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Objective To evaluate the effects of obesity-associated inflammation on influenza vaccine responses. Methods In young and elderly individuals, both lean and with obesity, antibody responses to influenza vaccination were measured. Results A decrease in in vivo vaccine responses, circulating switched memory, and transitional B cells and an increase in pro-inflammatory late/exhausted memory B cells were found. In vitro B cell function was measured by activation-induced cytidine deaminase and E47, markers of optimal antibody responses. Moreover, IL-6 production was increased, whereas IL-10 production was decreased in cultures of B cells from individuals with obesity. Markers of immune activation (TNF-α, TLR4, micro-RNAs) in unstimulated B cells were also found increased and were negatively correlated with B cell function. In order to reveal potential mechanisms, we stimulated B cells from lean individuals in vitro with leptin, the adipokine increased in obesity. Leptin increased phospho-STAT3, crucial for TNF-α production, and decreased phospho-AMPK, the energy sensing enzyme upstream of phospho-p38 MAPK and E47. Leptin-induced phospho-STAT3 and phospho-AMPK levels were similar to those in B cells from individuals with obesity. Conclusions These results demonstrate that leptin can be responsible for decreased B cell function in obesity.

Background Several studies documented abnormal nociceptive processing in PD patients. Pain central pathways are accessible by laser-evoked potentials (LEPs). LEPs recording show a N2/P2 complex mostly generated by the anterior cingulate cortex, preceded by an earlier negative component (N1), originating from the opercular cortex. Previous work demonstrated N2/P2 amplitude reduction in PD patients and suggested a centrally-acting pathomechanism for the genesis of pain. However, since a peripheral deafferentation has been recently demonstrated in PD, it is not clear if such LEP abnormalities reflect a mechanism acting centrally or not. Objective To assess whether abnormalities of nociceptive inputs occur at central and/or peripheral level in pain-free PD patients with hemiparkinson using Nd:YAP LEPs. Methods We recorded scalp Nd:YAP-LEPs to hand stimulation in 13 pain-free patients with unilateral PD and in 13 healthy subjects. Additionally, we collected laser pain-rating in both groups. Results PD patients and normal subjects showed comparable N1, N2 and P2 latencies. The N2/P2 amplitude was significantly lower in PD patients than in controls, regardless of the clinically affected side, whereas the N1/P1 amplitude was not different. PD patients had higher pain-rating, indicative of hyperalgesia. Conclusions These findings demonstrate that in the PD patients the abnormal processing of pain stimuli occurs at central rather than peripheral level. The co-existence of hyperalgesia and reduced amplitude of the N2/P2 complex, in spite of a normal N1/P1 component, suggests an imbalance between the medial and lateral pain systems. Such a dissociation might explain the genesis of central pain in PD.

Our research in the past few years has identified B cell–specific biomarkers able to predict optimal Ab responses in both young and elderly individuals. These biomarkers are activation-induced cytidine deaminase (AID), the enzyme of class switch recombination and somatic hypermutation; the transcription factor E47, crucial for AID expression; and the ability to generate optimal memory B cells. Moreover, we have found that the increased proinflammatory status of the elderly, both in sera and intrinsic to B cells, negatively impacts B cell function. We have now investigated whether particular inflammatory microRNAs (miRs) contribute to decreased E47 and AID in aged B cells. Our data indicate that E47 and AID mRNA stability is lower in stimulated B cells from elderly individuals. We measured the expression of two miRs crucial for class switch recombination, miR-155 and miR-16, in human unstimulated B cells from young and elderly individuals with the rationale that increases in these before stimulation would decrease E47/AID upon cell activation. We found these miRs and B cell–intrinsic inflammation upregulated in aged unstimulated B cells and negatively associated with AID in the same B cells after stimulation with CpG. We propose that the downregulation of AID in aged human B cells may occur through binding of miR-155 to the 3′-untranslated regions of AID mRNA and/or binding of miR-16 to the 3′-untranslated regions of E47 mRNA, as well as at the transcriptional level of less E47 for AID. Our results indicate novel molecular pathways leading to reduced B cell function with aging.

Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3β kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3β antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.

Summary Secretion of Yop effector proteins by the Yersinia pes- tis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environ- mental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37 ∞ C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN-dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino-acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA-bind- ing domain and TyeA to block secretion, but did not require an N-terminal secretion signal, an intact chap- erone-binding domain or the SycN/YscB chaperone. These results suggest that a C-terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothet- ical model for how the YopN/SycN/YscB/TyeA com- plex regulates Yop secretion is presented.

Summary Yersinia pestis , the causative agent of plague, exports a set of virulence proteins called Yops upon contact with eukaryotic cells. A subset of these Yops is trans- located directly into the cytosol of host cells. In this study, a novel protein tag-based reporter system is used to measure the translocation of Yops into cul- tured eukaryotic cells. The reporter system uses a small bipartite phosphorylatable peptide tag, termed the Elk tag. Translocation of an Elk-tagged protein into eukaryotic cells results in host cell protein kinase- dependent phosphorylation of the tag at a specific serine residue, which can subsequently be detected with phosphospecific antibodies. The YopN, TyeA, SycN, YscB and LcrG proteins function to prevent Yop secretion before host cell contact. The role of these proteins was investigated in the translocation of Elk- tagged YopE (YopE 129 -Elk) and YopN (YopN 293 -Elk) into HeLa cells. Y. pestis yopN , tyeA , sycN and yscB dele- tion mutants showed reduced levels of YopE 129 -Elk phosphorylation compared with the parent strain, indicating that these mutants translocate reduced amounts of YopE. We also demonstrate that YopN 293 - Elk is translocated into HeLa cells and that this pro- cess is more efficient in a Yersinia yop polymutant strain lacking the six translocated effector Yops. Y. pestis sycN and yscB mutants translocated reduced amounts of YopN 293 -Elk; however, tyeA and lcrG mutants translocated higher amounts of YopN 293 -Elk compared with the parent strain. These data suggest that TyeA and LcrG function to suppress the secretion

Gram-negative bacteria use type III secretion (TTS) systems to translocate proteins into the extracellular environment or directly into eukaryotic cells. These complex secretory systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway. Secretion substrates are directed to the TTS machinery via mRNA and/or amino acid secretion signals. TTS chaperones bind to select secretion substrates and assist in the export process. Recent progress in the understanding of TTS is reviewed.

