Mast cell clones: a model for the analysis of cellular maturation.

Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc epsilon R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance lg secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of 1 mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50-fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35SO4 into chondroitin sulfates rather than heparin. These findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc epsilon R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage

Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells.

We have examined the morphology, cytochemistry, and biochemistry of mouse leukocyte subsets by analyzing cloned leukocyte populations specialized to perform different immunologic functions. Cloned cells expressing high-affinity plasma membrane receptors for IgE and mediating natural killer (NK) lysis and cloned antigen-specific suppressor T cells contained prominent osmiophilic cytoplasmic granules similar by ultrastructure to those of mouse basophils. Both clones also incorporated 35SO4 into granule-associated sulfated glycosaminoglycans, expressed a characteristic ultrastructural pattern of nonspecific esterase activity, incorporated exogenous [3H]5-hydroxytryptamine, and contained cytoplasmic deposits of particulate glycogen. By contrast, cloned inducer T cells lacked cytoplasmic granules and glycogen, incorporated neither 35SO4 nor [3H]5-hydroxytryptamine, and differed from the other clones in pattern of nonspecific esterase activity. These findings establish that certain cloned cells with NK activity and cloned suppressor T cells express morphologic and biochemical characteristics heretofore associated with basophilic granulocytes. However, these clones differ in surface glycoprotein expression and immunologic function, and the full extent of the similarities and differences among these populations and basophils remains to be determined

Involvement of Bruton's tyrosine kinase in FcepsilonRI-dependent mast cell degranulation and cytokine production.

We investigated the role of Bruton's tyrosine kinase (Btk) in FcepsilonRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcepsilonRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcepsilonRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcepsilonRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed by the improvement in the ability of btk mutant mast cells to degranulate and to secrete cytokines after the retroviral transfer of wild-type btk cDNA, but not of vector or kinase-dead btk cDNA. Retroviral transfer of Emt (= Itk/Tsk), Btk's closest relative, also partially improved the ability of btk mutant mast cells to secrete mediators. Taken together, these results demonstrate an important role for Btk in the full expression of FcepsilonRI signal transduction in mast cells

Atomic Scale Dynamics Drive Brain-like Avalanches in Percolating Nanostructured Networks.

Self-assembled networks of nanoparticles and nanowires have recently emerged as promising systems for brain-like computation. Here, we focus on percolating networks of nanoparticles which exhibit brain-like dynamics. We use a combination of experiments and simulations to show that the brain-like network dynamics emerge from atomic-scale switching dynamics inside tunnel gaps that are distributed throughout the network. The atomic-scale dynamics emulate leaky integrate and fire (LIF) mechanisms in biological neurons, leading to the generation of critical avalanches of signals. These avalanches are quantitatively the same as those observed in cortical tissue and are signatures of the correlations that are required for computation. We show that the avalanches are associated with dynamical restructuring of the networks which self-tune to balanced states consistent with self-organized criticality. Our simulations allow visualization of the network states and detailed mechanisms of signal propagation

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals

Down regulation of the high-affinity IgE receptor associated with successful treatment of chronic idiopathic urticaria with omalizumab

Chronic idiopathic urticaria is a condition that is often controllable with antihistamine therapy. However, some patients have disease burden that is difficult to manage, non-responsive to antihistamines and often requires immunosuppressive medications such as corticosteroids or cyclosporine. We present here a study that demonstrates the effectiveness of omalizumab in treating this condition and the temporal relationship between improvement and down regulation of the high affinity IgE receptor (FcεRI). For this, blood samples were obtained from a symptomatic patient before each treatment and processed for flow cytometric analysis of FcεRI levels on the surface of blood basophils. Down regulation of FcεRI was observed in association with significant clinical improvement and discontinuation of immunosuppressive medications

Migraine and psychiatric comorbidity: a review of clinical findings

Migraine is an extremely common disorder. The underlying mechanisms of this chronic illness interspersed with acute symptoms appear to be increasingly complex. An important aspect of migraine heterogeneity is comorbidity with other neurological diseases, cardiovascular disorders, and psychiatric illnesses. Depressive disorders are among the leading causes of disability worldwide according to WHO estimation. In this review, we have mainly considered the findings from general population studies and studies on clinical samples, in adults and children, focusing on the association between migraine and psychiatric disorders (axis I of the DSM), carried over after the first classification of IHS (1988). Though not easily comparable due to differences in methodology to reach diagnosis, general population studies generally indicate an increased risk of affective and anxiety disorders in patients with migraine, compared to non-migrainous subjects. There would also be a trend towards an association of migraine with bipolar disorder, but not with substance abuse/dependence. With respect to migraine subtypes, comorbidity mainly involves migraine with aura. Patients suffering from migraine, however, show a decreased risk of developing affective and anxiety disorders compared to patients with daily chronic headache. It would also appear that psychiatric disorders prevail in patients with chronic headache and substance use than in patients with simple migraine. The mechanisms underlying migraine psychiatric comorbidity are presently poorly understood, but this topic remains a priority for future research. Psychiatric comorbidity indeed affects migraine evolution, may lead to chronic substance use, and may change treatment strategies, eventually modifying the outcome of this important disorder

Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos

<p>Abstract</p> <p>Background</p> <p>The use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. With the advent of more accessible and sophisticated imaging technologies as well as an abundance of fluorescent proteins with different spectral characteristics, the dynamic processes taking place <it>in situ </it>in living cells and tissues can now be probed. Photomodulatable fluorescent proteins are one of the emerging classes of genetically-encoded fluorescent proteins.</p> <p>Results</p> <p>We have compared PA-GFP, PS-CFP2, Kaede and KikGR four readily available and commonly used photomodulatable fluorescent proteins for use in ES cells and mice. Our results suggest that the green-to-red photoconvertible fluorescent protein, Kikume Green-Red (KikGR), is most suitable for cell labeling and lineage studies in ES cells and mice because it is developmentally neutral, bright and undergoes rapid and complete photoconversion. We have generated transgenic ES cell lines and strains of mice exhibiting robust widespread expression of KikGR. By efficient photoconversion of KikGR we labeled subpopulations of ES cells in culture, and groups of cells within <it>ex utero </it>cultured mouse embryos. Red fluorescent photoconverted cells and their progeny could be followed for extended periods of time.</p> <p>Conclusion</p> <p>Transgenic ES cells and mice exhibiting widespread readily detectable expression of KikGR are indistinguishable from their wild type counterparts and are amenable to efficient photoconversion. They represent novel tools for non-invasive selective labeling specific cell populations and live imaging cell dynamics and cell fate. Genetically-encoded photomodulatable proteins such as KikGR represent emergent attractive alternatives to commonly used vital dyes, tissue grafts and genetic methods for investigating dynamic behaviors of individual cells, collective cell dynamics and fate mapping applications.</p

Endotoxin tolerance and cross-tolerance in mast cells involves TLR4, TLR2 and FcεR1 interactions and SOCS expression: perspectives on immunomodulation in infectious and allergic diseases

<p>Abstract</p> <p>Background</p> <p>The study of the endotoxin tolerance phenomenon in light of the recently defined roles of mast cells and toll-like receptors as essential components of the innate immune response and as orchestrators of acquired immunity may reveal potentially useful mechanisms of immunomodulation of infectious and allergic inflammatory responses, such as sepsis or asthma. Here we evaluated the phenomenon of direct tolerance of endotoxins, as well as the induction of cross-tolerance and synergism by stimulation with toll-like receptor-2 (TLR2) and FcεR1 agonists, in murine mast cells prestimulated with lipopolysaccharide (LPS). Additionally, we evaluated some stimulatory and inhibitory signaling molecules potentially involved in these phenomena.</p> <p>Methods</p> <p>MC/9 cells and primary bone marrow-derived mast cells obtained from C57BL/6 and TLR4^-/-knock-out mice were sensitized to DNP-HSA (antigen) by incubation with DNP-IgE and were prestimulated with LPS for 18 hr prior to stimulation. Cultures were stimulated with LPS or Pam3Cys-Ser-(Lys)4 3HCl (P3C), a TLR2 agonist, individually or in combination with antigen. The production of IL-6 and TNFα, the phosphorylation of NFκB and p38 MAPK, and the expression of TLR4 and SOCS-1 and -3 were analyzed.</p> <p>Results</p> <p>We found that production of TNFα and IL-6 in murine mast cells that have been pretreated with LPS and challenged with TLR4 (LPS) or -2 (P3C) agonists was reduced, phenomena described as endotoxin tolerance (LPS) and cross-tolerance (P3C), respectively. The expression of TLR4 was not affected by LPS pretreatment. Our results show that the FcεR1 agonist DNP-HSA (antigen) interacts synergistically with LPS or P3C to markedly enhance production of cytokines (TNFα and IL-6). This synergistic effect with LPS and P3C was also attenuated by LPS pretreatment and was mediated by TLR4. These results may be attributed to the reduction in phosphorylation of the mitogen-activated protein kinase (MAPK), p38, and the transcription factor NFκB, as well as to an increase in the expression of the suppressors of cytokine signaling (SOCS)-1 and -3 proteins in LPS-pretreated mast cells.</p> <p>Conclusions</p> <p>These findings can be explored with respect to the modulation of inflammatory responses associated with infectious and allergic processes in future studies.</p

Psychomotor impairments and therapeutic implications revealed by a mutation associated with infantile Parkinsonism-Dystonia

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD