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In today’s competitive global marketplace wholesalers have to deal with increasingly complex supply networks. They have to sell their goods via e-commerce and their customers demand more choices, faster fulfilment, and lower prices.Wholesalers also try to expand the services that they offer and to improve customer satisfaction. Oracle JD Edwards EnterpriseOne is a very robust ERP solution, which can help you to optimize your supply chain and logistics operations in order to reduce your cost. It increases your sales profitability and manages your customer relations. This userfriendly and flexible solution has a robust functionality for financial, distribution, sales and marketing processes.
JD Edwards EnterpriseOne is state of the art, fully web-based and runs on a choice of databases, operating systems, and hardware. According to several analysts JD Edwards has a low Total Cost of Ownership (TCO) and can be implemented quickly, without any risks and at predictable costs.
Do you want to know more about the possibilities of JD Edwards ERP in the wholesale and distribution industry? Download the industry snapshot and check out how JD Edwards ERP can help you prepare your business for the future.
Corporate, tax, and governmental reporting standards require that you account for your inventory’s value. With JD Edwards EnterpriseOne Advanced Stock Valuation you accommodate the many stock valuation methods throughout distribution industries. You get inventory accounting at the level and precision you need for targeted analysis. Do you want upscale your stock’s valuation? Download the data sheet.
Your bulk stock inventory is unique. You want access to bulk stock inventory data across your enterprise. Controlling the storage, measurement, and movement of bulk inventory is a complex supply chain issue. JD Edwards EnterpriseOne Bulk Stock Inventory offers a solution that can effectively manage high-volume bulk inventory transactions. If you want to know more, download the data sheet.
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Residents and tourists will soon be able to rent paddle boards and kayaks at the Parksville Community Park for the first time since 2019.
City council voted to issue Island View Kayak Rentals a licence to operate at the former Hovercraft Base Site in the park’s north end. Six kayaks and six stand-up paddle boards will be available to rent, along with life jackets and other safety equipment, according to Amanda Weeks, the city’s manager of administrative services.
The applicant plans to use a transport van to carry and store equipment, as well as commercial white tent to help with day-to-day operations.
Island View Kayak Rentals was the only applicant that met the city’s stipulations after it advertised the opening for several weeks.
The site has not been leased since 2019 and before that it was only leased in the 2013 season and again in 2007 (when it was only leased for two months before the agreement was terminated) and prior to 2007 it was leased in 1994 according to Weeks’s report to council.
In 2007, the main complaint from the lessee was the lack of people making their way to the site. With the improvements to the community park since that time, including the boardwalk and walkway that ends at the Hovercraft site, foot traffic has improved in that section of the community park, according to the report.


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NEW YORK (CBSNewYork) -- A state senator from Long Island is pushing for regulations following concerns over a probable carcinogen in the water.
The man-made chemical 1,4-dioxane – which is listed by the Environmental Protection Agency as a "probable carcinogen" -- was found in nearly all the water districts on Long Island.
State Sen. Todd Kaminsky is has proposed a bill that would set regulations of the chemical.
"This is a chemical that should be watched, it's likely carcinogenic, meaning it could lead to the causing of cancer, but for a lot of reasons it's taking a long way to have that information wind its way up to the public and to different agencies that are supposed to regulate it," Kaminsky said. "A few states are doing something about it, Washington has not."
Kaminsky wants to set a maximum level allowed by law that water districts must follow.
"What you have on the one hand is a lot of reports telling us that we should be concerned about this chemical, yet not a lot of action limiting it or figuring out how to get it out of the water," Kaminsky said. "We need to step up to the plate... We need to start taking action."
The chemical was used in aviation, manufacturing and agricultural operations and is now being used as a stabilizer in solvents, and personal care products including shampoos and detergents.
The chemical can leech through the groundwater and into drinking water, where the sole source for Long Island is a network of underground aquifers.
The Suffolk County Water Authority is building a nearly $1 million ultraviolet reactor – the first of its kind in the state – that can neutralize 1,4-dioxane.
Gov. Andrew Cuomo announced in his State of the State address last week that there will be further testing for 1,4-dioxane on Long Island this year.
Cuomo also proposed a $2 billion fund to pay for clean water infrastructure, helping communities install treatment systems to get rid of contaminants.
Effects of 1,4-dioxane vary, depending on a person's exposure, health and age.
for more features.

domain: law


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The historic flooding has also displaced crocodiles and snakes, according to officials who issued a warning of wayward reptiles in floodwaters.
Over the past week, record levels of rain have deluged Queensland in northeastern Australia, prompting emergency evacuations – as well as some surreal sights.
Hundreds of people have been forced to leave their homes in and around Townsville, a city on the coast of Queensland that has suffered the bulk of the unprecedented flooding. Nearly two feet of rain have fallen in the region since Jan. 26, the New York Times reported.
Residents have been spotted kayaking down veritable rivers where pavement used to be. Others caught off guard by the incessant downpour had to flee to their rooftops for immediate safety. Throughout the region, whole blocks of houses are half-submerged in murky water.
And then there are the animals.
The historic flooding has also displaced crocodiles and snakes, according to officials who issued a warning Monday of wayward reptiles in floodwaters. Already, some residents had posted pictures of crocodiles spotted in distinctly human environments.
“Cannot stress it enough to stay out of the water,” Townsville resident Erin Hahn wrote on Facebook Sunday, beneath a picture of a crocodile at the end of her father’s driveway.
Several other Townsville residents spotted a crocodile seeking refuge above water on the trunk of a toppled tree.
Queensland officials warned residents Monday to expect crocodiles and snakes to turn up in “unexpected places” in the wake of the flooding. “Crocodiles prefer calmer waters and they may move around in search of a quiet place to wait for floodwaters to recede,” Queensland environment minister Leeanne Enoch said in a statement. “Crocodiles may be seen crossing roads, and when flooding recedes, crocodiles can turn up in unusual places such as farm dams or waterholes where they have not been seen before.”

