Loss Of Target Cells Research Articles

Single-Cell Proliferation Microfluidic Device for High Throughput Investigation of Replicative Potential and Drug Resistance of Cancer Cells.

Cell proliferation represents a major hallmark of cancer biology, and manifests itself in the assessment of tumor growth, drug resistance and metastasis. Tracking cell proliferation or cell fate at the single-cell level can reveal phenotypic heterogeneity. However, characterization of cell proliferation is typically done in bulk assays which does not inform on cells that can proliferate under given environmental perturbations. Thus, there is a need for single-cell approaches that allow longitudinal tracking of the fate of a large number of individual cells to reveal diverse phenotypes. We fabricated a new microfluidic architecture for high efficiency capture of single tumor cells, with the capacity to monitor cell divisions across multiple daughter cells. This single-cell proliferation (SCP) device enabled the quantification of the fate of more than 1000 individual cancer cells longitudinally, allowing comprehensive profiling of the phenotypic heterogeneity that would be otherwise masked in standard cell proliferation assays. We characterized the efficiency of single cell capture and demonstrated the utility of the SCP device by exposing MCF-7 breast tumor cells to different doses of the chemotherapeutic agent doxorubicin. The single cell trapping efficiency of the SCP device was found to be ~ 85%. At the low doses of doxorubicin (0.01µM, 0.001µM, 0.0001µM), we observed that 50-80% of the drug-treated cells had undergone proliferation, and less than 10% of the cells do not proliferate. Additionally, we demonstrated the potential of the SCP device in circulating tumor cell applications where minimizing target cell loss is critical. We showed selective capture of breast tumor cells from a binary mixture of cells (tumor cells and white blood cells) that was isolated from blood processing. We successfully characterized the proliferation statistics of these captured cells despite their extremely low counts in the original binary suspension. The SCP device has significant potential for cancer research with the ability to quantify proliferation statistics of individual tumor cells, opening new avenues of investigation ranging from evaluating drug resistance of anti-cancer compounds to monitoring the replicative potential of patient-derived cells. The online version contains supplementary material available at 10.1007/s12195-023-00773-z.

Abstract 6089: Novel workflow enables enhanced tissue dissociation and gentle clean-up for single cell applications

Abstract With genomic instability being a hallmark of cancer, heterogeneity within solid tumors is prevalent. Intra-tumoral heterogeneity contributes strongly to clinical outcomes. In fact, heterogeneity is often cited as a driver of therapeutic failure, chemo-resistance, and recurrence of disease. Current bulk profiling analysis of tumors is limited in its ability to provide the level of granularity necessary for uncovering aspects of heterogeneity that contribute to poor outcomes. Thus, it is imperative to gain a deeper understanding of tumors at the single-cell level. To properly analyze tumor heterogeneity at the single-cell level it is critical that quality methods for tissue dissociation and cell purification be utilized. While the most frequently used workflows rely on a combination of enzymatic and mechanical dissociation of the tissue followed by RBC lysis to remove contaminating red blood cells, these approaches tend to be harsh on cells leading to activation of cell stress pathways or even the loss of target cells due to their death during processing. The analysis of tumor heterogeneity has long been hampered by issues with low yield and sample preparation-induced alterations in cell physiology. To alleviate these issues, we present a workflow that couples Cellsonics’ SimpleFlow™ with Akadeum’s Buoyancy Activated Cell Sorting (BACS™) Microbubbles for gentle and effective tumor dissociation and clean-up that does not compromise precious samples. Cellsonics’ SimpleFlow™ system features the proprietary Bulk Lateral Ultrasonic™ (BLU™) energy to generate single cells from solid tissue with unparalleled speed, quality, and sample integrity. SimpleFlow™ is engineered to enable any lab to obtain the full spectrum of heterogeneous cell populations while maintaining the native gene expression patterns across all cell types. Single cell samples can be generated from solid tissues in 4-10 minutes under cold temperature without the use of enzymes. Akadeum’s BACS™ Microbubble technology features low-density microbubbles that are functionalized to capture an array of analytes including particular cell types. Akadeum’s BACS™ Microbubbles enable the purification of cell types of interest quickly, easily, and without sacrificing cell health and physiology. In this study, The SimpleFlow™ system was used to dissociate fresh tissues from mice to obtain a heterogeneous suspension of single cells. Following the dissociation, up to 99% of RBCs were removed from samples using Akadeum’s Mouse RBC Depletion. In subsequent studies, cancer cells were spiked into tissue samples prior to processing to assess their recovery and physiology. The novel workflow presented here combing SimpleFlow™ and microbubbles demonstrates the recovery of the full spectrum of cells and did not harm tumor cell recovery or health. Citation Format: Chananat Klomsiri, Edward Grimley, Jon Roussey, Carolina Livi, Casey Wegner, Brandon H. McNaughton, Steve Levers, Vibhu Vivek, Dan Snyder, Doug Summers, Ma Phiengsai. Novel workflow enables enhanced tissue dissociation and gentle clean-up for single cell applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6089.

