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A novel cyclic chalcone fluorescent probe C-PN was synthesized to detect ONOO - . After reaction with peroxynitrite, the double bond of C-PN in the cyclic chalcone structure was disconnected, which caused the change of intramolecular charge transfer (ICT) effect, emitting blue fluorescence and quenching orange red fluorescence. Visible to the naked eye, the color of the probe solution changed. The probe showed low sensitivity (detection limit = 20.2 nm), short response time (less than 60 s) at low concentration of ONOO - , good visibility, and good selectivity and stability for ONOO - ., (© 2024. The Author(s), under exclusive licence to the European Photochemistry Association, European Society for Photobiology.)

Objective: To investigate the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) combined with autologous Meek microskin transplantation on patients with extensive burns. Methods: The prospective self-controlled study was conducted. From May 2019 to June 2022, 16 patients with extensive burns admitted to the 990 th Hospital of PLA Joint Logistics Support Force met the inclusion criteria, while 3 patients were excluded according to the exclusion criteria, and 13 patients were finally selected, including 10 males and 3 females, aged 24-61 (42±13) years. A total of 20 trial areas (40 wounds, with area of 10 cm×10 cm in each wound) were selected. Two adjacent wounds in each trial area were divided into hUCMSC+gel group applied with hyaluronic acid gel containing hUCMSCs and gel only group applied with hyaluronic acid gel only according to the random number table, with 20 wounds in each group. Afterwards the wounds in two groups were transplanted with autologous Meek microskin grafts with an extension ratio of 1∶6. In 2, 3, and 4 weeks post operation, the wound healing was observed, the wound healing rate was calculated, and the wound healing time was recorded. The specimen of wound secretion was collected for microorganism culture if there was purulent secretion on the wound post operation. In 3, 6, and 12 months post operation, the scar hyperplasia in wound was assessed using the Vancouver scar scale (VSS). In 3 months post operation, the wound tissue was collected for hematoxylin-eosin (HE) staining to observe the morphological changes and for immunohistochemical staining to observe the positive expressions of Ki67 and vimentin and to count the number of positive cells. Data were statistically analyzed with paired samples t test and Bonferronni correction. Results: In 2, 3, and 4 weeks post operation, the wound healing rates in hUCMSC+gel group were (80±11)%, (84±12)%, and (92±9)%, respectively, which were significantly higher than (67±18)%, (74±21)%, and (84±16)% in gel only group (with t values of 4.01, 3.52, and 3.66, respectively, P <0.05). The wound healing time in hUCMSC+gel group was (31±11) d, which was significantly shorter than (36±13) d in gel only group ( t =-3.68, P <0.05). The microbiological culture of the postoperative wound secretion specimens from the adjacent wounds in 2 groups was identical, with negative results in 4 trial areas and positive results in 16 trial areas. In 3, 6, and 12 months post operation, the VSS scores of wounds in gel only group were 7.8±1.9, 6.7±2.1, and 5.4±1.6, which were significantly higher than 6.8±1.8, 5.6±1.6, and 4.0±1.4 in hUCMSC+gel group, respectively (with t values of -4.79, -4.37, and -5.47, respectively, P <0.05). In 3 months post operation, HE staining showed an increase in epidermal layer thickness and epidermal crest in wound in hUCMSC+gel group compared with those in gel only group, and immunohistochemical staining showed a significant increase in the number of Ki67 positive cells in wound in hUCMSC+gel group compared with those in gel only group ( t =4.39, P <0.05), with no statistically significant difference in the number of vimentin positive cells in wound between the 2 groups ( P >0.05). Conclusions: The application of hyaluronic acid gel containing hUCMSCs to the wound is simple to perform and is therefore a preferable route. Topical application of hUCMSCs can promote healing of the autologous Meek microskin grafted area in patients with extensive burns, shorten wound healing time, and alleviate scar hyperplasia. The above effects may be related to the increased epidermal thickness and epidermal crest, and active cell proliferation.

