Over the past several years, Dr. Bloom's labora- 
tory, in collaboration with Dr. William R.Jacobs, Jr. 
(HHMI, Albert Einstein College of Medicine), has 
developed genetic tools for introducing and ex- 
pressing immunological foreign antigens in myco- 
bacteria, including BCG vaccine strains. Because 
BCG takes 3-4 weeks to produce colonies, the strat- 
egy has been to develop shuttle vectors in which 
foreign genes are introduced and manipulated in 
Escherichia coli and then transferred to the myco- 
bacteria. 
Initial studies were carried out using temperate 
phages, for which in the past year the chromosomal 
and phage attachment sites have been defined that 
allow site-specific integration of single gene copies 
into the chromosome of BCG. In addition, a shuttle 
plasmid vector system has been developed that pro- 
duces 10-15 gene copies per BCG cell. For both 
systems, in collaboration with Drs. Charles K. Stover 
and Vidal De la Cruz (Medlmmune, Inc.), vectors 
have been created that utilize major heat-shock pro- 
moters, HSP60 and HSP70, to facilitate high-level 
expression of the introduced foreign antigens upon 
infection of culture cells or animals. 
For the initial studies, |0-galactosidase was used as 
a marker antigen, and later GP120 of human immu- 
nodeficiency virus (HIV) and the C fragment of teta- 
nus toxin were tested as recombinant antigens. The 
genes were expressed at varying levels in heat- 
shocked BCG in vitro. The recombinant BCGs were 
then used to immunize mice. The results of the first 
such experiments have been encouraging. All 
three types of potentially protective immune re- 
sponses — production of humoral antibodies, induc- 
tion of T cell-derived lymphokines including 
IFN-7, and generation of cytotoxic T lymphocytes 
— were produced in immunized mice. As few as 
100 bacilli were capable of immunizing, and im- 
mune responses from a single inoculation persisted 
for more than 20 weeks. 
The Killing of Mycobacterium tuberculosis 
by Activated Macrophages 
Mycobacterium tuberculosis is among the micro- 
organisms most resistant to killing. It can remain 
viable in air droplet nuclei for extended periods. In 
the body it grows primarily in mononuclear phago- 
cytes — specialized cells for ingesting and destroy- 
ing microorganisms. It is well established that re- 
active oxygen intermediates — superoxide anion, 
hydrogen peroxide, and the hydroxyl radical — rep- 
resent a major cytocidal mechanism in activated 
macrophages. Yet experiments in Dr. Bloom's and 
other laboratories have indicated that virulent M. 
tuberculosis is highly refractory to killing by reac- 
tive oxygen intermediates. 
In recent years a novel macrophage cytocidal 
mechanism appeared — namely, the production of 
reactive nitrogen intermediates, particularly ni- 
tric oxide (NO), within macrophages. In vitro 
macrophage-produced NO has been found to retard 
the growth of a number of intracellular pathogens. 
Mouse macrophages are very susceptible to infec- 
tion and intracellular growT:h of M. tuberculosis. 
However, when they are activated — and in this con- 
text both interferon-7 and tumor necrosis factor-a 
are required — they produce NO and exert a cytoci- 
dal and cytostatic effect on M. tuberculosis. The NO 
derives from an active pathway in which the N of the 
guanido group of arginine is oxidized to NO. The 
fact that NO is responsible for killing M. tuberculo- 
sis was established by showing that treatment of the 
macrophages with the inhibitor A^-monomethylar- 
ginine (NMMA) abrogated the killing and that the 
organism could be killed by pure NO in solution. 
The mechanisms by which the cytokines activate 
this pathway, the effectiveness of NO-mediated cyto- 
toxicity in vivo, and the question whether human 
mononuclear phagocytes possess this capability re- 
main important areas for future research. 
Dr. Bloom is also Weinstock Professor of Micro- 
biology and Immunology and Professor of Cell Bi- 
ology at the Albert Einstein College of Medicine of 
Yeshiva University, Bronx. 
Articles 
Barletta, R.G., Kim, D.D., Snapper, S B., Bloom, 
B.R., and Jacobs, W.R., Jr. 1992. Identification 
of expression signals of the mycobacteriophages 
Bxbl, LI and TM4 using the Escherichia- 
Mycobacterium shuttle plasmids pYUB75 and 
pYUB76 designed to create translational fusions 
to the lacZ gene. J Gen Microbiol 138:23-30. 
Barnes, P.F., Mehra, V., Rivoire, B., Fong, S.-J., Bren- 
nan, PJ., Voegtline, M.S., Minden, P., Houghten, 
R.A., Bloom, B.R., and Modlin, R.L. 1992. Immu- 
noreactivity of 1 0-kDa antigen of Mycobacterium 
tuberculosis. J Immunol 148:1835-1 840. 
Bloom, B.R. 1992. Tuberculosis. Back to a frighten- 
ing future. Nature 358:538-539. 
Bloom, B.R., Modlin, R.L., and Salgame, P. 1992. 
Stigma variations: observations on suppressor T 
cells and leprosy. Annu Rev Immunol 10:453- 
488. 
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