The co-infection plate was synchronised for 5 min at 21 °C and subjected for 1 h incubation at 37 °C in a humidified CO2 incubator. After 1 h, the phagocytosis was stopped by washing with ice-cold PBS. Counter-staining of spores that are not phagocytosed was performed with 0.5 mg ml−1 CFW (calcofluorwhite; Sigma) in PBS for 15 min at room temperature. The cells were washed twice with PBS then fixed with 3.7% (vol/vol) formaldehyde/PBS for 15 min followed by another two washes NVP-LDE225 research buy with PBS. Microscopic photographs were taken with Leica DM 4500B at a magnification of 40×. For statistical reproducibility, three biological replicates and in each case two technical replicates were performed

and analysed for each strain. The automated image analysis was performed by an algorithm that was previously implemented and rigorously validated in the context of phagocytosis assays for A. fumigatus conidia[16] and of invasion assays for Candida albicans.[20] The algorithm was developed within the Definiens Developer XD framework where the ruleset comprising all commands is written in a meta-language. Processing

the current image data of phagocytosis assays at a high level of performance was achieved by modifying this algorithm with regard to the second of its three main steps: (i) preprocessing, (ii) segmentation and (iii) classification. Each image is built of three distinct layers, one for each fluorescent label, and a schematic https://www.selleckchem.com/products/fg-4592.html representation of the ruleset acting on the three colour layers containing all spores (green layer), non-phagocytosed spores (blue layer) and macrophages (red layer)

is depicted in Fig. 1. Apart from a modification in the segmentation step, the original algorithm was applied for parameters values summarised in Table 1 that were adjusted to the images of size of 1600 × 1200 pixels with a pixel area of 0.0246 μm2 and a corresponding pixel-to-pixel ZD1839 mw distance of 0.157 μm. After the ruleset-based image data analysis was performed, features obtained for all four labelled classes (macrophages, phagocytosed spores, non-phagocytosed spores that can be either adherent or non-adherent to macrophages), e.g. area in pixel, layer intensity and number of neighbours of each object as well as class membership of every object, were exported and used for subsequent analyses. Finally, the number of cells per class was calculated to perform statistical analyses and validation procedures. Images were preprocessed by smoothing the three distinct layers with a Gauss filter to reduce noise (split point 1 in Fig. 1). Afterwards an edge-detection filter was applied to enhance object boundaries. This filter assigns to every pixel the maximal intensity value of its pixel neighbourhood. No further preprocessing was necessary at split point 2 in Fig. 1 to optimise the segmentation and classification of regions of interest (ROIs) in the subsequent steps. As depicted in Fig.

g. plasmacytoid DC (pDC) peculiarly require the E2-2 transcription factor for their development 12, 13. A major gap in this aspect of DC science relates to Flt3-independent development from monocytes. Monocytes are bipotential. selleckchem They can differentiate into macrophages with numerous scavenging and effector capacities. Alternatively, monocytes can develop into poorly phagocytic but highly immunostimulatory

DC. This differentiation of monocytes to DC has been studied mainly in vitro for years, using monocytes from human blood 14, 15. What about in vivo? During inflammation in mice, several recent reports describe how monocytes acquire some properties of DC, i.e. expression of MHC II and CD11c 16–19. Now it is important to determine whether monocytes fully differentiate into authentic DC in vivo. By authentic, I mean the VX-809 molecular weight monocytes must acquire such DC properties as distinctive motility, localization to T-cell areas, loss of responsiveness to M-CSF, and efficient capture and presentation of antigens for display

on both MHC I and II in vivo. Most research on DC development involve mice; the study of DC in the human system is needed. The expansion of DC numbers with Flt3L could have medical benefit. For example, Flt3L administration suppresses autoimmune diabetes in NOD mice 20, probably by expanding both DC and Treg as part of a homeostatic circuit 21. Different types of DC in the steady state, prior to the introduction of an infection or other stimulus, are called “subsets”. This field was initiated with mouse spleen 22, 23 and human blood 24, but now other organs are increasingly being scrutinized. Guilliams et al. 25 summarize studies in

