Modulation of sHLA-G, PD-1, and PD-L1 Expression in Cervical Lesions Following Imiquimod Treatment and Its Association with Treatment Success.

(1) Background: Cervical intraepithelial neoplasia (CIN) is a precancerous condition linked to human papillomavirus (HPV) infection, often necessitating surgical interventions carrying the risk of subsequent preterm births. This study explores the potential of imiquimod (IMQ), as a non-invasive alternative treatment. The focus is on understanding IMQ impact on immune checkpoint molecules, particularly PD-1, PD-L1, and sHLA-G, which play pivotal roles in shaping immune responses and cancer progression. (2) Methods: Forty-three patients diagnosed with a high-risk squamous intraepithelial lesion (HSIL, p16-positive) self-applied 5% IMQ encapsulated in sachets containing 250 g of cream into the vaginal cavity three times a week for 16 weeks. The impact of IMQ therapy on cervical lesion regression was assessed through immunohistochemistry (IHC), examining changes in sHLA-G, PD-L1, and PD-1 levels. The antiviral activity of IMQ was evaluated through HPV-E7 immunofluorescence. Ethical considerations were adhered to, and the research methods were based on a previously approved clinical trial (clinicaltrials.gov Identifier: NCT04859361). (3) Results: IMQ treatment demonstrated efficacy, leading to lesion regression. sHLA-G levels in CIN before starting IMQ application were associated with unsuccessful treatment (p = 0.0036). IMQ did not significantly alter the expression of PD-1. We observed a decrease in PD-L1 levels in those who were successfully treated (p = 0.0509) and a reduction in HPV burden. (4) Conclusions: IMQ exhibits promise as a non-invasive treatment for CIN, emphasising its potential to modulate the immune microenvironment. Baseline sHLA-G levels emerge as potential predictors of treatment response. Understanding the nuanced dynamics of immune checkpoints sheds light on IMQ mechanism of action. Further exploration is warranted to decipher the intricate mechanisms underlying IMQ treatment in the context of cervical lesions.

Contribution of HLA-G and FOXP3 genes and proteins in the severity of cervical intraepithelial neoplasia during HPV infection.

Human Papillomavirus (HPV) persistence leads to the chronification of cervical inflammation, where HLA-G and Foxp3; immunomodulatory molecules, may contribute to the aggravation of the lesion and cancerization. Here, we evaluated the synergic effect of these two molecules in the worsening of the lesion in presence of HPV infection. Hundred and eighty (180) women cervical cells and biopsies were collected for (i) HLAG Sanger sequencing and gene expression, and (ii) HLA-G and Foxp3 molecule expressions by immunohistochemistry. 53 women were HPV+ against 127 women HPV-. HPV+ women were more at risk of having cytological changes (p ≤ 0.0123), histological changes (p < 0.0011), and cervical lesion (p = 0.0004). The HLA-G + 3142CC genotype predisposed women to infection (p = 0.0190), while HLA-G + 3142C and +3035 T alleles were associated with HLA-G5 transcript expression. Both sHLA-G (p = 0.030) and Foxp3 (p = 0.0002) proteins were higher in cervical lesion as well as in high-grade lesion. In addition, sHLA-G+ cells were positively correlated to Foxp3+ cells in presence of HPV infection and in cervical grade II/III injuries. In conclusion, HPV may use HLA-G and Foxp3 as a way of host immune escape contributing to the persistence of infection and inflammation, leading to the cervical lesion and the worsening of lesions.

The antigen processing-associated transporter gene polymorphism: Role on gene and protein expression in HPV-infected pre-cancerous cervical lesion.

Human papillomavirus (HPV) is the major pathogen for cervical lesions. The evasion mechanism of the immune response and persistence of HPV infection can be influenced by polymorphisms (SNPs) in genes associated with transporter associated with antigen processing (TAP), which may change the peptide binding affinity or the TAP expression impacting the efficiency of peptide transport in the secretory pathway, and the presentation of peptides to cytotoxic T lymphocytes. This study aimed to evaluate the role of the TAP1 and TAP2 polymorphisms, TAP1, and TAP2 genes expressions, and protein levels in cervical cells presenting different degrees of pre-cancerous lesions in 296 immunocompetent women infected or not by HPV. TAP SNPs were genotyped by Sanger sequencing, and gene expression by real-time PCR. Aneuploidy was determined by DNA index using flow cytometry. TAP-1 and TAP-2 tissue expressions were evaluated by immunohistochemistry. The Asp697Gly SNP of TAP1 presented a risk for cellular aneuploidy (P=0.0244). HPV+ women had higher TAP-2 mRNA (P=0.0212) and protein (P<0.0001) levels. The TAP2D and TAP2E haplotypes were associated with the risk for aneuploidy and pre-cancerous lesions. In conclusion, nucleotide variability at the peptide binding region of peptide transporter genes, particularly of the TAP2 gene, may influence the HPV-peptide transportation from the cytosol to the endoplasmic reticulum, increasing the susceptibility to the development of high-grade cervical lesions.

