Infected Transgenic Mice Research Articles

Over-expression of CD163, CD169, and CD151 is not sufficient to improve the susceptibility to porcine reproductive and respiratory syndrome virus infection in transgenic mice

Over-expression of CD163, CD169, and CD151 is not sufficient to improve the susceptibility to porcine reproductive and respiratory syndrome virus infection in transgenic mice

Rhinovirus Infection of ORMDL3 Transgenic Mice Is Associated with Reduced Rhinovirus Viral Load and Airway Inflammation.

Orosomucoid like 3 (ORMDL3), a gene localized to chromosome 17q21, has been linked in epidemiologic studies to childhood asthma and rhinovirus (RV) infections. As the single nucleotide polymorphisms linking ORMDL3 to asthma are associated with increased expression of ORMDL3, we have used hORMDL3zp3-Cre mice (which have universal increased expression of human ORMDL3) to determine whether infection of these transgenic mice with RV influences levels of airway inflammation or RV viral load. RV infection of hORMDL3zp3-Cre mice resulted in reduced RV viral load assessed by quantitative real-time PCR (lung and airway epithelium), as well as reduced airway inflammation (total bronchoalveolar lavage cells, neutrophils, macrophages, and lymphocytes) compared with RV-infected wild-type mice. Levels of the antiviral pathways including IFNs (IFN-α, IFN-β, IFN-λ) and RNAse L were significantly increased in the lungs of RV-infected hORMDL3zp3-Cre mice. Levels of the antiviral mouse oligoadenylate synthetase (mOas)1g pathway and RNAse L were upregulated in the lungs of unchallenged hORMDL3zp3-Cre mice. In addition, levels of mOas2, but not mOas1 (mOas1a, mOas1b, mOas1g), or mOas3 pathways were significantly more upregulated by IFNs (IFN-α, IFN-β, IFN-λ) in epithelial cells from hORMDL3zp3-Cre mice compared with RV-infected wild-type mouse epithelial cells. RNAse L-deficient mice infected with RV had increased RV viral load. Overall, these studies suggest that increased levels of ORMDL3 contribute to antiviral defense to RV infection in mice through pathways that may include IFNs (IFN-α, IFN-β, IFN-λ), OAS, and RNAse L.

The Phosphocarrier Protein HPr Contributes to Meningococcal Survival during Infection.

Neisseria meningitidis is an exclusively human pathogen frequently carried asymptomatically in the nasopharynx but it can also provoke invasive infections such as meningitis and septicemia. N. meningitidis uses a limited range of carbon sources during infection, such as glucose, that is usually transported into bacteria via the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), in which the phosphocarrier protein HPr (encoded by the ptsH gene) plays a central role. Although N. meningitidis possesses an incomplete PTS, HPr was found to be required for its virulence. We explored the role of HPr using bioluminescent wild-type and ΔptsH strains in experimental infection in transgenic mice expressing the human transferrin. The wild-type MC58 strain was recovered at higher levels from the peritoneal cavity and particularly from blood compared to the ΔptsH strain. The ΔptsH strain provoked lower levels of septicemia in mice and was more susceptible to complement-mediated killing than the wild-type strain. We tested whether meningococcal structures impacted complement resistance and observed that only the capsule level was decreased in the ΔptsH mutant. We therefore compared the transcriptomic profiles of wild-type and ΔptsH strains and identified 49 differentially expressed genes. The HPr regulon contains mainly hypothetical proteins (43%) and several membrane-associated proteins that could play a role during host interaction. Some other genes of the HPr regulon are involved in stress response. Indeed, the ΔptsH strain showed increased susceptibility to environmental stress conditions. Our data suggest that HPr plays a pleiotropic role in host-bacteria interactions most likely through the innate immune response that may be responsible for the enhanced clearance of the ΔptsH strain from blood.

