In Vivo Anergized T Cells Form Altered Immunological Synapses In Vitro

T cells contact allogeneic antigen presenting cells (APCs) and assemble, at their contact interface, a molecular platform called the immunological synapse. Synapse-based molecules provide directional signals for the T cell--either positive signals, resulting in T-cell activation, or negative signals causing T-cell inactivation or anergy. To better understand the molecular basis of in vivo T-cell anergy we analyzed the contacts made between in vivo anergized T cells and APCs, and determined which signaling molecules were included or excluded from their immunological synapses. Anergy was induced in TCR transgenic mice by the intravenous injection of semiallogeneic donor spleen cells. T cells from anergized mice were mixed with APCs, the T-cell/APC synapses imaged using deconvolution microscopy, and their molecular compositions were determined. T cells from anergic mice formed unstable immunological synapses in vitro with allogeneic APCs and failed to recruit the signaling proteins necessary to initiate T-cell activation. These findings suggest that T-cell anergy induced by exposure to semiallogeneic donor cells is associated with defects in the earliest events of T-cell activation, immunological synapse formation and recruitment of TCR-mediated signaling proteins.     

A new method for application of force to cells via ferric oxide beads

 We describe a new method that uses straightforward physics to apply force to substrate-attached cells. In this method, collagen-coated magnetic ferric oxide beads attach to the dorsal surface of cells via receptors of the integrin family, and a magnetic field gradient is applied to produce a force. In this paper we present a complete characterization of the method in a configuration that is easy to use, in which a permanent magnet provides a fairly uniform gradient over a relatively large area. This allows a fairly uniform average force that can be controlled in magnitude, direction, and duration to be applied to a large number of cells. We show how to determine the applied force per cell by measuring the force per unit volume of magnetic bead, the distribution of bead diameters, and the distribution of beads per cell. We also show how to calculate the force per unit volume of bead in a three-dimensional region near the permanent magnet on the basis of field measurements, and present results for three of the magnets. An upward force applied to fibroblasts by this method produces a measurable time-dependent increase in attachment of cytoskeletal actin filaments to the force application points, and an increase in actin cross-linking. This is accompanied by an actin-dependent retraction of the force-induced upward movement of the dorsal surface of the cells. Received: 27 February 1997 / Received after revision: 10 August 1997 / Accepted: 1 September 1997     

Modulation of infection-induced inflammation and locomotive deficit and longevity in senescence-accelerated mice-prone (SAMP8) model by the oligomerized polyphenol Oligonol.

Oligonol is produced from the oligomerization of polyphenols (typically proanthocyanidin from a variety of fruits such as lychees, grapes, apples, persimmons, etc.) and contains catechin-type monomers and oligomers of proanthocyanidins. The ability of Oligonol to affect infection-dependent eye inflammation, locomotion and longevity in senescence-accelerated prone mice (SAMP8) (a model of senescence acceleration and geriatric disorders with increased oxidative stress and neuronal deficit) was investigated. Oligonol (60mg/kg) significantly modulated the extent of inflammation scores in the eye of SAMP8 mice. Examination of the mice indicated infection with mouse hepatitis virus and pinworm (Syphacia obvelata) in both males and females and with the intestinal protozoa (trichomonad) in males. A comparison of the two groups (using log-rank test) and the difference in the mean life span between groups (using Student's t-test) indicated significant differences in survival (p=0.043) and the mean life span (p=0.033) in male SAMP8 mice. Oligonol increased the mean life span and this was statistically significant. In the open-field locomotive test, the 7-week-old SAMP8 mice crossed more than 40 partitioned lines in 1min. At 48-week-old control untreated male SAMP8 crossed 2 lines. The Oligonol-treated 48-week-old male SAMP8 mice crossed 17 lines however. The improved locomotive activity was statistically significant even after 36weeks in the Oligonol-treated male SAMP8 but this was not the case throughout the time course of the study in the Oligonol-treated female SAMP8. Thus Oligonol treatment to SAMP8 mice modulated the severity of infection-dependent inflammation, prolonged life-span and significantly improved locomotive activity indicating potential benefit to aging-associated diseases such as Alzheimer's or Parkinson's diseases. This presents potential for further research to define infection-dependent inflammation associated with degenerative conditions and the molecular mechanism of dietary antioxidant protection.     

Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells

During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide–major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide–MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide–MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide–MHC complexes.     

Differential regulation of cytokine genes in gingival epithelial cells challenged by Fusobacterium nucleatum and Porphyromonas gingivalis

IL-8 mRNA in human gingival epithelial cells (HGECs) is up-regulated by Fusobacterium nucleatum, and up-/down-regulated by Porphyromonas gingivalis in a complex interaction in the early stages (< or = 4 h) after infection. The mechanisms involved in this regulation in response to F. nucleatum and/or P. gingivalis infection, and identification of co-regulated cytokine genes, are the focus of this investigation. Heat, formalin or protease treatment of F. nucleatum cells attenuated the IL-8 mRNA up-regulation. NF-kappaB, mitogen-activated protein kinase (MAPK) p38 and MAPK kinase/extracellular signal-regulated kinase (MEK/ERK) pathways were involved in IL-8 mRNA induction by F. nucleatum. Pretreatment of P. gingivalis with heat, formalin or protease enhanced IL-8 mRNA induction. NF-kappaB, MARK p38, and MEK/ERK pathways were also involved in this induction. In contrast, down-regulation of IL-8 mRNA by P. gingivalis involved MEK/ERK, but not NF-kappaB or MAPK p38 pathways. cDNA arrays analysis revealed that mRNA down-regulation by P. gingivalis is a specific reaction that only a number of genes, e.g. IL-1beta, IL-8, macrophage inflammatory protein-2alpha, and migration inhibitory factor-related protein-14, are affected based on examination of 278 cytokine/receptor genes. These data indicate that F. nucleatum and P. gingivalis trigger specific and differential gene regulation pathways in HGECs.     

Guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction

Summary:  Members of the cytohesin protein family, a group of guanine nucleotide exchange factors for adenosine diphosphate ribosylation factor (ARF) guanosine triphosphatases, have recently emerged as important regulators of signal transduction in vertebrate and invertebrate biology. These proteins share a modular domain structure, comprising carboxy-terminal membrane recruitment elements, a Sec7 homology effector domain, and an amino-terminal coiled-coil domain that serve as a platform for their integration into larger signaling complexes. Although these proteins have a highly similar overall build, their individual biological functions appear to be at least partly specific. Cytohesin-1 had been identified as a regulator of β2 integrin inside-out regulation in immune cells and was subsequently shown to be involved in mitogen-associated protein kinase signaling in tumor cell proliferation as well as in T-helper cell activation and differentiation. Cytohesin-3, which had been discovered to be strongly associated with T-cell anergy, was very recently described as an essential component of insulin signal transduction in Drosophila and in human and murine liver cells. Future work will aim to dissect the mechanistic details of the modes of action of the cytohesins as well as to define the precise roles of these versatile proteins in vertebrates at the genetic level.     

Activated Ras/JNK driven Dilp8 in imaginal discs adversely affects organismal homeostasis during early pupal stage in Drosophila,a new checkpoint for development

Background

Dilp8-mediated inhibition of ecdysone synthesis and pupation in holometabolous insects maintains developmental homeostasis through stringent control of timing and strength of molting signals. We examined reasons for normal pupation but early pupal death observed in certain cases.

Results

Overexpression of activated Ras in developing eye/wing discs inhibited Ptth expression in brain via upregulated JNK signaling mediated Dilp8 secretion from imaginal discs, which inhibited ecdysone synthesis in prothoracic gland after pupariation, leading to death of ~25- to 30-hour-old pupae. Inhibition of elevated Ras signaling completely rescued early pupal death while post-pupation administration of ecdysone to organisms with elevated Ras signaling in eye discs partially rescued their early pupal death. Unlike the earlier known Dilp8 action in delaying pupation, hyperactivated Ras mediated elevation of pJNK signaling in imaginal discs caused Dilp8 secretion after pupariation. Ectopic expression of certain other transgene causing pupal lethality similarly enhanced pJNK and early pupal Dilp8 levels. Suboptimal ecdysone levels after 8 hours of pupation prevented the early pupal metamorphic changes and caused organismal death.

Conclusions

Our results reveal early pupal stage as a novel Dilp8 mediated post-pupariation checkpoint and provide further evidence for interorgan signaling during development, wherein a peripheral tissue influences the CNS driven endocrine function.

     

Physiological mechanisms of TRPC activation

TRPC (canonical transient receptor potential) channels are vertebrate homologs of the Drosophila photoreceptor channel, TRP. Considerable research has been brought to bear on the seven members of this family, especially with regard to their possible role in calcium entry. Unfortunately, the current literature presents a confusing picture, with different laboratories producing widely differing results and interpretations. It appears that ectopically expressed TRPC channels can be activated by phospholipase C products (generally, diacylglycerols), by stimulation of trafficking to the plasma membrane, or by depletion of intracellular Ca²⁺ stores. Here, I discuss the possibility that these diverse experimental findings arise because TRPC channels can, under both experimental as well as physiological conditions, be activated in three distinct ways, possibly depending on their subunit composition and/or signaling complex environment. The TRPCs may be unique among ion-channel subunit families in being able to participate in the assembly and function of multiple types of physiologically important ion channels.     

A new family with epiphyseal chondrodysplasia type Miura

Epiphyseal chondrodysplasia, Miura type (ECDM) is a skeletal dysplasia with tall stature and distinctive skeletal features caused by heterozygous NPR2 pathogenic variants. Only four families have been reported. We present a family with five affected individuals (mother, three sons, and daughter). The mother's phenotype was relatively mild: borderline tall stature and elongated halluces operated during childhood. The children were remarkably more severely affected with tall stature, scoliosis, and elongated toes and fingers leading to suspicion of Marfan syndrome. Progressive valgus deformities (at the hips, knees, and ankles) were the main complaints and necessitated orthopedic investigations and surgery. Radiographs showed coxa valga, scoliosis, multiple pseudoepiphyses of the fingers and toes with uneven elongation of the digits and ankle valgus. The two older brothers underwent osteotomies and guided growth for axial deformities and arthrodesis for elongated halluces. Genetic testing confirmed the clinical diagnosis of ECDM: all affected individuals had a heterozygous c.2647G>A (p.Val883Met) NPR2 variant in a highly conserved region in the carboxyl‐terminal guanylyl cyclase domain. This two‐generation family elucidates the clinical and radiological variability of the disease. These rare cases are important to gain further understanding of the fundamental processes of growth regulation.