Spleen Cultures Research Articles

Cardiac Tamponade in Largemouth Bass (Micropterus salmoides)

A public aquarium with a 4-mo history of occasional fish mortalities submitted for necropsy an adult female largemouth bass (Micropterus salmoides) that died unexpectedly. Gross necropsy revealed that the pericardial cavity was markedly distended with partially coagulated blood. Examination of the heart revealed multiple nodular masses in the area of the atrium and two small perforations on the surface of one of the nodular masses. Histopathologic exam of the atrium revealed severe fibrinonecrotic endocarditis and transmural myocarditis with intralesional bacteria. A pure culture of Edwardsiella tarda was obtained from culture of posterior kidney and spleen. An area of stagnant water that may serve as the source of E. tarda was identified, and steps to rectify this problem were taken. Low-level supersaturation was also a significant stressor; the source of the supersaturation was not identified. To our knowledge, this is the first report of cardiac tamponade in a largemouth bass.

Count of Splenic Stromal Precursor Cells in Mice and Expression of Cytokine Genes in These Cells in Primary Cultures during Different Periods after Immunization of Animals with S. typhimurium Antigens

Injection of S. typhimurium antigens significantly (9-fold) increased cloning efficiency and, hence, the content of stromal precursor cells in the spleen as soon as after 24 h. These parameters returned to normal by days 6-15 after immunization. Cultured splenocytes collected from immune (but not intact) animals expressed the genes of proinflammatory cytokines IL-1β (on days 1, 6, 15) and IL-6 (on days 1 and 6), TNF-α (on days 6 and 15), and of IFN-α and IL-18 (on days 6 and 15). The expression of IL-4 gene was suppressed on day 6 after immunization, of IL-10 gene on days 1 and 6, of IL-6 gene on day 15. Hence, no signs of immune response suppression by stromal cells were found in this system. The spectrum and dynamics of the expression of pro- and anti-inflammatory cytokine genes in stromal cell cultures from the spleen of immunized mice seemed to correspond to those needed for support of the immune response to S. typhimurium antigens, observed in immunized animals. The results indicate possible involvement of stromal cells in the realization of immune response in vivo. The increase of stromal precursor cells cloning efficiency in response to antigen injection could not be reproduced in vitro: the presence of S. typhimurium antigens in primary cultures of intact mouse bone marrow and spleen throughout the entire period of culturing ≈ 20-fold reduced cloning efficiency in cultures.

Increased Small Intestinal Permeability and Liver Damage After Exhaustive Physical Exercise in Healthy Individuals

Purpose: TNF-α is a pro-inflammatory cytokine believed to be an important mediator of inflammatory bowel disease (IBD). TNF is known to induce cell shedding and increase permeability of the small intestine. We hypothesize that TNF induced intestinal cell shedding contributes to barrier dysfunction and increased antigen presentation. In this study, we used confocal laser endomicroscopy (CLE) and light microscopy (LM), to quantitate TNF induced epithelial cell shedding in the small intestine, and correlated cell shedding with dextran absorption and translocation of commensal bacteria. Methods: 129 Sv/Ev mice were divided into two groups: the treatment group received intraperitoneal injection of TNF-α (0.175μg/ g), while the control group received an equivalent volume of normal saline injection. CLE were performed using Optiscan confocal endomicroscope on exteriorized intestine stained with acriflavine. Epithelial cell shedding was quantitated using density of epithelial gaps created by shedding of intestinal cells. Epithelial density was defined as the total number of epithelial gaps per 1000 cells counted in 5-10 villi per mouse. For LM, frozen sections of the intestine were stained with alcian blue and nuclear fast red. 3D reconstructed CLE images and 2D LM sections were manually counted for epithelial gaps and cells. For FITCdextran absorption study, mice were gavaged with 0.6 mg/g dextran after an overnight fast, blood samples were collected after 4 hr for serum FITC-dextran determination. For translocation studies, mice were gavaged after an overnight fast with 10^10 GFP labelled E. coli JM 109 suspended in 0.17mL LB broth. After 24 hours, liver, spleen and blood samples were obtained for culture. Results: For saline and TNF treated mice (n=6), the CLEdetermined epithelial gap density (mean+SD) was 10.6 ± 2.4 and 47.1 ± 7.4 gaps/1000 cells (P = 0.001), respectively. The LM-determined epithelial gap density was 34.8 ± 6.0 and 64.2 ± 7.4 gaps/1000 cells (P <0.0001), respectively. The discrepancy in the gap density observed between CLE and LM were due to the 3D and 2D nature of image analysis. Serum dextran concentration in the saline and TNF treated mice were 0.59 ± 0.05, and 3.3 ± 0.5 μg/mL (P <0.001), respectively. The liver, spleen and blood cultures from the saline treated mice were all negative, while the TNF treated mice grew 1.4 ± 0.94 E 3 and 1.1 ± 0.5 E 3 cfu/mL of GFP labelled E. colifrom liver and spleen cultures, confirming translocation of commensal bacteria. Conclusion: TNF induced epithelial cell shedding in the small intestine as observed by significant increases in the epithelial gap density of 129 Sv/Ev mice using CLE and LM. This increase in gap density was associated with increased permeability to macromolecules as measured by dextran absorption and translocation of commensal bacteria.

