D. Radzioch

Genetic control of innate resistance to mycobacterial infections

The Mendelian segregation of resistance to infection in different strains of mice infected with mycobacteria, Salmonella and Leishmania spp, all of which live in macrophages, is currently under close scrutiny. Here, Erwin Schurr and colleagues review the nature and function of the Bcg gene in controlling innate resistance to mycobacterial infection in mice and speculate on the occurrence of a possible human equivalent.

Cutting Edge: STAT1 and T-bet Play Distinct Roles in Determining Outcome of Visceral Leishmaniasis Caused by Leishmania donovani

T-bet and STAT1 regulate IFN-γ gene transcription in CD4+ T cells, which mediate protection against Leishmania. Here we show that T-bet and STAT1 are required for the induction of an efficient Th1 response during Leishmania donovani infection, but they play distinct roles in determining disease outcome. Both STAT1−/− and T-bet−/− mice failed to mount a Th1 response, but STAT1−/− mice were highly resistant to L. donovani and developed less immunopathology, whereas T-bet−/− mice were highly susceptible and eventually developed liver inflammation. Adoptive cell transfer studies showed that RAG2−/− recipients receiving STAT1+/+ or STAT1−/− T cells developed comparable liver pathology, but those receiving STAT1−/− T cells were significantly more susceptible to infection. These unexpected findings reveal distinct roles for T-bet and STAT1 in mediating host immunity and liver pathology during visceral leishmaniasis.

PCR-analyzed microsatellites for the inbred mouse strain 129/Sv, the strain most commonly used in gene knockout technology.

The generation of gene-deficient (or knockout) mice is an important tool in the analysis of unknown gene function and the modeling of human disease (Camper et al. 1995; Gali-Taliadoros et al. 1995; Vidal et al. 1995). In addition, the development of genedeficient mice on different genetic backgrounds will help elucidate the roles of secondary genetic ~hctors on disease phenotype (Dorin et al. 1992; Sibilia and Wagner 1995; Threadgill et al. 1995). Unfortunately, crossing a gene knockout onto various inbred backgrounds is laborious and can take several years before acceptable levels of homozygosity are achieved. The bottleneck step in standard breeding protocols is the appropriate selection of backcross progeny. The advantage of microsatellite typing in gene-deficient mouse development is that it allows the easy selection of mice with the highest levels of background strain homozygosity (Delhaise et al. 1993). This technique not only expediates the breeding process, but reduces the total number of progeny mice (and backcrosses) required for the transfer of a gene knockout onto different genetic backgrounds. Because embryonic stem cell lines derived from strain 129/Sv are most commonly used for gene targeting experiments, it is essential to characterize microsatellites that distinguish this strain (Ledermann and Burki 1991; Stewart 1993). The mouse genetic map, reported on the Whitehead Institute/ MIT Center for Genome Research (WI/MIT CGR) internet site, contains 6,183 polymorphic mouse microsatellites (or simple sequence length polymorphisms) characterized for 12 inbred strains of mice (Fowlis, et al., 1992, Copeland et al., 1993, Dietrich et al. 1993; Suppl., 1995). However, microsatellite size analysis has not been reported for strain 129/Sv, the inbred strain most frequently employed in gene knockout technology. In an effort to develop 129/Sv strain-specific markers that can be used to monitor the level of homozygosity in backcross progeny, 123 microsateUites were tested for polymorphism among four inbred mouse strains: BALB/cJ, C57BL/6J, DBA/2J, and 129/SvJ. Microsatellites were selected to cover all 19 autosomes and the X chromosome with a mean frequency of five and a half markers per chromosome (range = two to eight). Of the 123 microsatellites analyzed, 109 microsatellites showed polymorphism between strain 129/SvJ and at least one of three control strains (identified above). Our results are summarized in Table 1. High-molecular-weight genomic DNA was prepared from tails collected from BALB/cJ, C57BL/6J, DBA/2J, and 129/SvJ inbred strains of mice. DNA was prepared in the absence of phenol by modification of the salting out procedure as previously described by Miller et al. (1988). Briefly, one-half inch of mouse tails were digested overnight at 55~ with proteinase K (0.5 mg/ml; Boehringer Mannheim, Laval, Quebec). Proteins were then pre-

Assessment of mycobacterial infection and multiplication in macrophages by polymerase chain reaction

The amplification of mycobacterium-specific DNA sequences from samples obtained from infected patients by the polymerase chain reaction (PCR) has been useful in the clinical diagnosis of mycobacterial diseases. Using 20 bp oligonucleotide primers that recognize a 123 bp repeated sequence present in M. bovis and M. tuberculosis DNA, we describe in detail the conditions of the PCR reaction that allow an assessment of the mycobacterial content of infected macrophages. The results of the highly reproducible, time-efficient PCR technique show good correlation with the widely used colony forming unit (CFU) and [3H]uracil incorporation methods for the detection of Mycobacterium. Our method allows an assessment of the level of M. bovis BCG infection from a variety of sources, including peritoneal macrophages and macrophage lines, within a few hours, making it the assay of choice for rapid determination of the level of mycobacterial growth in infected cells, in experimental models of mycobacterial infection.

Toll-Like Receptors, Cytokines and the Immunotherapeutics of Asthma

Asthma is a complex disease caused by a poorly characterized set of genetic and environmental factors whose pathology is a result of immune dysregulation. Toll-like receptors are pathogen associated molecular pattern receptors expressed by many airway and pulmonary tissues as well as cells of the innate and adaptive immune system. Ligation of toll-like receptors can lead to a change in the expression levels of multiple inflammatory and anti-inflammatory mediators which are involved in the pathogenesis of asthma. These ligands and their receptors are therefore prime candidates in the search for immunotherapeutic treatments of asthma. The use of murine models of allergic asthma as tools for the genetic dissection of this disease should allow the molecular mechanisms underlying asthma to be identified and possibly used as further immunotherapeutic targets.