[Unintended Observations Leading to Macrophage Growth and Neutrophil Factor Research].

My research area in the pharmaceutical industry is innate immunity, especially in phagocytic cells. First, I studied the heat-stable growth factor of peripheral macrophages in tumorous ascitic fluid and found that lipoproteins are an influencing factor. Later, my colleagues and I found that lipid-containing substances, namely, oxidized low-density lipoprotein, dead neutrophils, or purified lipids that could be scavenged by macrophages, induce their growth. From the series of this study, I concluded that phagocytic substances induce macrophage growth by autocrine stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). During the study, we found that neutrophils have growth-inhibitory effects against a variety of cells. Then, I elucidated that the primary factor is a zinc-binding protein, calprotectin, an abundant protein complex in the neutrophil cytosol. I found that calprotectin induces apoptosis in many cell types, including tumor cells and normal fibroblasts, and that the zinc-binding capacity is essential for its activity. Microscopic observations revealed that neutrophil extract contains factor-inducing three-dimensional cell aggregation of human mammary carcinoma, MCF-7. I elucidated that cathepsin G is responsible for this activity and that its effect is dependent on the activation of insulin-like growth factor-1. I believe that this modest, albeit novel, observation was crucial to my thirty-nine-year-long career researching phagocytic cells.

Peritoneal malignant mesothelioma: Slippery like an eel to diagnose on cytology-case series of 3 cases.

INTRODUCTION: Peritoneal malignant mesothelioma is an extremely rare tumor and is a difficult diagnosis to be made on cytology alone. We report 3 cases where the cytologic features were misdiagnosed as carcinoma/lymphoma but histopathology and immunohistochemistry (IHC) established the diagnosis of malignant mesothelioma. CLINICAL DETAILS: Case 1 was a 60-year-old man with multiloculated ascites and omental caking. Peritoneal fluid was reported as malignant on cytology but was misclassified as adenocarcinoma. Case 2, a 45-year-old man with ascites and peritoneal nodularity, radiologically mimicking peritoneal carcinomatosis, was also reported positive for malignancy on ascitic fluid cytology. Fine-needle aspiration (FNAC) from omental fat revealed signet ring cells, thus misleading to cytologic diagnosis of adenocarcinoma. Case 3 was a 63-year-old man with perisplenic mass with extensive omental caking and peritoneal nodularity that was also suspected to be peritoneal carcinomatosis on radiology. FNAC smears from perisplenic mass showed sheets of plasmacytoid cells. On cytology, the differential diagnoses offered were neuroendocrine tumor or non-Hodgkin lymphoma. The diagnosis of malignant mesothelioma was established only after IHC on histopathologic sections in all these cases. None of our patients had history of prior asbestos exposure. CONCLUSION: In such clinical scenarios, with radiology suggesting peritoneal carcinomatosis, the cytologic features need corroboration by IHC/fluorescence in situ hybridization on cell block or biopsy to correctly identify malignant mesothelioma and differentiate it from metastatic carcinomatous deposits and benign mesothelial proliferation.

Mesothelial-to-Mesenchymal Transition and Exosomes in Peritoneal Metastasis of Ovarian Cancer.

Most patients with ovarian cancer (OvCA) present peritoneal disseminated disease at the time of diagnosis. During peritoneal metastasis, cancer cells detach from the primary tumor and disseminate through the intraperitoneal fluid. The peritoneal mesothelial cell (PMC) monolayer that lines the abdominal cavity is the first barrier encountered by OvCA cells. Subsequent progression of tumors through the peritoneum leads to the accumulation into the peritoneal stroma of a sizeable population of carcinoma-associated fibroblasts (CAFs), which is mainly originated from a mesothelial-to-mesenchymal transition (MMT) process. A common characteristic of OvCA patients is the intraperitoneal accumulation of ascitic fluid, which is composed of cytokines, chemokines, growth factors, miRNAs, and proteins contained in exosomes, as well as tumor and mesothelial suspended cells, among other components that vary in proportion between patients. Exosomes are small extracellular vesicles that have been shown to mediate peritoneal metastasis by educating a pre-metastatic niche, promoting the accumulation of CAFs via MMT, and inducing tumor growth and chemoresistance. This review summarizes and discusses the pivotal role of exosomes and MMT as mediators of OvCA peritoneal colonization and as emerging diagnostic and therapeutic targets.

Ruthenium (II)/allopurinol complex inhibits breast cancer progression via multiple targets.

