C3H/HeN Research Articles

Impaired neutrophil migration underpins host susceptibility to infectious colitis

Citrobacter rodentium models infection with enteropathogenic E. coli and ulcerative colitis (UC). While C57BL/6 (C57) mice recover, C3H/HeN (C3H) mice succumb to infection, partially due to increased colonic neutrophil elastase activity, also seen in UC patients; however, the underlying cause was unknown. Here we found that bone marrow, blood and colonic C57 neutrophils expressed CD11bHi and reached the infected colonic lumen where they underwent productive NETosis. In contrast, while the number of C3H neutrophils increased in the bone marrow, blood, and colon, they remained CD11bLo and got trapped in the submucosa, away from C. rodentium, where they underwent harmful NETosis. CD11bLo neutrophils in C3H mice infected with CRi9, which triggers expression of neutrophil chemoattractants, reached the colonisation site resulting in host survival. UC patient neutrophils also displayed decreased levels of the activation/differentiation markers CD16/CXCR4. These results, suggesting that neutrophil malfunction contributes to exacerbated colitis, provide insight for future therapeutic prospects.

Genetic Deciphering of Prepulse Inhibition Reveals the Putative Role of an Atypical Cadherin in Schizophrenia Pathogenesis

Prepulse inhibition (PPI) of the startle response reflects a sensory-motor gating mechanism, and impaired PPI is a characteristic endophenotype of schizophrenia. We have previously localized quantitative trait loci (QTL) for PPI in chromosome 10 by analyzing 1,010 F2 mice derived by crossing C57BL/6N (B6) animals that showed high PPI with C3H/HeN (C3) animals that showed low PPI. However, large number of genes in the QTL hinders delineating precise role of genes in defining PPI.

Carbaryl Modulates Chronobiological Behaviors via Melatonin Receptors

Humans are exposed to low levels of carbaryl and other carbamate insecticides found in household products, food, and water due to persistent and widespread use. Environmental and occupational exposure to carbamate pesticides is linked to metabolic disorders and sleep apnea, though it is not clear which biological targets contribute to disease pathology (Montgomery et al. 2008; James‐Todd et al., 2016; Baumert et al. 2017). Carbaryl binds to MT1 and MT2 G protein‐coupled receptors with varying efficacies and potencies as demonstrated by our team via in‐silico molecular modeling and in‐vitro bioassays (Popovska‐Gorevski et al. 2017; Jones et al. 2017 EB Abst; Glatfelter et al. 2017 EB Abst). We hypothesize that carbaryl may produce alterations in circadian rhythms through interactions with melatonin receptors that could contribute to the aforementioned disease symptomology, progression, and/or pathologies. Previous in‐vitro (1–100μM) and ex‐vivo (10 mg/kg, i.p.) experiments demonstrated specificity for competition of carbaryl for 2‐[125I]‐iodomelatonin binding to brain melatonin receptors and its ability to reach target brain areas controlling circadian rhythms in‐vivo (e.g. suprachiasmatic nucleus: SCN; Glatfelter et al. 2017 EB Abs). We tested the ability of carbaryl (10 mg/kg, i.p.) to phase shift onset of circadian running wheel activity when given at circadian time (CT) 10 (CT12 = onset of running wheel activity in constant dark) for 3 days in wild‐type C3H/HeN (C3H) mice. Our results indicate that carbaryl (0.81h ± 0.1; n=12; p<0.0001) and positive control melatonin (3mg/kg s.c.; 0.97h ± 0.2; n=4; p<0.001) significantly phase advance running wheel activity onset compared to vehicle (0.03h ± 0.04; n=7) treated controls (F2,20=18.46; p<0.05; Dunnet's Post Test). In the same paradigm, carbaryl (10 mg/kg i.p.) given 10 hrs before onset of running wheel activity in constant dark at CT 2 showed a phase delay (−1.2h ± 0.11; n=14; p<0.0001) similar to positive control melatonin (3mg/kg s.c. 1.3h ± 0.14; n=4; p<0.001) compared to vehicle (−0.12h ± 0.02; n=19) treated controls (F2,34=75.96; p<0.05; Dunnet's Post Test). These data demonstrate that carbaryl has the same periods of circadian sensitivity for phase shift at CT2 & CT10 as melatonin (Benloucif & Dubocovich 1996). A dose response curve for carbaryl (0.01 – 10 mg/kg i.p.) to phase advance circadian running wheel activity rhythms at CT10 reveals an ED50 value of 0.019 mg/kg, i.p. which is similar in magnitude to melatonin (ED50=0.024 mg/kg s.c. Dubocovich et al. 1998). Carbaryl (10 mg/kg i.p.) did not phase advance circadian running wheel activity rhythms in C3H MT1KO mice (−0.04h ± 0.04; n=18; n.s.) at CT10 compared to vehicle controls (−0.04h ± 0.03; n=19; t=0.12; n.s.) suggesting an MT1 dependent mechanism. We conclude that carbaryl phase shifts circadian activity rhythms via activation of MT1 melatonin receptors suggesting environmental or occupational exposure to this insecticide in the tested dose range could influence disease pathologies via modulation of circadian biology.Support or Funding InformationSupported by ES 023684 and UB Jacobs School of Medicine funds to MLD and RVR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of Interleukin 10 by Borrelia burgdorferi Is Regulated by the Action of CD14-Dependent p38 Mitogen-Activated Protein Kinase and cAMP-Mediated Chromatin Remodeling.

