Utility of SOX11 for the diagnosis of solid pseudopapillary neoplasm of the pancreas on cytological preparations.

INTRODUCTION: The diagnosis of solid pseudopapillary neoplasm (SPN) on fine needle aspiration specimens can be challenging because of morphological overlap with other pancreatic neoplasms, including pancreatic neuroendocrine tumour (PanNET). SRY-related high-mobility group box 11 (SOX11) is a recently described sensitive and specific marker for SPN diagnosis. However, SOX11 immunocytochemistry on cytological smears has not been reported. We evaluated the utility of SOX11 for diagnosis of SPN on cytological preparations. METHODS: SOX11 immunocytochemistry was performed on Papanicolaou-stained smears and/or corresponding cell blocks from aspirates of 7 SPN and 10 PanNET cases identified between 2005 and 2020. Findings were compared with those for beta-catenin, a frequently used diagnostic marker for SPN. RESULTS: Six smears and 6 cell blocks from SPN cases and 8 smears and 10 cell blocks from PanNET cases were available for immunostaining. For SPN, nuclear staining for SOX11 was seen in 6 of 6 (100%) smears and 5 of 6 (83%) cell blocks, with equivocal staining in 1 cell block. In contrast, 7 of 8 (88%) smears and 9 of 10 (90%) cell blocks were negative for SOX11 for PanNet, with equivocal staining seen in 1 case. Beta-catenin immunocytochemistry showed nuclear staining in 6 of 7 (86%) SPN cases and no staining in all 10 (100%) PanNET cases. CONCLUSIONS: SOX11 detected by immunocytochemistry can serve as a useful diagnostic marker for SPN, in addition to beta catenin, and can be performed on cytological smears in cases without a cell block preparation.

A decade of change: Trends in the practice of cytopathology at a tertiary care cancer centre.

OBJECTIVES: The practice of cytopathology has evolved over the past decade with a growing need for doing more with less tissue. Changes in clinical practice guidelines and evolving needs in tissue acquisition for diagnosis and treatment have affected various areas of cytopathology in different ways. In this study, we evaluated the changing trends in cytopathological practice at our institution over the past decade. METHODS: We performed a retrospective review of our institutional database for cytopathology cases from calendar years 2009 (n = 28038) and 2019 (n = 31386) to evaluate the changing trends in practice. RESULTS: The overall number of exfoliative cases decreased 10% over the past decade, primarily due to a 64% decrease in gynaecological Pap testing. However, the volume of serous body cavity and cerebrospinal fluids increased 125% and 44%, respectively. The overall volume of fine needle aspiration (FNA) cases increased 38% from 2009 to 2019. The number of FNA cases increased across most body sites, driven primarily by a 180% increase in endobronchial ultrasound-guided transbronchial needle aspiration cases. In contrast, breast FNA volume decreased 43%. Ancillary studies increased substantially over the past decade, including immunostains (476%) and molecular testing (250%). CONCLUSIONS: The trends in our cytopathological practice showed an increased volume of cases, especially in non-gynaecological specimens. As expected, the number of FNA cases used for immunostains and molecular testing increased substantially, indicating an upward trend in ancillary studies in cytopathological practice.

Plasmodium falciparum histidine rich protein HRPII inhibits the anti-inflammatory function of antithrombin.

