RANKL induces organized lymph node growth by stromal cell proliferation

RANK and its ligand RANKL play important roles in the development and regulation of the immune system. We show that mice transgenic for Rank in hair follicles display massive postnatal growth of skin-draining lymph nodes. The proportions of hematopoietic and nonhematopoietic stromal cells and their organization are maintained, with the exception of an increase in B cell follicles. The hematopoietic cells are not activated and respond to immunization by foreign Ag and adjuvant. We demonstrate that soluble RANKL is overproduced from the transgenic hair follicles and that its neutralization normalizes lymph node size, inclusive area, and numbers of B cell follicles. Reticular fibroblastic and vascular stromal cells, important for secondary lymphoid organ formation and organization, express RANK and undergo hyperproliferation, which is abrogated by RANKL neutralization. In addition, they express higher levels of CXCL13 and CCL19 chemokines, as well as MAdCAM-1 and VCAM-1 cell-adhesion molecules. These findings highlight the importance of tissue-derived cues for secondary lymphoid organ homeostasis and identify RANKL as a key molecule for controlling the plasticity of the immune system.     

Effect of phospholipase A<Subscript>2</Subscript>inhibitory peptide on inflammatory arthritis in a TNF transgenic mouse model: a time-course ultrastructural study

We evaluated the therapeutic effect of secretory phospholipase A₂ (sPLA₂)-inhibitory peptide at a cellular level on joint erosion, cartilage destruction, and synovitis in the human tumor necrosis factor (TNF) transgenic mouse model of arthritis. Tg197 mice (N = 18) or wild-type (N = 10) mice at 4 weeks of age were given intraperitoneal doses (7.5 mg/kg) of a selective sPLA₂ inhibitory peptide, P-NT.II, or a scrambled P-NT.II (negative control), three times a week for 4 weeks. Untreated Tg197 mice (N = 10) were included as controls. Pathogenesis was monitored weekly for 4 weeks by use of an arthritis score and histologic examinations. Histopathologic analysis revealed a significant reduction after P-NT.II treatment in synovitis, bone erosion, and cartilage destruction in particular. Conspicuous ultrastructural alterations seen in articular chondrocytes (vacuolated cytoplasm and loss of nuclei) and synoviocytes (disintegrating nuclei and vacuoles, synovial adhesions) of untreated or scrambled-P-NT.II-treated Tg197 mice were absent in the P-NT.II-treated Tg197 group. Histologic scoring and ultrastructural evidence suggest that the chondrocyte appears to be the target cell mainly protected by the peptide during arthritis progression in the TNF transgenic mouse model. This is the first time ultrastructural evaluation of this model has been presented. High levels of circulating sPLA₂ detected in untreated Tg197 mice at age 8 weeks of age were reduced to basal levels by the peptide treatment. Attenuation of lipopolysaccharide- and TNF-induced release of prostaglandin E₂ from cultured macrophage cells by P-NT.II suggests that the peptide may influence the prostaglandin-mediated inflammatory response in rheumatoid arthritis by limiting the bioavailability of arachidonic acid through sPLA₂ inhibition.     

Lactobacillus rhamnosus GG secreting an antigen and Interleukin-2 translocates across the gastrointestinal tract and induces an antigen specific immune response

Lactobacillus rhamnosus strain GG (LGG) is a probiotic organism. In this present study, LGG that express the green fluorescence protein (LGG-GFP) and IL-2 and GFP as a fusion protein (LGG-IL-2-GFP) were used to examine bacterial uptake and the immune response induced by oral immunization. Using TEM to examine the intestinal tissue, the Lactobacilli were localized in M cells and in venules. After oral immunization, most of the bacteria were excreted in feces only a small fraction (0.15%) was retained in the intestine at 48 hr. However, more LGG-IL-2-GFP was found in the MLN and spleen than LGG-GFP. The loop ligation method was used to evaluate LGG uptake and both LGG-GFP and LGG-IL-2-GFP were found to translocate at the same rate. Analysis of LGG internalization in J774 macrophage cells indicated that IL-2 increased survival of LGG and this may explain the increased presence of these bacteria in the MLN for a longer period. After oral immunization, specific mucosal antibody production as well as GFP specific CTL activity was demonstrated. IL-2 co-expression with GFP further enhanced antibody production and CTL activity. In conclusion, Lactobacillus rhamnosus GG expressing an antigen could generate an effective immune response to the antigen and IL-2 improved the response generated probably by increasing LGG expressing antigen survival in immune cells.     

