Serial Paraffin Sections Research Articles

Tumor Vascular Interaction in Melanomas and Neurogenesis: A Review

Background: Melanomas arise from multipotent neural crest derivative, the melanocytes. At the tumor-stroma interphase, melanoma cells show organized neural differentiation forming tumor-vascular complexes. The present study explores the sequence of neurogenesis from aurophilic radial glial like cells in relation to angiogenesis, utilizing the tumor-vascular-complex (TVC) as a 3D model. Methods: Serial frozen and paraffin sections stained with HE, reticulin-gold impregnation for aurophilia; dopa oxidase; immunopositivity for: neural differentiation (nestin, Glial Fibrillary Acidic Protein (GFAP), Neural Fibrillary Protein (NFP), synaptophysin); indoleamines: (serotonin and melatonin), catecholamines (dopamine (DA), Noradrenalin (NA), pigment; Dopa Oxidase (DO)); hormones: ((PRL), Prolactin; (HGH), Human Growth Hormone;) and mitosis. The pattern of neural differentiation in tumor-vascular-complexes (TVC) is assessed by positivity in layer1- layer 5 and cell counts in each layer of the perimantle zone (PMZ). Statistical Analysis: ANOVA: Kruskal-Wallis One Way Analysis of Variance; All Pairwise Multiple Comparison Procedures [Tukey Test]. [t-test or Mann-Whitney U- test]. Results: A TVC is formed during angiogenic tumor-vascular interaction. Nes +ve angiogenic tubes enter the tumor margins. Periluminar cells show aurophilia, and extend dendritic arbors into the outer layers of the mantle zone. Cells and dendritic processes in layer 1/ layer 2 show Nes, Auro and GFAP positivity. NFP and Syn positivity is seen in layer 4/ layer 5 with a transition zone between layer 2/ layer 3. As two layers accrue, a wave of mitotic activity is seen and cells acquire PRL and HGH and indoleamine positivity. Catecholamine positivity is in layer 4/ layer 5 thus establishing a polarity. Dopamine is positive in layer 3/ layer 4 coinciding with dopa-oxidase which peaks in layer 4 with NA, ACTH positivity in the outer layers is in association with pigmentation in layer 4/ layer 5. Discussion: Thus during tumor-vascular interaction, melanoma cells differentiate into aurophilic radial glial like cells, as during embryonic neurogenesis, Nes, a marker of multi-lineage progenitor cells, identifies them as MASC, which differentiate into neuronal cells. The angiogenic vessel confers polarity and an embryonal microenvironment in the perivascular mantle zone of the TVC, inducing aggressive melanoma cells to function as neuronal stem cells recapitulating neurogenesis of bio-aminergic cells. The cell cycle is orchestrated by the three pituitary hormones, PRL, HGH and ACTH. The expression of PRL and HGH is related to mitotic activity while ACTH and pigment indicate differentiated function.

Upper terminal of the inferior vena cava and development of the heart atriums: a study using human embryos.

In the embryonic heart, the primitive atrium is considered to receive the bilateral sinus horns including the upper terminal of the inferior vena cava (IVC). To reveal topographical anatomy of the embryonic venous pole of the heart, we examined horizontal serial paraffin sections of 15 human embryos with crown-rump length 9-31 mm, corresponding to a gestational age of 6-7 weeks or Carnegie stage 14-16. The IVC was often fixed to the developing right pulmonary vein by a mesentery-like fibrous tissue. Rather than the terminal portion of the future superior vena cava, the IVC contributed to form a right-sided atrial lumen at the stage. The sinus venosus or its left horn communicated with the IVC in earlier specimens, but in later specimens, the left atrium extended caudally to separate the sinus and IVC. In contrast, the right atrium consistently extended far caudally, even below the sinus horn, along the IVC. A small (or large) attachment between the left (or right) atrium and IVC in adult hearts seemed to be derived from the left (or right) sinus valve. This hypothesis did not contradict with the incorporation theory of the sinus valves into the atrial wall. Variations in topographical anatomy around the IVC, especially of the sinus valves, might not always depend on the stages but partly in individual differences.

Human Embryonic and Hepatic Stem Cell Differentiation Visualized in Two and Three Dimensions Based on Serial Sections.

