SARS-CoV-2 Vaccination in Kidney Transplant Recipients-Stratified Analysis of the Humoral Immune Response.

Kidney transplant recipients are at increased risk of SARS-CoV-2 infection and a more severe course of COVID-19. Methods: We conducted a quantitative serologic testing of antibodies specific for the wild type of SARS-CoV-2 and the Omicron variant of concern before and after a third-dose vaccination, either mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) in a cohort of 103 stable kidney transplant recipients (median [range] age, 58 [22-84] y, 57 men [55.3%]). Results: Third-dose vaccination increased the seroconversion rate from 57.3% to 71.8%. However, despite a marked rise of the antibody concentrations after the booster, 55.4% and 11.6% only formed neutralizing antibodies against the SARS-CoV-2 wild type and Omicron, respectively. Treatment with mycophenolic acid/mycophenolate mofetil (in strata of the dose quartiles), advanced age, and' above all' impaired renal function (eGFR <60 mL/min) adversely influenced the humoral immunity regarding seroconversion and inhibition of the wild type of SARS-CoV-2. Conclusions: Apart from immunosuppressive therapy, the humoral vaccination response is largely affected by nonmodifiable factors in kidney transplant recipients. With the currently leading and clinically easier Omicron variant, this puts into perspective the strategy to significantly enhance the protective efficacy of the available vaccines by reducing or temporarily stopping proliferation inhibitors, not least considering the inherent rejection risk with a possible deterioration of graft function.

Heterologous immunization with BNT162b2 followed by mRNA-1273 in dialysis patients: seroconversion and presence of neutralizing antibodies.

INTRODUCTION: The vital renal replacement therapy makes it impossible for dialysis patients to distance themselves socially. This results in a high risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and developing coronavuris disease 2019, with excess mortality due to disease burden and immunosuppression. We determined the efficacy of a 100-µg booster of mRNA-1273 (Moderna, Cambridge, MA, USA) 6 months after two doses of BNT162b2 (BioNTech/Pfizer, Mainz, Germany/New York, USA) in 194 SARS-CoV-2-naïve dialysis patients. METHODS: Anti-SARS-CoV-2 spike antibodies were measured with the Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics, Mannheim, Germany) 4 and 10-12 weeks after two doses of BNT162b2 as well as 4 weeks after the mRNA-1273 booster. The presence of neutralizing antibodies was measured by the SARS-CoV-2 Surrogate Virus Neutralization Test (GenScript Biotech, Piscataway, NJ, USA). Two different cut-offs for positivity were used, one according to the manufacturer's specifications and one correlating with positivity in a plaque reduction neutralization test (PRNT). Receiver operating characteristics analyses were performed to match the anti-SARS-CoV-2 spike antibody cut-offs with the cut-offs in the surrogate neutralization assay accordingly. RESULTS: Any level of immunoreactivity determined by the anti-SARS-CoV-2 spike antibody assay was found in 87.3% (n = 144/165) and 90.6% (n = 164/181) of patients 4 and 10-12 weeks, respectively, after two doses of BNT162b2. This was reduced to 68.5% or 60.6% 4 weeks and 51.7% or 35.4% 10-12 weeks, respectively, when using the ROC cut-offs for neutralizing antibodies in the surrogate neutralization test (manufacturer's cut-off ≥103 U/mL and cut-off correlating with PRNT ≥196 U/mL). Four weeks after the mRNA-1273 booster, the concentration of anti-SARS-CoV-2 spike antibodies increased to 23 119.9 U/mL and to 97.3% for both cut-offs of neutralizing antibodies. CONCLUSION: Two doses of BNT162b2 followed by one dose of mRNA-1273 within 6 months in patients receiving maintenance dialysis resulted in significant titres of SARS-CoV-2 spike antibodies. While two doses of mRNA vaccine achieved adequate humoral immunity in a minority, the third vaccination boosts the development of virus-neutralizing quantities of SARS-CoV-2 spike antibodies (against wild-type SARS-CoV-2) in almost all patients.

Multi-Level Computational Modeling of Anti-Cancer Dendritic Cell Vaccination Utilized to Select Molecular Targets for Therapy Optimization.

