BACKGROUND

Identification of disease-associated single nucleotide polymorphisms (SNPs) in seasonal allergic rhinitis (SAR) may be facilitated by focusing on genes in a disease-associated pathway.

OBJECTIVE

To search for SNPs in genes that belong to the T-cell receptor (TCR) pathway and that change in expression in allergen-challenged CD4+ cells from patients with SAR.

METHODS

CD4+ cells from patients with SAR were analysed with gene expression microarrays. Allele, genotype and haplotype frequencies were compared in 251 patients and 386 healthy controls.

RESULTS

Gene expression microarray analysis of allergen-challenged CD4+ cells from patients with SAR showed that 25 of 38 TCR pathway genes were differentially expressed. A total of 62 SNPs were analysed in eight of the 25 genes; ICOS, IL4, IL5, IL13, CSF2, CTLA4, the inducible T-cell tyrosine kinase (ITK) and CD3D. Significant chi-squared values were identified for several markers in the ITK kinase gene region. A total of five SNPs were nominally significant at the 5% level. Haplotype analysis of the five significant SNPs showed increased frequency of a haplotype that covered most of the coding part of ITK. The functional relevance of ITK was supported by analysis of an independent material, which showed increased expression of ITK in allergen-challenged CD4+ cells from patients, but not from controls.

CONCLUSIONS

Analysis of SNPs in TCR pathway genes revealed that a haplotype that covers a major part of the coding sequence of ITK is a risk factor for SAR.

Events in the preimplantation period can have long-term consequences that affect embryo competence to establish and maintain pregnancy and which can extend into fetal and postnatal life. One of the molecules responsible for maternal modulation of embryonic development during this time is colony stimulating factor 2, also termed granulocyte-macrophage colony stimulating factor. This cytokine is produced by the oviduct and endometrium and can act on the preimplantation embryo to improve competence of the embryo to establish pregnancy and develop to term. Actions of CSF2 on the embryo include changes in gene expression (particularly for genes related to apoptosis and differentiation), inhibition of apoptosis, and an increase in numbers of cells in the inner cell mass. Female embryos respond to CSF2 differently than male embryos. Alterations in maternal environment during the preimplantation period can affect subsequent development in a sex-specific manner and CSF2 may be one of the maternal signals responsible for this phenomenon.

Obesity-related changes in adipose tissue leukocytes, in particular adipose tissue macrophages (ATMs) and dendritic cells (ATDCs), are implicated in metabolic inflammation, insulin resistance, and altered regulation of adipocyte function. We evaluated stromal cell and white adipose tissue (WAT) expansion dynamics with high fat diet (HFD) feeding for 3-56 days, quantifying ATMs, ATDCs, endothelial cells (ECs), and preadipocytes (PAs) in visceral epididymal WAT and subcutaneous inguinal WAT. To better understand mechanisms of the early response to obesity, we evaluated ATM proliferation and lipid accumulation. ATMs, ATDCs, and ECs increased with rapid WAT expansion, with ATMs derived primarily from a CCR2-independent resident population. WAT expansion stimulated proliferation in resident ATMs and ECs, but not CD11c+ ATMs or ATDCs. ATM proliferation was unperturbed in Csf2- and Rag1-deficient mice with WAT expansion. Additionally, ATM apoptosis decreased with WAT expansion, and proliferation and apoptosis reverted to baseline with weight loss. Adipocytes reached maximal hypertrophy at 28 days of HFD, coinciding with a plateau in resident ATM accumulation and the appearance of lipid-laden CD11c+ ATMs in visceral epididymal WAT. ATM increases were proportional to tissue expansion and adipocyte hypertrophy, supporting adipocyte-mediated regulation of resident ATMs. The appearance of lipid-laden CD11c+ ATMs at peak adipocyte size supports a role in responding to ectopic lipid accumulation within adipose tissue. In contrast, ATDCs increase independently of proliferation and may be derived from circulating precursors. These changes precede and establish the setting in which large-scale adipose tissue infiltration of CD11c+ ATMs, inflammation, and adipose tissue dysfunction contributes to insulin resistance.

Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein-protein and protein-RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR-Cas systems and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided effector complexes.