Pluripotent human embryonic stem cells (hESCs) are characterized by two defining properties, self-renewal and differentiation. Self-renewing hESCs express transcription factors OCT4, SOX2, and NANOG, and surface markers SSEA-4 and TRA-1-60 and TRA-1-81 and their ability to differentiate into derivatives of the three germ layers show the differentiating potential. Studies suggest a certain microheterogeneity of the hESC colonies, in which not all cells in one colony of apparently undifferentiated cells express all the expected markers. We describe a technique to paraffin embed an entire hESC colony, and prepare 3-5μm thick serial sections. Immunohistochemistry applied to individual sections produces a 2-dimensional survey of the developing hESC colony. Based on serial paraffin sections of the 2D-expression pattern, a new and useful 3D-visualization can be modeled. The actual 3D rendering of an entire colony is accomplished using 3D image processing software such as Mimics(®) or Amira(®). An extended version of this technique even allows for a high-magnification 3D-reconstruction of, e.g., hepatic stem cells in developing liver. These techniques combined allow for both a 2- and a 3-dimensional visualization of hESC colonies and stem cells in organs, which leads to new insights into and information about the interaction of stem cells with their surroundings.

Expression of Cold-Inducible RNA-Binding Protein in Normal Skin, Actinic Keratosis and Squamous Cell Carcinoma.

Dear Editor: Cold-inducible RNA-binding proteins (CIRP) are a kind of RNA binding protein associated with diverse cellular responses including cell growth, proliferation, and apoptosis1. CIRP are induced by various cellular stresses in addition to cold exposure such as ultraviolet (UV) radiation and hypoxia1,2. Recently, their proto-oncogenic functions have been suggested, but a detailed expression pattern in skin tumor has not been reported3,4,5. Therefore, we aimed to simply observe the expression pattern of CIRP in photo-damaged epidermis of actinic keratosis (AK), squamous cell carcinoma (SCC) and normal skin by immunohistochemical staining. Five samples of normal skin were obtained from 5 healthy individuals undergoing cosmetic surgery. Among them, 3 were taken from the back and 2 from the face. Specimens of AK and SCC were obtained from the faces of 5 patients who underwent excisional surgery. SCC samples were confined to that which developed after long-standing AK. Clinically and pathologically active lesions were taken as the samples. In all cases, informed consent was obtained from patients according to the ethics committee of the Chonnam National University Hospital. Serial paraffin sections of each specimen were stained with monoclonal antibodies specific for CIRP (Proteintech Group, Chicago, IL, USA) at a dilution of 1 : 100 according to the manufacturers' protocols. The expression of CIRP in epidermal keratinocytes was scored semi-quantitatively by two dermatologists. Nuclear and cytoplasmic staining were assessed separately. We considered both staining intensity and the ratio of positively stained cells in comparison with adjacent stromal cells, lymphocytes, and sebaceous and eccrine glands. No staining was cited as 0, weaker staining than stromal cells as 1, similar to stromal cells as 2, and stronger as 3. Statistical analysis was performed using the chi-square test and Wilcoxon's rank sum test to compare the expression pattern (SPSS ver. 17.0; SPSS Inc., Chicago, IL, USA). In normal skin specimens, CIRP expression was more evident in nuclei than in cytoplasm throughout the epidermal keratinocytes (Fig. 1). However, in specimens from the face, the most sun exposed area, cytoplasmic CIRP staining intensity was increased compared to that in specimens from the back, a less-sun exposed area. In the case of AK, nuclear CIRP expression was decreased while cytoplasmic expression was maintained or rather increased. And in the most pathologic spots of SCC, CIRP expression was significantly decreased both in the nuclei and cytoplasm. Statistical analysis revealed significantly decreased expression of CIRP in nuclei of AK and consequent SCC compared with normal skin. There was no statistically significant difference in cytoplasmic staining intensity among them (Fig. 2). Fig. 1 Cold-inducible RNA-binding protein (CIRP) expression is evident in the nuclei of epidermal keratinocytes in normal skin from back (CIRP immunohistochemical stain, ×200). Fig. 2 Expression patterns of Cold-inducible RNA-binding protein (CIRP). Representative pictures of five cases in each normal skin from face (A), actinic keratosis (AK) (B), and squamous cell carcinoma (SCC) (C) (A~C: CIRP immunohistochemical stain, ×200). ... In previous studies of human cancer, the majority of endometrial carcinoma showed decreased staining intensity4. However, staining intensity was increased in several other human tumors, such as colon and prostate cancer5. These conflicting results may come from the early inducing mechanism of CIRP. CIRP regulates gene expression at the level of translation1,3. Therefore, the exact cellular function of CIRP remains unknown at the moment and the expression pattern in cancer cells could vary according to the state of the tumor. The increased cytoplasmic CIRP expression we observed in sun-exposed areas might be explained by relocalization of CIRP that was triggered by UV exposure2. We observed a significant decrease of nuclear CIRP expression in AK and SCC compared with normal skin. Further studies are needed to elucidate the relationship between CIRP and UV radiation and consequent tumorigenesis in the skin.