Objective: To investigate the effect of Compound Qingqin Liquid (CQL) on the expression level of angiotensin II (Ang II) and COX-2 mRNA transcription and protein expression in the renal tissue of rats with uric acid nephropathy., Methods: SD rats were randomly divided into the blank control group, the model group, the positive drug group, the high, moderate, and low dose CQL group according to number randomization principle. The model was established by gastrogavage of adenine, accompanied with yeast feeding. Distilled water was given by gastrogavage to rats in the blank control group and the model group. Allopurinol at the daily dose of 9.33 mg/kg was given by gastrogavage to rats of the positive control group. CQL at the daily dose of 3.77 g/kg, 1.89 g/kg, and 0.09 g/kg was respectively given by gastrogavage to rats in the high, moderate, and low dose CQL groups. All treatment lasted for 6 weeks. Rats were randomly divided at week 4 (3 in the blank control group, and 6 in the rest groups), and the rest rats were killed at week 6. The renal tissue was extracted. The expression level of Ang II and COX-2 mRNA transcription were detected by RT-PCR. The expression level of Ang II was detected by ELISA. The expression level of COX-2 protein was detected by Western blot and immunohistochemical assay., Results: Compared with the blank control group, except the mRNA expression of Ang II at week 4, the mRNA and protein expression of Ang II and COX-2 obviously increased at week 4 and 6 in the model group (P < 0.01, P < 0.05). The COX-2 protein expression at week 4 was obviously lower in the high and moderate dose CQL groups than in the model group and the low dose CQL group (P < 0.05); the average integral of optical density value was obviously lower in the positive control group than in the model group. Except the mRNA expression of Ang II in the high dose CQL group at week 6, the mRNA and protein expression of Ang II obviously decreased in the positive control group and each dose CQL group (P < 0.01, P < 0.05). Of them, the effects were better in the high and moderate dose CQL groups than in the positive control group and the low dose CQL group (P < 0.05, P < 0.01). Besides, the mRNA expression of COX-2, the average integral of optical density value were obviously lower in the positive control group and each dose CQL group than in the model group (P < 0.05). The protein expression of COX-2 was obviously lower in the high and moderate dose CQL groups than in the model group (P < 0.05). Of them, the mRNA expression of COX-2 was better in the moderate dose CQL group than in the positive control group (P < 0.05); the protein expression of COX-2 was better in the high dose CQL group than in the low dose CQL group (P < 0.05)., Conclusion: CQL was capable of lowering the expression level of Ang II, COX-2 mRNA transcription and protein expression, thus suppressing the inflammatory pathological injury of the renal tissue.

IgE plays a critical role in the pathogenesis of allergy and asthma. Therefore, suppression of IgE production would provide therapeutic benefits to patients suffering from these diseases. We have reported that the production of IgE is regulated differently in the spleen vs. the draining lymph nodes (LN). IgE isotype switch and IgE producing B cell expansion occur in the draining LN after antigen (Ag) immunization, but do not happen in the spleen. In addition, a population of pre-existing IgE+ cells is observed in the spleen of normal or sham immunized mice, but is not present in the draining LN. To further understand the regulation of IgE production in different lymphoid organs, and the potential inhibitory factors of IgE isotype switch in the spleen, the involvement of IL-21 and IFN-gamma in regulating IgE production was investigated by using the IL-21 and the IFN-gamma deficient mice. We found that in the absence of IL-21 IgE isotype switch and IgE+ cell clonal expansion were dramatically enhanced in the spleen and IgE isotype switch was partially increased in the draining LN. In addition, IgE production of the pre-existing CD19-CD5+B220(low) IgE+ cells in the spleen was also increased in the absence of IL-21 under physiological conditions. In contrast, using the IFN-gamma deficient mice, we did not observe a negative impact of IFN-gamma on either IgE isotype switch or IgE production. Our data suggest that IL-21 appears to be a critical cytokine to keep low IgE levels under physiological and pathological conditions.

IgE is critical in the pathogenesis of allergic disorders. In this report, we investigated the differential regulation of antigen-specific and by-stander IgE. Ovalbumin (OVA) immunization did not increase IgE producing cells in the spleen, but significantly enhanced the intracellular IgE content of all IgE+ cells. In contrast, OVA induced a significant increase of IgE+ cells in the draining lymph nodes (LN). Furthermore, OVA-specific IgE was detected only in the ex vivo cultures of the draining LN but not the spleen cells, while total IgE was increased in both cultures. These results indicated that antigen-specific IgE was mainly produced in the draining LN, while the spleen was a major source for by-stander IgE. Anti-IL-4, but not anti-IL-13, antibody blocked the expansion of IgE producing cells in the draining LN as well as systemic OVA-specific and total IgE levels, indicating IL-4 was important in both antigen-specific IgE generation and total IgE upregulation.

