RESUMO
Intrathecal synthesis of central nervous system (CNS)-reactive autoantibodies is observed across patients with autoimmune encephalitis (AE), who show multiple residual neurobehavioral deficits and relapses despite immunotherapies. We leveraged two common forms of AE, mediated by leucine-rich glioma inactivated-1 (LGI1) and contactin-associated protein-like 2 (CASPR2) antibodies, as human models to comprehensively reconstruct and profile cerebrospinal fluid (CSF) B cell receptor (BCR) characteristics. We hypothesized that the resultant observations would both inform the observed therapeutic gap and determine the contribution of intrathecal maturation to pathogenic B cell lineages. From the CSF of three patients, 381 cognate-paired IgG BCRs were isolated by cell sorting and scRNA-seq, and 166 expressed as monoclonal antibodies (mAbs). Sixty-two percent of mAbs from singleton BCRs reacted with either LGI1 or CASPR2 and, strikingly, this rose to 100% of cells in clonal groups with ≥4 members. These autoantigen-reactivities were more concentrated within antibody-secreting cells (ASCs) versus B cells (P < 0.0001), and both these cell types were more differentiated than LGI1- and CASPR2-unreactive counterparts. Despite greater differentiation, autoantigen-reactive cells had acquired few mutations intrathecally and showed minimal variation in autoantigen affinities within clonal expansions. Also, limited CSF T cell receptor clonality was observed. In contrast, a comparison of germline-encoded BCRs versus the founder intrathecal clone revealed marked gains in both affinity and mutational distances (P = 0.004 and P < 0.0001, respectively). Taken together, in patients with LGI1 and CASPR2 antibody encephalitis, our results identify CSF as a compartment with a remarkably high frequency of clonally expanded autoantigen-reactive ASCs whose BCR maturity appears dominantly acquired outside the CNS.
Assuntos
Doenças Autoimunes do Sistema Nervoso , Encefalite , Glioma , Doença de Hashimoto , Humanos , Leucina , Peptídeos e Proteínas de Sinalização Intracelular , Recidiva Local de Neoplasia , Autoanticorpos , AutoantígenosRESUMO
LoxP-Cre technology was used to remove the selenocysteine tRNA gene, trsp, in either endothelial cells or myocytes of skeletal and heart muscle to elucidate the role of selenoproteins in cardiovascular disease. Loss of selenoprotein expression in endothelial cells was embryonic lethal. A 14.5-day-old embryo had numerous abnormalities including necrosis of the central nervous system, subcutaneous hemorrhage and erythrocyte immaturity. Loss of selenoprotein expression in myocytes manifested no apparent phenotype until about day 12 after birth. Affected mice had decreased mobility and an increased respiratory rate, which proceeded rapidly to death. Pathological analysis revealed that mice lacking trsp had moderate to severe myocarditis with inflammation extending into the mediastinitis. Thus, ablation of selenoprotein expression demonstrated an essential role of selenoproteins in endothelial cell development and in proper cardiac muscle function. The data suggest a direct connection between the loss of selenoprotein expression in these cell types and cardiovascular disease.
Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Coração/crescimento & desenvolvimento , Coração/fisiologia , Miocárdio/metabolismo , Selenoproteínas/biossíntese , Animais , Animais Recém-Nascidos/fisiologia , Feminino , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Gravidez , RNA de Transferência de Cisteína/genética , RNA de Transferência de Cisteína/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Selenocisteína/metabolismo , Comportamento Sexual Animal/fisiologiaRESUMO
Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system causing defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections. We identified a novel gram-negative rod in excised lymph nodes from a patient with CGD. Gram-negative rods grew on charcoal-yeast extract, but conventional tests could not identify it. The best 50 matches of the 16S rRNA (using BLAST) were all members of the family Acetobacteraceae, with the closest match being Gluconobacter sacchari. Patient serum showed specific band recognition in whole lysate immunoblot. We used mouse models of CGD to determine whether this organism was a genuine CGD pathogen. Intraperitoneal injection of gp91(phox -/-) (X-linked) and p47 (phox -/-) (autosomal recessive) mice with this bacterium led to larger burdens of organism recovered from knockout compared with wild-type mice. Knockout mouse lymph nodes had histopathology that was similar to that seen in our patient. We recovered organisms with 16S rRNA sequence identical to the patient's original isolate from the infected mice. We identified a novel gram-negative rod from a patient with CGD. To confirm its pathogenicity, we demonstrated specific immune reaction by high titer antibody, showed that it was able to cause similar disease when introduced into CGD, but not wild-type mice, and we recovered the same organism from pathologic lesions in these mice. Therefore, we have fulfilled Koch's postulates for a new pathogen. This is the first reported case of invasive human disease caused by any of the Acetobacteraceae. Polyphasic taxonomic analysis shows this organism to be a new genus and species for which we propose the name Granulobacter bethesdensis.
