Effects of Schistosomal mansoni infection on Calomys callosus coelom-associated lymphomyeloid tissue (milky spots)

Calomys callosus Rengger, 1830 (Rodentia: Cricetidae) is a mouse-like South American wild rodent, which is permissive to Schistosoma mansoni infection. In this paper we studied the effect of schistosomal infection in C. callosus mesenteric and omental milky spots (MS), subsidiary foci of coelom-associated lymphomyeloid tissue (CALT), during the acute, transitional (acute to chronic), and chronic phases of the infection. MS were morphologically analyzed by histological methods, using brigthfield and confocal laser scanning microscopies. The MS of infected animals were mainly of lymphomyelocytic (42 to 90 days) and lymphoplasmacytic (160 days of infection) types and showed frequent presence of lymphoid follicles with germinal centers, plasmacytogenesis and plasmacytosis, mastocytosis, megakaryopoiesis, erythropoiesis and less pronounced eosinopoiesis. These results indicate that MS are a preferential site of germinal-center-dependent and independent plasmacytogenesis, and a bone marrow-like organ, committed with various cellular lineages. The consequence of C. callosus MS reactivity for schistosomal infection is still unknown and is under investigation.

Solubility of cytoskeletal proteins in immunohistochemistry and the influence of fixation.

For accurate and quantitative immunohistochemical localization of antigens it is crucial to know the solubility of tissue proteins and their degree of loss during processing. In this study we focused on the solubility of several cytoskeletal proteins in cat brain tissue at various ages and their loss during immunohistochemical procedures. We further examined whether fixation affected either solubility or immunocytochemical detectability of several cytoskeletal proteins. An assay was designed to measure the solubility of cytoskeletal proteins in cryostat sections. Quantity and quality of proteins lost or remaining in tissue were measured and analyzed by electrophoresis and immunoblots. Most microtubule proteins were found to be soluble in unfixed and alcohol fixed tissues. Furthermore, the microtubule proteins remaining in the tissue had a changed cellular distribution. In contrast, brain spectrin and all three neurofilament subunits were insoluble and remained in the tissue, allowing their immunocytochemical localization in alcohol-fixed tissue. Synapsin I, a protein associated with the spectrin cytoskeleton, was soluble, and aldehyde fixation is advised for its immunohistochemical localization. With aldehyde fixation, the immunoreactivity of some antibodies against neurofilament proteins was reduced in axons unveiling novel immunogenic sites in nuclei that may represent artifacts of fixation. In conclusion, protein solubility and the effects of fixation are influential factors in cytoskeletal immunohistochemistry, and should be considered before assessments for a quantitative distribution are made.

Drug uptake in a rodent sarcoma model after intravenous injection or isolated lung perfusion of free/liposomal doxorubicin.

The distribution of free and liposomal doxorubicin (Liporubicin) administered by intravenous injection (IV) or isolated lung perfusion (ILP) was compared in normal and tumor tissues of sarcoma bearing rodent lungs. A single sarcomatous tumor was generated in the left lung of 35 Fischer rats, followed 10 days later by left-sided ILP (n=20) or IV drug administration (n=12), using 100 microg and 400 microg free or liposomal doxorubicin, respectively. The tumor and lung tissue drug concentration was measured by HPLC. Free doxorubicin administered by ILP resulted in a three-fold (100 microg) and 10-fold (400 microg) increase of the drug concentration in the tumor and normal lung tissue compared to IV administration. In contrast, ILP with Liporubicin resulted in a similar drug uptake in the tumor and lung tissue compared to IV injection. For both drug formulations and dosages, ILP resulted in a higher tumor to lung tissue drug ratio but also in a higher spatial heterogeneity of drug distribution within the lung compared to IV administration. ILP resulted in a higher tumor to lung tissue drug ratio and in a more heterogeneous drug distribution within the lung compared to IV drug administration.

