Loss of CFTR Reverses Senescence Hallmarks in SARS-CoV-2 Infected Bronchial Epithelial Cells.

SARS-CoV-2 infection has been recently shown to induce cellular senescence in vivo. A senescence-like phenotype has been reported in cystic fibrosis (CF) cellular models. Since the previously published data highlighted a low impact of SARS-CoV-2 on CFTR-defective cells, here we aimed to investigate the senescence hallmarks in SARS-CoV-2 infection in the context of a loss of CFTR expression/function. We infected WT and CFTR KO 16HBE14o-cells with SARS-CoV-2 and analyzed both the p21 and Ki67 expression using immunohistochemistry and viral and p21 gene expression using real-time PCR. Prior to SARS-CoV-2 infection, CFTR KO cells displayed a higher p21 and lower Ki67 expression than WT cells. We detected lipid accumulation in CFTR KO cells, identified as lipolysosomes and residual bodies at the subcellular/ultrastructure level. After SARS-CoV-2 infection, the situation reversed, with low p21 and high Ki67 expression, as well as reduced viral gene expression in CFTR KO cells. Thus, the activation of cellular senescence pathways in CFTR-defective cells was reversed by SARS-CoV-2 infection while they were activated in CFTR WT cells. These data uncover a different response of CF and non-CF bronchial epithelial cell models to SARS-CoV-2 infection and contribute to uncovering the molecular mechanisms behind the reduced clinical impact of COVID-19 in CF patients.

Ultrastructural Characterization of Human Bronchial Epithelial Cells during SARS-CoV-2 Infection: Morphological Comparison of Wild-Type and CFTR-Modified Cells.

SARS-CoV-2 replicates in host cell cytoplasm. People with cystic fibrosis, considered at risk of developing severe symptoms of COVID-19, instead, tend to show mild symptoms. We, thus, analyzed at the ultrastructural level the morphological effects of SARS-CoV-2 infection on wild-type (WT) and F508del (ΔF) CFTR-expressing CFBE41o- cells at early and late time points post infection. We also investigated ACE2 expression through immune-electron microscopy. At early times of infection, WT cells exhibited double-membrane vesicles, representing typical replicative structures, with granular and vesicular content, while at late time points, they contained vesicles with viral particles. ∆F cells exhibited double-membrane vesicles with an irregular shape and degenerative changes and at late time of infection, showed vesicles containing viruses lacking a regular structure and a well-organized distribution. ACE2 was expressed at the plasma membrane and present in the cytoplasm only at early times in WT, while it persisted even at late times of infection in ΔF cells. The autophagosome content also differed between the cells: in WT cells, it comprised vesicles associated with virus-containing structures, while in ΔF cells, it comprised ingested material for lysosomal digestion. Our data suggest that CFTR-modified cells infected with SARS-CoV-2 have impaired organization of normo-conformed replicative structures.

Immunolocalization of leptin and leptin receptor in colorectal mucosa of ulcerative colitis, Crohn's disease and control subjects with no inflammatory bowel disease.

The expression of leptin and leptin receptor (Ob-R) has been partially elucidated in colon of patients with inflammatory bowel diseases (IBDs), even though leptin is involved in angiogenesis and inflammation. We previously reported overexpression of GLUT5 fructose transporter, in aberrant clusters of lymphatic vessels in lamina propria of IBD and controls. Here, we examine leptin and Ob-R expression in the same biopsies. Specimens were obtained from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls who underwent screening for colorectal cancer, follow-up after polypectomy or with a history of lower gastrointestinal symptoms. Immunohistochemistry revealed leptin in apical and basolateral membranes of short epithelial portions, Ob-R on the apical pole of epithelial cells. Leptin and Ob-R were also identified in structures and cells scattered in the lamina propria. In UC, a significant correlation between leptin and Ob-R in the lamina propria was found in all inflamed samples, beyond non-inflamed samples of the proximal tract, while in CD, it was found in inflamed distal samples. Most of the leptin and Ob-R positive areas in the lamina propria were also GLUT5 immunoreactive in inflamed and non-inflamed mucosa. A significant correlation of leptin or Ob-R expression with GLUT5 was observed in the inflamed distal samples from UC. Our findings suggest that there are different sites of leptin and Ob-R expression in large intestine and those in lamina propria do not reflect the status of mucosal inflammation. The co-localization of leptin and/or Ob-R with GLUT5 may indicate concomitance effects in colorectal lamina propria areas.

