A Tour de Force: The Discovery, Properties, and Function of Piezo Channels.

Mechanical transducers appear throughout cell biology and are used to convert mechanical stress into chemical or electrical signals that allow the cell to respond to environmental changes. In the past six years, a eukaryotic mechanical channel family with two members, Piezo1 and Piezo2, has been identified. Piezo1 was shown to be a cation-selective channel that does not require ancillary proteins for activity. Mouse Piezo1 is large, with over 2500 amino acids, and is not homologous to other ion channels. Both piezo channels have rapid voltage-dependent inactivation with a reversal potential near 0mV. The CryoEm structure of Piezo1 at 4.8Å shows trimer stoichiometry. Since the discovery of the piezo channels, their roles in the physiological response of cells have started to emerge. Significant progress has been made in understanding the intrinsic properties of the channels and how these properties are modulated by cytoskeletal elements. Specific diseases, such as hereditary xerocytosis affecting red blood cells, have mutations in Piezo1 that alter the cell's response to force, typically slowing inactivation and introducing a latency for activation. A number of physiological functions for piezo channels have been identified. These range from sensing the stiffness of surrounding substrate, to the response to light touch, to serotonin release from the gut. This review provides a general overview of the properties and roles of Piezo1 and Piezo2 in eukaryotic mechanotransduction.

The Kinetics and the Permeation Properties of Piezo Channels.

Piezo channels are eukaryotic, cation-selective mechanosensitive channels (MSCs), which show rapid activation and voltage-dependent inactivation. The kinetics of these channels are largely consistent across multiple cell types and different stimulation paradigms with some minor variability. No accessory subunits that associate with Piezo channels have been reported. They are homotrimers and each ∼300kD monomer has an N-terminal propeller blade-like mechanosensing module, which can confer mechanosensing capabilities on ASIC-1 (the trimeric non-MSC, acid-sensing ion channel-1) and a C-terminal pore module, which influences conductance, selectivity, and channel inactivation. Repeated stimulation can cause domain fracture and diffusion of these channels leading to synchronous loss of inactivation. The reconstituted channels spontaneously open only in asymmetric bilayers but lack inactivation. Mutations that cause hereditary xerocytosis alter PIEZO1 kinetics. The kinetics of the wild-type PIEZO1 and alterations thereof in mutants (M2225R, R2456K, and DhPIEZO1) are summarized in the form of a quantitative model and hosted online. The pore is permeable to alkali ions although Li+ permeates poorly. Divalent cations, notably Ca2+, traverse the channel and inhibit the flux of monovalents. The large monovalent organic cations such as tetramethyl ammonium and tetraethyl ammonium can traverse the channel, but slowly, suggesting a pore diameter of ∼8Å, and the estimated in-plane area change upon opening is around 6-20nm2. Ruthenium red can enter the channel only from the extracellular side and seems to bind in a pocket close to residue 2496.

A Microfluidic Approach for Studying Piezo Channels.

Microfluidics is an interdisciplinary field intersecting many areas in engineering. Utilizing a combination of physics, chemistry, biology, and biotechnology, along with practical applications for designing devices that use low volumes of fluids to achieve high-throughput screening, is a major goal in microfluidics. Microfluidic approaches allow the study of cells growth and differentiation using a variety of conditions including control of fluid flow that generates shear stress. Recently, Piezo1 channels were shown to respond to fluid shear stress and are crucial for vascular development. This channel is ideal for studying fluid shear stress applied to cells using microfluidic devices. We have developed an approach that allows us to analyze the role of Piezo channels on any given cell and serves as a high-throughput screen for drug discovery. We show that this approach can provide detailed information about the inhibitors of Piezo channels.

Development of Autoimmune-Mediated ß Cell Failure After Total Pancreatectomy With Autologous Islet Transplantation.

