Revisiting the human umbilical cord epithelium. An atypical epithelial sheath with distinctive features.

The umbilical cord epithelium (UCE) is the surface tissue that covers the umbilical cord (UC). It is widely considered a single-layered epithelium composed of squamous or cuboidal cells, which are in constant contact with amniotic fluid. The objective of this study was to elucidate the distinctive structural characteristics and abundance of specific proteins in this unique epithelium, many of which have not been previously demonstrated. Samples of the UC were obtained from term pregnancies (n = 12) and processed for examination using stereo, light, electron, and 3D high-resolution confocal microscopy. Sections displayed a range of stratification, ranging from a single squamous layer to 4-5 layers of round/cuboid cells, challenging the notion of considering it as a single-layered structure. Cells are located on a well-developed basement membrane (BM), as evidenced by the expression of BM-specific proteins and PAS staining. The cells possess distinctive cytoplasmic domains that are tightly bound to each other by desmosomes and interdigitating anchoring surfaces. Desquamations and limited organelles suggest that the cells have reached the final stages of differentiation and are no longer actively synthesizing proteins, despite maintaining stratification-specific expression levels of cytoskeletal, junctional, receptor, and stem cell proteins. Although definitive keratinization was not observed, the distribution of proteins and the distinctive structural organization of the single/multi-layered cells suggest that they exhibit plasticity, likely due to adaptive mechanisms in response to chemical and/or mechanical stimuli during fetal development. These structural alterations may facilitate the active transportation of soluble ingredients between the amniotic fluid and cord blood through an intercellular route.

What are the common downstream molecular events between alcoholic and nonalcoholic fatty liver?

Liver fat storage, also called hepatic steatosis, is increasingly common and represents a very frequent diagnosis in the medical field. Excess fat is not without consequences. In fact, hepatic steatosis contributes to the progression toward liver fibrosis. There are two main types of fatty liver disease, alcoholic fatty liver disease (AFLD) and nonalcoholic fatty liver disease (NAFLD). Although AFLD and NAFLD are similar in their initial morphological features, both conditions involve the same evolutive forms. Moreover, there are various common mechanisms underlying both diseases, including alcoholic liver disease and NAFLD, which are commonalities. In this Review, the authors explore similar downstream signaling events involved in the onset and progression of the two entities but not completely different entities, predominantly focusing on the gut microbiome. Downstream molecular events, such as the roles of sirtuins, cytokeratins, adipokines and others, should be considered. Finally, to complete the feature, some new tendencies in the therapeutic approach are presented.

Expression Patterns of Cytokeratins (CK7, CK20, CK19, CK AE1/AE3) in Atypical Endometrial Hyperplasia Coexisting with Endometrial Cancer.

Few studies have evaluated cytokeratin's (CK) staining patterns in atypical endometrial hyperplasia (AEH) coexisting with early-stage endometrial cancer (EC). We aimed to assess the staining patterns of selected CKs (CK7, CK19, CK20, CK AE1/AE3) in 74 patients with coexisting AEH and EC by independently analyzing both morphological variables. Specimens were collected from women with AEH and EC who underwent surgical interventions between 2012 and 2019 at the Department of Obstetrics and Gynecology of Vilnius University Hospital "Santaros Klinikos" in Vilnius, Lithuania. Immunostaining was also qualitatively classified as being heterogeneous or intense. The results revealed heterogeneous CK7 expression in all AEH cases and intense staining in 95.95% cases of AEH. The heterogeneous expression of CK7 was detected in all EC specimens. Intense CK7 expression was observed in 95.09% cases of EC G1 and in all G2 ECs. Heterogenous CK19 expression was present in all AEH specimens with intense staining in 92.42% of cases. Heterogeneous CK19 expression was observed in all EC samples with intense expression in 86.27% cases of EC G1 and 100% cases of EC G2. Interestingly, a significant relationship was found when comparing the heterogeneous expression of CK19 between AEH and well-differentiated EC. A significant difference was reported in the intense expression of CK AE1/AE3 (p = 0.031; p = 0.029) between AEH and G2 ECs and in the intense expression of CK AE1/AE3 between G1 and G2 ECs. CK20 staining was not a characteristic feature for AEH and early-stage EC. CK staining is present either in AEH or in early-stage endometrioid-subtype EC in different manners. Heterogeneous CK19 expression was significantly more common in AEH than in EC. CK20 expression was not associated with either AEH nor early-stage EC. An intense expression of CK AE1/AE3 was mainly present in moderately differentiated ECs, whereas the intense reactivity of AE1/AE3 showed a significant difference in well to moderately differentiated uterine tumors. The clinical implication of CK staining may aid in the more accurate diagnosis of AEH and early-stage EC as well as detect micrometastases leading to better oncological outcomes.

