Neutrophil extracellular trap formation during surgical procedures: a pilot study.

Neutrophils can release neutrophil extracellular traps (NETs) containing DNA fibres and antimicrobial peptides to immobilize invading pathogens. NET formation (NETosis) plays a vital role in inflammation and immune responses. In this study we investigated the impact of surgical trauma on NETosis of neutrophils. Nine patients undergoing "Transcatheter/percutaneous aortic valve implantation" (TAVI/PAVI, mild surgical trauma), and ten undergoing "Aortocoronary bypass" (ACB, severe surgical trauma) were included in our pilot study. Peripheral blood was collected before, end of, and after surgery (24 h and 48 h). Neutrophilic granulocytes were isolated and stimulated in vitro with Phorbol-12-myristate-13-acetate (PMA). NETosis rate was examined by microscopy. In addition, HLA-DR surface expression on circulating monocytes was analysed by flow-cytometry as a prognostic marker of the immune status. Both surgical procedures led to significant down regulation of monocytic HLA-DR surface expression, albeit more pronounced in ACB patients, and there was a similar trend in NETosis regulation over the surgical 24H course. Upon PMA stimulation, no significant difference in NETosis was observed over time in TAVI/PAVI group; however, a decreasing NETosis trend with a significant drop upon ACB surgery was evident. The reduced PMA-induced NETosis in ACB group suggests that the inducibility of neutrophils to form NETs following severe surgical trauma may be compromised. Moreover, the decreased monocytic HLA-DR expression suggests a post-operative immunosuppressed status in all patients, with a bigger impact by ACB, which might be attributed to the extracorporeal circulation or tissue damage occurring during surgery.

Flow cytometry-based high-throughput RNAi screening for miRNAs regulating MHC class II HLA-DR surface expression.

HLA-DR isotype is a MHC-II cell-surface receptor found on APCs and plays a key role in initiating immune responses. In severely immunocompromised patients with conditions like sepsis, the number of HLA-DR molecules expressed on leukocytes is considered to correlate with infectious complications and patients' probability of survival. The underlying regulatory mechanisms of HLA-DR expression remain largely unknown. One probable path to regulation is through microRNAs (miRNAs), which have been implicated as regulatory elements of both innate and adaptive immune system development and function. In our study, flow cytometry-based high-throughput miRNA screening was performed in a stable HLA-DR-expressing human melanoma cell line, MelJuSo, for either up- or downregulating miRNAs of the surface HLA-DR expression. By the end of the screening, the top ten upregulators and top five downregulators were identified, and both the HLA-DR protein and mRNA regulations were further verified and validated. In-silico approaches were applied for functional miRNA-mRNA interaction prediction. The potential underlying gene regulations of different miRNAs were proposed. Our results promote the study of miRNA-mediated HLA-DR regulation under both physiological and pathological conditions, and may pave the way for potential clinical applications.

Development and Evaluation of a Droplet Digital PCR Assay for 8p23 ß-Defensin Cluster Copy Number Determination.

