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The human endometrium is unique in that it has a high potential for regeneration after menstruation without scarring. Although growth factors are thought to be responsible for scar formation, it has recently been shown for foetal skin that CD26-negative fibroblasts are essential. Thus, we investigated whether CD26 might be involved in scar formation. Primary human endometrial stromal cells (HPESCs) were stimulated with interleukin-1 alpha (IL1α) to induce CD26 protein expression, and secretion of the scar-associated proteins collagen 1 alpha 1 (COL1A1) and TGF-β3 was measured using ELISAs. The contribution of CD26 to wound closure was analysed using a wound healing assay. The CD26 inhibitor diprotin A (DPA) was used to attenuate CD26 activity. Immunohistochemistry of human uterine samples showed negligible stromal staining of CD26, but CD26 was abundant in the endometrial glands. Treatment of CD26-negative HPESCs with IL1α induced CD26 protein expression, strongly stimulated wound healing in vitro, and increased secretion of COL1A1, but decreased TGF-β3 secretion. DPA effectively attenuated all IL1α-induced effects. We suggest that the stromal non-expression of the scar-associated protein CD26 might contribute to non-scarring during endometrial wound healing.

The immunofluorescence assay is widely used for cellular biology and diagnosis applications. Such an antigen–antibody detection system enables the assessment and visualization of the expression and localization of target proteins. In the classical indirect immunofluorescence assay, secondary antibodies are conjugated to fluorophores. However, conventional secondary antibodies have limited applications due to their large size (150 kDa). Moreover, as animal-derived products, secondary antibodies are associated with ethical concerns and batch-to-batch variability. In this study, we developed fluorescence-labeled recombinant nanobodies as secondary antibodies by utilizing previously established anti–mouse and anti–rabbit IgG secondary nanobodies in combination with the self-labeling SNAP-tag. Nanobodies, which are significantly smaller (15 kDa), are capable to detect primary antibodies produced in mice and rabbits. The SNAP-tag (20 kDa) enables site-specific binding of various O6-benzylguanine (BG)-modified fluorophores to the recombinant nanobodies. These recombinant nanobodies were produced using mammalian cell expression system, and their specific binding to mouse or rabbit antibodies was validated using flow cytometry and multi-color fluorescence microscopy. The low cost, easy of expression, purification and site-specific conjugation procedures for these anti–mouse and anti–rabbit IgG secondary nanobodies make them an attractive alternative to traditional secondary antibodies for indirect immunofluorescence assays.

PurposeClaudins as the major components of tight junctions are important in maintaining cell–cell integrity and thus function as a barrier. Dysregulation of the claudins is often associated with loss of the epithelial phenotype, a process called epithelial–mesenchymal transition (EMT), which most often results in gain of migrative and invasive properties. However, the role of claudins in the endometrium or endometriosis has only rarely been examined.MethodsIn this study, we investigated localization of claudin-2 and claudin-3 in the eutopic and ectopic endometrium with immunohistochemistry. A detailed quantification with HSCORE was performed for claudin-2 and claudin-3 in endometrium without endometriosis and in cases with endometriosis compared to the three endometriotic entities: peritoneal, ovarian, and deep-infiltrating endometriosis.ResultsWe found a preferential localization of both claudins in the glandular and the luminal epithelial cells in the endometrium with and without endometriosis. Quantification of localization of both claudins showed no differences in eutopic endometrium of control cases compared to cases with endometriosis. Furthermore, both claudins are localized highly similar in the ectopic compared to the eutopic endometrium, which is in clear contrast to previously published data for claudin-3.ConclusionFrom our results, we conclude that localization of claudin-2 and claudin-3 is highly stable in eutopic and ectopic endometrium without any loss of the epithelial phenotype and thus do not contribute to the pathogenesis of endometriosis.

Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is limited to detecting a single marker per tissue section. Since immunotherapy has revolutionized the antineoplastic therapy, developing new immunohistochemistry strategies to detect multiple markers simultaneously to better understand tumor environment and predict or assess response to immunotherapy is necessary and urgent. Multiplex immunohistochemistry (mIHC)/multiplex immunofluorescence (mIF), such as multiplex chromogenic IHC and multiplex fluorescent immunohistochemistry (mfIHC), is a new and emerging technology to label multiple biomarkers in a single pathological section. The mfIHC shows a higher performance in cancer immunotherapy. This review summarizes the technologies, which are applied for mfIHC, and discusses how they are employed for immunotherapy research.

The epidermal growth factor receptor (EGFR) family contains four transmembrane tyrosine kinases (EGFR1/ErbB1, Her2/ErbB2, Her3/ErbB3 and Her4/ErbB4) and 13 secreted polypeptide ligands. EGFRs are overexpressed in many solid tumors, including breast, pancreas, head-and-neck, prostate, ovarian, renal, colon, and non-small-cell lung cancer. Such overexpression produces strong stimulation of downstream signaling pathways, which induce cell growth, cell differentiation, cell cycle progression, angiogenesis, cell motility and blocking of apoptosis.The high expression and/or functional activation of EGFRs correlates with the pathogenesis and progression of several cancers, which make them attractive targets for both diagnosis and therapy. Several approaches have been developed to target these receptors and/or the EGFR modulated effects in cancer cells. Most approaches include the development of anti-EGFRs antibodies and/or small-molecule EGFR inhibitors. This review presents the state-of-the-art and future prospects of targeting EGFRs to treat breast cancer.

Triple-negative breast cancer (TNBC) is a clinically and molecularly heterogeneous subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. In this study, tumor specimens from 104 TNBC patients were analyzed to characterize molecular and clinicopathological features and to assess the expression and therapeutic potential of four key surface markers: epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), tissue factor (TF), and trophoblast cell surface antigen (TROP2). Multiplex immunofluorescence (mIF) demonstrated elevated EGFR and TROP2 expression in the majority of samples. Significant positive correlations were observed between EGFR and TF, as well as between TROP2 and both TF and EpCAM. Expression analyses revealed increased EGFR and TF levels with advancing tumor stage, whereas EpCAM expression declined in advanced-stage tumors. TROP2 and TF expression were significantly elevated in higher-grade tumors. Additionally, EGFR and EpCAM levels were significantly higher in patients with elevated Ki-67 indices. Binding specificity assays using single-chain variable fragment (scFv-SNAP) fusion proteins confirmed robust targeting efficacy, particularly for EGFR and TROP2. These findings underscore the therapeutic relevance of EGFR and TROP2 as potential biomarkers and targets in TNBC.

Background Fatty acid synthase (FASN) is crucial to de novo long-chain fatty acid synthesis, needed to meet cancer cells’ increased demands for membrane, energy, and protein production. Methods We investigated FASN overexpression as a therapeutic and chemosensitization target in ovarian cancer tissue, cell lines, and primary cell cultures. FASN expression at mRNA and protein levels was determined by quantitative real-time polymerase chain reaction and immunoblotting and immunohistochemistry, respectively. FASN inhibition’s impact on cell viability, apoptosis, and fatty acid metabolism was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide assay, cell death detection enzyme-linked immunosorbent assay, immunoblotting, and 18  F-fluoromethylcholine uptake measurement, respectively. Results Relative to that in healthy fallopian tube tissue, tumor tissues had 1.8-fold average FASN protein overexpression; cell lines and primary cultures had 11-fold–100-fold mRNA and protein overexpression. In most samples, the FASN inhibitor cerulenin markedly decreased FASN expression and cell viability and induced apoptosis. Unlike concomitant administration, sequential cerulenin/cisplatin treatment reduced cisplatin’s half maximal inhibitory concentration profoundly (up to 54%) in a cisplatin-resistant cell line, suggesting platinum (re)sensitization. Cisplatin-resistant cells displayed lower 18  F-fluoro-methylcholine uptake than did cisplatin-sensitive cells, suggesting that metabolic imaging might help guide therapy. Conclusions FASN inhibition induced apoptosis in chemosensitive and platinum-resistant ovarian cancer cells and may reverse cisplatin resistance.

