Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Degradation and Erosion of Metal-Organic Frameworks: Comparative Study of a NanoMIL-100 Drug Delivery System.

To make a drug work better, the active substance can be incorporated into a vehicle for optimal protection and control of the drug delivery time and space. For making the drug carrier, the porous metal-organic framework (MOF) can offer high drug-loading capacity and various designs for effective drug delivery performance, biocompatibility, and biodegradability. Nevertheless, its degradation process is complex and not easily predictable, and the toxicity concern related to the MOF degradation products remains a challenge for their clinical translation. Here, we describe an in-depth molecular and nanoscale degradation mechanism of aluminum- and iron-based nanoMIL-100 materials exposed to phosphate-buffered saline. Using a combination of analytical tools, including X-ray photoelectron spectroscopy, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and electron microscopy, we demonstrate qualitatively and quantitatively the formation of a new coordination bond between metal(III) and phosphate, trimesate release, and correlation between these two processes. Moreover, the extent of material erosion, i.e., bulk or surface erosion, was examined from the transformation of nanoparticles' surface, morphology, and interaction with water. Similar analyses show the impact of drug loading and surface coating on nanoMIL-100 degradation and drug release as a function of the metal-ligand binding strength. Our results indicate how the chemistry of nanoMIL-100(Al) and nanoMIL-100(Fe) drug carriers affects their degradation behaviors in a simulated physiological medium. This difference in behavior between the two nanoMIL-100s enables us to better correlate the nanoscale and atomic-scale mechanisms of the observed phenomena, thus validating the presented multiscale approach.

A protective multiple gene-deleted African swine fever virus genotype II, Georgia 2007/1, expressing a modified non-haemadsorbing CD2v protein.

African swine fever virus is a complex DNA virus that causes high fatality in pigs and wild boar and has a great socio-economic impact. An attenuated genotype II strain was constructed by replacing the gene for wildtype CD2v protein with versions in which single or double amino acid substitutions were introduced to reduce or abrogate the binding to red blood cells and reduce virus persistence in blood. The mutant CD2v proteins were expressed at similar levels to the wildtype protein on the surface of infected cells. Three recombinant viruses also had K145R, EP153R, and in one virus DP148R genes deleted. Following immunization of pigs, the virus with a single amino acid substitution in CD2v, Q96R, induced moderate levels of replication, and 100% protection against virulent ASFV. Two additional recombinant viruses had two amino acid substitutions in CD2v, Q96R, and K108D, and induced no binding to red blood cells in vitro. In immunized pigs, reduced levels of virus in blood and strong early ASFV-specific antibody and cellular responses were detected. After challenge low to moderate replication of challenge virus was observed. Reduced clinical signs post-challenge were observed in pigs immunized with the virus from which DP148R gene was deleted. Protection levels of 83-100% were maintained across a range of doses. Further experiments with virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D showed low levels of virus dissemination in tissue and transient clinical signs at high doses. The results support further evaluation of GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D as a vaccine candidate.

Degradation of Polymer-Drug Conjugate Nanoparticles Based on Lactic and Itaconic Acid.

Tuberculosis (TB) is still a significant threat to human health. A promising solution is engineering nanoparticulate drug carriers to deliver anti-TB molecules. Itaconic acid (ITA) potentially has anti-TB activity; however, its incorporation in nanoparticles (NP) is challenging. Here we show an approach for preparing polymer-ITA conjugate NPs and a methodology for investigating the NP degradation and ITA release mechanism. The conjugate was synthesized by the two-directional growing of polylactic acid (PLA) chains, followed by capping their extremities with ITA. The poly(lactate)-itaconate PLA-ITA was then used to formulate NPs. The degradation and drug release processes of the polymer conjugate NPs were studied qualitatively and quantitatively. The molecular structures of released species were characterized by using liquid NMR spectroscopy and mass spectrometry. We discovered a complex NP hydrolysis process forming diverse oligomers, as well as monomeric lactic acid (LA) and drug ITA. The slow degradation process led to a low release of free drugs, although raising the pH from 5.3 to 7.4 induced a slight increase in the amounts of released products. TEM images showed that bulk erosion is likely to play the primary role in the degradation of PLA-ITA NPs. The overall results and methodology can be of interest for understanding the mechanisms of NP degradation and drug release of this new polymer-drug conjugate system.

