Diane E. Griffin

The role of antibody in recovery from alphavirus encephalitis

Alphaviruses infect neurons in the brain and spinal cord and cause acute encephalomyelitis in a variety of mammals. The outcome of infection is determined by whether the neurons survive infection and this, in turn, is determined by the virulence of the virus and the age of the host at the time of infection. We have been studying Sindbis virus (SV) infection i of mice as a model system for alphavirus‐induced encephalomyelitis. Investigation of intracerebral infection of weanling mice with two different strains of SV bas allowed us to analyze the role of the immune response in protection from fatal disease virulent NSV strain) and in CLEARANCE of virus from the nervous system during non‐fatal disease (less virulent SV AR339 strain). Neutralizing and non‐neutralizing antibodies to the El and E2 surface glycoproteins can protect mice from fatal NSV infection when given before or after infection, while T cells are not protective, The mechanism of antibody‐mediated protection is not known, but it is likely that more than one mechanism Is involved and that different mechanisms are involved in pre‐infection and post‐infection treatment protection. Clearance of infectious virus from the nervous system of mice during recovery from non‐fatal disease is accomplished by antibodies to the E2 glycoprotein. The process does not involve damage to the infected neurons and is independent of complement and mononuclear cells. Bivalent antibody is required and binds to the surface of the infected cell. Initially, release of virus by budding from the cell surface is prevented and, subsequently, intracellular virus replication is inhibited possibly through antiviral mechanisms induced in co‐operation with interferon. This non lytic mechanism for control of virus infection results in the prolonged presence of viral RNA in tissue and the need for prolonged intrathecal synthesis of antiviral antibody by B cells within the central nervous system.

Cytokine production in vitro and the lymphoproliferative defect of natural measles virus infection

In natural measles virus infection, evidence of intense immune system activation is present simultaneously with clinically relevant immune suppression. While evidence of activation is most prominent early in the disease, skin test responses and in vitro lymphoproliferation are depressed for weeks after the onset of the rash. It is not known whether the prolonged period of reduced immune responsiveness results from a single defect or a succession of different abnormalities. To gain further insight into measles-induced immune suppression we studied the production of soluble IL-2 receptor (sIL-2R), interferon-gamma (IFN-gamma), IL-1 beta, and tumor necrosis factor (TNF alpha) by peripheral blood mononuclear cells (PBMC) isolated from measles patients at various times after the onset of the rash. Studies included addition of supplemental recombinant IL-1 beta (rIL-1 beta) or recombinant IL-2 (rIL-2) or suppression of prostaglandin synthesis by indomethacin (IM). Proliferation in response to phytohemagglutin (PHA) was abnormal at all stages of disease. During the acute phase (first week after the onset of the rash) spontaneous production of sIL-2R was increased (76 +/- 54 vs. controls 4 +/- 4; P less than 0.03), suggesting in vivo T cell activation while PHA-induced sIL-2R was decreased (228 +/- 43 vs. control 582 +/- 127; P less than 0.002), suggesting that the capacity to produce IL-2 in response to mitogen was limited. Supplementation of PHA-stimulated cultures with rIL-2 improved but did not normalize both proliferation (58,600 +/- 4900 to 70,700 +/- 4400 vs. control 97,700 +/- 15,500; P less than 0.03) and sIL-2R levels (114 +/- 58 to 309 +/- 87 vs. control 582 +/- 127; P less than 0.003). Both spontaneous (25 +/- 18 vs. control 237 +/- 92; P less than 0.002) and PHA-induced (20 +/- 20 vs. control 604 +/- 129; P less than 0.004) TNF alpha levels were subnormal and were not improved with rIL-2, rIL-1 beta, or IM, suggesting a block in monocyte TNF alpha production. Spontaneous and PHA-induced IFN-gamma and IL-1 beta levels were normal. During the convalescent phase (greater than 2 weeks after the onset of the rash), spontaneous levels of sIL-2R were normal and PHA-induced levels were completely normalized with supplemental rIL-2 but proliferation remained below normal.(ABSTRACT TRUNCATED AT 400 WORDS)

Selection of a fixative for identifying T cell subsets, B cells, and macrophages in paraffin-embedded mouse spleen.