Host genotype influences the severity of murine Lyme borreliosis, caused by the spirochetal bacterium Borrelia burgdorferi C57BL/6 (B6) mice develop mild Lyme arthritis, whereas C3H/HeN (C3H) mice develop severe Lyme arthritis. Differential expression of interleukin 10 (IL-10) has long been associated with mouse strain differences in Lyme pathogenesis; however, the underlying mechanism(s) of this genotype-specific IL-10 regulation remained elusive. Herein we reveal a cAMP-mediated mechanism of IL-10 regulation in B6 macrophages that is substantially diminished in C3H macrophages. Under cAMP and CD14-p38 mitogen-activated protein kinase (MAPK) signaling, B6 macrophages stimulated with B. burgdorferi produce increased amounts of IL-10 and decreased levels of arthritogenic cytokines, including tumor necrosis factor (TNF). cAMP relaxes chromatin, while p38 increases binding of the transcription factors signal transducer and activator of transcription 3 (STAT3) and specific protein 1 (SP1) to the IL-10 promoter, leading to increased IL-10 production in B6 bone marrow-derived monocytes (BMDMs). Conversely, macrophages derived from arthritis-susceptible C3H mice possess significantly less endogenous cAMP, produce less IL-10, and thus are ill equipped to mitigate the damaging consequences of B. burgdorferi-induced TNF. Intriguingly, an altered balance between anti-inflammatory and proinflammatory cytokines and CD14-dependent regulatory mechanisms also is operative in primary human peripheral blood-derived monocytes, providing potential insight into the clinical spectrum of human Lyme disease. In line with this notion, we have demonstrated that cAMP-enhancing drugs increase IL-10 production in myeloid cells, thus curtailing inflammation associated with murine Lyme borreliosis. Discovery of novel treatments or repurposing of FDA-approved cAMP-modulating medications may be a promising avenue for treatment of patients with adverse clinical outcomes, including certain post-Lyme complications, in whom dysregulated immune responses may play a role.

