Sequential recruitment of neutrophils into lung and bronchoalveolar lavage fluid in LPS-induced acute lung injury

Infiltration of activated neutrophils [polymorphonuclear leukocytes (PMN)] into the lung is an important component of the inflammatory response in acute lung injury. The signals required to direct PMN into the different compartments of the lung have not been fully elucidated. In a murine model of LPS-induced lung injury, we investigated the sequential recruitment of PMN into the pulmonary vasculature, lung interstitium, and alveolar space. Mice were exposed to aerosolized LPS and bronchoalveolar lavage fluid (BAL), and lungs were harvested at different time points. We developed a flow cytometry-based technique to assess in vivo trafficking of PMN in the intravascular and extravascular lung compartments. Aerosolized LPS induced consistent PMN migration into all lung compartments. We found that sequestration in the pulmonary vasculature occurred within the first hour. Transendothelial migration into the interstitial space started 1 h after LPS exposure and increased continuously until a plateau was reached between 12 and 24 h. Transepithelial migration into the alveolar air space was delayed, as the first PMN did not appear until 2 h after LPS, reaching a peak at 24 h. Transendothelial migration and transepithelial migration were inhibited by pertussis toxin, indicating involvement of Galphai-coupled receptors. These findings confirm LPS-induced migration of PMN into the lung. For the first time, distinct transmigration steps into the different lung compartments are characterized in vivo.     

Diverse compounds mimic Alzheimer disease-causing mutations by augmenting Abeta42 production

Increased Abeta42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Abeta42. Among the more potent Abeta42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2-selective NSAID. Many COX-2-selective NSAIDs tested raised Abeta42, including multiple COX-2-selective derivatives of two Abeta42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Abeta42. These compounds seem to target the gamma-secretase complex, increasing gamma-secretase-catalyzed production of Abeta42 in vitro. Short-term in vivo studies show that two Abeta42-raising compounds increase Abeta42 levels in the brains of mice. The elevations in Abeta42 by these compounds are comparable to the increases in Abeta42 induced by Alzheimer disease-causing mutations in the genes encoding amyloid beta protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Abeta42 production in humans.     

Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI

In this study, the uptake of Na+ into the cytosol of rice (Oryza sativa L. cvs Pokkali and BRRI Dhan29) protoplasts was measured using the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate, SBFI-AM, and fluorescence microscopy. By means of inhibitor analyses the mechanisms for uptake and sequestration of Na+ in the salt-sensitive indica rice cv. BRRI Dhan29 and in the salt-tolerant indica rice cv. Pokkali were detected. Less Na+ was taken up into the cytosol of Pokkali than into BRRI Dhan29. The results indicate that K+-selective channels do not contribute to the Na+ uptake in Pokkali, whereas they are the major pathways for Na+ uptake in BRRI Dhan29 along with non-selective cation channels. However, non-selective cation channels seem to be the main pathways for Na+ uptake in Pokkali. Protoplasts from Pokkali leaves took up Na+ only transiently in the presence of extracellular Na+ at 5-100 mM. Therefore, it is likely that the protoplasts have a mechanism for fast extrusion of Na+ out of the cytoplasm. Experiments with protoplasts pretreated with NH4NO3 and NH4VO3 suggest that the salt-tolerant Pokkali extrudes Na+ mainly into the vacuole. After cultivation of both cultivars in the presence of 10 or 50 mM NaCl for 72 h, the isolated protoplasts from Pokkali took up less Na+ than the control protoplasts. The results suggest that the salt-tolerance in Pokkali depends on reduced uptake through K+-selective channels and a fast extrusion of Na+ into the vacuoles.     

Polymorphisms in HLA class I genes associated with both favorable prognosis of human immunodeficiency virus (HIV) type 1 infection and positive cytotoxic T-lymphocyte responses to ALVAC-HIV recombinant canarypox vaccines

