Immunological Staining Research Articles

Intrarenal vascular resistance and prothrombotic state in non-diabetic hypertensive patients

The enzyme dipeptidyl peptidase-IV (DPP-4) inactivates the incretin hormone glucagon-like peptide-1 (GLP-1). GLP-1 has therapeutic effects in patients with type 2 diabetes, but its potential is limited by a short half-life, DPP-4 inhibition is a promising approach to diabetes treatment. This study examined chronic (once-a-day dosing for 8 weeks) effects of the DPP-4 inhibitor PKF-275-055 (1, 3, and 10 mg/kg) on β-cell regeneration and plasma DPP-IV activity, intact GLP-1, glucose, and insulin after an oral glucose load in neonatal wistar rats injected with streptozotocin (STZ) (n2-STZ model), a recognized model of type 2 diabetes. In streptozotocin induced diabetic rats, PKF-275-055 (3, and 10 mg/kg) significantly reduced glucose excursion during the oral glucose tolerance test conducted 2 h and 10 h after administration, with increases in plasma insulin and active glucagon-like peptide-1 (GLP-1) levels and significantly inhibited (> 50% inhibition) plasma DPP-IV activity during both the 1st and 2nd OGTT in diabetic rats. In contrast, PKF-275-055 (1–10 mg/kg) did not cause hypoglycemia in fasted normal rats. Furthermore, PKF-275-055 significantly inhibited advance glycation end product (HbA1c), HOMA-Index, gastric emptying and small intestinal transit rates, with significance at doses of 1 mg/kg or higher. Immunological staining showed PKF-275-055 stimulates β-cell regeneration and reduces pancreatic cell apoptosis in diabetic treated rats. The present preclinical studies indicate that PKF-275-055 is a novel selective DPP-IV inhibitor with long-acting antidiabetic effect that might be a potential agent for type 2 diabetes

Evaluating Permeability Surface-Area Product as a Measure of Blood-Brain Barrier Permeability in a Murine Model.

Permeability surface-area product has been suggested as a marker for BBB permeability with potential applications in clinical care and research. However, few studies have demonstrated its correlation with actual quantitative measurements of BBB permeability. Our aim was to demonstrate the correlation of quantitative permeability surface-area product and BBB permeability in a murine model by histologic confirmation. Coronal MR imaging was performed on mice treated with mannitol (n = 6) for disruption of the BBB and controls treated with saline (n = 5). Permeability surface-area product was determined by ROI placement and was compared between saline- and mannitol-treated mice. Correlation was made with contrast-enhancement measurements and immunohistologic-stained sections of tripeptidyl peptidase-1 distribution in mice treated with mannitol and saline followed by injection of a viral vector containing the CLN2 gene, which directs production of tripeptidyl peptidase-1. Significantly increased permeability surface-area product was seen in mannitol- compared with saline-treated mice in the whole brain (P = .008), MCA territory (P = .014), and mixed vascular territories (P = .008). These findings were compared with contrast-enhancement measurements of BBB permeability and were correlated with immunohistologic-stained sections demonstrating BBB permeability to a large vector. Permeability surface-area product is increased in situations with known disruptions of the BBB, as evidenced by immunologic staining of large-vector passage through the BBB and concordance with contrast-enhancement measurements in a murine model. Quantitative permeability surface-area product has potential as an imaging marker of BBB permeability.

Decreased TUSC3 Promotes Pancreatic Cancer Proliferation, Invasion and Metastasis.

