Privileged Niche Research Articles

Tunable control of B. infantis abundance and gut metabolites by co-administration of human milk oligosaccharides

ABSTRACT Precision engineering of the gut microbiome holds promise as an effective therapeutic approach for diseases associated with a disruption in this microbial community. Engrafting a live biotherapeutic product (LBP) in a predictable, controllable manner is key to the consistent success of this approach and has remained a challenge for most LBPs under development. We recently demonstrated high-level engraftment of Bifidobacterium longum subsp. infantis (B. infantis) in adults when co-dosed with a specific prebiotic, human milk oligosaccharides (HMO). Here, we present a cellular kinetic-pharmacodynamic approach, analogous to pharmacokinetic-pharmacodynamic-based analyses of small molecule- and biologic-based drugs, to establish how HMO controls expansion, abundance, and metabolic output of B. infantis in a human microbiota-based model in gnotobiotic mice. Our data demonstrate that the HMO dose controls steady-state abundance of B. infantis in the microbiome, and that B. infantis together with HMO impacts gut metabolite levels in a targeted, HMO-dependent manner. We also found that HMO creates a privileged niche for B. infantis expansion across a 5-log range of bacterial inocula. These results demonstrate remarkable control of both B. infantis levels and the microbiome community metabolic outputs using this synbiotic approach, and pave the way for precision engineering of desirable microbes and metabolites to treat a range of diseases.

Insights and Strategies of Melanoma Immunotherapy: Predictive Biomarkers of Response and Resistance and Strategies to Improve Response Rates

Despite the recent successes and durable responses with immune checkpoint inhibitors (ICI), many cancer patients, including those with melanoma, do not derive long-term benefits from ICI therapies. The lack of predictive biomarkers to stratify patients to targeted treatments has been the driver of primary treatment failure and represents an unmet medical need in melanoma and other cancers. Understanding genomic correlations with response and resistance to ICI will enhance cancer patients' benefits. Building on insights into interplay with the complex tumor microenvironment (TME), the ultimate goal should be assessing how the tumor 'instructs' the local immune system to create its privileged niche with a focus on genomic reprogramming within the TME. It is hypothesized that this genomic reprogramming determines the response to ICI. Furthermore, emerging genomic signatures of ICI response, including those related to neoantigens, antigen presentation, DNA repair, and oncogenic pathways, are gaining momentum. In addition, emerging data suggest a role for checkpoint regulators, T cell functionality, chromatin modifiers, and copy-number alterations in mediating the selective response to ICI. As such, efforts to contextualize genomic correlations with response into a more insightful understanding of tumor immune biology will help the development of novel biomarkers and therapeutic strategies to overcome ICI resistance.

Regulation of hematopoietic and leukemia stem cells by regulatory T cells.

Adult bone marrow (BM) hematopoietic stem cells (HSCs) are maintained in a quiescent state and sustain the continuous production of all types of blood cells. HSCs reside in a specialized microenvironment the so-called HSC niche, which equally promotes HSC self-renewal and differentiation to ensure the integrity of the HSC pool throughout life and to replenish hematopoietic cells after acute injury, infection or anemia. The processes of HSC self-renewal and differentiation are tightly controlled and are in great part regulated through cellular interactions with classical (e.g. mesenchymal stromal cells) and non-classical niche cells (e.g. immune cells). In myeloid leukemia, some of these regulatory mechanisms that evolved to maintain HSCs, to protect them from exhaustion and immune destruction and to minimize the risk of malignant transformation are hijacked/disrupted by leukemia stem cells (LSCs), the malignant counterpart of HSCs, to promote disease progression as well as resistance to therapy and immune control. CD4+ regulatory T cells (Tregs) are substantially enriched in the BM compared to other secondary lymphoid organs and are crucially involved in the establishment of an immune privileged niche to maintain HSC quiescence and to protect HSC integrity. In leukemia, Tregs frequencies in the BM even increase. Studies in mice and humans identified the accumulation of Tregs as a major immune-regulatory mechanism. As cure of leukemia implies the elimination of LSCs, the understanding of these immune-regulatory processes may be of particular importance for the development of future treatments of leukemia as targeting major immune escape mechanisms which revolutionized the treatment of solid tumors such as the blockade of the inhibitory checkpoint receptor programmed cell death protein 1 (PD-1) seems less efficacious in the treatment of leukemia. This review will summarize recent findings on the mechanisms by which Tregs regulate stem cells and adaptive immune cells in the BM during homeostasis and in leukemia.

