Identification of ß2 microglobulin, the product of B2M gene, as a Host Factor for Vaccinia Virus Infection by Genome-Wide CRISPR genetic screens.

Genome-wide genetic screens are powerful tools to identify genes that act as host factors of viruses. We have applied this technique to analyze the infection of HeLa cells by Vaccinia virus, in an attempt to find genes necessary for infection. Infection of cell populations harboring single gene inactivations resulted in no surviving cells, suggesting that no single gene knock-out was able to provide complete resistance to Vaccinia virus and thus allow cells to survive infection. In the absence of an absolute infection blockage, we explored if some gene inactivations could provide partial protection leading to a reduced probability of infection. Multiple experiments using modified screening procedures involving replication restricted viruses led to the identification of multiple genes whose inactivation potentially increase resistance to infection and therefore cell survival. As expected, significant gene hits were related to proteins known to act in virus entry, such as ITGB1 and AXL as well as genes belonging to their downstream related pathways. Additionally, we consistently found ß2-microglobulin, encoded by the B2M gene, among the screening top hits, a novel finding that was further explored. Inactivation of B2M resulted in 54% and 91% reduced VV infection efficiency in HeLa and HAP1 cell lines respectively. In the absence of B2M, while virus binding to the cells was unaffected, virus internalization and early gene expression were significantly diminished. These results point to ß2-microglobulin as a relevant factor in the Vaccinia virus entry process.

Enhancing SARS-CoV-2 Surveillance through Regular Genomic Sequencing in Spain: The RELECOV Network.

Millions of SARS-CoV-2 whole genome sequences have been generated to date. However, good quality data and adequate surveillance systems are required to contribute to meaningful surveillance in public health. In this context, the network of Spanish laboratories for coronavirus (RELECOV) was created with the main goal of promoting actions to speed up the detection, analyses, and evaluation of SARS-CoV-2 at a national level, partially structured and financed by an ECDC-HERA-Incubator action (ECDC/GRANT/2021/024). A SARS-CoV-2 sequencing quality control assessment (QCA) was developed to evaluate the network's technical capacity. QCA full panel results showed a lower hit rate for lineage assignment compared to that obtained for variants. Genomic data comprising 48,578 viral genomes were studied and evaluated to monitor SARS-CoV-2. The developed network actions showed a 36% increase in sharing viral sequences. In addition, analysis of lineage/sublineage-defining mutations to track the virus showed characteristic mutation profiles for the Delta and Omicron variants. Further, phylogenetic analyses strongly correlated with different variant clusters, obtaining a robust reference tree. The RELECOV network has made it possible to improve and enhance the genomic surveillance of SARS-CoV-2 in Spain. It has provided and evaluated genomic tools for viral genome monitoring and characterization that make it possible to increase knowledge efficiently and quickly, promoting the genomic surveillance of SARS-CoV-2 in Spain.

