A tractable covalent linker strategy for the production of immunogenic antigen-TLR7/8L bioconjugates.

Despite the ease of production and improved safety profiles of recombinant vaccines, the inherently low immunogenicity of unadjuvanted proteins remains an impediment to their widespread adoption. The covalent tethering of TLR agonists to antigenic proteins offers a unique approach to co-deliver both constituents to the same cell-enhancing vaccine efficacy while minimizing reactogenicity. However, the paucity of simple and effective linker chemistries continues to hamper progress. Here, we present a modular, PEG-based linker system compatible with even extremely lipophilic and challenging TLR7/8 agonists. To advance the field and address previous obstacles, we offer the most straightforward and antigen-preserving linker system to date. These antigen-adjuvant conjugates enhance antigen-specific immune responses in mice, demonstrating the power of our approach within the context of modern vaccinology.

Bronchodilator responsiveness in patients with COPD.

The degree of acute improvement in spirometric indices after bronchodilator inhalation varies among chronic obstructive pulmonary disease (COPD) patients, and depends upon the type and dose of bronchodilator and the timing of administration. Acute bronchodilator responsiveness at baseline was examined in a large cohort of patients with moderate-to-very-severe COPD participating in the Understanding Potential Long-term Impacts on Function with Tiotropium (UPLIFT) trial, a 4-yr randomised double-blind trial evaluating the efficacy of 18 mug tiotropium daily in reducing the rate of decline in lung function. After wash-out of respiratory medications, patients received 80 mug ipratropium followed by 400 mug salbutamol. Spirometry was performed before and 90 min following ipratropium administration. The criteria used for forced expiratory volume in one second (FEV(1)) responsiveness were: >or=12% increase over baseline and >or=200 mL; >or=15% increase over baseline; and >or=10% absolute increase in the percentage predicted value. Of the patients, 5,756 had data meeting the criteria for analysis (age 64.5 yrs; 75% male; baseline FEV(1) 1.10 L (39.3% predicted) and forced vital capacity (FVC) 2.63 L). Compared with baseline, mean improvements were 229 mL in FEV(1) and 407 mL in FVC. Of these patients, 53.9% had >or=12% and >or=200 mL improvement in FEV(1), 65.6% had >or=15% improvement in FEV(1), and 38.6% had >or=10% absolute increase in FEV(1) % pred. The majority of patients with moderate-to-very-severe chronic obstructive pulmonary disease demonstrate meaningful increases in lung function following administration of inhaled anticholinergic plus sympathomimetic bronchodilators.

Chronic central infusion of cocaine- and amphetamine-regulated transcript (CART 55-102): effects on body weight homeostasis in lean and high-fat-fed obese rats.

BACKGROUND: Cocaine- and amphetamine-regulated transcript (CART) is expressed within hypothalamic nuclei implicated in the regulation of feeding behaviour. It is up-regulated by leptin, and CART-derived peptides acutely inhibit food intake. OBJECTIVE: The present study was designed to assess the long-term effects of central CART administration on food intake, body weight, plasma levels of glucose, insulin, leptin, free fatty acids and triglycerides, and on fuel utilisation in normal and high-fat-fed obese rats. DESIGN: Normal and high-fat-fed obese rats were cannulated intracerebroventricularly (i.c.v.) and infused for 6 days with CART (55-102) or its vehicle. At day 4, animals were placed in an indirect calorimeter for a 24 h period during which the respiratory quotient and the energy expenditure were determined hourly. RESULTS: In both normal and obese animals, the chronic i.c.v. infusion of CART (55-102) had marked, sustained inhibitory effects on food intake and body weight gain that were accompanied by decreases in plasma insulin and leptin levels. Using indirect calorimetry, it was observed that CART infusion promoted an increase in lipid oxidation in normal and in obese animals, although this increase reached statistical significance only in the obese group. The hypothalamic CART mRNA expression was found to be higher in obese rats (displaying hyperleptinaemia) than in normal animals. CONCLUSION: The data together show that chronic i.c.v. CART infusion is effective in inhibiting food intake, favouring lipid oxidation and limiting fat storage, both in normal and high-fat-diet-induced obese rats. The CART pathway thus seems to be an important determinant of body weight homeostasis in normal animals as well as in a model of nutritionally induced obesity.