Glyphosate damages blood-testis barrier via NOX1-triggered oxidative stress in rats: Long-term exposure as a potential risk for male reproductive health.

Blood-testis barrier (BTB) creates a privileged niche indispensable for spermatogenesis. Glyphosate (GLY), the most commonly used herbicide worldwide, has been reported to decrease sperm quality. However, whether and how GLY destroys the BTB to affect sperm quality remains to be elucidated. Herein, this study was designed to investigate the influence of GLY on the BTB in vivo and in vitro experiments. The results showed that male rats exposed to GLY for 4months exhibited a decrease in sperm quality and quantity, accompanied by BTB integrity disruption and testicular oxidative stress. Additionally, GLY-induced reactive oxygen species (ROS) contributed to the downregulation of BTB-related proteins in primary Sertoli cells (SCs). Intriguingly, we identified a marked upregulation of oxidative stress-related gene NOX1 in GLY-exposed testis based on transcriptome analysis. NOX1 knockdown blocked the GLY-induced oxidative stress, as well as prevented BTB-related protein decrease in SCs. Furthermore, the estrogen receptor (ER)-α was significantly upregulated in vivo and in vitro models. An ER-α inhibitor decreased the expression levels of both ER-α and NOX1. Mechanistically, GLY directly interacted with ER-α at the site of Pro39 and Lys401 to promote ER-α activation, which boosted NOX1 expression to trigger ROS accumulation. Collectively, these results demonstrate that long-term GLY exposure adversely affects BTB integrity, which disrupts spermatogenesis via activation of ER-α/NOX1 axis. This study presents a better understanding of the risk of long-term GLY exposure to male fertility.