The human hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The mechanisms of liver cell oncogenic transformation are still unknown. The beta-catenin mutations are identified in up to 30% of HCC and 80% of hepatoblastoma, suggesting a potential role of beta-catenin in the pathogenesis of liver cancers. To define the biological role of the stabilized beta-catenin in liver cell growth and transformation, we examined the effect of mutant beta-catenin on an immortalized murine hepatocyte cell line, AML12. A cell line that stably expresses mutant beta-catenin was established. The cell proliferation, apoptosis, and cell transformation of this cell line were characterized. Our data indicate that the stabilized beta-catenin enhances hepatocyte proliferation, suppresses TNFalpha/Act D-induced cell apoptosis, and causes weak anchorage-independent cell growth. The stabilized beta-catenin-containing cells did not develop tumor in immune-deficient mice. The target genes, c-myc and cyclin D1, were activated by beta-catenin in the hepatocytes. Our study suggests that mutant beta-catenin can promote cell proliferation and cell survival ability, but the stabilized beta-catenin alone is insufficient for completely oncogenic transformation.

Chemokine receptor transcripts were defined among CD4+ T cells in lymph nodes of mice with type-1 and type-2 inflammation, respectively, elicited by mycobacterial and schistosomal Ag. CXCR3 and CCR6 transcripts were biased to type-1, and CCR4 transcripts increased in type-1 and type-2 populations. CCR3 and CCR5 signals were too weak to establish differences. CCR8 transcripts were not increased among unstimulated populations. Compared to naïve, type-1 and type-2 populations had reduced CCR7 and enhanced CXCR5 transcripts, consistent with a shift to memory cells. Subset depletion revealed that transcript expression was induced among CD44+ memory T cells. Surprisingly, CCR3 transcripts were enriched among CD44lo fractions. Ag stimulation augmented CXCR3, CCR4, and CCR8 but down-regulated CCR6 and CXCR5. CCR4 showed association with IFN-gamma- and IL-4-producing cells, but other receptor transcripts were expressed among IFN-gamma/IL-4 negative memory T cells. These studies provide several novel findings regarding Th cell chemokine receptor expression in vivo.

Monocyte chemotactic protein-3 (MCP-3/CCL7) has potent eosinophil chemoattractant properties. The present study determined its relative contribution to the formation of Th2 cytokine-mediated (type-2) eosinophil-rich interstitial lung granulomas induced by antigens of Schistosoma mansoni eggs. Both MCP-3 transcripts and protein levels were more strongly expressed in lungs with type-2 than with type-1 (mycobacterial antigen-elicited Th1-mediated) granulomas. In vivo treatment with neutralizing antibodies demonstrated that MCP-3 abrogated eosinophil accumulation in type-2 lesions by 40 to 50%. Immunohistochemical staining revealed that MCP-3 localized to vessels in or near granulomas suggesting that endothelial cells were an important in situ source of MCP-3. Maximal MCP-3 transcript expression was abrogated by anti-interleukin-4 treatment. Furthermore, cultured mouse lung endothelial cells displayed augmented MCP-3 production in response to interleukin-4. Together, these results suggest that MCP-3 contributes to a significant component of eosinophil recruitment in the type-2 interstitial granuloma formation and Th2 cytokines promote its production.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances and primes monocyte functions, but its role in monocyte migration is poorly understood. We examined monocyte migration across human umbilical vein endothelial cells (HUVEC) grown on filters. GM-CSF had no chemotactic or chemokinetic effect. However, GM-CSF enhanced monocyte transendothelial migration (TEM) through unstimulated and IL-1-activated (5 h) HUVEC in response to C5a or monocyte chemoattractant protein-1 in a dose-dependent fashion, increasing the migration from 28.7 +/- 5.3% to 41.8 +/- 6.2% (n = 8, p < 0.05) and from 34.8 +/- 6% to 50.3 +/- 3.1%, p < 0.05), respectively. The enhanced TEM was inhibited by monoclonal antibodies (mAb) to LFA-1, but not by mAb to Mac-1 or to VLA-4. Furthermore, GM-CSF up-regulated and activated LFA-1, as assessed by NKI-L16 neoepitope expression. The results indicate that: (1) GM-CSF can prime monocytes for increased TEM, (2) GM-CSF enhances LFA-1-mediated monocyte TEM and (3) this effect is in part mediated by increasing LFA-1 expression and activation. Thus, increased GM-CSF production may promote monocyte accumulation in inflammation not only by inducing monocytosis, but also enhancing migration.

