SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of ß-thalassaemia.

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in ß-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in ß-thalassaemia mice (Hbbth3/+ ). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of ß-thalassaemia and related disorders.

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic, with increasing deaths worldwide. To date, documentation of the histopathological features in fatal cases of the disease caused by SARS-CoV-2 (COVID-19) has been scarce due to sparse autopsy performance and incomplete organ sampling. We aimed to provide a clinicopathological report of severe COVID-19 cases by documenting histopathological changes and evidence of SARS-CoV-2 tissue tropism. METHODS: In this case series, patients with a positive antemortem or post-mortem SARS-CoV-2 result were considered eligible for enrolment. Post-mortem examinations were done on 14 people who died with COVID-19 at the King County Medical Examiner's Office (Seattle, WA, USA) and Snohomish County Medical Examiner's Office (Everett, WA, USA) in negative-pressure isolation suites during February and March, 2020. Clinical and laboratory data were reviewed. Tissue examination was done by light microscopy, immunohistochemistry, electron microscopy, and quantitative RT-PCR. FINDINGS: The median age of our cohort was 73·5 years (range 42-84; IQR 67·5-77·25). All patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease including diabetes and obesity. The major pulmonary finding was diffuse alveolar damage in the acute or organising phases, with five patients showing focal pulmonary microthrombi. Coronavirus-like particles were detected in the respiratory system, kidney, and gastrointestinal tract. Lymphocytic myocarditis was observed in one patient with viral RNA detected in the tissue. INTERPRETATION: The primary pathology observed in our cohort was diffuse alveolar damage, with virus located in the pneumocytes and tracheal epithelium. Microthrombi, where observed, were scarce and endotheliitis was not identified. Although other non-pulmonary organs showed susceptibility to infection, their contribution to the pathogenesis of SARS-CoV-2 infection requires further examination. FUNDING: None.

Immunolocalization of protease-activated receptors in endothelial cells of splenic sinuses.

The immunolocalization of protease-activated receptors (PARs) and related proteins in splenic sinus endothelial cells was examined using immunofluorescence and electron microscopy. Immunofluorescence microscopy showed that PAR1 colocalized with PAR2, PAR3, and PAR4. PAR4 colocalized with PAR3 and P2Y12. Myosin heavy chain IIA localized to the outer shape and at the base of cells, but did not colocalize with α-catenin. The localization of di-phosphorylated myosin regulatory light chains (ppMLC) was partially detected on the outer circumference and conspicuously at the base of cells. Macrophage migration inhibitory factor (MIF) also localized in cells. Immunogold electron microscopy revealed the localization of PAR1 on the caveolar membrane, plasma membrane, and junctional membrane of cells. PAR2 and PAR3 localized to the plasma membrane of cells. PAR4 localized to the plasma membrane, depressions in the plasma membrane, and cytoplasmic vesicles. PpMLC was detected in stress fibers, but rarely near the adherens junctions of neighboring cells. MIF localized in vesicles on the apical and basal sides of the Golgi apparatus. Electron microscopy of endothelial cells with saponin extraction showed the depression of many coated pits formed by clathrin from the plasma membrane. Stress fibers developed at the base of cells; however, few actin filaments were observed near adherens junctions. These results indicate that PARs play important roles in splenic sinus endothelial cells, such as in endothelial barrier protection and the maintenance of firm adhesion to ring fibers.

Toxicity evaluation of magnetic iron oxide nanoparticles reveals neuronal loss in chicken embryo.

Magnetic iron oxide nanoparticles (IONs) display the ability to cross blood - brain barrier and are envisioned as diagnostic and therapeutic applications, but there are few studies on their potential embryonic toxicity in higher vertebrates. This study investigates interaction of IONs with egg albumen and its subsequent toxicity on chicken embryo. Physicochemical interactions of IONs with egg albumen revealed alterations in friccohesity and secondary structural changes due to weak Vander Waals forces. Toxicity assessment of IONs (10, 25, 50, 100, and 200 µg/ml doses) on chicken embryo accounted for 100% mortality at 200 µg/ml dose due to Fe2+ ions overload. However, lower doses (50 and 100 µg/ml) recorded decrement in whole weights and crown-rump lengths of chicken embryo possibly due to ION-albumen interactions. Histology of brain tissue revealed degeneration of neurons (50-60%) at 10-100 µg/ml dose range of IONs. Toxicity studies of IONs with diverse animal models are needed to set a toxicity benchmark for preventing embryonic toxicity prior to its use in biomedical applications. This is the first study on toxicity assessment of IONs in chicken embryo.