the skin that likely extend to other tissues. They provide a useful proposal in which there are at least five types of DC in the steady state: two types of classical DC, pDC, Langerhans cells, and monocyte-derived triclocarban DC. Five subsets are in fact less complex than some previous descriptions. Pabst and Bernhardt 26 discuss myeloid cells in the intestinal lamina propria. Pabst and Bernhardt concentrate on recent studies in which they examined for the first time some fundamental properties of CX3CR1high and CX3CR1low populations 27. CX3CR1high cells, or at least a sizeable fraction of them, derive from blood monocytes 28, 29 and are in a state where they do not present antigens effectively or migrate to the T-cell areas of mesenteric lymph node. In contrast, CX3CR1low/neg cells, which can express CD103, behave like bona fide DC, are able to present antigens effectively and also migrate to the T-cell areas. Swiecki and Colonna 30 focus on pDC and consider the increasing examples in which pDC are involved in immunosuppression and tolerance. Swiecki and Colonna 30 also provide a valuable outline of the consequences of high type I interferon production upon nucleic acid signaling, a hallmark of these DC; these include resistance to viral infection and development of autoinflammatory diseases 31–33.

, 2009). However, very little is known about the interaction of PMN with vaccine strains of mycobacteria. As neutrophils are the first cells to get exposed to any antigen and generate early immune response, their interaction with vaccine strains will help us to understand the exact nature of protective immune response. Hence, we studied in vitro modulation of neutrophil functions like phenotypic changes, apoptosis rate, and inflammatory

cytokines after infection with vaccine strains (BCG and Mw) and compared with standard laboratory strain H37Rv. To understand the paracrine Navitoclax role of neutrophils and their influence on mononuclear cell recruitment, we also studied the expression profile of the activation markers and chemokine receptors on T cells and monocytes. The study protocol was approved by the institutional ethical committee and followed the institute Idelalisib nmr ethical guidelines. Written informed consents were obtained from blood donors, and 10 mL of heparinized blood was collected through venipuncture. The study group consisted of normal healthy volunteers (N = 11) (mean age 24 years, range 22–28 years) who received BCG vaccination in childhood, but their tuberculin skin test status was unknown. They showed no clinical signs and symptoms of tuberculosis or any other immunosuppressive

diseases at the time of blood sampling. Two vaccine strains, namely BCG and Mw, available in India were used. The standard laboratory strain live H37Rv was used for comparison. Live, attenuated BCG vaccine was purchased from Serum institute of India, Chennai. Heat-killed Mw vaccine was purchased from Cadila

pharmaceuticals Limited, Ahmedabad. Colonies of H37Rv from Lowenstein-Jensen-slopes were inoculated in Sauton’s medium and grown as standing cultures at 37 °C. Log-phase cultures were centrifuged and washed with phosphate-buffered saline (PBS) (Biowhittaker, Belgium), and bacterial clumps were dispersed by passing them through 26-gauge needle. The bacterial suspension was centrifuged check to remove the remaining clumps, and the supernatant containing the single-cell suspension was adjusted to 5 × 107 cells mL−1 in sterile, endotoxin-free PBS and stored in aliquots at −70 °C until use. The viability of bacilli was enumerated by CFU values. Human neutrophils were isolated by standard protocol (Böyum, 1968). Briefly, heparinized venous blood was layered over Ficoll-Hypaque (Amersham Biosciences) for gradient centrifugation followed by sedimentation in 3% dextran (Sigma Chemicals). The PMN rich supernatant was collected, and the residual RBCs were lysed by hypotonic lysis. The cells were washed and resuspended in RPMI 1640 (Gibco BRL, CA) supplemented with 1% fetal bovine serum (FBS) (Gibco BRL). The viability of the cells was assessed to be > 95% by the trypan blue exclusion test, and the purity was always found to be > 90%. The cell density was adjusted to 0.5 × 106 cells mL−1.

In a recent study, using the same technique,

the metabolic and vascular effects of the nitric oxide vasodilator metacholine were investigated in a group of obese, insulin-resistant and insulin-sensitive individuals during glucose-stimulated physiological hyperinsulinemia [85]. The results demonstrated that, in obesity, even in the absence of measurable increments in total forearm blood flow, capillary recruitment (i.e., PSglucose) and forearm glucose disposal increased in response to a glucose challenge, which effect was blunted in the insulin-resistant individuals. Subsequently, it was demonstrated that in the obese, insulin-resistant subjects, an intrabrachial MK-2206 mw metacholine infusion attenuated the impairment of muscle microvascular recruitment and the kinetic defects in insulin action. To date, there is one study where the hypothesis that insulin increases delivery to muscle has been challenged [118]. During hyperinsulinemic euglycemic clamps, transport parameters and distribution volumes of [14C]inulin (a polymer of d-fructose of similar molecular size to insulin) were determined in healthy, non-obese subjects. The results suggest that, in contrast to earlier findings of the same group performed in a canine model [26,27], physiological hyperinsulinemia does not augment access of macromolecules Dabrafenib supplier to insulin-sensitive tissues

