Detectable tumor cells in the blood and bone marrow: smoke or fire?

This study was designed to test whether, using curved beam theory, a structural model of the proximal femur derived from two-dimensional dual energy x-ray absorptiometry could be used to predict femoral strength in an experimental simulation of a fall on the greater trochanter. A set of 22 fresh cadaveric femoral specimens were scanned with use of two-dimensional dual energy x-ray absorptiometry and then were tested to failure in a materials testing system, under three-point loading, with the ground impact vector aligned within the plane and along the bisector of the femoral neck-shaft angle. Failure locations generally corresponded to stress peak locations predicted by the curved beam model. Predicted failure loads correlated well with measured failure loads for femoral neck fractures (r=0.89; percent SE of estimate=23%) and some-what less well for intertrochanteric fractures (r=0.83; percent SE of estimate=29%). Overall predictions for failure load calculated from the maximum stress peak value over both locations corresponded to measured failure loads with an r value of 0.91 (percent SE of estimate=21%). This kind of structural approach to the analysis of data for hip bone mass has the potential to provide mechanistic interpretations of the statistical associations frequently shown between conventional bone mineral measures and either hip fracture risk in vivo or bone strength in vitro.     

Assessment of valvular regurgitation with Doppler echocardiography

Echocardiography is routinely performed for the evaluation of valvular regurgitation. Different applications of Doppler echocardiography have been successfully applied to detect and quantify valvular regurgitation. Recent advances in color Doppler made possible the study of the dynamic behavior of the regurgitant orifice and, along with continuous wave Doppler, can provide data on the regurgitant volume and fraction. Doppler echocardiography can also be used to follow serial changes in these hemodynamically important parameters after medical or surgical therapy.     

Attractin (DPPT-L), a member of the CUB family of cell adhesion and guidance proteins, is secreted by activated human T lymphocytes and modulates immune cell interactions

Attractin is a normal human serum glycoprotein of 175 kDa that is rapidly expressed on activated T cells and released extracellularly after 48-72 hr. We have cloned attractin and find that, as in its natural serum form, it mediates the spreading of monocytes that become the focus for the clustering of nonproliferating T lymphocytes. There are two mRNA species with hematopoietic tissue-specific expression that code for a 134-kDa protein with a putative serine protease catalytic serine, four EGF-like motifs, a CUB domain, a C type lectin domain, and a domain homologous with the ligand-binding region of the common gamma cytokine chain. Except for the latter two domains, the overall structure shares high homology with the Caenorhabditis elegans F33C8.1 protein, suggesting that attractin has evolved new domains and functions in parallel with the development of cell-mediated immunity.     

The myxoma virus M-T4 gene encodes a novel RDEL-containing protein that is retained within the endoplasmic reticulum and is important for the productive infection of lymphocytes

To investigate the contribution of the myxoma virus M-T4 gene to viral virulence, both copies of the M-T4 gene were inactivated by disruption and insertion of the Escherichia coli guanosine phosphoribosyltransferase gene. Infection of European rabbits with the recombinant M-T4-deleted virus, vMyxlacT4, resulted in disease attenuation. In contrast, infection of rabbits with vMyxlac elicited the classical features of lethal myxomatosis. A notable decrease in the number of secondary lesions in animals infected with vMyxlacT4 suggested an inability of the virus to disseminate in vivo. Infection of either a rabbit CD4+ T cell line, RL-5, or primary rabbit peripheral blood lymphocytes with vMyxlacT4- resulted in the rapid induction of apoptosis. Sequence analysis of M-T4 revealed both an N-terminal signal sequence and a C-terminal -RDEL sequence, suggesting that M-T4 resides in the endoplasmic reticulum. The M-T4 protein was found to be sensitive to endo H digestion and confocal fluorescence microscopy demonstrated that M-T4 colocalized with calreticulin, indicating that M-T4 is retained within the endoplasmic reticulum. Our results indicate that M-T4 is the first example of an intracellular virulence factor in myxoma virus that functions from within the endoplasmic reticulum and is necessary for the productive infection of lymphocytes.     

Cytotoxicity and apoptosis in human renal allografts: identification, distribution, and quantitation of cells with a cytotoxic granule protein GMP-17 (TIA-1) and cells with fragmented nuclear DNA

