Organ-dependent differences in composition and function observed in hepatic and intestinal granulomas isolated from mice with Schistosomiasis mansoni

In murine schistosomiasis mansoni, eggs deposited in the liver and intestines induce a cell-mediated granulomatous reaction. Previous studies have shown that maximal granuloma size differs in the liver and various segments of the gastrointestinal tract. The objective of this investigation was to isolate intestinal granulomas and to determine whether organ-dependent differences in cell composition and granuloma function exist. Intestinal granulomas representative of those in tissue were isolated by a combination of chemical and mechanical techniques. When dissociated by collagenase, these lesions yielded a viable heterogeneous population of inflammatory cells. Granulomas from the liver, colon, and ileum showed differences in cellular composition. Liver lesions contained the largest number of T and B lymphocytes, eosinophils, and mast cells whereas ileal granulomas comprised mostly macrophages. Immunofluorescence studies on frozen tissue sections revealed that T and B lymphocytes also displayed different patterns of distribution within granulomas from different tissues. In contrast to isolated cultured liver granulomas that produced MIF-like substances, isolated colonic and ileal granulomas had weak or no MIF activity. It thus appears that granuloma formation in various organs is influenced by local factors that could affect the ultimate resolution of the lesions.     

Tandem duplications of the lac region of the Escherichia coli chromosome

Tandem duplications are caused by unequal crossing over between homologous sequences. Duplications in the lac region of the Escherichia coli chromosome were isolated by two methods. Duplication frequency using a method involving P1 transduction increased from 0.4% with no UV to 2.0% following UV irradiation at 35 J/m2. Duplication frequency in lac using a second generalizable method that does not involve P1 transduction increased from 0.7 to 12% at 35 J/m2 UV. In both cases the duplication frequency began to plateau at UV doses of 12 J/m2 and 24 J/m2. According to segregation analysis of sixteen duplications there may be at least seven classes of duplications isolated by each method. Pulsed-field gel electrophoresis was used to measure the duplications isolated without P1 transduction. The minimum size of the duplications ranged from 30 to 320 kb but could be much larger.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Inactivation of SAM-Methyltransferase is the Mechanism of Attenuation of a Historic Louse Borne Typhus Vaccine Strain

Louse borne typhus (also called epidemic typhus) was one of man''s major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.     

Substance P is associated with heart enlargement and apoptosis in murine dilated cardiomyopathy induced by Taenia crassiceps infection

Dilated cardiomyopathy (degeneration of heart muscle and heart enlargement) is an important cause of heart failure among young adults. Dilated cardiomyopathy may be a complication during or after various viral, bacterial, or parasitic diseases. Substance P (SP) is a neurotransmitter that is involved in the pathogenesis of various diseases. To determine whether SP is associated with cardiac changes in murine cysticercosis, we compared heart-weight to body-weight ratio, cardiac pathology, cardiomyocyte size, and cardiac-apoptosis (TUNEL assay) in hearts from Taenia crassiceps-infected (wild-type vs. SP-knockout) mice. We noted that, as compared with control uninfected wild-type mice, elevated protein levels of SP and its receptor as studied by ELISA or immunohistochemistry, respectively, were elevated in the hearts of parasite-infected wild-type mice. The heart-weight to body-weight ratios were significantly higher in the parasite-infected wild-type mice versus those of the infected SP-knockout mice. Furthermore, wild-type infected mice developed dilated cardiomyopathy with increased chamber size of both ventricles, decreased ventricular wall thickness, compensatory cardiomyocyte hypertrophy, and increased cardiac apoptosis. This cardiac pathology did not develop in mice lacking SP activity (i.e., in infected SP knockout mice) or in uninfected mice. These data indicate that SP is associated with cardiac changes in an animal model of parasitic dilated cardiomyopathy.     

Vasoactive intestinal peptide stimulates T lymphocytes to release IL-5 in murine schistosomiasis mansoni infection.