in healthy humans. The study is somewhat hampered by the fact that microvascular perfusion was not assessed at the same time, in contrast to earlier

mentioned studies [38,85,104]. Insulin’s effect on capillary recruitment are considered to be caused by insulin-mediated effects on precapillary arteriolar tone and/or on arteriolar vasomotion [6,14,97]. Vasomotion is a spontaneous rhythmic change of arteriolar diameter that almost certainly plays an important role in ensuring that tissue such as muscle is perfused sufficiently to sustain the prevailing metabolic demand by periodically redistributing blood from one region of the muscle to another Cyclin-dependent kinase 3 [92]. It is an important determinant of the spatial and temporal heterogeneity of microvascular perfusion and, therefore, most likely of the number of perfused capillaries [19,92]. It has been suggested that vasomotion is regulated by both local vasoactive substances and influences of the central nervous system. The contribution of different regulatory mechanisms can be investigated by analyzing the contribution of different frequency intervals to the variability of the laser Doppler signal. Stefanovska et al. have analyzed the reflected laser Doppler signal from skin to provide indirect assessment of vasomotion [65,105]. In humans, they have interpreted the spectrum as follows: (1) 0.01–0.02 Hz, which is thought to contain local endothelial activity; (2) 0.02–0.06 Hz, which is thought to contain neurogenic activity; (3) 0.06–0.

One important facet is the circulatory system dysfunction, which includes capillary bed plugging. This review addresses the mechanisms of capillary plugging and highlights our recent discoveries on the roles of NO, ROS, and activated coagulation in platelet adhesion

and blood flow stoppage in septic mouse capillaries. We show that sepsis increases platelet adhesion, fibrin deposition and flow stoppage in capillaries, check details and that NADPH oxidase-derived ROS, rather than NO, play a detrimental role in this adhesion/stoppage. P-selectin and activated coagulation are required for adhesion/stoppage. Further, platelet adhesion in capillaries (i) strongly predicts capillary flow stoppage, and (ii) may explain why severe sepsis is associated with a drop in platelet count in systemic blood. Significantly, we also show that a single bolus of the antioxidant ascorbate (injected intravenously at clinically relevant dose of 10 mg/kg) inhibits adhesion/stoppage. Our data suggest that eNOS-derived NO at the platelet-endothelial interface is anti-adhesive and required RG-7388 in vivo for the inhibitory

effect of ascorbate. Because of the critical role of ROS in capillary plugging, ascorbate bolus administration may be beneficial to septic patients whose survival depends on restoring microvascular perfusion. “
“Please cite this paper as: Wijnstok N, Hoekstra T, Eringa E, Smulders Y, Twisk J, Serne E. The relationship of body fatness and body fat distribution with microvascular recruitment: The Amsterdam Growth and Health Longitudinal Study. Microcirculation 19: 273–279, 2012. Introduction:  Microvascular function has been proposed to link body fatness to CVD and DM2. Current knowledge of these relationships is mainly based on studies in selected populations of extreme phenotypes. Whether these findings can be translated to the general population remains to be investigated. Aim:  To assess the relationship of body fatness and body fat distribution with microvascular function in a healthy population-based cohort. Methods:  Body fatness parameters were obtained by anthropometry and whole-body dual-X-ray absorptiometry (DEXA) in 2000 and 2006. Microvascular

recruitment (i.e., absolute increase in perfused capillaries after arterial occlusion, using nailfold capillaroscopy) was measured in 2006. Linear regression analysis was used to examine the Dynein relationship of (changes in) body fatness and body fat distribution with microvascular recruitment. Results:  Data were available for 259 participants (116 men). Capillary density was higher in women than in men (difference 7.3/ mm2; p < 0.05). In the total population, the relationship between total body fatness and microvascular recruitment was positive (β = 0.43; p = 0.002), whereas a central pattern of fat distribution (trunk-over-total fatness) showed a negative relationship (β = −26.2; p = 0.032) with microvascular recruitment. However, no association remained apparent after adjustment for gender.

9 and 17.1%; 85.7 and 14.3%; 80.5 and 19.5%; and 90.8 and 9.2% respectively, in women with endometriosis (P = 0.004), women with minimal/mild endometriosis (P = 0.148), women with moderate/severe endometriosis (P = 0.002) and control group. Conclusion  The data suggest that in Brazilian women polymorphism PTPN22 (C1858T) may be an important genetic predisposing factor for endometriosis,

especially, in advanced disease. “
“Anaplasma phagocytophilum is an emerging tick-borne pathogen. Great genetic diversity of A. phagocytophilum has been described in animals and ticks. The present study is focused on the genetic variability of the groESL operon of A. phagocytophilum in human patients in Slovenia. During 1996–2008, there were 66 serologically confirmed patients with human granulocytic anaplasmosis. Nutlin-3a in vitro Of these, 46 were tested with a screening PCR for a small part of the 16S rRNA gene of A. phagocytophilum and 28 (60.9%) were positive. Positive samples were additionally tested with a PCR

targeting the groESL operon and a larger fragment of the 16S rRNA gene. All amplicons were further sequenced and analyzed. The homology p38 MAPK pathway search and the alignment of the groESL sequences showed only one genetic variant. Sequence analysis of the 16S rRNA gene revealed 100% identity among amplicons. Slovenia is a small country with diverse climate, vegetation, and animal representatives. In previous studies in deer, dogs, and ticks, great diversity of the groESL operon was found. In contrast, in wild boar and in human patients from this study, only one genetic variant was detected. The results suggest that only one genetic variant might be pathogenic for humans or is competent enough to replicate in humans. To support this theory, other genetic markers and further studies need to be performed. Anaplasmosis comprises a group of emerging tick-borne diseases. It is mostly mild and self-limiting

disease. The causative agent Anaplasma phagocytophilum is a pathogen known to cause disease not only in humans but also in ruminants, horses, and dogs. Anaplasma phagocytophilum shows differences in clinical severity, disease manifestation, reservoir competency, and antigenic diversity. Deer have been suggested as a reservoir animal. The heterogeneity Mannose-binding protein-associated serine protease of the groESL operon, as well as other genes of A. phagocytophilum, in animals and in a tick vector Ixodes ricinus has been described elsewhere. Only few studies report PCR-confirmed human cases of anaplasmosis. The present study is focused on the genetic variability of the groESL operon of A. phagocytophilum in human patients in Slovenia. Between the years 1996 and 2008, blood samples of human patients with clinical signs of anaplasmosis were tested for the presence of anaplasmal DNA. DNA was extracted from acute blood samples of patients that seroconverted or had at least a fourfold rise in antibody titer against A. phagocytophilum antigen. For initial screening of all samples, PCR for a small part of the 16S rRNA gene of A.

The exact cause of lack of HDL-C protection in the INCB018424 order dialysis population is still obscure. Methods:  A total of 89 stable non-diabetic haemodialysis patients were recruited. Fasting serum biochemical parameters, complete blood counts and inflammatory markers were obtained before the mid-week

dialysis. Insulin resistance was assessed by the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). Results:  The mean age was 58.2 ± 13.1 years, 37 (41.6%) patients were male. The mean HDL-C level was 56.3 ± 17.1 mg/dL. By bivariate correlation analysis, a lower serum HDL-C level was related to higher body mass index (r = −0.425; P < 0.001), higher triglyceride (r = −0.479; P < 0.001) and higher HOMA-IR (r = −0.211; P < 0.05) levels. The serum HDL-C level was also inversely related to high-sensitivity C-reactive protein (hsCRP)

