Red Pulp Of Spleen Research Articles

Site-Specific Carboxypeptidase B1 Tyrosine Nitration and Pathophysiological Implications following Its Physical Association with Nitric Oxide Synthase-3 in Experimental Sepsis

LPS-induced sepsis results in oxidative modification and inactivation of carboxypeptidase B1 (CPB1). In this study, immunoprecipitated CPB1 was probed for tyrosine nitration using monoclonal nitrotyrosine-specific Abs in a murine model of LPS-induced sepsis. Tyrosine nitration of CPB1 was significantly reduced in the presence of NO synthase (NOS) inhibitors and the xanthine oxidase (XO) inhibitor allopurinol and in NOS-3 knockout (KO) mice. CPB1 tyrosine nitration and loss of activity by the concerted action of NOS-3 and XO were also confirmed in vitro using both the NO donor 3-morpholinosydnonimine and peroxynitrite. Liquid chromatography/tandem mass spectrometry data indicated five sites of tyrosine nitration in vitro including Tyr(248), the tyrosine at the catalytic site. The site- and protein-specific nitration of CPB1 and the possible high nitration yield to inactivate it were elucidated by confocal microscopy. The studies indicated that CPB1 colocalized with NOS-3 in the cytosol of sinus-lining cells in the red pulp of the spleen. Further analysis of CPB1-immunoprecipitated samples indicated immunoreactivity to a monoclonal NOS-3 Ab, suggesting protein complex formation with CPB1. XO and NOS inhibitors and NOS-3 KO mice injected with LPS had decreased levels of C5a in spleens of septic mice, indicating peroxynitrite as a possible cause for CPB1 functional alteration. Thus, CPB1 colocalization, coupling, and proximity to NOS-3 in the sinus-lining cells of spleen red pulp could explain the site-specific tyrosine nitration and inactivation of CPB1. These results open up new avenues for the investigation of several enzymes involved in inflammation and their site-specific oxidative modifications by protein-protein interactions as well as their role in sepsis.

Atipical immunophenotype in a littoral cell angioma

Littoral-cell angioma (LCA) is a recently described benign vascular tumor of the spleen, whose imaging and pathologic characteristics have been discussed only by a few authors. The tumor is characterized by a mixture of papillary and cystic areas lined by neoplastic cells deriving from normal splenic lining--littoral cells. The neoplastic LCA cells express both endothelial and histiocytic antigens associated with CD8 negativity, compared with the normal endothelium of the venous sinuses of the spleen red pulp that only expresses endothelial antigens and CD8 positivity. Therefore, the typical and characteristic immunohistochemical pattern of the LCA is as follows: CD31, CD68, CD163, CD21, FVIII antigen positive; CD34, CD8 negative. We reported a 60-year-old male with moderate nodular splenomegaly with one large hypoechogenic solid lesion and mild thrombocytopenia in whom the diagnosis of LCA was made after the elective splenectomy. Namely, histopathological and immunohistochemical data allowed a final diagnosis of classical LCA in spite of CD21 negativity. As far as we know this is the first reporeted CD21-negative LCA patient. Histological specimens were presented and differential diagnoses discussed. Littoral-cell angioma is a very rare benign splenic neoplasm that should be considered in the differential diagnosis of multinodular splenomegaly, particularly if the patient has the signs of hypersplenism.

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.

