Ultrastructural study of liver and lead tissue concentrations in young mallard ducks (Anas platyrhynchos) after ingestion of single lead shot.

Lead (Pb) represents a serious threat to wildlife and ecosystems. The aim of this study was to examine the subcellular effects of dietary Pb pellet ingestion on mallard (Anas platyrhynchos) livers. After ingestion of a single Pb shot (LS4 size class: 0.177 ± 0.03 g) in 41 mallard ducks (22 males and 19 females) versus 10 controls (5 males and 5 females), all 7-week old, a morphologic study was conducted by TEM (transmission electron microscopy) of liver at the subcellular level. The results in treated mallards showed at a magnification of 2500 X that hepatic parenchyma was altered as evidenced by intralysosomal electron-dense deposits, which are compatible with Pb deposits. Further, at a higher magnification (15,000 X) in both genders, deterioration of mitochondria was observed in which the crests and, to a lesser extent, outer membrane were lysed. While the rough endoplasmic reticulum was fragmented, intracytoplasmic electron-dense material compatible with Pb deposits was maximally visible, thereby underscoring the deeply destructive effect of this metal on the subcellular architecture of the liver. In addition, applying an optimized and validated method in a clean room using electrothermal atomic absorption spectrophotometer (ETAAS) with Zeeman background correction, the objective was to improve and refine certain indispensable measurements pertaining to Pb impregnation in tissues other than liver such as kidneys, bones, and feathers of mallards. Data demonstrated show that compared with controls, Pb accumulation increases significantly, not only in the liver (3-fold), but also in the bones and the feathers (14-fold). No significant difference was noted between males and females. Bearing in mind the marked subcellular toxicity attributed to Pb, this study reinforces present-day arguments advocating limitation of game consumption.

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits.

Free-living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.

Impairment of autophagy in the central nervous system during lipopolysaccharide-induced inflammatory stress in mice.

BACKGROUND: Current evidence suggests a central role for autophagy in many neurodegenerative diseases including Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis. Furthermore, it is well admitted that inflammation contributes to the progression of these diseases. Interestingly, crosstalks between autophagy and inflammation have been reported in vitro and at the peripheral level such as in Crohn's disease. However, the impact of systemic inflammation on autophagic components in the brain remains to be documented. Therefore, this study monitored autophagy markers after acute and chronic lipopolysaccharide (LPS)-induced inflammatory stress in mice. RESULTS: We showed that acute inflammation, 24 h post-intraperitoneal 10 mg/kg LPS, substantially increased cytokine production (Interleukin(IL)-1ß, Tumor necrosis factor (TNF)-α and IL-6), decreased the levels of autophagy markers (Beclin-1, p62 and LC3 II) and reduced p70S6K activation in cortex and hippocampus. In hippocampus, IL-1ß levels and LC3 II expression were positively and highly correlated and a negative correlation was noted between TNF-α levels and p70S6K activation. Chronic inflammation by injection of 0.5 mg/kg LPS every three days during three months led to a moderate IL-1ß production and decreased TNF-α levels. Interestingly, Beclin-1 and LC3 II levels decreased while those of p62 increased. Cortical IL-1ß levels positively correlated with Beclin-1 and LC3 II and on the contrary inversely correlated with p62. CONCLUSION: The present study is the first showing links between IL-1ß-mediated inflammation and autophagy in the brain. It could open to new therapeutic strategies in brain diseases where regulation impairment of inflammation and autophagy progress with the severity of diseases.

Longitudinal follow-up of autophagy and inflammation in brain of APPswePS1dE9 transgenic mice.

BACKGROUND: In recent years, studies have sought to understand the mechanisms involved in the alteration of autophagic flux in Alzheimer's disease (AD). Alongside the recent description of the impairment of lysosomal acidification, we wanted to study the relationships between inflammation and autophagy, two physiological components deregulated in AD. Therefore, a longitudinal study was performed in APPswePS1dE9 transgenic mice at three, six and twelve months of age. METHODS: Autophagic markers (Beclin-1, p62 and LC3) and the activation of mammalian Target of Rapamycin (mTOR) signaling pathway were quantified by western blot. Cytokine levels (IL-1ß, TNF-α and IL-6) were measured by ELISA. Transmission electron microscopy was performed to detect autophagic vacuoles. Mann-Whitney tests were used to compare wild-type (WT) versus APPswePS1dE9 mice. Longitudinal changes in parameters were analyzed with a Kruskal-Wallis test followed by a post-hoc Dunn's test. Correlation between two parameters was assessed using a Spearman test. RESULTS: Compared to 12-month old WT mice, 12-month old APPswePS1dE9 mice had higher levels of IL-1ß and TNF-α, a greater inhibition of the mTOR signaling pathway and lower levels of Beclin-1 expression both in cortex and hippocampus. Regarding the relationship of the various parameters in 12-month old APPswePS1dE9 mice, Beclin-1 rates were positively correlated with IL-1ß and TNF-α levels. And, on the contrary, TNF-α levels were inversely correlated with the levels of mTOR activation. Altogether, these results suggest that inflammation could induce autophagy in APPswePS1dE9 mice. However, these transgenic mice displayed a large accumulation of autophagic vesicles within dystrophic neurons in cortex and hippocampus, indicating a terminal failure in the autophagic process. CONCLUSIONS: This first demonstration of relationships between inflammation and autophagy in in vivo models of AD should be taken into account in new therapeutic strategies to prevent inflammation and/or stimulate autophagy in advanced neurodegenerative process such as AD.