Implication of ICOSLG on Relapse in Infant T(4;11) Acute Lymphoblastic Leukemia

Infant t(4;11) acute lymphoblastic leukemia (ALL) is associated with a high relapse rate with 4-year event-free survival (EFS) of only 36%. Relapse has been shown to be the major cause of death as 83% of relapsed infant t(4;11) ALL patients die within three years of diagnosis. Therefore, it is of utmost therapeutic interest to elucidate molecular mechanisms of relapse.Here we show that t(4;11) ALL cells upregulate the inducible T-cell costimulator ligand (ICOSLG) in an early growth response 3 (EGR3) dependent manner thereby promoting the development of regulatory T-cells (T regs). We propose that short EFS and high ICOSLG expression are causally linked, in that ICOSLG-mediated induction of T regs interferes with immune recognition of leukemia cells. According to that hypothesis, ICOSLG would not only be a novel and independent prognostic biomarker but a potential therapeutic target.We investigated the function of EGR3 in infant ALL since EGR3 has been described as an indirect target of the iroquois homeobox 1 (IRX1) transcription factor. For that purpose we created a stable sleeping beauty transposon-based SEM cell line expressing EGR3 in a Doxycycline-inducible manner. Gene expression and western blot analysis revealed strong upregulation of ICOSLG 48h after Doxycycline induction when compared to an empty vector control. Additionally, chromatin immunoprecipitation (ChIP) experiments depicted that EGR3 directly binds to the promoter of the ICOSLG gene.The expression of ICOSLG in mesenchymal stem cells has been shown to foster the induction of T regs and ICOSLG-mediated T reg expansion has been identified as a driver of acute myeloid leukemia and glioblastoma. In the bone marrow (BM) hematopoietic stem cells (HSC) colocalize with T regs which provide an immune privilege to the stem cell niche. Based on these observations, we hypothesize that ICOSLG expressing ALL cells could create an immune privileged niche appearing to be necessary for HSC maintenance and sanctuary from immune attack. This could create independence from the BM immune privilege enabling migration of the ALL cells. If so, this could contribute to minimal residual disease (MRD) formation after induction therapy and subsequently to higher probability of relapse.To evaluate our hypothesis, we cocultured the EGR3-SEM and control cells with primary human CD4 + and CD8 + T-cells. The T-lymphocytes were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors and stimulated with coated α-CD2, -CD3 and -CD28 beads. We observed 16% more CD4 +CD25 ++FOXP3 + T regs after 48h of coculture with EGR3-SEM cells compared to the control. The addition of a neutralizing monoclonal α-ICOSLG antibody to the coculture led to a reduction of the T reg frequency in EGR3-SEM coculture. Taken together, these results strongly suggest that ALL cells expressing EGR3 induce the formation of T regs via ICOSLG expression.To confirm our results with patient-derived material, we investigated the gene expression of 50 infant t(4;11) pro-B phenotypic ALL patients. Pearson correlation testing confirmed that ICOSLG expression strongly correlates with EGR3 and IRX1 expression. Furthermore, we were able to classify the patients considering their ICOSLG expression level into an ICOSLG-high (ICOSLG-hi) and an ICOSLG-low (ICOSLG-lo) group. Outcome data for a 5-year follow-up were available for 35 of 50 patients, n=5 ICOSLG-hi and n=30 ICOSLG-lo. 100% (5/5) of the ICOSLG-hi patients failed within 17 months of diagnosis whereas 53% (16/30) of the ICOSLG-lo patients failed within 56 months of diagnosis. The remaining 14 ICOSLG-lo patients were alive after 60 months. These data underscore the role of overexpressed ICOSLG in relapse development. However, a verification of these findings in a larger cohort is needed.In conclusion, our study identifies ICOSLG as a promising prognostic marker and novel therapeutic target in infant t(4;11) ALL. Furthermore, our findings implicate the interaction between T-cells and leukemia stem cells as contributory to disease progression. DisclosuresCario: Novartis: Other: Lecture Fee.

Abstract NG13: Decoding the stem cells-immune cell dialogues for cancer immunotherapy

Abstract NG13: Decoding the stem cells-immune cell dialogues for cancer immunotherapy

HIV-Associated Disseminated Histoplasmosis and Rare Adrenal Involvement: Evidence of Absence or Absence of Evidence.

Adrenal histoplasmosis and primary adrenal insufficiency are mostly described in immunocompetent patients. This particular tropism is attributed to the presence of cortisol within the adrenal gland, a privileged niche for Histoplasma growth. In French Guiana, disseminated histoplasmosis is the main opportunistic infection in HIV patients. Our objective was to search in our HIV-histoplasmosis cohorts to determine how frequent adrenal insufficiency was among these patients. Between January 1, 1981 and October 1, 2014, a multicentric retrospective, observational study of histoplasmosis was conducted. Patients co-infected by HIV and histoplasmosis were enrolled in French Guiana’s histoplasmosis and HIV database. Among 349 cases of disseminated histoplasmosis between 1981 and 2014, only 3 had adrenal insufficiency (0.85%). Their respective CD4 counts were 10, 14 and 43 per mm3. All patients had regular electrolyte measurements and 234/349 (67%) had abdominal ultrasonography and 98/349 (28%) had abdominopelvic CT scans. None of these explorations reported adrenal enlargement. Overall, these numbers are far from the 10% reports among living patients and 80-90% among histoplasmosis autopsy series. This suggests 2 conflicting hypotheses: First, apart from acute adrenal failure with high potassium and low sodium, less advanced functional deficiencies, which require specific explorations, may have remained undiagnosed. The second hypothesis is that immunosuppression leads to different tissular responses that are less likely to incapacitate the adrenal function. Furthermore, given the general immunosuppression, the adrenal glands no longer represent a particular niche for Histoplasma proliferation.

