A TCR affinity threshold regulates memory CD4 T cell differentiation following vaccination.

Diverse Ag-specific memory TCR repertoires are essential for protection against pathogens. Subunit vaccines that combine peptide or protein Ags with TLR agonists are very potent at inducing T cell immune responses, but their capacity to elicit stable and diverse memory CD4 T cell repertoires has not been evaluated. In this study, we examined the evolution of a complex Ag-specific population during the transition from primary effectors to memory T cells after peptide or protein vaccination. Both vaccination regimens induced equally diverse effector CD4 TCR repertoires, but peptide vaccines skewed the memory CD4 TCR repertoire toward high-affinity clonotypes whereas protein vaccines maintained low-affinity clonotypes in the memory compartment. CD27-mediated signaling was essential for the maintenance of low-affinity clonotypes after protein vaccination but was not sufficient to promote their survival following peptide vaccination. The rapid culling of the TCR repertoire in peptide-immunized mice coincided with a prolonged proliferation phase during which low-affinity clonotypes disappeared despite exhibiting no sign of enhanced apoptosis. Our study reveals a novel affinity threshold for memory CD4 T cell differentiation following vaccination and suggests a role for nonapoptotic cell death in the regulation of CD4 T cell clonal selection.

Cutting edge: MHC class II-restricted peptides containing the inflammation-associated marker 3-nitrotyrosine evade central tolerance and elicit a robust cell-mediated immune response.

Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-E(K)-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC(88-103)) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC(88-103) with nitrotyrosine abrogates recognition by the MCC(88-103)-specific T cell hybridoma 2B4. CBA (H2(K)) mice immunized with MCC(88-103) or nitrated MCC(88-103) peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC(88-103), but exhibited a robust immune response against nitrated MCC(88-103). Analysis of T cell hybridomas specific for nitrated-MCC(88-103) indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.

[Lipid peroxidation and enzymes of the antioxidant defense system in patients with bronchial asthma]. / Perekisnoe okislenie lipidov i fermenty antioksidantnoi sistemy u bol'nykh bronkhial'noi astmoi.

In 71 patient with bronchial asthma (BA), lipid peroxidation (LPO) was studied together with the condition of the antioxidant system (AOS) as related to the degree of severity of the illness, with results evaluated of conventional therapy versus complex treatment involving the use of cytochrome C. Established in the above series was intensification of LPO in the presence of inhibition of activity of SOD enzymes and catalase, their degree dependent on severity of the condition. Unlike basic therapy, employment of cytochrome C has been found to be associated with a more complete restoration of balance in the LPO/AOS system, especially so in steroid-independent form of BA.

A method for functional evaluation of caspase activation pathways in intact lymphoid cells using electroporation-mediated protein delivery and flow cytometric analysis.

The purpose of the study was to develop a rapid technique for determining the functional status of caspase activation pathways in intact lymphocytes. Proteins known to activate caspase-family cell death proteases (cytochrome c; granzyme-B; caspase-8) were introduced into human leukemia and lymphoma cell lines, as well as freshly isolated lymphocytes and leukemia cells, by electroporation. Fluorochrome-labeled proteins with a wide range of molecular weights (from 15 to 150 kDa) were used to evaluate electroporation efficiency by flow cytometry and to compare the efficiency of protein delivery using various electroporation conditions. Caspase activity was monitored using a cleavable, cell-permeable fluorogenic substrate. Conditions were identified for efficient delivery of proteins of +150 kDa into lymphoid cells. Caspase activation induced by various proteins was compared in normal and leukemic lymphocytic cells, revealing impaired caspase activation pathways in some malignant cells. We conclude that electroporation of apoptotic proteins into intact lymphoid cells can be used to contrast the status of various caspase activation pathways, thereby providing insights into the pathological defects in apoptosis regulation that exist in individual patient specimens.

Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

Ligation of retinoic acid receptor alpha regulates negative selection of thymocytes by inhibiting both DNA binding of nur77 and synthesis of bim.

