Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding.

We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.

Absent semicircular canals in CHARGE syndrome: radiologic spectrum of findings.

BACKGROUND AND PURPOSE: This paper describes the CT findings that characterize the middle and inner ear anomalies in coloboma, heart defects, choanal atresia, mental retardation, genitourinary, and ear anomalies (CHARGE) syndrome. With this information, neuroradiologists will be better prepared to provide clinically relevant information to their referring physicians regarding this rare syndrome. MATERIALS AND METHODS: CT studies from 13 patients were reviewed by 2 neuroradiologists with Certificate of Additional Qualification. Each ear was counted separately for a total of 26 ears. Middle and inner ear anomalies associated with CHARGE syndrome were categorized. Investigational review board approval was obtained. RESULTS: Twenty of 26 (77%) ears demonstrated cochlear aperture atresia. Four of these ears were evaluated with MR imaging and were found to lack a cochlear nerve. Twenty-one of 26 (81%) cochlea had some form of dysplasia. Six of 26 (23%) round windows were aplastic. Three of 26 (12%) round windows were hypoplastic. Twenty-one of 26 (81%) oval windows were atretic or aplastic. Fifteen of 26 (58%) vestibules were hypoplastic or dysplastic. There were 5 of 26 (19%) enlarged vestibular aqueducts. Twelve of 26 (46%) vestibular aqueducts had an anomalous course. All cases demonstrated absent semicircular canals. Twenty-three of 26 (88%) facial nerve canals had an anomalous course. Four of 26 (15%) tympanic segments were prolapsed. Three of 26 (12%) temporal bones had an anomalous emissary vein referred to as a petrosquamosal sinus. Twenty-one of 26 (81%) middle ear cavities were small. Twenty-three of 26 (93%) ossicles were dysplastic with ankylosis. Three of 26 (12%) internal auditory canals were small. CONCLUSION: The CT findings that correlate to the anomalies of CHARGE syndrome affect conductive as well as sensorineural hearing. Stenosis of the aperture for the cochlear nerve aperture on CT is suggestive of hypoplasia or absence of the cochlear nerve, which has been demonstrated in some cases by MR. Absence of the cochlear nerve would be a contraindication to cochlear implantation.

Limitations of T2*-Gradient Recalled-Echo and Susceptibility-Weighted Imaging in Characterizing Chronic Subdural Hemorrhage in Infant Survivors of Abusive Head Trauma.

A possible misconception among radiologists is that chronic subdural hemorrhage should show some degree of blooming on T2*-gradient recalled-echo or susceptibility-weighted sequences such as SWI and susceptibility-weighted angiography, which is not necessarily true. We present 5 cases of chronic subdural hemorrhages in infants, demonstrating intensity near or greater than that of CSF with variable amounts of hemosiderin staining along the neomembranes. We review the physiology and MR imaging physics behind the appearance of a chronic subdural hemorrhage, highlighting that the absence of a BBB can allow hemosiderin to be completely removed from the subdural compartment. Finally, we stress the importance of reviewing all multiplanar sequences for the presence of neomembranes, which can be quite subtle in the absence of hemosiderin staining and are critical for making the diagnosis of chronic subdural hemorrhage.

Parenchymal Brain Laceration as a Predictor of Abusive Head Trauma.

BACKGROUND AND PURPOSE: Accurate differentiation of abusive head trauma and accidental head injury in infants and young children is critical and impacts clinical care, patient prognosis, forensic investigations, and medicolegal proceedings. No specific finding seen on cross-sectional brain imaging has been reported to distinguish abusive head trauma from accidental injury. Our study investigated whether a specific imaging finding, parenchymal brain laceration, is unique to children diagnosed with abusive head trauma. MATERIALS AND METHODS: We retrospectively identified 137 patients with abusive head trauma and 28 patients who incurred moderate to severe accidental brain injury. Brain MR imaging represented the imaging standard for characterizing intracranial injuries. RESULTS: Among the abusive head trauma cohort, parenchymal brain lacerations were identified in 18 patients, while none were identified in any patients with accidental injury. CONCLUSIONS: Our findings are in concurrence with the existing forensic, pathology, and imaging literature, which suggests that parenchymal brain lacerations may be related to abusive injury mechanisms.

