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The size distribution of adipocytes in fat tissue provides important information about metabolic status and overall health of patients. Histological measurements of biopsied adipose tissue can reveal cardiovascular and/or cancer risks, to complement typical prognosis parameters such as body mass index, hypertension or diabetes. Yet, current methods for adipocyte quantification are problematic and insufficient. Methods such as hand-tracing are tedious and time-consuming, ellipse approximation lacks precision, and fully automated methods have not proven reliable. A semi-automated method fills the gap in goal-directed computational algorithms, specifically for high-throughput adipocyte quantification. Here, we design and develop a tool, AdipoCyze, which incorporates a novel semi-automated tracing algorithm, along with benchmark methods, and use breast histological images from the Komen for the Cure Foundation to assess utility. Speed and precision of the new approach are superior to conventional methods and accuracy is comparable, suggesting a viable option to quantify adipocytes, while increasing user flexibility. This platform is the first to provide multiple methods of quantification in a single tool. Widespread laboratory and clinical use of this program may enhance productivity and performance, and yield insight into patient metabolism, which may help evaluate risks for breast cancer progression in patients with comorbidities of obesity. ABBREVIATIONS BMI: body mass index.

OBJECTIVE The principal objective of this investigation was to identify novel cytokine associations with BMI and type 2 diabetes (T2D). METHODS Cytokines were profiled from African American women with obesity who donated plasma to the Komen Tissue Bank. Multiplex bead arrays of analytes were used to quantify 88 cytokines and chemokines in association with clinical diagnoses of metabolic health. Regression models were generated after elimination of outliers. RESULTS Among women with obesity, T2D was associated with breast adipocyte hypertrophy and with six plasma analytes, including four chemokines (chemokine [C-C motif] ligand 2, chemokine [C-C motif] ligand 16, chemokine [C-X-C motif] ligand 1, and chemokine [C-X-C motif] ligand 16) and two growth factors (interleukin 2 and epidermal growth factor). In addition, three analytes were associated with obesity independently of diabetes: interleukin 4, soluble CD40 ligand, and chemokine (C-C motif) ligand 3. CONCLUSIONS Profiling of inflammatory cytokines combined with measures of BMI may produce a more personalized risk assessment for obesity-associated disease in African American women.

// Dongzhi Yang 1, 2, * , Gregory W. Severin 3, 4, * , Casey A. Dougherty 1, 5 , Rachel Lombardi 1 , Daiqin Chen 1 , Marcian E. Van Dort 1 , Todd E. Barnhart 6 , Brian D. Ross 1 , Andrew P. Mazar 7 , Hao Hong 1 1 Center for Molecular Imaging, Department of Radiology, University of Michigan, Ann Arbor, Michigan 48109, United States 2 Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China 3 Center for Nuclear Technologies, Technical University of Denmark, 4000 Roskilde, Denmark 4 Department of Chemistry, Facility for Rare Isotope Beams, Michigan State University, East Lansing, Michigan 48824, United States 5 Department of Chemistry, Iona College, New Rochelle, New York 10801, United States 6 Department of Medical Physics, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States 7 Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, United States * These authors contributed equally to this work Correspondence to: Andrew P. Mazar, email: a-mazar@northwestern.edu Hao Hong, email: hahong@med.umich.edu Keywords: urokinase plasminogen activator (uPA), uPAR, immunoPET, quantitative analysis, cancer biomarkers Received: July 13, 2016 Accepted: October 03, 2016 Published: October 08, 2016 ABSTRACT Mounting evidence suggests that the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a central role in tumor progression. The goal of this study was to develop an 89 Zr-labeled, antibody-based positron emission tomography (PET) tracer for quantitative imaging of the uPA/uPAR system. An anti-uPA monoclonal antibody (ATN-291) was conjugated with a deferoxamine (Df) derivative and subsequently labeled with 89 Zr. Flow cytometry, microscopy studies, and competitive binding assays were conducted to validate the binding specificity of Df-ATN-291 against uPA. PET imaging with 89 Zr-Df-ATN-291 was carried out in different tumors with distinct expression levels of uPA. Biodistribution, histology examination, and Western blotting were performed to correlate tumor uptake with uPA or uPAR expression. ATN-291 retained uPA binding affinity and specificity after Df conjugation. 89 Zr-labeling of ATN-291 was achieved in good radiochemical yield and high specific activity. Serial PET imaging demonstrated that, in most tumors studied (except uPA- LNCaP), the uptake of 89 Zr-Df-ATN-291 was higher compared to major organs at 120 h post-injection, providing excellent tumor contrast. The tumor-to-muscle ratio of 89 Zr-Df-ATN-291 in U87MG was as high as 45.2 ± 9.0 at 120 h p.i. In vivo uPA specificity of 89 Zr-Df-ATN-291 was confirmed by successful pharmacological blocking of tumor uptake with ATN-291 in U87MG tumors. Although the detailed mechanisms behind in vivo 89 Zr-Df-ATN-291 tumor uptake remained to be further elucidated, quantitative PET imaging with 89 Zr-Df-ATN-291 in tumors can facilitate oncologists to adopt more relevant cancer treatment planning.