Management of acute gastroenteritis in children.

Acute gastroenteritis is a common and costly clinical problem in children. It is a largely self-limited disease with many etiologies. The evaluation of the child with acute gastroenteritis requires a careful history and a complete physical examination to uncover other illnesses with similar presentations. Minimal laboratory testing is generally required. Treatment is primarily supportive and is directed at preventing or treating dehydration. When possible, an age-appropriate diet and fluids should be continued. Oral rehydration therapy using a commercial pediatric oral rehydration solution is the preferred approach to mild or moderate dehydration. The traditional approach using "clear liquids" is inadequate. Severe dehydration requires the prompt restoration of intravascular volume through the intravenous administration of fluids followed by oral rehydration therapy. When rehydration is achieved, an age-appropriate diet should be promptly resumed. Antiemetic and antidiarrheal medications are generally not indicated and may contribute to complications. The use of antibiotics remains controversial.

Cis-acting human ApoE tissue expression element is associated with human pattern of intraneuronal ApoE in transgenic mice.

Apolipoprotein E polymorphic variants (ApoE-epsilon2, epsilon3, and epsilon4) are associated with the age of onset distribution and risk of Alzheimer disease. The question of whether ApoE is expressed at a comparatively low level in human neurons compared to astrocytes, or whether ApoE enters neuronal cytoplasm via altered endosomal metabolism is important to understanding potential pathogenic roles for ApoE as a susceptibility gene in Alzheimer disease. ApoE deficient ("knock-out") mice received large human genomic DNA fragment transgenes for each of the three common apoE alleles. All transgenic mice demonstrated glial/astrocytic (normal rodent pattern), as well as cortical intraneuronal ApoE immunoreactivity with all three human isoforms and at multiple ApoE human allele doses (Xu et al. (32)). To test whether ApoE intraneuronal immunoreactivity was due to ApoE gene sequences between mouse and human, we examined another set of mice constructed using targeted replacement so that the human ApoE gene was placed under mouse gene promoters. Current analyses show that targeted replacement recombinant mice show normal rodent glial expression pattern, but no ApoE neuronal immunoreactivity through six months of age compared to large human genomic DNA fragment transgenic mice, which show neuronal content of ApoE throughout adult life. We conclude that cis-acting DNA sequences, rather than the specific sequence of the ApoE gene, may be responsible for low levels of transcription activity in cortical neurons.

Cholinergic axonal dystrophy and mitochondrial pathology in prosimian primates.

Progressive cholinergic axonal dystrophy, cholinergic denervation, and generalized gliosis begin in the prosimian primate species Otolemur at 10% of maximum life span. In these same animals, extensive cerebral beta-amyloidosis follows relatively more abruptly at 50% of maximum life span. In contrast, even at maximum life span, the prosimian primate species Galago senegalensis Moholi, Microcebus murinus, and Eulemur fulvus collaris and insectivore species T. belangeri are either spared or much less affected. In this report, we further document this progressive cholinergic denervation in Otolemur which involves first projections of the pedunculopontine nucleus (PPN, CH5-6) and later projections of CH1-4 cholinergic nuclei, as well as other noncholinergic pathways. Affected cholinergic cell bodies and axons contain abnormal mitochondria with increased content of manganese superoxide dismutase (MnSOD). This syndrome correlates with moderate copper deficiency marked by diminished liver copper levels and cuproenzyme activities, carnitine deficiency possibly secondary to renal Fanconi syndrome, and evidence for stress inflammatory response activation. Mitochondrial pathology was observed in pancreatic islet cells, proximal renal tubule epithelial cells, and choroid plexus epithelial cells, and it involved central cholinergic neurons. In Otolemur garnetti, the degree of central cholinergic injury directly correlated to depression of liver copper stores. The Otolemur syndrome involves "sentinel" central cholinergic injury and selective mitochondrial pathology in cell classes defined by high mitochondrial content and/or metabolic activity and high content of nitric oxide synthetase and MnSOD. Environmental factors affecting copper and carnitine metabolism could interact with genetic defects or traits to produce abnormal and aggressive aging of Otolemur. Subclinical, cell-class specific mitochondrial dysfunction in these prosimian primates may be a model for human neurodegenerative diseases.

