Quinolinic Acid Levels in a Murine Retrovirus-Induced Immunodeficiency Syndrome

Abstract: Mice infected with the retrovirus mixture designated LP-BM5 murine leukemia virus (MuLV) develop an immunosuppressive disease. Quinolinic acid (QUIN) is an endogenous neurotoxic N -methyl- d -aspartate agonist that may contribute to the pathogenesis of HIV-associated neurologic disease. In the present study, the levels of QUIN in brain and blood were measured in mice infected with LP-BM5 MuLV and compared with those in uninfected mice and mice infected with the nonpathogenic strain of ecotropic MuLV (helper component of LP-BM5 MuLV). Infection with LP-BM5 MuLV resulted in progressive increases in blood QUIN levels beginning 2 weeks after inoculation that peaked by 16 weeks postinfection. QUIN levels were also increased in cerebral cortex, hippocampus, and striatum. In systemic tissues, QUIN levels were increased in lung, liver, and spleen. In contrast, infection with the ecotropic viral component of the LP-BM5 MuLV mixture was not associated with any changes in brain, blood, or systemic tissue QUIN levels, even though helper virus burdens were comparable to those in mice infected with LP-BM5 MuLV. Treatment of LP-BM5 MuLV-infected mice with the antiretroviral agent zidovudine (azidothymidine) significantly reduced blood and brain QUIN levels in association with reductions in viral load in brain and spleen. These observations suggest that elevated QUIN production is not attributable to productive infection with retrovirus per se but occurs in response to an agent or agents, such as cytokines, that are produced by the host in response to virus infection.     

Infectivity of scrapie prions bound to a stainless steel surface.

BACKGROUND: The transmissible agent of Creutzfeldt-Jakob disease (CJD) is not readily destroyed by conventional sterilization and transmissions by surgical instruments have been reported. Decontamination studies have been carried out thus far on solutions or suspensions of the agent and may not reflect the behavior of surface-bound infectivity. MATERIALS AND METHODS: As a model for contaminated surgical instruments, thin stainless-steel wire segments were exposed to scrapie agent, washed exhaustively with or without treatment with 10% formaldehyde, and implanted into the brains of indicator mice. Infectivity was estimated from the time elapsing to terminal disease. RESULTS: Stainless steel wire (0.15 x 5 mm) exposed to scrapie-infected mouse brain homogenate and washed extensively with PBS retained the equivalent of about 10(5) LD50 units per segment. Treatment with 10% formaldehyde for 1 hr reduced this value by only about 30-fold. CONCLUSIONS: The model system we have devised confirms the anecdotal reports that steel instruments can retain CJD infectivity even after formaldehyde treatment. It lends itself to a systematic study of the conditions required to effectively inactivate CJD, bovine spongiform encephalopathy, and scrapie agent adsorbed to stainless steel surfaces such as those of surgical instruments.     

Cell-mediated immune responses in mice infected with fonsecaea pedrosoi

Time course of cellular and humoral immune responses in mice infected with Fonsecaea pedrosoi was investigated by using an antigen prepared from culture filtrate of this fungus. Mice were infected by intravenous injection with yeast-like cells of the fungus. Viable fungus was recovered from the brain of the infected mice until the 36th day after inoculation, and from the other organs examined until 14th to 16th day. Inflammatory lesions were observed in the brain, lung, heart, liver, spleen, kidney and intestine during the first 30 days after inoculation. Macrophage migration inhibition factor response in these mice was insignificant until 8 days after inoculation. A significant response was developed at day 10 and persisted until day 63. This response returned negative by 95 days after inoculation. Lymphocyte transformation response of these mice was negative until 4 days after inoculation. At day 6 blastogenic index increased to 1.5, and at day 10, 14 and 16 the indices were 1.8, 2.4 and 1.7 respectively. Precipitin response to this fungus could not be detected in these mice until 16 days after inoculation. Positive results were obtained at day 21 and lasted until 51 days after inoculation. The precipitin titers, however, did not exceed one fold in any of these mice.     

Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD

Background

The evolution of the variant Creutzfeldt-Jakob disease (vCJD) epidemic is hazardous to predict due to uncertainty in ascertaining the prevalence of infection and because the disease might remain asymptomatic or produce an alternate, sporadic-like phenotype.

