RESUMEN

AIMS: Multiple sclerosis (MS) is the leading cause of non-traumatic neurological disability in young adults, and its diagnosis is often delayed due to the lack of diagnostic markers. Initiation of disease -modifying therapy in the early stages of MS is especially critical because currently available therapy mostly target relapsing-remitting MS, and is less effective as disease progresses into the more chronic form of secondary-progressive MS. Therefore, exploring specific and sensitive biomarkers will facilitate an expedited and more accurate diagnosis to allow currently available therapies to be more effective. MAIN METHODS: Western blotting was conducted to detect the expression of neurolymphatic proteins in human brain endothelial cells in culture. Additionally, using a cohort of 150 patients with relapsing remitting MS, 26 with secondary progressive MS, and 60 healthy control samples, neurolymphatic protein expression was detected in serum samples using dot blot analysis. KEY FINDINGS: Human brain microvascular endothelial cells express neurolymphatic markers. Neurolymphatic protein abundance increases with tumor necrosis factor (TNF)-α stimulation but decreases with interferon (IFN)- γ or combined (TNF + IFN) treatment. Circulating neurolymphatic protein levels is significantly lower in MS patients. Further, one of the markers, FOXC2, is associated with the clinical stages of MS, with significantly lower expression in secondary progressive MS compared to relapsing remitting MS. SIGNIFICANCE: Our findings describe brain endothelial expression of neurolymphatic proteins, which is altered under inflammatory stress, and provide a possibility of using a collective pool of circulating neurolymphatic proteins as a diagnostic and prognostic biomarker of MS.

Asunto(s)

Biomarcadores/sangre , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Inflamación/sangre , Esclerosis Múltiple Crónica Progresiva/sangre , Esclerosis Múltiple Recurrente-Remitente/sangre , Esclerosis Múltiple/sangre , Adulto , Estudios de Casos y Controles , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/inmunología , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Femenino , Humanos , Inflamación/diagnóstico , Inflamación/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/inmunología , Esclerosis Múltiple Crónica Progresiva/diagnóstico , Esclerosis Múltiple Crónica Progresiva/inmunología , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Esclerosis Múltiple Recurrente-Remitente/inmunología

RESUMEN

BACKGROUND: Although multiple sclerosis (MS) is thought to represent an excessive and inappropriate immune response to several central nervous system (CNS) autoantigens, increasing evidence also suggests that MS may also be a neurovascular inflammatory disease, characterized by endothelial activation and shedding of cell membrane microdomains known as 'microparticles' into the circulation. OBJECTIVE: To investigate the relationships between these endothelial biomarkers and MS. METHODS: We examined the relative abundance of CD31(+)/PECAM-1, CD51(+)CD61(+) (αV-ß3) and CD54(+) (ICAM-1) bearing microparticles in sera of healthy individuals, patients with relapsing-remitting MS, and secondary-progressive MS. We also investigated the correlation among circulating levels of different microparticle species in MS with conventional MRI (T2- and T1-lesion volumes and brain atrophy), as well as novel MR modalities [assessment of iron content on susceptibility-weighted imaging (SWI)-filtered phase]. RESULTS: Differences in circulating microparticle levels were found among MS groups, and several microparticle species (CD31(+)/CD51(+)/CD61(+)/CD54(+)) were found to correlate with conventional MRI and SWI features of MS. CONCLUSION: These results indicate that circulating microparticles' profiles in MS may support mechanistic roles for microvascular stress and injury which is an underlying contributor not only to MS initiation and progression, but also to pro-inflammatory responses.

Asunto(s)

Antígenos CD/sangre , Encéfalo/patología , Esclerosis Múltiple Crónica Progresiva/sangre , Esclerosis Múltiple Crónica Progresiva/patología , Esclerosis Múltiple Recurrente-Remitente/sangre , Esclerosis Múltiple Recurrente-Remitente/patología , Adulto , Estudios de Casos y Controles , Estudios Transversales , Femenino , Citometría de Flujo , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad

RESUMEN

BACKGROUND AND PURPOSE: The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, 'lymphangions'. Here we have studied the effects of the inflammatory cytokine IL-1ß on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC). EXPERIMENTAL APPROACH: We measured IL-1ß in colon-conditioned media (CM) from acute (AC-CM, dextran sodium sulfate) and chronic (CC-CM, T-cell transfer) colitis-induced mice and corresponding controls (Con-AC/CC-CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h-IL-1ß or h-TNF-α (5, 10, 20 ng·mL(-1) ), with or without COX inhibitors [TFAP (10(-5) M), diclofenac (0.2 × 10(-5) M)], PGE2 (10(-5) M)], IL-1-receptor antagonist, Anakinra (5 µg·mL(-1) ), or a selective prostanoid EP4 receptor antagonist, GW627368X (10(-6) and 10(-7) M). KEY RESULTS: Tonic contractility of RMLMC was reduced by AC- and CC-CM compared with corresponding control culture media, Con-AC/CC-CM. IL-1ß or TNF-α was not found in Con-AC/CC-CM, but detected in AC- and CC-CM. h-IL-1ß concentration-dependently decreased RMLMC contractility, whereas h-TNF-α showed no effect. Anakinra blocked h-IL-1ß-induced RMLMC relaxation, and with AC-CM, restored contractility to RMLMC. IL-1ß increased COX-2 protein and PGE2 production in RMLMC.. PGE2 induced relaxations in RMLMC, comparable to h-IL-1ß. Conversely, COX-2 and EP4 receptor inhibition reversed relaxation induced by IL-1ß. CONCLUSIONS AND IMPLICATIONS: The IL-1ß-induced decrease in RMLMC tonic contraction was COX-2 dependent, and mediated by PGE2 . In experimental colitis, IL-1ß and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC-CM and pharmacological blockade of IL-1ß restored tonic contraction.

Asunto(s)

Ciclooxigenasa 2/fisiología , Dinoprostona/fisiología , Interleucina-1beta/farmacología , Células Musculares/efectos de los fármacos , Animales , Células Cultivadas , Colitis/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Ganglios Linfáticos/citología , Masculino , Mesenterio/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Células Musculares/metabolismo , Células Musculares/fisiología , Ratas , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Theiler's murine encephalomyelitis virus (TMEV) belongs the family Picornaviridae. TMEV not only replicates in the gastrointestinal tract but also spreads to the central nervous system (CNS) either by a hematogenous or a neural pathway during natural infection. The DA strain of TMEV infects neurons during the acute phase, and glial cells and macrophages during the chronic phase, leading to a demyelinating disease similar to multiple sclerosis. Different virus-host receptor interactions in the peripheral and the neuronal cells could explain the pathways of viral spread from the peripheral to the CNS and neurons to glial cells. However, the receptor for TMEV remains unknown. P0 protein, a 28-31 kD glycoprotein, belongs to the immunoglobulin superfamily and constitutes 50% of the total myelin protein in the peripheral nerve. Other picornaviruses use members of the immunoglobulin superfamily as receptors. Thus we hypothesized P0 protein could act as a receptor for TMEV. In a virus overlay assay, radiolabeled TMEV bound to a 28-30 kD protein from the peripheral nerve of wild-type C57BL/6, but no binding was found in the peripheral nerve from P0-knockout mice. TMEV replicated fourfold higher in P0-transfected BW5147.G.1.4 cells than in mock-transfected cells. The increase in virus replication in the P0-transfected cell line was blocked by preincubation of the cells with anti-P0 antibody. A virus binding study showed that TMEV bound to P0-transfected cells but not to mock-transfected cells. The use of the P0 protein in Schwann cells as a receptor may be one mechanism by which TMEV spreads from the gastrointestinal tract to the CNS.