Automated video analysis system reveals distinct diurnal behaviors in C57BL/6 and C3H/HeN mice

Advances in rodent behavior dissection using automated video recording and analysis allows detailed phenotyping. This study compared and contrasted 15 diurnal behaviors recorded continuously using an automated behavioral analysis system for a period of 14 days under a 14/10 light/dark cycle in single housed C3H/HeN (C3H) or C57BL/6 (C57) male mice. Diurnal behaviors, recorded with minimal experimental interference and analyzed using phenotypic array and temporal distribution analysis showed bimodal and unimodal profiles in the C57 and C3H mice, respectively. Phenotypic array analysis revealed distinct behavioral rhythms in Activity-Like Behaviors (i.e. walk, hang, jump, come down) (ALB), Exploration-Like Behaviors (i.e. dig, groom, rear up, sniff, stretch) (ELB), Ingestion-Like Behaviors (i.e. drink, eat) (ILB) and Resting-Like Behaviors (i.e. awake, remain low, rest, twitch) (RLB) of C3H and C57 mice. Temporal distribution analysis demonstrated that strain and time of day affects the magnitude and distribution of the spontaneous homecage behaviors. Wheel running activity, water and food measurements correlated with timing of homecage behaviors. Subcutaneous (3mg/kg, sc) or oral (0.02mg/ml, oral) melatonin treatments in C57 mice did not modify either the total 24h magnitude or temporal distribution of homecage behaviors when compared with vehicle treatments. We conclude that C3H and C57 mice show different spontaneous activity and behavioral rhythms specifically during the night period which are not modulated by melatonin.

Different patterns of expression and of IL-10 modulation of inflammatory mediators from macrophages of Lyme disease-resistant and -susceptible mice.

C57BL/6J (C57) mice develop mild arthritis (Lyme disease-resistant) whereas C3H/HeN (C3H) mice develop severe arthritis (Lyme disease-susceptible) after infection with the spirochete Borrelia burgdorferi. We hypothesized that susceptibility and resistance to Lyme disease, as modeled in mice, is associated with early induction and regulation of inflammatory mediators by innate immune cells after their exposure to live B. burgdorferi spirochetes. Here, we employed multiplex ELISA and qRT-PCR to investigate quantitative differences in the levels of cytokines and chemokines produced by bone marrow-derived macrophages from C57 and C3H mice after these cells were exposed ex vivo to live spirochetes or spirochetal lipoprotein. Upon stimulation, the production of both cytokines and chemokines was up-regulated in macrophages from both mouse strains. Interestingly, however, our results uncovered two distinct patterns of spirochete- and lipoprotein-inducible inflammatory mediators displayed by mouse macrophages, such that the magnitude of the chemokine up-regulation was larger in C57 cells than it was in C3H cells, for most chemokines. Conversely, cytokine up-regulation was more intense in C3H cells. Gene transcript analyses showed that the displayed patterns of inflammatory mediators were associated with a TLR2/TLR1 transcript imbalance: C3H macrophages expressed higher TLR2 transcript levels as compared to those expressed by C57 macrophages. Exogenous IL-10 dampened production of inflammatory mediators, especially those elicited by lipoprotein stimulation. Neutralization of endogenously produced IL-10 increased production of inflammatory mediators, notably by macrophages of C57 mice, which also displayed more IL-10 than C3H macrophages. The distinct patterns of pro-inflammatory mediator production, along with TLR2/TLR1 expression, and regulation in macrophages from Lyme disease-resistant and -susceptible mice suggests itself as a blueprint to further investigate differential pathogenesis of Lyme disease.

Immunotoxicologic effects of cyclosporine on tumor progression in models of squamous cell carcinoma and B-cell lymphoma in C3H mice