TNF Alpha Induced Epithelial Cell Shedding in the Small Intestine Correlates With Increased Permeability to Macromolecules and Translocation of Commensal Bacteria

Purpose: TNF-α is a pro-inflammatory cytokine believed to be an important mediator of inflammatory bowel disease (IBD). TNF is known to induce cell shedding and increase permeability of the small intestine. We hypothesize that TNF induced intestinal cell shedding contributes to barrier dysfunction and increased antigen presentation. In this study, we used confocal laser endomicroscopy (CLE) and light microscopy (LM), to quantitate TNF induced epithelial cell shedding in the small intestine, and correlated cell shedding with dextran absorption and translocation of commensal bacteria. Methods: 129 Sv/Ev mice were divided into two groups: the treatment group received intraperitoneal injection of TNF-α (0.175μg/ g), while the control group received an equivalent volume of normal saline injection. CLE were performed using Optiscan confocal endomicroscope on exteriorized intestine stained with acriflavine. Epithelial cell shedding was quantitated using density of epithelial gaps created by shedding of intestinal cells. Epithelial density was defined as the total number of epithelial gaps per 1000 cells counted in 5-10 villi per mouse. For LM, frozen sections of the intestine were stained with alcian blue and nuclear fast red. 3D reconstructed CLE images and 2D LM sections were manually counted for epithelial gaps and cells. For FITCdextran absorption study, mice were gavaged with 0.6 mg/g dextran after an overnight fast, blood samples were collected after 4 hr for serum FITC-dextran determination. For translocation studies, mice were gavaged after an overnight fast with 10^10 GFP labelled E. coli JM 109 suspended in 0.17mL LB broth. After 24 hours, liver, spleen and blood samples were obtained for culture. Results: For saline and TNF treated mice (n=6), the CLEdetermined epithelial gap density (mean+SD) was 10.6 ± 2.4 and 47.1 ± 7.4 gaps/1000 cells (P = 0.001), respectively. The LM-determined epithelial gap density was 34.8 ± 6.0 and 64.2 ± 7.4 gaps/1000 cells (P <0.0001), respectively. The discrepancy in the gap density observed between CLE and LM were due to the 3D and 2D nature of image analysis. Serum dextran concentration in the saline and TNF treated mice were 0.59 ± 0.05, and 3.3 ± 0.5 μg/mL (P <0.001), respectively. The liver, spleen and blood cultures from the saline treated mice were all negative, while the TNF treated mice grew 1.4 ± 0.94 E 3 and 1.1 ± 0.5 E 3 cfu/mL of GFP labelled E. colifrom liver and spleen cultures, confirming translocation of commensal bacteria. Conclusion: TNF induced epithelial cell shedding in the small intestine as observed by significant increases in the epithelial gap density of 129 Sv/Ev mice using CLE and LM. This increase in gap density was associated with increased permeability to macromolecules as measured by dextran absorption and translocation of commensal bacteria.