A soluble form of Siglec-9 provides a resistance against Group B Streptococcus (GBS) infection in transgenic mice

Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), possessing terminal sialic acid residues that suppress host immune response and provide a survival advantage to the pathogen. CPS binds to Siglec-9 expressed on neutrophils, which is expected to down-regulate the immune responsiveness of neutrophils. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of CPS to Siglec-9 on immune cells, leading to provide antibacterial benefit against GBS infection in the transgenic mouse line expressing sSiglec-9 (sSiglec-9 Tg). The sSiglec-9 in the sera of sSiglec-9 Tg bound to the sialylated-GBS strains belonging to serotypes Ia, Ib, II, III, IV and V in whole GBS cell ELISA. When GBS cells of serotype III that is a common serotype in late-onset GBS disease (LOD) were intraperitoneally inoculated into sSiglec-9 Tg, sSiglec-9 Tg showed a significant resistance as compared with non-transgenic littermates. Furthermore, GBS serotype III organisms were not detected in cultures of the blood from surviving mice (<1 × 103 CFU/ml). These results indicated that sSiglec-9 Tg mice were more efficient in eliminating GBS and survived better after the intraperitoneal challenge with GBS serotype III bacteria.

Abstract 82: Mnsodtg Mice Exhibit Improved Heart and Mitochondrial Function During Chronic Chagas Disease

Background: We observed that mitochondrial reactive oxygen species (mtROS) plays very important roles in the pregression of chagesic disease (CD). In this study, we utilized genetically-modified mice to scavenge mtROS to investigate the impact of improved ROS scavenging capacity on heart function in CD. Methods and Results: C57BL/6 mice (wild-type, MnSODtg, MnSOD+/-) were infected with Trypanosoma cruzi(Tc). Chronically infected mice (≥120dpi) exhibited a substantial decrease in heart tissue MnSOD gene expression, protein level, enzyme activity and antioxidant level; decrease of heart dysfunction via lower of SV, CO, EF, FS and LVPW,s, and increase of ESV/EDS and LVID;s; enhancement of hypertrophy by increase of IVS, LV mass and areas duo to augmentation of collagen expressions. One of our novel observations was that sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) lost its role of maintenance of low cytoplasm free calcium and mediated calcium uptake to intracellular store in Tc-induced chronic chagasic disease. Studies of fresh heart slices using O2K confirmed that Tc diminished heart mitochondrial function like decrease of oxygen flux and respiratory control ratio (RCR), which were caused by enhancements of ROS. Myocardial mitochondrial damage was pronounced and associated with a &gt;x% decline in mitochondrial oxygen flux in chronically infected wild-type and MnSOD transgenic mice. Imaging of intact heart for cardiomyocytes and collagen by the nonlinear optical microscopy techniques showed significant increase in collagen (&gt;x0-fold) in chronically infected wild-type mice; while MnSODtg mice exhibited a basal increase in collagen that did not change during chronic phase. Chronically infected MnSODtg mice exhibited a marginal decline in Tc-induced heart function, heart hypertrophy, mitochondrial dysfunction Conclusions: Overexpression of MnSOD inhibited Tc-induced oxidative damage od heart tissue. , suggesting that enhancing the mitochondrial ROS scavenging capacity was beneficial in controlling the inflammatory and oxidative pathology, and cardiac remodeling responses that are hallmarks of chronic Chagas disease.

Transgenic over-expression of mammalian heparanase delays prion disease onset and progression

Cellular heparan sulfate (HS) has a dual role in scrapie pathogenesis; it is required for PrPSc (scrapie prion protein) formation and facilitates infection of cells, mediating cellular uptake of prions. We examined the involvement of heparanase, a mammalian endoglycosidase degrading HS, in scrapie infection. In cultured cells, heparanase treatment or over-expression resulted in a profound decrease in PrPSc. Moreover, disease onset and progression were dramatically delayed in scrapie infected transgenic mice over-expressing heparanase. Together, our results provide direct in vivo evidence for the involvement of intact HS in the pathogenesis of prion disease and the protective role of heparanase both in terms of susceptibility to infection and disease progression.

Impact of corticosteroids on experimental meningococcal sepsis in mice

Neisseria meningitidis is responsible for septicemia and meningitis with high fatality that is associated with an excessive inflammatory reaction particularly with hyperinvasive isolates of the clonal complex ST-11 (cc11). However, anti-inflammatory adjuvant treatment remains controversial and difficult to assess in patients. We addressed this topic in a well-defined experimental meningococcal infection in transgenic mice expressing the human transferrin. Mice were infected by intra-peritoneal challenge with bioluminescent serogroup C/cc11 strain. After 3h of infection mice were differentially treated every 6h by saline, amoxicillin alone or amoxicillin and dexamethasone (DXM). Infected mice were scored for clinical status, temperature and weight. Biological markers of inflammation were also quantified.Significant clinical improvement was observed in mice treated with amoxicillin and DXM compared to the two other groups. A significant reduction of the inflammatory reaction assessed by CRP and Lipocalin 2 (two acute phase proteins) was also observed with this treatment. DXM significantly increased blood levels of IL-10 at 6h post-infection. DXM/amoxicillin treated mice, compared to the two other groups, also showed lower levels of TNF-α and lower bacterial blood load assessed by serial dilutions of blood and bioluminescence dynamic imaging. Our results suggest that DXM, added to an appropriate antibiotic therapy, has a beneficial effect on experimental sepsis with a hyperinvasive meningococcal strain in transgenic mice expressing human transferrin. This is most likely due to the reduction of inflammatory response by an early induction of IL-10 cytokine. These data may allow better decision-making to use or not corticotherapy during meningococcal sepsis.

Reduction of regulatory T cells during acute Toxoplasma gondii infection is caused by IFN-γ (MPF2P.759)

Abstract Toxoplasmosis is an infection disease caused by the protozoan parasite Toxoplasma gondii. A TH1 immune response is essential for protection against the parasite, IFN-γ being the major mediator of resistance. CD4+Foxp3+ Regulatory T cells (Treg) maintain immune homeostasis by actively suppressing pathological and physiological immune responses. Intraperitoneal infection of BALB/c mice with T. gondii cysts leads to high mortality rate and exacerbated IFN-γ levels in sera both of which parallel a decreased Treg number in blood and spleen. Since we observed an inverse correlation between high IFN-γ levels and low Treg cell number in infected mice, we analyzed if this cytokine was involved in Treg reduction. Since STAT-1 is involved during signaling by IFN-γ, we infected STAT-1-/- KO mice with T. gondii and found that the Treg cell number was unchanged in blood and increased in spleen, although all mice died; IFN-γ levels in sera in these mice were lower than those observed in infected WT mice, and CD4+ - and CD8+-producing IFN-γ cells were also reduced. These results strongly suggested that IFN-γ has a major role in the reduction of Treg cells. Thus we injected a neutralizing mAb against IFN-γ to infected Foxp3EGFP transgenic mice and unchanged Treg cell number was detected in blood. Our results thus demonstrate that exacerbated IFN-γ levels during acute toxoplasmosis are responsible of collapse of Treg cell number in this model.

Induction of protection against foot-and-mouth disease virus in cell culture and transgenic suckling mice by miRNA targeting integrin αv receptor

Foot-and-mouth disease virus (FMDV) is an RNA virus that causes a highly contagious disease in domestic and wild cloven-hoofed animals. Although vaccination has been used to protect animals against FMDV, there are shortcomings in the efficacy of the available vaccines. RNA interference (RNAi) is triggered by small RNA molecules, including short interfering RNAs and microRNAs (miRNAs), and the use of RNAi-based methods have demonstrated promise as an alternative method of controlling the transmission of FMDV. However, the method of delivery, short duration of siRNA and miRNA in vivo, and the genetic variability of FMDV confound the use of RNAi-based strategies for FMDV control. FMDV has been shown to exploit host-cell integrins as cell-surface receptors to initiate infection. We selected the gene for the integrin αv subunit as an RNAi target, and constructed three αv-specific miRNA expression plasmids. The effects of these miRNAs on FMDV infection were examined in PK-15 cells and transgenic suckling mice. In PK-15 cells, the expression of the αv-specific miRNAs significantly inhibited the expression of integrin αv receptor and decreased FMDV infection. The transgenic mice were generated by integrating the αv-specific miRNA expression cassette using pronuclear microinjection. When challenged with a dose of FMDV ten times greater than the LD50, the survival rate of transgenic suckling mice was approximately six-fold higher than that of their non-transgenic littermates, indicating that the interference of the miRNAs significantly reduced FMDV infection in the transgenic mice. This is the first report of limiting FMDV attachment to cellular receptors using miRNA-mediated gene knock down of cell-surface receptors to significantly reduce FMDV infection in cell culture and transgenic suckling mice.