A Founder Effect Led Early SARS-CoV-2 Transmission in Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome analysis has identified five large clades worldwide which emerged in 2019 (19A and 19B) and in 2020 (20A, 20B, and 20C). This study aimed to analyze the diffusion of SARS-CoV-2 in Spain using maximum-likelihood phylogenetic and Bayesian phylodynamic analyses. The most recent common ancestor (MRCA) of the SARS-CoV-2 pandemic was estimated to have emerged in Wuhan, China, around 24 November 2019. Phylogenetic analyses of the first 12,511 SARS-CoV-2 whole-genome sequences obtained worldwide, including 290 from 11 different regions of Spain, revealed 62 independent introductions of the virus in the country. Most sequences from Spain were distributed in clades characterized by a D614G substitution in the S gene (20A, 20B, and 20C) and an L84S substitution in ORF8 (19B) with 163 and 118 sequences, respectively, with the remaining sequences branching in 19A. A total of 110 (38%) sequences from Spain grouped in four different monophyletic clusters of clade 20A (20A-Sp1 and 20A-Sp2) and 19B clade (19B-Sp1 and 19B-Sp2) along with sequences from 29 countries worldwide. The MRCAs of clusters 19A-Sp1, 20A-Sp1, 19A-Sp2, and 20A-Sp2 were estimated to have occurred in Spain around 21 and 29 January and 6 and 17 February 2020, respectively. The prevalence of clade 19B in Spain (40%) was by far higher than in any other European country during the first weeks of the epidemic, probably as a result of a founder effect. However, this variant was replaced by G614-bearing viruses in April. In vitro assays showed an enhanced infectivity of pseudotyped virions displaying the G614 substitution compared with those having D614, suggesting a fitness advantage of D614G.IMPORTANCE Multiple SARS-CoV-2 introductions have been detected in Spain, and at least four resulted in the emergence of locally transmitted clusters that originated not later than mid-February, with further dissemination to many other countries around the world, and a few weeks before the explosion of COVID-19 cases detected in Spain during the first week of March. The majority of the earliest variants detected in Spain branched in the clade 19B (D614 viruses), which was the most prevalent clade during the first weeks of March, pointing to a founder effect. However, from mid-March to June 2020, G614-bearing viruses (clades 20A, 20B, and 20C) overcame D614 variants in Spain, probably as a consequence of an evolutionary advantage of this substitution in the spike protein. A higher infectivity of G614-bearing viruses than D614 variants was detected, suggesting that this substitution in SARS-CoV-2 spike protein could be behind the variant shift observed in Spain.

Cryptococcus neoformans can form titan-like cells in vitro in response to multiple signals.

Cryptococcus neoformans is an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain them in vitro. In this work, we describe in vitro conditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2 enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and it was independent of the cryptococcal serotype/species. Transition to titan-like cell was impaired by pharmacological inhibition of PKC signaling pathway. Analysis of the gene expression profile during the transition to titan-like cells showed overexpression of enzymes involved in carbohydrate metabolism, as well as proteins from the coatomer complex, and related to iron metabolism. Indeed, we observed that iron limitation also induced the formation of titan cells. Our gene expression analysis also revealed other elements involved in titan cell formation, such as calnexin, whose absence resulted in appearance of abnormal large cells even in regular rich media. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation.

Frequency of low-level and high-level mosaicism in sporadic retinoblastoma: genotype-phenotype relationships.

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene.

Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.

Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae.

Streptococcus pneumoniae is the main etiological agent of community-acquired pneumonia and a major cause of mortality and morbidity among children and the elderly. Genome sequencing of several pneumococcal strains revealed valuable information about the potential proteins and genetic diversity of this prevalent human pathogen. However, little is known about its transcriptional regulation and its small regulatory noncoding RNAs. In this study, we performed deep sequencing of the S. pneumoniae TIGR4 strain RNome to identify small regulatory RNA candidates expressed in this pathogen. We discovered 1047 potential small RNAs including intragenic, 5'- and/or 3'-overlapping RNAs and 88 small RNAs encoded in intergenic regions. With this approach, we recovered many of the previously identified intergenic small RNAs and identified 68 novel candidates, most of which are conserved in both sequence and genomic context in other S. pneumoniae strains. We confirmed the independent expression of 17 intergenic small RNAs and predicted putative mRNA targets for six of them using bioinformatics tools. Preliminary results suggest that one of these six is a key player in the regulation of competence development. This study is the biggest catalog of small noncoding RNAs reported to date in S. pneumoniae and provides a highly complete view of the small RNA network in this pathogen.

Monkeypox virus genomic accordion strategies.

The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome's low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established "genomic accordion" evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.

Genomic Analysis of West Nile Virus Lineage 1 Detected in Mosquitoes during the 2020-2021 Outbreaks in Andalusia, Spain.