Modeling Within-Host Dynamics of SARS-CoV-2 Infection: A Case Study in Ferrets.

The pre-clinical development of antiviral agents involves experimental trials in animals and ferrets as an animal model for the study of SARS-CoV-2. Here, we used mathematical models and experimental data to characterize the within-host infection dynamics of SARS-CoV-2 in ferrets. We also performed a global sensitivity analysis of model parameters impacting the characteristics of the viral infection. We provide estimates of the viral dynamic parameters in ferrets, such as the infection rate, the virus production rate, the infectious virus proportion, the infected cell death rate, the virus clearance rate, as well as other related characteristics, including the basic reproduction number, pre-peak infectious viral growth rate, post-peak infectious viral decay rate, pre-peak infectious viral doubling time, post-peak infectious virus half-life, and the target cell loss in the respiratory tract. These parameters and indices are not significantly different between animals infected with viral strains isolated from the environment and isolated from human hosts, indicating a potential for transmission from fomites. While the infection period in ferrets is relatively short, the similarity observed between our results and previous results in humans supports that ferrets can be an appropriate animal model for SARS-CoV-2 dynamics-related studies, and our estimates provide helpful information for such studies.

A flow cytometric assay for the quantification of MSC lysis by peripheral blood mononucleated cells

Mesenchymal stromal cells (MSC) are attractive candidates for the treatment of acute graft versus host disease (aGvHD) or autoimmune disorders. However, mechanisms of MSC recognition remain unclear and there are evidences that MSC are not totally immunoprivileged. Data suggest that MSC undergo apoptosis after infusion in presence of cytotoxic cells and their death could drive immunosuppression. In GvHD patients, that activity was associated with clinical response. It is mandatory to develop an in vitro potency testing predictor of the "in vivo" response to the therapy.We describe a flow cytometric assay based on differential immunostaining of target and effector cells where BM MSC are enumerated with fluorospheres to determine the loss of target cells after co-culture with PB MNC.6/13 (46%) of BM MSC lots were lysed by PB MNC and the lysis was proportional to the E/T cell ratio.The method overcomes the problems linked to the use of dyes or radioactive, evidencing the limitations linked to the use of a single vital dye and proposing a precise gating strategy based on absolute cell counts where cells are left untouched. The assay is easy and could be used to predict the response of the patients to the therapy.

Improvements in Flow Cytometry-Based Cytotoxicity Assay.

