The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation.

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types (including virus infected and uninfected cells) into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

Presence of leucocytes in prostatic secretions correlates with the severity of lower urinary tract symptoms.

We investigated the correlation between the presence of leucocytes in expressed prostatic secretion and the lower urinary tract symptom severity by retrospectively reviewing 699 men with lower urinary tract symptoms. The patients were evaluated by the International Prostate Symptoms Score and the Overactive Bladder Symptoms Score and underwent expressed prostatic secretion testing. Patients were classified into groups 1 and 2 based on the expressed prostatic secretion leucocyte count. The mean total and storage score of the International Prostate Symptoms Score, and mean total Overactive Bladder Symptoms Score were higher in group 1. Urine flow metrics showed that voided volume and maximum flow rate were lower in group 1. The scores for International Prostate Symptoms Score questions 4, 6 and 7 and Overactive Bladder Symptoms Score question 2 were higher in group 1 and showed a weak positive correlation with expressed prostatic secretion. Voided volume and maximum flow rate showed the strongest correlation, although International Prostate Symptoms Score question 7 and Overactive Bladder Symptoms Score question 2 were the only independent predictors of expressed prostatic secretion. Therefore, leucocytes in expressed prostatic secretion are associated with the lower urinary tract symptom severity, particularly nocturnal urination symptoms.

Boosting the Biogenesis and Secretion of Mesenchymal Stem Cell-Derived Exosomes.

A limitation of using exosomes to their fullest potential is their limited secretion from cells, a major bottleneck to efficient exosome production and application. This is especially true for mesenchymal stem cells (MSCs), which can self-renew but have a limited expansion capacity, undergoing senescence after only a few passages, with exosomes derived from senescent stem cells showing impaired regenerative capacity compared to young cells. Here, we examined the effects of small molecule modulators capable of enhancing exosome secretion from MSCs. The treatment of MSCs with a combination of N-methyldopamine and norepinephrine robustly increased exosome production by three-fold without altering the ability of the MSC exosomes to induce angiogenesis, polarize macrophages to an anti-inflammatory phenotype, or downregulate collagen expression. These small molecule modulators provide a promising means to increase exosome production by MSCs.

Is microductectomy still necessary to diagnose breast cancer: a 10-year study on the effectiveness of duct excision and galactography.

PURPOSE: Patients with spontaneous nipple discharge (SND) who have neither clinically palpable masses nor evidence of disease on imaging with mammogram and/or ultrasound are traditionally investigated with galactogram and duct excision. As breast imaging improves, it has raised the question whether galactography and microductectomy are necessary to diagnose breast cancer. The purpose of this study was to determine the incidence of malignancy in patients presenting with SND who underwent microductectomy and to evaluate the utility of duct excision and galactography in patients whose initial clinical and radiological evaluation were negative. METHODS: A 10-year retrospective study was conducted in British Columbia's largest tertiary breast referral center examining the clinical, radiological and pathological results for all patients who underwent a microductectomy procedure for SND between 2008 and 2017. RESULTS: A total of 231 microductectomies were performed and the overall incidence of malignancy was 13% (n = 32). Following initial work up, 155 patients (67%) had only discharge on exam and no radiologically suspicious findings of malignant disease. Of these patients, 14% (n = 21) were diagnosed with cancer by duct excision. Galactography yielded a sensitivity and specificity of 63% and 36%, respectively (PPV 15% and NPV 85%). Lastly, we found that 3% of patients (n = 8) initially diagnosed with benign disease later developed breast cancer. CONCLUSIONS: Patients with SND should continue to be evaluated with microductectomy to prevent missing a breast cancer. Moreover, we do not recommend performing galactography for diagnosing breast cancer due to poor sensitivity and specificity though it may assist in preoperative planning.

Membrane protein CD9 is repositioned and released to enhance uterine function.

Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.

Detection of tumor spread through airspaces by airway secretion cytology from resected lung cancer specimens.

It currently remains unclear whether tumor spread through airspaces (STAS) actually exist in vivo or are an artifact. The morphologies of STAS and tumor cell clusters in airway secretions collected from the segmental or lobar bronchus of resected lung adenocarcinomas and squamous cell carcinomas were compared among 48 patients. The EGFR status of tumor cell clusters in airway secretions was also compared with that of the main tumor in EGFR mutant adenocarcinomas. Tumor cell clusters were observed in the airway secretion cytology of ten patients (20.8%), and eight patients were adenocarcinoma (20.0% of adenocarcinoma). The morphology of STAS closely resembled that of tumor cell clusters detected in airway secretion cytology. The positive rates of airway secretion cytology were 83.3%, 100%, and 50% in papillary adenocarcinoma, micropapillary adenocarcinoma, and invasive mucinous adenocarcinoma, respectively. Among three EGFR mutant adenocarcinomas, the EGFR mutation subtypes of the main tumors in FFPE sections and tumor cell clusters in airway secretions were identical. These indicate that STAS may be detected in the airway secretion cytology. STAS is common in papillary or micropapillary adenocarcinoma and may spread as far as the segmental or lobar bronchus at the time of surgery.

