Activated status of tumour-infiltrating lymphocytes and apoptosis in testicular seminoma.

Testicular seminoma is characterized by a prominent lymphoid infiltrate and an excellent prognosis. Cytotoxic T-lymphocytes (CTLs) infiltrating seminoma tumour nests constitute a major subset of the lymphoid infiltrate. The objective of this study was to determine whether CTLs express markers of cytotoxic potential and activity and whether the number of activated CTLs correlates with the extent of apoptosis in testicular seminomas, as opposed to non-seminomatous testicular germ cell tumours (NSTGCTs). Twenty cases of pure seminoma as well as 20 cases of NSTGCTs including 16 mixed germ cell tumours (MGCTs) were studied. Immunohistochemistry for the cytotoxic markers TIA-1 (cytotoxic potential) and granzyme B (cytotoxic activity) and the T-cell markers CD3 and CD8 was performed on formalin-fixed, paraffin-embedded sections. The apoptotic index (AI) was determined by the TUNEL method. The number of CD3(+), CD8(+), TIA-1(+), and granzyme B(+) cells in tumour cell nests was markedly increased in testicular seminomas, compared with NSTGCTs (p<0.01). Activated granzyme B(+) cells numbered 25.6+/-5.2 per high power field in seminomas and 8.9+/-3.2, 8.1+/-3.9, and 0.4+/-0.2 for embryonal carcinomas, yolk sac tumours, and immature teratomas, respectively. Double immunohistochemical staining for granzyme B and CD8 revealed that 82.6+/-8.5% of granzyme B-expressing cells were CD8(+). The tumour cell AI was significantly increased in embryonal carcinoma, compared with the seminoma, yolk sac tumour, and immature teratoma subgroups (6.7+/-1.3, 2.3+/-0.3, 3.0+/-1.1, and 2.3+/-1.1, respectively, p<0.001). TUNEL/CD3 double immunostaining revealed that a significant proportion of the apoptotic seminomatous tumour cells were in direct contact with one or more CD3(+) lymphocytes (47.2+/-6.2%). The number of activated granzyme B(+) CTLs showed a strong linear correlation with the AI in the seminoma group (r=0.71, p<0.0001) but not in other subgroups. TUNEL/granzyme B double immunolabelling revealed that a proportion of activated granzyme B(+) lymphocytes (20%) were often seen in close contact with apoptotic tumour cells. The presence of increased numbers of activated cytotoxic lymphocytes in testicular seminomas suggests that apoptotic tumour cell death in this neoplasm may be triggered by cytotoxic granule effectors. This phenomenon may be one of the key host immune mechanisms leading to the excellent prognosis in this tumour.     

Apoptosis, proliferation, and Fas (APO-1, CD95)/Fas ligand expression in medullary carcinoma of the breast

Medullary carcinoma (MC) of the breast is a unique subtype of infiltrating ductal carcinoma that is characterized by a prominent lymphoid infiltrate and improved prognosis. Activated granzyme B(+)/CD8(+) cytotoxic T-lymphocytes (CTLs) infiltrating tumour cell nests constitute a major subset within the lymphoid infiltrate. As CTLs destroy target tumour cells by triggering apoptosis, it would be of interest to determine whether the apoptotic rate in MC is increased. This study evaluates the extent of apoptosis in relation to Fas (APO-1, CD95)/Fas ligand (FasL) expression in MC. Fourteen cases of typical MC (TMC) and 15 cases of atypical MC (AMC) classified according to the Ridolfi criteria, as well as 19 cases of poorly differentiated infiltrating ductal carcinoma (PDC) were evaluated. The apoptotic index (AI) was assessed by the TUNEL method on paraffin-embedded tissue. Cell proliferation was evaluated immunohistochemically by PCNA staining. The level of Fas/FasL expression was determined semiquantitatively by immunohistochemistry using a four-grade scoring system. The AI was significantly increased in TMC and AMC as opposed to the PDC subgroup (2.2+/-0.8, 2.1+/-0.8, and 1.3+/-0.6, respectively; p<0.05). A significant proportion (31.8+/-7.9% in TMC and 25.8+/-9.7% in AMC) of the apoptotic tumour cells within tumour nests were in close contact with CD3(+) lymphocytes. Increased apoptosis was not accompanied by increased proliferation of tumour cells. The extent of Fas expression did not differ between the three subgroups. FasL was expressed both by tumour infiltrating lymphocytes in MC and by tumour epithelium in all three subgroups. The observation that the majority of MCs express Fas and are infiltrated by lymphocytes expressing FasL suggests that increased apoptosis in MC is mediated by Fas/FasL. However, our observation that the majority of MCs also express FasL and the fact that tumours co-expressing Fas and FasL did not show increased apoptosis suggest that there may be additional cytotoxic pathways that lead to tumour apoptosis in MC.     

Microarray analysis of VEGF-responsive genes in myometrial endothelial cells

There is evidence that the vasculature of different organs display different functional characteristics in response to cytokines and growth factors. The aim of this study was to use cDNA gene expression microarray to analyse changes in gene expression following stimulation of myometrial microvascular endothelial cells (MMECs) with vascular endothelial growth factor (VEGF). Primary isolates of MMECs were obtained from fresh hysterectomy specimens and purified with magnetic beads. Cells were stimulated with 15 ng/ml VEGF for 3, 6 and 12 h, and two unstimulated experiments served as controls. A total of six arrays was performed over these time-points. A total of 110 genes were identified as up-regulated by VEGF, 19% of which (21 genes) have previously been reported as up-regulated by VEGF or by angiogenesis. Among the novel genes to be up-regulated by VEGF were brain-derived growth factor, oxytocin receptor and estrogen sulphotransferase. The significance of the genes identified in the physiological and pathological functioning of the myometrial vasculature is discussed.     

