Large Cell Clusters Research Articles

Npc1 deficiency in the C57BL/6J genetic background enhances Niemann–Pick disease type C spleen pathology

Niemann–Pick type C (NPC) disease is an autosomal recessive neurovisceral lipid storage disorder. The affected genes are NPC1 and NPC2. Mutations in either gene lead to intracellular cholesterol accumulation. There are three forms of the disease, which are categorized based on the onset and severity of the disease: the infantile form, in which the liver and spleen are severely affected, the juvenile form, in which the liver and brain are affected, and the adult form, which affects the brain. In mice, a spontaneous mutation in the Npc1 gene originated in the BALB/c inbred strain mimics the juvenile form of the disease. To study the influence of genetic background on the expression of NPC disease in mice, we transferred the Npc1 mutation from the BALB/c to C57BL/6J inbred background. We found that C57BL/6J -Npc1 −/− mice present with a much more aggressive form of the disease, including a shorter lifespan than BALB/c -Npc1 −/− mice. Surprisingly, there was no difference in the amount of cholesterol in the brains of Npc1 −/− mice of either mouse strain. However, Npc1 −/− mice with the C57BL/6J genetic background showed striking spleen damage with a marked buildup of cholesterol and phospholipids at an early age, which correlated with large foamy cell clusters. In addition, C57BL/6J Npc1 −/− mice presented red cell abnormalities and abundant ghost erythrocytes that correlated with a lower hemoglobin concentration. We also found abnormalities in white cells, such as cytoplasmic granulation and neutrophil hypersegmentation that included lymphopenia and atypias. In conclusion, Npc1 deficiency in the C57BL6/J background is associated with spleen, erythrocyte, and immune system abnormalities that lead to a reduced lifespan.

Poly(acrylic acid)/poly(vinyl alcohol) compositions coaxially electrospun with poly(ɛ-caprolactone) and multi-walled carbon nanotubes to create nanoactuating scaffolds

Skeletal muscle regeneration usually causes scar tissue formation and loss of function, an alternative method is needed. In this study, poly(ɛ-caprolactone), multi-walled carbon nanotubes, and (83/17, 60/40, 50/50, and 40/60) poly(acrylic acid)/poly(vinyl alcohol) (PCL-MWCNT-PAA/PVA) were coaxially electrospun to create scaffolds. All four were conductive; however, not all scaffolds actuated when electrically stimulated. The best response occurred when 20 V was applied. A biocompatibility study where skeletal muscle cells were exposed to 0, 0.14%, and 0.7% MWCNT showed that these concentrations were low enough to not cause harm over a four week period. All scaffolds were biocompatible but, the 40/60 scaffolds had more cells. Fluorescent staining showed large clusters of multinucleated cells with actin interaction. Although scaffold tensile properties are greater than skeletal muscle, our other results show that with more modification to cause contraction instead of bending this combination of materials may show promise as components in an artificial muscle.

TCF/Lef1 activity controls establishment of diverse stem and progenitor cell compartments in mouse epidermis

Mammalian epidermis consists of the interfollicular epidermis, hair follicles (HFs) and associated sebaceous glands (SGs). It is constantly renewed by stem and progenitor cell populations that have been identified and each compartment features a distinct mechanism of cellular turnover during renewal. The functional relationship between the diverse stem cell (SC) pools is not known and molecular signals regulating the establishment and maintenance of SC compartments are not well understood. Here, we performed lineage tracing experiments to demonstrate that progeny of HF bulge SCs transit through other SC compartments, suggesting a hierarchy of competent multipotent keratinocytes contributing to tissue renewal. The bulge was identified as a bipotent SC compartment that drives both cyclic regeneration of HFs and continuous renewal of SGs. Our data demonstrate that aberrant signalling by TCF/Lef1, transcription factors crucial for bulge SC activation and hair differentiation, results in development of ectopic SGs originating from bulge cells. This process of de novo SG formation is accompanied by the establishment of new progenitor niches. Detailed molecular analysis suggests the recapitulation of steps of tissue morphogenesis.

Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables: Cabbage and lettuce

The present study was designed to investigate the ability of six Bacillus cereus strains to attach and form biofilm on cabbage and lettuce surfaces. These six strains were; a reference strain DSMZ 345 and five biofilm-producing strains (aquatic strains; TUB8, TUB30, TUB31, TUB32 and TUB33) isolated from drinking-water distribution network. Hydrophobicity, biofilm formation ability, attachment strength (SR) of spores and vegetative cells of the six B. cereus strains were also determined. Due to their high hydrophobicity, spores of all strains had high ability to attach polystyrene and did not affect by dilution of tryptone soy broth (TSB, 1:20 v/v) in the in vitro experiment. Significant (p < 0.05) enhancement in vitro biofilm formation by vegetative cells of B. cereus was recorded in the diluted TSB. The highest biofilm formation on cabbage and lettuce surfaces was obtained by spores and vegetative cells of all tested strains on the 4th hour of the incubation period. These populations were significantly (p < 0.05) increased by elongating incubation time from 4 h to 24 h except DSMZ 345 and TUB8. Biofilm formation behavior obtained by B. cereus spores and vegetative cells on the polystyrene surface was different compared with that recorded on produce surface. The SR of both spores and vegetative cells of the studied strains to the lettuce surface was higher than that of the cabbage surface. The hydrophobicity, biofilm formation and SR of spores and vegetative cells of the biofilm-producing strains were higher than that of the reference strain DSMZ 345. Scanning electron microscopy (SEM) exposed random distribution of cells either on the surface or cut edge, without clear obvious affinity for the surface structures. Increasing in the presence of large clusters of cells on leaf surfaces was demonstrated after 4 and 24 h.In conclusion, use of aquatic environmental isolates is more useful for studying biofilm formation than the reference strain. Lettuce surface supported the attachment of B. cereus spores and vegetative cells compared with the cabbage surface. Further investigations are required to improve our knowledge of biofilm formation mechanisms by the human pathogenic microorganisms, especially by using the environmental and clinical isolates. To ensure safety level of green-leafy vegetables, biofilm formation after harvest should be considered as critical control point during handling of these vegetables.

Reactions of periodontal ligament epithelial cell clusters and OX6-immunopositive cells to experimental tooth movement and periodontitis

The aim of this study was to investigate reactions of periodontal ligament epithelial cell clusters and major histocompatibility complex class II (OX6)-immunopositive cells to simultaneously induced tooth movement and periodontitis employing Waldo's method. Elastic gums were inserted between the right upper first and second molars of rats. Animals were killed by intracardiac perfusion on days 1, 3, 7 and 14 after the experimental procedures, and maxillary molars were decalcified and processed for OCT compound. Cytokeratin and OX6 antibodies to detect epithelial and immunocompetent cells were used for double-fluorescence immunohistochemistry. Immunostained sections of rat upper molar regions were examined with a fluorescence microscope. Large periodontal ligament epithelial cell clusters appeared and became contiguous with each other, and OX6-immunopositive cells surrounded the clusters over time in the periodontal ligament near the gum insertion site. In the periodontal ligament distant from the gum insertion site, epithelial cell clusters and OX6-immunopositive cells were scattered. After 14 d, thickened epithelium and elongated rete pegs were found close to large epithelial cell clusters in the periodontal ligament near the gum insertion site. These findings suggest proliferation and/or aggregation of periodontal ligament epithelial cells, and interaction between OX6-immunopositive cells and the periodontal ligament epithelial cells, in response to tooth movement and periodontal inflammation. This method may be a useful experimental model to elucidate the relationship between rete pegs and periodontal ligament epithelial cell clusters in inflammatory conditions.

Postnatal vasculogenesis: Identification, growth and function of endothelial progenitor cells from peripheral blood mononuclear cells

To identify best growth conditions for the in vitro differentiation of human peripheral blood mono- nuclear cells (PBMNCs) into endothelial progenitor cells (EPCs), PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, and IGF-I; M199 with bovine retina-derived growth sup- plement (RDGS); human umbilical vein endothelial cell (HUVEC) conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in M199 with RDGS (cocul- tures system); PHA burst (10lL/10 6 cells) for 24 h and culture in M199 with RDGF. EPCs were identified by FACS using mAbs for endothelial markers. EPCs mi- gration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. Matrigel was used to assess in vitro angiogenesis capability. Spindle-shaped and attached cells sprouted with growth factors, differentiating in EPCs within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDGS, numerous large cell clusters appeared within 1 week but the number of clusters decreased during culture. After stimulation with PHA, many cells clusters and some adherent EC-like cells were observed within 7 days; their number and size increased within 14 days. With HUVEC conditioned medium, spindle- shaped cells were observed after 10 days. FACS con- firmed the endothelial phenotype. Cocultures induced the appearance of double-labeled EPCs expressing endothelial antigens starting from 7 days. EPCs were able to migrate in response to VEGF and to incorporate in capillary-like structures formed by HUVECs on Matrigel. PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions. The contact with mature endothelial cells (ECs) makes this process quicker.