Metal complexes based on ruthenium have established excellent activity with less toxicity and great selectivity for tumor cells. This study aims to assess the anticancer potential of ruthenium(II)/allopurinol complexes called [RuCl2(allo)2(PPh3)2] (1) and [RuCl2(allo)2(dppb)] (2), where allo means allopurinol, PPh3 is triphenylphosphine and dppb, 1,4-bis(diphenylphosphino)butane. The complexes were synthesized and characterized by elemental analysis, IR, UV-Vis and NMR spectroscopies, cyclic voltammetry, molar conductance measurements, as well as the X-ray crystallographic analysis of complex 2. The antitumor effects of compounds were determined by cytotoxic activity and cellular and molecular responses to cell death mechanisms. Complex 2 showed good antitumor profile prospects because in addition to its cytotoxicity, it causes cell cycle arrest, induction of DNA damage, morphological and biochemical alterations in the cells. Moreover, complex 2 induces cell death by p53-mediated apoptosis, caspase activation, increased Beclin-1 levels and decreased ROS levels. Therefore, complex 2 can be considered a suitable compound in antitumor treatment due to its cytotoxic mechanism.

A comparison of the analysis of 3 types of body fluids using the XN-350 hematology analyzer versus light microscopy assessment.

ABSTRACT: We evaluated the capacity of the XN-350 instrument to analyze 3 different types of body fluid samples under "body fluid mode."The performance of XN-350 was evaluated in terms of precision, carryover, limit of blank, limit of detection, limit of quantification, and linearity. Cell enumeration and differential data produced by the XN-350 were compared to manual chamber counting results in 63 cerebrospinal fluid (CSF), 51 ascitic fluid, and 51 pleural fluid (PF) samples. Comparisons between XN-350 versus Cytospin data were also performed in PF samples.The precision, carry-over, limit of blank, and linearity of the XN-350 were acceptable. The limits of detection for white blood cells (WBCs) and red blood cells were 1.0/µL, and 1,000.0/µL, respectively; the corresponding limits of quantitation (LOQs) were 5.0/µL and 2,000.0/µL, respectively. The XN-350's cell enumeration and differential counting correlated well with those of manual chamber counting for all 3 sample types (except for differential counting in CSF samples), particularly parameters involving monocytes (r = 0.33) and mononuclear cells (MO- body fluid [BF]; r = 0.26), as well as total cell (TC-BF) enumeration (r = 0.50) and WBC-BF (r = 0.50) in PF samples. The MO-BF in CSF samples differed significantly from manual chamber counting results, but neither TC-BF nor WBC-BF in PF samples did. The XN-350 also showed good correlations with Cytospin analyses for differential counting of neutrophils, lymphocytes, and monocytes in PF samples. The differential counting of eosinophils via the XN-350 and Cytospin were not significantly correlated, but the difference between them was not significant.The XN-350 is an acceptable alternative to manual fluid analysis. Samples with low cellularity around the LOQ should be checked manually. Moreover, manual differential counting should be performed on CSF samples, particularity those with low cell numbers.

Cisplatin effect on digital cytomorphometric and bioinformatic tumor cell characteristics in rat ovarian cancer model-a preliminary study.

BACKGROUND: Ovarian cancer is one of the most common diseases of the female reproductive system. The aim of this study was the investigation of the antitumor cisplatin effect on ascitic fluid tumor cells in the ovarian cancer experimental model by digital cytomorphometry and cell bioinformatic analysis. METHODS: Female Wistar rats were inoculated by ovarian cancer strain. After ovarian cancer transplantation rats were divided into two groups: control group-without cisplatin treatment, the experimental group-under cisplatin treatment. The ascitic fluid was taken on the 9-th day after tumor cell inoculation. Digital cytomorphometric and cytobioinformatic analysis were used to study ascitic fluid cancer cell morphofunctional changes under cisplatin treatment. RESULTS: Digital cytomorphometric characteristics of rat ovarian cancer ascitic cells were obtained. Tumor cells were classified into four groups according to their geometric size: small, medium, large, "gigantic". The algorithm and the computer program based on tumor cell morphometric characteristics were created to calculate such cell bioinformatic characteristic as information redundancy coefficient R. Three parameters were determined as the criteria of cisplatin effect on cancer cells: cell number, nuclear/cytoplasmic ratio, R-value. CONCLUSIONS: Data obtained suggest that cisplatin reduces the total cell number, the nuclear/cytoplasmic ratio and R-value thus indicates a decrease in cellular resistance and adaptive potential. The digital cytomorphometry and bioinformatics could be recommended as a testing system in the experimental or clinical study.

Predictive and Prognostic Value of Ascitic Fluid Mannose Binding Lectin in Patients with Spontaneous Bacterial Peritonitis.

