RESUMO
Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4(+) iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4(+) iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4(+) iNKT cells from third-party mice were as protective as CD4(+) iNKT cells from donor mice although third-party CD4(+) iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4(+) iNKT cells resulted in a robust expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/transplante , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/prevenção & controle , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/fisiologia , Células Matadoras Naturais/transplante , Animais , Proliferação de Células , Células Cultivadas , Citoproteção/imunologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Auxiliares-Indutores/fisiologiaRESUMO
Dysregulated donor T cells lead to destruction of host tissues resulting in graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We investigated the impact of highly purified (>95%) donor CD4(+) invariant natural killer T (iNKT) cells on GVHD in a murine model of allogeneic HCT. We found that low doses of adoptively transferred donor CD4(+) iNKT cells protect from GVHD morbidity and mortality through an expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). These Tregs express high levels of the Ikaros transcription factor Helios and expand from the Treg pool of the donor graft. Furthermore, CD4(+) iNKT cells preserve T-cell-mediated graft-versus-tumor effects. Our studies reveal new aspects of the cellular interplay between iNKT cells and Tregs in the context of tolerance induction after allogeneic HCT and set the stage for clinical translation.
Assuntos
Transferência Adotiva , Linfócitos T CD4-Positivos/fisiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Células T Matadoras Naturais/fisiologia , Linfócitos T Reguladores/imunologia , Animais , Contagem de Linfócito CD4 , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/mortalidade , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
CD4(+) natural killer T (NKT) cells, along with CD4(+)CD25(+) regulatory T cells (Tregs), are capable of controlling aberrant immune reactions. We explored the adoptive transfer of highly purified (> 95%) CD4(+)NKT cells in a murine model of allogeneic hematopoietic cell transplantation (HCT). NKT cells follow a migration and proliferation pattern similar to that of conventional T cells (Tcons), migrating initially to secondary lymphoid organs followed by infiltration of graft-versus-host disease (GVHD) target tissues. NKT cells persist for more than 100 days and do not cause significant morbidity or mortality. Doses of NKT cells as low as 1.0 × 10(4) cells suppress GVHD caused by 5.0 × 10(5) Tcons in an interleukin-4 (IL-4)-dependent mechanism. Protective doses of NKT cells minimally affect Tcon proliferation, but cause significant reductions in interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production by donor Tcons and in skin, spleen, and gastrointestinal pathology. In addition, NKT cells do not impact the graft-versus-tumor (GVT) effect of Tcons against B-cell lymphoma-1 (BCL-1) tumors. These studies elucidate the biologic function of donor-type CD4(+)NKT cells in suppressing GVHD in an allogeneic transplantation setting, demonstrating clinical potential in reducing GVHD in HCT.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Interleucina-4/imunologia , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Doença Aguda , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/imunologia , Proliferação de Células , Mediadores da Inflamação/metabolismo , Interferon gama/biossíntese , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-4/biossíntese , Camundongos , Especificidade de Órgãos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We report the isolation of a novel helicobacter isolated from the caecum of the Siberian hamster (Phodopus sungorus). Sequence analysis showed 97% sequence similarity to Helicobacter ganmani. In addition, we report the co-infection of these Siberian hamsters with a Campylobacter sp. and a second Helicobacter sp. with 99% sequence similarity to Helicobacter sp. flexispira taxon 8 (Helicobacter bilis), a species isolated previously from patients with bacteraemia. Gross necropsy and histopathology did not reveal any overt pathological lesions of the liver and gastrointestinal tract that could be attributed to the Helicobacter or Campylobacter spp. infections. This is the first helicobacter to be identified in the Siberian hamster and the first report of co-infection of Helicobacter spp. and Campylobacter sp. in asymptomatic Siberian hamsters.
