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The accurate execution of DNA replication requires a strict control of the replication licensing factors hCdt1 and hCdc6. The role of these key replication molecules in carcinogenesis has not been clarified. To examine how early during cancer development deregulation of these factors occurs, we investigated their status in epithelial lesions covering progressive stages of hyperplasia, dysplasia, and full malignancy, mostly from the same patients. Abnormal accumulation of both proteins occurred early from the stage of dysplasia. A frequent cause of unregulated hCdc6 and hCdt1 expression was gene amplification, suggesting that these components can play a role per se in cancer development. Overexpression of hCdt1 and hCdc6 promoted rereplication and generated a DNA damage response, which activated the antitumor barriers of senescence and apoptosis. Generating an inducible hCdt1 cellular system, we observed that continuous stimulus by deregulated hCdt1 led to abrogation of the antitumor barriers and resulted in the selection of clones with more aggressive properties. In addition, stable expression of hCdc6 and hCdt1 in premalignant papilloma cells led to transformation of the cells that produced tumors upon injection into nude mice depicting the oncogenic potential of their deregulation. [Cancer Res 2007;67(22):10899–909]

Supplementary Figures 1-7, Tables 1-2 from Deregulated Overexpression of hCdt1 and hCdc6 Promotes Malignant Behavior

(English) This is a unique case of retinoschisis as an ocular manifestation of Brucellosis. A 38-year-old male presented with recurrent episodes of bilateral eye redness predominately on his left eye. His visual acuity was not affected, and he did not report any other symptoms. On slit-lamp examination, binocular Koeppe nodules of the iris and cells in the left anterior chamber were observed. Fundoscopy followed by meticulous multimodal imaging confirmed left inferior retinoschisis. The patient was diagnosed with panuveitis and a series of laboratory examinations revealed positive anti-IgM Brucella antibodies. Ocular brucellosis can cause variable, atypical and serious presentations, hence early diagnosis is paramount to avoid complications. Keywords: Retina; Panuveitis; Choroidal/Retinal Inflammation; Techniques of Retinal Examination; Microbiology Zusammenfassung Dies ist ein einzigartiger Fall von Retinoschisis als Augenmanifestation einer Brucellose. Ein 38-jähriger Mann stellte sich mit wiederkehrenden Episoden beidseitiger Augenrötung, vorwiegend am linken Auge, vor. Seine Sehschärfe war nicht beeinträchtigt, und er berichtete über keine anderen Symptome. Bei der Spaltlampenuntersuchung wurden binokulare Koeppe-Knötchen der Iris und Zellen in der linken Vorderkammer beobachtet. Eine Fundoskopie gefolgt von einer sorgfältigen multimodalen Bildgebung bestätigte eine linke untere Retinoschisis. Bei dem Patienten wurde eine Panuveitis diagnostiziert, und eine Reihe von Laboruntersuchungen ergab positive Anti-IgM-Brucella-Antikörper. Okuläre Brucellose kann variable, atypische und schwerwiegende Erscheinungsformen verursachen, daher ist eine frühzeitige Diagnose von größter Bedeutung, um Komplikationen zu vermeiden. Schlüsselwörter: Netzhaut; Panuveitis; Aderhaut-/Netzhautentzündung; Techniken der Netzhautuntersuchung; Mikrobiologie

Paragangliomas are rare lesions of the endocrine system that can be treated either by embolization preoperatively followed by surgical excision or by surgical excision or radiotherapy. In this report, we present an extremely rare location of a nonfunctional paraganglioma in the head and neck region, located in the right supraclavicular fossa, which was misdiagnosed as a thyroid tumor, in a 72-year-old female patient. Imaging revealed a 2.5 × 4.5 × 2 cm well-defined vascularized mass. Fine-needle aspiration (FNA) on the tumor was not diagnostic initially; however, a third attempt revealed thyroid cells suggesting the existence of an ectopic thyroid tumor. FNA was additionally performed on the right thyroid lobe, revealing atypical follicular colloid cells of the Bethesda 3 category. Therefore, the excision of the ectopic thyroid tumor along with right lobectomy was planned. No embolization was initiated preoperatively in this case. Histopathology revealed that the supraclavicular mass was a paraganglioma. Paragangliomas supplied by the subclavian, innominate, and common carotid artery are rarely reported, and to our knowledge, this is the third case to be reported worldwide.

SummaryImmune responses against SARS-CoV-2 have been vigorously analyzed. It has been proposed that a subset of mild or asymptomatic cases with undetectable antibodies may clear the virus in a T-cell cytotoxic-dependent manner, albeit recent data revealed the importance of B-cells in that regard. We hypothesized that underdiagnosed antigenemia/viremia may conceal humoral response possibly through immunocomplex formation. We report the first case of late-onset seroconversion detected following decline in antigenemia/viremia levels. Consequently, classification of at least a subset of COVID-19 cases as non-responders might not represent a true immunobiological phenomenon, rather reflect antibody masking due to prolonged antigenemia/viremia.