BACKGROUND: Spontaneous bacterial peritonitis is a common bacterial infection of ascitic fluid, mainly in ascites due to liver cirrhosis. Mannose-binding lectin (MBL) can activate phagocytosis and the complement system. Spontaneous bacterial peritonitis was detected to be higher in MBL deficiency. This study aimed to assess ascitic fluid MBL in liver cirrhosis and spontaneous bacterial peritonitis. METHODS: Ninety patients with cirrhotic ascites were included. Forty five of them had SBP. Child- Pugh score, Model for End Stage Liver Disease (MELD) and its update (uMELD) scores were used to assess the severity of liver cirrhosis. Ascitic fluid samples were obtained for differentiation of leucocytic count, estimation of albumin, protein, glucose, and serum-ascitic albumin gradient. Ascitic fluid levels of MBL were measured for all patients. SBP was documented if polymorphonuclear leucocytic count ≥250/mm in ascitic fluid. RESULTS: Ascitic fluid MBL level was significantly lower in patients with SBP. MBL had a significant negative correlation with ascitic total leukocytic count (TLC), also with serum creatinine, bilirubin, PT, INR and MELD score among SBP patients. However, it had a significant positive correlation with ascitic protein and with platelets. According to multivariate analysis, fever, TLC, platelets, creatinine, MBL, glucose and polymorphs were independent predictors for SBP development. CONCLUSION: Ascitic fluid MBL could be a good predictive and prognostic marker in patients with cirrhosis and spontaneous bacterial peritonitis.

Discrimination of ascitic fluid infection in malignant ascites by polymorphonuclear neutrophil ratio and count: Investigating the validity of count ≥250/mm3 as the one-size-fits-all criterion.

AIM: Neutrocytic ascites, traditionally defined as a polymorphonuclear neutrophil count ≥250/mm3 , is infrequently reported during paracenteses to relieve malignant ascites (MA). This study aims to explore new potential diagnostic criteria to discriminate ascitic fluid infection associated with MA and to examine the clinical and laboratory characteristics of neutrocytic ascites. METHODS: The investigators retrospectively collected data on paracenteses to relieve MA at the Emergency Department of National Cancer Center, Korea, from January 2014 to February 2017. We analyzed the patients whose ascites fulfilled the traditional criteria for classification as neutrocytic ascites; polymorphonuclear neutrophils ≥250/mm3 with no history of either hepatocellular carcinoma or liver cirrhosis. RESULTS: In total, 1467 patients underwent paracentesis to relieve MA. Excluding 98 follow-up paracenteses cases, 112 cases (8.2%) showed neutrocytic ascites. Of these 112 patients, 27 (24.1%) had positive culture results. Receiver-operating characteristic analysis indicated that the area under the curve (AUC) values were 0.90 (95% CI 0.82-0.95) and 0.86 (95% CI 0.78-0.92) for polymorphonuclear neutrophil ratio and count, respectively. The difference between the two AUCs was not statistically significant (P = .29). Moreover, the best cutoff points were 70% and 1500/mm3 for polymorphonuclear neutrophil ratio and count, respectively. In addition, extensive liver metastasis was a significant independent risk factor of MA associated with ascitic fluid infection. CONCLUSIONS: Both polymorphonuclear neutrophil ratio and count had good discriminative abilities for culture results in MA. Polymorphonuclear neutrophil ratio was somewhat better despite lacking statistical significance compared to polymorphonuclear neutrophil count, with 70% as best cutoff.

Case Report: Molecular Identification of Larval Taenia martis Infection in the Pouch of Douglas.

Taenia martis is a tapeworm dwelling in the intestine of mustelids and a rare zoonotic cysticercosis pathogen in its larval stage. The metacestode is morphologically very similar to more prevalent cysticercosis parasites, such as the larvae of Taenia solium and Taenia crassiceps, and may be indistinguishable from other metacestodes on histological sections. However, the epidemiology of human T. martis infections is different, and for prognosis, prevention, and detection of natural parasite reservoirs, the species should be identified. We here report the molecular identification of a T. martis larva located in the pouch of Douglas in a female German patient who underwent surgery for endometriosis. This case represents the fifth human infection described worldwide; all previous cases were also in European women, involving the eye, brain, and the peritoneum.

High neutrophil incorporation rate of ascitic fluid cytology as an indicator of cancerous ascites.