Recently we reported that monocyte migration through a barrier of human synovial fibroblasts (HSF) is mediated by the CD11/CD18 (beta2) integrins, and the beta1 integrins VLA-4 and VLA-5 on monocytes. Here we investigated in parallel the role of beta2 integrin family members, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on monocytes, and the immunoglobulin supergene family members, ICAM-1 and ICAM-2 on HSF and on human umbilical vein endothelial cells (HUVEC), in monocyte migration through HSF and HUVEC monolayers. Using function blocking monoclonal antibodies (mAb), when both VLA-4 and VLA-5 on monocytes were blocked, treatment of monocytes with mAb to both LFA-1 and to Mac-1 completely inhibited monocyte migration across HSF barriers, although blocking either of these beta2 integrins alone had no effect on migration, even when VLA-4 and VLA-5 were blocked. This indicates that optimal beta2 integrin-dependent monocyte migration in synovial connective tissue may be mediated by either LFA-1 or Mac-1. Both ICAM-1 and ICAM-2 were constitutively expressed on HSF and on HUVEC, although ICAM-2 was only minimally expressed on HSF. Based on results of mAb blockade, ICAM-1 appeared to be the major ligand for LFA-1-dependent migration through the HSF. In contrast, both ICAM-1 and ICAM-2 mediated LFA-1-dependent monocyte migration through HUVEC. However, neither ICAM-1 nor ICAM-2 was required for Mac-1 -dependent monocyte migration through either cell barrier, indicating that Mac-1 can utilize ligands distinct from ICAM-1 and ICAM-2 on HSF and on HUVEC during monocyte transmigration.

Monocytes migrate through vascular endothelium, and then in connective tissue. As a model of this process, we investigated adhesion molecules involved in monocyte migration through HUVEC and a barrier of human synovial fibroblasts (HSF). Minimal spontaneous monocyte migration (6-7%) occurred through either cell barrier, but this increased markedly (27-35% of added monocytes) when a C5a chemotactic gradient was present. Migration across unstimulated HUVEC was partially inhibited (40%) by mAb to CD18 (beta2 integrin) and completely blocked by anti-CD18 plus anti-alpha4 (CD49d; very late Ag-4 (VLA-4)) mAbs. In contrast, migration across HSF induced by C5a or monocyte chemoattractant protein-1 was not inhibited by mAb to CD18 and was only partially inhibited (33%) in combination with anti-alpha4 mAb. The CD18- and VLA-4-independent migration across HSF was completely inhibited by mAb to alpha5 of VLA-5. The inhibitory effect of mAbs to VLA-4 and VLA-5 was on the monocyte and required blockade of CD11/CD18 to be observed. In contrast to HSF, no role for VLA-5 in monocyte transendothelial migration was detected. Both HSF and IL-1-stimulated HUVEC expressed vascular cell adhesion molecule-1 (VCAM-1). However, VLA-4-mediated monocyte migration across HSF was only partially dependent on VCAM-1, in contrast to transendothelial migration, which was completely blocked by anti-VCAM-1 mAbs. In conclusion, unlike transendothelial migration, for which VLA-4 is the alternative mechanism to CD11/CD18 on monocytes, both VLA-4 and VLA-5 can mediate monocyte migration through fibroblast barriers. In addition to VCAM-1, other ligand(s) on HSF are also involved in the VLA-4-mediated migration.