랫드를 이용한 Acetanilide의 반복투여 및 생식/발생독성 병행시험

The study was conducted to assess the repeated dose and reproduction and developmental toxicities of acetanilide, an intermediate for drug production, as a part of OECD Screening Information Data Set (SIDS) program. The test agent was administered by gavage at dose levels of 0, 22, 67, 200 and 600 mg/kg to Sprague-Dawley rats (12/group/sex) during pre-mating and mating period for males(up to 30 days) and females and pregnancy and early lactation period for females (up to 39-50 days). At 22 mg/kg, decreases in HGB, HCT (males) and MCHC (females), hyperplasia of spleen red pulp, hyperplasia of femur bone marrow (both sexes) were observed. At 67 mg/kg, salivation (males), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and MCHC (males), increases in MCV (males) and spleen weight (males), hyperplasia of spleen red pulp and femur bone marrow (both sexes) were observed. At 200 mg/kg, decreases in locomotor activity and salivation (both sexes), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and increases in MCV, MCH, BUN, T-BIL (males), enlargement of spleen (both sexes), increased weight of spleen (males), hyperplasia of spleen red pulp and femur bone marrow and extramedullary hematopoiesis of liver (both sexes), atrophy of thymus and corpus luteum hyperplasia of ovary (females) were observed. At 600 mg/kg, decreases in locomotor activity, cyanosis (both sexes), reddish tear, and salivation (males), mortality (4 out of 12 females), decreased body weight (females), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and MCHC and increases in WBC, MCV, MCH, reticulocyte, neutrophil, lymphocyte, monocyte, AST, ALT, BUN, T-BIL, ALB, Ca and A/G ratio (males), enlargement of spleen, increased weights of spleen (both sexes), liver (males), kidney and ovary, decreased weights of thymus (females), hyperplasia of spleen red pulp, hyperplasia of femur bone marrow and extramedullary hematopoiesis of liver (both sexes), and atrophy of thymus and corpus luteum hyperplasia of ovary (females) were observed. Regarding the reproduction and development toxicities, there were no treatment-related changes in precoital time, mating index, fertility index and pregnancy index at all doses tested. At 22 and 67 mg/kg, there were no adverse effects on all the parameters observed. At 200 mg/ kg, decreased body weight of pups (day 4 p.p.) were observed. At 600 mg/kg, decreased body weight of pups (day 0 and 4 p.p.) and viability index (day 4 p.p.), increased incidence of newborns dead or with abnormal clinical signs were observed. The results suggest that the NOAELs for general toxicity are < 22 mg/kg, LOAELs are 22 mg/kg and the NOAELs for reproductive toxicity are 67 mg/kg.

Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes >> heart > cerebrum > or = ileum > stomach = liver = jejunum > or = epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.

Murine Norovirus 1 Infection Is Associated with Histopathological Changes in Immunocompetent Hosts, but Clinical Disease Is Prevented by STAT1-Dependent Interferon Responses

Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease.

Autoimmune response in MRL+/+ mice following treatment with dichloroacetyl chloride or dichloroacetic anhydride

Dichloroacetyl chloride (DCAC) is formed from trichloroethene (TCE), which is implicated in inducing/accelerating autoimmune response. Due to its potent acylating activity, DCAC may convert proteins to neo-antigens and thus could induce autoimmune responses. Dichloroacetic anhydride (DCAA), which is a similar acylating agent, might also induce autoimmune responses. To evaluate if chloroacylation plays a role in the induction of autoimmunity, we have measured the autoimmune responses following treatment with DCAC or DCAA in autoimmune-prone MRL+/+ mice. Five-week-old female mice were injected intraperitoneally (twice weekly) with 0.2 mmol/kg of DCAC or DCAA in corn oil for 6 weeks. Total serum IgG, IgG1, and IgE levels were significantly increased in DCAC-treated mice as compared to controls. These increases corresponded with increases in DCAC-specific IgG and IgG1 levels. Total serum IgM was decreased in both DCAC- and DCAA-treated mice. Antinuclear antibodies, measured as an indication of systemic autoimmune responses, were increased in both DCAC- and DCAA-treated mice. Of eight Th1/Th2 cytokines measured in the serum, only IL-5 was significantly decreased in both treatment groups. The cytokine secretion patterns of splenic lymphocytes after stimulation with antibodies against CD3 (T cell receptor-mediated signal) and CD28 (costimulatory signal) differed between treatment and control groups. Levels of IL-1, IL-3, IL-6, IFN-gamma, G-CSF, and KC were higher in cultures of stimulated splenocytes from either DCAC- or DCAA-treated mice than from controls. The level of IL-17 was only increased in cultures from DCAC-treated mice. Increased lymphocytic populations were found in the red pulp of spleens following treatment with either DCAC or DCAA. In addition, thickening of the alveolar septa in the lungs of DCAC- or DCAA-treated mice was observed. The lung histopathology in exposed mice was consistent with the symptomology observed in welders exposed to DCAC/phosgene. Thickening was more pronounced in DCAC-treated mice. Our data suggest that DCAC and DCAA elicit autoimmune responses in MRL+/+ mice that might be reflective of their chloroacylation potential in vivo.