The cell­in­cell phenomenon (CiCP) involves the incorporation of a viable cell by other cells (host cells) and includes two concepts: Emperipolesis and cell cannibalism. The former involves the incorporation of hematopoietic cells as the incorporated cells, while the latter involves cell incorporation by tumor cells as host cells. A total of 239 peritoneal cavity fluid cytology specimens were evaluated for CiCP and the number of singly detectable nuclei (SDN) were measured by examining virtual slide image files. The rates of CiCP­positive cases (RCPCs) and CiCP emergence rate (CER)/SDN were significantly higher in ascites samples than in peritoneal washing samples (P<0.0001 and P=0.0026, respectively), although the numbers of SDN were not significantly different between the groups (P=0.8063). Both the RCPCs and CER/SDN were significantly higher in tumor­positive specimens than in tumor­negative specimens (P=0.0220 and P=0.0312, respectively), although the numbers of SDN were not significantly different between the samples (P=0.2471). Most of the incorporated cells were lymphocytes and the host cells were macrophages; however, the rate of neutrophil incorporation (NI) by host cells in the total CiCP cells in a sample was significantly higher in tumor­positive specimens than in tumor­negative specimens (P=0.0288). NI was mainly performed via emperipolesis by macrophages, with only six examples not by macrophages observed among all CiCP samples. The threshold NI rate/total CiCP (NI/CiCP) between tumor­positive and tumor­negative groups was 11.1% (P=0.0115). Using this threshold, the peripheral blood leukocyte count was significantly higher in the high­NI/CiCP group than in the low­NI/CiCP group (P=0.0022). The present findings revealed novel aspects of less frequently observed CiCP in ascitic fluid cytology by utilizing combined manual and computer assisted image analysis evaluation of samples. Notably, the present study indicated the importance of increased NI as an indicator of cancerous ascites.

Evaluation of cell blocks from effusion specimens in Gynecologic Oncopathology: An experience of 220 cases, diagnosed at a Tertiary Cancer Referral Center.

One of the common indications of ascitic fluid examination in gynecological oncopathology is the detection and classification of malignant cells, especially in cases of clinically suspicious tubo-ovarian masses. The present study was undertaken to assess and validate the diagnostic utility of cell blocks (CBs) and compare its results with the corresponding conventional smears, prepared from effusion samples. CBs were prepared by thromboplastin technique in 220 cases. In 208 cases, diagnostic concordance between results obtained from smears and corresponding CBs was evaluated. Various antibody markers were tested, as per individual case. The average age of patients was 52.2 years. Positive immunohistochemical (IHC) staining for various markers was observed in 182 cases (82.7%) The most frequently positive antibody marker was PAX8 (101/134), followed by p53 (85/92) [mutation type (either diffusely positive or completely negative)], WT1 (tumor cells) (80/112), calretinin (2/87) (diffuse), BerEP4 (21/49), CA125 (21/24), CK7 (31/39) and CK20 and CDX2, together (5/16). Various other IHC markers utilized, including their positive expression, were TTF1 (1/10), p40 (3/3), p63 (2/4), ER (21/29), HBME1 (1/7), GATA3 (1/4), and MIC2 (1/1). Complete diagnostic concordance between CBs and smears was observed in 170/208 cases (81.7%). There were 20 major discordances, 10 minor and 8 cases with sampling errors. IHC was useful in classifying 158/182 (86.8%) cases, including serous or Müllerian adenocarcinoma (n = 123), mostly high-grade (121); metastatic squamous carcinoma (3); gastrointestinal-type adenocarcinoma (8); pulmonary adenocarcinoma (1); breast adenocarcinoma (1); Ewing sarcoma (1); and mesothelioma (2). CBs are complementary to smears in the detection of gynecological malignancies, mostly high-grade serous adenocarcinomas. These provide an opportunity for testing several IHC markers, for a precise diagnosis, including in various uncommon case scenarios, associated with significant therapeutic implications.

Predictors of immediate and short-term mortality in spontaneous bacterial peritonitis.

BACKGROUND: There is scarce data from the Indian subcontinent on the outcomes following spontaneous bacterial peritonitis (SBP). AIM: To study the immediate (within 30 days) and short-term mortality (31-90 days) associated with SBP and to determine the predictors of the same. METHODS: This prospective observational study was done among patients with liver cirrhosis who underwent paracentesis. Patient data included age, gender, co-morbidity, cirrhosis-related complications, model of end-stage liver disease (MELD), and Child-Turcotte-Pugh (CTP) scores. SBP was diagnosed based on ascitic fluid polymorphonuclear leukocyte count > 250/mm3 with or without ascitic fluid culture positivity. RESULTS: Of the 870 patients with cirrhosis and ascites registered during the study period, 610 fulfilled the criteria for inclusion. Altogether, 122 patients with SBP were identified: 52 (42.6%) died, 40 (32.8%) survived without liver transplant, and 30 (24.6%) underwent liver transplantation within 3 months. Thirty-two patients (26.2%) were blood culture posi tive for bacteria and 7 (5.7%) demonstrable bacterial growth in ascitic fluid. Blood culture positivity was significantly higher in the group with immediate mortality (p < 0.0001) and was also significantly associated (p 0.005) with mortality at 3 months. CONCLUSION: Nearly two-fifths (42.6%) of the study cohort died within 3 months of an episode of SBP. Four-fifths of these patients died within 30 days. Blood culture positivity was significantly associated with immediate and short-term mortality.