Accumulation of leucocytes in inflamed lung tissue and alveolar space involves their migration through vascular endothelium and then lung connective tissue. As a model of this process, we investigated human polymorphonuclear leucocyte (PMNL) and monocyte migration through a biological barrier of human lung fibroblasts (HLF) grown on polycarbonate filters. Very few PMNL (1-2%) or monocytes (3-8%) migrated through the HLF barriers spontaneously. Migration increased to 48-53% of added PMNL and 17-24% of added monocytes, when a C5a chemotactic gradient was present. The monocyte migration induced by C5a was not inhibited by monoclonal antibodies (mAb) to CD18 (beta 2 integrins). This CD18-independent migration was partially inhibited (35%) by mAb to gamma 5 of VLA-5 and completely inhibited by the combination of mAb to gamma 4 of VLA-4 with mAb to VLA-5, in the presence of mAb to CD18. In contrast, PMNL migration across HLF induced by C5a was partially inhibited by mAb to CD18 alone, but even with the addition of mAb to VLA-4, VLA-5 beta 1 and VLA-6, the greatest degree of inhibition was only 60%. Blocking the function of CD18 was not required to observe the inhibition by mAb to VLA-4, although the inhibitory effect of mAb to VLA-5 and VLA-6 alone or in combination was only observed when CD18 mechanisms were also blocked with anti-CD18 mAb. These results demonstrate that (a) both monocytes and PMNL can use either CD11/CD18 (beta 2 integrin) or beta 1 (CD49/CD29) integrins to migrate through HLF barriers; (b) in the case of monocytes, the VLA-4 and VLA-5 integrins account for essentially all the CD11/CD18-independent migration mechanisms; and (c) in contrast to monocytes, PMNL CD18-independent migration is mediated not only by VLA-4 and VLA-5, but also by VLA-6, and up to 40% of the migration appears to be via yet to be defined PMNL surface molecules.

Islet cell antigens have been administered orally and intravenously (I.V.) to NOD mice to assess their abilities to protect from or delay the onset of diabetes, and thereby provide insights that may have therapeutic implications in human trials. Whereas we and others have observed a delay in the onset of diabetes in NOD mice that have been fed with insulin from early life, we report here for the first time that feedings with porcine GAD65 alone (p = 0.226) or in combination with insulin (p = 0.011), have anti-diabetic effects in a prolonged study period (>400 days). While antigen-specific inhibitions of in vitro lymphocytic proliferation responses were seen (p < 0.05), antibody levels were unaffected by oral antigen treatments. IFN-gamma mRNA levels were downregulated in the islet infiltrates following oral antigen treatments while IL-2 and TNF-beta were expressed in all instances. We also observed that I.V. human recombinant GAD65, and porcine GAD given at weaning, delayed diabetes onset (p = 0.004) while similar treatments with a variety of inactive insulin preparations were generally ineffective. These findings thus indicate varying effects of oral and I.V. autoantigen administrations on the development of diabetes in NOD mice, and describe the immunological processes induced by oral autoantigen treatments.

Interventional approaches that have been successful in delaying insulin-dependent diabetes mellitus (IDDM) using antigen-based immunotherapies include parenteral immunization. It has potential for clinical application provided that effective adjuvants suitable for human use can be found. We have previously shown that immunization with insulin and insulin B chain but not A chain in incomplete Freund's adjuvant (IFA) prevented diabetes by reducing IFN-gamma mRNA in the insulitis lesions. In this paper we show that the insulin B chain peptide (p9-23) contain the most protective epitope. Immunization with selected GAD peptides was ineffective. Immunization with B chain but not A chain using alum as adjuvant delayed diabetes onset (P = 0.012), whereas administration of alum alone was not protective. When Diphtheria-Tetanus toxoid-Acellular Pertussis (DTP) vaccine was used as the adjuvant vehicle, DTP itself induced significant protection (P < 0.003) which was associated with a Th2-like cytokine producing insulitis profile, IL-4 driven IgG1 antibody responses to insulin, GAD in the periphery and an augmentation of the autoimmune response to GAD. The anti-diabetic effect of DTP was enhanced when given with insulin B chain. These results encourage consideration of an approach using alum/DTP and insulin B chain immunization in clinical trials.