Assessment of Concentration KRT6 Proteins in Tumor and Matching Surgical Margin from Patients with Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) are one of the most frequently detected cancers in the world; not all mechanisms related to the expression of keratin in this type of cancer are known. The aim of this study was to evaluate type II cytokeratins (KRT): KRT6A, KRT6B, and KRT6C protein concentrations in 54 tumor and margin samples of head and neck squamous cell carcinoma (HNSCC). Moreover, we examined a possible association between protein concentration and the clinical and demographic variables. Protein concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Significantly higher KRT6A protein concentration was found in HNSCC samples compared to surgical margins. An inverse relationship was observed for KRT6B and KRT6C proteins. We showed an association between the KRT6C protein level and clinical parameters T and N in tumor and margin samples. When analyzing the effect of smoking and drinking on KRT6A, KRT6B, and KRT6C levels, we demonstrated a statistically significant difference between regular or occasional tobacco and alcohol habits and patients who do not have any tobacco and alcohol habits in tumor and margin samples. Moreover, we found an association between KRT6B and KRT6C concentration and proliferative index Ki-67 and HPV status in tumor samples. Our results showed that concentrations of KRT6s were different in the tumor and the margin samples and varied in relation to clinical and demographic parameters. We add information to the current knowledge about the role of KRT6s isoforms in HNSCC. We speculate that variations in the studied isoforms of the KRT6 protein could be due to the presence and development of the tumor and its microenvironment. It is important to note that the analyses were performed in tumor and surgical margins and can provide more accurate information on the function in normal and cancer cells and regulation in response to various factors.

Comparative Characterization of Human Meibomian Glands, Free Sebaceous Glands, and Hair-Associated Sebaceous Glands Based on Biomarkers, Analysis of Secretion Composition, and Gland Morphology.

Meibomian gland dysfunction (MGD) is one of the main causes of dry eye disease. To better understand the physiological functions of human meibomian glands (MGs), the present study compared MGs with free sebaceous glands (SGs) and hair-associated SGs of humans using morphological, immunohistochemical, and liquid chromatography-mass spectrometry (LCMS)-based lipidomic approaches. Eyelids with MGs, nostrils, lips, and external auditory canals with free SGs, and scalp with hair-associated SGs of body donors were probed with antibodies against cytokeratins (CK) 1, 8, 10, and 14, stem cell markers keratin 15 and N-cadherin, cell-cell contact markers desmoglein 1 (Dsg1), desmocollin 3 (Dsc3), desmoplakin (Dp), plakoglobin (Pg), and E-cadherin, and the tight junction protein claudin 5. In addition, Oil Red O staining (ORO) was performed in cryosections. Secretions of MGs as well as of SGs of nostrils, external auditory canals, and scalps were collected from healthy volunteers, analyzed by LCMS, and the data were processed using various multivariate statistical analysis approaches. Serial sections of MGs, free SGs, and hair-associated SGs were 3D reconstructed and compared. CK1 was expressed differently in hair-associated SGs than in MGs and other free SGs. The expression levels of CK8, CK10, and CK14 in MGs were different from those in hair-associated SGs and other free SGs. KRT15 was expressed differently in hair-associated SGs, whereas N-cadherin was expressed equally in all types of glands. The cell-cell contact markers Dsg1, Dp, Dsc3, Pg, and E-cadherin revealed no differences. ORO staining showed that lipids in MGs were more highly dispersed and had larger lipid droplets than lipids in other free SGs. Hair-associated SGs had a smaller number of lipid droplets. LCMS revealed that the lipid composition of meibum was distinctively different from that of the sebum of the nostrils, external auditory canals, and scalp. The 3D reconstructions of the different glands revealed different morphologies of the SGs compared with MGs which are by far the largest type of glands. In humans, MGs differ in their morphology and secretory composition and show major differences from free and hair-associated SGs. The composition of meibum differs significantly from that of sebum from free SGs and from hair-associated SGs. Therefore, the MG can be considered as a highly specialized type of holocrine gland that exhibits all the histological characteristics of SGs, but is significantly different from them in terms of morphology and lipid composition.