Oxygenator Is the Main Responsible for Leukocyte Activation in Experimental Model of Extracorporeal Circulation: A Cautionary Tale.

In order to assess mechanisms underlying inflammatory activation during extracorporeal circulation (ECC), several small animal models of ECC have been proposed recently. The majority of them are based on home-made, nonstandardized, and hardly reproducible oxygenators. The present study has generated fundamental information on the role of oxygenator of ECC in activating inflammatory signaling pathways on leukocytes, leading to systemic inflammatory response, and organ dysfunction. The present results suggest that experimental animal models of ECC used in translational research on inflammatory response should be based on standardized, reproducible oxygenators with clinical characteristics.

Chronic overcirculation-induced pulmonary arterial hypertension in aorto-caval shunt.

Pulmonary arterial hypertension is a common complication of congenital heart defects with left-to-right shunts. Current preclinical models do not reproduce clinical characteristics of shunt-related pulmonary hypertension. Aorto-caval shunt was firstly described as a model of right ventricle volume overload. The pathophysiology and the possible determination of pulmonary arterial hypertension of different periods of shunt exposure are still undefined. A method to create standardized, reproducible aorto-caval shunt was developed in growing rats (260±40 g). Three groups of animals were considered: shunt exposure for 10 weeks, shunt exposure for 20 weeks and control (sham laparotomy). Echocardiography and magnetic resonance revealed increased right ventricular end diastolic area in shunt at 10 weeks compared to control. Hemodynamic analysis demonstrated increased right ventricular afterload and increased effective pulmonary arterial elastance (Ea) in shunt at 20 weeks compared to control (1.29±0.20 vs. 0.14±0.06 mmHg/µl, p=0.004). At the same time point, the maximal slope of end-systolic pressure-volume relationship (Ees) decreased (0.5±0.2 mmHg/ml vs. 1.2±0.3, p<0.001). Consequently, right ventricular-arterial coupling was markedly deteriorated with a ≈50% decrease in the ratio of end-systolic to pulmonary artery elastance (Ees/Ea). Finally, left ventricular preload diminished (≈30% decrease in left ventricular end-diastolic volume). Histology demonstrated medial hypertrophy and small artery luminal narrowing. Chronic exposure to aorto-caval shunt is a reliable model to produce right ventricular volume overload and secondary pulmonary arterial hypertension. This model could be an alternative with low mortality and high reproducibility for investigators on the underlying mechanisms of shunt-related pulmonary hypertension.

Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter's syndrome.

Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

Glucose transporter/T1R3-expressing cells in rat tracheal epithelium.

Glucose transport plays an important role in maintaining low sugar concentration in airway surface liquid (ASL), which is critical for mucociliary clearance and bacterial colonization. Experimental evidence indicates that glucose/hexose uptake in lung/airway cells occurs by means of two structurally distinct glucose transporter pathways: the Na(+) -dependent glucose transporters (SGLT family) and the facilitative glucose transporters (GLUT family). In this study, we examined the expression of the major glucose transporters of the intestine, GLUT2, GLUT5, SGLT1 and T1R3 taste receptor subunit, in the trachea of rats using immunohistochemistry and immunoelectron microscopy, and compared them using double-labeled confocal microscopy. We found that GLUT2, GLUT5, SGLT1 and T1R3 are selectively expressed in different cell types. T1R3 and GLUT2 are predominantly expressed in subsets of solitary chemoreceptor cells (SCCs) and ciliated cells, GLUT5 is present in subsets of SCCs and in secretory cells, and SGLT1 is exclusively expressed in a unique cell type, SCCs. Furthermore, we demonstrated that T1R3 is colocalized with SGLT1 in SCCs and with GLUT2 transporter in ciliated cells. In conclusion, these findings reveal that different cell types are associated with the uptake of glucose in ASL and that, due to their T1R3 expression, SCCs and ciliated cells are most likely to participate in the chemosensory process in ASL.

CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells.

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19.

The transcriptional profile of adipose-derived stromal cells (ASC) mirrors the whitening of adipose tissue with age.

