Profiling of inflammatory cytokines in patients with caustic gastrointestinal tract injury.

INTRODUCTION: Study of inflammatory cytokines in patients with caustic gastrointestinal tract injury is sketchy. This study investigated the cytokine profiling of patients with caustic substance ingestion, and analyzed the differences between patients with severe and mild injury. METHODS: This prospective, cross-sectional study enrolled 22 patients admitted to Chang Gung Memorial Hospital between March and October 2018. All patients underwent esophagogastroduodenoscopy in 24 hours. Patients were categorized into two subgroups, as mild (<2b, n = 11) or severe (≥2b, n = 11) group. RESULTS: The neutrophil count was higher in severe than mild group (P = 0.032). Patients in mild and severe groups exhibited significantly higher circulating inflammatory cytokines than healthy control, including interleukin (IL)-2, IL-5, IL-8, IL-9, IL-12, IL-13, interferon-gamma inducible protein-10, macrophage inflammatory protein-1 beta, regulated upon activation, normal T cell expressed and presumably secreted and tumor necrosis factor-alpha. Furthermore, the levels of IL-2 and tumor necrosis factor-alpha were significantly higher in patients with severe group than mild group. Although there was no difference in cumulative survival between both groups (P = 0.147), the severe group received more operations (P = 0.035) and suffered more gastrointestinal complications (P = 0.035) than mild group. CONCLUSION: Caustic substance ingestion produces mucosal damages and leads to excessive neutrophils and inflammatory cytokines in peripheral blood.

Combination of dexamethasone and Avastin® by supramolecular hydrogel attenuates the inflammatory corneal neovascularization in rat alkali burn model.

Corneal neovascularization (CNV) is one of the leading causes of vision loss and a high-risk factor for transplant rejection. The present study proposed a supramolecular hydrogel comprised of MPEG-PCL micelles and α-cyclodextrin (α-CD) for co-delivery of dexamethasone sodium phosphate (Dexp) and Avastin® (Ava), and further evaluated its therapeutic efficacy in rat alkali burn model. A physical mixing of Dexp/Ava, MPEG-PCL micelles, and α-CD aqueous solution leads to a spontaneous formation of the supramolecular hydrogel via a "host-guest" recognition between MPEG and α-CD. The supramolecular hydrogel provides a relatively quick release of Dexp over Ava during the study of the 5-day in vitro release. The results of in vitro cytotoxicity test and wound healing assay illustrated that the proposed supramolecular hydrogel was non-toxic against L-929 and HCEC cells and did not significantly affect the migration of HCEC cells after 24h incubation. The corneal distribution test suggested that the precorneal duration of Ava was significantly extended by the supramolecular hydrogel with respect to its solution formulation. Moreover, the supramolecular hydrogel showed high ocular biocompatibility and was a non-irritant after topical instillation. Furthermore, the Dexp-Ava hydrogel medication, but not by Ava solution and Ava hydrogel medication, could greatly attenuate the alkali burn-induced corneal inflammation and remarkably suppress the corneal neovascularization via the downregulation of VEGF, CD31, and α-SMA expression in the rat alkali burn model. As a result, the combined Dexp and Ava by supramolecular hydrogel exhibited an advantage over Ava monotherapy approach, which might be a promising alternative therapy for inflammatory CNV.

Comprehensive Modeling of Corneal Alkali Injury in the Rat Eye.

PURPOSE: To characterize the molecular, clinical, and histopathological profiles in the rat cornea after alkali injury over a 21-day period. METHODS: Alkali injury was induced in one eye of male Lewis rats. Corneal opacity and corneal neovascularization were assessed daily. Real-time qRT-PCR analysis and immunohistochemical staining were conducted to examine inflammation, neovascularization, and fibrosis. RESULTS: We found that within 2 hours of chemical exposure, corneal opacification rapidly developed with an acute increase in various cytokine expressions, while several cytokines demonstrated a secondary peak by day 7. Early neutrophil infiltration peaked at day 1 post-injury while macrophage infiltration peaked at day 7. Throughout the time course of the study, corneal opacity persisted and neovascularization, lymphangiogenesis, and fibrosis progressed. CONCLUSIONS: This study highlights the molecular, clinical, and histopathological changes throughout the progression of alkali injury in the rat cornea. These profiles will assist in the development of new strategies and therapies for ocular alkali injury.

Angiogenin ameliorates corneal opacity and neovascularization via regulating immune response in corneal fibroblasts.