Total pancreatectomy with islet autotransplantation (TPIAT) is performed for definitive treatment of chronic pancreatitis; patients are not diabetic before surgery, or have C-peptide positive pancreatogenous diabetes. Thus, TPIAT recipients are not traditionally considered at risk for autoimmune loss of the islet graft. We describe a 43-year-old female who underwent TPIAT with high mass islet graft of 6031 IEQ/kg, with no evidence of presurgical ß cell autoimmunity who developed type 1 diabetes within the first year after TPIAT, resulting in complete loss of beta cell function. The patient had positive GAD and insulin autoantibodies at 1 year and 18 months after TPIAT, not present prior, and undetectable C-peptide after mixed meal and intravenous glucose tolerance testing at 18 months. Glucagon secretion was preserved, suggesting the transplanted alpha cell mass was intact. HLA typing revealed a DR3/DR4 class II haplotype. This case highlights the need to consider de novo type 1 diabetes in patients with unexpected islet graft failure after TPIAT.

Teplizumab treatment may improve C-peptide responses in participants with type 1 diabetes after the new-onset period: a randomised controlled trial.

AIMS/HYPOTHESIS: Type 1 diabetes results from a chronic autoimmune process continuing for years after presentation. We tested whether treatment with teplizumab (a Fc receptor non-binding anti-CD3 monoclonal antibody), after the new-onset period, affects the decline in C-peptide production in individuals with type 1 diabetes. METHODS: In a randomised placebo-controlled trial we treated 58 participants with type 1 diabetes for 4-12 months with teplizumab or placebo at four academic centres in the USA. A central randomisation centre used computer generated tables to allocate treatments. Investigators, patients, and caregivers were blinded to group assignment. The primary outcome was a comparison of C-peptide responses to a mixed meal after 1 year. We explored modification of treatment effects in subgroups of patients. RESULTS: Thirty-four and 29 subjects were randomized to the drug and placebo treated groups, respectively. Thirty-one and 27, respectively, were analysed. Although the primary outcome analysis showed a 21.7% higher C-peptide response in the teplizumab-treated group (0.45 vs 0.371; difference, 0.059 [95% CI 0.006, 0.115] nmol/l) (p = 0.03), when corrected for baseline imbalances in HbA(1c) levels, the C-peptide levels in the teplizumab-treated group were 17.7% higher (0.44 vs 0.378; difference, 0.049 [95% CI 0, 0.108] nmol/l, p = 0.09). A greater proportion of placebo-treated participants lost detectable C-peptide responses at 12 months (p = 0.03). The teplizumab group required less exogenous insulin (p < 0.001) but treatment differences in HbA(1c) levels were not observed. Teplizumab was well tolerated. A subgroup analysis showed that treatment benefits were larger in younger individuals and those with HbA(1c) <6.5% at entry. Clinical responders to teplizumab had an increase in circulating CD8 central memory cells 2 months after enrolment compared with non-responders. CONCLUSIONS/INTERPRETATIONS: This study suggests that deterioration in insulin secretion may be affected by immune therapy with teplizumab after the new-onset period but the magnitude of the effect is less than during the new-onset period. Our studies identify characteristics of patients most likely to respond to this immune therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00378508 FUNDING: This work was supported by grants 2007-502, 2007-1059 and 2006-351 from the JDRF and grants R01 DK057846, P30 DK20495, UL1 RR024139, UL1RR025780, UL1 RR024131 and UL1 RR024134 from the NIH.

Immunology of Diabetes Society T-Cell Workshop: HLA class I tetramer-directed epitope validation initiative T-Cell Workshop Report-HLA Class I Tetramer Validation Initiative.

BACKGROUND: Identification of T-cell reactivity to ß-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). METHODS: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545). We also compared the readout of TMrs and PMrs with a CD8+ T-cell interferon-γ enzyme-linked immunospot assay. RESULTS: Despite the minute frequencies of autoreactive cells detected by TMrs/PMrs, most (73-77%) T1D patients had responses to one or more of the epitopes used. All four epitopes were recognized by T1D patients, with a prevalence ranging from 5 to 25%. TMrs and PMrs detected more positive responses to the ß-cell epitopes than CD8+ T-cell interferon-γ enzyme-linked immunospot. However, concordance between positive responses to TMrs and PMrs was limited. CONCLUSIONS: Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.