Inverse relationship between p53 and phospho-Chk1 (Ser317) protein expression in UVB-induced skin tumors in SKH-1 mice

Immunohistochemical evaluation of serial stored paraffin sections from 42 keratoacanthomas and 11 squamous cell carcinomas demonstrated that skin tumors from UVB-exposed mice showed an inverse relationship (>95%) between p53 protein expression and phospho-Chk1 (Ser317), but not phospho-Chk1 (Ser345) protein expression. Tumors expressing high levels and large areas of p53 protein had no detectable phospho-Chk1 (Ser317), whereas tumors expressing high levels and large areas of phospho-Chk1 (Ser317) protein had no detectable p53. Squamous cell carcinomas that demonstrated heterogeneous p53 and phospho-Chk1 (Ser317) protein expression within the same tumor showed that areas expressing p53 were negative for phospho-Chk1 (Ser317) immunostaining while areas expressing phospho-Chk1 (Ser317) were negative for p53. Similar patterns were observed for keratoacanthomas. These findings were also observed in epidermal areas distant from tumors that demonstrated no detectable phospho-Chk1 (Ser317), but appreciable p53 protein in the basal layer. Tumors from congenic hairless p53 knockout mice had elevated levels of phospho-Chk1 (Ser317) compared to tumors from p53 wild-type SKH-1 controls. After a single exposure to UVB, normal epidermal cells from a p53 knockout mouse expressed a relatively high level of phospho-Chk1 (Ser317) whereas epidermal cells from a p53 wild-type littermate induced p53 protein and expressed a relatively low level of phospho-Chk1 (Ser317). These data illustrate the dynamic regulation of checkpoint function, suggesting that phosphorylation of Chk1 on Serine 317 is regulated by p53 status and that p53 may act as a molecular on/off switch for phosphorylation at this site.

Lack of Epstein-Barr virus infection in Chinese myasthenia gravis patients

There are three recent contradictory reports on the incidence of Epstein-Barr virus in the pathogenesis of myasthenia gravis, with all studies carried out in Caucasian patients. The current study evaluated whether Epstein-Barr virus infection had a role in the pathogenesis of myasthenia gravis in a cohort of 30 Chinese patients. Serial paraffin sections of thymic hyperplasia obtained from myasthenia gravis patients were analyzed for the presence of Epstein-Barr virus-encoded small RNA -1 and Epstein-Barr virus latent membrane protein 1 by in situ hybridization and immunohistochemistry, respectively. Epstein-Barr virus(+) cervical lymph nodes from lymphoma patients and Epstein-Barr virus(-) thymus specimens obtained during cardiac surgery served as the positive and negative control groups, respectively. All the 30 myasthenia gravis specimens were negative for both Epstein-Barr virus-encoded small RNA -1 and Epstein-Barr virus latent membrane protein 1 tests. However, we obtained well-characterized membrane and cytoplasmic immunohistochemical and in situ hybridization staining for both Epstein-Barr virus latent membrane protein 1 and Epstein-Barr virus-encoded small RNA -1, respectively, in the positive control samples. Our results therefore do not support a role of thymic Epstein-Barr virus infection in myasthenia gravis pathogenesis and calls for an integration of methodological and interpretation issues in detecting Epstein-Barr virus incidence in myasthenia gravis patients.