Hierarchical evaluation of histology and p16-labeling can improve the risk assessment on cervical intraepithelial neoplasia progression.

OBJECTIVE: High-grade cervical lesions (HSIL) are associated with the presence of high-risk HPV types, tissue expression of p16, and increased chance of malignant progression, requiring surgical intervention. To improve risk evaluation, we assessed the discriminatory power of the histological findings associated with p16 immunohistochemistry (IHC) staining to classify the low-grade cervical lesion (LSIL) and HSIL. METHODS: We collected cervical biopsies from colposcopy-visible lesions and non-affected tissue (adjacent to the lesions) of 62 Brazilian women and labeled them with anti-p16 antibodies. In addition to the observational pattern and labeling to define the latent classes (affected vs. non-affected), a computational tool was used for semi-quantitative analysis of p16 expression. The intensity of staining of the nucleus or cytoplasm was captured using the Gimp 2.10 software. ROC curves were used to determine cutoff values for p16 expression in patients classified as LSIL and HSIL by latent class statistics for each labeling stratum. RESULTS: p16 nuclear labeling showed the best sensitivity and specificity to discriminate LSIL with low p16 expression (62%) and HSIL with high p16 expression (37%). Many patients whose lesions had intermediate levels of p16 nuclear staining were subsequently stratified according to the expression of p16 in the cytoplasm, indicating that five of 21 LSIL were at risk of progression, and 13 of 41 HSIL at risk of regression. CONCLUSIONS: We suggest a hierarchical analysis, with histology at the first level, followed by a labeling analysis in the nucleus and then in the cytoplasm to increase the accuracy of the HPV cervical lesion stratification.

Intraurethral injection with LPS: an effective experimental model of prostatic inflammation.

OBJECTIVE: Chronic inflammation has been recognized as having a prominent role pathogenesis of benign prostatic hyperplasia (BPH) and cancer. It is believed that chronic inflammation induces prostatic fibromuscular growth. This correlation has been clearly illustrated by both in vivo and in vitro studies; however, current experimental models of BPH require complex surgery or hormonal treatment. Therefore, the aim of the present study was to propose a new murine model of BPH/prostatitis induced by intraurethral injection of LPS. METHODS: Male Swiss and C57Bl/6 mice were then sacrificed 3, 7, 10, and 14 days after intraurethral injection of LPS. The prostates were quickly dissected and fixed for morphological and immunohistochemical analyses. RESULTS: The results showed that LPS played an important role in the cell proliferation of the prostate. Histological and ultrastructural analysis showed epithelial hyperplasia, clear stromal cells, little inflammatory infiltration, and heavy bleeding. Treatment with LPS also promoted the increase of growth factor (FGF-7 and TGF-ß), α-actin, and proinflammatory cytokines (IL-1, IL-6, IL-17), both in the stroma and epithelium. CONCLUSION: According to the present findings, it can be concluded that the intraurethral administration of LPS promotes tissue remodeling, as well as stimulating the pattern of pro-inflammatory cytokines, and therefore, constitutes an effective experimental model of BPH/inflammation.

Chronic treatment with Sildenafil has no effect on folliculogenesis or fertility in C57BL/6 and C57BL/6 knockout for iNOS mice.

Sildenafil is an important phosphodiesterase inhibitor used to treat a range of diseases, including cardiovascular disease, prostatic hyperplasia and pulmonary hypertension. Its main mechanism of action is the inhibition of phosphodiesterase 5, leading to increased intracellular cyclic guanosine 3',5'-monophosphate. This second messenger plays an interesting role in the reproductive tract. The aim of the present study was to evaluate the effect of Sildenafil on folliculogenesis and fertility in mice. To do so, C57BL/6 wild-type mice and inducible nitric oxide synthase knockout (iNOS(-/-)) mice were treated with Sildenafil, and reproductive variables were evaluated. The treated and control animals underwent estrous cycle and fertility assay. Lipid profile, serum nitric oxide levels and the expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and guanylate cyclase were evaluated. Additionally, ovaries were submitted to histological and morphological analysis. The findings demonstrated that chronic treatment with Sildenafil had no effect on folliculogenesis or fertility in C57BL/6 mice, suggesting that this drug can be safely used by women of childbearing age.