Leptospira Interrogans induces fibrosis in the mouse kidney through Inos-dependent, TLR- and NLR-independent signaling pathways.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.

Kidney protection by hypothermic total liquid ventilation after cardiac arrest in rabbits.

BACKGROUND: Total liquid ventilation (TLV) with perfluorocarbons has been shown to induce rapid protective cooling in animal models of myocardial ischemia and cardiac arrest, with improved neurological and cardiovascular outcomes after resuscitation. In this study, the authors hypothesized that hypothermic TLV can also limit kidney injury after cardiac arrest. METHODS: Anesthetized rabbits were submitted to 15 min of untreated ventricular fibrillation. After resuscitation, three groups of eight rabbits each were studied such as (1) life support plus hypothermia (32°-33 °C) induced by cold TLV (TLV group), (2) life support without hypothermia (control group), and (3) Sham group (no cardiac arrest). Life support was continued for 6 h before euthanasia and kidney removal. RESULTS: Time to target esophageal temperature was less than 5 min in the TLV group. Hypothermia was accompanied by preserved renal function in the TLV group as compared with control group regarding numerous markers including creatinine blood levels (12 ± 1 vs. 16 ± 2 mg/l, respectively; mean ± SEM), urinary N-acetyl-ß-(D)-glucosaminidase (1.70 ± 0.11 vs. 3.07 ± 0.10 U/mol of creatinine), γ-glutamyltransferase (8.36 ± 0.29 vs. 12.96 ± 0.44 U/mol of creatinine), or ß2-microglobulin (0.44 ± 0.01 vs. 1.12 ± 0.04 U/mol of creatinine). Kidney lesions evaluated by electron microscopy and conventional histology were also attenuated in TLV versus control groups. The renal-protective effect of TLV was not related to differences in delayed inflammatory or immune renal responses because transcriptions of, for example, interferon-γ, tumor necrosis factor-α, interleukin-1ß, monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, and vascular endothelial growth factor were similarly altered in TLV and control versus Sham. CONCLUSION: Ultrafast cooling with TLV is renal protective after cardiac arrest and resuscitation, which could increase kidney availability for organ donation.

Al amyloidosis.

UNLABELLED: DEFINITION OF THE DISEASE: AL amyloidosis results from extra-cellular deposition of fibril-forming monoclonal immunoglobulin (Ig) light chains (LC) (most commonly of lambda isotype) usually secreted by a small plasma cell clone. Most patients have evidence of isolated monoclonal gammopathy or smoldering myeloma, and the occurrence of AL amyloidosis in patients with symptomatic multiple myeloma or other B-cell lymphoproliferative disorders is unusual. The key event in the development of AL amyloidosis is the change in the secondary or tertiary structure of an abnormal monoclonal LC, which results in instable conformation. This conformational change is responsible for abnormal folding of the LC, rich in ß leaves, which assemble into monomers that stack together to form amyloid fibrils. EPIDEMIOLOGY: AL amyloidosis is the most common type of systemic amyloidois in developed countries with an estimated incidence of 9 cases/million inhabitant/year. The average age of diagnosed patients is 65 years and less than 10% of patients are under 50. CLINICAL DESCRIPTION: The clinical presentation is protean, because of the wide number of tissues or organs that may be affected. The most common presenting symptoms are asthenia and dyspnoea, which are poorly specific and may account for delayed diagnosis. Renal manifestations are the most frequent, affecting two thirds of patients at presentation. They are characterized by heavy proteinuria, with nephrotic syndrome and impaired renal function in half of the patients. Heart involvement, which is present at diagnosis in more than 50% of patients, leading to restrictive cardiopathy, is the most serious complication and engages prognosis. DIAGNOSTIC METHODS: The diagnosis relies on pathological examination of an involved site showing Congo red-positive amyloid deposits, with typical apple-green birefringence under polarized light, that stain positive with an anti-LC antibody by immunohistochemistry and/or immunofluorescence. Due to the systemic nature of the disease, non-invasive biopsies such as abdominal fat aspiration should be considered before taking biopsies from involved organs, in order to reduce the risk of bleeding complications. DIFFERENTIAL DIAGNOSIS: Systemic AL amyloidosis should be distinguished from other diseases related to deposition of monoclonal LC, and from other forms of systemic amyloidosis. When pathological studies have failed to identify the nature of amyloid deposits, genetic studies should be performed to diagnose hereditary amyloidosis. MANAGEMENT: Treatment of AL amyloidosis is based on chemotherapy, aimed at controlling the underlying plasma clone that produces amyloidogenic LC. The hematological response should be carefully checked by serial measurements of serum free LC. The association of an alkylating agent with high-dose dexamethasone has proven to be effective in two thirds of patients and is considered as the current reference treatment. New agents used in the treatment of multiple myeloma are under investigation and appear to increase hematological response rates. Symptomatic measures and supportive care is necessary in patients with organ failure. Noticeably, usual treatments for cardiac failure (i.e. calcium inhibitors, ß-blockers, angiotensin converting enzyme inhibitors) are inefficient or even dangerous in patients with amyloid heart disease, that should be managed using diuretics. Amiodarone and pace maker implantation should be considered in patients with rhythm or conduction abnormalities. In selected cases, heart and kidney transplantation may be associated with prolonged patient and graft survival. PROGNOSIS: Survival in AL amyloidosis depends on the spectrum of organ involvement (amyloid heart disease being the main prognosis factor), the severity of individual organs involved and haematological response to treatment.