Abnormal deposition of collagen around hepatocytes in Wilson's disease is associated with hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2

BACKGROUND/AIMS: Lysyl-oxidases catalyze the oxidation of lysine residues in collagen and elastin thereby promoting their polymerization. We have studied here the expression of four lysyl-oxidases in normal and diseased human liver. METHODS: The expression of the different lysyl-oxidases in paraffin embedded liver sections was studied using in-situ hybridization and immunohistochemistry. The enzymatic activity of lysyl-oxidase like protein-2 (Loxl2 or LOR-1) using a previously described lysyl-oxidase assay. RESULTS: We have found that the four lysyl-oxidases which we examined are not significantly expressed in the normal liver. By contrast, Wilson's disease and primary biliary cirrhosis (PBC) patients express lysyl-oxidase (Lox) and lysyl-oxidase like protein-2 (Loxl2 or LOR-1) in hepatocytes, and the expression is accompanied by collagen deposition around the hepatocytes. Lysyl-oxidases are also expressed in additional fibrotic liver diseases such as hepatitis B and C but in these diseases the expression is confined to the fibrotic lesions and collagen does not accumulate around hepatocytes. We have found that Loxl2 is able to oxidize lysine residues of collagen, and behaves in that respect similarly to Lox. The copper chelator D-penicillamine inhibits Loxl2 induced oxidation of collagen but the Lox inhibitor beta-aminopropionitrile did not inhibit the oxidation using a BAPN concentration at which Lox activity was completely inhibited. Loxl2 also catalyzed the oxidation of cell surface proteins on HepG2 hepatoblastoma cells and inhibited their proliferation. CONCLUSIONS: Upregulation of Lox and Loxl2 in hepatocytes of Wilson's disease and PBC patients may contribute to liver damage by various mechanisms. The upregulation of Lox and Loxl2 in Wilson's disease could perhaps be utilized for diagnostic purposes since their expression is up-regulated in hepatocytes even before the onset of fibrosis.     

Correlation between body mass index and chondral lesions in isolated medial meniscus tears

Background:

Chondral lesions of the knee are commonly found during arthroscopic partial meniscectomy. The literature advises against arthroscopic medial meniscectomy in the presence of advanced chondral derangement because of unfavorable outcome. Recent studies have shown an association between obesity and chondropathy in patients with meniscal tears. The aim of this study was to assess whether body mass index (BMI) correlates with the severity of chondral lesions in patients with isolated medial meniscus tears (i.e. without ligamentous or lateral meniscal injury).

Materials and Methods:

837 knee arthroscopies were performed in a regional referral center of arthroscopic surgery between January 2011 and December 2012. Of these 168 (109 males, 59 females) patients with no axial knee deformity and no radiological signs of osteoarthritis who have had arthroscopic debridement for isolated torn medial meniscus were included in the study. The correlation between different demographic factors and the level of chondral damage reported at surgery was evaluated. The mean age of patient was 50 years (range 13-82 years) and an average BMI was 28.2 kg/m² (range17.5-42.5 kg/m²).

Results:

Overall, regression analysis showed both age and BMI to be linearly correlated to chondral score (r = 0.53, P < 0.04); however, there were no advanced chondral lesions found in patients younger than 40 years of age and all severe lesions were at age 50 years or more. Therefore, further analysis was performed for age subgroups: patients were grouped as younger than 40, between the age of 40 and 50 (middle age) and older than 50 years. The BMI was linearly correlated to the severity of chondral score exclusively in the middle aged group (i.e. 40-50 years old). There was no correlation between activity level and chondral damage. Women had worse chondral lesions than men in all age groups.

Conclusion:

Higher BMI in middle aged patients with isolated medial meniscus tears and unremarkable radiographs may predict more advanced chondral lesions at arthroscopy.     

Reconstruction of acne keloidalis nuchae using osmotic tissue expanders—a case series

Background

Acne keloidalis nuchae (AKN) is a benign condition of keloid-like papules on the occipital scalp area. Treatment for AKN is divided into conservative and surgical. The use of tissue expander enables preservation of the hirsute area and thereby achieves a good cosmetic result. Osmotic tissue expander is a self-filling device, which absorbs tissue fluids in order to increase skin volume gradually. So far, the use of osmotic expander for reconstruction of AKN has not been described. We present our experience with five consecutive cases of tissue reconstruction using osmotic expanders in AKN.

Methods

Five patients suffering from AKN, median age of 43 years (range 35–50), who were admitted to our department between April 2010 and December 2011 underwent reconstruction using an osmotic tissue expander. All patients had the lesions for a median of 12 years (range 7–15).

Results

In three of the five patients, one expander was used per patient. In the remaining two, two expanders were used. In all the cases, the operative and postoperative management were uneventful with no major complications. A minor complication included partial extrusion of the expander (one patient), which caused an earlier conclusion of the reconstruction, nevertheless with a pleasing result. The average expansion period was 7 weeks (range 4–9). During that time, there was a median of one follow-up visit (range 1–2). Final aesthetic results were satisfactory in all the cases.

Conclusions

Osmotic expander is a reliable tool for tissue expansion. The main advantages of this device make it especially suitable for AKN reconstruction. Its main disadvantages include the inability to control the filling rate and the need to remove it in case of tissue damage. Level of Evidence: Level V, therapeutic study.