Fractionated External Beam Radiotherapy as a Suitable Preparative Regimen for Hepatocyte Transplantation After Partial Hepatectomy

Hepatocyte transplantation is strongly considered to be a promising option to correct chronic liver failure through repopulation of the diseased organ. We already reported on extensive liver repopulation by hepatocytes transplanted into rats preconditioned with 25-Gy single dose selective external beam irradiation (IR). Herein, we tested lower radiation doses and fractionated protocols, which would be applicable in clinical use. Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with partial liver external beam single dose IR at 25 Gy, 8 Gy, or 5 Gy, or fractionated IR at 5 × 5 Gy or 5 × 2 Gy. Four days after completion of IR, a partial hepatectomy (PH) was performed to resect the untreated liver section. Subsequently, 12 million wild-type (DPPIV(+)) hepatocytes were transplanted via the spleen into the recipient livers. The degree of donor cell integration and liver repopulation was studied 16 weeks after transplantation by means of immunofluorescence and DPPIV-luminescence assay. Donor hepatocyte integration and liver repopulation were more effective in the irradiated livers following pretreatment with the IR doses 1 × 25 Gy and 5 × 5 Gy (formation of large DPPIV-positive cell clusters) than single-dose irradiation at 8 Gy or 5 Gy (DPPIV-positive clusters noticeably smaller and less frequent). Quantitative analysis of extracted DPPIV revealed signals exceeding the control level in all transplanted animals treated with IR and PH. Compared with the standard treatment of 1 × 25 Gy, fractionation with 5 × 5 Gy was equally efficacious, the Mann-Whitney U test disclosing no statistically significant difference (p = 0.146). The lower doses of 1 × 5 Gy, 1 × 8 Gy, and 5 × 2 Gy were significantly less effective with p < 0.05. This study suggests that fractionated radiotherapy in combination with PH is a conceivable pretreatment approach to prime the host liver for hepatocyte transplantation, thus bringing the experimental model a step closer to clinical application.

Cytopathologic features and differential diagnostic considerations of primary lymphoepithelioma‐like carcinoma of the lung

Primary lymphoepithelioma-like carcinoma (LELC) of the lung is an extremely rare disease that occurs more commonly in Asians, and is composed of undifferentiated carcinoma with prominent lymphoid stroma. LELC is reported to be closely associated with Epstein-Barr virus (EBV) infection. A case is presented here in which bronchial brushing smears in a 70-year-old man, revealed large clusters of neoplastic cells with scant cytoplasm. The nuclei were large, hyperchromatic, of irregular contour and with prominent nucleoli. Also identified were prominent intratumoral lymphoid infiltration and brisk mitotic figures. We detected EBV-coded small RNA in situ hybridization in smears. A cytologic diagnosis of a LELC was suggested. Further evaluation and immunohistochemical studies were conducted on formalin-fixed, paraffin-embedded material. Cords or nests of large neoplastic cells with enlarged nuclei and prominent nucleoli with marked lymphoid infiltration and lymphoid stroma were identified on H&E sections. Immunohistochemically, the tumor cells showed diffuse and strong membranous staining for CK(AE1/AE3), CK5/6, CK34βE12, Napsin A and Bcl-2 but were negative for CK7, CK14, CK20, EMA, TTF-1, chromogranin A, synaptophysin and CD56. The proliferative index with MIB-1 was around 60%, and the p53 positive cells around 20%. The diagnosis of primary LELC of the lung was confirmed based on cytopathologic, histopathologic, immunohistochemical and EBER results, and a detailed systemic examination to exclude possible extrapulmonary (nasopharyngeal) origin. We report the cytopathological features of LELC of the lung and demonstrate here for the first time the positivity of the EBER with RNA-ISH method in smears with emphasis on differential diagnostic considerations.

