Michael P. McGarry

Molecular markers near the mouse brachymorphic (bm) gene, which affects connective tissues and bleeding time

Several inherited skeletal/connective tissue defects are associated with hemorrhagic disorders in humans. Accordingly, three mouse mutants (brachymorphic [bm], hemimelic extra toes [Hx], and ulnaless [Ul]), with inherited skeletal abnormalities, were analyzed for hemorrhagic tendencies. All three had prolonged bleeding times. Platelet numbers, size, and function, as well as common soluble plasma clotting factors, were not measurably affected. To further define the bm mutation, its chromosomal location relative to 19 other molecular markers was determined to a high resolution in a large interspecific backcross. Several microsatellite markers were found to be very closely linked to bm and should provide useful entry points for the eventual identification of this gene by positional/candidate cloning techniques. These results suggest that inherited skeletal abnormalities and bleeding tendencies are associated more frequently in both humans and animal models than is commonly recognized. Identification of these genes may reveal novel relationships between osteogenesis and hemostasis.

Hyper-resistance to infection in TIMP-1-deficient mice is neutrophil dependent but not immune cell autonomous.

The TIMP/MMP axis has been postulated to influence both acquired and innate immune responses in vivo . 1 Among the experimental results consistent with this possibility are the observations that macrophages from mice deficient for MMP-12 have reduced kinetics of migration both in vitro and in vivo 2 and that the complement factor C1 inhibitor can be degraded in vitro by MMP-8 and -9. 3 However, reports documenting a direct influence of the TIMP/MMP axis on responses to infection have been lacking. We have observed that loss of TIMP-1 had a dramatic effect on the course of corneal infection by Pseudomonas aeruginosa . Infections became established and bacteria underwent a burst of growth identically in both groups of mice. However, coincident with massive neutrophil accumulation in corneas between 24 and 48 hours postinfection, the bacterial burden in mutant mice dropped to a level 1,600-fold lower than in wild-type mice. This was shown to be reversible by treatment of mutant mice with BB-94. Furthermore, the mechanism underlying the hyper-resistance to infection in mutant mice was shown to be complement dependent since depletion of the complement system from mutant mice suppressed their phenotype (Osiewicz et al. , submitted for publication). The complement branch of the innate immune system consists of approximately 30 plasma proteins that can be activated by a proteolytic cascade to generate two bactericidal activities. Bacteria may be killed directly by the formation of a pore in bacterial membranes by the membrane attack complex of complement. Alternatively, activated complement factor C3b can bind to bacterial surfaces targeting bacteria for phagocytosis by complement receptor-containing neutrophils. In order to study the contribution of neutrophils to the hyper-resistance phenotype seen in TIMP-1–deficient mice, infections were done in neutropenic mice, and the bacterial burdens were measured 48 hours after infection. Neutropenia was induced by cyclophosphamide administered intraperitoneally on day 0 and day 5 at a dose of 150 μ g/kg. Infections were done on day 4 using an inoculum of 3 × 10 6 CFU/eye, and bacterial burdens were measured on day 6 by homogenization of whole eyes followed by plate count assays. During the course of the infection, cyclophosphamide treatment reduced the number of circulating neutrophils by approximately 90% (data not shown). Macrophage numbers were reduced by 50%; however, these cells were very rare in infected eyes during the first 48 hours of infection and are unlikely to be significant to the mutant phenotype. The results showed that in neutropenic mice, the

Incidence of Friend Virus-Induced Polycythemia in Splenectomized Mice

Summary Polycythemia (HCT 58%) has been observed in splenectomized Ha/ICR (18%) and DBA/2 (16%) mice infected with a polycythemia-inducing strain (Mirand) of Friend virus. Data presented indicate that hepatic erythropoiesis, perhaps stimulated by the observed elevations in plasma ESF during the postinfection anemia, contributes significantly to the polycythemia. The polycythemic response appears more contingent on the presence of an expandable erythropoietic tissue than on the presence of the spleen per se. The participation of J. Jividen, A. Bulba, R, Kirchmyer, D. Mickens, and D. Mason is gratefully acknowledged.