Negative selection refers to the selective deletion of autoreactive thymocytes. Its molecular mechanisms have not been well defined. Previous studies in our laboratory have demonstrated that retinoic acids, physiological ligands for the nuclear retinoid receptors, selectively inhibit TCR-mediated death under in vitro conditions, and the inhibition is mediated via the retinoic acid receptor (RAR) alpha. The present studies were undertaken to investigate whether ligation of RARalpha leads to inhibition of TCR-mediated death in vivo and to identify the molecular mechanisms involved. Three models of TCR-mediated death were studied: anti-CD3-mediated death of thymocytes in wild-type mice, and Ag- and bacterial superantigen-driven thymocyte death in TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c in the context of the E(k) (class II MHC) molecule. Our data demonstrate that the molecular program of both anti-CD3- and Ag-driven, but not that of superantigen-mediated apoptosis involves up-regulation of nur77, an orphan nuclear receptor, and bim, a BH3-only member of the proapoptotic bcl-2 protein family, proteins previously implicated to participate in the negative selection. Ligation of RARalpha by the synthetic agonist CD336 inhibited apoptosis, DNA binding of nur77, and synthesis of bim induced by anti-CD3 or the specific Ag, but had no effect on the superantigen-driven cell death. Our data imply that retinoids are able to inhibit negative selection in vivo as well, and they interfere with multiple steps of the T cell selection signal pathway.

Ferricytochrome c encapsulated in silica hydrogels: correlation between active site dynamics and solvent structure.

Ferricytochrome c encapsulated in silica hydrogels has been prepared by the sol-gel technique following, with some modifications, the procedure originally developed by Zink et al. A suitable preparation of hydrogels enables to have both 'wet' and 'dry' samples. Wet samples have a high water content: as the temperature is lowered below approximately 260 K water freezes and the samples crack. On the contrary, dry samples have a low water content (hydration h approximately 0.35): in these conditions water does not freeze even at cryogenic temperatures and the samples remain transparent and non-cracking. The dynamics of ferricytochrome c and its dependence on the surrounding medium have been studied by optical absorption spectroscopy in the temperature range 10-300 K. At each temperature, spectra were collected both in the Soret region and in the near infrared at approximately 1.45 microm (the water overtone band); this enables to probe the local dynamics of the protein active site as well as the 'structure' of water molecules present in the sample. The data show that sol-gel encapsulation 'per se' does not alter the protein active site dynamics, but rather introduces an increased local heterogeneity. At difference, we find a correlation between active site dynamics and water structure: in the wet hydrogel, freezing of water quenches the ensemble of soft modes linearly coupled to the Soret transition; while, in the dry hydrogel, water does not freeze, and an active site dynamic behavior-similar to the non-freezing water/glycerol solution-is observed.

Human CD4 expression at the late single-positive stage of thymic development supports T cell maturation and peripheral export in CD4-deficient mice.

Positive selection of developing thymocytes is initiated at the double-positive (DP) CD4(+)CD8(+) stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient mice. However, it is unclear whether later onset CD4 expression would still allow such a restoration. To investigate this issue, we used transgenic mice in which a hCD4 transgene is not expressed on DP, but only on single-positive cells. By crossing these animals with CD4-deficient mice, we show that late hCD4 expression supports the maturation of T cell precursors and the peripheral export of mature TCRalphabeta(+) CD8(-) T cells. These results were confirmed in two different MHC class II-restricted TCR transgenic mice. T cells arising by this process were functional in the periphery because they responded to agonist peptide in vivo. Interestingly, thymocytes of these mice appeared refractory to peptide-induced negative selection. Together, these results indicate that the effect of CD4 on positive selection of class II-restricted T cells extends surprisingly late into the maturation process by a previously unrecognized pathway of differentiation, which might contribute to the generation of autoreactive T cells.

Evidence that nerve growth factor mediates the formation of sensory pericellular baskets in the rat trigeminal ganglion.

