Spleen Sections Research Articles

Structure, organization and expression of common carp ( Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.

Immunohistochemical Detection of Brucella Melitensis Antigens in Cases of Naturally Occurring Abortions in Sheep

Brucella melitensis, a worldwide zoonotic pathogen, is a significant cause of abortion in sheep and goats in some countries. The present study was carried out to determine, by immunohistochemistry, the presence of B. melitensis antigens in 110 naturally occurring aborted sheep fetuses. Sections of lung, liver, kidney, and spleen of each fetus were stained with immunoperoxidase to detect Brucella antigens. Brucella melitensis antigens were detected in 33 of 110 fetuses (30%). In the 33 positive cases, Brucella antigens were found in lung (25 [22.7%]), liver (21 [19%]), spleen (13 [11.8%]), and kidney (6 [5.4%]). Microscopic studies demonstrated that Brucella antigens were mainly located in the cytoplasm of macrophages and neutrophils of the lung, and in the cytoplasm of macrophages in the portal infiltrates and Kupffer cells of the liver. It was concluded that immunohistochemistry in formalin-fixed, paraffin-embedded tissues is a useful tool for the diagnosis of spontaneous ovine abortion caused by B. melitensis.

Chagas disease in extremely very low birth weight: Vertical transmission

This study evaluates the effect of Trypanosoma cruzi biotherapy 17dH (BIOT) on mice of different ages, infected with the protozoa concerned.Method: Performing a blind, controlled, randomized by drawing experiment, 110 animals four or eight-week-old, Swiss, male mice were divided into infected control treated hydroalcoholic 7% (CI-4 = 34 or CI-8 = 21 animals) and infected control treated with biotherapy 17dH–0.2 mL/animal/20 consecutive days/oral regimen (BIOT-4 = 33 or BIOT-8 = 21 animals). Animals were inoculated intraperitoneally with 1400 trypomastigote, T. cruzi Y-strain. Parasitological, immunological and histopathologic parameters were evaluated statistically, using Statistica-8.0 and R 3.0.2 program to analysis of survival. The study was approved by the Ethics Committee for Animal Experimentation/UEM.Results: Four-week-old mice showed no statistical difference in parasitemia (P = 0.5718) between the treated and control group. Eight-week-old mice from the treated group had a higher parasite peak (P = 0.0424) and higher parasitemia (P < 0.005) than the control. To both groups of 4 and 8 weeks of age, treated or untreated, survival of mice was higher in the treated group than in the control, although it was not statistically significant (p-value = 0.32, 0.55 respectively). Four-week-old mice displayed a spleen section with a number of amastigote nests significantly higher in BIOT-4 than CI-4 (P = 0.01). In eight-week-old mice the number of amastigote nests (P < 0.001) and inflammatory foci (P < 0.06–10% significance) in the liver section were smaller in BIOT-8 than CI-8. Spleen giant cells were significantly higher in CI-8 than in BIOT-8 (P < 0.01). Eight-week-old animals treated with biotherapy showed higher parasitemia and lower tissue parasitism. Opposite pattern was observed in four-week-old animals.Conclusion: There is a difference of high diluted medication effect in four and eight-week-old mice. In the group of animals 8 weeks the immunomodulatory effect seems to have been higher. Hence, treatment with the medicine produced from T. cruzi modulates the inflammatory response with increased apoptosis and decreased serum levels of TGF-β.

Novel monoclonal antibodies recognizing different subsets of lymphocytes from the common marmoset ( Callithrix jacchus)

Callithrix jacchus, the common marmoset, is a small new world primate that is considered effective as an experimental animal model for various human diseases. In this study, we generated monoclonal antibodies (mAbs) against common marmoset lymphocytes for immunological studies on the common marmoset. We established five hybridoma clones, 6C9, 10D7, 6F10, 7A4 and 5A1, producing anti-marmoset mAbs against cell surface antigens on marmoset T and/or B lymphocytes. We confirmed that 6C9 and 10D7 antibodies recognized CD45 antigen, and 6F10 antibody recognized CD8 antigen by flow cytometry using marmoset cDNA transfectants. We also tested them for application of immunoprecipitation, Western blot analysis and immunohistochemistry. We found that immunohistochemistry using marmoset spleen sections could be applied with all established mAbs but immunoprecipitation and the Western blot analysis could be applied with 6F10 and 10D7 antibodies but not with the other three mAbs. These results show that these monoclonal antibodies are useful for advancing immunological research on the common marmoset.