Contribution of NFI-C to TGF-β1 signalling and tissue regeneration

Summary : Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor-ß (TGF-ß) are two crucial growth factors in tissue repair and regeneration. They control migration and proliferation of macrophages and fibroblasts, as well as myofibroblast differentiation and synthesis of the new connective tissue. The transcription factor Nuclear Factor I-C (NFI-C) has been implicated in the TGF-ß pathway and regulation of extracellular matrix proteins in vitro. This suggests a possible implication of NFI-C in tissue repair. In this study, our purpose was to identify the NFI-C target genes in TGF-ß1 pathway activation and define the relationship between these two factors in cutaneous wound healing process. High-throughput genomic analysis in wild-type and NFI-C knock-out embryonic fibroblasts indicated that NFI-C acts as a repressor of the expression of genes which transcriptional activity is enhanced by TGF-ß. Interestingly, we found an over representation of genes involved in connective tissue inflammation and repair. In accordance with the genomic analysis, NFI-C-/- mice showed an improvement of skin healing during the inflammatory stage. Analysis of this new phenotype indicated that the expression of PDGFA and PDGF-Ra genes were increased in the wounds of NFI-C-/- mice resulting in early recruitment of macrophages and fibroblasts in the granulation tissue. In correlation with the stimulation effect of TGF-ß on myofibroblast differentiation we found an increased differentiation of these cells in null mice, providing a rationale for rapid wound closure. Thus, in the absence of NFI-C, both TGF-ß and PDGF pathways may be activated, leading to enhanced healing process. Therefore, the inhibition of NFI-C expression could constitute a suitable therapy for healing improvement. In addition, we identified a delay of hair follicle cycle initiation in NFI-C-/- mice. This prompted us to investigate the role of NFI-C in skin appendage. The transition from a quiescent to a proliferative phase requires a perfect timing of signalling modulation, leading to stem cell activation. As a consequence of cycle initiation delay in null mice, the activation of signalling involved in cell proliferation was also retarded. Interestingly, at the crucial moment of cell fate determination, we identified a decrease of CD34 gene in mutant mice. Since CD34 protein is involved in migration of multipotent cells, we suggest that NFI-C may be involved in stem cell mobilisation required for hair follicle renewal. Further investigations of the role of NFI-C in progenitor cell activation will lead to a better understanding of tissue regeneration and raise the possibility of treating alopecia with NFI-C-targeting treatment. In summary, this study demonstrates new regenerative functions of NFI-C in adult mice, which regulates skin repair and hair follicle renewal. Résumé : PDGF et TGF-ß sont des facteurs important du mécanisme de défense immunitaire. Ils influencent la prolifération et migration des macrophages et des fibroblastes, ainsi que la différenciation des myofibroblastes et la formation du nouveau tissu conjonctif. Le facteur de transcription NFI-C a été impliqué dans la voie de signalisation de TGF-ß et dans 1a régulation de l'expression des protéines de la matrice extracellulaire in vitro. Ces études antérieures laissent supposer que NFI-C serait un facteur important du remodelage tissulaire. Cependant le rôle de NFI-C dans un tissu comme la peau n'a pas encore été étudié. Dans ce travail, le but a été de d'identifier la relation qu'il existe entre I~1FI-C et TGF-ßl à un niveau transcriptionnel et dans le processus de cicatrisation cutanée in vivo. Ainsi, une analyse génétique à grande échelle, a permis d'indiquer que NFI-C agit comme un répresseur sur l'expression des gènes dont l'activité transcriptionnelle est activée par TGF-ß. De plus nous avons identifié un groupe de gènes qui controlent le développement et l'inflammation du tissue conjonctif. En relation avec ce résultat, l'absence de NFI-C dans la peau induit une cicatrisation plus rapide pendant la phase inflammatoire. Durant cette période, nous avons montré que les expressions de PDGFA et PDGFRa seraient plus élevées en absence de NFI-C. En conséquence, l'activation de la voie de PDGF induit une infiltration plus importante des macrophages et fibroblastes dans le tissue granuleux des souris mutantes. De plus, en corrélation avec le rôle de TGF-ßl dans la différenciation des myofibroblasts, nous avons observé une différenciation plus importante de ces cellules chez les animaux knock-out, ce qui peut expliquer une contraction plus rapide de la plaie. De plus, nous avons découvert que NFI-C est impliqué dans l'initiation du cycle folliculaire. La caractérisation de ce nouveau phénotype a montré un ralentissement de la transition telogène-anagène des souris NFI-C-/-. Or, un événement clé de cette transition est la modulation de plusieurs signaux moléculaires aboutissant à' l'activation des cellules souches. En corrélation avec le decalage du cycle, l'activation de ces signaux est également décalée dans les souris NFI-C-/-. Ainsi, au commencement de l'anagène, la prolifération des keratinocytes,NFI-C-/- est retardée et corrèle avec une diminution de l'expression de CD34, une protéine responsable de la détermination du migration des cellules multipotentes. Ainsi, NFI-C semble être impliqué dans la mobilisation des cellules souches qui sont nécessaires au renouvellement folliculaire. En résumé, NFI-C est impliqué dans la régulation des signaux moléculaires nécessaires à la réparation tissulaire et son inhibition pourrait constituer un traitement de la cicatrisation. L'analyse de son rôle dans l'activation des cellules souches permettrait de mieux comprendre le renouvellement tissulaire et, à long terme, d'améliorer les techniques de greffe des cellules souches épithéliales ou consituter une cible pour le traitement de l'alopecie.