PHYSICOCHEMICAL CHARACTERIZATION OF FRUIT AND VEGETABLE FIBER SUSPENSIONS. I: EFFECT OF HOMOGENIZATION

ABSTRACTDifferent physicochemical properties and the chemical composition of soluble and insoluble fiber were studied. The influence of fiber source (apple, tomato, carrot and potato pulp), concentration and homogenization were investigated. The fiber suspensions respond in different ways to homogenization. This can, for most physicochemical properties investigated, be due to the fundamentally different microstructure. Carrot and potato pulp suspensions were found to consist of large cell clusters and aggregates, respectively, which were degraded to smaller cell clusters when homogenized. Apple and tomato suspensions were found to consist of large, single cells and cell fragments before homogenization. Tomato suspensions were easily degraded by homogenization, affording a high water‐holding capacity. Apple suspensions were only slightly affected by homogenization, but had the highest elasticity. However, no change in the composition of soluble and insoluble fiber was detected in the homogenized fiber suspensions.PRACTICAL APPLICATIONSProcessing of dietary fiber before use as an additive in food products can change its physicochemical properties. These changes must be understood in order to predict the changes in the product resulting from the addition of dietary fiber. Processing in the form of pressure homogenization of the fiber suspensions gave rise to smaller particles and an increased elasticity. Furthermore, it was shown that the more aggregated the microstructure of the fiber suspension the lower its water‐holding capacity and elastic modulus. This knowledge represents a great potential for the product development process which can then be less time consuming. Thus more products fortified with dietary fiber can appear on the market, increasing the consumption of dietary fiber.

Persistence of functional hepatocyte-like cells in immune-compromised mice

Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte-like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver. In this study, we assess the therapeutic value of HLCs in long-term cell-based therapies in vivo. hESC-derived HLCs were injected into the spleen of acutely injured NODscid(IL-2Rγ) null mice and analysed at various time points post-transplantation up to 3 months. Large clusters of human cells engrafted in the spleen after 3 days and had expanded considerably by 31 days. At these time points, we identified human cells expressing parenchymal hepatocyte markers, exhibiting biliary duct-like structures and expressing myofibroblast markers. Three months after transplantation, we could detect human HLCs that were positive for albumin and CK18 by immunostaining and human DNA by fluorescent in situ hybridisation. Moreover, we could detect secretion of human serum albumin by enzyme-linked immunoabsorbant assay. We observed the persistence, engraftment and function of HLCs in vivo up to 3 months post-translation; however, all murine recipients developed large splenic and liver tumours that contained endodermal and mesodermal cell types. Although our studies demonstrate that hESC-derived HLCs have the potential to play an important role in cell-based therapies, current methodologies and transplantation strategies require substantial refinement before they can be deployed safely.

114 BOVINE EMBRYO-MATERNAL RECOGNITION MAY OCCUR EARLIER IN AI THAN IN EMBRYO TRANSFER (ET)

Embryo-maternal recognition is known to be mediated by the trophoblast and/or its secretory product during the preimplantation period. However, it was recently proposed that embryo-maternal recognition (along with the maternal immune system) could be activated by zona pellucida-degradation products including oligosaccharide chains from very early stage embryos (Fujiwara et al. 2009 J. Reprod. Immunol. 81, 1–8). If this is true, we predict that mammalian mothers recognise the presence of an embryo immediately after fertilization in vivo. The purpose of the present study was to compare the expressions of immune tolerance genes in bovine endometrial-luminal lymphoid (ELL) cells collected from animals after AI or embryo transfer (ET). ELL cells were collected from recipient cows inseminated with frozen–thawed semen on Day 0 (=oestrus, n = 8) and from cows that received a transferred embryo on Day 7 (n = 8) of the oestrous cycle. On Days 9 and 11, ELL cells ipsilateral to the corpus luteum were flushed non-surgically from four cows of each group using Ringer's solution (500 mL). To eliminate large cell clusters of endometrium and the embryo (blastocyst), the flushing medium was passed through an embryo collection filter (EmCon filter, 75 μm stainless steel screen). A single viable blastocyst was collected from each recipient cow. The recovered flushing medium containing ELL cells was centrifuged. The ELL cells were suspended in PBS and separated by Lympholyte-H (Cedarlane, Ontario, Canada) gradient centrifugation. Using RT-real-time PCR, transcripts for four immune tolerance genes in ELL cells from AI or ET examined were transforming growth factor-β (TGF-β), macrophage-colony stimulating factor (M-CSF), interleukin-10 (IL-10), and forkhead box P3 (Foxp3). Data were normalized to that of an endogenous control (glyceraldehyde-3-phosphate dehydrogenase) and analysed using ANOVA followed by Scheffe′s test. Transcripts for TGF-β, M-CSF, IL-10, and Foxp3 were detected in all RT-samples derived from ELL cells on Days 9 and 11 of the oestrous cycle. Transcript levels of TGF-β, M-CSF and IL-10 in the AI group were the same as those of the ET group (P &gt; 0.05), although the transcript level of Foxp3, a master regulator of the function of regulatory T-cells, in the AI group was significantly higher than that of the ET group on Days 9 and 11 (P &lt; 0.05). Furthermore, the transcript level of Foxp3 in the ET group on Day 11 was significantly higher than that of the ET group on Day 9 (P &lt; 0.05). These results indicate that the embryo-maternal recognition in AI occurs earlier than in ET, and suggest that cows recognise an embryo earlier than those generally believed.