Methodology/Principal Findings

Transgenic mice were produced that overexpress human prion protein with methionine at codon 129, the only allele found so far in vCJD-affected patients. These mice were infected with prions derived from variant and sporadic CJD (sCJD) cases by intracerebral or intraperitoneal route, and transmission efficiency and strain phenotype were analyzed in brain and spleen. We showed that i) the main features of vCJD infection in humans, including a prominent involvement of the lymphoid tissues compared to that in sCJD infection were faithfully reproduced in such mice; ii) transmission of vCJD agent by intracerebral route could lead to the propagation of either vCJD or sCJD-like prion in the brain, whereas vCJD prion was invariably propagated in the spleen, iii) after peripheral exposure, inefficient neuroinvasion was observed, resulting in an asymptomatic infection with life-long persistence of vCJD prion in the spleen at stable and elevated levels.

Conclusion/Significance

Our findings emphasize the possibility that human-to-human transmission of vCJD might produce alternative neuropathogical phenotypes and that lymphoid tissue examination of CJD cases classified as sporadic might reveal an infection by vCJD-type prions. They also provide evidence for the strong propensity of this agent to establish long-lasting, subclinical vCJD infection of lymphoreticular tissues, thus amplifying the risk for iatrogenic transmission.     

Feline Toxoplasmosis from Acutely Infected Mice and the Development of Toxoplasma Cysts*

SYNOPSIS. The development of Toxoplasma cysts was studied in mice inoculated with tachyzoites by several routes. After 1–30 days of infection, murine tissues were examined microscopically, and portions or whole carcasses were fed to mice and cats. The feces of the cats were examined for oocyst shedding. Cyst-like structures containing distinct PAS-positive granules were first seen after 3 days of infection with tachyzoites, and became numerous by 6 days. Argyrophilic walls were first seen after 6 days, and became numerous by 16 days of infection with tachyzoites. Prepatent periods to oocyst shedding (PPO) were either “short” (3–10 days) or “long” (19–48 days). The “short” PPO was found only in cats that had ingested mice infected for 3 days or longer, and was related to the development of PAS-positive granules in T. gondii, and to high, 60–100%, oral infectivity rates for cats. The “long” PPO followed the ingestion of mice infected for only 1–2 days, and was related to tachyzoites without distinct PAS-positive granules and low, 32% or less, infectivity for cats. The “long” PPO followed also the ingestion of oocysts and the parenteral inoculation of tachyzoites, bradyzoites, or sporozoites. Using the “short” PPO as a criterion for detecting cysts in tissues, it was shown that (a) numerous cysts developed in mice 5 days after inoculation with tachyzoites, 7–9 days after inoculation with cysts, and 9–10 days after inoculation with oocysts, and (b) cysts developed faster and more frequently in the brain and muscle than in lungs, liver, spleen, and kidneys of mice inoculated with tachyzoites.     

Susceptibility of chickens to avian encephalomyelitis virus. II. Behavior of the virus in day-old chicks.

The VR strain of avian encephalomyelitis virus, which had been adapted to embryonated hen's eggs, was inoculated into 2-day-old chicks by the subcutaneous route (10(2.5) approximately 10(3.0) EID50) or by the oral route (10(4.8) EID50). The chicks were examined chronologically for the distribution of the virus in the body. As a result, minute amounts of the virus were detected from the liver, spleen, pancreas, and muscle at the site of inoculation one day after inoculation and various amounts from almost all the organs 3 days and more after inoculation. The virus titer could nearly reach a maximum 7 to 9 days after inoculation. Above all, such high virus titers as ranging from 10(4.3) to 10(5.8) EID50/0.1 g were demonstrated in the brain, heart, liver, spleen, and pancreas. After that, there was a tendency for virus titer to decrease in most organs and for virus to multiply persistently in the pancreas, brain, and eyeball. Virus titer was maintained at a level of 10(2.3) approximately 10(2.8) EID50/0.1 g in these three organs even 21 days after inoculation. In the group of subcutaneous inoculation, all the chicks manifested clinical signs of infection 5 to 10 days after inoculation. On the other hand, no chicks were involved in clinical infection in the group of oral inoculation. Multiplication of the virus was delayed in the body of these chicks. Small amounts of the virus were detected from the spleen and pancreas 11 days after inoculation. Low titers (10(2.7) EID50/0.1 g at the highest) of the virus were only detected from the brain, spinal cord, spleen, pancreas, esophagus, and other organs 14 and 21 days after inoculation.     

Localization of Mycoplasma pulmonis in mice.

