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These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Background: Rapid galactomannan tests, such as the sõna Aspergillus GM Lateral Flow Assay (GM‐LFA) and the Aspergillus Galactomannan Ag VIRCLIA® Monotest (GM‐Monotest), which are suitable for the analysis of single samples, have the potential to accelerate diagnosis of invasive aspergillosis (IA). Objectives: To compare the performance of the GM‐Monotest and the GM‐LFA for the diagnosis of IA. Patients/Methods: Two patient cohorts were analysed: adults who had received an allogeneic haematopoietic stem‐cell transplant (alloHSCT‐cohort) and patients with proven/probable IA from a 5‐year period (cross‐sectional IA‐cohort). In the alloHSCT‐cohort, weekly serum samples were tested, whereas in the cross‐sectional IA‐cohort sera and bronchoalveolar lavage fluids were analysed. The diagnostic performance was calculated using two definitions for positivity: (1) a single positive GM result and (2) at least two positive GM results from consecutive samples. IA classification followed EORTC/MSG 2019. Results: The alloHSCT‐cohort included 101 patients. Four had proven/probable IA, 26 possible IA and 71 no IA. The specificity for one positive serum and two consecutively positive sera was 88.7% and 100% (GM‐Monotest) and 85.9% and 98.6% (GM‐LFA). Comparison of ROC curves in the alloHSCT‐cohort showed no significant difference. The cross‐sectional IA‐cohort included 59 patients with proven/probable IA. The sensitivity for one positive sample and two consecutively positive samples was 83.1% and 55.1% (GM‐Monotest) and 86.4% and 71.4% (GM‐LFA). Conclusions: Both assays showed comparable diagnostic performance with a higher sensitivity for the GM‐LFA if two consecutive positive samples were required for positivity. However, due to poor reproducibility, positive GM‐LFA results should always be confirmed. [ABSTRACT FROM AUTHOR]

The DAMIC-M (DArk Matter In CCDs at Modane) experiment employs thick, fully depleted silicon charged-coupled devices (CCDs) to search for dark matter particles with a target exposure of 1 kg-year. A novel skipper readout implemented in the CCDs provides single electron resolution through multiple non-destructive measurements of the individual pixel charge, pushing the detection threshold to the eV-scale. DAMIC-M will advance by several orders of magnitude the exploration of the dark matter particle hypothesis, in particular of candidates pertaining to the so-called "hidden sector." A prototype, the Low Background Chamber (LBC), with 20g of low background Skipper CCDs, has been recently installed at Laboratoire Souterrain de Modane and is currently taking data. We will report the status of the DAMIC-M experiment and first results obtained with LBC commissioning data.

Arginase 1 (Arg1), which converts L-arginine into ornithine and urea, exerts pleiotropic immunoregulatory effects. However, the function of Arg1 in inflammatory bowel disease (IBD) remains poorly characterized. Here, we found that Arg1 expression correlated with the degree of inflammation in intestinal tissues from IBD patients. In mice, Arg1 was upregulated in an IL-4/ IL-13 and intestinal microbiota-dependent manner. Tie2-Cre [Arg1.sup.fl/fl] mice lacking Arg7 in hematopoietic and endothelial cells recovered faster from colitis than Arg7-expressing([Arg1.sup.fl/fl]) littermates. This correlated with decreased vessel density, compositional changes in intestinal microbiota, diminished infiltration by myeloid cells, and an accumulation of intraluminal polyamines that promote epithelial healing. The proresolving effect of Arg1 deletion was reduced by an L-arginine- free diet, but rescued by simultaneous deletion of other L-arginine-metabolizing enzymes, such as Arg2 or Alos2, demonstrating that protection from colitis requires L-arginine. Fecal microbiota transfers from Tie2-Cre [Arg1.sup.fl/fl] mice into WT recipients ameliorated intestinal inflammation, while transfers from WT littermates intoArg7-deficient mice prevented an advanced recovery from colitis. Thus, an increased availability of L-arginine as well as altered intestinal microbiota and metabolic products accounts for the accelerated resolution from colitis in the absence of Arg1. Consequently, L-arginine metabolism may serve as a target for clinical intervention in IBD patients., Introduction Ulcerative colitis (UC) and Crohn's disease (CD) are characterized by an immune-mediated inflammation of the gastrointestinal (GI) tract. Both inflammatory bowel diseases (IBDs) are thought to result from dysregulated [...]