Direct Exchange of Electrons Within Aggregates of an Evolved Syntrophic Coculture of Anaerobic Bacteria

Microbial consortia that cooperatively exchange electrons play a key role in the anaerobic processing of organic matter. Interspecies hydrogen transfer is a well-documented strategy for electron exchange in dispersed laboratory cultures, but cooperative partners in natural environments often form multispecies aggregates. We found that laboratory evolution of a coculture of Geobacter metallireducens and Geobacter sulfurreducens metabolizing ethanol favored the formation of aggregates that were electrically conductive. Sequencing aggregate DNA revealed selection for a mutation that enhances the production of a c-type cytochrome involved in extracellular electron transfer and accelerates the formation of aggregates. Aggregate formation was also much faster in mutants that were deficient in interspecies hydrogen transfer, further suggesting direct interspecies electron transfer.

Particle Size Reduction Leading to Cell Wall Rupture Is More Important for the β-Carotene Bioaccessibility of Raw Compared to Thermally Processed Carrots

The amount of nutrients that can be released from food products (i.e., nutrient in vitro bioaccessibility) is often studied as it is a starting point for investigating nutrient bioavailability, an indicator for the nutritional value of food products. However, the importance of mastication as a particle size reduction technique is poorly understood and is often neglected during in vitro procedures determining bioaccessibility. Therefore, the aim of the present work was to study the effect of mechanical breakdown on the β-carotene bioaccessibility of carrot samples, having different textural/structural characteristics (as a result of thermal processing). In the first part of this study, the all-E-β-carotene bioaccessibility of carrot particles of different sizes (ranging from cell fragments up to large cell clusters), generated from raw as well as from gently and intensely cooked carrot samples, was determined. In the second part of the study, the effect of human mastication on the particle size reduction of raw as well as of gently and intensely cooked carrot samples was investigated in order to allow identification and validation of a technique that could mimic mastication during in vitro procedures. Results showed a strong dependency of the all-E-β-carotene bioaccessibility on the particle size for raw and gently cooked carrots. After intense cooking, on the other hand, a considerable amount of all-E-β-carotene could be released from cell fragments (smaller than a cell) as well as from small and large cell clusters. Hence, the importance of mechanical breakdown, and thus also of (in vitro) mastication, is dependent on the carrot sample that is considered (i.e., the extent to which the carrot sample has been thermally processed prior to the particle size reduction). Structural changes occurring during mechanical and thermal processing are hereby key factors determining the all-E-β-carotene bioaccessibility. The average particle size distribution curves of raw and cooked carrots, which were chewed by 15 persons, could be mimicked by mixing 50 g of carrots using a Grindomix (Retsch) at 2500 rpm during 5 s. Based on this scientific knowledge, the identified in vitro mastication technique was successfully integrated in the in vitro digestion procedure determining the all-E-β-carotene bioaccessibility of carrot samples.

