Low Proliferative Activity Research Articles

A new in vitro wound model based on the co‐culture of human dermal microvascular endothelial cells and human dermal fibroblasts

Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored. The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in co-cultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds. On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.

Gene expression shift towards normal B cells, decreased proliferative capacity and distinct surface receptors characterize leukemic blasts persisting during induction therapy in childhood acute lymphoblastic leukemia

In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. In the present study, we address molecular and cell biologic features of blasts persisting after 1 week of induction glucocorticoid therapy. Genome-wide gene expression analysis of leukemic samples from precursor B-cell ALL patients (n=18) identified a set of genes differentially expressed in blasts at diagnosis day 0 (d0) and persisting on day 8 (d8). Expression changes indicate a shift towards mature B cells, inhibition of cell cycling and increased expression of adhesion (CD11b/ITGAM) and cytokine (CD119/IFNGR1) receptors. A direct comparison with normal B cells, which are largely therapy resistant, confirmed the differentiation shift at the mRNA (n=10) and protein (n=109) levels. Flow cytometric analysis in independent cohorts of patients confirmed both a decreased proliferative activity (n=13) and the upregulation of CD11b and CD119 (n=29) in d8 blasts. The differentiation shift and low proliferative activity in d8 blasts may account for the persistence of blasts during therapy and affect their sensitivity to further therapeutic treatment. CD11b and CD119 are potential specific markers for d8 blast persistence and detection of minimal residual disease, which warrant further investigation.

99mTc-EDDA/HYNIC-Octreotate in Detection of Atypical Bronchial Carcinoid

Pulmonary carcinoids cause serious difficulties in imaging diagnostics in all stages of the disease. SRS holds great promise for detecting occult primary tu and metastatic lesions. (99m)Tc-EDDA/HYNIC-octreotate, a new scintigraphic agent, should significantly improve sensitivity of the diagnostics of carcinoids due to better affinity to SSR2 than (111)In-Octreoscan and the higher count rate obtained from (99m)Tc over (111)In. We present a case of a 40-year-old women operated on because of lung carcinoid tumour in 2002. The symptoms did not resolve after the operation and 5-OHIAA was still elevated. The thorax spiral CT revealed the focal lesion beneath carina. (111)In-Octreoscan and (99m)Tc-EDDA/HYNIC-octreotate SRS revealed two focal lesions in the mediastinum. (99m)Tc-EDDA/HYNIC-octreotate detected two additional lesions in the lower part of the right lung. Target/non-target count ratios of the lesions were as follows: (99m)Tc-EDDA/HYNIC-octreotate scans - 2,9, (111)In-Octreoscan- 2,1. PET-FDG examination revealed no pathology. Owing to severe bone pains and carcinoid symptoms the patient was referred for the 90Y-DOTA-octreotate treatment. SRS with a new 99mTc marked somatostatin analogue - octreotate allows for a more sensitive detection of metastatic leasions in carcinoid tumours. The usefulness of 18F-FDG PET, widely used as a powerful imaging technique in clinical oncology, is limited in detection of carcinoid tumours due to the low proliferative activity.