Innate immune responses in hepatitis B virus (HBV) infection

Hepatitis B virus (HBV) infection has a low rate of chronicity compared to HCV infection, but chronic liver inflammation can evolve to life threatening complications. Experimental data from HBV infected chimpanzees and HBV transgenic mice have indicated that cytotoxic T cells are the main cell type responsible for inhibition of viral replication, but also for hepatocyte lysis during chronic HBV infection. Their lower activation and impaired function in later stages of infection was suggested as a possible mechanism that allowed for low levels of viral replication. The lack of an interferon response in these models also indicated the importance of adaptive immunity in clearing the infection. Increased knowledge of the signalling pathways and pathogen associated molecular patterns that govern activation of innate immunity in the early stages of viral infections in general has led to a re-evaluation of the innate immune system in HBV infection. Numerous studies have shown that HBV employs active strategies to evade innate immune responses and induce immunosuppression. Some of the immune components targeted by HBV include dendritic cells, natural killer cells, T regulatory cells and signalling pathways of the interferon response. This review will present the current understanding of innate immunity in HBV infection and of the challenges associated with clearing of the HBV infection.

Point Mutations in FimH Adhesin of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Enhance Intestinal Inflammatory Response

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH LF82 (expressing FimH with an AIEC-associated mutation) with fimH K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.

Gingival Lymphatic Drainage Protects Against Porphyromonas gingivalis–Induced Bone Loss in Mice

Periodontitis is characterized by tissue destruction and bone loss mainly due to inflammatory responses after bacterial challenge of the gingiva. Gingiva is supplied with lymphatics that drain interstitial fluid and transport immune cells to the lymph nodes for antigen presentation; yet, the role of lymphatics in periodontal disease development is unknown. To investigate the lymphatic function after periodontal infection, we used K14-VEGF receptor 3-Ig (K14) mice that lack lymphatics in gingiva. Mice were orally infected with human Porphyromonas gingivalis and observed for 42 days. The infected K14 mice developed significantly more bone loss than the wild-type mice, and were associated with an increased number of macrophages and major histocompatibility complex class II antigen-presenting cells in the bone resorptional areas. The infected transgenic mice expressed a significant higher periodontal level of several proinflammatory cytokines, whereas the plasma level of P. gingivalis IgG was significantly lower than in the wild-type mice. No differences were found in immune cell distribution in draining lymph nodes between the strains. Our results show that a strong periodontal inflammatory response and a weakened systemic humoral B-cell response took place in K14 mice after infection. We conclude that gingival lymphatics protect against P. gingivalis-induced periodontitis, and we speculate that they are critical in the protection by clearance of infection and by promotion of humoral immune responses.

Integrity of Helix 2-Helix 3 Domain of the PrP Protein Is Not Mandatory for Prion Replication

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.

The Toll-Like Receptor 3-Mediated Antiviral Response Is Important for Protection against Poliovirus Infection in Poliovirus Receptor Transgenic Mice

RIG-I-like receptors and Toll-like receptors (TLRs) play important roles in the recognition of viral infections. However, how these molecules contribute to the defense against poliovirus (PV) infection remains unclear. We characterized the roles of these sensors in PV infection in transgenic mice expressing the PV receptor. We observed that alpha/beta interferon (IFN-α/β) production in response to PV infection occurred in an MDA5-dependent but RIG-I-independent manner in primary cultured kidney cells in vitro. These results suggest that, similar to the RNA of other picornaviruses, PV RNA is recognized by MDA5. However, serum IFN-α levels, the viral load in nonneural tissues, and mortality rates did not differ significantly between MDA5-deficient mice and wild-type mice. In contrast, we observed that serum IFN production was abrogated and that the viral load in nonneural tissues and mortality rates were both markedly higher in TIR domain-containing adaptor-inducing IFN-β (TRIF)-deficient and TLR3-deficient mice than in wild-type mice. The mortality rate of MyD88-deficient mice was slightly higher than that of wild-type mice. These results suggest that multiple pathways are involved in the antiviral response in mice and that the TLR3-TRIF-mediated signaling pathway plays an essential role in the antiviral response against PV infection.