Differential survival of AML subpopulations in NOD/SCID mice

Leukemia-initiating cells can retrospectively be defined by tumorigenicity in immunodeficient mice and be characterized by surface markers. The latter still being discussed for acute myeloid leukemia (AML), nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were used to evaluate long-time reconstitution and expansion of AML subpopulations. Bone marrow cells from patients with AML were separated according to CD34 expression, aldehyde dehydrogenase (ALDH) activity, and divisional kinetics in comparison to cord blood-derived CD34(+) hematopoietic stem cells, evaluating survival and expansion in NOD/SCID mice. The AML long-term surviving capacity of subpopulations recovered from NOD/SCID mice was confirmed by ex vivo survival. AML mononuclear cells were detected in bone marrow and spleen of NOD/SCID mice 12 weeks after transplantation. The majority of recovered cells were CD34(+) and significantly more CD34(+) cells were recovered after application of ALDH(bright) (high ALDH activity), CD34(+), or slowly dividing (PKH(bright)) than after ALDH(dim), CD34(-), or fast dividing (PKH(dim)) cell application. CD123(+), CD63(+), and CD44v7(+) cells were also more abundant after the transfer of ALDH(bright) or CD34(+) AML mononuclear cells. In the spleen, large AML cell clusters were only recovered after ALDH(bright), CD34(+), or PKH(bright) cell transfer. Importantly, in secondary long-term in vitro cultures, quite exclusively CD34(+) AML mononuclear cells survived and expanded. Separation of ALDH(bright), CD34(+), or PKH(bright) cells enriches for AML long-term surviving capacity, which reside in the CD34(+) subpopulation, as rather exclusively CD34(+) cells survived and expanded in vivo and ex vivo. Long-term survival capacity may be supported by CD44v7 expression.

Cytological findings of intrathyroidal epithelial thymoma/carcinoma showing thymus‐like differentiation: A study of eight cases

The purpose of this article is to describe the cytologic findings of intrathyroidal epithelial thymoma/carcinoma showing thymus-like differentiation (ITET/CASTLE) in detail and discuss its differential diagnoses. We examined cytologic specimens taken from eight ITET/CASTLE cases, who underwent fine needle aspiration. Cytologic features of ITET/CASTLE include (1) hypercellularity, (2) large cell clusters without papillary or follicular pattern, (3) round or spindle tumor cells with distinct nucleoli and cell border, (4) few keratinized cells and intracytoplasmic lumina (ICL), and (5) lymphocytic background. The differential diagnoses included poorly differentiated carcinoma, metastatic lymphoepithelioma, squamous cell carcinoma, and mucoepidermoid carcinoma. The presence of individual keratinizing cells and ICL and the location of the tumor may be helpful in indicating ITET/CASTLE.

Isolated Ganglioneuroma of the Colon

Purpose: Ganglioneuromas (GN) are neuroectodermal tumors consisting of ganglion cells, nerve fibers and Schwann sheath elements. When found in the gastrointestinal tract, they are of uncommon occurrence. We report a case of an isolated ganglioneuroma of the colon. Case: A 43 year old Ecuadorian female who was otherwise healthy, underwent surveillance colonoscopy that revealed a 6mm polyp at the hepatic flexure and a 4mm polyp at the sigmoid colon. Histology of the resected polyps from the hepatic flexure and sigmoid colon showed tubular adenoma and ganglioneuroma respectively. Discussion: GI ganglioneuromas are rare, benign, and well differentiated reported as 3 subtypes: (1) solitary polypoid GN, (2) ganglioneuromatous polyposis and (3) diffuse ganglioneuromatosis. Polypoid GNs are small, sessile polyps that resemble juvenile polyps, adenomas, or hyperplastic polyps. Gangioneuromatous polyposis is characterized by small multiple polyps (20-40 lesions) with mature ganglion cells in the mucosa and submucosa. Diffuse ganglioneuromatosis (GNM) have band-like enlargement of the submucosal plexus and myenteric nerve plexus caused by hyperplasia of nerve fibers and ganglion cells. There are no specific symptoms eluding toward the clinical diagnosis of ganglioneuromas of the GI tract. Isolated ganglioneuromas are usually asymptomatic. Ganglion cells are not normally found within the GI mucosa. Diagnosis can be facilitated through immunostaining; S-100 protein, neuronase specific enolase (NSE) and neurofilament protein (NFP). In our patient, stains were not performed in order to make the final diagnosis. Mature ganglion cells infiltrating the submucosa as seen in the clusters of large cells with vesicular eccentrically placed nuclei and abundant eosinophilic cytoplasm are seen in high power field (See Figure 1). In the background stroma, spindle and Schwann cells are present. There is no indication for resection in asymptomatic patients. Isolated colonic ganglioneuromas have no reported predilection toward malignancy and there is no indication for long term follow-up in this patient population.Figure