Induction of pathological changes and impaired expression of cytokines in developing female rat spleen after chronic excess fluoride exposure.

This study was designed to investigate the effects of excessive fluoride on spleen toxicity. Twenty-four healthy female rats were randomly divided into two groups, each of 12 rats. Each group of female rats was given a control diet and either F- = 0 mg/L or an excessive F- = 150 mg/L in the drinking water for 120 days. The histomorphological and ultrastructural changes in their splenic tissues were observed under light and transmission electron microscopes. DNA damage and splenocyte apoptosis were examined using the micronucleus (MN) assay, single-cell gel electrophoresis (SCGE), and flow cytometry. The expression levels of cytokines, including interleukin (IL)-1ß, IL-2, IL-6, and tumor necrosis factor (TNF)-α, were determined through immunohistochemistry and Western-blot analysis. Results demonstrated that the histomorphological characteristics and ultrastructure of the splenic tissues were affected by excessive fluoride. Nuclear dying, nuclear membrane dissolution, mitochondrial vacuolation, and endoplasmic reticulum dilation were observed. SCGE and MN assays showed that the nuclear DNA of splenocytes was damaged by fluoride treatment, and splenocyte apoptosis was exacerbated in the fluoride group. With damage to the splenocyte structure and DNA, the protein expression levels of IL-1ß, IL-2, IL-6, and TNF-α were significantly downregulated by exposure to fluoride. Excessive fluoride ingestion caused splenic pathological damage and abnormal cytokine expression in female rats.

Isolation and Characterization of a Ranavirus Associated with Disease Outbreaks in Cultured Hybrid Grouper (♀ Tiger Grouper Epinephelus fuscoguttatus × â™‚ Giant Grouper E. lanceolatus) in Guangxi, China.

An outbreak of suspected iridovirus disease in cultured hybrid grouper (♀Tiger Grouper Epinephelus fuscoguttatus × â™‚ Giant Grouper Epinephelus lanceolatus) occurred in the Guangxi Province in July, 2018. In this study, grouper iridovirus Guangxi (SGIV-Gx) was isolated from diseased hybrid grouper that were collected from Guangxi. Cytopathic effects were observed and identified in grouper spleen cells that were incubated with diseased tissue homogenates after 24 h, and the effects increased at 48 h postinfection. The transmission electron microscopy results showed that viral particles that were about 200 nm in diameter with hexagonal profiles were present in the cell cytoplasm of suspected virus-infected cells. The presence of SGIV-Gx (accession number: MK107821) was identified by polymerase chain reaction (PCR) and amplicon sequencing, which showed that this strain was most closely related to Singapore grouper iridovirus (AY521625.1). The detection of SGIV-Gx infection was further supported by novel aptamer (Q2c)-based detection technology. The effects of temperature and pH on viral infectivity were analyzed by using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and cell culture. The results indicated that SGIV-Gx was resistant to exposure to pH levels 5, 7, and 7.5 for 1 h, but its infectivity was remarkably lower at pH levels 3 and 10 after 1 h. The analyses showed that SGIV-Gx was stable for 1 h at 4°C and 25°C but was inactivated after 1 h at 40, 50, and 60°C.

Analytical High-resolution Electron Microscopy Reveals Organ-specific Nanoceria Bioprocessing.

This is the first utilization of advanced analytical electron microscopy methods, including high-resolution transmission electron microscopy, high-angle annular dark field scanning transmission electron microscopy, electron energy loss spectroscopy, and energy-dispersive X-ray spectroscopy mapping to characterize the organ-specific bioprocessing of a relatively inert nanomaterial (nanoceria). Liver and spleen samples from rats given a single intravenous infusion of nanoceria were obtained after prolonged (90 days) in vivo exposure. These advanced analytical electron microscopy methods were applied to elucidate the organ-specific cellular and subcellular fate of nanoceria after its uptake. Nanoceria is bioprocessed differently in the spleen than in the liver.

Detection of titanium particles in human liver and spleen and possible health implications.

