Anti-alkaline Phosphatase Research Articles

Osteoblastogenesis and osteolysis in the Zucker Diabetic Sprague Dawley rat humerus head.

The endocrinology of type 2 diabetes (T2D) and its predisposing factors have been studied extensively while its skeletal effects have received negligible research despite this being a global disease. The cellular and molecular association between proximal humeral fractures and T2D has not been fully elucidated. We aimed to study bone cell quantities and immunolabel osteogenic and antiosteogenic cytokines. The study used 12-week-old rats (23 males) consisting of 8 Sprague Dawley (SD) and 15 Zucker Diabetic Sprague Dawley (ZDSD). Weekly mass measurements were taken while fasting blood glucose levels were recorded every 2 weeks with oral glucose tolerance tests conducted once every 4 weeks. Upon termination at the age of 28 weeks, humeri were fixed in 10% buffered formalin, prior to decalcification in ethylenediaminetetraacetic acid. The bone samples were then processed in ascending grades of alcohol using an automatic processor before embedding in paraffin wax. Sections were cut at 5 µm thickness in a series for Haematoxylin and Eosin stain, and immunohistochemistry was performed with the anti-tartrate-resistant acid phosphatase (TRAP), anti-alkaline phosphatase (ALP), anti-bone morphogenetic protein 3 (BMP3), anti-transforming growth factor beta 1 (TGFβ1), anti-aged glycation end product (AGE) antibodies in the sequence. ZDSD rats had more adipocytes, BMP3 and AGEs expression with higher numbers of TRAP positive osteocytes and fewer ALP cells although no differences were found in TGFβ1 immunopositivity. We also found that T2D increases the number of AGEs immuno-positive cells, as well as its extracellular expression, thus providing a conducive environment for the interaction of the osteogenic cytokine and its antagonist to suppress osteoblastogenesis. ZDSD groups had higher adipocyte numbers therefore increased marrow adiposity in T2D.

The administration of bovine hydroxyapatite: Alendronate implant accelerates bone defect healing in an osteoporotic rat.

Bone fracture is the main consequence of osteoporosis, which may become a neglected disease. This study aims to fabricate bovine hydroxyapatite-gelatine (BHA-GEL) based bone-implant with alendronate (ALE) in vivo. Wistar rats were used for an osteoporotic animal model induced by ovariectomy. There were three groups: negative control, BHA-GEL implant, and BHA-GEL-ALE implant. Each group performed a defect by drilling the femur (diameter of 2.2mm and depth of 2mm). Observations on the closure of bone defects were performed by X-ray radiography at the second and sixth week after surgery. The mechanism of bone healing was observed by using hematoxylin-eosin (HE) staining and immunohistochemical technique with anti-vascular endothelial growth factor (VEGF) and anti-alkaline phosphatase (ALP) antibodies. The radiograph examination showed the implanted group had accelerated bone growth. In addition, the osteoblast, osteoclast and osteocyte had accelerated migration to the defect area. Moreover, the immunoreactive score (IRS) of VEGF at the sixth week in the BHA-GEL-ALE group was lower than the other groups. Meanwhile, the IRS of ALP in BHA-GEL-ALE was higher compared to other groups. The BHA-GEL-ALE implant accelerates the healing of bone defect in the osteoporotic rat by increasing the ALP expression and the total number of cells.

Application of carbon-coated nanomultilayer sintered porous titanium microprosthesis for the repair of osteochondral defects of the knee joint

We constructed a carbon-coated nanomultilayer (CNM) sintered porous titanium (Ti) microprosthesis for the optimal treatment of osteochondral defects of the knee joint. Physicochemical property tests revealed that the porosity of the constructed porous Ti-CNM prosthesis was ∼75%, while the pore size ranged 300–400 μm. The prosthesis had an elastic modulus (9.18 GPa) and compressive strength (98.3 MPa) similar to bone tissue. Furthermore, the CNM effectively reduced the roughness of the prosthesis surface. In animal experiments, the porous Ti-CNM improved the mineralization ability of osteoblasts in bone defects, as indicated by antialkaline phosphatase and osteocalcin, and improved bone mineral density, trabecular thickness, average trabecular number, and cortical bone thickness in the defective regions, thereby shortening the repair time. In addition, porous Ti-CNM effectively improved the production of extracellular matrix components in cartilage defects, such as collagen I, collagen II, and aggrecan, thereby enhancing the repair of cartilage defects and effectively preventing the degeneration of the opposite joint. The porous Ti-CNM prosthesis thus demonstrates great therapeutic potential for middle-aged patients with osteoarthritis.