The flow cytometry-based assay has been increasingly used to assess the cell-mediated cytotoxicity since the 1980s due to its advantages over the conventional radioactive 51 Cr release assay (CRA), such as higher sensitivity at the single-cell level and nonradioactivity. The basic principle of this assay is the usage of two dyes, one nontoxic dye for labeling targets or effector cells to distinguish one from another, one viability dye for discrimination of dead from live cells. Due to the problem of spontaneous release or leakage of the nontoxic dye, the concern about the cross-staining has not yet been clearly elucidated. In this study, carboxyfluorescein diacetate succinimidyl ester (CFSE) was utilized to label target cells and Hoechst 33258 was used as the viability dye. We confirmed that no cross-staining occurred between the effector and target cells after 4h of coculture. We also found that the cytotoxicity would be overestimated if effector cells instead of target cells were labeled due to the exclusion of viable targets in effector-target conjugates. Using EDTA at the end of culture or labeling targets can solve this problem. Furthermore, the gating strategy could be improved by plotting CFSE against forward scatter (FSC) to discriminate some early apoptotic events. Due to the loss of target cells lysed by effector cells, counting beads are normally preferable in this assay. Here, we found an alternative to the use of beads in standardizing the flow cytometry-based assay. Instead of using beads, sample acquisition in a fixed time was shown to have the same effect in specific lysis evaluation as the beads application but have a greater stability than the latter. With a good quality control, the acquisition time for each sample could be shortened to 15 s, thus making this work to be done efficiently, especially in the case of larger sample sizes. Collectively, the findings in this study can improve the flow cytometric cytotoxicity assay to be carried out in a more accurate, efficient, and cost-effective way. © 2020 International Society for Advancement of Cytometry.

Isolation of proliferating cells from whole blood using Human Transferrin Receptor in a two-stage separation system

Blood is a routinely tested biological fluid for diagnosis and monitoring of diseases as many diseases would trigger a change in white blood cell count. Thus, several methods have been established to isolate or enrich white blood cells from patient blood samples for such analyses. One method of preparing an enriched white blood cell sample is through the selective lysis of red blood cells by hypotonic shock and restoration of osmolarity to maintain viability of target white blood cells. An inherent problem with this approach is the loss of target cells during sample handling. We report a two-stage separation system that can perform lysis and restoration of osmolarity of blood on-chip and direct the resultant sample to the second step of the analysis. Hence, there is no loss of sample. The post-lysis makeup features a protein-rich buffer to help stabilize cells. As proof of concept, we spiked HL-60 cells into a whole blood and a pre-lysed blood sample and compared capture metrics of each method using a downstream affinity separation. The capture efficiency of the whole blood sample ranged between 40 and 80% using <7 μL of sample compared to 10–52% from 60 μL of pre-lysed blood required for similar analysis. In addition, both pre-lysed and whole blood samples showed no significant difference in purity and viability. This two-stage separation system has demonstrated the capacity to replace centrifugation and wash steps required for the preparation of lysed blood, for white blood cell analyses.

Progress in Circulating Tumor Cell Research Using Microfluidic Devices.

Circulating tumor cells (CTCs) are a popular topic in cancer research because they can be obtained by liquid biopsy, a minimally invasive procedure with more sample accessibility than tissue biopsy, to monitor a patient’s condition. Over the past decades, CTC research has covered a wide variety of topics such as enumeration, profiling, and correlation between CTC number and patient overall survival. It is important to isolate and enrich CTCs before performing CTC analysis because CTCs in the blood stream are very rare (0–10 CTCs/mL of blood). Among the various approaches to separating CTCs, here, we review the research trends in the isolation and analysis of CTCs using microfluidics. Microfluidics provides many attractive advantages for CTC studies such as continuous sample processing to reduce target cell loss and easy integration of various functions into a chip, making “do-everything-on-a-chip” possible. However, tumor cells obtained from different sites within a tumor exhibit heterogenetic features. Thus, heterogeneous CTC profiling should be conducted at a single-cell level after isolation to guide the optimal therapeutic path. We describe the studies on single-CTC analysis based on microfluidic devices. Additionally, as a critical concern in CTC studies, we explain the use of CTCs in cancer research, despite their rarity and heterogeneity, compared with other currently emerging circulating biomarkers, including exosomes and cell-free DNA (cfDNA). Finally, the commercialization of products for CTC separation and analysis is discussed.