Anogenital distance (AGD) is regarded as a potential biomarker for endometriosis, and a measurement on MRI images has been found to be promising. This study aimed to evaluate the measurement of AGD on MRI to predict the surgical diagnosis of endometriosis. We included 127 patients who received an MRI for endometriosis between October 2018 and February 2023. AGD was measured on MRI by two readers (MRI-AGD-AC: clitoris to anus; MRI-AGD-AF: posterior fourchette to anus). The feasibility and interobserver reliability of AGD measurements were evaluated. Differences in AGD between patient groups were analyzed. The intraclass correlation coefficient estimates indicated a good to excellent reliability of MRI-AGD-AC (0.92; 95% CI: 0.83–0.95) and a poor to good reliability of MRI-AGD-AF (0.68; 95% CI: 0.27–0.83). No statistically significant differences in the mean MRI-AGD-AC and MRI-AGD-AF in patients with and without surgical diagnosis of DIE ( p  = 0.413; p  = 0.110), peritoneal endometriosis with and without DIE ( p  = 0.641; p  = 0.323), and ovarian endometriosis ( p  = 0.155; p  = 0.150) were found. The AUC ranged from 0.475 (95% CI: 0.365–0.584) to 0.586 (95% CI: 0.454–0.718). Thus, AGD does not constitute a valuable biomarker for patients with clinically suspected endometriosis.

BackgroundPostoperative pain remains a common problem in gynecologic laparoscopy, especially in head zone-related regions, triggered by intra-abdominal pressure during capnoperitoneum. Humidified and prewarmed insufflation gas may ameliorate pain and be beneficial.MethodsThis prospective randomized controlled parallel group multi-arm single-center study investigated the effects of temperature and humidity of insufflation gas on postoperative pain during gynecologic laparoscopy with a duration ≥ 60 min. Female participants (18—70 years) were blinded and randomly assigned—computer generated—to either insufflation with dry cold CO2 with forced air warming blanket (“AIR”), humidified warm gas without forced air warming blanket (“HUMI”), or humidified warm gas with forced air warming blanket (“HUMI +”). We hypothesized that using humidified warm gas resulted in lower pain scores and less analgesic consumption. The primary endpoint postoperative pain was assessed for different pain localizations every 12 h during 7 days after surgery. Secondary endpoints were demand for painkillers and epidural anesthetics, length of stay in recovery room, and hospital stay. (Registration: ClinicalTrials.gov NCT02781194—completed).Results150 participants were randomized. Compared to group “AIR” (n = 48), there was significantly less pain in group “HUMI +” (n = 48) in the recovery room (− 1.068; 95% CI − 2.08 to − 0.061), as well as significantly less ibuprofen use at day two (− 0.5871 g ± 0.258; p-value = 0.0471). Other variables did not change significantly. Stratification for presence of endometriosis or non-previous abdominal surgery in patient history revealed significantly less pain in both groups “HUMI” (n = 50) and “HUMI +” versus group “AIR.” Related side effects were not noted.ConclusionIn the overall population, the use of warm, humidified insufflation gas did not yield clinically relevant effects; however, in predisposed patients with endometriosis and who could otherwise expect high pain levels, warm and humidified gas may be beneficial.

In the last few decades, antibody-based diagnostic and therapeutic applications have been well established in medicine and have revolutionized cancer managements by improving tumor detection and treatment. Antibodies are unique medical elements due to their powerful properties of being able to recognize specific antigens and their therapeutic mechanisms such as blocking specific pathways, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Furthermore, modification techniques have paved the way for improving antibody properties and to develop new classes of antibody-conjugate-based diagnostic and therapeutic agents. These techniques allow arming antibodies with various effector molecules. However, these techniques are utilizing the most frequently used amino acid residues for bioconjugation, such as cysteine and lysine. These bioconjugation approaches generate heterogeneous products with different functional and safety profiles. This is mainly due to the abundance of lysine and cysteine side chains. To overcome these limitations, different site-direct conjugation methods have been applied to arm the antibodies with therapeutic or diagnostics molecules to generate unified antibody conjugates with tailored properties. This review summarizes some of the enzyme-based site-specific conjugation approaches.