Identifying immune signatures of sepsis to increase diagnostic accuracy in very preterm babies.

Bacterial infections are a major cause of mortality in preterm babies, yet our understanding of early-life disease-associated immune dysregulation remains limited. Here, we combine multi-parameter flow cytometry, single-cell RNA sequencing and plasma analysis to longitudinally profile blood from very preterm babies (<32 weeks gestation) across episodes of invasive bacterial infection (sepsis). We identify a dynamically changing blood immune signature of sepsis, including lymphopenia, reduced dendritic cell frequencies and myeloid cell HLA-DR expression, which characterizes sepsis even when the common clinical marker of inflammation, C-reactive protein, is not elevated. Furthermore, single-cell RNA sequencing identifies upregulation of amphiregulin in leukocyte populations during sepsis, which we validate as a plasma analyte that correlates with clinical signs of disease, even when C-reactive protein is normal. This study provides insights into immune pathways associated with early-life sepsis and identifies immune analytes as potential diagnostic adjuncts to standard tests to guide targeted antibiotic prescribing.

Perinatal inflammation influences but does not arrest rapid immune development in preterm babies.

Infection and infection-related complications are important causes of death and morbidity following preterm birth. Despite this risk, there is limited understanding of the development of the immune system in those born prematurely, and of how this development is influenced by perinatal factors. Here we prospectively and longitudinally follow a cohort of babies born before 32 weeks of gestation. We demonstrate that preterm babies, including those born extremely prematurely (<28 weeks), are capable of rapidly acquiring some adult levels of immune functionality, in which immune maturation occurs independently of the developing heterogeneous microbiome. By contrast, we observe a reduced percentage of CXCL8-producing T cells, but comparable levels of TNF-producing T cells, from babies exposed to in utero or postnatal infection, which precedes an unstable post-natal clinical course. These data show that rapid immune development is possible in preterm babies, but distinct identifiable differences in functionality may predict subsequent infection mediated outcomes.

The nonhomologous end joining factor Artemis suppresses multi-tissue tumor formation and prevents loss of heterozygosity.

Nonhomologous end joining (NHEJ) is a critical DNA repair pathway, with proposed tumor suppression functions in many tissues. Mutations in the NHEJ factor ARTEMIS cause radiation-sensitive severe combined immunodeficiency in humans and may increase susceptibility to lymphoma in some settings. We now report that deficiency for Artemis (encoded by Dclre1c/Art in mouse) accelerates tumorigenesis in several tissues in a Trp53 heterozygous setting, revealing tumor suppression roles for NHEJ in lymphoid and non-lymphoid cells. We also show that B-lineage lymphomas in these mice undergo loss of Trp53 heterozygosity by allele replacement, but arise by mechanisms distinct from those in Art Trp53 double null mice. These findings demonstrate a general tumor suppression function for NHEJ, and reveal that interplay between NHEJ and Trp53 loss of heterozygosity influences the sequence of multi-hit oncogenesis. We present a model where p53 status at the time of tumor initiation is a key determinant of subsequent oncogenic mechanisms. Because Art deficient mice represent a model for radiation-sensitive severe combined immunodeficiency, our findings suggest that these patients may be at risk for both lymphoid and non-lymphoid cancers.

Linkage of the angiotensinogen gene locus to human essential hypertension in African Caribbeans.

The renin-angiotensin system regulates blood pressure and sodium balance. The angiotensinogen gene which encodes the key substrate within this system has been linked to essential hypertension in White Europeans. It has been suggested that people of West African ancestry may have a different genetic basis for hypertension. In this study we have tested whether there is linkage of the angiotensinogen gene to essential hypertension in African Caribbeans from St. Vincent and the Grenadines. DNA from 63 affected sibling pairs with hypertension was tested for linkage by analyzing whether there was excess allele sharing among siblings genotyped using an angiotensinogen dinucleotide repeat sequence. There was significant support for linkage (T = 3.07, P = 0.001) and association of this locus to hypertension (chi 2 = 50.2, 12 degrees of freedom, P << 0.001). A DNA polymorphism which alters methionine to threonine at position 235 (M235T) within the angiotensinogen peptide has been associated previously with hypertension. However, we found no association of this variant with hypertension in this study. These findings provide support for linkage and association of the angiotensinogen locus to hypertension in African Caribbeans and suggest some similarities in the genetic basis of essential hypertension in populations of different ethnicity.

Tumor-derived products induce Il-1 a, Il-1 b, Tnf a, and Il-6 gene expression in murine macrophages: distinctions between tumor- and bacterial endotoxin-induced gene expression.