Up-regulation of systemic and local BDNF in non-allergic Nasal Polyps in China.

Objective: Although the role of brain-derived neurotrophic factor (BDNF) in allergic rhinitis and/or nasal polyps (NPs) development has been studied, the contribution of BDNF in non-allergic NPs has not been evaluated yet. This study was to investigate the possible role of BDNF in non-allergic NPs pathogenesis. Methods: The study was carried out at The Second Hospital of Shandong University from December 2020 to November 2021. The non-allergic NPs patients (n=26) and the control group (n=22) were included. Lund-Mackay CT scores, nasal endoscopy scores, and pulmonary function testing were evaluated before surgery. Tissue and serum levels of BDNF, eosinophil cationic protein (ECP), and cytokeratins 5 (CK5) were assessed between different groups. Result: The BDNF level in serum and tissue, CK5 count, and eosinophil infiltration in tissue were higher in non-allergic NPs. The eosinophils infiltration, ECP mRNA expression level, as well as BDNF mRNA level were increased in the BDNFhigh subgroup compared with BDNFlow subgroup. Significantly negative correlations between BDNF count and the situation of airway obstruction were found in non-allergic NPs. Conclusion: BDNF may have both local and systemic effects in non-allergic NPs pathogenesis. BDNF may be a possible therapeutic target or an indicator for eosinophilic NPs management.

Detection of micro-metastasis using cytokeratins (AE1/AE3) in haematoxylin & eosin-stained N0 lymph nodes of oral squamous cell carcinoma.

Background & objectives: Oral squamous cell carcinoma (OSCC) is one of the most common malignancies affecting the head-and-neck region, regional lymph nodes being an important prognostication factor dictating the survival rate. Despite an array of modalities used, clinically, radiographically and routine histopathologically, the detection of micro-metastasis (2-3 mm tumour cell deposits) in the lymph nodes often escapes identification. The presence of few of these tumour epithelial cells in the lymph nodes drastically increases mortality and alters treatment plan. Hence, the identification of these cells is of major prognostic significance for a patient. Thus, the present study was aimed to evaluate and detect the efficacy of the immunohistochemical (IHC) marker [cytokeratin (CK) AE1/AE3] over routine Hematoxylin & eosin (H & E) staining in detecting micro-metastasis in the lymph nodes of OSCC cases. Methods: Hundred H & E-stained N0 lymph nodes of OSCC cases treated with radical neck dissection were subjected to IHC with marker AE1/AE3 antibody cocktail for detecting micro-metastasis. Results: The IHC marker CK cocktail (AE1/AE3) did not demonstrate any positive reactivity for the target antigen in all the 100 H & E stained lymph node sections evaluated in the present study. Interpretation & conclusions: This study was undertaken to check the efficacy of IHC (CK cocktail AE1/AE3) in the detection of micro-metastasis in lymph nodes that are found to be negative in routine H&E stained sections. The findings of this study suggest that the IHC marker AE1/AE3 did not prove to be useful to detect micro-metastasis in this study population.

The potential of organoids in toxicologic pathology: Histopathological and immunohistochemical evaluation of a mouse normal tissue-derived organoid-based carcinogenesis model.

Recently, we introduced an organoid-based chemical carcinogenesis model using mouse normal tissue-derived organoids. In the present review article, the histopathological and immunohistochemical characteristics of mouse normal tissue-derived organoids and tumors derived from these organoids after their in vitro treatment with genotoxic carcinogens and injection into nude mouse are reviewed. In organoids treated in vitro with genotoxic carcinogens, we confirmed macroscopic tumorigenicity and histopathological findings, including neoplastic characteristics, such as multilayered epithelia and/or invasion of epithelia into the surrounding interstitium. In contrast glandular/cystic structures with monolayered epithelia were clearly demarcated from the surrounding Matrigel/interstitium in the untreated control groups. In addition to macroscopic tumorigenicity, these microscopic epithelial changes, which are characteristic of the early stages of carcinogenesis, are included in the requirements for carcinogenicity-positive judgement of the organoid-based carcinogenesis model. Immunohistochemistry of cytokeratins (CKs), used to determine the origin of epithelia and distribution of extraductal invasive lesions, or oncogenic kinases, which reflect molecular activation in epithelia following chemical treatment, is helpful for accurate diagnosis and molecular evaluation in the early stages of carcinogenesis. This information improves our biological understanding of organoid-based chemical carcinogenesis models.