Dendritic cells (DCs) can be used for therapeutic vaccination against cancer. The success of this therapy depends on efficient tumor-antigen presentation to cytotoxic T lymphocytes (CTLs) and the induction of durable CTL responses by the DCs. Therefore, simulation of such a biological system by computational modeling is appealing because it can improve our understanding of the molecular mechanisms underlying CTL induction by DCs and help identify new strategies to improve therapeutic DC vaccination for cancer. Here, we developed a multi-level model accounting for the life cycle of DCs during anti-cancer immunotherapy. Specifically, the model is composed of three parts representing different stages of DC immunotherapy - the spreading and bio-distribution of intravenously injected DCs in human organs, the biochemical reactions regulating the DCs' maturation and activation, and DC-mediated activation of CTLs. We calibrated the model using quantitative experimental data that account for the activation of key molecular circuits within DCs, the bio-distribution of DCs in the body, and the interaction between DCs and T cells. We showed how such a data-driven model can be exploited in combination with sensitivity analysis and model simulations to identify targets for enhancing anti-cancer DC vaccination. Since other previous works show how modeling improves therapy schedules and DC dosage, we here focused on the molecular optimization of the therapy. In line with this, we simulated the effect in DC vaccination of the concerted modulation of combined intracellular regulatory processes and proposed several possibilities that can enhance DC-mediated immunogenicity. Taken together, we present a comprehensive time-resolved multi-level model for studying DC vaccination in melanoma. Although the model is not intended for personalized patient therapy, it could be used as a tool for identifying molecular targets for optimizing DC-based therapy for cancer, which ultimately should be tested in in vitro and in vivo experiments.

Contrasting co-occurrence patterns of photobiont and cystobasidiomycete yeast associated with common epiphytic lichen species.

The popular dual definition of lichen symbiosis is under question with recent findings of additional microbial partners living within the lichen body. Here we compare the distribution and co-occurrence patterns of lichen photobiont and recently described secondary fungus (Cyphobasidiales yeast) to evaluate their dependency on lichen host fungus (mycobiont). We sequenced the nuclear internal transcribed spacer (ITS) strands for mycobiont, photobiont, and yeast from six widespread northern hemisphere epiphytic lichen species collected from 25 sites in Switzerland and Estonia. Interaction network analyses and multivariate analyses were conducted on operational taxonomic units based on ITS sequence data. Our study demonstrates the frequent presence of cystobasidiomycete yeasts in studied lichens and shows that they are much less mycobiont-specific than the photobionts. Individuals of different lichen species growing on the same tree trunk consistently hosted the same or closely related mycobiont-specific Trebouxia lineage over geographic distances while the cystobasidiomycete yeasts were unevenly distributed over the study area - contrasting communities were found between Estonia and Switzerland. These results contradict previous findings of high mycobiont species specificity of Cyphobasidiales yeast at large geographic scales. Our results suggest that the yeast might not be as intimately associated with the symbiosis as is the photobiont.

Epiphytes in wooded pastures: Isolation matters for lichen but not for bryophyte species richness.

Sylvo-pastoral systems are species-rich man-made landscapes that are currently often severely threatened by abandonment or management intensification. At low tree densities, single trees in these systems represent habitat islands for epiphytic cryptogams. Here, we focused on sycamore maple (Acer pseudoplatanus) wooded pastures in the northern European Alps. We assessed per tree species richness of bryophytes and lichens on 90 sycamore maple trees distributed across six study sites. We analysed the effects of a range of explanatory variables (tree characteristics, environmental variables and isolation measures) on the richness of epiphytic bryophytes and lichens and various functional subgroups (based on diaspore size, habitat preference and red list status). Furthermore, we estimated the effect of these variables on the occurrence of two specific bryophyte species (Tayloria rudolphiana, Orthotrichum rogeri) and one lichen species (Lobaria pulmonaria) of major conservation concern. Bryophytes and lichens, as well as their subgroups, were differently and sometimes contrastingly affected by the variables considered: tree diameter at breast height had no significant effect on bryophytes but negatively affected many lichen groups; tree phenological age positively affected red-listed lichens but not red-listed bryophytes; increasing isolation from neighbouring trees negatively affected lichens but not bryophytes. However, the high-priority bryophyte species T. rudolphiana was also negatively affected by increased isolation at small spatial scales. Orthotrichum rogeri was more frequent on young trees and L. pulmonaria was more frequent on trees with thin stems and large crowns. The results indicate that local dispersal is important for lichens, whereas long distance dispersal seems to be more important for colonisation by bryophytes. Furthermore, our study highlights that different conservation measures need to be taken depending on the taxonomic and functional species group or the individual species that is addressed. In practice, for the conservation of a high overall richness in sylvo-pastoral systems, it is crucial to sustain not only old and large trees but rather a wide range of tree sizes and ages.