Multipotent stem cells persist within the stromal vascular fraction (SVF) of adipose tissue during adulthood. These cells, commonly referred to as adipose-derived stromal cells (ASC), have been extensively investigated over the past years as a promising therapeutic tool based on their regenerative and immunomodulatory properties. However, how ASC might mirror the age-related alteration of the fat they reside in remains unclear. Herein, we show that inguinal adipose tissue in mice turns from brown/beige- to white-like with age and resident ASC readily mirror these changes both at mRNA and microRNA transcriptional level. Mechanistically, our data suggest that these brown/age-related changes in ASC transcription rely on changes in the activity of E2F1 and NFkB transcription factors.

Glucose transporters are expressed in taste receptor cells.

In the intestine, changes of sugar concentration generated in the lumen during digestion induce adaptive responses of glucose transporters in the epithelium. A close matching between the intestinal expression of glucose transporters and the composition and amount of the diet has been provided by several experiments. Functional evidence has demonstrated that the regulation of glucose transporters into enterocytes is induced by the sensing of sugar of the enteroendocrine cells through activation of sweet taste receptors (T1R2 and T1R3) and their associated elements of G-protein-linked signaling pathways (e.g. α-gustducin, phospholipase C ß type 2 and transient receptor potential channel M5), which are signaling molecules also involved in the perception of sweet substances in the taste receptor cells (TRCs) of the tongue. Considering this phenotypical similarity between the intestinal cells and TRCs, we evaluated whether the TRCs themselves possess proteins of the glucose transport mechanism. Therefore, we investigated the expression of the typical intestinal glucose transporters (i.e. GLUT2, GLUT5 and SGLT1) in rat circumvallate papillae, using immunohistochemistry, double-labeling immunofluorescence, immunoelectron microscopy and reverse transcriptase-polymerase chain reaction analysis. The results showed that GLUT2, GLUT5 and SGLT1 are expressed in TRCs; their immunoreactivity was also observed in cells that displayed staining for α-gustducin and T1R3 receptor. The immunoelectron microscopic results confirmed that GLUT2, GLUT5 and SGLT1 were predominantly expressed in cells with ultrastructural characteristics of chemoreceptor cells. The presence of glucose transporters in TRCs adds a further link between chemosensory information and cellular responses to sweet stimuli that may have important roles in glucose homeostasis, contributing to a better understanding of the pathways implicated in glucose metabolism.

Early versus late GD-DTPA MRI enhancement in experimental glioblastomas.

PURPOSE: To compare early versus late enhancement in two glioblastoma models characterized by different infiltrative/edematous patterns. MATERIALS AND METHODS: Three weeks after inoculation into nude mice of U87MG and U251 cells, T1-weighted images were acquired early (10.5 min), intermediate (21 min) and late (30.5 min) after a bolus injection of Gd-DTPA at 300 µ mol/kg dosage. EARLY(TH) and LATE(TH) were the corresponding volumes with an enhancement higher than a threshold TH, defined by the mean (µ) and standard deviation (σ) on a contralateral healthy area. ADD(TH) was the enhancing volume found in LATE(TH) but not in EARLY(TH). T2 imaging of both tumors was performed, and T2 mapping of U251. RESULTS: In all tumors, LATE(TH) was significantly higher than EARLY(TH) for TH ranging from µ+σ to µ+5σ. The ADD(TH) /EARLY(TH) ratio was not significantly different when U251 and U87MG tumors were compared. In the U87MG tumors, some enhancement was observed outside the regularly demarcated T2-hyperintense area. In the U251 tumors, irregularly T2 demarcated, a large portion of ADD(µ+3σ) had normal T2 values. At histology, U251 showed a higher infiltrative pattern than U87MG. CONCLUSION: In these models, the increase over time in the enhancing volume did not depend on the different infiltrative/edematous patterns and was not closely related with edema.

MRI characterization of rat brain aging at structural and functional level: Clues for translational applications.