T cell activation pathways: B7, LFA-3, and ICAM-1 shape unique T cell profiles.

Two signals are required for induction of cell proliferation and cytokine production in resting T cells. Occupancy of the T cell receptor by antigen/MHC complexes delivers the first signal to the T cell, while the second signal is provided by interaction with costimulatory ligands on APC. CD2, LFA-1, and CD28 are the major costimulatory and adhesive molecules on T cells and bind to the LFA-3, ICAM-1 and B7 ligands, respectively, on APC. LFA-3 plays a central role for naive and memory T helper cells during the early phase of an immune response. The LFA-3/CD2 pathway initiates strong antigen-independent cell adhesion, substantial expansion of naive T helper cells, and induction of large amounts of IFN-gamma in memory cells. The release of IFN-gamma may upregulate expression of ICAM-1 and B7 on APC and allows multiple adhesion pathways to amplify the immune response. The LFA-1/ICAM-1 pathway stimulates adhesion and cell proliferation more efficiently in memory T helper cells than in naive cells. Further, the results suggest that naive T helper cells express functionally inactive LFA-1 molecules on the cell surface, which may have a physiological role in keeping these cells in a resting state. B7 costimulation superinduces IL-2 production in both naive and memory T helper cells and generates long-lasting cell proliferation. This permits transition from an autocrine to a paracrine immune response. Coexpression of B7/LFA-3 provides an optimal APC function and enables a vigorous T cell response to minute amounts of antigen. AP-1 and NF-kappa B transcription factors are involved in the induction of several cytokine gene promoters and play a central role in the regulation of IL-2 gene transcription. LFA-3 costimulation only moderately enhances AP-1 DNA-binding activity and does not influence the NF-kappa B activity induced by TCR engagement, whereas B7 costimulation induces large amounts of NF-kappa B and AP-1 activity in T helper cells. The costimulatory ligands represent a family of adhesion molecules with considerable redundancy. Interfamily redundancy of LFA-3, B7, and ICAM ligands offers an opportunity to regulate distinct T cell response profiles in various microenvironments at separate time points of an immune response.

Linomide abolishes leukocyte adhesion and extravascular recruitment induced by tumor necrosis factor alpha in vivo.

The immunomodulator Linomide (roquinimex) ameliorates the development of numerous inflammatory and immunological diseases, including sepsis, arthritis, and encephalomyelitis. However, the mechanism underlying this protective effect of Linomide remains unclear. In this study, we wanted to evaluate the effect of Linomide treatment on the different steps in the extravasation process of leukocytes stimulated by tumor necrosis factor alpha (TNF-alpha) in vivo. For this purpose, we used intravital microscopy in the mouse cremaster muscle microcirculation. We found that pretreatment with Linomide dose-dependently (3-300 mg/kg) reduced TNF-alpha-induced leukocyte adhesion and tissue recruitment. Notably, at 300 mg/kg response to TNF-alpha was nearly abolished, i.e. leukocyte adhesion was decreased by 83% and recruitment by 86%. In fact, the anti-inflammatory effect of this dose of Linomide corresponded in magnitude to the potency of 10 mg/kg of dexamethasone. Moreover, administration of Linomide did not alter the systemic leukocyte counts. On the other hand, 1-10 mg/kg of dexamethasone decreased the circulating number of mononuclear leukocytes by 77%. Taken together, our novel findings demonstrate that Linomide is a potent inhibitor of leukocyte adhesion and recruitment in cytokine-activated tissues. These data may help explain the documented protection provided by Linomide in inflammatory diseases characterized by cytokine and leukocyte accumulation.

Enlarged parietal foramina: association with cerebral venous and cortical anomalies.

OBJECTIVE: To evaluate a series of patients with enlarged parietal foramina for associated brain anomalies. BACKGROUND: Enlarged parietal foramina are usually considered a benign calvarial defect. METHODS: Ten patients with enlarged parietal foramina were identified. Seven patients were evaluated with neuroimaging: two by cranial CT and five by CT and/or MRI. Three patients who underwent MRI also underwent MR angiography or MR venography. RESULTS: Six of seven patients had cranial imaging showing a persistent falcine venous sinus. Three of six patients had variations of occipital cortical infolding. One patient had focal encephalomalacia in close proximity to the persistent falcine venous sinus and one had a previously undiagnosed atretic occipital encephalocele. CONCLUSION: This constellation of findings suggests that aberrant vascular evolution during fetal development may affect cerebrovascular, brain, or skull development. Individuals with enlarged parietal foramina (>5 mm) warrant imaging of underlying brain parenchyma and vasculature.

