Sterile Phosphate-buffered Saline Research Articles

Transforming Growth Factor-β1 Induces Heparan Sulfate 6-O-Endosulfatase 1 Expression in Vitro and in Vivo

Transforming growth factor (TGF)-beta1 plays an important role in the development of pulmonary fibrosis. In this study we examined the relationship between TGF-beta1 stimulation and the expression of heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) in cultured normal human lung fibroblasts (NHLFs) and in murine lungs in vivo. By removing 6-O-sulfates from specific HS intrachain sites on the cell surface, Sulf1 has been shown to modulate the activities of many HS binding growth factors and morphogens including fibroblast growth factor (FGF)-2. Real time reverse transcription-PCR analysis revealed that TGF-beta1 increased Sulf1 expression in NHLFs in a dose- and time-dependent manner which was accompanied by a decrease in 6-O-sulfated disaccharides as revealed by high performance liquid chromatography analysis. Decreased ERK activation after FGF-2 stimulation was observed in TGF-beta1-treated NHLFs compared with control cells without changes in HS-dependent FGF-2 binding or FGF-2.FR1c complex formation. To study the function of Sulf1, negative control or Sulf1-specific small interference RNA (siRNA)-transfected NHLFs were stimulated with TGF-beta1. Enhanced Smad2/3 phosphorylation and elevated total Smad2 protein level were observed in Sulf1 siRNA-transfected cells and were accompanied by enhanced expression of alpha-smooth muscle actin and fibronectin. In addition, Sulf1 siRNA transfection enhanced the anti-proliferative effect of TGF-beta1. Finally Sulf1 expression was up-regulated in the lungs of mice treated with adenovirus encoding active TGF-beta1. Taken together, our data indicate that Sulf1 is a TGF-beta1-responsive gene both in vitro and in vivo and may function as a negative regulator of TGF-beta1-induced fibrogenesis.

Plant-Derived Compounds Inactivate Antibiotic-Resistant Campylobacter jejuni Strains

Sixty-three Campylobacter jejuni isolates were screened for their resistance to the antibiotics ampicillin, cefaclor, ciprofloxacin, erythromycin, gentamycin, tetracycline, and trimethoprim-sulfamethoxazole. Based on this screen, the resistant strains D28a and H2a and the nonresistant strain A24a were selected for evaluation of their resistance and susceptibility to inactivation by cinnamaldehyde and carvacrol, the main constituents of plant-derived cinnamon and oregano oils, respectively. Different concentrations (0.05, 0.1, and 0.2% [vol/vol] in sterile phosphate-buffered saline) of cinnamaldehyde and carvacrol were added to C. jejuni cultures with initial populations of 104 CFU/ml. The samples were then mixed thoroughly and incubated at 37°C. Viable bacterial populations were enumerated at incubation periods of 0, 30, 60, and 120 min. The results indicate that the extent of inhibition of microbial survival was related to both the nature and concentration of antimicrobials and the incubation time. Both cinnamaldehyde and carvacrol exhibited rapid antimicrobial activity against both antibiotic-resistant and non-resistant C. jejuni strains, at concentrations of ~0.1% and higher. The antimicrobial efficacy of cinnamaldehyde was greater than that of carvacrol. The possible significance of the results for microbiological food safety is discussed.

Interleukin-13 Protects Against Experimental Autoimmune Myocarditis by Regulating Macrophage Differentiation