The polarization of macrophages is regulated by transcription factors, such as NF-[kappa]B and activator protein 1 (AP-1). In this manuscript, we delineated the role of the transcription factor Fos-related antigen 1 (Fra-1) during macrophage activation and development of arthritis. Network level interaction analysis of microarray data derived from Fra-1- or Fra-2-deficient macrophages revealed a central role of Fra-1, but not of Fra-2, in orchestrating the expression of genes related to wound response, Toll-like receptor activation, and interleukin signaling. ChIP sequencing and standard ChIP analyses of macrophages identified arginase 1 (Arg1) as a target of Fra-1. Luciferase reporter assays revealed that Fra-1 downregulated Arg1 expression by direct binding to the promoter region. Using macrophage-specific Fra-1- or Fra-2-deficient mice, we observed enhanced expression and activity of Arg1 and a reduction of arthritis in the absence of Fra-1, but not of Fra-2. This phenotype was reversed by treatment with the arginase inhibitor [N.sup.[omega]]-hydroxy-nor-l-arginine, while L-arginine supplementation increased arginase activity and alleviated arthritis, supporting the notion that reduced arthritis in macrophage-specific Fra-1-deficient mice resulted from enhanced Arg1 expression and activity. Moreover, patients with active rheumatoid arthritis (RA) showed increased Fra-1 expression in the peripheral blood and elevated Fra-1 protein in synovial macrophages compared with RA patients in remission. In addition, the Fra-1/ARG1 ratio in synovial macrophages was related to RA disease activity. In conclusion, these data suggest that Fra-1 orchestrates the inflammatory state of macrophages by inhibition of Arg1 expression and thereby impedes the resolution of inflammation., Introduction Macrophages are remarkably diverse in their plasticity, allowing adaptation to a broad range of environmental stimuli. They participate in various processes essential for homeostatic functions and development, but also [...]

To allow an efficient protection against viruses like the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), it is important to avoid their spreading by using filtering face pieces (FFP), which are categorized by different standards according to their filtration efficiency. In this study, we subjected six brands of FFP2 standard masks to three different conditions and subsequently analysed them for their filtration performance to evaluate potentials for reusability. The conditions comprised changes of temperature and air humidity, an exposure to isopropyl alcohol (IPA) and an autoclave sterilization. While four of six masks consisted of electrostatically treated melt blown non-wovens, two masks were fabricated using a nanofibrous multilayer system. Due to the absence of prior electrostatic treatment, the nano-masks did not show a significant change in filtration efficiency when discharged by IPA, unlike the melt blown nonwoven masks showing a significant decrease of filtration efficiency down to around 50% at a particle size of 0.3 μm. However, most melt blown masks maintained a sufficient filtration efficiency after all other treatments with even better results than the nanofibrous masks. This was particularly the case for the capacity to filter smallest particles/droplets with a size of around 0.1 μm, which is below the range of typical filtering standards and important for the retention of virally contaminated nano-aerosols or unattached viruses. After temperature/humidity variation and autoclave sterilization, melt blown masks were able to retain a filtration efficiency up to over 90% at 0.1 μm contrary to nano-masks showing a decrease down to around 70%. Based on their better filtration performance, lower price and potential reusability, we conclude that electret melt blown masks are the preferable type of FFP2 masks. [ABSTRACT FROM AUTHOR]