Asunto(s)

Infecciones por Cardiovirus/metabolismo , Proteína P0 de la Mielina/metabolismo , Nervio Ciático/virología , Theilovirus , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Cápside/genética , Proteínas de la Cápside , Células Cultivadas , Cricetinae , Expresión Génica/fisiología , Riñón/citología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteína P0 de la Mielina/genética , Proteína P0 de la Mielina/inmunología , Nervio Ciático/citología , Transfección

RESUMEN

Although many viruses have been isolated from patients with multiple sclerosis (MS), as yet, no one agent has been demonstrated to cause MS. In contrast, epidemiological data indicate that viral infections are associated with exacerbations of MS. Here, we present data showing that virus infections can subclinically prime animals for central nervous system (CNS) autoimmune disease; long after the original infection has been eradicated, a nonspecific challenge/infection can trigger an exacerbation. The priming infectious agent must show molecular mimicry with self-CNS antigens such as glial fibrillary acidic protein (GFAP), myelin associated glycoprotein (MAG) or myelin proteolipid protein (PLP). The subsequent challenge, however, may be nonspecific; complete Freund's adjuvant (CFA), or infection with a recombinant vaccinia virus encoding an irrelevant protein, could trigger CNS disease. In the CNS, we could detect a mononuclear cell infiltration, but no demyelination was found. However, if the pathogenesis of MS is similar to that of this novel animal model for CNS autoimmune disease, our findings could help explain why exacerbations of MS are often associated with a variety of different viral infections.

Asunto(s)

Enfermedades Desmielinizantes/virología , Encefalomielitis Autoinmune Experimental/virología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Animales , División Celular/inmunología , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Linfocitos/citología , Linfocitos/virología , Ratones , Ratones Endogámicos , Imitación Molecular/inmunología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Esclerosis Múltiple/virología , Proteína Proteolipídica de la Mielina/genética , Proteína Proteolipídica de la Mielina/inmunología , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Ubiquitinas/genética , Ubiquitinas/inmunología , Vaccinia/patología , Virus Vaccinia/genética

RESUMEN

Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups based on neurovirulence. During the acute phase, DA virus infects cells in the gray matter of the central nervous system (CNS). Throughout the chronic phase, DA virus infects glial cells in the white matter, causing demyelinating disease. Although GDVII virus also infects neurons in the gray matter, infected mice developed a severe polioencephalomyelitis, and no virus is detected in the white matter or other areas in the CNS in rare survivors. Several sequence differences between the two viruses are located in VP2 puff B and VP1 loop II, which are located near each other, close to the proposed receptor binding site. We constructed a DA virus mutant, DApBL2M, which has the VP1 loop II of GDVII virus and a mutation at position 171 in VP2 puff B. While DApBL2M virus replicated less efficiently than DA virus during the acute phase, DApBL2M-induced acute polioencephalitis was comparable to that in DA virus infection. Interestingly, during the chronic phase, DApBL2M caused prolonged gray matter disease in the brain without white matter involvement in the spinal cord. This is opposite what is observed during wild-type DA virus infection. Our study is the first to demonstrate that conformational differences via interaction of VP2 puff B and VP1 loop II between GDVII and DA viruses can play an important role in making the transition of infection from the gray matter in the brain to the spinal cord white matter during TMEV infection.

Asunto(s)

Cápside/genética , Enfermedades Desmielinizantes/virología , Theilovirus/genética , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside , Corteza Cerebral/patología , Corteza Cerebral/virología , Enfermedades Desmielinizantes/etiología , Enfermedades Desmielinizantes/patología , Ratones , Datos de Secuencia Molecular , Mutación , Theilovirus/patogenicidad , Virulencia/genética

RESUMEN

Multiple sclerosis (MS) can be divided into 4 clinical forms: relapsing-remitting (RR), primary progressive (PP), secondary progressive (SP), and progressive relapsing (PR). Since PP-MS is notably different from the other forms of MS, both clinically and pathologically, the question arises whether PP-MS is immunologically similar to the other forms. The pathogenesis of the PP-MS remains unclear, partly due to a lack of highly relevant animal models. Using an encephalitogenic peptide from myelin oligodendrocyte glycoprotein (MOG)92-106, we have established animal models that mimic different forms of MS in 2 strains of H-2s mice, SJL/J and A.SW. We induced experimental allergic encephalomyelitis (EAE) using MOG92-106 in the presence or absence of supplemental Bordetella pertussis (BP). Although, SJL/J mice developed RR-EAE whether BP was given or not, A.SW mice developed PP-EAE without BP and SP-EAE with BP. Histologically, SJL/J mice developed mild demyelinating disease with T cell infiltration, while A.SW mice developed large areas of plaque-like demyelination with immunoglobulin deposition and neutrophil infiltration, but with minimal T cell infiltration. In A.SW mice without BP, high titer serum anti-MOG antibody was detected and the anti-MOG IgG2a/IgG1 ratio correlated with survival times of mice. We hypothesized that, in A.SW mice, a Th2 response favors production of myelinotoxic antibodies, leading to progressive forms with early death. Our new models indicate that a single encephalitogen could induce either RR-, PP-, or SP- forms of demyelinating disease in hosts with immunologically different humoral immune responses.