[Nonspecific IgE potential in nasal discharge for diagnosing allergic rhinitis].

Given that the nasal discharge of subjects with allergic rhinitis contains IgE, we studied nonspecific IgE potential in nasal discharge as a diagnostic marker for allergic rhinitis. We divided 38 adults into 2 groups, one with 22 with Japanese cedar pollinosis and one with 16 with watery rhinorrhea negative to MAST33. The pollinosis group was exposed to Japanese cedar pollen in an environmental exposure unit, and eosinophils in nasal discharge, serum total IgE, pollen-specific IgE, and local IgE in nasal discharge were examined for the 2 groups to determine IgE levels in nasal discharge using the Allerwatch rapid test. In the pollinosis group, nasal discharge IgE correlated significantly with the number of eosinophils. The nasal discharge IgE had higher specificity than the eosinophil examination, whereas nasal discharge eosinophils had higher sensitivity than the IgE examination. We thus found that measuring IgE and eosinophils in nasal discharge is useful for clinically diagnosing allergic rhinitis.

Correlation of ductal lavage cytology with ductoscopy-directed duct excision histology in women at high risk for developing breast cancer: a prospective, single-institution trial.

OBJECTIVES: The study was designed to determine which histological lesions produce cellular atypia in lavage specimens and whether ductoscopy adds useful information for the evaluation of high-risk patients with atypical lavage cytology. METHODS: We prospectively recruited women ≥35 years at high risk for developing breast cancer. All underwent ductal lavage. Women found to have atypia underwent ductoscopy-directed duct excision (group 1). Women without atypia were observed (group 2). Data included patient demographics, risk assessment, cytologic and histologic findings, and outcomes. Descriptive statistics were utilized for data summary and were compared using Fisher's exact test. RESULTS: We enrolled 102 women; 93 (91%) were Caucasian. Their median age was 49 (range 34-73) years with a median follow-up of 80 (range 5-90) months. Overall, 27 (26%) had atypical lavage cytology (group 1), and 75 (74%) had benign cytology (group 2). Subsequent duct excision in group 1 revealed benign histology in 11 (44%), papillomas in 9 (36%), atypical hyperplasia (AH) in 4 (16%), and ductal carcinoma in situ (DCIS) in 1 (4%). At follow-up, three patients developed breast cancer, including one group 1 patient and two group 2 patients. There were no differences between groups 1 and 2 according to patient demographics, Gail scores, or risk for subsequent breast cancer (P > 0.05). CONCLUSIONS: Although 20% of high-risk women with ductal lavage atypia have AH or malignancy on subsequent excision, the majority do not. Atypia identified by ductal lavage is not associated with a higher risk of developing subsequent breast cancer, even in this high-risk population.

[Efficacy of using vitaprost in patients with infertility caused by chronic prostatitis].

The article provides the results of Vitaprost efficacy in 40 infertile men with chronic prostatitis. Vitaprost treatment was found effective, safe and well tolerated.

Mammary ductoscopy and ductal washings for the evaluation of patients with pathologic nipple discharge.

The majority of breast diseases result from lesions of the ductal epithelium. Mammary ductoscopy allows for visualization of intraductal abnormalities, and ductoscopic lavage provides thousands of cells for analysis. We reviewed our experience of 89 cases of patients with pathologic nipple discharge (PND) undergoing ductoscopy-directed duct excision and collection of ductal washings. Patients undergoing ductoscopy-directed duct excision with ductal washings had an 88% abnormal pathology rate. Most abnormalities were benign (71% papillomas), but the atypia rate for this group was 62%. The combination of visualization and pathologic analysis of washings provided the highest predictive value for the diagnosis of papilloma. Cellular yields for this technique were excellent with most specimens yielding >5,000 epithelial cells per high powered field and with evaluable ductal cells in 82% of specimens. Mammary ductoscopy offers the advantage of a high lesion localization rates with intraoperative guidance. The most accurate tool was the combination of ductal washings and ductoscopic visualization, but preoperative use of these techniques is not helpful in most cases. Greater than 90% of patients with PND are found to have a lesion on pathologic examination when using this technique for directed duct excision. Of interest, ductal washings obtained from symptomatic patients with benign diseases are often atypical.