Uncertain histological origin of "glia-like" cells in adult human brain tissue cultures.

OBJECTIVES: Most brain cells studies come from cultured rodent brain tissue, so basic questions about the behaviour of cultured adult human glial cells may remain unanswered. BAGROUND: Cells cultured from adult human brain have been poorly defined until now and are often termed "glia-like" based on some morphological similarities with astrocytes. However, the cells in question fail to express glial markers and may be well be of non-glial origin. METHODS: We examined adult human brain and cultures from 10 patients with non-malignant diagnoses. Immunofluorescence methods were used for glial and non-glial cell type identifications. RESULTS: Confluent cultures contained the following: 0.1 % astrocytes, ≤ 0.01 % oligodendrocytes, 2-5 % microglial and 95-98 % "glia-like" cells. Astrocytes tested as followed: GFAP+/Vim+, microglia: Ferr+Vim+, "glia-like" cells: Vim+/Fn+/CK- or CK+. In the brain tissue, astrocytes were GFAP+/Vim+, microglia Ferr+/Vim-, fibronectin expression was restricted to brain vessels. CONCLUSION: This report demonstrates considerable morphological and cytoskelatal dedifferentiation of cultured brain cells. Cytokeratins, specific markers for epithelial cell differentiation, were absent in the brain tissue. However, they were expressed in "glia-like" cells. This finding could be considered glial dedifferentiation given the ectodermal origin of the brain tissue. We suggest that "glia-like" cells come from currently unknown glial progenitor cells scattered through the brain tissue (Tab. 1, Fig. 4, Ref. 19).

Increased circulating cytokeratin 19 fragment levels in preterm neonates receiving mechanical ventilation are associated with poor outcome.

Invasive mechanical ventilation and oxygen toxicity are postnatal contributors to chronic lung disease of prematurity, also known as bronchopulmonary dysplasia (BPD). Cyfra 21-1 is a soluble fragment of cytokeratin 19, which belongs to the cytoskeleton stabilizing epithelial intermediate filaments. As a biomarker of structural integrity, Cyfra 21-1 might be associated with airway injury and lung hypoplasia in neonates. Serum Cyfra 21-1 concentrations for 80 preterm and 80 healthy term newborns were measured within 48 h after birth. Preterm infants with the combined endpoint BPD/mortality had significantly higher Cyfra 21-1 levels compared with those without fulfilling BPD/mortality criteria (P = 0.01). Also, severe RDS (>grade III) was associated with higher Cyfra levels (P = 0.01). Total duration of oxygen therapy was more than five times longer in neonates with high Cyfra 21-1 levels (P = 0.01). Infants with higher Cyfra 21-1 values were more likely to receive mechanical ventilation (50% vs. 17.5%). However, the duration of mechanical ventilation was similar between groups. The median Cyfra value was 1.93 ng/mL (IQR: 1.68-2.53 ng/mL) in healthy term neonates and 8.5 ng/mL (IQR: 3.6-16.0 ng/mL) in preterm infants. Using ROC analysis, we calculated a Cyfra cutoff > 8.5 ng/mL to predict BPD/death with an AUC of 0.795 (P = 0.004), a sensitivity of 88.9%, and a specificity of 55%. Mortality was predicted with a cutoff > 17.4 ng/mL (AUC: 0.94; P = 0.001), a sensitivity of 100%, and a specificity of 84%. These findings suggest that Cyfra 21-1 concentration might be useful to predict poor outcome in premature infants.

Technical note: double immunohistochemical stain (anti-CD31 and anti-cytokeratins) as a tool for a confident forensic post-mortem diagnosis of amniotic fluid embolism.