Fine needle aspiration cytology diagnosis of ductal lesions of breast

To find out the most effective and combined cytomorphologic criteria trying to set up an effective diagnostic model for breast ductal lesion in fine needle aspiration cytology (FNAC). A total of 400 breast FNAC cases were collected with follow-up information of more than six years. A retrospective analysis including 104 non-proliferative breast diseases, 163 proliferative breast diseases and 133 carcinomas basing on the diagnostic results of surgical biopsies. Altogether, 60 cytomorphologic variables were counted for the evaluation of each case, including 4 main categories: the cellularity and components, natures of background, cellular arrangements and the cellular features. According to the quantity or the classification stage, the variables were semi-quantitatively scored. Multiple step-wise logistic regression (SPSS) and classification tree model (SAS) were performed to determine the significant and combined variables predictive for the diagnosis of non-proliferative lesion, proliferative breast diseases and carcinoma, respectively. (1) Among 400 benign and malignant cases studied, and basing on the result of analyses of multiple step-wise logistic regression system, intermingling of myoepithelial cells within the epithelial cluster (P < 0.05), presence of large epithelial cell cluster (P < 0.05), presence of small epithelial cell cluster (P < 0.05), cytoplasmic vacuoles (P < 0.05) and figures of "progressive intussusception" of cells (P < 0.1) were selected as the effectively differential diagnostic criteria for the benign and malignant lesions. However, according to the classification tree model, the most useful variable selected associating with the benign lesion was intermingling of myoepithelial cells within epithelial cluster. The diagnostic accuracy will be increased to 94.4%, if another criterion, presence of a big amount of large epithelial clusters, was used as the second useful variable in combination. Presence of a moderate to large amount of small epithelial cell clusters were indicative of proliferative lesion. If the criterion of myoepithelial cells intermingling within epithelial cluster was not found in the sample and associating with presence of small epithelial cell clusters, cytoplasmic vacuoles and figures of "progressive intussusception" of cells, mostly (81.3%), it would be considered as a case of carcinoma. (2) Among 267 benign non-proliferative and proliferative breast diseases studied, both the multiple step-wise logistic regression and classification tree model, presence of irregular intercellular spaces within the epithelial clusters (P = 0.001), loose epithelial clusters (P < 0.05) and hyperchromasia (P < 0.1) were selected as the significant differential diagnostic criteria for the proliferative lesion. The architectural variables and the amount of the abnormal cell features such as cell cluster formation were considered to be more important. A high frequency of presence of irregular intercellular spaces within the epithelial clusters and the amount of loose epithelial clusters indicated a higher possibility of a proliferative lesion. Presence of a single variable of irregular intercellular spaces within the epithelial clusters had the possibility of a benign lesion diagnosis up to 70.1% in all the proliferative breast disease cases collected in this series. If the frequency of irregular intercellular spaces increased to a moderate degree or even higher, the possibility of a benign lesion would be increased to 82.7%. The possibility of a proliferative breast disease would be reached to 87.5%, if both the criteria of irregular intercellular spaces and loosely arranged epithelial cell clusters were counted in combination. (3) The histological results of 35 lesions with atypical cytological features in FNAC specimens were predominantly a proliferative lesion of the breast (26 cases), and most of them were fibroadenoma with ductal hyperplasia. Occasionally, there might be a few benign cases complicating with lesions of atypical hyperplasia or carcinoma. In breast FNAC diagnosis, a combined evaluation of significant variables and the amount of the variable involved are effective for the differential diagnosis between benign/malignant and non-proliferative/proliferative lesions. Lesion accompanying with atypical cellular features should avoid to be overdiagnosed as carcinoma, and biopsy for a histological diagnosis is indicative.

Discriminant Model for Cytologic Distinction of Large Cell Neuroendocrine Carcinoma from Small Cell Carcinoma of the Lung