Assuntos
Infecções Bacterianas/complicações , Bactérias Gram-Negativas/isolamento & purificação , Doença Granulomatosa Crônica/microbiologia , Linfadenite/microbiologia , Adulto , Sequência de Aminoácidos , Animais , Infecções Bacterianas/patologia , Infecções Bacterianas/terapia , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Doença Granulomatosa Crônica/patologia , Doença Granulomatosa Crônica/terapia , Humanos , Linfonodos/microbiologia , Linfonodos/patologia , Linfadenite/patologia , Linfadenite/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , RNA Ribossômico 16S/genéticaRESUMO
The protein encoded by growth arrest and DNA damage-inducible transcript 34 (Gadd34) is associated with translation initiation regulation following certain stress responses. Through interaction with the protein phosphatase 1 catalytic subunit (PP1c), Gadd34 recruits PP1c for the removal of an inhibitory phosphate group on the alpha subunit of elongation initiation factor 2, thereby reversing the shutoff of protein synthesis initiated by stress-inducible kinases. In the absence of stress, the physiologic consequences of Gadd34 function are not known. Initial analysis of Gadd34-null mice revealed several significant findings, including hypersplenism, decreased erythrocyte volume, increased numbers of circulating erythrocytes, and decreased hemoglobin content, resembling some thalassemia syndromes. Biochemical analysis of the hemoglobin-producing reticulocyte (an erythrocyte precursor) revealed that the decreased hemoglobin content in the Gadd34-null erythrocyte is due to the reduced initiation of the globin translation machinery. We propose that an equilibrium state exists between Gadd34/PP1c and the opposing heme-regulated inhibitor kinase during hemoglobin synthesis in the reticulocyte.
Assuntos
Antígenos de Diferenciação/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hemoglobinas/biossíntese , Animais , Antígenos de Diferenciação/genética , Domínio Catalítico , Proteínas de Ciclo Celular/genética , Células Cultivadas , Dieta , Eritrócitos/patologia , Meia-Vida , Heme/metabolismo , Hemoglobinas/genética , Deficiências de Ferro , Camundongos , Camundongos Mutantes , Iniciação Traducional da Cadeia Peptídica , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 1 , Subunidades Proteicas , Reticulócitos/metabolismo , Talassemia/sangue , Talassemia/patologia , Talassemia/fisiopatologia , eIF-2 Quinase/metabolismoRESUMO
Smith-Lemli-Opitz syndrome (SLOS) is a genetic syndrome caused by mutations in the 3beta-hydroxysterol Delta(7)-reductase gene (DHCR7). SLOS patients have decreased cholesterol and increased 7-dehydrocholesterol (7-DHC) levels. Dietary cholesterol supplementation improves systemic biochemical abnormalities; however, because of the blood-brain barrier, the central nervous system (CNS) is not treated. Simvastatin therapy has been proposed as a means to treat the CNS. Mice homozygous for a null disruption of Dhcr7, Dhcr7(Delta3-5/Delta3-5), die soon after birth, thus they cannot be used to study postnatal development or therapy. To circumvent this problem, we produced a hypomorphic SLOS mouse model by introducing a mutation corresponding to DHCR7(T93M). Both Dhcr7(T93M/T93M) and Dhcr7(Delta3-5/T93M) mice are viable. Phenotypic findings in Dhcr7(T93M/Delta3-5) mice include CNS ventricular dilatation and two to three syndactyly. Biochemically, both Dhcr7(T93M/T93M) and Dhcr7(T93M/Delta3-5) mice have elevated tissue 7-DHC levels; however, the biochemical defect improved with age. This has not been observed in human patients, and is due to elevated Dhcr7 expression in mouse tissues. Dietary cholesterol therapy improved sterol profiles in peripheral, but not CNS tissues. However, treatment of Dhcr7(T93M/Delta3-5) mice with simvastatin decreased 7-DHC levels in both peripheral and brain tissues. Expression of Dhcr7 increased in Dhcr7(T93M/Delta3-5) tissues after simvastatin therapy, consistent with the hypothesis that simvastatin therapy improves the biochemical phenotype by increasing the expression of a Dhcr7 allele with residual enzymatic activity. We conclude that simvastatin treatment is efficacious in improving the SLOS-associated sterol abnormality found in the brain, and thus has the potential to be an effective therapeutic intervention for behavioral and learning problems associated with SLOS.
