Kent T. HayGlass

Human IP-10 selectively promotes dominance of polyclonally activated and environmental antigen-driven IFN-γ over IL-4 responses

Human interferon‐inducible protein 10 (IP‐10) differs from most chemokines in its apparent specificity for activated T lymphocytes. We hypothesized that IP‐10 was relevant not only for recruiting T cells to inflammatory sites, but also for regulating cytokine synthesis patterns. We examined the effect of recombinant human IP‐10 (rhIP‐10) on human interferon γ (IFN‐γ) and interleukin 4 (IL‐4) production by fresh peripheral blood mononuclear cells. We demonstrate for the first time that this CXC chemokine selectively up‐regulates human T cell cytokine synthesis, with enhancement selectively targeted to promotion of Th1‐like dominance. Superantigen (TSST‐1), soluble anti‐CD3 mAb, and phytohemagglutinin were used to activate distinct intracellular signaling pathways, thereby inducing quantitatively different IFN‐γ:IL‐4 ratios. Selective enhancement of IFN‐γ responses was consistently observed, with median increases of 105–470%. Environmental antigens (Ag) were used to evaluate IP‐10s effect on CD4‐dependent, chloroquine‐sensitive cytokine synthesis. Ag‐driven IFN‐γ responses exhibited median 19‐ to 30‐fold increases in the presence of nanomolar concentrations of rhIP‐10. IL‐4 responses were neither enhanced nor inhibited under any of the conditions tested. These findings suggest a potential role for this T cell‐focused chemokine in maintenance of the default Th1‐like responses usually seen to environmental Ag and indicate a potential application in the modulation of Ag‐driven responses in vivo.—Gangur, V., Simons, F.E.R., Hayglass, K. T. Human IP‐10 selectively promotes dominance of polyclonally activated and environmental antigen‐driven IFN‐γ over IL‐4 responses. FASEB J. 12, 705–713 (1998)

Comparison of [3H]thymidine incorporation with MTT- and MTS-based bioassays for human and murine IL-2 and IL-4 analysis Tetrazolium assays provide markedly enhanced sensitivity

The sensitivity of [3H]thymidine incorporation and MTT/MTS colorimetric bioassays for detection and quantitation of murine and human IL-4 and IL-2 were compared in CT.4S, CT.h4S and HT-2 bioassays respectively. We reasoned that low levels of cytokine, insufficient to induce cell proliferation (thus, DNA synthesis and [3H]thymidine incorporation), may be sufficient to maintain the viability of the bioassay cells in culture. Because colorimetric assays such as those employing MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-( 4-sulfonyl)-2H- tetrazolium) measure conversion of these salts to intensely colored formazan products by mitochondrial enzymes independent of whether proliferation is induced, we reasoned that such assays could be superior for detection of low levels of cytokine protein. Direct comparison of these approaches demonstrated that the MTT- and MTS-based assays were consistently able to detect 2-16-fold lower cytokine levels than methods based on [3H]thymidine incorporation. Moreover, the MTT and MTS assays exhibited higher precision with standard deviations of < 1-4% vs. 5-15% for thymidine incorporation. This finding is of particular importance in approaches such as limiting dilution analysis, or primary bulk culture of antigen-stimulated human lymphocytes, where levels of cytokine production may be extremely low.

Infant gut microbiota and food sensitization: associations in the first year of life

The gut microbiota is established during infancy and plays a fundamental role in shaping host immunity. Colonization patterns may influence the development of atopic disease, but existing evidence is limited and conflicting.

Mass spectrometry-based proteomic analysis of urine in acute kidney injury following cardiopulmonary bypass: a nested case-control study.

BACKGROUND The early evolution of acute kidney injury (AKI) in humans is difficult to study noninvasively. We hypothesized that urine proteomics could provide insight into the early pathophysiology of human AKI. STUDY DESIGN A prospective nested case-control study (n = 250) compared serial urinary proteomes of 22 patients with AKI and 22 patients without AKI before, during, and after cardiopulmonary bypass surgery. OUTCOMES AKI was defined as a greater than 50% increase in serum creatinine level, and non-AKI, as less than 10% increase from baseline. MEASUREMENTS Serum creatinine, urine protein-creatinine ratio, neutrophil gelatinase-associated lipocalin (NGAL), alpha1-microglobulin, interferon-inducible protein-10 (IP-10), monokine induced by interferon gamma (Mig), interferon-inducible T cell alpha chemoatractant (I-TAC), interleukin 6 (IL-6), IL-1beta, and IL-10. Urine protein profiling by means of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). RESULTS SELDI-TOF-MS showed intraoperative tubular stress in both groups on arrival to the intensive care unit, evidenced by beta2-microglobulinuria. Non-AKI proteomes returned toward baseline postoperatively. In contrast, AKI proteomes showed a second phase of tubular injury/stress with the reappearance of beta2-microglobulin and multiple unidentified peaks (3 to 5 and 6 to 8 kDa) and the appearance of established tubular injury markers: urinary protein, alpha1-microglobulin, and NGAL. Furthermore, 2 novel peaks (2.43 and 2.78 kDa) were found to be dominant in postoperative non-AKI urine samples. The 2.78-kDa protein was identified as the active 25-amino acid form of hepcidin (hepcidin-25), a key regulator of iron homeostasis. Finally, an inflammatory component of reperfusion injury was evaluated by means of enzyme-linked immunosorbent assay analysis of candidate chemokines (IP-10, I-TAC, and Mig) and cytokines (IL-6, IL-1beta, and IL-10). Of these, IP-10 was upregulated in patients with versus without AKI postoperatively. LIMITATIONS This is an observational study. SELDI-TOF-MS is a semiquantitative technique. CONCLUSIONS Evaluation of human AKI revealed early intraoperative tubular stress in all patients. A second phase of injury observed in patients with AKI may involve IP-10 recruitment of inflammatory cells. The enhancement of hepcidin-25 in patients without AKI may suggest a novel role for iron sequestration in modulating AKI.