INTRODUCTION: It has been proposed that the copy number (CN) variation (CNV) in ß-defensin genes (DEFB) on human chromosome 8p23 determines phenotypic differences in inflammatory diseases. However, no method for accurate and easy DEFB CN quantification is yet available. OBJECTIVE: Droplet digital polymerase chain reaction (ddPCR) is a novel method for CNV quantification and has been used for genes such as CCL4L, CCL3L1, AMY1, and HER2. However, to date, no ddPCR assay has been available for DEFB CN determination. In the present study, we aimed to develop and evaluate such a ddPCR assay. METHODS: The assay was designed using DEFB4 and RPP30 as the target and the reference gene, respectively. To evaluate the assay, 283 DNA samples with known CNs previously determined using the multiple ligation-dependent probe amplification (MLPA) method, the current gold standard, were used as standards. To discover the optimal DNA template amount, we tested 80 to 2.5 ng DNA by a serial of one to two dilutions of eight samples. To evaluate the reproducibility of the assay, 31 samples were repeated to calculate the intra- and inter-assay variations. To further validate the reliability of the assay, the CNs of all 283 samples were determined using ddPCR. To compare results with those using quantitative PCR (qPCR), DEFB CNs for 48 samples were determined using qPCR with the same primers and probes. RESULTS: In a one-dimensional plot, the positive and negative droplets were clearly separated in both DEFB4 and RPP30 detection channels. In a two-dimensional plot, four populations of droplets were observed. The 20 ng template DNA proved optimal, with either high (80 ng) or low (10, 5, 2.5 ng) template input leading to ambiguous or inaccurate results. For the 31 standard samples, DEFB CNs were accurately determined with small intra- and inter-assay variations (coefficient of variation < 0.04 for both). In the validation cohort, ddPCR provided the correct CN for all 283 samples with high confidence. qPCR measurements for the 48 samples produced noisy data with high uncertainty and low accuracy. CONCLUSIONS: ddPCR is an accurate, reproducible, easy-to-use, cheap, high-throughput method for DEFB CN determination. ddPCR could be applied to DEFB CN quantification in large-scale case-control studies.

A serotonin transporter polymorphism is associated with postoperative nausea and vomiting: An observational study in two different patient cohorts.

BACKGROUND: Clinical risk factors for postoperative nausea and vomiting (PONV) are well described, whereas genetic findings are conflicting. OBJECTIVE: The aim of this study was to investigate a possible association of genetic variants and nongenetic variables with the incidence and severity of PONV. DESIGN: A prospective observational study in two independent and different patient cohorts. SETTING: Two independent patient cohorts differing in surgical procedures were enrolled in two tertiary care hospitals between 2008 and 2016. PATIENTS: Consecutive patients of European origin undergoing elective surgery in two university hospitals. Clinical data were collected up to 24 h after surgery, and blood was drawn for genotyping. Of 2773 patients enrolled, 918 (Cohort A) and 1663 (Cohort B) with complete data sets were analysed. MAIN OUTCOME MEASURE: Patients were allocated to one of three groups (No PONV, Intermediate PONV or Severe PONV) depending on the frequency of vomiting, the severity of nausea and the need for antiemetics. Clinical variables and 13 genetic variants of seven candidate genes were evaluated for association with these three phenotypes. The cohorts were analysed separately by ordinal logistic regression analysis, treating PONV as a dependent ordinal three-stage variable. Odds ratios (ORs) with 95% confidence intervals were calculated. RESULTS: In Cohort A, the main predictors for PONV were female sex [OR (95% CI): 3.6 (2.7 to 4.8), P < 0.0001], nonsmoking status 1.8 (1.3 to 2.5), P < 0.001, the SS genotype (5-HTTLPR, rs4795541) of the promoter polymorphism in the serotonin transporter 1.5 (1.1 to 2.1), P = 0.019, and patient age 0.99 (0.98 to 0.99), P = 0.013. Analysis of Cohort B was consistent with these findings [5-HTTLPR: 1.8 (1.4 to 2.3), P < 0.00001]. Sex-specific regression models confirmed this 5-HTTLPR association in women and men. CONCLUSION: In two independent cohorts, in addition to the well known clinical factors, a polymorphism of 5-HTTLPR in the serotonin transporter was independently associated with PONV. A possible evaluation of this biomarker to improve risk prediction within the scope of precision medicine should be considered. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT03490175.

ERK and p38 contribute to the regulation of nociceptin and the nociceptin receptor in human peripheral blood leukocytes.