Fixation techniques were investigated for their ability to preserve morphology, esterase activity and cell surface antigens in paraffin-embedded mouse lymphoid tissue. The avidin-biotin-peroxidase system was used to stain antigens Thy-1.2, Lyt-1, Lyt-2, RA32C2 and F4/80. Conventional fixatives were compared with fixatives containing periodate and lysine plus paraformaldehyde and/or glutaraldehyde. Conventional fixatives preserved esterase activity but not many cell surface antigens. Periodate-lysine fixatives allowed better preservation of membrane antigens, but esterase activity was often lost at antigen-preserving concentrations of paraformaldehyde or glutaraldehyde. However, a periodate-lysine fixative containing both paraformaldehyde and glutaraldehyde preserved esterase and showed good to excellent staining of Lyt-1, Thy-1.2, RA32C2, and F4/80. Lyt-2 could not be stained with any fixative, but was well preserved in frozen material post-fixed with periodate-lysine based fixatives. We conclude that with proper fixation immune cell surface markers can be identified in paraffin-embedded tissue.

Human leukocyte antigens and cytokine expression in cerebral inflammatory demyelinative lesions of X-linked adrenoleukodystrophy and multiple sclerosis

The two most common forms of X-linked adrenoleukodystrophy (X-ALD) are the cerebral forms (CER) with an inflammatory demyelinating reaction that resembles multiple sclerosis, and adrenomyeloneuropathy (AMN) which involves primarily the spinal cord and in which the inflammatory reaction is mild or absent. We found no significant association between the childhood cerebral form (CCER) or AMN and the human leukocyte (HLA) class I and Class II antigens including the class II DR2 haplotypes associated with multiple sclerosis. Inflammatory cytokine (tumor necrosis factor-alpha, interleukin-1 beta, interleukin-4, interleukin-6 and interferon-gamma) gene expression was increased in multiple sclerosis brain lesions, as has been reported previously, but much less so in CER brain lesions. These findings suggest that the pathogenesis of the inflammatory response in X-ALD differs from that in multiple sclerosis.

Cytokine dysregulation in HIV-associated neurological disease.

AIDS is associated with three major neurological syndromes: dementia (HIVD), vacuolar myelopathy (VM) and plainful sensory neuropathy (PSN). The pathogenesis of these conditions remains unclear although they all demonstrate a marked increase in macrophage number and activation despite systemic immunosuppression. It was therefore of interest to determine the profile of cytokine and HIV expression in brain, spinal cord and peripheral nerves of AIDS patients with AD, VM and PSN, as compared to AIDS patients without neurological disease and seronegative controls. RNA was extracted from brain, spinal cord and peripheral nerve and RT/PCR for cytokine and HIV mRNA was performed. In situ RT/PCR was performed to determine the number and type of cells expressing cytokine message and this was compared to the number of cells containing HIV DNA detected with in situ PCR. We found a consistent profile of increased TNF alpha and decreased IFN gamma and IL4 in all three syndromes compared to AIDS patients without neurological disease. IL1 did not increase in parallel with TNF alpha IL10 was decreased in the VM tissue. HIV transcripts were increased in the AD brains compared to non-demented controls but were detected only occasionally in spinal cord and not at all in peripheral nerve. Preliminary data from in situ RT/PCR suggests that a large number of cells are expressing. TNF alpha but only a small number are infected with HIV.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of endoneurial T-cells in human sural nerve biopsies

We used immunocytochemical methods on sural nerve biopsies from 42 patients with peripheral neuropathy to identify mononuclear cells, determine whether lymphocytic infiltration occurs in a variety of neuropathies, and identify the subtypes of lymphocytes. Immunostained cells were present in 76% of nerve biopsies. CD3+ cells (T lymphocytes) were greatest in density (cells/mm2). In patients whose CD4:CD8 T cell ratio was measured also in blood and cerebrospinal fluid, the CD4:CD8 T cell ratio was similar in all three compartments. These findings suggest that T lymphocytes are frequently present in nerves obtained from patients with various types of neuropathies and raise questions about factors that attract T lymphocytes into nerve that may be important in pathogenesis.