BACKGROUND: Angiogenin (ANG), a component of tears, is involved in the innate immune system and is related with inflammatory disease. We investigated whether ANG has an immune modulatory function in human corneal fibroblasts (HCFs). METHODS: HCFs were cultured from excised corneal tissues. The gene or protein expression levels of interleukin (IL)-1beta (ß), IL-4, IL-6, IL-8, IL-10, complements, toll-like receptor (TLR)4, myeloid differentiation primary response gene (MYD)88, TANK-binding kinase (TBK)1, IkappaB kinase-epsilon (IKK-ε) and nuclear factor-kappaB (NF-κB) were analyzed with or without ANG treatment in tumor necrosis factor-alpha (TNF-α)- or lipopolysaccharide (LPS)-induced inflammatory HCFs by real-time polymerase chain reaction (PCR), Western blotting and immunocytochemistry. Inflammatory cytokine profiles with or without ANG were evaluated through immunodot blot analysis in inflammatory HCFs. Corneal neovascularization and opacity in a rat model of corneal alkali burn were evaluated after application of ANG eye drops. RESULTS: ANG decreased the mRNA levels of IL-1ß, IL-6, IL-8, TNF-α receptor (TNFR)1, 2, TLR4, MYD88, and complement components except for C1r and C1s and elevated the mRNA expression of IL-4 and IL-10. Increased signal intensity of IL-6, IL-8 and monocyte chemotactic protein (MCP)-1 and MCP-2 induced by TNF-α or LPS was weakened by ANG treatment. ANG reduced the protein levels of IKK-ε by either TNF-α and LPS, and decreased TBK1 production induced by TNF-α, but not induced by LPS. The expression of NF-κB in the nuclei was decreased after ANG treatment. ANG application lowered corneal neovascularization and opacity in rats compared to controls. CONCLUSION: These results demonstrate that ANG reduces the inflammatory response induced by TNF-α or LPS in HCFs through common suppression of IKK-ε-mediated activation of NF-κB. This may support the targeting of immune-mediated corneal inflammation by using ANG.

Bone marrow-derived mesenchymal stem cells affect immunologic profiling of interleukin-17-secreting cells in a chemical burn mouse model.

PURPOSE: This study investigated interleukin (IL)-17-secreting cell involvement in sterile inflammation, and evaluated the effect of mesenchymal stem cells (MSCs) on IL-17-secreting cell immunologic profiling. METHODS: Twenty mice were sacrificed at time points of 6 hours, 1 day, 1 week, and 3 weeks (each group, n = 5) after the cornea was chemically injured with 0.5N NaOH; IL-17 changes in the cornea were evaluated using enzyme-linked immunosorbent assay. Further, IL-17 secreting cells were assessed in the cervical lymph nodes by a flow cytometer. Rat MSCs were applied intraperitoneally in a burn model (n = 10), IL-17-secreting T helper 17 (Th17) cell and non-Th17 cell changes were checked using a flow cytometer in both cornea and cervical lymph nodes at 1 week, and compared with those in the positive control (n = 10). RESULTS: IL-17 was highest in the cornea at 1 week, while, in the cervical lymph nodes, IL-17-secreting cells showed early increase at 6 hours, and maintained the increase through 1 day to 1 week, and levels returned to the basal level at 3 weeks. Specifically, the non-Th17 cells secreted IL-17 earlier than the Th17 cells. When the MSCs were applied, IL-17 secretion was reduced in CD3(+)CD4(-)CD8(-), CD3(+)CD4(+)CD8(-), and CD3(+) CD4(-)CD8(+) cells of the cervical lymph nodes by 53.7%, 43.8%, and 50.8%, respectively. However, in the cornea, IL-17 secretion of CD3(+)CD4(-)CD8(-) cells was completely blocked. CONCLUSIONS: The results indicated that both IL-17-secreting non-Th17 and Th17 cells were involved in the chemical burn model, and MSCs appeared to mainly modulate non-Th17 cells and also partially suppress the Th17 cells.

Combined treatment with antioxidants and immunosuppressants on cytokine release by human peripheral blood mononuclear cells - chemically injured keratocyte reaction.