Clara cells drive eosinophil accumulation in allergic asthma.

Development of allergic asthma is a complex process involving immune, neuronal and tissue cells. In the lung, Clara cells represent a major part of the "immunomodulatory barrier" of the airway epithelium. To understand the contribution of these cells to the inflammatory outcome of asthma, disease development was assessed using an adjuvant-free ovalbumin model. Mice were sensitised with subcutaneous injections of 10 μg endotoxin-free ovalbumin in conjunction with naphthalene-induced Clara cell depletion. Clara epithelial cell depletion in the lung strongly reduced eosinophil influx, which correlated with decreased eotaxin levels and, moreover, diminished the T-helper cell type 2 inflammatory response, including interleukin (IL)-4, IL-5 and IL-13. In contrast, airway hyperresponsiveness was increased. Further investigation revealed Clara cells as the principal source of eotaxin in the lung. These findings are the first to show that Clara airway epithelial cells substantially contribute to the infiltration of eotaxin-responsive CCR3+ immune cells and augment the allergic immune response in the lung. The present study identifies Clara cells as a potential therapeutic target in inflammatory lung diseases such as allergic asthma.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.

Delayed mortality and attenuated thrombocytopenia associated with severe malaria in urokinase- and urokinase receptor-deficient mice.

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (-/-) mice. The mortality resulting from Plasmodium berghei ANKA infection was delayed in uPA(-/-) and uPAR(-/-) mice but was similar to that of the wild type (+/+) in tPA(-/-) mice. Parasitemia levels were similar in uPA(-/-), uPAR(-/-), and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR(-/-) mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR(-/-) mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA(-/-) and uPAR(-/-) mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR(-/-) mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR(-/-) mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR(-/-) mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.

Repeated injections of 131I-rituximab show patient-specific stable biodistribution and tissue kinetics.

PURPOSE: It is generally assumed that the biodistribution and pharmacokinetics of radiolabelled antibodies remain similar between dosimetric and therapeutic injections in radioimmunotherapy. However, circulation half-lives of unlabelled rituximab have been reported to increase progressively after the weekly injections of standard therapy doses. The aim of this study was to evaluate the evolution of the pharmacokinetics of repeated 131I-rituximab injections during treatment with unlabelled rituximab in patients with non-Hodgkin's lymphoma (NHL). METHODS: Patients received standard weekly therapy with rituximab (375 mg/m2) for 4 weeks and a fifth injection at 7 or 8 weeks. Each patient had three additional injections of 185 MBq 131I-rituximab in either treatment weeks 1, 3 and 7 (two patients) or weeks 2, 4 and 8 (two patients). The 12 radiolabelled antibody injections were followed by three whole-body (WB) scintigraphic studies during 1 week and blood sampling on the same occasions. Additional WB scans were performed after 2 and 4 weeks post 131I-rituximab injection prior to the second and third injections, respectively. RESULTS: A single exponential radioactivity decrease for WB, liver, spleen, kidneys and heart was observed. Biodistribution and half-lives were patient specific, and without significant change after the second or third injection compared with the first one. Blood T(1/2)beta, calculated from the sequential blood samples and fitted to a bi-exponential curve, was similar to the T(1/2) of heart and liver but shorter than that of WB and kidneys. Effective radiation dose calculated from attenuation-corrected WB scans and blood using Mirdose3.1 was 0.53+0.05 mSv/MBq (range 0.48-0.59 mSv/MBq). Radiation dose was highest for spleen and kidneys, followed by heart and liver. CONCLUSION: These results show that the biodistribution and tissue kinetics of 131I-rituximab, while specific to each patient, remained constant during unlabelled antibody therapy. RIT radiation doses can therefore be reliably extrapolated from a preceding dosimetry study.