Assuntos
Infecções por Campylobacter/veterinária , Campylobacter/isolamento & purificação , Coinfecção/veterinária , Infecções por Helicobacter/veterinária , Helicobacter/isolamento & purificação , Doenças dos Roedores/microbiologia , Animais , Campylobacter/classificação , Campylobacter/genética , Infecções por Campylobacter/complicações , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/patologia , Ceco/microbiologia , Análise por Conglomerados , Coinfecção/complicações , Coinfecção/microbiologia , Coinfecção/patologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Feminino , Trato Gastrointestinal/patologia , Helicobacter/classificação , Helicobacter/genética , Infecções por Helicobacter/complicações , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Histocitoquímica , Fígado/patologia , Masculino , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Phodopus , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNAAssuntos
Doenças do Gato/patologia , Cistadenocarcinoma/veterinária , Glândula Parótida/patologia , Neoplasias Parotídeas/veterinária , Animais , Doenças do Gato/cirurgia , Gatos , Cistadenocarcinoma/patologia , Cistadenocarcinoma/cirurgia , Masculino , Glândula Parótida/cirurgia , Neoplasias Parotídeas/patologia , Neoplasias Parotídeas/cirurgiaRESUMO
An 10-y-old, intact male rhesus macaque (Macaca mulatta) presented for bilateral scrotal swelling and a distended abdomen. A soft mass in the left upper quadrant of the abdomen was palpated. A barium study did not reveal any gastrointestinal abnormalities. Exploratory laparotomy revealed a large (1.25 kg, 15.0 × 13.0 × 9.5 cm), red and tan, soft, circumscribed, spherical mass within the greater omentum and 10 to 20 smaller (diameter, 1 to 4 cm), soft to firm masses in the mesentery and greater omentum. The resected mass was a self-strangulating abdominal lipoma, a pedunculated neoplasm composed of white adipocytes arising from peritoneal adipose tissue undergoing secondary coagulation necrosis after strangulation of the blood supply due to twisting of the mass around the peduncle. The smaller masses were histologically consistent with simple or self-strangulating pedunculated abdominal lipomas. The macaque presented again 9 mo later with a firm, 5.0-cm mass in the midabdomen, with intestinal displacement visible on radiographs. Given this animal's medical history and questionable prognosis, euthanasia was elected. Necropsy revealed numerous, multifocal to coalescing, 1.0- to 15.0-cm, pale tan to yellow, circumscribed, soft to firm, spherical to ellipsoid, pedunculated masses that were scattered throughout the mesentery, greater omentum, lesser omentum, and serosal surfaces of the gastrointestinal tract. All of the masses were pedunculated abdominal lipomas, and most demonstrated coagulation necrosis due to self-strangulation of the blood supply. To our knowledge, this report is the first to describe abdominal lipomatosis with secondary self-strangulation of masses in a rhesus macaque.
Assuntos
Animais de Laboratório , Lipomatose/veterinária , Macaca mulatta , Doenças dos Macacos/patologia , Neoplasias/veterinária , Neoplasias Peritoneais/veterinária , Animais , Técnicas Histológicas/veterinária , Lipomatose/patologia , Masculino , Necrose/patologia , Necrose/veterinária , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Omento/patologia , Neoplasias Peritoneais/patologiaRESUMO
To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCHs) from engineered proteins, here we develop protein-polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20kDa and 40kDa star-shaped PEG variants were investigated, and all four PEG-peptide variants were able to undergo a sol-gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C-P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.
Assuntos
Células Progenitoras Endoteliais/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Isquemia/terapia , Músculo Esquelético/irrigação sanguínea , Polietilenoglicóis/química , Proteínas Recombinantes/química , Alicerces Teciduais , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Forma Celular , Células Cultivadas , Química Farmacêutica , Preparações de Ação Retardada , Modelos Animais de Doenças , Módulo de Elasticidade , Células Progenitoras Endoteliais/metabolismo , Membro Posterior , Humanos , Hidrogéis , Células-Tronco Pluripotentes Induzidas/metabolismo , Injeções Intramusculares , Isquemia/patologia , Isquemia/fisiopatologia , Cinética , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Peso Molecular , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Necrose , Ligação Proteica , Regeneração/efeitos dos fármacos , Solubilidade , Tecnologia Farmacêutica/métodos , Fator A de Crescimento do Endotélio Vascular/química , ViscosidadeRESUMO
Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3(-/-) Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3(-/-) Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3(-/-) Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3(-/-) and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation.
Assuntos
Antígenos CD/imunologia , Transplante de Medula Óssea/efeitos adversos , Doença Enxerto-Hospedeiro/imunologia , Linfócitos T/imunologia , Transplante Homólogo/efeitos adversos , Análise de Variância , Animais , Antígenos CD/genética , Proliferação de Células , Citometria de Fluxo , Fluoresceínas , Medições Luminescentes , Camundongos , Camundongos Knockout , Succinimidas , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Biologic samples from 18 (12 female, 6 male) Siberian hamsters (Phodopus sungorus) representing an aged colony (17 to 27 mo) were examined. Values for CBC and serum biochemical parameters were determined, and macroscopic and microscopic pathologic evaluations were performed. Blood urea nitrogen levels were significantly higher in male (54.2 ± 14 mg/dL) compared with female (35.3 ± 22 mg/dL) hamsters and correlated histologically with a higher incidence of chronic glomerulonephropathy in males (5 of 6 males; 0 of 12 females). All 18 hamsters had histologic evidence of follicular mite infestation. Half (6 of 12) of the female hamsters showed cystic rete ovarii. Other histologic findings included thymic or thyroid branchial cysts (3 of 18), focal enteritis (2 of 18), and single cases of hepatic hemangiosarcoma, renal adenoma, subcutaneous mast cell tumor, cutaneous sebaceous adenoma, cutaneous trichofolliculoma, squamous papilloma of the nonglandular stomach, epididymal cholesteatoma, pyometra, and pituitary craniopharyngeal cyst. This study is the first published report of hematologic and serum chemical values for any population of Siberian hamsters and the first published report showing a potential male predisposition for chronic progressive glomerulonephropathy and a potential female predisposition for cystic rete ovarii.