Magnetic carbohydrate nanoparticles for affinity cell separation.

Magnetically responsive nanoparticles were prepared from enzymatically hydrolysed starch and magnetite. Two different monoclonal antibodies were covalently coupled to the particles. The antibody-coupled particles were in the size range of 100-300 nm and had an iron content of about 60%. Using 100 micrograms of magnetic particles (coupled with monoclonal mouse anti-rat Ig kappa light chain antibody) a very high depletion of surface Ig positive cells (mostly B-cells) from one million rat peripheral blood mononuclear cells could be achieved. The separation efficiency was evaluated by flow cytofluorometric analysis. This technique permits the detection of a small number of surface Ig positive cells among 10,000 negative cells.

Suppression of experimental autoimmune neuritis by ABR-215062 is associated with altered Th1/Th2 balance and inhibited migration of inflammatory cells into the peripheral nerve tissue.

The therapeutic effects of ABR-215062, which is a new immunoregulator derived from Linomide, have been evaluated in experimental autoimmune neuritis (EAN), a CD4(+) T cell-mediated animal model of Guillain-Barré syndrome in man. In previous studies, we reported that Linomide suppressed the clinical EAN and myelin antigen-reactive T and B cell responses. Here EAN induced in Lewis rats by inoculation with peripheral nerve myelin P0 protein peptide 180-199 and Freund's complete adjuvant was strongly suppressed by ABR-215062 administered daily subcutaneously from the day of inoculation. ABR-215062 dose-dependently reduced the incidence of EAN, ameliorated clinical signs and inhibited P0 peptide 180-199-specific T cell responses as well as also the decreased inflammation and demyelination in the peripheral nerves. The suppression of clinical EAN was associated with inhibition of the inflammatory cytokines IFN-gamma and TNF-alpha, as well as the enhancement of anti-inflammatory cytokine IL-4 in lymph node cells and periphery nerve tissues, respectively, in a dose-dependent manner. These effects indicate that ABR-215062 may mediate its effects by regulation of Th1/Th2 cytokine balance and suggest that ABR-215062 is potentially a new chemical entity for effective treatment of autoimmune diseases.

Activation of alloreactive natural killer cells is resistant to cyclosporine.

BACKGROUND: We have previously shown that cytotoxic T lymphocytes (CTL) with alloreactivity were induced when Wistar Furth (WF; RT1u) rats were immunized with allogeneic Brown Norway (BN; RT1n) cells. In contrast, when BN rats were immunized with WF cells, the allospecific response was confined to alloreactive natural killer (NK) cells, and no CTL activity was observed. In this study, the effect of cyclosporine (CsA) on the activation of alloreactive NK cells in vivo was analyzed. METHODS: Distinct peritoneal effector cells from rats immunized with allogenic cells with or without concomitant CsA and/or interleukin (IL) 2 treatment were tested for specific cytolytic activity. Furthermore, the presumptive role of NK cells in rejection immunity was addressed in a cardiac graft model. RESULTS: The results showed that doses of CsA that completely inhibited the activation of alloreactive CTL, only marginally affected the activation of alloreactive NK cells. We also showed that CsA treatment failed to prolong graft survival in BN recipients of WF hearts. Treatment of BN rats with CsA/IL-2 during immunization with allogeneic WF cells resulted in concomitant induction of alloreactive NK cells and alloreactive CTL. CONCLUSIONS: We have demonstrated that CsA failed to suppress the activation of alloreactive NK cells. Consequently, the cardiac graft survival in the donor-recipient combination known to activate alloreactive NK cells was not significantly prolonged by CsA treatment, emphasizing the involvement of NK cells as effectors in organ rejection. Furthermore, the parallel emergence of alloreactive NK cells and CTL only in the presence of CsA/IL-2 indicated that CsA interfered with alloreactive NK cell-associated suppression of CTL activated by allogeneic tissue.

