ABSTRACT
BACKGROUND: The incidence of epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. OBJECTIVES: To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC. METHODS: The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real-time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s. RESULTS: Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa-associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal-related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo. CONCLUSIONS: These results provide evidence for the role of tumour-cell-derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally. Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.
Subject(s)
Carcinoma, Squamous Cell , Keratosis, Actinic , Skin Neoplasms , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Humans , Keratinocytes , Skin Neoplasms/geneticsABSTRACT
BACKGROUND: Tumour-specific expression of matrix metalloproteinase (MMP)-7 has been noted in cutaneous squamous cell carcinomas (SCCs) in patients with recessive dystrophic epidermolysis bullosa (RDEB). OBJECTIVES: To examine the potential role of MMP-7 in shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in RDEB-associated and sporadic SCCs. METHODS: Tissue microarrays of RDEB-associated SCC (n = 20), non-EB SCC (n = 60) and Bowen disease (n = 28) were immunostained for MMP-7, CD44 variant 3 (CD44v3) and HB-EGF. Shedding of HB-EGF was studied in vitro using two cutaneous SCC cell lines. RESULTS: Immunohistochemical analysis showed that HB-EGF was absent in tumour cells when MMP-7 and CD44v3 colocalized, and that the absence of HB-EGF was more pronounced in RDEB-associated SCCs than in non-EB SCCs. The loss of HB-EGF in MMP-7-CD44v3 double-positive areas was interpreted to indicate shedding and activation of HB-EGF; this was also detected in Bowen disease indicating its importance in the early phase of SCC development. Specific knockdown of MMP-7 expression in human cutaneous SCC cells by small interfering RNA inhibited shedding of HB-EGF and resulted in diminished activation of the EGF receptor (EGFR) and ERK1/2, and in reduced proliferation of SCC cells. CONCLUSIONS: These findings provide evidence for the role of MMP-7 in promoting the growth of cutaneous SCCs by shedding HB-EGF, and identify EGFR signalling as a potential therapeutic target in RDEB-associated SCC and unresectable sporadic cutaneous SCC.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Matrix Metalloproteinase 7/physiology , Skin Neoplasms/metabolism , Adult , Carcinoma, Squamous Cell/pathology , Cell Proliferation/drug effects , Dipeptides/pharmacology , Enzyme Activation , ErbB Receptors/physiology , Female , Gene Knockdown Techniques , Heparin-binding EGF-like Growth Factor , Humans , Hyaluronan Receptors/metabolism , Male , Matrix Metalloproteinase Inhibitors , Middle Aged , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Neoplasm Proteins/metabolism , Protease Inhibitors/pharmacology , RNA, Small Interfering/genetics , Signal Transduction/physiology , Skin Neoplasms/pathology , Tumor Cells, Cultured , Young AdultABSTRACT
BACKGROUND: Patients with recessive dystrophic epidermolysis bullosa (RDEB) have an increased risk of developing rapidly progressive and metastatic cutaneous squamous cell carcinomas (SCC). It is unclear why these SCC behave more aggressively than sporadic SCC. Matrix metalloproteinases (MMP) are a family of endopeptidases that contribute to growth, invasion and metastasis of SCC. The role of MMP in RDEB-associated SCC is not known. OBJECTIVES: To investigate the expression of MMP-7, MMP-13 and MMP-9 in RDEB-associated SCC in comparison with sporadic SCC and Bowen's disease. METHODS: Immunohistochemical analysis of 25 RDEB-associated SCC, 61 sporadic SCC and 28 sporadic lesions of Bowen's disease was carried out using monoclonal antibodies for MMP-7, MMP-9, MMP-13 and E-cadherin and syndecan-1. RESULTS: MMP-7 was detected in all RDEB-associated SCC, in tumour cells within the invasive edge, where E-cadherin and syndecan-1 were markedly diminished or absent. MMP-7 expression was also observed in 98% of sporadic SCC and in 68% of Bowen's diseases. MMP-7 staining was significantly stronger in RDEB-associated SCC than in sporadic SCC, and was most abundant in poorly differentiated tumours. MMP-13 was detected in tumour cells in 96% of RDEB-associated SCC and in all sporadic cutaneous SCC. MMP-9 was detected in the inflammatory cells in all SCC examined. CONCLUSIONS: These results identify MMP-7 and MMP-13 as tumour cell-specific markers for SCC progression and as potential therapeutic targets in RDEB-associated SCC. The pattern of immunolabelling suggests that MMP-7 may shed E-cadherin and syndecan-1 from the SCC cell surface.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/metabolism , Epidermolysis Bullosa Dystrophica/metabolism , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 7/metabolism , Skin Neoplasms/metabolism , Adolescent , Adult , Carcinoma, Squamous Cell/etiology , Cell Line, Tumor , Epidermolysis Bullosa Dystrophica/complications , Female , Gene Expression , Humans , Male , Matrix Metalloproteinase 13/therapeutic use , Matrix Metalloproteinase 7/therapeutic use , Middle AgedABSTRACT
Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n=42) compared to HEKs (n=8) revealed that p38alpha and p38delta isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38alpha or p38delta activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38alpha and p38delta inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38alpha activity also compromised survival of SCC cells. p38alpha and p38delta were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38alpha and p38delta by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38alpha or p38delta in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38alpha and p38delta specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.
