Use of cytological samples of metastatic melanoma for ancillary studies.

OBJECTIVE: Fine needle aspiration (FNA) is widely used in the diagnosis of metastatic melanoma, both at initial presentation and in the setting of recurrent disease. The purpose of this study was to evaluate the performance of confirmatory immunohistochemistry (IHC) and molecular analysis of the BRAF mutation in cytological preparations of metastatic melanoma. METHODS: A 2-year retrospective review of pathology reports was performed on cytological samples of metastatic melanoma at the University Health Network (Toronto, Canada) and the Santa Casa Medical School (Sao Paulo, Brazil). IHC was performed on cell block sections prepared from formalin-fixed, fresh samples or residuum of CytoLyt/PreservCyt post-fixed in formalin. BRAF V600E/K mutations were assessed by amplification refractory mutation system (ARMS) analysis. RESULTS: A total of 104 samples (94 FNAs and 10 fluids) from 83 patients (20 women, 63 men) were included. IHC was attempted in 43 cases (41.3%) and successful in 41 (95.3%). The panel number of antibodies ranged from 1 to 15 (median 3). The most frequently used melanoma markers included HMB-45, melanoma cocktail and S100 protein, used in 25 (58.1%), 23 (53.5%) and 18 samples (41.9%). Thirty cases (69.8%) used three or fewer markers. The BRAF V600E/K mutation was tested in eight samples, being successful in seven (87.5%) and positive in three (37.5%). CONCLUSIONS: Cytological samples are a reliable and sufficient source for IHC and subsequent molecular analysis, allowing a reduced diagnostic time and rapid, appropriate treatment options in patients with advanced melanoma.

Upregulation of relaxin receptors in the PDL by biophysical force.

OBJECTIVES: We have previously reported that relaxin (Rln) expression from the ovary is upregulated by orthodontic tooth movement. This study was performed to test the hypothesis that Rln family peptides (Rxfps), the G-protein-coupled Rln receptor, is induced in periodontal ligament (PDL) cells to modulate the molecules involved in periodontal tissue remodeling while applying biophysical force. MATERIALS AND METHODS: Rats were implanted with orthodontic appliances to investigate changes to Rxfps in vivo. An in vitro biophysical force analysis was performed to measure the level of Rxfp 1 messenger RNA (mRNA) in primary human PDL cells. RESULTS: The levels of Rxfp 2 transcription and translation increased in a time-dependent manner during tooth movement. Rxfp 2 was localized in the PDL by immunofluorescence. In vitro analyses revealed that the level of Rxfp 1 mRNA in PDL cells increased significantly with both compression and tension force. The levels of matrix metalloproteinase (MMP)-1, MMP-2, interleukin-6, and vascular endothelial growth factor mRNA, which are important for periodontal tissue remodeling, also changed under force application and Rln treatment. CONCLUSIONS: PDL cells responded to Rln to modulate effector molecules for periodontal tissue remodeling by upregulating Rxfps expression under a biophysical force. CLINICAL RELEVANCE: Rln and Rxfps may serve as a PDL turnover molecule complex to control orthodontic tooth movement.

Cytomorphological and clinicopathological spectrum of pulmonary marginal zone lymphoma: the utility of immunophenotyping, PCR and FISH studies.