Asunto(s)

Anticuerpos/análisis , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple Crónica Progresiva/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Animales , Sistema Nervioso Central/patología , ADN Bacteriano/fisiología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Inmunoglobulinas/metabolismo , Etiquetado Corte-Fin in Situ , Linfocitos/patología , Ratones , Ratones Mutantes , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Sistema Nervioso/patología , Fragmentos de Péptidos/inmunología , Plásmidos/genética

RESUMEN

OBJECTIVE: To compare selective salpingography and balloon tuboplasty for the treatment of tubal obstruction. DESIGN: A retrospective evaluation of results of women treated for tubal obstruction by outpatient methods at a single center. SETTING: Tertiary-care, university-affiliated hospital. PATIENT(S): A total of 3,424 infertile women, of whom 418 had bilateral tubal obstruction by hysterosalpingography, treated at Nihon Medical Center from 1982 to 1997. INTERVENTION(S): Women with tubal obstructions who had visual evidence of an intact uterine tubal ostium at hysteroscopy were treated by selective salpingography. If selective salpingography could not establish patency, then transcervical balloon tuboplasty was performed with one of three catheter systems. Patients were followed expectantly for 1 year after treatment. MAIN OUTCOME MEASURE(S): Postoperative tubal patency and overall pregnancy rates (PRs) at 1-year of follow-up. RESULT(S): The overall patency rate was 67.5%, with 30% of these patients conceiving (20.2% of all subjects). Selective salpingography was associated with a 35. 7% patency rate, and 27.3% of these patients conceived. Of the subjects who failed selective salpingography and underwent balloon tuboplasty, 66.2% achieved patency, of whom 33% spontaneously conceived. Balloon tuboplasty was effective in restoring patency in many cases after selective salpingography had failed. Statistically significant differences were found between selective salpingography and balloon tuboplasty and for the different balloon tuboplasty catheters in terms of patency rates, while a trend was seen for PRs. CONCLUSION(S): Many women diagnosed as having tubal obstruction can be treated by outpatient methods that do not require general anesthesia. Achieving patency by these methods is associated with high PRs and avoids the need for assisted reproductive technologies in some cases. Balloon tuboplasty is a more effective treatment than selective salpingography. The choice of balloon tuboplasty catheter system may affect success rates.

Asunto(s)

Enfermedades de las Trompas Uterinas/terapia , Pacientes Ambulatorios , Adulto , Cateterismo , Enfermedades de las Trompas Uterinas/diagnóstico por imagen , Femenino , Humanos , Histerosalpingografía , Infertilidad Femenina/terapia , Embarazo , Índice de Embarazo , Estudios Retrospectivos

RESUMEN

DA, GDVII and H101 are neurovirulent strains of Theiler's murine encephalomyelitis virus that cause very different neuropathology and CNS disease when inoculated into SJL/J mice. DA virus causes a chronic demyelinating disease, GDVII virus causes an acute fatal polioencephalomyelitis, and H101 virus causes an acute pachymeningitis with hydrocephalus. Performing RNase protection assays, we detected the same pattern of chemokine (RANTES, MCP-1, IP-10, MIP-1beta, MIP-1alpha and MIP-2) mRNA expression in brain and spinal cord during all three infections. In contrast, IFN-beta and IL-6 mRNA were highly expressed only in GDVII virus infection, whereas high levels of LT-alpha mRNA were only found during DA virus infection. Our study demonstrates that proinflammatory cytokines are involved in the neuropathogenesis of CNS disease and modulate the acute and chronic process underlying different pathologic features of disease.