Clinically non-functioning pituitary macroadenomas in the elderly

The aim of the present study was to evaluate the clinical presentation, characteristics and post-surgical outcome of non-functioning pituitary macroadenomas (NFPM) in elderly patients. 27 patients (65-81 years; 13 Males, 14 Females) with NFPM (20-45 mm in diameter) were studied. The symptoms prompting neuroradiological studies were vision alterations in 52%, and dizziness, loss of memory, confusion, headache and depression in 29%; in 19% of patients, the disease was incidentally discovered during computed tomography (CT) or magnetic resonance imaging (MRI) for head trauma or cerebral ischemic attacks. Endocrinological evaluation on diagnosis showed global anterior hypopituitarism in 33% and partial hypopituitarism in 37% of patients. Immunohistochemistry showed signs of neurosecretion in most NFPM (chromogranin-A in 55%, gonadotropins in 19%, ACTH in 3.7%). Ki-67 antigen expression was indicative of low proliferative activity. Surgery was highly effective in improving alterations in vision and compressive symptoms, but was unable to restore normal pituitary function in established hypopituitarism in most cases. Eight patients (31%) were free of disease on subsequent MRI (follow-up 1-6 years). In 18 (69%) patients, a post-surgical residue was present. Of these, 6 (33%) underwent radiotherapy in the following years, owing to an increase in the volume of the remnants, and six (33%) underwent additional surgical treatment, followed by radiotherapy for further signs of growth in two. In the remaining patients, a small intrasellar remnant was stable on yearly MRI. in elderly patients, the development of hypopituitarism is often overlooked and the initial diagnosis of NFPM may be delayed. This can expose patients to the risks of unrecognized hypopituitarism and jeopardize post-surgical outcome.

Nuclear β-catenin accumulation associates with epithelial morphogenesis in craniopharyngiomas

Activation of the Wnt/wingless signalling cascade is a key mechanism in developmental morphogenesis, whereas aberrant nuclear accumulation of beta-catenin in adult tissues seems to be associated with neoplastic transformation and tumour progression. Adamantinomatous craniopharyngiomas carry activating mutations in exon 3 of the beta-catenin gene, which results in a distinct pattern of nuclear beta-catenin accumulation in up to 95% of respective tumour specimens. To better characterise the impact of nuclear beta-catenin aggregation in these neoplasms, we systematically examined epithelial differentiation and cell cycle-associated molecules in accumulating compared to non-accumulating tumour cell clusters using a cohort of 65 adamantinomatous craniopharyngiomas. Monoclonal antibodies directed against cytokeratins 5/6 (CK5/6) were utilised to differentiate squamous from simple epithelium, the latter being identified by immunoreactivity for cytokeratins 8 and 18 (CK8/CK18). Intriguingly, nuclear beta-catenin accumulation in whorl-like tumour cell clusters was always associated with a distinct CK8 and CK18 immunoreactivity, whereas surrounding non-accumulating tumour cells showed exclusively squamous differentiation indicated by CK5/6 expression. In addition, a low proliferation activity combined with an increased expression of p21(WAF1/CIP1), a key control protein of the cell cycle, was observed in beta-catenin accumulating cells. Our data support an impact of nuclear beta-catenin on different cytoarchitectural and epithelial differentiation patterns in adamantinomatous craniopharyngiomas.

Umbilical cord blood mesenchymal stem cells

We studied umbilical cord blood mesenchymal stem cells and compared mesenchymal stem cells derived from umbilical cord blood, adipose tissue, and skin. Umbilical cord blood mesenchymal stem cells were characterized morphologically, cytofluorometrically, and by their differentiation potential. Umbilical cord blood mesenchymal stem cells did not differ from cells isolated from adipose tissue and skin by the main parameters (by morphology, expression of surface markers, and differentiation potential). A specific feature of umbilical cord blood mesenchymal stem cells is their low count per volume of the initial material and very low proliferative activity.

Prognostic value of serum angiogenic activity in colorectal cancer patients

Angiogenesis, resulting from an imbalance between angiogenic activator factors and inhibitors, is required for tumour growth and metastasis. The determination of the circulating concentration of all angiogenic factors (activators and inhibitors) is not feasible at present. We have evaluated diagnostic and prognostic values of the measurement of serum angiogenic activity in colorectal carcinoma (CRC) patients. Serum proliferative activity (PA) on human umbilical vein endothelial cells (HUVEC) in vitro, and serum vascular endothelial growth factor (VEGF) levels were determined by ELISA in 53 patients with primary CRC, 16 subjects with non-neoplastic gastrointestinal disease (SC) and 34 healthy individuals. Data were compared with clinical outcome of the patients. Although serum from CRC patients significantly increased the PA of HUVEC, compared to culture control (HUVEC in medium + 10% foetal bovine serum (FBS); P < 0.001); our results indicate that serum PA in CRC patients was similar to that of SC or healthy individuals. There was no correlation between serum PA and circulating VEGF concentrations. Surgery produced a decrease of PA at 8 hrs after tumour resection in CRC patients compared to pre-surgery values (186 ± 47 versus 213 ± 41, P < 0.001). However, an increase in serum VEGF values was observed after surgery (280 [176–450] versus 251 [160–357] pg/ml, P = 0.004). Patients with lower PA values after surgery showed a worse outcome that those with higher PA values. Therefore, this study does not support a diagnostic value for serum angiogenic activity measured by proliferative activity on HUVEC but suggests it could have a prognostic value in CRC patients.