AIMS: Accumulation of carbon dioxide (CO₂) in cucumber fermentations is known to cause hollow cavities inside whole fruits or bloaters, conducive to economic losses for the pickling industry. This study focused on evaluating the use of a malic acid decarboxylase (MDC)‐deficient starter culture to minimize CO₂ production and the resulting bloater index in sodium chloride‐free cucumber fermentations brined with CaCl₂. METHODS AND RESULTS: Attempts to isolate autochthonous MDC‐deficient starter cultures from commercial fermentations, using the MD medium for screening, were unsuccessful. The utilization of allochthonous MDC‐deficient starter cultures resulted in incomplete utilization of sugars and delayed fermentations. Acidified fermentations were considered, to suppress the indigenous microbiota and favour proliferation of the allochthonous MDC‐deficient Lactobacillus plantarum starter cultures. Inoculation of acidified fermentations with L. plantarum alone or in combination with Lactobacillus brevis minimally improved the conversion of sugars. However, inoculation of the pure allochthonous MDC‐deficient starter culture to 10⁷ CFU per ml in acidified fermentations resulted in a reduced bloater index as compared to wild fermentations and those inoculated with the mixed starter culture. CONCLUSIONS: Although use of an allochthonous MDC‐deficient starter culture reduces bloater index in acidified cucumber fermentations brined with CaCl₂, an incomplete conversion of sugars is observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Economical losses due to the incidence of bloaters in commercial cucumber fermentations brined with CaCl₂ may be reduced utilizing a starter culture to high cell density.

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotype.

Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.

The evidence for HLA-Cw antigens' involvement in the modulation of the immune response in bone marrow transplantation, NK alloreactivity and the susceptibility and follow-up for different diseases has been growing in the recent years, but very few data on HLA-Cw distribution in healthy Italian subjects are available to date. This report presents an updated description of HLA-Cw frequencies in Italy, comparing data from the northern (Lombardia) and southern (Campania and Puglia) parts of the country. A total of 1101 healthy subjects of Italian origin were genotyped, and the results showed that HLA-Cw⁎04, Cw⁎07, Cw⁎12, and, in particular, Cw⁎0401, Cw⁎0701, Cw⁎1203, were the most frequent alleles found in all three regions analysed. Nevertheless, statistically significant differences were observed in Cw⁎07 distribution, which was more frequent in the southern than in the northern part of Italy (28.8% vs 22.4%; p = 0.001; OR: 1.4; 95%CI: 1.14–1.73), and in Cw⁎12 distribution, which was more frequent in the north than the south (17.0% vs 12.4%; p = 0.007, OR: 1.4; 95%CI: 1.10–1.91). These results, which give an improved pattern of distribution of HLA-Cw alleles in the Italian population, would be useful in bone marrow transplantation and anthropological studies. Moreover, due to the important role of HLA-Cw antigens in modulation of the immune response and NK alloreactivity, these data would be of interest in studies on susceptibility, follow-up and/or protection against different diseases.

Summary The effect of several variables on the drying rate of sugar beet root slices has been investigated. The standard solution of the nonstationary-state diffusion equation was used to analyse the experimental results during the initial phase of the falling rate period of drying. It was found that Fick's law can be used to predict average drying time, internal moisture distributions and sample temperature during dehydration with reasonable accuracy, despite the complex nature of the mechanisms of moisture transport.

Summary The purpose of the present study is to verify if the water sorption isotherms of raw sugar beet root could be predicted from knowledge of sorption behaviour of its main components, namely sucrose and the water insoluble fraction. For this purpose, a study of water sorption in amorphous sucrose at 35 and 47°C was undertaken. These results were used along with previously reported isotherms of raw sugar beet root and its water insoluble fraction. Good agreement between the predicted and experimental isotherms was observed at low relative humidities (up to 25% RH); at higher relative humidities poor agreement was obtained, probably because phase transitions of the sucrose.

Summary The water sorption isotherms of raw sugar beet root and its water insoluble components, at various temperatures, were determined. An attempt is made to describe the experimental equilibrium moisture content data using some existing theories on physical adsorption.

Continuous cell movement requires the coordination of protrusive forces at the leading edge with contractile forces at the rear of the cell. Myosin II is required to generate the necessary contractile force to facilitate retraction; however, Dictyostelium cells that lack myosin II (mhcA–) are still motile. To directly investigate the role of myosin II in contractility we used a gelatin traction force assay to measure the magnitude and dynamic redistribution of traction stresses generated by randomly moving wild-type, myosin II essential light chain null (mlcE–) and mhcA– cells. Our data show that for each cell type, periods of rapid, directed cell movement occur when an asymmetrical distribution of traction stress is present, in which traction stresses at the rear are significantly higher than those at the front. We found that the major determinants of cell speed are the rate and frequency at which traction stress asymmetry develops, not the absolute magnitude of traction stress. We conclude that traction stress asymmetry is important for rapid, polarized cell movement because high traction stresses at the rear promote retraction, whereas low traction at the front allows protrusion. We propose that myosin II motor activity increases the rate and frequency at which traction stress asymmetry develops, whereas actin crosslinking activity is important for stabilizing it.