domain: geography


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18 Apr 2014: The PLOS Neglected Tropical Diseases Staff (2014) Correction: Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways. PLOS Neglected Tropical Diseases 8(4): e2802. https://doi.org/10.1371/journal.pntd.0002802 View correction
Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Rodents carry L. interrogans asymptomatically in their kidneys and excrete bacteria in the urine, contaminating the environment. Humans get infected through skin contact and develop a mild or severe leptospirosis that may lead to renal failure and fibrosis. L. interrogans provoke an interstitial nephritis, but the induction of fibrosis caused by L. interrogans has not been studied in murine models. Innate immune receptors from the TLR and NLR families have recently been shown to play a role in the development and progression of tissue fibrosis in the lung, liver and kidneys under different pathophysiological situations. We recently showed that TLR2, TLR4, and NLRP3 receptors were crucial in the defense against leptospirosis. Moreover, infection of a human cell line with L. interrogans was shown to induce TLR2-dependent production of fibronectin, a component of the extracellular matrix. Therefore, we thought to assess the presence of renal fibrosis in L. interrogans infected mice and to analyze the contribution of some innate immune pathways in this process.
Here, we characterized by immunohistochemical studies and quantitative real-time PCR, a model of Leptospira-infected C57BL/6J mice, with chronic carriage of L. interrogans inducing mild renal fibrosis. Using various strains of transgenic mice, we determined that the renal infiltrates of T cells and, unexpectedly, TLR and NLR receptors, are not required to generate Leptospira-induced renal fibrosis. We also show that the iNOS enzyme, known to play a role in Leptospira-induced interstitial nephritis, also plays a role in the induction of renal fibrosis.
To our knowledge, this work provides the first experimental murine model of sustained renal fibrosis induced by a chronic bacterial infection that may be peculiar, since it does not rely on TLR or NLR receptors. This model may prove useful to test future therapeutic strategies to combat Leptospira-induced renal lesions.
Leptospirosis is a bacterial disease transmitted by asymptomatic rodents to humans. The symptoms may be mild, or severe with kidney failure. Renal fibrosis, occurring during inflammatory situations, is characterized by the pathological accumulation of extra-cellular matrix components and can compromise the kidney functions of patients with leptospirosis. Recent research revealed that both innate and adaptive immune responses are involved in the establishment of fibrosis, in several organs and in different pathophysiological situations. In the present study, we characterized a mouse model of chronic infection with Leptospira that provokes mild renal fibrosis. We show that fibrogenesis requires the presence of live Leptospira in the kidney and that B and T cells from the adaptive immune response do not participate in the induction of renal fibrosis. Unexpectedly, we also found that innate immune receptors, TLRs and NLRs, are not involved in the Leptospira-induced fibrosis. Finally, we show that the enzyme responsible for NO production, iNOS, known to participate in renal inflammatory lesions induced by Leptospira, is also involved in renal fibrosis. Our work provides a novel mouse model to study fibrosis occurring due to leptospirosis.
Citation: Fanton d'Andon M, Quellard N, Fernandez B, Ratet G, Lacroix-Lamandé S, Vandewalle A, et al. (2014) Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways. PLoS Negl Trop Dis 8(1): e2664. https://doi.org/10.1371/journal.pntd.0002664
Editor: Jessica N. Ricaldi, Universidad Peruana Cayetano Heredia, Peru
Received: July 19, 2013; Accepted: December 12, 2013; Published: January 30, 2014
Copyright: © 2014 Fanton d'Andon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by Institut Pasteur, Paris, France, by an ERC grant (PGNfromSHAPEtoVIR 202283) to IGB, and by an ANR grant (ANR-08-MIEN-000)to AV and CW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declared that they have no competing interests.
Leptospira interrogans (L. interrogans) are spirochetal bacteria responsible for a worldwide reemerging zoonosis . Rodents asymptomatically carry the bacteria in their kidneys and excrete them in the urine, contaminating the environment. Humans become infected from contaminated ponds, through direct contact with the bacteria via broken skin or mucosa. Leptospirosis can be mild or severe with respiratory, liver and kidney failure, and constitutes a health problem is East Asia, especially among paddy workers. Chronic kidney disease (CKD) is a common feature of numerous renal diseases. A key component of CKD is renal fibrosis, a complex process involving different resident and infiltrated cell types and signaling pathways. Fibrosis results in structural and functional renal alterations characterized by an excessive accumulation of extracellular matrix proteins in scarring tissues, which may lead to organ dysfunction . From human and canine biopsy studies, leptospirosis has been shown to be associated with chronic interstitial nephritis and fibrosis . Recently, it was reported that leptospirosis led to irreversible tubule-interstitial fibrosis in a young male, requiring continuous hemodialysis . In Taiwan, 10% of patients with CKD were seropositive for L. interrogans, although most of them did not have any history record of leptospirosis . Therefore, underestimated leptospirosis could be one of the reasons for the high prevalence of kidney disease in Taiwan  and more generally in East Asia.
The physiopathology of leptospirosis has been studied in several animal models including rats, gerbils, guinea pigs, and hamsters, and revealed several decades ago that kidney tubulo-interstitial lesions were a hallmark of infection with L. interrogans. A recent study highlighted the importance of iNOS in tubulo-interstitial lesions in mice . However, to our knowledge, the long-term pathophysiological consequences of L. interrogans infection in mice, and in vivo studies about Leptospira-induced fibrosis, have not yet been investigated. Nonetheless, in vitro studies showed that outer membrane components of L. interrogans, among them LipL32, the major lipoprotein of L. interrogans and TLR2 agonist , activate human cells  to produce extracellular matrix components , .
We recently developed a mouse model of acute leptospirosis. We showed that, in contrast to C57BL/6J mice that are asymptomatic, mice deficient for both Toll-like Receptor (TLR)-2 (TLR2) and -4 (TLR4) (TLR2/4dko) are susceptible, and can die from L. interrogans infection with all the features of the severe, acute human disease. We demonstrated that B cells, through both TLR2- and TLR4-mediated signaling, play a crucial role in clearance of the bacteria. Moreover, infected TLR2/4dko mice developed a deleterious inflammation within a few days, associated with renal tubulo-interstitial infiltrates of T cells . We also recently showed that L. interrogans infection triggers a NLRP3-dependent IL1ß secretion in the mouse kidney, as a result of a synergistic effect of two cell wall components, leptospiral LPS and glycolipoprotein, through its downregulation of the Na/K ATPase pump . Preliminary data obtained in surviving mice, several weeks after L. interrogans infection, suggested the presence of fibrotic lesions in mouse kidneys.
Innate immune receptors, TLRs and Nod-like receptors (NLRs) such as the inflammasome receptor NLRP3, have recently been shown to play a crucial role in the development and progression of tissue fibrosis of the lung , liver  and in a mouse model of kidney fibrosis induced by unilateral ureteral obstruction . Whether innate receptors also play a role in murine L. interrogans-induced renal fibrosis and whether infiltrated inflammatory cells such as T cells or macrophages, already known to promote the renal fibrosis , , are important players in the Leptospira induced fibrosis is currently unknown.
Here, we characterized a novel murine model of renal fibrosis induced by bacterial infection, and showed that Leptospira infection of C57BL/6J mice led to a sustained fibrosis, associated with chronic carriage of Leptospira. Using several strains of transgenic mice, we determined that T cells and, unexpectedly, TLR and NLR receptors, were not required to generate Leptospira-induced fibrosis. However, we show that the iNOS enzyme, known to play a role in the interstitial nephritis due to Leptospira, also plays a role in the Leptospira-induced renal fibrosis.
Materials and Methods
Female C57BL/6J mice (8- to 10-wk old) were purchased from Janvier (Le Genest, France) and used as control mice. Mice deficient for TLR2 (TLR2ko), TLR3 (TLR3ko), TLR4 (TLR4ko), TLR5 (TLR5ko), TLR9 (TLR9ko) and MyD88 (MyD88ko), originally given by Shizuo Akira (Osaka University, Osaka, Japan), have been further backcrossed eight times into C57BL/6J mice and kindly provided by Michel Chignard (Institut Pasteur, Paris). Double TLR2/TLR4 deficient mice (TLR2/4ko) have been previously described . Mice deficient for Nod1 (Nod1ko) or Nod2 (Nod2ko), respectively given by John Bertin (Millenium, Cambridge, MA) and Jean-Pierre Hugot (Hôpital Robert Debré, Paris, France) to Dana Philpott (University of Toronto, Toronto), have been further backcrossed eight times into C57BL/6J mice, before being crossed and genotyped to get double Nod1/Nod2 deficient mice (Nod1/2dko). CD3 deficient mice (CD3ko), B cell deficient mice (μMT), and caspase-1 deficient mice (Casp1ko) were kindly provided by Armelle Phalipon (Institut Pasteur, Paris), Claude Leclerc (Institut Pasteur, Paris), and Mathew Alberts (Institut Pasteur, Paris), respectively. iNOS deficient mice (iNOSko) in the C57BL/6J background were obtained from the Jackson Laboratory.
All protocols were reviewed by the Institut Pasteur, the competent authority, for compliance with the French and European regulations on Animal Welfare and with Public Health Service recommendations. This project has been reviewed and approved (# 2013-0034) by the Institut Pasteur ethic committee (CETEA #89).
L. interrogans and in vivo infection experiments
L. interrogans serovar Copenhageni strain Fiocruz L1–130 and L. interrogans serovar Manilae strain L495 were used in this study as described earlier . Just before infection, bacteria in early stationary phase (around 5×108 Leptospira per ml), grown in liquid EMJH medium at 28°C, were centrifuged, resuspended in endotoxin-free PBS, and counted using a Petroff-Hauser chamber. The LD50 of the Fiocruz strain in C57BL6/J WT mice is above 109 bacteria/mouse, and WT mice are considered resistant to L. interrogans infection, as are CD3ko, iNOSko, TLR3ko, Casp1ko and Nod1/2ko mice. However, the LD50 of the Fiocruz strain in sensitive MyD88ko, TLR4ko and TLR2/4ko mice is around 107 bacteria/mouse. Therefore, to ensure survival of all mice, Leptospira-resistant mice were infected with 2×108 Fiocruz strain in 200 µl of PBS by the intraperitoneal (IP) route, whereas Leptospira-sensitive mice were infected with a lower dose of 2×106 Fiocruz/mouse. Since the LD50 of the Manilae strain in WT mice is around 108 bacteria/mouse, WT mice were infected with 107 Manilae strain/mouse. Mice were sacrificed at different days post-infection (p.i.). Liver, kidneys, and lungs of infected and naive mice were removed. Organs were either rapidly frozen in liquid nitrogen, then stored at −80°C for nucleic acid preparations, or fixed for histology and immunohistochemical studies.
Within the first days after experimental IP infection, Leptospira disseminate through blood circulation and reach all the organs, including the kidneys. Then, within one week post-infection, the bacteria disappear from the circulation and settle in and colonize their renal niche, then begin to be shed in the urine. Therefore, penicillin G (Sigma), at the equivalent human dose of 9 million units/60 kg, was administered in 100 µl of endotoxin free PBS (Biowhittaker) to 20 g C57BL/6J mice via IP route once a day for 5 consecutive days, either beginning one day p.i. to clear disseminating bacteria, or 3 days p.i. to stop the infection after the renal colonization has started.
Generation of mouse bone marrow-derived macrophages and in vitro stimulation