Magnetic resonance imaging (MRI) paradigms, using non-invasive approaches, can provide relevant findings about brain aging. The attention has been primarily focused on neurodegenerative diseases, while little or nothing has been done to differentiate physiology from pathology. The present study aimed to test diffusion tensor imaging (DTI) and functional MRI (fMRI) metrics to analyze physiological age-related changes in rats at myelin structure and activation level; findings were validated by ex vivo histology. The purpose is to find comparable biomarkers in rodents and humans to allow a reliable translation from pre-clinical to clinical settings. Data evidenced: i) a significantly higher cerebrospinal fluid volume in middle-aged and aged vs. young rats; ii) a progressive alteration of white matter; iii) a significant reduction of evoked activity in aged animals. These results partially mirror the age-related changes in humans and may represent a preliminary step to find reliable tools for a lifelong monitoring with a value for the clinical practice (e.g., to provide support to the early diagnosis of dementia in asymptomatic subjects).

Ultrastructural Analysis of Rehydrated Human Donor Corneas After Air-Drying and Dissection by Femtosecond Laser.

Purpose: To evaluate the efficiency of femtosecond laser (FSL) incision of rehydrated human donor corneas after air-drying and its effects on corneal structure. Methods: We compared the rehydrated and fresh-preserved corneas by microscopy following Victus-Tecnolas FSL treatment for straight-edge anterior lamellar keratoplasty (ALK). The corneas were dehydrated at room temperature under a laminar-flow hood. Results: To obtain the horizontal cut in rehydrated corneas, we increased the FSL pulse energy to 1.2 µJ from 0.80 µJ applied for the fresh corneas and obtained a clear-cut separation of the lamellar lenticule cap from the corneal bed. Light microscopy showed regular arrangement of stromal collagen lamellae, with spaces in between the fibers in the corneal stroma in the fresh and the rehydrated corneas, but the uppermost epithelial layers in the rehydrated corneas were lost. Transmission electron microscopy (TEM) revealed no signs of thermal or mechanical damage to the corneal structure. The epithelial basal membrane and Bowman's layer maintained their integrity. The epithelial basal layer and cells were separated by large spaces due to junction alteration in the rehydrated corneas. There were gaps between the lamellar layers in the stroma, especially in the rehydrated corneas. Keratocytes displayed normal structure in the fresh corneas but were devoid of microorganules in the rehydrated corneas. Minor irregularities were observed in the vertical incision and the horizontal stroma appeared smooth on scanning electron microscopy. Conclusion: The corneal stroma of rehydrated corneas maintained morphology and integrity, while corneal cellular components were generally altered. When corneas are intended for FSL-assisted ALK, effective stromal bed incision is best achieved at a laser power higher than that currently adopted for fresh corneas.

Hippocampal FGF-2 and BDNF overexpression attenuates epileptogenesis-associated neuroinflammation and reduces spontaneous recurrent seizures.

Under certain experimental conditions, neurotrophic factors may reduce epileptogenesis. We have previously reported that local, intrahippocampal supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) increases neurogenesis, reduces neuronal loss, and reduces the occurrence of spontaneous seizures in a model of damage-associated epilepsy. Here, we asked if these possibly anti-epileptogenic effects might involve anti-inflammatory mechanisms. Thus, we used a Herpes-based vector to supplement FGF-2 and BDNF in rat hippocampus after pilocarpine-induced status epilepticus that established an epileptogenic lesion. This model causes intense neuroinflammation, especially in the phase that precedes the occurrence of spontaneous seizures. The supplementation of FGF-2 and BDNF attenuated various parameters of inflammation, including astrocytosis, microcytosis and IL-1ß expression. The effect appeared to be most prominent on IL-1ß, whose expression was almost completely prevented. Further studies will be needed to elucidate the molecular mechanism(s) for these effects, and for that on IL-1ß in particular. Nonetheless, the concept that neurotrophic factors affect neuroinflammation in vivo may be highly relevant for the understanding of the epileptogenic process.

Artificial dermal substitutes for tissue regeneration: comparison of the clinical outcomes and histological findings of two templates.