Mucinous carcinoma originating in the peritoneum diagnosed by an ascites cell block: a case report.

BACKGROUND: Peritoneal carcinoma is a rare disease that is diagnosed and treated in a manner similar to ovarian cancer. In most cases, the histological type is serous carcinoma, and chemotherapy is effective. However, there are a few case reports of mucinous peritoneal carcinoma. We inferred the histological type before surgery using an ascites cell block sample, which was useful for determining the treatment plan. CASE PRESENTATION: Our patient was a 60-year-old Japanese woman. She presented with a feeling of fullness in the abdomen. A computed tomographic scan showed a large quantity of ascitic fluid and thickening of the greater omentum, as well as thickening of the peritoneum at the pouch of Douglas and diaphragm. Hence, peritoneal carcinoma was suspected. The tumor markers carcinoembryonic antigen, cancer antigen 19-9, and cancer antigen 125 were all increased, and no malignant findings were observed in the uterus or ovaries. Cells suggestive of carcinoma were found in the ascitic fluid, and immunostaining by the cell block method suggested the possibility of mucinous carcinoma. The preoperative chemotherapy strategy was changed to short courses, and tumor reduction surgery was planned. Similar to the suspicion before surgery, the pathology results indicated mucinous carcinoma, and the therapeutic effect of chemotherapy was grade 0. CONCLUSIONS: Determining whether peritoneal carcinoma is serous carcinoma is important for therapy and prognostic prediction. In this case, we encountered a patient for whom surgery was chosen because of drug therapy resistance inferred through histological type estimation using the cell block method. Inferring the histological type by cell block preparation is useful for diagnosis and treatment selection.

Characterization of lymphocyte subsets in ascitic fluid and peripheral blood of decompensated cirrhotic patients with chronic hepatitis C and alcoholic liver disease: A pivotal study.

Hepatitis C virus and alcoholic liver disease are major causes of chronic liver diseases worldwide. Little is known about differences between chronic hepatitis C and alcoholic liver disease in terms of lymphocytes' sub-population. Aim of the present study was to compare the sub-populations of lymphocytes in both ascitic compartment and peripheral blood in patients with decompensated liver cirrhosis due to chronic hepatitis C and alcoholic liver disease. Patients with decompensated liver cirrhosis due to hepatitis C virus or alcoholic liver disease evaluated from April 2014 to October 2016 were enrolled. Whole blood and ascitic fluid samples were stained with monoclonal antibodies specific for human TCRɑß, TCRɣδ, CD3, CD4, CD8, CD19, CCR6, CD16, CD56, CD25, HLA-DR, Vɑ24. Sixteen patients with decompensated liver cirrhosis were recruited (9 with hepatitis C virus and 7 with alcoholic liver disease). In ascitic fluid, the percentage of both CD3+CD56- and CD3+CD56+iNKT cells resulted higher in hepatitis C virus patients than in alcoholic liver disease patients (1.82 ± 0.35% vs 0.70 ± 0.42% (p < 0.001) and 1.42 ± 0.35% vs 0.50 ± 0.30% (p < 0.001), respectively). Conversely, in peripheral blood samples, both CD3+CD56- and CD3+CD56+iNKT cells resulted significantly higher in alcoholic liver disease than in hepatitis C virus patients (4.70 ± 2.69% vs 1.50 ± 1.21% (p < 0.01) and 3.10 ± 1.76% vs 1.00 ± 0.70% (p < 0.01), respectively). Both elevation of iNKT cells in ascitic fluid and reduction in peripheral blood registered in hepatitis C virus but not in alcoholic liver disease patients might be considered indirect signals of tissutal translocation. In conclusion, we described relevant differences between the two groups. Alcoholic liver disease patients displayed lower number of CD3+CD4+ cells and a higher percentage of CD3-CD16+, Vα24+CD3+CD56- and Vα24+CD3+CD56+iNKT cells in ascitic fluid than hepatitis C virus positive subjects. Further studies might analyze the role of immune cells in the vulnerability toward infections and detect potential targets for new treatments especially for alcoholic liver disease patients.