The microenvironmental niche in classic Hodgkin lymphoma is enriched for CTLA-4–positive T cells that are PD-1–negative

AbstractClassic Hodgkin lymphoma (cHL) is a tumor composed of rare, atypical, germinal center–derived B cells (Hodgkin Reed-Sternberg [HRS] cells) embedded within a robust but ineffective inflammatory milieu. The cHL tumor microenvironment (TME) is compartmentalized into “niches” rich in programmed cell death-1 ligand (PD-L1)–positive HRS cells and tumor-associated macrophages (TAMs), which associate with PD-1–positive T cells to suppress antitumor immunity via PD-L1/PD-1 signaling. Despite the exquisite sensitivity of cHL to PD-1 checkpoint blockade, most patients eventually relapse and need therapeutic alternatives. Using multiplex immunofluorescence microscopy with digital image analysis, we found that cHL is highly enriched for non–T-regulatory, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)–positive T cells (compared with reactive lymphoid tissues) that outnumber PD-1–positive and lymphocyte-activating gene-3 (LAG-3)–positive T cells. In addition, T cells touching HRS cells are more frequently positive for CTLA-4 than for PD-1 or LAG-3. We further found that HRS cells, and a subset of TAMs, are positive for the CTLA-4 ligand CD86 and that the fractions of T cells and TAMs that are CTLA-4–positive and CD86-positive, respectively, are greater within a 75 μm HRS cell niche relative to areas outside this region (CTLA-4, 38% vs 18% [P = .0001]; CD86, 38% vs 24% [P = .0007]). Importantly, CTLA-4–positive cells are present, and focally contact HRS cells, in recurrent cHL tumors following a variety of therapies, including PD-1 blockade. These results implicate CTLA-4:CD86 interactions as a component of the immunologically privileged niche surrounding HRS cells and raise the possibility that patients with cHL refractory to PD-1 blockade may benefit from CTLA-4 blockade.

Abstract A100: Downregulation of type 1 interferon receptor (IFNAR1) regulates the balance of regulatory T cells (Tregs) and cytotoxic T lymphocytes (CTLs) in tumor microenvironment

Abstract Solid tumors are infiltrated by not only CD8+ effector T cells (CTLs) exerting tumoricidal function, but also by regulatory T cells (Tregs), which are a key barrier to antitumor immunity (1). In the tumor microenvironment, the type 1 interferon (IFN) signaling is downregulated due to proteolytic degradation of IFNAR1 (2). Reduced IFNAR1 levels in tumor stroma are important for establishing the immune privileged niche to support tumor development and progression (3). IFN pathway plays complex and controversial roles in regulating immune responses ([4-7). In this study, we show that loss of IFNAR1 leads to changes in the activities of Tregs and CTLs. Genetic stabilization of IFNAR1 (Ifnar1S526A, SA) recruits more CTLs in the tumor microenvironment; however, it does not change the frequency of Tregs. In addition, in SA tumor tissues, the CTLs appear to exhibit a greater tumoricidal activity. Furthermore, we find that SA Treg lost their inhibitory function and SA CTLs are resistant to Treg-mediated immune suppression. In all, we discuss putative mechanisms underlying these phenotypes that IFN pathway regulates the balance of Treg and CTL activities in the tumor microenvironment. Citation Format: Hongru Zhang, Jun Gui, Angelica Ortiz, Serge Fuchs. Downregulation of type 1 interferon receptor (IFNAR1) regulates the balance of regulatory T cells (Tregs) and cytotoxic T lymphocytes (CTLs) in tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A100.

Bone Marrow Stromal Cells Drive Key Hallmarks of B Cell Malignancies.