Localization of Mycoplasma pulmonis was examined in mice infected by direct contact with previously infected mice. After contact with infected animals, the organisms were shown to become detectable first in the nasal and oral cavities and trachea on the next day, and then they were recovered from the middle ear and brain after 3 and 4 days, respectively. After 7 days of contact, isolation rates retained to be 100% in the nasal, oral and tracheal cavities, while 95% in the middle ear and brain, 25% in the lung and 20% in the vagina and uterus. The number of colonies was the most numerous from the nasal, uterine and vaginal cavities, followed by the trachea, middle ear, oral cavity, brain and lung in this order, except for a few mice having pneumonic lesions giving a large number of the organisms. The isolation rates with these organs were not changed even after 6 weeks of contact and organisms were never detected from the liver, spleen, kidney and heart. Mice from a naturally infected breeding colony showed similar finding to those sacrificed after 6 weeks of experimental contact.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

The evolution of immunosuppressive cell populations in experimental mycobacterial infection.

Immunosuppressor activity of considerable potency and complexity was generated during the course of chronic, progressive infection of C3H/Anf mice by Mycobacterium lepraemurium. From the 5th through 10th week after inoculation, spleen cells from infected mice mildly but reproducibly suppressed the direct plaque-forming cell response of normal spleen cell cultures to sheep erythrocytes. Suppression at this stage of infection was mediated by cells with macrophage-like characteristics. A marked increase in splenic suppressor activity at 10 to 11 weeks was associated with the appearance of a second suppressor cell subpopulation composed of T lymphocytes. The appearance of these cells was closely related in time to the onset of rapid splenic enlargement and a loss of cutaneous delayed type hypersensitivity to antigens of M. lepraemurium in mice at 10 to 11 weeks of infection. Suppressor cells were not present in peripheral lymph nodes until terminal infection at 22 to 25 weeks. Suppressor spleen cells depressed the T-dependent antibody response most severely, but there was also a direct effect upon B cells as shown by moderate suppression of responses to TNP-LPS and DNP-Ficoll. Spleen cells from 14-week-infected mice generated a soluble suppressor factor(s) that induces depression of moderate severity, however, the immunosuppression by intact cells was far greater.     

Experimental infection of Peromyscus californicus with Toxoplasma gondii

Eight female Peromyscus californicus were infected with 10(2) or 10(4) Toxoplasma gondii culture-derived tachyzoites (Type II or X) isolated from southern sea otters. All but 2 mice survived infection and developed antibodies to T. gondii. The 2 fatally infected mice were inoculated with 10(4) tachyzoites of the Type X strain. Parasite detection by immunohistochemistry (IHC) and DNA amplification with 2 polymerase chain reaction (PCR) methods was compared for brain, heart, lung, liver, spleen, biceps muscle, and tongue, at a mean of 41 days postinfection. Parasites were detected most commonly by IHC in spleen (8/8) and brain (6/8). DNA amplification by PCR was most successful from brain, heart, and spleen.     

Immunological analysis of host and agent effects on Creutzfeldt-Jakob disease and scrapie prion proteins.

Creutzfeldt-Jakob disease (CJD) and scrapie are degenerative neurological diseases caused by unusual infectious pathogens. The term prion has been introduced to underscore the apparent distinctness of these agents from viruses and viroids. The only macromolecule shown to be associated with the infectious agent, the CJD or scrapie prion protein (PrPCJD or PrPSc, respectively), is encoded by the same gene as a normal cellular protein. In several studies biochemical differences have been reported in PrPScs derived from a common host species infected with different putative strains of the scrapie agent, suggesting agent-specific characteristics independent of the host. We analyzed various agent-host combinations by Western blotting of PrPs that were separated by size or charge. The profile of immunoreactive proteins for CJD prions isolated from mice, guinea pigs, and humans appeared distinct. Importantly, PrPCJDS purified from a human brain and from the corresponding first-passage mouse brains were clearly distinguishable. PrPCJDs isolated from CJD prions propagated in NAMRU or B10.Q mice, which are homozygous for a short-incubation-time gene; from the short-incubation-time backcross progeny of (B10.Q x I/LnJ)F1 x B10.Q; or from NAMRU mice inoculated with I/LnJ prions were identical to each other but distinguishable from those of I/LnJ mice, which are homozygous for the long-incubation-time gene. The PrPs from human CJD and ovine scrapie propagated in the same mouse strain appeared the same, but they were distinct from the same isolate of scrapie passaged in hamsters. Lastly, PrPScs purified from five different strains of scrapie propagated in C57BL mice were identical, including strains, ME7 and 139A, which were previously reported to be distinct. This evidence does not support, although it does not exclude, agent-mediated characteristics independent of host-mediated ones for scrapie and CJD.     

Pathogenesis of acute murine cytomegalovirus infection in resistant and susceptible strains of mice.