Acanthamoeba sp. promotes the survival and growth of Acinetobacter baumanii.

Acinetobacter baumanii, which may be found in water, is an important emerging hospital-acquired pathogen. Free-living amoebae can be recovered from the same water networks, and it has been shown that these protozoa may support the growth of other bacteria. In this paper, we have studied potential relationships between A. baumanii and Acanthamoeba species. Two strains of A. baumanii isolated from hospital water were co-cultivated with the trophozoites or supernatants of two free-living amoebae strains: Acanthamoeba castellanii or Acanthamoeba culbertsoni. Firstly, the presence of the amoebae or their supernatants induced a major increase in A. baumanii growth, compared with controls. Secondly, A. baumanii affected only the viability of A. culbertsonii, with no effect on A. castellanii. Electron microscopy observations of the cultures investigating the bacterial location in the protozoa showed persistence of the bacteria within cyst wall even after 60 days of incubation. In our study, the survival and growth of A. baumanii could be favored by Acanthamoeba strains. Special attention should consequently be paid to the presence of free-living amoebae in hospital water systems, which can promote A. baumanii persistence.

TLR4 facilitates translocation of bacteria across renal collecting duct cells.

Uropathogenic Escherichia coli (UPEC) are the most frequent causes of urinary tract infections and pyelonephritis. Renal medullary collecting duct (MCD) cells are the intrarenal site to which UPEC strains prefer to adhere and initiate an inflammatory response, but the ability of UPEC strains to translocate across impermeant MCD cells has not been demonstrated definitively. Here, several UPEC strains adhered to the apical surface and translocated across confluent murine inner MCD cells grown on filters. UPEC strains expressing cytolytic and vacuolating cytotoxins disrupted the integrity of cell layers, whereas noncytolytic UPEC strains passed through the cell layers without altering tight junctions. Apical-to-basal transcellular translocation was dramatically reduced after extinction of Toll-like receptor 4 (TLR4) and the lipid raft marker caveolin-1 by small interfering RNA. Furthermore, disruption of lipid raft integrity by filipin III and methyl-beta-cyclodextrin significantly reduced both the transcellular translocation of UPEC across murine inner MCD cell layers and the stimulation of proinflammatory mediators. Bacterial translocation was also significantly reduced in primary cultures of TLR4-deficient mouse MCD cells compared with MCD cells from wild-type mice. Benzyl alcohol, an anesthetic that enhances membrane fluidity, favored the recruitment of caveolin-1 in lipid rafts and increased the translocation of UPEC across cultured TLR4-deficient MCD cells. These findings demonstrate that the transcellular translocation of UPEC strains across impermeant layers of MCD cells may occur through lipid rafts via a TLR4-facilitated process.

Role of the monoclonal kappa chain V domain and reversibility of renal damage in a transgenic model of acquired Fanconi syndrome.

Acquired Fanconi syndrome (FS) is a complication of monoclonal gammopathies featuring a generalized dysfunction of the proximal tubule of the kidney, due to the storage within proximal tubular cells of a monoclonal immunoglobulin light chain. We engineered transgenic mice in which the endogenous mouse Jkappa cluster was replaced by a human VkappaJkappa rearranged gene cloned from a patient with smoldering myeloma-associated FS. The V region belonged to the VkappaI subgroup and was related to the O2-O12 germ-line gene, a V segment previously found associated with FS and light-chain crystallization in several patients with myeloma. Association of the human VkappaI domain with a mouse kappa constant domain in transgenic animals yielded a nephrotoxicity pattern similar to that observed in patients, strongly suggesting that the whole pathogenic effect of FS light chains can be ascribed to a peculiar structure of the V domain. Morphologic alterations of the kidney tubular cells, which contained rhomboid-shape crystals, were observed in mice, together with alterations of the proximal tubule reabsorption function. Moreover, the number of renal crystalline inclusions was dramatically reduced after conditional deletion of the human VkappaI transgene, showing that proximal tubular lesions are reversible upon suppression of the nephrotoxic light chain secretion.