Assuntos
Modelos Animais de Doenças , Sinvastatina/uso terapêutico , Síndrome de Smith-Lemli-Opitz/tratamento farmacológico , Síndrome de Smith-Lemli-Opitz/genética , Células 3T3 , Alelos , Substituição de Aminoácidos/genética , Animais , Células Cultivadas , Cruzamentos Genéticos , Células-Tronco Embrionárias/transplante , Feminino , Espectroscopia de Ressonância Magnética , Metionina/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Fenótipo , Síndrome de Smith-Lemli-Opitz/enzimologia , Treonina/genéticaRESUMO
Sphingosine-1-phosphate (S1P), an important sphingolipid metabolite, regulates diverse cellular processes, including cell survival, growth, and differentiation. Here we show that S1P signaling is critical for neural and vascular development. Sphingosine kinase-null mice exhibited a deficiency of S1P which severely disturbed neurogenesis, including neural tube closure, and angiogenesis and caused embryonic lethality. A dramatic increase in apoptosis and a decrease in mitosis were seen in the developing nervous system. S1P(1) receptor-null mice also showed severe defects in neurogenesis, indicating that the mechanism by which S1P promotes neurogenesis is, in part, signaling from the S1P(1) receptor. Thus, S1P joins a growing list of signaling molecules, such as vascular endothelial growth factor, which regulate the functionally intertwined pathways of angiogenesis and neurogenesis. Our findings also suggest that exploitation of this potent neuronal survival pathway could lead to the development of novel therapeutic approaches for neurological diseases.
Assuntos
Vasos Sanguíneos/embriologia , Lisofosfolipídeos/metabolismo , Neovascularização Fisiológica , Sistema Nervoso/embriologia , Defeitos do Tubo Neural/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Esfingosina/análogos & derivados , Animais , Apoptose/genética , Vasos Sanguíneos/anormalidades , Vasos Sanguíneos/enzimologia , Camundongos , Camundongos Knockout , Mitose/genética , Neovascularização Fisiológica/genética , Sistema Nervoso/enzimologia , Defeitos do Tubo Neural/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/metabolismoRESUMO
Bacillus anthracis edema toxin (ET), an adenylyl cyclase, is an important virulence factor that contributes to anthrax disease. The role of ET in anthrax pathogenesis is, however, poorly understood. Previous studies using crude toxin preparations associated ET with subcutaneous edema, and ET-deficient strains of B. anthracis showed a reduction in virulence. We report the first comprehensive study of ET-induced pathology in an animal model. Highly purified ET caused death in BALB/cJ mice at lower doses and more rapidly than previously seen with the other major B. anthracis virulence factor, lethal toxin. Observations of gross pathology showed intestinal intralumenal fluid accumulation followed by focal hemorrhaging of the ileum and adrenal glands. Histopathological analyses of timed tissue harvests revealed lesions in several tissues including adrenal glands, lymphoid organs, bone, bone marrow, gastrointestinal mucosa, heart, and kidneys. Concomitant blood chemistry analyses supported the induction of tissue damage. Several cytokines increased after ET administration, including granulocyte colony-stimulating factor, eotaxin, keratinocyte-derived cytokine, MCP-1/JE, interleukin-6, interleukin-10, and interleukin-1beta. Physiological measurements also revealed a concurrent hypotension and bradycardia. These studies detail the extensive pathological lesions caused by ET and suggest that it causes death due to multiorgan failure.