Because of their persistence in aquatic environments and ability to impair reproduction and other critical physiological processes, organochlorine (OC) contaminants pose significant health risks to marine organisms. Despite such concerns, few studies have investigated levels of OC exposure in sharks, which are fish particularly threatened by anthropogenic pollution because of their tendency to bioaccumulate and biomagnify environmental contaminants. The present study examined concentrations of 29 OC pesticides and total polychlorinated biphenyls (PCBs) in the bonnethead shark (Sphyrna tiburo), an abundant species for which evidence of reproductive impairment has been observed in certain Florida populations. Quantifiable levels of PCBs and 22 OC pesticides were detected via gas chromatography and mass spectrometry in liver of 95 S. tiburo from four estuaries on Florida's Gulf coast: Apalachicola Bay, Tampa Bay, Florida Bay, and Charlotte Harbor. In general, OC concentrations were significantly higher in Apalachicola Bay, Tampa Bay, and Charlotte Harbor S. tiburo in relation to the Florida Bay population. Because the rate of infertility has been shown to be dramatically higher in Tampa Bay versus Florida Bay S. tiburo, the present findings allude to a possible relationship between OC exposure and reproductive health that requires further investigation. Pesticide and PCB concentrations did not appear to significantly increase with growth or age in S. tiburo, suggesting limited potential for OC bioaccumulation in this species compared with other sharks for which contaminant data are available. Concentrations of OCs in serum and muscle were not correlated with those in liver, indicating that these tissues are poor surrogates for measuring internal OC burden in this species via nonlethal sampling procedures.

Deletions, translocations, or point mutations in the CREB-binding protein (CBP) gene have been associated with Rubinstein-Taybi Syndrome; a human developmental disorder characterized by retarded growth and reduced mental function. To examine the role of CBP in memory, transgenic mice were generated in which the CaMKIIα promoter drives expression of an inhibitory truncated CBP protein in forebrain neurons. Examination of hippocampal long-term potentiation (LTP), a form of synaptic plasticity thought to underlie memory storage, revealed significantly reduced late-phase LTP induced by dopamine-regulated potentiation in hippocampal slices from CBP transgenic mice. However, four-train induced late-phase LTP is normal. Behaviorally, CBP transgenic mice exhibited memory deficits in spatial learning in the Morris water maze and deficits in long-term memory for contextual fear conditioning, two hippocampus-dependent tasks. Together, these results demonstrate that CBP is involved in specific forms of hippocampal synaptic plasticity and hippocampus-dependent long-term memory formation.

Puberty is a critical, hormone-mediated event during which an animal acquires the ability to breed and propagate. Despite the importance of this stage in animal reproduction, little is known regarding the physiological factors that regulate and/or accompany puberty in several vertebrate groups including elasmobranchs. To address the need for such information, the present study investigated morphological and hormonal changes that occur during puberty in male bonnethead sharks (Sphyrna tiburo). Serial changes in development of claspers, paired copulatory organs in male elasmobranchs, and serum steroid concentrations during puberty were evaluated in captive-held male S. tiburo. Captive-animal studies were supplemented by observations on gonadal development, gonaduct morphology, and serum steroid concentrations in feral, peripubertal male S. tiburo. Changes in size and histological architecture of testes and gonaducts of peripubertal sharks mirrored the seasonal progression of events that occur in these structures in mature males. Claspers grew in length continuously during puberty, but sharks did not reach functional maturity until a short period before mating activity commences in the mature population. Clasper growth appeared to be strictly regulated in S. tiburo, perhaps to ensure growth of these organs to sizes deemed critical for reproductive success. Serum concentrations of testosterone, dihydrotestosterone, progesterone, and 17β-estradiol increased in both captive and feral sharks during pubertal development, and may be associated with development of the gonads and gonaducts. Differences in hormone profiles of captive and feral sharks were observed at certain periods during puberty, but their origin remains unclear.

Anticardiolipin antibodies (aCL) of the immunoglobulin (Ig) G isotype have been significantly associated with neurological manifestations of antiphospholipid syndrome (APS). In a previous study we described the direct pathogenic effects of IgG aCL on living neurons in culture. Therefore, we studied the IgG aCL titre as a factor influencing the extent of this effect. Seventeen patients with a history of primary antiphospholipid syndrome were grouped according to their IgG aCL titre into low positive (GPLor = 40), high positive (40GPL100) and very high positive (GPL100). IgG from these patients were incubated with cerebellar neurons in primary culture for 24h and the effect was evaluated by using the tetrazolium salt (MTT) assay. We found that almost all patients' IgGs reduced cell viability in vitro, but the differences in the extent of the effect were statistically significant only for patients with40 GPL. Our results reinforce the causal association between increasing level of IgG aCL and clinical features of aPS.

