltrating CSF cells produce TNF-alpha, which plays a role in host immune defenses against causative agents including HTLV-I and in lesion formation within the central nervous system in inflammatory diseases.

HIV-1 Heterogeneity and Cytokines

Mild manifestations (HIV-1 associated minor cognitive/motor disorder), severe manifestations (HIV-1 associated dementia complex and HIV-1 associated myelopathy), and sensory neuropathy are consequences of HIV-1 infection. Our goal is to elucidate the role of HIV-1 in the complications of AIDS including cytokine immunopathology and HIV-1 DNA sequence variants. We have examined the brain and sensory ganglia from 60 AIDS patients and 20 seronegative controls using PCR, DNA sequencing of the HIV-1 envelope protein (env), in situ hybridization (ISH), and immunohistochemistry (IHC). Using our combined ISH-IHC technique, we could identify different types of cells and HIV-1 simultaneously in cryostat and paraffin sections. We found HIV-1 predominantly in macrophage/microglia in brain. In dorsal root ganglia (DRG) we found rare macrophages infected with HIV-1 and neurons and interstitial cells (including macrophages) which were apoptotic. Cytokines were detected in mononuclear and endothelial cells near neurons. We achieved single copy sensitivity detecting HIV-1 in nervous tissue using nested PCR. We sequenced HIV-1, DNA from 3 intravenous drug users (IDUs): from brain, CSF, and blood. PCR amplification was followed by cloning and then sequencing the HIV-1 insert: V1-V5 regions of the envelope (env) gene. We found that the env genes had increased sequence variation compared to the literature, cDNA sequences derived from RNA were less heterogeneous than clones derived from DNA from the same specimens, clones derived from brain are more closely related (show restricted heterogeneity) compared to clones from blood and CSF from the same patients. Patient 149 clones we examined to date did not correspond to any of the designated subtypes (A-F) of HIV-1 based on the DNA sequences of the C2-V3 regions. Finally, the HIV-1 RNA produced in these tissues is derived from a minority of DNA clones. Although HIV-1 infected macrophages are not entirely responsible for pathology in the brain and less so in sensory ganglia, some of the products of infection, cytokines, are more widespread in these tissues. Furthermore, HIV-1 strains infecting the brain appear to exhibit restricted heterogeneity compared to autologous CSF and blood and these strains may be associated with cytokines and pathology. HIV-1 strains that infect nervous tissue and cytokines produced in this tissue may effect neuropathogenesis, in vivo, in spite of low levels of local HIV-1 infection. We attempt to delineate, here, common sequence variations in HIV-1 isolates in the hope of developing future therapeutic strategies.

Clinical features and a mutation with late onset of limb girdle muscular dystrophy 2B

Objective and methods Dysferlin encoded by DYSF deficiency leads to two main phenotypes, limb girdle muscular dystrophy (LGMD) 2B and Miyoshi myopathy. To reveal in detail the mutational and clinical features of LGMD2B in Japan, we observed 40 Japanese patients in 36 families with LGMD2B in whom dysferlin mutations were confirmed. Results and conclusions Three mutations (c.1566C>G, c.2997G>T and c.4497delT) were relatively more prevalent. The c.2997G>T mutation was associated with late onset, proximal dominant forms of dysferlinopathy, a high probability that muscle weakness started in an upper limb and lower serum creatine kinase (CK) levels. The clinical features of LGMD2B are as follows: (1) onset in the late teens or early adulthood, except patients homozygous for the c.2997G>T mutation; (2) lower limb weakness at onset; (3) distal change of lower limbs on muscle CT at an early stage; (4) impairment of lumbar erector spinal muscles on muscle CT at an early stage; (5) predominant involvement of proximal upper limbs; (6) preservation of function of the hands at late stage; (7) preservation of strength in neck muscles at late stage; (8) lack of facial weakness or dysphagia; (9) avoidance of scoliosis; (10) hyper-Ckaemia; (11) preservation of cardiac function; and (12) a tendency for respiratory function to decline with disease duration. It is important that the late onset phenotype is found with prevalent mutations.

Efficacy and tolerance of gastrostomy feeding in Japanese muscular dystrophy patients.