Bone marrow derived macrophages (BMDM) were isolated as described previously and cultured for 7 days in 10% L929-conditioned medium . Mouse BMDM (2×105 cells in 200 µl) were seeded in 96-well plate and stimulated 3 h later with live or killed (56°C 30 min) L. interrogans, at different multiplicities of infection (MOI). Stimulations were stopped 24 h later, and nitric oxide (NO) formation was evaluated in supernatants by the measure of nitrites (NO2−) via the Griess reaction.
Plasma biochemical analysis
Before sacrifice, blood samples (200 µl) were collected by retromandibular puncture into tubes containing 20 µl of heparin (Choay). Samples were centrifuged (1500 g, 5 min), and the plasma was stored at −80°C. Total serum creatinine was measured in plasma samples using an Olympus AU400 autoanalyzer.
Leptospiral load in urine
The leptospiral burden in urine was determined by quantitative real-time DNA PCR (qPCR). Total DNA was extracted from a drop of urine (5 to 100 µl) using the Maxwell 16 instrument and Cell LEV DNA purification kit (Promega). The qPCR reaction was calibrated using a known number of heat-killed L. interrogans. The DNA concentration was adjusted to around 100 ng in the qPCR reaction. Primers were designed in the peculiar lpxA gene of L. interrogans Fiocruz strain  to specifically detect pathogenic Leptospira spp but not other spirochetes or bacteria, using Primer Express 3 software (Forward (Fw): 5′-TTTTGCGTTTATTTCGGGACTT-3′; Reverse primer (Rv): 5′-CAACCATTGAGTAATCTCCGACAA-3′; Probe: 5′-TGCTGTACATCAGTTTTG -3′). qPCR reactions were run on a Step one Plus real-time PCR apparatus using the absolute quantification program (Applied Biosystems), with the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 15 s and annealing temperature 60°C for 1 min, according to the manufacturer's instructions. Results were expressed as the number of Leptospira in 100 µl of urine. Observation and subcultures of Leptospira from fresh urines showed that shed bacteria were mobile and alive (data not shown).
Morphological and immunohistochemical studies
Thin transversal sections of kidneys were collected and fixed in Dubosq-Brazil for 2 h then post-fixed in 10% formalin in PBS and embedded in paraffin. Tissue sections (5 µm thickness) were stained with Hematoxylin-Eosin, to evaluate inflammatory changes by light microscopy, or labeled for 30 min with a solution of 0.1% (W/V) Red Sirius in saturated picric acid, for evaluation of the fibrosis. Picro-Sirius is currently used to stain collagens I and III deposited within the interstitial areas, and is recommended for the diagnosis of chronic renal injury. All sections were examined by two pathologists blinded to the experimental conditions. The degree of interstitial inflammation was graded on a 6-point scale as follows: 0- no inflammation, 1- scattered interstitial mononuclear inflammatory cells, 2- mild diffuse mononuclear cell infiltration, 3- focal nodular mononuclear cell infiltration, 4- diffuse and nodular mononuclear cell infiltration without tubulitis 5- diffuse and nodular mononuclear cell infiltration with significant tubulitis. The degree of interstitial fibrosis was determined using a semi-quantitative scale as previously established  as follows: 0- no abnormality, 1- slight increase of interstitial fibrosis affecting less than 25% of kidney samples, with almost normal tubule, 2- moderate interstitial fibrosis affecting less than 25–50% of kidney samples with focal tubular atrophy, 3- severe interstitial fibrosis affecting more than 50% of kidney samples with diffuse extensive tubular atrophy. Morphometry was performed using computerized automatic scan (Visilog, VFG, Paris) and expressed as the mean of Red Sirius positive labeling per surface area (104 µm2), counted on five different kidney tissue sections for each condition tested.
Immunohistochemical studies were performed using avidin-biotin coupled to peroxidase substrate kits (Vector Laboratories) according to the manufacturer's instructions. Peroxidase activity was revealed with diaminobenzidine (brown staining) (Dako REAL detection System). Antibodies used in this study were: a polyclonal antibody against LipL32 (a kind gift from David Haake, 1/2000), a monoclonal antibody against CD3 (Santa Cruz Sc-20047, 1/100), a rat monoclonal anti-mouse-Gr1 (Ly-6G/C) antibody (CliniSciences 1/100) and a monoclonal antibody, anti CD11b (Clinisciences, 1/100). The number of labeled cells (T cells with anti CD3 antibody, neutrophils with anti Gr1 antibody, and macrophages/monocytes with anti CD11b) per surface area (104 µm2) was counted on five different kidney tissue sections for each of the experimental condition tested.
Kidney samples were fixed for 30 min in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, embedded in Epon, and processed for transmission electronmicroscopy by standard procedures.
Real-time and reverse transcription PCR (qRT-PCR)
Total RNA was extracted from kidneys using the RNeasy mini kit (Qiagen). RNA concentration was determined by measuring optical density at 260 nm. 2 µg total RNA was incubated in a final volume of 20 µl containing 1 µl oligo dT (100 µM) (Fermentas), 1 µl dNTP (10 mM each), 2 µl DTT (0.1 M), 0.5 µl Superscript II reverse transcriptase (200 U/ µl) and 4 µl 5×first strand buffer (Invitrogen). RNA with oligo dT was first denatured at 65°C for 5 min, then the enzyme and other reagents were added and maintained at 42°C for 1 h, followed by heat-inactivation at 70°C for 15 min. The generated cDNA was stored at −20°C. After RT, qPCR was performed using cDNA combined with primers, probes and mixed according to the manufacturer's recommendations (Applied Biosystems). qPCR reactions were run on a Step one Plus real-time PCR apparatus (Applied Biosystems), with the following conditions : 50°C for 2 min, 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 15 s, and annealing temperature 60°C for 1 min. Data were analyzed according to the method of relative gene expression using the comparative CT method also referred to as the 2−ΔΔCT method. PCR data were reported as the relative increase in mRNA transcripts versus that found in kidneys from naive mice, corrected by the respective levels of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA used as an internal standard. The sequences of primers and probes for iNOS, IL6, TNF, and RANTES have already been described . Primers for TGFß (NM_011577) were Fw:5′-TGACGTCACTGGAGTTGTACGG-3′ (nt1461–1482), Rv: 5′-GGTTCATGTCATGGATGGTGC-3′ (nt 1610–1630), probe 5′-TTCAGCGCTCACTGCTCTTGTGACAG-3′ (nt 1522–1547). Validated primers and probes for Mmp2, ACTA-2 and fibronectin (Mn_00439498, Mn_01546133, Mn_01256744, respectively) were from Applied Biosystems.
Rescue of μMT mice with serum
μMT mice lacking B cells were infected IP with 2×107 L. interrogans Fiocruz strain and rescued from death by passive transfer of protective serum, as described . To obtain the protective serum, ten C57BL/6J mice were infected with 5×107 L. interrogans serovar Fiocruz and bled 20 days p.i. After overnight coagulation at 4°C, the sera were collected and heat-inactivated at 56°C for 30 min and kept frozen at −80°C. To be sure the Leptospira will reach the kidney, we let the infection develop for two days before injecting IP 200 µl of protective pooled sera to both infected and naive μMT mice. Survival was monitored and all surviving mice were sacrificed at 15-days p.i.
Statistical analysis was performed using GraphPad Prism software. The unpaired t test, (two-tailed P values) was used to compare two groups. The distribution of three or more groups was analyzed by One-Way ANOVA. Values are expressed as means, or means + standard deviation (SD). A p value<0.05 was considered significant.
Leptospira infection triggers a sustained fibrosis in the mouse kidney
C57BL/6J mice infected with 2×108 L. interrogans serovar Copenhageni strain Fiocruz are resistant, and survive infection . One month p.i., WT mice were sacrificed and their liver, lungs, and kidneys studied by immunohistochemistry. Neither inflammation nor fibrosis could be observed in the livers or lungs of infected mice (data not shown), although infected mice displayed kidney inflammation and fibrosis (Fig. 1A). Indeed, hematoxylin-eosin staining revealed from 1 to 4 inflammatory foci, composed of around one hundred infiltrating cells per kidney section in infected mice, compared to naive mice injected with PBS, that showed no inflammation (Fig. 1A and Fig. 1B). The cellular composition of the infiltrates in naïve and infected mice was investigated by immunohistochemistry. One month p.i., the renal infiltrates were mostly composed of CD3 positive (+) T cells and CD11b+ macrophages/monocytes. In contrast, the few Gr1+ cells (mostly neutrophils) detected at day-3 p.i. were no longer detected in kidneys one month p.i. (Fig. 1C). Red Sirius labels collagens I and III, known to accumulate upon fibrosis. The observation and morphometry of Red Sirius staining one month p.i., revealed mild focal fibrosis in both the cortex and medulla regions of the kidney in Leptospira-infected mice (Fig. 1A and Fig. 1D). To ensure that fibrosis was not peculiar to the Fiocruz strain, C57BL/6J mice were infected with another pathogenic L. interrogans, serovar Manilae strain L495. One month p.i., similar mild fibrosis was observed in kidneys from mice infected with the serovar Manilae strain L495 (data not shown).
(A) Light microscopy of nodular infiltrates stained with hematoxylin-eosin (HE), infiltrating CD11b+ macrophages and CD3+ T cells and collagen deposition stained with Red Sirius (RS) in kidneys from C57BL/6J mice 30 days (D30) after the inoculation of 2×108 L. interrogans strain Fiocruz. As controls, mice were injected with PBS. Magnification, ×100. (B) Score of kidney inflammation of interstitial nodular infiltrates per surface areas from five different renal tissue sections in control (PBS) and L. interrogans strain Fiocruz infected mice (n = 8 per group). (C) Quantification of the number of CD11b+ macrophages, Gr1+ neutrophils and CD3+ T Lymphocytes per surface area in kidneys from day-3 (D3) and day-30 post-infected mice. (D) Fibrosis quantification by Red Sirius morphometry, expressed as percent of surface area and (E) Inflammation evaluation by mRNA expression of proinflammatory mediators in kidneys of 10 infected mice sacrificed at different time points. Values are means ± SD of counts (C and D) and mRNA quantification (E) from 5 different tissue sections from n = 2 separate mice in each group tested. ***P<0.001.
We next thought to establish the kinetics of fibrosis appearance and potential healing in mice. C57BL/6J mice were infected with L. interrogans copenhageni strain Fiocruz and sacrificed at 15, 30, 60, 90, and 180 days p.i, and their kidneys analyzed by Red Sirius morphometry and qRT-PCR to detect fibrosis and inflammation, respectively. The fibrosis was already present at day-15 p.i., and persisted at the same mild level for the next 3 months (Fig. 1D). Fibrosis was still present after 6 months p.i., although a tendency to decrease was observed. Because fibrosis usually occurs upon inflammation, pro-inflammatory chemokines (RANTES) and cytokines (IL6, TNF) were monitored by qRT-PCR. The leptospiral infection triggered a marked inflammatory response in day-15 p.i kidneys that decreased over time, but unexpectedly reappeared at day-180 p.i. (Fig. 1E). We also measured the expression of TGF-ß that is usually upregulated upon fibrosis, but we did not find any significant upregulation in the kidneys of infected mice (data not shown). These findings suggest that Leptospira infection triggers an early inflammation, associated with a sustained renal fibrosis for at least 3 months p.i.
Early antibiotic treatment abolishes the Leptospira-induced fibrosis