Human apolipoprotein E2, E3, and E4 isoform-specific transgenic mice: human-like pattern of glial and neuronal immunoreactivity in central nervous system not observed in wild-type mice.

Apolipoprotein E (apoE) and its three major alleles (APOE2, E3, and E4) have been implicated in Alzheimer's disease and other neurological disorders. Little is known of the role apoE plays in normal brain function and pathology. To create a model to study apoE in brain, we have generated APOE transgenic mice using microinjection of allele-specific human genomic fragments to establish founders which were then bred to APOE knockout mice lacking a functional mouse apoE protein. This allows the study of apoE without interference from the endogenous mouse APOE gene. Results demonstrate that transgenic lines have been established that transcribe and express apoE appropriately in brain, liver, and other tissues. High cholesterol levels found in APOE knockout mice are substantially corrected in the APOE transgenic lines. ApoE immunoreactivity has been detected in glial cells and selected classes of neurons in all three isoform-specific transgenics. This pattern of immunoreactivity is similar to that observed in nonhuman primates and man, and contrasts with the strictly glial staining pattern of normal rodents.

Angiotensin converting enzyme inhibitor-associated angioedema: higher risk in blacks than whites.

We compared the incidence of angioedema during exposure to angiotensin converting enzyme inhibitors (ACEIs) in blacks with that in whites in a retrospective cohort study of Medicaid recipients from Michigan, Ohio and Tennessee during 1986- 1992. Medicaid administrative files were used to identify filled prescriptions for ACEIs and calcium channel blockers (CCBs), and to identify first episodes of angioedema that occurred during exposure to an ACEI or CCB. There were 48,776 black and 106,482 white patients with 43,989 and 114,448 person-years of exposure to an ACEI, respectively. For comparison, there were 49,004 black and 110,129 white patients with 43,064 and 117,684 person-years of exposure to a CCB, respectively. The incidence of angioedema in blacks ranged from 3.3 to 4.6 per 1000 person-years of ACEI exposure--three to four times higher than that for whites in each state. Controlling for a slightly greater incidence of angioedema in blacks and other covariates, black users of ACEIs had a 3.1-fold greater incidence than white users. First exposure to ACEIs was associated with a higher risk of angioedema than chronic exposure in blacks, but not whites. Among blacks, the rate of angioedema during the first 30 days of exposure was 11.4 times greater (95% confidence interval [CI], 3.5 to 37.0) among ACEI users than CCB users; for those with at least 1 year of exposure, the rate was 4.5 times higher (95% CI, 2.6 to 8.0). Among whites, the increased risk of angioedema was more modest (rate ratio 1.7, 95% CI, 1.3 to 2.4 compared to CCB users) and did not differ by duration of ACEI use. In an analysis of angioedema cases and randomly selected controls in a subset of the full cohort, the greater risk of ACEI-associated angioedema in blacks was not explained by concurrent exposure to diuretics or antibiotics. Blacks may be at greater risk of angioedema when taking ACEIs than whites. Further study is necessary to confirm these findings, to examine effects of underlying disease on angioedema incidence, and to describe any racial differences in the severity of angioedema occurring with ACEIs.

Searching for an AIDS cure.

PIP: At least 5 major groups of HIV strains are known to exist. HIV and AIDS have been notoriously hard to fight due to the complexity of the virus and its ability to mutate into new strains at unusually high rates. For example, at least one type of vaccine may be required for each strain of HIV. Vaccine and drug research is nonetheless under way on many fronts. The qualities of more than 20 experimental HIV vaccines are being investigated in small, preliminary studies in human volunteers, with many more agents being tested in laboratories. Successful vaccines would be used prophylactically and/or therapeutically, although the majority of vaccines is being developed for prophylactic use. While phase 1 and 2 trials suggest that most experimental vaccines cause no serious adverse effects and can stimulate infection-fighting antibodies, it will be several years before large-scale phase 3 trials may confirm whether vaccines really work. The new 3-drug treatment therapy of AZT, ddI, and pyridinone shows promise in the lab, but remains to be proven in vivo. Most antiretrovirals are also prone to side effects with the host ultimately developing resistance to the therapy. Even if and when a cure and/or vaccine are developed, logistical obstacles must be overcome to widely disseminate them around the world. For now and well into the future, the important necessity of preventing HIV transmission can not be overstated.^ieng

