Uptake and transport of novel amphiphilic polyelectrolyte-insulin nanocomplexes by caco-2 cells - towards oral insulin

“The original publication is available at www.springerlink.com”. Copyright SpringerPurpose: The influence of polymer architecture on cellular uptake and transport across Caco-2 cells of novel amphiphilic polyelectrolyte-insulin nanocomplexes was investigated. Method: Polyallylamine (PAA) (15 kDa) was grafted with palmitoyl chains (Pa) and subsequently modified with quaternary ammonium moieties (QPa). These two amphiphilic polyelectrolytes (APs) were tagged with rhodamine and their uptake by Caco-2 cells or their polyelectrolyte complexes (PECs) with fluorescein isothiocyanate-insulin (FITC-insulin) uptake were investigated using fluorescence microscopy. The integrity of the monolayer was determined by measurement of transepithelial electrical resistance (TEER). Insulin transport through Caco-2 monolayers was determined during TEER experiments. Result: Pa and insulin were co-localised in the cell membranes while QPa complexes were found within the cytoplasm. QPa complex uptake was not affected by calcium, cytochalasin D or nocodazole. Uptake was reduced by co-incubation with sodium azide, an active transport inhibitor. Both polymers opened tight junctions reversibly where the TEER values fell by up to 35 % within 30 minutes incubation with Caco-2 cells. Insulin transport through monolayers increased when QPa was used (0.27 ngmL-1 of insulin in basal compartment) compared to Pa (0.14 ngmL-1 of insulin in basal compartment) after 2 hours. Conclusion: These APs have been shown to be taken up by Caco-2 cells and reversibly open tight cell junctions. Further work is required to optimise these formulations with a view to maximising their potential to facilitate oral delivery of insulin.Peer reviewe

Epitope Mapping of HIV-Specific CD8+ T cells in a Cohort Dominated by Clade A1 Infection

CD8+ T cell responses are often detected at large magnitudes in HIV-infected subjects, and eliciting these responses is the central aim of many HIV-1 vaccine strategies. Population differences in CD8+ T cell epitope specificity will need to be understood if vaccines are to be effective in multiple geographic regions.In a large Kenyan cohort, we compared responsive CD8+ T cell HIV-1 Env overlapping peptides (OLPs) to Best Defined Epitopes (BDEs), many of which have been defined in clade B infection. While the majority of BDEs (69%) were recognized in this population, nearly half of responsive OLPs (47%) did not contain described epitopes. Recognition frequencies of BDEs were inversely correlated to epitopic sequence differences between clade A1 and BDE (P = 0.019), and positively selected residues were more frequent in "new" OLPs (without BDEs). We assessed the impact of HLA and TAP binding on epitope recognition frequencies, focusing on predicted and actual epitopes in the HLA B7 supertype.Although many previously described CD8 epitopes were recognized, several novel CD8 epitopes were defined in this population, implying that epitope mapping efforts have not been completely exhausted. Expansion of these studies will be critical to understand population differences in CD8 epitope recognition

Effect of Baseline HIV Disease Parameters on CD4+ T Cell Recovery After Antiretroviral Therapy Initiation in Kenyan Women

Antiretroviral therapy (ART) for HIV infection reconstitutes the immune system and improves survival. However, the rate and extent of CD4+ T cell recovery varies widely. We assessed the impact of several factors on immune reconstitution in a large Kenyan cohort.HIV-infected female sex workers from a longitudinal cohort, with at least 1 year of pre-ART and 6 months of post-ART follow-up (n = 79), were enrolled in the current study. The median pre-ART follow-up was 4,040 days. CD4 counts were measured biannually and viral loads where available. The median CD4 count at ART initiation was 180 cells/ul, which increased to 339 cells/ul at the most recent study visit. The rate of CD4+ T cell increase on ART was 7.91 cells/month (mean = 13, range -25.92 to 169.4). LTNP status prior to ART initiation did not associate with the rate of CD4 recovery on ART. In univariate analyses, associations were observed for CD4 recovery rate and duration of pre-ART immunosuppression (r = -0.326, p = 0.004) and CD4 nadir (r = 0.284, p = 0.012). In multivariate analysis including age, CD4 nadir, duration of HIV infection, duration of pre-ART immunosuppression, and baseline viral load, only CD4 nadir (p = 0.007) and not duration of immunosuppression (p = 0.87) remained significantly associated with the rate of CD4 recovery.These data suggest that prior duration of immune suppression does not predict subsequent recovery once ART is initiated and confirm the previous observation that the degree of CD4 depletion prior to ART initiation is the most important determinant of subsequent immune reconstitution

Search for the Θ+\Theta^+ pentaquark in the reaction γd→pK−K+n\gamma d \to p K^- K^+ n