Characterization of glycoinositol phospholipids in the amastigote stage of the protozoan parasite Leishmania major.

The major macromolecules on the surface of the parasitic protozoan Leishmania major appear to be down-regulated during transformation of the parasite from an insect-dwelling promastigote stage to an intracellular amastigote stage that invades mammalian macrophages. In contrast, the major parasite glycolipids, the glycoinositol phospholipids (GIPLs), are shown here to be expressed at near-constant levels in both developmental stages. The structures of the GIPLs from tissue-derived amastigotes have been determined by h.p.l.c. analysis of the deaminated and reduced glycan head groups, and by chemical and enzymic sequencing. The deduced structures appear to form a complete biosynthetic series, ranging from Man alpha 1-4GlcN-phosphatidylinositol (PI) to Gal alpha 1-3Galf beta 1-3Man alpha 1-3Man alpha 1-4GlcN-PI (GIPL-2). A small proportion of GIPL-2 was further extended by addition of a Gal residue in either alpha 1-6 or beta 1-3 linkage. From g.c.-m.s. analysis and mild base treatment, all the GIPLs were shown to contain either alkylacylglycerol or lyso-alkylglycerol lipid moieties, where the alkyl chains were predominantly C18:0, with lower levels of C20:0, C22:0 and C24:0. L. major amastigotes also contained at least two PI-specific phospholipase C-resistant glycolipids which are absent from promastigotes. These neutral glycolipids were resistant to both mild acid and mild base hydrolysis, contained terminal beta-Gal residues and were not lost during extensive purification of amastigotes from host cell membranes. It is likely that these glycolipids are glycosphingolipids acquired from the mammalian host. The GIPL profile of L. major amastigotes is compared with the profiles found in L. major promastigotes and L. donovani amastigotes.

JNK inhibition and inflammation after cerebral ischemia.

The c-Jun-N-terminal kinase signaling pathway (JNK) is highly activated during ischemia and plays an important role in apoptosis and inflammation. We have previously demonstrated that D-JNKI1, a specific JNK inhibitor, is strongly neuroprotective in animal models of stroke. We presently evaluated if D-JNKI1 modulates post-ischemic inflammation such as the activation and accumulation of microglial cells. Outbred CD1 mice were subjected to 45 min middle cerebral artery occlusion (MCAo). D-JNKI1 (0.1 mg/kg) or vehicle (saline) was administered intravenously 3 h after MCAo onset. Lesion size at 48 h was significantly reduced, from 28.2+/-8.5 mm(3) (n=7) to 13.9+/-6.2 mm(3) in the treated group (n=6). Activation of the JNK pathway (phosphorylation of c-Jun) was observed in neurons as well as in Isolectin B4 positive microglia. We quantified activated microglia (CD11b) by measuring the average intensity of CD11b labelling (infra-red emission) within the ischemic tissue. No significant difference was found between groups. Cerebral ischemia was modelled in vitro by subjecting rat organotypic hippocampal slice cultures to oxygen (5%) and glucose deprivation for 30 min. In vitro, D-JNKI1 was found predominantly in NeuN positive neurons of the CA1 region and in few Isolectin B4 positive microglia. Furthermore, 48 h after OGD, microglia were activated whereas resting microglia were found in controls and in D-JNKI1-treated slices. Our study shows that D-JNKI1 reduces the infarct volume 48 h after transient MCAo and does not act on the activation and accumulation of microglia at this time point. In contrast, in vitro data show an indirect effect of D-JNKI1 on the modulation of microglial activation.