We report here that interleukin (IL)-13 protects BALB/c mice from myocarditis, whether induced by peptide immunization or by viral infection. In contrast to mild disease in IL-4 knockout (KO) BALB/c mice, IL-13 KO BALB/c mice developed severe coxsackievirus B3 (CVB3)-induced autoimmune myocarditis and myocarditogenic peptide-induced experimental autoimmune myocarditis. Such severe disease was characterized by increased cardiac inflammation, increased total intracardiac CD45(+) leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis. Echocardiography revealed that IL-13 KO mice developed severe dilated cardiomyopathy with impaired cardiac function and heart failure. Hearts of IL-13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL-1beta, IL-18, interferon-gamma, transforming growth factor-beta1, and IL-4 as well as histamine. The hallmark of the disease in IL-13 KO mice was the up-regulation of T-cell responses. CD4(+) T cells were increased in IL-13 KO hearts both proportionally and in absolute number. Splenic T cells from IL-13 KO mice were highly activated, and myosin stimulation additionally increased T-cell proliferation. CD4(+)CD25(+)Foxp3(+) regulatory T-cell numbers were decreased in the spleens of IL-13 KO mice. IL-13 deficiency led to decreased levels of alternatively activated CD206(+) and CD204(+) macrophages and increased levels of classically activated macrophages. IL-13 KO mice had increased caspase-1 activation, leading to increased production of both IL-1beta and IL-18. Therefore, IL-13 protects against myocarditis by modulating monocyte/macrophage populations and by regulating their function.

First Report of Fire Blight Caused by Erwinia amylovora on Pear, Apple, and Quince in Morocco.

In the spring of 2006, symptoms similar to those of fire blight were observed on pear (Pyrus communis), apple (Malus pumila), and quince (Cydonia oblonga) trees at the flowering and early fruit set stages in an orchard in the Meknès Region, 140 km east of Rabat. Symptoms consisted of i) water-soaked flowers that became wilted, shrivelled, and then turned brown to black; ii) "shepherd's crook" of the shoots; iii) dark green pedicles that became brown to black; iv) wilted and shrivelled leaves that turned brown but remained attached; and v) water-soaked fruits that became brown to black with droplets of exudates on the surface. In an effort to eradicate the disease, 42 ha of pears were dug up and burned in October 2006. In the spring of 2007, fire blight reappeared in the same orchard and was encountered in five other orchards with disease incidences from 1 to 60%. Three hectares of pears were removed and burned. Samples of diseased young shoots and fruits were collected. Bacteria were isolated either from washed tissues or directly from bacterial ooze on the host with King's B (KB) and semiselective CCT media (1,4). Colonies with morphology similar to that of Erwinia amylovora were purified through repetitive plating on KB medium. The isolates were first characterized based on colony morphology and biochemical and physiological tests (1). All isolates showed typical colony morphology on both media as compared with the reference strains of E. amylovora. They produced white colonies on KB medium, were gram negative, did not produce fluorescent pigment on KB, did not grow at 36°C, were levan positive, facultative anaerobes, and induced a hypersensitive reaction when infiltrated on tobacco leaves (cv. Xanthi). The isolates reacted negatively for oxidase, urease, indol, H2S production, and potato rotting tests, but they reacted positively for catalase and citrate tests. More than 87% of the isolates induced gelatine liquefaction, did not reduce nitrate, and produced acid from sorbitol but not from inositol, raffinose, and salicin. Thirty-three isolates were identified as E. amylovora by immunofluorescence microscopy with a polyclonal antibody (IF), double-antibody sandwich indirect-ELISA (3), and PCR (2). Pathogenicity was performed with a detached-leaf test tube assay (1). Each isolate was inoculated by wounding three young leaves of pears (cv. William's) along the main vein with a scalpel dipped in a 109 CFU/ml bacterial suspension. The inoculated detached leaves were kept at 25°C and 80% relative humidity in tubes with sterile 1% agar. Positive controls consisted of reference strains of E. amylovora. For negative controls, detached leaves were wounded with a scalpel dipped in sterile phosphate-buffered saline and the leaves were kept separate from the inoculated ones. Within a week, symptoms similar to those observed on leaves inoculated with the reference strains of E. amylovora, including discoloration, browning, and production of bacterial ooze along inoculated vein leaf, appeared. No symptoms developed on negative controls. Reisolation of bacteria produced colonies with characteristics like E. amylovora. Representative colonies were confirmed by IF. The occurrence of fire blight in Morocco creates a serious threat to the pome fruit industry.