Pancreatic cancer is an aggressive disease with dismal prognosis. It is of paramount importance to understand the underlying etiological mechanisms and identify novel, consistent, and easy-to-apply prognostic factors for precision therapy. TUSC3 (tumor suppressor candidate 3) was identified as a potential tumor suppressor gene and previous study showed TUSC3 is decreased in pancreatic cancer at mRNA level, but its putative tumor suppressor function remains to be verified. In this study, TUSC3 expression was found to be suppressed both at mRNA and protein levels in cell line models as well as in clinical samples; decreased TUSC3 expression was associated with higher pathological TNM staging and poorer outcome. In three pairs of cell lines with different NF-κB activity, TUSC3 expression was found to be reversely correlated with NF-κB activity. TUSC3-silenced pancreatic cancer cell line exhibited enhanced potential of proliferation, migration and invasion. In an orthotopic implanted mice model, TUSC3 silenced cells exhibited more aggressive phenotype with more liver metastasis. In conclusion, the current study shows that decreased immunological TUSC3 staining is a factor prognostic of poor survival in pancreatic cancer patients and decreased TUSC3 promotes pancreatic cancer cell proliferation, invasion and metastasis. The reverse correlation between NF-κB activity and TUSC3 expression may suggest a novel regulation pattern for this molecule.

Alzheimer’s disease: A Commentary on Biofilms, Beta Amyloid and their Locations

The pathological study of brains affected by Alzheimer’s disease (AD) has shown the presence of spirochetal infection. These bacteria produce a detectible biofilm as a defense mechanism, impenetrable by both the immune system and antibiotic medications. The failure of the innate immune system at the site of biofilm deposition ultimately leads to the pathognomonic beta-amyloid (Aβ) plaques of AD at the exact same location as visualized by immunological staining. Furthermore, recent studies show that senile plaques are partially composed of bacterial Aβ in addition to that of the host. The bacterial and neuropathology similarities continue with similar staining results using all of the following: silver impregnation techniques, green thioflavin S fluorescence, anti-AβPP, and anti-AβPP antibodies. TUNEL assay of senile plaques further support the DAPI visualization and in situ hybridization detection of bacterial DNA with extracellular DNA fragmentation that corresponds to spirochetal apoptosis in the plaque’s biofilms, confirming the plaques are composed of spirochetes, Aβ, and biofilm. These significant similarities of biofilms and Aβ substantiate our hypothesis that amyloid of AD is located in the extracellular space.

Immunological impression cytology of the conjunctival epithelium in patients with thyroid orbitopathy-related dry eye

Thyroid orbitopathy (TO) is an autoimmune disease that is complicated by ocular surface disorders, leading to discomfort. Dry eye is very prevalent in patients with TO. Recent studies on the pathogenesis of dry eye have focused on the inflammatory process, and some supporting evidence has been discovered. Because TO is a disorder of autoimmune origin, we assumed that the association between TO and dry eye is related to inflammation. Inflammation of the ocular surface in TO-related dry eye has not been well studied. In this study, we assessed cellular inflammation of the ocular surface and the cytokine profiles in patients with TO-related dry eye. Conjunctival impression cytology (CIC) was assessed with an immunofluorescent assay. TO-related dry eye was diagnosed by using the Schirmer test, tear break-up time, thyroid function, and clinical signs. CIC was combined with immunological staining of interleukin-1a (IL-1a), IL-1b, and IL- 6. The immunological impression cytology (IC) grade was compared to the clinical activity score of TO. All TO patients with dry eye were positive for IL-1a, IL-1b, and IL-6. However, the normal controls were also positive for IL-1a. A trend was observed between the clinical inflammatory score and immunological IC grade. This study was the first to delineate the immunological IC of TO-related dry eye. Our study aimed to investigate the pathogenesis of dry eye in TO. Our findings suggest that the conjunctival cytokines IL-1a, IL-1b, and IL-6 may play a role. The results of this study will be useful for future studies of additional inflammatory cytokines, and the levels of these cytokines could be used as an outcome to assess the efficacy of treatment, such as anti-cytokine or immunosuppression therapy, in patients with TO-related dry eye or other ocular surface inflammatory disorders.

Visualizing Long Noncoding RNAs on Chromatin.