A role for nerve growth factor (NGF) in the remodeling of sensory neurons in the trigeminal ganglion was examined. Intracerebroventricular NGF infusion and/or bilateral removal of the sympathetic superior cervical ganglia, both of which are believed to increase the availability of NGF to primary sensory neurons, resulted in a significant increase in the frequency of calcitonin gene-related peptide immunoreactive pericellular baskets. The results of this study suggest that increased NGF is sufficient to enhance the formation of sensory baskets in this ganglion, and provide evidence that NGF may mediate the formation of sensory baskets in the sensory ganglia following injury.

Smac is required for cytochrome c-induced apoptosis in prostate cancer LNCaP cells.

Release of cytochrome c from mitochondria to cytosol has been identified as one of the central events of apoptosis. Direct injection of cytochrome c induces apoptosis in some but not in all cell types. We observed that LNCaP prostate cancer cells failed to undergo apoptosis induced by cytochrome c microinjections. Microinjection of cytochrome c with another mitochondrial protein, Smac, was sufficient to activate caspases, however. Smac is believed to function as a neutralizer of caspase inhibitors, and mass spectrometry analysis identified XIAP as a predominant Smac binding protein in LNCaP cells. These findings are consistent with a requirement for a release of Smac from mitochondria to enable caspase activation in prostate cells. Indeed, translocation of Smac from mitochondria to cytosol was observed in LNCaP cells that undergo apoptosis and was inhibited by epidermal growth factor, which is a survival factor for these cells. These results further emphasize the central role of mitochondria in the regulation of apoptosis in prostate cancer cells.

Resting membrane potential as a marker of apoptosis: studies on Xenopus oocytes microinjected with cytochrome c.

Observation of the electrical potential difference across the cell membrane is described as a new method for monitoring apoptosis of a single cell. The resting membrane potential (DeltaPsi) of Xenopus oocytes has been recorded in real time following microinjection of cytochrome c. Soon after microinjection, DeltaPsi becomes less negative and attains a new constant value with a half time, t(m), of about 35 (+ /- 5) min at all cytochrome c concentrations greater than 1 microM. The cytosol extract of cytochrome c-injected oocytes shows DEVD proteolytic activity characteristic of aspartate specific proteases, implicating an apoptotic death pathway. In response to the delivery of cytochrome c into the cytosol, caspases are activated within 7 min while the changes in DeltaPsi begin to occur after about 30 min. The method described here will be potentially useful to assess the effectiveness of cell death regulators and modulators of synthetic and biological origin, and the results presented shed light on the currently debated issue of the importance of the redox state of cytochrome c in the initiation of apoptosis.

CD80 costimulation is required for Th2 cell cytokine production but not for antigen-specific accumulation and migration into the lung.

The CD28 ligands CD80 and CD86 are expressed on APC, and both provide costimulatory function. However, the reason for the expression of two separate CD28 ligands remains unclear. We have previously shown that blockade of CD80 costimulation by Y100F-Ig, a CTL-associated Ag-4 (CTLA4)-Ig mutant that does not bind CD86, inhibits the development of lung inflammatory immune responses, but does not affect blood eosinophilia or Ab production. Each of those responses was inhibited by treatment with CTLA4-Ig, which binds both CD80 and CD86. To clarify the mechanism underlying these observations we have developed a model of lung inflammation using adoptively transferred CD4(+) T cells expressing a Valpha11(+)Vbeta3(+) transgenic TCR specific for I-E(k) and moth cytochrome c. Treatment with Y100F-Ig inhibited the induction of lung eosinophilia in adoptively transferred mice. However, Y100F-Ig did not detectably affect the accumulation of Ag-specific T cells at the site of peptide deposit or in the draining lymphoid tissues. Acquisition of an activated phenotype and expression of adhesion molecules required for migration into the lung were modestly affected. Importantly, treatment with Y100F-Ig diminished the ability of T cells to produce the cytokines IL-4 and IL-5 following intranasal challenge with Ag. All the responses examined were severely inhibited by treatment with CTLA4-Ig. We conclude that T cells require CD80 costimulation for the optimal production of IL-5 following intranasal administration of Ag. Decreased IL-5 production is the most likely explanation for the diminished airway eosinophilia observed.