Assuntos
Antígenos de Bactérias/toxicidade , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/toxicidade , Adenilil Ciclases/toxicidade , Glândulas Suprarrenais/patologia , Animais , Medula Óssea/patologia , Osso e Ossos/patologia , Citocinas/biossíntese , Mucosa Gástrica/patologia , Hemorragia , Íleo/patologia , Mucosa Intestinal/patologia , Rim/patologia , Tecido Linfoide/patologia , Camundongos , Camundongos Endogâmicos BALB C , Insuficiência de Múltiplos Órgãos , Miocárdio/patologia , Fatores de Virulência/toxicidadeRESUMO
The perineal or perineal and facial skin were evaluated on 53 rhesus macaques as part of a necropsy protocol. Microscopic evaluation of H & E stained skin sections revealed 19 animals positive for Demodex spp. Mites were seen within all portions of the hair follicles. Infestation varied from minimal to severe. Mites were found in macaques of all ages and in both sexes. Reaction to the mites ranged from no reaction, to minimal follicular epidermal hyperplasia to furunculosis. Immune status of the animal did not determine infestation but immune compromised macaques had more severe lesions. This is the first known report of Demodex spp. in rhesus macaques.
Assuntos
Folículo Piloso/parasitologia , Macaca mulatta , Infestações por Ácaros/veterinária , Ácaros/crescimento & desenvolvimento , Doenças dos Macacos/parasitologia , Dermatopatias/veterinária , Animais , Feminino , Histocitoquímica , Masculino , Infestações por Ácaros/parasitologia , Dermatopatias/parasitologiaRESUMO
Muscarinic acetylcholine receptors (mAChRs) expressed by pancreatic acinar cells play an important role in mediating acetylcholine-dependent stimulation of digestive enzyme secretion. To examine the potential roles of M(1) and M(3) mAChRs in this activity, we used M(1) and M(3) receptor single knockout (KO) and M(1)/M(3) receptor double KO mice as novel experimental tools. Specifically, we examined the ability of the muscarinic agonist carbachol to stimulate amylase secretion in vitro, using dispersed pancreatic acini prepared from wild-type and mAChR mutant mice. Quantitative reverse transcription-polymerase chain reaction studies using RNA prepared from mouse pancreatic acini showed that deletion of the M(1) or M(3) mAChR genes did not lead to significantly altered mRNA levels of the remaining mAChR subtypes. Moreover, immunoprecipitation studies with M(1) and M(3) mAChR-selective antisera demonstrated that both mAChR subtypes are expressed by mouse pancreatic acini. Strikingly, carbachol-induced stimulation of amylase secretion was significantly impaired in acinar preparations from both M(1) and M(3) receptor single KO mice and abolished in acinar preparations from M(1)/M(3) receptor double KO mice. However, another pancreatic secretagogue, bombesin, retained its ability to fully stimulate amylase secretion in acinar preparations from M(1)/M(3) receptor double KO mice. Together, these studies support the concept that cholinergic stimulation of pancreatic amylase secretion is mediated by a mixture of M(1) and M(3) mAChRs and that other mAChR subtypes do not make a significant contribution to this activity. These findings clarify the long-standing question regarding the molecular nature of the mAChR subtypes mediating the secretion of digestive enzymes from the exocrine pancreas.
Assuntos
Amilases/metabolismo , Pâncreas Exócrino/enzimologia , Receptor Muscarínico M1/fisiologia , Receptor Muscarínico M3/fisiologia , Amilases/sangue , Animais , Carbacol/farmacologia , Imunoprecipitação , Masculino , Camundongos , Camundongos Knockout , Receptor Muscarínico M1/análise , Receptor Muscarínico M1/genética , Receptor Muscarínico M3/análise , Receptor Muscarínico M3/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cryptococcal meningoencephalitis develops as a result of hematogenous dissemination of inhaled Cryptococcus neoformans from the lung to the brain. The mechanism(s) by which C. neoformans crosses the blood-brain barrier (BBB) is a key unresolved issue in cryptococcosis. We used both an in vivo mouse model and an in vitro model of the human BBB to investigate the cryptococcal association with and traversal of the BBB. Exposure of human brain microvascular endothelial cells (HBMEC) to C. neoformans triggered the formation of microvillus-like membrane protrusions within 15 to 30 min. Yeast cells of C. neoformans adhered to and were internalized by the HBMEC, and they crossed the HBMEC monolayers via a transcellular pathway without affecting the monolayer integrity. The histopathology of mouse brains obtained after intravenous injection of C. neoformans showed that the yeast cells either were associated with endothelial cells or escaped from the brain capillary vessels into the neuropil by 3 h. C. neoformans was found in the brain parenchyma away from the vessels by 22 h. Association of C. neoformans with the choroid plexus, however, was not detected during up to 10 days of observation. Our findings indicate that C. neoformans cells invade the central nervous system by transcellular crossing of the endothelium of the BBB.
Assuntos
Barreira Hematoencefálica/microbiologia , Sistema Nervoso Central/microbiologia , Cryptococcus neoformans/fisiologia , Cryptococcus neoformans/patogenicidade , Células Endoteliais/microbiologia , Animais , Encéfalo/irrigação sanguínea , Encéfalo/microbiologia , Células Cultivadas , Circulação Cerebrovascular , Criptococose/microbiologia , Endotélio Vascular/citologia , Endotélio Vascular/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microcirculação/microbiologia , Microscopia Eletrônica de VarreduraRESUMO
Identification of the specific muscarinic acetylcholine receptor (mAChR) subtypes mediating stimulation of salivary secretion is of considerable clinical interest. Recent pharmacological and molecular genetic studies have yielded somewhat confusing and partially contradictory results regarding the involvement of individual mAChRs in this activity. In the present study, we re-examined the roles of M(1) and M(3) mAChRs in muscarinic agonist-mediated stimulation of salivary secretion by using M(1) and M(3) receptor single-knockout (KO) mice and newly generated M(1)/M(3) receptor double-KO mice. When applied at a low dose (1 mg/kg, s.c.), the muscarinic agonist pilocarpine showed significantly reduced secretory activity in both M(1) and M(3) receptor single-KO mice. However, when applied at higher doses, pilocarpine induced only modestly reduced (5 mg/kg, s.c.) or unchanged (15 mg/kg, s.c.) salivation responses, respectively, in M(1) and M(3) receptor single-KO mice, indicating that the presence of either M(1) or M(3) receptors is sufficient to mediate robust salivary output. Quantitative reverse transcriptase-polymerase chain reaction studies with salivary gland tissue showed that the inactivation of the M(1) or M(3) mAChR genes did not lead to significantly altered mRNA levels of the remaining mAChR subtypes. Strikingly, the sialagogue activity of pilocarpine was abolished in M(1)/M(3) receptor double-KO mice. However, salivary glands from M(1)/M(3) receptor double-KO mice remained responsive to stimulation by the beta-adrenergic receptor agonist, (S)-isoproterenol. Taken together these studies support the concept that a mixture of M(1) and M(3) receptors mediates cholinergic stimulation of salivary flow.