BACKGROUND: Titanium dioxide (TiO2) is produced at high volumes and applied in many consumer and food products. Recent toxicokinetic modelling indicated the potential of TiO2 to accumulate in human liver and spleen upon daily oral exposure, which is not routinely investigated in chronic animal studies. A health risk from nanosized TiO2 particle consumption could not be excluded then. RESULTS: Here we show the first quantification of both total titanium (Ti) and TiO2 particles in 15 post-mortem human livers and spleens. These low-level analyses were enabled by the use of fully validated (single particle) inductively coupled plasma high resolution mass spectrometry ((sp)ICP-HRMS) detection methods for total Ti and TiO2 particles. The presence of TiO2 in the particles in tissues was confirmed by Scanning Electron Microscopy with energy dispersive X-ray spectrometry. CONCLUSIONS: These results prove that TiO2 particles are present in human liver and spleen, with ≥24% of nanosize (< 100 nm). The levels are below the doses regarded as safe in animals, but half are above the dose that is deemed safe for liver damage in humans when taking into account several commonly applied uncertainty factors. With these new and unique human data, we remain with the conclusion that health risks due to oral exposure to TiO2 cannot be excluded.

Protection of Spleen Tissue of γ-ray Irradiated Mice against Immunosuppressive and Oxidative Effects of Radiation by Adenosine 5'-Monophosphate.

The immune system is very sensitive to radiation. This study revealed that adenosine 5′-monophosphate (5′-AMP) increased the DNA contents of the spleen and the spleen index of irradiated mice. Moreover, the exogenous 5′-AMP could significantly repair the ultra-structure of the damaged spleen through transmission electron microscopy. When indicators of the mouse immune system were assessed, the flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that the administration of exogenous 5′-AMP could reduce the apoptosis in the splenic cells. It could also regulate the transition of cells towards S phase, increase the proportion of CD4⁺ and CD8⁺ cellular subsets, and enhance the secretion of interleukin-2 (IL-2), IL-4, IL-10, and interferon-γ (IFN-γ). These effects were associated with a decrease in oxidative stress, as evidenced by changes in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), reduced glutathione (GSH), and malondialdehyde (MDA) levels of spleen tissues. These results suggested that exogenous 5′-AMP could repair the damaged spleen, increase the spleen index, and regulate the cell cycles and apoptosis. There was an increase in the production of various cytokines and play a protective role on the immune system of irradiated mice by dynamically adjusting the REDOX balance.

[Ultramicroscopic features of cells and vessels of the spleen (experimental study)].

OBJECTIVE: Introduction: The spleen is a multifunctional odd organ, which is a biological filter, but its structure in experimental animals is not fully understood. PATIENTS AND METHODS: Materials and methods: The ultramicroscopic structure of the cells and vessels of the spleen of amphibian animals (Pelophylax lessonae С. - the pool frog and Pelophylax ridibundus Р. - the marsh frog) are elucidated in the research. RESULTS: Results: The cytopupulation of the spleen is formed by lymphocytes, neutrophils, erythrocytes, macrophages, plasma cells etcetera. A feature of the cellular composition of the parenchyma of the spleen of the frogs is a significant number of neutrophilic eosinophils. The organelles, which situated in the cell cytoplasm, are characteristic of somatic cells. The endothelial lining and the structure of the capillary basement membrane, which has the form of a plate, formed from 8-12 endothelial cells, grouped in complexes and adjoining contacts in adjacent zones, are experimentally substantiated by the somatic type of vascular endothelium of the microcirculatory channel. Such cells are contained on the basal membrane, surrounded by a dense interlacing of collagen, elastic fibers and the main substance, and the sites of endothelial cell adhesion have the uneven contours. The nuclei of the endothelial cells are surrounded by a cytoplasm on all sides, which reveals foamy cysts. CONCLUSION: Conclusions: The results obtained are of great interest for evolutionary immunomorphology, the extrapolation of which improves understanding of microcirculation, transport of substances in the spleen, and mechanisms for formation of the immune response.

ULTRASTRUCTURAL CHANGES IN THE ORGANS OF THE IMMUNE SYSTEM UNDER THE INFLUENCE OF XENOBIOTICS.