Overexpression of PepT1 Increases the Susceptibility of Mice to Colitis in a Process Involving NOD2 Activity

Purpose: TNF-α is a pro-inflammatory cytokine believed to be an important mediator of inflammatory bowel disease (IBD). TNF is known to induce cell shedding and increase permeability of the small intestine. We hypothesize that TNF induced intestinal cell shedding contributes to barrier dysfunction and increased antigen presentation. In this study, we used confocal laser endomicroscopy (CLE) and light microscopy (LM), to quantitate TNF induced epithelial cell shedding in the small intestine, and correlated cell shedding with dextran absorption and translocation of commensal bacteria. Methods: 129 Sv/Ev mice were divided into two groups: the treatment group received intraperitoneal injection of TNF-α (0.175μg/ g), while the control group received an equivalent volume of normal saline injection. CLE were performed using Optiscan confocal endomicroscope on exteriorized intestine stained with acriflavine. Epithelial cell shedding was quantitated using density of epithelial gaps created by shedding of intestinal cells. Epithelial density was defined as the total number of epithelial gaps per 1000 cells counted in 5-10 villi per mouse. For LM, frozen sections of the intestine were stained with alcian blue and nuclear fast red. 3D reconstructed CLE images and 2D LM sections were manually counted for epithelial gaps and cells. For FITCdextran absorption study, mice were gavaged with 0.6 mg/g dextran after an overnight fast, blood samples were collected after 4 hr for serum FITC-dextran determination. For translocation studies, mice were gavaged after an overnight fast with 10^10 GFP labelled E. coli JM 109 suspended in 0.17mL LB broth. After 24 hours, liver, spleen and blood samples were obtained for culture. Results: For saline and TNF treated mice (n=6), the CLEdetermined epithelial gap density (mean+SD) was 10.6 ± 2.4 and 47.1 ± 7.4 gaps/1000 cells (P = 0.001), respectively. The LM-determined epithelial gap density was 34.8 ± 6.0 and 64.2 ± 7.4 gaps/1000 cells (P <0.0001), respectively. The discrepancy in the gap density observed between CLE and LM were due to the 3D and 2D nature of image analysis. Serum dextran concentration in the saline and TNF treated mice were 0.59 ± 0.05, and 3.3 ± 0.5 μg/mL (P <0.001), respectively. The liver, spleen and blood cultures from the saline treated mice were all negative, while the TNF treated mice grew 1.4 ± 0.94 E 3 and 1.1 ± 0.5 E 3 cfu/mL of GFP labelled E. colifrom liver and spleen cultures, confirming translocation of commensal bacteria. Conclusion: TNF induced epithelial cell shedding in the small intestine as observed by significant increases in the epithelial gap density of 129 Sv/Ev mice using CLE and LM. This increase in gap density was associated with increased permeability to macromolecules as measured by dextran absorption and translocation of commensal bacteria.

Dose Range Evaluation of Mycograb C28Y Variant, a Human Recombinant Antibody Fragment to Heat Shock Protein 90, in Combination with Amphotericin B-Desoxycholate for Treatment of Murine Systemic Candidiasis

Systemic candidiasis causes significant mortality in patients despite amphotericin B (AMB) therapy. Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, is closely related to Mycograb, which showed a survival advantage in combination with AMB in a phase III human trial. The Mycograb C28Y variant could potentially increase the antifungal effect of AMB. In our study, the interaction between AMB-desoxycholate (DAMB) and the Mycograb C28Y variant was characterized in vitro by using a checkerboard method. Quantitative cultures of kidneys, livers, and spleens of neutropenic mice with systemic Candida albicans infections were used to assess the in vivo interaction between 1.4 mg/kg of body weight/day of DAMB and 0.15, 1.5, and 15 mg/kg/day of the Mycograb C28Y variant after 1, 3, and 5 days of therapy. DAMB and Mycograb C28Y variant monotherapies, vehicle, and a no-treatment arm served as controls. Also, single- and multidose pharmacokinetics for the Mycograb C28Y variant were determined. Indifference or synergy between DAMB and the Mycograb C28Y variant was seen in two trials by the checkerboard method. The pharmacokinetics of the Mycograb C28Y variant was best described by a 2-compartment model with a median serum t(1/2)(α) of ~0.198 h and a t(1/2)(β) of ~1.77 h. In mice, DAMB together with the Mycograb C28Y variant was no more effective than AMB alone (P > 0.05 by analysis of variance). The Mycograb C28Y variant alone had no antifungal activity. We therefore conclude that the Mycograb C28Y variant in combination with DAMB offered no benefit over DAMB monotherapy in a neutropenic murine model of systemic candidiasis.