Asunto(s)

Infecciones por Cardiovirus/metabolismo , Quimiocinas/genética , Citocinas/genética , ARN Mensajero/metabolismo , Theilovirus , Animales , Femenino , Inmunohistoquímica , Ganglios Linfáticos/metabolismo , Ratones , Hibridación de Ácido Nucleico , Ratas , Ratas Endogámicas , Ribonucleasas , Especificidad de la Especie , Theilovirus/genética

RESUMEN

Ova are captured by the oviductal fimbria and rapidly transported to the ampullary-isthmic junction of the fallopian tube. Fertilized ova and oviductal fluids are then carried medially in the fallopian tube, while undergoing maturation in preparation for entering the uterine cavity, where nidation and further development take place. This movement of oviductal fluids was visualized in a rabbit model with human chorionic gonadotropin-induced ovulation, by injection of a contrast medium into the ampulla region of the oviduct. In the ampulla, the opaque medium was observed to oscillate at 0-85.4 mm/s. This medial transport of the fluid towards the uterus decreased to 0-9.6 mm/s in the isthmic portion of the tube. This decrease substantiates previous findings that the transport of material in the isthmic portion of the oviduct is more strongly under the control of ciliary action than under peristaltic activity.

Asunto(s)

Líquidos Corporales/fisiología , Trompas Uterinas/fisiología , Óvulo/fisiología , Animales , Gonadotropina Coriónica/farmacología , Femenino , Inducción de la Ovulación , Conejos

RESUMEN

PURPOSE: Our purpose was to elucidate the roles of the ampullar and isthmic portions of the oviduct and the effects of drugs on oviductal contractility. METHODS: Prostaglandin F2 alpha (PGF2 alpha; Ono Pharmaceuticals, Osaka) and oxytocin (Atonin-O; Teikoku Hormone Manufacturing Co. Ltd., Tokyo) were used to stimulate oviductal contractility, and ritodrine hydrochloride (Utemerin; Solvay-Duphar Corp., Denmark) to inhibit the contractility. RESULTS: Both PGF2 alpha and Atonin-O were involved in ovum capture by the ampullar oviduct by stimulating contractility, thus altering the intraductal pressures. Utemerin is effective in inhibiting the enhanced contractility induced by PGF2 alpha and Atonin-O. CONCLUSIONS: Variations in pressure of the ampullar portion of the oviduct seem necessary for the capture of ova expelled from the ovary. Once in the isthmic portion of the oviduct, transport appears to be under the influence of ciliary activity rather than variations in contractility.

Asunto(s)

Agonistas Adrenérgicos beta/farmacología , Dinoprost/farmacología , Trompas Uterinas/efectos de los fármacos , Trompas Uterinas/fisiología , Óvulo/fisiología , Animales , Femenino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Ovulación/efectos de los fármacos , Óvulo/efectos de los fármacos , Oxitocina/farmacología , Presión , Conejos , Receptores Adrenérgicos beta 2/efectos de los fármacos , Ritodrina/farmacología

RESUMEN

PURPOSE: Our purpose was to determine the efficacy of Surfacten, a bovine pulmonary surfactant, on the maturation of in vitro bovine ova. METHODS: We used Surfacten as a supplement to the coculture media both at the onset of coculture and after cleavage in bovine ova had been determined. The controls received no Surfacten. RESULTS: The maturation rate in bovine embryos to the blastocyst stage statistically improved (P < 0.05) in the series in which Surfacten was added to the media at the onset of coculture, compared with the controls and the series in which Surfacten was added after cleavage had been determined. CONCLUSIONS: Surfacten, a commercially available surfactant which is a naturally occurring phospholipid that dramatically increases in the cervical mucus and the ampullaris of the oviduct at or near the time of ovulation, improves the maturation of bovine embryos in vitro by making the coculture medium approach the conditions found in the oviducts.

