Integrative analysis of bulk and single-cell RNA-seq reveals the molecular characterization of the immune microenvironment and oxidative stress signature in melanoma.

Background: The immune microenvironment and oxidative stress of melanoma show significant heterogeneity, which affects tumor growth, invasion and treatment response. Single-cell and bulk RNA-seq data were used to explore the heterogeneity of the immune microenvironment and oxidative stress of melanoma. Methods: The R package Seurat facilitated the analysis of the single-cell dataset, while Harmony, another R package, was employed for batch effect correction. Cell types were classified using Uniform Manifold Approximation and Projection (UMAP). The Secreted Signaling algorithm from CellChatDB.human was applied to elucidate cell-to-cell communication patterns within the single-cell data. Consensus clustering analysis for the skin cutaneous melanoma (SKCM) samples was executed with the R package ConsensusClusterPlus. To quantify immune infiltrating cells, we utilized CIBERSORT, ESTIMATE, and TIMERxCell algorithms provided by the R package Immuno-Oncology Biological Research (IOBR). Single nucleotide variant (SNV) analysis was conducted using Maftools, an R package specifically designed for this purpose. Subsequently, the expression levels of PXDN and PAPSS2 genes were assessed in melanoma tissues compared to adjacent normal tissues. Furthermore, in vitro experiments were conducted to evaluate the proliferation and reactive oxygen species expression in melanoma cells following transfection with siRNA targeting PXDN and PAPSS2. Results: Malignant tumor cell populations were reclassified based on a comprehensive single-cell dataset analysis, which yielded six distinct tumor subsets. The specific marker genes identified for these subgroups were then used to interrogate the Cancer Genome Atlas Skin Cutaneous Melanoma (TCGA-SKCM) cohort, derived from bulk RNA sequencing data, resulting in the delineation of two immune molecular subtypes. Notably, patients within the cluster2 (C2) subtype exhibited a significantly more favorable prognosis compared to those in the cluster1 (C1) subtype. An alignment of immune characteristics was observed between the C2 subtype and unique immune functional tumor cell subsets. Genes differentially expressed across these subtypes were subsequently leveraged to construct a predictive risk model. In vitro investigations further revealed elevated expression levels of PXDN and PAPSS2 in melanoma tissue samples. Functional assays indicated that modulation of PXDN and PAPSS2 expression could influence the production of reactive oxygen species (ROS) and the proliferative capacity of melanoma cells. Conclusion: The constructed six-gene signature can be used as an immune response and an oxidative stress marker to guide the clinical diagnosis and treatment of melanoma.

Clinical and histopathological characteristics, diagnosis and treatment, and comorbidities of Bowen's disease: a retrospective study.

Background: Bowen's disease (BD) is a slow-growing precancerous skin condition, often concurrent with other diseases, with a high misdiagnosis rate. Previous studies show that patients with BD in different populations have differentiated characteristics. Materials and methods: A retrospective study was conducted in a tertiary hospital in Shenzhen, China. Data about demographic information, diagnosis and treatment, clinical and pathological characteristics, and comorbidities of 50 patients with BD were collected and analyzed. Results: Clinical data of onset age and disease course of 43 patients with BD were available, the average onset age of male and female patients are 55.1 (standard deviation (SD) = 15.29) and 58.2 (SD = 15.59) years old, respectively; the average disease course of male and female patients are 25.3 (SD = 28.63) and 33.9 (SD = 49.65) months, respectively. The onset age (p = 0.52) and disease course (p = 0.49) between male and female patients are not significantly different. Interestingly, there is a negative correlation between onset age and disease course (r = -0.245, p = 0.11). The correct rate of clinical diagnosis is relatively low (54.00%); Some patients with BD are misdiagnosed as Bowenoid papulosis (10.00%), actinic keratosis (8.00%), basal cell carcinoma (8.00%), seborrheic keratosis (6.00%), and pigmented naevus (4.00%). Trunk and limbs are the most common distribution sites of BD lesions, and 94.00% patients with BD are treated with surgical resection; 66.00% patients with BD had comorbidities, including skin diseases (48.48%), cardiovascular diseases (39.39%), gastrointestinal diseases (30.30%), respiratory diseases (27.27%), and tumors (18.18%). The most commonly observed histopathological characteristics of BD are squamous-cell hyperplasia (86.00%), disordered maturation with atypical keratinocytes (74.00%), atypical mitoses (60.00%), hyperkeratosis with hypokeratosis (48.00%), dermal inflammatory cell infiltration (36.00%), and koilocytosis (22.00%). Conclusion: BD often occurs in middle-aged and elderly people and is easily misdiagnosed. The onset age and disease course of patients with BD are not significantly different between males and females, whereas there is a negative correlation between the onset age and disease course. BD is more likely to occur in trunk and limbs in the Chinese population, and most patients with BD are concurrent with comorbidities.

Acitretin enhances the cytotoxic effect of 5-aminolevulinic acid photodynamic therapy on squamous cell carcinoma cells.