OBJECTIVE: Artificial dermal substitutes (DSs) are fundamental in physiological wound healing to ensure consistent and enduring wound closure and provide a suitable scaffold to repair tissue. We compared the clinical and histological features of two DSs, Pelnac and Integra, in the treatment of traumatic and iatrogenic skin defects. METHODS: This prospective observational study involved 71 randomly selected patients from our hospital. Wound healing was analyzed using the Wound Surface Area Assessment, the Vancouver Scar Scale, and a visual analog scale. Histological and immunohistochemical evaluations were also performed. RESULTS: At 2 weeks, greater regeneration with respect to proliferation of the epidermis and renewal of the dermis was observed with Pelnac than with Integra. At 4 weeks, the dermis had regenerated with both DSs. Both templates induced renewed collagen and revascularization. Differences in the Vancouver Scar Scale score were statistically significant at 4 weeks and 1 year. Pelnac produced a significant increase in contraction at 2 weeks with increasing effectiveness at 4 weeks. Integra produced a higher percentage reduction in the wound surface area and a shorter healing time than Pelnac for wounds >1.5 cm deep. CONCLUSION: Our observational data indicate that both DSs are effective and applicable in different clinical contexts.

Cocoa Flavonoids Reduce Inflammation and Oxidative Stress in a Myocardial Ischemia-Reperfusion Experimental Model.

: Consumption of flavonoid-rich nutraceuticals has been associated with a reduction in coronary events. The present study analyzed the effects of cocoa flavonols on myocardial injury following acute coronary ischemia-reperfusion (I/R). A commercially available cocoa extract was identified by chromatographic mass spectrometry. Nineteen different phenolic compounds were identified and 250 mg of flavan-3-ols (procyanidin) were isolated in 1 g of extract. Oral administration of cocoa extract in incremental doses from 5 mg/kg up to 25 mg/kg daily for 15 days in Sprague Dawley rats (n = 30) produced a corresponding increase of blood serum polyphenols and become constant after 15 mg/kg. Consequently, the selected dose (15 mg/kg) of cocoa extract was administered orally daily for 15 days in a treated group (n = 10) and an untreated group served as control (n = 10). Both groups underwent surgical occlusion of the left anterior descending coronary artery and reperfusion. Cocoa extract treatment significantly reversed membrane peroxidation, nitro-oxidative stress, and decreased inflammatory markers (IL-6 and NF-kB) caused by myocardial I/R injury and enhanced activation of both p-Akt and p-Erk1/2. Daily administration of cocoa extract in rats is protective against myocardial I/R injury and attenuate nitro-oxidative stress, inflammation, and mitigates myocardial apoptosis.

Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.

OBJECTIVES: Among the factors that could determine neurological outcome after hypothermic circulatory arrest (HCA) rewarming is rarely considered. The optimal rewarming rate is still unknown. The goal of this study was to investigate the effects of 2 different protocols for rewarming after HCA on neurological outcome in an experimental animal model. METHODS: Forty-four Sprague Dawley rats were cooled to 19 ± 1°C body core temperature by cardiopulmonary bypass (CPB). HCA was maintained for 60 min. Animals were randomized to receive slow (90 min) or fast (45 min) assisted rewarming with CPB to a target temperature of 35°C. After a total of 90 min of reperfusion in both groups, brain samples were collected and analysed immunohistochemically and with immunofluorescence. In 10 rats, magnetic resonance imaging was performed after 2 and after 24 h to investigate cerebral perfusion and cerebral oedema. RESULTS: Interleukin 6, chemokine (C-C motif) ligand 5, intercellular adhesion molecule 1 and tumour necrosis factor α in the hippocampus are significantly less expressed in the slow rewarming group, and microglia cells are significantly less activated in the slow rewarming group. Magnetic resonance imaging analysis demonstrated better cerebral perfusion and less water content in brains that underwent slow rewarming at 2 and 24 h. CONCLUSIONS: Slow rewarming after HCA might be superior to fast rewarming in neurological outcome. The present experimental study demonstrated reduction in the inflammatory response, reduction of inflammatory cell activation in the brain, enhancement of cerebral blood flow and reduction of cerebral oedema when slow rewarming was applied.

Amylase expression in taste receptor cells of rat circumvallate papillae.