Nuclear medicine in the treatment of neuroendocrine tumours—problems and perspectives

Neuroendocrine tumours (NETs) constitute a large group of neoplasms originating from neuroepithelial crest-derived pluripotent stem cells or differentiated endocrine cells. NET cells and their ancestors are characterised by the ability to take up and decarboxylate the amine precursors (APUD system), the production of several bioactive peptides and the demonstration of particular histopathological staining [1]. The majority of NETs are well differentiated, displaying relatively low proliferative activity. Their ability to produce and release biologically active substances (amines and neuropeptides) results in the appearance of characteristic clinical symptoms [2]. On the other hand, some poorly differentiated and highly malignant NET display very aggressive behaviour, presumably as a result of the overexpression of various cytokines, including transforming growth factor (TGF)-α, nerve growth factor (NGF) and vascular endothelial growth factor (VEGF). Owing to the widespread distribution of APUD cells, the primary NET foci may develop in almost every organ, including gastrointestinal and respiratory tracts, pancreas, adrenal, thyroid and parathyroid glands, thymus, etc. In addition to local infiltration, NET cells form metastases in distant organs at early stages of tumour development. Thus, a radical surgical treatment, which could be curative, is unfortunately impossible [2]. Therefore, NET management requires a multidisciplinary approach that involves nuclear medicine methods as a key constituent of diagnosis, providing data reflecting the biological status of the tumour cells. These methods offer an opportunity to analyse tumour metabolism, stage of differentiation, proliferation rate, expression of various receptors and radiotracer uptake and accumulation. These crucial parameters may be further used to select the most effective form of NET therapy, e.g. surgery, chemotherapy, immunomodulation, external radiotherapy or endoradiotherapy [3]. Since endoradiotherapy is aimed at both the primary tumour and distant metastatic foci, it may be useful either early after surgery, to eliminate occult disease, or in later stages to destroy disseminated neoplasm, with radiation delivery targeting tumour cells while ensuring relative sparing of normal tissue.

Differential Regulation of D-Type Cyclins by Insulin-Like Growth Factor-I and Serum in Multiple Myeloma Cells.