Immunohistochemical Evaluation of the Effects of Paraffin Section Storage on Biomarker Stability

Environmental stresses can alter immunoreactivity of biomarkers in stored tissue sections. The effect of temperature and lighting on 49 cellular or microbial antigens was evaluated in 4 serial paraffin sections, cut 12 months, 10 months, 8 months, 5 months, 3 months, 1 month, 3 days, and 1 day before immunohistochemistry. Slides were stored at room temperature (RT) in the dark, at 4°C in the dark, at RT under fluorescent light, or at RT with windowpane exposure to sunlight. Immunohistochemistry was performed simultaneously in an automated immunostainer. Immunoreactivity was compared with that in the corresponding 1-day-old section and scored as 4 (<10% reduction), 3 (10%-25% reduction), 2 (26%-60% reduction), 1(>60% reduction), or 0 (no reactivity). Any loss of immunoreactivity was proportional to the tissue section age and was least in sections stored in the dark. Immunoreactivity was only completely lost in light-exposed sections and as early as 1 month for CD45. Other markers with complete loss of immunoreactivity were bovine viral diarrhea virus, CD18 (only with fluorescent light), CD31, CD68, canine parvovirus, chromogranins, and thyroid transcription factor-1. Markers with complete loss after light exposure also had reduced immunoreactivity when stored in the dark, as early as day 3. Eight markers (Bartonella spp, CD11d, high molecular weight cytokeratins, feline coronavirus, GATA-4, insulin, p63, progesterone receptor) had minimal decrease in immunoreactivity, regardless of treatment. In conclusion, light-induced antigen decay (tissue section aging) is antigen dependent and could explain unexpectedly weak or negative immunohistochemical reactions in stored paraffin sections.

The Efficacy of Using the Tissue Fragments Present in Cervical Scrapes for the Histologic Diagnosis of Cervical Neoplasia

Cervical scrapes to diagnose cervical neoplasia, collected by the clinician with brushes, are sent to the Leiden Cytology and Pathology Laboratory (LCPL) in vials containing BoonFix, a noncross-linking coagulant fixative. Because the residual material left in the vials contains tissue fragments with important diagnostic information, we stored the residual material in our archives. The tissue fragments can be mummified and archived in commercially available histology cassettes. We can produce paraffin sections thereof. Immunostaining is beautiful on serial paraffin sections cut from these blocks. We experienced that it is important to leave the brush in the vial such that all tissue fragments can be used for histologic diagnosis. In order to optimize the system, tissue fragments left in the endocervical part of the brush are removed in a paint shaker. We illustrate this principle of recovering mummified tissue fragments in a false negative case with cytology containing many undiagnosable collapsed tissue fragments. This case shows clearly the efficacy of using the tissue fragments present in cervical scrapes for the histologic diagnosis of cervical neoplasia.

Effect of Sympathetic ganglionectomy on the Pineal Gland in Rhesus Monkey

The light microscopic structure and the effect of superior cervical ganglionectomy on the pineal gland of Rhesus monkey was studied in 24 animals. The animals were divided into three groups. Group I normal controls, Group II after unilateral and Group III after bilateral ganglionectomy. The animals in group II were sacrificed 15 days and group III were sacrificed 21 days after the operation. Every seventh slide of serial paraffin sections were stained with H&E stain, Red Holzer’s stain for glial fibers, Weigert’s and Van Gieson’s stain, Weil’s stain for myelin sheath, Peter’s protein silver stain for nerve fibers and Glee’s stain for degenerating nerve fibers . A close relationship between the processes of the pinealocytes and interstitial cells and capillaries was suggestive of an exchange of material between the two types of cells and blood vessels. Two types of acervulei in the control group, type I comprising of concentric whorl around a central heterochromatic nucleus and the type II made up of superimposed droplets were noted in the control group. Degenerating nerve fibers, cell free areas and acervulei of superimposed secretions were seen in group II and III suggesting an autonomic nervous control on the secretions of the gland.