Structure of a fully assembled tumor-specific T cell receptor ligated by pMHC.

The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαß/CD3δγε2ζ2 complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements.

Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells.

To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

Amantadine Associated Myoclonus: Case Report and Review of the Literature.

PURPOSE: Amantadine is commonly used to treat Parkinson's disease. A case of myoclonus and asterixis was associated with amantadine is reported. CASE SUMMARY: An 80-year-old man with Parkinson's disease diagnosed in 2015 was started on amantadine for treatment of progressive tremor and orofacial dyskinesias induced by levodopa. He took amantadine 100mg orally daily for 7 days, then increased to 100mg twice a day thereafter. The patient complained of "worsening tremor" after 9 days and amantadine was decreased to 100mg daily. After 1 month on this dose, the patient reported that his "tremor" persisted and experienced visual hallucinations. His examination demonstrated diffuse myoclonus throughout his extremities and trunk, as well as asterixis when attempting to stand or holding his arms antigravity. Laboratory testing for renal and hepatic failure was unrevealing. Amantadine was reduced to 50mg daily for 4 days and then discontinued. Myoclonus resolved 3 days after discontinuation of amantadine. CONCLUSION: While amantadine-induced myoclonus is rare, health care providers should be vigilant in monitoring for signs and symptoms of myoclonus following amantadine initiation.

New insights on the supramolecular structure of highly porous core-shell drug nanocarriers using solid-state NMR spectroscopy.

Nano-sized metal-organic frameworks (nanoMOFs), with engineered surfaces to enhance the targeting of the drug delivery, have proven efficient as drug nanocarriers. To improve their performances a step further, it is essential to understand at the molecular level the interactions between the nanoMOF interfaces and both the surface covering groups and the drug loaded inside the micropores. Here we show how solid-state NMR spectroscopy allows us to address these issues in an aluminum-based nanoMOF coated and loaded with phosphorus-containing species.

Focal electrical stimulation as an effective sham control for active rTMS and biofeedback treatments.

A valid sham control is important for determining the efficacy and effectiveness of repetitive transcranial magnetic stimulation (rTMS) as an experimental and clinical tool. Given the manner in which rTMS is applied, separately or in combination with self-regulatory approaches, and its intended impact on brain states, a valid sham control of this type may well serve as a meaningful control for biofeedback studies, where efforts to develop a credible control have often been less than ideal. This study examined the effectiveness of focal electrical stimulation of the frontalis muscle as a sham technique for blinding participants to high-frequency rTMS over the dorso-lateral prefrontal cortex (DLPFC) at durations, intensities, and schedules of stimulation similar to many clinical applications. In this within-subjects single blind design, 19 participants made guesses immediately after receiving 54 counterbalanced rTMS sessions (sham, 10 Hz, 20 Hz); 7 (13 %) of the guesses were made for sham, 31 (57 %) were made for 10 Hz, and 16 (30 %) were made for 20 Hz. Participants correctly guessed the sham condition 6 % (CI 1, 32 %) of the time, which is less than the odds of chance (i.e., of guessing at random, 33 %); correctly guessed the 10 Hz condition 66 % (CI 43, 84 %) of the time, which was greater than chance; and correctly guessed the 20 Hz condition 41 % (CI 21, 65 %) of the time, which was no different than chance. Focal electrical stimulation therefore can be an effective sham control for high-frequency rTMS of the DLPFC, as well as for active biofeedback interventions. Participants were unaware that electrical stimulation was, in fact, sham rTMS.

Protection from oxidative and electrophilic stress in the Gsta4-null mouse heart.