Assuntos
Agonistas Muscarínicos/farmacologia , Pilocarpina/farmacologia , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/metabolismo , Glândulas Salivares/metabolismo , Salivação/efeitos dos fármacos , Animais , Sítios de Ligação , Relação Dose-Resposta a Droga , Expressão Gênica , Camundongos , Camundongos Knockout , N-Metilescopolamina/metabolismo , Reação em Cadeia da Polimerase , Receptor Muscarínico M1/deficiência , Receptor Muscarínico M1/genética , Receptor Muscarínico M3/deficiência , Receptor Muscarínico M3/genética , Receptores Colinérgicos/metabolismo , Glândulas Salivares/efeitos dos fármacos , Fatores de Tempo , TrítioRESUMO
Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein-coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein-coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.
Assuntos
Evolução Molecular , Geografia , Histoplasma/classificação , Histoplasma/genética , Modelos Genéticos , Filogenia , Análise por Conglomerados , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Cryptococcus neoformans is the etiologic agent of cryptococcosis. Two mating types exist in this fungus, MAT alpha and MATa. The CPRa gene of C. neoformans is a MATa strain-specific gene and encodes a putative seven-transmembrane domain pheromone receptor. Unlike the other reported fungal pheromone receptors, CPRa shows functional diversity. Deletion of CPRa drastically affects mating efficiency but does not abolish mating. CPRa expression is developmentally regulated and is not affected by deletion of the transcriptional regulator STE12a. The expression of CPRa is markedly increased by shifting cultures from liquid to solid media. CPRa also plays a significant role in virulence. Delta cpra cells produce smaller capsules in the brains of mice than the wild-type cells, and the mice infected with Delta cpra survive significantly longer than those receiving the wild-type strain. Our results suggest that the MATa pheromone receptor of C. neoformans is not only required for mating but also important for survival and growth of the fungus in host tissue.
Assuntos
Cryptococcus neoformans/fisiologia , Cryptococcus neoformans/patogenicidade , Proteínas Fúngicas/fisiologia , Animais , Sequência de Bases , Encéfalo/microbiologia , Encéfalo/patologia , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , DNA Fúngico/genética , Feminino , Proteínas Fúngicas/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Meningite Criptocócica/etiologia , Meningite Criptocócica/microbiologia , Meningite Criptocócica/patologia , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Virulência/genética , Virulência/fisiologiaRESUMO
Impaired immune reconstitution has moved to the forefront of clinical problems limiting progress in allogeneic bone marrow transplantation (BMT). The identification of therapies that can enhance immune reconstitution by increasing thymopoiesis is critical to solving this problem. Interleukin 7 (IL-7) is the most potent thymopoietic cytokine identified thus far. To study the effects of IL-7 on immune reconstitution and graft-versus-host disease (GVHD) following allogeneic BMT, we administered recombinant human IL-7 (rhIL-7) in a murine parent into an F1 model. Results showed that rhIL-7 therapy lowered the "threshold" T-cell dose required to induce both clinical signs of GVHD as well as lethal GVHD. Histologic analysis of GVHD target tissues revealed that rhIL-7 increased the degree of inflammation and tissue damage observed at all T-cell doses studied, but did not change the pattern of organs affected or the histologic appearance of the GVHD within target organs. In addition, we evaluated the capacity for rhIL-7 to enhance thymopoiesis in the setting of allogeneic T cell-depleted (TCD) and T-cell-replete BMT. We observed that rhIL-7 therapy enhanced thymic function in TCD allogeneic BM transplant recipients, but not in animals that received even modest doses of T cells presumably due to thymic toxicity of the graft-versus-host reaction. Thus, caution must be exercised as IL-7 is developed clinically as an immunorestorative agent for use in the setting of allogeneic BMT. These results suggest that use of IL-7 should be limited to the setting of TCD BMT to obtain the greatest benefit on immune competence with the least toxicity.