Many immunosuppressive drugs are associated with an increased risk of neoplasia, principally non-melanoma skin cancers and B-cell lymphomas. However, only 6 of the 13 immunosuppressive drugs tested in 2 year bioassays increased the incidence of neoplasia. For example, the 2-year bioassays conducted with cyclosporine (CSA), an International Agency for Research on Cancer (IARC) Group 1 human carcinogen, were negative. The purpose of these investigations was to use transplanted tumor models in immunocompetent, syngeneic mice to gain insight into the failure of the 2-year bioassay to show an increased incidence of neoplasia with CSA. C3H HeN mice were used in a battery of assays with a transplanted squamous cell carcinoma (SCC VII cells) or a B-cell, lymphoma (38C13 cells) cells to study effects of CSA on local growth and metastases, experimental metastases, and progression of established metastases. Mice received CSA twice weekly by subcutaneous (SC) injection at doses of 0.5, 5, or 50 mg/kg; controls received the CSA vehicle. CSA had a modest inhibitory effect on SC tumors initiated by 38C13 cells and on intramuscular tumors initiated by SCC VII cells. CSA also decreased the number of lung colonies and decreased the size, growth fraction and vascularity of established lung metastases initiated by SCC VII cells. In contrast, CSA increased progressive growth of metastases to the sentinel lymph node from an intramuscular SCC VII tumor, but had no effect cellular traffic to the node. In conclusion, CSA at doses up to 50 mg/kg did not facilitate tumor progression and it partially inhibited tumor growth, suggesting that suppression of tumor progression may partially explain the failure of CSA to act as a carcinogen in 2 year bioassays.

Identification of novel immune factors regulating outcome to viral encephalitis. (170.12)

Abstract Viewed as a public health and zoonotic threat, Venezuelan equine encephalitis virus (VEEV) causes morbidity and mortality in both human and equine populations during naturally occurring epizootic outbreaks. The specific immune factors contributing to lethality or survival following viral invasion of the brain remain largely unknown. Using intranasal infection of the vaccine strain of VEEV (TC83), we established three models of brain infection: (1) Lethal infection in C3H/HeN (C3H) mice (2) Non-lethal infection in C57BL/6 (C57) mice (3) Non-lethal infection followed by chronic viral persistence in α/β T cell-receptor deficient mice (TCR KO) on a C57 background. The three models display similar viral kinetics with each developing a high level viral load in the brain. While absence of a functional T-cell compartment contributes to persistent brain infection, profiling the host response using genomics and multiplex cytokine array identified natural killer (NK) cells and type I interferon (IFN) as potential prognosticators of outcome. The systemic depletion of NK cells enabled C3H mice to tolerate brain infection without developing lethal disease. The trend toward lower INF-β levels in the brains of lethally infected animals compared to surviving strains corroborates a protective role for IFN in the brain. The novel knowledge generated in this model system enabled identification of intervention points that influence a positive outcome to viral invasion of the brain.

Innate immune cells are the major determinants of host-related susceptibility differences to experimental spotted fever rickettsiosis (42.24)

Abstract Infection of C3H/HeN (C3H) mice with 3 × 105 PFU of R. conorii resulted in 100% mortality, while all C57BL/6 (B6) mice survived the same inoculum. To unravel the strategy by which R. conorii establishes acute fatal disease, we explored its early interaction with the different immune systems in these two mouse strains. Compared to sublethal infection in C3H mice, rickettsiae induced a greater cytotoxic T cell response in infected B6 mice. Lethal infection suppressed antigen-specific proliferation of CD4+ and CD8+ T cells on day 5 post infection, which correlated with upregulated expression levels of PD-1/PD-L1 on T cells and dendritic cells (DCs), respectively. Resistant mice completely cleared the bacteria in four days with greatly expanded cytotoxic NK cells, IFN-γ(+) CD8+ T cells, DCs, but not neutrophils, and enhanced production of MCP-1, MCP-5, RANTES, and IL-12p70. The high resistance of RAG -/- mice on a B6 background to rickettsial infection suggested that the host resistance was determined at the innate immune cell level. Finally, R. conorii induced altered expression of TLR2 and TLR9 in bone marrow derived DCs from C3H and B6 mice, respectively, suggesting that differential TLR signaling pathway was involved in the innate immune responses to rickettsial infection in these two mouse strains. These data suggest that NK cell activation by DCs and their migration to infected sites may play a critical role in innate resistance against Rickettsia.