Immunology of Diabetes Society T-Cell Workshop: HLA class II tetramer-directed epitope validation initiative.

BACKGROUND: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study. METHODS: A tetramer-based assay was used in five participating centres to measure T-cell reactivities to DR0401-restricted epitopes. Three participating centres concurrently performed ELISPOT or immunoblot assays. Each centre used blind-coded, centrally distributed peptide and tetramer reagents. RESULTS: All participating centres detected responses to auto-antigens and the positive control antigen, and in some cases cloned the corresponding T cells. However, response rates varied among centres. In total, 74% of patients were positive for at least one islet epitope. The most commonly recognized epitope was GAD270-285. Only a minority of the patients tested by tetramer and ELISPOT were concordant for both assays. CONCLUSIONS: This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course.

Comparison of cryopreservation methods on T-cell responses to islet and control antigens from type 1 diabetic patients and controls.

BACKGROUND: Type 1 diabetes (T1D) is a cell-mediated autoimmune disease characterized by destruction of the pancreatic islet cells. The use of cryopreserved cells is preferable to the use of freshly isolated cells to monitor clinical trials to decrease assay and laboratory variability. METHODS: The T-Cell Workshop Committee of the Immunology of Diabetes Society compared two widely accepted T-cell freezing protocols (warm and cold) to freshly isolated peripheral blood mononuclear cells from patients with T1D and controls in terms of recovery, viability, cell subset composition, and performance in functional assays currently in use in T1D-related research. Nine laboratories participated in the study with four different functional assays included. RESULTS: The cold freezing method yielded higher recovery and viability compared with the warm freezing method. Irrespective of freezing protocol, B cells and CD8+ T cells were enriched, monocyte fraction decreased, and islet antigen-reactive responses were lower in frozen versus fresh cells. However, these results need to take in to account that the overall response to islet autoantigens was low in some assays. CONCLUSIONS: In the current study, none of the tested T-cell functional assays performed well using frozen samples. More research is required to identify a freezing method and a T-cell functional assay that will produce responses in patients with T1D comparable to responses using fresh peripheral blood mononuclear cells.

Genetic correlation of a mouse light chain variable region marker with a thymocyte surface antigen.

The inbred and congenic strain distribution of the I(H)-peptide marker in the variable region of mouse immunoglobulin light chains has been compared with other known genetic markers. A positive correlation was noted between the I(H)-peptide marker and expression of the Ly-3.1 thymocyte cell surface antigen. This suggests that the locus responsible for I(H)-peptide expression is genetically linked to the Ly-2 and Ly-3 loci in linkage group XI on chromsome 6 of the mouse.

The Ly-3 antigens on mouse thymocytes: immune precipitation and molecular weight characterization.

Specific anti-Ly sera were employed to precipitate Ly antigens from Nonidet P-40 extracts of mouse thymocytes labeled with 125I using lactoperoxidase and with NaB3H4 using galactose oxidase. Thymocytes from mice of the congenic strains C57BL/6J (Ly-2.2, Ly-3.2 positive), C57BL/6Ly-2a, Ly-3a (Ly-2.1, Ly-3.1 positive) and C57BL/6-Ly-2a (Ly-2.1, Ly-3.2-positive) were used as sources of labeled antigens and as immune adsorbants to permit evaluation of the specificity of each anti-Ly serum employed. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions are consistent with the Ly-3.1 antigen containing a glycoprotein subunit with an apparent mol wt of 35,000 daltons. Specific precipitates obtained using anti-Ly-2.1 serum yielded SDS-PAGE profiles identical to that obtained with anti-Ly-3.1 serum, suggesting that the Ly-2 and Ly-3 antigens have the same molecular weight distribution. The relationships of these results to the observed close genetic and topological linkage of Ly-2 and Ly-3 and to the genetic linkage of these loci with the IB-peptide marker, a mouse Bk-region polymorphism, are discussed.