Amniotic fluid embolism (AFE) is a rare cause of unexpected late maternal gestational death. The forensic post-mortem diagnosis is rendered upon the histological recognition of fetal "foreign" material inside maternal lung vasculature. The authors propose a double immunohistochemical (anti-CD31 plus anti-cytokeratin AE1/AE3) stain in order to assess accurate amniotic fluid pulmonary embolic burden in a highly reproducible fashion based on the fact that such technique allows to detect an impressive amount of scales within lung vasculature, thereby offering further evidence that pulmonary embolic obstructive microangiopathy, rather than anaphylactoid reaction, is major determinant in AFE-related death.

Cutaneous Spindle Cell Squamous Cell Carcinoma in Cats: Clinical, Histological, and Immunohistochemical Study.

This study describes the clinical and pathological characteristics of cutaneous spindle cell squamous cell carcinoma (SCSCC) in 18 cats. The average age of the cats was 11.8 ± 2.7 years, and all tumors were located in the facial skin, mainly affecting the pinna (13/18, 72%), followed by the periorbital area (4/18, 22%) and the dorsal muzzle (1/18, 6%). Tumors were composed of fusiform neoplastic cells with moderate atypia arranged in solid sheets or fascicles with foci of squamous differentiation. A panel of antibodies against cytokeratins, vimentin, S-100 protein, NSE, GFAP, Melan A, SMA, desmin, CD18, CD31, and p63 was used to help differentiate SCSCC from other spindle cell malignancies. SCSCCs expressed CK5/6 (17/18, 94%), AE1/AE3 (15/18, 83%), and p63 protein (18/18, 100%), but there was no immunolabeling for CK8/18. A role for sunlight exposure in the pathogenesis of the tumors was suggested by changes indicative of actinic keratosis, the location of the tumors in dorsal areas, and the absence of histomorphologic features of papillomavirus infection. Recurrence was not recorded in 14/18 cases (78%) during a follow-up period of 7 to 25 months. Three of 18 (17%) tumors recurred or led to humane euthanasia due to local progression, and one case (5%) had regional lymph node metastasis. Clinical outcome varied with cutaneous location, mitotic count, and invasion of surgical margins; thus, SCSCCs with a more aggressive behavior were located in the periorbital area (4/4 cases), had ≥14 mitoses in 10 high-power fields (2.37 mm2) (4/4 cases), and showed invasion of surgical margins (3/4 cases).

Nonalcoholic Fatty Liver Disease: Focus on New Biomarkers and Lifestyle Interventions.

Nonalcoholic fatty liver disease (NAFLD) is considered a hepatic manifestation of metabolic syndrome, characterized from pathological changes in lipid and carbohydrate metabolism. Its main characteristics are excessive lipid accumulation and oxidative stress, which create a lipotoxic environment in hepatocytes leading to liver injury. Recently, many studies have focused on the identification of the genetic and epigenetic modifications that also contribute to NAFLD pathogenesis and their prognostic implications. The present review is aimed to discuss on cellular and metabolic alterations associated with NAFLD, which can be helpful to identify new noninvasive biomarkers. The identification of accumulated lipids in the cell membranes, as well as circulating cytokeratins and exosomes, provides new insights in understanding of NAFLD. This review also suggests that lifestyle modifications remain the main prevention and/or treatment for NAFLD.

Bile ductular reactions in the liver: similarities are only skin deep.

Extensive bile ductular reactions (DRs) accompany many cholestatic liver diseases such as primary biliary cholangitis and primary sclerosing cholangitis (PSC) as well as parenchymal liver cell diseases such as alcoholic liver disease, non-alcoholic steatohepatitis and HCV and HBV infections. DRs originate from bile ducts or hepatocytes after damage and can be identified by expression of markers associated with cholangiocytes, often being associated with disease progression and fibrosis. In a recent issue of The Journal of Pathology, Govaere et al employed high-throughput RNA sequencing to compare the transcriptomic profiles of DR cells from liver diseases of different aetiology; HCV infection affecting hepatocytes and PSC initially affecting biliary epithelial cells. Both DR transcriptomes were markedly different from that of their neighbouring hepatocytes and 330 genes were significantly differently expressed between the DRs of the HCV and PSC liver diseases. Exploring such gene expression profiles could enable therapeutic targeting of DRs, on the one hand to inhibit liver fibrosis and inflammation and conversely to promote hepatocyte and cholangiocyte regeneration. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gingival Response to Dental Implant: Comparison Study on the Effects of New Nanopored Laser-Treated vs. Traditional Healing Abutments.