AA-Amyloidosis Can Be Transferred by Peripheral Blood Monocytes

Spongiform encephalopathies have been reported to be transmitted by blood transfusion even prior to the clinical onset. Experimental AA-amyloidosis shows similarities with prion disease and amyloid-containing organ-extracts can prime a recipient for the disease. In this systemic form of amyloidosis N-terminal fragments of the acute-phase reactant apolipoprotein serum amyloid A are the main amyloid protein. Initial amyloid deposits appear in the perifollicular region of the spleen, followed by deposits in the liver. We used the established murine model and induced AA-amyloidosis in NMRI mice by intravenous injections of purified amyloid fibrils (‘amyloid enhancing factor’) combined with inflammatory challenge (silver nitrate subcutaneously). Blood plasma and peripheral blood monocytes were isolated, sonicated and re-injected into new recipients followed by an inflammatory challenge during a three week period. When the animals were sacrificed presence of amyloid was analyzed in spleen sections after Congo red staining. Our result shows that some of the peripheral blood monocytes, isolated from animals with detectable amyloid, contained amyloid-seed that primed for AA-amyloid. The seeding material seems to have been phagocytosed by the cells since the AA-precursor (SAA1) was found not be expressed by the monocytes. Plasma recovered from mice with AA amyloidosis lacked seeding capacity. Amyloid enhancing activity can reside in monocytes recovered from mice with AA-amyloidosis and in a prion-like way trigger amyloid formation in conjunction with an inflammatory disorder. Human AA-amyloidosis resembles the murine form and every individual is expected to be exposed to conditions that initiate production of the acute-phase reactant. The monocyte-transfer mechanism should be eligible for the human disease and we point out blood transfusion as a putative route for transfer of amyloidosis.

Rituximab-Induced Changes in Hematolymphoid Tissues Found at Autopsy

The effect of rituximab on malignant B cells and normal circulating B cells has been previously studied. In contrast, data on the degree of depletion of nonneoplastic B cells induced by rituximab in lymph nodes and spleen is limited. For this purpose, clinical charts, autopsy records, lymph node and spleen sections, and immunoperoxidase stains were reviewed from 10 patients who had received 1 to 40 doses of rituximab before death. The percentage of nonneoplastic B cells was lower in the lymph node and spleen in rituximab-treated patients when compared with cyclophosphamide, doxorubicin, vincristine, and prednisone-treated patients and patients without lymphoma. The effect of rituximab on nonneoplastic B cells was observed as soon as 1 month after administration and with as few as 3 doses. Reappearance of normal numbers of B cells was not observed 1 to 12 months after the last dose of rituximab was administered. We conclude that rituximab induces prompt, consistent, profound, and prolonged depletion of B lymphocyte populations in human lymphoid tissue.

Macrophages contribute to the cellular uptake of von Willebrand factor and factor VIII in vivo

AbstractVon Willebrand factor (VWF) and factor VIII (FVIII) circulate in a tight noncovalent complex. At present, the cells that contribute to the removal of FVIII and VWF are of unknown identity. Here, we analyzed spleen and liver tissue sections of VWF-deficient mice infused with recombinant VWF or recombinant FVIII. This analysis revealed that both proteins were targeted to cells of macrophage origin. When applied as a complex, both proteins were codirected to the same macrophages. Chemical inactivation of macrophages using gadolinium chloride resulted in doubling of endogenous FVIII levels in VWF-null mice, and of VWF levels in wild-type mice. Moreover, the survival of infused VWF was prolonged almost 2-fold in VWF-deficient mice after gadolinium chloride treatment. VWF and FVIII also bound to primary human macrophages in in vitro tests. In addition, radiolabeled VWF bound to human THP1 macrophages in a dose-dependent, specific, and saturable manner (half-maximal binding at 0.014 mg/mL). Binding to macrophages was followed by a rapid uptake and subsequent degradation of the internalized protein. This process was also visualized using a VWF–green fluorescent protein fusion protein. In conclusion, our data strongly indicate that macrophages play a prominent role in the clearance of the VWF/FVIII complex.

