The common marmoset as a model for the study of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of metabolic syndrome. The more clinically concerning form of the disease, nonalcoholic steatohepatitis (NASH), is characterized by steatosis, lobular inflammation, and ballooning degeneration. Here we describe a naturally occurring syndrome in the common marmoset that recapitulates the pathologic findings associated with NAFLD/NASH in humans. Hepatomegaly determined to result from NAFLD was observed in 33 of 183 marmosets. A comprehensive histopathologic assessment performed in 31 marmosets demonstrated that NAFLD was characterized by variably sized, Oil Red O staining cytoplasmic vacuoles and observed primarily in animals with evidence of obesity and insulin resistance. A subset of marmosets (16 of 31) also demonstrated evidence of NASH characterized by multifocal inflammation combined with ballooning hepatocellular degeneration. Marmosets with NASH demonstrated an increase in immunostaining with an antibody targeted against the human leukocyte antigens (HLA)-DP, HLA-DQ, and HLA-DR compared with marmosets without NASH (38.89 cells/10× field vs 12.05 cells/10× field, P = .05). In addition, marmosets with NASH demonstrated increased Ki-67 immunopositive cellular proliferation compared with those without (5.95 cells/10× field vs 1.53 cells/10× field, P = .0002). Finally, animals with NASH demonstrated significantly increased mean circulating serum iron levels (160.47 µg/dl, P = .008) and an increase in numbers of Prussian blue-positive Kupffer cells (9.28 cells/40× field, P = .005) relative to marmosets without NASH (97.75 µg/dl and 1.87 cells/40×, respectively). This study further characterizes the histopathology of NAFLD/NASH and suggests that the marmoset may be a valuable animal model with which to investigate the host and environmental factors contributing to the progression of NAFLD/ NASH.

[Imaging diagnostics of bone sarcomas]. / Bildgebende Diagnostik der Knochensarkome.

BACKGROUND: Bone tumors and especially bone sarcomas are rare lesions of the skeletal system in comparison to the much more frequently occurring bone metastases. Despite the relative rarity they are important differential diagnoses of bone lesions. OBJECTIVE: The aim of this article is to give the reader an insight into the fundamentals of the primary imaging of bone sarcomas and to illustrate this with the help of two examples (e.g. osteosarcoma and chondrosarcoma). RESULTS: The foundation of the imaging of bone sarcomas is the radiograph in two planes. This method delivers important information on bone tumors. This information should be analyzed with the help of the Lodwick classification, the configuration of periosteal reactions and a possible reaction of the cortex. A possible tumor matrix and the localization within the skeleton or within long bones also provide important information for differential diagnostic delimitation. Magnetic resonance imaging (MRI) with specific adapted bone tumor sequences allows an exact local staging of a bone sarcoma. In addition to local imaging a compartmental MRI which illustrates the entire extent of tumor-bearing bone and the adjacent joints should be performed to rule out possible skip lesions. The most common distant metastases of osteosarcoma and chondrosarcoma occur in the lungs; therefore, a computed tomography (CT) of the chest is part of staging. Other imaging methods, such as CT of the tumor, positron emission tomography CT (PET-CT), bone scan and whole body MRI supplement the imaging depending on tumor type.

Risk stratification of patients with locally aggressive differentiated thyroid cancer. Results of the MSDS trial.

UNLABELLED: The Multicentre Study Differentiated Thyroid Cancer (MSDS) collective represents a well defined group of patients with locally aggressive thyroid carcinomas (pT4; AJCC/UICC 1997). The aim of the present study was to compare the survival of patients with minimum and extensive extrathyroidal growth according to the new AJCC/UICC TNM staging system 2009. PATIENTS, METHODS: The follow-up data of 347 patients were analysed. Patients were reclassified according to the current AJCC/UICC 2009 classification. The event-free and overall survival was evaluated using Kaplan-Meier analysis. In addition, postoperative complications and status of disease were documented. RESULTS: 327 patients were assigned to stage pT3 and 20 patients to stage pT4a, respectively. Median follow-up was 6.1 years (range 0.04-9.8 years). 92.5% of patients reached complete remission. There were 7.8 % recurrences in the thyroid bed, in locoregional lymph nodes and/or in distant sites. The overall survival was >98% both in pT3 and pT4a patients (p = n. s.). In contrast, the event-free survival was significantly less favourable in pT4a patients (p < 0.001). Using multivariate analysis the following parameters were significant predictors of event-free survival: histological tumour type, degree of extrathyroidal extension and nodal metastasis (p < 0.05). CONCLUSIONS: The MSDS patients with locally aggressive differentiated thyroid cancer showed an excellent overall survival during a median follow-up of 6.1 years. According to the current AJCC/UICC 2009 classification, pT3 patients with minimal extrathyroidal extension revealed a significantly better event-free survival than pT4a patients with extensive extrathyroidal growth.