Pathogenesis of Chronic Wasting Disease in Cervidized Transgenic Mice

Chronic wasting disease (CWD) is a fatal, endemic prion disease of wild and captive cervids, including deer, elk, and moose. Typical of prion diseases, CWD is characterized by the conversion of the native, protease-sensitive protein PrP(C) to a protease-resistant isoform, denoted as PrP(RES). Here we have studied the expression of cervid PrP(C) and the pathogenesis of CWD infection in transgenic mice expressing the normal cervid prion protein (Tg[CerPrP] mice). Using tissue-based in situ immunohistochemistry protocols, we first identified cervid PrP(C) expression in the lymphoid, nervous, hemopoietic, endocrine, and certain epithelial tissues of Tg[CerPrP] mice. Tg[CerPrP] mice were then inoculated with CWD via one of four routes (intracerebral, intravenous, intraperitoneal, or oral); all groups developed spongiform encephalopathy, although the oral route required a larger infecting dose. Incubation periods were 184 +/- 13, 218 +/- 15, 200 +/- 7, and 350 +/- 27 days after inoculation, respectively. In longitudinal studies, we tracked the appearance of PrP(RES) in the brain, spleen, Peyer's patches, lymph nodes, pancreatic islets of Langerhans, bone marrow, and salivary glands of preclinical and terminal mice. In addition, we documented horizontal transmission of CWD from inoculated mice and to un-inoculated cohabitant cage-mates. This work documents the multiroute susceptibility, pathogenesis, and lateral transmission of CWD infection in Tg[CerPrP] mice, affirming this model as a robust system to study this cervid transmissible spongiform encephalopathy.

Fatal Transmissible Amyloid Encephalopathy: A New Type of Prion Disease Associated with Lack of Prion Protein Membrane Anchoring

Prion diseases are fatal neurodegenerative diseases of humans and animals characterized by gray matter spongiosis and accumulation of aggregated, misfolded, protease-resistant prion protein (PrPres). PrPres can be deposited in brain in an amyloid-form and/or non-amyloid form, and is derived from host-encoded protease-sensitive PrP (PrPsen), a protein normally anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). Previously, using heterozygous transgenic mice expressing only anchorless PrP, we found that PrP anchoring to the cell membrane was required for typical clinical scrapie. However, in the present experiments, using homozygous transgenic mice expressing two-fold more anchorless PrP, scrapie infection induced a new fatal disease with unique clinical signs and altered neuropathology, compared to non-transgenic mice expressing only anchored PrP. Brain tissue of transgenic mice had high amounts of infectivity, and histopathology showed dense amyloid PrPres plaque deposits without gray matter spongiosis. In contrast, infected non-transgenic mice had diffuse non-amyloid PrPres deposits with significant gray matter spongiosis. Brain graft studies suggested that anchored PrPsen expression was required for gray matter spongiosis during prion infection. Furthermore, electron and light microscopic studies in infected transgenic mice demonstrated several pathogenic processes not seen in typical prion disease, including cerebral amyloid angiopathy and ultrastructural alterations in perivascular neuropil. These findings were similar to certain human familial prion diseases as well as to non-prion human neurodegenerative diseases, such as Alzheimer's disease.