LPSF/GQ-02 inhibits the development of hepatic steatosis and inflammation in a mouse model of non-alcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD) defines a wide spectrum of liver diseases that extends from simple steatosis to non-alcoholic steatohepatitis. Although the pathogenesis of NAFLD remains undefined, it is recognized that insulin resistance is present in almost all patients who develop this disease. Thiazolidinediones (TZDs) act as an insulin sensitizer and have been used in the treatment of patients with type 2 diabetes and other insulin-resistant conditions, including NAFLD. Hence, therapy of NAFLD with insulin-sensitizing drugs should ideally improve the key hepatic histological changes, while also reducing cardiometabolic and cancer risks. Controversially, TZDs are associated with the development of cardiovascular events and liver problems. Therefore, there is a need for the development of new therapeutic strategies to improve liver function in patients with chronic liver diseases. The aim of the present study was to assess the therapeutic effects of LPSF/GQ-02 on the liver of LDLR-/- mice after a high-fat diet. Eighty male mice were divided into 4 groups and two different experiments: 1-received a standard diet; 2-fed with a high-fat diet (HFD); 3-HFD+pioglitazone; 4-HFD+LPSF/GQ-02. The experiments were conducted for 10 or 12 weeks and in the last two or four weeks respectively, the drugs were administered daily by gavage. The results obtained with an NAFLD murine model indicated that LPSF/GQ-02 was effective in improving the hepatic architecture, decreasing fat accumulation, reducing the amount of collagen, decreasing inflammation by reducing IL-6, iNOS, COX-2 and F4 / 80, and increasing the protein expression of IκBα, cytoplasmic NFκB-65, eNOS and IRS-1 in mice LDLR -/-. These results suggest a direct action by LPSF/GQ-02 on the factors that affect inflammation, insulin resistance and fat accumulation in the liver of these animals. Further studies are being conducted in our laboratory to investigate the possible mechanism of action of LPSF/GQ-02 on hepatic lipid metabolism.

Effect of new thiazolidine derivatives LPSF/GQ-02 and LPSF/GQ-16 on atherosclerotic lesions in LDL receptor-deficient mice (LDLR(-/-)).

BACKGROUND: Atherosclerotic cardiovascular disease is a chronic inflammatory condition. Thiazolidinediones (TZDs) are used to enhance sensitivity to insulin and have demonstrated a protective effect over a variety of cardiovascular markers and risk factors. Controversially, the TZDs are associated with the development of heart failure. Thus, lines of research have invested in the search for new molecules in order to obtain more selective and less harmful treatment alternatives for the pathogenesis of atherosclerosis and its risk factors. METHODS: Animals were fed a diet rich in fat for 10 weeks. In the last 2 weeks, animals received either pioglitazone, LPSF/GQ-02, or LPSF/GQ-16 daily through gavage. At the end of the treatment, blood was collected for biochemical analysis and the aortas were dissected for subsequent analyses. RESULTS: No changes in the blood lipid profile were found following the use of the drugs in comparison to the control. However, the new thiazolidine derivatives were more efficient in improving insulin resistance in comparison to pioglitazone and the control group. Morphometric analyses revealed that neither pioglitazone nor LPSF/GQ16 led to satisfactory effects over atherosclerosis. However, LPSF/GQ-02 led to a reduction in area of the atherosclerotic lesions. Ultrastructural analyses revealed extensive degeneration of the endothelium and an increase in apoptotic cells in the subendothelial space following the use of pioglitazone and LPSF/GQ-16. However, LPSF/GQ-02 caused minimal cell alterations in the aortic endothelium. Regarding markers, endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase 9 (MMP-9), LPSF/GQ-16, and pioglitazone exerted similar effects, increasing the expression of MMP-9, and had no effect on the expression of eNOS compared with the control group. On the other hand, LPSF/GQ-02 was effective in reducing the expression of MMP-9 and increased eNOS significantly. CONCLUSIONS: The results suggest that the new thiazolidine derivative LPSF/GQ-02 is a promising candidate for the treatment of atherosclerosis.

New imidazolidine derivatives as anti-Trypanosoma cruzi agents: structure-activity relationships.

Imidazolidine derivatives are key components for the development of bioactive compounds for the treatment of many diseases, especially Chagas. In fact, others studies showed that the imidazolidine-2,4-dione has stood out by presenting a wide spectrum of pharmacological activities including anticonvulsants, antiarrhythmic, and antiparasitic. In the present study, we investigated the morphological alterations induced by imidazolidine derivates LPSF/NN-52 and LPSF/NN-100 on trypomastigotes forms of Trypanosoma cruzi through ultrastructural analysis by electron microscopy. Many concentrations were used to measure the antiparasitic propriety promoted by imidazolidine derivatives, and our study indicates that parasites treated with 13 µg mL(-1) of the imidazolidine derivates for 24 h revealed severe damage to the parasite's mitochondrial complex. Beyond that, also observed in treated parasites were the following: myelin bodies, enlargement of cytoplasm vacuole, fragmentation of endoplasmic reticulum, and some treated samples clearly showed signs of necrosis. To confirm the ultrastructural results, some assays were performed for knowledge cellular death induction promoted by imidazolidine derivates against immune spleen cells. The induction of the necrotic process through derivatives LPSF/NN-52 and LPSF/NN-100 showed similar results in relation to nifurtimox and benznidazole. In the last assays, it was demonstrated that NN-100 was efficient against epimastigotes and trypomastigotes forms and these results reinforce the mechanisms of action of both new imidazolidine derivatives against T. cruzi.