Determination of the therapeutic activity of caspofungin compared with the amphotericin B in an animal experimental model of histoplasmosis in hamster (Mesocrisetus auratus)

Background Treatment with amphotericin B is highly effective in histoplasmosis. Caspofungin has shown good activity against Candida and Aspergillus spp . In vitro studies have demonstrated that Histoplasma capsulatum is inhibited by caspofungin. Objectives The purpose of this study was to evaluate the effectiveness of caspofungin in the treatment of histoplasmosis in an animal experimental model. Methods Three strains of Histoplasma capsulatum var. capsulatum were used. Treatment started one week post-inoculation and the animals were randomly assigned to six groups: amphotericin B 6 mg/Kg/d, caspofungin 2 mg/Kg/d, 4 mg/Kg/d, 8 mg/Kg/d and the other two groups received saline solution and dextrose solution. Blood samples for culture were obtained once a week, from day 7 to 35 post-inoculation. One week after the end of the treatment the animals were sacrificed and spleen cultures were performed. Results Blood cultures were negative in all the hamsters which received amphotericin B (100%, P < 0.001); those treated with caspofungin and the control animals presented 30 and 32% of positive cultures respectively ( P = 0.59). Spleen cultures were negative in the animals treated with amphotericin B, while the percentage of positive spleen cultures in the caspofungin groups varied from 25 to 100%, and in the control groups from 35 to 94.8% ( P = 0.07). The statistical analysis of the undiluted cultures showed the use of amphotericin B as the only independent predictor of negative culture ( P < 0.001). Conclusions The efficacy of amphotericin B is well known for the treatment of histoplasmosis, though we could not demonstrate that caspofungin is better than control.

Extraction and activity of regulatory peptides from sea urchin eggs

Experiments were performed to extract low molecular weight peptides from the eggs of the sea urchin Strongylocentrotus pallidus using sterilizing microfiltration and solid-phase extraction, along with analytical gel chromatography and spectroscopy. The peptides extracted had molecular weights of 360 – 1200 Dal and were divided into two fractions: acidic peptides contained a predominance of glutamic and aspartic acid residues, along with aromatic amino acid residues; alkaline peptides contained mainly lysine and arginine residues and lacked aromatic amino acids. Assessment of the biological activity of these extracts demonstrated high specificity in stimulating the development of explants in organotypic cultures of spleen, testicle, myocardium, and cerebral cortex tissues.

Effect of antigens on colony forming efficiency of stromal clonogenic cells and expression of cytokine genes in primary cultures of bone marrow and spleen of mice

Presence of microbial cells preparation (HCL – extract of group A streptococcus) in primary cell cultures of bone marrow (BMCC) and spleen (SCC) of CBA mice induced the expression mRNA of proinflammatory cytokines genes: IL-2, IL-6 and IL-8 in BMCC; in SCC cultures mRNA IL-1 β and IL-6 appeared and mRNA IL-4 disappeared. Injection of S. typhimurium antigen complex to mice CBA increased 5 times colony forming efficiency (CFE) and, respectively, content of stromal precursor cells (CFU-F) in femur bone marrow and 9 times in spleen of these animals with maximum at the first day. In both primary BMCC and SCC from immunized animals expression of proinflammatory cytokines genes IL-1 β, IL-6, IL-8 and TNF- α was revealed - in BMCC on 1 - 3 day after immunization, and in SCC – up to 15 days. In CBA mice injected with polyvinylpyrrolidone (PVP) CFE and number of CFU-F in BMCC and SCC increased 1, 9 and 1, 8 times only and expression of genes IL-6, IL-8 and TNF- was observed only on the first day. In СВА/N xid mice there was neither increase CFU-F in the corresponding organs, nor expression of proinflammatory cytokines genes in primary cultures. The data suggest the possibility of positive participation of stromal cells in the development on of immune response in organism. Degree of activation of stromal tissue in immunized mice, apparently, correlates with the degree of immune response, supposing a close relationship between stromal tissue and immune system.