Subject(s)
Carcinoma, Squamous Cell/pathology , Cell Division/physiology , Head and Neck Neoplasms/pathology , Isoenzymes/physiology , Neoplasm Invasiveness , p38 Mitogen-Activated Protein Kinases/physiology , Base Sequence , Blotting, Western , Carcinoma, Squamous Cell/enzymology , Cell Line, Tumor , DNA Primers , Enzyme Activation , Flow Cytometry , Head and Neck Neoplasms/enzymology , Humans , Immunohistochemistry , Isoenzymes/metabolism , Keratinocytes/enzymology , Matrix Metalloproteinases/metabolism , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
OBJECTIVE: To evaluate the usefulness of immunohistochemical staining of cyclin A and Ki-67 together with DNA content in the classification of benign prostatic hyperplasia, prostatic intraepithelial neoplasm (PIN) and prostatic carcinoma foci and to compare these parameters with each other and with parameters obtained from conventional histopathology. STUDY DESIGN: We selected 37 carcinoma, 18 PIN and 8 hyperplastic foci from prostatectomies done during 1996 and 1997 at Turku University Central Hospital. Cyclin A and Ki-67 staining was assessed by immunohistochemistry and DNA content by image cytometry. RESULTS: The hyperplastic, PIN and carcinoma foci differed clearly in their 2.5c exceeding rates, image cytometric proliferation indices and staining indices for cyclin A and Ki-67. No significant differences were found between these histologic entities in their modal DNA ploidy values. In carcinomas, cyclin A and Ki-67 indices differed between low, intermediate and high Gleason and World Health Organization grading groups. Diploid and tetraploid carcinomas had similar cyclin A and Ki-67 indices, which differed from those of aneuploid carcinomas. CONCLUSION: The 2.5c exceeding rate and image cytometric proliferation index as well as the cyclin A and Ki-67 indices differed significantly between different types of prostatic lesions. Cyclin A and Ki-67 had good correlations with the histologic grade of carcinoma.
Subject(s)
Cyclin A/analysis , Ki-67 Antigen/analysis , Prostatic Diseases/pathology , Biomarkers, Tumor , DNA, Neoplasm/analysis , Diagnosis, Differential , Humans , Image Cytometry , Immunohistochemistry , Male , Ploidies , Prostatic Diseases/genetics , Prostatic Hyperplasia/genetics , Prostatic Hyperplasia/pathology , Prostatic Intraepithelial Neoplasia/chemistry , Prostatic Intraepithelial Neoplasia/genetics , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/chemistry , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathologyABSTRACT
We investigated the role of cardiomyocyte apoptosis in the remodeling of the left ventricle from 24 h to 12 wk after myocardial infarction in the rat. Infarct size planimetry, quantification of cardiomyocyte apoptosis, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) methodology, and echocardiography (left ventricular diastolic diameter and ejection fraction) were performed. Sham-operated animals showed low rates of cardiomyocyte apoptosis (0.03%) and no change in diastolic diameter or ejection fraction during the study. Twenty-four hours after infarction, TUNEL positivity was high in the infarct areas (1.4%) and border zones (4.9%). It declined to 0.34% (P < 0.01 vs. sham) at 4 wk and 0.10% at 12 wk in the border zones. In the remote myocardium, cardiomyocyte apoptosis increased to 0.07% (P = 0.03 vs. sham) on day 1 and remained on the same level up to 4 wk. The increase in diastolic diameter 1-4 wk after infarction correlated (r = 0.60, P < 0.01) with cardiomyocyte apoptosis in the noninfarcted myocardium, which quantitatively contributed most (>50%) to the apoptotic cell loss by 4 wk.