Small intestinal adenocarcinoma in common marmosets (Callithrix jacchus).

Small intestinal adenocarcinomas are uncommon neoplasms that are rarely reported in nonhuman primates. These neoplasms are also rare in humans, although they are thought to share a similar pathogenesis with the more common colorectal carcinoma. Herein the authors report the clinical, histologic, immunohistochemical, and molecular characteristics of small intestinal adenocarcinoma in 10 common marmosets (Callithrix jacchus). Retrospective analysis of necropsy records revealed small intestinal carcinoma to be the most common neoplastic cause of morbidity and mortality in aged common marmosets. The average age of affected animals was 6.6 years old, and there was no sex predilection. Nine of 10 (90%) tumors arose within the proximal small intestine near the interface with the duodenum. All cases were characterized by disorganization, loss of polarity, and proliferation of neoplastic epithelial cells along the crypt to midvillous interface. Two of 10 (20%) were defined as carcinoma in situ. Eight of 10 (80%) had some degree of invasion, with lymphatic invasion and lymph node metastasis present in 6 of 10 (60%) animals. Immunohistochemically, 10 of 10 (100%) expressed cytokeratin; 7 of 9 (77%) expressed E-cadherin; and 8 of 9 (88%) expressed beta-catenin. The expression of E-cadherin and beta-catenin was decreased in the cell membrane and increased in the cytoplasm. No Helicobacter-like bacteria were observed via silver stain, and callitrichine herpesvirus 3 was detected by polymerase chain reaction with equal frequency from neoplastic and nonneoplastic intestinal sections. The tumors described in this population illustrate comparable features to human cases of small intestine carcinoma and may serve as a potential animal model for small intestinal carcinomas.

[B lymphocyte differentiation in granulomatous tissues of the lung and the nasal mucosa in Wegener's granulomatosis: origin of anti-neutrophil cytoplasmic antibody formation?]. / B-Lymphozyten-Differenzierung in granulomatösen Geweben der Lunge und der Nasenschleimhäute beim Morbus Wegener: Ursprungsort der ANCA-Bildung?

Wegener's granulomatosis (WG) starts with granulomatous inflammation of the respiratory tract before it converts into a potentially organ and life threatening systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA). The site of formation of the highly specific ANCA directed against "Wegener's autoantigen" proteinase 3 (PR3) is still unknown. Previously, we have shown that follicle-like B lymphocytic infiltrates in the vicinity to PR3 expressing cells in WG-granulomata. We characterized the immunoglobulin-VH repertoire in lung and nasal granulomata (paraffin embedded) from four WG patients. A total of 115 individual VH genes were characterized and compared to 84 VH genes from the peripheral blood of a healthy donor. We found an increased frequency of mutations with a bias to amino acid exchanges within the antigen binding sites (CDR) 1 and 2 in WG tissue. A large number of mutations led to negatively charged amino acids and may increase affinity to the positively charged PR3. Furthermore, the occurrence of differently mutated members of one B cell clone indicates clonal expansion and intraclonal diversification by an antigen, e.g. PR3. Several WG tissue derived genes displayed similarities to published sequences from peripheral PR3 ANCA producing B cells. Thus, granulomata of the lower and upper respiratory tract contain follicle-like B cell clusters with a selected VH repertoire infiltrate in WG. WG granulomata could be the place of autoantigen presentation and formation of high-affinity ANCA within neoformed ectopic or tertiary lymphoid-like tissue areas.

The ELF -97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets.