Inefficiency of C3H/HeN Mice to Control Chlamydial Lung Infection Correlates with Downregulation of Neutrophil Activation During the Late Stage of Infection

We previously reported that massive infiltration of neutrophils in C3H/HeN (C3H) mice could not efficiently control Chlamydia muridarum (Cm) infection and might contribute to the high susceptibility of these mice to lung infection. To further define the nature of neutrophil responses in C3H mice during chlamydial infection, we examine the expression of adhesion molecules and CD11b related to neutrophils infiltration and activation, respectively, following intranasal Cm infection. The results showed that the expression of selectins (E-selectin, P-selectin and L-selectin), and intercellular cell adhesion molecule-1 (ICAM-1) in the lung of C3H mice increased more significantly than in C57BL/6 (B6) mice, the more resistant strain. These results correlated well with the massive neutrophils infiltration in C3H mice. In contrast, CD11b expression on peripheral blood and lung neutrophils in C3H mice exhibited a significant reduction compared with B6 mice during the late phage of infection (day 14). These findings suggest that the high-level expression of adhesion molecules in C3H mice may enhance neutrophils recruitment to the lung, but the decline of CD11b expression on neutrophils may attenuate neutrophil function. Therefore, CD11b down-regulation on neutrophils may contribute to the failure of C3H mice to control chlamydial lung infection.

Differences in gene expression and benzo[a]pyrene‐induced DNA adduct formation in the liver of three strains of female mice with identical AhRb2 genotype treated with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin and/or benzo[a]pyrene

To search for genes whose products modify aryl hydrocarbon receptor (AhR)-dependent toxicity caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), gene expression profiles in the liver were surveyed using microarrays 24 h after the administration of TCDD to three strains of female mice, BALB/cAnN (BALB), C3H/HeN (C3H) and CBA/JN (CBA) all of identical AhR genotype. The BALB/cAnN strain had a more marked induction of a number of glutathione S-transferase (GST) sub-families, particularly the GSTmicro gene family, compared with the other two strains. To assess the effects of GSTs induction to metabolize carcinogens, TCDD (40 microg kg(-1)) was administered to BALB and CBA strains, followed 24 h later by an i.p. injection of low or high dose of benzo[a]pyrene (B[a]P, 50 or 200 mg kg(-1)). The 32P-postlabelling analysis showed that administration of TCDD alone failed to induce DNA adduct formation in both BALB and CBA strain mouse livers. The low dose of B[a]P alone produced DNA adduct in the liver of both strains to a similar extent. Treatment with TCDD 24 h before the low dose of B[a]P suppressed the formation of B[a]P-induced DNA-adduct more markedly in the BALB strain compared with the CBA strain. Taken together, these findings show that TCDD treatment causes strain-specific alterations in gene expression and B[a]P-induced DNA adduct formation in the liver of female mice of the same AhRb2 genotype. Furthermore, it suggests that TCDD-treated female mice of the BALB strain may have genes whose products modify the toxicity of B[a]P as evidenced by TCDD-induced alterations in B[a]P-DNA adduct formation.

Tissue tropism and neuroinvasion of West Nile virus do not differ for two mouse strains with different survival rates

West Nile virus (WNV) is a mosquito-borne flavivirus that infects the central nervous system of humans and other animals. In this study, we found that C3H/HeN (C3H) mice exhibited a higher morbidity and mortality than C57BL/6 (B6) mice. We compared tissue tropism, viral replication and kinetics for C3H and B6 mice during acute viral infection. WNV was detected in multiple tissues, including novel sites such as the skin, duodenum and pancreas, but the tropism was identical for the two strains. Additionally, viral load and kinetics of spread did not differ substantially between strains. Neuroinvasion occurred in both strains by day 3 post-inoculation with early detection in the olfactory bulbs and spinal cord, suggesting that WNV neuroinvades at specific sites. Furthermore, neuroinvasion and viral load in the CNS did not predict disease outcome. Our data suggest that the disparities in morbidity and mortality between C3H and B6 mice are not due to differences in tropism, viral load or kinetics during acute WNV infection.