The efficacy of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on invasive cutaneous squamous cell carcinoma (cSCC) remains to be improved due to the limited penetration of this treatment. Previous study showed that acitretin and ALA-PDT had synergistic effect on cSCC, but whether acitretin can enhance the cytotoxic effect of ALA-PDT on cSCC is unclear. OBJECTIVE: To investigate whether acitretin can enhance the cytotoxic effect of ALA-PDT on SCL-1 cells, as well as the possible mechanism involved. METHODS: Inverted microscopy, trypan blue exclusion assay, and flow cytometry were used to studied the morphology, viability and apoptosis of SCL-1 cells treated with acitretin, ALA-PDT and acitretin followed by ALA-PDT treatment, respectively. Confocal microscopy was applied to detect the ROS formation of SCL-1 cells treated with acitretin of four different concentrations. The ROS formation of SCL-I cells treated with acitretin of four different concentrations followed by ALA-PDT treatment was detected using confocal microscopy and flow cytometry. RESULTS: SCL-1 cells exhibited a significant morphological alteration when treated with acitretin followed by ALA-PDT. The combination of acitretin and ALA-PDT induced a higher cell death rate and apoptosis than that with acitretin or ALA-PDT treatment alone. ROS could be induced when incubated with acitretin at a concentration of 6.4 × 10-4mg /mL or above. However, a higher level of ROS formation was observed when SCL-1 cells were treated with acitretin followed by ALA-PDT than that with ALA-PDT or acitretin alone. CONCLUSION: Acitretin can enhance the cytotoxic effect of ALA-PDT on SCL-1 cells, possibly via the ROS pathway.

5-aminolevulinic acid photodynamic therapy inhibits invasion and metastasis of SCL-1 cells probably via MTSS1 and p63 gene related pathways.

OBJECTIVE: To investigate the effect of 5-aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) on the invasion and metastasis in cutaneous squamous cell carcinoma (cSCC) cell line(SCL-1) and to study whether the effect was via the MTSS1 gene and p63 gene related pathways. METHODS: SCL-1 cells were cultured and submitted to ALA-PDT treatment (ALA-PDT group), ALA treatment alone (ALA group), LED illumination alone (LED group) and remains untreated (control group). Scratch test, Transwell migration chamber assay and Matrigel cell invasion assay were used to detect the ability of migration and invasion of SCL-1 cells after treatment. The mRNA levels and protein expressions of tumor metastasis suppressor gene (MTSS1) and p63 gene were further detected by using quantitative real-time PCR and flow cytometry assay respectively after treatment. RESULTS: The migration and invasion abilities of SCL-1 cells after treatment were significantly reduced in the ALA-PDT groups than that in ALA group, LED group and control group (P＜0.05). Both the mRNA and protein expression levels of MTSS1 gene were up-regulated, while the mRNA and protein expression levels of p63 gene were down-regulated after ALA-PDT treatment. CONCLUSION: ALA-PDT suppressed the migration and invasion of human cSCC cell line, probably via the MTSS1 gene and p63 gene related pathways. This study put forward a possible mechanism of invasion in SCL-1 cell, also providing a potential target for the therapy of cSCC.

5-aminolevulinic acid photodynamic therapy enhance the effect of acitretin on squamous cell carcinoma cells: An in vitro study.

Squamous cell carcinoma (SCC) remains the second most common nonmelanoma skin cancer (NMSC) worldwide. Both acitretin and 5-Aminolevulinic acid mediated photodynamic therapy (ALA-PDT) have validated effect on SCC. However, the effects of both treatmens remain limited, and there has been no report concerning the potential synergistic effect of both treatments for SCC. OBJECTIVE: To investigate the cytotoxic effect of acitretin on SCL-1 cells, and whether ALA-PDT enhances this effect. METHODS: CCK-8 and trypan blue exclusion array were used to detect the cell cytotoxicity after acitretin treatment with different concentrations (1.6 × 10-4mg/mL, 1.6 × 10-3 mg/mL, 1.6 × 10-2mg/mL and 1.6 × 10-1mg/mL) for 24 h, 48 h and 72 h. Flow cytometry and trypan blue exclusion assay were used to detect the apoptosis and viability of SCL-1 cells after treated with acitretin, ALA-PDT and ALA-PDT immediately followed by acitretin. Independent sample t test was used to analyze the different incubation time of acitretin and acitretin combined with ALA-PDT on SCL-1 cells. Bonferroni Test One-way Anova method was used to analyze the effect of different treatment on the SCL-1 cells. RESULTS: A significant cytotoxic effect was observed after acitretin treatment, in an acitretin concentration-dependent manner within the range of 1.6 × 10-4mg/mL to 1.6 × 10-1mg/mL and an acitretin incubation time-dependent manner within 24 h-72 h. The total apoptosis rate and dead cells rate in group of ALA-PDT combined with acitretin were both significantly higher than that of acitretin, ALA-PDT group. A stronger apoptotic and cytotoxic effect detected 24 h after treated with acitretin than that of 12 h was observed in this study. CONCLUSION: Acitretin has a cytotoxic effect on SCL-1 cells, and ALA-PDT treatment enhances the the cytotoxic effect of acitretin.