Effects of diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected immunological and histological measurements in the spleen of gilts1,2

Feeding experiments with diets containing Fusarium toxin-contaminated wheat were conducted to clarify the pathogenesis of immunological effects of Fusarium toxins to porcine spleen cells. Contaminated diets were fed to 36 Landrace prepubertal gilts for 35 d. Concentrations (as-fed basis) of the indicator toxins deoxynivalenol (DON) and zearalenone (ZON), respectively, were 210 and 4 (control--group I), 3,070 and 88 (group II), 6,100 and 235 (group III), and 9,570 and 358 microg/kg (group IV). No signs of hyperestrogenism or uterotrophic effects were observed because of dietary treatments. The feeding of mycotoxin-contaminated diets did not cause gross pathological findings in the spleens of animals. In vivo, no inhibitory effects were detected on concanavalin A-stimulation of blood lymphocytes; however, the proliferation rate of splenocytes was inhibited (P < 0.05) in pigs fed the diet with the highest DON/ZON concentration. With in vitro studies, lower proliferation rates of blood lymphocytes and splenocytes preexposed to DON were detected. Serum IgA concentrations of pigs in group II were increased (P < 0.05) compared with the baseline value before feeding the DON/ZON diet. The histopathological data indicated elevated (P < 0.05) iron staining in the red pulp of spleens in gilts from groups I to IV after 35 d of feeding. The presence of hemosiderin particles in the spleen sections was confirmed by transmission electron microscopic investigation. Together, the results provide evidence of spleen dysfunction (hemosiderosis) in the absence of clinical signs, especially in pigs fed higher concentrations (groups III and IV) of Fusarium toxin-contaminated wheat.

Identification of B and T cells in human spleen sections by infrared microspectroscopic imaging

Infrared spectroscopy probes the chemical composition and molecular structure of complex systems such as tissue and cells. Infrared spectroscopic imaging combines this spectral information with lateral resolution near the single-cell level. We analyzed whether this method is competitive with classic immunohistochemical methods for immunologic tissue and cells. We recorded infrared microspectroscopic mapping datasets with a 90- x 90-microm2 aperture from a 3- x 3-mm2 unstained tissue area of human spleen. A secondary follicle containing a germinal center and a T zone were studied in more detail by infrared microspectroscopic imaging with lateral resolution near 5 mum. The results were compared with consecutive sections stained by immunoglobulin D antibodies. T and B lymphocytes were extracted from human blood and served as independent test samples. Cluster analysis of infrared datasets produced images that distinguished anatomical features such as primary and secondary follicles, T zones, arteries, and spleen red pulp. The assignments could be confirmed in consecutive sections by immunohistochemical staining. Main spectral variances between T and B lymphocytes in high-resolution measurements were attributed to specific spectral contributions of DNA and cytosol. Sensitivity and specificity of the infrared based methods are comparable to those of standard staining procedures for identification of B and T cells. However, infrared spectroscopic imaging can offer advantages in velocity, data throughput, and standardization because of minimal sample preparation. The results emphasize the potential of infrared spectroscopy as an innovative tool for the distinction of cell types, in particular in immunologic tissue.

Soluble glucocorticoid-induced TNF receptor (sGITR) induces inflammation in mice.

Glucocorticoid-induced TNF receptor (GITR) was a new member of the TNF/nerve growth factor receptor (TNFR/ NGFR) family and induced in murine T cells by dexamathasone. Recombinant soluble GITR (sGITR) induced an inflammation in peritoneal membrane and changes in spleen after i.p. injection of 3 mg/kg in C57BL/6 mice. Spleen was enlarged and percentage of neutrophils and monocytes were increased. The area of red pulp in spleen was increased, while that of white pulp was decreased after GITR injection. The thickening of membrane and neutrophil infiltration was observed in peritoneal membrane with increased myeloperoxidase activity. At later time, neutrophil infiltration moved to inside the tissue with tissue damage. GITR ligand and GITR were expressed constitutively on the surface of spleen cells and cells from peritoneal fluid. In contrast, no significant change in the spleen and in peritoneal membrane was observed in mice treated with LPS. GITR may play a role in body's inflammatory processes.

Burn injury-mediated alterations in cell cycle progression in lymphoid organs of mice.

A key event in the cellular and molecular pathogenesis of multiple organ failure (MOF) after burn injury may be the change in profiles of the cell cycle progression in affected organs. We investigated the effects of burn injury on cell cycle progression in immune organs. Cell cycle analysis in the lymphoid tissues of mice after 18% burn injury revealed that S phase entry was temporarily arrested in the thymus 1 day after injury, whereas the spleen had substantially increased S phase entry at day 8. This mode of cell cycle regulation was reproduced in different age groups and strains of mice. Furthermore, the reactivity to the Ki-67 antibody (indicative of proliferation) was markedly reduced in the thymic cortex at day 1. There was a distinct pattern of hematopoietic foci formation and increased reactivities to the Ki-67 antibody in myelogenous cells in the red pulp of spleen at day 7, consistent with the elevated S phase entry. These data suggest that differential regulation of cell cycle progression may play a crucial role in the phenotypic changes in immune organs after burn injury.

Rat neonatal intestinal wall-free graft.

An experimental study was carried out in order to evaluate the regeneration capacity of the neonatal intestinal wall in ischaemia and its repercussion over organs of the immune system such as the spleen. We isolated a reservoir of small intestine in adult Sprague-Dawley rats that, after having been everted and placed at a subcutaneous level, was grafted with a free small intestine segment of neonatal Sprague-Dawley rats and analysed 3, 15 and 30 days after grafting. Samples obtained were stained with Martin's trichromic stain and studied at the light microscopic level. A total regeneration of the crypt architecture, formed by absorptive enterocytes, was observed in the interior of the reservoirs. A quantitative immunohistochemical study with monoclonal antibodies against CD4, CD8, MHC I and MHC II was carried out in the spleen of these animals. An additional immunohistochemical study was also performed in the small intestine reservoirs and spleen of transplanted animals using nitric oxide synthase (NOS) antibodies. Three days after transplantation NOS immunoreactive cells were observed in the reservoirs with transplanted neonatal small intestine. Antigenic stimulation produced, in the spleen red pulp of transplanted animals, a significant increase (P < 0.001) in the percentage of NOS, CD8 and MHC I immunoreactive cells in relation with values observed in control animals. These morphometric changes could be related with the stimulation that nitric oxide produces in the proliferation of the cytotoxic T cells.

Molecular Cloning and Characterization of Human Chondrolectin, a Novel Type I Transmembrane Protein Homologous to C-Type Lectins

CHODL, a novel human gene encoding chondrolectin, was isolated by PCR screening. It is localized at chromosome 21q21 and consists of six exons and five introns. The open reading frame of CHODL encodes a type I transmembrane protein containing a single carbohydrate recognition domain (CRD) of C-type lectins in its extracellular portion. CHODL was detected as a 2.6-kb transcript by northern blot using enriched human testis RNA. RT-PCR analysis revealed preferential expression of CHODL in testis, prostate, and spleen. Immunohistochemistry demonstrated that the expression of CHODL is mainly limited to vascular muscle of testis, smooth muscle of prostate stroma, heart muscle, skeletal muscle, crypts of small intestine, and red pulp of spleen. Western blot analysis revealed that CHODL is an N-glycosylated protein with a molecular weight of approximately 36 kDa. In transiently transfected COS1 cells, CHODL shows a predominantly perinuclear localization. Although the predicted CHODL protein shares a significant homology (45% overall and 60% within the CRD) with layilin, a recently identified hyaluronan receptor, we failed to detect a specific interaction between CHODL and hyaluronan using cetylpyridinium chloride precipitation.