In murine schistosomiasis, granulomas form around ova deposited in the liver and intestines of infected mice. The granulomas have eosinophils that produce vasoactive intestinal peptide (VIP) and T cells that display VIP receptors. IL-5 is a lymphokine important for the development and maturation of eosinophils. It seemed plausible that VIP, released from eosinophils, may interact with lymphocyte VIP receptors and modulate IL-5 production as part of a feedback regulatory circuit. Thus, we determined whether granuloma T cells make IL-5 and whether VIP modulates IL-5 production. Isolated granuloma cells enriched for T lymphocytes spontaneously released IL-5. Culture of these cells in the presence of VIP increased IL-5 secretion. Spleen cells were also studied. Spleen cells from infected mice did not spontaneously release IL-5 or express IL-5 mRNA and VIP did not stimulate these resting spleen cells to produce this IL. However, these cells did express IL-5 mRNA and secreted IL-5 in response to Con A or soluble egg Ag. VIP could not appreciably modulate IL-5 release when cells were cultured with VIP and the Ag or mitogen. Spleen cells washed free of Con A ceased IL-5 secretion within 24 h. These preactivated splenic T cells resumed vigorous IL-5 secretion in response to either Con A or VIP. Yet only Con A prominently induced IL-5 mRNA expression. VIP was an effective stimulus at concentrations equal to or above the kDa of the VIP receptor on both splenic and granuloma T cells (10(-8) M). It is concluded that, in murine schistosomiasis, VIP invokes IL-5 release from activated T cells that are not undergoing immediate TCR stimulation.     

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

Production of haploids and doubled haploids in oil palm

Background

Studies on host-pathogen interactions in a range of pathosystems have revealed an array of mechanisms by which plants reduce the efficiency of pathogenesis. While R-gene mediated resistance confers highly effective defense responses against pathogen invasion, quantitative resistance is associated with intermediate levels of resistance that reduces disease progress. To test the hypothesis that specific loci affect distinct stages of fungal pathogenesis, a set of maize introgression lines was used for mapping and characterization of quantitative trait loci (QTL) conditioning resistance to Setosphaeria turcica, the causal agent of northern leaf blight (NLB). To better understand the nature of quantitative resistance, the identified QTL were further tested for three secondary hypotheses: (1) that disease QTL differ by host developmental stage; (2) that their performance changes across environments; and (3) that they condition broad-spectrum resistance.

Results

Among a set of 82 introgression lines, seven lines were confirmed as more resistant or susceptible than B73. Two NLB QTL were validated in BC₄F₂ segregating populations and advanced introgression lines. These loci, designated qNLB1.02 and qNLB1.06, were investigated in detail by comparing the introgression lines with B73 for a series of macroscopic and microscopic disease components targeting different stages of NLB development. Repeated greenhouse and field trials revealed that qNLB1.06 _Tx303 (the Tx303 allele at bin 1.06) reduces the efficiency of fungal penetration, while qNLB1.02 _B73 (the B73 allele at bin 1.02) enhances the accumulation of callose and phenolics surrounding infection sites, reduces hyphal growth into the vascular bundle and impairs the subsequent necrotrophic colonization in the leaves. The QTL were equally effective in both juvenile and adult plants; qNLB1.06 _Tx303 showed greater effectiveness in the field than in the greenhouse. In addition to NLB resistance, qNLB1.02 _B73 was associated with resistance to Stewart's wilt and common rust, while qNLB1.06 _Tx303 conferred resistance to Stewart's wilt. The non-specific resistance may be attributed to pleiotropy or linkage.

Conclusions

Our research has led to successful identification of two reliably-expressed QTL that can potentially be utilized to protect maize from S. turcica in different environments. This approach to identifying and dissecting quantitative resistance in plants will facilitate the application of quantitative resistance in crop protection.     

IL-12 induction of mRNA encoding substance P in murine macrophages from the spleen and sites of inflammation

Substance P (SP), a neuropeptide, interacts with the neurokinin 1 receptor (NK-1R) on immune cells to help control IFN-gamma production. In murine schistosomiasis mansoni, schistosome worms produce ova that incite focal Th2-type granulomatous inflammation within the liver and intestines. Normal gut is characterized by a controlled state of inflammation. IL-10 knockout mice develop chronic Th1-type colitis spontaneously. Both schistosome granulomas and gut mucosa display an SP immune regulatory circuit. However, the origin and regulation of SP production at these sites of inflammation are poorly understood. Macrophages are a potential source of SP. We therefore studied macrophages (F4/80(+)) from these models of inflammation. SP mRNA (preprotachykinin A (PPT A)) was detected within the schistosome granuloma, spleen, and lamina propria macrophages. Compared with those from wild-type mice, granuloma macrophages from STAT6(-/-) mice had 10-fold higher PPT A mRNA expression, whereas in STAT4(-/-) animals, PPT A mRNA expression was nearly abolished. IL-12 signals via STAT4 to induce Th1-type inflammation. It was demonstrated that IL-12, but not IL-18, induces SP mRNA expression in resting splenic macrophages from Schistosoma-infected mice and in wild-type lamina propria mononuclear cells. Thus, macrophages are a source for SP at these sites of chronic inflammation, and IL-12 and STAT4 are regulators of macrophage SP mRNA expression.