Loss of p21ras, p53, and Mdm2 Protein Expression Is Indicative of Worse Prognosis in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a group of clonal disorders of the hematopoietic stem cell characterized by ineffective hematopoiesis and refractory cytopenias that can progress to acute leukemia. In order to identify molecular events implicated in the pathogenesis of MDS, we analyzed the bone marrow expression and mutational status of ras, p53 and mdm2 genes in a cohort of 25 patients with MDS. The expression of p21ras, p53, and Mdm2 proteins was studied in marrow cytopins stained by APAAP (alkaline phosphatase anti-alkaline phosphatase procedure) using monoclonal antibodies Y13-219, PAb 1801 and 2A10 to detect p21ras, p53 and mdm2 proteins, respectively. N-, K-, H-ras and p53 gene mutations were assessed by PCR-SSCP (polymerase chain reaction/single strand conformation polymorphism) and DNA sequencing to exclude the presence of gene mutations. The quantitative wild-type expression of p21ras, p53 and mdm2 proteins in patient samples (combination of number of cells and staining intensity) was compared to the values of expression of these proteins in 7 normal bone marrow samples. We found overexpression of p21ras and p53 proteins in 12/22 (55%) and 7/23 (30%) of the patients. Five of 7 patients who overexpressed wt-p53 also co-overexpressed wt-p21ras protein. We observed absence of expression of p21ras, p53 and mdm2 in 4/22, 4/23 and 9/19 cases, respectively. Overall survival of patients with absence of p21ras and p53 expression was considerably worse than that of patients with normal expression of these proteins (p=0.0006 to p21ras and p=0.004 to p53 by Log-Rank test; p=0.02 to p21ras and p=0.01 to p53 by Cox proportional hazards model after age, neutrophil and platelet count, and hemoglobin adjustment). We also observed that the patients who exhibited hypoexpression (p=0.055) and absence of mdm2 expression (p=0.07) demonstrated a tendency to have worse prognosis (Log-Rank). There was no correlation between variables p21ras, p53 and mdm2 versus hemoglobin, platelet and neutrophil counts and no association between FAB SMD subcategories and absence of p53 and mdm2 expression by Fisher test (two-tailed), but there was a strong over-representation between absence of p21ras expression and MDS with excess of blasts (p=0.0006). There was a significant correlation between p53 and mdm2 gene expression (p=0.03, Pearson). Only one patient harbored one transversion mutation in codon 12 of K-ras gene and did not correlate with p53 overexpression, but with worse prognosis. No p53 mutation was found. Overall, these findings suggest that the via of signal transduction p21ras/p53 and the auto-regulatory genes p53/mdm2 may be active and play a pivotal role in the pathogenesis of MDS. Hence, loss of bone marrow expression of p21ras, p53 and/or mdm2 proteins is an indicator of worse prognosis in MDS and may serve as early markers of leukemic transformation.

Bovine Hydroxyapatite-Based Bone Scaffold with Gentamicin Accelerates Vascularization and Remodeling of Bone Defect.

Osteomyelitis is an infectious disease which is also a major complication of bone defects. This study aims to determine the effect of bovine hydroxyapatite-gelatin-based bone implants with gentamicin as an antibiotic (BHA-GEL-GEN implant) on the regeneration of bone defects in vivo. The BHA-GEL-GEN and BHA-GEL implants were made by direct compression. In vivo study was carried out with Wistar rats. The rats were divided into three groups: negative control, BHA-GEL implant, and BHA-GEL-GEN implants. The defect model used was the burr hole defect model with diameter 2.2 mm and 2 mm deep. After 2, 7, 14, and 28 days, the rats were sacrificed. Bone integrity was carried out using X-ray radiography. Radiological examination was performed using haematoxylin and eosin (HE) staining and immunohistochemical techniques with anti-vascular endothelial growth factor (VEGF) and anti-alkaline phosphatase (ALP) antibodies. Based on the radiograph, the implanted group had accelerated bone growth in the defect area. Semiquantitative data from HE staining showed that the implanted group had accelerated migration of osteoclasts, osteoblasts, and osteocytes in the defect area. The immunoreactive score showed that the BHA-GEL-GEN group had higher VEGF expression compared to two other groups. The three groups did not provide a significant difference in ALP expression. In conclusion, the BHA-GEL-GEN implant causes accelerated bone defects repair by accelerating tissue vascularity and does not interfere with the bone remodeling process. Therefore, the BHA-GEL-GEN implant is potentially a biomedical material for osteomyelitis therapy.

Clasmatosis in Sea Star Immune Responses Observed in T.E.M Invertebrate Antibody. Biosynthesis in Anti-Hrp Responses And In Anti-Alkaline Phosphatase Responses

Anti-HRP (Horse-radish peroxydase) and Anti-Alkalin phosphatase immunocytochemical responses occur in Asterina gibbosa (Asterid Echinodermata) after immunizations to HRP and, on the other hand to Alkalin phosphatase enzymes. Classical TEM observations from ergastoplasmic labelling to Golgi apparatus one were done : they shown also “clasmatosis phenomenon” and recall , in a general way invertebrate antibody biosynthesis.

Alkaline phosphatase detection using electrochemical impedance of anti-alkaline phosphatase antibody (Ab354) functionalized silicon-nanowire-forest in phosphate buffer solution

This article presents an electrochemical impedance study on the detection of Alkaline Phosphatase (ALP) in phosphate buffer saline (PBS) solution by using anti-ALP functionalized Si nanowires (SiNWs). Electrochemical impedance was measured using a three-electrode cell where the working electrode was a very dense and highly disordered array of Si NWs, e.g. SiNW-forest. This array was grown using plasma enhanced chemical vapor deposition (PECVD) at 350 °C. The NW surface was modified by immobilizing the anti-ALP. A very efficient detection of very low ALP concentrations in PBS solution, ranging from 0.03 U/L to 0.3 U/L (1 nM–10 nM respectively), as well as a high selectivity due to the antibody/antigen interaction were demonstrated. The remarkable sensitivity and selectivity achieved through the combination of SiNW-forest combined electrochemical impedance spectroscopy suggest an effective strategy for real time and quantitative sensing of ALP.

The clinical significance of T-cell subset, cytokine levels in bronchoalveolar lavage fluid of children with mycoplasma pneumoniae pneumonia

Objective To explore the levels and clinical significances of T-cell subset, interleukin(IL)-6, interferon(IFN)-γ, and monocyte chemotactic protein(MCP)-1 in bronchoalveolar lavage fluid(BALF) of children with mycoplasma pneumoniae pneumonia(MPP). Methods Thirty-six children with pneumonia were admitted in our hospital from October 2014 to March 2016.They were divided into refractory mycoplasma pneumoniae pneumonia (RMPP) group (n=18) and MPP group(n=18). Sixteen cases with bronchial foreign body were selected as the control group.The levels of IL-6, MCP-1, IFN-γ and T-cell subset in BALF were detected by ELISA and alkaline phosphatase-anti-alkaline phosphatase(APAAP) bridging enzyme linked immunosorbent assay. Results (1)The levels of CD3+ and CD8+ T cells in BALF of MPP group and RMPP group were higher than those in the control group (P 0.05). (2) The level of IL-6 in acute phase of RMPP group was higher than that in the control group (P<0.05). The levels of IL-6 in acute phase of RMPP group and MPP group were both higher than that in recovery period (P<0.05, respectively). (3)The levels of IFN-γ in the acute phase of RMPP group and MPP group were higher than that in the control group, and the differences were statistically significant (P<0.05). The IFN-γ levels in acute and recovery phases of RMPP group were both significantly higher than those in MPP group respectively(P< 0.05). (4)The level of MCP-1 in BALF in control group was lower than those in the acute phase of RMPP group and MPP group(P<0.05). The level of MCP-1 in RMPP acute group was significantly higher than that in MPP acute group, and higher in acute phase than those in recovery phase both in two groups(P<0.05). Conclusion There are cell immune regulation and function disorders when children are infected mycoplasma pneumoniae.IL-6, IFN-γ and MCP-1 may participate in the pathogenesis of MPP, and more importantly, IFN-γ and MCP-1 may be the important indicator to forecast the severity of MPP. Key words: Mycoplasma pnuemoniae; Bronchoalveolar lavage fluid; T-cell subset; Cytokine,