Two novel cell lines (JURL-MK1 and JURL-MK2) have been established from the peripheral blood of a patient in the blastic phase of chronic myelogenous leukemia. The cells grow in a single cell suspension with doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2). Cytogenetic analysis has shown that JURL-MK1 is hypodiploid whereas JURL-MK2 is near triploid and that both cell lines retain t(9;22). Moreover, JURL-MK1 and JURL-MK2 have a bcr/abl-fused gene with the same junction found in the patient's fresh cells, and both cell lines express the b3/a2 type of hybrid bcr/abl mRNA. The morphology and immunophenotype of these cell lines are reminiscent of megakaryoblasts. In both lines, a limited but consistent percentage of cells expresses gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gplb (CD42b), glycophorin A, hemoglobin and CD34. Both cell lines are clearly positive for CD33, CD43, CD45RO and CD63, while CD13, CD44, CD54, CD30 and CD40 are specific features of JURL-MK2. Among cytokine receptors, CD117/SCF-R is strongly displayed by a large fraction of JURL-MK1 cells but is hardly detectable on about 20% JURL-MK2 cells. Both cell lines are clearly positive for CD25/IL2R alpha, while a marked expression of CD116/GM-CSF-R and CDw123/IL3R alpha is restricted to JURL-MK2. Induction of cell differentiation in vitro has demonstrated that TPA is able to modulate the JURL-MK1 phenotype, causing an increased expression of platelet-associated antigens. The JURL-MK2 phenotype is easily modulated by both TPA and DMSO, which cause an increased expression of CD41a and CD117 accompanied by a decreased expression of CD30. Proliferation studies demonstrated that JURL-MK1 cell growth is enhanced by stem cell factor, while JURL-MK2 proliferation is unaffected by this cytokine. JURL-MK1 and JURL-MK2 are two novel cell lines with divergent biological features, representing a 'two-sided' model for investigating new aspects of megakaryocytopoiesis.

We herein have described a case of de novo gastric cancer in a renal transplant recipient with a concomitant diagnosis of gastrointestinal cytomegalovirus (CMV) disease. We hypothesize that CMV, through causing an imbalance between cell proliferation and cell death, functions as the causative agent for the progression of the gastric tumor in this case after gastric colonization. To the best of our knowledge, this is the second such case ever reported of such kind and may represent a platform for investigations aimed at understanding the possible interplay between CMV and gastric cancer.

The present study was designed to characterize the adhesive interaction of c-myc-transfected B-lymphoblastoid cell lines with resting and interleukin-1-activated endothelial cells. The transfected cell lines expressed lower levels of leukocyte function-associated antigen-1 compared with control non-transfected lines, while no reduction of expression of other surface structures, including the beta 1 integrin very late antigen-4, was observed. The transfected cell lines adhered to resting or activated endothelial cells less than control cells. Anti-CD18 monoclonal antibody inhibited binding of control cell lines but had a modest or no effect on adhesion of transfected cell lines. Anti-very late antigen-4 monoclonal antibody effectively inhibited binding of both transfected and control cell lines; this was more pronounced in the presence of anti-CD18, suggesting a cooperative interaction between these adhesion pathways. Transfected cell lines also had an impaired ability to penetrate endothelial cell monolayers in a transmigration assay. Our results indicate that activation of the c-myc oncogene in B-cells causes alterations in the adhesive interaction with endothelial cells. This may be relevant in the localization and malignant behavior of B-cell lymphomas carrying an activated c-myc oncogene.

Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.(1) On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.(2) Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.(3) To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

Killer Immunoglobulin-like Receptor (KIR) genes may affect both resistance and susceptibility to autoimmune disorders, but their role in the pathogenesis of Multiple Sclerosis (MS) is still unclear. To evaluate the involvement of KIRs and their HLA ligands in the development of MS we performed genotyping of HLA -A, -B, -Cw, -DRB1 and KIRs loci in 121 RRMS patients and 103 healthy controls (HC). Results evidenced a possible protective role of the activating KIR2DS1 gene (p(y)=0.001; OR:0.38), enhanced in the presence of its ligand group HLA-C2 (p(y)=0.0001; OR:0.23). Our data suggest that the presence of functional compounds of activating KIR receptors together with their HLA ligands, allowing the immunomodulatory function of NK cells, may have a protective role against the disease.