Magnetic Resonance Imaging Versus Ultrasound as the Initial Imaging Modality for Pediatric and Young Adult Patients With Suspected Appendicitis.

BACKGROUND: While ultrasound (US), given its lack of ionizing radiation, is currently the recommended initial imaging study of choice for the diagnosis of appendicitis in pediatric and young adult patients, it does have significant shortcomings. US is time-intensive and operator dependent and results in frequent inconclusive studies, thus necessitating further imaging and admission for observation or repeat clinical visits. A rapid focused magnetic resonance imaging (MRI) for appendicitis has been shown to have definitive sensitivity and specificity, similar to computed tomography but without radiation and offers a potential alternative to US. OBJECTIVE: In this single-center prospective cohort study, we sought to determine the difference in total length of stay and charges between rapid MRI and US as the initial imaging modality in pediatric and young adult patients presenting to the emergency department (ED) with suspected appendicitis. We hypothesized that rapid MRI would be more efficient and cost-effective than US as the initial imaging modality in the ED diagnosis of appendicitis. METHODS: A prospective randomized cohort study of consecutive patients was conducted in patients 2 to 30 years of age in an academic ED with access to both rapid MRI and US imaging modalities 24/7. Prior to the start of the study, the days of the week were randomized to either rapid MRI or US as the initial imaging modality. Physicians evaluated patients with suspected appendicitis per their usual manner. If the physician decided to obtain radiologic imaging, the predetermined imaging modality for the day of the week was used. All decisions regarding other diagnostic testing and/or further imaging were left to the physician's discretion. Time intervals (minutes) between triage, order placement, start of imaging, end of imaging, image result, and disposition (discharge vs. admission), as well as total charges (diagnostic testing, imaging, and repeat ED visits) were recorded. RESULTS: Over a 100-day period, 82 patients were imaged to evaluate for appendicitis; 45 of 82 (55%) of patients were in the US-first group, and 37 of 82 (45%) patients were in the rapid MRI-first group. There were no differences in patient demographics or clinical characteristics between the groups and no cases of missed appendicitis in either group. Eleven of 45 (24%) of US-first patients had inconclusive studies, resulting in follow-up rapid MRI and five return ED visits contrasted with no inconclusive studies or return visits (p < 0.05) in the rapid MRI group. The rapid MRI compared to US group was associated with longer ED length of stay (mean difference = 100 minutes; 95% confidence interval [CI] = 35-169 minutes) and increased ED charges (mean difference = $4,887; 95% CI = $1,821-$8,513). CONCLUSIONS: In the diagnosis of appendicitis, US-first imaging is more time-efficient and less costly than rapid MRI despite inconclusive studies after US imaging. Unless the process of obtaining a rapid MRI becomes more efficient and less expensive, US should be the first-line imaging modality for appendicitis in patients 2 to 30 years of age.

Predictors of Nondiagnostic Ultrasound for Appendicitis.

BACKGROUND: Ionizing radiation and cost make ultrasound (US), when available, the first imaging study for the diagnosis of suspected pediatric appendicitis. US is less sensitive and specific than computed tomography (CT) or magnetic resonance imaging (MRI) scans, which are often performed after nondiagnostic US. OBJECTIVES: We sought to determine predictors of nondiagnostic US in order to guide efficient ordering of imaging studies. METHODS: A prospective cohort study of consecutive patients 4 to 30 years of age with suspected appendicitis took place at an emergency department with access to 24/7 US, MRI, and CT capabilities. Patients with US as their initial study were identified. Clinical (i.e., duration of illness, highest fever, and right lower quadrant pain) and demographic (i.e., age and sex) variables were collected. Body mass index (BMI) was calculated based on Centers for Disease Control and Prevention criteria; BMI >85th percentile was categorized as overweight. Patients were followed until day 7. Univariate and stepwise multivariate logistic regression analysis was performed. RESULTS: Over 3 months, 106 patients had US first for suspected appendicitis; 52 (49%) had nondiagnostic US results. Eighteen patients had appendicitis, and there were no missed cases after discharge. On univariate analysis, male sex, a yearly increase in age, and overweight BMI were associated with nondiagnostic US (p < 0.05). In the multivariate model, only BMI (odds ratio 4.9 [95% CI 2.0-12.2]) and age (odds ratio 1.1 [95% CI 1.02-1.20]) were predictors. Sixty-eight percent of nondiagnostic US results occurred in overweight patients. CONCLUSION: Overweight and older patients are more likely to have a nondiagnostic US or appendicitis, and it may be more efficient to consider alternatives to US first for these patients. Also, this information about the accuracy of US to diagnose suspected appendicitis may be useful to clinicians who wish to engage in shared decision-making with the parents or guardians of children regarding imaging options for children with acute abdominal pain.