We have characterized the progress of acute murine cytomegalovirus (MCMV) infection in the spleen, liver, and salivary gland of susceptible (BALB/c) and resistant (C3H) strains of mice after intraperitoneal inoculation. Viral replication was analyzed by virus titration, infectious-center assays, and in situ cytohybridization with cloned subgenomic fragments of the MCMV genome. The most striking differences between strains were observed in the spleen. At 24 h postinfection (p.i.), both strains had a similar number of infected spleen cells. At 48 h p.i., BALB/c mice showed marked dissemination of the splenic infection which continued until 96 h p.i. In contrast, the number of infected C3H spleen cells did not increase from the 24-h level but declined later on. This early block in dissemination of MCMV infection in C3H mouse spleens was not a result of the H-2k haplotype, as BALB.K (H-2k) mice, which show an intermediate level of resistance to MCMV infection, exhibited dissemination of the infection between 24 and 48 h p.i., albeit at a reduced level. However, between 72 and 96 h p.i., we observed a decline in the number of infected spleen cells in BALB.K mice similar to that observed in C3H mice. We also demonstrated by Southern blot analysis of DNA from the infected spleen cells that the termini of the MCMV genome fuse after in vivo infection.     

Prolonged replication in the mouse central nervous system of reoviruses isolated from persistently infected cell cultures.

We examined pathogenic characteristics of plaque-purified reoviruses isolated from persistently infected L-cell cultures (PI viruses) after intracranial inoculation into newborn mice. The PI viruses were isolated from independent cultures initiated with high-passage stocks of the wild-type (wt) strain, type 3 Dearing. The virulence of most PI viruses was equivalent to that of the wt strain. However, replication of PI viruses in the central nervous system of infected mice was prolonged to 25 (but not 50) days postinoculation. Thirty-eight percent (n = 186) of mice inoculated with the PI viruses had residual virus detectable in brain tissue 25 days after inoculation, in contrast to only 16% (n = 57) of mice inoculated with wt virus (P = 0.009). Mean residual brain titers were more than 20-fold higher in mice inoculated with PI viruses compared with wt virus (4.3 x 10(4) versus 2.1 x 10(3); P = 0.006). Tropism of PI virus within the brain resembled that of wt virus, and the distribution of PI virus antigen in the brain did not change over time. The extent of necrosis in the brains of mice harboring PI virus 25 days after inoculation was minimal, despite continued presence of high titers of infectious virus. The latter observation resembles the absence of cytopathicity seen in L-cell cultures persistently infected with reovirus. These observations suggest that the interaction of PI viruses with cells can be altered in vivo as well as in cell culture, but virus is eventually cleared from the infected animal.     

Modulation of immune response by interleukin-10 in systemic <Emphasis Type="Italic">Corynebacterium kutscheri</Emphasis> infection in mice

Interleukin (IL)-10 is an anti-inflammatory cytokine that modulates sepsis by decreasing pro-inflammatory cytokine production and chemokine expression. In this study, IL-10-deficient and wild-type (WT) mice were infected with Corynebacterium kutscheri to determine if the absence of IL-10 altered the protective immunity and pathogenesis. After infection, IL-10 knockout (KO) mice had a higher survival rate than WT mice. The decrease of body weight and the increased weight of organs such as liver and spleen were greater in WT mice. Bacterial counts were significantly increased after inoculation in WT mice over those in IL-10 KO mice. WT mice had more granulomatous inflammation and coagulative necrosis in the liver and spleen, lymphocyte depletion in lymphoid follicles, and apoptosis of immune cells in the spleen. WT mice had significantly higher plasma concentrations of aspartate aminotransferase and alanine aminotransferase. Furthermore, more upregulation of tumor necrosis factor-α and IL-4 in the plasma, macrophage inflammatory protein-2, keratinocyte-derived chemokine, inducible nitric oxide synthase, and interferon-inducible protein 10 mRNA in the spleen were observed in WT mice after inoculation. These results suggest that the lack of IL-10 contributes to an increase in the systemic clearance of C. kutscheri, and that IL-10 plays a detrimental role in controlling systemic C. kutscheri infection.     

Persistent infection with mouse hepatitis virus of low virulence in nude mice.

A persisting type of infection with wasting syndrome was established in congenitally athymic nude mice after intraperitoneal inoculation with a mouse hepatitis virus which was not fully pathogenic for heterozygous haired littermates. From the liver, spleen, lymph nodes, and brain of most infected nude mice, the virus was detected at high titers during aperiod from 6 to 35 days postinfection, occurrence of degenerative and necrotic lesions being correlated with virus titers in these organs. The titer of serum neutralizing antibody remained undetectable or very low in most diseases nude mice, whereas some animals resisting the infection could produce antibody at a later stage. In heterozygous haired mice, some lesions were detectable at a very early stage of infection in the spleen and liver, but they seemed to disappear with a marked elevation of the neutralizing antibody titer. Nude mice were able to resist the virus infection when they had previously received transfer of thymocytes from weanling heterozygous littermates.     