Subject(s)
Apoptosis , Myocardial Infarction/pathology , Myocardium/pathology , Ventricular Remodeling , Animals , Cell Count , Electrocardiography , In Situ Nick-End Labeling , Male , Myocardial Infarction/physiopathology , Necrosis , Rats , Rats, Wistar , Stroke Volume , Ventricular FunctionABSTRACT
OBJECTIVE: To assess the feasibility of MR (magnetic resonance)-guided bone biopsies. DESIGN AND PATIENTS: Thirty-six consecutive patients with known or suspected benign or malignant bone lesions underwent comprehensive MR imaging. A dynamic contrast-enhanced sequence followed by stationary T1-weighted sequences were obtained and MR-guided bone biopsy of the tumor at the site with fastest enhancement was performed using an open 0.23 T MR imager. RESULTS: All MR-guided bone biopsies samples were estimated to be sufficient by the pathologists. The biopsy specimens were diagnostic in 34 of 36 cases. CONCLUSION: MR-guided bone biopsies combined with dynamic contrast-enhanced imaging are feasible and safe for the diagnostic investigation of equivocal bone lesions.
Subject(s)
Biopsy, Needle , Bone Neoplasms/pathology , Bone and Bones/pathology , Magnetic Resonance Imaging , Adolescent , Adult , Aged , Aged, 80 and over , Contrast Media/administration & dosage , Female , Follow-Up Studies , Gadolinium DTPA/administration & dosage , Humans , Male , Middle AgedABSTRACT
OBJECTIVES: The expression of endothelial adhesion molecules and their functional significance in leukocyte adhesion to human myocardial blood vessels in acute myocardial infarction (AMI) were studied. BACKGROUND: Leukocyte extravasation, mediated by specific adhesion molecules, exacerbates tissue injury after restoration of blood supply to an ischemic tissue. Experimental myocardial reperfusion injury can be alleviated with antibodies that block the function of adhesion molecules involved in leukocyte emigration, but the relevant molecules remain poorly characterized in human AMI. METHODS: Semiquantitative immunohistochemistry and in vitro adhesion assays were used to study the expression and granulocyte binding abilities of different endothelial adhesion molecules in human AMI. Changes in the molecular nature of vascular adhesion protein-1 (VAP-1) were evaluated using immunoblotting. RESULTS: Certain endothelial adhesion molecules (intercellular adhesion molecule [ICAM-2], CD31 and CD73) were expressed in myocardial blood vessels homogeneously in normal and ischemic hearts, whereas others (E-selectin and peripheral lymph node addressin) were completely absent from all specimens. The synthesis of ICAM-1 was locally, and that of P-selectin regionally, upregulated in the infarcted hearts when compared with nonischemic controls. Vascular adhesion protein-1 showed ventricular preponderance in expression and alterations in posttranslational modifications during ischemia-reperfusion. Importantly, P-selectin, ICAM-1 and VAP-1 mediated granulocyte binding to blood vessels in the ischemic human heart. CONCLUSIONS: Human P-selectin, ICAM-1 and VAP-1 appear to be the most promising targets when antiadhesive interventions preventing leukocyte-mediated tissue destruction after myocardial ischemia are planned.
Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Cell Adhesion Molecules/metabolism , Coronary Vessels/metabolism , Intercellular Adhesion Molecule-1/metabolism , Leukocytes/physiology , Myocardial Infarction/metabolism , P-Selectin/metabolism , 5'-Nucleotidase/metabolism , Antibodies, Monoclonal , Antigens, CD/metabolism , Antigens, Surface/metabolism , Cell Adhesion , Cell Movement/physiology , Coronary Vessels/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Female , Granulocytes/physiology , Humans , Immunoenzyme Techniques , Male , Membrane Proteins , Middle Aged , Myocardial Infarction/pathology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Receptors, Lymphocyte Homing/metabolismABSTRACT
Despite well-documented cardiotoxic effects, doxorubicin remains a major anticancer agent. To study the role of myocardial apoptosis following doxorubicin administration, male Wistar rats were exposed to 1.25, 2.5, and 5 mg/kg of i.p. doxorubicin and terminated on days 1-7 in groups of five. Doxorubicin caused a significant (P < 0.001) and dose-dependent induction of cardiomyocyte apoptosis at 24-48 h after the injection. Repeated injections of 2.5 mg/kg given every other day resulted in peaks of apoptosis at 24 h after each injection. However, no additive effect of repeated dosing was noted. In histological samples, alterations in the cytoskeletal apparatus with focal loss of contractile elements were seen after a single injection. Myocyte necrosis was absent. Thus, acute doxorubicin-induced cardiotoxicity involves cardiomyocyte apoptosis, a potentially preventable form of myocardial tissue loss.
Subject(s)
Apoptosis/drug effects , Doxorubicin/toxicity , Heart/drug effects , Myocardium/pathology , Animals , Dose-Response Relationship, Drug , In Situ Nick-End Labeling , Male , Rats , Rats, Wistar , Time FactorsABSTRACT
Nuclear mitotic apparatus protein (NuMA) has an indispensable function in normal mitosis as an organizer of the mitotic spindle. NuMA is a prominent component of interphase cell nuclear matrix but its role during interphase is largely unknown. We examined the presence of NuMA in several human tissues. The majority of cells were positive for NuMA but a few negative cell types were found, including spermatozoa, superficial keratinocytes, neutrophil granulocytes, syncytiotrophoblasts, and some neurons, fibroblasts, and smooth and skeletal muscle cells. We further investigated the presence of NuMA in a cultured estrogen-dependent human breast cancer cell line and observed the disappearance of nuclear NuMA in the quiescent cells. The percentage of NuMA-positive cells diminished from an initial approximately 100 to 60% during 6 days of culture. The presence of NuMA correlated positively with the presence of proliferation marker Ki-67 antigen and negatively with the culture time, confluence, and size of the cell islets. These results show that some nonproliferating, highly differentiated cell types lack NuMA and that cells may lose their NuMA without dramatic effects on the nuclear shape. This suggests that NuMA may be a nonessential component of the interphase nucleus.
Subject(s)
Cell Division/physiology , Cell Nucleus/physiology , Cell Nucleus/ultrastructure , Nuclear Proteins/analysis , Antigens, Nuclear , Breast Neoplasms , Cell Cycle Proteins , Cell Differentiation , Female , HeLa Cells , Humans , Ki-67 Antigen/analysis , Male , Mitosis , Nuclear Matrix-Associated Proteins , Organ Specificity , Reference Values , Spindle Apparatus/ultrastructure , Tumor Cells, CulturedABSTRACT
During normal placentation trophoblast cells invade maternal tissues and remodel the uterine arteries into low-resistance channels. In pre-eclampsia, trophoblast invasion is impaired and this, along with endothelial dysfunction, has been suggested to play a role in the pathogenesis of pre-eclampsia. We studied the expression of adhesion molecules important for leukocyte extravasation in the placental bed with immunohistochemistry and compared the expression in pre-eclampsia to that in normal pregnancy. Our major finding was that only invasive trophoblasts expressed cutaneous lymphocyte antigen-1 (CLA-1) in the third trimester of pregnancy, whereas villous trophoblasts did not. In the first trimester both villous trophoblasts and invasive trophoblast cells in decidua remained negative for CLA-1. Pre-eclampsia did not change the expression of leukocyte-endothelium adhesion or lymphocyte homing-associated antigens, ICAM-1, ICAM-2, VCAM, P-selectin, E-selectin, L-selectin, CLA-1, CD73, VAP-1 and alphaEbeta7 in the placental bed. Furthermore, pre-eclampsia was not associated with an aberrant accumulation of lymphocytes carrying antigens of any particular known organ-specific homing systems. The results on the unchanged pattern of adhesion molecule expression in pre-eclampsia suggests that there is no major change in the adhesive properties of the endothelium of the placental bed in pre-eclampsia.
Subject(s)
Cell Adhesion Molecules/metabolism , Placenta/immunology , Pre-Eclampsia/immunology , Antigens, Differentiation, T-Lymphocyte , Antigens, Neoplasm , Case-Control Studies , Endothelium, Vascular/immunology , Endothelium, Vascular/pathology , Female , Humans , Immunohistochemistry , Lymphocytes/immunology , Lymphocytes/pathology , Membrane Glycoproteins/metabolism , Placenta/pathology , Pre-Eclampsia/etiology , Pre-Eclampsia/pathology , Pregnancy , Trophoblasts/immunology , Trophoblasts/pathologyABSTRACT
BACKGROUND: Cardiomyocyte apoptosis has been found in congestive heart failure, but its clinical significance has been difficult to study. We compared the occurrence of cardiomyocyte apoptosis in explanted hearts with the progression of severe heart failure until the need for transplantation. DESIGN: Using the TUNEL assay, apoptotic cardiomyocytes were quantified in explanted failing hearts from patients with either idiopathic dilated cardiomyopathy (n = 21) or ischaemic heart disease (n = 14). The percentage was compared with the clinical severity and progression of endstage heart failure. Samples obtained at autopsy and during open heart surgery served as controls. RESULTS: The number of apoptotic cardiomyocytes was significantly increased in failing hearts regardless of aetiology (medians 0.075% in ischaemic heart disease and 0.119% in dilated cardiomyopathy) compared with control myocardium. In patients with dilated cardiomyopathy, apoptotic cardiomyocytes were more numerous in subjects with a rapidly deteriorating clinical course (0.192%, n = 10) than in patients with intermediate (0.093%, n = 6, P = 0.03) or slow (0.026%, n = 5, P = 0.003) progression. No such association was observed in patients with ischaemic heart disease, in whom we found significantly increased cardiomyocyte apoptosis adjacent to scars of previous infarctions (0.576%) in contrast to the diffuse distribution seen in dilated cardiomyopathy. Expression of Bcl-2, an antiapoptotic protein, was increased in all failing hearts by immunohistochemistry. CONCLUSION: Cardiomyocyte apoptosis is a consistent feature of end-stage heart failure in man and appears to be quantitatively related to the clinical severity of deterioration in dilated cardiomyopathy. Increased expression of Bcl-2 in cardiomyocytes indicates activation of an antiapoptotic response. These observations suggest that cardiomyocyte apoptosis is a clinically relevant and potentially modifiable pathophysiological phenomenon in severe heart failure.
Subject(s)
Apoptosis , Heart Failure/pathology , Heart Transplantation , Myocardium/pathology , Adult , Aged , Aged, 80 and over , Cardiomyopathy, Dilated/pathology , Cardiomyopathy, Dilated/surgery , Disease Progression , Heart Failure/surgery , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Middle Aged , Myocardial Ischemia/pathology , Myocardial Ischemia/surgery , Myocardium/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Time FactorsABSTRACT
Medical records and archival myocardial specimens of 33 children and adolescents with end-stage idiopathic dilated cardiomyopathy (IDCM) were collected to evaluate retrospectively the potential role of enteroviral persistence in the pathogenesis of IDCM. The clinical history and laboratory assessment of each patient were reviewed carefully in order to obtain information on the nature and etiology of infections in the past and at the time of diagnosis of cardiomyopathy. Sixty-four formaldehyde-fixed, paraffin-embedded myocardial specimens, obtained from endomyocardial biopsies (n = 5), explanted hearts (n = 10), or autopsies (n = 49), were studied by the polymerase chain reaction (PCR) and by in situ hybridization to detect enteroviral RNA in the specimens. Control specimens included 34 formaldehyde-fixed, paraffin-embedded myocardial specimens from children with other cardiomyopathies, metabolic diseases, structural heart defects, or various noncardiac malignancies. The presence of cellular RNA in the specimens was confirmed by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA or beta-actin mRNA as positive controls. Only one specimen from the 32 IDCM patients with appropriate myocardial specimens was positive for enteroviral RNA by PCR. Sequence analysis of the amplified viral segment showed a significant degree of homology between the viral sequence and echovirus 1. One specimen from the control patients also appeared positive by PCR, but sequence analysis of the amplified viral segment revealed it as rhinovirus 16. The results do not indicate any significant role for enteroviral persistence in end-stage childhood IDCM, although they need to be confirmed using a prospective study with fresh frozen specimens. However, mechanisms other than viral persistence may be more important in the progression of IDCM to end-stage heart failure in this age group.