OBJECTIVE: To review cytomorphological criteria and clinicopathological findings in combination with ancillary tests for the specific diagnosis of pulmonary marginal zone lymphoma (MZL) in fine needle aspiration (FNA) specimens. METHODS: Cases of pulmonary MZL diagnosed using cytological specimens from 2005 to 2012 were retrieved and reviewed by three cytopathologists. Results of immunophenotypic analysis, interphase fluorescence in situ hybridization (FISH) and molecular assays were collated, together with clinical information and imaging data. Concurrent surgical biopsies were also retrieved. RESULTS: Fifteen lung FNA specimens were identified. The smears consisted predominantly of small centrocyte-like cells. Marked plasma cell differentiation was evident in 11 cases. All cases with slides available showed tissue fragments with lymphoid tangles (TFLTs). Multinucleated giant cells were present in nine cases, two of which showed granulomas. Immunophenotyping confirmed B-cell clonality in all cases. B-cell clonality was detected by polymerase chain reaction (PCR) in two samples. FISH identified MALT1 translocation in four of 10 cases tested and trisomy 3 in three of four cases. Concurrent surgical biopsies were diagnosed independently as MZL in seven cases. CONCLUSIONS: Cytology smears from lung FNA samples consisting of small lymphoid cells with a relative abundance of plasma cells or plasmacytoid cells and large TFLTs should prompt immunophenotyping and other ancillary studies, even if multinucleated giant cells and poorly formed granulomas are also identified. Specific diagnosis of pulmonary MZL in FNA samples can be rendered on the basis of morphological features coupled with the demonstration of B-cell clonality by immunophenotyping or PCR and cytogenetic abnormalities by FISH.

Epstein-Barr virus encoded RNA detected by in situ hybridization using cytological preparations.

OBJECTIVE: Detection of Epstein-Barr virus (EBV) status might help in the diagnosis of EBV-related neoplasms. The rate of successful assays for the detection of EBV-infected cells in cytological preparations has not been fully explored. Our aims were to examine the rate of successful in situ hybridization (ISH) assays for EBV-encoded RNA (EBER) in cytological specimens and to explore reasons for failure. METHODS: An electronic search selected cases with ISH-EBER assays performed on cytological preparations during a 10-year period. Data regarding patient age, gender and immune status, sample type and site, type of preparation, ISH-EBER results, immunophenotyping and immunohistochemistry results, final diagnosis and correspondent histopathological samples were retrieved. RESULTS: Sixty specimens from 58 patients with diagnoses of lymphoproliferative disorder (n = 35), carcinoma (n = 24) and sarcoma (n = 1) were identified. ISH-EBER assays were performed on 50 cell block sections and on 10 cytospin preparations, with 22 positive and 32 negative results. Six tests (four cytospins and two cell block sections) failed owing to loss of material during the assay and background staining, with an overall failure rate of 10% and 4% if cytospins were excluded. Assays were performed on 13 cytology and surgical specimens from the same site, with only one discrepant result. CONCLUSIONS: Cell block sections had more successful ISH-EBER assays when compared with cytospins. Reasons for failure were loss of material on the slide and background staining. A high concordance rate with surgical specimens emphasizes the usefulness of cytological samples for determining EBV status in patients with exhausted or no histological material available.

Lipopolysaccharide-induced indoleamine 2,3-dioxygenase expression in the periodontal ligament.

BACKGROUND AND OBJECTIVE: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-oxidizing enzyme with immune-inhibitory effects. The aim of this study was to investigate the expression of IDO by lipopolysaccharide (LPS), a component of gram-negative bacteria, in human periodontal ligament (PDL) cells. MATERIAL AND METHODS: Human PDL cells and gingival fibroblasts (GFs) were prepared from explants of human PDLs and from gingival tissues of clinically healthy donors, respectively. Real-time RT-PCR, western blotting and the IDO enzyme assay were performed to determine the expression of IDO following LPS treatment of cells. LPS was injected into mice tail veins to evaluate the effects of LPS in vivo in the maxillary first molar. Immunofluorescence staining and histological analysis were followed to localize IDO in mouse PDL. RESULTS: The level of expression of IDO mRNA in primary human PDL cells after LPS treatment was increased in a dose-dependent manner, reaching a peak 8 h after LPS treatment. The expression and activities of IDO protein were significantly increased in comparison with those of the control. In addition, the increased production of kynurenine in culture medium was observed 72 h after LPS treatment. In the immunofluorescence findings, stronger immunoreactivities were shown in PDL than in gingival tissues in the maxillae. In accordance with the immunofluorescence findings, LPS treatment induced a strong up-regulation of IDO mRNA in human PDL cells, whereas human GFs showed only a weak response to LPS. CONCLUSION: These results clearly show that IDO was induced by LPS in primary human PDL cells, suggesting that PDL might be involved in the regulation of oral inflammatory disease.

