Electron microscopy of frozen biological suspensions

The methodology for preparing specimens in the frozen, hydrated state has been assessed using crystals and T4 bacteriophages. The methods have also been demonstrated with lambda bacteriophages, purple membrane of Halobacterium halobium and fibres of DNA. For particles dispersed in an aqueous environment, it is shown that optimum structural preservation is obtained from a thin, quench-frozen film with the bulk aqueous medium in the vitreous state. Crystallization of the bulk water may result in solute segregation and expulsion of the specimen from the film. Contrast measurements can be used to follow directly the state of hydration of a specimen during transition from the fully hydrated to the freeze-dried state and permit direct measurement of the water content of the specimen. By changing the concentration and composition of the aqueous medium the contrast of particles in a vitreous film can be controlled and any state of negative, positive or zero contrast may be obtained. At 100 K, frozen-hydrated, freeze-dried or sugar embedded crystals can withstand a three- to four-fold increase in electron exposure for the same damage when compared with similar sugar-embedded or freeze-dried samples at room temperature

Hall, Seebeck, and Nernst Coefficients of Underdoped HgBa2CuO4+d: Fermi-Surface Reconstruction in an Archetypal Cuprate Superconductor

Charge density-wave order has been observed in cuprate superconductors whose crystal structure breaks the square symmetry of the CuO2 planes, such as orthorhombic YBa2Cu3Oy (YBCO), but not so far in cuprates that preserve that symmetry, such as tetragonal HgBa2CuO4+d (Hg1201). We have measured the Hall (R_H), Seebeck (S), and Nernst coefficients of underdoped Hg1201 in magnetic fields large enough to suppress superconductivity. The high-field R_H(T) and S(T) are found to drop with decreasing temperature and become negative, as also observed in YBCO at comparable doping. In YBCO, the negative R_H and S are signatures of a small electron pocket caused by Fermi-surface reconstruction, attributed to charge density-wave modulations observed in the same range of doping and temperature. We deduce that a similar Fermi-surface reconstruction takes place in Hg1201, evidence that density-wave order exists in this material. A striking similarity is also found in the normal-state Nernst coefficient, further supporting this interpretation. Given the model nature of Hg1201, Fermi-surface reconstruction appears to be common to all hole-doped cuprates, suggesting that density-wave order is a fundamental property of these materials

Evidence for a small hole pocket in the Fermi surface of underdoped YBa2Cu3Oy

The Fermi surface of a metal is the fundamental basis from which its properties can be understood. In underdoped cuprate superconductors, the Fermi surface undergoes a reconstruction that produces a small electron pocket, but whether there is another, as yet undetected portion to the Fermi surface is unknown. Establishing the complete topology of the Fermi surface is key to identifying the mechanism responsible for its reconstruction. Here we report the discovery of a second Fermi pocket in underdoped YBa2Cu3Oy, detected as a small quantum oscillation frequency in the thermoelectric response and in the c-axis resistance. The field-angle dependence of the frequency demonstrates that it is a distinct Fermi surface and the normal-state thermopower requires it to be a hole pocket. A Fermi surface consisting of one electron pocket and two hole pockets with the measured areas and masses is consistent with a Fermi-surface reconstruction caused by the charge-density-wave order observed in YBa2Cu3Oy, provided other parts of the reconstructed Fermi surface are removed by a separate mechanism, possibly the pseudogap.Comment: 23 pages, 5 figure

Fermi Surface of the Electron-doped Cuprate Superconductor Nd_{2-x}Ce_xCuO_{4} Probed by High-Field Magnetotransport

We report on the study of the Fermi surface of the electron-doped cuprate superconductor Nd$_{2-x}$Ce$_x$CuO$_{4}$ by measuring the interlayer magnetoresistance as a function of the strength and orientation of the applied magnetic field. We performed experiments in both steady and pulsed magnetic fields on high-quality single crystals with Ce concentrations of $x=0.13$ to 0.17. In the overdoped regime of $x > 0.15$ we found both semiclassical angle-dependent magnetoresistance oscillations (AMRO) and Shubnikov-de Haas (SdH) oscillations. The combined AMRO and SdH data clearly show that the appearance of fast SdH oscillations in strongly overdoped samples is caused by magnetic breakdown. This observation provides clear evidence for a reconstructed multiply-connected Fermi surface up to the very end of the overdoped regime at $x\simeq 0.17$. The strength of the superlattice potential responsible for the reconstructed Fermi surface is found to decrease with increasing doping level and likely vanishes at the same carrier concentration as superconductivity, suggesting a close relation between translational symmetry breaking and superconducting pairing. A detailed analysis of the high-resolution SdH data allowed us to determine the effective cyclotron mass and Dingle temperature, as well as to estimate the magnetic breakdown field in the overdoped regime.Comment: 23 pages, 8 figure