Structural Characterization of Amyloids Comprised of Anchorless Prion Proteins

Prion diseases are fatal, neurodegenerative diseases that afflict sheep, cows, and humans. The key event in prion diseases is the conversion of the α-helical, cellular isoform of the prion protein (PrPC) to an insoluble, β-sheet-rich, infectious isoform (PrPSc). Host-encoded PrPC is anchored to the cell membrane by a glycosylphosphatidyl inositol (GPI) anchor and converts to GPI-anchored PrPSc during prion infection. Here we describe prion infection in transgenic mice (Tg8015) that lack the signal sequence for attachment of the GPI anchor and therefore express non-anchored, free-floating, PrPC (ΔGPI-PrP). The lack of the GPI anchor leads to incomplete modification of PrP, resulting in an unglycosylated molecule. Therefore, Tg8015 mice produce unglycosylated ΔGPI-PrPSc upon infection with the Rocky Mountain Laboratory (RML) prion strain. These mice did not show clinical signs of disease until >300 days post-infection (dpi), but their brains harbored ΔGPI-PrPSc and macroscopic amyloid deposits. In contrast, RML infection of wild-type (wt) mice resulted in clinical signs of disease within 120 dpi but no macroscopic amyloid formation. ΔGPI-PrPSc and wt PrPSc in the brains of Tg8015 and wt mice, respectively, were purified by two different protocols, both involving N-terminal truncation by proteinase K. The resulting, highly concentrated ΔGPI-PrP 27-30 and wt PrP 27-30 preparations were analyzed and compared by negative-stain electron microscopy to characterize possible differences in fibril morphology. Additional analyses were performed using Fourier transform infrared spectroscopy and X-ray fiber diffraction to compare the structures of ΔGPI-PrP 27-30 amyloid and wt PrP 27-30 amyloid preparations. Additionally, we tested whether ΔGPI-PrPSc could self-propagate in vitro by evaluating its seeding capacity in a prion-specific amyloid seeding assay and examined its biological activity in vivo using mouse-based bioassays.

Helicobacter pylori Eradication Prevents Progression of Gastric Cancer in Hypergastrinemic INS-GAS Mice

Helicobacter pylori infection results in chronic gastritis, which may progress to gastric cancer. In this study, we investigated the efficacy of H. pylori eradication in preventing the progression of gastritis to gastric cancer in H. pylori-infected transgenic INS-GAS mice. H. pylori infection induced severe dysplasia and gastric cancer classified as high-grade and low-grade gastrointestinal intraepithelial neoplasia (GIN) in INS-GAS mice at 28 weeks postinfection (WPI). H. pylori eradication therapy using omeprazole, metronidazole, and clarithromycin was administered p.o. at 8, 12, or 22 WPI. Compared with untreated infected mice, H. pylori eradication at 8, 12, and 22 WPI significantly reduced the severity of dysplasia (P < 0.01). Moreover, H. pylori eradication at 8 WPI completely prevented the development of GIN (P < 0.001). Although not as effective as early antimicrobial treatment, prevention of progression to high-grade GIN was achieved by H. pylori eradication at 12 and 22 WPI (P < 0.05). Consistent with reduced gastric pathology, H. pylori eradication at all time points significantly down-regulated gastric Interferon-gamma, tumor necrosis factor-alpha, inducible nitric oxide synthase, and Reg 1 mRNA levels (P < 0.05) and reduced epithelial proliferation in the corpus (P < 0.01) compared with untreated infected mice. We concluded that H. pylori eradication prevented gastric cancer to the greatest extent when antibiotics are given at an early point of infection, but that eradication therapy given at a later time point delayed the development of severe dysplastic lesions.

Disease‐associated prion protein is not detectable in human systemic amyloid deposits

Cerebral and cardiac amyloid deposits have been reported after scrapie infection in transgenic mice expressing variant prion protein (PrP(C)) lacking the glycophosphatidylinositol anchor. The amyloid fibril protein in the systemic amyloid deposits was not characterized, and there is no clinical or pathological association between prion diseases and systemic amyloidosis in humans. Nevertheless, in view of the potential clinical significance of these murine observations, we tested both human amyloidotic tissues and isolated amyloid fibrils for the presence of PrP(Sc), the prion protein conformation associated with transmissible spongiform encephalopathy (TSE). We also sequenced the complete prion protein gene, PRNP, in amyloidosis patients. No specific immunohistochemical staining for PrP(Sc) was obtained in the amyloidotic cardiac and other visceral tissues of patients with different types of systemic amyloidosis. No protease-resistant prion protein, PrP(res), was detectable by Western blotting of amyloid fibrils isolated from cardiac and other systemic amyloid deposits. Only the complete normal wild-type PRNP gene sequence was identified, including the usual distribution of codon 129 polymorphisms. These reassuringly negative results do not support the idea that there is any relationship of prions or TSE with human systemic amyloidosis, including cardiac amyloid deposition.

Structural Abnormalities in Neurons Are Sufficient To Explain the Clinical Disease and Fatal Outcome of Experimental Rabies in Yellow Fluorescent Protein-Expressing Transgenic Mice

Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resin-embedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies.