Geometry of the vapor layer under a leidenfrost drop.

In the Leidenfrost effect, liquid drops deposited on a hot surface levitate on a thin vapor cushion fed by evaporation of the liquid. This vapor layer forms a concave depression in the drop interface. Using laser-light interference coupled to high-speed imaging, we measured the radius, curvature, and height of the vapor pocket, as well as nonaxisymmetric fluctuations of the interface for water drops at different temperatures. The geometry of the vapor pocket depends primarily on the drop size and not on the substrate temperature.

Suppression of the development of experimental allergic encephalomyelitis by suramin.

Suramin was tested for its ability to suppress experimental allergic encephalomyelitis. Prophylactic administration caused significant reduction in the severity of the disease, incidence of paralysis and cellular infiltration in nervous tissue. Therapeutic treatment with suramin also caused a reduction in the severity of the disease, the incidence of paralysis and cellular infiltration, but to a lesser extent. Significantly fewer animals were paralysed for more than two days on therapeutic treatment.

Encephalitogenic Th1 cells are inhibited by Th2 cells with related peptide specificity: relative roles of interleukin (IL)-4 and IL-10.

Cytokines secreted by T-helper type 2 (Th2) cells inhibit the antigen-induced stimulation of type 1 (Th1) helper T cells. To study this form of regulation in an autoimmune disease model, the cytokines secreted by a Th2 clone specific for the encephalitogenic proteolipid protein (PLP) peptide 139-151 were tested for their ability to inhibit proliferation of an encephalitogenic Th1 clone specific for an epitope contained within the same peptide. Cytokines, produced by stimulation of the Th2 clone with CD3-specific monoclonal antibodies (mAbs), inhibited proliferation of the Th1 clone when stimulated by antigen and splenic antigen-presenting cells (APC). Inhibition was, however, not antigen-specific since cytokines released upon stimulation of an unrelated Th2 clone were also inhibitory. Inhibition was found to be caused by effects on either antigen presentation or co-stimulatory activity of the APC and not by direct effects on the Th1 cells. MAbs for the two major regulatory Th2 cytokines were used to identify the inhibitory component secreted by activated Th2 cells. Interleukin-10 (IL-10)-specific mAb abolished the inhibitory effect, while mAb specific for IL-4 had no effect on inhibition. The addition of recombinant IL-4 (rIL-4) and rIL-10 confirmed that inhibition of Th1 proliferation was due to secretion of IL-10 by the Th2 clone and its subsequent effects on APC. The studies described here demonstrate that PLP-specific Th2 cells which recognize peptide 139-151 inhibit encephalitogenic Th1 cells which respond to an epitope on the same peptide. This phenomenon may be important for local, antigen-specific regulation of inflammation in the central nervous system.

Contrasting roles for nitric oxide and peroxynitrite in the peroxidation of myelin lipids.

Peroxynitrite is formed by the reaction of nitric oxide (NO) and superoxide. Since widespread peroxynitrite activity was observed during experimental allergic encephalomyelitis (EAE), the effect of this strong lipid-peroxidizing agent on myelin integrity was examined. Incubation of myelin suspensions with the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) resulted in the formation of the lipid peroxidation product, malondialdehyde (MDA). MDA formation was inhibited in the presence of butylated hydroxytoluene, which interrupts the progression of the lipid peroxidation chain reaction. Superoxide dismutase inhibited the effect of SIN-1, which indicates a role for superoxide, and contradicts a role for its dismutation product, hydrogen peroxide. The latter was confirmed by the failure of the catalase to inhibit MDA formation. Neither NO nor superoxide alone induced significant MDA formation in myelin, indicating that peroxynitrite formation is required for myelin-lipid peroxidation. Interestingly, NO actually inhibited lipid peroxidation in myelin, as demonstrated using simple NO donors. On the other hand, the simultaneous production of superoxide, as achieved with the NO-donor SIN-1, negated the inhibitory effect of NO. Finally, the production of isoprostanes, novel products generated during lipid peroxidation, was examined. Peroxynitrite-induced peroxidation of myelin resulted in isoprostane formation. Furthermore, increased levels of F2-isoprostanes and neuroprostanes were observed in spinal cords of mice during early progressive stages of autoimmune encephalomyelitis.

Serum cytokine levels in chronic progressive multiple sclerosis: interleukin-2 levels parallel tumor necrosis factor-alpha levels.

Serum levels of the cytokines interleukin-1 alpha (IL-I alpha), IL-1 beta, IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and the soluble IL-2 receptor were measured in chronic progressive multiple sclerosis patients (CPMS) and normal, inflammatory, and noninflammatory disease controls. Serum IL-2 levels displayed the most consistent abnormalities in the group of tests for the CPMS group, and were the only cytokine levels to achieve significance in statistical group analyses. However, several patients with CPMS had normal serum IL-2 levels. An incidental finding was a statistical correlation between serum IL-2 and TNF-alpha levels among all groups tested. This finding was supported on analysis of serial serum samples from CPMS patients. These results suggest a linkage of IL-2 and TNF-alpha production, especially in pathological conditions.

Chronic experimental allergic encephalomyelitis and antibody responses in rabbits immunized with bovine proteolipid apoprotein.

A chronic form of experimental allergic encephalomyelitis can be produced by sensitization of rabbits with bovine myelin proteolipid apoprotein (PLP). To investigate the humoral immune response in this model, serum PLP antibodies were determined by enzyme-linked immunosorbent and dot immunobinding assays. In an initial experiment, 3 PLP-sensitized rabbits with severe chronic experimental allergic encephalomyelitis had a positive antibody response whereas 3 with mild disease, or with no visible clinical disease, had no detectable antibodies against PLP. In a second experiment, 3 rabbits were preimmunized with PLP in incomplete Freund's adjuvant, followed by a single immunization with PLP in complete Freund's adjuvant. These animals developed chronic experimental allergic encephalomyelitis with different progression rates, although all eventually became severely paralyzed. In both experiments the anti-PLP response was maximal before or immediately after disease onset and tended to decline during disease progression. The degree of the anti-PLP response correlated with clinical and histologic disease severity. These data suggest a possible role for humoral factors in the modulation of the chronic EAE induced in PLP-immunized rabbits.

Immune processing of proteolipid protein by subsets of antigen-presenting spleen cells.

Myelin proteolipid protein (PLP) contains one established antigenic epitope within the 139-151 amino acid sequence, with encephalitogenic activity for SJL mice (PLP139-151). In the current study, the processing and presentation of PLP by subsets of splenic antigen-presenting cells (APC) were examined by comparing their capacity to stimulate PLP-responsive T-cell clones, two of which are specific for PLP139-151, and one which is not specific for this peptide. In order to study whether PLP requires processing before its presentation by APC, PLP-pulsed and fixed APC were shown to stimulate PLP-specific T cells. However, the addition of PLP to unpulsed, fixed APC resulted in the absence of T-cell stimulation, while the ability of these fixed APC to bind antigenic peptide and efficiently present it to T cells, was demonstrated by their ability to use a synthetic peptide for the stimulation of the T cells. In order to study potentially different processing efficiencies among APC subsets, spleen cells were fractionated by adherence to plastic, and their respective APC activities were studied separately. The non-adherent (NAd) APC were unable to stimulate PLP139-151 specific T-cell clones with PLP as antigen. In contrast, a T-cell clone specific for a separate, but unidentified epitope on PLP was stimulated by NAd APC efficiently. In addition, stimulation of PLP139-151-specific T-cell clones by NAd APC did occur when the synthetic peptide instead of intact PLP was used as antigen, indicating a defect in PLP processing by the NAd APC.(ABSTRACT TRUNCATED AT 250 WORDS)

Serial studies of serum interleukin-2 in chronic progressive multiple sclerosis patients: occurrence of 'bursts' and effect of cyclosporine.

Serum levels of immunoreactive interleukin-2 (IL-2) were determined at monthly intervals from a group of placebo- and drug-treated chronic progressive multiple sclerosis patients before and during a cyclosporine A therapeutic trial. Significantly elevated levels of the lymphokine in active patients confirmed earlier studies. The magnitude of the initial levels varied inversely with the duration of disease and predicted subsequent worsening in chronic progressive patients. In addition, the occurrence of periodic bursts of serum IL-2 was noted. Although in some patients there appeared to be a sudden drop in serum IL-2 levels with the onset of cyclosporine A medication, no effect of this drug was noted on group analysis.

Fine-specificity differences in the recognition of an encephalitogenic peptide by T helper 1 and 2 cells.