Brote de histoplasmosis en la Escuela de Cadetes de la Base Aérea de Morón, Provincia de Buenos Aires, República Argentina

A histoplasmosis outbreak affecting 6 previously healthy Air Force cadets is herein presented. The patients suffered from fever and respiratory symptoms after having cleaned an abandoned hangar soiled with pigeons and bat droppings. They all presented fever, myalgia, tachypnea, and nonproductive cough. Chest X-ray and CT scan studies showed disseminated reticulonodular images affecting both lungs. Hiliar adenomegalies were also observed. All patients achieved a favourable outcome without antifungal treatment. Both serologic tests searching for specificic antibodies (immunodiffusion and counterimmunoelectrophoresis) and histoplasmin skin tests were positive in all cases. Five soil samples mixed with pigeons and bat droppings were collected from the hangar. Suspensions of these samples were inoculated into 20 hamsters by intraperitoneal injection; mycelial phase of H. capsulatum was isolated from liver and spleen cultures. The genetic profile of this strain was compared with 12 isolates obtained from Argentinean patients, and a great degree of homogeneity was observed (> 96% similarity). Although histoplasmosis is endemic in the wet Pampas, this is the first epidemic outbreak reported south of the 34th parallel.

Efficacy of doripenem in the treatment of Pseudomonas aeruginosa experimental pneumonia versus imipenem and meropenem

The aim of this study was to compare doripenem with imipenem and meropenem in an experimental rabbit model of Pseudomonas aeruginosa pneumonia and then to compare different doripenem doses and methods of intravenous administration. Using a rabbit experimental model of pneumonia, efficacy was assessed following 2 days of treatment by colony counts of different tissues (lung, spleen and blood culture). Mean pulmonary bacterial loads were 3.17 ± 0.53, 3.42 ± 0.61 and 2.75 ± 0.59 log(10) cfu/g for imipenem, doripenem (0.5 g three times daily) and meropenem, respectively, compared with 7.57 ± 0.99 cfu/g for control animals. At a higher dose (1 g three times daily), doripenem showed significantly better efficacy (2.70 ± 0.65 log(10) cfu/g) than the standard regimen of doripenem. Sterilization of spleen cultures was achieved with standard regimens of imipenem (1 g three times daily) and a higher dose of doripenem. In this model of P. aeruginosa pneumonia, doripenem had an efficacy equivalent to that of meropenem and imipenem at a high dose of 1 g three times a day and lower efficacy at a standard dose (0.5 g three times daily) than the other two agents in terms of bacteria cultivated from spleens. Doripenem is a new drug that offers new therapeutic options, especially for difficult-to-treat infections such as pneumonia due to non-fermenting Gram-negative bacteria.

Delineation of precursors in murine spleen that develop in contact with splenic endothelium to give novel dendritic-like cells

AbstractHematopoietic cell lineages are best described in terms of distinct progenitors with limited differentiative capacity. To distinguish cell lineages, it is necessary to define progenitors and induce their differentiation in vitro. We previously reported in vitro development of immature dendritic-like cells (DCs) in long-term cultures (LTCs) of murine spleen, and in cocultures of spleen or bone marrow (BM) over splenic endothelial cell lines derived from LTCs. Cells produced are phenotypically distinct CD11bhiCD11cloCD8−MHC-II− cells, tentatively named L-DCs. Here we delineate L-DC progenitors as different from known DC progenitors in BM and DC precursors in spleen. The progenitor is contained within the lineage-negative (Lin)−c-kit+ subset in neonatal and adult spleen. This subset has multipotential reconstituting ability in mice. In neonatal spleen, the progenitor is further enriched within the c-kitlo and CD34+ subsets of Lin−c-kit+ cells. These cells seed cocultures of splenic endothelial cells, differentiating to give L-DCs that can activate T cells. L-DC progenitors are distinguishable from described splenic CD11clo DC precursors and from Fms-like tyrosine kinase 3+ DC progenitors in BM. Overall, this study confirms that LTCs are a physiologically relevant culture system for in vitro development of a novel DC type from spleen progenitors.