Correlation of Inflammatory Cells in Adventitia and Formation and Extending of Atherosclerotic Lesions in Coronary Artery of Apolipoprotein E Gene Knockout Mice

Accumulating evidence shows that adventitial inflammation contributes to the development of atherosclerotic lesions. The aim of this study was to investigate the relationship between atherosclerotic lesions in coronary artery (CA) and accumulation of inflammatory cells at local adventitia in apolipoprotein E gene knockout (apoE-/-) mice. Modified Movat's pentachrome staining, HE staining, immunohistochemistry and transmission electron microscopy were used to observe and to identify serial paraffin sections of aortic foot and inflammatory cells in CA adventitia of apoE-/- mice of 60 weeks old. There was always accumulation of inflammatory cells in the adventitia of CA with extending lesions from aortic orifice to CA trunks. The CA samples were divided into type I: infiltration of inflammatory cells in CA adventitia without lesions extending in the intima, type II: infiltration of inflammatory cells in CA adventitia with the top of extending lesions in the intima and type III: infiltration of inflammatory cells at CA adventitia with lesions covering all the face of intima. The three types of CA sample represent the different developmental processes of atherosclerotic lesions, respectively. No extending lesions were found in the CA trunks without inflammatory cells in adventitia. In type I samples, 60% of infiltrated inflammatory cells were macrophages 57% of infiltrated cells were neutrophils in type II samples; 67% of infiltrated cells were lymphocytes in type III samples. Our studies revealed that adventitial inflammation may be an early event in the development of atherosclerotic lesions. Different cell types predominate in different stages of CA adventitia. The neutrophils are closely related to the extending of atherosclerotic lesions.

Adenocarcinoma of gallbladder: an immunohistochemical profile and comparison with cholangiocarcinoma

AimCholangiocarcinomas display intestinal and pyloric gland metaplasia-cell phenotypes. Those that arise in chronically inflamed (fluke infested) bile ducts more frequently express the intestinal metaplasia-cell phenotype and p53 than sporadic cholangiocarcinomas....

Immunohistochemical expression of apoptotic factors, cytokeratins, and metalloproteinase-9 in periapical and epithelialized gingival lesions

The aim of the study was to assess the involvement of apoptotic factors, cytokeratins and metalloproteinase- 9 in the histogenesis of both Epithelialized Gingival Lesions (EGL) and Periapical Lesions (PAL). 55 consecutive patients, 30 with PAL and 25 with EGL, were selected for the study after clinical and radiological examinations. The PAL patients had severe periapical lesions and tooth decay with exposure of the pulp chamber. All PAL and EGL biopsies were surgically extracted, fixed in 10% buffered formalin, and processed for routine light microscopy. Ten biopsies of each category were processed for immunohistochemistry (IHC). Serial paraffin sections were stained by IHC with appropriate antibodies to detect cytokeratins (CKs) 1, 5, 8, 10 and 14, caspase-3 and -9, metalloproteinase-9, and for PCNA and TUNEL assays. Both PAL and EGL showed a high expression of the cytokeratin 1, 5 and 8 with higher expression in EGL. Moreover, CK10 was markedly less intense expressed in EGL compared to PAL, while CK14 was almost three times stronger expressed in EGL. The expression of caspase-3 and -9 was stronger in PAL compared to EGL, however, the difference was only significant for caspase-9. In PAL apoptosis detected by TUNNEL method and the expression of MMP-9 were higher than in EGL, whereas PCNA was significantly more expressed in EGL. The results clearly suggest that both lesions have exclusively an epithelial origin and that epithelial proliferation was correlated with the degree of apoptosis in both entities. PAL and EGL presented mostly similar cytokeratin expression except for CK10 and CK14, though with marked differences in the distribution and intensity of IHC reactions. Finally, the degradation of extracellular matrix in both lesions could be partially attributed to the strong presence of MMP-9.

Immunohistochemical expression of apoptotic factors, cytokeratins, and metalloproteinase-9 in periapical and epithelialized gingival lesions