Expression of an idiotype (Id-460) during in vivo anti-dinitrophenyl antibody responses. I. Mapping of genes for Id-460 expression to the variable region of immunoglobulin heavy-chain locus and to the variable region of immunoglobulin kappa-light-chain locus.

The genetic contro of the expression of an idiotype (Id-460) associated with the 2,4-dinitrophenyl (DNP)-binding BALB/c myeloma protein MOPC 460 was studied using congenic strains of mice. It was shown that the expression of high levels of Id-460 during secondary in vivo anti-DNP-ovalbumin responses was determined by genes governing immunoglobulin heavy-chain variable and kappa-light chain variable regions (V kappa). Appropriate alleles at both loci were required for the expression of Id-460. Genes in the major histocompatability complex and the X-linked immune deficiency gene found in strain CBA/N did not greatly affect Id-460 expression. The V kappa gene controlling Id-460 expression can be differentiated from Lyt-3, and it is the first instance in which expression of an idiotype subdivides the V kappa genes associated with the Lyt-3a allele. Although it is likely that the V kappa gene(s) involved are structural, the involvememt of a regulatory gene linked to the structural gene can not be excluded.

Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis.

Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full-length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain-specific expression of V kappa Ser-associated phenotypic markers are discussed.

Nonexistence of large mascons at mare marginis and mare orientale.

The analysis of line-of-sight residual accelerations from Lunar Orbiters 3 and 5 does not show any evidence for large mascons near the lunar limbs. Although unfavorable geometry reduces the acceleration effect due to any mascon near the limb, simulations show that large masses at Mare Orientale and Mare Marginis would produce substantial accelerations, in complete disagreement with the actual Doppler tracking data obtained from a Lunar Orbiter experiment.

Lunar Gravity via Apollo 14 Doppler Radio Tracking.

Gravity measurements at high resolution were obtained over a 100-kilometer band from + 70 degrees to -70 degrees of longitude during the orbits of low periapsis altitude (approximately 16 kilometers). The line-of-sight accelerations are plotted on Aeronautical Chart and Information Center mercator charts (scale 1 : 1,000,000) as contours at 10-milligal intervals. Direct correlations between gravity variations and surface features are easily determined. Theophilus, Hipparchus, and Ptolemaeus are negative features, whereas Mare Nectaris is a large positive region. The acceleration profiles over Mare Nectaris are suggestive of a broad disk near the surface rather than a deeply buried spherical body. These data are in good agreement with the short arc of Apollo 12 lunar module descent data.

Lunar Gravity over Large Craters from Apollo 12 Tracking Data.

The Doppler residuals from the Apollo 12 lunar module radio tracking data indicate large negative accelerations over the craters Ptolemaeus and Albategnius. The mass deficienicies required to produce these accelerations are approximately equivalent to the removal of the surface material to a depth of 1 kilometer over the entire area of these craters. Several other features of the gravity fine structure can also be correlated with topography.

A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin.

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.

Identification of a rel-related protein in the nucleus during the S phase of the cell cycle.

The c-rel proto-oncogene encodes a 75-kDa protein (p75c-rel) which is present in the cytosol of chick embryo fibroblasts (CEF) associated with a distinct set of cellular proteins with molecular masses of 40, 115, and 124 kDa. CEF cultures arrested in S phase of the cell cycle, or enriched for G2 or mitotic cells, were examined to determine whether the expression of c-rel was altered during the cell cycle. Levels of p75c-rel remained constant in all portions of the cell cycle examined; however, a Rel-related protein with an apparent molecular mass of 64 kDa was detected in nuclei of S-phase cells. As cells enter G2, the level of this protein in the nucleus decreases. This protein reacts with antiserum generated against the carboxy terminus of p75c-rel in radioimmunoprecipitations and Western immunoblot experiments and was also detected in a Western immunoblot with antiserum generated against the first 161 amino acids of pp59v-rel. Thus, unlike other Rel/NF-kappa B family members, p64 has carboxy-terminal homology with c-Rel. The majority of peptides generated by partial proteolytic cleavage of p64 are shared with peptides generated by digestion of p75c-rel and/or pp59v-rel. We suggest that this protein represents a new member of the Rel family of transcription factors and is located in the nucleus of avian fibroblasts during S phase of the cell cycle.