Dysregulation of a D-type cyclin gene is an early and universal event in multiple myeloma (MM), but given the low proliferative activity in this disease, the functional significance of these genetic lesions is unclear. In this study we first examined the expression and regulation of D-type cyclins and other cell-cycle regulatory proteins in a panel of human myeloma cell lines (HMCLs) and in primary normal and malignant CD138+ plasma cells by Western blotting. D-type cyclins, cyclin dependent kinase-4 (CDK4), CDK6, p27, the retinoblastoma protein (pRb) and proliferating cell nuclear antigen (PCNA) were absent in normal bone marow (BM) CD138+ cells (n=5) and heterogeneously expressed in both HMCLs (n=11) and in primary CD138+ MM cells (n=20; 16 from BM aspirates and 4 extra-medullary). Furthermore, expression of these proteins was positively associated with disease progression. Nine of the primary malignant samples were, like normal CD138+ cells, negative for D-type cyclins, their associated kinases or pRB. Of these, one was from a newly diagnosed patient, six were from patients with stable disease and two from patients on treatment. The remaining eleven MM patient samples had heterogeneous expression of D-type cyclins and other cell cycle regulators, four of which had detectable CDK 4/6-phosphorylated pRb. All four of these were from patients with progressive disease, and three were extra-medullary. Furthermore, we demonstrate for the first time in both HMCLs and primary CD138+ MM cells, that cyclins D1 and -D2 are positively regulated by IGF-I and foetal calf serum leading to increased phosphorylation of pRb on CDK4/6 specific sites and an increase in cells in S (DNA synthesis) phase of the cell cycle. In addition, p27 was down-regulated by IGF-I and FCS in some HMCLs suggesting that this cyclin dependent kinase inhibitor also contributes to cell cycle regulation in myeloma cells. However, at low stoichiometric concentrations observed in primary malignant plasma cells, p27 was up-regulated by mitogenic stimuli, consistent with its role in stabilising cyclin D/CDK complexes. Immunoprecipitation analysis revealed that cyclins D1 and -D2 were present in complexes with both CDKs −4 and −6, suggesting that both of these kinases mediate the effect of cyclin D1 or cyclin D2 on pRb phosphorylation and cell cycle entry in these cells. Unlike HMCLs in which cyclin D2 was the primary controller of cell cycle exit/entry (MM1-S and NCI-H929), HMCLs harbouring an 11q13 IgH translocation (KMS12-BM and U266) were refractory to serum withdrawal, suggesting that 11q13 may confer growth factor independent expression of cyclin D1. To determine if cyclin D1 expression via 11q13 was sufficient to promote cell cycle progression, we functionally inactivated it using siRNA in KMS12-BM. Knock-down of cyclin D1 coincided with decreased CDK4/6-specific pRb phosphorylation and an increase in cells arrested in G1 phase of the cell cycle, confirming that cyclin D1 expression via 11q13 leads to cell cycle progression in MM cells, independent of exogenous growth factors. In summary, we show that D-type cyclins are functional in MM, differentially responsive to exogenous growth factors and that their expression is positively associated with aggressive disease.

Histopathologic and Molecular Features of Persistent Polyclonal B-Cell Lymphocytosis Challenging the Benign Nature of the Disorder.

Persistent polyclonal B-cell lymphocytosis (PPBL) is an unusual expansion of B lymphocytes, whose immunophenotype resembles that of memory B cells. The physiopathology of PPBL is unknown and whether this syndrome represents a pre-malignant or benign disease is unclear. Aiming at further defining the histological features of BM involvement and at challenging the polyclonal nature of PPBL, we reviewed 6 cases of PPBL who underwent BM biopsy. There were 3 males and 3 females, with a median age at diagnosis of 35 years (range 17–46). The median lymphocyte count was 5.3 × 109/L (5.1–8.3). One patient had been splenectomized 10 years earlier and 2 were splenomegalic. During the follow-up (median 20 months, range 5–138), 1 showed progressive spleen enlargement and 2 additional patients developed splenomegaly. Screening for hepatitis B, C and HIV was negative. PB morphology showed 2 to 32% of cleaved lymphocytes. Immunophenotype documented 21 to 70% B lymphocytes in both PB and BM aspirate, all positive for CD19, CD22, CD79b, FMC7, CD18 and CD11a, negative for CD5, CD23, CD10, with no Ig light chain restriction. CD25 was expressed in 4 cases. PCR analysis of the IgH gene configuration of PB and BM cells showed a polyclonal picture in all cases. On Giemsa-stained sections of BM trephines, BM was normocellular in 5 cases and slightly hypocellular in 1. A mild lymphoid infiltrate was visible, mainly interstitial with occasional small lymphoid aggregates. An intrasinusoidal pattern was seen in 4/6 cases. Immunohistochemistry confirmed the interstitial infiltration by B cells (CD79a+, CD20+, IgM+, IgD+, DBA44−/+, CD5−, AnnexinD1−) in all cases and highlighted the intrasinusoidal pattern in 4 in which a splenic marginal zone lymphoma (MZL)-like morphology was described. By IgH gene PCR on BM tissue, a clonal rearrangement was detected in 2/4 cases with intrasinusoidal infiltration; in 2 cases DNA quality was poor. The 4 cases showed splenomegaly at diagnosis or during the follow-up. Splenectomy was performed in 1 patient due to progressive enlargement of the spleen over 9 years. White pulp was normal and red pulp was expanded by an infiltration of small lymphocytes with an intrasinusoidal distribution. Splenic B lymphocytes were CD79a+, CD20+, IgM+, IgD+, DBA44−, as in the BM biopsy, with low proliferation activity and proven clonality by IgH-PCR. In the remaining 2 cases, in which no intrasinusoidal lymphoid pattern was evident, clonal IgH rearrangement was absent and no splenomegaly has developed. We confirm the interstitial lymphoid infiltration of BM trephine in all cases of PPBL, with an evident intrasinusoidal component in some. The detection of IgH rearrangements on lymphocytes infiltrating the BM from bioptic samples and the absence of clonality in PB lymphocytes and in the BM aspirate, possibly due to the intrasinusoidal distribution, have not been described. These findings suggest that at least some of the cases described as PPBL are pre-malignant disorders close to splenic MZL, justifying the occurrence of progressive splenomegaly over time. Prolonged follow-up of patients with PPBL and an accurate biologic work-up of the BM, are therefore warranted.