The aim of the study was to assess the involvement of apoptotic factors, cytokeratins and metalloproteinase-9 in the histogenesis of both Epithelialized Gingival Lesions (EGL) and Periapical Lesions (PAL). 55 consecutive patients, 30 with PAL and 25 with EGL, were selected for the study after clinical and radiological examinations. The PAL patients had severe periapical lesions and tooth decay with exposure of the pulp chamber.All PAL and EGL biopsies were surgically extracted, fixed in 10% buffered formalin, and processed for routine light microscopy. Ten biopsies of each category were processed for immunohistochemistry (IHC). Serial paraffin sections were stained by IHC with appropriate antibodies to detect cytokeratins (CKs) 1, 5, 8, 10 and 14, caspase-3 and -9, metalloproteinase-9, and for PCNA and TUNEL assays. Both PAL and EGL showed a high expression of the cytokeratin 1, 5 and 8 with higher expression in EGL. Moreover, CK10 was markedly less intense expressed in EGL compared to PAL, while CK14 was almost three times stronger expressed in EGL. The expression of caspase-3 and -9 was stronger in PAL compared to EGL, however, the difference was only significant for caspase-9. In PAL apoptosis detected by TUNNEL method and the expression of MMP-9 were higher than in EGL, whereas PCNA was significantly more expressed in EGL. The results clearly suggest that both lesions have exclusively an epithelial origin and that epithelial proliferation was correlated with the degree of apoptosis in both entities. PAL and EGL presented mostly similar cytokeratin expression except for CK10 and CK14, though with marked differences in the distribution and intensity of IHC reactions. Finally, the degradation of extracellular matrix in both lesions could be partially attributed to the strong presence of MMP-9.

Comparative study of laser-induced choroidal neovascularization in rats by paraffin sections, frozen sections and high-resolution optical coherence tomography

The purpose of this study was to demonstrate the value of a quantitative study concerning experimental choroidal neovascularization (CNV) tissue by correlating the paraffin sections and frozen sections with optical coherence tomography (OCT) images. After OCT imaging was performed, four right eyes from the normal Brown Norway (BN) rats were made into paraffin sections and four left eyes were made into frozen sections. Laser photocoagulation was performed on the right eyes of the other 32 BN rats to induce CNV; these rats then were divided into two groups. After the OCT imaging was performed, four eyes were made into serial paraffin sections in Group A or frozen sections in Group B at 1 week after laser photocoagulation, and weekly for 4 weeks. A quantitative comparison of histopathological images and OCT images was performed. The thickness of the sensory retina and choroid of normal BN rats in paraffin sections was perceptibly less than that in the frozen sections, and the thickness of the sensory retina in the frozen sections was similar to that measured in the OCT images. The maximum thickness of the CNV tissue in the frozen sections was greater than that in the paraffin sections, but similar to that measured in the OCT images from week 1 to week 4. Only the frozen sections accurately matched the high-resolution OCT images because of the processing artifacts in the paraffin sections. In further quantitative studies on experimental CNV, the frozen sections may be more preferable if the thickness of the CNV tissue is to be measured and compared.

Human melanoma cells in the rhombencephalon of the chick embryo: a novel model for brain metastasis

Malignant melanoma has the highest propensity to metastasize to the brain of all primary neoplasms in adults. Here, we describe invasive growth and the development of melanoma metastases from suspensions of human melanoma cells in the brain of the chick embryo. Patient-derived melanoma cells and established melanoma cell lines were injected into the rhombencephalic brain vesicle of the two-day-chick embryo. After 48 and 96 h, tumor formation was studied in serial paraffin sections with melanoma-specific HMB45 and human-specific MIB1 proliferation markers. The majority of the cells injected into the embryonic liquor cavity perished. Only melanoma cells in newly formed aggregates or when attached to the dorsal roof plate escaped apoptosis. Local invasion occurred not in the ventral differentiating neural epithelium but only in the roof plate. Although after 48 h melanoma cells invaded the rhombencephalic roof plate profusely at both sides, after 96 h typically one large tumor developed in the midline between roof plate and the dorsal surface epithelium. From the tumor, single cells invaded the mesenchyme and blood vessels. Cell lines with different invasive properties retained their graded invasive behaviour. Maximally invasive cells formed continuous tracks via vessels and along nerve fibres. The central tumor in the roof plate of the chick embryo rhombencephalon resembles a metastatic nodule in the patients with melanoma. Penetration of the roof plate epithelium, tumor formation and invasion of surrounding tissues by single cells can reliably be reproduced. The chick embryo model can be used for molecular studies of early phases of melanoma brain metastasis.