Linomide-induced suppression of experimental autoimmune neuritis is associated with down-regulated macrophage functions.

Experimental autoimmune neuritis (EAN) is a T-cell mediated autoimmune disease of the peripheral nervous system, in which macrophages and T-cells feature prominently in nerve lesions. EAN represents a counterpart to Guillain-Barré syndrome in humans. In the present study, we investigated the in vitro and in vivo effects of Linomide (LS-2616, quinoline-3-carboxamide), a synthetic immunomodulatory compound, on macrophages in relation to EAN. Linomide strongly suppressed IFN-gamma and lipopolysaccharide (LPS)-induced IL-1 beta, TNF-alpha and IL-6 mRNA expression in macrophages in vitro as demonstrated by in situ hybridisation. Linomide administered daily subcutaneously from the day of inoculation completely prevented the development of clinical symptoms of EAN. Linomide administered from day 9 post immunisation (p.i.) significantly suppressed clinical EAN. Macrophages from Linomide-treated EAN rats showed decreased IL-1 beta, TNF-alpha and IL-6 mRNA expression in response to IFN-gamma and LPS. LPS-induced nitric oxide production by macrophages was also suppressed by Linomide in vitro. Linomide, however, does not affect macrophage death and release of lactate dehydrogenase. We conclude that Linomide may exert its actions in EAN and perhaps also in other autoimmune disease models, by suppressing macrophage functions.

Linomide suppresses acute experimental autoimmune encephalomyelitis in Lewis rats by counter-acting the imbalance of pro-inflammatory versus anti-inflammatory cytokines.

Linomide (quinoline-3-carboxamide) is a synthetic immunomodulator that suppresses several experimental autoimmune diseases. Here we report the effects of Linomide on experimental autoimmune encephalomyelitis (EAE), a CD4+ T cell-mediated animal model of multiple sclerosis (MS). EAE induced in Lewis rats by inoculation with homogenized guinea pig spinal cord and Freund's complete adjuvant was strongly suppressed by Linomide administered daily subcutaneously from the day of inoculation. Linomide dose-dependently delayed the interval between immunization and onset of clinical EAE, and reduced severity of EAE symptoms. These clinical effects were associated with dose-dependent down-modulation of myelin antigens-induced T cell responses and by suppression of the proinflammatory cytokines IFN-gamma and TNF-alpha, and upregulation IL-4, IL-10 and TGF-beta as evaluated by in situ hybridization for mRNA expression in spleen mononuclear cells and spinal cord sections. These findings suggest that Linomide could be useful in certain T cell dependent autoimmune diseases.

Linomide suppresses both Th1 and Th2 cytokines in experimental autoimmune myasthenia gravis.

Suppressive effects of the synthetic immunomodulatory drug Linomide have been shown in several autoimmune models, including antibody-mediated experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia gravis (MG). To define the mechanisms underlying EAMG suppression, we injected Linomide subcutaneously at different doses into Lewis rats immunized with Torpedo acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA), and investigated AChR-specific T and B cell responses, and the levels of lymph node cells expressing mRNA of different cytokines after AChR stimulation in vitro. Both 160 and 16, but not 1.6, mg/kg/day of Linomide effectively suppressed clinical muscle weakness, accompanied by decreased AChR-induced T and B cell responses. Linomide also suppressed the mRNA expression of the Th1 cytokines IFN-gamma, IL-12 and TNF-alpha as well as the Th2 cytokines IL-4 and IL-10, which are important in the immunopathogenesis of EAMG by promoting antibody production. There were no differences for IL-1beta, IL-6, lymphotoxin or TGF-beta expression in Linomide-treated vs nontreated control EAMG rats. We conclude that Linomide suppresses clinical EAMG as well as B and T cell responses to AChR by counteracting the production of AChR-induced Th1 and Th2 cytokines.

Use of a monoclonal rat anti-mouse Ig light chain (RAMOL-1) antibody reduces background binding in immunohistochemical and fluorescent antibody analysis.