Barcoding lichen-forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation.

Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database-currently the most complete database for lichen-forming fungi-can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).

The Effects of Increased Protein Intake on Fullness: A Meta-Analysis and Its Limitations.

BACKGROUND: Higher protein intake has been implicated in weight management because of its appetitive properties. However, the effects of protein intake on appetitive sensations such as fullness have not been systematically assessed. Meta-analysis is a useful technique to evaluate evidence of an intervention's effect on testable outcomes, but it also has important limitations. OBJECTIVE: The primary aim of this study was to synthesize the available evidence on the effect of protein intake on fullness using a quantitative meta-analysis and a secondary directional analysis using the vote-counting procedure. A tertiary aim was to address limitations of meta-analyses as they pertain to findings from this meta-analysis. DESIGN: We searched multiple databases for interventional studies that evaluated the effect of increased protein intake on fullness ratings. Inclusion criteria for both analyses were as follows: healthy human participants, preload studies that utilized intact dietary protein, delivery of protein load orally, and studies reporting fullness as an outcome. For the meta-analysis, an additional criterion was that the studies also needed to report 2- to 4-hour area under the curve value for fullness. RESULTS: Five studies met all criteria for the meta-analysis. Twenty-eight studies met all criteria for the directional analysis. The meta-analysis indicated higher protein preloads have a greater effect on fullness than lower protein preloads (overall effect estimate: 2,435.74 mm.240 min, (95% CI 1,375.18 to 3,496.31 mm.240 min; P<0.0001). The directional analysis also revealed a positive effect on fullness with higher protein preloads (P<0.01). Many related scientifically rigorous studies were excluded from the analysis because analytical criteria required a narrowly focused research question. CONCLUSIONS: The present analyses show that higher protein preloads increase fullness ratings more than lower protein preloads under tightly defined conditions. Extrapolation of findings to common conditions outside the specified criteria of this analysis must be made cautiously, as must speculation about the influence of fullness sensations on ingestive behavior, body weight, and various health outcomes.

Effects of Management on Lichen Species Richness, Ecological Traits and Community Structure in the Rodnei Mountains National Park (Romania).

Lichens are valuable bio-indicators for evaluating the consequences of human activities that are increasingly changing the earth's ecosystems. Since a major objective of national parks is the preservation of biodiversity, our aim is to analyse how natural resource management, the availability of lichen substrates and environmental parameters influence lichen diversity in Rodnei Mountains National Park situated in the Eastern Carpathians. Three main types of managed vegetation were investigated: the transhumance systems in alpine meadows, timber exploitation in mixed and pure spruce forests, and the corresponding conserved sites. The data were sampled following a replicated design. For the analysis, we considered not only all lichen species, but also species groups from different substrates such as soil, trees and deadwood. The lichen diversity was described according to species richness, red-list status and substrate-specialist species richness. The variation in species composition was related to the environmental variables. Habitat management was found to negatively influence species richness and alter the lichen community composition, particularly for threatened and substrate-specialist species. It reduced the mean level of threatened species richness by 59%, when all lichen species were considered, and by 81%, when only epiphytic lichens were considered. Management-induced disturbance significantly decreased lichen species richness in forest landscapes with long stand continuity. The diversity patterns of the lichens indicate a loss of species richness and change in species composition in areas where natural resources are still exploited inside the borders of the national park. It is thus imperative for protected areas, in particular old-growth forests and alpine meadows, to receive more protection than they have received in the past to ensure populations of the characteristic species remain viable in the future.

European phylogeography of the epiphytic lichen fungus Lobaria pulmonaria and its green algal symbiont.

In lichen symbiosis, fungal and algal partners form close associations, often codispersed by vegetative propagules. Due to the particular interdependence, processes such as colonization, dispersal or genetic drift are expected to result in congruent patterns of genetic structure in the symbionts. To study the population structure of an obligate symbiotic system in Europe, we genotyped the fungal and algal symbionts of the epiphytic lichen Lobaria pulmonaria at eight and seven microsatellite loci, respectively, and analysed about 4300 L. pulmonaria thalli from 142 populations from the species' European distribution range. Based on a centroid approach, which localizes centres of genetic differentiation with a high frequency of geographically restricted alleles, we identified the South Italy-Balkan region as the primary glacial refugial area of the lichen symbiosis. Procrustean rotation analysis and a distance congruence test between the fungal and algal population graphs indicated general concordance between the phylogeographies of the symbionts. The incongruent patterns found in areas of postglacial recolonization may show the presence of an additional refugial area for the fungal symbiont, and the impact that horizontal photobiont transmission and different mutation rates of the symbionts have on their genotypic associations at a continental scale.

Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments.

Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis in macrophages. However, mechanisms by which M. tuberculosis adapts to acidic environments are poorly understood. In this study, we analysed the physiological functions of OmpATb, a surface-accessible protein of M. tuberculosis. OmpATb did not complement the permeability defects of a Mycobacterium smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of M. tuberculosis indicating that OmpATb is not a general porin. Chemical analysis of low-pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of M. tuberculosis. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which M. tuberculosis neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that M. tuberculosis has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of M. tuberculosis to acidic environments in vitro but not in mice.

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest.

The success of Mycobacterium tuberculosis depends on its ability to survive within host macrophages. Here, M. tuberculosis avoids the acidic, hydrolytically competent environment of the phagolysosome by arresting phagosome maturation. Having shown previously that a M. tuberculosis mutant deficient in lipoprotein signal peptidase (LspA) is strongly attenuated in vivo in a mouse model of infection, we now studied putative mechanisms involved in attenuation of the lspA : : aph mutant at a cellular level. In this work we investigated the ability of the mutant to interfere with two host defence mechanisms, i.e. Toll-like receptor (TLR)2-dependent immune response and phagosome maturation. While mycobacterial lipoproteins have been reported to trigger a TLR2 signalling pathway critical for innate immune responses, we found that growth control of the lspA : : aph mutant was independent of TLR2. In addition, the lspA : : aph mutant arrested phagosome maturation to an extent similar to that of the wild-type, as measured by lysosomal-associated membrane protein 1 (LAMP1) co-localization and intraphagosomal pH. These observations demonstrate severe attenuation even in the presence of arrested phagosome maturation, and point to a role for the early phagosome in growth restriction of the M. tuberculosis lspA mutant.

Mycobacterium tuberculosis prevents inflammasome activation.

Mycobacterium tuberculosis (Mtb) parasitizes host macrophages and subverts host innate and adaptive immunity. Several cytokines elicited by Mtb are mediators of mycobacterial clearance or are involved in tuberculosis pathology. Surprisingly, interleukin-1beta (IL-1beta), a major proinflammatory cytokine, has not been implicated in host-Mtb interactions. IL-1beta is activated by processing upon assembly of the inflammasome, a specialized inflammatory caspase-activating protein complex. Here, we show that Mtb prevents inflammasome activation and IL-1beta processing. An Mtb gene, zmp1, which encodes a putative Zn(2+) metalloprotease, is required for this process. Infection of macrophages with zmp1-deleted Mtb triggered activation of the inflammasome, resulting in increased IL-1beta secretion, enhanced maturation of Mtb containing phagosomes, improved mycobacterial clearance by macrophages, and lower bacterial burden in the lungs of aerosol-infected mice. Thus, we uncovered a previously masked role for IL-1beta in the control of Mtb and a mycobacterial system that prevents inflammasome and, therefore, IL-1beta activation.

Anaerobic arginine metabolism of Mycobacterium tuberculosis is mediated by arginine deiminase (arcA), but is not essential for chronic persistence in an aerogenic mouse model of infection.

In many pathogens, degradation of arginine via the arginine deiminase pathway supports anaerobic metabolism. Here we show by deletion of Rv1001 (arcA) in Mycobacterium tuberculosis that this gene functions as an arginine deiminase. Arginine metabolism in the presence of oxygen was not affected by the mutation, indicating a separate pathway for arginine degradation under aerobic conditions. Following aerosol infection in mice, the DeltaarcA mutant and wild-type strain of M. tuberculosis multiplied and persisted in infected organs in a similar fashion.

Characterization of a Mycobacterium tuberculosis mutant deficient in pH-sensing adenylate cyclase Rv1264.

Mycobacterium tuberculosis open reading frame Rv1264 encodes an adenylate cyclase that exhibits its highest enzymatic activity at an acidic pH of 6.0. This is the pH M. tuberculosis encounters in the phagosome. Consequently Rv1264 has been suggested to sense the phagosomal milieu resulting in adaption of M. tuberculosis to its intracellular niche. A targeted knock-out mutant deficient in Rv1264, however, exhibits wild-type virulence.