Identification of the Hyaluronan Receptor for Endocytosis (HARE)

Rat liver sinusoidal endothelial cells (LECs) express two hyaluronan (HA) receptors, of 175 and 300 kDa, responsible for the endocytic clearance of HA. We have characterized eight monoclonal antibodies (mAbs) raised against the 175-kDa HA receptor partially purified from rat LECs. These mAbs also cross-react with the 300-kDa HA receptor. The 175-kDa HA receptor is a single protein, whereas the 300-kDa species contains three subunits, alpha, beta, and gamma at 260, 230, and 97 kDa, respectively (Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831-33834). The 97-kDa subunit was not recognized by any of the mAbs in Western blots. Based on their cross-reactivity with these mAbs, the 175-, 230-, and 260-kDa proteins appear to be related. Two of the mAbs inhibit (125)I-HA binding and endocytosis by LECs at 37 degrees C. All of these results confirm that the mAbs recognize the bone fide LEC HA receptor. Indirect immunofluoresence shows high protein expression in liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Because the tissue distribution for this endocytic HA receptor is not unique to liver, we propose the name HARE (HA receptor for endocytosis).

Indicators of erythrocyte formation and degradation in rats with either vitamin A or iron deficiency

Vitamin A deficiency produces anemia and altered iron status. In this study with rats we tested two hypotheses regarding vitamin A deficiency: (1) that it impairs erythropoiesis, leading to an increased red cell turnover, and (2) that it inhibits the glycosylation of transferrin. Erythropoietic activity was assessed indirectly by determining the myeloid:erythroid ratio in bone marrow smears, the number of erythroid colonies in the red pulp of spleen, the blood reticulocyte index, and zinc protoporphyrin and plasma transferrin receptor concentrations. Transferrin glycosylation was assessed by measuring the sialic acid content of transferrin. The effects of vitamin A deficiency were compared with those of iron deficiency. Iron deficiency produced anemia and low iron levels in organs. Vitamin A deficiency produced low levels of plasma and hepatic retinol, and it induced decreased plasma total iron-binding capacity and raised iron levels in tibia and spleen. Short- but not long-term iron deficiency reduced the number of erythroid colonies in spleen; vitamin A deficiency had no influence. Neither iron nor vitamin A deficiency influenced the myeloid:erythroid ratio in bone marrow smears and the blood reticulocyte production. Plasma transferrin receptor and erythrocyte zinc protoporphyrin concentrations were not affected by vitamin A deficiency but increased with iron deficiency. Vitamin A deficiency did not stimulate erythrocyte breakdown, as indicated by unaltered plasma lactate dehydrogenase activity and reduced plasma total bilirubin levels. Both vitamin A and iron deficiencies raised the proportion of multiple sialylated transferrins in plasma. Thus, we have not found evidence that vitamin A deficiency affects erythropoiesis and erythrocyte turnover. The iron accumulation in spleen and bone marrow may be related to reduced iron transport due to inhibition of transferrin synthesis rather than inhibition of transferrin sialylation.

Characterization and distribution of B cells in the lymphoid organs of goats.