Nowadays scientific achievements in various areas of lives have caused the creation of more and more «foreign body substances¼ known as xenobiotics. As it is widely accepted that human health is a product of both genetics and the environment; and premise that also holds true for the immune system with unclear morphogenetic aspect, so we selected the purpose of our work as detection of ultrastructural changes in the spleen and thymus under the influence of tryglycidyl ether of polyoxypropylenetriol (TEPPT) and propylene glycol (PP). Subacute experiment has been performed on the matured male rat's with administration of 1/10 LD50 and 1/100 LD50 of TEPPT and PP during 7 days, 15 days, 30 days and 45 days. Obtained materials of spleen and thymus have been investigated with ultramicroscopic and histological examination. Detection of cellular density has been performed. On the base of obtained results we can conclude that structure of spleen and thymus is susceptible to influence of TEPPT and PP. Ultrastructural changes in those organs of the immune system are characterized by margination of chromatin in nuclei, appearance of pronounced invaginations of karyolemma till fragmentation of nuclei; condensed, wrinkled cytoplasm, dilatation of mitochondria, vacuolization of cytoplasm. Such changes are manifestation of hydropic dystrophy and apoptosis development with resulting in reducing of cellular density in 45 days more pronounced under TEPPT influence with 1/10 LD50 dose: in mantle zone of spleen follicle from 171.1±4.1to 123.7±10.8 cells/104 µm2, in marginal zone of spleen follicle from 104.6±3.8 to 79.4±9.7, in cortical zone of thymus from 180.1±3.9 to 128.3±9.1, in medullar zone of thymus from 137.4±3.7 to 98.6±8.3.

MICROSCOPIC FEATURES OF THE SPLEEN UNDER THE INFLUENCE OF LAPROXIDES.

Rapid technology growth and its implementation in all spheres of the people's lives dictates the necessity for thorough study of the influence of different chemicals on human's health. This study was undertaken to elucidate the structural changes that occur in the matured rats' spleen experimentally induced by selected xenobiotic, so, purpose of our work was detection of microscopic peculiarities of the spleen under the influence of laproxides. In subacute experiment were uncovered organometric alterations of the matured male rat's spleen after the administration of 1/10 LD50 of polyether-tryglycidyl ether of polyoxypropylene triol (TEPPT). The study was performed on 72 outbreed WAG male matured rats with the weight 200±10g. Histological slides were studied with performing morphometric and statistical methods. We revealed changes of morphologiс data in comparison to control data which shows reactivity of the spleen in response to the induced xenobiotic. The received and analyzed data demonstrate the morphological changes of the spleen, specifically changes of the linear dimensions and weight of the spleen due to the influence of the TEPPT. The spleen is very sensitive to the effects of xenobiotics, in particular, TEPPT that is even reflected in its grossly (weight and linear dimensions) and histological features (reliable changes of the of the white pulp area of the spleen from 17.87±1.04% to 27.37±1.71%, diameter of lymphatic follicles from 426.59±11.18 µm to 382.31±11.73 µm, width of the mantle zone from 45.73±1.08 µm to 37.18±2.29 µm, width of the marginal zone from 81.32±1.79 µm to 74.63±2.08 µm, width of the periarterial zone from 88.73±2.69 µm to 97.24±2.61 µm).

Telocytes: a potential defender in the spleen of Npc1 mutant mice.

Niemann-Pick disease, type C1 (Npc1), is an atypical lysosomal storage disorder caused by autosomal recessive inheritance of mutations in Npc1 gene. In the Npc1 mutant mice (Npc1-/- ), the initial manifestation is enlarged spleen, concomitant with free cholesterol accumulation. Telocytes (TCs), a novel type of interstitial cell, exist in a variety of tissues including spleen, presumably thought to be involved in many biological processes such as nursing stem cells and recruiting inflammatory cells. In this study, we found that the spleen is significantly enlarged in Npc1-/- mice, and the results from transmission electron microscopy examination and immunostaining using three different TCs markers, c-Kit, CD34 and Vimentin revealed significantly increased splenic TCs in Npc1-/- mice. Furthermore, hematopoietic stem cells and macrophages were also elevated in Npc1-/- spleen. Taken together, our data indicate that splenic TCs might alleviate the progress of splenic malfunction via recruiting hematopoietic stem cells and macrophages.