Seven new prenylated flavonoids (1-7) and one new prenylated phenylpropiophenone (8) were isolated from roots and rhizomes of Sophora tonkinensis, along with nine known compounds (9-17). The structures 1-8 were elucidated by spectroscopic data analysis and comparison with reported values. Compounds 8 and 12 (7-methoxyebenosin) showed inhibitory activities against nitric oxide production in lipopolysaccharide-induced RAW264.7 cells, with IC₅₀ values of 8.1 and 6.2 μM, respectively. They also significantly lowered expression of CSF2, TNF, and IL-1β. Lonchocarpol A (10) and erybraedin D (16) at concentrations of 20 μM downregulated proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA expression in HepG2 cells. Moreover, erybraedin D (16) inhibited PCSK9 protein synthesis (IC₅₀ 7.8 μM), while simultaneously activating AMP-activated protein kinase and acetyl-CoA carboxylase.

Memory and mood dysfunction are the key symptoms of Gulf war illness (GWI), a lingering multi-symptom ailment afflicting >200,000 veterans who served in the Persian Gulf War-1. Research probing the source of the disease has demonstrated that concomitant exposures to anti-nerve gas agent pyridostigmine bromide (PB), pesticides, and war-related stress are among the chief causes of GWI. Indeed, exposures to GWI-related chemicals (GWIR-Cs) and mild stress in animal models cause memory and mood impairments alongside reduced neurogenesis and chronic low-level inflammation in the hippocampus. In the current study, we examined whether exposure to GWIR-Cs and stress causes chronic changes in the expression of genes related to increased oxidative stress, mitochondrial dysfunction, and inflammation in the hippocampus. We also investigated whether GWI is linked with chronically increased activation of Nrf2 (a master regulator of antioxidant response) in the hippocampus, and inflammation and enhanced oxidative stress at the systemic level. Adult male rats were exposed daily to low-doses of PB and pesticides (DEET and permethrin), in combination with 5 min of restraint stress for 4 weeks. Analysis of the hippocampus performed 6 months after the exposure revealed increased expression of many genes related to oxidative stress response and/or antioxidant activity (Hmox1, Sepp1, and Srxn1), reactive oxygen species metabolism (Fmo2, Sod2, and Ucp2) and oxygen transport (Ift172 and Slc38a1). Furthermore, multiple genes relevant to mitochondrial respiration (Atp6a1, Cox6a1, Cox7a2L, Ndufs7, Ndufv1, Lhpp, Slc25a10, and Ucp1) and neuroinflammation (Nfkb1, Bcl6, Csf2, IL6, Mapk1, Mapk3, Ngf, N-pac, and Prkaca) were up-regulated, alongside 73-88% reduction in the expression of anti-inflammatory genes IL4 and IL10, and nuclear translocation and increased expression of Nrf2 protein. These hippocampal changes were associated with elevated levels of pro-inflammatory cytokines and chemokines (Tnfa, IL1b, IL1a, Tgfb, and Fgf2) and lipid peroxidation byproduct malondialdehyde in the serum, suggesting the presence of an incessant systemic inflammation and elevated oxidative stress. These results imply that chronic oxidative stress, inflammation, and mitochondrial dysfunction in the hippocampus, and heightened systemic inflammation and oxidative stress likely underlie the persistent memory and mood dysfunction observed in GWI.