Impact of Dietary Gluten on Regulatory T Cells and Th17 Cells in BALB/c Mice

Dietary gluten influences the development of type 1 diabetes (T1D) and a gluten-free (GF) diet has a protective effect on the development of T1D. Gluten may influence T1D due to its direct effect on intestinal immunity; however, these mechanisms have not been adequately studied. We studied the effect of a GF diet compared to a gluten-containing standard (STD) diet on selected T cell subsets, associated with regulatory functions as well as proinflammatory Th17 cells, in BALB/c mice. Furthermore, we assessed diet-induced changes in the expression of various T cell markers, and determined if changes were confined to intestinal or non-intestinal lymphoid compartments. The gluten-containing STD diet led to a significantly decreased proportion of γδ T cells in all lymphoid compartments studied, although an increase was detected in some γδ T cell subsets (CD8+, CD103+). Further, it decreased the proportion of CD4+CD62L+ T cells in Peyer's patches. Interestingly, no diet-induced changes were found among CD4+Foxp3+ T cells or CD3+CD49b+cells (NKT cells) and CD3−CD49b+ (NK) cells. Mice fed the STD diet showed increased proportions of CD4+CD45RBhigh+ and CD103+ T cells and a lower proportion of CD4+CD45RBlow+ T cells in both mucosal and non-mucosal compartments. The Th17 cell population, associated with the development of autoimmunity, was substantially increased in pancreatic lymph nodes of mice fed the STD diet. Collectively, our data indicate that dietary gluten influences multiple regulatory T cell subsets as well as Th17 cells in mucosal lymphoid tissue while fewer differences were observed in non-mucosal lymphoid compartments

Apoptosis of Purified CD4+ T Cell Subsets Is Dominated by Cytokine Deprivation and Absence of Other Cells in New Onset Diabetic NOD Mice

BACKGROUND: Regulatory T cells (Treg) play a significant role in immune homeostasis and self-tolerance. Excessive sensitivity of isolated Treg to apoptosis has been demonstrated in NOD mice and humans suffering of type 1 diabetes, suggesting a possible role in the immune dysfunction that underlies autoimmune insulitis. In this study the sensitivity to apoptosis was measured in T cells from new onset diabetic NOD females, comparing purified subsets to mixed cultures. PRINCIPAL FINDINGS: Apoptotic cells are short lived in vivo and death occurs primarily during isolation, manipulation and culture. Excessive susceptibility of CD25(+) T cells to spontaneous apoptosis is characteristic of isolated subsets, however disappears when death is measured in mixed splenocyte cultures. In variance, CD25(-) T cells display balanced sensitivity to apoptosis under both conditions. The isolation procedure removes soluble factors, IL-2 playing a significant role in sustaining Treg viability. In addition, pro- and anti-apoptotic signals are transduced by cell-to-cell interactions: CD3 and CD28 protect CD25(+) T cells from apoptosis, and in parallel sensitize naïve effector cells to apoptosis. Treg viability is modulated both by other T cells and other subsets within mixed splenocyte cultures. Variations in sensitivity to apoptosis are often hindered by fast proliferation of viable cells, therefore cycling rates are mandatory to adequate interpretation of cell death assays. CONCLUSIONS: The sensitivity of purified Treg to apoptosis is dominated by cytokine deprivation and absence of cell-to-cell interactions, and deviate significantly from measurements in mixed populations. Balanced sensitivity of naïve/effector and regulatory T cells to apoptosis in NOD mice argues against the concept that differential susceptibility affects disease evolution and progression

Key Role of the GITR/GITRLigand Pathway in the Development of Murine Autoimmune Diabetes: A Potential Therapeutic Target