Since fibrosis is a complex mechanism observed in many inflammatory conditions, most of them unrelated to infectious processes, we wondered whether the presence of Leptospira or their antigens in the kidney were required for inducing fibrosis. Therefore, C57BL/6J mice were infected with L. interrogans serovar Manilae and then treated daily for five consecutive days with penicillin G, the antibiotic used to treat most patients with leptospirosis. Infected mice were either treated from day-1 to day-5 p.i. (D1–D5), allowing sufficient time for the bacteria to disseminate and reach the kidneys, but be cleared before colonization, or treated from day-3 until day-7 (D3–D7) to allow some Leptospira to colonize the kidneys before the antibiotic treatment. Thereafter mice were sacrificed at 24-day p.i., and renal fibrosis and inflammation quantified by Red Sirius morphometry and scoring (data not shown). In parallel, the carriage of Leptospira was detected by qPCR of leptospiral DNA in the urine. High amounts of Leptospira were detected in the urine of non penicillin-treated infected mice, and to a much lesser extent in the urine of D3–D7 antibiotic-treated mice, whereas no Leptospira were detected in the urine of D1–D5 antibiotic-treated mice (Fig. 2A, left panel). In parallel, the presence in the kidneys of leptospiral antigens was monitored by immunohistochemistry using an antibody directed against LipL32, the major lipoprotein of Leptospira (Fig. 2A, right panel). Interestingly, LipL32 was detected in all infected mice, with a more pronounced labeling in the infected, non-antibiotic treated mice, confirming that the timing of penicillin treatment was adequate to allow Leptospira to reach the kidneys. Histological scoring also revealed that the inflammation was only present in infected, non-treated mice and was not observed in D1–D5 or D3–D7 antibiotic-treated mice (Fig. 2B, left panel). Also, qRT-PCR analysis revealed less up-regulation of the inflammatory RANTES mRNA in antibiotic-treated mice compared to the infected mice, not treated with antibiotics. D3–D7 penicillin-treated mouse kidneys exhibited significantly greater levels of RANTES mRNA expression than D1–D5 penicillin-treated mouse kidneys (Fig. 2B, right panel). This suggests that the inflammation observed in the kidney is related to the Leptospira load in the urine, reflecting the renal burden (data not shown). Red Sirius staining (Fig. 2C, right panel) and quantification (Fig. 2C, left panel) revealed a slight renal fibrosis in non-treated mice compared to naïve mice, although no fibrosis was observed in D1–D5 antibiotic-treated mice, and 2 out of 3 mice exhibited fibrosis in the group of D3–D7 penicillin-treated mice. These results suggest that renal fibrosis occurs in mice colonized by Leptospira in their kidneys and excreting live Leptospira. However, no correlation between the number of Leptospira in the urine and the extent of fibrosis could be observed. Of note, no inflammation or renal fibrosis were observed in the kidneys of mice that cleared the Leptospira infection, but still harbored leptospiral antigens.
C57BL/6J mice were infected with 107 L. interrogans serovar Manilae (n = 3) or PBS (n = 1) and injected (IP) daily for 5 days with penicillin G (Pen) from day-1 p.i. until day-5 (D1–D5) or from day-3 p.i. until day-7 (D3–D7). Thereafter, mice were sacrificed at day-24 p.i. (A) Leptospiral loads in 100 µl urine determined by qRT-PCR, and imaging of the LipL32 leptospiral major antigen immunostaining in kidney sections. (B) Inflammation score (left panel) and pro-inflammatory RANTES mRNA expression (right panel). (C) Quantification (left panel) and microscopy (right panels) of fibrosis by Red Sirius staining. Values are counts (The bars represent the mean value in each group) or means of mRNA quantification ± SD from n = 3 mice per group and are from one representative experiment of two. *P<0.05; ***P<0.001 between groups.
T and B lymphocytes cells are not required for Leptospira-induced renal fibrosis
We next thought to assess the role of the inflammation in Leptospira-induced renal fibrosis. Because inflammatory infiltrates observed one month p.i. with L. interrogans in kidneys were mostly composed of T cells (see Fig. 1C), we questioned whether T cells could be involved in the Leptospira-induced fibrosis. Indeed, T cells have been recently shown to promote renal fibrosis induced by unilateral ureteral obstruction in mice . First, semi-quantitative evaluation by immunohistochemistry of the number of T cells in kidneys of infected C57BL/6J mice revealed that the number of T cell infiltrates decreased over time (Fig. 3A). This suggests that T cells are not directly associated with the observed sustained renal fibrosis. However, to be sure that T cells infiltrates were not the initial trigger of fibrosis, mice deficient for T cells (CD3ko mice) and their C57BL/6J counterparts were infected with L. interrogans strain Fiocruz. Mice were sacrificed at day-15 p.i. and their kidneys prepared for immunohistochemistry. Red Sirius morphometric quantification was equivalent in infected WT and CD3ko mouse kidneys, which were both significantly greater compared to the respective naive kidneys (Fig. 3B). Electron microscopy analysis of the kidney of a 4 month post-infected CD3ko mouse revealed a marked interstitial fibrosis, in contrast to the morphological aspect of the kidney of a naive mouse (Fig. 3C). Altogether, these results strongly suggest that T cells do not participate in the induction of renal fibrosis caused by Leptospira.
(A) Quantification of the number of infiltrating CD3+ cells in Leptospira-infected kidneys from WT C57BL/6J mice at different time points post p.i.. Values are means ± SD from five kidney sections per surface area from (n = 2) mice at each different time point tested. (B) Percentage of Red Sirius labeling per surface area in kidney sections from WT and CD3ko mice at day-15 p.i. (D15) and in naive mice. The bars represent the mean value in each group. (C) Electron microscopy (magnification ×100) of kidneys sections from a naïve mouse and CD3ko mouse at 4 months p.i. (D120) showing fibrosis. (D) Survival curves and (E) images of renal Red Sirius staining from B cell deficient naïve μMT mice or infected with 107 L. interrogans strain Fiocruz, rescued by immune serum from Leptospira-infected WT mice at day-15 p.i. (D15). *P<0.05; ***P<0.001 between groups.
B cells from the adaptive immune response have also been reported to be involved in renal fibrosis through IgG deposition . We already showed that B cells were crucial for clearance of Leptospira, and that transgenic mice deficient for B cells (μMT mice) were lethally susceptible to experimental leptospirosis . To test the role of B cells in Leptospira-induced renal fibrosis, μMT mice were infected with Leptospira and rescued with passive transfer of protective serum obtained from Leptospira-infected C57BL/6J mice 20 days p.i. (Fig. 3D), as previously described . Serum-treated naïve and infected μMT mice were sacrificed at day-15 p.i. Kidneys of rescued mice, stained with Red Sirius, showed some mild fibrosis compared to kidneys from the non-infected mice treated with protective serum (Fig. 3E), suggesting that B cells are not involved in Leptospira-induced fibrosis. Collectively, these results suggest that the adaptive immune response to Leptospira from both T and B cells is not directly involved in the induction of renal fibrosis.
Leptospira-induced renal fibrosis does not depend on TLR2 and TLR4
Innate immunity receptors have recently been linked to fibrosis. Since TLR2 and TLR4 are crucial in mouse defense against Leptospira , and since in vitro experiments suggest that TLR2 stimulation by outer membrane components of Leptospira is important for expression of fibronectin and extra cellular matrix components , we aimed at testing whether TLR2 and/or TLR4 could be involved in Leptospira-induced renal fibrosis in mice. We previously showed that TLR2/4dko mice are sensitive to pathogenic Leptospira and die from the infection. Therefore, groups of WT, TLR2ko, and TLR2/4dko mice were infected with a sub-lethal dose (2×106) of L. interrogans strain Fiocruz. Mice were then sacrificed at day-90 p.i., and their kidneys analyzed by Red Sirius morphometry. Unexpectedly, Leptospira-infected kidneys from all genotypes, including TLR2/4dko mice, presented fibrosis when compared to their naïve counterparts (Fig. 4A). Consistent with the marked Red Sirius staining in kidneys from all tested mice, mRNA expression of typical markers classically up-regulated in fibrotic conditions, such as metalloprotease 2 (Mmp2)  which is important for the degradation of extracellular matrix components, smooth muscle actin (ACTA-2) over-expressed by activated myofibroblasts  and fibronectin, an extracellular matrix glycolipoprotein that binds other fibrillar components such as collagens, were all up-regulated in the kidneys from mice at day-90 p.i., with no significant statistical differences between the four 4 different mouse genotypes (Fig. 4B). Up-regulation of mRNA expression of the inflammatory RANTES chemokine was also detected in kidneys from all 4 mouse groups, with no statistical differences between groups (Fig. 4B). These rather unexpected findings, showing no differences between Leptospira-induced fibrosis and inflammation in WT versus TLR deficient mice, led us to measure the leptospiral loads in the urine of infected mice. Results from q-PCR of the leptospiral DNA showed that 90 days p.i. WT and TLR2ko mice excreted around 105 and 107 Leptospira per 100 µl of urine respectively, whereas susceptible TLR4ko and TLR2/4dko mice were more heavily infected, with around 108 and 109 Leptospira per 100 µl of urine respectively (Fig. 4C). Of note, quantification of renal burden in the corresponding kidneys by q-PCR of the leptospiral DNA gave the same trend (data not shown). These data are consistent with our previous results obtained at day-3 p.i., showing an important role of TLR2 and TLR4 in the mouse defense and clearance of Leptospira , and further suggest that the extent of fibrosis is not directly proportional to bacterial excretion, reflecting the renal bacterial loads. To evaluate whether Leptospira infection, and subsequent renal colonization and fibrosis could be deleterious to renal function, the level of serum creatinine, used as a marker of renal function, was measured in the serum of mice at day-90 p.i. Interestingly, the infected WT mice did not show any statistically significant elevation of the serum creatinine when compared to naïve WT mice (Fig. 4D). In contrast, all TLRko mice presented a slight but significant elevation of the serum creatinine levels compared to those of WT mice and corresponding naïve TLRko mice (Fig. 4D). These results indicate that neither TLR2 nor TLR4 were required for Leptospira-induced renal fibrosis, and suggest that the chronic carriage of Leptospira can be associated with a slight alteration of the kidney function, if the Leptospira load is not restricted by the presence of TLR2 and/or TLR4.
(A–D) Renal fibrosis, levels of inflammatory mediators, bacterial loads, and serum creatinine levels in WT or TLR2 and/or TLR4 deficient mice infected with 2×106 L. interrogans Fiocruz at 3 months p.i. (D90) (A) Red Sirius staining. (B) Fibrosis and inflammation evaluation by mRNA expression of fibronectin, Mmp2, ACTA-2 and RANTES, in kidneys of WT, TLR2ko, TLR4ko and TLR2/4dko mice at 3 months p.i. Values are means ± SD from n = 5 mice per genotype group. No statistical difference between genotypes was found for the different markers by One-Way Anova. (C) Bacterial loads in 100 µl of urine from mice at 3 months p.i. The bars represent the mean values in each group tested. (D) Serum creatinine levels in naïve and Leptospira-infected mice at 3 months p.i. *P<0.05; **P<0.01; ***P<0.001 between groups.
TLR and NLR independent Leptospira-induced fibrosis
Aside from TLR2 and TLR4, other TLRs that have not yet been studied in the context of leptospirosis, such as TLR5 that senses flagellin, and TLR9, the receptor of bacterial DNA, could in theory be involved in the murine defense against L. interrogans. MyD88 is the adaptor of most TLRs, except TLR3. To get insight in the putative role of TLRs other than TLR2 and TLR4 in the Leptospira-induced fibrosis, MyD88ko mice were infected with a sub-lethal dose (2×106 bacteria) of L. interrogans strain Fiocruz. Fifteen days p.i., a greater burden of Leptospira was detected in the urine (Fig. 5A), and an increased renal inflammatory response measured in kidneys from MyD88ko mice compared to WT mice (Fig. 5B). Red Sirius staining also revealed a fibrosis in kidneys from both infected WT and MyD88ko mice (Fig. 5C, left panel), which was confirmed by qRT-PCR of different markers of fibrosis, whose up-regulation was not statistically different between WT and MyD88ko kidneys from infected mice (Fig. 5C, right panels). Infection of both TLR5ko and TLR9ko mice also confirmed that the Leptospira-induced fibrosis was independent of these TLRs (data not shown).
WT and Myd88ko mice infected with 2×106 L. interrogans Fiocruz were sacrificed at 15 days p.i. (A) Bacterial loads in 100 µl of urine. (B) Inflammation score in kidneys. (C) Red Sirius staining (C, left panel) and mRNA expression of ACTA-2, fibronectin and Mmp2, in the infected WT and Myd88ko mice kidneys (C, right panel). Data are means ± SD from WT mice (n = 3) and MyD88ko mice (n = 5). The images in C are representative of three separate experiments. (D) Percentage of Red Sirius staining in kidneys from WT mice, TLR3ko, Casp1ko and double Nod1/2ko mice infected with 2×108 L. interrogans Fiocruz and sacrificed at day-15 p.i. (n = 4 per genotype group). Three naïve kidneys of each genotype were used as controls. *P<0.05; **P<0.01 between groups.