Hormone Expression in Melanomas Related to Tumor Angiogenesis

Background: Vicarious production of pituitary hormones by malignant tumors is a well known phenomenon. Prolactin [PRL], human growth hormone [HGH], adenocorticotropic hormone [ACTH] and alpha-melanocyte stimulating hormone [α-MSH] expression has been reported in malignant melanoma tumor cell lines. The present study examines the pattern of expression of the hormones ACTH, PRL and HGH in melanoma with regard to tumor growth and differentiation in relation to angiogenesis. Methods: During angiogenesis the tumor cells encircle a central vessel in layers to form a tumor-vascular-complex [TVC] with a central vessel and a mantle of 5 to 6 layers. Hormone expression is assessed by immunopositivity for PRL, HGH, & ACTH in the tumor-vascular complexes, on serial frozen and paraffin sections and subjected to statistical analysis [t-test or Mann-Whitney U- test]. Results: As two layers accrue the tumor cells acquire PRL & HGH positivity and are associated with a wave of mitotic activity in these layers. Further addition of layers shows ACTH positivity in the outer layers in association with pigmentation. Discussion: There is a coordinated growth of melanoma cells around neovascular channels, the cell cycle of malignant melanocytes being orchestrated by the three pituitary hormones, PRL, HGH & ACTH. The expression of PRL & HGH is related to mitotic activity while ACTH & pigment indicate differentiated function.

A Shared Vision to Prevent Heart Disease and Stroke Through United Action

Cardiovascular diseases are the second and fourth leading causes of death in North Carolina, and when combined become the leading killers of North Carolinians. A key strategy for reducing this disease burden and saving lives is to focus on prevention through a combined approach of policy change and program implementation.

Loss of keratin 13 in oral carcinoma in situ: a comparative study of protein and gene expression levels using paraffin sections

AbstractImmunohistochemical loss of keratin (K)13 is one of the most valuable diagnostic criteria for discriminating carcinoma in situ (CIS) from non-malignancies in the oral mucosa while K13 is stably immunolocalized in the prickle cells of normal oral epithelium. To elucidate the molecular mechanism for the loss of K13, we compared the immunohistochemical profiles for K13 and K16 which is not expressed in normal epithelia, but instead enhanced in CIS, with their mRNA levels by in-situ hybridization in formalin-fixed paraffin sections prepared from 23 CIS cases of the tongue, which were surgically removed. Reverse transcriptase-PCR was also performed using RNA samples extracted from laser-microdissected epithelial fragments of the serial paraffin sections in seven of the cases. Although more enhanced expression levels for K16 were confirmed at both the protein and gene levels in CIS in these seven cases, the loss of K13 was associated with repressed mRNA levels in four cases, but not in the other three cases. The results suggest that the loss of K13 is partly due to its gene repression, but may also be due to some unknown post-translational events.

The migration and loss of human primordial germ stem cells from the hind gut epithelium towards the gonadal ridge

Human primordial germ cells (PGCs) can be recognized in the yolk sac wall, from 3-4 weeks post conception (wpc), in the hind gut epithelium from week 4 and in the gonadal area from early week 5. The objective of this study was to map the migration route of PGCs and elucidate the role of the nervous system in this process. Sixteen human specimens, 5-14 wpc obtained from legal abortions were included. On serial paraffin sections, PGCs were detected immunohistochemically by expression of OCT4 and c-Kit, nerve fibers by β-III-tubulin and stem cell factor (SCF) as a possible chemoattractive cue for PGC migration. PGCs were present in the hind gut epithelium, in the mesenchyme of the dorsal mesentery and in the developing gonadal ridge of 4-6 wpc embryos, prior to connections between the enteric and the sympathetic nervous system. From 6 wpc onwards, the PGCs travelled along the developing nerve fibers from the wall of the hind gut via the dorsal mesentery to the midline of the dorsal wall and laterally into the gonads. Numerous PGCs were still present in the nervous system by 14 wpc. PGCs in 4-5 wpc embryos are suggested to leave the gut epithelium by EMT-like transition. SCF may facilitate further migration, but after establishment of connections between the enteric and sympathetic nervous systems. PGCs follow sympathetic nerve fibers towards the gonads. PGCs failing to exit the nerve branches at the gonadal site, may continue along the sympathetic trunk ending up in other organs where they may form germ cell tumors if not eliminated by apoptosis.