Subject(s)
Cardiomyopathy, Dilated/virology , Enterovirus Infections/virology , Enterovirus/isolation & purification , RNA, Viral/isolation & purification , Adolescent , Adult , Base Sequence , Blotting, Southern , Child , Child, Preschool , Enterovirus/genetics , Female , Heart/virology , Humans , In Situ Hybridization , Infant , Male , Molecular Sequence Data , Retrospective Studies , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Group II phospholipase A2 (PLA2) is a lipolytic enzyme suggested to play a role in inflammation and antibacterial defence. In seminal fluid, the concentration of PLA2 is exceedingly high under normal circumstances (about 1,000 times the concentration in blood plasma of healthy humans). To elucidate the origin of the enzyme present in seminal plasma, we investigated the expression of group II PLA2 in male reproductive organs both at protein and mRNA levels. In addition, the presence of the enzyme was studied in common male genital tumors. METHODS: The methods used were immunocytochemistry, in situ hybridization, and Northern blotting. RESULTS: Northern blotting gave positive results for group II PLA2 mRNA in normal prostate, whereas other normal genital tissues gave negative results. Immunohistochemistry and in situ hybridization of group II PLA2 gave identical results. The enzyme was produced exclusively by the secretory epithelial cells of the prostatic gland. Surprisingly, expression was restricted to the posterior lobe and paraurethral glands of the prostate. Cells of prostatic adenocarcinoma expressed group II PLA2, whereas cells of other male genital tumors contained neither the enzyme protein nor the mRNA of group II PLA2. In some cases prostatic cancer cell seemed to express group II PLA2 at a higher rate than normal prostatic gland cells. CONCLUSIONS: The high content of group II PLA2 in seminal plasma is due to the local production and secretion of the enzyme by the epithelial cells of the prostatic glands. Group II PLA2 is expressed focally, suggesting that specialized prostatic glands secrete this enzyme. All prostatic adenocarcinomas tested expressed group II PLA2 in variable amounts.
Subject(s)
Gene Expression Regulation, Enzymologic , Genital Neoplasms, Male/enzymology , Genitalia, Male/enzymology , Phospholipases A/biosynthesis , Prostate/enzymology , Prostatic Hyperplasia/enzymology , Prostatic Neoplasms/enzymology , Epididymis/enzymology , Genital Neoplasms, Male/pathology , Genital Neoplasms, Male/surgery , Genitalia, Male/cytology , Genitalia, Male/pathology , Humans , Male , Phospholipases A2 , Prostate/pathology , Prostate/surgery , Prostatic Neoplasms/pathology , Prostatic Neoplasms/surgery , RNA, Messenger/biosynthesis , Reference Values , Semen/enzymology , Seminal Vesicles/enzymology , Testicular Neoplasms/enzymology , Testis/enzymology , Transcription, Genetic , Urethra/enzymologyABSTRACT
Four cases of ossifying fibromyxoid tumour of soft parts are described. One of them was in the mediastinum, a hitherto unreported location of this rare neoplasm. Another was removed from the subcutaneous tissue of the head of a two-year-old girl, the youngest patient so far described. A peculiar feature of this tumour was haphazard spindle cell groups showing smooth muscle differentiation. One tumour was remarkably proliferative with 20 mitotic figures per 10 high power fields and 50% of cells positive for Ki-67 antigen. Immunohistochemical analysis revealed that all the tumours were diffusely positive for vimentin, and focally for S-100-protein. In addition to this the infantile tumour expressed focal alpha-smooth muscle actin, desmin and glial fibrillary acidic protein, while the mediastinal tumour expressed only alpha-smooth muscle actin and the highly proliferative one expressed none of these antigens. Background cells, including histiocytes, lymphocytes and mast cells were numerous. DNA cytometry analysis using both static and flow methods showed that the mediastinal tumour contained two cell clones, while the others were diploid. The proliferative fraction of cells (S plus G2 phases) was prominent in the proliferative and infantile tumours.