Different Expression Pattern of TIM-3 and Galectin-9 Molecules by Peripheral and Peritoneal Lymphocytes in Women with and without Endometriosis.

Endometriosis is a gynecological condition that is associated with chronic pelvic inflammation, pain, and infertility. Although substantial evidence supports that immunological alterations contribute to its pathogenesis and we previously posed a pivotal role of Galectin-9 (Gal-9) in this disorder, the involvement of the TIM-3/Gal-9 pathway in the development of endometriosis-associated immunological abnormalities is not yet known. In the present study, multicolor flow cytometry was used to compare the immunophenotype and cell surface expression of TIM-3 and Gal-9 molecules on peripheral blood (PB) and peritoneal fluid (PF) lymphocytes of women with and without endometriosis. We found an altered distribution of different lymphocyte subpopulations, a markedly decreased TIM-3 labeling on all T and NK subsets and a significantly increased Gal-9 positivity on peripheral CD4+ T and Treg cells of the affected cohort. Furthermore, a significantly increased TIM-3 expression on CD4+T-cells and elevated Gal-9 labeling on all T and NK subsets was also revealed in the PF of the examined patients. In conclusion, our results suggest a persistent activation and disturbed TIM-3/Gal-9-dependent regulatory function in endometriosis, which may be involved in the impaired immune surveillance mechanisms, promotes the survival of ectopic lesions, and aids the evolution of reproductive failures in endometriosis.

Peritoneal fluid collection in healthy cattle and evaluation of changes in cell morphology during storage in refrigerated and non-refrigerated samples / Colheita do fluido peritoneal de bovinos sadios e verificação das alterações na morfologia celular quando mantido refrigerado ou à temperatura ambiente

This study aimed to evaluate the appropriate sites of abdominocentesis for peritoneal fluid collection in cattle and to investigate the time of cell viability in vitro, comparing three methods of sample conservation. Twenty-one healthy cattle (19 females and 2 males) were subjected to a laparocentesis procedure to obtain peritoneal fluid, with punctures in three defined sites: left cranial, right cranial, and right caudal. The total peritoneal fluid collected was divided into three aliquots and maintained under three preservation conditions: room temperature (26°C), refrigeration (4°C), and room temperature (26°C) with the addition of 1µL of 10% formaldehyde per 1mL of peritoneal fluid. The peritoneal fluid analysis performed immediately after collection consisted of: physical examination (color, appearance, volume, and specific gravity), biochemical measures (pH, total protein, fibrinogen, creatinine, and glucose), and cellularity (total and differential counts). The determination of proteins and the examination of cells were repeated in each separate aliquot at two, four, six, and eight hours after harvest. Data were analyzed through repeated measures ANOVA or Friedman test. The harvest was productive in 67% of cattle. The left cranial and the right cranial puncture sites were the most appropriate. Peritoneal fluid analyzed after collection, the total protein concentration ranged from 1.4 to 3.6g/dL, and number of leukocytes ranged from 54 to 1,322 cells/µL; 60 to 95% of leukocytes were lymphocytes. The protein concentration decreased, but the absolute values of leukocytes, lymphocytes, and segmented neutrophils did not change up to eight hours after collection, independent of the maintenance method. Cell lysis was delayed by cooling, and the addition of formaldehyde did not help preserve the integrity of cellular morphology. Laparocentesis is a safe and secure procedure in cattle and maybe more productive when performed in specific sites on the left or right sides of the cranial abdominal wall. Peritoneal fluid samples may be analyzed with reliable results for up to eight hours after collection when kept refrigerated and for up to six hours when kept at room temperature.(AU)

