Footpad Research Articles

Optimization of murine CD8 + cytotoxic T-lymphocyte responses to pseudorabies virus

The aim of this work was to optimize the procedures used to elicit a cellular immune response to pseudorabies virus (PrV) in mice, using various immunization schedules and routes. An Eu-labeling-based cytotoxic T-lymphocyte (CTL) test was developed to measure the response. This necessitated optimization of numerous steps. In suspension, Eu labeling required high concentrations of dextran-sulfate (DXS) and Eu with a 30-min labeling time at room temperature. For anchored cells, the labeling time was 1 to 48 h, and the labeling efficiency depended strongly on the Eu concentration, but only marginally on the DXS concentration. In vivo and in vitro stimulation protocols were also optimized for the CTL test. For in vitro stimulation, spleen cells were cultured in T-25 flasks at a multiplicity of infection (m.o.i.) of 2. The CTL test was validated by specific depletion of CD8 + CTL, FACS analysis, and by comparing Eu and 51Cr labeling. Then groups of mice were vaccinated once or twice by various routes (intraperitoneal (i.p.), intravenous (i.v.), subcutaneous (s.c.) and in the rear footpads (FP)) and according to different time schedules. CTL activity was detected only in boosted animals immunized FP, i.p. or i.v. That the cellular immune response contributes to protection was further suggested by the observation that i.p. immunization conferred better protection against challenge than s.c. immunization.

Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection

Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.

Reactivation of latent Herpes simplex virus-1 (HSV) from mouse footpad cells demonstrated by in situ hybridization

HSV-1 specific RNA sequences became detectable by in situ hybridization 5-6 days after culture of footpads (FP) explanted from latently infected mice. HSV-specific RNA first appeared in basal cells of hair follicles and cells of the hair root sheath, in epithelial cells of sebaceous glands and in cells within the epidermis. When first detected graining was light and usually present over individual cells, subsequently graining became heavy and present over large groups of cells. HSV reactivation from latency could be demonstrated in individual cells and therefore identified those cells in which the virus has been latent. The heavy graining over clumps of cells characteristic of a lytic infection was seen at later times after explantation; we infer that the infection had spread progressively from the initial foci of reactivation from latency.

Rifampicin-resistant strains of Mycobacterium leprae may have reduced virulence.

A strain of Mycobacterium leprae resistant to rifampicin (RMP) failed to infect normal mice when injected into the foot pads (FP) at a dose of 10 or 100 bacilli/FP, although it could be maintained by serial passage in mice by the use of inocula of 10(4) bacilli/FP; normal mice can be infected by RMP-sensitive M. leprae at a dose of 10 bacilli/FP. By contrast, nude (athymic) mice could be infected with an inoculum of 10 bacilli/FP of the RMP-resistant strain. It is suggested that the strain concerned possessed reduced virulence for normal mice, and the implications of this for the probability of occurrence of human disease caused by RMP-resistant strains of M. leprae are discussed.

Viral genes modify herpes simplex virus latency both in mouse footpad and sensory ganglia.

Herpes simplex virus type 2 wild-type and 13 temperature-sensitive mutants have been examined for their ability to be recovered from latently infected Biozzi mice. After inoculation into the rear footpad, virus could be recovered from both the dorsal root ganglia (DRG) and the footpad (FP) at the site of inoculation (wt, ts 1, ts 2, ts 4, ts 6, ts 7 and ts 8), or from the DRG only (ts 11 and ts 13), or from FP only (ts 3, ts 9, ts 12). Two mutants (ts 5 and ts 10) have not been recovered from either site. Both DNA-positive and DNA-negative mutants have been shown to be capable of establishing latent infection.

Survival of mice injected with Plasmodium vinckei or Plasmodium yoelii XL by the footpad route.

NIH-white mice were injected with varying doses of Plasmodium vinckei or Plasmodium yoelii lethal via the hind footpad (FP), intraperitoneal (IP), subcutaneous (SC) routes. Survival was dependent on the dose of the inoculum and route of injection. Injection via the IP and SC routes led to higher mortality than the FP route. The results showed that at a dose of 10 4 parasitized erythrocytes injected IP led to 100% mortality, whereas the same dose injected via the FP route gave protection.

Production of large quantities of ascitic fluid for kidney allograft enhancement in rats.

A method is presented for the production of large amounts of rat alloantibody in ascitic fluid (AF) to be used for the enhancement of renal allografts. AF significantly prolonged allograft survival and reduced the rise of 7-day blood urea nitrogen. AF was induced in Lewis (LEW) rats by multiple i.p. injections of Dark Agouti (DA) rat spleen cells emulsified in complete Freund's adjuvant (CFA). A large volume ratio of adjuvant to antigen was utilized (1 part spleen cells:9 parts CFA). Various groups of rats underwent different presensiti-zation protocol prior to AF induction. The method of presensitization significantly affected the cumulative volume of AF produced per rat. Rats that were presen-sitized by footpad (FP) injections of DA spleen cells emulsified with an equal volume of CFA produced the largest mean cumulative volume of AF per rat and the highest mean total antibody units per rat. The method of presensitization did not significantly alter AF antibody titers among the various rat groups. Antibody titers and IgG concentrations of serum and corresponding AF samples from individual rats were not significantly different.

Age at infection as a determinant of the behavioral effects of herpes encephalitis in mice

The influence of age at infection on the nature of the behavioral effects of herpes simplex type 1 encephalitis in mice was examined. Weanling and adult mice were initially immunized with a footpad (FP) inoculation of virus, followed 2 weeks later by an intracerebral (IC) inoculation. Subsequent open-field testing 2 weeks later revealed that weanling mice were hypoactive and showed a reduced proportion of center-field entries. In contrast, adult mice were hyperactive. No significant effects on Y-maze alternation or activity were observed. These results indicate that the qualitative nature of the effects of herpes encephalitis on behavior depends upon characteristics of the host.

Induction of cell-mediated immunity to Actinomyces viscosus in mice

Immune reactivity following multiple encounters with Actinomyces viscosus was studied in C57B1/6 mice. Delayed hypersensitivity (DH) was evaluated in vivo by the foot-pad (FP) assay and in vitro by splenic lymphocyte stimulation; antibody titres were determined by agglutination. Induction of DH was by multiple subcutaneous (SQ) injections. FP reactivity increased with subsequent injections and became maximal after 4 injections whereas the lymphocyte stimulation peaked after two. Intraperitoneal (IP) inoculation induced neither FP reactivity nor lymphocyte stimulation. The induction of DH was inhibited by both anti-thymo-cyte serum (ATS) and cyclophosphamide (Cy). Sensitized mice treated with ATS or Cy prior to FP challenge also showed suppressed reactivity. Both SQ and IP inoculation stimulated formation of agglutinating antibodies. In both cases, antibody levels increased with successive injections.