Proteolytically Inactive Per a 10 Allergen of Periplaneta americana Modulates Th2 Response and Enhances IL-10 in Mouse Model

Purified allergens with reduced IgE reactivity are required to improve the safety and efficacy of allergen-specific immunotherapy (IT). The present study investigates the efficacy of purified cockroach allergen immunotherapy with proteolytically active and inactive Per a 10 in allergic mouse model. Balb/c mice were sensitized intraperitoneally with cockroach extract (CE) and purified allergen Per a 10 in separate groups. Mice were treated subcutaneously with phosphate-buffered saline (PBS), CE, active and inactive Per a 10 and challenged intranasally. Antigen specific IgE, IgG1 and IgG2a in serum and cytokines IL-4, IL-13, IFN-gamma, IL-10, TGF-beta in bronchoalveolar lavage (BAL) fluid and spleen culture supernatant (CS) were estimated by enzyme-linked immunosorbent assay. Lung histology was analyzed by hematoxylin and eosin staining. IT with Per a 10 demonstrated significant reduction in IgE levels in serum, IL-4 levels in BAL fluid, CS, and eosinophilic infiltration in lungs than PBS-treated mice. This was associated with significantly increased IL-10 secretion in BAL fluid and CS. IT with Per a 10 effectively suppressed T-helper type 2 (Th2) response in mice sensitized with Per a 10 than CE group. Further, IT with inactive Per a 10 showed maximum reduction in systemic and airway inflammation and induced maximum IL-10 release in BAL fluid and CS than other antigens. IT with Per a 10 effectively suppressed Th2 response and lung inflammation in Per a 10- or CE-sensitized mice. The beneficial effects of IT with inactive Per a 10 are more pronounced than active Per a 10.

Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells.

Dendritic cells (DC) are known to develop from macrophage dendritic progenitors (MDP) in bone marrow (BM), which give rise to conventional (c)DC and monocytes, both dominant antigen presenting cell (APC) subsets in spleen. This laboratory has however defined a distinct dendritic-like cell subset in spleen (L-DC), which can also be derived in long-term cultures of spleen. In line with the restricted in vitro development of only L-DC in these stromal cultures, we questioned whether self-renewing HSC or progenitors exist in spleen with restricted differentiative capacity for only L-DC. Neonatal spleen and BM were compared for their ability to reconstitute mice and to give rise to L-DC, as well as other splenic APC. Neonatal spleen cells were transplanted into allotype-distinct lethally irradiated hosts along with host-type competitor BM cells, and assayed over 8 to 51 weeks for haematopoietic reconstitution of L-DC and cDC subsets, along with other lymphoid and myeloid cells. In this study, neonatal spleen showed multilineage haematopoietic reconstitution in mouse chimeras, rather than specific or restricted ability to differentiate into L-DC. However, the representation of individual APC subsets was found to be unequal in chimeras partially reconstituted with donor cells, such that more donor-derived progeny were seen for L-DC than for myeloid and cDC subsets. The ability of HSC in spleen to develop into L-DC was indicated by a strong bias in the subset size of these cells over other splenic APC subsets. This type of evidence supports a model whereby spleen represents an important site for haematopoiesis of this distinct DC subset. The conditions under which haematopoiesis of L-DC occurs in spleen, or the progenitors involved, will require further investigation.

Paraparesis in a Polar Bear (Ursus maritimus) Associated with West Nile Virus Infection

A polar bear (Ursus maritimus) housed at the Toronto Zoo presented with acute-onset, nonambulatory paraparesis. Physical examination 24 hr after onset was otherwise unremarkable, spinal radiographs looked normal, and blood tests indicated mild dehydration. With continued deterioration in its general condition, euthanasia was elected a day later. Necropsy did not reveal a cause for the major presenting clinical signs. Serum collected at the time of initial examination was positive for West Nile virus (WNV) antibodies in a serum neutralization assay and at the time of euthanasia was positive in both a competitive enzyme-linked immunosorbent assay and in a plaque reduction neutralization assay. The major microscopic finding was a mild-to-moderate nonsuppurative meningoencephalomyelitis. WNV was not detected by immunohistochemistry in brain or spinal cord or by real-time reverse transcription-polymerase chain reaction (RT-PCR) and cell culture of brain and kidney, but it was isolated and identified by RT-PCR in second passage cell culture of spleen. Retrospective immunohistochemistry on spleen revealed rare antigen-positive cells, probably macrophages. Prevention of exposure to potentially WNV-infected mosquitoes or vaccination of captive bears against WNV should be considered.