The effect of feeding a diet naturally contaminated with deoxynivalenol (DON) and zearalenone (ZON) on the spleen and liver of sow and fetus from day 35 to 70 of gestation

Pregnant sows were fed a control diet (CON, 0.15 mg deoxynivalenol (DON) and 0.0035 mg zearalenone (ZON) per kg diet) or diet containing 15% of Fusarium toxin contaminated triticale (MYCO, 4.42 mg DON and 0.048 mg ZON per kg diet) during days 35–70 of gestation. All sows were fed in a restricted feeding regimen with the same amount of feed (2000 g/d) over the whole study. At the end of the experiment, fetuses were delivered by Caesarian section and samples of spleen and liver of euthanized sows and fetuses were analyzed. At terminal necropsy, no macroscopic lesion was observed in any organ of either sows or fetuses. The histopathological data indicated significant alteration only in elevated iron staining in the red pulp of spleens in sows of MYCO group after 35 days of feeding. The presence of hemosiderin particles in the spleen sections was confirmed by transmission electron microscopical investigation and by an enhanced Fe 2+ concentration in spleen. A glycogen increase ( p < 0.05) was found in liver cells of fetuses in the experimental group. Together, the results provide evidence of spleen dysfunction (hemosiderosis) in sows fed a Fusarium toxin-contaminated wheat, however, with absence of clinical signs. Enhanced glycogen and an impairment of mitochondria in liver of fetuses was present when their mothers consumed the MYCO diet.

Development of monoclonal antibodies to rohu [ Labeo rohita] immunoglobulins for use in immunoassays

Serum immunoglobulins [Ig] of rohu [ Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [ Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [±SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [±2.34] and 51.84% [±2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [±1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.

An Unusual Case of Severe Hypertriglyceridemia and Splenomegaly

A 49-year-old man of Japanese and British ancestry was referred to a metabolic diseases clinic for evaluation 5 months after nontraumatic spleen rupture requiring splenectomy. Prior history included hypertension and mild frontal headaches, but no other neurological or cardiovascular symptoms. The patient did not smoke and used alcohol infrequently. His mother had coronary artery disease, and his father had mild hypertension. There was no family history of consanguinity, splenomegaly, diabetes, or developmental delay. The ruptured spleen weighed 727 g, and splenomegaly was associated with marked sinus histiocytosis spreading apart the lymphoid component. The overwhelming majority of histiocytes were foamy (Fig. 1a⇓ ), and only a few had sea-blue appearance and reacted positively with periodic acid-Schiff (PAS), PAS and diastase, and May-Giemsa stains. A lipid storage disorder was suspected, but the histiocytes did not have the cytoplasmic linearity appearance of Gaucher cells and were otherwise nonspecific. Before splenic rupture, the patient’s lipoprotein profile was reported as being normal, with no past recorded triglyceride measurement exceeding 2 mmol/L. Figure 1. Pathology and genomic studies of the index patient. (a), Hematoxylin and eosin–stained section (40×) of spleen, showing expanded white pulp displacing Malpighian bodies. The pallor is due to the abundance of foamy histiocytes in the sinusoids, seen in inset (400X). (b), LightCycler (Roche Diagnostics) analysis of genomic DNA with curve C representing the patient’s APOE melting curve at codon 158, showing heterozygosity at codon 158 for 1 TGC trinucleotide (cysteine) with Tm of 56.4 °C and 1 indeterminate trinucleotide coding sequence with a melting temperature of 62.99 °C, which was outside the Cys158 acceptable range of 63.5–68.5 °C, indicating another amino acid encoded by this residue. Melting curves for normal E2/E4 (A) and E2/E3 (B) controls are shown for reference. (c), Direct sequencing of the patient’s plasmid cloned APOE alleles showing inframe deletion …

Exacerbation of DSS induced intestinal inflammation following inoculation of BALB/c mice with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (M. ptb) DNA has been detected in lesions of human patients with Crohn's disease, a distinct clinical and histologic form of inflammatory bowel disease. However, the significance of this observation remains unresolved. The purpose of the following study was to determine if infection of mice with M. ptb would influence the development of DSS induced intestinal inflammation. BALB/c mice were inoculated intraperitoneally with 1x109 CFU of either live or heat‐killed (HK) M. ptb or saline. At 4–7 months post inoculation, the mice were administered dextran sulfate sodium (DSS) at a suboptimal concentration of 2% in the drinking water for 6 days. After addition of DSS to the drinking water, mouse weights were recorded daily. When the mice were euthanized, sections of small intestine, colon, cecum, liver, and spleen, were collected for histopathology, M. ptb isolation, acid‐fast staining, and for cytokine analysis via qRT‐PCR. Histologic lesion scores were generally more severe and the cecal lengths were generally shorter in the BALB/c mice inoculated with HK M. ptb relative to mice inoculated with live M. ptb or saline. Mice inoculated with either HK or live M. ptb expressed higher levels of IL‐6 and IL‐1 alpha. This data suggests that prior exposure of mice in this model system to M. ptb enhances the colitis that develops after acute exposure to DSS.This study was funded by an Iowa State University research grant.