Subject(s)
Mediastinal Neoplasms/pathology , Soft Tissue Neoplasms/pathology , Child, Preschool , DNA, Neoplasm/analysis , Female , Flow Cytometry , Humans , Immunohistochemistry , Ki-67 Antigen/analysis , Male , Mediastinal Neoplasms/chemistry , Middle Aged , Soft Tissue Neoplasms/chemistryABSTRACT
BACKGROUND: It has been suggested that phospholipase A2 (PLA2) has an essential role in the pathogenesis of inflammatory bowel diseases. AIMS: This study aimed at identifying cells in intestinal and mesenteric tissue samples that might express group II phospholipase A2 (PLA2-II) at the mRNA and enzyme protein levels in patients with ulcerative colitis. PATIENTS AND TISSUE SAMPLES: Tissue samples were obtained from the intestine, mesentery, skeletal muscle, and subcutaneous fat of six patients who underwent panproctocolectomy for severe ulcerative colitis. Mucosal biopsy specimens were obtained from the colon of another group of six patients with ulcerative colitis during routine diagnostic colonoscopies. Tissues from six patients without intestinal inflammatory diseases served as controls. METHODS: Tissue samples were studied by light microscopy, immunohistochemistry for PLA2-II enzyme protein, and in situ hybridisation and northern hybridisation for PLA2-II mRNA. RESULTS: PLA2-II mRNA and PLA2-II protein were detected in metaplastic Paneth cells in six patients and in the columnar epithelial cells of colonic mucosa in four out of six patients with active ulcerative colitis. Positive findings were less numerous in patients with mild ulcerative colitis. Only two out of six control patients had a weak positive signal for PLA2-II mRNA and one of these two patients had a weak positive immunoreaction for PLA2-II in columnar epithelial cells in the colonic mucosa. None of the control patients had metaplastic Paneth cells. CONCLUSIONS: Metaplastic Paneth cells and colonic epithelial cells synthesise PLA2-II in ulcerative colitis. The activity of the PLA2-II synthesis seems to be related to the degree of inflammation in the diseased bowel.
Subject(s)
Colitis, Ulcerative/enzymology , Phospholipases A/genetics , Adult , Blotting, Northern , Case-Control Studies , Female , Gene Expression Regulation, Enzymologic , Group II Phospholipases A2 , Humans , Immunohistochemistry , In Situ Hybridization , Intestinal Mucosa/enzymology , Intestinal Mucosa/pathology , Male , Metaplasia/complications , Middle Aged , Phospholipases A2 , RNA, MessengerABSTRACT
BACKGROUND: After reopening of the infarct-related coronary artery, cardiomyocytes continue to die during reperfusion. The mechanisms of cell death have been subject to debate. We studied whether an apoptotic type of cell death occurs in human acute myocardial infarction (AMI). METHODS AND RESULTS: We studied myocardial samples of eight patients who died of AMI and had patent infarct-related arteries at autopsy. Six of the patients had received initially successful thrombolysis. Extensive formation of DNA strand breaks, the typical biochemical feature of apoptosis, was detected with the use of the in situ DNA end-labeling method. Apoptotic cardiomyocytes were observed particularly in the border zones of histologically infarcted myocardium, whereas very few apoptotic cells were present in the remote noninfarcted myocardium. Internucleosomal fragmentation was confirmed by agarose gel electrophoresis of DNA isolated from the representative myocardial areas. CONCLUSIONS: This study provides evidence that in addition to overt necrosis, a subset of myocytes undergo apoptosis during ischemia-reperfusion injury. Apoptosis may provide a new target for cardioprotection during evolving AMI in humans.