Glial Cell Line-Derived Neurotrophic Factor Increases β-Cell Mass and Improves Glucose Tolerance

Pancreatic beta-cell mass increases in response to increased demand for insulin, but the factors involved are largely unknown. Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that plays a role in the development and survival of the enteric nervous system. We investigated the role of GDNF in regulating beta-cell survival. Studies were performed using the beta-TC-6 pancreatic beta-cell line, isolated mouse pancreatic beta cells, and in vivo in transgenic mice that overexpress GDNF in pancreatic glia. GDNF receptor family alpha1 and c-Ret receptor expression were assessed by reverse-transcription polymerase chain reaction and immunofluorescence microscopy. Apoptosis was evaluated by assessing caspase-3 cleavage. Phosphoinositol-3-kinase signaling pathway was analyzed by Akt phosphorylation. Glucose homeostasis was assessed by performing intraperitoneal glucose tolerance tests. Insulin sensitivity was assessed using intraperitoneal injection of insulin. We demonstrate the presence of receptors for GDNF, GFRalpha1, and c-Ret on beta cells. GDNF promoted beta-cell survival and proliferation and protected them from thapsigargin-induced apoptosis (P<.0001) in vitro. Exposure of beta-cells to GDNF also resulted in phosphorylation of Akt and GSK3beta. Transgenic mice that overexpress GDNF in glia exhibit increased beta-cell mass, proliferation, and insulin content. No differences in insulin sensitivity and c-peptide levels were noted. Compared with wild-type mice, GDNF-transgenic mice have significantly lower blood glucose levels and improved glucose tolerance (P<.01). GDNF-transgenic mice are resistant to streptozotocin-induced beta-cell loss (P<.001) and subsequent hyperglycemia. We demonstrate that over expression of GDNF in pancreatic glia improves glucose tolerance and that GDNF may be a therapeutic target for improving beta-cell mass.

Sickle RBCs Stimulate Human Monocyte Cytokine Expression.

Sickle red cells (SS RBCs) express numerous adhesion molecules, including LW, CD44, CD47, and Lutheran (LU) proteins, as well as phosphatidylserine. Proadhesive SS RBCs bind to endothelial cells, leading to their activation. Sickle monocytes have also been shown to express increased surface CD11b, as well as the cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), indicating their activated state. Chronic inflammation and high plasma levels of IL-1β can stimulate leukocyte proliferation and leukocytosis. An association between leukocytosis and increased morbidity and mortality of sickle cell disease (SCD) has been observed. However, the mechanism of monocyte activation in SCD is still unknown. We therefore sought to determine if SS RBCs played a role in monocyte activation. Blood samples were obtained from six normal adult volunteers, and SS RBCs were obtained from 12 adult patients homozygous for hemoglobin S. Plasma and buffy coat were removed, and 6 μl of SS RBCs were washed with sterile phosphate-buffered saline. After incubation with one million cells of the human promonocytic cell line U937 for 2 hours at 37°C, RBCs were lysed and total U937 RNA was isolated. Following reverse transcription, real-time PCR was performed using 200 nM primers for IL-1β, IL-6 or TNF-α, 50 ng cDNA and 12.5 μl of SYBR green 2× Super Mix in a 25 μl reaction volume. The amplification reaction was carried out over 40 cycles (10 min at 95°C, followed by a two-step cycling program of 15s at 95°C and 30s at 60°C). GAPDH was analyzed in parallel as an internal control. IL-1β, IL-6 and TNF-α U937 mRNA was significantly up-regulated after incubation with SS RBCs vs incubation with normal RBCs (relative to control without RBCs, relative mRNA levels: IL-1β: 2.77 ± 1.79 vs 0.82 ± 0.19, p=0.012; IL-6: 2.17 ± 0.94 vs 0.74 ± 0.34, p=0.003; and TNF-α: 3.01 ± 1.30 vs 1.09 ± 0.17, p=0.003). To identify the adhesion molecules involved in the interaction between SS RBCs and monocytes, 10 μg/ml soluble recombinant Lutheran (srLU) was incubated with U937 cells at 37°C for 2h. After incubation, total RNA was isolated from untreated and treated U937 cells. Real time PCR results showed that srLU treatment activated U937 cell TNF-α expression, (relative mRNA level 1.63 ± 0.02 vs 1.00 ± 0.01, p<0.0001, n=3) but not IL-1β or IL-6 expression (relative mRNA levels sLU treated vs untreated control, n=3-IL-1β: 1.24 ± 0.09 vs 1.00 ± 0.02, p=0.07; IL-6: 1.35 ± 0.30 vs 1.00 ± 0.01, p = 0.31). Neither soluble LW nor soluble CD44 had a similar effect. To verify that LU was involved in the interaction between SS RBCs and monocytes, inhibition assays were also performed. SS RBCs were incubated with anti-LU Ab for 1 h at 4°C to block RBC surface LU before incubation with U937 cells. Data from five paired treated and untreated samples showed decreased TNF-α mRNA expression after anti-LU antibody treatment (1.16 ± 0.14 vs 1.40 ± 0.12, p=0.005). Overall, these data suggest that monocytes can be activated by cell-cell interactions with SS RBCs to express the cytokines IL-1β, IL-6 and TNF-α. This activation is at least partially mediated through SS RBC Lutheran protein, which is over-expressed by these cells and is known to bind to leukocyte α4β1 integrin. These studies thus reveal that intact SS RBCs likely play a direct role in monocyte activation in SCD patients.