We compared fluorescent signals obtained with fluorescein conjugates and the ELF-97 (enzyme-labelled fluorescence) phosphatase substrate [2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF-97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright yellow-green, with maximal excitation at approximately 360 nm and maximal emission at approximately approximately 530 nm. The ELF substrate was used with streptavidin-alkaline phosphatase, to fluorescently label site-specific probes bound to their targets, including cell-surface sites, cytoplasmic organelles, nuclear antigens and cytoskeletal networks. All targets were labelled successfully with both the ELF substrate and fluoresceinated probes or protein conjugates. However, the ELF method was frequently more sensitive, with lower background fluorescence, allowing detection of more lysosomes, actin filaments, microtubules and nuclear targets than were visible with corresponding fluoresceinated probes. The ELF substrate was also used with antifluorescein-alkaline phosphatase to amplify fluorescein signals. We found that the ELF signal was in all cases brighter and more photostable than fluorescein signals, permitting shorter film exposures and allowing more time for examining samples. Surprisingly, relative brightness and photostability depended on the target, rather than being a general phenomenon related to the choice of dye alone.

Omiga: a PC-based sequence analysis tool.

Computer-based sequence analysis, notation, and manipulation are a necessity for all molecular biologists working with any but the most simple DNA sequences. As sequence data become increasingly available, tools that can be used to manipulate and annotate individual sequences and sequence elements will become an even more vital implement in the molecular biologist's arsenal. The Omiga DNA and Protein Sequence Analysis Software tool, version 2.0 provides an effective and comprehensive tool for the analysis of both nucleic acid and protein sequences that runs on a standard PC available in every molecular biology laboratory. Omiga allows the import of sequences in several common formats. Upon importing sequences and assigning them to various projects, Omiga allows the user to produce, analyze, and edit sequence alignments. Sequences may also be queried for the presence of restriction sites, sequence motifs, and other sequence features, all of which can be added into the notations accompanying each sequence. This newest version of Omiga also allows for sequencing and polymerase chain reaction (PCR) primer prediction, a functionality missing in earlier versions. Finally, Omiga allows rapid searches for putative coding regions, and Basic Local Alignment Search Tool (BLAST) queries against public databases at the National Center for Biotechnology Information (NCBI).

Estrogen protects against hypertension in the spontaneously hypertensive rat, but its protective mechanism is unrelated to impaired arterial muscle relaxation.

OBJECTIVES: To determine if estrogen acutely and directly alters arterial muscle relaxation, if estrogen is responsible for gender dichotomy in hypertension, and if arterial muscle from female spontaneously hypertensive rats (SHR) is slow to relax as is muscle from male SHR compared with arterial muscle of normotensive Wistar-Kyoto rats (WKY). METHODS: Relaxation rates of isometrically contracted arterial muscle from male rats were measured before and after addition of beta-estradiol. Blood pressure (BP) was monitored in intact male and female SHR and WKY and in ovariectomized and estrone-treated ovariectomized SHR and WKY. Relaxation rates of maximum isometric contractions of arterial muscle excised from male SHR and WKY and female SHR and WKY with varying chronic estrogen status were measured. RESULTS: Beta-estradiol had no direct, acute effect on arterial muscle force or relaxation. Intact and estrone-implanted SHR females had significantly lower BP than males. Ovariectomized SHR developed high BP equivalent to that of males. Arterial relaxation was slower in both male and female SHR compared with WKY. CONCLUSIONS: Estrogen lowers BP in female SHR. Strain differences in relaxation rates are independent of gender and estrogen status. Estrogen has no effect on arterial muscle relaxation, suggesting another mechanism for the protective effect of estrogen in hypertension.

Randomised trial of paclitaxel versus doxorubicin as first-line chemotherapy for advanced breast cancer: quality of life evaluation using the EORTC QLQ-C30 and the Rotterdam symptom checklist.

The aim of the study was to compare the quality of life (QL) of patients treated with single-agent paclitaxel versus doxorubicin as first-line chemotherapy for advanced breast cancer. 331 patients with advanced breast cancer were randomised, with 294 eligible for analysis. Patients completed both the EORTC QLQ-C30 questionnaire and the Rotterdam Symptom Checklist (RSCL) with six additional items, at baseline and after the third, fifth and seventh cycles of chemotherapy. A significant difference in progression-free survival in favour of doxorubicin caused a bias in the data with differences in expected completion rates of questionnaires beyond cycle three. Therefore, statistical comparisons were performed only for the first three cycles. Baseline compliance was 64% and 61% for the QLQ-C30 and RSCL questionnaires, respectively. Doxorubicin was associated with significantly more nausea/vomiting (P=0.001), loss of appetite (P=0.010) and a greater burden of disease and treatment (P=0.044), but with less bone pain (P=0.042) and rash (P=0.045) than paclitaxel. Both treatments were associated with improved emotional function and reduction in psychological distress at cycle 3. Longitudinal data suggested that doxorubicin was associated with less pain, specifically bone pain. Doxorubicin was more active but may have had more side-effects during the first three cycles. Long-term QL outcomes could not be assessed.