Little is known about the mechanisms involved in the regulation of nociceptin and its receptor (nociceptin opioid peptide receptor, NOP) in response to inflammation and pain in humans. In this study, specific signaling pathways contributing to the regulation of nociceptin and NOP in human peripheral blood leukocytes were investigated. After approval by the ethics committee, peripheral blood obtained from healthy donors was cultured with or without phorbol-12-myristate-13-acetate (PMA). Prepronociceptin (ppNOC) and NOP mRNA were analyzed by real-time quantitative polymerase chain reaction, and nociceptin concentrations in culture supernatants by fluorescent enzyme immunoassay. Nociceptin and NOP protein levels in blood leukocyte subsets were determined using flow cytometry. To examine the contribution of signaling pathways to ppNOC and NOP regulation, blood was pre-treated with kinase inhibitors specific for ERK, JNK, p38, and NFκB pathways prior to culturing with or without PMA. PMA dose-dependently upregulated ppNOC mRNA but downregulated NOP mRNA in human peripheral blood leukocytes. PMA 10 ng/ml increased ppNOC after 6 h and suppressed NOP after 3 h compared to controls (both P <0.005). Nociceptin concentrations were increased in supernatants of PMA-induced blood samples after 24 h ( P <0.005), whereas expression of cell-membrane NOP was decreased by PMA in blood leukocyte subsets (all P <0.05). Blockade of ERK or p38 pathways partially prevented PMA effects on ppNOC and NOP mRNA (all P <0.05). The combination of ERK and p38 inhibitors completely reversed the effects of PMA ( P <0.05). ERK and p38 are two major signaling pathways regulating nociceptin and its receptor in human peripheral blood leukocytes under inflammatory conditions.

RNA-Seq analysis during the life cycle of Cryptosporidium parvum reveals significant differential gene expression between proliferating stages in the intestine and infectious sporozoites.

Cryptosporidium parvum is a major cause of diarrhoea in humans and animals. There are no vaccines and few drugs available to control C. parvum. In this study, we used RNA-Seq to compare gene expression in sporozoites and intracellular stages of C. parvum to identify genes likely to be important for successful completion of the parasite's life cycle and, thereby, possible targets for drugs or vaccines. We identified 3774 protein-encoding transcripts in C. parvum. Applying a stringent cut-off of eight fold for determination of differential expression, we identified 173 genes (26 coding for predicted secreted proteins) upregulated in sporozoites. On the other hand, expression of 1259 genes was upregulated in intestinal stages (merozoites/gamonts) with a gene ontology enrichment for 63 biological processes and upregulation of 117 genes in 23 metabolic pathways. There was no clear stage specificity of expression of AP2-domain containing transcription factors, although sporozoites had a relatively small repertoire of these important regulators. Our RNA-Seq analysis revealed a new calcium-dependent protein kinase, bringing the total number of known calcium-dependent protein kinases (CDPKs) in C. parvum to 11. One of these, CDPK1, was expressed in all stages, strengthening the notion that it is a valid drug target. By comparing parasites grown in vivo (which produce bona fide thick-walled oocysts) and in vitro (which are arrested in sexual development prior to oocyst generation) we were able to confirm that genes encoding oocyst wall proteins are expressed in gametocytes and that the proteins are stockpiled rather than generated de novo in zygotes. RNA-Seq analysis of C. parvum revealed genes expressed in a stage-specific manner and others whose expression is required at all stages of development. The functional significance of these can now be addressed through recent advances in transgenics for C. parvum, and may lead to the identification of viable drug and vaccine targets.

Technical Approach Determines Inflammatory Response after Surgical and Transcatheter Aortic Valve Replacement.