To establish cytologic criteria for pulmonary large cell neuroendocrine carcinoma (LCNEC), we developed and evaluated a discriminant model for cytologic differential diagnosis between LCNEC and small cell lung carcinoma (SCLC). Aspiration cytologic and/or imprint smears from 29 LCNEC cases were reviewed in comparison with 26 SCLC cases. We selected the following parameters for assessment: background, cellular arrangement, cell clusters, cell cohesion, arrangements, cell dimensions areas, the presence of cytoplasm and/or prominent nucleoli, nuclear features, mitosis, naked nuclei, and nuclear streaking. To demonstrate the utility of differences in frequencies of cytologic parameters for LCNECs and SCLCs, a discriminant model was developed and evaluated. Among the cytologic parameters investigated, large clusters (consisting of >or=60 tumor cells) with tight cohesion and small tumor cells (showing <or=120 microm) without prominent nucleoli on each case were particular focuses of attention, because statistically significant differences with good power were evident between the LCNEC and SCLC groups for their frequencies (p < 0.0001). On the basis of variation in plotted location on scatter plots, a discriminant model for LCNEC and SCLC was made and evaluated by logistic discriminant analysis. Sensitivity, specificity, and accuracy were all 100%. With leave-one-out cross validation, the predicted error rate of the discriminant model for new cases was 0.00545. Our model based on the cytologic features of large cell clusters with tight cohesion and of small tumor cells without prominent nucleoli should be a useful aid for distinction between LCNECs and SCLCs.

Expression of aberrant β-catenin and impaired p63 in craniopharyngiomas

Craniopharyngiomas are rare, histologically benign, non-neuroepithelial epithelial tumors arising from the sellar region, the molecular pathogenesis of CPs is yet not understood. The aim of the present study was to assess expression of aberrant β-catenin and impaired p63 in 66 craniopharyngiomas included 51 adamantinomatous craniopharyngiomas and 15 squamous papillary craniopharyngiomas. On immunohistochemistry, 47out of 51 adamantinomatous craniopharyngiomas, but not squamous papillary craniopharyngiomas, showed strong nuclear/cytoplasmic expression for β-catenin predominantly in compactly cohesive epithelial cells within the whorl-like arrays where ki-67 was almost absent and rarely in palisaded cells where ki-67 was mainly present. P63 overexpression was observed in 45 out of 51 adamantinomatous craniopharyngiomas and 14 out of 15 squamous papillary craniopharyngiomas. P63 stained not only in the nuclei of basal layer cells but also within the whorl-like arrays in adamantinomatous craniopharyngiomas and uniformly in squamous papillary craniopharyngiomas. Using quantitative real time polymerase chain reaction techniques to correlate p63 protein expression with p63 mRNA levels, TAp63 isoforms mRNA was reduced, whereas ▵Np63 mRNA elevated at levels in 5 snap frozen tissue samples with multiple large p63 positive cell clusters compared with normal tissues. In conclusion, the present study confirmed that the two variants of CPs have genetically not only distinctive but also common feature. It demonstrated that cytoplasm/nuclear β-catenin accumulation is an exclusively characteristic morphology of adaCPs. P63 immunohistochemical overexpression were found in both adaCPs and spCPs variant when analyzed in the same study. Taken together, the impaired p63 expression may be attributed to elevated ▵Np63 mRNA and reduced TAp63mRNA in CPs.

Rhesus monkey derived „cardiospheres“ (CSps) for cardiac regenerative therapy in a mouse infarct model

Resident cardiac stem cells have been isolated and expanded in vitro. They form large cell clusters termed cardiospheres (CSps), differentiate into cardiomyocytes in vitro and might be a promising autologous cell source for cardiac restoration. The aim of this study was to determine cell survival and functional effects of Rhesus monkey cardiosphere derived cells (CDCs) in a mouse myocardial infarction model (MI). Cardiospheres derived from Rhesus monkeys were expanded in vitro and either CDCs (1.0×106) or placebo were injected intramyocardially into immunodeficient SCID beige mice after LAD ligature (each group N=10). All animals underwent conductance catheter (CC) evaluation to determine functional cardiac outcome (each group N=5). LAD ligature resulted in a significantly reduced myocardial function compared to non-infarct controls (LV-EF 64% vs. 23%, p<0.05). CDC grafts could be identified in all animals after cell transplantation. Cell proliferation was detectable until one week as determined by immunohistochemical staining for Ki67. The characterization of the CDC grafts (e.g. cardiospecific markers) is currently under investigation. Functional parameters determined by CC evaluation tended to improve with cell therapy after 4weeks (LV-EF placebo 21% vs. LV-EF CDC 30%; n.s.). For the first time, Rhesus CDCs have been used to restore infarcted myocardium in an animal model. The cells proliferated in vivo, survived until 4 weeks after grafting and apparently improved heart function as determined by conductance catheter evaluation. Cardiosphere derived cells appear to be a promising cell source for myocardial regeneration. Further investigation is warranted to develop therapeutic applications in patients.