Phytic Acid Exposure Alters AflatoxinB1‐Induced Reproductive and Oxidative Toxicity in Albino Rats (Rattus norvegicus)

The increased use of feed in Egypt's aquaculture and animal industries raises concerns about the possible presence of mycotoxins in feedstuffs. The use of alternative medicine, such as botanicals and nutritional supplements, has become popular with inflammatory cases. The present study aimed to testify the role played by phytic acid (IP6) in enhancing the reproductive and oxidative toxicity induced in aflatoxinB1 (AFB1) treated white male albino rats (Rattus norvegicus) throughout treatment and withdrawal periods. One hundred and twenty white male albino rats were grouped into four groups. Group 1, was injected with 300 μg kg−1 body wt of AFB1 once every 3 days for 15 days and left uninjected for another 15 days to study the withdrawal effect. Group 2, was injected with 300 μg kg−1 body wt of AFB1 once every 3 days for 15 days and treated simultaneously with IP6 daily for another 15 days. Group 3, was treated daily with IP6 (40 mg kg−1 body wt) for 15 days and with no treatment for other 15 days. Group 4, injected with equivalent volume of sterile phosphate buffer saline solution as a control group. Sera were taken at the experimental intervals and assayed for testosterone hormone, follicular-stimulating hormone (FSH) and luteinizing hormone (LH) to determine the toxicological impact of AFB1 and the possibility of amelioration by phytic acid on the reproductive performance of the studied animal. The effects of AFB1 treatment on the absolute and relative weight of testis as well as its histopathologic effect on the testis and the possibility of amelioration by IP6 treatment were evaluated. The activities of enzymatic and non-enzymatic anti-oxidants, in addition to lipid peroxidation were measured in the testis’ homogenate of AFB1-treated rats. A decrease in sex hormone levels, an increase in testicular lipid peroxidation product levels and a significant decrease in testicular glutathione content, catalase and total peroxidase and superoxide dismutase activities were recorded. The histopathologic alterations revealed a degeneration and highly mitotic division within the spermatogenic nuclei, in addition to some karyomegaly and nuclear pyknosis. It is concluded that the reduction in the toxicity of free radicals by phytic acid might be responsible for the protective influence observed.

First Report of a Leaf Spot Disease of Wild Rocket (Diplotaxis tenuifolia) in Florida Caused by Xanthomonas campestris pv. raphani.