Chromosome Banding Analysis Allows a More Precise Classification of CLL Than Interphase FISH: A Study on 446 Cases.

In CLL data from chromosome banding analysis have been scarce due to the low proliferative activity in vitro. We improved the cultivation technique using an immunostimulatory CpG-oligonucleotide DSP30 and IL-2 leading to a high success rate of chromosome banding analysis in routine diagnostics. Since August 2005 446 CLL were analyzed in parallel with chromosome banding analysis (CBA) and interphase-FISH. Diagnosis of CLL was established by standard criteria based on cytomorphology and immunophenotyping. The FISH panel included probes for the detection of trisomy 12, IGH-rearrangements and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). 440/446 (98.7%) cases could be successfully stimulated for metaphase generation and are the basis of this study. 370/440 (84.0%) cases showed chromosomal aberrations in CBA while abnormalities were detected by FISH in 353 of 440 (80.2%) successfully evaluated cases. Overall 452 abnormalities were detected by FISH and 788 abnormalities by CBA. Based on FISH results 277 cases showed 1, 67 cases 2, 8 cases 3 and 1 case 4 abnormalities, respectively. In CBA at least 1 aberration was detected in 177, 2 in 98, 3 in 45, 4 in 19, and 5 or more aberrations in 31 patients. In 31 of 87 cases (35.6%) showing no aberrations in FISH abnormalities were detected in CBA. On the other hand 14 of 70 cases (20.0%) with a normal karyotype demonstrated abnormalities using FISH. In 7 of these cases CBA missed the abnormalities due to the small size of the aberrant clone or insufficient proliferation of the aberrant clone in vitro and in another 7 cases due to the small size of the 13q deletion not visible in CBA. Using CBA, in total 97 balanced translocations, 169 unbalanced translocations leading to gain and/or loss of genetic material, 368 deletions, 77 gains of whole chromosomes, 40 losses of whole chromosomes, and 37 other aberrations were observed. Only 17 of 97 balanced translocations involved the IGH gene. In 28 cases balanced translocations involved the breakpoint 13q14. Although these translocations were reciprocal and seemed balanced in CBA FISH demonstrated a 13q14 deletion in the breakpoint region. Therefore, based on CBA cases with 13q deletion could be subdivided into 3 different categories: 1. del(13q) sole, 2. del(13q) with additional abnormalities and 3. del(13q) due to a reciprocal translocation. This genetic heterogeneity might account for differences in clinical outcome. In cases with TP53 deletions the number of chromosome abnormalities was higher compared to cases without TP53 deletion (mean 5.0 vs 1.5, p<0.0001). In conclusion, CBA offers important information in addition to interphase FISH in CLL. 1) CBA detects chromosome abnormalities which can not be detected with a standard interphase FISH panel. These additional abnormalities could explain heterogeneous clinical outcome. 2) CBA provides new biological insights into different CLL subclasses based on a much more heterogeneous pattern of cytogenetic abnormalities as assumed so far for CLL. Therefore, prospective clinical trials should evaluate the prognostic impact of these additional abnormalities that now can be identified by chromosome banding analysis.