Rationale SARS-CoV-2 infection of the respiratory system can progress to a life threatening multi-systemic disease, mediated via an excess of cytokines (“cytokine storm”), but the molecular mechanisms are poorly understood. Objectives To investigate whether SARS-CoV-2 may induce cellular senescence in lung epithelial cells, leading to secretion of inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). Methods Autopsy lung tissue samples from eleven COVID-19 patients and sixty age-matched non-infected controls were analysed by immunohistochemistry for SARS-CoV-2 and markers of cellular senescence (SenTraGor, p16INK4A) and key SASP cytokines (interleukin-1β, interleukin-6). We also investigated whether SARS-CoV-2 infection of an epithelial cell line induces senescence and cytokine secretion. Measurements and Main Results SARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in alveolar type-2 (AT2) cells, which also expressed the angiotensin-converting-enzyme 2 (ACE2), a critical entry receptor for this virus. In COVID-19 samples, AT2 cells displayed increased markers of senescence [p16INK4A, SenTraGor staining positivity in 12±1.2% of cells compared to 1.7±0.13% in non-infected controls (p Conclusions We demonstrate that in severe COVID-19 patients, AT2 cells are infected with SARS-CoV-2 and show senescence and expression of proinflammatory cytokines. We also show that SARS-CoV-2 infection of epithelial cells may induce senescence and inflammation, indicating that cellular senescence may be an important molecular mechanism of severe COVID-19.

Onychomycosis is considered as one of the major public health problems with a global distribution associated with geographic, demographic and environmental factors, underlying comorbidities and immunodeficiency disorders. This study was conducted to investigate the etiological agents of onychomycosis, in Northwestern Greece during a 7-year period. The study population included 1095 outpatients with clinically suspected onychomycosis that presented to the University Hospital of Ioannina, NW Greece (2011&ndash, 2017). Samples were examined for causative fungi, and mycological identification was established using standard mycological methods. Demographic data of each patient, comorbidities, localization of infection and history of previous fungal infection were collected. Onychomycosis was diagnosed in 317 of the 1095 suspected cases (28.9%) and the most frequently isolated pathogens were yeasts (50.8%) followed by dermatophytes (36.9%) and non-dermatophyte molds (NDMs) (12.3%). Dermatophytes were mostly involved in toenail onychomycosis (90.6%) and more commonly affected males than females (57.3% vs. 42.7%), while the predominantly isolated pathogen was &Tau, richophyton rubrum (74.4%) followed by &Tau, richophyton interdigitale (21.4%). Candida albicans was the most prevalent isolated yeast (82%), whereas among the cases with onychomycosis due to NDMs, Aspergillus spp. were isolated as the principal species (59%). Continuous monitoring should be performed in order to identify possible trends and shifts in species isolation rates and to evaluate the impact of onychomycosis among the general population and high-risk groups.

Background: Pancreatic ductal adenocarcinoma (PDAC) is resistant to single-agent immunotherapies. To understand the mechanisms leading to the poor response to this treatment, a better understanding of the PDAC immune landscape is required. The present work aims to study the immune profile in PDAC in relationship to spatial heterogeneity of the tissue microenvironment (TME) in intact tissues. Methods: Serial section and multiplex in situ analysis were performed in 42 PDAC samples to assess gene and protein expression at single-cell resolution in the: a) tumor center (TC), b) invasive front (IF), c) normal parenchyma adjacent to the tumor, and d) tumor positive and negative draining lymph nodes (LNs). Results: We observed: a) enrichment of T cell subpopulations with exhausted and senescent phenotype in the TC, IF and tumor positive LNs, b) a dominant type 2 immune response in the TME, which is more pronounced in the TC, c) an emerging role of CD47-SIRP&alpha, axis, and d) a similar immune cell topography independently of the neoadjuvant chemotherapy. Conclusion: This study reveals the existence of dysfunctional T lymphocytes with specific spatial distribution, thus opening a new dimension both conceptually and mechanistically in tumor-stroma interaction in PDAC with potential impact on the efficacy of immune-regulatory therapeutic modalities.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory system can progress to a multisystemic disease with aberrant inflammatory response. Cellular senescence promotes chronic inflammation, named senescence-associated secretory phenotype (SASP). We investigated whether coronavirus disease 2019 (COVID-19) is associated with cellular senescence and SASP.MethodsAutopsy lung tissue samples from 11 COVID-19 patients and 43 age-matched non-COVID-19 controls with similar comorbidities were analysed by immunohistochemistry for SARS-CoV-2, markers of senescence and key SASP cytokines. Virally induced senescence was functionally recapitulated in vitro, by infecting epithelial Vero-E6 cells and a three-dimensional alveosphere system of alveolar type 2 (AT2) cells with SARS-CoV-2 strains isolated from COVID-19 patients.ResultsSARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in AT2 cells. Infected AT2 cells expressed angiotensin-converting enzyme 2 and exhibited increased senescence (p16INK4A and SenTraGor positivity) and interleukin (IL)-1β and IL-6 expression. In vitro, infection of Vero-E6 cells with SARS-CoV-2 induced senescence (SenTraGor), DNA damage (γ-H2AX) and increased cytokine (IL-1β, IL-6, CXCL8) and apolipoprotein B mRNA-editing (APOBEC) enzyme expression. Next-generation sequencing analysis of progenies obtained from infected/senescent Vero-E6 cells demonstrated APOBEC-mediated SARS-CoV-2 mutations. Dissemination of the SARS-CoV-2-infection and senescence was confirmed in extrapulmonary sites (kidney and liver) of a COVID-19 patient.ConclusionsWe demonstrate that in severe COVID-19, AT2 cells infected by SARS-CoV-2 exhibit senescence and a proinflammatory phenotype. In vitro, SARS-CoV-2 infection induces senescence and inflammation. Importantly, infected senescent cells may act as a source of SARS-CoV-2 mutagenesis mediated by APOBEC enzymes. Therefore, SARS-CoV-2-induced senescence may be an important molecular mechanism of severe COVID-19, disease persistence and mutagenesis.