Alphavirus Pathogenesis and Immunity

Most alphavirus infections in nature occur in wild birds, rodents, and occasionally reptiles and amphibians. Some of these natural hosts sustain a relatively asymptomatic, prolonged, high-titered viremia, facilitating transmission of the virus by mosquitoes (Chamberlain, 1980). Although there is undoubtedly mortality and morbidity in the wild due to alphavirus infections, many native species have inapparent infection, and epizootic, epidemic, or endemic disease is usually recognized after mosquito-mediated transmission to a domestic host (e.g., horses, pigeons, captive pheasants) or to man. Affected individuals develop a wide range of symptoms, including fever, skin rashes, arthritis, myositis, and/or encephalitis. Depending on the virus and on the host, the infection may be clinically inapparent or cause fulminant disease and death (Shope, 1980).

Natural killer cell activity during measles.

Natural killer cells are postulated to play an important role in host anti‐viral defences. We measured natural killer cell activity in 30 individuals with acute measles (73 ± 21 lytic units (LU)/107 cells) and 16 individuals with other infectious diseases (149 ± 95 LU) and found it reduced compared with values for adults (375 ± 70 LU; P<0.001) or children (300 ± 73 LU, P<0.01) without infection. Reduced natural killer cell activity was found in measles patients with (84 ± 30 LU) and without (55 ± 18 LU) complications and was present for at least 3 weeks after the onset of the rash. Activity was increased by in vitro exposure of cells to interleukin‐2. Depressed natural killer cell activity parallels in time the suppression of other parameters of cell‐mediated immunity that occurs during measles.

Changes in plasma IgE levels during complicated and uncomplicated measles virus infections

Plasma IgE levels were measured in 214 samples from 182 Peruvian patients with acute measles virus infections. Plasma IgE levels were significantly elevated early in infection compared to later time points. Plasma levels of IgG from the same patients rose during the same time period, whereas levels of IgA and IgM did not change. In patients with postmeasles encephalomyelitis, IgE remained elevated longer than it did in patients either with uncomplicated measles or measles complicated by pneumonia. It is proposed that the elevation of IgE is another manifestation of the altered immunoregulatory function in patients with measles.

Spontaneous proliferation of peripheral mononuclear cells in natural measles virus infection: Identification of dividing cells and correlation with mitogen responsiveness

Spontaneous proliferation of peripheral mononuclear cells is pronounced following measles virus infection at a time when patients mount effective humoral and cell-mediated immune responses and manifest a range of poorly understood immunologic abnormalities. We found spontaneous activity (measles 8000 +/- 1200 cpm vs control 1900 +/- 350 cpm; P less than 0.05) to wax and wane abruptly during the first week after the rash in parallel with expression of the lymphocyte activation marker OKT10. At peak activity, approximately 10% of circulating mononuclear cells were actively synthesizing DNA. Double labeling of individual mononuclear cells with autoradiography and immunoperoxidase demonstrated that B and T lymphocytes as well as monocytes participate in the spontaneous activity. Proliferative activity was increased 3- to 20-fold over control levels in all PBMC subsets such that close to one-third of circulating B cells and monocytes and 5-10% of CD4- and CD8-positive T cells were preparing to divide. Mitogen responsiveness was generally decreased in measles patients (58,800 +/- 4600 cpm vs control 97,700 +/- 15,500 cpm; P less than 0.002). Neither spontaneous proliferation nor mitogen responsiveness was correlated with age, sex, or the presence of complications. Patients with the lowest mitogen responses, however, had the greatest increases in B cell (P less than 0.03) and CD8-positive T cell (P less than 0.05) proliferation. These data demonstrate that all major immunologic cell types proliferate in response to measles virus infection. Mechanisms by which spontaneous proliferative activity in individual mononuclear subsets could contribute to depressed mitogen responsiveness are discussed.