Mouse spleen lymphoblasts generated in vitro. Their replication and differentiation in vitro

Mouse spleen lymphoblasts induced with lipopolysaccharide and fetal calf serum were obtained in high yield and purity in their first proliferative cell cycle by floatation in dense bovine plasma albumin columns (3). The blasts were maintained in vitro for 3 more days. The cultures were examined in bulk on each day, and in addition, those cells in S phase initially were tagged with [(3)H]thymidine and followed continuously in vitro. Grain count dilution data indicated that most blasts divided but twice over a 2- to 3-day interval in vitro. [(3)H]Thymidine pulse radiolabeling and flow microfluorometry suggested that at least 50-70 percent of the proliferating blasts withdrew from proliferative activity after 2-3 days of culture. Morphologic studies demonstrated that lymphoblasts persisted as such for 1-2 days in vitro and then matured into typical plasma cells. Many of the blastprogeny had small nuclei and considerable basophilic cytoplasm on Giemsa-stained cell smears; abundant rough endoplasmic reticulum by electron microscopy; and readily detectable cytoplasmic Ig by immunocytochemistry. Reversion of blasts to small lymphocytes could not be detected; however, some blasts persisted even after 3 days of culture. The viability of the cultured lymphoblast was followed by initially tagging the cells with [(3)H]thymidine as well as several other techniques. Little cell death was documented during the first day of culture. The number of labeled progeny increased twofold whereas the grain count halved. But 40- 50 percent of the cell-associated label was lost during each of the second and third days, and fewer labeled progeny than predicted by grain count dilution were identified. The culture medium could not be implicated in this loss of lymphoblast progeny, and we suggest that the maturation of the lymphoblast to a short-lived plasma cell was responsible. Therefore mitogen-stimulated B blasts seem to mature into typical plasma cells after just two cycles of cell division. The plasma cells resemble those produced in situ during an immune response in their cytologic features, withdrawal from active proliferative activity, and short life-span

Dendritic cells are accessory cells for the development of anti-trinitrophenyl cytotoxic T lymphocytes.

This study establishes that dendritic cells (DC) are the critical accessory cells for the development of anti-trinitrophenol (TNP) cytotoxic T lymphocytes (CTL) in vitro. We developed a model in which nylon wool-nonadherent spleen cells were used both as the responding and stimulating cells, the latter having been TNP-modified and x-irradiated. Thy-1-bearing CTL developed in C57BL/6, B6D2F1, and CBA mice only when small numbers of DC were added. Maximal responses in 5-d cultures were achieved with 0.5-1 DC/100 responding T cells. The DC did not have to be TNP modified directly. Anti-Ia and complement inactivated accessory cells, whereas similar treatment of the responders had no effect. DC exposed to ultraviolet radiation were ineffective, but x-irradiated DC were fully active. Culture media from DC, or from DC-nylon wool-passed spleen T cell cocultures that contained abundant CTL, would not substitute for viable DC. Enriched preparations of macrophages (MΦ) were obtained from blood, peritoneal cavity, and spleens of BCG-immune and unprimed mice. MΦ added at doses of 0.2-4% were weak or inactive as accessory cells. The level of Ia antigens on test MΦ populations was quantitated and visualized by binding of a radioiodinated monoclonal anti-I-A(b,d) antibody, clone B-21. MΦ that bore substantial amounts of Ia from all organs were weak accessory cells. Addition of MΦ to DC-T cell cocultures produced inhibitory effects, usually at a dose of 2% MΦ. In contrast, 0.5% Ia-bearing MΦ from BCG-immune boosted mice inhibited \u3e80% of the DC-mediated CTL response. Addition of indomethacin reversed MΦ inhibition, and 10-9M prostaglandin E2 in turn blocked the indomethacin effect. Indomethacin also restored a low level of accessory cell function in immune-boosted adherent peritoneal cells, but not in preparations of monocytes and spleen MΦ. Small numbers of DC were identified in preparations of immune-boosted peritoneal cells and may have accounted for the observed accessory activity. We conclude that the development of anti-TNP CTL is an immune response in which (a) DC are the critical accessory cells; (b) Ia-bearing MΦ are weak or inactive; and (c) MΦ can inhibit DC-mediated response by an indomethacin-sensitive mechanism

Roughening of the (1+1) interfaces in two-component surface growth with an admixture of random deposition

We simulate competitive two-component growth on a one dimensional substrate of $L$ sites. One component is a Poisson-type deposition that generates Kardar-Parisi-Zhang (KPZ) correlations. The other is random deposition (RD). We derive the universal scaling function of the interface width for this model and show that the RD admixture acts as a dilatation mechanism to the fundamental time and height scales, but leaves the KPZ correlations intact. This observation is generalized to other growth models. It is shown that the flat-substrate initial condition is responsible for the existence of an early non-scaling phase in the interface evolution. The length of this initial phase is a non-universal parameter, but its presence is universal. In application to parallel and distributed computations, the important consequence of the derived scaling is the existence of the upper bound for the desynchronization in a conservative update algorithm for parallel discrete-event simulations. It is shown that such algorithms are generally scalable in a ring communication topology.Comment: 16 pages, 16 figures, 77 reference

Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells

Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size <40 nm) resonant laser scattering and Raman scattering cross-sections are too small to allow single nanoparticle observation. Nanodiamonds can however be rendered photoluminescent with a perfect photostability at room temperature. Such a remarkable property allows easier single-particle tracking over long time-scales. In this work we use photoluminescent nanodiamonds of size <50 nm for intracellular labeling and investigate the mechanism of their uptake by living cells . By blocking selectively different uptake processes we show that nanodiamonds enter cells mainly by endocytosis and converging data indicate that it is clathrin mediated. We also examine nanodiamonds intracellular localization in endocytic vesicles using immunofluorescence and transmission electron microscopy. We find a high degree of colocalization between vesicles and the biggest nanoparticles or aggregates, while the smallest particles appear free in the cytosol. Our results pave the way for the use of photoluminescent nanodiamonds in targeted intracellular labeling or biomolecule deliver

Automated Batch Characterization of Icf Shells With Vision-Enabled Optical Microscope System

OAK-B135 Inertial Confinement Fusion (ICF) shells are mesoscale objects with nano-scale dimensional and nano-surface finish requirements. Currently, the shell dimensions are measured by white-light interferometry and an image analysis method. These two methods complement each other and give a rather complete data set on a single shell. The process is, however, labor intensive. They have developed an automation routine to fully characterize a shell in one shot and perform unattended batch measurements. The method is useful to the ICF program both for production screening and for full characterization. It also has potential for Inertial Fusion Energy (IFE) power plant where half a million shells need to be processed daily

Geriatric pharmacotherapy : optimisation through integrated approach in the hospital setting

Since older patients are more vulnerable to adverse drug-related events, there is a need to ensure appropriate prescribing in these patients in order to prevent misuse, overuse and underuse of drugs. Different tools and strategies have been developed to reduce inappropriate prescribing; the available measures can be divided into medication assessment tools, and specific interventions to reduce inappropriate prescribing. Implicit criteria of inappropriate prescribing focus on appropriate dosing, search for drug-drug interactions, and increase adherence. Explicit criteria are consensus-based standards focusing on drugs and diseases and include lists of drugs to avoid in general or lists combining drugs with clinical data. These criteria take into consideration differences between patients, and stand for a medication review, by using a systematic approach. Different types of interventions exist in order to reduce inappropriate prescribing in older patients, such as: educational interventions, computerized decision support systems, pharmacist-based interventions, and geriatric assessment. The effects of these interventions have been studied, sometimes in a multifaceted approach combining different techniques, and all types seem to have positive effects on appropriateness of prescribing. Interdisciplinary teamwork within the integrative pharmaceutical care is important for improving of outcomes and safety of drug therapy. The pharmaceutical care process consists offour steps, which are cyclic for an individual patient. These steps are pharmaceutical anamnesis, medication review, design and follow-up of a pharmaceutical care plan. A standardized approach is necessary for the adequate detection and evaluation of drug-related problems. Furthermore, it is clear that drug therapy should be reviewed in-depth, by having full access to medical records, laboratory values and nursing notes. Although clinical pharmacists perform the pharmaceutical care process to manage the patient’s drug therapy in every day clinical practice, the physician takes the ultimate responsibility for the care of the patient in close collaboration with nurses

A Double-Blind Randomized Phase I Clinical Trial Targeting ALVAC-HIV Vaccine to Human Dendritic Cells

BACKGROUND: We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone. METHODOLOGY/PRINCIPAL FINDINGS: Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (i.m.), intradermal injection (i.d.) or subcutaneous injection (s.q.) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the i.m. arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production. CONCLUSIONS/SIGNIFICANCE: ALVAC-HIV delivered i.m., i.d. or s.q. with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00013572

Accounting Problems Under the Excess Profits Tax

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV- 1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8(+) T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.Funding Agencies|Research Council of Norway; Odd Fellow</p

The Imperative to Share Clinical Study Reports: Recommendations from the Tamiflu Experience

Peter Doshi and colleagues describe their experience trying and failing to access clinical study reports from the manufacturer of Tamiflu and challenge industry to defend their current position of RCT data secrecy

Treatment of refractory epilepsy with natalizumab in a patient with multiple sclerosis. Case report

Background. Multiple sclerosis (MS) is considered an autoimmune disease of the central nervous system and therapeutic inhibition of leukocyte migration with natalizumab, an anti-alpha4 integrin antibody, is highly effective in patients with MS. Recent studies performed in experimental animal models with relevance to human disease suggested a key role for blood-brain barrier damage and leukocyte trafficking mechanisms also in the pathogenesis of epilepsy. In addition, vascular alterations and increased leukocyte accumulation into the brain were recently documented in patients with refractory epilepsy independently on the disease etiology. Case report. Here we describe the clinical course of a 24-year-old patient with MS in whom abrupt tonic-clonic generalized seizures manifested at disease onset. Although MS had a more favorable course, treatment with glatiramer acetate and antiepileptic drugs for 7 years had no control on seizure generation and the patient developed severe refractory epilepsy. Interestingly, generalized seizures preceded new MS relapses suggesting that seizure activity may contribute to MS worsening creating a positive feedback loop between the two disease conditions. Notably, treatment with natalizumab for 12 months improved MS condition and led to a dramatic reduction of seizures. Conclusion. Our case report suggests that inhibition of leukocyte adhesion may represent a new potential therapeutic approach in epilepsy and complement the traditional therapy with anti-epileptic drugs