Bone regeneration enhancement by ultra-violet (UV) treatment for uHA/PLLA absorbable mesh

PurposeThe purpose of this study was to evaluate the effect of bone regeneration enhancement by ultra-violet (UV) treatment of an unsintered hydroxyapatite (u-HA)/poly-l-lactic acid (PLLA) mesh in rabbitnasal bone. Materials and methodsThirty adult male Japanese white rabbits (12–16 weeks, 2.5–3.0 kg) were used in this study. After incising along the nasal bone, 6 × 6 × depth 1 mm two bone defects were made on both sides. In the UV group (n = 30), the defects were covered with uHA/PLLA mesh, treated by UV (wavelength 172 nm, tube wall illumination 13 mW/cm2, period 8 min, intensity 6.26 J/cm2) and screwed. In the control group (n = 30), untreated uHA/PLLA was applied in a similar manner. The rabbits were sacrificed at 1, 2, 3, 4 and 6 weeks postoperatively, and formalin-fixed specimens were frozen. The specimens were stained with haematoxylin and eosin. For immunohistochemical analysis, the specimens were treated with anti-alkaline phosphatase (ALP). Finally, bone ratio and ALP expression were evaluated microscopically. ResultsThe UV group had a significantly higher number of ALP stained cells than the control group after 1, 2, and 3 weeks (P < 0.05). The bone ratio was also significantly higher in the UV group than in the control group after 1, 2, 3, and 4 weeks (P < 0.05). ConclusionThis study suggests that bone regeneration can be enhanced by UV treatment using an uHA/PLLA mesh.

Effects of shilajit on the bone tissue of alcohol administered rats

Purpose: The purpose of the current study was to examine the effect of Shilajit; a herbomineral, supplementation on the mechanical strength of alcohol treated rat bone. Methods: Experimental animals each were assigned to six groups: group A(control): control rats were given water orally for a period of ten weeks; group B (treated): Animals were given processed shilajit (PS; 100mg/kg/day); group C (treated): Animals were given processed shilajit (PS; 200mg/kg/day); group D: animals were given 30% alcohol; group E: animals were given 30% alcohol and shilajit (100mg/kg/day) orally; group F: animals were given 30% alcohol and shilajit (200mg/kg/day) orally for ten weeks. Bone tissue mechanical strength along with bone weight, liver antioxidative enzymes and alkaline phosphatase (ALP) were assessed for all the treatment groups. Results: Mechanical strength of the bone tissue (both femur as well as tibia) was found to be significantly enhanced upon shilajit supplementation to alcohol treated group. Also, the activities of anti oxidant enzymes and alkaline phosphatase in the liver of alcohol administered groups were restored upon shilajit administration. Conclusion: These findings suggest that shilajit is very efficacious and competent in the maintenance of bone health.

Localization of and #945; - Smooth Muscle Actin in the Jejunum of the Camel (Camelus dromedarius)

The antibody specific for human alpha smooth muscle actin is known to be useful for detection of similar antigen in several species, but not tested for the camel. The present study tested this antibody for cross-reactivity with the smooth muscle of the small intestine of the camel using alkaline phosphatase anti-alkaline phosphatase technique. Formalin fixed specimens were obtained from the small intestine of 5 male dromedary camels (8-10 years old). The specimens were processed and paraffin sections were prepared. Paraffin Sections from human pharyngeal tonsils were stained in parallel with camel sections as a positive control. The monoclonal mouse anti-human alpha smooth muscle actin IgG2a, Kappa (Dako Gloostrup, Denmark) was applied on the sections. The reactivity was developed using fast red. The antibody reacted with the smooth muscles in the camel small intestine. No non specific reactions were observed. The intensity of the reaction was variable in the same section. The smooth muscles of the lamina muscularis mucosae, as well as, those in the wall of the blood vessels of the tunica submucosa showed more intense reaction than that of the tunica muscularis. The variation in the intensity of the reaction referred to the amount of alpha actin in the smooth muscle. The degree of reactivity in the tunica muscularis was variable. Thin layer of alpha smooth muscle actin positive cells observed between the intestinal crypts and in the core of intestinal villi. The striated border (microvilli) of the columnar epithelium covering the intestinal villi expressed alpha smooth muscle actin positive reaction. The camel smooth muscle actin and that of the human may show some similarity in their antigenic structure so that the monoclonal mouse anti- human alpha smooth muscle actin can also identify the alpha smooth muscle actin in the camel.