The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions

Studies were performed to elucidate the possible relationship between microvessel density, proliferative activity and angiogenesis in eutopic endometrium from women with and without endometriosis and peritoneal endometriotic lesions. The question whether changes in these parameters in endometriotic lesions were reflected by the level of vascular endothelial growth factor-A (VEGF-A) in serum and peritoneal fluid was also studied. Biopsy specimens of both eutopic endometrium and peritoneal endometriotic lesions from women with endometriosis (n = 25) as well as eutopic endometrium from women without endometriosis (n = 14) were analysed immunohistochemically regarding microvessel density, proliferative activity, and expression of VEGF-A and its receptors vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2) in stroma, glands and blood vessels. The VEGF-A concentration was measured in peritoneal fluid and serum. Secretory phase eutopic endometrium from women with endometriosis had significantly higher microvessel density, expression of VEGF-A in glandular epithelium and VEGFR-2 in endometrial blood vessels than those from women without endometriosis. Endometriotic lesions with high proliferative activity had a higher microvessel density and showed higher vascular expression of VEGFR-2 as well as being accompanied by higher levels of VEGF-A in peritoneal fluid and serum, compared with lesions with low proliferative activity. In conclusion, there seems to be a dysregulation of angiogenic activity in the eutopic endometrium of women with endometriosis and endometriotic lesions with high proliferative activity were accompanied by higher local angiogenic activity and higher levels of VEGF in serum and peritoneal fluid.

Konzept und Problematik der lobulären Neoplasie

The term lobular neoplasia (LN) includes lobular carcinoma in situ (LCIS) and atypical lobular neoplasia (ALH). It is generally considered to be a risk lesion and a non-obligatory precursor for the subsequent development of an invasive carcinoma in the ipsilateral or contralateral breast. LN has also been termed lobular intraepithelial neoplasia (LIN). A grading system (LIN 1-LIN 3) has been suggested as a tool for a more precise estimation of the individual risk. When LN is the most significant finding in a core biopsy, the probability of a higher grade lesion is about 17% in the follow-up surgical biopsy, justifying follow-up surgery in the majority of cases. A higher risk of progression is attributed to LIN 3 (pleomorphic LN, extensive LN, and signet ring cell LN) compared to LIN 1 or LIN 2. These special forms of LN may have an unusual presentation clinically or histologically. Using immunohistology, LN are characterized by the loss of E-cadherin, low proliferative activity and by positive hormone receptor status. The molecular characteristics of LN are similar to those of invasive lobular carcinomas, indicating the nature of LN as a precursor lesion.

Clinical added-value of 18FDG PET in neuroendocrine-merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare and highly malignant skin cancer with neuroendocrine differentiation. We studied the potential value of 18FDG PET in the management of MCC. Eleven patients with MCC were examined by 18FDG PET and PET-CT for staging purpose (n=4) or for detection of recurrence (n=7). Qualitative and quantitative interpretation of PET studies was performed routinely. 18FDG PET observations were compared to clinical and radiological findings. In 6 patients, PET findings were also compared to histology. In 7 patients, the 18FDG tumor uptake was compared to the MCC proliferative activity expressed by the Ki-67 index. 18FDG PET was contributive in 10/11 MCC patients. In 7 patients, 18FDG PET detected focal lesions or a disseminated stage of the disease including dermal, nodal and visceral metastases. In 3 patients, a normal 18FDG PET confirmed complete remission of disease. Most MCC patients exhibited highly 18FDG-avid sites suggestive of increased glucose metabolism. This imaging pattern was related to a high proliferative activity (Ki-67 index >50%). In 1 patient with a weakly proliferative nodal MCC (Ki-67<10%), a false negative result was yielded by metabolic imaging. In 4/11 patients, 18FDG PET revealed an unsuspected second neoplasm in addition to MCC. It is concluded that whole-body 18FDG PET may be useful in the management of MCC patients. However, a normal 18FDG PET aspect cannot rule out MCC with low proliferative activity.

Cytokine production profile of splenocytes derived from zymosan A-treated SKG mice developing arthritis

SKG mice have a point mutation of the zeta-associated protein of 70 kD (ZAP-70) and spontaneously develop a severe polyarthritis in the conventional condition, whereas they are healthy under the specific pathogen free (SPF) condition. The purpose of this study was to investigate the cytokine production from splenocytes in SKG mice developing arthritis under the SPF condition. SKG and BALB/c mice were intraperitoneally injected with zymosan A under the SPF condition. Spleen was isolated 1, 2 or 8 weeks after the intraperitoneal injection of saline or zymosan A. Splenocytes were cultured with concanavalin A. Cytokine production and proliferation were measured 48 and 72 h after the culture. An intraperitoneal injection of zymosan A induced severe polyarthritis with increased levels of rheumatoid factor and interleukin 6 (IL-6) only in SKG mice. Splenocytes from SKG mice did not proliferate well maybe because of less productivity of IL-2. The IL-4 production from splenocytes of SKG mice was higher, while interferon-gamma production was lower than those of BALB/c mice. An injection of zymosan A reduced the IL-4 production only in SKG mice. SKG mice do not develop arthritis under the SPF condition possibly because of a low proliferative activity of T cells and Th2-predominance.

Consequences of Verteporfin Photodynamic Therapy on Choroidal Neovascular Membranes

To examine the impact of photodynamic therapy (PDT) on angiogenesis in human choroidal neovascular membranes with respect to vascular endothelial growth factor (VEGF) expression, proliferation, and vascularization. Retrospective review of an interventional case series of 50 patients (50 eyes) who underwent removal of choroidal neovascular membranes. Choroidal neovascularization was secondary to age-related macular degeneration. Twenty patients were treated with PDT 3 to 655 days before surgery. Choroidal neovascular membranes were stained for CD34, CD105, Ki-67, cytokeratin 18, and VEGF. Thirty choroidal neovascular membranes secondary to age-related macular degeneration without previous treatment were used as controls. Specimens without pretreatment disclosed varying degrees of vascularization, proliferative activity, and VEGF expression by different cells. Specimens treated with PDT 3 days earlier showed mostly occluded vessels, damaged endothelial cells, and low proliferative activity. In contrast, specimens excised at later time points after PDT were highly vascularized and proliferating. This chronology was associated with an impressive VEGF immunoreactivity unique to retinal pigment epithelial cells shortly after PDT that also shifted to other cells at later time points. Photodynamic therapy induces selective vascular damage in choroidal neovascular membranes. The effectiveness and selectivity of this treatment, however, seem to be jeopardized by a rebound effect initiated by enhanced VEGF expression in retinal pigment epithelial cells.

Bcl-2 and Bax in congenital naevi

Melanocytes represent a static component of the epidermis, and the role of apoptosis in basal melanocyte function and melanocytic tumour formation has not been fully elucidated. The aim of this study was to investigate the expression of Bcl-2 anti-apoptotic and Bax apoptotic proteins in congenital naevi in correlation with p-27 protein and Ki-67 proliferative index. Our material comprised 30 congenital naevi (eight giant) excised from children aged from 15 days to 14 years old. The immunohistochemical streptavidin-biotin method was performed on paraffin sections for the detection of Bcl-2 (cl100/D5), Bax (cl2D2) , Ki-67 (MIB-1) and p-27 (1B4) proteins with monoclonal antibodies. Bcl-2 protein was detected in all cases showing a strong diffuse cytoplasmic expression in >70% of the naevocytes and was preserved in the deeper parts of the naevi. On the other hand, Bax was detected in 13 of the cases, showing a fainter cytoplasmic expression in 40-50% of the naevocytes without any particular topographic distribution. Ki-67 was detected in all cases showing a limited expression in 1-2% of the nuclei mainly in the junctional and upper dermal components. p-27 protein showed a broad diffuse nuclear expression (>70% of the nuclei) in all cases with a particular increase in the deeper parts of the naevi. Bcl-2 expression showed a parallel correlation with p-27 protein. Broad Bcl-2 expression in congenital naevi suggests that suppression of apoptosis may play an important role in the maintenance of naevocytes despite the low proliferative activity.