Assuntos
Transplante de Medula Óssea/imunologia , Doença Enxerto-Hospedeiro/patologia , Interleucina-7/toxicidade , Redução de Peso , Animais , Peso Corporal/efeitos dos fármacos , Transplante de Medula Óssea/patologia , Colo/patologia , Progressão da Doença , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
TGF-beta1 null mice die by 3 to 4 weeks of age due to a severe autoimmune-mediated multifocal inflammation resulting in multi-organ failure. To assess the therapeutic potential of circulating levels of active TGF-beta1, we generated mice with endocrine expression of active TGF-beta1 on a TGF-beta1 null background (TGF-beta1 (-/-/TG)) by crossing TGF-beta1(+/-) mice with transgenic mice (TG) that express recombinant TGF-beta1 specifically in the liver and secrete it in the blood. The TGF-beta1 (-/-/TG) mice exhibit a survival profile similar to the TGF-beta1 (-/-) mice indicating a failure to rescue the lethal phenotype. However, serum TGF-beta1 levels in the TGF-beta1 (-/-/TG) mice were restored to near normal levels with expression in all the tissues, notably in the kidney and spleen. Histopathology showed reduced inflammation in the target tissues, especially in the heart. Interestingly, unlike TGF-beta1 (-/-) mice, the TGF-beta1 (-/-/TG) mice have glomerulonephritis in their kidneys similar to the TG mice. Thus, the phenotype of TGF-beta1 (-/-/TG) animal model indicates the potential role of circulating active-TGF-beta1 in reducing inflammation, but its failure to rescue lethality in TGF-beta1 null mice indicates a critical autocrine role of TGF-beta1.
Assuntos
Sistema Endócrino/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Peso Corporal , Cruzamentos Genéticos , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Glomerulonefrite/etiologia , Glomerulonefrite/genética , Heterozigoto , Inflamação , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/patologia , Fenótipo , Fatores de Tempo , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta1RESUMO
The only proven requirement for ascorbic acid (vitamin C) is in preventing scurvy, presumably because it is a cofactor for hydroxylases required for post-translational modifications that stabilize collagen. We have created mice deficient in the mouse ortholog (solute carrier family 23 member 1 or Slc23a1) of a rat ascorbic-acid transporter, Svct2 (ref. 4). Cultured embryonic fibroblasts from homozygous Slc23a1(-/-) mice had less than 5% of normal ascorbic-acid uptake. Ascorbic-acid levels were undetectable or markedly reduced in the blood and tissues of Slc23a1(-/-) mice. Prenatal supplementation of pregnant females did not elevate blood ascorbic acid in Slc23a1(-/-) fetuses, suggesting Slc23a1 is important in placental ascorbic-acid transport. Slc23a1(-/-) mice died within a few minutes of birth with respiratory failure and intraparenchymal brain hemorrhage. Lungs showed no postnatal expansion but had normal surfactant protein B levels. Brain hemorrhage was unlikely to be simply a form of scurvy since Slc23a1(-/-) mice showed no hemorrhage in any other tissues and their skin had normal skin 4-hydroxyproline levels despite low ascorbic-acid content. We conclude that Slc23a1 is required for transport of ascorbic acid into many tissues and across the placenta. Deficiency of the transporter is lethal in newborn mice, thereby revealing a previously unrecognized requirement for ascorbic acid in the perinatal period.