The distribution of B cells in the lymphoid organs of the goat was studied using a panel of 13 monoclonal antibodies (mAbs) developed against different markers for bovine B cells. Samples of mesenteric lymph nodes, jejunal and ileal Peyer's patches, duodenum, jejunum, ileum, colon, caecum and rectum were taken from four 7-month-old male Murciano-granadina goats using the avidin-biotin-peroxidase (ABC) method on frozen sections as described by Hsu et al. (1981). The mAbs against immunoglobulins (Ig) recognized a large number of cells, particularly in the light zones of the germinative centres of the lymphoid follicles, regardless of the isotype against which they were directed. However, the greatest numbers of B cells in the germinative centres and outer coronas of the lymphoid follicles of the lymph node, spleen and Peyer's patches were recognized by mAbs against the L lambda chain of Ig and against IgM. This was also the case in other locations where B cells were abundant, such as the medulla of the lymph node and the dome of the Peyer's patches. These mAbs recognized not only B lymphocytes but also plasma cells, showing an intracytoplasmatic reaction (numerous in the spleen red pulp and the intestinal lamina propria when mAbs were used against the L lambda chain of the Ig, scarce in the intestinal lamina propria when used against IgM and scarce in spleen red pulp and numerous in the intestinal lamina propria when mAbs against IgA were used). The mAbs BAQ44 A, GC65 A and GB25 A are of interest because, besides marking cells in the B areas where lymphocytes show surface Ig, they give a positive reaction in areas where there are Ig-cells (the dark zone of the germinative centre) and do not immunostain plasma cells. Thus, these mAbs recognize a surface marker which is not an Ig.

Pharmacokinetics, Tissue Distribution, and Expression Efficiency of Plasmid [33P]DNA Following Intravenous Administration of DNA/Cationic Lipid Complexes in Mice: Use of a Novel Radionuclide Approach

The pharmacokinetics, tissue distribution, and efficacy of a systemic gene transfer method were examined in male BALB/c mice (6–8weeks old) using 33P-labeled plasmid DNA for luciferase. The DNA was delivered via tail vein injection in saline ([33P]DNA) or in a cationic lipid formulation ([33P]DNA/lipid). One group of mice received ≈1–3 µCi (45 µg of DNA) of either formulation, and mice were euthanized at 2 and 20 min, and 1 and 24h postdose (2 mice/time point). Blood and plasma radioactivity were quantified, and whole body autoradiographic (WBAR) images were obtained from 20-µm whole body sections. A tissue distribution (TD) study was conducted in a second group of mice, which received ≈4–6 µCi (45–60 µg of DNA) of [33P]DNA or [33P]DNA/lipid. Mice were euthanized at 1.5h (1 mouse; [33P]DNA/lipid) or 24h (2 mice/group), and organ radioactivity and luciferase expression were measured in lung, liver, kidney, spleen, thymus, and parotid salivary gland by direct quantitation methods. Microautoradiography (MAR) was performed on a third group of mice (n=2), which received 3 µCi (45 µg of DNA) of [33P]DNA/lipid and were euthanized at 24h postdose. For WBAR, the [33P]DNA/lipid tissue distribution (% dose equiv/g) at 2min was lung ≫ liver>spleen (red pulp)>kidney (cortex); at 24h the ranking was spleen (red pulp)>liver>lung, kidney (cortex). The [33P]DNA organ distribution observed at 2min was liver ≫ spleen (red pulp)>lung, blood>kidney (cortex); at 24h the ranking was liver, spleen (red pulp)>kidney (cortex)>lung, blood. High levels of radioactivity in bone (cortical, marrow, growth plate) in both groups may represent uptake of the 33P-labeled test articles by the cellular component of the bone marrow, particularly macrophages, as well as deposition of [33P]phosphate in the bone matrix following metabolism of the [33P]DNA. In the luciferase component of the study, no expression was observed in the [33P]DNA group at 24h. The [33P]-DNA/lipid group exhibited expression as early as 1.5h in the lung; at 24 h, expression was seen in all the organs examined. Microautoradiography of 24-h tissue samples revealed radioactivity in hepatic Kupffer cells, reticuloendothelial system cells in the marginal zone of the spleen, and diffusely along alveolar septae with scattered accumulations in alveolar macrophages. The results of the WBAR, TD, MAR, and luciferase assay show that the use of cationic lipids significantly altered the biodistribution and resulting expression of the DNA plasmid. Further, 33P (0.25MeV β, half-life=25days) was shown to be an excellent radionuclide for quantitative WBA and MAR, providing sharp images with less personal hazard and greater ease of handling than 32P (1.71MeV β, half-life=14.3days).