The chemical composition of the luminal content is now accepted to have a profound influence on the performance of chemosensory receptors. Gustatory and intestinal chemoreceptors have in common their expression of molecules involved in taste sensing and signal transduction pathways. The recent finding that enterocytes of the duodenal epithelium are capable of expressing luminal pancreatic amylase suggests that taste cells of the gustatory epithelium might, in the same way, express salivary amylase in the oral cavity. Therefore, we investigated amylase expression in rat circumvallate papillae by using analyses involving immunohistochemistry, Western blot, and reverse transcription with the polymerase chain reaction. In addition, we used double-labeling confocal laser microscopy to compare amylase immunolabeling with that of the following markers: protein gene product 9.5 (PGP 9.5) and chromogranin A (CgA) for endocrine cells, alpha-gustducin and phospholipase C beta 2 (PLC beta 2) as taste-signaling molecules, and cystic fibrosis transmembrane regulator (CFTR) and Clara-cell-specific secretory protein of 10-kDa (CC10) as secretory markers. The results showed that amylase was present in some taste bud cells; its immunoreactivity was observed in subsets of cells that expressed CgA, alpha-gustducin, PLC beta 2, CFTR, or CC10. PGP 9.5 immunoreactivity was never colocalized with amylase. The data suggest that amylase-positive cells constitute an additional subset of taste receptor cells also associated with chemoreceptorial and/or secretory molecules, confirming the occurrence of various pathways in taste buds.

Immunohistochemical localization of Clara cell secretory proteins (CC10-CC26) and Annexin-1 protein in rat major salivary glands.

The oral cavity is continuously bathed by saliva secreted by the major and minor salivary glands. Saliva is the first biological medium to confront external materials that are taken into the body as part of food or drink or inhaled volatile substances, and it contributes to the first line of oral defence. In humans, it has been shown that sputum and a variety of biological fluids contain Clara cell secretory proteins (CC10-CC26). Various studies of the respiratory apparatus have suggested their protective effect against inflammatory response and oxidative stress. Recently, CC10 deficiency has been related to the protein Annexin-1 (ANXA1), which has immunomodulatory and anti-inflammatory properties. Considering the defensive role of both Clara cell secretory proteins and ANXA1 in the respiratory apparatus, and the importance of salivary gland secretion in the first line of oral defence, we decided to evaluate the expression of CC10, CC26 and ANXA1 proteins in rat major salivary glands using immunohistochemistry. CC10 expression was found only in the ductal component of the sublingual gland. Parotid and submandibular glands consistently lacked CC10 immunoreactivity. In the parotid gland, both acinar and ductal cells were always CC26-negative, whereas in the submandibular gland, immunostaining was localized in the ductal component and in the periodic acid Schiff (PAS)-positive area. In the sublingual gland, ductal cells were always positive. Acinar cells were not immunostained at all. ANXA1 was expressed in ductal cells in all three major glands. In parotid and sublingual glands, acinar cells were negative. In submandibular glands, immunostaining was present in the mucous PAS-positive portion, whereas serous acinar cells were consistently negative. The existence of some CC10-CC26-ANXA1-positive cells in rat salivary glandular tissue is an interesting preliminary finding which could support the hypothesis, suggested for airway tissue, that these proteins have a defensive and protective role. Protein expression heterogeneity in the different portions of the glands could be an important clue in further investigations of their role.

Washout of small molecular contrast agent in carcinoma-derived experimental tumors.

The use of contrast-enhanced magnetic resonance imaging (MRI) for the assessment of breast carcinomas reveals satisfactory sensitivity, but due to low specificity, it does not obviate the need for subsequent tissue sampling. Its capability to differentiate benign from malignant lesion is under continuous investigation. Dynamic contrast-enhanced MRI (DCE-MRI) could improve specificity of MRI through the analysis of the kinetic of contrast enhancement. In particular, the study of the washout pattern is considered a promising tool to improve in vivo diagnosis and even to evaluate the response under chemotherapy. To provide a comprehensive characterization of this parameter in malignant tumor models, in vivo mapping of the washout of small molecular contrast agent (Gd-DTPA, molecular weight 0.57 kDa) was carried out in three transplanted/spontaneous mammary tumors, which differed in their histopathological and microvascular features. It resulted that in all models around 40% of tumor volume lacks efficient washout; washout areas are frequently, but not always, restricted to the tumor periphery and that non-washout areas are not restricted to necrotic regions. Difference in the distribution of lymphatic vessels characterized spontaneous vs. transplanted tumors but did not produce a corresponding different washout pattern, confirming that Gd-DTPA drainage does not mainly depend on lymphatic architecture. Finally, the efficiency of washout is correlated with parameters obtainable during the earlier phases of the enhancement curve and in malignant tumors it could be indirectly estimated from them.