PURPOSE: To investigate the effect of antioxidants and immunosuppresants on mixed peripheral blood mononuclear cells (PBMC) - chemically injured keratocytes reaction (MLKR). METHODS: The PBMC stimulation assay was performed using chemically injured keratocytes treated with 0.05 N NaOH for 90 s (MLKR). MLKR were treated with various drugs including rapamycin, dexamethasone, mycophenoleic acid (MPA), alpha lipoic acid (ALA), and N-acetyl cysteine (NAC). Matrix metalloprotease-9 (MMP-9), transforming growth factor-beta 1 (TGF-ß1), interleukin-6 (IL-6), and macrophage migration inhibitory factor (MIF) secretion profiles of activated PBMCs stimulated by NaOH-treated keratocytes were determined by ELISA. RESULTS: Anti-oxidants as well as immunosuppressants suppressed PBMC proliferation. MMP-9 levels were lower in antioxidants group. IL-6 levels decreased in dexamethasone group and anti-oxidants group. Combination of immunosuppressants and antioxidants suppressed more PBMC proliferation except for rapamycin + ALA group, suppressed MMP-9 production except for MPA + ALA group, decreased IL-6 levels and increased MIF levels except for rapamycin + ALA group. TGF-ß1 levels were elevated in rapamycin group and rapamycin + ALA group. CONCLUSIONS: Cytokine production was different depending on combination of drugs.Our results suggest that the different drugs should be selected for treatment according to the phases of corneal chemical burn.

Lymphatic vessels correlate closely with inflammation index in alkali burned cornea.

PURPOSE: To study the relationship between corneal lymphangiogenesis and inflammation in alkali burned corneas. METHODS: Rat corneal lymphatic and blood vessels were labeled and distinguished by whole mount immunofluorescence and 5'-nase-alkaline phosphatase (5'-NA-ALP) double enzyme-histochemistry. Then, lymphatic vessel areas (LVA) and lymphatic vessel counting (LVC) were examined. Corneal inflammation was evaluated by inflammation index (IF) grading, histopathology, electron microscope, and polymorphonuclear leukocyte (PMN) infiltration. The relationship between LVC, LVA, IF, and PMN was examined, respectively. In addition, corneal lymphatic vessels of eleven human alkali burned corneas were examined by lymphatic vessel endothelial receptor (LYVE-1) immunohistochemistry. RESULTS: Corneal lymphangiogenesis occurred on Day 3, reached the peak at the end of two weeks, and disappeared five weeks after alkaline burns. Both LVA and LVC were strongly and positively correlated with IF after corneal alkaline burns. However, the relationship between LVC and PMN, between LVA and PMN were significant but converse. Among eleven human alkali burned corneas, corneal lymphangiogenesis was present in three corneas. CONCLUSIONS: Corneal lymphagiogenesis develops after alkaline burns and correlates closely with corneal inflammation.

Suppression of alkali burn-induced corneal neovascularization by dendritic cell vaccination targeting VEGF receptor 2.

PURPOSE: To investigate whether the induction of cytotoxic T lymphocytes (CTLs) targeting VEGF receptor 2 inhibits corneal neovascularization caused by alkali injury. METHODS: H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGF receptor 2 (VEGFR2(400-408)) was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of VEGFR2(400-408)- or gp70-pulsed mature DCs three times at 6-day intervals. After the third immunization, corneal neovascularization was induced by alkali injury. Two weeks after the injury, the corneal vascularized area was evaluated by lectin angiography. To confirm the peptide-specific CTL activities in C57BL/6 mice, CD8(+) T cells from immunized mice were subjected to ELISA for interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and (51)Cr-release cytotoxicity assay. To determine the in vivo effector T cells, the immunized mice were intraperitoneally injected with an anti-CD4 or -CD8 depletion antibody. RESULTS: Corneal neovascularization was significantly attenuated in mice immunized with VEGFR2(400-408) compared with those not immunized or immunized with gp70. VEGFR2(400-408) or gp70, but not beta-gal(96-103), application led to dose-dependent induction of IFN-gamma and TNF-alpha in the CD8(+) T cells cocultured with stimulator cells. Cytotoxicity assays showed the specific lysis of major histocompatibility complex-matched cells expressing VEGFR2, but not beta-gal(96-103). In vivo depletion of CD8(+), but not CD4(+), T cells significantly reversed the suppressive effect of VEGFR2(400-408) immunization on corneal neovascularization to the level observed in nonimmunized or gp70-immunized animals. CONCLUSIONS: These results indicate the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for corneal chemical injury.

Accelerated wound healing of alkali-burned corneas in MRL mice is associated with a reduced inflammatory signature.