Humoral immunity has emerged as a vital immune component against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nevertheless, a subset of recovered Coronavirus Disease-2019 (COVID-19) paucisymptomatic/asymptomatic individuals do not generate an antibody response, constituting a paradox. We assumed that immunodiagnostic assays may operate under a competitive format within the context of antigenemia, potentially explaining this phenomenon. We present a case where persistent antigenemia/viremia was documented for at least 73 days post-symptom onset using ‘in-house’ methodology, and as it progressively declined, seroconversion took place late, around day 55, supporting our hypothesis. Thus, prolonged SARS-CoV-2 antigenemia/viremia could mask humoral responses, rendering, in certain cases, the phenomenon of ‘non-responders’ a misnomer.

Cellular senescence is a stress response mechanism ensuring homeostasis. Its temporal activation during embryonic development or normal adult life is linked with beneficial properties. In contrast, persistent (chronic) senescence seems to exert detrimental effects fostering aging and age-related disorders, such as cancer. Due to the lack of a reliable marker able to detect senescence in vivo, its precise impact in age-related diseases is to a large extent still undetermined. A novel reagent termed GL13 (SenTraGorTM) that we developed, allowing senescence recognition in any type of biological material, emerges as a powerful tool to study the phenomenon of senescence in vivo. Exploiting the advantages of this novel methodological approach, scientists will be able to detect and connect senescence with aggressive behavior in human malignancies, such as tolerance to chemotherapy in classical Hodgkin Lymphoma and Langerhans Cell Histiocytosis. The latter depicts the importance of developing the new and rapidly expanding field of senotherapeutic agents targeting and driving to cell death senescent cells. We discuss in detail the current progress of this exciting area of senotherapeutics and suggest its future perspectives and applications.

The Ebstein Barr virus(EBV), herpes virus 5 has been associated with lymphoproliferative disordrers. Age-related EBV+ B-LPD is defined as an EBV+ clonal B-cell lymphoid proliferation or EBV+-DLBCL developing in patients over the age of 40 years in the absence of any known immunodeficiency and without an underlying T-cell lymphoma1. We present a case of EBV+ clonal B-cell lymphoid proliferation. Key words:Oral mucosa ulcer, EBV+-DLBCL, age related.

The mammalian E2F family of transcription factors comprises a group of 8 proteins, which either activate or repress transcription of numerous target genes, playing a role in cell-cycle progression and apoptosis. We have collectively investigated the immunohistochemical expression of E2F1, E2F2, and E2F4 transcription factors and their relation to cell kinetic parameters using serial section analysis in a series of 100 cases of human colorectal adenocarcinomas. E2F1 and E2F4 expressed nuclear immunopositivity in all cases. The range of their expression was 2% to 80% (mean 21% ± 15%) and 2% to 90% (mean 66% ± 20%), respectively. E2F2 was expressed in 41 cases at low levels (range, 1% to 5%, mean 2% ± 9%). A statistically significant direct association between E2F4 and cell proliferation, as expressed by high levels of Ki-67 labeling index, was shown. A mutually exclusive immunostaining pattern between E2F1 and E2F4 and a direct correlation of E2F1 and apoptosis were also highlighted. Our results point to a possible direct tumor-promoting role for E2F4 in the context of colorectal carcinogenesis. The inverse immunohistochemical relationship between E2F1 and E2F4 indicates a possible mechanistic interlink in colorectal cancer. Low expression of E2F2 may reflect functional redundancy between members of the E2F family, in this case between E2F1 and E2F2.

Angiogenesis leads to new blood vessel formation and is implicated in both physiological and pathological situations. The vascular endothelial growth factor (VEGF) family is the major mediator of this process. The aim of our study was to evaluate the expression of VEGF-A, vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 and their correlation with clinicopathological parameters and prognosis in patients with classical Hodgkin lymphoma (cHL), since the role of angiogenesis in this tumor still remains unclear. The immunohistochemical expression of VEGF-A, VEGFR-1 and VEGFR-2 was examined in 194 patients with cHL. The neoplastic Hodgkin Reed-Sternberg (HRS) cells expressed VEGF-A, VEGFR-1 and VEGFR-2 in 90.3%, 97.2% and 94.1% of cases, respectively. Only the expression of VEGFR-2 was positively correlated with serum albumin levels ≥ 4 g/dL. No correlation with patient outcome was observed. All three molecules were statistically correlated with ramifications of blood vessels. Summarizing, our results are not sufficient to consider VEGF-A and/or VEGF receptors as prognosticators in cHL.

The expression of c-myc oncogene in neoplastic and non-neoplastic lymphoid tissue was studied using the specific monoclonal antibody myc 1-9E10 and the 3 step streptavidin-biotin immunoperoxidase method in paraffin sections. Twelve samples of non-neoplastic reactive lymph nodes of various etiology, 181 of non-Hodgkin's lymphomas (NHL), and five of hairy cell leukemia (HCL) were studied. The staining pattern was mainly nuclear but cytoplasmic reactions were also occasionally observed. The percentage of positive cells per total, cell population was estimated and all cases were classified into four groups: 0.-15%, 16-.30%, 31-45% and more than 45% positive cells. In the reactive lymph nodes, mainly germinal center cells were stained, but the percentage in eleven of the 12 cases was less than 15%. All cases of HCL were negative. In NHL lymphoma, only malignant cells were evaluated. Fifty-six per cent of cases of NHL had more than 15% positive cells. The percentage of positive cells was greater in high grade than in intermediate and low grade NHL (80.6x, 47.6%, and 10.6 respectively had more than 30:< positive cells). This correlation proved to be statistically significant (p < 0.001). The above findings suggest a relation between the expression of the c-myc oncogene and lymphomagenesis and indicate that (evaluation of the expression of c-myc in NHL may be helpful in determining the grade of malignancy.

Integrins mediate cell adhesion to the extracellular matrix. Integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site and has been associated with high malignant potential in breast cancer cells, signaling the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including RGD peptides, have been used as potential anti-cancer agents. In the present work, the effect of the linear RGD hexapeptide GRGDSP was studied, for the first time, on breast tumor explants, as well as on well-spread human breast cancer cells from primary cultures, using the explant technique, to clarify the role of this peptide in the suppression of breast cancer cell migration. The results showed that incubation of breast tumor explants with RGD peptide at the beginning of culture development inhibited completely the migration of cancer cells out of the tissue fragment as revealed by electron microscopy. RGD incubation of well-spread breast cancer cells from primary culture resulted in rounding and shrinkage of the cells accompanied by altered distribution of integrin alphavbeta3 and concomitant F-actin cytoskeletal disorganization, as revealed by immunofluorescence. Electron immunocytochemistry showed aggregation of integrin alphavbeta3 at the cell periphery and its detection in noncoated vesicles. However, Western immunoblotting showed no change in beta3 subunit expression, despite the altered distribution of the integrin alphavbeta3. In light of the above, it appears that the RGD peptide plays an important role in the modulation of cell motility and in the perturbation of cell attachment affecting the malignant potential of breast cancer cells in primary cultures.

The expression of growth hormone-releasing hormone (GHRH) splice variant 1 (SV1) receptor in neoplastic lesions of the oral cavity was assessed. The sensitivity of HaCaT keratinocytes to GHRH analogs was also evaluated. Thirty-three benign precancerous oral lesions and 27 squamous cell carcinomas of the oral cavity were evaluated by immunohistochemistry for SV1 expression. SV1 expression in HaCaT keratinocytes was assessed by western blot. HaCaT proliferation was evaluated by cell counting. Anti-SV1 immunoreactivity was detected in only 9% (three of 33) precancerous lesions (one hyperplasia and two dysplasias), while 44% (12 of 27) carcinomas were positive for SV1 (p

Background: Concerted alterations between stromal fibroblasts and neoplastic cells underline the carcinogenic process. Activation of alpha-smooth muscle actin (SMA) expression, a cytoskeleton protein normally expressed only in myoepithelial cells, is considered a landmark for the activation of stromal fibroblasts with little however being known regarding the mechanism governing the expression of SMA in the stroma.Methods: We have evaluated by immunohistochemistry the expression of SMA in the stroma of oral malignant and pre-malignant lesions, in association with the expression of p53 and p21 tumor suppressors that were shown previously to be deregulated and/or mutated in stromal fibroblasts of various cancers. The effects of p21 knockdown in SMA expression and cell migration and the mRNA levels of endogenous p21 in fibroblasts co-cultured with cancer cells were also assessed.Results: We found that both p21 and SMA expression was elevated in the stroma, but not the epithelium, of malignant as compared to pre-malignant lesions. We also noted that the expression of both was positively correlated, implying that SMA expression may be regulated by p21. Consistently with this notion we found that siRNA-mediated p21 suppression resulted in the reduction of SMA levels and also inhibited cell migration.Conclusion: Our results show that p21 deregulation is associated with the activation of stromal fibroblasts of oral cancers by a mechanism that involves the stimulation of SMA expression.

Leptin, adiponectin, and resistin are adipokines linked to the development of insulin resistance, which plays a central role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We aimed to define adipokine serum levels in severely obese patients undergoing bariatric surgery and to correlate these with anthropometric and metabolic variables, liver function tests, and histopathological parameters of NAFLD and nonalcoholic steatohepatitis (NASH). Surgical liver biopsies were obtained from 50 bariatric patients with no history of liver disease or significant alcohol consumption. Serum leptin, adiponectin, and resistin levels were measured, and histology was assessed using Brunt’s and Kleiner’s scoring systems. Waist/hip ratio was significantly higher in men (p = 0.0001), and leptin (p = 0.036) and adiponectin (p = 0.0001) serum levels were higher in women. Forty-one of 50 patients (82%) had histological NAFLD, including 10 (20%) with NASH. Nine patients (18%) had normal liver histology (obese control subgroup). In NAFLD patients, serum adiponectin was negatively correlated with activity grade and fibrosis stage, resistin was negatively correlated with steatosis grade (p = 0.033), while leptin was not related to histology. Leptin/adiponectin ratio showed positive association with stage (p = 0.044). In the subgroup of NASH patients, adiponectin was negatively correlated only with stage (p = 0.01), while there was no correlation between leptin, resistin, or leptin/adiponectin and histology. Serum adiponectin and resistin levels are related to liver histology in bariatric patients and may be indicative of the histological severity of NAFLD and the extent of hepatic steatosis, respectively. Serum leptin levels are not informative of underlying liver histology in severely obese patients.

We examined the relationship between the PLA2 polymorphism of the platelet GPIIIa receptor and major adverse cardiac events (MACE) after percutaneous coronary intervention (PCI).PLA2 polymorphism has been associated with increased thrombosis and myocardial infarction. The association of PlA2 with MACE post-PCI has not been determined.200 patients with normal baseline CKMB undergoing non-urgent PCI for symptomatic coronary artery disease were tested for the PLA2 polymorphism and followed for 1 year while on aspirin and clopidogrel. MACE were recorded and adjudicated by an independent, blinded committee.Baseline demographic and lesion characteristics, platelet aggregation, activated clotting time and use of GP Ilb/llla blockers were similar between the 2 groups. The normal (A1A1), heterozygous (A1A2), and homozygote (A2A2) variants were found in 144 (72%), 55 (27.5%), and 1 (0.5%) patients, respectively. The presence of the PLA2 genetic polymorphism had no influence on 1-year MACE: 7.1% for the A1A1 group versus 6.5% for the A1A2 group (P=NS). The rate of any CKMB elevation post-PCI was 39% vs. 38% respectively (P=NS).In this study, the GPIIIa PlA2 polymorphism was frequent (27.5%), but the homozygous variant was very infrequent (0.5%). The presence of PLA2 had no influence on peri-procedural or one-year clinical outcomes.

The aim of the present study was to observe possible qualitative and quantitative expression differences between nuclear matrix proteins (NMPs) of human colon adenocarcinoma and their mirror biopsies, using the technique of two-dimensional gel electrophoresis, in order to identify the existence of specific NMP fingerprints for colon cancer. Colon tissues were examined ultrastructurally and NMPs were isolated biochemically, by serial extraction of lipids, soluble proteins, DNA, RNA, and intermediate filaments and were separated according to their isoelectric point (pI) and their molecular weight (MW) by high-resolution two-dimensional electrophoresis (2D). By comparing the 2D electropherograms of colon cancer tissues and mirror biopsy tissues we observed qualitative and quantitative expression differences between their NMPs but also a differentiation of NMP composition between the stages of malignancy. Moreover, despite the similarities between mirror biopsy samples, a highlight percentage of exception was observed. Electrophoretic results provided in this study demonstrated that the examined NMPs could be further investigated as potential markers for detection of colorectal cancer in an early stage, for the assessment of the disease progression, as well as useful tools for individual therapy and for preventing a possible recurrence of cancer and metastasis.

Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies.

The use of genomic DNA rather than cDNA or mini-gene constructs in gene therapy might be advantageous as these contain intronic and long-range control elements vital for accurate expression. For gene therapy of cystic fibrosis though, no bacterial artificial chromosome (BAC), containing the whole CFTR gene is available. We have used Red homologous recombination to add a to a previously described vector to construct a new BAC vector with a 250.3-kb insert containing the whole coding region of the CFTR gene along with 40.1 kb of DNA 5′ to the gene and 25 kb 3′ to the gene. This includes all the known control elements of the gene. We evaluated expression by RT-PCR in CMT-93 cells and showed that the gene is expressed both from integrated copies of the BAC and also from episomes carrying the oriP/EBNA-1 element. Sequencing of the human CFTR mRNA from one clone showed that the BAC is functional and can generate correctly spliced mRNA in the mouse background. The BAC described here is the only CFTR genomic construct available on a convenient vector that can be readily used for gene expression studies or in vivo studies to test its potential application in gene therapy for cystic fibrosis.

Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and upon binding to specific GHRH receptors in the pituitary stimulates growth hormone production and release. In addition to its neuroendocrine action GHRH plays a role in tumorigenesis. Consistently with this latter role, the splice variant 1 (SV1) of GHRH receptor, which is widely expressed in non-pituitary normal tissues and cancers, can mediate the proliferative effects of GHRH and even in the absence of GHRH is capable of eliciting mitogenic signals in the tissues in which it is expressed. The aim of the present study was to investigate the expression of GHRH and its tumoral receptor SV1 in primary human melanomas and dysplastic nevi by immunohistochemistry. None of the specimens tested expressed GHRH. Only 1 of 12 (8%) dysplastic nevi expressed SV1 but 14 of 23 (61%) melanomas showed moderate or strong staining for SV1 (association p

Purpose: To correlate the immunohistochemical expression of topoisomerase IIα (topoIIα) in Hodgkin's lymphoma (HL) with clinicopathological parameters, the expression of Ki-67 and the outcome of patients, who had been homogenously treated with ABVD or equivalent regimens. Experimental Design: Immunohistochemistry using the monoclonal antibody Ki-S1 (topoIIα) was performed in 238 HL patients. MiB1 (Ki-67) expression was evaluated in 211/238. Results: The mean ± SD percentage of topoIIα- and Ki-67–positive Hodgkin-Reed-Sternberg (HRS) cells was 63 ± 19% (5%-98%) and 73 ± 19% (8%-99%), respectively. The median percentage of topoIIα-positive HRS cells was 64% (interquartile range, 51-78%). There was no correlation between topoIIα expression and patient characteristics. TopoIIα and Ki-67 expression were correlated (Spearman's Rho 0.255, P < 0.001). TopoIlα expression within the highest quartile of this patient population was predictive of failure free survival (FFS) (10-year rates 82 ± 3% vs 68 ± 7%, P = 0.02 for patients falling into the quartiles 1-3 and 4 respectively). In multivariate analysis topoIIα expression was independently predictive of FFS. Conclusion: TopoIIα was expressed in all cases of HL showing a correlation with Ki-67 expression. Under current standard therapy including drugs inhibiting its activity, topoIIα was an independent adverse predictor of FFS with no statistically significant correlation with other established prognostic factors.

Summary The present study aimed at evaluating the modulation of insulinlike growth factor I receptor (IGF-IR) and estrogen receptor β (ER- β ) expression and their correlation during tumorigenesis of sporadic colorectal cancer, with particular interest in the insulin resistance syndrome. In a series of 100 individuals (54 men and 46 women; mean age, 67.3 ± 9.4 years) with colorectal neoplasms, classified as early adenomas (n = 25), advanced adenomas (n = 44), and adenocarcinomas (n = 31), IGF-IR and ER- β expression was quantified in formalin-fixed, paraffin-embedded biopsy specimens, using confocal laser scanning microscopy and a computer-based method for assessment of immunofluorescent staining. All individuals were evaluated for insulin resistance markers (hyperglycemia, dyslipidemia, central obesity, and arterial hypertension), and 50 (26 men and 24 women; mean age, 68.2 ± 9.0 years) were diagnosed with the insulin resistance syndrome. For the sequence of early adenoma-advanced adenoma-adenocarcinoma, a gradual increase in IGF-IR expression and a gradual decrease in ER- β expression were observed. The partial correlation coefficient between IGF-IR and ER- β expression, controlled for age, sex, insulin resistance, type of lesion, and location of lesion was 0.295 ( P = .004, 2-tailed significance). Analysis of variance demonstrated that the effect of the insulin resistance syndrome on IGF-IR and ER- β expression was significant ( P = .007 and P = .018, respectively). The results suggest the combined effect of IGF-I and estrogens in colorectal cancer, with a distinctive role in individuals with the insulin resistance syndrome.

Intact cerebellum cytoarchitecture and cellular communication are indispensable for successful motor coordination and certain forms of memory. Cytosine arabinoside (AraC), often used as an anti-neoplastic agent in humans, can have cerebellum-targeting adverse effects. In order to characterize the nature of AraC-induced cerebellar lesions in an adult rodent model, we have administered AraC (400 mg/kg b.w., i.p.) in adult male Wistar rats for 5 days. The animals' walking pattern, motor coordination, locomotion, spatial navigation and cognition were evaluated, along with neurofilament- and calbindin-like distribution in the cerebellum. AraC-treated rats demonstrated a disturbed walking pattern and a reduced ability of motor learning and coordination, indicative of a mild cerebellar deficit. Although the general locomotion and spatial cognition of AraC-treated rats was not significantly altered, their navigation into the water, in terms of swimming velocity, was irregular, compared to vehicle-treated animals. Neurofilament-like immunostaining was reduced in the molecular cerebellar layer, while calbindin D 28 kDa levels were increased in Purkinje neurons, following AraC treatment. Administration of the antioxidant N-acetylcysteine (NAC) (200 mg/kg b.w., p.o.), for 14 days (prior to and during AraC treatment) largely prevented the AraC-induced behavioral deficits. Our in vivo model of neurotoxicity provides data on the AraC-induced behavioral and cellular alterations concerning the adult rat cerebellum. Furthermore, it provides evidence of a possible neuroprophylactic role of the antioxidant N-acetylcysteine in this model of chemotherapy-induced toxicity.