Localization of low-abundant cancer cells in a sharply expanded microfluidic step-channel using dielectrophoresis

An efficient method for cell trapping onto a designated location and reduction of the sample volume is required to reduce the risk of sample losses during the discrimination and molecular characterization of low-abundant cells suspended in a large volume of the sample. In this study, we propose a novel microfluidic device that allows for highly efficient trapping of the cancer cells onto a designated location and correction of the trapped cells with a significantly reduced sample volume. The microfluidic device, which utilizes dielectrophoresis cell trapping, consists of an attraction-zone to control the vertical position of the cells and a trap-zone to capture the target cells by drastically decreasing the flow velocity around the cells with a sharply increased channel height. The device showed that 92 ± 9% of the introduced cancer cells were trapped on a designated location (500-μm behind the step) with a high inlet flow rate of 100 μl min−1. The trapped cells were corrected with a one-hundredth volume by introducing reagents for the downstream analysis to the outlet of the device. The feasibility of the system with regard to the localization and recovery of target cells was successfully demonstrated by trapping a mixture of cancer cells and white blood cells and detecting single nucleotide variants from the collected samples. We believe that our system is suitable for the sample preparation of low-abundant cells suspended in a large sample volume, owing to its ability to perform the localization and recovery of trapped cells with minimal target cell loss.

Efficient analysis of a small number of cancer cells at the single-cell level using an electroactive double-well array.

Analysis of the intracellular materials of a small number of cancer cells at the single-cell level is important to improve our understanding of cellular heterogeneity in rare cells. To analyze an extremely small number of cancer cells (less than hundreds of cells), an efficient system is required in order to analyze target cells with minimal sample loss. Here, we present a novel approach utilizing an advanced electroactive double-well array (EdWA) for on-chip analysis of a small number of cancer cells at the single-cell level with minimal loss of target cells. The EdWA consisted of cell-sized trap-wells for deterministic single-cell trapping using dielectrophoresis and high aspect ratio reaction-wells for confining the cell lysates extracted by lysing trapped single cells via electroporation. We demonstrated a highly efficient single-cell arraying (a cell capture efficiency of 96 ± 3%) by trapping diluted human prostate cancer cells (PC3 cells). On-chip single-cell analysis was performed by measuring the intracellular β-galactosidase (β-gal) activity after lysing the trapped single cells inside a tightly enclosed EdWA in the presence of a fluorogenic enzyme substrate. The PC3 cells showed large cell-to-cell variations in β-gal activity although they were cultured under the same conditions in a culture dish. This simple and effective system has great potential for high throughput single-cell analysis of rare cells.

Abstract B16: Development of a high-throughput imaging screen for the functional assessment of cell-linking moieties using effector and target cells in a cell kill assay

Abstract Cell Linking Moieties (CLMs) are bi-specific antibody fragments that are capable of recognizing epitopes on tumor cells and specific effector cells via unique cell surface receptors. They are comprised of 2 scFv elements recognizing unique epitopes, bound by a linking element. CLMs offer a distinct advantage in that they do not rely on the expression of MHC class I molecules, generation of specific antigenic peptide, or ex vivo manipulation of cells to exert their tumor cell killing effect. To identify the proper orientation of scFv elements and to define the most potent and selective CLM, we have established an image-based screening platform capable of interrogating a large number of CLMs based on their ability to activate effectors and specifically kill tumor cells. We demonstrate the ability to screen a number of CLMs targeting wild type EGFR and HER2 receptors on fluorescently-tagged tumor cell lines for their ability to recruit CD3+ T-cells from unstimulated PBMC pools and mediate selective cell killing. CLMs were created by transient transfection of HEK293 cells with PCR-generated DNA fragments in 96 well plates. Supernatant was harvested 3 days post transfection. Dilutions of crude supernatant were applied to A549, MDA-MB-468, and SW620 (negative) target cells expressing EGFR or HER2 receptors in the presence of unstimulated PBMCs at 10:1 PBMC:Target ratio in 384 well plates. PBMC to target ratios were pre-optimized by running a titration from 2:1 through 1:30 Target:PBMCs in a kill assay. Resulting target cell loss (cell death) was compared to control wells (mock transfected supernatant). In the screening campaign, a primary screen first selects CLM hits based on killing ability, and then a secondary screen is employed to provide dose-responses, IC50s, and additional information, such as non-specific killing of negative lines. Using this image-based screen, consistent Z-scores above 0.5 were obtained, which corresponds to 12 standard deviations between the positive and negative controls, indicating an excellent dynamic range and low variability in the assay. In a proof of concept study using this platform, two EGFR-CD3 CLMs were selected from a larger list and characterized further in the secondary screen. In the top candidate, secondary screening resulted in IC50 average values of 1632 and 12,971 - fold dilution of CLM-containing HEK293 supernatant in A549 cells, and MDA-MB-468 cells, respectively. At high doses of top-candidate CLMs, no target cells remained in PBMC co-cultures, representing a near 100% killing effect. No killing was observed in the negative control line, SW620. Consistent ranking of CLM candidates was observed among 4 donors of PBMCs evaluated. In conclusion, this image-based screening platform allows for well- and cell-level killing information to be obtained with specific loss of target cells in complex mixtures to be accurately calculated without the presence of radioactive compounds like 51Cr. Furthermore, this method allows for kinetic analysis where the same well can be scanned and analyzed at multiple timepoints in the assay to understand the dynamics of killing activity. Lastly, the development of a specific and selective high throughput functional screening assay using non-terminal high content fluorescent imaging to rapidly select CLM-T cell hits results in a shortening of the developmental time line. Citation Format: Jeffrey Rosenbloom, Jason Isaacson, Srinivas Rengarajan, Paul Szymanski, Jennifer Buenviaje, Kristi Elliott, Francois Lebel, John A. Barrett, Richard Einstein. Development of a high-throughput imaging screen for the functional assessment of cell-linking moieties using effector and target cells in a cell kill assay. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B16.