Introduction: Ischemic stroke remains one of the most debilitating diseases and is the fifth leading cause of death in the US. The ability to predict stroke outcomes within the acute period of stroke would be essential for care planning and rehabilitation. The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC; clinicaltrials.gov NCT03153683) study collects arterial blood immediately distal and proximal to the intracranial thrombus at the time of mechanical thrombectomy. These blood samples are an innovative resource in evaluating acute gene expression changes at the time of ischemic stroke. The purpose of this study was to identify inflammatory genes and important immune factors during mechanical thrombectomy for emergent large vessel occlusion (ELVO) and which patient demographics were predictors for stroke outcomes (infarct and/or edema volume) in acute ischemic stroke patients. Methods: The BACTRAC study is a non-probability sampling of male and female subjects (≥18 year old) treated with mechanical thrombectomy for ELVO. We evaluated 28 subjects (66 ± 15.48 years) relative concentrations of mRNA for gene expression in 84 inflammatory molecules in arterial blood distal and proximal to the intracranial thrombus who underwent thrombectomy. We used the machine learning method, Random Forest to predict which inflammatory genes and patient demographics were important features for infarct and edema volumes. To validate the overlapping genes with outcomes, we perform ordinary least squares regression analysis. Results: Machine learning analyses demonstrated that the genes and subject factors CCR4, IFNA2, IL-9, CXCL3, Age, T2DM, IL-7, CCL4, BMI, IL-5, CCR3, TNFα, and IL-27 predicted infarct volume. The genes and subject factor IFNA2, IL-5, CCL11, IL-17C, CCR4, IL-9, IL-7, CCR3, IL-27, T2DM, and CSF2 predicted edema volume. The overlap of genes CCR4, IFNA2, IL-9, IL-7, IL-5, CCR3, and IL-27 with T2DM predicted both infarct and edema volumes. These genes relate to a microenvironment for chemoattraction and proliferation of autoimmune cells, particularly Th2 cells and neutrophils. Conclusions: Machine learning algorithms can be employed to develop prognostic predictive biomarkers for stroke outcomes in ischemic stroke patients, particularly in regard to identifying acute gene expression changes that occur during stroke.

Asthma is a heterogeneous airway inflammatory disease characterized by increased airway hyperreactivity (AHR) to specific and unspecific stimuli. Group 2 innate lymphoid cells (ILC2)s are type-2 cytokine secreting cells capable of inducing eosinophilic lung inflammation and AHR independent of adaptive immunity. Remarkably, reports show that ILC2s are increased in the blood of human asthmatics as compared to healthy donors. Nevertheless, whether ILC2 expression of adhesion molecules regulates ILC2 trafficking remains unknown. Our results show that IL-33-activated ILC2s not only express LFA-1 but also strikingly LFA-1 ligand ICAM-1. Both LFA-1^-/- and ICAM-1^-/- mice developed attenuated AHR in response to IL-33 intranasal challenge, associated with a lower airway inflammation and less lung ILC2 accumulation compared to controls. Our mixed bone marrow chimera studies however revealed that ILC2 expression of LFA-1 - but not ICAM-1 - was required for their accumulation in the inflamed lungs. Importantly, we found that LFA-1 remarkably controlled ILC2 homing to the lungs, suggesting that LFA-1 is involved in ILC2 trafficking to the lungs. Our exploratory transcriptomic analysis further revealed that ICAM-1 deficiency on ILC2s significantly affects their effector functions. While it downregulated pro-inflammatory cytokines such as Il5, Il9, Il13, and Csf2, it however notably also upregulated cytokines including Il10 both at the transcriptomic and protein levels. These findings provide novel avenues for future investigations, as modulation of LFA-1 and/or ICAM-1 represents an unappreciated regulatory mechanism for ILC2 trafficking and cytokine production respectively, potentially serving as therapeutic target for ILC2-dependent diseases such as allergic asthma.

Keywords: ICAM-1; IL-10; ILC2; LFA-1; airway hyperreactivity; asthma; trafficking.

Bone is one of the most common metastatic sites for breast cancer. In this study, we observed a promoting effect of osteoblast-conditioned medium (OCM) on the migration of MCF-7, a noninvasive cell line of breast cancer cells. Cytokine antibody array was used to compare the cytokines of OCM with the conditioned medium of non-differentiated osteoblast cells, which consequently revealed factors related to migration, such as IL8, IL6, CSF2 (G-CSF), CSF3 (GM-CSF), and TNFRSF11B (osteoprotegerin). The expression of genes related to migration was also estimated with a PCR array, which showed that 9 genes were upregulated and 26 genes downregulated. Moreover, activated p38, ERK, and AKT pathways were found in the OCM treatment group. This finding indicated the migration ability of breast cancer cells, which move toward the bone depending on the presence of specific cytokines in its surrounding microenvironment.

OBJECTIVE

Persistent infection with oncogenic human papillomavirus (HPV) is known to be the necessary cause of cervical cancer and a majority of vulvar cancers. Persistent HPV infections must evade host immune responses, including cytokines released by activated T-helper (Th) cells. In this study, we investigated the risk of cervical and vulvar cancers associated with common genetic variations in 560 tagging single-nucleotide polymorphisms (SNPs) in candidate cytokine genes.

METHODS

The study included 399 invasive squamous cell carcinomas (SCCs) and 502 in situ or invasive adenocarcinomas (AC) of the cervix; 357 in situ or invasive vulvar SCC; and 1109 controls from the Seattle-area case-control studies of HPV-related cancers. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) using a log additive model, with adjustment for multiple testing.

RESULTS

Statistically significant risks were observed for HPV16-containing SCC of the cervix with the variant allele rs879576 in IL17RA and rs2229094 in TNF [OR, 95% CI and multiple-testing corrected p: 1.91 (1.30-2.79), p=0.018 and 0.61 (0.45-0.83), p=0.02, respectively]. We also observed significantly increased risk of HPV-positive vulvar cancers associated with variant alleles in CSF2 (rs25882 and rs27438, 26-28% increased risk) and IL-12B (rs2569254 and rs3181225, 40-41% increased risk) genes.

CONCLUSIONS

We found that variation in several Th-cytokine genes is significantly associated with cervical and vulvar cancer risk. The strong association between these HPV-related cancers and common variation in cytokine genes in the Th1 and Th17 pathways may be important for development of new therapies.

Myeloid-derived suppressor cells (MDSCs) are known to contribute to tumour immune escape, studies have verified that MDSCs can induce cancer stem cells (CSCs) and promote tumour immune evasion in breast cancers, cervical cancers and glioblastoma. However, the potential function of MDSCs in regulating CSCs in epithelial ovarian cancer (EOC) progression is unknown. Our results indicated that compared to nonmalignant ovarian patients, EOC patients showed a significantly increased proportion of MDSCs in the peripheral blood. In addition, MDSCs dramatically promoted tumour sphere formation, cell colony formation and CSC accumulation, and MDSCs enhanced the expression of the stemness biomarkers NANOG and c-MYC in EOC cells during co-culture. Moreover, the mechanisms by which MDSCs enhance EOC stemness were further explored, and 586 differentially expressed genes were found in EOC cells co-cultured with or without MDSCs; during co-culture, the expression level of colony stimulating factor 2 (CSF2) was significantly increased in EOC cells co-cultured with MDSCs. Furthermore, the depletion of CSF2 in EOC cells was successfully performed, the promotive effects of MDSCs on EOC cell stemness could be markedly reversed by downregulating CSF2 expression, p-STAT3 signalling pathway molecules were also altered, and the p-STAT3 inhibitor could markedly reverse the promotive effects of MDSCs on EOC cell stemness. In addition, the CSF2 expression level was correlated with EOC clinical staging. Therefore, MDSCs enhance the stemness of epithelial ovarian cancer cells by inducing the CSF2/p-STAT3 signalling pathway. Targeting MDSCs or CSF2 may be a reasonable strategy for enhancing the efficacy of conventional treatments.

Aging and dysregulation of immune responds are closely associated through a complicated but unclear mechanism. Although many theories have been proposed as overall dysregulation involved in aging, mechanisms such as efficiency of DNA repairing, over-expression of transcription factors (such as NF-κB family), and shift of cell types, are among many factors that contribute to and affect aging process. It is of great interests to understand the possible mechanism that is involved in aging immune system. Here, we report that the inducible genes Il2 and Csf2 are increased as T cells undergo activation and aging. Of particular note were the findings that the relative composition of the circulating CD4(+) T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells. In addition, mRNA levels of NF-κB family genes that are essential elements for cytokine activation in adult mice and activated T cells are significantly increased. We have demonstrated that the expression of inducible genes is accompanied by increased memory/effector type cells and by increased expression level of NF-κB family genes during cell activation and development.

Chungkookjang is a Korean fermented soybean containing microorganisms, proteinase, and diverse bioactive compounds, including a high concentration of isoflavones and peptides. Growth of breast cancer MCF7 cells decreased dependent on the concentration of fermented soybean extracts. The effect of fermented soybean on cellular gene expression was determined in a systematic manner comprehensively. DNA microarray analysis was performed using 25,804 probes. Ninety one genes whose expression levels were significantly changed were selected. TGFβI and Smad3 were upregulated. Downregulation of inflammation-related CSF2, CSF2RA, and CSF3 was found. Differential expression of chemokines CCL2, CCL3, CCL3L3, CXCL1, and CXCL2 were observed. Network analysis identified ERβ in the network. Based on the experimental results, taking fermented soybean might be helpful for preventing breast cancer by a mechanism activating TGFβ pathway and depressing inflammation.

Aspirin, one of the most widely used non-steroidal anti-inflammatory drugs, has extensively studied effects on the cardiovascular system. To reveal further pleiotropic, beneficial effects of aspirin on a number of pro- and anti-inflammatory microglial mechanisms, we performed morphometric and functional studies relating to phagocytosis, pro- and anti-inflammatory cytokine production (IL-1β, tumor necrosis factor-α (TNF-α) and IL-10, respectively) and analyzed the expression of a number of inflammation-related genes, including those related to the above functions, in pure microglial cells. We examined the effects of aspirin (0.1mM and 1mM) in unchallenged (control) and bacterial lipopolysaccharide (LPS)-challenged secondary microglial cultures. Aspirin affected microglial morphology and functions in a dose-dependent manner as it inhibited LPS-elicited microglial activation by promoting ramification and the inhibition of phagocytosis in both concentrations. Remarkably, aspirin strongly reduced the pro-inflammatory IL-1β and TNF-α production, while it increased the anti-inflammatory IL-10 level in LPS-challenged cells. Moreover, aspirin differentially regulated the expression of a number of inflammation-related genes as it downregulated such pro-inflammatory genes as Nos2, Kng1, IL1β, Ptgs2 or Ccr1, while it upregulated some anti-inflammatory genes such as IL10, Csf2, Cxcl1, Ccl5 or Tgfb1. Thus, the use of aspirin could be beneficial for the prophylaxis of certain neurodegenerative disorders as it effectively ameliorates inflammation in the brain.

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r^+/- mouse model of ALSP hypothesized a central role of elevated cerebral Csf2 expression. Here, we show that monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r^+/- mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in humans. Our data provide insights into the mechanisms underlying ALSP. Because increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions.

Protein kinase CK1alpha regulates several fundamental cellular processes including proliferation and differentiation. Up to four forms of this kinase are expressed in vertebrates resulting from alternative splicing of exons; these exons encode either the L-insert located within the catalytic domain or the S-insert located at the C terminus of the protein. Whereas the L-insert is known to target the kinase to the nucleus, the functional significance of nuclear CK1alphaLS has been unclear. Here we demonstrate that selective L-insert-targeted short hairpin small interfering RNA-mediated knockdown of CK1alphaLS in human vascular endothelial cells and vascular smooth muscle cells impairs proliferation and abolishes hydrogen peroxide-stimulated proliferation of vascular smooth muscle cells, with the cells accumulating in G(0)/G(1). In addition, selective knockdown of CK1alphaLS in cultured human arteries inhibits vascular activation, preventing smooth muscle cell proliferation, intimal hyperplasia, and proteoglycan deposition. Knockdown of CK1alphaLS results in the harmonious down-regulation of its target substrate heterogeneous nuclear ribonucleoprotein C and results in the altered expression or alternative splicing of key genes involved in cellular activation including CXCR4, MMP3, CSF2, and SMURF1. Our results indicate that the nuclear form of CK1alpha in humans, CK1alphaLS, plays a critical role in vascular cell proliferation, cellular activation, and hydrogen peroxide-mediated mitogenic signal transduction.

Interleukin 3 (encoded by the IL3 gene) and granulocyte-macrophage colony-stimulating factor (encoded by the CSF2 gene) are small secreted polypeptides that bind to specific cell surface receptors and regulate the growth, gene expression, and differentiation of many of the hematopoietic cell lineages, particularly nonlymphoid cells. The IL3 and CSF2 genes have been cloned and mapped to human chromosome bands 5q23-31. Only 10 kilobases of DNA separates the two genes, suggesting that they have a common origin and/or regulation. We have cloned 70 kilobases of genomic DNA that includes the IL3 and CSF2 genes, as well as flanking sequences, and report a physical map of this region. Several unique-sequence DNA segments have been identified in this region, and one of these fragments detects two restriction fragment length polymorphisms in DNA from unrelated Caucasians. Segregation of these DNA polymorphisms was followed in the Centre Etudé du Polymorphisme Humaine (CEPH) panel of 40 large three-generation pedigrees, and linkage was detected with 17 genetic markers previously typed in these families. Multipoint linkage analysis permits the placement of the region containing the IL3 and CSF2 structural genes on the recombination-genetic linkage map of chromosome 5q and thereby allows the role of these genes in leukemogenesis to be more critically examined.