Unusual hepatic mitochondrial arginase in an Indian air-breathing teleost,Heteropneustes fossilis: Purification and characterization

A functional urea cycle with both cytosolic (ARG I) and mitochondrial (ARG II) arginase activity is present in the liver of an ureogenic air-breathing teleost, Heteropneustes fossilis. Antibodies against mammalian ARG II showed no cross-reactivity with the H. fossilis ARG II. ARG II was purified to homogeneity from H. fossilis liver. Purified ARG II showed a native molecular mass of 96 kDa. SDS–PAGE showed a major band at 48 kDa. The native enzyme, therefore, appears to be a homodimer. The pI value of the enzyme was 7.5. The purified enzyme showed maximum activity at pH 10.5 and 55 °C. The K_m of purified ARG II for l-arginine was 5.25 ± 1.12 mM. l-Ornithine and N^ω-hydroxy-l-arginine showed mixed inhibition with K_i values 2.16 ± 0.08 and 0.02 ± 0.004 mM respectively. Mn^+ 2 and Co^+ 2 were effective activators of arginase activity. Antibody raised against purified H. fossilis ARG II did not cross-react with fish ARG I, and mammalian ARG I and ARG II. Western blot with the antibodies against purified H. fossilis hepatic ARG II showed cross reactivity with a 96 kDa band on native PAGE and a 48 kDa band on SDS–PAGE. The molecular, immunological and kinetic properties suggest uniqueness of the hepatic mitochondrial ARG II in H. fossilis.     

Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia

Recent studies have shown that Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named “Focused Screening and Treatment” (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as “high risk” and “low risk” based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow.     

Entomological outcomes of cluster-randomised,community-driven dengue vector-suppression interventions in Kampong Cham province,Cambodia

Cambodia has one of the highest dengue infection rates in Southeast Asia. Here we report quantitative entomological results of a large-scale cluster-randomised trial assessing the impact on vector populations of a package of vector control interventions including larvivorous guppy fish in household water containers, mosquito trapping with gravid-ovitraps, solid waste management, breeding-container coverage through community education and engagement for behavioural change, particularly through the participation of school children. These activities resulted in major reductions in Container Index, House Index, Breteau Index, Pupal Index and Adult Index (all p-values 0.002 or lower) in the Intervention Arm compared with the Control Arm in a series of household surveys conducted over a follow-up period of more than one year, although the project was not able to measure the longer-term sustainability of the interventions. Despite comparative reductions in Adult Index between the study arms, the Adult Index was higher in the Intervention Arm in the final household survey than in the first household survey. This package of biophysical and community engagement interventions was highly effective in reducing entomological indices for dengue compared with the control group, but caution is required in extrapolating the reduction in household Adult Index to a reduction in the overall population of adult Aedes mosquitoes, and in interpreting the relationship between a reduction in entomological indices and a reduction in the number of dengue cases. The package of interventions should be trialled in other locations.     

Lactobacilli secreting a tumor antigen and IL15 activates neutrophils and dendritic cells and generates cytotoxic T lymphocytes against cancer cells

Lactobacillus rhamnosus GG (LGG) has been used to successfully induce tumor regression in an orthotopic model of bladder cancer. Increased infiltration of neutrophils and macrophages into the tumor mass was observed after therapy. This study evaluates the potential of LGG to induce a directed anti-tumor response. Lactobacilli were modified to secrete the prostate specific antigen (PSA) or IL15 and PSA (IL-15-PSA). Neutrophils and DC were exposed to LGG for 2 h as in clinical therapy for bladder cancer. Recombinant LGG activated neutrophils (elevated MHC class I expression) induced DC maturation (increased expression of CD86, CD80, CD40, MHC II and CD83), T cell proliferation and PSA specific cytotoxic T lymphocytes (CTL) activity. IL15 enhanced direct DC activation of CTL. Thus LGG secreting tumor antigens may activate antigen specific immune responses when instilled intravesically and IL15 could enhance this response.     

Phospholipid composition and metabolism in mouse muscular dystrophy

1. The uptake of monosaccharides and polyols in the obligatory aerobic yeast Rhodotorula gracilis (glutinis) was accompanied by proton uptake. 2. The half-saturation constant of transport, KT, depended on pH, changing from about 2mM at pH 4.5 to 80mM at pH8.5 for D-xylose; this change of the effective carrier affinity was reversible. 3. The apparent dissociation constant of the monosaccharide carrier was estimated at pKa 6.75. 4. At pH8.5, when the pH gradient across the cell membrane vanished, no sugar accumulation was demonstrable. 5. The half-saturation constants of sugar uptake and H+ co-transport were very similar to each other, the latter obviously being controlled by the former. 6. The H+/sugar stoicheiometry remained constant under various physiological conditions; it amounted to one H+ ion per sugar molecule taken up. 7. The data are interpreted as a strong piece of evidence in favour of the active monosaccharide transport in R. gracilis (glutinis) being an H+-symport energized by the electrochemical gradient of H+ across the plasma membrane of the yeast.