Analysis of Neonatal T Cell and Antigen Presenting Cell Functions

Neonates are more susceptible to infection than adults and exhibit more intense or prolonged clinical symptoms. The extent to which deficiencies in T cell or antigen presenting cell (APC) function underlie hyporesponsiveness is incompletely understood. Here, immune function of cord blood mononuclear cells (CBMC), from healthy, full-term neonates was compared with adult PBMC. As widely reported, polyclonally-stimulated T cell proliferation was found to be equivalent, while IFN gamma responses were markedly lower amongst neonates. Reasoning that such stimuli may elicit responses qualitatively different from those that would be obtained following MHC-dependent, cognate T cell activation, alloantigen-specific responses were evaluated. Strikingly, neonates exhibited IFN gamma, IL-4 and IL-10 production equal to adults in short term primary culture. Both the frequency (Fishers p < 0.0004) and intensity (< 7.5 vs 36.5 pg/ml; Wilcoxon P = 0.005) of alloantigen stimulated IL-5 responses were elevated among neonates, a finding equally evident using irradiated adult or neonatal cells as stimulators. Finally, the relative capacity of neonatal APC as stimulators of cytokine synthesis was assessed by a novel approach using CBMC as both responders and stimulators in MLR. Irradiated neonatal cells consistently stimulated similar proliferative but substantially lower IFN gamma responses than did adult APC, independent of responder origin. The data argue; (i) T cells are largely immunocompetent at birth, (ii) accessory cell function is not fully mature, and (iii) the widely observed hyporesponsiveness to pathogenes may be primarily due to immaturity of APC function or costimulator molecule expression.

Role of the phosphoinositide 3‐kinase p110δ in generation of type 2 cytokine responses and allergic airway inflammation

Phosphoinositide 3‐kinases (PI3K) regulate immune activation via their roles in signal transduction of multiple classes of receptors. Here, we examined the effect of genetic inactivation of the hemopoietic cell‐restricted PI3K isoform p110δ on systemic cytokine and chemokine responses and allergic airway inflammation. We found that type 2 cytokine responses (IL‐4, IL‐5 and IL‐13) are significantly decreased in p110δ mutants, whereas type 1 cytokine responses (IFN‐γ and CXCL10) were robust. Elevated IFN‐γ production during the primary response to ovalbumin (OVA) was associated with reduced production of the regulatory cytokine IL‐10. IFN‐γ and IL‐10 production normalized after secondary OVA immunization; however, type 2 cytokine production was persistently reduced. Type 2 cytokine‐dependent airway inflammation elicited by intranasal challenge with OVA was dramatically reduced, with reduced levels of eosinophil recruitment and mucus production observed in the lungs. Induction of respiratory hyper‐responsiveness to inhaled methacholine, a hallmark of asthma, was markedly attenuated in p110δ‐inactivated mice. Adoptive transfer of OVA‐primed splenocytes from normal but not p110δ‐inactivated mice could induce airway eosinophilia in naive, airway‐challenged recipient mice. These data demonstrate a novel functional role for p110δ signaling in induction of type 2 responses in vivo and may offer a new therapeutic target for Th2‐mediated airway disease.

RanBPM interacts with psoriasin in vitro and their expression correlates with specific clinical features in vivo in breast cancer

BackgroundPsoriasin has been identified as a gene that is highly expressed in pre-invasive breast cancer, but is often downregulated with breast cancer progression. It is currently unknown whether psoriasin influences epithelial cell malignancy directly or by affecting the surrounding environment. However the protein is found in the nucleus, cytoplasm as well as extracellularly. In the present study we have sought to identify potential psoriasin-binding proteins and to describe their expression profile in breast tumors.MethodsThe yeast two-hybrid method was used to identify potential binding partners for psoriasin. The interaction of psoriasin with RanBPM was confirmed in-vitro by co-immunoprecipitation. The expression of RanBPM and psoriasin was measured by RT-PCR in a series of breast cell lines, breast tumors and primary lymphocytes.ResultsWe have identified RanBPM as an interacting protein by the yeast two-hybrid assay and confirmed this interaction in-vitro by co-immunoprecipitation. RT-PCR analysis of RanBPM mRNA expression in cell lines (n = 13) shows that RanBPM is widely expressed in different cell types and that expression is higher in tumor than in normal breast epithelial cell lines. RanBPM expression can also be induced in peripheral blood mononuclear cells by treatment with PHA. RanBPM mRNA is also frequently expressed in invasive breast carcinomas (n = 64) and a higher psoriasin/RanBPM ratio is associated with both ER negative (p < 0.0001) and PR negative status (p < 0.001), and inflammatory cell infiltrates (p < 0.0001) within the tumor.ConclusionsThese findings support the hypothesis that psoriasin may interact with RanBPM and this may influence both epithelial and stromal cells and thus contribute to breast tumor progression.