Elevated levels of fibroblast growth factor-2 in serum from women with endometriosis

The purpose of this study was to determine whether serum and peritoneal fluid levels of fibroblast growth factor-2 (FGF-2) differ between women with and without endometriosis and to examine the relationship between proliferative activity in the endometriotic lesions and the levels of FGF-2 in serum and peritoneal fluid. Biopsy specimens of peritoneal endometriotic lesions (n = 25) were analyzed immunohistochemically regarding proliferative activity in stroma, glands, and blood vessels. The FGF-2 concentration was measured in peritoneal fluid and serum from 25 women with endometriosis and 14 healthy controls. Patients with endometriosis had higher serum concentrations of FGF-2 during both the proliferative and secretory menstrual phases than healthy women. Peritoneal fluid levels of FGF-2 differed significantly between women with high and low proliferative activity in their endometriotic lesions. Women with endometriosis have a higher serum level of FGF-2 than healthy controls.

Comparative genomic hybridization analysis on male breast cancer

The spectrum of genetic alterations in primary male breast cancer is not well established. We analyzed chromosomal imbalances in 39 tumor samples from primary male breast cancer by comparative genomic hybridization (CGH) and correlated CGH findings with clinicopathological factors. Chromosomal gains were most frequent at 1q (46%), 8q (46%), 16p (36%), 17q (36%), Xq (28%), 20q (26%) and Xp (18%). Losses were most commonly observed at 8p (36%), 16q (28%), 13q (28%), 6q (18%), 11q (18%) and 22q (18%). Gains at 16p, 20q and Xq and losses at 13q correlated significantly with higher degree of cytogenetic complexity. Significant associations with clinicopathological factors were observed for +8q and -16q with larger tumor size and -16q with lower proliferative activity and lower grade of malignancy. A comparison with reported CGH data from female breast cancer showed a similar pattern of chromosomal imbalances, including +1q, -8p, +8q, -13q, +16p, -16q, +17q and +20q. Our results indicate that male breast cancer shares a common pattern of imbalances with female breast cancer, suggesting that similar genetic events may underlie the development and progression of male and female breast cancer.

Resting smooth muscle cells as a model for studying vascular cell activation

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions. This capacity plays a major role in the inception and progression of such cardiovascular diseases as atherosclerosis, hypertension and restenosis. Porcine coronary smooth muscle (PCSM) cells exhibit a synthetic phenotype (ON cells) under standard culturing conditions, but they can be reverted to a contractile phenotype (OFF cells) in a serum-free medium. However, OFF cells can also reacquire a synthetic phenotype (OFF/ON cells) upon serum administration. In the present study, proliferative and contractile behaviors were characterized by expression of specific differentiation markers. Taken together, these results demonstrate that porcine vascular smooth muscle cells can retain their phenotypic plasticity in culture, and thus mimick in vitro their in vivo differentiation states. OFF cells may thus provide a suitable model system in studying the mechanism(s) by which either known or unknown serum factors may trigger vascular smooth muscle activation. In the present study, this possibility was actually tested by exposing OFF cells to fetal bovine serum (FBS), PDGF-BB and IGF-I. Data show that only FBS could induce a synthetic phenotype in OFF cells, while both PDGF-BB and IGF-I failed to induce any VSM activation.