All B cell leukaemias and a substantial fraction of lymphomas display a natural niche residency in the bone marrow. While the bone marrow compartment may only be one of several sites of disease manifestations, the strong clinical significance of minimal residual disease (MRD) in the bone marrow strongly suggests that privileged niches exist in this anatomical site favouring central elements of malignant transformation. Here, the co-existence of two hierarchical systems, originating from haematopoietic and mesenchymal stem cells, has extensively been characterised with regard to regulation of the former (blood production) by the latter. How these two systems cooperate under pathological conditions is far less understood and is the focus of many current investigations. More recent single-cell sequencing techniques have now identified an unappreciated cellular heterogeneity of the bone marrow microenvironment. How each of these cell subtypes interact with each other and regulate normal and malignant haematopoiesis remains to be investigated. Here we review the evidences of how bone marrow stroma cells and malignant B cells reciprocally interact. Evidently from published data, these cell–cell interactions induce profound changes in signalling, gene expression and metabolic adaptations. While the past research has largely focussed on understanding changes imposed by stroma- on tumour cells, it is now clear that tumour-cell contact also has fundamental ramifications for the biology of stroma cells. Their careful characterisations are not only interesting from a scientific biological viewpoint but also relevant to clinical practice: Since tumour cells heavily depend on stroma cells for cell survival, proliferation and dissemination, interference with bone marrow stroma–tumour interactions bear therapeutic potential. The molecular characterisation of tumour–stroma interactions can identify new vulnerabilities, which could be therapeutically exploited.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in <em>Chlamydia trachomatis</em>

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Agrin Mediates Angiogenesis in the Tumor Microenvironment

Angiogenesis represents a hallmark of cancer. Several proteoglycans associate with cell surface receptors and regulate angiogenesis within the tumor microenvironment (TME). We highlight the recent discovery that the proteoglycan Agrin cross talks between the tumor and the endothelium to promote an angiogenesis privileged niche during cancer progression.

Mycobacterium tuberculosis Rv0426c promotes recombinant mycobacteria intracellular survival via manipulating host inflammatory cytokines and suppressing cell apoptosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is still a leading cause of death worldwide. M. tuberculosis has evolved multipronged strategies to subvert host immune defenses and establish an immunologically privileged niche in macrophages. Rv0426c has been predicted to be an effector involved in the Mtb-host interactions. To investigate the potential role played by Rv0426c, we constructed recombinant M. smegmatis strains with heterologous expression of Rv0426c. We observed that Rv0426c recombinants became more susceptible to various stresses by increasing cell wall permeability, however with elevated early survival rate within macrophages. This was accompanied by decreased levels of pro-inflammatory cytokines and host cell apoptosis. The data suggested that Rv0426c was a new player involved in the interactions between Mtb and macrophages.

Application of rice (Oryza sativa L.) root endophytic diazotrophic Azotobacter sp. strain Avi2 (MCC 3432) can increase rice yield under green house and field condition

Use of plant-associated beneficial microbes, especially endophytes are getting popular day by day as they occupy a relatively privileged niche inside different plant tissues with lesser competition for food and shelter than rhizosphere. The effects of different physical factors like temperature, rainfall, and seasonal variation and UV radiation on plant growth promoting endophytic communities are less pronounced than those on the rhizospheric and phylloplane microbes. This present work has been compromised with further utilization of an indigenous rice (Oryza sativa L.) root endophytic Azotobacter sp. strain Avi2 (MCC 3432) (AzA) as a bio-formulation for sustainable rice production based on several physiological parameters (plant height, root length/weight, leaf area, yield, chlorophyll contain), in-vitro comparative plant growth promoting assays, greenhouse and field experiments (dry and wet season). Treatments with AzA exhibited higher yield as well as maximal chlorophyll fluorescence (Fm) of flag leaves in flowering and grain filling stages indicating higher photosynthetic rates. Scanning electron microscopic image of rice roots demonstrated accumulation of bacterial biofilm at the junction of primary and lateral root confirming the root-colonizing ability of the bacterium. The results of the study were quite encouraging as AzA exhibited better vegetative and reproductive growth of rice in pot and field experiment compared to formulated rhizospheric Azotobacter sp. (commercial product). Apart from that plants treated with AzA (supplemented 50% nitrogenous fertilizer of recommended dose) exhibited similar yield parameters when it was compared with the recommended dose of fertilizer (RDF; 120:60:60 mg N:P:K kg−1 soil/ without any bacterial). Therefore, it can be concluded that application of this plant growth promoting endophyte can reduce a substantial amount of N-fertilizer for field application.

Alveolar and monocyte-derived macrophages differentially engage antibacterial programs during adaptive immunity to Mycobacterium tuberculosis.