Martin Röllinghoff, a prominent member of the immunological community in Germany, passed away on November 22, 2022, after being diagnosed with cancer only five months earlier. Born in 1941 in Hamburg, Röllinghoff began his medical studies in Freiburg and later pursued a career in immunology under the guidance of Professor Paul Klein. He made significant contributions to the field, particularly in tumor immunology and host-pathogen interactions. Röllinghoff served as a professor at the Friedrich‐Alexander‐University Erlangen‐Nürnberg and was recognized for his scientific achievements with various honors and awards. He was known for his dedication to mentoring and supporting young scientists. Röllinghoff's impact on the German research landscape will be remembered with respect and gratitude. [Extracted from the article]

RNA vaccines are efficient preventive measures to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. High levels of neutralizing SARS-CoV-2 antibodies are an important component of vaccine-induced immunity. Shortly after the initial two mRNA vaccine doses, the immunoglobulin G (IgG) response mainly consists of the proinflammatory subclasses IgG1 and IgG3. Here, we report that several months after the second vaccination, SARS-CoV-2–specific antibodies were increasingly composed of noninflammatory IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. IgG4 antibodies among all spike-specific IgG antibodies rose, on average, from 0.04% shortly after the second vaccination to 19.27% late after the third vaccination. This induction of IgG4 antibodies was not observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. Single-cell sequencing and flow cytometry revealed substantial frequencies of IgG4-switched B cells within the spike-binding memory B cell population [median of 14.4%; interquartile range (IQR) of 6.7 to 18.1%] compared with the overall memory B cell repertoire (median of 1.3%; IQR of 0.9 to 2.2%) after three immunizations. This class switch was associated with a reduced capacity of the spike-specific antibodies to mediate antibody-dependent cellular phagocytosis and complement deposition. Because Fc-mediated effector functions are critical for antiviral immunity, these findings may have consequences for the choice and timing of vaccination regimens using mRNA vaccines, including future booster immunizations against SARS-CoV-2. Anti-spike IgG4 rises from obscurity: The four human IgG subclasses have distinct effector properties due to differences in binding Fc receptors and activating complement. The serum concentration of human IgG4 is normally lower than either IgG1, IgG2, or IgG3. Irrgang et al. did a longitudinal analysis of the level of spike-specific antibodies from each IgG subclass in recipients of the SARS-CoV-2 BNT162b2 mRNA vaccine. Anti-spike IgG4 as a fraction of total anti-spike IgG rose by 6 months after the second vaccination and increased further after a third vaccine dose. Serum antibody effector activity assessed by antibody-dependent phagocytosis or complement deposition was less after the third dose than after the second dose. Further studies are needed to determine how emergence of an IgG4 anti-spike response influences vaccine-induced protection from SARS-CoV-2 infection. —IRW [ABSTRACT FROM AUTHOR]

Orofacial clefts (OFC) present different phenotypes with a postnatal challenge for oral microbiota development. In order to investigate the impact of OFC on oral microbiota, smear samples from 15 neonates with OFC and 17 neonates without OFC were collected from two oral niches (tongue, cheek) at two time points, i.e. after birth (T0: Ø3d OFC group; Ø2d control group) and 4–5 weeks later (T1: Ø32d OFC group; Ø31d control group). Subsequently, the samples were analyzed using next-generation sequencing. We detected a significant increase of alpha diversity and anaerobic and Gram-negative species from T0 to T1 in both groups. Further, we found that at T1 OFC neonates presented a significantly lower alpha diversity (lowest values for high cleft severity) and significantly higher levels of Enterobacteriaceae (Citrobacter, Enterobacter, Escherichia-Shigella, Klebsiella), Enterococcus, Bifidobacterium, Corynebacterium, Lactocaseibacillus, Staphylococcus, Acinetobacter and Lawsonella compared to controls. Notably, neonates with unilateral and bilateral cleft lip and palate (UCLP/BCLP) presented similarities in beta diversity and a mixture with skin microbiota. However, significant differences were seen in neonates with cleft palate only compared to UCLP/BCLP with higher levels of anaerobic species. Our findings revealed an influence of OFC as well as cleft phenotype and severity on postnatal oral microbiota maturation. [ABSTRACT FROM AUTHOR]

Vector‐borne infections of humans with the protozoan parasite Leishmania (L.) infantum can cause a systemic and potentially lethal disease termed visceral leishmaniasis. In the corresponding mouse model, an intravenous infection with L. infantum leads to the persistence of parasites in various organs, including bone marrow (BM). Considering the anatomical proximity between the BM and the cortical bone, we investigated whether a chronic infection with L. infantum affected bone homeostasis. Unexpectedly, chronic infection with L. infantum caused an increase in bone mass in mice. In vivo, an increased number of osteoblasts and osteocytes and a decreased maturation of osteoclasts characterized the phenotype. Confocal laser scanning fluorescence microscopy confirmed the infection of BM macrophages but also revealed the presence of parasites in osteoclasts. In vitro, mature osteoclasts took up L. infantum parasites. However, infection of osteoclast progenitors abolished their differentiation and function. In addition, secretory products of infected BM–derived macrophages inhibited the maturation of osteoclasts. Both in vitro and in vivo, infected macrophages and osteoclasts showed an enhanced expression of the anti‐osteoclastogenic chemokine CCL5 (RANTES). Neutralization of CCL5 prevented the inhibition of osteoclast generation seen in the presence of culture supernatants from L. infantum‐infected macrophages. Altogether, our study shows that chronic infection with Leishmania increases bone mass by inducing bone formation and impairing osteoclast differentiation and function. © 2022 American Society for Bone and Mineral Research (ASBMR). [ABSTRACT FROM AUTHOR]

The duration of protection after a single dose of yellow fever vaccine is a matter of debate. To summarize the current knowledge, we performed a systematic literature review and meta-analysis. Studies on the duration of protection after 1 and ≥2 vaccine doses were reviewed. Data were stratified by time since vaccination. In our meta-analysis, we used random-effects models. We identified 36 studies from 20 countries, comprising more than 17 000 participants aged 6 months to 85 years. Among healthy adults and children, pooled seroprotection rates after single vaccination dose were close to 100% by 3 months and remained high in adults for 5 to 10 years. In children vaccinated before age 2 years, the seroprotection rate was 52% within 5 years after primary vaccination. For immunodeficient persons, data indicate relevant waning. The extent of waning of seroprotection after yellow fever vaccination depends on age and immune status at primary vaccination. [ABSTRACT FROM AUTHOR]

Gram+ bacteria are very common in clinical medicine and responsible for a large number of infectious diseases. For example, Gram+ bacteria play a major role in causing bloodstream infections and sepsis. Therefore, the detection of Gram+ bacteria is of great importance for the diagnosis and treatment of infectious diseases. Furthermore, these bacteria are often present in biofilms that cover implants. Recent research work has mainly focused on the biologic activity and removal of Gram-negative bacteria or bacterial components such as lipopolysaccharides (LPS). In contrast, the effects of lipoteichoic acid (LTA) have been less well studied so the relevance of their removal from body fluids is possibly underestimated. To address this topic, we evaluated superparamagnetic iron oxide particles (SPION) carrying different peptides derived from the innate immune receptor (GP-340) for their ability to bind and remove Gram+ bacteria and LTA from different media. Our results show that, beyond S. aureus, effective agglutinating and removing of S. pneumoniae was possible. Furthermore, we were able to show for the first time that this was possible with LTA alone and that the magnetic removal of bacteria was also efficient under flow conditions. We also found that this method was able to capture Stapyhylococcus aureus from platelet concentrates, which can help to enhance the sensitivity of microbiological diagnostics, quality control measures, and blood product safety. [ABSTRACT FROM AUTHOR]