4-Hydroxynonenal (4-HNE) mediates many pathological effects of oxidative and electrophilic stress and signals to activate cytoprotective gene expression regulated by NF-E2-related factor 2 (Nrf2). By exhibiting very high levels of 4-HNE-conjugating activity, the murine glutathione transferase alpha 4 (GSTA4-4) helps regulate cellular 4-HNE levels. To examine the role of 4-HNE in vivo, we disrupted the murine Gsta4 gene. Gsta4-null mice exhibited no cardiac phenotype under normal conditions and no difference in cardiac 4-HNE level as compared to wild-type mice. We hypothesized that the Nrf2 pathway might contribute an important compensatory mechanism to remove excess cardiac 4-HNE in Gsta4-null mice. Cardiac nuclear extracts from Gsta4-null mice exhibited significantly higher Nrf2 binding to antioxidant response elements. We also observed responses in critical Nrf2 target gene products: elevated Sod2, Cat, and Akr1b7 mRNA levels and significant increases in both cardiac antioxidant and anti-electrophile enzyme activities. Gsta4-null mice were less sensitive and maintained normal cardiac function following chronic doxorubicin treatment, known to increase cardiac 4-HNE levels. Hence, in the absence of GSTA4-4 to modulate both physiological and pathological 4-HNE levels, the adaptive Nrf2 pathway may be primed to contribute to a preconditioned cardiac phenotype in the Gsta4-null mouse.

Genetic evidence for involvement of adaptive immunity in the development of IgA nephropathy: MHC class II alleles are protective in a Caucasian population.

There is evidence suggesting that IgA nephropathy (IgAN) is an immunological disease. The role of HLA class II DR beta 1 (DRB1) has previously not been well studied. The aim of our study was to investigate the association of HLA-DRB1 variants with IgAN in a Swedish Caucasian cohort. Our study consisted of 213 patients with biopsy proven IgAN, all of self-reported Caucasian ancestry. As a control cohort, 1569 healthy subjects from the same population in Sweden were included. HLA-DRB1 low-resolution genotyping was performed and odds ratios were calculated to assess the risk. In an allelic model the HLA-DRB1(*)03 and (*)10, demonstrated association for IgAN after correction for multiple comparison, with subsequent OR=0.54 (95% CI 0.37-0.78) and 3.44 (95% CI 1.67-7.07). When the influence of risk allelic groups was adjusted for protective allelic groups and vice versa, only a protective effect of HLA-DRB1(*)03 remained significant. In conclusion, the variants of HLA-DRB1 were associated with IgAN of which the HLA-DRB1(*)03 revealed a strong protective effect for IgAN. Our data replicates finding from other Caucasian populations and suggest that involvement of adaptive immunity may be of importance in the development of the disease.

T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements.

Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.

Cost effectiveness of tobacco control policies in Vietnam: the case of population-level interventions.

BACKGROUND: Tobacco smoking is one of the leading public health problems in the world. It is also possible to prevent and/or reduce the harm from tobacco use through the use of cost-effective tobacco control measures. However, most of this evidence comes from developed countries and little research has been conducted on this issue in developing countries. OBJECTIVE: The objective of this study was to analyse the cost effectiveness of four population-level tobacco control interventions in Vietnam. METHODS: Four tobacco control interventions were evaluated: excise tax increase; graphic warning labels on cigarette packs; mass media campaigns; and smoking bans (in public or in work places). A multi-state life table model was constructed in Microsoft® Excel to examine the cost effectiveness of the tobacco control intervention options. A government perspective was adopted, with costing conducted using a bottom-up approach. Health improvement was considered in terms of disability-adjusted life-years (DALYs) averted. All assumptions were subject to sensitivity and uncertainty analysis. RESULTS: All the interventions fell within the definition of being very cost effective according to the threshold level suggested by the WHO (i.e.

Association of soluble CD89 levels with disease progression but not susceptibility in IgA nephropathy.