TLR3 is the only TLR using TRIF, but not MyD88, as an adaptor. TLR3 is known as a viral RNA sensor and is not expected to be involved in Leptospira defense. However, to be sure that none of the TLRs were involved in Leptospira-induced fibrosis, TLR3ko mice were infected with 2×108 L. interrogans Fiocruz and sacrificed at day-15 p.i. Compared to naïve mice, Leptospira infection induced mild renal fibrosis in most of the infected TLR3ko (Fig. 5D). Collectively, these data indicate that none of the TLRs are critically involved in renal fibrosis caused by Leptospira.
Since TLRs seem not to be involved in Leptospira-induced renal fibrosis, we wondered whether other innate immune receptors from the NLR family, such as the cytosolic Nod1, Nod2, and NLR3 receptors, could be involved. Nod1 and Nod2 are receptors of muropeptides of bacterial peptidoglycan, but their role in defense against Leptospira remains unknown. We recently showed that L. interrogans activates the NLRP3 inflammasome in the mouse kidney . When activated, the NLRP3 inflammasome induces caspase 1 cleavage, which in turns cleaves pro-IL1ß, allowing for its maturation and secretion. To test the role of these NLRs in the induction of renal fibrosis, WT, Nod1/2dko and Casp1ko mice were infected with 2×108 L. interrogans strain Fiocruz (Fig. 5D). Compared to infected WT mice, no reduction in Leptospira-induced fibrosis was observed in kidneys of either Nod1/2dko or Casp1ko mice, showing that Nod1, Nod2 and NLRP3 are not involved in the Leptospira-induced renal fibrosis. As a whole, these results indicate that the fibrosis induced by Leptospira does not directly rely on TLR and NLR activation.
iNOS participates in the Leptospira-induced fibrosis
Infiltrating CD11b+ macrophages were detected in kidneys of WT mice one month p.i. (see Fig. 1C). Their role in leptospirosis is difficult to assess since transgenic mice devoid of macrophages were not available. Apart from their phagocytic role, another defense mechanism of macrophages is the production of reactive oxygen species derived from nitric oxide (NO), which is toxic for bacteria and is produced by different nitric oxide synthase enzymes, among them the inducible iNOS. We previously showed that infection with L. interrogans strain Fiocruz induces iNOS mRNA upregulation in WT mouse kidneys at day-3 p.i. . Here, BMDM from C57BL/6J mice were stimulated with L. interrogans strain Fiocruz, and nitrite (NO2−) production measured in cell supernatants 24 h later. Both live bacteria and heat-killed Leptospira induced dose-dependent production of NO2− (Fig. 6A), showing that macrophages could be a potential source of NO induced by Leptospira infection. NO production constitutes an innate defense mechanism, but is also known to be responsible for cell toxicity. Recently iNOS was shown to play a deleterious role in Leptospira-induced interstitial nephritis . WT and iNOSko mice were therefore infected with 2×108 L. interrogans Fiocruz strain to test the role of iNOS in the induced fibrosis. Mice were sacrificed at day-15 p.i. and kidneys were processed for immunohistochemistry. No difference could be observed in bacterial loads in urine or inflammatory scores in kidneys between the infected WT and iNOSko mice (Fig. 6B and 6C), although renal fibrosis was slightly reduced, but not abolished, in the infected iNOSko compared to WT kidneys (Fig. 6D). Since the fibrosis at day-15 p.i. was mild, we also compared by qRT-PCR the expression of ACTA-2, fibronectin and Mmp2. These markers were statistically slightly less up-regulated in the kidneys of infected iNOSko mice compared to those of WT mice (Fig. 6E, upper panel), although expression levels of transcripts in kidneys of naïve iNOSko mice can not account for the observed down-regulation (Fig. 6E, lower panel). Altogether, these results suggest that upregulation of iNOS mRNA in response to Leptospira infection is deleterious and participates in induction of renal fibrosis. Nevertheless, additional mechanisms appear to be required for Leptospira-induced renal fibrosis.
NO2− production in supernatants from bone marrow macrophages derived from C57BL/6J mice, stimulated for 24 h with live (Lepto) or heat-killed (HKLepto) L. interrogans strain Fiocruz at a multiplicity of infection of 1 (1∶1) 10 (10∶1) and 100 (100∶1), as measured by the Griess reaction. Values are means +SD from 3 separate experiments. (B–E) Bacterial loads in urine (B), renal inflammatory scores (C), percentage of Red Sirius staining (D) and mRNA expression of ACTA-2, fibronectin and Mmp2 (E, upper panel) in kidneys from WT and iNOSko mice infected with 2×108 L. interrogans Fiocruz and sacrificed at day-15 p.i.. Values are means ± SD from WT (n = 3) and iNOSko (n = 5) mice. (E, lower panel), comparison of mRNA expression levels of fibrosis markers in kidneys of naïve WT and iNOSko mice, measured as ΔCT compared to HPRT. Values are means ± SD from WT (n = 3) and iNOSko (n = 3) mice. *P<0.05; **P<0.01 between groups.
Wistar rats are currently used as a model of chronic leptospirosis. After being infected, rats carry Leptospira in their kidneys, and persistently excrete them in their urine , . Histopathology studies have revealed the presence of renal tubulo-interstitial lesions in all experimental animal models of acute and chronic leptospirosis . Naturally susceptible C3H/HeJ mice develop acute renal nephritis after experimental infection with Leptospira, but, to our knowledge, chronic carriage of Leptospira for several months in mice has not been described . Despite a few previous studies mentioning renal fibrosis in dogs  and rats  infected with Leptospira, the putative mechanisms leading to renal fibrosis induced by chronic carriage of Leptospira have not been investigated. In the present study, we characterized a mild, but sustained, renal interstitial fibrosis occurring upon experimental infection of C57BL/6J mice with L. interrogans.
A number of immunohistochemical studies have revealed the presence of Leptospira antigens including lipopolysaccharide (LPS), glycolipoprotein and lipoproteins in the renal tubulo-interstitial lesions, although the presence of live Leptospira was difficult to assess because of their long generation time and fastidious in vitro growth. Recent availability of quantitative real-time PCR techniques to evaluate the DNA presence in urine and organs eased the monitoring of Leptospira carriage. Visual and morphometric analysis of the kidneys of infected mice also permitted analysis of the degree of renal inflammation and fibrosis as described earlier . Because fibrosis was found to be highly focal by visual examination, morphometric analysis revealed only an average mild fibrosis. However, this mild fibrosis was reproducibly measured and correlated with the mRNA upregulation of key markers of renal fibrosis. Although Leptospira-induced renal fibrosis appears to be correlated with inflammation as assessed by semi-quantitative scoring and upregulation of inflammatory cytokines, we did not find any significant up-regulation of TGF-ß (not shown), which is considered a key pro-fibrotic factor, usually produced by CD11b+ macrophages infiltrating the infected kidneys. In this line, a recent work showed that TGF-ß, mostly produced by infiltrating macrophages was not mandatory to ischemia/reperfusion induced fibrosis .
Acute leptospirosis is characterized by multiple organ failure, including liver, lung, and kidney dysfunctions, and marked inflammation and dissemination of Leptospira in all these organs , , , . Interestingly, fibrosis was not found in the liver and lungs of infected mice that, contrary to the kidneys, were devoid of bacteria (not shown). This suggests that it is neither the initial phase of hematogenous dissemination of Leptospira, nor the initial inflammation, but the leptospiral colonization of the kidneys that triggers the fibrosis. In agreement with this hypothesis, mice that received the early antibiotic treatment (day-1 to -5) were cleared of Leptospira and did not develop renal fibrosis. In contrast, when the antibiotic treatment began later at day-3 p.i., it did not succeed in totally eliminating the Leptospira, perhaps due to their potential intracellular location in renal epithelial cells or protected niche in the lumen of renal tubules, and some of the mice developed renal fibrosis. These findings suggest that a direct correlation may exist between renal fibrosis and the presence of live Leptospira in the urine.
An unexpected and puzzling finding of this study was the lack of correlation between the levels of colonization and the extent of renal fibrosis. Indeed, the sensitive TLR4ko and TLR2/4dko mice, more heavily infected than the WT and TLR2ko mice, did not show any enhanced fibrosis. Together with our results on the antibiotic treated mice, the number of bacteria colonizing the kidney did not correlate with the extent of fibrosis. Therefore, we hypothesize that the initial endothelial insult of live Leptospira penetrating in the kidney may trigger the fibrosis, and that once within its niche in the kidney, colonization by Leptospira would not affect the fibrosis course.
Interestingly, one month p.i., the early antibiotic-treated mice developed neither renal fibrosis nor inflammation, although they harbored LipL32 antigens in the kidneys. LipL32 is the major lipoprotein of Leptospira and was demonstrated to be a TLR2 agonist , and an important component of the outer membrane, involved in vitro in the production of extracellular matrix components by human renal cell lines , –. Our present finding, together with the fact that TLR2 is not involved in Leptospira induced renal fibrosis, strongly suggests that in vivo, LipL32 is not involved in the fibrogenesis process. This discrepancy between the in vivo and in vitro results is striking and may emphasize that the complex phenomenon of fibrogenesis cannot be fully mimicked in vitro, and/or that species specificities of the TLRs may be involved. However, if differences in human and mouse TLR4 specificity towards leptospiral lipid A have already been shown , with only mouse TLR4 recognizing the lipid A, we never noticed such a differential recognition of leptospiral lipoproteins between human and mouse TLR2.
Tissue fibrosis is a very complex dynamic process leading to excessive and pathologic accumulation of matrix components that involves many different cells, differentiation and signaling pathways . In a former study, we showed at day-3 p.i. a protective role of T cells, producing IFN-γ and helping macrophages to fight Leptospira in the mouse kidney . This finding is in accordance with the earlier study of Martha Pereira who showed that depletion of CD4+ and CD8+ T cells in mice worsened interstitial nephritis . A role of CD4+ T cells in promoting the renal fibrosis has been recently described in a mouse model of renal fibrosis induced by unilateral ureteral obstruction . This model generates a progressive fibrosis in the kidney with interstitial infiltrations of macrophages. The question arises as to whether recruited T cells in kidneys of Leptospira-infected mice could, beside their early protective role, have also an adverse effect in promoting the fibrosis at later time points. The use of transgenic mice devoid of T cells (CD3ko mice) showed unambiguously that T cells do not take part of the renal fibrotic process in Leptospira-infected mice.
Other cells from the adaptive immunity such as B lymphocytes have been associated with renal fibrotic processes by deposition of immunoglobulins, such as IgG4-related disease, showing high level of serum IgG4 and abundant IgG4-positive plasma cell infiltration into the renal interstitium with fibrosis . We previously showed the crucial and protective role of B cells in leptospirosis through early protective, specific IgM production and later IgG production . The experiment using μMT transgenic mice devoid of B cells and rescued from leptospirosis by administration of immune sera, showed that these mice also exhibit some renal fibrosis, suggesting that B cells and/or related antibody production are not involved in the renal fibrotic process.