Abstract Cognate interactions between antigen-specific CD4 T cells and Mycobacterium tuberculosis (Mtb)-infected cells are critical for mucosal immunity to tuberculosis (TB). However, Mtb resides intracellularly within a variety of myeloid cell populations, and the relative impact of CD4 T cells on distinct infected cell types remains unknown. Here we report that intratracheally-transferred, Mtb-specific T cells are unable to reduce the bacterial burden in the first week after aerosol infection, when Mtb resides almost exclusively within alveolar macrophages (AMs). However, transferred T cells do mediate protection by day 14, at which point Mtb has disseminated to monocyte-derived macrophages (MDMs). Using an Mtb “replication clock” strain, which loses plasmid at a fixed rate with bacterial division, we observe that Mtb continues to replicate in AMs even after the onset of adaptive immunity, whereas replication in MDMs is curtailed. We also find that MDMs are less reliant on oxidative metabolism and express more NOS2 than AMs, which may explain their superior antibacterial activity. Finally, RNA-Seq analysis of these two macrophage populations reveals enriched expression of proinflammatory pathways (i.e. NFkB signaling) in Mtb-infected MDMs compared to AMs. Together, our results indicate that AMs may serve as a privileged niche for Mtb, despite the presence of Mtb-specific T cells. As many current TB vaccine candidates seek to induce lung-resident T cells that can recognize and control Mtb early after aerosol infection, the relative resistance of Mtb-infected AMs to T cell-mediated immunity could represent a previously unappreciated barrier to this strategy.

Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line

ABSTRACT Objective: Staphylococcus aureus is a particularly difficult pathogen to eradicate from the respiratory tract. Previous studies have highlighted the intracellular capacity of S.aureus in several phagocytic and non-phagocytic cells. The aim of this study was to define S.aureus interaction within a murine alveolar macrophage cell line. Methods: Cell line MH-S was infected with Newman strain. Molecular mechanisms involved in phagocytosis were explored. To assess whether S.aureus survives intracellularly quantitative (gentamicin protection assays and bacterial plating) and qualitative analysis (immunofluorescence microscopy) were performed. Bacterial colocalization with different markers of the endocytic pathway was examined to characterize its intracellular trafficking. Results: We found that S.aureus uptake requires host actin polymerization, microtubule assembly and activation of phosphatidylinositol 3-kinase signaling. Time course experiments showed that Newman strain was able to persist within macrophages at least until 28.5 h post infection. We observed that intracellular bacteria are located inside an acidic subcellular compartment, which co-localizes with the late endosome/lysosome markers Lamp-1, Rab7 and RILP. Colocalization counts with TMR-dextran might reflect a balance between bacterial killing and intracellular survival. Conclusions: This study indicates that S.aureus persists and replicates inside murine alveolar macrophages, representing a privileged niche that can potentially offer protection from antimicrobial activity and immunological host defense mechanisms.

High-level expression and purification of soluble form of human natural killer cell receptor NKR-P1 in HEK293S GnTI− cells

Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the surface of NK cells, where it serves as inhibitory receptor; and on T and NKT cells functioning as co-stimulatory receptor promoting secretion of IFNγ. Most notably, it is expressed on Th17 and Tc17 lymphocytes where presumably promotes targeting into LLT1 expressing immunologically privileged niches. We tested effect of different protein tags (SUMO, TRX, GST, MsyB) on expression of soluble NKR-P1 in E. coli. Then we optimized the expression construct of soluble NKR-P1 by preparing a library of expression constructs in pOPING vector containing the extracellular lectin-like domain with different length of the putative N-terminal stalk region and tested its expression in Sf9 and HEK293 cells. Finally, a high-level expression of soluble NKR-P1 was achieved by stable expression in suspension-adapted HEK293S GnTI− cells utilizing pOPINGTTneo expression vector. Purified soluble NKR-P1 is homogeneous, deglycosylatable, crystallizable and monomeric in solution, as shown by size-exclusion chromatography, multi-angle light scattering and analytical ultracentrifugation.

A retrospective cross-sectional quantitative molecular approach in biological samples from patients with syphilis.