OBJECTIVE: To investigate the periprocedural inflammatory response in patients with isolated aortic valve stenosis undergoing surgical aortic valve replacement (SAVR) or transcatheter aortic valve implantation (TAVI) with different technical approaches. MATERIAL AND METHODS: Patients were prospectively allocated to one of the following treatments: SAVR using conventional extracorporeal circulation (CECC, n = 47) or minimized extracorporeal circulation (MECC, n = 15), or TAVI using either transapical (TA, n = 15) or transfemoral (TF, n = 24) access. Exclusion criteria included infection, pre-procedural immunosuppressive or antibiotic drug therapy and emergency indications. We investigated interleukin (IL)-6, IL-8, IL-10, human leukocyte antigen (HLA-DR), white blood cell count, high-sensitivity C-reactive protein (hs-CRP) and soluble L-selectin (sCD62L) levels before the procedure and at 4, 24, and 48 h after aortic valve replacement. Data are presented for group interaction (p-values for inter-group comparison) as determined by the Greenhouse-Geisser correction. RESULTS: SAVR on CECC was associated with the highest levels of IL-8 and hs-CRP (p<0.017, and 0.007, respectively). SAVR on MECC showed the highest descent in levels of HLA-DR and sCD62L (both p<0.001) in the perioperative period. TA-TAVI showed increased intraprocedural concentration and the highest peak of IL-6 (p = 0.017). Significantly smaller changes in the inflammatory markers were observed in TF-TAVI. CONCLUSION: Surgical and interventional approaches to aortic valve replacement result in inflammatory modulation which differs according to the invasiveness of the procedure. As expected, extracorporeal circulation is associated with the most marked pro-inflammatory activation, whereas TF-TAVI emerges as the approach with the most attenuated inflammatory response. Factors such as the pre-treatment patient condition and the extent of myocardial injury also significantly affect inflammatory biomarker patterns. Accordingly, TA-TAVI is to be classified not as an interventional but a true surgical procedure, with inflammatory biomarker profiles comparable to those found after SAVR. Our study could not establish an obvious link between the extent of the periprocedural inflammatory response and clinical outcome parameters.

Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes.

BACKGROUND: The apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into. RESULTS: To determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites. CONCLUSIONS: The distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.

RNA Seq analysis of the Eimeria tenella gametocyte transcriptome reveals clues about the molecular basis for sexual reproduction and oocyst biogenesis.

BACKGROUND: The protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite's sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission. RESULTS: Comparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered. CONCLUSIONS: The need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.

Evaluation of putative anti-cryptosporidial drugs in an in vitro culture system.

Cryptosporidium species are major pathogens severely affecting the health of neonate animals, in particular calves, but also cause life-threatening infection of immunocompromised humans. Currently, only halofuginone is approved for prophylactic and metaphylactic treatment of calves but this drug suffers from its limited safety margins. For treatment of immunodeficient human patients, only nitazoxanide and paramomycin are used but data regarding their efficacy are controversial. Aim of the present study was to test a substituted benzimidazole (BAY-AF76184) and a heterocyclic substituted 1,2,4-triazin (BAY-AB24992), both drugs with known anti-protozoal activity, for their potential ability to interfere with Cryptosporidium parvum development in vitro. Development of C. parvum in HCT-8 cells treated with these compounds was compared to negative controls and parasites treated with paromomycin or halofuginone using real-time PCR targeting the 18S rDNAs of parasites and host cells as template. Potential cytotoxic and anti-proliferative effects of drugs were analysed using a lactate dehydrogenase and a WST-1 assay, respectively. BAY-AF76184 and paromomycin dose-dependently inhibited development of C. parvum with EC50 values of 2.37 µM and 69.5 µM, respectively. Although high concentrations of halofuginone and BAY-AB24992 also significantly inhibited parasite development, effects of lower concentrations were very heterogeneous preventing calculation of EC50 values. Halofuginone and BAY-AF76184 dose-dependently interfered with host cell proliferation (EC50 values of 0.35 µM and 9.07 µM, respectively) and the latter also had direct cytotoxic effects (EC50=48.78 µM). However, drug concentrations required for cytopathic were higher than those for effects against C. parvum. Therefore, both BAY-AB24992 and BAY-AF76184 should be considered for further evaluation, e.g. using in vivo models.

Direct visualization of peptide/MHC complexes at the surface and in the intracellular compartments of cells infected in vivo by Leishmania major.

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.

A dynamic map of antigen recognition by CD4 T cells at the site of Leishmania major infection.

