Three-dimensional culture model to study the biology of vacuolated notochordal cells from mouse nucleus pulposus explants.

Intervertebral disc degeneration (IDD) involves cellular changes in the nucleus pulposus (NP) characterised by a decline of the large vacuolated notochordal cells (vNCs) and a rise of smaller vacuole-free mature chondrocyte-like NP cells. An increasing number of studies demonstrate that notochordal cells (NCs) exert disease-modifying effects, establishing that NC-secreted factors are essential for the maintenance of a healthy intervertebral disc (IVD). However, understanding the role of the NCs is hampered by a restricted reserve of native cells and the lack of robust ex vivo cell model. A precise dissection enabled the isolation of NP cells from 4 d post-natal stage mouse spines and their culture into self-organised micromasses. The maintenance of cells' phenotypic characteristics was demonstrated by the presence of intracytoplasmic vacuoles and the immuno-colocalisation of the NC-markers (brachyury; SOX9) after 9 d of culture both in hypoxic and normoxic conditions. A significant increase of the size of the micromass was observed under hypoxia, consistent with a higher level of Ki-67+ immunostained proliferative cells. Furthermore, several proteins of interest for the study of vNCs phenotype (CD44; caveolin-1; aquaporin 2; patched-1) were successfully detected at the plasma membrane of NP-cells cultured in micromasses under hypoxic condition. IHC was performed on mouse IVD sections as control staining. An innovative 3D culture model of vNCs derived from mouse postnatal NP is proposed, allowing future ex vivo exploration of their basic biology and of the signalling pathways involved in IVD homeostasis that may be relevant for disc repair.

Management of pregnancy sensitized with anti-Inb with monocyte monolayer assay and maternal blood donation.

CONCLUSIONS: Maternal red blood cell (RBC) alloantibodies can cause hemolytic disease of the fetus and newborn (HDFN). Although much is described about common antibodies associated with HDFN, management of a pregnancy complicated by a maternal rare antibody presents several challenges related to assessment of fetal anemia risk, availability of blood for transfusion to the mother and/or the fetus or newborn if needed, and planning for delivery in the case of maternal hemorrhage. Here we report the laboratory medicine workup of a patient who presented for obstetrical care in the United States in the third trimester and had a rare antibody (anti-Inb). Prenatal antibody detection testing demonstrated maternal anti-Inb in a 28-year-old woman (gravida 4 para 1021). Ultrasound could not rule out fetal anemia. Monocyte monolayer assay was performed to assess for the clinical significance of the anti-Inb and revealed that the antibody may be capable of causing accelerated clearance of antigen-positive RBCs. A local and national query revealed that no appropriate units of RBCs were available for either the mother or neonate. Given this information, serial maternal autologous blood donations were performed, and a comprehensive care plan with a multidisciplinary approach for delivery, neonatal management, and preparation for hemorrhage was developed. Published data and our experience suggest that maternal blood donation appears to be a safe and effective way to manage mothers who cannot safely use the community blood supply. Involvement of obstetric, transfusion medicine, anesthesia, and neonatology providers was imperative for a favorable outcome. The antibody did not cause clinically significant anemia in this infant.

Dictyostelium, a microbial model for brain disease.

BACKGROUND: Most neurodegenerative diseases are associated with mitochondrial dysfunction. In humans, mutations in mitochondrial genes result in a range of phenotypic outcomes which do not correlate well with the underlying genetic cause. Other neurodegenerative diseases are caused by mutations that affect the function and trafficking of lysosomes, endosomes and autophagosomes. Many of the complexities of these human diseases can be avoided by studying them in the simple eukaryotic model Dictyostelium discoideum. SCOPE OF REVIEW: This review describes research using Dictyostelium to study cytopathological pathways underlying a variety of neurodegenerative diseases including mitochondrial, lysosomal and vesicle trafficking disorders. MAJOR CONCLUSIONS: Generalised mitochondrial respiratory deficiencies in Dictyostelium produce a consistent pattern of defective phenotypes that are caused by chronic activation of a cellular energy sensor AMPK (AMP-activated protein kinase) and not ATP deficiency per se. Surprisingly, when individual subunits of Complex I are knocked out, both AMPK-dependent and AMPK-independent, subunit-specific phenotypes are observed. Many nonmitochondrial proteins associated with neurological disorders have homologues in Dictyostelium and are associated with the function and trafficking of lysosomes and endosomes. Conversely, some genes associated with neurodegenerative disorders do not have homologues in Dictyostelium and this provides a unique avenue for studying these mutated proteins in the absence of endogeneous protein. GENERAL SIGNIFICANCE: Using the Dictyostelium model we have gained insights into the sublethal cytopathological pathways whose dysregulation contributes to phenotypic outcomes in neurodegenerative disease. This work is beginning to distinguish correlation, cause and effect in the complex network of cross talk between the various organelles involved. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Early divergence in lymphoid tissue apoptosis between pathogenic and nonpathogenic simian immunodeficiency virus infections of nonhuman primates.