Muscle recovery after repair of short and long peripheral nerve gaps using fibrin conduits.

Peripheral nerve injuries with loss of nervous tissue are a significant clinical problem and are currently treated using autologous nerve transplants. To avoid the need for donor nerve, which results in additional morbidity such as loss of sensation and scarring, alternative bridging methods have been sought. Recently we showed that an artificial nerve conduit moulded from fibrin glue is biocompatible to nerve regeneration. In this present study, we have used the fibrin conduit or a nerve graft to bridge either a 10 mm or 20 mm sciatic nerve gap and analyzed the muscle recovery in adult rats after 16 weeks. The gastrocnemius muscle weights of the operated side were similar for both gap sizes when treated with nerve graft. In contrast, muscle weight was 48.32 ± 4.96% of the contra-lateral side for the 10 mm gap repaired with fibrin conduit but only 25.20 ± 2.50% for the 20 mm gap repaired with fibrin conduit. The morphology of the muscles in the nerve graft groups showed an intact, ordered structure, with the muscle fibers grouped in fascicles whereas the 20 mm nerve gap fibrin group had a more chaotic appearance. The mean area and diameter of fast type fibers in the 20 mm gap repaired with fibrin conduits were significantly (P<0.01) worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. Furthermore, the 10 mm gaps repaired with either nerve graft or fibrin conduit showed similar muscle fiber size. These results indicate that the fibrin conduit can effectively treat short nerve gaps but further modification such as the inclusion of regenerative cells may be required to attain the outcomes of nerve graft for long gaps.

Comparison of polymerase chain reaction on fresh tissue samples and fecal drops on filter paper for detection of Trypanosoma cruzi in Rhodnius prolixus

PCR detection of Trypanosoma cruzi in Rhodnius prolixus using fresh tissue or fecal drops on filter paper showed comparable results: 38.7% infection rate using the fresh tissue sample and 37.9% by dried fecal drop.

Detection of coronary stenoses with contrast enhanced, three-dimensional free breathing coronary MR angiography using the gadolinium-based intravascular contrast agent gadocoletic acid (B-22956).

PURPOSE: To determine the diagnostic value of the intravascular contrast agent gadocoletic acid (B-22956) in three-dimensional, free breathing coronary magnetic resonance angiography (MRA) for stenosis detection in patients with suspected or known coronary artery disease. METHODS: Eighteen patients underwent three-dimensional, free breathing coronary MRA of the left and right coronary system before and after intravenous application of a single dose of gadocoletic acid (B-22956) using three different dose regimens (group A 0.050 mmol/kg; group B 0.075 mmol/kg; group C 0.100 mmol/kg). Precontrast scanning followed a coronary MRA standard non-contrast T2 preparation/turbo-gradient echo sequence (T2Prep); for postcontrast scanning an inversion-recovery gradient echo sequence was used (real-time navigator correction for both scans). In pre- and postcontrast scans quantitative analysis of coronary MRA data was performed to determine the number of visible side branches, vessel length and vessel sharpness of each of the three coronary arteries (LAD, LCX, RCA). The number of assessable coronary artery segments was determined to calculate sensitivity and specificity for detection of stenosis > or = 50% on a segment-to-segment basis (16-segment-model) in pre- and postcontrast scans with x-ray coronary angiography as the standard of reference. RESULTS: Dose group B (0.075 mmol/kg) was preferable with regard to improvement of MR angiographic parameters: in postcontrast scans all MR angiographic parameters increased significantly except for the number of visible side branches of the left circumflex artery. In addition, assessability of coronary artery segments significantly improved postcontrast in this dose group (67 versus 88%, p < 0.01). Diagnostic performance (sensitivity, specificity, accuracy) was 83, 77 and 78% for precontrast and 86, 95 and 94% for postcontrast scans. CONCLUSIONS: The use of gadocoletic acid (B-22956) results in an improvement of MR angiographic parameters, asssessability of coronary segments and detection of coronary stenoses > or = 50%.