Oral non-Hodgkin’s lymphomas (O-NHLs) are a rare group of diverse lymphoid tissue malignancies and represent less than 5% of the oral cavity malignancies and 2% of all extra-nodal NHLs. Oral-NHLs affect the Waldeyer’s-ring, the salivary glands, the bone of the jaws and the oral mucosa, their clinical appearance is very heterogeneous. Among the risk factors for NHLs are immunosuppression (primary or secondary), autoimmunity and inflammation. O-NHLs share the same risk factors. This case report describes a patient with O-NHL which was possibly linked to the combination of methotrexate and etanercept for the treatment of her rheumatoid arthritis. To our knowledge this is probably among the first cases of O-NHL with possible relation to the use of a Tumor Necrosis Factor (TNF) antagonist biological agent (etanercept). This case could contribute to the sensitization of the dentists for the signs and symptoms of this rare malignancy. It also underlines the need for thorough medical history and medication recording for all the dental patients. Key words:Lymphoma (oral) methotrexate, etanercept.

Udgivelsesdato: nov 30 Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression.

In a previous study, the authors have shown cytokeratin 8 (CK8) and epitope H ultrastructural localization in breast cancer cell nuclei. Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H. In this study, double immunogold labeling of CK8 and epitope H combined with the EDTA regressive staining method was applied in biopsy material from infiltrating ductal breast carcinomas and fibroadenomas, to localize both antigens in correlation to RNPs distribution in the nuclear subcompartments of cancer cells. CK8 and epitope H were localized mostly over condensed chromatin, whereas staining was weaker over interchromatin granule clusters and perichromatin fibers. These results revealed, the distribution of CK8 in the nucleus as MAR-binding protein, contributing in the organization of the nuclear DNA in the neoplastic cell, as well as the distribution of O-GlcNAc glycosylated polypeptides bearing the epitope H. The latter finding indicates that these polypeptides might play a significant role in the neoplastic behavior of breast cancer cells because they colocalize in the same nuclear subcompartments with proteins modified by O-GlcNAc, such as hnRNPs G and A1, RNA polymerase II, its transcription factors, and the oncogene product of c-myc. These proteins are known to participate in coordinated transcription/RNA processing events, contributing in the neoplastic behavior of breast cancer cells.

DNA damage checkpoint genes, such as p53, are frequently mutated in human cancer, but the selective pressure for their inactivation remains elusive. We analysed a panel of human lung hyperplasias, all of which retained wild-type p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and Chk2 phosphorylation, p53 accumulation, focal staining of p53 binding protein 1 (53BP1) and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). We propose that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability and selective pressure for p53 mutations.

Administration of anti-tumor necrosis factor (anti-TNF) agents is beneficial in a variety of chronic inflammatory conditions, including psoriasis. We describe 5 patients in whom psoriasiform skin lesions developed 6-9 months after the initiation of anti-TNF therapy for longstanding, seropositive rheumatoid arthritis (etanercept or adalimumab), typical ankylosing spondylitis (infliximab), and Adamantiades-Behçet's disease (infliximab). In all 5 patients, the underlying disease had responded well to anti-TNF therapy. Four patients developed a striking pustular eruption on the palms and/or soles accompanied by plaque-type psoriasis at other skin sites, while 1 patient developed thick erythematous scaly plaques localized to the scalp. In 3 patients there was nail involvement with onycholysis, yellow discoloration, and subungual keratosis. Histologic findings from skin biopsies were consistent with psoriasis. None of these patients had a personal or family history of psoriasis. In all patients, skin lesions subsided either with topical treatment alone, or after discontinuation of the responsible anti-TNF agent. The interpretation of this paradoxical side effect of anti-TNF therapy remains unclear but may relate to altered immunity induced by the inhibition of TNF activity in predisposed individuals.

Twenty-one days of restraint stress has been shown to affect hippocampal plasticity, neurogenesis, and spatial memory. Hippocampal glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) are the main mediators of stress response and learning/memory processes. We studied the performance of male and female rats on a hippocampal-dependent spatial task after 21 days of restraint in relation to the stress-induced changes of GR and MR status in their hippocampi. Reduced GR immunostaining was detected in the dentate gyrus and CA1 area of stressed male rats. Stressed male rats performed worse than the male control rats on the Morris water maze. In contrast, unaltered (in the dentate gyrus) or increased (in CA1) GR immunoreactivity was seen in the hippocampus of stressed female rats. Stressed female rats had an improved memory score in the task compared with the female control rats. In addition, stressed female rats showed increased MR immunostaining in the CA3 area, which is known to be severely affected by stress. The observed sexually dimorphic effects of 21-day restraint in spatial learning and memory may be associated with the sex-dependent changes in hippocampal corticosteroid receptor status after stress.