In some definite patients, a standard neurophysiological tool may not solve a complete differential diagnosis in common nerve peroneal neuropathy. In this study we have assessed a new simple procedure to study the orthodromic sensory conduction of both the superficial peroneal nerves (SPN) and deep peroneal nerves (DPN) in a heterogeneous group of 55 normal subjects. The mean sensory orthodromic conduction velocity of the SPN was 58.35 m/s. The mean sensory orthodromic conduction velocity of the mixed nerve action potential (MNAP) of the DPN was 55.27 m/s. The sensory conduction velocity, the amplitude of sensory-evoked potentials of SPN and DPN across the fibular head and the normative values are discussed. Our results confirm that these recording methods are easy to repeat and reliable in identifying peroneal neuropathy.

The localization and membrane topology of the Yersinia pestis YscJ lipoprotein, an essential component of the type III secretion apparatus, was investigated. YscJ was demonstrated to be an inner membrane (IM) lipoprotein that is anchored to the periplasmic face of the IM via an N-terminal lipid moiety and via a C-terminal transmembrane (TM) domain. Localization of the N-terminal lipid moiety to the IM occurred regardless of the amino-acid residues found in the +2 or +3 positions. IM localization was dependent upon an intact N-terminal domain (amino acids +1 to +61), suggesting that this region plays a role in YscJ localization. In contrast, the YscJ C-terminal domain and TM domain were not required for IM localization. N-terminal sequence analysis demonstrated that a significant proportion of membrane-localized YscJ lacks N-acylation, the final modification required for Lol-dependent transport of a lipoprotein to the OM. Interestingly, attachment of the N-terminus to the IM was required for YscJ function; however, the YscJ secretion signal and lipo-box could be functionally replaced by the first TM domain of the YscV protein, suggesting that the mechanism of attachment to the IM was not critical.

To identify the target of IgG autoimmune response in Hashimoto's encephalopathy (HE), we studied the binding of IgG present in serum and cerebro-spinal fluid (CSF) from six patients with HE and 15 controls to human central nervous system (CNS) white matter antigens by 2D-PAGE and immunoblotting and by immunohistochemistry. We found that CSF IgG from HE patients specifically recognized 3 spots, which were identified as dimethylargininase-I (DDAHI) and aldehyde reductase-I (AKRIAI). DDAHI was present in two isoforms recognized respectively by five and four HE patients; immunohistochemistry with anti-DDAHI antiserum depicted endothelial cells in normal human CNS. AKRIAI was recognized by three HE CSF and this enzyme was widely distributed on neurons and endothelia by immunohistochemistry. IgG from HE CSF immunostained both neuronal and endothelial cells in mouse CNS. The presence of these autoantibodies selectively in the CSF of HE patients may have important diagnostic and pathogenetic implications, since the autoimmune response to these enzymes may lead to vascular and/or neuronal damage, two major mechanisms involved in the pathogenesis of HE.

Secretion of Yop effector proteins by the Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environmental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37 degrees C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN-dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino-acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA-binding domain and TyeA to block secretion, but did not require an N-terminal secretion signal, an intact chaperone-binding domain or the SycN/YscB chaperone. These results suggest that a C-terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothetical model for how the YopN/SycN/YscB/TyeA complex regulates Yop secretion is presented.

YopN is a secreted protein that prior to secretion directly interacts with the cytosolic SycN/YscB chaperone complex and TyeA. This study identifies a secreted YopN-TyeA hybrid protein that is expressed by Yersinia pestis , but not by Yersinia enterocolitica . DNA sequence analysis and site-directed mutagenesis studies demonstrate that the hybrid protein is the result of a +1 translational frameshift event.

To report and discuss the neuropsychological deficits and neuroimaging findings in a patient with probable Marchiafava-Bignami disease.A right-handed woman with chronic alcoholism demonstrated mutism, impaired comprehension of spoken language, alexia, and right-handed agraphia. The syndrome of interhemispheric disconnection was manifested by left-handed deficit of constructional ability and agraphia. The patient underwent brain computed tomographic scans, technetium 99 hexylmethylpropylene amineoxime-single photon emission computed tomography, and magnetic resonance imaging (MRI) that also included fluid attenuated inversion recovery images.Clinical neurology department.The patient's symptoms were related to scattered lesions of the corpus callosum and to extensive symmetrical lesions of the centrum semiovale. Only the latter were detected by computed tomographic scans. Results of single photon emission computed tomography did not show areas of focal hypoperfusion. Results of fast spinecho MRI showed all lesions were hyperintense in T1-weighted images and hypointense in T2-weighted images. Fluid attenuated inversion recovery images revealed that periventricular lesions had a hypointense core surrounded by a hyperintense rim; callosal lesions were still hyperintense.We believe that our patient's symptoms are due to the discontinuous affection of the corpus callosum and to the bilateral cutting of the outflow from the cortex. The MRI findings may be interpreted as indicating central necrosis and peripheral demyelination of periventricular lesions and demyelination of the corpus callosum. The combined use of fast spin echo and fluid attenuated inversion recovery MRI reproduced with more accuracy than fast spin echo MRI alone some features of Marchiafava-Bignami disease known from observations at autopsy.