The ability of progressing tumors to regulate host physiology is an important consideration in our understanding of tumor-host relationships. Previous data indicated that several lines of murine sarcoma cells produced one or more activities that were able to regulate both Il-1 a and Il-1 b gene transcription in macrophages (MO). We now describe an indepth analysis using Northern analysis and bioassays and show that two of these tumors produce one or more activities that when incubated with peritoneal MO result in the transcription of the Il-1a, Il-1 b, Tnf a, and Il-6 genes. Concordant with the Northern analyses was the finding that interleukin-1 (IL-1) and tumor necrosis factor (TNF) biological activities were detected in lysates of induced MO, fixed MO, and supernates of MO cultures. Induction of gene expression was shown to be distinct from that induced by bacterial endotoxin or lipopolysaccharide by a number of criteria. The data suggest that tumor cell products may play an important role in regulating several host physiological processes, particularly those involving Il-1a, Il-1 b, Tnf a, and Il-6 gene expression.

The potential role of the macrophage colony-stimulating factor, CSF-1, in inflammatory responses: characterization of macrophage cytokine gene expression.

In this report we report that recombinant human monocyte-macrophage colony-stimulating factor-1 (CSF-1) induces resident murine peritoneal cells (PCs) to transcribe several inflammatory cytokine genes, including interleukin (IL)-1 alpha, IL-1 beta, IL-6, and granulocyte-macrophage CSF in a dose-dependent and time-related manner. Peak mRNA levels were seen between 4 and 6 h. CSF-1 did not modulate the expression of tumor necrosis factor-alpha mRNA. The serum content of the culture medium appeared to regulate both the extent of CSF-1-induced gene transcription and the adherence properties of the cells. Decreasing the serum concentration significantly reduced CSF-1-induced transcription and was associated with the rapid spreading of the majority of the adherent cells. This reduced sensitivity to CSF-1 was paralleled by a markedly lower levels of c-fms mRNA encoding the CSF-1 receptor. Induced gene transcription was followed by the release of large quantities of IL-6 only. IL-1 activity remained associated with the cells. Neither supernatant nor cell lysate granulocyte-macrophage CSF activity was inducible above the low levels associated with control cultures. Evidence that the mononuclear phagocytes, as opposed to B or T cells, were the targets of CSF-1 was obtained in two ways: (1) PCs from B6 scid/scid and NOD scid/scid mice consisting of 78-86% MAC-1+, F4/80+ cells and few B or T cells, as shown by flow cytometry analysis, released 5- to 10-fold more IL-6 in response to CSF-1 stimulation than B6 PCs, which contained approximately 30% double-positive cells, and (2) pretreatment of B6 PCs with antibodies to the CSF-1 receptor blocked the CSF-1-induced secretion of IL-6. These data suggest that CSF-1 primes noninflammatory mononuclear phagocytes for a role in inflammatory responses but does not provide the necessary signals for either secretion or translation of all cytokines equally.

Synergistic interaction of bacterial lipopolysaccharide and the monocyte-macrophage colony-stimulating factor: potential quantitative and qualitative changes in macrophage-produced cytokine bioactivity.

In this brief definitive report, we show that over a 6-h period and under serum-free conditions, recombinant monocyte-macrophage colony-stimulating factor 1 (rCSF-1) and lipopolysaccharide (LPS) synergize and induce macrophages to express higher levels of mRNA for interleukin 1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 and to release more bioactivity than macrophages treated with LPS alone. This synergy was regulated by the amount of LPS in the culture medium. Paraformaldehyde-fixed macrophages like-wise showed augmentation of IL-1 activity, but whereas all of the bioactivity associated with the fixed macrophages could be neutralized by anti-IL-1 alpha antibody only approximately 40% of the supernate activity could be attributed to IL-1 alpha. Preliminary data suggest that the augmenting effect induced by CSF-1 cannot be explained solely on a quantitative basis because the addition of rIL-1 alpha to supernates of macrophages treated with LPS alone or with the combination of LPS and CSF-1 resulted in an increase in thymocyte mitogenic activity to a level that could not be explained on an additive basis. Although the supernates contained TNF and IL-6, antibody neutralization assays made it unlikely that these were directly responsible for the augmenting effect. These results suggest that CSF-1 not only enhances basic genetic responses induced by LPS alone but also may induce a mechanism that amplifies cytokine bioactivity.

CSF-1 (M-CSF) differentially sensitizes mononuclear phagocyte subpopulations to endotoxin in vivo: a potential pathway that regulates the severity of gram-negative infections.

CSF-1 is known to prime mononuclear phagocytes (MNP) for inflammatory stimuli in vitro. We hypothesized that CSF-1 in vivo can sensitize the host to the increased production of endotoxic shock mediators such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Indeed, when CSF-1-primed mice were challenged with lipopolysaccharide (LPS), increased levels of serum IL-6 and TNF-alpha were detected. Both intravenous and intraperitoneal injections of CSF-1 resulted in increased sensitivity to LPS challenge, which induced maximum increases in serum IL-6 when administered via the intraperitoneal route. The peak serum IL-6 production in control and CSF-1-primed mice occurred 2-3 h after LPS injection, whereas that of TNF-alpha occurred by 1-2 h. When peripheral blood leukocytes, spleen cells, and resident peritoneal cells (PC) were isolated from CSF-1-primed mice injected with LPS, only the PC were shown to release IL-6 constitutively and none released TNF-alpha. A comparison of mRNA isolated from various cells and tissues after intraperitoneal CSF-1 priming indicated that only PC expressed IL-6 mRNA, whereas PC, liver, and spleen expressed TNF-alpha mRNA. All tissues showed increased levels of IL-6 and TNF-alpha mRNA in response to LPS challenge. Only liver and kidney showed an enhanced level of IL-6 expression in CSF-1-primed mice challenged with LPS, whereas liver, lung, and kidney showed enhanced TNF-alpha expression. These data indicate that CSF-1 primes tissue MNP but not circulating MNP to transcribe mRNA and release IL-6 and TNF-alpha. Overall, the data suggest that CSF-1 plays an important role in regulating the sensitivity of the host to the pathophysiological effects of endotoxin.

CSF-1 regulation of Il6 gene expression by murine macrophages: a pivotal role for GM-CSF.

We test the hypothesis that the monocyte-macrophage colony-stimulating factor (CSF-1 or M-CSF) plays a major role in the inflammatory responses of Mphi by acting as a priming agent that heightens their responsiveness to secondary stimulation by other mediators. We previously reported that CSF-1 induced peritoneal Mphi (PMphi) to transcribe several genes including interleukin-6 (Il6) and granulocyte-macrophage colony-stimulating factor (Csfgm). It was reported that the Il6 and Csfgm genes were individually regulated by different pathways but it was not clear to what extent the two genes interacted during Mphi-mediated inflammatory responses. We now show that CSF-1 induces the release of bioactive GM-CSF from mouse resident PMphi. GM-CSF induces Il6 gene expression and synergizes with CSF-1 to induce the release of large amounts of IL-6. PMphi from C57BL/6J-Csfgm(null) mice were shown to release minimal IL-6 in response to CSF-1 and to express a much reduced response to the highly stimulatory combination of CSF-1 and lipopolysaccharide (LPS). Exogenous recombinant GM-CSF restored the IL-6 response of GM-CSF null PMphi to a great extent but not completely. As controls, three other recombinant proteins were tested but of these only tumor necrosis factor alpha (TNF-alpha) was shown to synergize with both CSF-1 and GM-CSF. Using PMphi from mice deficient in the expression of the Il6 gene, it was shown that they released two- to threefold more GM-CSF in response to CSF-1 than their control counterparts. However, an exogenous supply of recombinant IL-6 had no effect on GM-CSF release. The data indicate that the pathways regulating Il6 gene expression are under the control of a complex network of cytokine interactions involving at least CSF-1, GM-CSF, and TNF-alpha, with the added possibility that IL-6 may exert modulatory activity within this network.

Tumor cell IL-6 gene expression is regulated by IL-1 alpha/beta and TNF alpha: proposed feedback mechanisms induced by the interaction of tumor cells and macrophages.

In the present report, we show that progressive growth of the immunogenic C57BL/6J sarcoma, MCA/76-9, was accompanied by an increase in serum interleukin-6 (IL-6) activity. The possible pathways leading to the induction of IL-6 release by the tumor cells are described. It was shown that macrophage products IL-1 alpha, IL-1 beta, and to a lesser extent, TNF alpha, induced the tumor cells in vitro to transcribe the IL-6 gene and release the gene product. IL-1 induced significantly more IL-6 mRNA and bioactivity than TNF alpha, although both cytokines induced a cumulative increase of bioactivity in the supernates over a period of 24 h. The tumor cells were shown to express receptors for IL-1 alpha, which could be blocked with anti-IL-1 receptor antibody. Given the previous reports that tumor-associated macrophages expressed both IL-1 alpha/beta and TNF alpha, the data suggest, first, that the mutual interaction of tumor cells and macrophages in situ may contribute to the observed increase in circulating IL-6 activity, and second, that the release of IL-6 in vivo may serve to regulate both anti-tumor immune responses and suppressor mechanisms.

Angiotensinogen in human essential hypertension.

The candidacy of angiotensinogen for a role in the genetic basis of hypertension is supported by the observation that plasma angiotensinogen levels track with raised blood pressure through families. In addition, transgenic mice with overexpression of a rat angiotensinogen gene develop hypertension, and knockout mice with a disrupted gene and absent angiotensinogen production develop low blood pressure. There are now two studies in populations of white European origin and one in African Caribbeans providing support for a role of the angiotensinogen gene locus in human essential hypertension.

Essential hypertension in African Caribbeans associates with a variant of the beta2-adrenoceptor.

Populations of West African ancestry dwelling in Western communities exhibit greater prevalence of human essential hypertension and higher rates of end-organ damage. The sympathetic nervous system influences cardiac output, vascular tone, renal sodium reabsorption, and renin release and could be implicated in enhanced vascular responsiveness observed in African hypertensives. Such an effect could arise from genetic variants that alter agonist response of alpha-adrenoceptors, leading to enhanced vasoconstriction, or attenuate beta2-adrenoceptor-mediated vasodilatation. Indeed, there is evidence of a blunted vasodilator response to the beta-agonist isoprenaline in African Americans. A variant of the beta2-adrenoceptor gene that encodes glycine rather than arginine at position 16 (Arg16-->Gly) has been shown to confer exaggerated agonist-mediated receptor downregulation, which might attenuate vasodilator response. One hundred thirty-six unrelated hypertensives and 81 unrelated normotensives of African Caribbean origin were identified from primary care on the island of St Vincent. Genomic DNA from these subjects was analyzed for the presence of the Gly16 and Arg16 alleles by using an allele-specific polymerase chain reaction method. We report strong support for association of the prodownregulatory glycine 16 variant of the beta2-adrenoceptor gene with hypertension in African Caribbeans from St Vincent and the Grenadines (chi2=18.9, P=.000014, 1 df). This observation, coupled with reports of attenuated vasodilator responses to beta-agonists among people of West African ancestry, may provide a mechanism for enhanced vascular reactivity and identify a candidate gene for hypertension in this ethnic group.

Stability of RNA isolated from macrophages depends on the removal of an RNA-degrading activity early in the extraction procedure.

Ubiquitous RNases are the usual causes of RNA degradation on its isolation from mammalian cells. Using guanidine hydrochloride for the extraction of RNA from mouse peritoneal macrophages, we identify a major source of RNA-degrading activity, the stage of the extraction procedure at which this activity may be detected and show that its removal early in the extraction leads to a more dependable method for the recovery of high quality RNA.

Modifications of the guanidine hydrochloride procedure for the extraction of RNA: isolation from a variety of tissues and adherent/nonadherent cell types.

In a previous report dealing with the guanidine hydrochloride protocol for the extraction of RNA from mouse peritoneal macrophages, we identified a major source of RNA-degrading activity and showed that its removal early in the extraction procedure resulted in a more dependable method for the recovery of high-quality RNA. This report extends these findings and demonstrates the general applicability of the technique to a variety of fresh or frozen adherent cell types, cell suspensions and tissues, further highlighting stages at which degradation is most likely to occur and how to avoid a variety of pitfalls associated with the extraction procedure.

The atrial natriuretic peptide gene and essential hypertension in African-Caribbeans from St Vincent and the Grenadines.

Atrial natriuretic peptide (ANP) which alters sodium balance, blood volume and vascular tone represents an important candidate for investigating the genetic basis of essential hypertension (EH). Accordingly, we have studied Bgl1 and Xho1 restriction fragment length polymorphisms (RFLPs) of the ANP gene in 147 hypertensive, 141 normotensive and 67 population-based control subjects from a homogenous population of West African origin from St Vincent and the Grenadines. We found no association of either Bgl1 and Xho1 RFLPs with EH. This study suggests that the ANP locus may not exert a major gene effect on EH amongst the black people of St Vincent and the Grenadines.

The therapeutic efficacy of murine anti-tumor T cells: freshly isolated T cells are more therapeutic than T cells expanded in vitro.