The health of peri-implant soft tissues is important for the long-term success rate of dental implants and the surface topography is pivotal in influencing it. Thus, the aim of this study was to evaluate, in human patients, the inflammatory mucosal microenvironment in the tissue surrounding a new, nanoscale, laser-treated healing abutment characterized by engineered nanopores versus a standard machined-surface. Analyses of anti- and pro-inflammatory markers, cytokeratins, desmosomal proteins and scanning electron microscopy were performed in 30 soft-tissue biopsies retrieved during second-stage surgery. The results demonstrate that the soft tissue surrounding the laser-treated surface was characterized by a lower grade of inflammation than the one facing the machined-surface, which, in turn, showed a disrupted epithelium and altered desmosomes. Moreover, higher adhesion of the epithelial cells on the laser-treated surface was detected compared to the machined one. In conclusion, the laser-treated surface topography seems to play an important role not only in cell adhesion, but also on the inflammatory makers' expression of the soft tissue microenvironment. Thus, from a clinical point of view, the use of this kind of topography may be of crucial importance not only on healing abutments but also on prosthetic ones.

Double-positive in triple-negative? How significant is basal cytokeratin expression in breast cancer?

BACKGROUND: Basal-like breast cancer has an unfavorable prognosis. Immunohistochemically, they are predominantly estrogen receptor (ER), progesterone receptor (PR) and CerbB2 receptor (HER2)-negative, show expression of Cytokeratins (CKs) 5/6, CK14, CK 17 and P-cadherin and are associated with germline BRCA1 mutations. Immunohistochemistry (IHC) is an easily available and relatively inexpensive technique that can detect this cancer subtype, and patients can benefit from aggressive management protocols. The aim of this study was to evaluate the expression of CK 5/6 and CK14 in breast cancer and its correlation with age, tumor grade, tumor size, histomorphological pattern, nodal status, ER, PR, HER2/neu, and Ki-67 index. METHODS: Fifty treatment-naїve patients of breast carcinoma who underwent surgery constituted the study group. No core cut biopsy specimens were considered. Histopathological examination along with IHC was performed for CK5/6, CK14, ER, PR, HER2/neu, and Ki-67. Comparison between the expression of CK5/6 and CK14 with age, tumor size, tumor grade, histological subtype, nodal status, ER, PR, HER2/neu, and Ki-67 expression was performed using SPSS 20 version software. RESULTS: Twenty-six percent of cases showed expression of CK5/6 and CK14. CK5/6 and CK14 expression correlated strongly with ER/PR negativity, young age, and Ki-67 proliferative index greater than 15%. No significant association with HER2/neu negativity was demonstrated. Contrasting results were obtained between CK5/6 and CK14 expression with respect to tumor grade and lymph node status. CONCLUSION: IHC can be used to identify patients with basal phenotype breast cancer with good sensitivity and specificity, and such patients can benefit from aggressive management.

Dysplastic urothelial changes accompany empagliflozin administration in urinary bladder of experimental diabetes.

Empagliflozin (EMPA) is a promising novel antidiabetic drug; however, doubts have been raised regarding its use and the increased risk of urinary bladder carcinoma. In this study, we evaluated urothelium expression of cytokeratins (CKs) and Ki-67 proliferative activity in the urinary bladder of diabetic (DM + EMPA) and non-diabetic rats after EMPA administration. By routine histology, dysplastic changes were detected in the urothelium of diabetic as well as non-diabetic animals after EMPA administration. Moreover, the expression of CK-7 and CK-8 was significantly decreased (P < .05) while that of CK-20 as well as Ki-67 was significantly increased (P < .05) in EMPA per se and DM + EMPA urothelium groups compared to that of control and diabetics. The dysplastic changes together with the increased proliferative activity in urothelium after EMPA administration provide a cellular evidence that supports the former clinical concerns.

CD44+ cytokeratin-positive tumor cells in blood and bone marrow are associated with poor prognosis of patients with gastric cancer.