Akt regulates cell survival and apoptosis at a postmitochondrial level.

Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. We previously showed that transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Here we show that stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c induced caspase activation in a cell-free assay while lysates from Bcl-2-expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c-induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9.

Controlled delivery of drugs from a novel injectable in situ formed biodegradable PLGA microsphere system.

A novel method for in situ preparation of injectable biodegradable microspheres from the copolymer, poly(lactide-co-glycolide) (PLGA), without incorporating unacceptable organic solvents is described. The delivery system is a dispersion of PLGA microglobules ('premicrospheres' or 'embryonic microspheres') in an acceptable vehicle mixture (continuous phase) and whose integrity is maintained by the use of appropriate stabilizers. A solution of PLGA, triacetin, a model protein (cytochrome c), PEG 400, and Tween 80 (oil phase 1) is added dropwise with continuous homogenization to Miglyol 812-Span 80 solution (oil phase 2), thereby inducing phase separation (coacervation) of PLGA and forming PLGA microglobules (containing cytochrome c) dispersed in the continuous phase. This novel drug delivery system (NDDS) is a dispersion and has a viscous consistency, but is sufficiently syringeable. When injected, it comes in contact with water from an aqueous buffer or physiological fluid and, as a result, the microglobules harden to form solid matrix type microparticles entrapping cytochrome c (in situ formed microspheres). Cytochrome c is then released from these microspheres in a controlled fashion. The composition, rationale, and optimization of the NDDS are described here. Various formulation variables such as the PLGA concentration and type and the substitution of the continuous phase by a fresh oil phase 2 influenced the characteristics of this system. A preliminary investigation of the reproducibility and stability of the NDDS, as well as the physical stability of the encapsulated cytochrome c, revealed that these characteristics were not adversely affected.

Comparison of various injectable protein-loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices: in-situ-formed implant versus in-situ-formed microspheres versus isolated microspheres.

The purpose of this research was to prepare various injectable, protein (cytochrome c)-loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices by a novel microencapsulation method and to compare their characteristics. Syringeable mixtures of polymer and protein solidified upon injection when coming in contact with water, and formed a solid matrix-type implant or microspheres (in-situ-formed implant or in-situ-formed microspheres, respectively) with cytochrome c entrapped. These devices exhibited different characteristics in terms of in vitro cytochrome c release profile, percentage cytochrome c encapsulation efficiency, and particle size. The burst effect from these devices exhibited the following trend: in-situ-formed implant > in-situ-formed microspheres > isolated microspheres. The in-situ-formed microspheres were larger in size than the isolated microspheres. Also, the isolated microspheres exhibited the slowest release of cytochrome c, whereas the in-situ-formed implant exhibited the fastest release. The microencapsulation process can produce various drug-loaded injectable biodegradable PLGA devices having different characteristics.

Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria.

Growth factors signaling through the phosphoinositide 3-kinase/Akt pathway promote cell survival. The mechanism by which the serine/threonine kinase Akt prevents cell death remains unclear. We have previously shown that Akt inhibits the activity of DEVD-targeted caspases without changing the steady-state levels of Bcl-2 and Bcl-x(L). Here we show that Akt inhibits apoptosis and the processing of procaspases to their active forms by delaying mitochondrial changes in a caspase-independent manner. Akt activation is sufficient to inhibit the release of cytochrome c from mitochondria and the alterations in the inner mitochondrial membrane potential. However, Akt cannot inhibit apoptosis induced by microinjection of cytochrome c. We also demonstrated that Akt inhibits apoptosis and cytochrome c release induced by several proapoptotic Bcl-2 family members. Taken together, our results show that Akt promotes cell survival by intervening in the apoptosis cascade before cytochrome c release and caspase activation via a mechanism that is distinct from Bad phosphorylation.