(r = −0.297; P = 0.005) and tumour necrosis factor-α (TNF-α) (r = −0.295; P = 0.005) and directly correlated with adiponectin (r = 0.560; P < 0.001). In multivariate linear regression analysis, HDL-C was found to be directly correlated with adiponectin (β-coefficient = 0.569; P < 0.001) and inversely correlated Selleck Venetoclax with TNF-α (β-coefficient = −0.292; P = 0.001). Conclusion:  A strong association between HDL-C, inflammatory surrogates, and insulin resistance in this non-diabetic, non-obese haemodialysis patient group is demonstrated. The HDL-C level is still a good parameter to screen high-risk patients. “
“Chronic kidney disease (CKD), and its associated cardiovascular events, is one of the major causes of morbidity and recurrent hospitalization in Asian Pacific region. The subtotal nephrectomy (STNx) model has remained the state-of-the-art prototype Rucaparib concentration which closely mimics human CKD and cardiac-renal syndrome. In this article, we comprehensively outline the procedure and methodology required to develop the rat model 5/6 nephrectomy

and the associated procedures involved in assessing cardiac and renal functional outcomes. In addition, the expected functional outcomes from our own experience, and those of others, have been described. The STNx model in the rat is an established model of CKD and displays all the functional and structural hallmarks observed in the human condition. Lesser known are the cardiac effects of this model which make it ideal for studying cardiorenal syndrome. “
“Renal primary cilia are microscopic sensory organelles found on the apical surface of epithelial cells of the nephron and collecting duct. They are based upon a microtubular cytoskeleton, bounded by a specialized membrane, and contain an array of proteins that facilitate their assembly, maintenance and function. Cilium-based signalling is important for the control of epithelial differentiation and has been implicated in the pathogenesis of various cystic kidney diseases and in renal repair.

One of the most important aspects of subcellular proteomics is the inference of hypothetical protein function based on its subcellular localization. Many proteomic studies in trypanosomatids have focused on specific subcellular compartments or fractions, simply to increase the probability of detecting those proteins and/or improving their functional characterization. The subproteomic studies on specific compartments such as the glycosome, an organelle involved in the first part of glycolytic pathway (62), the LDK378 acidocalcisome, an organelle mediating calcium homoeostasis and pH homoeostasis

(61), the reservosome, a storage organelle (63,64), the flagellum (65), the nucleus (66), plasma membrane (67) and mitochondrion (68) provided an opportunity to improve the functional annotation of hypothetical proteins and search for the candidate drug targets. In addition, glycoproteome (69), GPI-anchored proteome (GPIome) (70) and secreted proteome (secretome) studies (71–74) are of great interest because of their potential role in the interaction with host proteins. Among the many possible post-translational modifications (PTMs), cellular protein phosphorylation is a key mechanism of controlling development in trypanosomatids. The trypanosomatid phosphoproteome (kinome) was recently

characterized (75–77). Studies of other frequent PTMs, such as glycosylation GW-572016 price (69), histone acetylation and deamidation (78), have been recently reported. Mass spectrometry coupled with 2-D PAGE has been the most efficient and popular approach to characterize trypanosomatid proteome profiles. With more extensive use of high-throughput proteomic Alanine-glyoxylate transaminase approaches (79–82), we will soon see the emergence of more complete and diverse proteomic datasets that should complement the transcriptome data and facilitate the unravelling of the pathobiology of trypanosomatids. With genomic data available, transcriptome profiles abundant, and cultivation methods for different life cycle stages well established, trypanosomatids are emerging as ideal model organisms

for metabolomic studies (83). Ultrahigh resolution metabolomic studies in trypanosomatids are offering new tools to identify biomarkers of disease, comprehensively characterize cellular responses to perturbations, and identify novel potential drug targets (84–86). Currently available databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) (87–89) and Pathway Tools software (90) allow mapping the results onto reconstructed networks. The MetExplore web server (91) offers the tools to link metabolites identified in untargeted metabolomics surveys within the context of genome scale reconstructed metabolic networks. Genome-wide metabolic networks in Leishmania spp.

“
“Microcirculation (2010) 17, 226–236. doi: 10.1111/j.1549-8719.2010.00022.x Tissue blood flow is controlled by a branching network of resistance arteries coupled in series and parallel with one another. To alter organ perfusion

during periods of elevated metabolic demand, the arterial segments comprising these networks must dilate in a coordinated manner. Gap junctions are intercellular www.selleckchem.com/products/torin-1.html pores that facilitate arterial coordination by enabling electrical stimuli to conduct among and between endothelial and/or smooth muscle cells. Through this novel perspective, readers will be introduced to the vascular communication field, the process of intercellular conduction, and how key cellular properties influence charge flow. This overview will begin with a brief historical review