Subject(s)
Apoptosis , Myocardial Infarction/pathology , Adult , Aged , Aged, 80 and over , Autoradiography , Cadaver , DNA Damage , DNA Fragmentation , Electrophoresis , Female , Humans , Male , Middle Aged , Myocardial Infarction/genetics , Myocardial Reperfusion Injury/pathology , Myocardium/pathologyABSTRACT
PURPOSE: The aim of this study was to demonstrate the synthesis and cellular distribution of group II phospholipase A2 and lysozyme in the main and accessory lacrimal glands. METHODS: The authors studied samples of normal main lacrimal glands of seven autopsied subjects and accessory lacrimal glands of eight patients who underwent ptosis surgery. The specimens were immunostained with a rabbit antiserum against group II phospholipase A2 and a monoclonal antibody against lysozyme. Expression of group II phospholipase A2 gene was shown using Northern hybridization and in situ hybridization. RESULTS: Lysozyme was present in the secretory granules of most acini, whereas group II phospholipase A2 was seen in a minority of acinar cells, primarily in the central parts of lobules in the main and accessory lacrimal glands. Synthesis of group II phospholipase A2 in the glandular cells was confirmed by Northern hybridization and by in situ hybridization. CONCLUSIONS: There are two specialized cell types in the main and accessory lacrimal glands, one synthesizing group II phospholipase A2 and the other synthesizing lysozyme. These enzymes are important nonspecific antibacterial factors in tears.
Subject(s)
Lacrimal Apparatus/enzymology , Muramidase/biosynthesis , Phospholipases A/biosynthesis , Aged , Aged, 80 and over , Animals , Antibodies , Antibodies, Monoclonal , Blotting, Northern , Female , Humans , Immunohistochemistry , In Situ Hybridization , Lacrimal Apparatus/cytology , Male , Middle Aged , Muramidase/analysis , Phospholipases A/analysis , Phospholipases A2 , RNA Precursors/biosynthesis , RNA Probes , RNA, Messenger/biosynthesis , Rabbits , Transcription, GeneticABSTRACT
The concentration of group II phospholipase A2 was measured in blood plasma samples before and after liver transplantation in a 27-year-old woman who suffered from a massive epitheloid hemangioendothelioma of the liver. The pretransplantation concentration of group II phospholipase A2 was 830 microg/L, which is markedly above the upper limit of the reference interval (10.8 microg/L). After the transplantation, there was a dramatic decrease in the group II phospholipase A2 level (33 microg/L on the first posttransplantation day). In situ hybridization of mRNA and immunohistochemistry revealed synthesis of group II phospholipase A2 in hepatocytes in non-neoplastic areas of the liver. Tumor cells did not contain group II phospholipase A2. The results indicate that group II phospholipase A2 circulating in blood plasma originates in hepatocytes.
Subject(s)
Hemangioendothelioma, Epithelioid/enzymology , Liver Neoplasms/enzymology , Liver/enzymology , Phospholipases A/blood , Adult , Female , Hemangioendothelioma, Epithelioid/genetics , Hemangioendothelioma, Epithelioid/surgery , Humans , Immunohistochemistry , In Situ Hybridization , Liver Neoplasms/genetics , Liver Neoplasms/surgery , Liver Transplantation/physiology , Phospholipases A/classification , Phospholipases A/genetics , Phospholipases A2 , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolismABSTRACT
BACKGROUND: It has been suggested that group II phospholipase A2 (PLA2-II) plays an essential role in inflammation, but the cellular source(s) of the enzyme is (are) largely unknown. EXPERIMENTAL DESIGN: Expression of PLA2-II was analyzed in human gastrointestinal tract at both protein and mRNA levels. Both normal tissues and specimen from patients with a few different common gastrointestinal disorders were analyzed. RESULTS: Expression of PLA2-II mRNA was seen in the Paneth cells but not in any other cell type of the intestinal tract. Blood vessel walls showed PLA2-II immunoreactivity but were negative in in situ hybridization for PLA2-II mRNA. CONCLUSIONS: It was concluded that nonpancreatic group II PLA2 is synthesized and stored by Paneth cells, whereas other cell types of the gastrointestinal tract seem incapable of synthesis of this enzyme. The positive immunoreaction in vascular structures may reflect the entry of circulating PLA2-II into vessel walls rather than local production of the enzyme.