The lymphokine production of two T-cell clones, which both recognize epitopes within the encephalitogenic 139-151 sequence of myelin proteolipid protein, was examined after stimulation with immobilized antibodies to the CD3 moiety of the T-cell-receptor complex. Clone A1 produced interleukin (IL)-2 and interferon (IFN)-gamma, but no IL-4, while clone D5 produced IL-4, but no IL-2 or IFN-gamma. A1 therefore belongs to the T-helper type 1 (Th1) subset, while D5 is a Th2 clone. In addition, the Th1 clone induced severe experimental allergic encephalomyelitis (EAE), while the Th2 clone did not induce any signs of EAE. Synthetic peptides were used to demonstrate that these clones recognized slightly different epitopes within the 139-151 sequence. Histidine 139 was shown to be optimal for the stimulation of the Th2 clone, while the presence of this residue inhibited the stimulation of the Th1 clone. Th2 cells specific for an encephalitogenic peptide may be important in the regulation of encephalitogenic Th1 cells.

Extensive peroxynitrite activity during progressive stages of central nervous system inflammation.

Nitric oxide (NO) production has been associated with disease activity in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). This free radical can be transformed by superoxide to peroxynitrite, an extremely toxic oxidant which causes lipid peroxidation. In addition, peroxynitrite nitrates tyrosine residues, resulting in nitrotyrosine, which can be identified immunohistochemically. The results of this study indicate that peroxynitrite is formed very early during EAE development, correlating with clinical disease activity. Nitrotyrosine-positive cells display a widespread distribution in brain and spinal cord during severe disease and are associated with both perivascular infiltrates and parenchymal sites. Double-staining procedures demonstrated that a subpopulation of CD11b-positive cells (macrophages/microglia) reacted with nitrotyrosine antibodies. Immunostaining for inducible NO synthase demonstrated a similar distribution as nitrotyrosine staining. These experiments indicate that peroxynitrite is formed during progressive stages of disease activity.

The development and characterization of encephalitogenic cloned T cells specific for myelin proteolipid protein.

T-cell clones have been isolated from SJL/J mice after immunization with myelin proteolipid protein (PLP). The cloned cells responded strongly to PLP stimulation in vitro as well as to the synthetic PLP-related peptide 139-151. The response to PLP is Ia mediated, since it was inhibited by monoclonal antibodies to the matched I-As haplotype, but not with antibodies to other I-A haplotypes. Phenotypic analysis using immunofluorescence demonstrated the following characteristics of the clones: Thy-1+, CD4+, CD5+ and CD8-. Injection of 10-30 million PLP-activated cells from one clone induced severe experimental allergic encephalomyelitis in five mice, both clinically and histologically. This represents to our knowledge the first report of PLP-specific encephalitogenic cloned T cells.

The adoptive transfer of chronic relapsing experimental allergic encephalomyelitis with lymph node cells sensitized to myelin proteolipid protein.

In this report we describe the transfer of chronic relapsing experimental allergic encephalomyelitis (EAE) with in vitro-stimulated lymph node cells (LNC) from SJL/J mice immunized with human myelin proteolipid protein (PLP). No additional immune enhancing procedures were applied in the transfer recipients. Clinical and histological EAE was transferred with 10-30 X 10(6) LNC to 27/28 mice. The LNC proliferated in vitro to PLP, but not to myelin basic protein (MBP), and induced delayed-type hypersensitivity. Enrichment for lymphoblasts by Ficoll centrifugation was essential for the disease development. The clinical course usually showed an early episode of acute paralytic illness, followed by chronic relapsing disease, and resembled the transfer of EAE using MBP-specific cells, both clinically and histologically.

Peripheral blood mononuclear cells from multiple sclerosis patients recognize myelin proteolipid protein and selected peptides.

Myelin proteolipid protein (PLP) can induce a T cell-mediated chronic relapsing autoimmune encephalomyelitis in animals and therefore is a candidate for an antigen involved in the pathogenesis of multiple sclerosis. In this report, evidence is presented that peripheral blood mononuclear cells from certain multiple sclerosis (MS) patients recognize the intact PLP molecule as well as certain synthetic PLP peptides in proliferation assays. PLP-specific T cell lines could be obtained from six of ten MS patients with early relapsing-remitting disease. These lines recognized more than one PLP peptide and the relevant peptides differed among patients. The relevance of these observations to the pathogenesis of MS remains to be determined.

Nitric oxide and T helper cell immunity.

