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Inflammatory lymphangiogenesis is intimately linked to immune regulation and tissue homeostasis. However, current evidence has suggested that classic lymphatic vessels are physiologically absent in intraocular structures. Here, we show that neolymphatic vessels were induced in the iris after corneal alkali injury (CAI) in a VEGFR3-dependent manner. Cre-loxP-based lineage tracing revealed that these lymphatic endothelial cells (LECs) originate from existing Prox1+ lymphatic vessels. Notably, the ablation of iridial lymphangiogenesis via conditional deletion of VEGFR3 alleviated the ocular inflammatory response and pathological T cell infiltration. Our findings demonstrate that iridial neolymphatics actively participate in pathological immune responses following injury and suggest intraocular lymphangiogenesis as a valuable therapeutic target for the treatment of ocular inflammation.
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Lesiones de la Cornea , Linfangiogénesis , Humanos , Linfangiogénesis/fisiología , Células Endoteliales , Álcalis , Linfocitos T , Inflamación , IrisRESUMEN
PURPOSE: Ocular allergy leads to acute and chronic inflammation that may deteriorate the conjunctiva and other ocular tissue. The conjunctiva is covered with abundant lymphatic vessels but how the conjunctival lymphatic system patriciates in the development of allergic eye disease (AED) remains to be elucidated. METHODS AND RESULTS: By using ovalbumin (OVA)+pertussis toxin (PTX) as a sensitizer followed by daily OVA challenges, we induced optimized AED manifestations in mice. We show that conjunctival lymphatics underwent significant expansion after 28 days of chronic OVA challenge, and this process can be prevented by inducible genetic ablation of lymphatic Vegfr3. Through transcriptomic profile analysis in combination with histopathological examinations, we found that pro-lymphangiogenic VEGFR3 signal promoted allergy-induced activation of T helper 2 (Th2) type immune responses, including antigen presentation, and Th2 cells, B cells and mast cell-related pathways in the conjunctiva, thereby critically contributing to the immunoglobulin E (IgE) production and AED manifestations. As a result, ocular allergy can be alleviated by genetic inhibition of lymphatic Vegfr3. Interestingly, pro-lymphangiogenic VEGFR3 signal did not appear to affect the obstruction of meibomian glands (MGs) or the activation of Th17 type and neutrophil pathways that are associated with AED. CONCLUSIONS: These data reveal the key role of pro-lymphangiogenic VEGFR3 signaling in the development of AED and provide experimental evidence that VEGFR3 inhibition may be useful in treating ocular allergy in patients.
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Oftalmopatías , Hipersensibilidad , Animales , Ratones , Modelos Animales de Enfermedad , Linfangiogénesis/fisiología , Ovalbúmina , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
As a new generation of treatment, tumor immunotherapy targeting tumor-associated antigens (TAA) has attracted widespread attention. The survivin antigen belongs to TAA. It is a key inhibitor of apoptosis and a key regulator of cell cycle progression; furthermore, it may be a candidate target for tumor therapy. In addition, studies have confirmed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and CCL17 significantly affect local anti-tumor immunity in the tumor microenvironment. The mouse survivin gene was screened by BIMAS and SYFPEITHI to obtain the highest scored mouse survivin epitope peptide, which was synthesized into a peptide vaccine to immunize normal mice. Subsequently, spleen lymphocytes were isolated to induce survivin-specific cytotoxic T lymphocytes (CTL). Next, genetic engineering was used to construct the B16F10 cell line that stably expressed CCL17 and GM-CSF genes. A mouse melanoma model was used to observe the effects of the combination of the three on tumor volume and tumor weight. In-vitro survivin-specific CTL combined with CCL17 gene had a stronger inhibitory effect on B16F10 cells, while combined GM-CSF gene did not enhance the inhibitory effect of CTL on B16F10 cells. In-vivo experiments demonstrated that survivin-specific CTL combined with GM-CSF and CCL17 genes can inhibit the growth of mouse melanoma. HE staining and immunohistochemistry showed that the tumor had more necrotic cells and more infiltrating lymphocytes. The results showed that survivin-specific CTL combined with CCL17 and GM-CSF genes could inhibit tumor growth better.
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Quimiocina CCL17/genética , Melanoma/inmunología , Survivin/genética , Linfocitos T Citotóxicos/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer , Línea Celular Tumoral , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Inmunoterapia/métodos , Ratones , Carga Tumoral , Microambiente Tumoral/inmunologíaRESUMEN
OBJECTIVE: To compare the efficiency and complications of transrectal ultrasound (TRUS)-guided prostate biopsy with a 16-gauge (16G) or an 18G puncture needle in the diagnosis of PCa. METHODS: This prospective randomized controlled study included 142 male patients undergoing TRUS-guided prostate biopsy in our hospital, 71 with the 16G and the other 71 with the 18G puncture needle. We compared the post-puncture incidence rates of hematuria, bleeding and infection between the two groups of patients and classified the complications according to the Clavien-Dindo scores. RESULTS: The detection rate of PCa was significantly lower in the 18G than in the 16G group (12.68% vs 36.62%, χ2 = 10.958, P = 0.001), even with f/tPSA ≤ 0.15 (8.51% vs 44.44%, χ2 = 12.617, P = 0.001), but showed no statistically significant difference between the two groups with f/tPSA > 0.15 (P<0.05). No post-puncture infection was observed in any of the patients. There were no statistically significant differences between the 18G and 16G groups in the incidence rates of rectal bleeding (21.13% vs 15.49%, χ2 = 0.753, P = 0.385) and urethral bleeding (18.31% vs 16.90%, χ2 = 0.049, P = 0.826), nor in Clavien-Dindo grades (26 vs 20 cases of grade I; no grade II in either group; 2 vs 3 cases of grade III ; Z = ï¼0.698, P = 0.458). CONCLUSIONS: The 16G puncture needle can achieve a higher detection rate of PCa than the 18G needle in TRUS-guided prostate biopsy without increasing the incidence of complications.
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Biopsia/instrumentación , Agujas , Neoplasias de la Próstata , Ultrasonografía Intervencional , Humanos , Masculino , Estudios Prospectivos , Neoplasias de la Próstata/diagnóstico , PuncionesRESUMEN
The aim of the present study was to use The Cancer Genome Atlas (TCGA) database to identify tumor neoantigens, combined with a bioinformatics analysis to design and analyze antigen epitope peptides. Epitopes were screened using immunogenicity tests to identify the ideal epitope peptides to target tumor neoantigens, which can specifically activate the immune response of T cells. The high-frequency mutation loci (top 10) of colorectal, lung and liver cancer genes were screened using TCGA database. The antigenic epitope peptides with high affinity for major histocompatibility complex molecules were selected and synthesized using computer prediction algorithms, and were subsequently detected using flow cytometry. The cytotoxicity of specific cytotoxic T lymphocytes (CTLs) on peptide-loaded T2 cells was initially verified using interferon IFN-γ detection and a calcein-acetoxymethyl (Cal-AM) release assay. Tumor cell lines expressing point mutations in KRAS, TP53 and CTNNB1 genes were constructed respectively, and the cytotoxicity of peptide-induced specific CTLs on wild-type and mutant tumor cells was verified using a Cal-AM release assay and carboxyfluorescein succinimidyl ester-propidium iodide staining. The high-frequency gene mutation loci of KRAS proto-oncogene (KRAS) G12V, tumor protein p53 (TP53) R158L and catenin ß1 (CTNNB1) K335I were identified in TCGA database. A total of 3 groups of wild-type and mutant peptides were screened using a peptide prediction algorithm. The CTNNB1 group had a strong affinity for the human leukocyte antigen-A2 molecule, as determined using flow cytometry. The IFN-γ secretion of specific CTLs in the CTNNB1 group was the highest, followed by the TP53 and the KRAS groups. The killing rate of mutant peptide-induced specific CTLs on peptide-loaded T2 cells in the CTNNB1 group was higher compared with that observed in the other groups. The killing rate of specific CTLs induced by mutant peptides present on tumor cells was higher compared with that induced by wild-type peptides. However, when compared with the TP53 and KRAS groups, specific CTLs induced by mutant peptides in the CTNNB1 group had more potent cytotoxicity towards mutant and wild-type tumor cells. In conclusion, point mutant tumor neoantigens screened in the three groups improved the cytotoxicity of specific T cells, and the mutant peptides in the CTNNB1 group were more prominent, indicating that they may activate the cellular immune response more readily.