Glutamine preferentially inhibits T-helper type 2 cell-mediated airway inflammation and late airway hyperresponsiveness through the inhibition of cytosolic phospholipase A(2) activity in a murine asthma model.

BACKGROUND: The non-essential amino acid, l-glutamine (Gln), is abundant in the human body. Gln exhibits beneficial effects on endotoxic shock through the inhibition of cytosolic phospholipase A(2) (cPLA(2)) activity. cPLA(2) has been reported to be implicated in the pathogenesis of asthma, but the effects of Gln on asthma have not yet been defined. OBJECTIVE: To investigate the effects of Gln on allergic bronchial inflammation and airway hyperresponsiveness (AHR), and to determine the possible action mechanisms of Gln in a murine model of asthma. METHODS: cPLA(2) phosphorylation was assessed by immunoprecipitation and Western blotting. Smears of bronchoalveolar lavage cells were stained with Diff-Quik solution for differential cell counting. Airway levels of the proteins [T-helper type-1 (Th1) and Th2 cytokines, and mucin] were measured by ELISA. mRNA expression of cytokines was assessed by real-time RT-PCR. AHR was assessed as a change in airway resistance (RL). Histological studies were performed to assess the levels of mucin and pulmonary inflammation. RESULTS: Systemic Gln administration inhibited cPLA(2) phosphorylation and its enzymatic activity in the lungs. Additionally, Gln effectively suppressed the key features of Th2-dependent asthmatic features, such as airway eosinophilia, mucus formation, and airway type 2 cytokine production, as well as late AHR. CONCLUSION: Gln was found to be effective in the suppression of Th2-dependent phenotypes and late AHR, and this effect of Gln appeared to be at least partially attributable to its ability to suppress cLPA(2) activity in the airway. Our results suggest that clinical use of Gln for patients with asthma may be beneficial.

Liver-Wrapping, Nitric Oxide-Releasing Nanofiber Downregulates Cleaved Caspase-3 and Bax Expression on Rat Hepatic Ischemia-Reperfusion Injury.

BACKGROUND: Hepatic ischemia-reperfusion injury (IRI) is an important determinant of the outcome of hepatic surgery, including re-section and transplantation. Previous studies have shown that nitric oxide (NO) has a protective effect against IRI. Therefore, many studies have examined methods for supplying NO. In this study, we investigated the effect of NO-releasing nanofibers on hepatic IRI in a rat model. METHODS: Male Sprague-Dawley rats were divided into 4 groups: control, IRI only (n = 3); group 1, hepatic IRI and liver-wrapping with nanofiber lacking NO (n = 4); group 2, hepatic IRI and liver-wrapping with NO rapid-releasing nanofiber (n = 4); and group 3, hepatic IRI and liver-wrapping with NO slow-releasing nanofiber (n = 5). RESULTS: The levels of aspartate aminotransferase and alanine aminotransferase were not significantly different between groups. On the basis of Western blots, Bax/ß-actin levels were significantly lower in group 2 than in group 3 (P < .01). Cleaved Caspase-3/ß-actin levels were significantly lower in group 2 than in the control, group 1, and group 3 (P < .05, .01, and .01, respectively). However, there were no significant differences in Bcl-2/ß-actin between groups. CONCLUSIONS: The liver-wrapping NO rapid-releasing nanofiber downregulated cleaved Caspase-3 and Bax expression. It has a protective effect by reducing apoptosis in hepatic IRI in rats.

Augmentation of tumor metastasis by platelet-activating factor.

The effect of platelet-activating factor (PAF) on experimental pulmonary metastasis by the B16F10 murine melanoma and the possible involvement of PAF in the activities of tumor necrosis factor alpha (TNF-alpha) and interleukin 1alpha (IL-1alpha) in tumor metastasis were investigated. i.p. injection of PAF enhanced the lung colonization in a dose- and time-dependent manner. PAF enhanced lung colonization when it was administered after, but not before, B16F10 inoculation. Multiple injections of PAF were more effective than a single injection. Neutralization of endogenous PAF with PAF antagonist BN50739 decreased lung colonization, suggesting that endogenous PAF plays an important role in pulmonary metastases. A single i.p. injection of TNF-alpha or IL-1alpha caused a marked enhancement in lung colonization. TNF-alpha- and IL-1alpha-mediated enhancement in lung colonies was significantly inhibited by BN50739. These results demonstrate that PAF has a metastasis-enhancing effect and is a mediator of the metastatic activities of TNF-alpha and IL-1alpha.