Although muscular dystrophy patients often have feeding difficulty and need long-term enteral nutrition, only a few reports have described gastrostomy feeding in these patients. This study was designed to evaluate the efficacy and tolerance of gastrostomy feeding in patients with muscular dystrophy. We performed a retrospective, multicenter study on 144 patients with muscular dystrophy who received gastrostomy feeding between 2007 and 2009 in 25 neuromuscular centers in Japan. There were 77 Duchenne muscular dystrophy (median age at gastrostomy placement 26 years, range 13-47 years), 40 myotonic dystrophy (median age 54.5 years, range 13-70 years), 11 Fukuyama congenital muscular dystrophy (median age 22 years, range 13-29 years), 5 limb girdle muscular dystrophy (median age 62 years, range 43-78 years), and 5 facioscapulohumeral muscular dystrophy (median age 52 years, range 28-67 years) patients. Many benefits including amelioration of malnutrition, swallowing difficulty and respiratory status were observed after the introduction of gastrostomy feeding. Especially in patients with Duchenne muscular dystrophy, mean body weight significantly increased after gastrostomy placement. Although most complications, which are commonly observed in other populations, were tolerable, respiratory failure and peritonitis were important concerns. These findings suggest that gastrostomy placement at an appropriate time is advisable in patients with muscular dystrophy.

Immunocytochemical analysis of the cellular infiltrate in brain lesions in subacute sclerosing panencephalitis

We studied lymphocyte subsets and major histocompatibility complex (MHC) class II antigens in frozen brain tissues from three patients with subacute sclerosing panencephalitis (SSPE) using immunocytochemical techniques. Perivascular cuffs consisted predominantly of T cells and MHC class II-positive cells. In the parenchymal lesions, there were frequent B and CD4+ cells. MHC class II-positive cells were also diffusely distributed throughout the parenchyma. There were more CD4+ than CD8+ cells both in the parenchyma and in the perivascular cuffs. These findings suggest an overrepresentation of CD4+ cells in SSPE lesions and that CD4+ cells may be involved in the pathogenesis of SSPE.

Parvalbumin and calbindin D-28 k immunoreactivity in dorsal root ganglia in acquired immunodeficiency syndrome

Various degrees of neuronal degeneration have been found in lumbosacral dorsal root ganglia of patients with acquired immunodeficiency syndrome (AIDS). To characterize the subpopulations of primary sensory neurons affected in AIDS. we immunostained dorsal root ganglion tissues from 11 AIDS patients and six controls using antibodies to the calcium binding proteins, parvalbumin and calbindin D‐28 k. In controls, the proportion of neurons containing parvalbumin and calbindin was 18.0% and 22.4%, respectively. The majority of parvalbumin‐positive neurons, which are thought to be proprioceptive neurons, were of medium to large size, while calbindin was found in both large‐ and small‐sized neurons. The density of parvalbumin‐immunoreactive neurons was reduced by 7.3% in AIDS patients, but the density of calbindin‐immunoreactive neurons was preserved. Furthermore, in AIDS cases, the number of parvalbumin‐positive neurons was reduced more in dorsal root ganglia in which human immunodeficiency virus (HIV) antigen was detected than in HIV‐negative ganglia. These results suggest that specific subpopulations of sensory neurons positive for parvalbumin may be differentially affected over the course of AIDS, and that this could be related to peripheral neuropathy which frequently occurs in the late stages of AIDS.

Detection of vasoactive intestinal polypeptide messenger RNA in ganglioneuroblastoma by in situ hybridization

Using in situ hybridization with35S-labeled prepro-VIP cDNA probes, vasoactive intestinal polypeptide (VIP) mRNA was detected in tumor tissues from two cases of ganglioneuroblastoma associated with watery diarrhea syndrome. The distribution of VIP mRNA was confined to the cytoplasm of the cells showing ganglionic differentiation, whereas the undifferentiated neuroblastic cells failed to be labeled by the probe. These findings were consistent with the localization of VIP-like immunoreactivity in the same tumor tissues. Direct evidence is presented at the single cell level for the production of VIP by ganglionic cells in ganglioneuroblastoma.