We previously showed the important role of both TLR2 and TLR4 in the murine innate defense against Leptospira , . Surprisingly, we did not find any role of TLR2 nor TLR4 in the induction of renal fibrosis, although recent data indicate a role of TLRs in renal pathologies . Hence, TLR4 activation has been shown to favor kidney fibrosis in the mouse model of unilateral urinary obstruction . TLR2 has also been involved in renal fibrosis after unilateral ureteral obstruction , and has been suggested to be important for Leptospira-induced fibrosis . However, our in vivo results showed that renal fibrosis is still present in TLR2/4 double deficient mice, excluding any major contribution of these receptors, and confirmed the fact that TLR2 agonists such as LipL32 are not major players in triggering renal interstitial fibrosis. Moreover, the fact that Leptospira-infected kidneys from MyD88ko and TLR3ko mice were also fibrotic, excludes any important role for TLRs in the mechanism of Leptospira-induced renal fibrosis. These rather unexpected results are in accordance with the recent work of Anders's group showing that post obstructive renal fibrosis is independent of TLR2, TLR9 and MyD88 .
Apart from the trans-membrane TLR innate immune receptors, the cytosolic family of Nod-like receptors also sense cellular intrusion of pathogens and danger signals. For example, Nod1 and Nod2 detect distinct muropeptides of bacterial peptidoglycan . Leptospira species have a peptidoglycan whose chemical composition is close to the one of Gram-negative bacteria despite some peculiarities in their muropeptide composition . However, infected kidneys from Nod1/2dko mice exhibited interstitial fibrosis, therefore excluding a role for both Nod1 and Nod2 in the induction of renal fibrosis. On the other hand, the inflammasome receptor NLRP3, shown to participate in the lung fibrosis induced by uric acid , is activated in kidneys from day-3 p.i. with Leptospira . We also reported that the activation of the NLRP3 inflammasome does not occur through reactive oxygen species production, but rather more through the effect of the glycolipoprotein, an outer membrane toxin of Leptospira inhibiting the sodium/potassium pump (Na/KATPase) in macrophages . This leads to a potassium dysregulation and activation of NLRP3. NLRP3, like most other NLRPs, uses the adaptor ASC to activate caspase1 that in turn cleaves pro-IL1ß, allowing for the IL1ß secretion. Although drugs inhibiting Na/KATPase together with ROS production have been shown to promote renal fibrosis , we neither observed any decrease in fibrosis in Casp1ko mice nor in ASCko mice (not shown). Therefore, our results also strikingly exclude a role for NLRs in the Leptospira-induced fibrosis.
We previously reported that Leptospira induce up-regulation of the iNOS mRNA in kidneys at day-3 p.i. . Here we confirmed the production of NO upon stimulation of bone marrow macrophages with live or dead Leptospira. NO production is a potent innate mechanism to eliminate invading bacteria, but upregulation of renal iNOS has also been linked to kidney injury during systemic inflammation . The fact that iNOS deficient mice and antibiotic-treated WT mice were less fibrotic at day-15 p.i., suggests that early iNOS functions, in response to the initial phase of colonization of the kidneys by Leptospira, would be important for the fibrogenesis process. However, fibrosis is not abolished in the infected iNOSko mice, suggesting that other unknown mechanisms exist, promoting leptospiral-induced renal fibrosis. iNOS has recently been shown by others to participate in Leptospira-induced interstitial nephritis, as measured one month p.i. by a lower histopathological scoring of inflammation in kidneys from iNOSko mice, that were slightly more infected compared to WT mice . Here, we also found a slightly greater number of Leptospira in the urine of iNOSko mice, suggesting that NO participates in bacterial clearance. However, we did not find less inflammation in iNOSko mice, although we found less fibrosis, both by scoring and morphometry of Red Sirius, and, down-regulation of fibrosis markers. Although we demonstrated in a previous study that at day-3 p.i. the up-regulation of iNOS is decreased in TLR2/4dko mice , TLR2/4dko mice still developed renal fibrosis. One explanation for this apparent discrepancy could be that NO, which we previously found to be released from the parenchymal compartment at day 3 p.i. , would be produced by macrophages at later time points through other unknown TLR-independent pathway(s).
Analysis of serum creatinine levels, as a marker of renal function, reveals that WT mice do not show elevated levels of serum creatinine 3 months p.i., confirming their asymptomatic carrier status. However, TLR deficient mice, which harbored more bacteria in their urine, exhibited discretely increased levels of serum creatinine, suggesting that the renal function is slightly affected. Moreover, the renal inflammation in WT mice 6 months p.i., suggests that chronic carriage of Leptospira in the long-term could be deleterious. Interestingly, since humans do not sense leptospiral LPS through TLR4 , we may hypothesize that chronic carriage of Leptospira, already demonstrated in humans , , , could also be linked to a slightly impaired renal function further favoring development of other kidney diseases as previously suggested by Yang's group in Taiwan .
To summarize, the present study provides lines of evidences that renal colonization by Leptospira induces a mild renal fibrosis in mice through TLR- and NLR-independent pathways, and suggests that the activation of iNOS plays a role in the induction of the renal fibrosis. Our work also highlights the fact that future therapeutic strategies should aim at eliminating Leptospira very early after infection, before the renal colonization. Therefore, development of efficient human vaccines against pathogenic Leptospira would be extremely useful to prevent chronic carriage of Leptospira that may, in the long term, alter renal function.
We thank Richard Wheeler for critical reading of the manuscript and editing of English. We thank Stephanie Sasasinh, Hélène Chavigneau and Benjamin Paquet for their contribution to qRT-PCR and immunohistochemistry experiments.
Conceived and designed the experiments: CW JMG IGB AV . Performed the experiments: MF NQ BF GR CW . Analyzed the data: CW JMG AV. Contributed reagents/materials/analysis tools: SL AV. Wrote the paper: CW.
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- 10. Chassin C, Picardeau M, Goujon JM, Bourhy P, Quellard N, et al. (2009) TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans. J Immunol 183: 2669–2677.
- 11. Lacroix-Lamande S, d'Andon MF, Michel E, Ratet G, Philpott DJ, et al. (2012) Downregulation of the Na/K-ATPase pump by leptospiral glycolipoprotein activates the NLRP3 inflammasome. J Immunol 188: 2805–2814.
- 12. Gasse P, Riteau N, Charron S, Girre S, Fick L, et al. (2009) Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis. Am J Respir Crit Care Med 179: 903–913.
- 13. Aoyama T, Paik YH, Seki E (2010) Toll-like receptor signaling and liver fibrosis. Gastroenterol Res Pract 2010: 192543.
- 14. Campbell MT, Hile KL, Zhang H, Asanuma H, Vanderbrink BA, et al. (2011) Toll-like receptor 4: a novel signaling pathway during renal fibrogenesis. J Surg Res 168: e61–69.
- 15. Liu L, Kou P, Zeng Q, Pei G, Li Y, et al. (2012) CD4+ T Lymphocytes, especially Th2 cells, contribute to the progress of renal fibrosis. Am J Nephrol 36: 386–396.
- 16. Conway B, Hughes J (2012) Cellular orchestrators of renal fibrosis. QJM 105: 611–615.
- 17. Que-Gewirth NL, Ribeiro AA, Kalb SR, Cotter RJ, Bulach DM, et al. (2004) A methylated phosphate group and four amide-linked acyl chains in leptospira interrogans lipid A. The membrane anchor of an unusual lipopolysaccharide that activates TLR2. J Biol Chem 279: 25420–25429.
- 18. Goujon JM, Vandewalle A, Baumert H, Carretier M, Hauet T (2000) Influence of cold-storage conditions on renal function of autotransplanted large pig kidneys. Kidney Int 58: 838–850.
- 19. Tsubata Y, Akiyama F, Oya T, Ajiro J, Saeki T, et al. (2010) IgG4-related chronic tubulointerstitial nephritis without autoimmune pancreatitis and the time course of renal function. Intern Med 49: 1593–1598.
- 20. Cheng S, Pollock AS, Mahimkar R, Olson JL, Lovett DH (2006) Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury. FASEB J 20: 1898–1900.
- 21. Tucunduva de Faria M, Athanazio DA, Goncalves Ramos EA, Silva EF, Reis MG, et al. (2007) Morphological alterations in the kidney of rats with natural and experimental Leptospira infection. J Comp Pathol 137: 231–238.
- 22. Athanazio DA, Silva EF, Santos CS, Rocha GM, Vannier-Santos MA, et al. (2008) Rattus norvegicus as a model for persistent renal colonization by pathogenic Leptospira interrogans. Acta Trop 105: 176–180.
- 23. Monahan AM, Callanan JJ, Nally JE (2009) Review paper: Host-pathogen interactions in the kidney during chronic leptospirosis. Vet Pathol 46: 792–799.
- 24. Pereira MM, Andrade J, Marchevsky RS, Ribeiro dos Santos R (1998) Morphological characterization of lung and kidney lesions in C3H/HeJ mice infected with Leptospira interrogans serovar icterohaemorrhagiae: defect of CD4+ and CD8+ T-cells are prognosticators of the disease progression. Exp Toxicol Pathol 50: 191–198.
- 25. Farris AB, Colvin RB (2012) Renal interstitial fibrosis: mechanisms and evaluation. Curr Opin Nephrol Hypertens 21: 289–300.
- 26. Huen SC, Moeckel GW, Cantley LG (2013) Macrophage-specific deletion of transforming growth factor-beta1 does not prevent renal fibrosis after severe ischemia-reperfusion or obstructive injury. Am J Physiol Renal Physiol 305: F477–84.
- 27. Pereira MM, Da Silva JJ, Pinto MA, Da Silva MF, Machado MP, et al. (2005) Experimental leptospirosis in marmoset monkeys (Callithrix jacchus): a new model for studies of severe pulmonary leptospirosis. Am J Trop Med Hyg 72: 13–20.
- 28. Silva JJ, Dalston MO, Carvalho JE, Setubal S, Oliveira JM, et al. (2002) Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis. Rev Soc Bras Med Trop 35: 395–399.
- 29. Hung CC, Chang CT, Chen KH, Tian YC, Wu MS, et al. (2006) Upregulation of chemokine CXCL1/KC by leptospiral membrane lipoprotein preparation in renal tubule epithelial cells. Kidney Int 69: 1814–1822.
- 30. Hung CC, Chang CT, Tian YC, Wu MS, Yu CC, et al. (2006) Leptospiral membrane proteins stimulate pro-inflammatory chemokines secretion by renal tubule epithelial cells through toll-like receptor 2 and p38 mitogen activated protein kinase. Nephrol Dial Transplant 21: 898–910.
- 31. Yang CW, Hung CC, Wu MS, Tian YC, Chang CT, et al. (2006) Toll-like receptor 2 mediates early inflammation by leptospiral outer membrane proteins in proximal tubule cells. Kidney Int 69: 815–822.
- 32. Nahori MA, Fournie-Amazouz E, Que-Gewirth NS, Balloy V, Chignard M, et al. (2005) Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells. J Immunol 175: 6022–6031.
- 33. Saeki T, Nishi S, Imai N, Ito T, Yamazaki H, et al. (2010) Clinicopathological characteristics of patients with IgG4-related tubulointerstitial nephritis. Kidney Int 78: 1016–1023.
- 34. Goncalves GM, Castoldi A, Braga TT, Camara NO (2011) New roles for innate immune response in acute and chronic kidney injuries. Scand J Immunol 73: 428–435.
- 35. Braga TT, Correa-Costa M, Guise YF, Castoldi A, de Oliveira CD, et al. (2012) MyD88 signaling pathway is involved in renal fibrosis by favoring a TH2 immune response and activating alternative M2 macrophages. Mol Med 18: 1231–1239.
- 36. Skuginna V, Lech M, Allam R, Ryu M, Clauss S, et al. (2011) Toll-like receptor signaling and SIGIRR in renal fibrosis upon unilateral ureteral obstruction. PLoS One 6: e19204.
- 37. Bourhis LL, Werts C (2007) Role of Nods in bacterial infection. Microbes Infect 9: 629–636.
- 38. Slamti L, de Pedro MA, Guichet E, Picardeau M (2011) Deciphering morphological determinants of the helix-shaped Leptospira. J Bacteriol 193: 6266–6275.
- 39. Liu J, Kennedy DJ, Yan Y, Shapiro JI (2012) Reactive Oxygen Species Modulation of Na/K-ATPase Regulates Fibrosis and Renal Proximal Tubular Sodium Handling. Int J Nephrol 2012: 381320.
- 40. Heemskerk S, Pickkers P, Bouw MP, Draisma A, van der Hoeven JG, et al. (2006) Upregulation of renal inducible nitric oxide synthase during human endotoxemia and sepsis is associated with proximal tubule injury. Clin J Am Soc Nephrol 1: 853–862.
- 41. Chow E, Deville J, Nally J, Lovett M, Nielsen-Saines K (2012) Prolonged leptospira urinary shedding in a 10-year-old girl. Case Rep Pediatr 2012: 169013.
- 42. Ganoza CA, Matthias MA, Saito M, Cespedes M, Gotuzzo E, et al. (2010) Asymptomatic renal colonization of humans in the peruvian Amazon by Leptospira. PLoS Negl Trop Dis 4: e612.