BACKGROUND: It has been reported that histidine-rich protein II (HRPII), secreted by the malaria parasite, Plasmodium falciparum (Pf), inhibits the heparin-dependent anticoagulant activity of antithrombin (AT) in vitro and in plasma-based assay systems. OBJECTIVE: The objective of this study was to test the hypothesis that HRPII may also interact with the AT-binding vascular glycosaminoglycans (GAGs), thereby inhibiting the anti-inflammatory signaling function of the serpin. METHODS: We expressed HRPII in bacteria, purified it to homogeneity and studied its effect on endothelial cell signaling in the absence and presence of AT employing established signaling assays. RESULTS: We demonstrate that a low concentration of HRPII potently disrupts the barrier permeability function of endothelial cells. Moreover, HRPII competitively inhibits the protective effect of AT by a concentration-dependent manner. Similarly, AT inhibits the pro-inflammatory activity of HRPII by a concentration-dependent manner. The siRNA knockdown of 3-O-sulfotransferase 1 (3-OST-1), the enzyme responsible for the essential 3-O-sulfation of the AT-binding GAGs, downregulates the pro-inflammatory function of HRPII in endothelial cells, supporting the hypothesis that HRPII competitively inhibits the interaction of AT with 3-OS containing vascular GAGs. Histidine-rich protein II elicits its barrier-disruptive effect by the Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and AT counteracts this effect. We further demonstrate that inorganic polyphosphates bind HRPII with a high affinity to amplify the pro-inflammatory signaling function of HRPII in both cellular and in vivo permeability models. CONCLUSION: We postulate that Pf-derived HRPII and polyphosphate can contribute to the pathogenesis of malaria infection by downregulating the AT-dependent anti-inflammatory and anticoagulant pathways.

Familial Adenomatous Polyposis Syndrome: An Update and Review of Extraintestinal Manifestations.

CONTEXT.­: Familial adenomatous polyposis (FAP) is a rare genetic disorder with autosomal dominant inheritance, defined by numerous adenomatous polyps, which inevitably progress to colorectal carcinoma unless detected and managed early. Greater than 70% of patients with this syndrome also develop extraintestinal manifestations, such as multiple osteomas, dental abnormalities, and a variety of other lesions located throughout the body. These manifestations have historically been subcategorized as Gardner syndrome, Turcot syndrome, or gastric adenocarcinoma and proximal polyposis of the stomach. Recent studies, however, correlate the severity of gastrointestinal disease and the prominence of extraintestinal findings to specific mutations within the adenomatous polyposis coli gene (APC), supporting a spectrum of disease as opposed to subcategorization. Advances in immunohistochemical and molecular techniques shed new light on the origin, classification, and progression risk of different entities associated with FAP. OBJECTIVE.­: To provide a comprehensive clinicopathologic review of neoplastic and nonneoplastic entities associated with FAP syndrome, with emphasis on recent developments in immunohistochemical and molecular profiles of extraintestinal manifestations in the thyroid, skin, soft tissue, bone, central nervous system, liver, and pancreas, and the subsequent changes in classification schemes and risk stratification. DATA SOURCES.­: This review will be based on peer-reviewed literature and the authors' experiences. CONCLUSIONS.­: In this review we will provide an update on the clinicopathologic manifestations, immunohistochemical profiles, molecular features, and prognosis of entities seen in FAP, with a focus on routine recognition and appropriate workup of extraintestinal manifestations.

Protein C Deficiency.

Protein C (PC) deficiency is a heritable or acquired risk factor for thrombophilia, with presentations varying from asymptomatic to venous thromboembolism to neonatal purpura fulminans, a life-threatening disorder. Hereditary PC deficiency is caused by mutation in the PC (PROC) gene located on chromosome 2q14.3. Heterozygous and acquired PC deficiencies are more common than homozygous deficiency. The recommended initial laboratory test measures PC activity using either clot-based or chromogenic methods. There are numerous potential interferences in PC activity testing that may result in either false-positive (falsely low activity) or false-negative (falsely normal or elevated activity) results. In the present review, we discuss common clinical presentations; laboratory testing, with a focus on potential assay interferences; treatment options; and prognosis in patients with PC deficiency.

Solid Pseudopapillary Neoplasm of the Pancreas: A Rare Entity With Unique Features.