Cellular localization of messenger RNA encoding amyloid-beta-protein in normal tissue and in Alzheimer disease.

Amyloid precursor protein (APP) gene encodes the short peptide fragment amyloid-beta-protein present in senile plaque cores, cerebrovascular amyloid, and intracellular neurofibrillary tangles in Alzheimer disease (AD). Using in situ hybridization with biotin-labeled RNA probes, we found distinctive patterns of APP gene expression in different regions of postmortem human brain. Strong hybridization signal for APP messenger RNA (mRNA) was detected in specific classes of neurons, fascicular oligodendroglia, satellite glia, and presumptive microglia. Weaker signal was seen in other neuronal classes, fascicular astrocytes, and vascular endothelial cells, but no signal was seen in protoplasmic astrocytes. Human thymus also shows a restricted pattern of hybridization with high signal in reticular epithelial cells, and much lower signal in lymphocytes. In AD patients, neuronal hybridization for APP mRNA was specifically increased in hippocampus, but not cerebellar and visual cortex when compared to hybridization for neuron-specific enolase mRNA. Most neurons with neurofibrillary tangles had strong APP mRNA signal. These results suggest that APP gene expression is highly regulated in normal tissue, that many different cell classes in brain express the APP gene, and that neuronal expression may increase specifically in brain regions where widespread injury occurs in AD. Amyloid deposits in brains of AD patients might be explained by local production of precursor protein in endothelial cells, neurons or glia.

Localization of neuron-specific enolase (NSE) mRNA in human brain.

In situ hybridization with biotinylated DNA and RNA probes derived from the 3' untranslated region of human neuron-specific or gamma gamma-enolase (NSE) permits histochemical detection of NSE mRNA in autopsy samples of human brain. In both aged controls and Alzheimer's disease (AD) patients, NSE probe hybridized with variable intensity to nerve cells in visual cortex and cerebellum, but not to glial or liver cells. These results provide evidence that NSE mRNA is expressed in neurons in human brain tissue, pattern of NSE mRNA expression in unaffected brain regions in AD is qualitatively normal, and NSE mRNA expression may vary widely among different classes of neurons.

Uncoupling effects of 2,4-dinitrophenol on electron transfer reactions and cell bioenergetics in rat brain in situ.

We have examined the effects of uncoupling on electron transfer reactions and cell bioenergetics in rat brain in situ. The redox reactions of cyt aa3, cyt c and cyt b as well as the levels of phosphocreatine/inorganic phosphate ratio (PCr/Pi) were monitored in isolated perfused rat head, using optical and [31P]NMR techniques, respectively. In the first series of experiments anesthetized (pentobarbital) and mechanically ventilated rats underwent bilateral carotid arterial cannulation. The head (skull intact, muscle removed) was perfused with a perfluorochemical solution (FC-43), and the animal was then decapitated. By means of reflectance spectrophotometry, the redox reactions of cyt aa3, c and b were monitored before and after perfusing the head with uncoupler 2,4-dinitrophenol (DNP) as well as after complete anoxia. The second series of experiments was performed using [31P]NMR spectroscopy (24.33 MHz; 10.25 in. magnet). PCr/Pi ratios were first determined for living rats (in vivo) before and after exchange transfusion with FC-43 (Hct less than 0.5%), and then in isolated perfused head (in situ) before and after 2,4-DNP addition. Reduction of cyt aa3 (32 +/- 1.4%, mean +/- S.E.M.), cyt c (29 +/- 6%), and cyt b (19 +/- 2%) was induced by addition of 2,4-DNP. Compared to in vivo aerobic metabolism with a PCr/Pi ratio of 3.0-5.0, the value in the uncoupled state in situ was less than 0.1. We conclude that in contrast to in vitro, uncoupling of oxidative phosphorylation induces reduction of intramitochondrial cytochromes in situ.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of cyanide with cytochrome aa3 in isolated perfused rat head in situ.