Carriers of certain human leukocyte antigen class I alleles show favorable prognosis of human immunodeficiency virus type 1 (HIV-1) infection, presumably due to effective CD8(+) cytotoxic T-lymphocyte responses, but close relationships between class I variants mediating such responses to natural and to vaccine HIV-1 antigen have not been established. During 6 to 30 months of administration and follow-up in trials of ALVAC-HIV recombinant canarypox vaccines, cells from 42% of 291 HIV-1-negative vaccinated subjects typed at class I loci responded to an HIV-1 protein in a lytic bulk CD8(+) cytotoxic T-lymphocyte assay. By 2 weeks after the second dose, higher proportions of vaccinees carrying one of two alleles consistently associated with slower progression of natural HIV-1 infection reacted at least once: B*27 carriers reacted to Gag (64%; odds ratio [OR] = 10.3, P = 0.001) and Env (36%; OR = 4.6, P = 0.04), and B*57 carriers reacted to Env (44%; OR = 6.6, P < 0.05). By 2 weeks after the third or fourth dose, B*27 carriers had responded (two or more reactions) to Gag (33%; OR = 4.4, P < 0.05) and B*57 carriers had responded to both Gag (39%; OR = 5.3, P = 0.013) and Env (39%; OR = 9.5, P = 0.002). Homozygosity at class I loci, although conferring an unfavorable prognosis following natural infection, showed no such disadvantage for vaccine response. Individual class I alleles have not previously demonstrated such clear and consistent relationship with both the clinical course of an infection and cellular immunity to a vaccine against the infectious agent. This proof of principle that class I an alleles modulate both processes has implications for development of HIV-1 and presumably other vaccines.     

Effects of estrogen and progesterone treatment on rat hippocampal NMDA receptors: relationship to Morris water maze performance

Estrogen modulates NMDA receptors function in the brain. It increases both dendritic spine density and synapse number in the hippocampus, an effect that can be blocked by NMDA antagonist. In this study, we investigated the effect of 17beta-estradiol and progesterone treatment on NMDA receptors in ovariectomized rats. Two different doses were used for 10 weeks. Receptor autoradiography was done on brain sections using [(3)H] MK-801 as a ligand. Our results showed a significant increase in [(3)H] MK-801 binding in the dentate gyrus, CA3 and CA4 areas of the hippocampus of ovariectomized compared to sham operated rats. In addition, we observed similar changes in CA1. 17beta-estradiol treatment in both doses reduced the binding back to the normal level while progesterone treatment did not show any effect. Spatial reference memory was tested on Morris water maze task. Ovariectomy severely impaired spatial reference memory. Estradiol but not progesterone treatment significantly improved the memory performance of the ovariectomized rats. Low dose treatment showed better learning than high dose estrogen treatment. The decrease in the antagonist sites by estradiol treatment could result in an increase in the sensitivity of the hippocampus to the excitatory stimulation by glutamate system and hence the effect of estradiol on learning and memory. The changes of NMDA receptors in the hippocampus support the concept that estrogen-enhancing effect on spatial reference memory could be through the enhancing of NMDA function.     

Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses

BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.     

Expression of three mitochondrial solute carriers, citrin, aralar1 and ornithine transporter, in relation to urea cycle in mice

The present report describes the expression profiles of different tissues and developmental changes of mouse aspartate/glutamate carrier (AGC) genes, Slc25a13 and Slc25a12, and an ornithine transporter gene, Ornt1, in relation to urea cycle enzyme genes, carbamoylphosphate synthetase I (CPS) and argininosuccinate synthetase (ASS). Slc25a13 encodes citrin, recently found to be deficient in adult-onset type II citrullinemia and to function as AGC together with its isoform and product of Slc25a12, aralar1. Citrin was broadly distributed, but mainly in the liver, kidney and heart. Aralar1 was expressed in diaphragm, skeletal muscle, heart, brain and kidney, but not in the liver. These distribution profiles are different from the restricted of Ornt1, ASS and CPS. Citrin, ASS, CPS and Ornt1 showed similar patterns of developmental changes in the liver and small intestine, where they play a role in urea and arginine synthesis. Dietary, hormonal and physical manipulations caused varied changes of CPS, ASS and Ornt1 in the liver, but the change of citrin was not so marked as that of the others. Analysis using RT-PCR and restriction enzyme digestion revealed that the ornithine transporter most expressed is Ornt1, although Ornt2 is detectable at a minute level. All these results suggest that citrin as AGC plays a role in urea synthesis as well as many fundamental metabolic pathways in the liver, and shares metabolic functions with aralar1 in other tissues, and that Ornt1 is an important component in urea synthesis in the liver and in arginine synthesis in the small intestine during the neonatal period.