Solid pseudopapillary neoplasm of the pancreas is a rare entity with low malignant potential and excellent overall prognosis. It has nonspecific clinical presentations such as abdominal pain and nausea, with vague radiologic features. Histologic features of this neoplasm are usually specific. The tumor shows minimally cohesive, uniform, monotonous cells lining delicate capillary-sized blood vessels, described as pseudopapillary architecture. Other features including hyaline globules, cytoplasmic vacuoles, and nuclear grooving are frequently present. Use of a select panel of immunostains always helps pathologists to differentiate this tumor from other circumscribed tumors of the pancreas. Recently, ß-catenin, CD10, and E-cadherin have been shown to be very important in the diagnosis of solid pseudopapillary neoplasm. Nuclear staining of tumor cells by ß-catenin and membranous presentation of CD10 is seen in almost 100% of cases. Tumor cells can be partially positive for synaptophysin and chromogranin. This tumor has a low malignant potential, and definite treatment is surgical resection.

Gastric heterotopia in rectum: A literature review and its diagnostic pitfall.

OBJECTIVES: The term heterotopia, in pathology, refers to the presence of normal tissues at foreign sites. Gastric heterotopia has been reported anywhere in the gastrointestinal tract. However, the presence of gastric heterotopia in the rectum is very rare. METHODS: We, here, report a rare case of a localized 2-cm area of cratered mucosa with heaped-up borders in the rectum of a 51-year-old, asymptomatic woman who underwent screening colonoscopy. RESULTS: Histologic examination of the biopsy from the lesional tissue in rectum demonstrated fragments of rectal mucosa co-mingling with oxyntic- and antral-type gastric mucosa. No intestinal metaplasia or Helicobacter pylori is identified. CONCLUSION: Patients with gastric heterotopia in rectum usually present with bleeding and/or abdominal pain. Definite treatment of choice is surgical or endoscopic resection, although the lesions also respond to histamine-2 receptor blockers. In this article, most recent literature about gastric heterotopia in rectum is reviewed, following a case presentation about it.

Occupancy of human EPCR by protein C induces ß-arrestin-2 biased PAR1 signaling by both APC and thrombin.

Activation of protease-activated receptor 1 (PAR1) by activated protein C (APC) and thrombin elicits paradoxical cytoprotective and cytotoxic signaling responses in vascular endothelial cells through cleavage of the receptor at Arg-46 and Arg-41 protease recognition sites, respectively. It has been reported that unlike a disruptive G-protein-mediated PAR1 signaling by thrombin, APC induces a protective ß-arrestin-2 biased PAR1 signaling by unknown mechanisms. We hypothesize that the occupancy of endothelial protein C receptor (EPCR) by the Gla-domain of protein C/APC is responsible for the ß-arrestin-2 biased PAR1 signaling independent of the protease cleavage site. To test this hypothesis, we monitored the signaling specificity of thrombin in endothelial cells in response to lipopolysaccharide (LPS) with or without pretreatment of cells with protein C-S195A. The PAR1-dependent recruitment of ß-arrestin-2 in response to LPS by both APC and thrombin was analyzed by functional, gene silencing, and signaling assays. Results indicate that similar to APC, thrombin exerts cytoprotective effects via ß-arrestin-2 biased PAR1 signaling. Similar to APC, thrombin triggered ß-arrestin-2-dependent recruitment of disheveled 2 (Dvl-2) in PC-S195A pretreated cells. Further studies in HeLa cells transfected with PAR1 constructs revealed that EPCR occupancy initiates ß-arrestin-2 biased PAR1 signaling independent of the protease cleavage sites. We demonstrate that EPCR occupancy recruits G-protein coupled receptor kinase 5, thereby inducing ß-arrestin-2 biased PAR1 signaling by both APC and thrombin. In support of a physiological relevance for these results, intraperitoneal administration of PC-S195A conferred a cytoprotective effect for thrombin in an in vivo inflammatory model.

Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays.

Protein C (PC) is a vitamin K-dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband's negative VTE pedigree.

Intraperitoneal administration of activated protein C prevents postsurgical adhesion band formation.