Asunto(s)

Desarrollo Embrionario y Fetal , Fertilización In Vitro/métodos , Células de la Granulosa/citología , Óvulo/citología , Surfactantes Pulmonares , Células Tecales/citología , Animales , Bovinos , Técnicas de Cocultivo , Femenino , Líquido Folicular/química , Humanos , Fosfolípidos/análisis

RESUMEN

Theiler's murine encephalomyelitis virus (TMEV) infection and relapsing-remitting experimental allergic encephalomyelitis (R-EAE) have been used to investigate the viral and autoimmune etiology of multiple sclerosis (MS), a possible Th1-type mediated disease. DNA immunization is a novel vaccination strategy in which few harmful effects have been reported. Bacterial DNA and oligodeoxynucleotides, which contain CpG motifs, have been reported to enhance immunostimulation. Our objectives were two-fold: first, to ascertain whether plasmid DNA, pCMV, which is widely used as a vector in DNA immunization studies, could exert immunostimulation in vitro; and second, to test if pCMV injection could modulate animal models for MS in vivo. We demonstrated that this bacterially derived DNA could induce interleukin (IL)-12, interferon (IFN)gamma, (Th1-promoting cytokines), and IL-6 production as well as activate NK cells. Following pCMV injections, SJL/J mice were infected with TMEV or challenged with encephalitogenic myelin proteolipid protein (PLP) peptides. pCMV injection exacerbated TMEV-induced demyelinating disease in a dose-dependent manner. Exacerbation of the disease did not correlate with the number of TMEV-antigen positive cells but did with an increase in anti-TMEV antibody. pCMV injection also enhanced R-EAE with increased IFNgamma and IL-6 responses. These results caution the use of DNA vaccination in MS patients and other possible Th1-mediated diseases.

Asunto(s)

ADN Bacteriano/efectos adversos , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/microbiología , Animales , Anticuerpos Antivirales/sangre , Islas de CpG/inmunología , Citocinas/biosíntesis , Citomegalovirus/inmunología , ADN Bacteriano/inmunología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Vectores Genéticos/efectos adversos , Vectores Genéticos/inmunología , Inmunoglobulina G/sangre , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos , Esclerosis Múltiple/patología , Proteína Proteolipídica de la Mielina/inmunología , Médula Espinal/patología , Células TH1/inmunología , Theilovirus/inmunología , Vacunas de ADN/efectos adversos , Vacunas de ADN/inmunología

RESUMEN

Theiler's murine encephalomyelitis viruses are picornaviruses that can infect the central nervous system. The DA strain produces an acute polioencephalomyelitis followed by a chronic demyelinating disease in its natural host, the mouse. The ability of DA virus to induce a demyelinating disease renders this virus infection a model for human demyelinating diseases such as multiple sclerosis. Here we describe the generation and characterization of DA virus mutants that contain specific mutations in the viral capsid protein VP1 at sites believed to be important contact regions for the cellular receptor(s). A mutant virus with a threonine-to-aspartate (T81D) substitution in VP1 loop I adjacent to the putative virus receptor binding site exhibited a large-plaque phenotype but had a slower replication cycle in vitro. When this mutant virus was injected into susceptible mice, an altered tropism was seen during the acute stage of the disease and the chronic demyelinating disease was not produced. A virus with a threonine-to-valine substitution (T81V) did not cause any changes in the pattern or extent of disease seen in mice, whereas a virus with a tryptophan substitution at this position (T81W) produced a similar acute disease but was attenuated for the development of the chronic disease. A change in amino acids in a hydrophobic patch located in the wall of the pit, VP1 position 91, to a hydrophilic threonine (V91T) resulted in a profound attenuation of the acute and chronic disease without persistence of virus. This report illustrates the importance of the loop I of VP1 and a site in the wall of the pit in pathogenesis and that amino acid substitutions at these sites result in altered virus-host interactions.

Asunto(s)

Cápside/genética , Infecciones por Cardiovirus/virología , Theilovirus/genética , Animales , Proteínas de la Cápside , Línea Celular , Humanos , Inmunohistoquímica , Ratones , Modelos Moleculares , Mutación , Theilovirus/patogenicidad , Virulencia/genética , Replicación Viral/genética

RESUMEN

Using a bipalmitoylated lipopeptide consisting of an ovalbumin helper T-cell epitope covalently linked to an influenza virus cytotoxic T-lymphocyte (CTL) epitope, we addressed possible factors that may be critical for CTL induction. Antigen processing of lipopeptide appears to be required for T-cell induction since there was virtually no in vitro binding of lipopeptide to purified MHC molecules. A major portion of lipopeptide immunogenicity was due to its particulate nature inasmuch as CTL induction in mice correlated with insoluble lipopeptide constructs, whereas more soluble analogs were significantly less immunogenic. Immunohistological analysis of tissue from immunized animals revealed that lipopeptide migration from the s.c. injection site to the spleen could be detected as early as 1 h after immunization and cell-associated lipopeptide was observed on macrophages and dendritic cells, implicating both cell populations in the processing and presentation of lipopeptide particles to CTLs.