In the present study, we analyzed human renal allografts using immunohistochemical techniques to determine the site, identity, and frequency of (a) cytotoxic and apoptotic cells, as identified by staining for GMP-17 (TIA-1), a component of cytotoxic granules; and (b) DNA fragmentation in situ, as detected by the TUNEL method. In acute cellular rejection (n = 15), GMP-17+ mononuclear cells accounted for 29% +/- 12% of the infiltrating cells in the interstitium (341 +/- 164/mm2) and were significantly more concentrated in tubulitis lesions, where they amounted to 65% +/- 14% of the mononuclear cells (96 +/- 61/mm2) (p < 0.01 versus interstitium). GMP-17+ mononuclear cells were also found in sites of endothelialitis. An estimated 80% of the GMP-17+ lymphocytes expressed CD8, and 10% to 20% expressed either CD4 or the macrophage marker CD14. The latter finding led us to analyze normal peripheral blood monocytes by flow cytometry, all of which were found to contain GMP-17. NK cells and neutrophils, which are known to express GMP-17, were detected only rarely in allografts. Specimens with cyclosporine A toxicity (n = 7) or acute tubular necrosis (n = 13) showed fewer GMP-17+ cells in the interstitium (22 +/- 46/mm2 and 62 +/- 50/mm2, respectively) and tubules (2 +/- 6/mm2 and 10 +/- 10/mm2, respectively) (all p < 0.01 versus rejection). These differences were due largely to less intense mononuclear cell infiltration. In cyclosporine A toxicity, however, the percentages of GMP-17+ mononuclear cells within tubules and the interstitium were significantly lower than in rejection (p = 0.02), whereas in acute tubular necrosis significantly lower percentages were found in the tubules (p = 0.04) but not in the interstitium. Native kidneys with end-stage diabetic nephropathy (n = 5) had very low proportions of GMP-17+ cells in interstitial infiltrates (7% +/- 6%) and in tubules (11% +/- 15%), although the infiltrates were focally intense (517 +/- 355/mm2). TUNEL+ cells were found in acute cellular rejection, predominantly in areas with intense mononuclear infiltrates and also within lesions of tubulitis and endothelialitis. Although some TUNEL+ cells were intrinsic renal cells, most appeared to be infiltrating mononuclear cells, and we were able to detect CD3 in some. In areas of intense cellular infiltration, the percentages of TUNEL+ cells (range, 0.5% to 4.2%) were comparable to those seen in the rat thymus, indicating a high level of apoptosis. Overall, in the allograft samples, the numbers of GMP-17+ cells and TUNEL+ cells were significantly correlated (r = 0.79; p < 0.01). These data provide new evidence that T cell (and possibly macrophage)-mediated cytotoxicity plays an important role in acute renal allograft rejection, particularly in the case of tubular injury, and furthermore suggest that apoptosis may be a mechanism not only for graft cell destruction, but also for elimination of activated T cells in the infiltrate.     

Mycobacteremia in patients with the acquired immunodeficiency syndrome

BACKGROUND: Bacillemia is a key event in the pathogenesis of tuberculosis. Although current evidence indicates that Mycobacterium tuberculosis bacteremia is rare in patients seronegative for the human immunodeficiency virus, it has been increasingly reported in patients with the acquired immunodeficiency syndrome (AIDS). OBJECTIVE: To determine clinical and laboratory characteristics of patients with AIDS and tuberculosis with and without bacillemia. METHODS: Fifty patients with AIDS with clinical suspicion of disseminated mycobacterial disease were prospectively selected. Three consecutive blood samples were collected for culture using a standardized protocol. RESULTS: Mycobacterium was isolated from any body site in 42 patients (84%). Bacillemia was detected in 30 (71.4%) of these 42 patients: 11 (28.2%) caused by Mycobacterium avium-intracellulare complex and 19 (71.8%) caused by M tuberculosis. Blood culture was the only method used to confirm the diagnosis in 5 (15%) of the 33 tuberculosis cases. Tuberculosis in patients with AIDS developed with nonspecific insidious symptoms, a remarkable elevated alkaline phosphatase level, and without the classic miliary radiological pattern. We could demonstrate 2 previously unrevealed clinical characteristics of bacteremic tuberculosis in patients with AIDS: a shift to the left in the white blood cell count and abdominal lymph node enlargement. In patients with tuberculosis, the in-hospital mortality rate was higher among patients with bacillemia, although the posttreatment survival rate was comparable. CONCLUSIONS: Blood culture is a valuable tool to confirm the clinical diagnosis of disseminated tuberculosis in patients with AIDS and can distinguish patients with characteristic clinical findings and outcome. Abdominal ultrasonography may be an additional helpful tool to identify these patients.     

Evidence for a requirement for both phospholipid and phosphotyrosine binding via the Shc phosphotyrosine-binding domain in vivo

The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosine-binding (PTB) domain, and a role for the PTB domain in phosphotyrosine-mediated signaling has been well documented. The structure of the Shc PTB domain demonstrated a striking homology to the structures of pleckstrin homology domains, which suggested acidic phospholipids as a second ligand for the Shc PTB domain. Here we demonstrate that Shc binding via its PTB domain to acidic phospholipids is as critical as binding to phosphotyrosine for leading to Shc phosphorylation. Through structure-based, targeted mutagenesis of the Shc PTB domain, we first identified the residues within the PTB domain critical for phospholipid binding in vitro. In vivo, the PTB domain was essential for localization of Shc to the membrane, as mutant Shc proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane preceded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylation of Shc following receptor activation. Thus, Shc, through its interaction with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor via a single PTB domain.     

beta-Arrestin1 knockout mice appear normal but demonstrate altered cardiac responses to beta-adrenergic stimulation

beta-Arrestin1 knockout mice were studied to define the physiological role of beta-arrestin1 in the regulation of G protein-coupled receptors. beta-Arrestin1 is thought to be involved in the desensitization of many G protein-associated cell surface receptors, particularly beta-adrenergic receptors. Homozygous knockout mice are overtly normal. Resting cardiovascular parameters modulated by beta-adrenergic receptors such as heart rate, blood pressure, and left ventricular ejection fraction are not changed. However, homozygous mutants are more sensitive to beta-receptor agonist-stimulated increases in ejection fraction, consistent with a role of beta-arrestin1 in beta-adrenergic receptor desensitization. We conclude that beta-arrestin1 is important for in vivo G protein-coupled receptor desensitization and that this aspect of desensitization represents a mechanism for fine-tuning responses. However, beta-arrestin1 does not appear to be required for development or for other essential biological functions.