A search for the \thp in the reaction $\gamma d \to pK^-K^+n$ was completed using the CLAS detector at Jefferson Lab. A study of the same reaction, published earlier, reported the observation of a narrow \thp resonance. The present experiment, with more than 30 times the integrated luminosity of our earlier measurement, does not show any evidence for a narrow pentaquark resonance. The angle-integrated upper limit on \thp production in the mass range of 1.52 to 1.56 GeV/c$^2$ for the $\gamma d \to pK^-\Theta^+$ reaction is 0.3 nb (95% CL). This upper limit depends on assumptions made for the mass and angular distribution of \thp production. Using \lamstar production as an empirical measure of rescattering in the deuteron, the cross section upper limit for the elementary $\gamma n \to K^-\Theta^+$ reaction is estimated to be a factor of 10 higher, {\it i.e.}, $\sim 3$ nb (95% CL).Comment: 5 figures, submitted to PRL, revised for referee comment

Single pi+ Electroproduction on the Proton in the First and Second Resonance Regions at 0.25GeV^2 < Q^2 < 0.65GeV^2 Using CLAS

The ep -> e'pi^+n reaction was studied in the first and second nucleon resonance regions in the 0.25 GeV^2 < Q^2 < 0.65 GeV^2 range using the CLAS detector at Thomas Jefferson National Accelerator Facility. For the first time the absolute cross sections were measured covering nearly the full angular range in the hadronic center-of-mass frame. The structure functions sigma_TL, sigma_TT and the linear combination sigma_T+epsilon*sigma_L were extracted by fitting the phi-dependence of the measured cross sections, and were compared to the MAID and Sato-Lee models.Comment: Accepted for publication in PR

Photodisintegration of 4^4He into p+t

The two-body photodisintegration of $^4$He into a proton and a triton has been studied using the CEBAF Large-Acceptance Spectrometer (CLAS) at Jefferson Laboratory. Real photons produced with the Hall-B bremsstrahlung-tagging system in the energy range from 0.35 to 1.55 GeV were incident on a liquid $^4$He target. This is the first measurement of the photodisintegration of $^4$He above 0.4 GeV. The differential cross sections for the $\gamma$$^4$He$\to pt$ reaction have been measured as a function of photon-beam energy and proton-scattering angle, and are compared with the latest model calculations by J.-M. Laget. At 0.6-1.2 GeV, our data are in good agreement only with the calculations that include three-body mechanisms, thus confirming their importance. These results reinforce the conclusion of our previous study of the three-body breakup of $^3$He that demonstrated the great importance of three-body mechanisms in the energy region 0.5-0.8 GeV .Comment: 13 pages submitted in one tgz file containing 2 tex file and 22 postscrip figure

π0\pi^0 photoproduction on the proton for photon energies from 0.675 to 2.875 GeV

Differential cross sections for the reaction $\gamma p \to p \pi^0$ have been measured with the CEBAF Large Acceptance Spectrometer (CLAS) and a tagged photon beam with energies from 0.675 to 2.875 GeV. The results reported here possess greater accuracy in the absolute normalization than previous measurements. They disagree with recent CB-ELSA measurements for the process at forward scattering angles. Agreement with the SAID and MAID fits is found below 1 GeV. The present set of cross sections has been incorporated into the SAID database, and exploratory fits have been extended to 3 GeV. Resonance couplings have been extracted and compared to previous determinations.Comment: 18 pages, 48 figure

Exclusive ρ0\rho^0 electroproduction on the proton at CLAS

The $e p\to e^\prime p \rho^0$ reaction has been measured, using the 5.754 GeV electron beam of Jefferson Lab and the CLAS detector. This represents the largest ever set of data for this reaction in the valence region. Integrated and differential cross sections are presented. The $W$, $Q^2$ and $t$ dependences of the cross section are compared to theoretical calculations based on $t$-channel meson-exchange Regge theory on the one hand and on quark handbag diagrams related to Generalized Parton Distributions (GPDs) on the other hand. The Regge approach can describe at the $\approx$ 30% level most of the features of the present data while the two GPD calculations that are presented in this article which succesfully reproduce the high energy data strongly underestimate the present data. The question is then raised whether this discrepancy originates from an incomplete or inexact way of modelling the GPDs or the associated hard scattering amplitude or whether the GPD formalism is simply inapplicable in this region due to higher-twists contributions, incalculable at present.Comment: 29 pages, 29 figure

First Measurement of Beam-Recoil Observables Cx and Cz in Hyperon Photoproduction

Spin transfer from circularly polarized real photons to recoiling hyperons has been measured for the reactions $\vec\gamma + p \to K^+ + \vec\Lambda$ and $\vec\gamma + p \to K^+ + \vec\Sigma^0$. The data were obtained using the CLAS detector at Jefferson Lab for center-of-mass energies $W$ between 1.6 and 2.53 GeV, and for $-0.85<\cos\theta_{K^+}^{c.m.}< +0.95$. For the $\Lambda$, the polarization transfer coefficient along the photon momentum axis, $C_z$, was found to be near unity for a wide range of energy and kaon production angles. The associated transverse polarization coefficient, $C_x$, is smaller than $C_z$ by a roughly constant difference of unity. Most significantly, the {\it total} $\Lambda$ polarization vector, including the induced polarization $P$, has magnitude consistent with unity at all measured energies and production angles when the beam is fully polarized. For the $\Sigma^0$ this simple phenomenology does not hold. All existing hadrodynamic models are in poor agreement with these results.Comment: 28 pages, 18 figures, Submitted to Physical Review