Fluorescence in situ hybridization (FISH) enables the detection of specific nucleic acid sequences within single cells. For example, RNA FISH provides information on both the expression level and localization of RNA transcripts and, when combined with detection of associated proteins and chromatin modifications, can lend essential insights into long noncoding RNA (lncRNA) function. Epigenetic effects have been postulated for many lncRNAs, but shown for only a few. Advances in in situ techniques and microscopy, however, now allow for visualization of lncRNAs that are expressed at very low levels or are not very stable. FISH-based detections of RNA and DNA coupled with immunological staining of proteins/histone modifications offer the possibility to connect lncRNAs to epigenetic effects. Here, we describe an integrated set of protocols to detect, individually or in combination, specific RNAs, DNAs, proteins, and histone modifications in single cells at a high level of sensitivity using conventional fluorescence microscopy.

The Nanoscale Observation of the Three-Dimensional Structures of Neurosynapses, Membranous Conjunctions Between Cultured Hippocampal Neurons and Their Significance in the Development of Epilepsy.

The nanoscale three-dimensional structures of neurosynapses are unknown, and the neuroanatomical basis of epilepsy remains to be elucidated. Here, we studied the nanoscale three-dimensional synapses between hippocampal neurons, and membranous conjunctions between neurons were found with atomic force microscopy (AFM) and confirmed by transmission electron microscope (TEM), and their pathophysiological significance was primarily investigated. The neurons and dendrites were marked by MAP-2, axons by neurofilament 200, and synapses by synapsin I immunological staining. In the synapsin I-positive neurite ends of the neurons positively stained with MAP-2 and neurofilament 200, neurosynapses with various nanoscale morphology and structure could be found by AFM. The neurosynapses had typical three-dimensional structures of synaptic triplet including the presynaptic neurite end, synaptic cleft of 30 ∼ 40 in chemical synapses and 2 ∼ 6nm in electrical ones, the postsynaptic neurite or dendrite spine, the typical neurite end button, the distinct pre- and postsynaptic membranes, and the obvious thickening of the postsynaptic membranes or neurites. Some membranous connections including membrane-like junctions (MLJ) and fiber-tube links (FTL) without triplet structures and cleft were found between neurons. The development frequencies of the two membranous conjunctions increased while those of the synaptic conjunctions decreased between the neurons from Otx1 knock-out mice in comparison with those between the neurons from normal mice. These results suggested that the neuroanatomical basis of Otx1 knock-out epilepsy is the combination of the decreased synaptic conjunctions and the increased membranous conjunctions.

Abstract 16016: Role For Macitentan in Angiogenesis of Right Ventricle, Peripheral Muscle and Lungs in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a vascular disease characterized by remodeling of distal pulmonary arteries (PA) leading to increased mean PA pressure, subsequent right ventricular (RV) failure and ultimately death of patients. Impaired angiogenesis is now recognized as a major factor in PAH etiology. It contributes not only to the decrease in lung perfusion but also to a rarefaction of the capillary network within the skeletal muscle and RV leading to exercise intolerance and RV failure. Several studies have suggested that endothelin-1 (ET-1) contributes to decrease angiogenesis in PAH. In light of these observations, we hypothesized that macitentan, an ET-1 receptors blocker, improves PAH by promoting angiogenesis in lungs, RV and skeletal muscles. Methods/Results: In the Sugen/hypoxia rat model of PAH, we demonstrated by right catheterization in closed chest rats that compared to vehicle treated animals, daily treatment during two weeks with macitentan (30 mg/kg) decreases significantly mean PA pressure (n=8-20; p<0.01) and total pulmonary resistance (n=8-20; p<0.05) and improves cardiac output, stroke volume (n=7-17; p<0.05) and RV hypertrophy measured by echo (n=6-10; p<0.05). These effects were associated with an increase of 2.9 fold in lung blood perfusion (n=6), 4.1 fold in quadriceps perfusion (n=5-7) and 1.5 fold in right ventricle perfusion (n=5-6) measured in vivo by micro CT angiogram (p<0.05). Both in vivo lectin perfusion (n=4-10; p<0.05) and immunologic CD31 staining (n=4-5; p<0.05) confirmed these findings. In the lungs, the hemodynamic and perfusion improvements were associated with decreased vascular remodeling (H&E) (n=6-17; p<0.01). In vitro, using primary cultured human endothelial cells isolated from distal PA (PAEC) and RV (RVEC), we showed that macitentan decreases PAH-PAEC proliferation (Ki67 assay) and resistance to apoptosis (TUNEL assay), while increasing angiogenesis capacity of healthy PAEC and RVEC (matrigel assay). Conclusion: Our data provide structural and functional evidence that ET-1 receptor inhibition with macitentan may improve PAH by promoting angiogenesis.