An important area of genetic research is the identification of functional mechanisms in polymorphisms associated with diseases. A highly relevant functional mechanism is the influence of polymorphisms on gene expression levels (differential allelic expression, DAE). The coding single nucleotide polymorphisms (SNPs) CSF2(rs25882) and IL13(rs20541) have been associated with asthma. In this work, we investigated whether the mRNA expression levels of CSF2 or IL13 were correlated with these SNPs. Samples were analyzed by mass spectrometry-based quantification of gene expression. Both SNPs influenced gene expression levels (CSF2(rs25882): p(overall) = 0.008 and p(DAE samples) = 0.00006; IL13(rs20541): p(overall) = 0.059 and p(DAE samples) = 0.036). For CSF2, the expression level was increased by 27.4% (95% CI: 18.5%-35.4%) in samples with significant DAE in the presence of one copy of risk variant CSF2(rs25882-T). The average expression level of IL13 was increased by 29.8% (95% CI: 3.1%-63.4%) in samples with significant DAE in the presence of one copy of risk variant IL13(rs20541-A). Enhanced expression of CSF2 could stimulate macrophages and neutrophils during inflammation and may be related to the etiology of asthma. For IL-13, higher expression could enhance the functional activity of the asthma-associated isoform. Overall, the analysis of DAE provides an efficient approach for identifying possible functional mechanisms that link disease-associated variants with altered gene expression levels.

Inflammation is implicated in preterm birth, but genetic studies of inflammatory genes have yielded inconsistent results. Maternal DNA from 1,646 participants in the Pregnancy, Infection, and Nutrition Cohort, enrolled in Orange and Wake counties, North Carolina (1995-2005), were genotyped for 432 tag single-nucleotide polymorphisms (SNPs) in 30 candidate genes. Gene-level and SNP associations were modeled within strata of genetic ancestry. Six genes were associated with preterm birth among European Americans: interleukin 12A (IL12A); colony-stimulating factor 2 (CSF2); interferon γ receptor 2 (IFNGR2); killer cell immunoglobulin-like receptor, three domain, long cytoplasmic tail, 2 (KIR3DL2); interleukin 4 (IL4); and interleukin 13 (IL13). Of these, relatively strong single-SNP associations were seen in IFNGR2 and KIR3DL2. Among the 4 genes related to natural killer cell function, 2 (IL12A and CSF2) were consistently associated with reduced risk of prematurity for both European and African Americans. SNPs tagging a locus control region for IL4 and IL13 were associated with an increased risk of spontaneous preterm birth for European Americans (rs3091307; risk ratio = 1.9; 95% confidence interval: 1.4, 2.5). Although gene-level associations were detected only in European Americans, single-SNP associations among European and African Americans were often similar in direction, though estimated with less precision among African Americans. In conclusion, we identified novel associations between variants in the natural killer cell immune pathway and prematurity in this biracial US population.

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFβ2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most--except IL3 and IL4--showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024, and FJ89029.

Checkpoint inhibitors represent a new class of therapeutics in the treatment of cancer that has demonstrated remarkable clinical effectiveness. However, some patients have experienced serious immune-mediated adverse effects including pneumonitis, hepatitis, colitis, nephritis, dermatitis, encephalitis, and adrenal or pituitary insufficiency. These adverse events were not predicted by nonclinical studies. To determine if bone marrow-liver-thymus (BLT) immune humanized mice could demonstrate these adverse effects, we studied the effect of nivolumab on 2 strains of BLT-humanized mice, NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) and NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3, CSF2)10-7Jic/JicTac (NOG-EXL). Mice were treated with 2.5, 5.0, or 10.0 mg/kg nivolumab or saline twice weekly for 28 days. BLT-NOG mice had significantly reduced survival compared with BLT-NOG-EXL mice. In spite of the difference in survival, both BLT-humanized strains showed adverse reactions similar to those reported in humans, including pneumonitis and hepatitis, with nephritis, dermatitis and adrenalitis also noted in some individuals. Additional histopathologic findings included pancreatic atrophy, myositis, and osteomyelitis in some animals. T-cell activation increased with concomitant loss of PD-1 detection. These findings show that BLT immune humanized mice can demonstrate immune-mediated adverse effects of antiPD1 therapy, and may represent a model that can be used to better understand toxicity of this class of drugs.