O estudo teve como objetivo avaliar os locais adequados de laparocentese para a colheita de fluido peritoneal de bovinos e estabelecer o tempo de viabilidade celular in vitro, comparando três métodos de conservação. Vinte e um bovinos hígidos (19 fêmeas e 2 machos) foram submetidos ao procedimento de laparocentese para obtenção de fluido peritoneal, com punção em três pontos definidos: cranial esquerdo, cranial direito e caudal direito. O volume total do líquido peritoneal foi dividido em três alíquotas mantidas sob três métodos de conservação: temperatura ambiente (26°C); refrigeração (4°C); e temperatura ambiente (26°C) com adição de 1µL de formol 10% para cada 1mL de líquido peritonial. A análise do líquido peritoneal realizada imediatamente após sua obtenção consistiu em: exames físico (cor, aspecto, volume e densidade); bioquímicos (pH, proteína total, fibrinogênio, creatinina e glicose); e da celularidade (contagens total e diferencial). A determinação de proteínas e o exame da celularidade foram repetidos, em cada alíquota separada, as duas, quatro, seis e oito horas após a colheita. Análise de variâncias de medidas repetidas ou teste de Friedman foram empregados para avaliação ao longo do tempo. A colheita foi produtiva em 67% dos bovinos e os locais de punção craniais esquerdo e direito foram os mais adequados. A concentração de proteína total variou de 1,4 a 3,6g/dL e o número de leucócitos de 54 a 1.322 células/µL, com predomínio de linfócitos (60 a 95% das células) no fluido peritoneal analisado logo após a colheita. A concentração de proteínas diminuiu, mas os valores absolutos de leucócitos, de linfócitos e de neutrófilos segmentados não se modificaram até oito horas após a colheita, independente do método de manutenção das amostras. A lise celular foi retardada pela refrigeração e a adição de formol não contribuiu para preservar a integridade da morfologia celular. A laparocentese é um procedimento seguro e de execução fácil em bovinos sendo mais produtiva quando realizada em locais específicos à esquerda ou à direita craniais da parede abdominal. Amostras de fluido peritoneal podem ser analisadas com resultados confiáveis quando mantidas refrigeradas por até oito horas após a colheita e quando mantidas à temperatura ambiente por até seis horas.(AU)

Peritoneal Level of CD206 Associates With Mortality and an Inflammatory Macrophage Phenotype in Patients With Decompensated Cirrhosis and Spontaneous Bacterial Peritonitis.

BACKGROUND & AIMS: Peritoneal macrophages (PMs) regulate inflammation and control bacterial infections in patients with decompensated cirrhosis. We aimed to characterize PMs and associate their activation with outcomes of patients with spontaneous bacterial peritonitis (SBP). METHODS: We isolated PMs from ascites samples of 66 patients with decompensated cirrhosis (19 with SBP) and analyzed them by flow cytometry, quantitative real-time polymerase chain reaction, functional analysis, and RNA microarrays. We used ascites samples of a separate cohort of 111 patients with decompensated cirrhosis (67 with SBP) and quantified the soluble form of the mannose receptor (CD206) and tumor necrosis factor by enzyme-linked immunosorbent assay (test cohort). We performed logistic regression analysis to identify factors associated with 90-day mortality. We validated our findings using data from 71 patients with cirrhosis and SBP. Data from 14 patients undergoing peritoneal dialysis for end-stage renal disease but without cirrhosis were included as controls. RESULTS: We used surface levels of CD206 to identify subsets of large PMs (LPM) and small PMs (SPM), which differed in granularity and maturation markers, in ascites samples from patients with cirrhosis. LPMs vs SPMs from patients with cirrhosis had different transcriptomes; we identified more than 4000 genes that were differentially regulated in LPMs vs SPMs, including those that regulate the cycle, metabolism, self-renewal, and immune cell signaling. LPMs had an inflammatory phenotype, were less susceptible to tolerance induction, and released more tumor necrosis factor than SPMs. LPMs from patients with cirrhosis produced more inflammatory cytokines than LPMs from controls. Activation of PMs by Toll-like receptor agonists and live bacteria altered levels of CD206 on the surface of LPMs and release of soluble CD206. Analysis of serial ascites fluid from patients with SBP revealed loss of LPMs in the early phase of SBP, but levels increased after treatment. In the test and validation cohorts, patients with SBP and higher concentrations of soluble CD206 in ascites fluid (>0.53 mg/L) were less likely to survive for 90 days than those with lower levels. CONCLUSIONS: Surface level of CD206 can be used to identify mature, resident, inflammatory PMs in patients with cirrhosis. Soluble CD206 is released from activated LPMs and increased concentrations in patients with cirrhosis and SBP indicate reduced odds of surviving for 90 days.

Mucosal-Associated Invariant T Cells Redistribute to the Peritoneal Cavity During Spontaneous Bacterial Peritonitis and Contribute to Peritoneal Inflammation.