Syphilis is the sexually transmitted disease caused by Treponema pallidum, a pathogen highly adapted to the human host. As a multistage disease, syphilis presents distinct clinical manifestations that pose different implications for diagnosis. Nevertheless, the inherent factors leading to diverse disease progressions are still unknown. We aimed to assess the association between treponemal loads and dissimilar disease outcomes, to better understand syphilis. We retrospectively analyzed 309 DNA samples distinct anatomic sites associated with particular syphilis manifestations. All samples had previously tested positive by a PCR-based diagnostic kit. An absolute quantitative real-time PCR procedure was used to precisely quantify the number of treponemal and human cells to determine T.pallidum loads in each sample. In general, lesion exudates presented the highest T.pallidum loads in contrast with blood-derived samples. Within the latter, a higher dispersion of T.pallidum quantities was observed for secondary syphilis. T.pallidum was detected in substantial amounts in 37 samples of seronegative individuals and in 13 cases considered as syphilis-treated. No association was found between treponemal loads and serological results or HIV status. This study suggests a scenario where syphilis may be characterized by: i) heterogeneous and high treponemal loads in primary syphilis, regardless of the anatomic site, reflecting dissimilar duration of chancres development and resolution; ii) high dispersion of bacterial concentrations in secondary syphilis, potentially suggesting replication capability of T.pallidum while in the bloodstream; and iii) bacterial evasiveness, either to the host immune system or antibiotic treatment, while remaining hidden in privileged niches. This work highlights the importance of using molecular approaches to study uncultivable human pathogens, such as T.pallidum, in the infection process.

Human Screams Occupy a Privileged Niche in the Communication Soundscape

Screaming is arguably one of the most relevant communication signals for survival in humans. Despite their practical relevance and their theoretical significance as innate [1] and virtually universal [2, 3] vocalizations, what makes screams a unique signal and how they are processed is not known. Here, we use acoustic analyses, psychophysical experiments, and neuroimaging to isolate those features that confer to screams their alarming nature, and we track their processing in the human brain. Using the modulation power spectrum (MPS [4, 5]), a recently developed, neurally informed characterization of sounds, we demonstrate that human screams cluster within restricted portion of the acoustic space (between ∼30 and 150Hz modulation rates) that corresponds to a well-known perceptual attribute, roughness. In contrast to the received view that roughness is irrelevant for communication [6], our data reveal that the acoustic space occupied by the rough vocal regime is segregated from other signals, including speech, a pre-requisite to avoid false alarms in normal vocal communication. We show that roughness is present in natural alarm signals as well as in artificial alarms and that the presence of roughness in sounds boosts their detection in various tasks. Using fMRI, we show that acoustic roughness engages subcortical structures critical to rapidly appraise danger. Altogether, these data demonstrate that screams occupy a privileged acoustic niche that, being separated from other communication signals, ensures their biological and ultimately social efficiency.

Bone specific immunity and its impact on metastasis.

Bone is one of the most common sites of metastasis in solid malignancy. Contributing to this osteotropism are the dynamic interactions between tumor cells and the numerous cell types resident in the normal bone, particularly osteoclasts and osteoblasts, which create a tumor supporting microenvironment. However, disseminated cells are detected in the bone marrow long before evidence of metastatic outgrowth, and it is likely that prolonged survival is also reliant on immunoescape. Compared with other peripheral organs such as the lung and spleen, the bone marrow constitutes a unique immune cell compartment that likely provides an immune privileged niche for disseminated tumor cells. This includes the large proportions of immunosuppressive cells, including myeloid derived suppressor cells and regulatory T cells, that blunt the activity of cytotoxic lymphocytes involved in tumor immunosurveillance. This review highlights key aspects of the osteoimmune landscape and emerging mechanisms by which tumor cells create or co-opt an immunosuppressed niche to support their outgrowth in bone. Future studies in this field are likely to shed light on the differences in immunoregulation between the bone and other sites including the primary tumor, and the potential for immunotherapeutics in treating disseminated disease in the bone. However, more immunocompetent models, that recapitulate tumor heterogeneity and bone metastasis need to be developed to accelerate this field.