A Rare Presentation of Metastatic Merkel Cell Carcinoma

Introduction: Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignancy that typically affects elderly Caucasians and immunocompromised patients. It arises from primary neural cells and has a predilection for regional lymphatic metastasis as well as distant spread to the skin, lung, liver, brain and bones. Metastasis to the bowel is rare; there have only been 6 previous reports of spread to the stomach, small bowel or colon and 1 describing spread to the small bowel mesentery. This case reports a patient with MCC who was found to have metastasis to the duodenum presenting with melena and anemia. Case Presentation: A 79 year old male with a history of MCC, atrial fibrillation and an aortic valve replacement presented with four days of melena. He was diagnosed with MCC of the right upper extremity two years prior, treated with surgery and radiation. He had local recurrence, currently being treated with chemotherapy and radiation. A recent PET scan showed increased marrow uptake along with pancytopenia, suggesting bone marrow infiltration. Otherwise he was without known metastatic disease. The patient underwent an EGD which revealed a large, fungating, ulcerative mass in the first and second portions of the duodenum. Histopathology revealed sheets of small hyperchromatic cells with nuclear molding and scant cytoplasm filling the lamina propria. Immunologic staining was positive for AE1/AE3 cytokeratin, perinuclear dot-like expression of CK 20, CD 56 and chromogranin. These findings were consistent with a diagnosis of metastatic MCC. Discussion: MCC remains a rare cutaneous malignancy, however its incidence has tripled over the last two decades. Up to fifty percent of patients will eventually develop metastasic disease, most commonly to regional lymph nodes, other skin sites, lung, liver, brain and bone. Spread to the bowel is very unusual, with only six previous cases reported between 1985 and 2010 describing lesions in the upper and lower gastrointestinal tract. An additional case in 2011 described a lesion in the mesentery which infiltrated the small bowel causing vague abdominal pain. This case highlights the utility in knowing the type of cutaneous malignancy in a patient's history when evaluating abdominal symptoms, to determine if metastatic disease is a concern.

Glomus tumour masquerading as an aural polyp in chronic middle ear disease: A case report

Abstract Paraganglioma is an uncommon benign tumour of the temporal bones. It usually causes pulsatile tinnitus, recurrent ear bleeds, deafness or facial palsy and is rarely associated with chronic suppurative otitis media (CSOM). The latter may lead to false histopathological findings. We present an unusual case of a 45-year-old female with a right ear glomus tumour that was associated with CSOM and a large polyp protruding from the auditory canal. Despite preoperative investigations including computed tomography, diagnosis of the tumour could not be established. After taking a biopsy, a curative operation had to be abandoned because of a torrential intra-operative haemorrhage. The initial biopsy report suggested cholesteatoma; however, further histopathological studies including S-100 protein immune-staining revealed it to be paraganglioma. Large aural polyps and granulation tissues in CSOM can mask the characteristic histopathological features of these vascular tumours. We recommend including glomus tumour in the differential diagnosis of similar cases and performing optimum preoperative radiological investigations and immunological staining to confirm the diagnosis.

Human keratin 8 variants promote mouse acetaminophen hepatotoxicity coupled with c-jun amino-terminal kinase activation and protein adduct formation.