BACKGROUND & AIMS: Mucosal-associated invariant T (MAIT) cells are depleted from blood in patients with advanced liver disease and show features of immune dysfunction. Because circulating MAIT cells differ from organ-resident MAIT cells, we aimed to investigate the frequency, phenotype, and function of peritoneal MAIT cells from patients with cirrhosis and spontaneous bacterial peritonitis (SBP). METHODS: MAIT cells in blood and ascitic fluid from patients with cirrhosis were characterized using flow cytometry. Healthy individuals and noncirrhotic patients undergoing peritoneal dialysis served as controls. MAIT cell migration was studied in transwell assays. Cytokine release in response to infected ascitic fluid and bacterial products was assessed in vitro. RESULTS: Peritoneal CD3+ CD161hi Vα7.2+ T cells had an inflammatory, tissue retention phenotype, expressing the alpha E integrin, the chemokine receptors CCR5 and CXCR3, and the activation marker CD69 at higher levels than their circulating equivalents. Seventy-seven percent bound to MR1 tetramers loaded with the pyrimidine intermediate 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil. The ratio of peritoneal to blood MAIT cell frequency increased from 1.3 in the absence of SBP to 2.6 at diagnosis and decreased by day 3. MAIT cells migrated toward infected ascitic fluid containing CCL5 and CCL20 and released cytokines in an MR1-restricted fashion. Whereas the depleted circulating MAIT cell pool displayed features of immune exhaustion, peritoneal MAIT cells remained competent producers of inflammatory cytokines in response to bacterial products. Peritoneal MAIT activation correlated with systemic inflammation, suggesting a possible link between peritoneal and systemic immunity. CONCLUSIONS: Peritoneal MAIT cells phenotypically and functionally differ from circulating MAIT cells in decompensated cirrhosis and redistribute to the peritoneum during SBP.

Characterizing the Effect of Automated Cell Sorting Solutions on Cytomorphological Changes.

INTRODUCTION: Liquid-based cytology has become a widely adopted, automated screening system for gynecologic and nongynecologic cytology. Automated screening systems function by distinguishing atypical cells based on their cytoplasmic and nuclear areas, densitometric measurement, and so on. However, the morphological influence of the washing solution has not been fully considered. Here, we examined the morphological effect and temporal change resulting from saving the cytologic samples in various solutions. METHODS: Cytologic specimens were obtained from the ascites (AS) of patients with peritoneal cancer. Various solutions of a physiological saline, a Ringer's solution, a low-molecular dextran L injection, VOLUVEN 6% solution, MIXID L injection (ML), RPMI-1640 medium, and horse serum (HS) were added to aliquot sediments. All samples were refrigerated at 4°C, and aliquots were subsequently processed at specific time points (0, 1, 2, 4, 7, and 14 days). For all samples, cytoplasmic and nuclear size of the Papanicolaou-stained specimens were measured. RESULTS: In terms of cytoplasmic and nuclear areas, samples stored in ML and HS showed no significant difference compared to the AS sample; in contrast, the other samples were significantly larger in both cytoplasmic and nuclear areas than the AS sample. In examining the temporal change among the solutions, we found that the cytoplasms and nuclei became small over the time course for all of the tested solutions. CONCLUSION: We showed that cells swell in the solution after 1 h of storage and contract as time progresses. Together, our findings have important implications for how mathematical analysis is applied during the automated screening process.

It is all in the bag: collodion bag versus HistoGel cell block method.

INTRODUCTION: We performed a comparison of cell blocks prepared with the collodion bag and HistoGel to evaluate the ease of embedding and cutting, performance with low cellularity specimens, time and cost per specimen, and value to support immunohistochemistry and molecular diagnostics. MATERIALS AND METHODS: We processed 11 fresh, unfixed effusions using both the collodion bag and the HistoGel cell block preparation methods. Six immunohistochemistry stains were tested on 2 of the body fluids. DNA was extracted and quantified, and polymerase chain reaction cycle thresholds were evaluated from cell blocks prepared from 5 of the body fluids. The comparison parameters included embedding difficulty, cutting resistance, adequacy, cell yield, cell preservation, immunohistochemistry stain quality, DNA quantity, integrity, and purity. The time and cost to prepare each specimen was compared using normalized values for preparation of specimen, cost per year, and cost per specimen. RESULTS: Each parameter was assessed for both cell block preparation methods. All 3 of the samples with moderate or poor cell yield were low-volume (5-mL) samples prepared with the HistoGel method. In contrast, the collodion bag method produced a good yield with all three 5-mL samples. DNA recovery was greater in the collodion bag method. Similar crossing threshold values in purity reactions indicated equally high-quality matrix properties for the collodion bag and HistoGel preparations. Preparation of the specimen was 10 minutes faster with the collodion bag method, and the cost for the collodion bag method was $0.24 more expensive per cell block than using the HistoGel. CONCLUSIONS: The collodion bag method produced superior cell blocks for both morphologic and molecular studies more consistently, with lower volume specimens and with less time per specimen.