The Fc-α receptor (FcαR/CD89) is involved in IgA complex formation and may affect the development of IgA nephropathy (IgAN). In this study, we tested the genetic variations of the CD89 gene in relation to disease susceptibility in IgAN and the expression of soluble CD89 (sCD89) in sera of patients with IgAN and in controls. There was a significant difference between the levels of sCD89-IgA complexes, measured by sandwich enzyme-linked immunosorbent assay (ELISA), in 177 patients with IgAN with and without disease progression at the time of first diagnosis. No such difference was found in 42 patients with other renal diseases. The patients with IgAN without disease progression had stable but high levels of sCD89 over 5-15 years of follow-up in contrast to stable but low levels of sCD89 in the disease progression group. Moreover, levels of sCD89 complexes were correlated with one of the five CD89 genetic variants in 212 patients with IgAN and 477 healthy Caucasians; the single-nucleotide polymorphism (SNP) rs11084377 was significantly associated with a lower expression of sCD89. However, no association between CD89 gene polymorphisms and susceptibility to IgAN was detected. Thus, we found an association between the levels of sCD89-IgA complexes in serum and the severity of IgAN, and a possible genetic component in regulating the production or expression of sCD89.

Genetic risk factors in lupus nephritis and IgA nephropathy--no support of an overlap.

BACKGROUND: IgA nephropathy (IgAN) and nephritis in Systemic Lupus Erythematosus (SLE) are two common forms of glomerulonephritis in which genetic findings are of importance for disease development. We have recently reported an association of IgAN with variants of TGFB1. In several autoimmune diseases, particularly in SLE, IRF5, STAT4 genes and TRAF1-C5 locus have been shown to be important candidate genes. The aim of this study was to compare genetic variants from the TGFB1, IRF5, STAT4 genes and TRAF1-C5 locus with susceptibility to IgAN and lupus nephritis in two Swedish cohorts. PATIENTS AND METHODS: We genotyped 13 single nucleotide polymorphisms (SNPs) in four genetic loci in 1252 DNA samples from patients with biopsy proven IgAN or with SLE (with and without nephritis) and healthy age- and sex-matched controls from the same population in Sweden. RESULTS: Genotype and allelic frequencies for SNPs from selected genes did not differ significantly between lupus nephritis patients and SLE patients without nephritis. In addition, haplotype analysis for seven selected SNPs did not reveal a difference for the SLE patient groups with and without nephritis. Moreover, none of these SPNs showed a significant difference between IgAN patients and healthy controls. IRF5 and STAT4 variants remained significantly different between SLE cases and healthy controls. In addition, the data did not show an association of TRAF1-C5 polymorphism with susceptibility to SLE in this Swedish population. CONCLUSION: Our data do not support an overlap in genetic susceptibility between patients with IgAN or SLE and reveal no specific importance of SLE associated SNPs for the presence of lupus nephritis.

Genetic variation in the transforming growth factor-beta1 gene is associated with susceptibility to IgA nephropathy.

BACKGROUND: There is growing evidence of genetic risk for susceptibility to IgA nephropathy. Among several candidate genes related to immunological regulation in renal tissue, TGFB1 is known to be a contributor to proliferation and the development of fibrosis. METHODS: We analysed several SNPs in a region of this gene using 212 DNA samples from biopsy-proven IgA nephropathy patients, 146 men and 66 women and 477 healthy age-matched controls (321 men and 156 women) from the same population in Sweden. RESULTS: Frequencies of four out of five selected SNPs (rs6957, rs2241715, rs1800471, rs1982073 and rs1800469) were found to significantly differ between male patients and male controls in a co-dominant model (corrected P

Transcriptome-based identification of candidate membrane proteins.

A full understanding of leukocyte responses to external stimuli requires knowledge of the full complement of proteins found on their surfaces. Systematic examination of the mammalian cell surfaces at the protein level is hampered by technical difficulties associated with proteomic analysis of so many membrane proteins and the large amounts of starting material required. The use of transcriptomic analyses avoids challenges associated with protein stability and separation and enables the inclusion of an amplification step; thus allowing the use of cell numbers applicable to the study of sub populations of, for example, primary lymphocytes. Here we present a transcriptomic methodology based on Serial Analysis of Gene Expression (SAGE) to recover an essentially complete and quantitative profile of mRNA species in a particular cell. We discuss how, using bioinformatic tools accessible to standard desktop computers, plasma membrane proteins can be identified in silico, from this list. While we describe the use of this approach to characterise the cell surface protein complement of a resting CD8(+) T-cell clone, it is theoretically applicable to any cell surface, where a suitable pure population of cells is available.

Differential remodeling of a T-cell transcriptome following CD8- versus CD3-induced signaling.