Correlation of nipple aspiration and ductal lavage cytology with histopathologic findings for patients before scheduled breast biopsy examination.

BACKGROUND: The use of ductal lavage to obtain cells from within the breast ducts has been described for patients at high risk for breast cancer. The purpose of this study was to correlate ductal lavage cytologic findings with the corresponding histology. METHODS: Twenty-two patients were evaluated and 20 patients underwent ductal lavage followed by breast biopsy examination as a result of positive nipple aspiration fluid. Ductal lavage samples were classified by a cytopathologist as negative, mild atypia, marked atypia, or malignant. A different pathologist interpreted the histologic findings of the biopsy examination. RESULTS: Adequate specimens for cellularity were obtained in 12 of 22 (53%) patients: 6 of the 12 (50%) had both benign cytology and histology, 2 (16.7%) had benign cytology with atypical ductal hyperplasia or atypical lobular hyperplasia on histology, 2 (16.7%) had marked atypia on cytology and benign histology, and 2 (16.7%) had malignant cytology and benign histology. The specificity of the procedure was 83.4%. CONCLUSIONS: Ductal lavage yielding an adequate sample for analysis was successful in only 52% of patients. Of those, the cytologic-histologic correlation was discordant in 50%. The role of ductal lavage in accurately predicting lesions present on subsequent breast histologic evaluation of planned biopsy examinations requires further investigation.

Ductal lavage findings in women with mammographic microcalcifications undergoing biopsy.

BACKGROUND: We designed a prospective study to assess the likelihood that early lesions presenting as mammographic calcifications could be accessed for cytological diagnosis by ductal lavage (DL). METHODS: Consenting women with calcifications (Breast Imaging Reporting and Data System 4 or 5) underwent DL of fluid-yielding ducts (FYDs) before stereotactic core or excisional biopsy. The DL catheter was used to inject .2 to 1 mL of Isovue 300 into the duct to determine whether the FYD corresponded to the duct containing calcifications (designated overlap). Additional FYDs were injected, if possible, until overlap was identified. DL cytology was compared with histology. RESULTS: Twenty women were enrolled (mean age, 54.2 years); the mean size of the calcification-bearing area was 190 mm(2). The histological findings were as follows: 1 invasive cancer, 9 ductal carcinomas-in-situ (DCIS), 5 atypical hyperplasias, and 5 usual hyperplasias or fibrocystic changes. Four women had no FYD. In 15 women who underwent DL and ductography, overlap of dye and calcifications was seen in 4 (27%): 1 fibrocystic change, 1 hyperplasia, 1 atypical hyperplasia (cytological diagnosis mildly atypical), and 1 DCIS (cytological diagnosis benign). Of the remaining 8 DCIS lesions, 4 had no nipple aspiration fluid, 1 showed extravasation, and 3 were lavaged but the duct did not overlap. CONCLUSIONS: These results are consistent with data from women undergoing mastectomy for larger invasive cancer and DCIS and show that cancer-containing ducts do not yield nipple fluid in most cases.

The association of bloody nipple discharge with breast pathology.

BACKGROUND: It is believed that bloody spontaneous nipple discharge (SND) portends a greater chance of malignancy than nonbloody discharge, and that cytologic evaluation of SND assists in treatment planning. Our aims were to assess (1) the pathology of women with/without SND who require diagnostic breast surgery, (2) whether bloody SND is associated with a different spectrum of pathologic findings than nonbloody, and (3) whether SND cytology is influenced by pathologic findings. METHODS: One hundred seventy-five women who underwent breast operation were enrolled. Cytologic evaluation was performed on Papanicolaou-stained cytospin preparations of SND. RESULTS: Papilloma and hyperplasia (both without atypia) were more frequent in breasts with than without SND; breast cancer was more common in breasts without SND (P <.001 for all). All cases of breast cancer with SND, but only 33% without, contained a papilloma or papillary features. Seventy-five percent of cancers presenting with SND were nonbloody. Papilloma was the most common diagnosis in breasts with bloody SND and was more common (P=.017) than in breasts without, whereas hyperplasia was the most common diagnosis in breasts with nonbloody SND and was more common (P=.031) than in breasts with bloody SND. SND cytology was not significantly influenced by pathology. CONCLUSIONS: Breast cancer can present as unilateral nonbloody SND, indicating the importance of surgical intervention. Papilloma was more common in women with SND than without and most often presented as bloody SND.