BACKGROUND: The cross-talk between pathogenic T lymphocytes and regulatory T cells (Tregs) plays a major role in the progression of autoimmune diseases. Our objective is to identify molecules and/or pathways involved in this interaction and representing potential targets for innovative therapies. Glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand are key players in the T effector/Treg interaction. GITR is expressed at low levels on resting T cells and is significantly up-regulated upon activation. Constitutive high expression of GITR is detected only on Tregs. GITR interacts with its ligand mainly expressed on antigen presenting cells and endothelial cells. It has been suggested that GITR triggering activates effector T lymphocytes while inhibiting Tregs thus contributing to the amplification of immune responses. In this study, we examined the role of GITR/GITRLigand interaction in the progression of autoimmune diabetes. METHODS AND FINDINGS: Treatment of 10-day-old non-obese diabetic (NOD) mice, which spontaneously develop diabetes, with an agonistic GITR-specific antibody induced a significant acceleration of disease onset (80% at 12 weeks of age). This activity was not due to a decline in the numbers or functional capacity of CD4(+)CD25(+)Foxp3(+) Tregs but rather to a major activation of 'diabetogenic' T cells. This conclusion was supported by results showing that anti-GITR antibody exacerbates diabetes also in CD28(-/-) NOD mice, which lack Tregs. In addition, treatment of NOD mice, infused with the diabetogenic CD4(+)BDC2.5 T cell clone, with GITR-specific antibody substantially increased their migration, proliferation and activation within the pancreatic islets and draining lymph nodes. As a mirror image, blockade of the GITR/GITRLigand pathway using a neutralizing GITRLigand-specific antibody significantly protected from diabetes even at late stages of disease progression. Experiments using the BDC2.5 T cell transfer model suggested that the GITRLigand antibody acted by limiting the homing and proliferation of pathogenic T cells in pancreatic lymph nodes. CONCLUSION: GITR triggering plays an important costimulatory role on diabetogenic T cells contributing to the development of autoimmune responses. Therefore, blockade of the GITR/GITRLigand pathway appears as a novel promising clinically oriented strategy as GITRLigand-specific antibody applied at an advanced stage of disease progression can prevent overt diabetes

High Distribution of CD40 and TRAF2 in Th40 T Cell Rafts Leads to Preferential Survival of this Auto-Aggressive Population in Autoimmunity

CD40-CD154 interactions have proven critical in autoimmunity, with the identification of CD4(lo)CD40(+) T cells (Th40 cells) as harboring an autoaggressive T cell population shedding new insights into those disease processes. Th40 cells are present at contained levels in non-autoimmune individuals but are significantly expanded in autoimmunity. Th40 cells are necessary and sufficient in transferring type 1 diabetes in mouse models. However, little is known about CD40 signaling in T cells and whether there are differences in that signaling and subsequent outcome depending on disease conditions. When CD40 is engaged, CD40 and TNF-receptor associated factors, TRAFs, become associated with lipid raft microdomains. Dysregulation of T cell homeostasis is emerging as a major contributor to autoimmune disease and thwarted apoptosis is key in breaking homeostasis.Cells were sorted into CD4(hi) and CD4(lo) (Th40 cells) then treated and assayed either as whole or fractionated cell lysates. Protein expression was assayed by western blot and Nf-kappaB DNA-binding activity by electrophoretic mobility shifts. We demonstrate here that autoimmune NOD Th40 cells have drastically exaggerated expression of CD40 on a per-cell-basis compared to non-autoimmune BALB/c. Immediately ex-vivo, untreated Th40 cells from NOD mice have high levels of CD40 and TRAF2 associated with the raft microdomain while Th40 cells from NOR and BALB/c mice do not. CD40 engagement of Th40 cells induces Nf-kappaB DNA-binding activity and anti-apoptotic Bcl-X(L) expression in all three mouse strains. However, only in NOD Th40 cells is anti-apoptotic cFLIP(p43) induced which leads to preferential survival and proliferation. Importantly, CD40 engagement rescues NOD Th40 cells from Fas-induced death.CD40 may act as a switch between life and death promoting signals and NOD Th40 cells are poised for survival via this switch. This may explain how they expand in autoimmunity to thwart T cell homeostasis