Influence of intraperitoneal injection of three types of hydrogel beads on expression of carbohydrate-binding sites in spleen macrophages

The influence of the chemical structure of polymer implants on selected characteristics of macrophages was studied to improve our understanding of the mechanisms of non-self recognition of synthetic materials. Three types of polymers differing in net charge were prepared to compare In vivo responses. Beads from preparations of poly(2-hydroxyethyl methacrylate), a copolymer of 2-hydroxyethyl methacrylate with sodium methacrylate, and a copolymer of 2-hydroxyethyl methacrylate and dimethylaminoethyl methacrylate were injected intraperitoneally into rats and harvested 48h later. The effects of these polymers on the presence of inflammatory cells in the peritoneal exudate, on the adhesion of macrophages to individual batches of the different types of beads and on distinct molecular aspects of macrophages in the red pulp of spleen were evaluated. Beads from both types of copolymer caused an elevation in the number of macrophages in the exudate, in contrast to the situation in rats treated with poly(2-hydroxyethyl methacrylate) beads and physiological saline solution as control. The molecular design of the implant had no significant influence on the extent of macrophage adhesion to beads or on the expression of selected carbohydrate-binding sites. Since important cellular functions such as cell adhesion and glycoprotein routing depend on the sugar part of glycoconjugates, labelled neoglycoproteins were employed to analyse this aspect of macrophages in the tested animals. The beads of the copolymer of 2-hydroxyethyl methacrylate with dimethylaminoethyl methacrylate clearly led to an elevation of the expression of specific binding sites for β-galactoside-terminating structures which are presented by asialofetuin, for mannose, fucose, sialic acid and N-acetylgalactosamine, which had been used as the ligand parts of biotinylated neoglycoproteins, in spleen macrophages, whereas the levels of sites which recognize mannose-6-phosphate were unaffected. Expression of sites with specificity to N-acetylglu-cosamine was lessened. The effect of beads from the copolymer of 2-hydroxyethyl methacrylate with sodium methacrylate on the measured glycobiological features in the splenic macrophages was only negligible. These results suggest the possibility of systemic effects of implanted polymers on the distinct recognitive functions of macrophages.

Expression of Flt3 tyrosine kinase receptor gene in mouse hematopoietic and nervous tissues

The Flt3 gene encodes a tyrosine kinase receptor highly related to the Kit and Fms gene products. We have studied the expression of Flt3 by using in situ hybridization of mouse tissue sections. The results show that Flt3 RNAs are present in certain regions of lympho-hematopoietic organs, placenta and nervous system. Flt3 is expressed in the medullary area of fetal and newborn thymus, in the paracortical regions of lymph nodes and in the red pulp of spleen. In placenta, labyrinthine trophoblasts express Flt3. Finally, Flt3 RNAs are found in several regions of the brain and in cerebellar Purkinje cells. Western-blot analysis showed that the FLT3 protein is present in the tissues positive for Flt3 RNA expression. Our observations allow for a comparison with the distribution of the Kit gene and analysis of a possible redundancy between KIT and FLT3 receptors.

Presence of bacterial flora-derived antigen in synovial tissue macrophages and dendritic cells.

In previous studies using and animal model human bacterial flora-derived peptidoglycan Polysaccharides were shown to be arthropathic after a single subcutaneous injection. A prerequisite for proof of the hypothesis that bacterial products from the normal resident flora are involved in the immune reaction of human chronic polyarthritis of unknown aetiology is the presence of these antigens in synovial tissue. 2E9, a monoclonal antibody we developed against intestinal peptidoglycan polysaccharides was used in a histochemical study in rats and stained macrophages in the spleen red pulp. In this study human synovial tissues from 10 rheumatoid arthritis (RA) and 20 non-RA patients were stained with 2E9. We found that eight out of 10 RA patients had 2E9-positive macrophages and dendritic cells in their synovia. A significant difference was observed with the control group in which seven out of 20 were positive. No positive cells or staining of the matrix was found in the cartilage of six RA patients. These results show that exogenous bacterial antigens are present in synovial tissue macrophages and dendritic cells. It was concluded that the unknown antigen in the immune reaction in RA is not necessarily endogenous.