Wild rocket, also known as wall rocket or sand rocket (Diplotaxis tenuifolia (L.) DC, family Brassicaceae), is grown in Florida as a salad green and herb, especially for addition to "spring mix" and other bagged salad blends. It is similar in texture and flavor to the more widely known garden arugula (Eruca vesicatoria (L.) Cav. subsp. sativa). During the winter vegetable season of 2006-2007, a leaf spot disease causing severe economic loss was seen in commercial fields of wild rocket near the town of Sebastian in Indian River County, FL. Discrete water-soaked to greasy appearing spots were observed on leaf blades that rarely exceeded 1 mm in diameter with some surrounded by a narrow, yellow halo. There was no evidence of marginal V-shaped lesions suggestive of vascular black rot disease caused by Xanthomonas campestris pv. campestris. A bacterium that formed yellow colonies on nutrient agar was consistently isolated from these lesions. Four strains were isolated, purified, and characterized. All strains were strictly aerobic, gram-negative rods. Strains were positive for esculin hydrolysis and gelatin liquefaction and negative for oxidase, nitrate reduction, urease production, fluorescence on King's B medium, and utilization of asparagine as a sole source of carbon and nitrogen. Proteolysis and an alkaline reaction were observed in inoculated tubes of litmus milk. Colonies were bright yellow and mucoid on plates of yeast extract-glucose-calcium carbonate agar. Carbon source utilization, as revealed by the Biolog system, indicated a match to X. campestris. Fatty acid methyl ester analysis indicated a match with Florida library strains of X. campestris pv. raphani (similarity indices of 0.737 to 0.779). Suspensions (2 × 107 CFU/ml in sterile phosphate-buffered saline) of the four wild rocket strains and a strain isolated in 2003 as a cause of a discrete leaf spot disease of cabbage in southern Florida (1) were sprayed onto plants until runoff with a hand-held plastic mist bottle. Pathogenicity of strains was tested in the greenhouse on seven replicate plants of green cabbage cv. Copenhagen Market, salad arugula cv. Rocket, and wild rocket (an unnamed selection propagated from seed provided by a Florida grower). Symptoms appeared within 6 to 7 days. The wild rocket strains and the cabbage strain were pathogenic on all wild rocket and cabbage test plants, producing small, nonvascular leaf spots. No symptoms were seen on salad arugula or on control plants of wild rocket, cabbage, or arugula sprayed with phosphate-buffered saline. The bacterium was reisolated from infected plants and identified as X. campestris pv. raphani, thus completing Koch's postulates. We have classified the bacterial leaf spot pathogen of wild rocket as X. campestris pv. raphani using the nomenclature of Vincente et al. (2) for X. campestris strains producing nonvascular leaf spots on brassicas. This disease problem seems to be related to widespread use of overhead irrigation in the fields where the disease is prevalent. We have not observed the disease in seepage-irrigated fields of wild rocket.

Impaired Spine Stability Underlies Plaque-Related Spine Loss in an Alzheimer's Disease Mouse Model

Dendritic spines, the site of most excitatory synapses in the brain, are lost in Alzheimer's disease and in related mouse models, undoubtedly contributing to cognitive dysfunction. We hypothesized that spine loss results from plaque-associated alterations of spine stability, causing an imbalance in spine formation and elimination. To investigate effects of plaques on spine stability in vivo, we observed cortical neurons using multiphoton microscopy in a mouse model of amyloid pathology before and after extensive plaque deposition. We also observed age-matched nontransgenic mice to study normal effects of aging on spine plasticity. We found that spine density and structural plasticity are maintained during normal aging. Tg2576 mice had normal spine density and plasticity before plaques appeared, but after amyloid pathology is established, severe disruptions were observed. In control animals, spine formation and elimination were equivalent over 1 hour of observation ( approximately 5% of observed spines), resulting in stable spine density. However, in aged Tg2576 mice spine elimination increased, specifically in the immediate vicinity of plaques. Spine formation was unchanged, resulting in spine loss. These data show a small population of rapidly changing spines in adult and even elderly mouse cortex; further, in the vicinity of amyloid plaques, spine stability is markedly impaired leading to loss of synaptic structural integrity.