The effects of 21-day exposure to restraint stress on hippocampal corticosteroid receptors and on spatial performance of male and female rats were evaluated. Stressed male animals exhibited a decrease in glucocorticoid receptor immunoreactivity in the CA1 area and the dentate gyrus. At the same time, stressed males tested on Morris water maze showed delayed learning and worse memory scores, compared with the control males. By contrast, stressed females exhibited an increase in glucocorticoid receptor immunoreactivity in CA1, similar learning ability and improved memory scores, compared with control females. In addition, stressed females showed a significant increase in mineralocorticoid receptor immunoreactivity in the CA3 area compared with controls. These data show that 21 days of restraint stress affect hippocampal corticosteroid receptors and spatial performance in a gender-specific manner. The observed changes in corticosteroid receptor levels following stress, may be causatively linked to the stress-induced alterations on spatial learning and memory.

Bone marrow angiogenesis has recently been implicated in the pathophysiology and course of various haematological malignancies. Little is known, however, about the significance of this phenomenon in hairy cell leukaemia (HCL). We evaluated various morphometric characteristics of microvessels, highlighted by means of anti-CD34 immunohistochemistry, in the bone marrow of 44 patients with typical HCL, before and after treatment with interferon-alpha (IFN-alpha). Overall, bone marrow from 103 HCL patients and 20 controls was examined. Microvessel density (MVD) and several size- and shape-related parameters were quantified in the region of most intense vascularization using image analysis. MVD, size-related parameters and the percentage of branching microvessels were higher in HCL than in controls. Likewise, perimeter counts were higher in partial/non-responders than in complete responders. Achievement of complete response was accompanied by smaller calibre microvessels. IFN-alpha induced a decrease in MVD and branching values in cases with diffuse marrow involvement. In univariate analysis, progression-free survival was adversely affected by MVD, branching and major axis length. Multivariate analysis indicated that MVD/branching independently affected progression-free survival and the likelihood of complete response. Our data suggest that the generation of bone marrow microvessels indicated an increased risk of progression and IFN-alpha treatment failure in HCL. Furthermore, the prognostic significance of angiogenesis requires the concomitant assessment of MVD and the complexity of the microvascular network.

Waldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder (LPD) characterized by lymphoplasmacytic infiltration of the bone marrow (BM) and/or occasionally other tissues and by the presence of a serum monoclonal IgM. The disease belongs to the lymphoplasmacytic lymphoma (LPL) subtype. Whether WM is indeed a separate entity or is merely an IgM-secreting subtype of low-grade B-cell lymphoma is still controversial. In our series of 67 patients, WM has a long median overall survival of 110 months, and the male/female ratio is 1.2/1. Clinical features include a wide spectrum of manifestations, many of which may be common to other LPDs. Differential diagnosis is based on: (1) clinical and laboratory features (anemia, organomegaly, lymphadenopathy, IgM paraproteinemia), (2) cell morphology (lymphocytes, lymphoplasmacytes, few plasma cells), (3) histopathology of bone marrow or lymph node, (4) immunophenotype (CD5 expression and the intensity of CD5, CD20, and CD79b antigens may help in discrimination from other LPDs and atypical CLL), and (5) characteristic genetic features (present in other LPDs). Based on the former, diagnosis is usually easy. It may be harder in LPL cases not secreting IgM. We consider that WM should be, for the time being, handled as a separate entity. Further studies are necessary.

The monoclonal antibody H (mAbH) detects an epitope consisting of an O-linked N-acetyl glucosamine (O-GlcNAc) and neighboring amino acids. This epitope has been found by using extracts from the MCF-7 human breast carcinoma cell line in immunoblotting experiments, on cytokeratin 8 (CK8) and 5 other polypeptides. In the present study, a double immunogold method was applied for the colocalization of CK8 and mAbH epitope on epoxy thin sections in 18 cases of infiltrating ductal breast carcinomas (IDBC) and in 6 cases of fibroadenomas, to study the accurate subcellular distribution of CK8 in breast cancer cells, as compared to the 5 polypeptides, recognized by mAbH. Furthermore, a detailed quantitative evaluation of the double immunolocalization over the cellular compartments of cancer cells was undertaken with the aid of a computerized image analysis system and the results were assessed statistically. The distribution pattern of CK8 and the mAbH epitope in the neoplastic mammary epithelial cells was similar in IDBC as compared to fibroadenomas, while the gold labeling intensity of these epitopes differed over the cellular compartments between malignant and benign biopsies. The results reveal the significance of the role of CK8 and O-GlcNAc glycosylation in the biology of the neoplastic mammary cells in vivo, determining their malignant potential.