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OBJECTIVE: To investigate the effect of the AKT1 gene mutation hotspot E17K on the growth, proliferation, survival, and migration of breast cancer cells, based on the survival and prognosis of breast cancer patients with the AKT1 E17K mutation shown in TCGA database. METHODS: The survival and incidence rates of AKT1 E17K mutation hotspots in breast cancer and other cancers were extracted from the Cancer Genome Atlas (TCGA). The recombinant eukaryotic expression plasmid AKT1 E17K-pIRES2-EGFP was constructed and transfected into breast cancer MCF-7, and MDA-MB-231 cell lines. MCF-7 and MDA-MB-231 cell lines were randomly divided into blank control groups, empty plasmid groups, and recombinant plasmid groups. The growth curve was drawn using the cell counting method. The proliferation and division of breast cancer cells were detected by CFSE fluorescent dye tracking. Apoptosis was detected by Annexin V/PI double labeling and cell vitality was detected using MTT assays, and cell migratory ability was detected by cell scratch and transwell chamber tests. RESULTS: In breast cancer, and other cancers, the overall survival rate of patients with an AKT E17K mutation was higher than that of patients with non-point mutation, and this mutation was the most common found in breast cancer. Compared with the wild type, the growth function of mutant MCF-7 cells was inhibited (P < 0.05), as was the proliferation of MCF-7 cells expressing the AKT1 E17K mutation gene (P < 0.001). The late apoptosis rate of mutant breast cancer cells increased (P < 0.05) and the viability was lower than that of wild-type cells (P < 0.05). Mutant MDA-MB-231 cells showed increased migration ability when compared to wild-type MDA-MB-231 cells (P < 0.05). CONCLUSIONS: The expression of the AKT1 E17K mutation hotspot can inhibit the growth, proliferation, and survival ability of breast cancer cells, and promote apoptosis, while it also improves their migratory ability. The survival and prognosis of breast cancer patients with this mutation are good, which may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway.
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OBJECTIVE: To analyze the confusing factors and clinical and audiological characteristics of ABR and tympanometry in infants who failed the first and second hearing screening. METHOD: Between August 2005 and November 2007, 94 infants (144 ears) with detailed birth record and hearing screening record were reviewed in the study. The age of this series ranged from 48 days to 6 months. They received hearing screening with otoacoustic emissions (OAE), and all failed in the first and second hearing screening. The birth history, high-risk factors of hearing-impaired during newborn period and pregnancy history of subjects were fully detailed. Subjects were classified according to the age: 1 to 3 months old infants were considered as group 1, while 4 to 6 months old infants were considered as group 2. Auditory brainstem response (ABR), distortion product otoacoustic emissions (DPOAE) and acoustic immittance measurement were examined. RESULT: (1) The 226 Hz tympanograms of 144 ears showed type A of a single-peaked tympanogram in 77 ears (53.4%), a double-peaked tympanogram in 23 ears (16.0%), type Ad of a single-peaked tympanogram in 20 ears (13.9%), type As of a single-peaked tympanogram in 16 ears (11.1%), a flat-shaping tympanogram (type B) in 6 ears (4.2%), and others shapes (including C and D type) in 2 ears (1.4%). (2) The results of ABR showed that there were 64 ears (44.4%) with normal hearing (according to the threshold of ABR), 58 ears (40.3%) with mild hearing loss, 12 ears (8.3%) with moderate hearing loss, 3 ears (2.1%) with severe hearing loss, 7 ears (4.9%) with profound hearing loss. And the proportion of mild hearing loss was increased in the group, while the proportion of moderate and severe hearing loss was decreased. (3) The proportion of type A tympanogram was 50% (32 ears) in normal hearing subjects, which implied that the 226 Hz probe tones to record tympanogram would lead to a high false negative rate. And type proportion of type B tympanogram was higher in normal (4.7%) and mild hearing loss (3.4%) groups than in moderate and severe group. CONCLUSION: Middle ear function and development of auditory system in infants may be confusing factors in hearing screening. The 226 Hz probe tones to record tympanogram are unreliable for accurate assessment of middle ear status of infants. Therefore the results of hearing screening should be interpreted appropriately.