domain: literature


text:
(HYIPs are risky investment schemes managed by unscrupulous individuals with limited expertise in the industry. They lure investors with promises of high returns, so it's crucial to research before investing.)
High-Yield Investment Program Explanation:
The term HYIP stands for High Yield Investment Program. These programs are a gold mine for providing one of the highest-yielding investments you can make. There is no better way to benefit from a massive ROI (Return on Investment) offering than by taking advantage of market-leading interest rates ranging from 5% to triple-digit monthly figures. To maximize the return on their HYIP investments, they use smart investment sources derived from a portfolio of market leaders.
These methodologies are based on the concept of advanced monetary management. These policies have the advantage of allowing maximum interest payouts on short-term investment time frames that are not available anywhere else in the world.
These professionals have established all of the necessary industry relationships that can provide you with a massive return on investment. These experts have created strategies for trading traditional assets in the constantly expanding markets for bitcoin, altcoins, and other similar cryptocurrencies.
But. It sounds too good to be true. High-yield investment programs hyip or hyips are frequently run by seasoned con artists with little prior knowledge of the stock and bond markets or the online Forex trading environment. Within the HYIP case study, the alleged Ponzi schemes or speculation triangles were correctly categorized. Bitexa uses the same programs that are regarded as scams.
High-yield investment Programs are frequently only found on the web and can even offer a first-rate UX. These investment possibilities provide a fantastic entry point for those seeking a high rate of interest with a low entry threshold and high risk. With HYIPs, the buy-in can be as low as $10 to reserve your investment. This starting point may be even lower given the abundance of high-yield investment schemes available online. Given their features, high-yield investment programs might end up being a choice that is simply too appealing to pass up. HYIPs are among the best investments available today, but they are also the riskiest due to their low entry point, high volatility, and significant return on investment.
However, read through a few of our suggestions before you make your first investment in any risky plan: Be prepared to experience volatility and lose all of your invested funds. Decide on a strict smart investment policy and follow it. The general rule is to only spend money that you can afford to lose. Any investment you make should be based on research. Unless you enjoy overspending, avoid using credit cards. The DeFi (Decentralized Finance) component of the investment process is a feature of the contemporary HYIP environment. Cryptocurrency, which is distributed by decentralized money and offers excellent dividends, has high risk and high reward potential. Even though cryptocurrencies have a reputation for being unpredictable, volatility is a crucial component of their incredibly high return rate. Along with capitalization rates that are through the roof, bitcoin is demonstrating its ability to compete with high-yield investment programs in terms of interest returns.

domain: finance


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Raise a glass with us and celebrate the New Year in true Savannah style at the Savannah Waterfront Association’s “Up the Cup” countdown sponsored by Wet Willie’s. The New Year’s Eve celebration takes place Monday, December 31st beginning at 2PM on historic River Street.
Live bands and DJs will rock Rousakis Plaza with a fantastic light show while we countdown the final hours of 2018. G100’s Mia Mance and Stacy Scott from Hot 98.3 & Rewind 107.9 will be on hand to lead us into the New Year in style as the 6-foot “to-go” cup rises on River Street. Squash kicks-off the live music on the Rousakis Stage at 3:30PM Monday afternoon. Great music continues with The Emerald Empire Band at 8:00PM. DJ Gabe will also be spinning tunes throughout the day and evening.
“It’s going to be a lot of fun. We’ve got great bands performing and organized corn hole and beerpong tournaments with cash prizes. Up the Cup is our fun way of toasting the New Year. New York may have a crystal ball, and Atlanta has a big peach, but only in Savannah can you get a drink in a roadie cup and enjoy a beautiful evening on the waterfront,” says Victoria Smith, Executive Director for the Savannah Waterfront Association. “It is also a privilege to be helping our community partner, the Alzheimer’s Association, raise awareness for such a worthy cause,” continued Smith. The New Year’s Eve “Up the Cup” countdown clock will be displayed at 8:00 pm until it hits the midnight hour, followed by champagne toast. This year’s spectacular firework display is proudly sponsored by Hyatt Regency Savannah, Marriott Savannah Riverfront, The Alida Hotel, The Westin Savannah Harbor, Homewood Suites and Savannah Riverboat Cruises. The New Year’s Eve “Up the Cup” Countdown Celebration is hosted by the Savannah Waterfront Association and proudly sponsored by Wet Willie’s. For a detailed schedule of events, visit riverstreetsavannah.com.

domain: entertainment


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Just when you think you’ve seen it all, you come across something like this. ZEAL AND ARDOR is a one-man project by Swiss-born Manuel Gagneux, which he describes as spiritual black metal blues. Now, combining blues and metal is hardly a new idea but ZEAL AND ARDOR doesn’t stop at that. I came across the titular track “Devil is Fine” earlier this year whilst browsing upcoming releases and I was hooked after the first listen. It was an ingenious mix of African-American crooning and Luciferian black metal. It brings to mind the devil-infused early days of blues. It’s Gagneux‘s imagining of America’s slaves rebelling in the same way as Nordic black metallers, i.e. by turning to the Lord of Light.
Sure enough, the first track, “Devil is Fine,” starts with a howling Southern voice screaming the gospel of the adversary of the soul. His warbling is accented by the rhythmic chime of men in chains in the midst of hard labor. In this song, the familiar guitars and blast beats of black metal take a backseat to the power of wicked prayer. The idea becomes crystal clear, to these people Satan is the only salvation. He offers everything but demands very little. These people live in hell so the lake of fire is no deterrent. The sound is superbly distorted through reverb with just enough simple piano accompaniment to really drive the point home. This song alone is one of the most interesting things I’ve heard in a good while. The guitars kick off “In Ashes” but still remain mostly as a background element. The African-American round singing turns possibly even more ominous with this one. The line “Burn the young boy, burn him good” should send a chill down any goodly little lamb’s spine. The whole song expresses doubt in a god and begins to revel in the realization of his absence. The minimal use of guitars and growling vocals serve to illustrate this. It goes slow when it wants to be creepy and fast when it wants to embrace the flames. It’s incredible how effective it is. This being the first new song I’ve heard on the record, I’d say we’re off to a grand start.
At this point one would be forgiven for double-checking that they’re still listening to the same album. “Sacrilegium I” is a straight-up electronic house music song. In any other context I’d rule it as nothing more than a way to test my sub-woofer. I wouldn’t recommend it to fans of EDM, however, since it takes every opportunity to mess with the time signature and would prove very difficult to dance to. In its 2 minute runtime, it does manage to dish out one good DAFT PUNK-esque melody and a lot of what I believe today’s youth refer to as “sick beats.” It’s not entirely removed from the album since it does have some recordings thrown in which fit the larger tone. A strange interlude, no doubt but it keeps the listener on their toes.
A beautifully over-reverbed vocal melody gets us back on track; it’s very gospel-inspired and catchy, the cornerstone of “Come on Down.” If you came for fast guitars and growls – this one is for you. The guitar melodies are recognizably black metal with a few dips into modern melodic death metal and some more dulcet breaks. Here Gagneux gets to show off his pipes a bit more as the vocals get more powerful while still remaining properly soulful. The growling vocals, however, remain as mere occasional roars. That being said, they are perfectly placed and gorgeously nuanced, even when the song would’ve already been great as is it still dares to end in a melancholy and subdued note. As I’m analyzing it, I realize that on paper it shouldn’t work, but somehow it still does. A true testament to Gagneux‘s musicianship – it makes me want to listen to it again and again.
It seems “Children’s Summon” heard my prayers and not only brings even heavier and more ferocious guitars into the mix but also echoes elements of “Come on Down.” The guitars shred away in the background while an enthralling synth melody takes center stage. This is, at times, either somewhat downplayed or completely halted by Satanic chants (my Latin is a bit rusty, so forgive me if I classify anything with “hail” and “Satani” in it as Satanic.) “Children’s Summon” has a sense of urgency that no other song on the album has. It’s not just fast, it’s ridiculously fast. I can see why it takes breathers – I certainly would. “Sacrilegium II” sounds like the devil’s music box. No really, it’s a music box. But even this gentle lullaby has an underlying eeriness to it. Seeing as the last part of this song was a detour, it seems prudent for the continuation to also be a subversion of the formula. The logically illogical intermission has a very subtle and gradual build-up. Starting out with just the clear sound of thin strands of metal reverberating as they are plucked by tiny bumps on a rotating cylinder, it simulates the sensation of falling asleep by fading into a blurry, ethereal world. If anything on this album could be referred to as ‘lovely’, this would be it.
Going back to what I only hesitantly describe as the status quo, “Blood in the River” sees the return of the chains and pickaxes, as well as the blast beats. This is the point at which the sheer speed and precision of the kick-drum makes sure everyone can see that the drums are programmed. Though that may annoy some listeners, they are still at very least fitting for this record. The dark parish’s mantra of “a good god is a dead one” isn’t even the half of it for this one. I would recommend exercising caution in choosing the company in which one should listen to this. Positively evil and exhilarating – a high-speed barrage of defiant rage.
Not enough songs begin with a clear bass-line. When they do, however, it usually works. “What is a Killer Like You Gonna Do Here” has a smooth, slow, and low groove. Gagneux brings his vocals way down to a gravely baritone in order to issue some spoken-word prose. This is further accented by beatnik-y finger-snaps which make me want to rumble with The Sharks, but we don’t want to advocate musical violence so we suggest the listener simply nod in agreement whilst preferably in a turtleneck and beret. Halfway through the song, the guitar joins in with with a defiantly 1950s sound. An exemplary blues guitar solo brings the song home before it fades out in anticipation for the finale.
Bringing the album to a close is “Sacrilegium III.” It’s best seen as an outro or epilogue; it’s not a grandiose epic but simply a moody synth melody and not much else. It layers those synths expertly and always knows to what it wants to draw the listener’s attention. It invokes a feeling of dread and emptiness. It echoes as if in a chasm of despair and leaves us there wanting for more.
My hopes for this album were so lofty that I wasn’t sure it’d ever meet them. However, even on the first listen I knew this artist was far from a one-hit-wonder. It’s a bizarre record that doesn’t feel at home at either parties or lazy Sunday reading sessions. What it is though, is a fantastic, subversive, innovative, and memorable record. It’s not just a cheap mix of genres – each move seems deliberate and poignant. The Satanism never feels shallow or attention-seeking, but genuine and fresh. For its short runtime, this is an exceptionally well-put-together album with all the makings of a modern classic. I would be surprised if I heard a more interesting release all year.
Written by Vincent Parkkonen
- Devil is Fine
- In Ashes
- Sacrilegium I
- Come On Down
- Children’s Summon
- Sacrilegium II
- Blood In the River
- What is a Killer Like You Gonna Do Here?
- Sacrilegium III
GALLERY: 18.3.2023 Maaliskuun Musta Messu V @ Sammiosali, Lahti
Symphonic Metal Icons SIRENIA to Release New Album, “1977,“ on May 26, 2023
Finnish KAKSONEN release new music after 10 year hiatus
REVIEW: Shores of Null – The Loss of Beauty
18.3.2023 Maaliskuun Musta Messu V @ Sammiosali, Lahti
Interview with Marianas Rest — “It shouted more contrast and more black and white.”
PREMIERE: cadeu ft. spellsinmyhead – attention
Luna Killsiltä Tavastialla kuvattu deep cuts -video ja lisää keikkoja
Melodic Death/Doom Masters MARIANAS REST Unleash Music Video for Colossal New Single, “Light Reveals Our Wounds”
NE OBLIVISCARIS Share new song “Misericorde I – As The Flesh Falls”