Assuntos
Cricetinae/sangue , Glomerulonefrite/veterinária , Infestações por Ácaros/veterinária , Cistos Ovarianos/veterinária , Phodopus/sangue , Doenças dos Roedores , Envelhecimento/sangue , Envelhecimento/patologia , Animais , Animais de Laboratório/sangue , Nitrogênio da Ureia Sanguínea , Causalidade , Doença Crônica , Feminino , Glomerulonefrite/epidemiologia , Glomerulonefrite/patologia , Masculino , Infestações por Ácaros/epidemiologia , Cistos Ovarianos/epidemiologia , Cistos Ovarianos/patologia , Prevalência , Doenças dos Roedores/sangue , Doenças dos Roedores/patologia , Fatores SexuaisRESUMO
An adult female squirrel monkey (Saimiri sciureus) presented with a 3.0 x 2.5 cm firm mass palpable within the caudal abdomen. Differentiation of the organs or structures involved with the mass could not be achieved with radiography or ultrasonography. Exploratory laparotomy revealed a mass within the lumen of the uterus; the mass was removed by partial hysterectomy. On gross examination, the mass was a focally extensive, unencapsulated, firm, solitary tumor. Histologic examination revealed that the mass was composed of interlacing bundles of smooth muscle cells with little fibrous stroma. The cells were elongated with poorly delineated borders and cigar-shaped nuclei, each containing a single, small nucleolus. Fewer than 1 mitosis per 20 high-power (magnification, x 400) fields were present. These gross and histologic findings supported a diagnosis of uterine leiomyoma. Although leiomyomas are the most common tumor of the reproductive tract in nonhuman primates, to our knowledge the current lesion is the first uterine leiomyoma reported to occur in a squirrel monkey.
Assuntos
Leiomioma/veterinária , Doenças dos Macacos/diagnóstico , Saimiri , Neoplasias Uterinas/veterinária , Animais , Animais de Laboratório , Feminino , Histerectomia/métodos , Histerectomia/veterinária , Leiomioma/diagnóstico , Leiomioma/cirurgia , Doenças dos Macacos/patologia , Doenças dos Macacos/cirurgia , Músculo Liso/ultraestrutura , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/cirurgiaRESUMO
Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 µg/mL, distribution volume was 841.96 mL/kg, and area under the time-concentration curve was 57.59 µg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 µg/mL, 915.85 mL/kg, and 47.42 µg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs' habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing.
Assuntos
Antibacterianos/farmacocinética , Fluoroquinolonas/farmacocinética , Xenopus laevis/metabolismo , Doenças dos Animais/tratamento farmacológico , Animais , Antibacterianos/administração & dosagem , Antibacterianos/sangue , Antibacterianos/uso terapêutico , Área Sob a Curva , Ciprofloxacina/sangue , Ciprofloxacina/metabolismo , Enrofloxacina , Feminino , Fluoroquinolonas/administração & dosagem , Fluoroquinolonas/sangue , Fluoroquinolonas/uso terapêutico , Meia-Vida , Injeções Intramusculares/veterinária , Injeções Subcutâneas/veterinária , Xenopus laevis/sangueRESUMO
An adult, male, rhesus macaque presented with pruritus and a focal, exudative, inflamed, erythematous skin lesion of approximately 2 cm in diameter on the ventral aspect of the mandible. The lesion resolved after 10 d of treatment with 1% chlorhexidine solution and triple-antibiotic ointment. However, the skin lesion subsequently recurred several times over a 2-mo period. A punch biopsy was performed, and histological changes were most consistent with a diagnosis of atopic dermatitis. Treatment with topical tacrolimus ointment, an immunosuppressive drug, proved successful in the resolution of all clinical signs after 4 mo. According to a literature review, this article is the first report of the use of tacrolimus ointment as a topical treatment of atopic dermatitis in a rhesus macaque.