Postsurgical peritoneal adhesion bands are the most important causes of intestinal obstruction, pelvic pain, and female infertility. In this study, we used a mouse model of adhesion and compared the protective effect of activated protein C (APC) to that of the Food and Drug Administration-approved antiadhesion agent, sodium hyaluronate/carboxymethylcellulose (Seprafilm) by intraperitoneal administration of either APC or Seprafilm to experimental animals. Pathological adhesion bands were graded on day 7, and peritoneal fluid concentrations of tissue plasminogen activator (tPA), d-dimer, thrombin-antithrombin complex, and cytokines (IL-1ß, IL-6, interferon-γ, tumor necrosis factor-α, transforming growth factor-ß1) were evaluated. Inflammation scores were also measured based on histologic data obtained from peritoneal tissues. Relative to Seprafilm, intraperitoneal administration of human APC led to significantly higher reduction of postsurgical adhesion bands. Moreover, a markedly lower inflammation score was obtained in the adhesive tissues of the APC-treated group, which correlated with significantly reduced peritoneal concentrations of proinflammatory cytokines and an elevated tPA level. Further studies using variants of human APC with or without protease-activated receptor 1 (PAR1) signaling function and mutant mice deficient for either endothelial protein C receptor (EPCR) or PAR1 revealed that the EPCR-dependent signaling activity of APC is primarily responsible for its protective activity in this model. These results suggest APC has therapeutic potential for preventing postsurgical adhesion bands.

Coating of electrospun poly(lactic-co-glycolic acid) nanofibers with willemite bioceramic: improvement of bone reconstruction in rat model.

We have investigated the combination effects of bioceramics and poly(lactide-co-glycolide) (PLGA) on bone reconstruction in calvarial critical size defects using a rat model. Willemite (Zn2SiO4) ceramics were prepared and coated on the surface of electrospun fabricated scaffolds. After scaffolds and nanoparticles characterization, osteoconductivity of the construct was analyzed using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. Eight weeks after implantation, no sign of inflammation was observed at the site of the osseous defect. The results showed that the ceramics supported bone regeneration and highest bone reconstruction were observed in willemite-coated PLGA. This suggests that electrospun PLGA nanofibers coated with BG are potential candidate implants for bone tissue engineering applications.

Enhanced chondrogenesis of human nasal septum derived progenitors on nanofibrous scaffolds.

Topographical cues can be exploited to regulate stem cell attachment, proliferation, differentiation and function in vitro and in vivo. In this study, we aimed to investigate the influence of different nanofibrous topographies on the chondrogenic differentiation potential of nasal septum derived progenitors (NSP) in vitro. Aligned and randomly oriented Ploy (l-lactide) (PLLA)/Polycaprolactone (PCL) hybrid scaffolds were fabricated via electrospinning. First, scaffolds were fully characterized, and then NSP were seeded on them to study their capacity to support stem cell attachment, proliferation and chondrogenic differentiation. Compared to randomly oriented nanofibers, aligned scaffolds showed a high degree of nanofiber alignment with much better tensile strength properties. Both scaffolds supported NSP adhesion, proliferation and chondrogenic differentiation. Despite the higher rate of cell proliferation on random scaffolds, a better chondrogenic differentiation was observed on aligned nanofibers as deduced from higher expression of chondrogenic markers such as collagen type II and aggrecan on aligned scaffolds. These findings demonstrate that electrospun constructs maintain NSP proliferation and differentiation, and that the aligned nanofibrous scaffolds can significantly enhance chondrogenic differentiation of nasal septum derived progenitors.

Adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells.

The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A2A and A2B are expressed at the highest level among the four receptor subtypes (A2B > A2A > A1 > A3 ) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A2A receptor and by the A2A -specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1, and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1, and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation.

Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor.

