Effects of Alpha Interferon Treatment on Intrinsic Anti-HIV-1 Immunity In Vivo

Alpha interferon (IFN-α) suppresses human immunodeficiency virus type 1 (HIV-1) replication in vitro by inducing cell-intrinsic retroviral restriction mechanisms. We investigated the effects of IFN-α/ribavirin (IFN-α/riba) treatment on 34 anti-HIV-1 restriction factors in vivo. Expression of several anti-HIV-1 restriction factors was significantly induced by IFN-α/riba in HIV/hepatitis C virus (HCV)-coinfected individuals. Fold induction of cumulative restriction factor expression in CD4+ T cells was significantly correlated with viral load reduction during IFN-α/riba treatment (r2 = 0.649; P < 0.016). Exogenous IFN-α induces supraphysiologic restriction factor expression associated with a pronounced decrease in HIV-1 viremia.

Structural and functional studies of the human integrin $\alpha 4\beta 1$ (CD49d/CD29)

The integrin receptor $\alpha 4\beta 1$ is a cell surface heterodimer involved in a variety of highly regulated cellular interactions. The purpose of this dissertation was to identify and characterize unique structural and functional properties of the $\alpha 4\beta 1$ molecule that may be important for adhesion regulation and signal transduction. To study these properties and to establish a consensus sequence for the $\alpha 4$ subunit, cDNA encoding $\alpha 4$ was cloned and sequenced. A comparison with previously described human $\alpha 4$ sequences identified several substitutions in the $5\prime$ and $3\prime$ untranslated regions, and a nonsynonymous G to A transition in the coding region, resulting in a glutamine substitution for arginine. Further analysis of this single nucleotide substitution indicated that two variants of the $\alpha 4$ subunit exist, and when compared with three ancestrally-related species, the new form cloned in our laboratory was found to be evolutionarily conserved.^ The expression of $\alpha 4$ cDNA in transfected K562 erythroleukemia cells, and subsequent studies using flow cytofluorometric, immunochemical, and ligand binding/blocking analyses, confirmed $\alpha 4\beta 1$ as a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1), and provided a practical means of identifying two novel monoclonal antibody (mAb) binding epitopes on the $\alpha 4\beta 1$ complex that may play important roles in the regulation of leukocyte adhesion.^ To investigate the association of $\alpha 4\beta 1$-mediated adhesion with signals involved in the spreading of lymphocytes on FN, a quantitative method of analysis was developed using video microscopy and digital imaging. The results showed that HPB-ALL $(\alpha 4\beta 1\sp{\rm hi},\ \alpha 5\beta 1\sp-)$ cells could adhere and actively spread on human plasma FN, but not on control substrate. Many cell types which express different levels of the $\alpha 4\beta 1$ and $\alpha 5\beta 1$ FN binding integrins were examined for their ability to function in these events. Using anti-$\alpha 4$ and anti-$\alpha 5$ mAbs, it was determined that cell adhesion to FN was influenced by both $\beta 1$ integrins, while cell spreading was found to be dependent on the $\alpha 4\beta 1$ complex. In addition, inhibitors of phospholipase A$\sb2$ (PLA$\sb2$), 5-lipoxygenases, and cyclooxygenases blocked HPB-ALL cell spreading, yet had no effect on cell adhesion to FN, and the impaired spreading induced by the PLA$\sb2$ inhibitor cibacron blue was restored by the addition of exogenous arachidonic acid (AA). These results suggest that the interaction of $\alpha 4\beta 1$ with FN, the activation of PLA$\sb2,$ and the subsequent release of AA, may be involved in lymphocyte spreading. ^

Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men

BACKGROUND: Mental stress reliably induces increases in salivary alpha amylase (sAA), a suggested surrogate marker for sympathetic nervous system (SNS) reactivity. While stress-induced sAA increases correlate with norepinephrine (NE) secretion, a potential mediating role of noradrenergic mechanisms remains unclear. In this study, we investigated for the first time in humans whether a NE-stress-reactivity mimicking NE-infusion with and without alpha-adrenergic blockade by phentolamine would induce changes in sAA. METHODS: In a single-blind placebo-controlled within-subjects design, 21 healthy men (29-66 years) took part in three different experimental trials varying in terms of substance infusion with a 1-min first infusion followed by a 15-min second infusion: saline-infusion (trial-1), NE-infusion (5 μg/min) without alpha-adrenergic blockade (trial-2), and with phentolamine-induced non-selective blockade of alpha1- and alpha2-adrenergic receptors (trial-3). Saliva samples were collected immediately before, during, and several times after substance infusion in addition to blood pressure and heart rate readings. RESULTS: Experimental trials significantly differed in sAA reactivity to substance-infusion (p=.001) with higher sAA reactivity following NE-infusion with (trial-3; p=.001) and without alpha-adrenergic-blockade (trial-2; p=.004) as compared to placebo-infusion (trial-1); sAA infusion reactivity did not differ between trial-2 and trial-3 (p=.29). Effective phentolamine application was verified by blood pressure and heart rate infusion reactivity. Salivary cortisol was not affected by NE, either with or without alpha-adrenergic-blockade. CONCLUSIONS: We found that NE-infusion stimulates sAA secretion, regardless of co-administered non-selective alpha-adrenergic blockade by phentolamine, suggesting that the mechanism underlying stress-induced sAA increases may involve NE.

CD62L (L-selectin) shedding for assessment of functional blockade of TNF alpha in anti-TNF treated IBD patients: clinical feasibility and perspectives (DOP060)

Background Tumor necrosis factor (TNF) inhibition is central to the therapy of inflammatory bowel diseases (IBD). However, loss of response (LOR) is frequent and additional tests to help decision making with costly anti-TNF Therapy are needed. Methods Consecutive IBD Patients receiving anti-TNF therapy (Infliximab (IFX) or Adalimumab (after IFX LOR) from Bern University Hospital were identified and followed prospectively. Patient whole blood was stimulated with a dose-titration of two triggers of TLR receptors human: TNF and LPS. Median fluorescence intensity of CD62L on the surface of granulocytes was quantified by surface staining with specific antibodies (CD33, CD62L) and flow cytometry and logistic curves to these data permits the calculation of EC50 or the half maximal effective concentration TNF concentration to induce shedding [1]. A shift in the concentration were CD62L shedding occurred was seen before and after the anti-TNF agent administraion which permits to predict the response to the drug. This predicted response was correlated to the clinical evolution of the patients in order to analyze the ability of this test to identify LOR to IFX. Results We collected prospective clinical data and blood samples, before and after anti-TNF agent administration, on 33 IBD patients, 25 Crohn's disease and 8 ulcerative colitis patients (45% females) between June 2012 and November 2013. The assay showed a functional blockade of IFX (PFR) for 22 patients (17 CD and 5 UC) whereas 11 (8 CD and 3 UC) had no functional response (NR) to IFX. Clinical characteristics (e.g. diagnosis, disease location, smoking status, BMI and number of infusions) were no significantly different between predicted PFR and NR. Among the 22 Patients with PRF, only 1 patient was a clinical non responder (LOR to IFX), based on clinical prospective evaluation by IBD gastroenterologists (PJ, AM), and among the 11 predicted NR, 3 had no clinical LOR. Sensitivity of this test was 95% and specificity 73% and AUC adjusted for age and gender was 0.81 (Figure 1). During follow up (median 10 mo, 3–15) 8 “hard” outcomes occured (3 medic. flares, 4 resections and 1 new fistula) 2 in the PFR and 6 in the NR group (25% vs. 75%; p < 0.01). Correlation with clinical response is presented in Figure 2. Figure 1. Figure 2. Correlation clinical response - log EC50 changes: 1 No, 2 partial, 3 complete clinical response. Conclusion CD62L (L-Selectin) shedding is the first validated test of functional blockade of TNF alpha in anti-TNF treated IBD patients and will be a useful tool to guide medical decision on the use of anti-TNF agents. Comparative studies with ATI and trough level of IFX are ongoing. 1. Nicola Patuto, Emma Slack, Frank Seibold and Andrew J. Macpherson, (2011), Quantitating Anti-TNF Functionality to Inform Dosing and Choice of Therapy, Gastroenterology, 140 (5, Suppl. I), S689.

