Dynamics of T cells and TCR excision circles differ after treatment of acute and chronic HIV infection

We quantified T cell proliferation and thymic function in primary HIV infection (PHI; n = 19) and chronic HIV infection (CHI; n = 14) by measuring Ki67 staining and TCR excision circle (TREC) number. After antiretroviral therapy of PHI there is a profound decrease in the number and percentage of Ki67(+) T cells (<6% Ki67(+)) with no significant increase in TREC per million cells and a transient increase in TREC per milliliter. In contrast, after antiretroviral therapy of CHI there is a reduction in the percentage but little change in the total number of Ki67(+)CD4(+) T cells associated with increases in both TREC per million cells and TREC per milliliter. Using a mathematical model that accounts for proliferation, death, and redistribution of T cells, we find that redistribution is consistent with the TREC changes observed during treatment of PHI and that an increase in thymic output is needed to explain the increase in TREC during treatment of CHI. Consideration of TREC per milliliter shows that changes in proliferation alone cannot explain the changes in TREC. In addition, although increased proliferation of memory cells in HIV infection has been established, we find no difference in TREC per million CD45RA(-) "memory" T cells between healthy and infected individuals (p = 0.154 for CD4(+); p = 0.383 for CD8(+)). Finally, although the number of TREC per million cells is always much lower in memory T cells than in naive T cells, in the setting of HIV infection, given that memory cells make up a larger proportion of total T cells, we find that 50% of TREC per milliliter in CD4(+) T cells is harbored in the CD45RA(-) "memory" subset of our infected subjects.     

Platelet Recruitment Promotes Keratocyte Repopulation following Corneal Epithelial Abrasion in the Mouse

Corneal abrasion not only damages the epithelium but also induces stromal keratocyte death at the site of injury. While a coordinated cascade of inflammatory cell recruitment facilitates epithelial restoration, it is unclear if this cascade is necessary for keratocyte recovery. Since platelet and neutrophil (PMN) recruitment after corneal abrasion is beneficial to epithelial wound healing, we wanted to determine if these cells play a role in regulating keratocyte repopulation after epithelial abrasion. A 2 mm diameter central epithelial region was removed from the corneas of C57BL/6 wildtype (WT), P-selectin deficient (P-sel^-/-), and CD18 hypomorphic (CD18^hypo) mice using the Algerbrush II. Corneas were studied at 6h intervals out to 48h post-injury to evaluate platelet and PMN cell numbers; additional corneas were studied at 1, 4, 14, and 28 days post injury to evaluate keratocyte numbers. In WT mice, epithelial abrasion induced a loss of anterior central keratocytes and keratocyte recovery was rapid and incomplete, reaching ~70% of uninjured baseline values by 4 days post-injury but no further improvement at 28 days post-injury. Consistent with a beneficial role for platelets and PMNs in wound healing, keratocyte recovery was significantly depressed at 4 days post-injury (~30% of uninjured baseline) in P-sel^-/- mice, which are known to have impaired platelet and PMN recruitment after corneal abrasion. Passive transfer of platelets from WT, but not P-sel^-/-, into P-sel^-/- mice prior to injury restored anterior central keratocyte numbers at 4 days post-injury to P-sel^-/- uninjured baseline levels. While PMN infiltration in injured CD18^hypo mice was similar to injured WT mice, platelet recruitment was markedly decreased and anterior central keratocyte recovery was significantly reduced (~50% of baseline) at 4–28 days post-injury. Collectively, the data suggest platelets and platelet P-selectin are critical for efficient keratocyte recovery after corneal epithelial abrasion.     

Baroreceptor reflex control of heart rate in rats studied by induced and autogenic changes in arterial pressure

The function of the arterial baroreflex has traditionally been assessed by measurement of reflex changes in heart rate (HR) or sympathetic nerve activity resulting from experimenter-induced manipulation of arterial blood pressure (the Oxford method, also termed the pharmacological method). However, logistical and flexibility limitations of this technique have promoted the development of new methods for assessing baroreflex function such as the evaluation of changes in spontaneous arterial pressure and HR. Although this new spontaneous method has been validated in dogs and humans, it has not been rigorously tested in rats. In the present study, the method of correlating spontaneous changes in systolic blood pressure and HR was evaluated in resting, normotensive Sprague-Dawley rats. This technique was found to be neither reliable nor valid under the conditions employed in the present protocol. We also tested a variation of the spontaneous method that evaluates particular sequences of data during which arterial pressure and pulse interval are changing in the same direction for at least three consecutive heartbeats (the sequence method). The sequence method did not provide extra reliability or validity over the spontaneous method. We conclude that due to the restricted range of variability obtained by measuring spontaneous blood pressure fluctuations, the spontaneous and sequence techniques do not provide data that are comparable to the traditional method of assessing HR changes triggered by arterial blood pressure increases and decreases induced by vasoactive drugs. However, it is possible that surgical stress obscured the relationship between blood pressure and HR, and therefore additional studies are needed to determine whether the spontaneous and sequence methods can be applied to rats during different behavioral states.     

Bile acid-induced secretion in polarized monolayers of T84 colonic epithelial cells: Structure-activity relationships

Bile acid epimers and side-chain homologues are present in the human colon. To test whether such bile acids possess secretory activity, cultured T84 colonic epithelial cells were used to quantify the secretory properties of synthetic epimers and homologues of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). In our study, chloride secretion was measured as changes in short-circuit current (DeltaI(sc), in microA/cm2) with the use of voltage-clamped monolayers of T84 cells mounted in Ussing chambers. Bile acids were added at 0.5 mM, a concentration that did not alter transepithelial resistance. Data were expressed as peak DeltaI(sc) (means +/- SD). When added bilaterally, DCA stimulated a DeltaI(sc) response of 15.7 +/- 12.5 microA/cm2. The 12beta-OH epimer of DCA was less potent (DeltaI(sc) = 8.0 +/- 1.7 microA/cm2), whereas its 3beta-OH epimer had no effect. CDCA stimulated secretion (DeltaI(sc) = 8.2 +/- 5.5 microA/cm2), whereas both its 7beta-OH and 3beta-OH epimers were inactive, as was lithocholic acid. HomoDCA (1 additional side-chain carbon) was active (DeltaI(sc) = 7.8 +/- 4.8 microA/cm2), whereas norDCA (1 fewer carbon) and dinorDCA (2 fewer carbons) were not. Taurine conjugates of DCA and CDCA stimulated secretion (DeltaI(sc) = 12.3 +/- 7.5 and 8.8 +/- 4.8 microA/cm2, respectively) from the basolateral side but not the apical side. Uptake of taurine conjugates from the basolateral but not the apical side was shown by mass spectrometry. These studies indicate marked structural specificity for bile acid-induced chloride secretion and show that modification of bile acid structure by colonic bacteria modulates the secretory properties of these endogenous secretagogues.     

Evaluation of selective inhibitors of 11β-HSD1 for the treatment of hypertension

In an effort to understand the origin of blood-pressure lowering effects observed in recent clinical trials with 11β-HSD1 inhibitors, we examined a set of 11β-HSD1 inhibitors in a series of relevant in vitro and in vivo assays. Select 11β-HSD1 inhibitors reduced blood pressure in our preclinical models but most or all of the blood pressure lowering may be mediated by a 11β-HSD1 independent pathway.     

ApoC-III content of apoB-containing lipoproteins is associated with binding to the vascular proteoglycan biglycan

Retention of apolipoprotein (apo)B and apoE-containing lipoproteins by extracellular vascular proteoglycans is critical in atherogenesis. Moreover, high circulating apoC-III levels are associated with increased atherosclerosis risk. To test whether apoC-III content of apoB-containing lipoproteins affects their ability to bind to the vascular proteoglycan biglycan, we evaluated the impact of apoC-III on the interaction of [(35)S]SO(4)-biglycan derived from cultured arterial smooth muscle cells with lipoproteins obtained from individuals across a spectrum of lipid concentrations. The extent of biglycan binding correlated positively with apoC-III levels within VLDL (r = 0.78, P < 0.01), IDL (r = 0.67, P < 0.01), and LDL (r = 0.52, P < 0.05). Moreover, the biglycan binding of VLDL, IDL, and LDL was reduced after depletion of apoC-III-containing lipoprotein particles in plasma by anti-apoC-III immunoaffinity chromatography. Since apoC-III does not bind biglycan directly, enhanced biglycan binding may result from a conformational change associated with increased apo C-III content by which apoB and/or apoE become more accessible to proteoglycans. This may be an intrinsic property of lipoproteins, since exogenous apoC-III enrichment of LDL and VLDL did not increase binding. ApoC-III content may thus be a marker for lipoproteins characterized as having an increased ability to bind proteoglycans.     

Further observations on the ultrastructure of Cystosporogenes operophterae (Canning, 1960) (phylum Microsporidia) parasitic in Operophtera brumata L. (Lepidoptera, Geometridae)

Reinvestigation of Cystosporogenes operophterae [J. Parasitol. 46 (1960) 755] by electron microscopy confirmed that development in host cells takes place in a vacuole with a single membrane at its boundary. Although ribosomes were not clustered on this membrane, it is hypothesised that it originates from host endoplasmic reticulum. The dome-shaped anchoring disc, the morphology of the polaroplast and the separation of the polar tube coils from the ribosome-packed cytoplasm are newly described details of spore structure. The polaroplast consists of an outer region of compact lamellae forming 'arms' surrounding an inner region of widely spaced lamellae. The 'arms' extends back into the region of an elongate nucleus. The genera Cystosporogenes and Endoreticulatus were differentiated by their positions in a previously obtained 16S rDNA phylogeny and on the new ultrastructural data.     

The effect of membrane curvature on the conformation of antimicrobial peptides: implications for binding and the mechanism of action

Short cationic antimicrobial peptides (AMPs) are believed to act either by inducing transmembrane pores or disrupting membranes in a detergent-like manner. For example, the antimicrobial peptides aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, despite being closely related, appear to act by fundamentally different mechanisms depending on their length. Using molecular dynamics simulations, the structural properties of these four peptides have been examined in solution as well as in a variety of membrane environments. It is shown that each of the peptides has a strong preference for binding to regions of high membrane curvature and that the structure of the peptides is dependent on the degree of local curvature. This suggests that the shorter peptides aurein 1.2 and citropin 1.1 act via a detergent-like mechanism because they can induce high local, but not long-range curvature, whereas the longer peptides maculatin 1.1 and caerin 1.1 require longer range curvature to fold and thus bind to and stabilize transmembrane pores.     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

Circulating Insulin Concentrations,Smoking, and Alcohol Intake Are Important Independent Predictors of Leptin in Young Healthy Men

Objective : Leptin, an adipocyte-secreted hormone, has been shown to signal the status of energy stores to the brain, regulate energy homeostasis, and mediate the neuroendocrine response to food deprivation. Obesity is associated with increased leptin levels, and several hormones, including insulin and glucocorticoids, have been associated with leptin levels and expression in rodents. Although obesity has been strongly associated with increased leptin in humans, a significant percentage of leptin's variability remains unexplained. The role of endogenous hormones, demographic factors, or certain life-style factors in explaining the residual variability of leptin levels has not yet been clarified. We performed this cross-sectional study to document the relative importance of obesity, lifestyle factor, and endogenous hormones in determining serum leptin levels. Research Methods and Procedures : We measured serum concentrations of insulin, Cortisol, testosterone, growth hormone, and dehydroepiandrosterone sulfate; ascertained anthropometric, demographic, and lifestyle characteristics; and studied these variables in relationship to serum leptin concentrations in a sample of young healthy men. Results : Obesity and alcohol intake were independently and positively associated with circulating leptin concentrations. Additionally, cigarette smoking was negatively and independently associated with leptin concentrations. Finally, serum insulin concentration was an independent hormonal determinant of circulating leptin concentrations, whereas serum testosterone was negatively associated with leptin only by bivariate analysis. Discussion : We conclude that, in addition to obesity, cigarette smoking, alcohol intake, and serum insulin levels are associated with leptin levels in a population of healthy young men.