Evaluation of Zebrafish Danio rerio as a Model for Enteric Septicemia of Catfish (ESC)

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 x 10(3), 6 x 10(4), or 6 x 10(5) colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 x 10(4) CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 x 10(2) CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 x 10(5), 1.16 x 10(6), and 1.16 x 10(7) CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis.

The Role of Tumor Necrosis Factor Alpha in Lipopolysaccharide/Ranitidine-Induced Inflammatory Liver Injury

Exposure to a nontoxic dose of bacterial lipopolysaccharide (LPS) increases the hepatotoxicity of the histamine-2 (H2) receptor antagonist, ranitidine (RAN). Because some of the pathophysiologic effects associated with LPS are mediated through the expression and release of inflammatory mediators such as tumor necrosis factor alpha (TNF), this study was designed to gain insights into the role of TNF in LPS/RAN hepatotoxicity. To determine whether RAN affects LPS-induced TNF release at a time near the onset of liver injury, male Sprague-Dawley rats were treated with 2.5 x 10(6) endotoxin units (EU)/kg LPS or its saline vehicle (iv) and 2 h later with either 30 mg/kg RAN or sterile phosphate-buffered saline vehicle (iv). LPS administration caused an increase in circulating TNF concentration. RAN cotreatment enhanced the LPS-induced TNF increase before the onset of hepatocellular injury, an effect that was not produced by famotidine, a H2-receptor antagonist without idiosyncrasy liability. Similar effects were observed for serum interleukin (IL)-1beta, IL-6, and IL-10. To determine if TNF plays a causal role in LPS/RAN-induced hepatotoxicity, rats were given either pentoxifylline (PTX; 100 mg/kg, iv) to inhibit the synthesis of TNF or etanercept (Etan; 8 mg/kg, sc) to impede the ability of TNF to reach cellular receptors, and then they were treated with LPS and RAN. Hepatocellular injury, the release of inflammatory mediators, hepatic neutrophil (PMN) accumulation, and biomarkers of coagulation and fibrinolysis were assessed. Pretreatment with either PTX or Etan resulted in the attenuation of liver injury and diminished circulating concentrations of TNF, IL-1beta, IL-6, macrophage inflammatory protein-2, and coagulation/fibrinolysis biomarkers in LPS/RAN-cotreated animals. Neither PTX nor Etan pretreatments altered hepatic PMN accumulation. These results suggest that TNF contributes to LPS/RAN-induced liver injury by enhancing inflammatory cytokine production and hemostasis.

CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats

Seven healthy native goats in early lactation, weighing 30–40 kg, were used in this study. The right mammary gland of the seven does were infused with CpG-ODN at a dosage of 100 μg kg −1 body weight on the day 5 postpartum (PP). The left glands were used as controls and infused with sterile phosphate-buffered saline (PBS). On day 8 PP, the same dosage of CpG-ODN or PBS was again infused. On day 9 PP, the mammary glands (both right and left) of the seven does were infused with 6 × 10 6 colony-forming units (CFU) Escherichia coli and, at 0, 8, 16, 24, 48 and 72 h postinfection (PI), milk samples were collected from all glands. Goats were euthanized at 72 h PI and the mammary tissue harvested. Infusion with 6 × 10 6 CFU ml −1 E. coli induced acute mastitis. Histopathological evaluations showed that polymorphonuclear neutrophils (PMNs) were still present in alveoli at 72 h PI, but PMNs in the CpG-ODN-treated glands has disappeared. Bacteria counts in milk peaked at 16 h PI and CpG-ODN induced a significant decrease in viable bacteria from 16 h PI until the end of the experiment. This study showed that CpG-ODN promoted the expression of its specific receptor (TLR-9 mRNA) in mammary tissue, stimulated IL-6 production, reduced bacteria counts in milk, attenuated the impact of inflammation mediators on cells and significantly shortened the inflammation course. These results suggest that the CpG-ODN improved mammary gland defense and, thereby, had a beneficial effects against mastitis caused by E. coli infection in goats.