Emerging infectious diseases are one of the most important global health challenges because of their impact on human and animal health. The vector-borne West Nile virus (WNV) is transmitted between birds by mosquitos, but it can also infect humans and horses causing disease. The local circulation of WNV in Spain has been known for decades, and since 2010, there have been regular outbreaks in horses, although only six cases were reported in humans until 2019. In 2020, Spain experienced a major outbreak with 77 human cases, which was followed by 6 additional cases in 2021, most of them in the Andalusian region (southern Spain). This study aimed to characterize the genomes of the WNV circulating in wild-trapped mosquitoes during 2020 and 2021 in Andalusia. We sequenced the WNV consensus genome from two mosquito pools and carried out the phylogenetic analyses. We also compared the obtained genomes with those sequenced from human samples obtained during the outbreak and the genomes obtained previously in Spain from birds (2007 and 2017), mosquitoes (2008) and horses (2010) to better understand the eco-epidemiology of WNV in Spain. As expected, the WNV genomes recovered from mosquito pools in 2020 were closely related to those recovered from humans of the same outbreak. In addition, the strain of WNV circulating in 2021 was highly related to the WNV strain that caused the 2020 outbreak, suggesting that WNV is overwintering in the area. Consequently, future outbreaks of the same strain may occur in in the future.

Role of PatAB Transporter in Efflux of Levofloxacin in Streptococcus pneumoniae.

PatAB is an ABC bacterial transporter that facilitates the export of antibiotics and dyes. The overexpression of patAB genes conferring efflux-mediated fluoroquinolone resistance has been observed in several laboratory strains and clinical isolates of Streptococcus pneumoniae. Using transformation and whole-genome sequencing, we characterized the fluoroquinolone-resistance mechanism of one S. pneumoniae clinical isolate without mutations in the DNA topoisomerase genes. We identified the PatAB fluoroquinolone efflux-pump as the mechanism conferring a low-level resistance to ciprofloxacin (8 µg/mL) and levofloxacin (4 µg/mL). Genetic transformation experiments with different amplimers revealed that the entire patA plus the 5'-terminus of patB are required for levofloxacin-efflux. By contrast, only the upstream region of the patAB operon, plus the region coding the N-terminus of PatA containing the G39D, T43A, V48A and D100N amino acid changes, are sufficient to confer a ciprofloxacin-efflux phenotype, thus suggesting differences between fluoroquinolones in their binding and/or translocation pathways. In addition, we identified a novel single mutation responsible for the constitutive and ciprofloxacin-inducible upregulation of patAB. This mutation is predicted to destabilize the putative rho-independent transcriptional terminator located upstream of patA, increasing transcription of downstream genes. This is the first report demonstrating the role of the PatAB transporter in levofloxacin-efflux in a pneumoccocal clinical isolate.

Age-dependent nasal immune responses in non-hospitalized bronchiolitis children.

Bronchiolitis in children is associated with significant rates of morbidity and mortality. Many studies have been performed using samples from hospitalized bronchiolitis patients, but little is known about the immunological responses from infants suffering from mild/moderate bronchiolitis that do not require hospitalization. We have studied a collection of nasal lavage fluid (NLF) samples from outpatient bronchiolitis children as a novel strategy to unravel local humoral and cellular responses, which are not fully characterized. The children were age-stratified in three groups, two of them (GI under 2-months, GII between 2-4 months) presenting a first episode of bronchiolitis, and GIII (between 4 months and 2 years) with recurrent respiratory infections. Here we show that elevated levels of pro-inflammatory cytokines (IL1ß, IL6, TNFα, IL18, IL23), regulatory cytokines (IL10, IL17A) and IFNγ were found in the three bronchiolitis cohorts. However, little or no change was observed for IL33 and MCP1, at difference to previous results from bronchiolitis hospitalized patients. Furthermore, our results show a tendency to IL1ß, IL6, IL18 and TNFα increased levels in children with mild pattern of symptom severity and in those in which non RSV respiratory virus were detected compared to RSV+ samples. By contrast, no such differences were found based on gender distribution. Bronchiolitis NLFs contained more IgM, IgG1, IgG3 IgG4 and IgA than NLF from their age-matched healthy controls. NLF from bronchiolitis children predominantly contained neutrophils, and also low frequency of monocytes and few CD4+ and CD8+ T cells. NLF from infants older than 4-months contained more intermediate monocytes and B cell subsets, including naïve and memory cells. BCR repertoire analysis of NLF samples showed a biased VH1 usage in IgM repertoires, with low levels of somatic hypermutation. Strikingly, algorithmic studies of the mutation profiles, denoted antigenic selection on IgA-NLF repertoires. Our results support the use of NLF samples to analyze immune responses and may have therapeutic implications.