Rat monoclonal antibodies (MAb) directed to mouse Ig heavy and light chain determinants were produced. A rat anti-mouse light chain MAb (RAMOL-1) which bound to all (24/24) mouse Ig of the kappa light chain type and with varying strength to 4/4 lambda light chain-bearing Ig was evaluated as a general secondary reagent, together with two MAb that bound to the heavy chain of mouse IgG. They were conjugated with biotin or FITC and used in immunohistochemical and immunofluorescence assays to detect mouse monoclonal antibodies binding to antigens expressed in rat and human tissues and cells. As compared to commercially available polyclonal reagents, RAMOL-1 gave higher staining contrast by showing lower background staining and equal or higher staining of the primary MAb tested. This was a result of two main effects. First, crossreactivity with endogenous Ig and tissue type-specific determinants was eliminated. With polyclonal anti-mouse Ig reagents, binding to endogenous Ig was noted in vascular spaces and on Ig-bearing cells, and to rat gastric mucosa and epithelial tumor tissue in frozen tissue sections, even when diluted in high concentrations of serum homologous to the tissue. Second, binding of the secondary reagent was reduced to cells and tissues prone to have high nonspecific binding capability, such as monocytes/macrophages and formalin-fixed, paraffin-embedded tissue. Owing to unlimited and reproducible access to this homogeneous reagent, RAMOL-1 is used as second antibody to standardize the procedure used for immunohistochemical grading of human malignant tumors by determination of blood group antigen expression detected with mouse MAb.

Proliferation of human CD4+45R+ and CD4+45R- T helper cells is promoted by both IL-2 and IL-4 while interferon-gamma production is restricted to IL-2 activated CD4+45R- T cells.

Recombinant IL-2 (rIL-2) and IL-4 (rIL-4) promote proliferation of human CD4+ T cells activated in the presence of PHA, TPA or OKT-3 monoclonal antibody (MAb), whereas the production of interferon-gamma (IFN) can be induced only by rIL-2. rIL-4 induced strong proliferative responses both in accessory cell independent assays and in the presence of autologous monocytes, but has failed to induce IFN production in any of these systems. The ability of rIL-2 to induce IFN production was strongly enhanced by the addition of monocytes, although a similar proliferative response was recorded in the absence or presence of monocytes. The MAb anti-Tac inhibited the proliferative response and the production of IFN by CD4+ T cells activated in the presence of rIL-2, whereas the proliferative response to rIL-4 was unaffected. CD4+45R+ and CD4+45R- T helper cell subsets proliferated in response to both IL-2 and IL-4. A kinetic analysis demonstrated that the production of IFN throughout a five day activation period was restricted to stimulation of CD4+45R- T cells with rIL-2. This report clearly demonstrates a dissociation of IFN production and T cell proliferation in man. While proliferation can be induced by both IL-2 and IL-4 in both the helper T cell subsets studied, IFN production was induced only in the CD4+45R- subsets and only in response to IL-2.

Locally superantigen-activated peritoneal cytolytic T lymphocytes belong to the CD8+ CD45RC- subset and lyse MHC class II+ tumor cells.

Bacterial encoded superantigens (SA) are capable of activating and targeting cytolytic human and mouse T lymphocytes (CTL) to lyse major histocompatibility complex class II positive (MHC class II+) target cells. In this study both in vitro and in vivo activated rat CTL were directed against MHC II+ tumor targets by bacterial encoded SA. Polyclonal in vitro activation of rat peripheral blood T lymphocytes generated CTL capable of killing MHC class II+ human BSM cells coated by staphylococcal enterotoxin (SE) -A, -E, -D, and TSST-1 but not by SEB or SEC1-3. Allo selective peritoneal CTL generated by intraperitoneal stimulation with allogeneic spleen cells were directed against BSM cells by SEA, -D, and -E but not by SEB, SEC1-3 or TSST-1. Based on the above observations, and in order to locally activate CTL, SEA was chosen for in vivo priming of rats by intraperitoneal inoculation of the toxin. SEA injection generated highly cytolytic CTL, and maximum cytolytic responses were seen at 50-250 micrograms SEA per animal with a peak in response 48-72 hours after injection of the toxin. The cytolytic activity of peritoneal SEA reactive effector cells was confined to the TCR alpha beta+ CD4- CD8+ CD45RC- cell population. MHC class II- colon carcinoma cells were insensitive to lysis by SEA reactive CTL but colon carcinoma cells induced to express MHC class II by interferon-gamma (IFN-gamma) treatment were efficiently lysed in the presence of SEA. Comparison of rat and human MHC II+ colon carcinomas revealed a peak in sensitivity to lysis at 10-100 ng SEA/ml for both tumor targets. These findings suggest that superantigens can be used in local immunotherapy of peritoneal tumors such as ovarian and colorectal carcinomatosis, with inducible or constitutive expression of MHC class II.