Splenic stromal niches support hematopoiesis of dendritic-like cells from precursors in bone marrow and spleen

The aims of this study are to test the ability of stromal cells from murine spleen to support hematopoiesis, to define the tissue source of precursors that seed these hematopoietic niches, and to determine the type of cells produced. Cloned isolates of murine spleen stroma have been developed that support hematopoiesis. Analysis has been investigated in terms of tissue source of progenitors. Type and number of cells produced were analyzed by flow cytometry. Hematopoietic precursors that seed cocultures exist in spleen and bone marrow (BM), but not thymus. Cell production is highest if overlay cells are enriched for hematopoietic precursors. BM contains more precursors than spleen, but the cell types produced are different. Cocultures established from spleen maintain a high proportion of a distinct class of dendritic-like cells produced in only low numbers in BM cocultures. These reflect the immature myeloid dendritic cell (DC) produced continuously in long-term spleen cultures established previously in this laboratory. Stroma-conditioned medium alone does not support DC development, but does support early outgrowth of myelomonocytic cells from precursors in both spleen and BM. The outcome has been development of a coculture system that supports hematopoiesis of immature myeloid dendritic-like cells in vitro. Although production of monocytes can occur in the presence of stroma-conditioned medium alone, production of DC is dependent on stromal cell interaction. Results presented here raise questions about the role of spleen as a site for DC hematopoiesis from endogenous precursors.

Heat-Treated Lactobacillus crispatus KT Strains Reduce Allergic Symptoms in Mice

In the current study, we investigated the effects of heat-treated Lactobacillus crispatus KT strains on allergic response in mice. We found that the number of interferon (IFN)-gamma(+)CD4(+) cells was higher in C3H/HeN mouse spleen cultures incubated with L. crispatus KT strains than in those cultured with Lactobacillus JCM type cultures. The serum immunoglobulin E levels in NC/Nga mice that were administered KT strains were lower than those in the mice that were not given any bacterium. The ratio of spleen IFN-gamma(+)CD4(+)/interleukin-4(+)CD4(+) was highest in mice given L. crispatus KT-11. L. crispatus KT-11 also increased the expression of Toll-like receptor (TLR) 2, nucleotide-binding oligomerization domain (NOD) 1, and NOD2 in C3H/HeN mouse Peyer's patch cells. These results suggest that the L. crispatus KT-11 strain reduces allergic symptoms in NC/Nga mice via the adjustment of the type 1 helper T cell and type 2 helper T cell balance via TLR2, NOD1, and NOD2.

A Combined DNA Vaccine Provides Protective Immunity AgainstMycobacterium bovisandBrucella abortusin Cattle

We evaluated the immunogenicity and protective efficacy of a combined DNA vaccine containing six genes encoding immunodominant antigens from Mycobacterium bovis and Brucella abortus. The number of lymph node and spleen cultures positive for M. bovis and B. abortus from calves immunized with the combined DNA vaccine was significantly reduced (p < 0.01) compared with unvaccinated calves after challenge with virulent M. bovis and B. abortus 544. The combined DNA vaccine group displayed stronger antigen-specific interferon-gamma (IFN-gamma) responses and antigen-specific IFN-gamma ELISPOT activities 2 months after final immunization and after challenge. Antigen-specific CD4(+) and CD8(+) T cell responses in the combined DNA vaccine group were higher than either the Bacillus Calmette-Guerin (BCG)-positive or S19-positive control group. Likewise, more calves in the DNA vaccine group exhibited antigen-specific IgG titers and had higher IgG titers than those in the BCG- or S19-immunized groups 2 months after the final immunization. Moreover, two antigens in the combined DNA vaccine induced significant antigen-specific IFN-gamma responses 6 months after challenge (p < 0.05). Bacterial counts and pathological analyses of the challenged animals indicated that the combined DNA vaccine provided significantly better protection than the BCG vaccine against M. bovis, and the protection level induced by the combined DNA vaccine was comparable to S19 against B. abortus. This is the first report to demonstrate that a single combined DNA vaccine protects cattle against two infectious diseases.