The events that contribute to the progression to AIDS during the acute phase of a primate lentiviral infection are still poorly understood. In this study, we used pathogenic and nonpathogenic simian models of simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) and African green monkeys (AGMs), respectively, to investigate the relationship between apoptosis in lymph nodes and the extent of viral replication, immune activation, and disease outcome. Here, we show that, in SIVmac251-infected RMs, a marked increased in lymphocyte apoptosis is evident during primary infection at the level of lymph nodes. Interestingly, the levels of apoptosis correlated with the extent of viral replication and the rate of disease progression to AIDS, with higher apoptosis in RMs of Indian genetic background than in those of Chinese origin. In stark contrast, no changes in the levels of lymphocyte apoptosis were observed during primary infection in the nonpathogenic model of SIVagm-sab infection of AGMs, despite similarly high rates of viral replication. A further and early divergence between SIV-infected RMs and AGMs was observed in terms of the dynamics of T- and B-cell proliferation in lymph nodes, with RMs showing significantly higher levels of cycling cells (Ki67(+)) in the T-cell zones in association with relatively low levels of Ki67(+) in the B-cell zones, whereas AGMs displayed a low frequency of Ki67(+) in the T-cell area but a high proportion of Ki67(+) cells in the B-cell area. As such, this study suggests that species-specific host factors determine an early immune response to SIV that predominantly involves either cellular or humoral immunity in RMs and AGMs, respectively. Taken together, these data are consistent with the hypotheses that (i) high levels of T-cell activation and lymphocyte apoptosis are key pathogenic factors during pathogenic SIV infection of RMs and (ii) low T-cell activation and apoptosis are determinants of the AIDS resistance of SIVagm-infected AGMs, despite high levels of SIVagm replication.

TGF-beta in intestinal lymphoid organs contributes to the death of armed effector CD8 T cells and is associated with the absence of virus containment in rhesus macaques infected with the simian immunodeficiency virus.

SIV-infected macaques exhibit distinct rates of progression to AIDS and despite significant increases in CD8+ T cells, immune cells fail to control and eradicate SIV in vivo. Here, we investigated the interplay between viral reservoir sites, CD8+ T-cell activation/death and outcome. Our data provide strong evidence that mesenteric (Mes) lymph nodes represent major reservoirs not only for SIV-infected macaques progressing more rapidly toward AIDS but also in controllers. We demonstrate that macaques progressing faster display greater expression of TGF-beta and Indoleamine 2,3 dioxygenase in particular in intestinal tissues associated with a phosphorylation of the p53 protein on serine 15 in CD8+ T cells from Mes lymph nodes. These factors may act as a negative regulator of CD8+ T-cell function by inducing a Bax/Bak/Puma-dependent death pathway of effector/memory CD8+ T cells. Greater T-cell death and viral dissemination was associated with a low level of TIA-1+ expressing cells. Finally, we provide evidence that abrogation of TGF-beta in vitro enhances T-cell proliferation and reduces CD8+ T-cell death. Our data identify a mechanism of T-cell exhaustion in intestinal lymphoid organs and define a potentially effective immunological strategy for the modulation of progression to AIDS.

Immunopotentiation of the antibody response against influenza HA with apoptotic bodies generated by rabies virus G-ERA protein-driven apoptosis.

Apoptosis is considered to be a way of eliminating unwanted cells without causing major inflammation. Nevertheless, several lines of evidence show that apoptotic cell-derived antigens can be strong immunogens. The rabies virus glycoprotein G-ERA is an apoptotic molecule. We tested the ability of G-ERA to potentiate a B cell response against an exogenous antigen (influenza hemagglutinin, HA). We found that co-expression of G-ERA and HA in apoptotic bodies increased both the primary and memory HA-specific immune responses. The immunopotentiation of G-ERA is apoptosis-mediated but not necrosis-mediated. Our data indicate that G-ERA-mediated apoptosis might be useful to improve the immunogenicity of live vaccines.

Frequent infection of Hylobates pileatus (pileated gibbon) with species-associated variants of hepatitis B virus in Cambodia.