Antileukemic potency of CD19-specific T cells against chemoresistant pediatric acute lymphoblastic leukemia

Adoptive therapy with chimeric antigen receptor (CAR) T cells (CART cells) has exhibited great promise in clinical trials, with efficient response correlated with CART-cell expansion and persistence. Despite extensive clinical use, the mechanisms regulating CART-cell expansion and persistence have not been completely elucidated. We have examined the antileukemia potency of CART cells targeting CD19 antigen using second-generation CAR containing a CD28 co-stimulatory domain cloned into piggyBac-transposon vector and patient-derived chemoresistant pediatric acute lymphoblastic leukemia samples. In the presence of large numbers of target cells characteristic of patients with high leukemia burden, excessive proliferation of CART cells leads to differentiation into short-lived effector cells. Transient leukemia growth delay was induced by CART-cell infusion in mice xenografted with rapidly growing CD19+ acute lymphoblastic leukemia cells and was followed by rapid CART-cell extinction. Conditioning with the hypomethylating agent 5-aza-2'-deoxycytidine-activating caspase 3 and promotion of apoptosis in leukemia cells maximized the effect of CART cells and improved CART-cell persistence. These data suggest that the clinical use of 5-aza-2'-deoxycytidine before CART cells could be considered. Coculture of leukemia cells with bone marrow stroma cells reduced target cell loss, suggesting that leukemia cell mobilization into circulation may help to remove the protective effect of bone marrow stroma and increase the efficacy of CART-cell therapy.

Isolation and purification of rabbit mesenchymal stem cells using an optimized protocol.

Mesenchymal stem cells were first isolated and grown in vitro by Friedenstein over 40yr ago; however, their isolation remains challenging as they lack unique markers for identification and are present in very small quantities in mesenchymal tissues and bone marrow. Using whole marrow samples, common methods for mesenchymal stem cell isolation are the adhesion method and density gradient fractionation. The whole marrow sample adhesion method still results in the nonspecific isolation of mononuclear cells, and activation and/or potential loss of target cells. Density gradient fractionation methods are complicated, and may result in contamination with toxic substances that affect cell viability. In the present study, we developed an optimized protocol for the isolation and purification of mesenchymal stem cells based on the principles of hypotonic lysis and natural sedimentation.