P-Selectin Sustains Extramedullary Hematopoiesis in the Gata1 low Model of Myelofibrosis.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contributing to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakaryocytes (MK). These MK express high levels of P-selectin (P-sel) that, by triggering neutrophil emperipolesis, may cause TGF-ß release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1(low) mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-sel(null) Gata1(low) mice survived splenectomy and lived 3 months longer than P-sel(WT) Gata1(low) littermates and expressed limited fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-ß content, and corrected the HSC distribution that in Gata1(low) mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-ß reduced P-sel expression in MK and corrected HSC distribution. Spleens, but not marrow, of Gata1(low) mice contained numerous cKIT(pos) activated fibrocytes, probably of dendritic cell origin, whose membrane protrusions interacted with MK establishing niches hosting immature cKIT(pos) hematopoietic cells. These activated fibrocytes were not detected in spleens from P-sel(null) Gata1(low) or TGF-ß-inhibited Gata1(low) littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore, in Gata1(low) mice, and possibly in PMF, abnormal P-sel expression in MK may mediate the pathological cell interactions that increase TGF-ß content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen.

Sub-chronic inhalation of lead oxide nanoparticles revealed their broad distribution and tissue-specific subcellular localization in target organs.

BACKGROUND: Lead is well known environmental pollutant, which can cause toxic effects in multiple organ systems. However, the influence of lead oxide nanoparticles, frequently emitted to the environment by high temperature technological processes, is still concealed. Therefore, we investigate lead oxide nanoparticle distribution through the body upon their entry into lungs and determine the microscopic and ultramicroscopic changes caused by the nanoparticles in primary and secondary target organs. METHODS: Adult female mice (ICR strain) were continuously exposed to lead oxide nanoparticles (PbO-NPs) with an average concentration approximately 106 particles/cm3 for 6 weeks (24 h/day, 7 days/week). At the end of the exposure period, lung, brain, liver, kidney, spleen, and blood were collected for chemical, histological, immunohistochemical and electron microscopic analyses. RESULTS: Lead content was found to be the highest in the kidney and lungs, followed by the liver and spleen; the smallest content of lead was found in brain. Nanoparticles were located in all analysed tissues and their highest number was found in the lung and liver. Kidney, spleen and brain contained lower number of nanoparticles, being about the same in all three organs. Lungs of animals exposed to lead oxide nanoparticles exhibited hyperaemia, small areas of atelectasis, alveolar emphysema, focal acute catarrhal bronchiolitis and also haemostasis with presence of siderophages in some animals. Nanoparticles were located in phagosomes or formed clusters within cytoplasmic vesicles. In the liver, lead oxide nanoparticle exposure caused hepatic remodeling with enlargement and hydropic degeneration of hepatocytes, centrilobular hypertrophy of hepatocytes with karyomegaly, areas of hepatic necrosis, occasional periportal inflammation, and extensive accumulation of lipid droplets. Nanoparticles were accumulated within mitochondria and peroxisomes forming aggregates enveloped by an electron-dense mitochondrial matrix. Only in some kidney samples, we observed areas of inflammatory infiltrates around renal corpuscles, tubules or vessels in the cortex. Lead oxide nanoparticles were dispersed in the cytoplasm, but not within cell organelles. There were no significant morphological changes in the spleen as a secondary target organ. Thus, pathological changes correlated with the amount of nanoparticles found in cells rather than with the concentration of lead in a given organ. CONCLUSIONS: Sub-chronic exposure to lead oxide nanoparticles has profound negative effects at both cellular and tissue levels. Notably, the fate and arrangement of lead oxide nanoparticles were dependent on the type of organs.

Magnetic mapping of iron in rodent spleen.

Evaluation of iron distribution and density in biological tissues is important to understand the pathogenesis of a variety of diseases and the fate of exogenously administered iron-based carriers and contrast agents. Iron distribution in tissues is typically characterized via histochemical (Perl's) stains or immunohistochemistry for ferritin, the major iron storage protein. A more accurate mapping of iron can be achieved via ultrastructural transmission electron microscopy (TEM) based techniques, which involve stringent sample preparation conditions. In this study, we elucidate the capability of magnetic force microscopy (MFM) as a label-free technique to map iron at the nanoscale level in rodent spleen tissue. We complemented and compared our MFM results with those obtained using Perl's staining and TEM. Our results show how MFM mapping corresponded to sizes of iron-rich lysosomes at a resolution comparable to that of TEM. In addition MFM is compatible with tissue sections commonly prepared for routine histology.

Ultrastructural and biomolecular detection of Rickettsiales-like organisms in tissues of rainbow trout with Red Mark Syndrome.