CD8 engagement with class I major histocompatibility antigens greatly enhances T-cell activation, but it is not clear how this is achieved. We address the question of whether or not the antibody-mediated ligation of CD8 alone induces transcriptional remodeling in a T-cell clone, using serial analysis of gene expression. Even though it fails to induce overt phenotypic changes, we find that CD8 ligation profoundly alters transcription in the T-cell clone, at a scale comparable to that induced by antibody-mediated ligation of CD3. The character of the resulting changes is distinct, however, with the net effect of CD8 ligation being substantially inhibitory. We speculate that ligating CD8 induces weak, T-cell receptor (TCR)-mediated inhibitory signals reminiscent of the effects of TCR antagonists. Our results imply that CD8 ligation alone is incapable of activating the T-cell clone because it fails to fully induce NFAT-dependent transcription.

Impact and perspective on chronic kidney disease in an Asian developing country: a large-scale survey in North Vietnam.

BACKGROUND: The prevalence of chronic kidney disease (CKD) in Asia is expected to increase along with increases of hypertension and diabetes. Most cases are not treated and progress to end-stage renal disease (ESRD) with an increased risk for cardiovascular complications. Renal replacement therapies are so expensive that most ESRD patients die without treatment. Thus, countermeasures against early stages of CKD are urgently needed. Nevertheless, basic information for CKD has not been reported in Vietnam. METHODS: We conducted a survey of CKD in 8,505 inhabitants aged >40 years in Vietnam. Subjects with abnormal urinary findings were further examined, including serum creatinine levels. In this study, CKD was defined as <60 ml/min/1.73 m(2) of estimated creatinine clearance by the Cockcroft-Gault method. RESULTS: We identified 3.1% of subjects as CKD (stages 3-5) with positive findings in urine test. We also found that elderly hypertension and malnutrition were independent risk factors for CKD in this population. CONCLUSION: We found a significant number of CKD patients in Vietnam. To avoid a CKD pandemic in Asia including Vietnam, we strongly suggest further analyses of risk factors and prognosis of CKD in these populations, and the development of efficient management systems suitable for Asia.

Deep analysis of cellular transcriptomes - LongSAGE versus classic MPSS.

BACKGROUND: Deep transcriptome analysis will underpin a large fraction of post-genomic biology. 'Closed' technologies, such as microarray analysis, only detect the set of transcripts chosen for analysis, whereas 'open' e.g. tag-based technologies are capable of identifying all possible transcripts, including those that were previously uncharacterized. Although new technologies are now emerging, at present the major resources for open-type analysis are the many publicly available SAGE (serial analysis of gene expression) and MPSS (massively parallel signature sequencing) libraries. These technologies have never been compared for their utility in the context of deep transcriptome mining. RESULTS: We used a single LongSAGE library of 503,431 tags and a "classic" MPSS library of 1,744,173 tags, both prepared from the same T cell-derived RNA sample, to compare the ability of each method to probe, at considerable depth, a human cellular transcriptome. We show that even though LongSAGE is more error-prone than MPSS, our LongSAGE library nevertheless generated 6.3-fold more genome-matching (and therefore likely error-free) tags than the MPSS library. An analysis of a set of 8,132 known genes detectable by both methods, and for which there is no ambiguity about tag matching, shows that MPSS detects only half (54%) the number of transcripts identified by SAGE (3,617 versus 1,955). Analysis of two additional MPSS libraries shows that each library samples a different subset of transcripts, and that in combination the three MPSS libraries (4,274,992 tags in total) still only detect 73% of the genes identified in our test set using SAGE. The fraction of transcripts detected by MPSS is likely to be even lower for uncharacterized transcripts, which tend to be more weakly expressed. The source of the loss of complexity in MPSS libraries compared to SAGE is unclear, but its effects become more severe with each sequencing cycle (i.e. as MPSS tag length increases). CONCLUSION: We show that MPSS libraries are significantly less complex than much smaller SAGE libraries, revealing a serious bias in the generation of MPSS data unlikely to have been circumvented by later technological improvements. Our results emphasize the need for the rigorous testing of new expression profiling technologies.