Keratins 8 and 18 (K8/K18) are the intermediate filaments proteins of simple-type digestive epithelia and provide important cytoprotective function. K8/K18 variants predispose humans to chronic liver disease progression and poor outcomes in acute acetaminophen (APAP)-related liver failure. Given that K8 G62C and R341H/R341C are common K8 variants in European and North American populations, we studied their biological significance using transgenic mice. Mice that overexpress the human K8 variants, R341H or R341C, were generated and used together with previously described mice that overexpress wild-type K8 or K8 G62C. Mice were injected with 600 mg/kg of APAP or underwent bile duct ligation (BDL). Livers were evaluated by microarray analysis, quantitative real-time polymerase chain reaction, immunoblotting, histological and immunological staining, and biochemical assays. Under basal conditions, the K8 G62C/R341H/R341C variant-expressing mice did not show an obvious liver phenotype or altered keratin filament distribution, whereas K8 G62C/R341C animals had aberrant disulphide cross-linked keratins. Animals carrying the K8 variants displayed limited gene expression changes, but had lower nicotinamide N-methyl transferase (NNMT) levels and were predisposed to APAP-induced hepatotoxicity. NNMT represents a novel K8/K18-associated protein that becomes up-regulated after K8/K18 transfection. The more pronounced liver damage was accompanied by increased and prolonged JNK activation; elevated APAP protein adducts; K8 hyperphosphorylation at S74/S432 with enhanced keratin solubility; and prominent pericentral keratin network disruption. No differences in APAP serum levels, glutathione, or adenosine triphosphate levels were noted. BDL resulted in similar liver injury and biliary fibrosis in all mouse genotypes. Expression of human K8 variants G62C, R341H, or R341C in mice predisposes to acute APAP hepatotoxicity, thereby providing direct evidence for the importance of these variants in human acute liver failure.

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Autocrine pathways involving S100A8 and/or S100A9 that are postulated to regulate the immunological functions of macrophages in rats

The development of ulcerative colitis (UC) is closely associated with abnormally functioning macrophages. Rat S100A8 (r-S100A8) and r-S100A9 (S100 proteins) is abundantly expressed in immune cells of myeloid origin, macrophages; however, it remains unclear why r-S100A9 is dominantly expressed in the macrophages of UC rats (UCR). The purpose of this study was to verify the immunological roles of S100 proteins in UCR. We observed the distribution of S100 protein-positive macrophages in the large colons of UCR using a fluorescent immunological staining method, so that S100 protein-positive macrophages were restricted to the rectal tissues of the UCR, and that the mRNA levels of r-S100A8 and r-S100A9 were up-regulated by stimulation with recombinant rat S100A8 (rr-S100A8) alone and rr-S100A9 alone, respectively. When the changes in the mRNA levels of r-S100A8 and r-S100A9 in macrophages were examined in in vitro study by PCR and real-time PCR, the mRNA levels of anti-inflammatory and inflammatory cytokines increased selectively after stimulation with rr-S100A8 alone and rr-S100A9 alone, respectively. These results suggest that autocrine signal transduction pathways involving S100 proteins regulate the immunological functions of macrophages to maintain homeostasis in the gastrointestinal tract. This may be depended on expression balance of S100 proteins in macrophages. It is strongly suggested that in UCR the immune functions of macrophages are regulated in a complex manner by r-S100A8 and/or r-S100A9 through undefined autocrine pathways on the cells.

Co-Expression of Foreign Proteins Tethered to HIV-1 Envelope Glycoprotein on the Cell Surface by Introducing an Intervening Second Membrane-Spanning Domain

The envelope glycoprotein (Env) of human immunodeficiency virus type I (HIV-1) mediates membrane fusion. To analyze the mechanism of HIV-1 Env-mediated membrane fusion, it is desirable to determine the expression level of Env on the cell surface. However, the quantification of Env by immunological staining is often hampered by the diversity of HIV-1 Env and limited availability of universal antibodies that recognize different Envs with equal efficiency. To overcome this problem, here we linked a tag protein called HaloTag at the C-terminus of HIV-1 Env. To relocate HaloTag to the cell surface, we introduced a second membrane-spanning domain (MSD) between Env and HaloTag. The MSD of transmembrane protease serine 11D, a type II transmembrane protein, successfully relocated HaloTag to the cell surface. The surface level of Env can be estimated indirectly by staining HaloTag with a specific membrane-impermeable fluorescent ligand. This tagging did not compromise the fusogenicity of Env drastically. Furthermore, fusogenicity of Env was preserved even after the labeling with the ligands. We have also found that an additional foreign peptide or protein such as C34 or neutralizing single-chain variable fragment (scFv) can be linked to the C-terminus of the HaloTag protein. Using these constructs, we were able to determine the required length of C34 and critical residues of neutralizing scFv for blocking membrane fusion, respectively.

The predictive value of Gen-Probe's amplified Mycobacterium tuberculosis direct test compared with culturing in paraffin-embedded lymph node tissue exhibiting granulomatous inflammation and negative acid fast stain

The diagnosis of granulomatous inflammation with possible tuberculosis (TB) infection in histopathology is often difficult. There is a need for a rapid and reliable diagnostic test. Thus, we evaluated the performance of the Mycobacterium tuberculosis direct (MTD) test in specimens with granulomatous lymphadenitis and negative acid fast stains. The M. tuberculosis direct (MTD) test by Gen-Probe was performed on 45 formalin-fixed paraffin-embedded tissue samples including 34 lymph nodes. We measured the predictive values of the MTD test in specimens with granulomatous lymphadenitis and negative acid fast stains. The overall test sensitivity was 73.9%, and specificity was 95.4%. The MTD test sensitivity and specificity for lymph node tissue were 72.7% and 91.67%, respectively. In the presence of granulomatous inflammation, the MTD test sensitivity and specificity were higher than those for all tissue samples, at 75% and 100%, respectively. Based on this study, the MTD test should be used as a supportive test in addition to conventional histochemical or immunological staining methods when evaluating lymph node tissue with a granulomatous inflammation to deliver stronger evidence to support clinical decisions at a much earlier time than a culture would allow.

BMP-2 can promote the osteogenic differentiation of human endometrial stem cells

Abstract Background: Human endometrial-derived stem cells (hEnSCs) as multipotent accessible source of cells are known as useful cell candidates in the field of bone tissue engineering. However, the effect of bone morphogenic protein-2 (BMP-2) as an osteoinductive growth factor has not been clearly ascertained. Objective: To evaluate the effect of the remarkable osteoinductive growth factor BMP-2, on promotion of osteogenic differentiation in hEnSCs. Methods: Endometrial biopsies were obtained from healthy women referred to the hospital for infertility treatment. After tissue digestion in collagenase, the isolated endometrial cells were expanded in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS. The propagated cells were characterized based on the expression of endometrial (CD90, CD105), endothelial (CD31), and hematopoietic (CD34, CD133) stem cell markers. Cells were differentiated in osteogenic medium containing DMEM supplemented with 10% FBS, 10 nM dexamethasone, 50 μg/ml Ascorbic acid, and 10 mM β-glycerophosphate in the presence or absence of BMP-2 for 21 days. Alizarin red staining was performed to verify the matrix mineralization. Immunocytochemical staining was conducted to detect the expression of OCT-4, CD133, and osteopontin as well as osteocalcin. The expression of osteoblast transcripts, including osteopontin, osteonectin, and alkaline phosphatase (ALP) were analyzed by semi quantitative PCR. Results: The expanded EnSCs were spindle shaped. They were positive for the expression of Oct-4, CD90, and CD105, while they were negative for endothelial and hematopoietic markers. The matrix mineralization was confirmed by Alizarin red in both groups at day 21. Although the expression of osteopontin and osteocalcin was detected in both groups by immunological staining, the expression of osteocalcin was more intense in the presence of BMP-2. ALP, Osteonectin and osteopontin transcripts were expressed in all groups; however, the expression of ALP and osteopontin was upregulated in the presence of BMP-2. Conclusion: BMP-2 as an osteoinductive growth factor, could promote the osteogenic differentiation of EnSCs in vitro.