domain: artistic


I recently picked up some products from Greyman Tactical, honestly had no idea what to expect. I was just racking my brain trying to answer all the questions I had. Was the quality going to be top notch? How was a 0.188” thick piece of High Density Polyethylene going to hold a rifle and accessories? How would I mount it? What would I put on it? As you can see I had nothing but questions! I’m happy to tell you that I have all the answers for you!
I started by figuring out where in my vehicle I wanted to mount it. I decided to mount the RIP-M (Rigid Insert Panel – Molle) in the rear cargo area of my 2009 Chevrolet Tahoe LTZ. Once I figured this out, I busted out the tape measure. I skipped the old saying of “measure twice, cut once”. I went ahead and measured it about 10 times before I settled on the actual panel I wanted. I took the measurements from the area that I wanted to cover, and got on the inter-webs. As I went through each panel on the Greyman’s website until I found the one that was going to fit the best. I settled for the Insert Panel For made for the Pelican 1700 Case Lid (13in x 34.75in). I also picked out the "Hardware Mounting Kit - 6 Self Drilling Screws with Nylon Spacers" kit to secure the panel to my vehicle.
Once I had my panel picked out, I needed to figure out what I wanted to attach to it. The following is attached to the panel.
- 5.56 16” rifle
- 1 30round P-Mag with the rubber mag pull.
- 1 North American Rescue CAT Tourniquet.
- Howard Leight Impact Sport Electronic Earmuffs.
I attached my rifle via the Rifle Mounting Clamps sold on their website. Now I don’t want my gun getting ripped off in a “Smash and Grab” nor am I an irresponsible gun owner. I’m waiting for the “LOCKDOWN AR-15 Mag Well Lock” to be delivered. This is going to make sure the rifle stays in my truck at all times. I don’t need some jerk running off with it and using it on innocent people.
Since my rifles magwell will be occupied by an “Anti Jerk Device” I went ahead and mounted my Magpul P-Mag to the RIP-M with the “Toggle Quick Release Shock Cord - Rifle/Multi Purpose Strap” designed by S.T.R.Ap, which a Veteran/LEO owned company. What makes this multi purpose strap the right choice? It keeps perfect tension on what ever you put in between it, but still allows you to pull whatever it is out quick and easily. I also have the rubber Magpul pull-tab install on the magazine. This allows me to quickly rip out the magazine when I need it! A second magazine will be added when I pickup another Toggle Quick Release Shock Cord - Rifle/Multi Purpose Strap.
Next up a North American Rescue CAT Tourniquet. Holding this down is yet another awesome product from S.T.R.Ap. This product is the S.T.R.Ap (Shock cord Tourniquet Retention Apparatus) by S.T.R.Ap. This does an awesome job of holding CAT in there nice and tight, by easy to get out.
Lastly I have my Howard Leight Impact Sport Electronic Earmuffs. If I do find myself having to pull this rifle and use it, I will take the time to put on the Ear Pro, its sub 5 seconds. Without it, your hearing is toast the moment you pull that trigger. So beside the fact that its going to saving your hearing to help you hear the enemy coming, its going to amplify everything around you so you can definitely hear that enemy coming sooner! It’s held down with Greyman’s Velcro cinch strap.
All an all, this set up is pretty awesome and keeps me prepared for anything the world is going to bring me when I am outside the home. I am very surprised at the quality of every product I bought. And yes, the 0.188” thick piece of High Density Polyethylene is strong enough to hold everything I have on it and more! There are so many uses for these RIP-M panels. It doesn’t have to just be for gun stuff. If you’re an Off-road guy, this is a great way to mount them tools or spare parts you take out to the trail. The possibilities are endless with these things!
Greyman Tactical is a Veteran/LEO company and I am happy to do business with them. They are some great guys and they put out an amazing product. I HIGHLY recommend you go check them out!
- Guest Author Ben of GatOfTheDay.com


text:
Travelling with a wheelchair
We live to write and write to live ... professional writers talk about the craft and business of writing
Somewhere to speak my mind.......
THE BEST OF POETRY
A Photo Blog by Frank Rumpenhorst
Word Sprinting to the Finish Line
Building A Business While Having A Life
Something For Everyone's Needs
Site of author Eli Glasman
More than Common Tall
A journey from Atheism to Enlightenment
This WordPress.com site is the cat’s pajamas
be ye awesome
All posts copyright 2013–2018 by Mark Aldrich.
Welcome to your Near-Beth-Experience
A great WordPress.com site
Born in the UK with cerebral palsy. Living in Japan. Posts about disability and Japan.
Curiosity run amok . . .


text:
Mon 24 Oct, 2022
“There is nothing in the world so irresistibly contagious as laughter and good humor.” - A Christmas Carol, Charles Dickens
Bah Humbug! Christmas is coming to Shakespeare North Playhouse this winter with Dickens’ beloved classic A Christmas Carol to be staged this festive season. Lovingly adapted for the stage by writer Nick Lane and brought to life by director Ellie Hurt, this production is Dickens like you’ve never seen before! A fast, furious and whacky musical retelling of the original text but with a distinctly Prescot feel.
Originally produced for the Stephen Joseph Theatre, Nick’s eccentric adaptation has been uniquely transformed for Shakespeare North Playhouse to see the classic tale taking place in our very own Prescot town.
Christmas Carol Writer Nick Lane says, “The opportunity to write the first Christmas show at the Shakespeare North Playhouse was something I grasped with both hands – and, if my feet had any kind of grip strength I’d have grasped it with them too. It’s an amazing theatre in the heart of a town I felt instantly at home in; Prescotians are incredibly welcoming and a number of them really helped me get to know the place. I hope I’ve repaid their time and their care with something delightfully silly, and I look forward to seeing the Cockpit Theatre filled with excited kids – and yes, I’m going to say ‘of all ages’ because when it’s Christmas I’m just as big a kid as any of them!”
A Christmas Carol features a small but mighty quartet of actor/ musicians. Zoe West as Scrooge, Jessica Dives as Liza Grub, Abigail Middleton as Clara Winks and Eddy Westbury as Pod/Marley.
Known as the meanest man in Prescot, Ebeneezer Scrooge would cancel Christmas if he could! That is until one fateful night he is given the fright of his life, as three mysterious visitors make him see that there is more to life than counting out his pennies. What ensues is a wild and bonkers romp filled with musical nonsense and festive whimsy- there’s even a time worm thrown in for good measure! Experience a tale of friendship, compassion and humanity that’s jam packed with local references. Can the meanest man in Prescot change his ways before it’s too late? Or will he always be a ‘hard-faced bat, selfish, scrimpy and stingy?’
Director Ellie Hurt who’s recent directing credits include Hamlet at the National Theatre and associate director for the musical Fantastically Great Women who changed the world says, “We’re so excited to bring this brilliant and hilarious version of A Christmas Carol to Shakespeare North Playhouse this Christmas. This is Dickens’ classic story like you’ve never seen it before, in an outrageous and silly adaptation by Nick Lane, full to the brim with live music and with the people of Prescot at it’s heart. The resonance of this iconic story feels stronger now than ever, with the ever-growing divide between classes and the living crisis we are facing, the urgency of this story feels more potent than ever before. This is a story with an important message of compassion and humanity, and it’s told through an uplifting and bonkers show with hilarious characters and a whole host of instruments. Let’s celebrate the people of Prescot for this beautiful new theatre’s first Christmas!”
A Christmas Carol is written by Nick Lane and directed by Ellie Hurt alongside musical director Jessica Dives. Set design is by Hannah Sibai, sound design is by Ernest Acquah and lighting design is by Simisola Majekodunmi. Movement director is Grace Goulding. Rosie Pearson from Pearson Casting is casting director.
This will be the first Christmas production to be staged inside the Cockpit Theatre at Shakespeare North Playhouse, featuring familiar local references and a sprinkling of pantomime fun- it’s one for the whole family to enjoy!
Creative director of Shakespeare North Playhouse Laura Collier explains, “It’s our first Christmas here at Shakespeare North and I can’t think of a better production to kick off the festive season! Nick and Ellie’s version of A Christmas Carol encapsulates all the magic of Dickens’ beloved classic, but in true Prescot and Shakespeare North Playhouse style, does things a little differently!”
A Christmas Carol will be running at Shakespeare North Playhouse from the 25th of November 2022 to the 7th of January 2023.
For more information and to book tickets head to Shakespeare North Playhouse’s website https://shakespearenorthplayhouse.co.uk/event/a-christmas-carol/ or call our booking line 0300 303 4204.


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‘Why Buhari can’t deliver on lean bureaucracy’
The need to reward party loyalty and pander to political considerations are unarguably the main reasons why it has been impossible for President Muhammadu Buhari to deliver on the promise of a lean bureaucracy.
Yvonne Nelson Laments Ghana's Underdevelopment
Ghanaian film star, Yvonne Nelson is making it clear about the underdevelopment of her beloved country, Ghana. The multiple award-winning actress and mother of one took to her Twitter page to lament the lack of development in Ghana since the country attained her independence in 1957. According to her, the people need to sit out…

domain: sociology