Identification and interpretation of clinical and quality of life prognostic factors for survival and response to treatment in first-line chemotherapy in advanced breast cancer.

The aim of the project was to identify clinical and quality of life (QL) factors that together predict survival and response to chemotherapy in advanced breast cancer. Potential prognostic factors were studied in 187 women with baseline QL data from a trial of paclitaxel versus doxorubicin as first-line chemotherapy. Demographic and clinical factors studied were age, performance status, dominant site of disease and preceding disease-free interval (DFI). Factors from the EORTC QLQ-C30 were all function scales, fatigue, nausea/vomiting, pain, dyspnoea, insomnia, loss of appetite and global QL. The proportional hazards regression model with stratification for treatment, and the logistic regression model adjusting for treatment arm were used for univariate and multivariate analyses of survival and response to treatment, respectively. For survival, multiple sites of visceral disease, pain, global QL and fatigue were significant prognostic factors in the univariate analysis. The final multivariate model predicted poor survival with multiple sites of visceral disease (P=0.003), DFI

Human spermatogenesis as a model to examine gene potentiation.

The first tier of control over the expression of genic domains utilizes chromatin structure. Before the onset of transcription, the chromatin domain that encompasses the gene(s) must assume an open conformation. This renders large segments of the genome available to the tissue-specific and ubiquitous trans-factors necessary for proper expression of the genes present. This process has been termed potentiation. It is a necessary obligate, but alone it is not sufficient for gene expression. Spermatogenesis, the development of a viable fertile male gamete, provides a unique model to begin to address the underlying mechanism(s) governing differentiation and tissue-specific gene expression. Male gametogenesis is typified by the activation of numerous genes whose products have novel functions, as well as testis-specific forms of constitutively expressed somatic genes. We have shown that mouse spermatogenesis represents a selective potentiative process (Kramer et al., 1998: Development 125:4749-4655), but little is known about its human counterpart. To fill this void we have examined the potentiative state of several spermatid-expressed genes during the latter stages of human spermatogenesis. We have shown that spermatidexpressed genes are potentiated by the pachytene stage of differentiation. Furthermore, we establish that a chromatin domain functions as a discrete structural unit during differentiation. Interestingly, some of these open structures are maintained in the mature spermatozoon.

Reprogramming the male gamete genome: a window to successful gene therapy.

Hematopoiesis and spermatogenesis both initiate from a stem cell capable of renewal and differentiation. Each pathway reflects the expression of unique combinations of facultative, i.e. tissue-specific and constitutive, i.e. housekeeping, genes in each cell type. In spermatogenesis, as in hematopoiesis, commitment is mediated by the mechanism of potentiation whereby specific chromatin domains are selectively opened along each chromosome. Within each open chromatin domain, a unique battery of gene(s) is availed to tissue-specific and ubiquitous transacting factors that are necessary to initiate transcription. In the absence of an open domain, trans-factor access is denied, and the initiation of transcription cannot proceed. Cell-fate is thus ultimately defined by the unique series of open-potentiated cell-specific chromatin domains. Defining the mechanism that opens chromatin domains is fundamental in understanding how differentiation from stem cells is controlled and whether cell-fate can be modified. A recent examination of the mammalian spermatogenic pathway [Kramer, J.A., McCarrey, J.M, Djakiew, D., Krawetz, S.A., 1998. Differentiation: the selective potentiation of chromatin domains. Development 125, 4749-4755] supports the view that cell fate is mediated by global changes in chromatin conformation. This stride underscores the possibility of moderating differentiation through chromatin conformation. It is likely that gene therapeutics capable of selectively potentiating individual genic domains in populations of differentiating and/or replicating cells that modify cellular phenotype will be developed in the next millennium.

Temporal expression of the transgenic human protamine gene cluster.

OBJECTIVE: To ascertain the fidelity of expression of the genes from the transgenic human sperm-specific nuclear packaging protamine-1-->protamine-2-->transition protein-2 (PRM1-->PRM2-->TNP2) locus. DESIGN: Controlled human transgene study. SETTING: Basic science laboratory. ANIMAL(S): Age-matched transgenic and nontransgenic mice. INTERVENTION(S): Transgenic mice containing the human protamine locus were mated. One testis from each offspring was frozen at -80 degrees C and the other was preserved in formalin. MAIN OUTCOME MEASURE(S): The temporal expression of the human and mouse protamines was evaluated by Northern blot analysis. Orientation of the transgenic locus was determined by Southern blot analysis. Tissue morphology was assessed histologically. RESULT(S): Conservation of transgenic morphology was confirmed. Head-to-tail integration of the PRM1--> PRM2-->TNP2 locus was shown. Temporal expression of the mouse and human protamine genes was maintained in the transgenic state. CONCLUSION(S): These results show that the head-to-tail concatomer of the PRMI-->PRM2-->TNP2 locus contains all the necessary elements for appropriate temporal expression while maintaining testicular structure and function.

Genesis of a novel human sequence from the protamine PRM1 gene.

The members of the male haploid expressed protamine 1 (PRM1)-->protamine 2 (PRM2)-->transition protein 2 (TNP2) locus exist as a single, coordinately expressed genic domain. Previous analysis has revealed that the genes within the human PRM1-->PRM2-->TNP2 domain are inter-related, as they share significant sequence similarity at both the nucleotide and amino acid levels. Analysis described here supports the view that a fourth candidate coding region, gene4/Prm3, was derived from PRM1 during the genesis of the PRM1-->PRM2-->TNP2 domain. In some species, gene4 has diverged to a great extent, which can limit its expression.

Differentiation: the selective potentiation of chromatin domains.

Potentiation is requisite for the expression of our genome. It is the mechanism of opening chromatin domains to render genes accessible to tissue-specific and ubiquitous transacting-factors that enables transcription. The results presented in this study demonstrate that modulation of stage- and cell-type-specific gene expression during mammalian spermatogenesis involves selective potentiation of testis-expressed genes that reverses their repressive state when present in the spermatogonial stem cell. This directly contrasts hematopoiesis, which acts to selectively restrict lineage potential during differentiation from its permissive stem cell. These results are key to understanding how differentiative pathways are controlled and cellular phenotypes determined. A window to their modulation is presented.

Extended analysis of the region encompassing the PRM1-->PRM2-->TNP2 domain: genomic organization, evolution and gene identification.

The human male haploid expressed protamine 1 (PRM1)-->protamine 2 (PRM2)-->transition protein 2 (TNP2) locus comprises a coordinately regulated multigenic domain. This region of 16p13.13 has been used as a model to address how the organization of genes and genic domains within the human genome may influence tissue specific gene expression. Toward this goal, we have completed an extensive computational and biological analysis of the region encompassing the PRM1-->PRM2-->TNP2 domain. These analyses have revealed the likely genesis of this domain. Interestingly, the SOCS-1 gene and an hnRNPC-class pseudogene lies just 3' of this domain. Regions of nuclear matrix attachment also mark these newly identified genes.

Centromere sequences localize to the nuclear halo of human spermatozoa.

Chromatin is organized into a series of discrete nuclear matrix-associated and non-nuclear matrix-associated domains. The non-matrix-associated domains consist of loops of DNA that are attached to the proteinaceous nuclear matrix by matrix-associated regions (MARs). Although this organization is well characterized in somatic cells, comparatively little is known of this mode of organizing the genome in the human sperm nucleus. To define this relationship, the interaction of human sperm chromatin with the nuclear matrix was assessed by fluorescence in situ hybridization using specific alpha satellite probes directed to the centromeric regions of chromosomes 13 plus 21 and 18. Hybridization of the centromeric sequences was visualized as segmented, bundled structures that extended from the nuclear core into the halo.

A matrix associated region localizes the human SOCS-1 gene to chromosome 16p13.13.

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3' end of the human PRM1-->PRM2-->TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3' of the spermatid-expressed PRM1-->PRM2-->TNP2 domain at position 16p13.13.