As well as being distributed widely in human populations, hepatitis B virus (HBV) infections occur frequently in chimpanzee, gibbon and other ape populations in sub-Saharan Africa and South-East Asia. To investigate the frequency and genetic relationships of HBV infecting gibbons in Cambodia, pileated gibbons (Hylobates pileatus) that were originally wild-caught were screened for surface antigen. Twelve of 26 (46 %) were positive, of which 11 were positive for HBV DNA. Phylogenetic analysis of complete genome sequences revealed two distinct genetic groups in the gibbon/orangutan clade. Three were similar to previously described variants infecting H. pileatus in Thailand and eight formed a distinct clade, potentially representing distinct strains of HBV circulating in geographically separated populations in South-East Asia. Because of the ability of HBV to cross species barriers, large reservoirs of infection in gibbons may hamper ongoing attempts at permanent eradication of HBV infection from human populations in South-East Asia through immunization.

Adipocyte cholesterol balance in obesity.

Adipose tissue is specialized in the storage of energy in the form of triacylglycerol. Within the fat cell, triacylglycerols are found in a well-defined structural compartment called the lipid droplet, which occupies the vast majority of the fat cell volume. However, many other lipids are present in the lipid droplet. These include sterols, carotenoids, cholecalciferol and lipophilic toxic pollutants of the environment such as dioxins and tocopherols. The topic of this article is the role of fat cell cholesterol in adipose tissue physiology and its potential implication in pathological states such as obesity.

New insights into how adipocytes sense their triglyceride stores. Is cholesterol a signal?

In recent years, our view of adipose tissue has evolved from a passive sink for energy storage to an active tissue producing multiple molecules acting on various tissues in different aspects of energy homeostasis. The production of adipose-derived secretory products is tightly regulated as a function of adipocyte lipid accumulation, but the mechanisms by which fat cells are able to sense the levels of their triglyceride stores still remains largely unknown. This paper reviews new insights into this question taking cholesterol as a potential intracellular signaling molecule.

Human alpha 2A-adrenergic receptor gene expressed in transgenic mouse adipose tissue under the control of its regulatory elements.

Catecholamines regulate white adipose tissue function and development by acting through beta- and alpha2-adrenergic receptors (ARs). Human adipocytes express mainly alpha 2A- but few or no beta 3-ARs while the reverse is true for rodent adipocytes. Our aim was to generate a mouse model with a human-like alpha2/beta-adrenergic balance in adipose tissue by creating transgenic mice harbouring the human alpha 2A-AR gene under the control of its own regulatory elements in a combined mouse beta 3-AR-/- and human beta 3-AR+/+ background. Transgenic mice exhibit functional human alpha 2A-ARs only in white fat cells. Interestingly, as in humans, subcutaneous adipocytes expressed higher levels of alpha2-AR than perigonadal fat cells, which are associated with a better antilipolytic response to epinephrine. High-fat-diet-induced obesity was observed in transgenic mice in the absence of fat cell size modifications. In addition, analysis of gene expression related to lipid metabolism in isolated adipocytes suggested reduced lipid mobilization and no changes in lipid storage capacity of transgenic mice fed a high-fat diet. Finally, the development of adipose tissue in these mice was not associated with significant modifications of glucose and insulin blood levels. Thus, these transgenic mice constitute an original model of diet-induced obesity for in vivo physiological and pharmacological studies with respect to the alpha2/beta-AR balance in adipose tissue.

Decreased resistin expression in mice with different sensitivities to a high-fat diet.

The regulation of resistin, a new adipose-derived circulating factor, is the subject of controversy. In particular, the question of its modulation in obesity led to opposite results reported by two different groups. In the current study, we assayed adipocyte resistin mRNA using fluorescent real-time RT-PCR. We studied the expression of resistin in mice which are differently sensitive to diet-induced obesity: the FVB/n strain, which poorly responds to high-fat diet and transgenic mice that express human alpha 2A-AR in adipose tissue in the absence of beta 3-adrenergic receptor (AR) under the FVB genetic background which are highly sensitive to high-fat diet and develop hyperplastic obesity. We observed that FVB mice, which have no significant increased body weight after an 8-week high-fat diet period, exhibited no alteration of resistin expression. In contrast, the transgenic mice developing high-fat diet-induced obesity exhibited markedly downregulated adipocyte resistin mRNA. We also showed that obesity induced by gold thioglucose injection in FVB/n mice reduces the expression of resistin in isolated adipocytes. This argues for decreased expression of resistin as a hallmark of obesity. Moreover, our data show that feeding a high-fat diet is not a primary determinant of resistin regulation.

Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes.