BRONJ-related jaw bone is associated with increased Dlx-5 and suppressed osteopontin-implication in the site-specific alteration of angiogenesis and bone turnover by bisphosphonates.

Site-specific suppression of bone remodelling has been implicated in bisphosphonate-(BP)-related osteonecrosis of the jaws (BRONJ). Due to the origin of jaw bone from cranial neural crest, osseous differentiation is regulated specifically by the antagonizing BMP-2-downstream-transcription factors Msx-1 and Dlx-5. Osteopontin has been implicated in bone remodelling and angiogenesis. The osteoblast and osteoclast progenitor proliferation mediating Msx-1 has been demonstrated to be suppressed in BRONJ. In vitro BPs were shown to increase Dlx-5 and to suppress osteopontin expression. This study targeted Dlx-5 and osteopontin in BRONJ-related and BP-exposed jaw bone compared with healthy jaw bone samples at protein- and messenger RNA (mRNA) level, since increased Dlx-5 and suppressed osteopontin might account for impaired bone turnover in BRONJ. Fifteen BRONJ-exposed, 15 BP-exposed and 20 healthy jaw bone samples were processed for real-time reverse transcription polymerase chain reaction (RT-PCR) and for immunohistochemistry. Targeting Dlx-5, osteopontin and glyceraldehyde 3-phosphate dehydrogenase mRNA was extracted, quantified by the LabChip-method, followed by quantitative RT-PCR. For immunohistochemistry, an autostaining-based alkaline phosphatase antialkaline phosphatase (APAPP) staining kit was used. Semiquantitative assessment was performed measuring the ratio of stained cells/total number of cells (labelling index, Bonferroni adjustment). The labelling index was significant decreased for osteopontin (p < 0.017) and significantly increased for Dlx-5 (p < 0.021) in BRONJ samples. In BRONJ specimens, a significant fivefold decrease in gene expression for osteopontin (p < 0.015) and a significant eightfold increase in Dlx-5 expression (p < 0.012) were found. BRONJ-related suppression of bone turnover is consistent with increased Dlx-5 expression and with suppression of osteopontin. The BP-related impaired BMP-2-Msx-1-Dlx-5 axis might explain the jaw bone specific alteration by BP. The findings of this study help to explain the restriction of RONJ to craniofacial bones. BRONJ might serve as a model of disease elucidating the specific signal transduction of neural crest cell-derived bone structures in health and disease.

Improved Enzymatic Treatment for Accurate Cell Counting from Extracellular Matrix–Rich Periodontal Ligament Cell Sheets

The objective of this study was to establish a method for accurate cell counting from matrix-rich cell sheets in the clinical setting. Human periodontal ligament (HPDL) cells were obtained from healthy donors to prepare PDL cell sheets. To obtain single cell suspensions, the cell sheets were treated with three different enzymatic formulations: collagenase alone, trypsin-ethylenediaminetetraacetic acid (EDTA) alone, and a combination of collagenase and trypsin-EDTA. After cell dispersion, cell numbers and cell survival rates were measured. To evaluate damage to the cell surfaces from the enzymes, the dispersed cells were analyzed by a flow cytometer with an anti-alkaline phosphatase antibody. Treatment with collagenase alone or trypsin-EDTA alone dispersed few cells from HPDL cell sheets. In contrast, combined treatment with collagenase and trypsin-EDTA successfully produced a large amount of single cells from cell sheets. Flow cytometry analysis showed that single cells obtained by combined use of collagenase and trypsin-EDTA preserved alkaline phosphatase epitopes on the cell surfaces. Cell sheets rich with extracellular matrix were dispersed via combined treatment with collagenase and trypsin-EDTA without destroying the expression of cell surface markers. The results suggest that this method would be useful for determining the accurate cell number of cell sheets for cell therapies and should also be applicable for other kinds of matrix-rich cell sheets.