Kinetics of cytokine profile during intraperitoneal inoculation of Japanese encephalitis virus in BALB/c mice model

Infection with Japanese encephalitis virus (JEV) is mostly asymptomatic/subclinical in 90% of the individuals. Host immune response during subclinical JEV infection is poorly understood. We assessed iNOS, IFN-gamma, TNF-alpha, IL-10 and IL-4 production in spleen, brain and sera of intraperitoneally challenged BALB/c mice by RT-PCR and ELISA along with brain histopathology at different days post inoculation (d.p.i.). In spleen of virus infected mice, expression of all cytokines including iNOS mRNA were upregulated till 5d.p.i. followed by decline. At 5d.p.i., IL-10 expression outcompeted TNF-alpha, IFN-gamma and IL-4. However, in the virus infected mice sera, IL-4 production predominated over TNF-alpha and IL-10 at 5d.p.i. Conversely, cytokines expression and iNOS mRNA remained unchanged in the brain of virus infected mice from 1 to 7d.p.i. A significant increase in the cytokine expression was observed at 11d.p.i. (P<0.05) in virus infected mice brain, with the predominance of IL-10 along with the presence of meningeal inflammation and viral RNA by histology and RT-PCR, respectively. We report a biased pattern of cytokine production in sera, brain and spleen of mice intraperitoneally challenged with JEV. IL-10 exerts neuroprotective function during JEV and regulates deleterious effects of proinflammatory cytokines; however, its mechanism needs further investigation.     

An Adenovirus Isolated from the Feces of Mice

Experimental infection of an inbred strain of DK1 mice was carried out with a mouse adenovirus strain K87, isolated from the feces of apparently healthy DK1 mice. Strain K87 was orally administered to four-week-old mice and the virus was recovered from their feces for at least 3 weeks. The highest virus titers in the feces were observed between 1 and 2 weeks after inoculation. In 7-week-old mice the period of virus excretion was about one week shorter. When neonatal or 2-week-old mice were administered virus orally, somewhat irregular results but similar to those from the 4 or 7-week-old mice were obtained. In these infected mice, virus growth was detected in the intestinal tract but not in oropharyngeal washings, nasal tissue, lung, spleen or urine. After inoculation through several parenteral routes, the virus was also detected in the feces and virus growth seemed to be limited mainly to the intestinal tract. No clinical manifestations were observed in any infected mice. When the virus was almost undetectable in the mice infected either orally or parenterally, the virus was readministered orally, but no further virus was recovered in the feces. Neutralizing antibody in the serum was detected 3 weeks after primary inoculation. Induction of immunological tolerance was examined by inoculating mice in utero 2–4 days before birth, but no evidence of induced tolerance was obtained. The use of the adenovirus strain K87-mouse system as a model system for the study of the infectious process and immune mechanisms is suggested because strain K87 has a tissue tropism analogous to many human adenoviruses.     

Accumulation of proteinase K-resistant prion protein (PrP) is restricted by the expression level of normal PrP in mice inoculated with a mouse-adapted strain of the Creutzfeldt-Jakob disease agent.

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disease of humans caused by an unidentified infectious agent, the prion. To determine whether there was an involvement of the host-encoded prion protein (PrPc) in CJD development and prion propagation, mice heterozygous (PrP+/-) or homozygous (PrP-/-) for a disrupted PrP gene were established and inoculated with the mouse-adapted CJD agent. In keeping with findings of previous studies using other lines of PrP-less mice inoculated with scrapie agents, no PrP-/- mice showed any sign of the disease for 460 days after inoculation, while all of the PrP+/- and control PrP+/+ mice developed CJD-like symptoms and died. The incubation period for PrP+/- mice, 259 +/- 27 days, was much longer than that for PrP+/+ mice, 138 +/- 12 days. Propagation of the prion was barely detectable in the brains of PrP-/- mice and was estimated to be at a level at least 4 orders of magnitude lower than that in PrP+/+ mice. These findings indicate that PrPc is necessary for both the development of the disease and propagation of the prion in the inoculated mice. The proteinase-resistant PrP (PrPres) was undetectable in the brain tissues of the inoculated PrP-/- mice, while it accumulated in the affected brains of PrP+/+ and PrP+/- mice. Interestingly, the maximum level of PrPres in the brains of PrP+/- mice was about half of the level in the similarly affected brains of PrP+/+ mice, indicating that PrPres accumulation is restricted by the level of PrPc.