Genetically determined susceptibility to tuberculosis in mice causally involves accelerated and enhanced recruitment of granulocytes.

Classical twin studies and recent linkage analyses of African populations have revealed a potential involvement of host genetic factors in susceptibility or resistance to Mycobacterium tuberculosis infection. In order to identify the candidate genes involved and test their causal implication, we capitalized on the mouse model of tuberculosis, since inbred mouse strains also differ substantially in their susceptibility to infection. Two susceptible and two resistant mouse strains were aerogenically infected with 1,000 CFU of M. tuberculosis, and the regulation of gene expression was examined by Affymetrix GeneChip U74A array with total lung RNA 2 and 4 weeks postinfection. Four weeks after infection, 96 genes, many of which are involved in inflammatory cell recruitment and activation, were regulated in common. One hundred seven genes were differentially regulated in susceptible mouse strains, whereas 43 genes were differentially expressed only in resistant mice. Data mining revealed a bias towards the expression of genes involved in granulocyte pathophysiology in susceptible mice, such as an upregulation of those for the neutrophil chemoattractant LIX (CXCL5), interleukin 17 receptor, phosphoinositide kinase 3 delta, or gamma interferon-inducible protein 10. Following M. tuberculosis challenge in both airways or peritoneum, granulocytes were recruited significantly faster and at higher numbers in susceptible than in resistant mice. When granulocytes were efficiently depleted by either of two regimens at the onset of infection, only susceptible mice survived aerosol challenge with M. tuberculosis significantly longer than control mice. We conclude that initially enhanced recruitment of granulocytes contributes to susceptibility to tuberculosis.

Interaction of Rv1625c, a mycobacterial class IIIa adenylyl cyclase, with a mammalian congener.

The adenylyl cyclase Rv1625c from Mycobacterium tuberculosis codes for a protein with six transmembrane spans and a catalytic domain, i.e. it corresponds to one half of the pseudoheterodimeric mammalian adenylyl cyclases (ACs). Rv1625c is active as a homodimer. We investigated the role of the Rv1625c membrane domain and demonstrate that it efficiently dimerizes the protein resulting in a 7.5-fold drop in K(m) for ATP. Next, we generated a duplicated Rv1625c AC dimer by a head-to-tail concatenation. This produced an AC with a domain order exactly as the mammalian pseudoheterodimers. It displayed positive cooperativity and a 60% increase of v(max) compared with the Rv1625c monomer. Further, we probed the compatibility of mycobacterial and mammalian membrane domains. The second membrane anchor in the Rv1625c concatamer was replaced with membrane domain I or II of rabbit type V AC. The mycobacterial and either mammalian membrane domains are compatible with each other and both recombinant proteins are active. A M. tuberculosis Rv1625c knockout strain was assayed in a mouse infection model. In vitro growth characteristics and in vivo organ infection and mortality were unaltered in the knockout strain indicating that AC Rv1625c alone is not a virulence factor.

Common and unique gene expression signatures of human macrophages in response to four strains of Mycobacterium avium that differ in their growth and persistence characteristics.

Classification of pathogenic species according to the distinct host transcriptional responses that they elicit may become a relevant tool for microarray-based diagnosis of infection. Individual strains of Mycobacterium avium, an opportunistic pathogen in humans, have previously been shown to differ in terms of growth and persistence. In order to cover a wide spectrum of virulence, we selected four M. avium isolates (2151SmO, 2151SmT, SE01, TMC724) that have distinct intramacrophage replication characteristics and cause differential activation in human macrophages. Following infection with each of these strains, the expression of 12,558 genes in human macrophages was systematically analyzed by microarray technology. Fifty genes (including genes encoding proinflammatory cytokines, chemokines, signaling, and adhesion molecules) were differentially expressed more than twofold in response to all of the M. avium isolates investigated and therefore constitute a common macrophage signature in response to M. avium. The magnitude of regulation of most of these genes was directly correlated with the host cell-activating capacity of the particular M. avium strain. The regulation of a number of genes not previously associated with mycobacterial infections was apparent; these genes included genes encoding lymphocyte antigen 64 and myosin X. In addition, individual response patterns typical for some M. avium isolates could be defined by the pronounced upregulation of interleukin-12p40 (IL-12p40) (in the case of 2151SmO) or the specific upregulation of SOCS-1 and IL-10 (in the case of SE01) in macrophages. TMC724, a strain of avian origin, could not be classified by any one of these schemes, possibly indicating the limits of pathogen categorization solely by immune response signatures.