Mutagenicity of Ascaris chymotrypsin inhibitor in germ cells of mice

Chymotrypsin inhibitor isolated from Ascaris suum (ACHI) was tested for the induction of dominant lethal mutations in male mice. Dominant lethal effects of ACHI for the main stages of germ cell development were analyzed by mating at specific time points after dosing. Two groups of adult BALB/c males received 24 or 40 mg per kilogram body weight (BW) per day intraperitoneal (IP) injection of ACHI in sterile phosphate-buffered saline (PBS) for five consecutive days (subacute exposure). Males from a third group were administered single IP injections of ACHI-60 mg/kg BW (acute exposure). The control group received concurrent injections of PBS for five successive days. After the last dose, each male was mated with two untreated females. For fractionated examination with regard to successive germ cell stages (spermatozoa, spermatids, spermatocytes, spermatogonia), every second week, two other untreated virgin females were placed with each male for mating. The uteri of the females were inspected on the 15th day of gestation, and preimplantation loss and postimplantation loss determined from dominant lethal parameters. Exposure of mice germ cells to ACHI did not impair mating activity of males. Fertility index was reduced (P < 0.05) only for females mated at the third week with males exposed to the highest dose of ACHI. In the females bred to ACHI-treated males, significant (P < 0.05) increase in preimplantation loss was observed at postinjection weeks 1 (reflecting exposure to spermatozoa after single treatment and to spermatozoa or late spermatids after subacute dosing) and 3 (reflecting exposure to mid and early spermatids for acute dosing and to mid and early spermatids or late spermatocytes following acute treatment), regardless of dose and length of exposure to the inhibitor. At the 60-mg/kg-BW group, a significant increase of this parameter was also noted at week 5 (reflecting exposure to early spermatocytes). During mating days 15-21, a significant (P < 0.05) increase in postimplantation loss and dominant lethal effects were observed for all doses of ACHI. Acute ACHI exposure 5 weeks prior to mating resulted in dominant lethal effects in early spermatocytes. These preliminary data suggest that ACHI induces dominant lethal mutations at postmeiotic and meiotic stages of spermatogenesis, but spermatids are the most sensitive cell stage to the effect of ACHI. These results show that ACHI may be one of the factors causing disturbances in spermatogenesis leading to a reduction of host reproductive success.

A Key Role for Orphan Nuclear Receptor Liver Receptor Homologue-1 in Activation of Fatty Acid Synthase Promoter by Liver X Receptor

Liver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others.

Fatty Acid Transport Protein 4 Is the Principal Very Long Chain Fatty Acyl-CoA Synthetase in Skin Fibroblasts

Fatty acid transport protein 4 (FATP4) is a fatty acyl-CoA synthetase that preferentially activates very long chain fatty acid substrates, such as C24:0, to their CoA derivatives. To gain better insight into the physiological functions of FATP4, we established dermal fibroblast cell lines from FATP4-deficient wrinkle-free mice and wild type (w.t.) mice. FATP4 -/- fibroblasts had no detectable FATP4 protein by Western blot. Compared with w.t. fibroblasts, cells lacking FATP4 had an 83% decrease in C24:0 activation. Peroxisomal degradation of C24:0 was reduced by 58%, and rates of C24:0 incorporation into major phospholipid species (54-64% decrease), triacylglycerol (64% decrease), and cholesterol esters (58% decrease) were significantly diminished. Because these lipid metabolic processes take place in different subcellular organelles, we used immunofluorescence and Western blotting of subcellular fractions to investigate the distribution of FATP4 protein and measured enzyme activity in fractions from w.t. and FATP4 -/- fibroblasts. FATP4 protein and acyl-CoA synthetase activity localized to multiple organelles, including mitochondria, peroxisomes, endoplasmic reticulum, and the mitochondria-associated membrane fraction. We conclude that in murine skin fibroblasts, FATP4 is the major enzyme producing very long chain fatty acid-CoA for lipid metabolic pathways. Although FATP4 deficiency primarily affected very long chain fatty acid metabolism, mutant fibroblasts also showed reduced uptake of a fluorescent long chain fatty acid and reduced levels of long chain polyunsaturated fatty acids. FATP4-deficient cells also contained abnormal neutral lipid droplets. These additional defects indicate that metabolic abnormalities in these cells are not limited to very long chain fatty acids.

