Macrophage Depletion Suppresses Cardiac Allograft Vasculopathy in Mice

Cardiac allograft vasculopathy (CAV) is a major source of late posttransplant mortality. Although numerous cell types are implicated in the pathogenesis of CAV, it is unclear which cells actually induce the vascular damage that results in intimal proliferation. Because macrophages are abundant in CAV lesions and are capable of producing growth factors implicated in neointimal proliferation, they are leading end-effector candidates. Macrophages were depleted in a murine heterotopic cardiac transplant system known to develop fulminant CAV lesions. C57BL/6 hearts were transplanted into (C57BL/6 x BALB/c)F(1) recipients, which then received anti-macrophage therapy with intraperitoneal carrageenan or i.v. gadolinium. Intraperitoneal carrageenan treatment depleted macrophages by 30-80% with minimal effects upon T, B or NK cells as confirmed by flow cytometry and NK cytotoxicity assays. Carrageenan treatment led to a 70% reduction in the development of CAV, as compared to mock-treated controls (p = 0.01), which correlated with the degree of macrophage depletion. Inhibition of macrophage phagocytosis alone with gadolinium failed to prevent CAV. Macrophages may represent the end-effector cells in a final common pathway towards CAV independent of T-cell or B-cell alloreactivity and exert their injurious effects through mechanisms related to cytokine/growth factor production rather than phagocytosis.     

Quantitative (82)Rb PET/CT: development and validation of myocardial perfusion database.

The use of myocardial perfusion (82)Rb PET/CT studies continues to increase but its accuracy using database quantification methods for the diagnosis of coronary artery disease (CAD) has not been established. METHODS: A sex-independent normal database and criteria for abnormality for rest-stress (82)Rb PET/CT myocardial perfusion imaging were developed and validated by evaluation of 281 patients (136 females: mean age +/- SD, 63.3 +/- 13.3 y; 145 males: mean age +/- SD, 63.9 +/- 12.8 y) who underwent a rest-adenosine stress (82)Rb PET/CT study. These patients were divided into 3 groups: (a) healthy group: 30 patients, with <5% likelihood of CAD (low likelihood [LLK]) based on sequential Bayesian analysis; these patients were used to generate the normal distribution; (b) pilot group: 174 patients; these patients were used to determine the optimal criteria for detecting and localizing the perfusion abnormality; and (c) validation group: 76 patients (23 with LLK of CAD and 53 who underwent coronary angiography; these patients were used for prospective validation. RESULTS: Of the 53 patients who underwent coronary angiography, 8 had <50% stenosis and 45 patients had at least one stenosis > or =50% in one major artery. Fifteen patients had single-vessel disease, 17 had double-vessel disease, and 13 had triple-vessel disease. The prospective validation shows a normalcy rate of 78% (18/23) for global CAD. The analyses by individual arteries show a normalcy rate of 96% (22/23) for the left anterior descending coronary artery, 96% for the left circumflex coronary artery (22/23), and 100% for the right coronary artery (23/23). The overall sensitivity for detection of CAD (> or =50% stenosis) was 93% (42/45). The overall specificity for detection of the absence of CAD (< or =50% stenosis) was 75% (6/8). Also, the positive predictive value for global CAD was 95% (42/44), the negative predictive value was 67% (6/9), and the accuracy was 91% (48/53). CONCLUSION: The quantitative (82)Rb PET/CT database created and validated in this study is highly accurate for the detection and localization of CAD. Physicians should consider using the quantitative output of these algorithms as decision support tools to aid with image interpretation.     

Secretion of the mycobacterial 19-kilodalton protein by Escherichia coli, a novel method for the purification of recombinant mycobacterial antigens.

We have reported previously (R.G. Hewinson and W.P. Russell, J. Gen. Microbiol. 139:1253-1259, 1993) the secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis when the protein is translated from its native initiation codon. N-terminal sequence analysis of the purified protein revealed that the signal peptide of MPB70 was cleaved by an endopeptidase of E. coli at the same cleavage site as reported for the protein in M. bovis. Since both the B- and T-cell antigenicities of the purified recombinant protein were similar to that of the native protein, the 19-kDa antigen of M. bovis was used as a model to test whether the signal peptide of MPB70 could direct the secretion of heterologous proteins in E. coli and whether antigen produced in this way retained antigenicity superior to that of recombinant protein produced as a fusion to glutathione-S-transferase. A chimeric protein was produced in which the signal peptide of MPB70 was fused to the 19-kDa antigen of M. bovis at amino acid residue 23. This chimeric protein was found to be secreted into the periplasm and culture medium of recombinant E. coli, and the signal peptide was cleaved by an endopeptidase of E. coli during secretion. Secretion of the 19-kDa antigen facilitated purification of the antigen by two-stage preparative electrophoresis which gave yields of 2.5 mg of purified, soluble 19-kDa antigen from 2.5 g (wet weight) of E. coli. Antigen purified in this way retained both B- and T-cell antigenicities. Moreover, the nonspecific mitogenic activity of the purified 19-kDa antigen was low, while the magnitude of the T-cell response induced by the purified antigen was considerably higher than that observed with purified antigen produced as a fusion protein with glutathione-S-transferase.     

Incomplete reversibility of an experimentally induced hypocholinergic state: biochemical and physiological, but not behavioral, recovery.

In previous reports, we described the experimental development of a hypocholinergic state in rats following the total replacement of dietary choline by an artificial isostere, N-aminodeanol (NADe). NADe shares most of the physicochemical and biochemical characteristics of choline (Ch) but is utilized less efficiently in pathways leading to the formation of both acetylcholine and phospholipids. This experimental model mimics many of the features of human degenrative dementias. We now discuss the behavioral and physiological effects of restoring a normal diet after the hypocholinergic state has become well established. The procedure by which that state was induced has been described in detail in earlier publications. After replacing Ch in the diets of weanling rats for 270 days, NADe replaced 70-85% of the phospholipid-bound Ch in plasma, brain, and peripheral tissue. When dietary NADe was removed and Ch was restored in the diet, NADe disappeared and plasma and erythrocyte (RBC) choline levels returned to normal within 30-60 days. Quinuclidinyl benzilate (QNB) binding showed that muscarinic receptors continued to be depressed in animals remaining on the NADe diet, but returned to control levels in the reversal group. There were no differences in cholinesterase activity among the three treatments. Choline acetyltransferase activity returned to control levels, while continuing to be lower in the NADe animals. Liver lipids were elevated in the latter and not significantly different in the control and reversal groups. Among physiological functions, body weight increased more rapidly in the reversal group than in animals continuing on the NADe diet. Brain weights of the reversal animals were significantly greater than those of animals not reversed, but less than controls. Core body temperatures did not differ from controls at any time during the reversal period. Behaviorally, nociceptive thresholds indicative of sensory-reflexive and sensory-perceptual responses remained significantly below normal, that is, a hyperalgesic state. Reversal animals also remained hyperactive and displayed memory significantly poorer than those on the normal diet, that is, no improvement over animals continuing on NADe. In general, the results suggest that behavioral losses induced by NADe reflect persisting changes in the CNS, despite essentially complete recovery of biochemical parameters. The changes may be morphological or be associated with adaptive changes in other neurochemical events in the CNS.