The reaction of cyanide with cytochrome aa3 in intact mitochondria is known to differ significantly from the reaction with the isolated enzyme. To examine the cyanide reaction with cytochrome aa3 in situ, we studied the spectral characteristics and the reaction kinetics of cyanide with reduced brain cytochrome aa3 in an isolated perfused rat head preparation. Anaesthetized rats underwent bilateral carotid-arterial cannulation. The head (skull intact, muscle removed) was perfused with a crystalloid solution containing Na2S2O4, and the animal was then decapitated. By means of reflectance spectrophotometry the reaction of cyanide with cytochrome aa3 was continuously monitored with the use of the 590 nm-575 nm, 610 nm-575 nm and 590 nm-610 nm wavelength pairs. We found that: the kinetics of the absorbance change at 590 nm and 610 nm were similar, with almost identical apparent rate constants, suggesting that these spectral changes are the results of the formation of a single complex; the difference spectrum obtained on addition of cyanide to the fully reduced preparation showed a peak at 588 nm and a trough at 610 nm, consistent with spectral characteristics of the cyanide-ferrocytochrome aa3 complex in isolated enzyme and isolated mitochondria in vitro; this observation underscores the accuracy of monitoring the effects of inhibitors of mitochondrial function on cytochrome redox reactions in situ; the half-maximal (K0.5) effect was approx. 50 microM, significantly lower than that in vitro. The lower apparent K0.5 for cyanide in this preparation in situ may be due to a difference in the pH of the two systems. This approach provides the means to study the inhibitors of mitochondrial function in intact brain under a physiological environment.

Non-invasive in vivo spectrophotometric monitoring of brain cytochrome aa3 revisited.

Cytochrome aa3 (cyt aa3) is the main catalyst of cellular oxygen consumption. The properties of cyt aa3 will define the tissue oxygen requirements and provide an insight into energy supply and demand. Currently dual-wavelength (605-590 or 605-620 nm) reflectance spectrophotometry is used to monitor cyt aa3 redox state in vivo. We have experimentally demonstrated that the compensation for blood contamination in the surveyed tissue by these wavelength pairs is less than optimal. An alternative approach, similar to spectrophotometric analysis of multicomponent systems used in vitro, is presented in the triple wavelength equation as follows: delta cyt aa3 = 1.000 (delta A605) - 0.662 (delta A586.1) + 0.316 (delta A580) Based on a series of experiments performed in cuvette in vitro, isolated perfused rat head in situ, and living rat head in vivo, we have demonstrated that the cyt aa3 equation fully compensates for changes in cerebral blood volume and saturation. This non-invasive method of in vivo monitoring of cyt aa3 can provide the means to reliably and accurately determine tissue oxygen delivery under physiological conditions.

Heme-copper relationship of cytochrome oxidase in rat brain in situ.

The role of copper aa3 in relation to heme aa3 of cytochrome oxidase in electron transfer and oxygen utilization is poorly understood in vitro. In an attempt to study this in situ, we have simultaneously monitored the steady state redox changes of heme aa3 and copper aa3 in an isolated perfused rat head model (skull intact, muscle removed). By means of reflectance spectrophotometry the redox reactions of heme aa3 and copper aa3 were continuously monitored using 605-625 nm and 815-920 nm wavelength pairs, respectively. The reaction kinetics of these components in response to transient perfusion interruption in energized and de-energized preparations were then examined. We found that in response to perfusion interruption, soon after full reduction, the copper signal begins to change toward oxidation despite continuation of anoxic insult and progressive reduction of heme aa3. This phenomenon disappeared by pretreatment of the preparation with 2,4-dinitrophenol. A schematic sequence of electron transport in situ is proposed which emphasizes an active role for Cua in this sequence.