Contrast-Enhanced Ultrasonograpic Studies on Pancreatic Carcinoma with Special Reference to Staining and Muscular Arterial Vessels

Comparative study of contrast-enhanced ultrasonography (CE-US) and histopathology of surgically resected specimens in 13 patients with pancreatic carcinoma. A time intensity curve was used to determine the percentage brightness increase in cancerous and normal regions and the patients were divided into two groups, hyperperfusion, with a percentage brightness increase over 80% (n=6) and hypoperfusion, with an increase of less than 80% (n=7) on CE-US. The hyperperfusion group included well-differentiated tubular adenocarcinoma, adenosquamous cell carcinoma and acinar cell carcinoma, while all 7 patients in the hypoperfusion group had moderately differentiated tubular adenocarcinoma. Immunological staining (α-SMA and anti-CD34) of the resected specimens showed significantly higher microartery count (MAC) in the hyperperfusion group (p<0.005) than in the hypoperfusion group or normal pancreas. In the normal pancreas, the mean vessel diameter was significantly higher (over 100 μm) than in the hyperperfusion group (30 μm; p<0.005). It was concluded that a muscular arterial vessel density of less than 30 μm is an important factor in determining staining degree and carcinoma progression by CE-US in pancreatic carcinoma.

Whole-mount in situ hybridization (WISH) optimized for gene expression analysis in mouse embryos and embryoid bodies.

Whole-mount in situ hybridization (WISH) is a technique widely used in developmental biology to study the localization of RNA sequences in intact tissues or whole organisms. In this chapter we present a detailed protocol that was optimized for gene expression analysis in early stage mouse embryos (5.5-10.5 days post-coitum) and embryoid bodies formed by differentiating embryonic stem cells and can be used for the detection of up to two distinct RNA sequences simultaneously. The initial steps of the procedure are the generation of the labeled riboprobe(s) and the embryo or embryoid body preparation, which can be completed in less than 2 days. The actual WISH procedure, comprised of the hybridization, the post-hybridization washes, and the immunological staining, can be completed in 3 days.

Establishment of a Promising Human Nucleus Pulposus Cell Line for Intervertebral Disc Tissue Engineering

Low-back pain caused by intervertebral disc degeneration could be recovered by the regeneration of the nucleus pulposus (NP). This study aimed to establish a chondrogenic recovery model with promising a human NP (hNP) cell line, an immortalized hNP (ihNP), which could be a screening platform to identify regenerative drugs. The ihNP cells were created from primary human NP cells transfected with a retroviral vector-driven HPV16 E6/E7. Growth properties and characteristics of ihNP were evaluated by comparing with parental NP cells. Successful immortalization of ihNP cells stably expressed HPV 16 E6/E7 mRNA. The doubling time of ihNP was shortened to 53.16±2.63 h compared with parental hNP-P1. Cell cycle regulators, including p53, p21, and pRB were downregulated compared to parental hNP-P1. The in vivo neoplastic forming assay also demonstrated that the ihNP was nontumorigenic. After 25 generations of cell cultures, the ihNP cells, yet stably expressed chondrogenic genes, including (SOX9), type II collagen (Col II), aggrecan, decorin, biglycan, and versican. Higher expressions of chondrogenic proteins, including Col II, phosphorylated SOX9 (p-SOX9), and CD44 were also determined. Under the stressful inflammatory conditions induced by lipopolysaccharides (LPS), the regenerative and anti-inflammatory potentials of ihNP in two-dimensional culture with the presence of platelet-rich plasma (PRP) were evaluated by reverse transcriptase polymerase chain reaction. PRP showed significant effects on restoring diminished chondrogenic markers and deleterious inflammatory responses induced by LPS in ihNP. The therapeutic potentials of ihNP in three-dimensional neocartilage model could also be exerted by PRP using histological evaluation and immunological staining. Hence, the established ihNP cells can provide a chondrogenic recovery model as a regenerative drug screening tool for further regenerative drug discovery and development.

Epstein–Barr Virus Induces Activation-Induced Cytidine Deaminase Expression in T or NK Cells Leading to Mutagenesis and Development of Lymphoma

IntroductionEpstein–Barr virus (EBV) infects B cells and rarely T or NK cells, causing EBV-positive T/NK-cell lymphoproliferative neoplasms (EBV-T/NK-neoplasm), such as extranodal NK/T-cell lymphoma, aggressive NK-cell leukemia, and EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs). EBV-T/NK-LPDs are fatal disorders presenting sustained inflammation, such as infectious mononucleosis-like symptoms, hypersensitivity to mosquito bites, or hydroa vacciniforme-like eruption accompanied by clonal proliferation of EBV-infected T or NK cells. EBV infects B cells making them immortal and resulting in B-cell lymphomas. However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies has not been elucidated yet. Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin genes. Deregulated AID expression acts as a genomic mutator leading to the development of B-cell lymphoma. In addition, EBV infection induces AID expression in B cells. These findings indicate that AID has a role in EBV-induced lymphomagenesis in B cells. However, of interest, AID transgenic mice developed T-cell lymphoma (J. Exp. Med., 197, p.1173, 2003). Recently, Nakamura et al. reported that AID expression was upregulated in the peripheral blood mononuclear cells (PBMCs) of EBV-T/NK-LPDs patients (Eur. J. Dermatol., 21, p.780, 2011). Therefore, we hypothesized that EBV infection induces AID expression in T or NK cells contributing to the tumor development. ObjectivesWe designed this study to investigate AID expression and its contribution to EBV-T/NK-neoplasms development. Materials and MethodsEBV-positive T- and NK-cell lines, SNT8, SNK6, SNT13, SNT15, and SNT16, were examined. The EBV-negative T-cell line HPB-ALL, Jurkat and human primary T cells derived from cord blood were used as the negative controls. Clinical samples were obtained from EBV-T/NK-LPDs patients who were diagnosed according to the previously described criteria (Blood, 119, p.673, 2012). To detect and isolate EBV-infected cells, T and NK cells were separated using magnetic beads from PBMCs. AID expression in tissue lesions were examined in clinical samples and xenograft models of EBV-T/NK-LPDs generated by transplantation of PBMCs from the EBV-T/NK-LPDs patients to NOD/Shi-scid, IL-2R γKO mice. For in vitro EBV infection, EBV was prepared from the culture medium of B95-8 cells and added to HPB-ALL or Jurkat cells (Proc. Natl. Acad. Sci., 100, pp.7836-40, 2003). To determine AID-induced mutation of the gene, DNA was extracted from SNT13, SNK6, or EBV-infected Jurkat cells, and c-myc gene was cloned by TA cloning, which was then sequenced. We compared the number of the mutation with that of Jurkat cells. ResultsWe detected AID expression in EBV-positive T- or NK-cell lines by RT-PCR, western blotting, and immunological staining using confocal microscopy, whereas it was not detected in the control. Furthermore, we validated the results in EBV-infected T or NK cells derived from 12 EBV-T/NK-LPDs patients (infected cell types: CD4, 5; CD8, 3; and CD56, 4). Quantitative RT-PCR demonstrated that AID expression was upregulated in EBV-infected T or NK cells compared with the control. Expression was confirmed by immunological staining using confocal microscopy in 5 patients. We also detected AID expression by histopathological staining in EBV-infected cells in lesions of 4 patients and the EBV-T/NK-LPDs xenograft models. Subsequently, we examined the role of EBV for AID expression in T cells. It was demonstrated that AID expression was induced by in vitro EBV infection in the T-cell line HPB-ALL and primary T cells. Moreover, it was demonstrated by the luciferase assay that the viral protein LMP1 upregulated AID promoter activity in HPB-ALL cells. Finally, we found that SNT13, SNK6, and EBV-infected Jurkat cells had increased number of the mutation of c-myc gene compared with Jurkat cells. ConclusionsWe previously reported that EBV infection of T or NK cells enhances survival of the infected cells by the activation of NF-κB. The results of the present study suggest that EBV contributes to the development of EBV-T/NK-neoplasms through NF-κB-induced cell survival and AID-induced mutagenesis leading to clonal evolution and expansion. Disclosures:No relevant conflicts of interest to declare.