Vascularized bone marrow transplant (VBMT) serves as a novel surgical approach to stem cell transplantation and stimulates normal repopulation of lymphocytes in the spleens of irradiated (XRT) rat hosts

Our lab demonstrated that VBMT, the microsurgical transplantation of a femur with its vascular pedicle, can induce immune tolerance across a semi‐allogeneic barrier without graft versus host‐disease. We hypothesized that VBMT alone is potentially superior to cellular stem cell transplantation and sufficient to induce normal repopulation of the spleens of XRT rat recipients.Lewis Brown Norway rats received 9 Gy radiation. Rats then received either a conventional cellular (cBMT, n=5) or VBMT (n=4) bone marrow transplant from Lewis donors. Control groups were: non‐transplanted, non‐irradiated (CT, n= 5) or XRT only (n= 4). Stained histologic sections of spleens were analyzed with digital image analysis (DIA).XRT rats died 9±1 days post irradiation. Other groups were sacrificed: cBMT at 110 days; VBMT at 102 ±1 days. Compared to CT, XRT spleens showed white pulp atrophy; cBMT had less white pulp but appeared hypercellular and VBMT spleens appeared normal. DIA results can be seen in the table below:VBMT is capable of reconstituting spleens of XRT semi‐allogeneic recipients similar in appearance to normal rats. This model has clinical relevance for both stem cell transplantation and solid organ transplantation, in that VBMT induces chimerism and tolerance.

Biomedical effect of tissue contact with metallic material used for body piercing modified by DLC coatings

Body piercing is one of the most popular methods to decorate the body at present. All the time this metallic material is in contact with body fluids and living tissues. It is well documented that carbon coatings were examined and pointed as safe and biocompatible. In this connection the jewellery made of 316L steel was covered by diamond-like coatings using two methods of plasma activated chemical vapour deposition (PACVD). The biomedical effect of tissue contact with metallic material commonly used for body piercing modified by DLC coating was investigated in this paper. In vivo short- and long-term investigations were carried out on WISTAR-herd male rats. Modified samples were implanted subcutaneously for 24 h, 1, 4 and 26 weeks. After that time the animals were euthanized and the modified jewellery, surrounding tissue and spleen were harvested. Evaluation of two routine stained sections of tissues and spleen revealed that: 1) carbon coating protects tissues surrounding the metallic implants against metal ion penetrations, 2) immune reaction of tissue and spleen doesn't occur while the non-coated jewellery caused strong reaction, 3) statistically significant differences ( p < 0.05) are in the numbers and types of cellular components and degree of reaction with respect to modified and non-modified jewellery.

Splenic red pulp lymphoma with numerous basophilic villous lymphocytes: a distinct clinicopathologic and molecular entity?

The presence of circulating villous lymphocytes (VLs) in lymphoma patients usually points to splenic marginal zone B-cell lymphoma (SMZL), even if the VLs can be found occasionally in other small B-cell lymphomas. However, those cells are variably described, and detailed cytologic characterization is often lacking. We identified lymphoma cases with numerous basophilic VLs among the large group of splenic lymphoma with VLs, and for further delineation, 37 cases with this particular cytology were analyzed. Patients, predominantly older men, presented with moderate lymphocytosis and splenomegaly without pancytopenia. The monoclonal B cells expressed IgM + D, IgM + G, IgM or IgG, as well as CD76 and CD11c, frequently CD103, and rarely CD123. Spleen sections were peculiar, with atrophic white pulp and a monomorphic diffuse lymphoma infiltration in a congested red pulp. Bone marrow infiltration was interstitial and intrasinusoidal without extensive fibrosis. Cytogenetic analysis showed a frequent absence of clonal aberrations (68%). Most cases (79%) were IgH mutated, with an overrepresentation of V(H)3 and V(H)4 gene families. These results, as well as the clinical evolution, show that those lymphoma cases represent a homogeneous group distinct from SMZL and reminiscent of hairy cell leukemia variant, perhaps corresponding to a separate lymphoma entity.