Mucosal Addressin Cell Adhesion Molecule (MAdCAM-1) Expression Is Upregulated in the Cirrhotic Liver and Immunolocalises to the Peribiliary Plexus and Lymphoid Aggregates

Enhanced cell expression of MAdCAM-1 is critical in tissue recruitment of lymphocytes in response to stimuli expressing the α4β7 integrin. MAdCAM-1 is well characterized in gut mucosa with emerging evidence of hepatic expression. (i) Compare quantitative/semi-quantitatively MAdCAM-1 expression in relation to early and advanced liver diseases (ii) Define the fine structure of vascular plexuses/lymphatics in the portal tract on which MAdCAM-1 is expressed. Using alkaline phosphatase anti-alkaline phosphatase methodology on paraffin embedded tissue sections (n=28) from cirrhotic individuals who underwent orthotopic liver transplant, we evaluated MAdCAM-1 expression and compared with pre-cirrhotic, fulminant hepatitis B, and non-cirrhotic portal hypertension tissue sections. The positive controls included normal colon tissue with negative controls without primary antibody and isotype-matched purified IgG. We developed a real time PCR to quantify levels of MAdCAM-1 mRNA in our samples. MAdCAM-1 was expressed in 27/28 of the cirrhotic sections, localized primarily to septal areas within (i) endothelium of the peribiliary vascular plexus (PBP) (ii) lymphoid aggregates, with absence from normal, non-cirrhotic portal hypertension and pre-cirrhotic livers. There was significant upregulation of MAdCAM-1 mRNA in cirrhosis (p<0.011), consistent with immunohistochemical analysis. MAdCAM-1 is up-regulated in cirrhosis with expression on PBP and lymphoid aggregates. MAdCAM-1 is likely to contribute to the localization and recruitment of α4β7 lymphocytes during the pathogenesis of cirrhosis. MAdCAM-1 could be a useful marker of advanced liver disease. Further studies with respect to the expression of MAdCAM-1 in the presence of reversible and non-reversible stages of liver disease may be of merit.

Morphology Antibody Chromosome Technique for Determining Phenotype and Genetic Status of the Same Cell

The morphology antibody chromosome (MAC) technique is a combination of methods that permits analysis of both phenotypic and genetic evaluation on a single interphase or mitotic cell as a basis for lineage analysis of neoplastic and normal cells. This unit describes MAC with sequential phenotypic analysis using antibody and an alkaline phosphatase anti-alkaline phosphatase (APAAP) complex and genotypic analysis using in situ hybridization with either enzymatic or fluorescence detection. Alternate methods for phenotypic analysis are also described, which include the use of horseradish peroxidase-conjugated antibodies, fluorochrome-conjugated antibodies, May-Grunwald-Giemsa cytochemical staining, and Sudan black B cytochemical staining. An additional protocol describes G- and C-banding as alternatives to in situ hybridization (ISH) for genotyping MAC specimens. Support protocols describe methods for preparing specimens, cytospin preparations, in situ cultures, paraffin-embedded or cryostat sections, and blood and bone marrow smears. Also described is a procedure for chromosome painting of previously GTG-banded slides. An additional protocol is included for FISH analysis on sorted hematopoietic stem cells and its application in the detection of leukemic stem cells. For overcoming the drawbacks of scarcity and variability from case to case of malignant plasma cells in multiple myeloma FISH analyses, a protocol is included for the enrichment of plasma cells by immunomagnetic beads.