[Effects of modifications of lipoproteins by water soluble forms of lineoleic-hydroxamic acid on biochemical markers of development of atherosclerosis]. / Vplyv modyfikatsiï lipoproteïniv vodorozchynnoiu formoiu linoleïlgidroksamovoï kysloty na biokhimichni markery razvytku aterosklerozu.

Effects of water-soluble form of linoleic-hydroxamic acid, inhibitor of lipoxygenase, on process of atherogenesis (alimentary model) were studied at rabbits. It was shown that inhibition of activity of lipoxygenase during process of development of atherosclerosis led to considerable decrease of area of lipoid plaques and lipoidosis, decreased content of cholesterol in blood and improved free-radical modification of lipoproteins of blood.

[The status of lipid metabolism and free-radical processes in workers in a 30-kilometer exclusion zone]. / Stan lipidnoho obminu ta vil'noradykal'nykh protsesiv u pratsivnykiv 30-kilometrovoï zony vidchuzhennia.

Patterns were studied of lipid metabolism as was the state of free radical processes in different groups of those individuals working within a 30-km zone of estrangement depending on the length of time they stayed there. Atherogenically directed changes in blood lipid spectrum and processes of lipid peroxidation identified under working conditions in the zone of estrangement appeared to be more pronounced in subjects working at the object Ukryttia [correction of "Ukrytiye"] (Shelter) than in those in the zone working outside the object Ukryttia [correction of "Ukrytiye"].

Osteoprotegerin expressed by osteoclasts: an autoregulator of osteoclastogenesis.

Osteoprotegerin (OPG) is secreted by stromal and osteoblastic lineage cells and inhibits osteoclastogenesis by preventing the interaction of receptor activator of nuclear factor-κB ligand (RANKL) with receptor activator of nuclear factor-κB (RANK). In this study, the expression of OPG in osteoclasts themselves and its biological functions during osteoclastogenesis were investigated for the first time. OPG expression in vivo in the developing rat maxilla was examined by immunofluorescence analysis. OPG expression in osteoclasts during in vitro osteoclastogenesis was determined by reverse-transcription polymerase chain-reaction (RT-PCR), Western blot, and immunofluorescence staining. We determined the function of OPG produced by osteoclasts during osteoclastogenesis by silencing the OPG gene. The effects of OPG on bone-resorbing activity and apoptosis of mature osteoclasts were examined by the assay of resorptive pit formation on calcium-phosphate-coated plate and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. In the immunofluorescence findings, strong immunoreactivities were unexpectedly seen in multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts around the growing and erupting tooth germs in the rat alveolar bone. In vitro, OPG expression was significantly increased during the differentiation of osteoclasts from mouse bone-marrow-derived cells treated with a combination of macrophage colony-stimulating factor (M-CSF) and RANKL. Interestingly, it was found that OPG small interfering (si)RNA treatment during osteoclastogenesis enhanced the sizes of osteoclasts, but attenuated their bone-resorbing activity. Also, the increased chromosomal DNA fragmentation and caspase-3 activity in the late phase of osteoclastogenesis were found to be decreased by treatment with OPG siRNA. Furthermore, effects of OPG siRNA treatment on osteoclastogenesis and bone-resorbing activity were recovered by the treatment of exogenous OPG. These results suggest that OPG, expressed by the osteoclasts themselves, may play an auto-regulatory role in the late phase of osteoclastogenesis through the induction of apoptosis.

Alendronate affects cartilage resorption by regulating vascular endothelial growth factor expression in rats.