BACKGROUND: The phenotypic heterogeneity of circulating tumor cells (CTC) in peripheral blood and disseminated tumor cells (DTC) in bone marrow is an important constraint for clinical decision making. Here, we investigated the implications of two different subpopulations of these cells in gastric cancer (GC). METHODS: GC patients (n = 228) who underwent elective gastric resections were prospectively examined for CTC/DTC. The cells obtained from peripheral blood and bone marrow aspirates were sorted by flow cytometry and CD45- cells expressing cytokeratins (8, 18, and 19) and CD44 were identified by immunofluorescent double staining. RESULTS: Ninety-three (41%) patients had cytokeratin-positive tumor cells in either blood or bone marrow, while cells expressing CD44 were found in 22 (10%) cases. CK+CD44+ cells were significantly more common among patients with distant metastases (50 vs 19%, P = 0.001), while no such correlations were demonstrated for CK+CD44- cells. Detection of CK+CD44+ cells, but not CK+CD44-, was associated with significantly shortened survival. Moreover, the Cox proportional hazards model identified CK+CD44+ cells as a negative prognostic factor with an odds ratio of 2.38 (95% CI 1.28-4.41, P = 0.006). CONCLUSION: CD44+ phenotype of cytokeratin-positive cells in blood and bone marrow is an independent prognostic factor in patients with gastric cancer.

3D-spheroids: What can they tell us about pancreatic ductal adenocarcinoma cell phenotype?

We aimed at analyzing the effect of the 3D-arrangement on the expression of some genes and proteins which play a key role in pancreatic adenocarcinoma (PDAC) progression in HPAF-II, HPAC and PL45 PDAC cells cultured in either 2D-monolayers or 3D-spheroids. Cytokeratins 7, 8, 18, 19 were differently expressed in 3D-spheroids compared to 2D-monolayers. Syndecan 1 was upregulated in HPAF-II and PL45 3D-spheroids, and downregulated in HPAC. Heparanase mRNA levels were almost unchanged in HPAF-II, and increased in HPAC and PL45 3D-spheroids. Hyaluronan synthase (HAS) 2 and 3 mRNA increased in all 3D-spheroids compared to 2D-monolayers. CD44 and CD44s were expressed to a lower extent in HPAF-II and HPAC 3D-spheroids. By contrast, the CD44s/v3 and the CD44s/v6 ratio increased in HPAC and PL45 3D-spheroids, compared to 2D-monolayers. The expression of MMP-7 was strongly upregulated in 3D-spheroids. STAT3 was similarly expressed 3D-spheroids or 2D-monolayers, while pSTAT3 was almost undetectable in 2D-monolayers and strongly upregulated in 3D-spheroids. These results suggest that 3D-spheroids represent a cell culture model that allows the characterization of PDAC cell phenotype, adding new information that contributes to a better understanding of the biology and behavior of PDAC cells.

Does gene expression in laryngeal subsites differ between patients with laryngopharyngeal reflux and controls?

OBJECTIVE: To identify laryngeal mRNA gene changes in patients with laryngopharyngeal reflux (LPR). METHOD: Laryngeal biopsies from non-smoking LPR patients (n=10; Reflux Symptom Index (RSI) >12 and a Reflux Finding Score (RFS) >6) and controls (n=9; RSI <12 and RFS <6) were collected from four subsites (true vocal cord, false vocal cord, medial arytenoid and posterior commissure) of the larynx. qRT-PCR analyses were conducted on 20 reflux- and inflammation-related genes, including interleukins 6 and 8, cytokeratins 8 and 14, mucin genes MUC1, MUC2, MUC3B, MUC4, MUC5B, MUC6 and MUC7 and carbonic anhydrase III. Statistical analysis (Mann-Whitney U test) compared gene expression levels between LPR and control groups at each subsite. RESULTS: Site-specific differences in squamous metaplasia and gene expression were noted in LPR patients, with the majority present in the medial arytenoid region. Significant.differences were noted in genes related to mucosal defence and inflammation, including CRNN, CD1d, TGFß-1, MUC2, MUC5B and CDH1. CONCLUSION: Whilst the posterior commissure is commonly identified as the area demonstrating the most significant macroscopic change in LPR, the histological changes and genes assessed here showed more pronounced LPR associated differences in the medial arytenoid. We identified differences in expression of mucin genes, cytokeratin-14 and molecular markers of inflammation. Whilst some of these changes may be metaplasia-related, further evaluation of the mRNA expression of these genes may provide a useful biomarker panel for diagnosis and therapeutic monitoring of LPR.