Disseminated tumour cells in bone marrow of patients with uveal melanoma

Approximately 50% of patients with uveal melanomas develop metastases. Thus, it is important to improve our understanding of how melanoma metastases develop. As part of a uveal melanoma micrometastasis study, we compared the detection rates of immunomagnetically selected (IMS) tumour cells in bone marrow (BM) with positively stained tumour cells using immunocytochemistry (ICC). Bone marrow mononuclear cells were isolated. Immunocytochemistry cytospin preparations were immunocytochemically stained in parallel with two different melanoma antibodies, 9.2.27 and HMB45. Using IMS, melanoma cells were selected from BM mononuclear cell fractions using immunomagnetic beads coated with the 9.2.27 antibody and identified by light microscopy. In cytospin preparations from 226 patients, melanoma cells were detected in 24 (10.6%), 10 with 9.2.27 and 17 with the HMB45 antibody. In three cases, we found positive cells with both antibodies. Six of the 226 (2.6%) patients that stained positively with ICC died with metastatic disease, all also positive with IMS. Sixty-six (29.2%) patients had positive BM samples with IMS at the first examination. Immunomagnetic selection (IMS) was positive in 36.8% of the 57 patients who later developed clinical metastases. Twenty-one IMS-positive patients and 31 IMS-negative patients died of metastases, in total 52 of 226 patients (23.0%). The mortality rate of melanoma metastasis was 24% (6/24) after at least 4 ½ years in ICC-positive patients compared to 38.5% (20/52) in IMS-positive patients. The presence of melanoma cells in BM of patients with uveal melanoma is documented in our study with IMS and ICC. Immunomagnetically selected is more sensitive than ICC in detecting tumour cells in BM. However, statistically, we did not find any prognostic impact of the presence of melanoma cells in BM.

Low Expression of RPS14 Accompanied with p53-Independent Over-Apoptosis in Myelodysplastic Syndromes Patients with 5q- Aberration

Abstract 5046 PurposeIn this study, we focused on evaluating ribosomal protein S14 (RPS14) and p53 expression and their correlation analysis with the purpose of investigating the role of RPS14-MDM2-p53 pathway in hematopoietic cell apoptosis of myelodysplatic syndrome (MDS) patients with 5q- aberration. Patients and Methods:Real time quantitative PCR was used to detect the expression levels of RPS14, MDM2 and p53 in 18 MDS patients, among which 7 with isolated del (5q) and 11 del (5q) with other chromosomal abnormalities were included. Besides, alkaline phosphatase-anti-alkaline phosphatase (APAAP) and terminal deoxynucleotidyI transferase dUTP nick end labeling (TUNEL) techniques were carried out simultaneously for each sample to respectively examine the expression of p53 and the apoptosis level of bone marrow (BM) mononuclear cells (MNCs). 10 healthy controls were included in the study. Results:under-expression of RPS14 gene was seen in 15/18 patients' (83.3%), of which 6/7 were isolated 5q- and 9/11 were 5q- with other chromosomal abnormalities. RPS14 expression was negatively correlated with the proportion of 5q- clonal cell in BM while it showed a positive correlation with both of the expression of MDM2 and p53. The expression of p53 was not correlated to the high apoptosis level of BM in patients with del (5q). Furthermore, APAAP and TUNEL results demonstrated apoptotic cells in patients BM were p53-negative (see Fig 1). Conclusions: RPS14 gene was under-expressed in MDS patients with 5q- aberration, which did not result in a p53-dependent apoptosis. A p53-independent pathway may contribute to the over apoptosis of 5q- MDS patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Decrease in the Density of HLA-DR-Positive Cells Occurs Faster in Corneas Stored in Organ Culture than under Hypothermic Conditions

Aims: To compare the number of antigen-presenting cells (APC) at various locations in fresh human corneoscleral disks and in those that are stored for grafting under hypothermic conditions or in organ culture (OC) with the aim of determining the conditions under which the decline in APC numbers is most substantial. Methods: Cryosections obtained from fresh corneoscleral disks and disks stored under hypothermic (Optisol-GS) or OC conditions were used. The density of HLA-DR-positive cells was determined on cross sections using enzyme immunohistochemistry (alkaline phosphatase-antialkaline phosphatase technique). Longitudinal sections were used for detecting ATPase activity. Results: The densities of HLA-DR-positive cells in both the epithelium and stroma increased from the central (3.79 and 0.61/mm²) to the peripheral cornea (5.56 and 1.35/mm²) as well as to the limbus and conjunctiva. A marked decrease in the number of HLA-DR-positive cells occurred after 7 days of storage in all corneal areas, the limbus and conjunctiva, compared to fresh tissue. No positive cells were found in the epithelium of corneas after 14 days in OC and after 21 days in hypothermic storage. Twenty-eight days of storage in OC led to the complete absence of HLA-DR-positive cells in the epithelium of the limbus and conjunctiva, and to a significant reduction in the stroma. Conclusion: Corneas stored in OC longer than 14 days are likely to be less immunogenic than corneas stored under hypothermic conditions, thus resulting in a possible positive effect on prolonging graft survival.