Leptin in adipose tissue - Morphological aspects

Leptin is a peptide hormone encoded by the obgene and released by adipocytes. Over the past few years, the synthesis, regulation and effects of leptin have been extensively investigated in view of its pleiotropic role in human (patho)physiology. This work aimed at reviewing current morphological knowledge on leptin synthesis and secretion in adipocytes. It is concluded that the intracellular trafficking of leptin and the structural basis of leptin secretion remain to be defined. Further morphological work, especially at the ultrastructural level, is needed to firmly establish the intracellular distribution and the release pathways of the hormone (AU)

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Intraductal aspiration cytology and galactography for nipple discharge.

The objective of this study was to assess the diagnostic value of preoperative cytology and galactography in women with nipple discharge using a simple intraductal aspiration method. From May 1997 to February 2002, 172 patients with unilateral, spontaneous nipple discharge without palpable masses underwent intraductal aspiration cytology followed by galactography. Major duct excision was performed in 133 of 155 successful cases. Pathological findings showed solitary papilloma in 65 cases, breast cancer in 16 cases, fibrocystic disease in 17 cases, papillomatosis in 12 cases, ductal hyperplasia in 11 cases, and finally, duct ectasia in 12 cases. Our results showed sensitivity of 75.0% and 68.8%, specificity of 86.3% and 62.4%, and overall accuracy of 85.1% and 63.2%, respectively, for cytological analysis and galactography. This suggests that the intraductal aspiration method for preoperative cytology and galactography is a minimally invasive and well-tolerated procedure that seems to be useful in differentiating between benign and malignant lesions in patients with unilateral, spontaneous nipple discharge.

Does preejaculatory penile secretion originating from Cowper's gland contain sperm?

PURPOSE: To determine if spermatozoa are present in the preejaculatory penile secretion, originating from Cowper's gland. DESIGN: Prospective clinical and laboratory study. SETTING: Andrology and Sex Counseling Unit, Department of Obstetrics and Gynecology, Academic Teaching Hospital. PATIENTS: Five patients referred for premature ejaculation, three for excessive fluid secreted during foreplay and four normal healthy volunteers. INTERVENTION: Glass slide smears of preejaculatory Cowper's gland secretion obtained during foreplay from at least two different occasions, and semen samples after masturbation. MAIN OUTCOME MEASURES: Microscopic examination of air-dried smears, and routine semen analyses. RESULTS: None of the preejaculatory samples contained sperm. All the patients had sperm in routine sperm analyses. CONCLUSIONS: Preejaculatory fluid secreted at the tip of the urethra from Cowper's gland during sexual stimulation did not contain sperm and therefore cannot be responsible for pregnancies during coitus interruptus.

Comparison of microscopic methods for detecting inflammation in expressed prostatic secretions.

PURPOSE: We evaluated microscopic methods of detecting inflammation in expressed prostatic secretions. MATERIALS AND METHODS: Methods of counting expressed prostatic secretion leukocytes were compared in 251 samples from 159 patients with chronic prostatitis/chronic pelvic pain syndrome, including traditional wet mounts, hemocytometer derived concentrations and expressed prostatic secretion smears stained with Gram's method or DiffQuick stain (Dade International, Inc., Miami, Florida). RESULTS: Of 159 initial patient evaluations 84 (53%) showed inflammation by hemocytometer concentration at 500 leukocytes per mm.3 or greater but only 37 (23%) were considered inflammation by the traditional wet mount method (p <0.001). Inflammation was identified in 149 of 251 specimens (59%) by hemocytometer but in only 82 (33%) by wet mount (p <0.001). When inflammation was defined as 1,000 leukocytes per mm.3 or greater the hemocytometer still identified significantly more patients (41%) and specimens (48%) with inflammation than the wet mount. The hemocytometer method had a substantially lower interassay and intra-assay coefficient of variation than the wet mount method. Polymorphonuclear neutrophils and macrophages were the most common cells observed on stained smears, which detected inflammation in 147 specimens (59%) by DiffQuick but in only 98 (39%) by Gram's method. CONCLUSIONS: Detecting inflammation in expressed prostatic secretions is method dependent. Significantly more cases of inflammation were detected by hemocytometer than by the traditional wet mount technique. Because the wet mount method also proved more variable than the hemocytometer and highly sensitive to volume, its use is not recommended. These findings support the adoption of hemocytometer and staining methods for accurate evaluation of expressed prostatic secretion inflammation in men with chronic prostatitis/chronic pelvic pain syndrome.