In this article, the controversial role of nitric oxide (NO) in T helper (Th) cell activation and T-cell-dependent immunity will be discussed with an emphasis on immunosuppression by NO. NO is generated by antigen-presenting cells (APC) during the process of antigen presentation to T cells. In mouse models, activation of the inducible NO synthase (iNOS) in APC is triggered by Th1-cell-derived IFN-gamma, in combination with other soluble or membrane-associated T-cell factors. The NO so-produced inhibits T-cell proliferation, while it does not inhibit T cell cytokine production. NO blocks T-cell proliferation during G1/S transition. In mouse models of T-cell-mediated autoimmunity such as myelin antigen-induced EAE, the disease is exacerbated by genetic deletion of iNOS, indicating that NO suppresses T-cell-mediated immunity in vivo. Recent studies reveal that interaction with superoxide diminishes the T-cell regulatory activity of NO. The role for NADPH oxidase as a source for NO-inhibiting superoxide is discussed. In conclusion, NO plays an important regulatory role in the induction phase of T-cell-mediated immunity. Superoxide may enhance T-cell-mediated immunity by preventing the immunosuppressive activity of NO.

T cell expansion is regulated by activated Gr-1+ splenocytes.

CD4+ T cell proliferation depends on the balance between NO and extra-cellular superoxide (O2-). By reducing NO bio-availability, O2- promotes splenic T cell proliferation and immune response intensity. Here, we show that spleen cells from naïve mice produced neither NO nor O2- during T cell activation, but Gr-1+ splenocytes from primed mice regulated Ag-specific T cell expansion via production of both molecules. Purified splenic Gr-1+ cells included mostly granulocytes at various stages of maturation, as well as monocytes. Activation or recruitment of regulatory Gr-1+ cells was dependent on immunization with CFA. Importantly, these regulatory cells were not detected in draining lymph nodes. These data suggest that innate Gr-1+ splenic cells regulate adaptive immunity.

Immunogenicity of JHM virus proteins: characterization of a CD4+ T cell epitope on nucleocapsid protein which induces different T-helper cell subsets.

The CD4+ T-cell-restricted recognition of the structural proteins in the JHM strain of MHV (JHMV) was compared between two mouse strains. Following immunization with inactivated JHMV, all proteins elicited an immune response in both C57BL/6 and BALB/c mouse strains, except the nucleocapsid (N) protein, which was not immunogenic in C57BL/6 mice. T-cell lines and clones were derived from BALB/c mice immunized with UV-inactivated JHMV, to determine the epitope(s) for CD4+ T cells. The N protein appears immunodominant, since all T-cell lines and clones derived therefrom recognized this protein. To locate epitope-containing domains within the N protein, truncated N-protein fragments expressed in vaccinia constructs were used to stimulate the T-cell clones. Five independent T-cell clones recognized three separate N-protein domains: 1-133, 134-249, and 250-306. Since three of five clones recognized the last domain, its sequence was studied in more detail by constructing overlapping synthetic peptides covering this region. A single epitope was localized within the peptide comprising the N-protein residues 266-279. Its restriction element was identified as I-E(d) using mAb to I-E(d) and I-A(d). In addition, peptide N266-279 contains the motif for binding to I-E(d). This peptide elicited proliferative responses following immunization with JHMV, confirming its recognition in the complete virus. In addition, peptide N266-279 was recognized by T-cell clones that express differences in cytokine profile as well as in TCR Vbeta usage.

Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from the central nervous system.

Acute infection of the central nervous system by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces nucleocapsid protein specific cytotoxic T lymphocytes (CTL) not found in the periphery (S. Stohlman, S. Kyuwa, J. Polo, D. Brady, M. Lai, and C. Bergmann, J. Virol. 67:7050-7059, 1993). Peripheral induction of CTL specific for the nucleocapsid protein of JHMV by vaccination with recombinant vaccinia viruses was unable to provide significant protection to a subsequent lethal virus challenge. By contrast, the transfer of nucleoprotein-specific CTL protected mice from a subsequent lethal challenge by reducing virus replication within the central nervous system, demonstrating the importance of the CTL response to this epitope in JHMV infection. Transfer of these CTL directly into the central nervous system was at least 10-fold more effective than peripheral transfer. Histological analysis indicated that the CTL reduced virus replication in ependymal cells, astrocytes, and microglia. Although the CTL were relatively ineffective at reducing virus replication in oligodendroglia, survivors showed minimal evidence of virus persistence within the central nervous system and no evidence of chronic ongoing demyelination.