Differential infection patterns of CD4+ T cells and lymphoid tissue viral burden distinguish progressive and nonprogressive lentiviral infections

AbstractNonhuman primate natural hosts for simian immunodeficiency viruses (SIV) develop a nonresolving chronic infection but do not develop AIDS. Mechanisms to explain the nonprogressive nature of SIV infection in natural hosts that underlie maintained high levels of plasma viremia without apparent loss of target cells remain unclear. Here we used comprehensive approaches (ie, FACS sorting, quantitative RT-PCR, immunohistochemistry, and in situ hybridization) to study viral infection within subsets of peripheral blood and lymphoid tissue (LT) CD4+ T cells in cohorts of chronically SIV-infected rhesus macaques (RMs), HIV-infected humans, and SIVsmm-infected sooty mangabeys (SMs). We find: (1) infection frequencies among CD4+ T cells in chronically SIV-infected RMs are significantly higher than those in SIVsmm-infected SMs; (2) infected cells are found in distinct anatomic LT niches and different CD4+ T-cell subsets in SIV-infected RMs and SMs, with infection patterns of RMs reflecting HIV infection in humans; (3) TFH cells are infected at higher frequencies in RMs and humans than in SMs; and (4) LT viral burden, including follicular dendritic cell deposition of virus, is increased in RMs and humans compared with SMs. These data provide insights into how natural hosts are able to maintain high levels of plasma viremia while avoiding development of immunodeficiency.

On the Role of CD8 T Cells in the Control of Persistent Infections

The control of pathogen density during infections is typically assumed to be the result of a combination of resource limitation (loss of target cells that the pathogen can infect), innate immunity, and specific immunity. The contributions of these factors have been considered in acute infections, which are characterized by having a short duration. What controls the pathogen during persistent infections is less clear, and is complicated by two factors. First, specific immune responses become exhausted if they are subject to chronic stimulation. Exhaustion has been best characterized for CD8 T cell responses, and occurs through a combination of cell death and loss of functionality of surviving cells. Second, new nonexhausted T cells can immigrate from the thymus during the infection, and may play a role in the control of the infection. In this article, we formulate a partial-differential-equation model to describe the interaction between these processes, and use this model to explore how thymic influx and exhaustion might affect the ability of CD8 T cell responses to control persistent infections. We find that although thymic influx can play a critical role in the maintenance of a limited CD8 T cell response during persistent infections, this response is not sufficiently large to play a significant role in controlling the infection. In doing so, our results highlight the importance of resource limitation and innate immunity in the control of persistent infections.

Killing of Targets by CD8+ T Cells in the Mouse Spleen Follows the Law of Mass Action

It has been difficult to correlate the quality of CD8 T cell responses with protection against viral infections. To investigate the relationship between efficacy and magnitude of T cell responses, we quantify the rate at which individual CD8 effector and memory T cells kill target cells in the mouse spleen. Using mathematical modeling, we analyze recent data on the loss of target cells pulsed with three different peptides from the mouse lymphocytic choriomeningitis virus (LCMV) in mouse spleens with varying numbers of epitope-specific CD8 T cells. We find that the killing of targets follows the law of mass-action, i.e., the death rate of individual target cells remains proportional to the frequency (or the total number) of specific CD8 T cells in the spleen despite the fact that effector cell densities and effector to target ratios vary about a 1000-fold. The killing rate of LCMV-specific CD8 T cells is largely independent of T cell specificity and differentiation stage. Our results thus allow one to calculate the critical T cell concentration at which growth of a virus with a given replication rate can be prevented from the start of infection by memory CD8 T cell response.

CCR3 as a single selection marker compared to CD123/HLADR to isolate basophils in flow cytometry: Some comments

CCR3 as a single selection marker compared to CD123/HLADR to isolate basophils in flow cytometry: Some comments

Microsystems for the Capture of Low-Abundance Cells

Efficient selection and enumeration of low-abundance biological cells are highly important in a variety of applications. For example, the clinical utility of circulating tumor cells (CTCs) in peripheral blood is recognized as a viable biomarker for the management of various cancers, in which the clinically relevant number of CTCs per 7.5 ml of blood is two to five. Although there are several methods for isolating rare cells from a variety of heterogeneous samples, such as immunomagnetic-assisted cell sorting and fluorescence-activated cell sorting, they are fraught with challenges. Microsystem-based technologies are providing new opportunities for selecting and isolating rare cells from complex, heterogeneous samples. Such approaches involve reductions in target-cell loss, process automation, and minimization of contamination issues. In this review, we introduce different application areas requiring rare cell analysis, conventional techniques for their selection, and finally microsystem approaches for low-abundance-cell isolation and enumeration.