FDC-SP, a novel secreted protein expressed by follicular dendritic cells

To define better the molecular basis for follicular dendritic cell (FDC) function, we used PCR-based cDNA subtraction to identify genes specifically expressed in primary FDC isolated from human tonsils. In this work we report the discovery of a novel gene encoding a small secreted protein, which we term FDC-SP (FDC secreted protein). The FDC-SP gene lies on chromosome 4q13 adjacent to clusters of proline-rich salivary peptides and C-X-C chemokines. Human and mouse FDC-SP proteins are structurally unique and contain a conserved N-terminal charged region adjacent to the leader peptide. FDC-SP has a very restricted tissue distribution and is expressed by activated FDCs from tonsils and TNF-α-activated FDC-like cell lines, but not by B cell lines, primary germinal center B cells, or anti-CD40 plus IL-4-activated B cells. Strikingly, FDC-SP is highly expressed in germinal center light zone, a pattern consistent with expression by FDC. In addition, FDC-SP is expressed in leukocyte-infiltrated tonsil crypts and by LPS- or Staphylococcus aureus Cowan strain 1-activated leukocytes, suggesting that FDC-SP can also be produced in response to innate immunity signals. We provide evidence that FDC-SP is posttranslationally modified and secreted and can bind to the surface of B lymphoma cells, but not T lymphoma cells, consistent with a function as a secreted mediator acting upon B cells. Furthermore, we find that binding of FDC-SP to primary human B cells is markedly enhanced upon activation with the T-dependent activation signals such as anti-CD40 plus IL-4. Together our data identify FDC-SP as a unique secreted peptide with a distinctive expression pattern within the immune system and the ability to specifically bind to activated B cells.

The Canadian Healthy Infant Longitudinal Development (CHILD) Study: examining developmental origins of allergy and asthma

The Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort study recruited 3624 pregnant women, most partners and 3542 eligible offspring. We hypothesise that early life physical and psychosocial environments, immunological, physiological, nutritional, hormonal and metabolic influences interact with genetics influencing allergic diseases, including asthma. Environmental and biological sampling, innate and adaptive immune responses, gene expression, DNA methylation, gut microbiome and nutrition studies complement repeated environmental and clinical assessments to age 5. This rich data set, linking prenatal and postnatal environments, diverse biological samples and rigorous phenotyping, will inform early developmental pathways to allergy, asthma and other chronic inflammatory diseases.

Validation of Urinary CXCL10 As a Marker of Borderline, Subclinical, and Clinical Tubulitis

Background. Renal allograft injury secondary to subclinical and clinical tubulitis remains an important cause of allograft fibrosis and loss despite modern immunosuppression. The goal of this study was to validate the previously reported use of urinary CXCL10 (interferon-&ggr;-induced protein of 10 kDa) as a noninvasive marker of tubulitis in an independent clinical cohort. Methods. Urine samples (n=102) from 91 patients with protocol or indication biopsies were assayed for urinary CXCL10 using ELISA. The groups analyzed were as follows: normal histology (n=22); interstitial fibrosis and tubular atrophy (IFTA) (n=20); IFTA and borderline tubulitis (n=13); borderline (n=13), subclinical (n=17); and clinical tubulitis (n=17) without IFTA. Results. The ratio of urinary CXCL10 to creatinine (CXCL10: Cr) was found to distinguish borderline, subclinical and clinical tubulitis from normal histology, and IFTA. The area under the curve receiver operating characteristic curve to distinguish normal versus borderline and subclinical tubulitis was 0.845 (OR 1.407, P=0.0184); normal versus borderline, subclinical and clinical tubulitis was 0.835 (OR 1.400, P=0.0127). CXCL10: Cr demonstrated a sensitivity of 73.3% and specificity of 72.7% for normal versus borderline and subclinical tubulitis at a cut-off of 1.97 ng CXCL10/mmol Cr. Conclusion. This study validates urinary CXCL10 as a noninvasive, sensitive, and specific marker for tubulitis in an independent cohort. The straightforward urine processing is accessible to clinical laboratories. We propose that CXCL10 may be useful as a supplementary noninvasive screening test for tubulitis in renal transplant patients, with a level more than 1.97 ng CXCL10/mmol Cr being a threshold to consider biopsy.