This study was performed to determine effects of alendronate on the tibial proximal epiphyseal cartilage undergoing endochondral ossification and the expression of vascular endothelial growth factor (VEGF) from the cartilage. Alendronate was injected subcutaneously every other day in postnatal Day 1 Sprague Dawley rats. The rats were sacrificed 3, 5, 7, and 10 days after the first injection. The effect of alendronate treatment for 10 days was demonstrated from the morphological change that the area of the secondary ossification center in the epiphysis was significantly smaller in the alendronate group than that in the control group (P < 0.05). Strong immunoreactivity to VEGF was observed in the hypertrophied chondrocytes and some proliferating chondrocytes in the epiphyseal cartilage at postnatal Day 5 and was decreased after the alendronate treatment for 5 days. Immunoreactivity was observed in not only hypertrophied cells but also the peripheral cartilaginous matrix adjacent to the vascular canals invading into the central portion of the cartilage at postnatal Day 7. This reactivity was also reduced considerably by the alendronate treatment for 7 days. The level of VEGF expression was reduced by the alendronate treatment at both the transcription and translation levels. However, the transcriptional level of the flt-1 and flk-1 receptors was relatively unaltered by the treatment. These results suggest that VEGF expression is required for vascular invasion into the developing cartilage and alendronate can affect its resorption by downregulating VEGF expression.

Parasympathectomy induces morphological changes and alters gene-expression profiles in the rat submandibular gland.

OBJECTIVE: The chorda-lingual (CL) nerve carries parasympathetic fibers to the hilum of the sublingual and submandibular glands (SMGs) and evokes the secretion of saliva. The effect of cutting the CL nerve on the biological processes in SMGs was investigated by examining the gene-expression profiles in the SMGs after a surgical parasympathectomy. METHODS: At day 3 after the CL nerve cut, the changes in the SMGs at both the experimental cut and contralateral control sides were analysed by microarray and light microscopy. The expression levels of 6 selected genes were confirmed by real-time PCR, Western blot and immunofluorescence staining. RESULTS: The wet weight of the parasympathectomised SMGs decreased significantly compared to that of the contralateral side (p<0.05). Histological analyses after the parasympathectomy showed a widened interacinar space as well as some atropic changes to the acini of the SMGs in the cut side. Microarray analysis revealed that twofold differential expression in mRNA expression in the parasympathectomized SMGs were detected in 88 genes (0.004%): 41 genes were overexpressed, 11 were underexpressed and 36 were unknown. Changes of the expression of 6 selected genes detected by Western blot and/or real-time PCR were consistent with the microarray data. CONCLUSION: The important genes involved in biological processes for salivation were identified through a large-scale gene expression analysis.

Expression of DCC in differentiating ameloblasts from developing tooth germs in rats.

OBJECTIVE: This study examined the expression pattern of the Deleted-in-colorectal-carcinoma (DCC) gene in developing rat tooth germs. METHODS: Rat pups at 4, 7 and 10 d postpartum were used in this study. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent localization were used to determine the level of DCC expression during tooth development. RESULTS: There was more than 2-fold higher level of DCC mRNA in the rat 2nd maxillary molar tooth germs on 10 d postpartum, which was the root stage, than in the rat 3rd maxillary molar tooth germ, which was at the cap/early bell development stage. In addition, the levels of DCC mRNA in the 2nd maxillary molar germs at 4, 7 and 10 d postpartum increased gradually according to tooth development. Interestingly, immunoreactivity against DCC was specifically detected in the differentiating ameloblasts. DCC was observed in the lateral and apical sides of the newly differentiating and secretory stage ameloblasts. Afterwards, DCC was localized only in the apical side of the maturation stage ameloblasts, not in the lateral side. CONCLUSION: DCC is expressed in the differentiating ameloblasts, which suggests that this molecule plays a crucial role in amelogenesis.

Effects of bisphosphonate on the endochondral bone formation of the mandibular condyle.

The development of the mandibular condylar cartilage is important for the overall growth of the mandible. However, there have been a few researches into medical approaches aimed at controlling condylar growth. This study examined the effects of bisphosphonate on the growth of the condylar cartilage. Alendronate (3.5 mg/kg/week) was administered to postnatal day 1 SD rats for 7 and 10 days. The thickness of each chondrocyte layer and the level of MMP-9 expression were measured. The anteroposterior diameter of the developing condyle was unaffected by the alendronate treatment for 7 days (P > 0.05). The total thickness of the cartilage layers was also unaffected by the treatment for 7 days (P > 0.05). In particular, there was no change in the thickness of the perichondrium and reserve cell layer at the measured condylar regions (P > 0.05). However, the thickness of the proliferating cell layer was reduced significantly, whereas the thickness of hypertrophied cartilage layer was increased (P < 0.05). The number of chondroclasts engaged in hypertrophied cartilage resorption was reduced significantly by the alendronate treatment (P < 0.05). The level of MMP-9 expression was reduced at both the transcription and translation levels by the alendronate treatment for 7 and 10 days. These results indicate that alendronate (>3.5 mg/kg/week) inhibits the longitudinal growth of the mandibular condyle by inhibiting chondrocyte proliferation and the resorption of hypertrophied cartilage for ossification.

Effects of alendronate on a disintegrin and metalloproteinase with thrombospondin motifs expression in the developing epiphyseal cartilage in rats.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been reported to play a role in the degradation of aggrecan, a major component of cartilage. This study was performed to examine the effects of alendronate on the expression of ADAMTS in developing femoral epiphyseal cartilage. Primary cultured chondrocytes from this cartilage were treated with alendronate in vitro and postnatal day 1 rats were injected subcutaneously with alendronate (1 mg/kg) every second day in vivo. The number of cultured chondrocytes and their aggrecan mRNA levels were unaffected by the alendronate treatment at 10(-6) to 10(-4) M concentrations. The mRNA levels of ADAMTS-1, -2 and -9 in chondrocytes were also unaffected. However, the levels of ADAMTS-5 and -4 were reduced significantly by the same treatment. The thickness of the proliferating chondrocyte layers and the aggrecan mRNA levels in the epiphysis were unaffected by the alendronate treatment in vivo. However, the hypertrophied chondrocyte layers became significantly thicker, and the size of the secondary ossification centre was reduced significantly by the same treatment (P < 0.05). Both ADAMTS-4 and -5 mRNA expressions were also reduced significantly in vivo. The immunoreactivity against ADAMTS-4 was seen in hypertrophied chondrocytes and reduced significantly by the alendronate treatment. These results suggested that alendronate can inhibit the degradation of aggrecan in the articular cartilage by downregulating the expression of matrix enzymes such as ADAMTS-4 and -5.

[Effect of incorporated ionizing radiation by cesium-137 on the state of free radical processes and the lipoprotein spectrum in rat blood]. / Vplyv inkorporovanoho ionizuiuchoho oprominennia tseziiem 137Cs na stan vil'noradykal'nykh protsesiv i sklad lipoproteïniv u krovi shchuriv.

In vivo experiments it was shown that the prolonged incorporated low levels radiation by 137Cs leads to changes in free-radical processes in blood and in lipoprotein exchange. It is considered that such changes could be very important for postradiation atherogenesis, as possible distant nonstochastic consequences of Chernobyl accident.

Analysis of clonality by X chromosome inactivation in uterine cervix cancer.

The determination of a unicellular or a multicellular origin of a tumor is an important due for understanding its etiology. To investigate this issue, we performed clonality assay of cervix cancer using polymerase chain reaction based on highly polymorphic locus of the androgen receptor gene, in which methylation of DNA correlates with inactivation of X chromosome. DNA samples were obtained from formalin-fixed, paraffin-embedded tissue of 20 invasive epidermoid carcinomas and 10 carcinoma in situ. Seven of ten carcinoma in situ, heterozygous for the androgen receptor locus, were monoclonal pattern. Among twenty invasive epidermoid carcinomas, eighteen of which showed heterozygous, twelve were monoclonal pattern and six were polyclonal pattern. We conclude that carcinoma in situ arises from a single cell. In invasive epidermoid carcinoma, most cases were monoclonal, although some cases were polyclonal. These suggest that invasive carcinoma of the cervix does not always arise from a single cell, but may arise from several cells with different mechanisms.