Campylobacter jejuni induces transcellular translocation of commensal bacteria via lipid rafts

BackgroundCampylobacter enteritis represents a risk factor for the development of inflammatory bowel disease (IBD) via unknown mechanisms. As IBD patients exhibit inflammatory responses to their commensal intestinal microflora, factors that induce translocation of commensal bacteria across the intestinal epithelium may contribute to IBD pathogenesis. This study sought to determine whether Campylobacter induces translocation of non-invasive intestinal bacteria, and characterize underlying mechanisms.MethodsMice were infected with C. jejuni and translocation of intestinal bacteria was assessed by quantitative bacterial culture of mesenteric lymph nodes (MLNs), liver, and spleen. To examine mechanisms of Campylobacter-induced bacterial translocation, transwell-grown T84 monolayers were inoculated with non-invasive Escherichia coli HB101 ± wild-type Campylobacter or invasion-defective mutants, and bacterial internalization and translocation were measured. Epithelial permeability was assessed by measuring flux of a 3 kDa dextran probe. The role of lipid rafts was assessed by cholesterol depletion and caveolin co-localization.ResultsC. jejuni 81–176 induced translocation of commensal intestinal bacteria to the MLNs, liver, and spleen of infected mice. In T84 monolayers, Campylobacter-induced internalization and translocation of E. coli occurred via a transcellular pathway, without increasing epithelial permeability, and was blocked by depletion of epithelial plasma membrane cholesterol. Invasion-defective mutants and Campylobacter-conditioned cell culture medium also induced E. coli translocation, indicating that C. jejuni does not directly 'shuttle' bacteria into enterocytes. In C. jejuni-treated monolayers, translocating E. coli associated with lipid rafts, and this phenomenon was blocked by cholesterol depletion.ConclusionCampylobacter, regardless of its own invasiveness, promotes the translocation of non-invasive bacteria across the intestinal epithelium via a lipid raft-mediated transcellular process.

Regulation of pathogenic IL-17 responses in collagen-induced arthritis: roles of endogenous interferon-gamma and IL-4

IntroductionInterleukin (IL)-17 plays an important role in the pathogenesis of rheumatoid arthritis and the mouse model collagen-induced arthritis (CIA). Interferon(IFN)-γ and IL-4 have been shown to suppress Th17 development in vitro, but their potential immunoregulatory roles in vivo are uncertain. The goals of this study were to determine the relationship between Th17 responses and disease severity in CIA and to assess regulation of IL-17 by endogenous IFN-γ and IL-4.MethodsDBA1/LacJ mice were immunized with type II collagen in complete Freund's adjuvant (CFA) to induce arthritis, and treated with neutralizing antibody to IFN-γ and/or IL-4. Systemic IL-17, IFN-γ, and IL-4 were measured in serum. At the peak of disease, cytokine production was measured by ELISA of supernatants from spleen, lymph node and paw cultures. Paws were also scored for histologic severity of arthritis.ResultsJoint inflammation was associated with a higher ratio of systemic IL-17/IFN-γ. Neutralization of IFN-γ accelerated the course of CIA and was associated with increased IL-17 levels in the serum and joints. The IFN-γ/IL-4/IL-17 responses in the lymphoid organ were distinct from such responses in the joints. Neutralization of IL-4 led to increased arthritis only in the absence of IFN-γ and was associated with increased bone and cartilage damage without an increase in the levels of IL-17.ConclusionsIL-4 and IFN-γ both play protective roles in CIA, but through different mechanisms. Our data suggests that the absolute level of IL-17 is not the only determinant of joint inflammation. Instead, the balance of Th1, Th2 and Th17 cytokines control the immune events leading to joint inflammation.