Differential Interaction of Dendritic Cells withRickettsia conorii: Impact on Host Susceptibility to Murine Spotted Fever Rickettsiosis

Spotted fever group rickettsioses are emerging and reemerging infectious diseases, some of which are life-threatening. In order to understand how dendritic cells (DCs) contribute to the host resistance or susceptibility to rickettsial diseases, we first characterized the in vitro interaction of rickettsiae with bone marrow-derived DCs (BMDCs) from resistant C57BL/6 (B6) and susceptible C3H/HeN (C3H) mice. In contrast to the exclusively cytosolic localization within endothelial cells, rickettsiae efficiently entered and localized in both phagosomes and cytosol of BMDCs from both mouse strains. Rickettsia conorii-infected BMDCs from resistant mice harbored higher bacterial loads compared to C3H mice. R. conorii infection induced maturation of BMDCs from both mouse strains as judged by upregulated expression of classical major histocompatibility complex (MHC) and costimulatory molecules. Compared to C3H counterparts, B6 BMDCs exhibited higher expression levels of MHC class II and higher interleukin-12 (IL-12) p40 production upon rickettsial infection and were more potent in priming naïve CD4(+) T cells to produce gamma interferon. In vitro DC infection and T-cell priming studies suggested a delayed CD4(+) T-cell activation and suppressed Th1/Th2 cell development in C3H mice. The suppressive CD4(+) T-cell responses seen in C3H mice were associated with a high frequency of Foxp3(+) T regulatory cells promoted by syngeneic R. conorii-infected BMDCs in the presence of IL-2. These data suggest that rickettsiae can target DCs to stimulate a protective type 1 response in resistant hosts but suppressive adaptive immunity in susceptible hosts.

Characterization of differential innate and adaptive immunity against Rickettsia conorii in resistant and susceptible mice (52.14)

Abstract Spotted fever group (SFG) rickettsiae cause severe human diseases in many endemic areas of the world. In order to identify the critical factors in determining host susceptibilities or resistance to SFG rickettsiosis, we characterized cellular immunity against R. conorii in highly susceptible C3H/HeN (C3H) mice, relatively susceptible BALB/c mice and highly resistant C57BL/6 (B6) mice. A dose of rickettsiae lethal to C3H mice is cleared by all wild type (WT) B6 and BALB/c mice in 4 days with survival. Compared to C3H mice, significantly higher expansion of natural killer (NK) cells was detected in WT B6 mice. Similar to the respective WT mice, all RAG−/− and IL-12p40−/− knockout mice on B6 background and SCID on BALB/c background survived, whereas BALB/c SCID mice with deficiency of NK cells died. These findings suggest that NK cells play a major role in the innate immunity, which is IL-12p40- independent in the resistant host. Protection against sublethal rickettsial infection in C3H mice was associated with a CD4+ Th1 cell response, the presence of greater quantities of inducible CD4+CD25+ T regulatory cells and CTL than in B6 mice. These findings suggest that a cell-mediated Th1 immune response is critical for protection against rickettsial infection in the susceptible host and T regulatory cells may contribute to the outcome of rickettsial infection, most likely through balancing T cell mediated protective immunity and immunopathology. This work was supported by a grant (AI021242) from the National Institute of Allergy and Infectious Diseases.

Repeated Topical Challenge with Chemical Antigen Elicits Sustained Dermatitis in NC/Nga Mice in Specific-Pathogen-Free Condition

NC/Nga mice are known to develop skin lesions resembling to atopic dermatitis (AD) in conventional but not in specific-pathogen-free (SPF) condition. An epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) increased skin thickness in C3H as well as NC/Nga mice in SPF environment, and the response was enlarged by repeating the challenge at weekly intervals. Although the skin reaction in C3H mice was ameliorated when the challenge was discontinued after the fifth application, the reaction in NC/Nga mice was sustained at least for 3 wk. Analyses of cytokine production by CD4+ cells from the draining lymph node proximal to the lesions revealed that, unlike C3H mice, NC/Nga mice fail to induce T helper 2 (Th2) cytokine interleukin-4 (IL-4), whereas the level of Th1 cytokine interferon-gamma in NC/Nga mice is equivalent to that of C3H mice. In addition, NC/Nga mice highly expressed IL-12, a cytokine-preventing formation of Th2 response, whereas C3H mice did not. Administration of anti-IL-12 antibody reduced duration of dermatitis in DNFB-treated NC/Nga mice. Taken together, our data suggest that IL-12 plays a role in the persistent skin reaction in NC/Nga mice. The action of IL-12 might be mediated by the decrease in IL-4 production.

Less inhibition of interferon-gamma to organism growth in host cells may contribute to the high susceptibility of C3H mice to Chlamydia trachomatis lung infection.

T-helper-1-like cytokine response and cell-mediated immunity have been shown to be critical in host resistance to Chlamydia trachomatis infection. Using a murine pneumonia model, we compared the susceptibility of C3H/HeN (C3H) and C57BL/6 mice to C. trachomatis mouse pneumonitis (MoPn) infection. C3H mice exhibited significantly higher mortality, greater organism growth and much more severe pathological changes in the lung compared with C57BL/6 mice. However, the pattern of adaptive immune responses including organism-specific delayed-type hypersensitivity, antibody responses and cytokine [interferon-gamma (IFN-gamma), interleukin-12 (IL-12), IL-4, IL-10 and tumour necrosis factor alpha] production by spleen and local draining lymph node cells in these two strains of mice appeared comparable during the process of infection. Interestingly, MoPn growth in the cultured ex vivo macrophages from C3H mice was found to be significantly less inhibited by the exogenous IFN-gamma present in the culture compared to C57BL/6 mice. The lower inhibition of MoPn growth in C3H mice was associated with significantly lower nitric oxide production by the infected macrophages following IFN-gamma stimulation. The data suggest that the cellular events downstream of cytokine production in chlamydia host cells may be important in determining the different susceptibility of hosts to chlamydial infection.

Independent variation in susceptibilities of six different mouse strains to induction of pepsinogen-altered pyloric glands and gastric tumor intestinalization by N-methyl- N-nitrosourea

Strain differences in susceptibility regarding stomach carcinogenesis due to N-methyl- N-nitrosourea were examined in males of six strains of mice: BALB/cA (BALB), C57BL/6N (C57BL6), CBA/JN (CBA), C3H/HeN (C3H), DBA/2N (DBA/2), and CD-1 (ICR). The frequency of pepsinogen-altered pyloric glands (PAPGs), putative precancerous lesions, was highest (19.6±9.9%) in the BALB and lowest in the ICR (12.3±5.7%) mice ( P<0.05). Incidences of adenocarcinomas at week 52 were 59.3% (16 of 27) and 18.5% (5 of 27), respectively ( P<0.005). Invasion also tended to be deepest in BALB compared with the other strains. Intestinal alkaline phosphatase-positive intestinal type cells were observed heterogeneously in some hyperplasias, adenomas and adenocarcinomas consisting of gastric type cells. Thus, intestinalization appeared to occur at random in both non-neoplastic and monoclonal neoplastic lesions, making it unlikely that IAP-positive cells could be precursors of gastric tumors. In contrast, the data suggest a direct histogenetic role for the PAPG, a useful preneoplastic marker lesion in mouse strains.

Borrelia burgdorferi gene expression in vivo and spirochete pathogenicity.

Borrelia burgdorferi spirochetes that do not cause arthritis or carditis were developed and used to investigate Lyme disease pathogenesis. A clonal isolate of B. burgdorferi N40 (cN40), which induces disease in C3H/HeN (C3H) mice, was repeatedly passaged in vitro to generate nonpathogenic spirochetes. The passage 75 isolate (N40-75) was infectious for C3H mice but did not cause arthritis or carditis, and spirochetes were at low levels or absent in the joints or hearts, respectively. N40-75 could, however, cause disease in severe combined immunodeficient (SCID) mice, suggesting that the response in immunocompetent mice prevented effective spirochete dissemination and the subsequent development of arthritis and carditis. Administration of immune sera at 4 days after spirochete challenge aborted N40-75, but not cN40, infection in SCID mice. A B. burgdorferi genomic expression library was differentially probed with sera from cN40- and N40-75-infected mice, to identify genes that may not be effectively expressed by N40-75 in vivo. N40-75 was defective in the up-regulation of several genes that are preferentially expressed during mammalian infection, including dbpAB, bba64, and genes that map to the cp32 family of plasmids. These data suggest that adaptation and gene expression may be required for B. burgdorferi to effectively colonize the host, evade humoral responses, and cause disease.

Mouse strain-dependent variation in the course and outcome of chlamydial genital tract infection is associated with differences in host response.

Whether there is a pathogenic or protective outcome to chlamydial infection may be defined by the host response. We infected C57BL/6 (C57) and C3H/HeN (C3H) mice with the human biovar of Chlamydia trachomatis, serovar E, and, in select experiments, with the mouse pneumonitis agent of C. trachomatis (MoPn). We compared the courses of infection, histopathology, and host responses that resulted from these infections. The duration of infection with either chlamydial biovar was significantly increased in the C3H strain of mice. The intensity of infection was examined in mice infected with serovar E, and it was significantly increased in the C3H strain. Histopathology revealed the incidence of severe hydrosalpinx to be significantly greater in C3H mice than in C57 mice. In contrast, severe distention of the uterine horns was observed in all infected C57 mice compared to none of the C3H mice infected with serovar E and only 25% of those infected with MoPn. Acute inflammation was significantly increased in the uterine horns of C57 mice compared to that of C3H mice. Examination of antigen-specific responses revealed qualitatively similar responses in the two strains. Determination of gamma interferon- versus interleukin 4- producing cells revealed the predominance of a Th1 response in both strains. Serum enzyme-linked immunosorbent assays for immunoglobulin G1 (IgG1) and IgG2a revealed a predominance of IgG2a antibody in both strains, although the levels of antibody were significantly greater in C3H mice. Lymphocyte proliferation studies revealed increased proliferation in the iliac nodes of both strains at 1 to 3 weeks after infection. Because of the early eradication of infection observed in the C57 strain, we explored the relative production of tumor necrosis factor alpha (TNF-alpha) in the two strains. TNF-alpha levels were significantly increased in the genital tract secretions of C57 mice compared to that of C3H mice during the first week of infection. Increased TNF-alpha may be beneficial to the host by leading to earlier eradication of infection, thereby preventing infection of the oviduct and thus the major disease sequelae associated with chlamydial infection of the genital tract.

Reactivation of chlamydial genital tract infection in mice.

A model was developed to study chlamydial quiescence in C3H/HeN (C3H) and C57BL/6N (C57) mice following genital tract infection by Chlamydia trachomatis MoPn. Reactivation of chlamydial shedding following immunosuppression indicated that viable MoPn remained in the genital tract for up to 4 or 5 weeks after the apparent clearance of a primary infection. Either cyclophosphamide or cortisone acetate treatment could cause reactivation, but cyclophosphamide was more effective. However, the frequency of reactivation by either drug diminished with time in both mouse strains. Progesterone treatment prior to infection of C57 mice greatly reduced the frequency of reactivation by cyclophosphamide and also correlated with the development of marked fluid accumulation and distension of the uterine horns in the vast majority of those animals. This pathology was apparent by 5 to 7 weeks postinfection and was consistently seen through 110 days postinfection. Neither of these phenomena was observed in C57 mice that had not been treated with progesterone or in C3H mice under any conditions tested. The infecting dose of MoPn did not clearly influence the frequency of reactivation in either inbred strain as defined by this model.