Asunto(s)

Vacunas contra la Influenza/inmunología , Lipoproteínas/inmunología , Activación de Linfocitos/inmunología , Fragmentos de Péptidos/inmunología , Proteínas de Unión al ARN , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Femenino , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Sarcoma de Mastocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Proteínas de la Nucleocápside , Nucleoproteínas/inmunología , Ovalbúmina/inmunología , Tamaño de la Partícula , Células Tumorales Cultivadas , Proteínas del Núcleo Viral/inmunología

RESUMEN

Although the etiology of multiple sclerosis (MS) is not known, several factors play a role in this disease: genetic contributions, immunologic elements, and environmental factors. Viruses and virus infections have been associated with the initiation and/or enhancement of exacerbations in MS. Theiler's murine encephalomyelitis virus (TMEV) infection of mice is one of the animal models used to mimic MS. In other animal model systems, DNA vaccination has been used to protect animals against a variety of virus infections. To explore the utility of DNA vaccination, we have constructed eukaryotic expression vectors encoding the TMEV capsid proteins VP1, VP2, and VP3. SJL/J mice were vaccinated intramuscularly once, twice, or three times with the different capsid protein cDNAs. This was followed by intracerebral TMEV infection to determine the effects of DNA vaccination on the course of TMEV-induced central nervous system (CNS) demyelinating disease. We found that vaccination of mice three times with cDNA encoding VP2 led to partial protection of mice from CNS demyelinating disease as determined by a decrease in clinical symptoms and histopathology. Vaccination of mice with cDNA encoding VP3 also led to a decrease in clinical symptoms. In contrast, mice vaccinated with cDNA encoding VP1 experienced a more severe disease with an earlier onset of clinical signs and enhanced histopathology compared with control mice. There was no correlation between anti-TMEV antibody titers and disease course. These results indicate that DNA immunization can modify chronic virus-induced demyelinating disease and may eventually lead to potential treatments for illnesses such as MS.

Asunto(s)

ADN Viral/inmunología , Esclerosis Múltiple/prevención & control , Theilovirus/inmunología , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/análisis , Western Blotting , Encéfalo/patología , Cápside/genética , Proteínas de la Cápside , ADN Viral/administración & dosificación , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ratones , Esclerosis Múltiple/patología , Esclerosis Múltiple/fisiopatología , Músculos/patología , Médula Espinal/patología , Vacunación , Vacunas de ADN/administración & dosificación , Vacunas Virales/administración & dosificación

RESUMEN

The effect of hyaluronic acid-carboxymethylcellulose film (Seprafilm) in reducing postoperative adhesion formation was examined in a rabbit induced-adhesion model. During laparotomy, the ileocaecal region was mechanically and chemically abraded to induce lesions. After a 28-day recovery period, adhesions were lysed by microsurgery and Seprafilm was applied to the lysed lesion in 10 rabbits and six rabbits received physiological saline. A third laparotomy was performed 10-14 days later and the area of adhesion reformation was compared to that found prior to application of test materials. The area of adhesion reformation with Seprafilm decreased to (mean +/- standard deviation) 11.71 +/- 10.97% of the originally lysed lesion and, the area was significantly reduced compared with controls treated with physiological saline. These results suggest that the use of Seprafilm may be a valuable new anti-adhesion material for abdominal or pelvic surgery and may be superior to existing anti-adhesion materials and techniques.

Asunto(s)

Materiales Biocompatibles , Membranas Artificiales , Adherencias Tisulares/prevención & control , Animales , Femenino , Ácido Hialurónico , Laparotomía , Microcirugia , Complicaciones Posoperatorias , Conejos

RESUMEN

The effects of cross-linked hyaluronate hydrogel and liquid sodium hyaluronate on post-surgical adhesion reformation were examined using a rabbit model. Primary adhesions in the ileocaecal region of Japanese white rabbits were induced by mechanical and chemical irritants during laparotomy. After 1 month the primary adhesions were lysed by microsurgery and cross-linked hyaluronate hydrogel or liquid sodium hyaluronate was applied to the lysed lesions. After 10-14 days the area of adhesion reformation was measured to assess any inhibitory effect of the test materials. Rabbits treated with cross-linked hyaluronate hydrogel showed a significant reduction in adhesion reformation area compared with liquid sodium hyaluronate or physiological saline treatment, and the area reduced to (mean +/- standard deviation) 0.6 +/- 1.95% of the original lesion. In a separate study, histological evaluation of rabbits treated with cross-linked hyaluronate hydrogel revealed a better healing pattern and a lower inflammatory response compared with controls. All these findings suggest cross-linked hyaluronate hydrogel may be a valuable anti-adhesion material to prevent post-surgical adhesion in abdominal or pelvic surgery.

Asunto(s)

Ácido Hialurónico/farmacología , Complicaciones Posoperatorias/prevención & control , Adherencias Tisulares/prevención & control , Animales , Reactivos de Enlaces Cruzados , Femenino , Hidrogeles/farmacología , Laparotomía/efectos adversos , Conejos , Distribución Aleatoria

RESUMEN

TachoComb consists of equine collagen in a sponge-like form coated on one side with human fibrinogen and bovine thrombin. This product functions as a haemostatic and physical barrier to inhibit post-surgical adhesion. In this study, we investigated TachoComb to control oozing in 16 patients who required haemostasis. Evaluation of post-surgical adhesion by second-look laparoscopy was performed at 3 months and 7 months after initial surgery. Observation via laparotomy during Caesarean section was also performed at 13 months, 3 years and 4 years after initial surgery. In all but one patient, no macroscopic evidence of TachoComb persistence was found. Furthermore, no de novo adhesions were detected at the TachoComb application site. We have thus demonstrated that TachoComb can be used to control oozing haemorrhage effectively from surgical sites and can prevent adhesion formation at the application site, and may thus be an effective method of preventing adhesion-induced infertility.

Asunto(s)

Aprotinina/uso terapéutico , Fibrinógeno/uso terapéutico , Procedimientos Quirúrgicos Ginecológicos/efectos adversos , Hemostasis Quirúrgica/métodos , Complicaciones Posoperatorias/prevención & control , Trombina/uso terapéutico , Adherencias Tisulares/prevención & control , Adulto , Combinación de Medicamentos , Femenino , Humanos , Persona de Mediana Edad

RESUMEN

Relapsing-remitting experimental allergic encephalomyelitis (R-EAE) is an animal model for multiple sclerosis (MS). Many potential immunomodulatory strategies for MS have been used first in EAE to assess their effectiveness. Recently, the injection of plasmid DNA has been shown to induce potent humoral and cellular immune responses. The primary aim of our experiments reported here was to determine if vaccination with cDNAs encoding myelin proteolipid protein (PLP) could prime for a PLP-specific immune response and affect subsequent R-EAE. We constructed cDNAs encoding whole PLP (pPLP(all)) or encephalitogenic epitopes PLP(139-151) (pPLP(139-151)) and PLP(178-191) (pPLP(178-191)). Following DNA injection, we induced R-EAE in SJL/J mice using PLP(139-151) or PLP(178-191) peptides in adjuvant. All 3 plasmid constructs enhanced R-EAE induced with PLP(139-151), and injection of mice with pPLP(all) increased R-EAE induced with PLP(178-191). DNA immunization induced higher PLP peptide-specific lymphoproliferative responses than did vector alone following R-EAE induction with IgG1 or IgG2b antibody responses. These data suggest that DNA immunization of PLP can modulate immune responses, leading to enhancement of R-EAE.

Asunto(s)

Encefalomielitis Autoinmune Experimental/terapia , Inmunoterapia/métodos , Esclerosis Múltiple/terapia , Proteína Proteolipídica de la Mielina/genética , Plásmidos/genética , Animales , Formación de Anticuerpos , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Femenino , Isotipos de Inmunoglobulinas , Trastornos Linfoproliferativos/terapia , Ratones , Músculo Esquelético/patología