and then introduce two differing theories on how electrical phenomena moves among and between vascular cells. The basis of the “syncytium” and “differential” hypothesis will be critically discussed within a framework of biophysical and experimental observations. This foundational understanding will be used to extend our mechanistic insight of: (i) “local” and “global” blood flow control; and (ii) debilitating disorders such as arterial vasospasm. “
“Vascular smooth muscle contraction and relaxation play a preponderant role on the active (acute) and structural (long-term) control of vascular diameter. This editorial overview summarizes and highlights the opinions expressed in seven reviews contained in this special topic issue of Microcirculation. GPCR Compound Library research buy The reviews address diverse aspects of the mechanisms that influence cell adhesion, calcium homeostasis, and cytoskeletal

remodeling, and how these mechanisms affect vascular structure and function at different levels of the circulation. “
“Please cite this paper as: Bachmeier, Beaulieu-Abdelahad, Mullan, and Paris (2011). Epitope-Dependent Effects of Beta-Amyloid Antibodies on Beta-Amyloid Clearance in an In Vitro Model of the Blood–Brain Barrier. Microcirculation 18(5), 373–379. Objective:  To investigate the role of RAGE in the epitope-dependent effects of Aβ antibodies Fossariinae used as a peripheral sink therapy in AD. Methods:  An in vitro model of the BBB was used to examine the effect of various Aβ antibodies or Aβ peptide fragments on Aβ exchange across the BBB. Results:  An N-terminal Aβ antibody significantly enhanced the basolateral-to-apical transcytosis of fluorescein-Aβ(1–42) across the BBB model (41%), while no effect was apparent with a C-terminal Aβ antibody. Interestingly, modulation of RAGE in the presence of a C-terminal Aβ antibody resulted in a 65% increase in Aβ clearance across the BBB model, suggesting the C-terminal antibody–Aβ complex is susceptible to RAGE transport.

6B, do not always correlate well with the levels of Egr2 in normal thymocytes; notably, in population A, where Egr2 expression is lowest, there are substantial effects on Socs1 expression in Egr2f/fCD4Cre mice. We suggest that the regulation of Socs1 by Egr2 is biologically significant, as events previously documented as lying downstream of Socs1 signaling during selection were also affected in Egr2-deficient thymocytes. Egr2-deficient CD4+CD8lo cells were unable to correctly upregulate Bcl2 expression as would normally occur following resumption of cytokine

signaling 30, and this was linked to lowered levels of pStat5 and a reduced ability to survive in IL-7-supplemented medium. We note that survival might be further compromised by the loss of a small population of high-level expressors of IL-7R in Egr2-deficient CD4+CD8lo subsets. ABT-263 concentration Regulation of Socs1 might also provide an explanation for the increase in numbers of CD8SP thymocytes in Egr2-Tg animals, as this fits well with the observations that in the absence of Socs1, CD8SP T-cell differentiation

is enhanced 33. It would be of great interest to determine whether gain of Socs1 is able to rescue this aspect of the Egr2-Tg phenotype. Autophagy inhibitor order We and others have shown that following TCR ligation, both the MAPK and calcineurin signaling pathways are required for induction of Egr2

15, 22. The convergence of these pathways on Egr2 suggests it may lie at a crucial control point in the selection process. Previously, it has been suggested that the expression levels and activity of Egr proteins combine to modulate positive selection following TCR ligation 24. Where the signal is strong, as in the highest affinity TCR interactions with peptide-MHC, Egr2 and its relatives Egr1 and Egr3 are induced at high levels and are not the rate-limiting step in the selection process. However, where the TCR is weak enough for a thymocyte to be on the boundary between positive selection and death by neglect, increased amounts of Egr proteins permit positive selection to occur, and decreased amounts cause the cell to fail selection. Our data suggest Selleckchem RG7420 an extension of this model whereby titration of the levels of Egr2 by TCR signal strength could perhaps modulate the cytokine-mediated survival signal by regulating the level of Socs1, thus fine-tuning the process of positive selection. Egr2-Tg mice were made by microinjection into oocytes of Sfi1-linearised pVAhCD2 plasmid 40 containing LoxP-flanked DsRed and Egr2 cDNA. Details of construct are available upon request. Other strains used have been previously published as follows: CD4Cre 27; Egr2f/f28 MHC-deficient mice 41, 42, Rag2−/−43, Rag1−/−44, TCR-β F5 45, TCR-β HY 46 and TCR-β OTII 47.