Red mark syndrome (RMS) and US strawberry disease (US SD) are skin disorders affecting rainbow trout farmed in Europe and USA. The disease etiology has not yet been established. In spite of specific investigations, identifying Rickettsia-like organism (RLO)- and Midichloria-like organism (MLO)-related DNA in affected individuals, these pathogens have never been observed. We performed histological, ultrastructural and biomolecular analysis on skin and spleen samples of trout with RMS. Examination by TEM revealed the presence of intracytoplasmic microorganisms resembling Rickettsiales within macrophages, fibroblasts and erythrocytes. The microorganisms were oval or short rod shaped (400-800 nm in length and 100-200 nm in width) and often showed a cell wall similar to Gram-negative bacteria. PCR analysis for Rickettsiales supported these findings: 53% of affected trout were positive by both PCR and TEM The primers RiFCfw-RiFCrev were used to anneal both the RLO 16S DNA sequence and the MLO 16S DNA sequence. For this reason, and in agreement with previous studies confirming the presence of Rickettsiales-related DNA in trout with RMS, we assume that TEM detected microorganisms morphologically consistent with bacteria belonging to Rickettsiales order and could be considered as possible causative agents of RMS.

Systemic Amyloidosis Model on Young Mice.

Subcutaneous daily injection (with neglect of aseptics) of 0.5 ml solution of soybean cream substitute (10% volume in distilled water) during 30 days caused systemic amyloidosis in 30-day-old mice. All the known methods for induction of systemic amyloidosis are based on the use of old animals, as senile tissue bradytrophy allows effective simulation of amyloidosis.

Identification of Langerhans-like cells in the immunocompetent tissues of channel catfish, Ictalurus punctatus.

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) that have a critical role in bridging innate and adaptive immune responses in vertebrates. Dendritic cells have been characterized morphologically and functionally in the teleost fish models such as rainbow trout, salmonids, medaka, and zebrafish. The presence of DCs with remarkable similarities to human Langerhans cells (LCs) has been described in the spleen and anterior kidney of salmonids and rainbow trout. However, there is no evidence of the presence of DCs and their role in channel catfish immunity. In this study, we assessed DC-like cells in the immunocompetent tissues of channel catfish by immunohistochemistry (IHC), flow cytometry and transmission electron microscopy (TEM). We identified Langerin/CD207+ (L/CD207+) cells in the channel catfish anterior kidney, spleen and gill by IHC. Moreover, we described the cells that resembled mammal LC DCs containing Birbeck-like (BL) granules in channel catfish spleen, anterior and posterior kidneys and gill by TEM. Our data suggest that cells with DC-like morphology in the immune related organs of catfish may share morphological and functional properties with previously reported DCs in teleost fish and mammals. More detailed knowledge of the phenotype and the function of catfish DCs will not only help gain insight into the evolution of the vertebrate adaptive immune system but will also provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use.

Follicular B cells in thyroids of mice with spontaneous autoimmune thyroiditis contribute to disease pathogenesis and are targets of anti-CD20 antibody therapy.

B cells are required for development of spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice where they function as important APCs for activation of CD4(+) T cells. Depletion of B cells using anti-CD20 effectively inhibits SAT development. The goals of this study were to characterize the B cells that migrate to thyroids in SAT, and to determine whether anti-CD20 effectively targets those B cells in mice with established SAT. The results showed that most thyroid-infiltrating B cells in mice with SAT are follicular (FO) B cells. Expression of CD80, CD86, and CD40 was significantly increased on FO, but not marginal zone, splenic B cells after SAT development. Thyroid-infiltrating and peripheral blood B cells had lower expresion of CD20 and CD24 compared with splenic and lymph node FO B cells. Despite reduced CD20 expression, anti-CD20 depleted most B cells in thyroids of mice with established SAT within 3 d. B cell depletion in thyroids of mice given anti-CD20 was more complete and longer lasting than in spleen and lymph nodes and was comparable to that in blood. Circulation of B cells was required for effective and rapid removal of B cells in thyroids because preventing lymphocyte egress by administration of FTY720 abrogated the effects of anti-CD20 on thyroid B cells. Therefore, the FO subset of B cells preferentially contributes to SAT development and persistence, and anti-CD20 targeting of FO B cells effectively eliminates B cells in the target organ even though thyroid B cells have decreased CD20 expression.