Measurement of isotopic uranium in water for compliance monitoring by liquid scintillation counting with alpha/beta discrimination

A simple and inexpensive method is described for analysis of uranium (U) activity and mass in water by liquid scintillation counting using $\alpha$/$\beta$ discrimination. This method appears to offer a solution to the need for an inexpensive protocol for monitoring U activity and mass simultaneously and an alternative to the potential inaccuracy involved when depending on the mass-to-activity conversion factor or activity screen.^ U is extracted virtually quantitatively into 20 ml extractive scintillator from a 1-$\ell$ aliquot of water acidified to less than pH 2. After phase separation, the sample is counted for a 20-minute screening count with a minimum detection level of 0.27 pCi $\ell\sp{-1}$. $\alpha$-particle emissions from the extracted U are counted with close to 100% efficiency with a Beckman LS6000 LL liquid scintillation counter equipped with pulse-shape discrimination electronics. Samples with activities higher than 10 pCi $\ell\sp-1$ are recounted for 500-1000 minutes for isotopic analysis. Isotopic analysis uses events that are automatically stored in spectral files and transferred to a computer during assay. The data can be transferred to a commercially available spreadsheet and retrieved for examination or data manipulation. Values for three readily observable spectral features can be rapidly identified by data examination and substituted into a simple formula to obtain $\sp{234}$U/$\sp{238}$U ratio for most samples. U mass is calculated by substituting the isotopic ratio value into a simple equation.^ The utility of this method for the proposed compliance monitoring of U in public drinking water supplies was field tested with a survey of drinking water from Texas supplies that had previously been known to contain elevated levels of gross $\alpha$ activity. U concentrations in 32 samples from 27 drinking water supplies ranged from 0.26 to 65.5 pCi $\ell\sp{-1}$, with seven samples exceeding the proposed Maximum Contaminant Level of 20 $\mu$g $\ell\sp{-1}$. Four exceeded the proposed activity screening level of 30 pCi $\ell\sp{-1}$. Isotopic ratios ranged from 0.87 to 41.8, while one sample contained $\sp{234}$U activity of 34.6 pCi $\ell\sp{-1}$ in the complete absence of its parent, $\sp{238}$U. U mass in the samples with elevated activity ranged from 0.0 to 103 $\mu$g $\ell\sp{-1}$. A limited test of screening surface and groundwaters for contamination by U from waste sites and natural processes was also successful. ^

Expression of TNF-$\alpha$ protein and C -FOS and TNF mRNA during human peripheral blood monocyte maturation and activation

Human peripheral blood monocytes (HPBM) were isolated by centrifugal elutriation from mononuclear cell enriched fractions after routine plateletapheresis and the relationship between maturation of HPBM to macrophage-like cells and activation for tumoricidal activity determined. HPBM were cultured for various times in RPMI 1640 supplemented with 5% pooled human AB serum and cytotoxicity to $\sp{125}$IUDR labeled A375M, a human melanoma cell line, and TNF-$\alpha$ release determined by cytolysis of actinomycin D treated L929 cells. Freshly isolated HPBM or those exposed to recombinant IFN-$\gamma$(1.0 U/ml) were not cytolytic and did not release TNF-$\alpha$ into culture supernatants. Exposure to bacterial lipopolysaccharide (LPS, 1.0 $\upsilon$g/ml) stimulated cytolytic activity and release of TNF-$\alpha$. Maximal release of TNF-$\alpha$ protein occurred at 8 hrs and returned to baseline by 72 hrs. Expression of TNF-$\alpha$ protein was determined by Western blotting. Neither freshly isolated nor IFN-$\gamma$ treated HPBM expressed TNF protein at any time during in vitro culture. LPS treated HPBM maximally expressed the 17KD TNF-$\alpha$ protein at 8 hrs, and protein was not detected after 36 hrs of in vitro culture. Expression of TNF-$\alpha$ mRNA was determined by Northern blotting. Freshly isolated HPBM express TNF-$\alpha$ mRNA which decays to basal levels by 6 hrs of in vitro culture. IFN-$\gamma$ treatment maintains TNF-$\alpha$ mRNA expression for up to 48 hrs of culture, after which it is undetectable. LPS induces TNF-$\alpha$ mRNA after 30 minutes of exposure with maximal accumulation occurring between 4 to 8 hrs. TNF mRNA was not detected in control HPBM at any time after 6 hrs or IFN-$\gamma$ treated HPBM after 48 hrs of in vitro culture. A pulse of LPS the last 24 hrs of in vitro culture induces the accumulation of TNF-$\alpha$ mRNA in HPBM cultured for 3, 5, and 7 days, with the magnitude of induction decreasing approximately 10 fold between 3 and 7 days. Induction of TNF-$\alpha$ mRNA occurred in the absence of detectable TNF-$\alpha$ protein or supernatant activity. Maturation of HPBM to macrophage-like cells controls competence for activation, magnitude and duration of the activation response. ^