Monkeypox outbreak in Madrid (Spain): Clinical and virological aspects.

BACKGROUND: Monkeypox is the most prevalent Orthopoxvirus zoonosis infection since the eradication of smallpox. The current multi-country outbreak involves five WHO regions affecting mainly Europe. Accurate clinical and virological aspects of the disease outside endemic areas are needed. METHODS: We performed an observational study of cases diagnosed in Madrid (Spain) (May/June 2022). Confirmation from vesicular lesions swabs, Orthopoxvirus real-time PCR, sequencing, phylogenetic analysis, and direct detection by Electron microscopy was performed. In addition, a structured epidemiological questionnaire was completed systematically to gather sociodemographic, clinical, and behavioral data from all confirmed cases. FINDINGS: We extracted data from 48 patients, all cisgender men. The median age was 35 years (IQR 29 - 44), and 87.5% were MSM. The most prevalent symptoms were the presence of vesicular-umbilicated and pseudo-pustular skin lesions (93.8%), asthenia (66.6%), and fever (52.1%). In addition, the location of the lesions in the genital or perianal area was related to the role in sexual intercourse (p<0.001). Sequencing analysis indicated the virus circulating in Spain belongs to the western African clade. Like the other European cases in the outbreak, the Spanish isolates are a direct descendant of viruses previously detected in Nigeria, the UK, Singapore, and Israel in 2017-2018. CONCLUSIONS: Monkeypox is an emerging infectious disease in Europe where community transmission is reported, mainly in MSM. The first symptom was skin lesions instead of classical fever and rash. The disease follows a self-limited course, and there have been no cases with a serious presentation or severe complications.

p85beta phosphoinositide 3-kinase regulates CD28 coreceptor function.

CD28 is a receptor expressed on T cells that regulates their differentiation after antigen stimulation to long-term-survival memory T cells. CD28 enhances T-cell receptor signals and reduces expression of CBL ubiquitin ligases, which negatively control T-cell activation. In the absence of CD28 ligation during the primary stimulation, CBL levels remain high and T cells fail to mount an efficient secondary response. CD28 associates with p85alpha, one of the regulatory subunits of phosphoinositide-3-kinase (PI3K), but the relevance of this interaction is debated. We examined here the contribution of the other ubiquitous PI3K regulatory subunit, p85beta, in CD28 function. We describe that p85beta bound to CD28 and to CBL with greater affinity than p85alpha. Moreover, deletion of p85beta impaired CD28-induced intracellular events, including c-CBL and CBL-b down-regulation as well as PI3K pathway activation. This resulted in defective differentiation of activated T cells, which failed to exhibit an efficient secondary immune response. Considering that p85beta-deficient T cells fail in recall responses and that p85beta binds to and regulates CD28 signals, the presented observations suggest the involvement of p85beta in CD28-mediated activation and differentiation of antigen-stimulated T cells.

Tumour biology: senescence in premalignant tumours.

Oncogene-induced senescence is a cellular response that may be crucial for protection against cancer development, but its investigation has so far been restricted to cultured cells that have been manipulated to overexpress an oncogene. Here we analyse tumours initiated by an endogenous oncogene, ras, and show that senescent cells exist in premalignant tumours but not in malignant ones. Senescence is therefore a defining feature of premalignant tumours that could prove valuable in the diagnosis and prognosis of cancer.

Adaptation of the emerging pathogenic yeast Candida auris to high caspofungin concentrations correlates with cell wall changes.

Candida auris has emerged as a fungal pathogen that causes nosocomial outbreaks worldwide. Diseases caused by this fungus are of concern, due to its reduced susceptibility to several antifungals. C. auris exhibits paradoxical growth (PG; defined as growth at high, but not intermediate antifungal concentrations) in the presence of caspofungin (CPF). We have characterized the cellular changes associated with adaptation to CPF. Using EUCAST AFST protocols, all C. auris isolates tested showed PG to CPF, although in some isolates it was more prominent. Most isolates also showed a trailing effect (TE) to micafungin and anidulafungin. We identified two FKS genes in C. auris that encode the echinocandins target, namely ß-1,3-glucan synthase. FKS1 contained the consensus hot-spot (HS) 1 and HS2 sequences. FKS2 only contained the HS1 region which had a change (F635Y), that has been shown to confer resistance to echinocandins in C. glabrata. PG has been characterized in other species, mainly C. albicans, where high CPF concentrations induced an increase in chitin, cell volume and aggregation. In C. auris CPF only induced a slight accumulation of chitin, and none of the other phenomena. RNAseq experiments demonstrated that CPF induced the expression of genes encoding several GPI-anchored cell wall proteins, membrane proteins required for the stability of the cell wall, chitin synthase and mitogen-activated protein kinases (MAPKs) involved in cell integrity, such as BCK2, HOG1 and MKC1 (SLT2). Our work highlights some of the processes induced in C. auris to adapt to echinocandins.

Genomic Background and Phylogeny of cfiA-Positive Bacteroides fragilis Strains Resistant to Meropenem-EDTA.

BACKGROUND: Bacteroides fragilis shows high antimicrobial resistance (AMR) rates and possesses numerous AMR mechanisms. Its carbapenem-resistant strains (metallo-ß-lactamase cfiA-positive) appear as an emergent, evolving clade. METHODS: This work examines the genomes, taxonomy, and phylogenetic relationships with respect to other B. fragilis genomes of two B. fragilis strains (CNM20180471 and CNM20200206) resistant to meropenem+EDTA and other antimicrobial agents. RESULTS: Both strains possessed cfiA genes (cfiA14b and the new cfiA28), along with other AMR mechanisms. The presence of other efflux-pump genes, mexAB/mexJK/mexXY-oprM, acrEF/mdtEF-tolC, and especially cusR, which reduces the entry of carbapenem via the repression of porin OprD, may be related to meropenem-EDTA resistance. None of the detected insertion sequences were located upstream of cfiA. The genomes of these and other B. fragilis strains that clustered together in phylogenetic analyses did not meet the condition of >95% average nucleotide/amino acid identity, or >70% in silico genome-to-genome hybridization similarity, to be deemed members of the same species, although <1% difference in the genomic G+C content was seen with respect to the reference genome B. fragilis NCTC 9343T. CONCLUSIONS: Carbapenem-resistant strains may be considered a distinct clonal entity, and their surveillance is recommended given the ease with which they appear to acquire AMR.

Functional inactivation of CXC chemokine receptor 4-mediated responses through SOCS3 up-regulation.

Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca(2+) flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Galpha(i) pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system.

CCR6 regulates EAE pathogenesis by controlling regulatory CD4+ T-cell recruitment to target tissues.

The T-cell subsets, characterized by their cytokine production profiles and immune regulatory functions, depend on correct in vivo location to interact with accessory or target cells for effective immune responses. Differentiation of naive CD4(+) T cells into effectors is accompanied by sequentially regulated expression of the chemokine receptors responsible for cell recruitment to specific tissues. We studied CCR6 function in EAE, a CD4(+) T-cell-mediated CNS disease characterized by mononuclear infiltration and demyelination. CCR6(-/-) mice showed an altered time course of EAE development, with delayed onset, a higher clinical score, and more persistent symptoms than in controls. An imbalanced cytokine profile and reduced Foxp3(+) cell frequency characterized CNS tissues from CCR6(-/-) compared with CCR6(+/+) mice during the disease effector phase. Transfer of CCR6(+/+) Treg to CCR6(-/-) mice the day before EAE induction reduced the clinical score associated with an increased in infiltrating Foxp3(+) cells and recovery of the cytokine balance in CCR6(-/-) mouse CNS. Competitive assays between CCR6(+/+) and CCR6(-/-) Treg adoptively transferred to CCR6(-/-) mice showed impaired ability of CCR6(-/-) Treg to infiltrate CNS tissues in EAE-affected mice. Our data indicate a CCR6 requirement by CD4(+) Treg to downregulate the CNS inflammatory process and neurological signs associated with EAE.

Statins induce regulatory T cell recruitment via a CCL1 dependent pathway.

The statins, a group of inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, are reported to influence a variety of immune system activities through 3-hydroxy-3-methylglutaryl coenzyme A reductase-dependent and -independent mechanisms. How statin treatment regulates immune system function in vivo nonetheless remains to be fully defined. We analyzed the immunomodulatory effects of lovastatin in a Candida albicans-induced delayed-type hypersensitivity reaction in mice. In this model, lovastatin administration reduced the acute inflammatory response elicited by C. albicans challenge. This anti-inflammatory activity of lovastatin was associated with a shift from a Th1 to a Th2 immune response, as well as an increase in the percentage of regulatory T cells at the inflammation site and in the regional draining lymph node. The lovastatin-induced increase in regulatory T cells in the inflamed skin was dependent on expression of CCL1, a chemokine that is locally up-regulated by statin administration. The anti-inflammatory effect of lovastatin was abrogated in CCL1-deficient mice. These results suggest that local regulation of chemokine expression may be an important process in statin-induced modulation of the immune system.

Genetic Traces of the Francisella tularensis Colonization of Spain, 1998-2020.

More than 1000 humans have acquired the febrile disease tularemia in Spain since the first notification of human cases in 1997. We here aimed to study the recent molecular evolution of the causative bacterium Francisella tularensis during disease establishment in Spain. Single-nucleotide polymorphisms (SNPs) and variable-number tandem repeats (VNTRs) were analyzed in whole-genome sequences (WGS) of F. tularensis. Short-read WGS data for 20 F. tularensis strains from humans infected in the periods 2014-2015 and 2018-2020 in Spain were generated. These data were combined with WGS data of 25 Spanish strains from 1998 to 2008 and two reference strains. Capillary electrophoresis data of VNTR genetic regions were generated and compared with the WGS data for the 11 strains from 2014 to 2015. Evolutionary relationships among strains were analyzed by phylogenetic methods. We identified 117 informative SNPs in a 1,577,289-nucleotide WGS alignment of 47 F. tularensis genomes. Forty-five strains from Spain formed a star-like SNP phylogeny with six branches emerging from a basal common node. The most recently evolved genomes formed four additional star-like structures that were derived from four branches of the basal common node. VNTR copy number variation was detected in two out of 10 VNTR regions examined. Genetic clustering of strains by VNTRs agreed with the clustering by SNPs. The SNP data provided higher resolution among strains than the VNTRs data in all but one cases. There was an excellent correlation between VNTR marker sizing by capillary electrophoresis and prediction from WGS data. The genetic data strongly support that tularemia, indeed, emerged recently in Spain. Distinct genetic patterns of local F. tularensis population expansions imply that the pathogen has colonized a previously disease-free geographical area. We also found that genome-wide SNPs provide higher genetic resolution among F. tularensis genomes than the use of VNTRs, and that VNTR copy numbers can be accurately predicted using short-read WGS data.