The immunopathology of chronic experimental allergic encephalomyelitis induced in rabbits with bovine proteolipid protein.

The role of myelin proteolipid apoprotein (PLP) in the central nervous system (CNS) immune response of rabbits has been investigated by analyzing the immunopathology of chronic experimental allergic encephalomyelitis (EAE) induced by sensitization with PLP. Clinical disease occurred in seven out of nine rabbits sensitized with bovine PLP and monitored for up to 6 mo. Positive delayed hypersensitivity skin test reactions to PLP occurred in all but one of the PLP-sensitized animals. All PLP-sensitized animals had meningeal and CNS parenchymal inflammation that correlated with disease severity. Serial blood samples were stained with a panel of antibodies to rabbit T and B cells, as well as Ia, and large and small mononuclear cell populations were analyzed by flow cytometry. Peripheral leukocyte population staining did not correlate with clinical signs or sensitization to PLP. Cryostat CNS tissue sections were stained with the same set of antibodies by using an immunoperoxidase technique, and positive cells and vessels were counted. T cells and macrophages were numerous and in equal numbers in perivascular parenchymal inflammatory infiltrates, whereas B cells were less numerous (p less than 0.001). T cells also diffusely infiltrated the parenchyma. Most perivascular inflammatory cells and many scattered parenchymal cells were Ia+; Ia vascular expression was increased over controls (p less than 0.001), and also correlated with disease severity. The immunopathology of this chronic EAE model is the same as that of whole CNS tissue- and myelin basic protein-induced EAE in other species, and is similar to that of multiple sclerosis. Cellular immune responses to PLP may therefore contribute to systemic and in situ responses in CNS tissue demyelinating diseases.

Nitric oxide inhibits the proliferation of T-helper 1 and 2 lymphocytes without reduction in cytokine secretion.

To study the effect of nitric oxide (NO) on the activity of Th subsets, cloned Th1 and Th2 lymphocytes were stimulated in the presence of an NO donor. NO, when present from the start of incubation, inhibited the proliferation of both Th subsets dose-dependently, achieving complete inhibition at a relatively low level. The addition of NO 24 h after the onset of T cell stimulation also resulted in reduced proliferation of both Th subsets, suggesting that NO affects a late process during T cell activation. Stimulation of T cells in the presence of NO did not induce apoptosis at the concentrations that completely inhibited proliferation, although apoptosis became evident at higher NO concentrations. The secretion of several cytokines (i.e., IFN-gamma, IL-4, and IL-5) was slightly upregulated, while IL-2 production was modestly inhibited in the presence of NO. However, exogenous IL-2 did not reverse the NO-induced inhibition of T cell proliferation, nor did additional stimulation with phorbol esters. Finally, expression of IL-2R was modestly decreased in the presence of NO, although TCR expression was not affected. These studies demonstrate that relatively low concentrations of NO induce a strong and specific inhibition of T cell proliferation in both Th subsets, suggesting that local NO production may regulate Th-mediated tissue inflammation.

Myelin proteolipid protein-induced Th1 and Th2 clones express TCR with similar fine specificity for peptide and CDR3 homology despite diverse V beta usage.

Myelin-specific T-helper (Th) cells which induce encephalomyelitis belong to the inflammatory Th1 subset. Th2 cells recognizing similar epitopes potentially represent specific inhibitors of encephalitogenic Th1 cells. Since the differential stimulation of antigen-specific Th2 cells may be important in the regulation of autoimmune inflammatory disorders, we have examined the fine specificity of a Th1 and a Th2 clone, induced by immunization of SJL mice with native proteolipid protein (PLP) and specific for the PLP 139-151 sequence. Stimulation of the clones by synthetic peptides containing single alanine substitutions demonstrated that L141, W144, H147, and P148 represent critical residues. Surprisingly, this pattern was identical for both subsets. Competition studies indicated indirectly that L141 and P148 may be MHC-binding residues, whereas W144 and H147 contact the TCR. Sequencing of the TCR expressed by both Th subset clones demonstrated different V beta usage as well as variation in the D-region sequence and length. Interestingly, realignment of the sequence of the CDR3 regions showed striking homology. This study demonstrates that Th1 and Th2 subsets can express very similar peptide specificities, while utilizing very different TCR V beta chains. These results suggest that the therapeutic modalities based on either peptide antagonists or antibodies specific for CDR3 may have limited effectiveness in treating autoimmune disorders, since they may also target the beneficial arm of the immune response.