Will the hold of solid biodegradable implants be influenced by swelling during the degradation process?

Water uptake after implantation of biodegradable devices induces swelling, as mentioned in literature. The hold in bone of solid devices will increase if the swelling is substantial enough. The results of weighing six Meniscus Arrows (MAs) before and after immersion in a sterile phosphate buffered saline solution during different time intervals were compared with the outcome of measurements under a field emission scanning electron microscope of six other MAs, stored under comparable conditions. The data were statistically evaluated with the Wilcoxon's signed rank test. The weight increase of 2.1 mg or 9.16% was statistically significant in the first 2 h following immersion, remaining stable afterwards with an average weight gain of 1.7 mg or 7.18%. The core diameter of the MAs increased to 0.01 mm or 1.01% with time. Although this is statistically significant, it is not expected to have any consequences for the hold. However, a remarkable and statistically significant decrease in the outer inter-barb diameter of 0.15 mm or 8.6% was noted with time. Mechanical testing should reveal the clinical relevance of the results of this study.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Monoclonal antibody (mAb) delivery by gene transfer in vivo may be an attractive alternative to current mAb therapies for applications that require long-term therapy. This article describes a transfer system that allows inducible high-level expression of unmodified mAbs in vivo. A recombinant adeno-associated viral (rAAV) vector is used that comprises an expression cassette consisting of a dimerizer-regulated promoter that drives expression of the antibody heavy and light chains linked by a 2A self-processing peptide and a furin cleavage site. Following intravenous injection of the rAAV vector, serum mAb levels >1 mg/ml were attained by administration of the inducer, rapamycin. Antibody expression could be rapidly shut off by discontinuing treatment with rapamycin. By optimizing the furin cleavage sequence, this system generated native antibody in vivo, decreasing the likelihood of a host immune response to foreign sequences. In summary, this optimized mAb expression system allows regulated high-level expression of native full-length mAbs in vivo and may offer a new opportunity for delivery of therapeutic mAbs in the clinic.

Role of CrkII in Fcγ Receptor-mediated Phagocytosis

Phagocytosis of IgG-opsonized pathogens by Fcgamma receptors requires extensive remodeling of the actin cytoskeleton, a process regulated by the small GTPase Rac. Vav was thought to be the guanine nucleotide exchange factor responsible for the activation of Rac, but recent evidence indicates that Fcgamma receptor-mediated phagocytosis is unaffected in macrophages lacking all three isoforms of Vav. We therefore tested whether another GEF, DOCK180, participates in Fcgamma receptor-initiated phagocytosis. DOCK180 associates with the adaptor protein Crk, which mediates recruitment of the GEF to sites of tyrosine phosphorylation. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Knockdown of Crk or DOCK180 in murine macrophages using small interfering RNA inhibited phagocytosis of IgG-opsonized particles. Moreover, transfection of dominant negative CrkII prevented both recruitment of DOCK180 and the activation of Rac at the phagocytic cup. This is the first report of a role for either Crk or DOCK180 in Fcgamma receptor-mediated phagocytosis. The Crk-DOCK180 complex is involved in the clearance of apoptotic cells, which unlike the ingestion of IgG-opsonized particles, is an anti-inflammatory process. The finding that CrkII-DOCK180 is also responsible, at least in part, for the effects of Fcgamma receptors implies that additional, parallel pathways must account for the associated pro-inflammatory effect.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Secondary Acylation of Klebsiella pneumoniae Lipopolysaccharide Contributes to Sensitivity to Antibacterial Peptides

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055DeltalpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055DeltalpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.