Protein profiling of non-malignant and malignant ascites by SELDI-TOF MS: proof of principle.

Ascites is a common clinical symptom in liver cirrhosis, inflammatory disorders of the abdomen and a major late manifestation of metastatic malignancies. Standard cytopathological techniques and immunocytochemistry have specificities and sensitivities of approximately 95 and 60%, respectively for the presence of tumor cells. Development of faster and more accurate screening methods would be of great clinical utility. In this work we examined differential analysis of the unbound proteins in the supernatant of ascites fluid by Protein-Chip SELDI mass spectrometry. There were 21 tumor cell-positive and 34 tumor cell-negative samples. We used principal component analysis coupled with linear regression applied to the mass spectra of the samples to distinguish between the sample groups. Two sample sets for statistical analysis were created after randomization, a training set with 37 samples and a validation set with 18 samples resulting in a specificity of 93% and a sensitivity of 83% on the training set. The validation set yielded a specificity and sensitivity of 75%. This study suggests that SELDI-TOF mass spectrometry appears to have great potential as a surrogate diagnostic tool to evaluate effusion specimens.

The Werner syndrome protein contributes to induction of p53 by DNA damage.

Mutations in the p53 tumor-suppressor gene promote increased genomic instability and cancer. Mutations in the WRN gene, encoding a DNA helicase, underlie the segmental progeroid Werner syndrome (WS). WS is also associated with increased genomic instability and elevated cancer risk. The p53 and WRN proteins can engage in direct protein-protein interactions. We report that excess WRN elicits increased cellular p53 levels and potentiates p53-mediated apoptosis. Importantly, cells derived from WS patients exhibit an attenuated and delayed induction of p53 by UV or by the topoisomerase I inhibitor camptothecin. These results suggest that WRN may participate in the activation of p53 in response to certain types of DNA damage. Furthermore, the failure to induce p53 effectively may contribute to enhanced genomic instability and elevated cancer risk in WS patients.

Generation of oscillations by the p53-Mdm2 feedback loop: a theoretical and experimental study.

The intracellular activity of the p53 tumor suppressor protein is regulated through a feedback loop involving its transcriptional target, mdm2. We present a simple mathematical model suggesting that, under certain circumstances, oscillations in p53 and Mdm2 protein levels can emerge in response to a stress signal. A delay in p53-dependent induction of Mdm2 is predicted to be required, albeit not sufficient, for this oscillatory behavior. In line with the predictions of the model, oscillations of both p53 and Mdm2 indeed occur on exposure of various cell types to ionizing radiation. Such oscillations may allow cells to repair their DNA without risking the irreversible consequences of continuous excessive p53 activation.

Feedback mechanisms between T helper cells and macrophages in the determination of the immune response.

The interactions between macrophages and T helper (Th) cells are a complex interplay of positive and negative signals. Some of the mathematical models of interactions between T helpers have indeed taken the influence of macrophages into account. In this work the macrophage is not considered as an extrinsic agent, that is duly directed by the T cells to be cytotoxic, nor is there consideration of T helper cell populations that are dominantly regulated by extrinsic properties of antigens per se, or by certain classes of presenting cells that preferentially select certain classes of lymphocytes or bias their commitment. Rather, a simplified model of feedback loops between Th cells and macrophages is formulated and analyzed. It is suggested how the mutual influence between Th and macrophages can determine the cytokine secretion pattern of these populations. The model provides a feedback scenario to account for experimental findings concerning reversal in the dominance of a specific cytokine profile in the course of some infections. A possible scenario accounting for the difference between the stability of Th1 and Th2 cytokine pattern is put forward. The model suggests explanations for the variability in the outcome of the immune response according to different body compartments. A rationale is presented that accounts for paradoxical findings indicating that Th1 cytokines are sometimes responsible for the downregulation of a Th1 dominated response.

On the role of feedback in promoting conflicting goals of the adaptive immune system.

We explored here the implications of two premises. 1) In their response over days or weeks to pathogen invasion, cells of the immune system combine several overlapping and perhaps contradictory goals. 2) The immune system has ways to monitor progress toward these goals via receptors that bind chemicals whose concentrations are related to such progress. We illustrate with simple mathematical models how such monitoring can lead to feedbacks that improve the efficiency of a given effector type in accomplishing its specialized task, and also how feedbacks can shift the balance among a variety of effectors toward a preponderance of the more effective. Specific suggestions are given for feedback molecules.

On the role of a possible dialogue between cytokine and TCR-presentation mechanisms in the regulation of autoimmune disease.

Autoimmune diseases are thought to occur through some weakness in an active process of autoregulation. Two different regulatory mechanisms have been proposed separately during the years: a "non-specific" mechanism, via Th1-Th2 non-specific cytokines, and a "specific" one-on-one mechanism, via presentation of peptides, i.e., T cell receptor (TCR) peptides, by the T cells themselves. Several anti-idiotypic models rely on the latter to explain the effects of "T-cell-vaccination" therapy. We present and analyse a model for the interaction between both regulatory mechanisms within an ensemble composed of Th1 and Th2 cells. Our model shows how both TCR presentation and non-specific Th1/2 signals can cooperate in the choice of the prevailing Th1 or Th2 response. We show how TCR presentation can foster regulation, without necessitating a particular "suppressor" agent, of the type that some have assumed to play a central role in the regulation of autoimmunity. Our results suggest an important role for the cells' sensitivities to Th1 and Th2 derived cytokines; only for certain sensitivity ranges, is it possible to switch dominance between subtypes. It is argued that memory is sustained via modulation of sensitivities to cytokines, not only to antigens. The results and hypotheses also suggest one possible reason for the known correlation between standard and autoimmune diseases. Several therapies and informative experiments are suggested. We argue, for example, that administering a non-relevant peptide while increasing the ratio between the clones reactive to it and other clones in the pancreas, might cure autoimmune diabetes. Moreover, we predict that disease could be prevented by administering an autoimmune peptide at an early age while forcing the system to react in a Th2 fashion.

Theory of orientation tuning in visual cortex.

The role of intrinsic cortical connections in processing sensory input and in generating behavioral output is poorly understood. We have examined this issue in the context of the tuning of neuronal responses in cortex to the orientation of a visual stimulus. We analytically study a simple network model that incorporates both orientation-selective input from the lateral geniculate nucleus and orientation-specific cortical interactions. Depending on the model parameters, the network exhibits orientation selectivity that originates from within the cortex, by a symmetry-breaking mechanism. In this case, the width of the orientation tuning can be sharp even if the lateral geniculate nucleus inputs are only weakly anisotropic. By using our model, several experimental consequences of this cortical mechanism of orientation tuning are derived. The tuning width is relatively independent of the contrast and angular anisotropy of the visual stimulus. The transient population response to changing of the stimulus orientation exhibits a slow "virtual rotation." Neuronal cross-correlations exhibit long time tails, the sign of which depends on the preferred orientations of the cells and the stimulus orientation.