CRM197-Coupled Der p 2 Peptides Suppress Allergic Airway Inflammation in a Der p 2-Induced Asthma Mouse Model.

Allergic diseases affect more than 25% of the global population. Der p 2 is the major allergen of the house dust mite (HDM) Dermatophagoides pteronyssinus. Allergen-specific immunotherapy is the only treatment to change the course of allergic diseases. In this study, two synthesized Der p 2 peptides coupled to cross-reacting material 197 (CRM197) showed reduced IgE reactivity and allergenic activity. CRM197-coupled Der p 2 peptides induced rDer p 2-specific IgG1 antibodies in mice, which could inhibit HDM-allergic patients' IgE binding to rDer p 2. The immunity effects of CRM197-coupled Der p 2 peptides were studied in an rDer p 2-induced asthma mouse model. CRM197-coupled Der p 2 peptides can suppress asthmatic airway inflammation in this model. Analysis of IL-4, IL-5, and IFN-γ levels in bronchoalveolar lavage fluid revealed that the suppression was associated with a shift from a Th2 to a Th1 response. Thus, CRM197-bound Der p 2 peptides exhibited less allergenic activity than the rDer p 2 allergen, which preserved immunogenicity and may be candidates for mite allergy vaccines.

Susceptibility of Ureaplasma urealyticum to Methylene Blue-Mediated Photodynamic Antimicrobial Chemotherapy: An in vitro Study.

The aim of this study was to detect the susceptibility of Ureaplasma urealyticum to methylene blue-mediated photodynamic antimicrobial chemotherapy (PACT). Three U. urealyticum strains including the standard serotype 1 and 5, and a clinically collected strain were used in this study. Strains were first incubated in 96-well culture plates in the presence of methylene blue with decreasing concentrations (from 1 to 0.015625 mg mL(-1)) for 20 or 60 min, and then submitted to irradiation with a light-emitting diode laser with a power density of 100 mW cm(-2) for 8, 17, 34 or 68 min. Regrowth of the strains was performed soon after irradiation. A significant inactivation effect was observed after PACT. Longer incubation time induced more extensive inactivation of U. urealyticum. No difference in response to PACT was observed between the two biovars of U. urealyticum. It was concluded that PACT had a significant inactivation effect on U. urealyticum, and it might be a promising alternative treatment for resistant U. urealyticum infections.

Single-nucleotide polymorphisms of MAMDC1 are associated with rash and photosensitivity, but not disease risk, of systemic lupus erythematosus in Chinese mainland population.

Systemic lupus erythematosus (SLE) is a complex immune disease affected by both genetic dispositions and environmental factors. Recently, the polymorphisms in MAMDC1 gene have been reported to associate with disease risk of SLE in European population. However, whether this association is replicated in Chinese population is unknown yet. A total of 491 SLE patients and 533 controls were recruited. Unlabeled probe-based high-resolution melting analysis (HRMA) was used in genotyping. HRMA with unlabeled probe successfully distinguished all genotypes. SNP rs961616 was associated with rash [P = 0.015, odds ratio (OR) = 0.73, 95% confidence interval (CI) = 0.57-0.94] and photosensitivity (P = 0.001, OR = 0.63, 95%CI = 0.48-0.84), but not the disease risk (P = 0.133, OR = 0.88, 95%CI = 0.74-1.04), of SLE in Chinese population. Polymorphisms of rs961616 in MAMDC1 gene were associated with rash and photosensitivity, but not disease risk, of systemic lupus erythematosus in Chinese population.

The association between single-nucleotide polymorphisms of NCF2 and systemic lupus erythematosus in Chinese mainland population.

Systemic lupus erythematosus (SLE) is a complex immune disease. The genetic variation in the NCF2 gene was found to associate with SLE in US and European populations. However, the association of rs10911363 with SLE was not extensively studied in Chinese mainland population. A total of 488 SLE patients and 380 controls were recruited. Unlabeled probe-based high-resolution melting analysis (HRMA) was used in genotyping. HRMA with unlabeled probe successfully distinguished all genotypes. Neither genotype nor allele frequencies of single-nucleotide polymorphism (SNP) rs10911363 showed statistically significant differences between SLE patients and controls. The association of SNP rs10911363 with the diagnostic criteria of SLE was also examined. Minor allele (G) of rs10911363 was found to significantly associate with the incidence of arthritis (p = 0.024, odds ratio (OR) = 1.35, and 95% confidence interval (CI) = 1.04-1.75) and increased abnormalities of antinuclear antibody (p = 0.002, OR = 1.51, and 95%CI = 1.17-1.95) and anti-DNA (p = 0.013, OR = 1.40, and 95%CI = 1.07-1.82). Polymorphisms of rs13277113 in NCF2 gene were associated with arthritis and autoantibody production, but not disease risk, of SLE in Chinese population.