The inhibitory effect in experimental autoimmune encephalomyelitis by the immunomodulatory drug Linomide (PNU-212616) is not mediated via release of endogenous glucocorticoids.

The immunomodulatory drug Linomide (PNU-212616) is an efficient inhibitor of experimental autoimmune encephalomyelitis (EAE) and a variety of other models of autoimmunity. The mechanism of action of the drug is, however, incompletely resolved. It was recently suggested that Linomide might exert its immunomodulatory activity by stimulation of the hypothalamic-pituitary-adrenal axis. To investigate the relevance of this mechanism of action, we monitored the plasma levels of endogenous corticosterone after treatment with Linomide, and also directly compared the inhibitory activity of the drug on acute EAE induced in sham or adrenalectomized SJL/N mice. Treatment with Linomide resulted in a dose related inhibition of EAE in line with previously reported results. Upon onset of clinical signs of EAE, there was a 7-10 fold elevation of plasma corticosterone from the normal baseline level. Administration of Linomide did however not by itself result in any change in plasma corticosterone levels, neither at the pre-symptomatic phase of the disease nor during acute short term treatment. In adrenal ectomized animals immunized for EAE, paralytic disease developed rapidly and was of a more severe and fatal nature as compared to sham-operated controls. Treatment with Linomide had a profound inhibitory effect on development of paralytic disease in both the ectomized and sham-operated groups. These results strongly suggest that Linomide does not exert its immunomodulatory activity via the release of endogenous glucocorticoids.

Inhibition of autoimmune disease by the immunomodulator linomide correlates with the ability to activate macrophages.

Linomide is a potent immunomodulator that has been shown to inhibit autoimmunity in several animal models of autoimmune disease, including experimental autoimmune encephalomyelitis (EAE). Linomide's mechanism of action is unknown, however, it has been suggested to modulate the function of antigen presenting cells (APC) and that this may account for the inhibition of autoimmune disease. In this study we have been able to show that Linomide treatment of SJL/N mice upregulates the cell surface expression of several activation markers on macrophages and B cells. Thus, we found the following markers, expressed as a % of control, to be significantly upregulated following Linomide treatment; MHC class II (260%), Ly-6A/E (520%), CD11a (280%), CD54 (190%) and CD80 (200%) on macrophages and Ly-6A/E (250%) and CD11a (150%) on B cells. The duration and dosage of Linomide required to obtain these effects is similar to those required for EAE inhibition. Several Linomide analogues were made by the introduction of structural modifications into the Linomide molecule, resulting in a number of compounds with varying effects on EAE. We found a linear relationship between the compound's ability to inhibit EAE and its ability to upregulate MHC class II on macrophages (p<0.001), such that compounds which were able to inhibit EAE also upregulated MHC class II expression, whereas those that did not inhibit EAE were unable to do so. These results suggest that drug-mediated activation of distinct APC functions may be protective in autoimmunity.

The immune modulator Linomide prevents neuronal death in injured peripheral nerves of the mouse.

Neuronal death after injury or disease could result from imbalanced cytokine expression. Linomide (LS-2616, quinoline-3-carboxamide), a synthetic immunomodulator with effects on cytokine production, suppresses autoimmune diseases of the nervous system. Here adult mice were pre-treated with 200 mg/kg/day of Linomide for 9 days, after which the sciatic nerves were crushed. After another 10 days of Linomide treatment the dorsal root ganglia were dissected out and stained for apoptosis, either immediately or after 2 days in culture, which increases cell death. Superior cervical ganglia were also cultured for 2 days. The Linomide pretreatment profoundly reduced (approximately 60-80%) the injury-induced apoptotic death of neurons and satellite cells in both systems. The results suggest that modulation of the inflammatory cytokine cascade is a promising road to nerve cell rescue.