In eukaryotic cells, the nucleus is the largest and most rigid organelle. Therefore, its physical properties contribute critically to the biomechanical behavior of cells, e.g., during amoeboid migration or perfusion through narrow capillaries. Furthermore, it has been speculated that nuclear deformations could directly allow cells to sense mechanical stress, e.g., by modulating the access of specific transcription factors to their binding sites. Defects in nuclear mechanics have also been reported in a variety of muscular dystrophies caused by mutations in nuclear envelope proteins, indicating an important role in the maintenance of cells in mechanically stressed tissue. These findings have prompted the growing field of nuclear mechanics to develop advanced experimental methods to study the physical properties of the nucleus as a function of nuclear structure and organization, and to understand its role in physiology and disease. These experimental techniques include micropipette aspiration, atomic force microscopy of isolated nuclei, cellular strain and compression experiments, and microneedle manipulation of intact cells. These experiments have provided important insights into the mechanical behavior of the nucleus under physiological conditions, the distinct mechanical contributions of the nuclear lamina and interior, and how mutations in nuclear envelope proteins associated with a variety of human diseases can cause distinct alterations in the physical properties of the nucleus and contribute to the disease mechanism. Here, we provide a brief overview of the most common experimental techniques and their application and discuss the implication of their results on our current understanding of nuclear mechanics.

Purpose: The chemokine receptor CXCR4 was identified as an independent predictor of poor prognosis in primary melanoma. The aim of the study was to investigate the role of CXCR4 in human melanoma metastases. Experimental Design: CXCR4 expression was evaluated in melanoma metastases and in metastatic cell lines through immunohistochemistry, immunoblotting, immunofluorescence, and reverse transcription-PCR. The function of CXCR4 was tested in the presence of the ligand, CXCL12, through induction of extracellular signal-regulated kinase-1 and -2 (Erk-1 and -2) phosphorylation, proliferation, apoptosis, and migration capabilities. Results: CXCR4 expression was detected in 33 out of 63 (52.4%) metastases from cutaneous melanomas. Metastatic melanoma cell lines expressed cell surface CXCR4; PES 43, Alo 40, and COPA cell lines showed the highest levels of CXCR4 (>90% of positive cells); PES 41, Alo 39, PES 47, POAG, and CIMA cell lines showed low to moderate degrees of expression (5-65% of positive cells). Other chemokine receptors, CCR7 and CCR10, were detected on the melanoma cell lines; CXCL12 activated Erk-1 and Erk-2, the whose induction was specifically inhibited by AMD3100 treatment. CXCL12 increased the growth in PES 41, PES 43, and PES 47 cells under suboptimal (1% serum) and serum-free culture conditions; AMD3100 (1 μmol/L) inhibited the spontaneous and CXCL12-induced proliferation. No rescue from apoptosis was shown but PES 41, PES 43, and PES 47 cells migrate toward CXCL12. Conclusions: These findings indicate that CXCR4 is expressed and active in human melanoma metastases, suggesting that active inhibitors such as AMD3100 may be experienced in human melanoma.

Background: The myelin basic protein (MBP) gene may confer genetic susceptibility to multiple sclerosis (MS). The association of MS with alleles of the (TGGA)n variable number tandem repeat (VNTR) 5′ to the MBP gene is the subject of conflicting reports. Objective: To study possible MS association with VNTR alleles of MBP gene in ethnic Italians and ethnic Russians. Methods: Two hundred sixty-nine unrelated patients with definite MS and 385 unrelated healthy control subjects from Italy and Russia were genotyped for the MBP VNTR region and for the human leukocyte antigen (HLA) class II DRB1 gene. The phenotype, allele, and genotype frequencies for two groups of MBP alleles were determined. Patients and control subjects were stratified according to HLA-DRB1 phenotypes. Results: The distribution of MBP alleles and genotypes in the two ethnic groups, including both MS patients and control subjects, was very similar. When MS patients and healthy control subjects were stratified according to HLA-DRB1 phenotypes, a significant association of MS with MBP alleles was found only in the DR4- and DR5-positive subgroups. A significant association with MBP alleles was also observed in the nonstratified groups, owing mainly to the contribution of the DR4- and DR5-positive individuals. Conclusion: Polymorphism of the MBP or another gene in its vicinity appears to contribute to the etiology of MS for the subgroups of DR4- and DR5-positive Italians and Russians.

Summary HCV viral nucleocapsid protein (C), non-structural protein 3 (NS3) and the envelope glycoproteins E1 and E2 are candidate immune targets for developing anti-HCV DNA vaccine. Nevertheless, the immune response elicited by these antigens often appears weak and/or transient. Different approaches have been studied for enhancing and/or modulating the immune response of the DNA vaccine. On the basis of a prototype multigenic plasmid vector constituted of two different transcription cassettes (pRC100), we have developed a plasmid vector that allows the independent and simultaneous expression of murine IL2 and of an antigenic domain of the HCV NS3 C terminus (pRC112-HCV). The highly conserved NS3 region spans from nt 4403 to nt 4829 and contains two putative B and T epitopes. The development of this multigenic plasmid vector may combine the expression and local production of an immunomodulatory molecule (mIL2) together with the possibility of addressing the host immune response to the most immunogenic and conserved epitopes, specifically tailored in the plasmid vector.

The internalization of conidia of the opportunistic fungus Aspergillus fumigatus by primary cell cultures of nonprofessional phagocytes was investigated. This study is the first to show that A. fumigatus conidia were able to be engulfed by tracheal epithelial, alveolar type II, and endothelial cells.

Background: Bromodomain-containing coregulators of transcription have been identified recently as new epigenetic targets to treat a variety of hematologic malignancies, including acute myeloid leukemias. However, the bromodomain mechanisms that couple histone acetylation to transcription in lymphopoiesis, and that govern mature lymphocyte responsiveness to growth signals, are only partly understood. Brd2 is a transcriptional co-regulator that contains dual Bromodomains and an ExtraTerminal domain (defining the BET family) that couples chromatin modification to cell cycle progression. We previously reported the first functional characterization of a BET protein as an effector of mammalian mitogenic signal transduction: Eμ-Brd2 transgenic mice develop an ‘activated B cell’ type of diffuse large B cell lymphoma. There have been no other animal models reported for genetic or lentiviral expression of BET proteins, which hampers testing of novel anticancer drugs such as JQ1 and its derivatives. Methods: Here we use Brd2 lentivirus transduction of hematopoietic stem cells and reconstitution of lethally irradiated recipient mice to examine the function of Brd2 in hematopoiesis in the bone marrow and mitogenesis in the periphery. Results: Three-fold over-expression of Brd2 in hematopoietic stem cells selectively leads to a doubling of the B cell compartment and increases mature B cell mitogenic responsiveness ex vivo. Knockdown of Brd2 by shRNA totally ablates hematopoietic lineages, likely by inhibiting the lineage-committed progenitor expansion through down-regulation of cyclin A, which reinforces results from Eμ-Brd2 transgenic mice. The novel small molecule BET inhibitor JQ1 reduces B cell mitogenesis by about 60% in parallel experiments, but this effect likely extends to other related BET family members and lacks Brd2 specificity. Conclusion/Significance: The Brd2 pathway integrates lymphopoiesis, leukemogenesis and normal mitogenesis. Targeting this pathway with therapeutic drugs may therefore confer multiple, diverse benefits in the treatment of hematologic malignancy, B cell mediated disease and likely inflammation. Citation Format: Anna C. Belkina, Wanda P. Blanton, Frank L. Lombardi, Gerald V. Denis. The double bromodomain protein Brd2 controls B cell expansion and mitogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4730. doi:10.1158/1538-7445.AM2013-4730

Twenty-five rhabdomyosarcomas, histopathologically and immunocytochemically classified as embryonal (12 cases), alveolar (11 cases), and pleomorphic (two cases), were studied by routine and immunogold electron microscopy, to identify, in poorly differentiated cells, reproducible markers useful for diagnostic purposes. Rhabdomyoblasts with rudimentary sarcomeres were observed in 12 cases (48%). Large cells (named oval cells) with an oval shape and an eccentric nucleus, like rhabdomyoblasts but lacking organized myofilaments, were observed in 20 cases (80%). By immunogold electron microscopy, performed on four of 25 cases, both oval cells and rhabdomyoblasts showed abundant intermediate filaments positive for desmin. Oval cells may be used as markers of poorly differentiated rhabdomyosarcomas.

Cytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Ha-ras-activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine-phalloidin and with a fluorescinated anti-non-muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down-regulation of alpha-actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of alpha-actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between alpha-actin down-regulation and stress fiber loss.

Purpose Photoreceptor cells rapidly lose their structure and function when they are cultured, thus requiring an in vivo setting for their studies. Genetic manipulation of photoreceptor cells using traditional transgenic mouse techniques is time consuming and costly. We explored the recently described method of in vivo electroporation as an alternate means to introduce foreign DNA constructs into mouse retinas. Ultimately, we will use this method for future studies of wildtype and mutant rhodopsin synthesis and turnover in transfected photoreceptor cells. Such studies may help to better understand the effects of some of the rhodopsin mutations that are causal for autosomal dominant retinitis pigmentosa. Methods To test in vivo electroporation technique, a DNA plasmid containing GFP driven by the ubiquitin promoter was generated. Under a dissecting microscope, DNA was injected into the subretinal space of newborn CD1 mice using a Hamilton syringe. After injection, tweezer-type electrodes soaked in PBS were placed on the sides of heads of pups and used to create five 80-V pulses. Electroporated retinae were harvested 21 days after injection, fixed, and cut on a cryostat in sections of 20 μm. Cryosections were then examined with a Zeiss Axioplan fluorescent microscope. To examine turnover of mutant rhodopsin in photoreceptors, constructs were made that use the rhodopsin promoter to drive expression of chimeric proteins containing timer fluorescence (dsRed-E5) and either wildtype or mutant rhodopsin. Results When viewed under the fluorescent microscope, cryosections of retinae containing the ubiquitin-GFP construct demonstrated expression of GFP in outer nuclear layer of photoreceptor cells as well as cells in the inner nuclear layer. Plasmids containing mouse rhodopsin promoter driving expression of wildtype rhodopsin-dsRed-E5 and rhodopsin mutant-dsRed-E5 were constructed and verified by sequencing. Conclusions Expression of GFP in the retinae of P21 mice after DNA injection demonstrates in vivo electroporation is a possible means to study mutant rhodopsin in mice. Future experiments will use in vivo electroporation to study the turnover of the mutant rhodopsin-dsRed-E5 constructs.

Summary To understand the effect of some antibiotics on phagocytic activity, we analysed the influence of different antibiotics on the ingestion phase of human macrophages in vitro . Monolayer cultures were treated with the following drugs: penicillin G, ampicillin, methicillin, cefazolin, cephalothin, tetracycline, doxycycline, streptomycin, gentamicin and tobramycin. The phagocytic activity was assayed by the direct microscopic count of ingested zymosan particles. Penicillin G, gentamicin, tobramycin, doxycycline and tetracycline increased the phagocytic activity of human macrophages in vitro . On the other hand methicillin, ampicillin, cefazolin, and cephalothin appeared to exert an inhibitory effect on phagocytosis.

Two patients from two separate families were diagnosed as having type IIB von Willebrand disease, because they had lifelong bleeding tendencies, prolonged bleeding times, no large von Willebrand factor multimers, and low levels of ristocetin cofactor in plasma with heightened ristocetin-induced platelet aggregation. There was no history of bleeding, and no laboratory abnormalities were found in the parents and sibship of either propositi, in contrast with the autosomal dominant pattern of inheritance usually observed in type IIB von Willebrand disease. Abnormalities of ristocetin-induced von Willebrand factor-platelet interactions were less severe than in a patient from a previously reported family with type IIB von Willebrand disease studied in parallel. The peculiar features of these cases provide additional evidence of the existence of heterogeneity within this variant.

The characteristics of a human cell line (LP-1) derived from the peripheral blood of a patient with IgG-lambda myeloma in leukemic transformation are described. The cells resemble immature plasma cells in that they exhibit a membrane phenotype that is intermediate between late B lymphocytes and plasma cells, even though they secrete IgG- lambda chains. Treatment of LP-1 cells with 12–0 tetradecanoylphorbol- 13-acetate (TPA) or pokeweek mitogen (PWM) induces the appearance of surface markers and ultrastructural features typical of mature plasma cells but does not affect their proliferative activity. Molecular analysis of the cell line showed an increased expression of the c-myc protooncogene and the presence of abnormally sized transcripts. Conventional cytogenetics and pulsed-field gel electrophoresis showed no structural rearrangements of the c-myc gene, suggesting that the abnormal c-myc expression may be due to point mutations or small deletions within the gene. The LP-1 cell line is a useful model in which to study the process of B-cell maturation; such study may lead to the uncovering of unusual mechanisms of c-myc activation. Furthermore, the LP-1 cell is a potential partner in the generation of human hybridomas.

Immunostaining for Factor VIII-related antigen was seen in deparaffinized sections from 19 of 20 postmastectomy angiosarcomas and from four of four sarcomas that arose in chronically edematous tissue unrelated to breast carcinoma. Staining was also seen in sections from two malignant hemangioendotheliomas, four capillary hemangiomas, and one granulation tissue specimen. Sections from two lymphangiomas were immunonegative for Factor VIII-related antigen in the endothelium of lymphatic channels, whereas staining was observed in the surrounding normal blood vessels. Electron microscopic study of four postmastectomy angiosarcomas disclosed ultrastructural features (fenestrae, intense pinocytotic activity, cell junctions, and Weibel-Palade bodies) supporting the blood vascular endothelial nature of the neoplastic cells. It is concluded that a neoplastic blood vessel component is present in sarcomas that arise in chronically edematous tissues. It is questionable whether a lymphatic component is also present. These tumors, therefore, should be regarded as angiosarcomas rather than lymphangiosarcomas.

The production of hydrolytic enzymes by embryo-less barley seeds in response to various gibberellins and abscisic acid was investigated. The data support the hypothesis that plant growth substances may affect the mechanisms of hydrolase production and secretion in cereal seeds in different ways and at different point.

Aggregates of fibrillar material were observed within the meshes of the nucleolonema and at the periphery of hepatocyte nucleoli of 7-day-old untreated C3Hf and BUA mice. These aggregates had a lower density and smaller diameter than microspherules induced in mice of the same age and strains by urethane, a chemical that has been shown to inhibit RNA and DNA synthesis. However, aggregates shared some features with the microspherules since both structures had a similar fibrillar appearance and an identical localization, were easily digested by pronase and pepsin, and were bleached more than chromatin after being subjected to Bernhard's preferential staining. A close correlation between aggregates and microspherules is postulated.

Two cases, one of Pneumocystis carinii pneumonia and the other of Absidia pneumonitis, are presented. Both are documented by appropriate laboratory studies. Both ended fatally in spite of seemingly appropriate current treatment. The Pneumocystis pneumonia was associated with prolonged corticosteroid therapy for progressive myositis and the Absidia infection was associated with uremia and corticosteroid administration. Infections from these opportunistic organisms may be seen with increasing frequency in patients on prolonged corticosteroids, immunosuppressive drugs, or chemotherapeutic agents.

The A-myb gene encodes a transcription factor that is related both functionally and structurally to the v-myb oncogene. Following our observations that A-myb is expressed in a restricted subset of normal mature human B lymphocytes, with the phenotype CD38+, CD39-, slgM-, we have now investigated the pattern of A-myb expression in neoplastic B cells representating the whole spectrum of B-cell differentiation and compared it to that of c-myb and B-myb. In a panel of 32 B-cell lines, A-myb was very strongly expressed in most Burkitt's lymphoma (BL) cell lines, but weak or negative in 2 pre-B acute lymphoblastic leukemia (ALL), 4 non-Hodgkin's lymphoma (NHL), 6 Epstein-Barr virus- immortalized lymphoblastoid cell lines, and 6 myeloma lines. Protein expression paralleled that of the RNA. We have also investigated A-myb expression in 49 fresh cases of B leukemias. Among 24 ALL, 6 were of the null and 11 of the common type and all these were negative for A- myb expression; on the other hand, all 7 B-ALL cases (slg+), as well as one fresh BL case with bone marrow infiltration, expressed A-myb. A-myb was undetectable in 4 prolymphocytic leukemias (PLL) but was strongly expressed in 5/20 (25%) of chronic lymphocytic leukemia (CLL) samples. In the latter A-myb did not correlate with phenotype or clinical stage. Finally, we have studied the progression of one case of CLL into Richter's syndrome and have found that the Richter's cells expressed about 25-fold less A-myb RNA than the CLL cells from the same patient. The pattern of c-myb and B-myb was clearly distinct from that of A-myb. C-myb and B-myb were expressed in all neoplastic groups, except in CLL cells. Thus, A-myb expression, unlike that of c-myb and B-myb, is restricted to a subset of B-cell neoplasias (in particular BL and slg+B- ALL) representative of a specific stage of B-cell differentiation. This expression may in part reflect expression of A-myb by the normal germinal center B cells that are the normal counterpart of these transformed B cells. The data presented strongly support a role for this transcription factor in B-cell differentiation and perhaps in B- cell transformation in some neoplasias.

Objective-To investigate whether quantitative alterations of both beta(2)microglobulin (beta(2)mu) associated HLA class I heavy chains (sHLA-I) and beta(2), mu free class I heavy chains (sHLA-FHC) in sera of patients with hepatitis C virus (HCV) infection occur and whether they distinguish patients with mixed cryoglobulinaemia (MC). Methods-83 HCV infected patients were studied and divided into three groups: (A) without cryoglobulinaemia (n=21), (B) with polyclonal MC (n=20), (C) with monoclonal MC (n=12). Serum sHLA-I and sHLA-FHC were measured by double determinant radioimmunoassay using monoclonal antibodies: TP25.99 as catching antibody, and NAMB-1 and HC-10 as revealing antibodies. Western blot identified HLA-I isoforms. Results-The serum concentrations of sHLA-I and of sHLA-FHC in HCV infected patients versus controls were respectively 1.3(0.5) mu g/ml (mean (SD)) versus 0.8 (0.3) (p

Background: The loss of intercellular cohesion among keratinocytes (acantholysis) may be considered the histologic marker of pemphigus. Many drugs, especially thiol drugs, proved to be able to provoke in vitro acantholysis by biochemical mechanisms interfering with the disulfide and thiol group balance. As to nonthiol drugs, the pathomechanism of acantholysis is still unexplained. Objective: To explain the molecular mechanism of enalapril-induced acantholysis a potential link between transglutaminase (TGase) activity and the effects of this drug was investigated. Methods: TGase activity in extracts from human breast skin cultured in the presence of thiopronine, captopril and enalapril were evaluated in vitro. The acantholytic potential of cystamine, a known TGase inhibitor, was also investigated. Results: Enalapril, the most powerful acantholytic drug in vitro, was found to inhibit both the purified enzyme and the TGase activity in the extracts from cultured human breast skin explants. Kinetic studies showed that enalapril inhibition was competitive with respect to the amino acceptor substrate and uncompetitive with respect to the amino donor substrate. Moreover, an acantholytic effect of cystamine on explants of normal human skin was shown. Conclusions: These results suggest that acantholysis and the inhibition of TGase activity could be two related phenomena.