Assuntos
Ácido Ascórbico/metabolismo , Encéfalo/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores , Animais , Desenvolvimento Embrionário e Fetal , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Essenciais , Humanos , Camundongos , Camundongos Knockout , Transportadores de Ânions Orgânicos Dependentes de Sódio/deficiência , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Mapeamento por Restrição , Transportadores de Sódio Acoplados à Vitamina CRESUMO
Pooled human immunoglobulin (IgG) was evaluated as prophylaxis and treatment of HSV-1 infection in mice. We compared the effects of IgG on the course of acute infection and spread of virus through the nervous system, as well as on the establishment, maintenance, and reactivation of virus from latency. Balb/c mice received a single 3.75 mg intraperitoneal injection of IgG 24 h before or 24 h, 48 h, or 72 h after ocular infection with 10(6) pfu of HSV-1 strain McKrae. Treatment with IgG protected against death in a time-dependent manner (P < 0.001 for -24 h vs. +48 h and +72 h IgG treatment groups). Viral shedding from the eyes was reduced more in mice treated with IgG at -24 h or +24 h relative to animals treated at +48 h. Viral titers in the eyes were reduced in mice treated with IgG at +24 h, but not at +48 h. In ganglia, virus recovery was reduced (P < 0.05) in mice treated at -24 h, +24 h, or +48 h relative to untreated mice, or ones treated at +72 h. In brains, similar results were observed in mice treated at -24 h, +24 h, or +48 h relative to +72 h. Upon explantation, virus reactivated from all ganglia of all surviving mice regardless of treatment group. DNA quantitation showed that mice pretreated with IgG tended towards lower quantities of latent genome copies compared to +24 h treatment and +48 h treatment. UV irradiation induced reactivation in vivo in 16/40 pretreated mice, 20/29 mice treated at +24 h, and in 8/8 mice treated at +48 h (P = 0.03 and P = 0.004, for comparisons at -24 h vs. +24 h, and -24 h vs. +48 h, respectively). Histopathological studies revealed that mice pretreated and treated with IgG had milder encephalitis and reduced virus spread compared to untreated mice. Pooled human IgG attenuates the spread of, and morbidity from, HSV-1 if given before and within 2 days after ocular infection.
Assuntos
Encefalite por Herpes Simples/prevenção & controle , Encefalite por Herpes Simples/terapia , Herpes Simples/prevenção & controle , Herpes Simples/terapia , Herpesvirus Humano 1 , Doença Aguda , Animais , Anticorpos Antivirais/farmacologia , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Córnea/virologia , Encefalite por Herpes Simples/mortalidade , Encefalite por Herpes Simples/patologia , Feminino , Herpes Simples/mortalidade , Herpes Simples/patologia , Imunoglobulina G/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Prevenção Secundária , Taxa de Sobrevida , Gânglio Trigeminal/virologia , Raios Ultravioleta , Células Vero , Latência Viral/efeitos da radiaçãoRESUMO
Highly pathogenic simian/human immunodeficiency virus chimeric viruses are known to induce a rapid, irreversible depletion of CD4+ T lymphocytes in the peripheral blood of acutely infected macaque monkeys. To more fully assess the systemic effects of this primary virus infection, specimens were collected serially between days 3 and 21 postinfection from variety of lymphoid tissues (lymph nodes, thymus, and spleen) and gastrointestinal tract and examined by DNA and RNA PCR, in situ hybridization, and immunohistochemical assays. In addition, the lymphoid tissues were evaluated by fluorescence-activated cell sorting. Virus infection was initially detected by DNA PCR on day 3 postinfection in lymph node samples and peaked on day 10 in the T-lymphocyte-rich areas of this tissue. CD4+ T-cell levels remained stable through day 10 in several lymphoid tissue specimens examined but fell precipitously between days 10 and 21. In situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed the accumulation of apoptotic cells during the second week of infection in both lymph nodes and thymus, which colocalized, to a large extent, to sites of both virus replication and CD4+ T-lymphocyte loss.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1 , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia , Animais , Apoptose , Contagem de Linfócito CD4 , Contagem de Células , Modelos Animais de Doenças , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Imuno-Histoquímica , Tecido Linfoide/imunologia , Tecido Linfoide/virologia , Macaca mulatta , Recombinação Genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/patogenicidade , Fatores de TempoRESUMO
Spontaneous rhabdomyosarcoma is rare in laboratory rodents. Incidence was estimated as 2.4/100,000 in BALB/c mice; incidence of <0.5% for rodents is generally accepted. We describe spontaneous rhabdomyosarcoma in 2 aged, female ([A/J x CBA/ J]F1) mice. Part of a small breeding colony, these mice were experimentally naive; incidence was estimated as 12.5%. Tumors were located in the muscles of the thoracic wall and muscles of the dorsolumbar region. Cross-striations were detected, using phosphotung- stic acid hematoxylin stain; presence of cross-striations within the neoplasm are diagnostic for rhabdomyosarcoma.