CD4 T helper cells play a central role in the control of infection by intracellular parasites. How efficiently pathogen-specific CD4 T cells detect infected cells in vivo is unclear. Here, we employed intravital two-photon imaging to examine the behavior of pathogen-specific CD4 T cells at the site of Leishmania major infection. While activated CD4 T cells enter the inflamed tissue irrespective of their antigen specificity, pathogen-specific T cells preferentially decelerated and accumulated in infected regions of the dermis. Antigen recognition by CD4 T cells was heterogeneous, involving both stable and dynamic contacts with infected phagocytes. However, not all infected cells induced arrest or deceleration of pathogen-specific T cells, and dense clusters of infected cells were poorly accessible to migrating T cells. Thus, disparities in the dynamics of T cell contacts with infected cells and local variation in T cell access to infected cells are important elements of the host-pathogen interplay.

Direct antigen presentation and gap junction mediated cross-presentation during apoptosis.

MHC class I molecules present peptides from endogenous proteins. Ags can also be presented when derived from extracellular sources in the form of apoptotic bodies. Cross-presentation of such Ags by dendritic cells is required for proper CTL responses. The fate of Ags in cells initiated for apoptosis is unclear as is the mechanism of apoptosis-derived Ag transfer into dendritic cells. Here we show that novel Ags can be generated by caspases and be presented by MHC class I molecules of apoptotic cells. Since gap junctions function until apoptotic cells remodel to form apoptotic bodies, transfer and cross-presentation of apoptotic peptides by neighboring and dendritic cells occurs. We thus define a novel phase in classical Ag presentation and cross-presentation by MHC class I molecules: presentation of Ags created by caspase activities in cells in apoptosis.

Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice.

The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania-infected phagosomes uniformly pass through a Rab5(+) stage on their intracellular path to compartments with late endosomal/early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to antileishmanial drugs. Here, we generated a unique gfp-rab5 transgenic mouse model to visualize Rab5 recruitment to early parasite-containing phagosomes in primary host cells. Using real-time fluorescence imaging of phagosomes carrying Leishmania mexicana, we determined that parasite-infested phagosomes follow a uniform sequence of transient Rab5 recruitment. Residence in Rab5(+) compartments was much shorter compared with phagosomes harboring latex beads. Furthermore, a comparative analysis of parasite life-cycle stages and mutants deficient in lpg1, the gene encoding the enzyme required for synthesis of the dominant surface lipophosphoglycan, indicated that parasite surface ligands and host cell receptors modulate pathogen residence times in Rab5(+) phagosomes, but, as far as tested, had no significant effect on intracellular L. mexicana survival or replication.

Transgenic, fluorescent Leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes.

Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.

Two waves of antigen-containing dendritic cells in vivo in experimental Leishmania major infection.

Dendritic cells (DC) can induce Th1 cell differentiation by producing IL-12. In experimental infection with Leishmania major, DC could differently respond to infection and induce Th1 cells in C57BL/6 but not BALB/c mice, and thus determine the resistance or susceptibility of these mice. We characterized L. major antigen-containing DC in vivo in draining lymph nodes of both strains. Conventional experimental infection is shown to result in two waves of these DC and our data argue against a relevant genetic difference in the DC initiating the anti-parasite Th cell response in these mice. In both strains the first wave of DC presented L. major antigens but was not infected, produced IL-12 but induced disease-mediating Th2 cells upon adoptive transfer. In contrast to current belief, this response was therefore not initiated by infected DC, which were only detected in the second wave. The kinetics of the two waves suggests that DC turnover has an important impact on antigen presentation during infections with complex pathogens.

Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria.

A rapidly maturing variant of the red fluorescent protein DsRed was optimized for bacterial expression by random mutagenesis. The brightest variant contains six mutations, two of which (S4T and a silent mutation in codon 2) explain most of the fluorescence enhancement. The novel variants are expressed at 9-60-fold higher levels in Escherichia coli compared to DsRed.T3, but are not superior fluorophores on a per molecule basis. In contrast to previously available DsRed variants, DsRed.T3_S4T is sufficiently bright to monitor Salmonella gene expression in infected animals using flow cytometry. However, no fluorescence enhancement was observed in Leishmania or HeLa cells, indicating that these novel variants are specifically useful for bacteria.