STAT5 proteins are adaptor proteins for histone acetylation enzymes. Histone acetylation at promoter and enhancer chromosomal regions opens the chromatin and allows access of transcription enzymes to specific genes in rapid response cell signals, such as in inflammation. Histone acetylation-mediated gene regulation is involved in expression of 2 key inflammatory response genes: CSF2, encoding granulocyte-macrophage colony stimulating factor (GM-CSF), and PTGS2, encoding prostaglandin synthase 2/cyclooxygenase 2 (PGS2/COX2). Prolonged CSF2 expression, high GM-CSF production, and GM-CSF activation of PTGS2 gene expression all are seen in type 1 diabetes (T1D) monocytes. Persistent phosphorylation activation of monocyte STAT5 (STAT5Ptyr) is also found in individuals with or at-risk for T1D. To examine whether elevated T1D monocyte STAT5Ptyr may be associated with aberrant inflammatory gene expression in T1D, blood monocytes from non-autoimmune controls and T1D patients were analyzed by flow cytometry for STAT5Ptyr activation, and by chromatin immuno-precipitation (ChIP) analyses for STAT5Ptyr's ability to bind at CSF2 and PTGS2 regulatory sites in association with histone acetylation. In unstimulated monocytes, STAT5Ptyr was elevated in 59.65% of T1D, but only 2.44% of control subjects (p<0.0001). Increased STAT5Ptyr correlated with T1D disease duration (p = 0.0030, r(2) = 0.0784). Unstimulated (p = 0.140) and GM-CSF-stimulated (p = 0.0485) T1D monocytes, had greater STAT5Ptyr binding to epigenetic regulatory sites upstream of CSF2 than control monocytes. Increased STAT5Ptyr binding in T1D monocytes was concurrent with binding at these sites of STAT6Ptyr (p = 0.0283), CBP/P300 histone acetylase, acetylated histones H3, SMRT/NCoR histone deacetylase (p = 0.0040), and RNA Polymerase II (p = 0.0040). Our study indicates that in T1D monocytes, STAT5Ptyr activation is significantly higher and that STAT5Ptyr is found bound to CSF2 promoter and PTGS2 enhancer regions coincident with histone acetylation and RNA polymerase II. These findings suggest that the persistent activation of STAT5 by GM-CSF may be involved in altering the epigenetic regulation of these inflammatory response genes in T1D monocytes.

In this study, we identified potential serum biomarkers for the diagnosis of active tuberculosis (TB) and screening for latent TB infections (LTBIs). Peripheral blood samples from 40 healthy individuals, 40 patients with TB, and 40 LTBI individuals were stimulated with the TB-specific antigens ESAT-6 and CFP-10. Human inflammatory cytokine arrays were used to detect the expression of inflammatory cytokines. Cytokines with significant changes were screened to construct a cytokine regulation network. The levels of the cytokines CCL1 (I-309), CXCL9 (MIG), IL-10, IL-6, CSF2, CSF3, IL-8, IL-1α, IL-7, TGF-β1, CCL2, IL-2, IL-13, and TNFα were significantly upregulated in the active TB group. The levels of CCL3, IL-1β, CCL8, IFNγ, and CXCL10 were significantly increased in the TB groups compared to those in the healthy control group. sTNF RII was upregulated in the LTBI group. CCL4 and MIP1d were significantly increased in all groups.The upregulated cytokines were mainly found in the IFNγ and IL-1α regulatory networks. Importantly, we found that CXCL10 (IP-10), CCL3, CCL8, and IL-1β may be more suitable than IFNγ for active or latent TB infection screening. Furthermore, we found that levels of CCL1 (I-309), CXCL9 (MIG), IL-10, IL-6, CSF2, CSF3, IL-8, IL-1α, IL-7, TGF-β1, CCL2, IL-2, and IL-13 after TB antigen stimulation may help distinguish between active and latent TB.