The extracellular nuclear proteins, histone H4 (H4) and high mobility group box 1 (HMGB1), released by injured cells during the activation of inflammation and coagulation pathways provoke potent inflammatory responses through interaction with pathogen-related pattern recognition receptors (ie, Toll-like receptors [TLRs] and receptor for advanced glycation end products [RAGE]) present on vascular and innate immune cells. Inorganic polyphosphate (polyP) has emerged as a key modulator of coagulation and inflammation. Here, we demonstrate that polyP binds to both H4 and HMGB1 with high affinity, thereby dramatically potentiating their proinflammatory properties in cellular and in vivo models. By using small interfering RNA knockdowns, pharmacologic inhibitors and extracellular domains of the receptors TLR2, TLR4, RAGE, and P2Y1 as competitive inhibitors, we demonstrate that polyP amplifies H4- and HMGB1-mediated inflammatory signaling in human umbilical vein endothelial cells specifically through interaction with the RAGE and P2Y1 receptors, thereby eliciting intracellular Ca(2+) release. Finally, we demonstrate that the natural anticoagulant protease, activated protein C, potently inhibits polyP-mediated proinflammatory effects of both nuclear proteins in cellular and in vivo systems.

Enhanced reconstruction of rat calvarial defects achieved by plasma-treated electrospun scaffolds and induced pluripotent stem cells.

Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.

Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius).

Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well.

A comparison of pluripotency and differentiation status of four mesenchymal adult stem cells.

The self-renewal and differentiation status of a stem cell is very important in the applications concerning regenerative medicine. Proliferation capacity, differentiation potentials and epigenetic properties of stem cells differ between sources. Studies have shown the high potentials of stem cells in iPS reprogramming. To examine this; we have compared the stem-ness and differential potential of four adult stem cells from common sources. We show a correlation between pluripotency and differentiation status of each stem cell with available data on the reprogramming efficiency. Four human adult stem cells including, adipose tissue-mesenchymal stem cells (AT-MSC), bone marrow mesenchymal stem cells (BM-MSCs), nasal septum derived multipotent progenitors (NSP) and umbilical cord blood stem cells (USSCs) were isolated and characterized. The self- renewal and differentiation potentials of each stem cell were assessed. Stem-ness transcription factors and the propagation potentials of all cells were analyzed. Furthermore the differentiation potentials were evaluated using treatment with induction factors and specific MicroRNA profile. Real-time PCR results showed that our stem cells express innate differentiation factors, miR145 and Let7g, which regulate the stem-ness and also the reprogramming potentials of each stem cell. To complete our view, we compared the propagation and differentiation potentials by correlating the stem-ness gene expression with differentiation MicroRNAs, also the direct effect of these factors on reprogramming. Our results suggest that the potentials of adipose tissue stem cells for GMP (Good Manufacturing Practice) compliant starting material are adequate for clinical applications. Our results indicate a low risk potential for AT-MSCs as starting material for iPS production. Although let7g and mir145 are well known for their differentiation promoting effects, but function more of a fine tuning system between self-renewal and differentiation status.

Novel approach to reduce postsurgical adhesions to a minimum: administration of losartan plus atorvastatin intraperitoneally.

BACKGROUND: Intraperitoneal adhesions are the most important cause of intestinal obstruction, pelvic pain, and female infertility. MATERIALS AND METHODS: Losartan (1, 5, and 10 mg/kg), atorvastatin (1, 20, and 30 mg/kg), losartan (10 mg/kg) plus atorvastatin (20 mg/kg), and sodium hyaluronate/carboxymethylcellulose (HA/CMC) were administered intraperitoneally in 90 male NMRI mice. After 7 d, the grade of adhesions was scored by two scaling methods and the concentrations of TGF-ß1, tPA, and PAI-1 were also evaluated. RESULTS: Simultaneous intraperitoneal administration of losartan and atorvastatin led to a much higher reduction of adhesions compared with that in the HA/CMC group (P < 0.05). When losartan plus atorvastatin was administered, significant changes in the serum concentration and mRNA expression, including the increase of tPA and the decrease of TGF-ß1 and PAI-1, were observed compared with those in other groups. CONCLUSIONS: Our findings suggest that the simultaneous application of losartan and atorvastatin leads to an enhanced reduction in adhesion bands more than that of HA/CMC treatment, compared with the control group, possibly through balancing the expression of TGF-ß1, tPA, and PAI-1.