Cerebral artery remodeling in stroke‐prone spontaneously hypertensive rats

Stroke is a major cause of disability and presents a serious and growing threat to public health [1]. Epidemiological studies have shown that stroke is the second leading cause of death in the world [2]. Among the risk factors for stroke, hypertension is the most powerful modifiable factor and the second most powerful risk factor after age [3–5]. Hypertension eventually results in target organ damage such as myocardial hypertrophy and artery remodeling [6]. Over 70% of stroke cases can be attributed to hypertension [7]. Arterial baroreflex (ABR) is one of the most important physiological mechanisms controlling blood pressure (BP) regulation. Its function, manifested as baroreflex sensitivity (BRS), correlates to the degree of end-organ damage in spontaneously hypertensive rats (SHR), a genetic model for hypertension [8]. Stroke-prone SHR (SHR-SP) is a substrain of SHR that exhibits much higher incidence of stroke compared with age-matched SHR. The mechanisms underlying such a difference remain to be fully understood. Signs of stroke, including movement of limbs, respiration, diet, fur and consciousness, were observed in 25 SHR (male 13, female 12) and 26 SHR-SP (male 13, female 13) at the age of 28 weeks. The neuroethology assessment was performed by using the Zea-Longa score (maximum score: 5, higher score indicates more severe neurological impairment). Systolic blood pressure (SBP), diastolic blood pressure (DBP) and BRS were determined in conscious rats as previously described [9]. Serial transverse paraffin sections (5 μm thick separated by 100 μm) were prepared using routine techniques. Ten slides were stained with hematoxylin-eosin and observed under light microscope. Structure of cerebral arteries with external diameter of 0.07–0.09 mm and 0.7–0.9 mm were evaluated with a SDK-2000 system for image analysis. Data are expressed as mean ± SD. Differences between the two groups were evaluated by unpaired t-test. Stroke incidence was analyzed with chi-square test. The relationships of SBP and BRS with wall thickness of cerebral arteries were assessed by univariate regression analysis. P < 0.05 was considered statistically significant. The behavior of each rat was observed separately. Positive signs occurred in 9 of the 26 SHR-SP (incidence: 34.62%), and none out of the 25 SHR (Figure 1A). The Zea-Longa score was significantly higher in SHR-SP (Figure 1B, P < 0.001 vs. SHR). SBP and DBP were significantly higher in SHR-SP (Figure 1C, P < 0.01 vs. SHR). BRS was significantly lower in SHR-SP (Figure 1D, P < 0.01 vs. SHR). Comparison of incidence of stroke, neuroethology assessment, BP and BRS between SHR and SHR-SP. 28-week-old SHR (n = 25, male 13, female 12) and SHR-SP (n = 26, male 13, female 13) were used. (A) Positive symptoms occurred in 9 of 26 SHR-SP, with a stroke incidence of 34.62%, and no animal with positive symptom was found in SHR. (B) Zea-Longa score of SHR-SP was significantly higher than that of SHR. (C) BP of SHR-SP was significant higher than that of SHR. (D) BRS of SHR-SP was significantly lower than that of SHR. **P < 0.01, ***P < 0.001 versus SHR. There was no significant difference in wall thickness, lumen diameter, wall-to-lumen ratio and wall cross sectional area between SHR-SP and SHR in the cerebral arteries with external diameter of 0.07–0.09 mm (Table 1). For cerebral arteries with external diameter of 0.7–0.9 mm, wall thickness, wall-to-lumen ratio and wall cross sectional area were significantly larger (P < 0.001), and the lumen diameter was significantly smaller (P < 0.05) in SHR-SP compared with that in SHR. In SHR-SP, the wall thickness was positively correlated with SBP (r = 0.366, P < 0.05), and negatively correlated with BRS (r = 0.596, P < 0.01). ABR dysfunction has been repeatedly reported to be present in acute stroke [10]. A recent study from this laboratory in hypertensive rats indicated that BRS is an independent predictor for stroke [9]. Results from the current study showed that hypertension and ABR dysfunction were more severe in SHR-SP than in SHR. Consistent with the relatively high BP and low BRS in SHR-SP, cerebral artery remodeling, e.g., increased vascular wall thickness and decreased lumen diameter, was also more pronounced in SHR-SP. In conclusion, cerebral artery remodeling may represent a key pathological feature that makes SHR-SP much more vulnerable to stroke than SHR.