A model of HIV-1 infection with HAART therapy and intracellular delays

We consider a model of HIV-1 infection with triple drug therapy (HAART) and three delays: the first delay represents the time between the infection and the viral production, the second is associated with the loss of target cells by infection, and the third represents the time for the newly produced virions to become infectious. We show that the incorporation of these delays improves the critical efficacy of the treatment, and destabilizes the infected steady state or leads to an infected steady state with more healthy cells and less infected cells and viruses. Also, we considered the periodic treatment case. We analyze the stability of the viral free steady state and derive an effective strategy for reducing the viral load.

Providing heterogeneous quality of service bounds for correlated video traffic at a multiplexor

Network support for variable bit-rate video needs to consider (i) properties of workload induced (e.g., significant auto-correlations into far lags and heterogeneous marginal distributions), and (ii) application specific bounds on delay-jitter and statistical cell-loss probabilities. The objective of this paper is to present a quality-of-service solution for such traffic at each multiplexing point in a network. Heterogeneity in both offered workload and quality-of-service requirements are addressed. A per-virtual-circuit framing structure and a pseudo earliest-due-date cell dispatcher are introduced to provide guaranteed delay-jitter bounds. Heterogeneous jitter-bounds are supported through software controlled frame-sizes which may be independently set for each virtual-circuit. The framing structure is a generalization of per-link framing introduced by Golestani. The proposed framing structure eliminates correcting for phase mismatches between incoming frames and outgoing frames, necessary in per-link framing. This results in reduction in end-to-end delay bound and buffer requirements, and a simpler implementation. Strong auto-correlations typically seen in video traffic make equivalent bandwidth computations for heterogeneous cell-loss bounds intractable. To address this, the framing strategy is combined with an active cell-discard mechanism with prioritized cell-dropping, the latter utilizing the history of dropped cells and target cell-loss bounds for each virtual circuit. Upper bounds on the equivalent bandwidth needed to support a given workload with a target quality of service are developed. These are validated through numerical and simulation results from variable bit-rate MPEG-I video traces.

Is the lack of protein F1/GAP-43 rnRNA in granule cells target-dependent?

Protein Fl/GAP-43 is differentially expressed in brain with high levels present in regions associated with memory functions. However, in hippocampus the granule cells lack Fl/GAP-43 expression. To determine if this lack of expression is due to inhibitory signals from the target cells, we selectively destroyed CA3 pyramidal cells unilaterally using microinjections of excitotoxins. Kainate lesions induced Fl/GAP-43 mRNA expression bilaterally in granule cells at 24 h post-injection. Since the induction contralateral to the lesion was not due to loss of target cells, that induction may be ascribed to consequences of seizure activity. However, Fl/GAP-43 mRNA hybridization decreased by 3 d post-lesion and was at background levels by 6 d, indicating that the lack of Fl/GAP-43 expression in granule cells is restored despite a lack of target neurons. Unilateral lesions of CA3 cells using ibotenate, which are not as complete as kainate but do not cause seizures, did not induce Fl/GAP-43 mRNA in granule cells on either the contralateral or, in 4 of 5 cases, the ipsilateral side. Taken together, these data suggest that the CA3 target is not essential for the absence of Fl/GAP-43 expression in granule cells. To compare the extent of damage caused by the lesions, we investigated the location of astrocytes undergoing reactive gliosis, employing as a reporter glial fibrillary acidic protein (GFAP) gene expression. After both kainate and ibotenate injections GFAP hybridization increased in the lesioned area as well as in the contralateral hippocampus. These results indicate that injections of kainate, and possibly ibotenate to a lesser extent, may affect behavior not only by damaging cells at the injection site, but also by altering gene expression in cells at distant sites.