PURPOSE: The present study was conducted to investigate healing of alkali-burned corneas in MRL/MpJ (MRL) mice. METHODS: Gross, clinical, and histologic criteria were used to compare healing of alkali-burned corneas in MRL and control C57BL/6J (B6) mice. Effects of neutrophil depletion of B6 mice and allogeneic reconstitution of B6 mice with MRL bone marrow on wound healing were evaluated. Gene expression patterns in normal and wounded corneas were surveyed with array-based quantitative real-time RT-PCR (AQPCR). RESULTS: MRL mice showed accelerated reepithelialization and decreased corneal opacity compared with B6 mice after alkali wounding. Marked inflammatory cell infiltration and fibrosis were evident in the corneas and anterior chambers of B6 mice. MRL mice showed less severe lesions, except for stromal edema. Rapid reepithelialization and reduced keratitis/iritis were also observed in neutrophil-depleted B6 mice, but not in B6 mice reconstituted with MRL bone marrow. AQPCR showed transcriptional changes of fewer genes associated with inflammation and corneal tissue homeostasis in alkali-burned corneas from MRL mice. Increased expression of an anti-inflammatory gene, Socs1, and a gene associated with healing, Mmp1a, were evident in MRL corneas. CONCLUSIONS: Alkali-burned corneas heal faster and more completely in MRL mice than in B6 mice, by means of rapid reepithelialization, reduced inflammation, and reduced fibrosis. Reduced inflammation, including decreased neutrophil infiltrates and the lack of a robust proinflammatory gene expression signature correlates with the rapid healing. However, the rapid-healing phenotype is not intrinsic to MRL hematopoietic progenitor cells.

Differential effects of sustained inflation recruitment maneuvers on alveolar epithelial and lung endothelial injury.

OBJECTIVE: The role of recruitment maneuvers in mechanical ventilation for patients with the acute respiratory distress syndrome and acute lung injury remains uncertain in part due to a lack of data on the effects of specific recruitment maneuvers on lung injury severity. The primary objective of this study was to determine the effect of one type of recruitment maneuver--sustained inflation--on alveolar epithelial and lung endothelial injury in experimental acute lung injury. DESIGN: Randomized experimental study. SETTING: Academic research laboratory. SUBJECTS: Forty-nine Sprague-Dawley rats. INTERVENTIONS: Lung injury was induced in anesthetized, ventilated rats by instillation of acid (pH 1.5) into the airspaces. Rats were ventilated with a tidal volume of 6 mL/kg and a positive end-expiratory pressure of 5 cm H(2)O with or without a sustained inflation recruitment maneuver repeated every 30 mins. Each recruitment maneuver consisted of two 30-sec inflations to total lung capacity (30 cm H(2)O) 1 min apart. MEASUREMENTS AND MAIN RESULTS: The use of recruitment maneuvers significantly improved oxygenation, compliance, end-expiratory lung volume, functional residual capacity, and deadspace fraction. Recruitment maneuvers reduced extravascular lung water and lung endothelial injury as measured by protein permeability (217 +/- 28 vs. 314 +/- 70 extravascular plasma equivalents [microL], p < .05). However, recruitment maneuvers did not prevent alveolar epithelial injury. Epithelial permeability and bronchoalveolar lavage RTI40 levels, a marker of type I cell injury, were similar with or without recruitment maneuvers. Recruitment maneuvers decreased epithelial fluid transport, a functional marker of epithelial injury. Recruitment maneuvers did not reduce markers of airspace inflammation. CONCLUSIONS: Sustained inflation recruitment maneuvers improve respiratory mechanics and oxygenation and may protect the lung endothelium but do not reduce alveolar epithelial injury. Because of the differential effects of recruitment maneuvers on the lung endothelium and alveolar epithelium, the net effect in clinical acute lung injury may not be beneficial. Additional clinical studies will be needed to assess the net impact of recruitment maneuvers in patients with acute lung injury.

[The parameters of the body immune reactivity in applying the therapeutic keratoplasty for the treatment of ocular burn lesions of extra severity]. / Pokazateli immunologicheskoi reaktivnosti organizma pri primenenii lechebnoi keratoplastiki v lechenii ozhogovoi bolezni glaz osobo tiazheloi stepeni.

One hundred and two (128 eyes) patients with stage 4 ocular burns underwent examinations. One-stage therapeutic keratoplasty was used in 67 (67.7%) patients. Retransplantation was made in 35 (34.3%) of patients due to graft rejection. According to the study, the more severe the clinical course of burn lesions is (cases that necessitated transplantation in the present research), the more pronounced changes are observed in the body immune reactivity of victims. In particular, the below peculiarities were observed: a progressing reduction of the contents of lymphocytes' and of B-lymphocytes; and a diminishing phagocytic activity of neutrophils in the peripheral blood registered at the terminal treatment stage in the patients after retransplantation versus the 1st-group patients with a more favorable clinical course. The compensatory increase of T-lymphocytes, T-helpers and of IgA, IgG was found to be less pronounced in group 2. A notably more intense sensitization response of the body to corneal antigens was established in the patients after retransplantation, which was a basis for using this index as a prognostication test in respect to the graft rejection crisis.