Enlarged fat cells exhibit modified metabolic capacities, which could be involved in the metabolic complications of obesity at the whole body level. We show here that sterol regulatory element-binding protein 2 (SREBP-2) and its target genes are induced in the adipose tissue of several models of rodent obesity, suggesting cholesterol imbalance in enlarged adipocytes. Within a particular fat pad, larger adipocytes have reduced membrane cholesterol concentrations compared with smaller fat cells, demonstrating that altered cholesterol distribution is characteristic of adipocyte hypertrophy per se. We show that treatment with methyl-beta-cyclodextrin, which mimics the membrane cholesterol reduction of hypertrophied adipocytes, induces insulin resistance. We also produced cholesterol depletion by mevastatin treatment, which activates SREBP-2 and its target genes. The analysis of 40 adipocyte genes showed that the response to cholesterol depletion implicated genes involved in cholesterol traffic (caveolin 2, scavenger receptor BI, and ATP binding cassette 1 genes) but also adipocyte-derived secretion products (tumor necrosis factor alpha, angiotensinogen, and interleukin-6) and proteins involved in energy metabolism (fatty acid synthase, GLUT 4, and UCP3). These data demonstrate that altering cholesterol balance profoundly modifies adipocyte metabolism in a way resembling that seen in hypertrophied fat cells from obese rodents or humans. This is the first evidence that intracellular cholesterol might serve as a link between fat cell size and adipocyte metabolic activity.

A computer program for the analysis of protein complex formation.

MOTIVATION: We needed an efficient way to explore the binding reactions leading to protein complexes of known composition and structure. RESULTS: A new program is described that allows the user to define a set of protein elements and to link these elements into an oligomeric 'ball-and-stick' assembly in a graphical interface. Once the structure of the oligomer has been defined, the program then employs a novel algorithm to deduce the binding reactions and intermediate complexes needed to make the oligomer from its starting protein components. The program also finds the equilibrium state of the system, using either default starting concentrations and Kd values or data supplied by the user.

Computer-based analysis of the binding steps in protein complex formation.

Computer models were used to examine whether and under what conditions the multimeric protein complex is inhibited by high concentrations of one of its components-an effect analogous to the prozone phenomenon in precipitin tests. A series of idealized simple "ball-and-stick" structures representing small oligomeric complexes of protein molecules formed by reversible binding reactions were analyzed to determine the binding steps leading to each structure. The equilibrium state of each system was then determined over a range of starting concentrations and Kds and the steady-state concentration of structurally complete oligomer calculated for each situation. A strong inhibitory effect at high concentrations was shown by any protein molecule forming a bridge between two or more separable parts of the complex. By contrast, proteins linked to the outside of the complex by a single bond showed no inhibition whatsoever at any concentration. Nonbridging, multivalent proteins in the body of the complex could show an inhibitory effect or not depending on the structure of the complex and the strength of its bonds. On the basis of this study, we suggest that the prozone phenomenon will occur widely in living cells and that it could be a crucial factor in the regulation of protein complex formation.

Transcription termination by bacteriophage T3 and SP6 RNA polymerases at Rho-independent terminators.

Transcription termination of T3 and SP6 DNA-dependent RNA polymerases have been studied on the DNA templates containing the threonine (thr) attenuator and its variants. The thr attenuator is from the regulatory region of the thr operon of Escherichia coli. The DNA template, encoding the thr attenuator, contains specific features of the rho-independent terminators. It comprises a dG + dC rich dyad symmetry, encoding a stem-and-loop RNA, which is followed by a poly(U) region at the 3'-end. Thirteen attenuator variants have been analyzed for their ability to terminate transcription and the results indicated that the structure as well as the sequence in the G + C rich region of RNA hairpin affect termination of both RNA polymerases. Also, a single base change in the A residues of the hairpin failed to influence termination, whereas changes in the poly(U) region significantly reduced the termination of both T3 and SP6 RNA polymerases. The requirement of a poly(U) region for termination by T3 and SP6 RNA polymerases was studied with nested deletion mutants in this region. The minimum number of U residues required for termination of SP6 and T3 RNA polymerases was five and three, respectively. However, both RNA polymerases needed at least eight U residues to reach a termination efficiency close to that achieved by wild-type thr attenuator encoding nine U residues. In addition, the orientation of the loop sequences of the RNA hairpin did not affect the transcription termination of either of the bacteriophage RNA polymerases.

The cascade-correlation learning: a projection pursuit learning perspective.

Cascade-correlation (Cascor) is a popular supervised learning architecture that dynamically grows layers of hidden neurons of fixed nonlinear activations (e.g., sigmoids), so that the network topology (size, depth) can be efficiently determined. Similar to a cascade-correlation learning network (CCLN), a projection pursuit learning network (PPLN) also dynamically grows the hidden neurons. Unlike a CCLN where cascaded connections from the existing hidden units to the new candidate hidden unit are required to establish high-order nonlinearity in approximating the residual error, a PPLN approximates the high-order nonlinearity by using trainable parametric or semi-parametric nonlinear smooth activations based on minimum mean squared error criterion. An analysis is provided to show that the maximum correlation training criterion used in a CCLN tends to produce hidden units that saturate and thus makes it more suitable for classification tasks instead of regression tasks as evidenced in the simulation results. It is also observed that this critical weakness in CCLN can also potentially carry over to classification tasks, such as the two-spiral benchmark used in the original CCLN paper.

Rapid numerical integration algorithm for finding the equilibrium state of a system of coupled binding reactions.

We have adapted a simple method of numerical integration to predict the equilibrium state of a population of components undergoing reversible association according to the Law of Mass Action. Its particular application is to populations of protein molecules in aqueous solution. The method is based on Euler integration but employs an adaptive step size: the time increment being reduced if it would make the concentration of any component negative and increased while the concentration of any component changes at greater than a specified rate. Parameters of the algorithm have been optimized empirically using a model set of binding equilibria with dissociation constants ranging from 10(-5) M to 10(-9) M. The method obtains the solution to a set of binding equilibria more rapidly than the conventional initial value methods (simple Euler, 4th order Runge-Kutta and variable-step Runge-Kutta methods were tested) for the same accuracy. A computer code in standard C is presented.

Computer simulated evolution of a network of cell-signaling molecules.

We have trained a computer model of a simple cell-signaling pathway to give specified responses to a pulse of an extracellular ligand. The pathway consists of two initially identical membrane receptors, each of which relays the concentration of the ligand to the level of phosphorylation of an intracellular molecule. Application of random "mutational" changes to the rate constants of the pathway, followed by selection in favor of certain outputs, generates a variety of wave forms and dose-response curves. The phenotypic effect of mutations and the frequency of selection both affect the efficiency with which the pathway achieves its target. When the pathway is trained to give a maximal response at a specific concentration of the stimulating ligand, it gives a consistent pattern of changes in which the two receptors diverge, producing a high-affinity form with excitatory output and a low-affinity form with inhibitory output. We suggest that some high- and low-affinity forms of receptors found in present-day cells might have originated by a similar process.

Regression modeling in back-propagation and projection pursuit learning.

We study and compare two types of connectionist learning methods for model-free regression problems: 1) the backpropagation learning (BPL); and 2) the projection pursuit learning (PPL) emerged in recent years in the statistical estimation literature. Both the BPL and the PPL are based on projections of the data in directions determined from interconnection weights. However, unlike the use of fixed nonlinear activations (usually sigmoidal) for the hidden neurons in BPL, the PPL systematically approximates the unknown nonlinear activations. Moreover, the BPL estimates all the weights simultaneously at each iteration, while the PPL estimates the weights cyclically (neuron-by-neuron and layer-by-layer) at each iteration. Although the BPL and the PPL have comparable training speed when based on a Gauss-Newton optimization algorithm, the PPL proves more parsimonious in that the PPL requires a fewer hidden neurons to approximate the true function. To further improve the statistical performance of the PPL, an orthogonal polynomial approximation is used in place of the supersmoother method originally proposed for nonlinear activation approximation in the PPL.

The impact of mammography on breast biopsy.

To determine the efficacy of mammography in the detection of early breast carcinoma at an urban teaching hospital, the results of all breast biopsies performed between 1983 and 1987 that were preceded by mammographic examination were retrospectively reviewed. There were 503 women in this population. Malignancy was detected in 79 cases (15.7%); 21 were in situ and 58 were invasive. Among all nonpalpable malignancies, 53.0 per cent were in situ, while only 2.4 per cent of all palpable malignancies were in situ. An abnormality was found in 374 mammograms (74%), and 73 (19.5%) were malignant. The abnormality most likely to represent a malignancy (44% yield) was spiculated density, followed by clustered microcalcifications (25%), mass (22%), and asymmetric density (14%). Six malignancies were detected by biopsy for clinical indications, despite a negative mammogram (4.7% false- negative rate). The interpretation of mammograms by radiologists carried a 2.4 per cent false-negative rate. The mammographic features of mass, clustered microcalcifications, spiculations or asymmetric density should generally mandate breast biopsy, although the clinical examination should remain an important basis for management decisions. An aggressive approach toward screening mammography and breast biopsy based on mammographic criteria may enhance survival among women with breast carcinoma.