Anti-toxoplasmosis Effects of Oleuropein Isolated from Fraxinus rhychophylla

Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii, with very few therapeutic treatment options. Typically, the choices for treatment are pyrimethamine and sulfadiazine, however their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound oleuropein isolated from Fraxinus rhynchophylla showed anti-T. gondii effects in vitro and in vivo. In Madin-Darby bovine kidney cells, the selectivity of oleuropein was 8.9, which was higher than sulfadiazine and pyrimethamine (3.8 and 2.5, respectively). In infected mice, the inhibition ratio of T. gondii in the peritoneal cavity was 55.4% compared to the negative control group after treatment with 300 mg/kg oleuropein. In addition, inhibitory effects on granuloma, apoptosis, necrosis and cyst-formation were shown in sections of spleen and liver. Oleuropein is therefore a potentially useful anti-T. gondii candidate for clinical application.

Histological, Histochemical and Morphometric Changes of Splenic Melanomacrophage Centers (Smmcs) in Sparicotyle-Infected Cultured Sea Breams (Sparus aurata)

Diseases caused by parasites are much more frequently described in cultured fish, which suffer from artificial conditions and numerous stress factors. This study investigates the histological, histochemical and morphometric modifications of splenic melanomacrophage centers (SMMCs) infected by Sparicotyle chrisophrii (Monogenea, ectoparasite of the gills) in sea breams (Sparus aurata), cultured in floating cages in the Gulf of Gaeta (Italy). Infected fish swam near the water surface, showing severe signs of anemia. Several spleens were collected from both healthy and dead fish (70–100 gr. body weight). A spleen histopathology was evaluated by using traditional stainings, such as Haematoxylin and Eosin (HE), Periodic Acid-Schiff reaction (PAS), Perl's reaction for haemosiderin and Schmorl's reaction for lipofuscins. Furthermore, SMMCs morphometry was performed on PAS-stained sections to study 7 morphometric parameters [Mean SMMCs profile area (MPA), Mean SMMCs maximum diameter (Media), Mean SMMCs minimum diameter (media), Mean SMMCs diameter (Dia), Mean SMMCs Perimeter (P), Mean SMMCs Form Factor (FF) and Mean SMMCs number per square millimeter of spleen tissue (MN)]. A light microscope of HE stained sections of spleen revealed a dramatic increase in the size and number of SMMCs in parasitized animals. Morphometric data illustrated statistically significant differences (p < 0.01) of all studied parameters between healthy and diseased fish. This study emphasizes the importance of using histopathological investigations to unravel the complex biological host/parasite interaction, which results in systemic lesions affecting reared marine species.

Accelerated Proliferation and Limited Differentiation, Mediated through Jak2, Exacerbate Ineffective Erythropoiesis in β-Thalassemia.

Ineffective erythropoiesis (IE) in β-thalassemia has been attributed to erythroid cell death mediated by apoptosis or hemolysis during the maturation process. Historically, ferrokinetic studies in this disease suggested that 60%–80% of erythroid precursors die in the marrow or extramedullary sites. However, several observations have challenged this view. First, the number of apoptotic erythroid cells in patients is low compared to net expansion of the erythroid cell pool. Second, hemolytic markers in β-thalassemic patients are normal or only slightly increased, unless additional pathological conditions appear. Third, our most recent study (Blood, Gardenghi et al, 2007 Jun 1) demonstrated that GI iron absorption in β-thalassemia is increased by the dysregulation of genes such as hepcidin and ferroportin that control iron absorption, resulting in iron levels that exceed the amount required for erythropoiesis. We have undertaken a detailed investigation using cohorts of mice (n>30 per genotype) with β-thalassemia intermedia (th3/+) and major (th3/th3). Using these models, we have previously shown that the severity of anemia (as low as 1 g/dL) inversely correlates with the total number of nucleated erythroid cells (»100 fold compared to wild-type (wt) mice). Cytological analysis has clearly shown that thalassemic spleen specimens were comprised of a homogeneous pre-erythroblastic population. In contrast, the percentage of apoptotic cells and the level of hemolytic markers, such as bilirubin and lactic acid dehydrogenase, slightly increased or were not different compared to wt mice. While not excluding a role for apoptosis, our observations suggest that control of the cell cycle and maturation of erythroid precursors play an important role in IE. We then explored whether the erythroid cell cycle was dysregulated in our model system. We found that erythropoietin (Epo) levels were raised in thalassemic animals by as much as three orders of magnitude. Binding of Epo to its receptor (EpoR), activates antiapoptotic and cell cycle promoting genes, through activation of Jak2 and Stat5. By Western blot we demonstrated up-regulation of EpoR, Stat5 and the antiapoptotic protein BclXL, as well as that of proliferation promoting genes, such as CycA and Cdk2, in purified thalassemic erythroid cells compared to those of wt animals. This data was confirmed by staining both wt and thalassemic liver and spleen sections using the proliferation markers Ki67 and Mcm3, by clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after BrdU injection. In the latter case, we observed 22%, 30% and 44% BrdU+ cells from wt, th3/+ and th3/th3 mice, respectively. In addition, freshly purified thalassemic erythroid cells proliferate faster in vitro than normal cells, a phenomenon blocked by AG490, a Jak2 inhibitor. Significantly, we have been able to reproduce results from our animal studies in humans, comparing normal and thalassemic blood and spleen specimens. In conclusion, we propose that IE in β-thalassemia is likely to be the result of altered cell proliferation and impaired cell differentiation, which in turn limit apoptosis, thereby mimicking tumor-like behavior.

Human Herpesvirus 8 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia.

Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi's sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those tumors and the expression of K1 alone causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NFκ-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer's patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4±1% of splenocytes from K1 mice versus 17±2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11±0.6% vs. 50±6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 - induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.

Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins

Pannexins are mammalian orthologs of the invertebrate gap junction proteins innexins and thus have been proposed to play a role in gap junctional intercellular communication. Localization of exogenously expressed pannexin 1 (Panx1) and pannexin 3 (Panx3), together with pharmacological studies, revealed a cell surface distribution profile and life cycle dynamics that were distinct from connexin 43 (Cx43, encoded by Gja1). Furthermore, N-glycosidase treatment showed that both Panx1 (approximately 41-48 kD species) and Panx3 (approximately 43 kD) were glycosylated, whereas N-linked glycosylation-defective mutants exhibited a decreased ability to be transported to the cell surface. Tissue surveys revealed the expression of Panx1 in several murine tissues--including in cartilage, skin, spleen and brain--whereas Panx3 expression was prevalent in skin and cartilage with a second higher-molecular-weight species present in a broad range of tissues. Tissue-specific localization patterns of Panx1 and Panx3 ranging from distinct cell surface clusters to intracellular profiles were revealed by immunostaining of skin and spleen sections. Finally, functional assays in cultured cells transiently expressing Panx1 and Panx3 were incapable of forming intercellular channels, but assembled into functional cell surface channels. Collectively, these studies show that Panx1 and Panx3 have many characteristics that are distinct from Cx43 and that these proteins probably play an important biological role as single membrane channels.

Amyloid-specific fluorophores for the rapid, sensitive in situ detection of prion contamination on surgical instruments

Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of rare, transmissible and fatal neurodegenerative diseases associated with the protein agent (PrP(Sc)). As such, the sensitive and rapid detection of prion PrP(Sc) amyloid on the surface of suspect surgical instruments is of great importance and may even allow remedial action to be taken prior to any further operative intervention and possible iatrogenic transmission. However, conventional PrP(Sc) detection methodologies tend to rely on the inefficient and unreliable removal of suspect material from a surface using swabs or wipes prior to antibody analysis. Here we show how the combination of an advanced light microscope technique, episcopic differential interference contrast/epifluorescence (EDIC/EF) microscopy, and the application of beta-amyloid fluorescent thiazole markers (thioflavin T, thioflavin S) can be used to detect, in situ, submicron (attomole) levels of prion protein amyloid contamination in brain and spleen sections, smears and homogenate on surgical stainless steel surfaces and surgical instruments. This technique, although not specific to an amyloid type, can be used to verify that surgical instruments are substantially free from prion amyloid protein soiling and hence reduce the risk of iatrogenic transmission.