[Natural cytokine complex as a drug bioregulator in healing of alkaline burns of cornea (experimental morphological study)]. / Estestvennyi kompleks tsitokinov kak lekarstvennyi bioreguliator pri zazhivlenii shchelochnykh ozhogov rogovitsy (éksperimental'no-morfologicheskoe issledovanie).

Clinical morphological features of reparative processes of alkaline burns of the cornea are studied in 24 rabbits. Effects of the natural cytokine complex on this process are validated. Burn process is regarded as an immune inflammation. Morphological and morphometric data demonstrate the mechanism of the cytokine effect on the reparative processes in the cornea, based on the regulation of the inflammatory postburn and proliferative processes. The repair of the cornea after burn in experiment and control is described in detail.

Clinical and experimental studies concerning circulating antibodies to corneal epithelium antigens.

Corneal epithelium antibodies were detected in patients with corneal melting disease (55%), uveitis (42%), corneal transplantation (42%) and marginal furrow disease (20%). These antibodies were not found in herpetic keratitis patients. In control groups, consisting of ocular surgery patients (glaucoma, retinal detachment and cataract) and persons without a history of ocular disease, approximately 4% of the subjects had these antibodies. To investigate the possible role of trauma to the cornea as an initiator of corneal epithelium antibodies, these antibodies were determined in rabbits after alkaline burns were made on the cornea. These antibodies were detected one week later and disappeared after six weeks. Serum from three patients with corneal melting disease and corneal transplantation containing a high antibody titre against corneal epithelium were used to isolate corneal epithelium antigens. A 54 kD and a 17 kD corneal epithelium antigen were isolated. The incidence of autoantibodies directed against these antigens was investigated in patients with corneal melting disease, uveitis and corneal transplantation using an ELISA. 50% of the sera positive in the immunofluorescence test were positive in the ELISA.

Postburn immunosuppression in an animal model: monocyte dysfunction induced by burned tissue.

We studied cell-mediated immunity (CMI) in burned mice using an assay that involves the induction of contact sensitivity to dinitrofluorobenzene (DNFB). Subsequent painting of the ears with DNFB and measurement of ear swelling with calipers is a sensitive and quantifiable assay for CMI. Results may be expressed as mean ear swelling (MS) in units of 10(-4) inches +/- 2 standard errors of the mean. CMI was severely depressed in burned mice over a 2-week period following burn (control MS 48.3 +/- 1.0, 14 days after burn 29.0 +/- 1.0, P less than 0.01). Immediate postburn eschar removal resulted in avoidance of immunosuppression (MS 41.5 +/- 1.0, P less than 0.01) while transfer of burned tissue subcutaneously into unburned mice resulted in severe immunosuppression (MS 33.2 +/- 2.6, P less than 0.01). CMI was restored by intravenous infusion of peritoneal macrophages from unburned mice (MS 41.4 +/- 2.2), but not by infusion of lymphocytes or of macrophages taken from burned mice. This model should prove useful for further study of burn injury-induced immunosuppression.

Changes in humoral components of host defense following burn trauma.

Serum opsonic activity for E. coli 075, conversion of C3 by inulin, total hemolytic complement (CH(50)), levels of native C3, factor B, C3b inactivator (KAF), properdin (P), and immunoglobulins (Ig) were determined in 14 patients with burns involving 13% to 91% body surface during 6 to 8 weeks postburn. In the 12 uninfected patients, levels of IgG and IgA were reduced during the first 10 days postburn, and decreased concentrations of P and IgM were demonstrated from three to 6 weeks postburn. C3 conversion was reduced from 10 days to 6 weeks postburn. Levels of C3, factor B, and KAF were normal or elevated for the entire study period. No difference in the occurrence of humoral abnormalities was noted in patients with burns caused by flame, immersion scald, or acid contact. Reduction in C3 conversion and P concentration were the only abnormalities which correlated with increasing burn size. Bacteremia and/or fungemia was documented in the other two patients. In one of these patients, reduction in CH(50) occurred during septicemia due to S. aureus, and in the other, reduction in all measurements of complement was associated with candidemia and Pseudomonas septicemia and occurred prior to the development of shock. Serum opsonic activity was only reduced significantly during sepsis, suggesting that this abnormality occurred as a result rather than a cause of infection. These results indicate that consumption of components of the classical and/or alternative pathways of complement activation may be an important mechanism by which infection is perpetuated in the burn patient. They also emphasize the importance of the clinical management of the burn patient in preventing the development of septic complications.