Initial results of screening of nondiabetic organ donors for expression of islet autoantibodies.

CONTEXT: Type 1A diabetes is characterized by a long prodromal phase during which autoantibodies to islet antigens are present. Nevertheless, we lack data on the pancreatic pathology of subjects who are positive for islet autoantibodies (to islet autoantigens GAD65, insulin, and ICA512). OBJECTIVE: In this manuscript, we describe a novel strategy in obtaining pancreata and pancreatic lymph nodes from islet autoantibody-positive organ donors that involves careful coordination among the laboratory and the organ donor provider organization. DESIGN: We developed a rapid screening protocol for islet autoantibodies measurement of organ donors to allow identification of positive subjects before organ harvesting. In this way we were able to obtain pancreata and pancreatic lymph nodes from subjects with and without islet autoimmunity. SETTING: The organ donors used in this study were obtained from the general community. SUBJECTS: The population studied consisted of 112 organ donors (age range 1 month to 86 yr, mean age 39 yr). MAIN OUTCOME MEASURE: The main outcome measure of this study consisted of evaluating the pancreatic histology and identify T cells autoreactive for islet antigens in the pancreatic lymph nodes. RESULTS: To date we have identified three positive subjects and obtained the pancreas for histological evaluation from one of the autoantibody-positive donors who expressed ICA512 autoantibodies. Although this subject did not exhibit insulitis, lymphocytes derived from pancreatic lymph nodes reacted to the islet antigen phogrin. CONCLUSION: In summary, these results indicate that it is possible to screen organ donors in real time for antiislet antibodies, characterize pancreatic histology, and obtain viable T cells for immunological studies.

Structure in nascent RNA leads to termination of slippage transcription by T7 RNA polymerase.

T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago it was observed that in the presence of only GTP as a substrate, on a template encoding the initial sequence GGGA., T7 RNA polymerase will synthesize a 'ladder' of poly-G RNA products. At each step, the ratio of elongation to product release is consistently approximately 0.75 until the RNA reaches a length of approximately 13-14 nt, at which point this ratio drops precipitously. One model to explain this drop in complex stability suggests that the nascent RNA may be structurally hindered by the protein; the RNA may be exiting via a pathway not taken by normally synthesized RNA and therefore becomes sterically destabilized. The fact that the length of RNA at which this occurs is close to the length at which the transition to a stably elongating complex occurs might have led to other mechanistic proposals. Here we show instead that elongation falls off due to the cooperative formation of structure in the nascent RNA, most likely an intramolecular G-quartet structure. Replacement of GTP by 7-deaza-GTP completely abolishes this transition and G-ladder synthesis continues with a constant efficiency of elongation beyond the limit of detection. The polymerase-DNA complex creates no barrier to the growth of the nascent (slippage) RNA, rather termination is similar to that which occurs in rho-independent termination.

Inhibition by tuftsin of Rauscher virus leukemia development in mice.

The antitumor effect of tuftsin, the natural phagocytosis-stimulating peptide, on leukemia induced by Rauscher murine leukemia virus (R-MuLV) was studied in vivo in SWR inbred mice. Tuftsin was found capable of significantly increasing the survival of R-MuLV-infected mice. The peptide, when injected both ip and iv into mice, exerted its activity in a dose- and time-dependent manner. Optimal antitumor activity was achieved upon administration of 25 micrograms tuftsin 4 days before R-MuLV inoculation.