Differential impairment of vascularization and angiogenesis in bisphosphonate-associated osteonecrosis of the jaw-related mucoperiosteal tissue

Impaired vascularization in the etiopathology of aminobisphosphonate-associated osteonecrosis of the jaw (BONJ) is assumed, but evidence is lacking. This immunohistochemical study differentiated vascularization and angiogenesis in BONJ-adjacent mucoperiosteal tissue. Twenty BONJ (after zoledronate treatment) and 20 control mucoperiosteal tissue samples were processed with an autostaining-based alkaline phosphatase-antialkaline phosphatase staining kit. Vascularization was assessed by CD31 staining and angiogenesis-related neovessels by CD105 staining. The ratio of stained capillary area to total area of visible field was assessed. Statistics included Bonferroni adjustment. CD31-stained microvessels were detected in each section and CD105-stained neovessels in each control. BONJ-adjacent mucoperiosteal tissue showed significantly fewer CD105-positive vessels in capillary areas (P < .05) than control samples. CD31-stained capillary area was not significantly reduced in mucoperiosteal BONJ-samples. Angiogenesis is impaired in BONJ-related mucoperiosteal tissue, but vascularization remains unaffected. Vessel remodeling and neovessel formation is delayed in BONJ, resulting in impaired tissue regeneration of bisphosphonate-exposed oral mucosa.

Primary effusion lymphoma associated with Human Herpes Virus-8 and Epstein Barr virus in an HIV-infected woman from Kampala, Uganda: a case report

IntroductionPrimary effusion lymphoma is a recently recognized entity of AIDS related non-Hodgkin lymphomas. Despite Africa being greatly affected by the HIV/AIDS pandemic, an extensive MEDLINE/PubMed search failed to find any report of primary effusion lymphoma in sub-Saharan Africa. To our knowledge this is the first report of primary effusion lymphoma in sub-Saharan Africa. We report the clinical, cytomorphologic and immunohistochemical findings of a patient with primary effusion lymphoma.Case presentationA 70-year-old newly diagnosed HIV-positive Ugandan African woman presented with a three-month history of cough, fever, weight loss and drenching night sweats. Three weeks prior to admission she developed right sided chest pain and difficulty in breathing. On examination she had bilateral pleural effusions.Haematoxylin and eosin stained cytologic sections of the formalin-fixed paraffin-embedded cell block made from the pleural fluid were processed in the Department of Pathology, Makerere University, College of Health Sciences, Kampala, Uganda. Immunohistochemistry was done at the Institute of Haematology and Oncology "L and A Seragnoli", Bologna University School of Medicine, Bologna, Italy, using alkaline phosphatase anti-alkaline phosphatase method. In situ hybridization was used for detection of Epstein-Barr virus.The tumor cells were CD45+, CD30+, CD38+, HHV-8 LANA-1+; but were negative for CD3-, CD20-, CD19-, and CD79a- and EBV RNA+ on in situ hybridization. CD138 and Ki-67 were not evaluable. Our patient tested HIV positive and her CD4 cell count was 127/μL.ConclusionsA definitive diagnosis of primary effusion lymphoma rests on finding a proliferation of large immunoblastic, plasmacytoid and anaplastic cells; HHV-8 in the tumor cells, an immunophenotype that is CD45+, pan B-cell marker negative and lymphocyte activated marker positive. It is essential for clinicians and pathologists to have a high index of suspicion of primary effusion lymphoma when handling HIV positive patients who have effusions without palpable tumor masses. Basic immunohistochemistry is essential for definitive diagnosis.