Cytochrome c is dispensable for fas-induced caspase activation and apoptosis.

Cytochrome c is thought to play an important role in the initiation of apoptosis following its release from mitochondria. It is controversial whether such release is also involved in caspase activation and apoptotic cell death after ligation of the cell surface molecule Fas. We addressed this issue by investigating cells from the human cell lines Jurkat and SKW6 which had been treated with the inhibitor of the mitochondrial F0/F1-ATPase, oligomycin. Oligomycin-treatment led, over a wide range of concentrations, to ATP-depletion and, at similar concentrations, abrogated the appearance of caspase-3-like activity caused by stauroporine. Electroporation of cytochrome c protein into intact cells induced caspase activation in both cell lines and significant nuclear apoptosis in Jurkat cells. In ATP-depleted cells, electroporation of cytochrome c induced neither caspase activation nor nuclear fragmentation. Fas-induced caspase activation and nuclear apoptosis, however, were unaffected by the depletion of ATP. Thus, cytochrome c is unlikely to be an important factor in Fas-induced cell death.

Termination of peripheral tolerance to a T cell epitope by heteroclitic antigen analogues.

Treating mice with an immunodominant T cell epitope from moth cytochrome c (MCC(88-103)) can induce T cell unresponsiveness under certain conditions of administration. In this report, we determined whether T cell tolerance to MCC(88-103) in adult animals can be overcome by immunization with cross-reactive analogues of the tolerizing Ag. A panel of analogues of the tolerogen were tested for their capacity to terminate the tolerant state following in vivo immunization. As analyzed by their stimulatory capacity for a representative MCC(88-103)-specific T cell clone, this panel covered a wide range of cross-reactivity, including nonantigenic, antagonistic, weakly, and strongly antigenic peptides. Interestingly, only heteroclitic analogues, as measured in vitro by their enhanced antigenicity for the T cell clone that was specific for MCC(88-103), were capable of breaking tolerance. Thus, an immune response to the cross-reactive, heteroclitic analogues of tolerized self Ags may represent a mechanism by which Ag molecular mimicry operates.

Superoxide production after spinal injury detected by microperfusion of cytochrome c.

Highly reactive oxygen-containing species may form upon CNS injury and cause oxidative damage to important cellular components, thereby destroying cells. To test this hypothesis, free radical formation following such insults should be characterized first. In this study, we measured the time course of superoxide production following impact injury to the rat spinal cord using a novel microcannula perfusion technique developed by us. Cytochrome c (50 microM in artificial cerebrospinal fluid) was perfused into the rat spinal cord through the cannula inserted laterally into the gray matter of the cord, and reduced cytochrome c was measured from perfusates spectrophotometrically. We found that the levels of superoxide in the extracellular space increased to approximately twice the basal level and remained elevated for over 10 h. Superoxide dismutase (60 U/ml) significantly reduced the elevation of superoxide levels (p = .016) and ferric chloride (0.1 mM)/EDTA (0.25 mM) infused together with cytochrome c completely removed the superoxide measured, validating the measurement of superoxide. The relatively long-lasting formation of superoxide reported herein suggests that removal of superoxide may be a realistic treatment strategy for reducing injury caused by free radicals.

[The role of cytochrome c in preventing postresuscitation functional disorders of the central nervous system]. / Rol' tsitokhroma c v preduprezhdenii postreanimatsionnykh narushenii funktsional'nogo sostoianiia tsentral'noi nervnoi sistemy.

The effect of intracarotid injection of cytochrome c on the course of the early postresuscitation period was studied in experimental animals (albino rats) after 5-min clinical death from acute blood loss. It was shown that the drug normalizes the disturbed metabolic processes in the brain, reduces the structural changes consequent upon total ischemia, stabilizes the activity of the cardiovascular system and, as a result of this, contributes to restoration of functional activity of the central nervous system in the early postresuscitation period.