Adoptive immunotherapy (AIT) involving transfer of tumor-sensitized T lymphocytes in combination with cyclophosphamide (CY)-injection results in the eradication of the C57BL/6J (B6) rhabdomyosarcoma, 76-9 and is associated with the accumulation of a large number of tumor-infiltrating lymphocytes (TIL). Using immune spleen cells (ISC) from B6 and congenic B6. PL. Thy-1a mice, it was shown that most (> or = 97%) of the TIL were donor-derived. This in situ increase in donor-derived T cells was confirmed by using positively-selected Thy- 1.1+ and Thy- 1.2+ TIL for AIT after isolating them from regressing tumors and expanding them in rIL-2. The extent of CD8+ TIL expansion in vivo correlated with the numbers of TIL adoptively transferred and this in turn determined the degree of anti-tumor effects. It was evident, however, that these in vitro-expanded TIL expressing mRNA for TNF alpha and IFN gamma were qualitatively different and therapeutically less efficacious than the T cells associated with ISC or with freshly-isolated TIL. Unlike freshly isolated TIL that expressed specific cytotoxicity towards the 76-9 targets in vitro, IL-2 expanded TIL killed 76-9 cells and unrelated tumor targets to the same extent. A cytotoxic CD8+ T cell line derived from ISC and selected for activity against the 76-9 tumor cells showed no therapeutic efficacy. The data suggest that, in this tumor model, expansion of CD8+ T cells in vitro selects against anti-tumor efficacy.

Effect of antibiotics on polymorphonuclear function in iron deficiency anaemia patients & normal volunteers.

The effect of erythromycin and gentamicin on polymorphonuclear leukocyte (PMN) functions was assessed in normal individuals and in patients with iron deficiency anaemia (IDA) before and after treatment with iron. The PMN phagocytic function was investigated by the standard method. Erythromycin in vivo significantly increased the PMN phagocytic function from 44.18 +/- 2.08 to 57.0 +/- 1.5 at 8 h and the bactericidal activity from 48.33 +/- 1.97 to 56.7 +/- 0.89 at 8 h in the normal adult male volunteers. A significant increase in phagocytic and bactericidal function of PMNs from IDA patients was also observed after in vivo administration of erythromycin. Gentamicin in vitro reduced the bactericidal activity of PMN from normal volunteers (P less than 0.05) but increased the PMN phagocytic activity in normal volunteers and IDA patients.

In vitro effect of intravenous immunoglobulin on serum opsonic activity in normal & intrauterine growth retarded neonates.

In vitro effect of intravenous immunoglobulin (IVIG), Intraglobin F, on serum opsonic activity against Staphylococcus aureus was studied in 26 full term normal healthy neonates and 18 intrauterine growth retarded (IUGR) neonates by the polymorphonuclear leucocyte overlay method (requiring only a few drops of blood). Cord IgG and IgM levels were determined by single radial immunodiffusion. Serum opsonic activity against Staph. aureus was significantly lower in the IUGR neonates (49.1 +/- 0.89), as compared to that in normal neonates (61.96 +/- 0.73; P less than 0.001). Immunoglobulin supplementation in vitro at a concentration of 5 g/dl significantly enhanced the opsonic activity of IUGR neonate sera. Cord IgG levels of IUGR neonates were significantly lower (P less than 0.01) than IgG levels of normal neonates. No significant difference was observed in cord IgM levels between the normal and IUGR neonates.

Polymorphonuclear leukocyte functions in children with cyanotic and acyanotic congenital heart disease.

Eighteen cyanotic congenital heart disease (CCHD) and 17 acyanotic congenital heart disease (ACHD) patients in the age range of 2 months to 10 years along with their age and nutrition matched controls were studied for bactericidal, chemotactic and phagocytic functions. Bactericidal and phagocytic functions were significantly depressed in CCHD (p < 0.001) as well as ACHD group (p < 0.001) compared with controls. Chemotactic function was not significantly affected in either. Arterial oxygen content (as a measure of hypoxia) was calculated for each patient and correlated with each immune parameter by univariate linear regression analysis. In CCHD patients linear correlation of borderline significance (p = 0.07) was found between arterial oxygen content and bactericidal activity, but no correlation could be established with phagocytic and chemotactic functions. No correlation was obtained between hematocrit and any of the immune parameters. In ACHD patients no correlations were obtained between the immune parameters and arterial oxygen content or hematocrit. Iron deficiency anemia, known to affect bactericidal function, did not seem to affect the immune parameters in CCHD and ACHD groups. Altered oxygen content of the blood owing to hypoxia in CCHD patients may be an important etiological factor in the genesis of bacteremia and cerebral abscess. The affection of immune functions in ACHD cannot be adequately explained.