In vitro displacement by rat serum of adsorbed radiolabeled poloxamer and poloxamine copolymers from model and biodegradable nanospheres

Poloxamer 407 and poloxamine 908 have been used by many research groups to modify the surface of both model latex and biodegradable nanospheres, thereby producing nanospheres that have shown reduced protein adsorption in vitro and extended circulation times in vivo. A potential limitation of such systems is the desorption of the copolymer coating layer. We describe a two-stage process to radiolabel poloxamer 407 and poloxamine 908 that has facilitated an investigation into this potential desorption, in vitro. The first stage of the labeling procedure involved the substitution of the terminal hydroxyl groups in each poly(ethylene oxide) (PEO) chain of poloxamer 407 and poloxamine 908 with an amino group. The aminated copolymers were then radiolabeled with 125Iodine Bolton-Hunter reagent. The efficiency of labeling was calculated to be approximately 20% for the tetramine poloxamine 908 and approximately 33% for the diamine poloxamer 407. Remaining free amino groups were then either acetylated, using acetic anhydride, or left in the free amino form. Covalent linkage of the radiolabel to the copolymer was confirmed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. The stability of the link between radiolabel and copolymer to hydrolysis was also confirmed; <4% loss of radiolabel occurred from poloxamine 908 after incubation in phosphate-buffered saline (PBS) at 37 degrees C for 8 days. The radiolabeled copolymers (with the free amino groups acetylated) were then used in experiments that have given the first direct evidence that adsorbed copolymers can be displaced by serum proteins in significant amounts from the surface of model and biodegradable nanospheres. The displacement was highly dependent on copolymer-nanosphere compatibility, with up to 78% of 125I tetramine poloxamine 908 being displaced from poly(lactide-co-glycolide) (PLGA) nanospheres in 24 h, compared with 20% displacement of 125I tetramine poloxamine 908 in 24 h from polystyrene nanospheres. These results have direct implication for the future design of drug delivery systems based on coated nanospheres.     

Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells

Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-alpha-induced apoptosis. To address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or double transfected with Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibited apoptosis induced by either anti-Fas or TNF-alpha. In addition, Bcl-xL prevented the change in cytochrome c immunolocalization induced by anti-Fas or TNF-alpha treatment. Using antibodies that recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was detected in MCF7/F cells following anti-Fas or TNF-alpha, but not during UV-induced apoptosis. In MCF7/FB cells, caspase-8 was processed normally while processing of the downstream caspase-7 was markedly attenuated. Moreover, apoptosis induced by direct microinjection of recombinant, active caspase-8 was completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8 signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step.     

The role of Alx-4 in the establishment of anteroposterior polarity during vertebrate limb development

We have determined that Strong's Luxoid (lstJ) [corrected] mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ [corrected] mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and Fibroblast Growth Factor (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.     

Protection of mice against Plasmodium yoelii sporozoite challenge with P. yoelii merozoite surface protein 1 DNA vaccines

Immunization of mice with DNA vaccines encoding the full-length form and C and N termini of Plasmodium yoelii merozoite surface protein 1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titers after challenge. In C57BL/6 mice, two DNA vaccines provided protection comparable to that of recombinant protein consisting of the C terminus in Freund's adjuvant.     

Rupture recurrence after surgical repair of postinfarction ventricular septal rupture. Influence of early thrombolysis

OBJECTIVES: The aim of this study was to identify factors causing rupture recurrence after surgical repair of postinfarction ventricular septal rupture and to evaluate the indication for reoperation. PATIENTS: Recurrence of rupture was analysed in 25 out of a series of 109 patients who underwent surgical repair for postinfarction ventricular septal rupture between 1980 and 1992 in our institution. RESULTS: The mean interval between initial operation and recurrence was 3.6 days with a median of 2 days. Multivariate logistic regression analysis identified early thrombolysis after infarction (P = 0.0085) as a risk factor for recurrence of the rupture. Rupture recurrence occurred more in the anterior then in the posterior infarction site, although non-significant. Reoperation was indicated in 15 patients, in 13 for postrecurrent cardiac failure. The main determinant of cardiac failure was a large postrecurrent shunt (P = 0.05). The mean interval between initial operation and reoperation was 136 days with a median of 101 days. In 6 patients a combined apical ventricular septal rupture recurrence and anterior ventricular aneurysm was found, in 9 patients the recurrent rupture was proximally located, without concomitant aneurysm formation. Of 15 patients who were reoperated, one died in hospital and three after the in-hospital period. Of 10 patients treated conservatively, one died in hospital and two after the in-hospital period. One residual ventricular septal rupture closed spontaneously. CONCLUSIONS: Rupture recurrence is mainly determined by early thrombolysis. Postrecurrent cardiac failure, as the main indication for reoperation, is dependent on postrecurrent shunt size.     

Synergy between T cell Receptor and Fas (CD95/APO-1) signaling in mouse thymocyte death

Both extracellular and intracellular calcium (Ca2+) play important roles in hypoxic pulmonary vasoconstriction (HPV) and the vasoconstrictor responses to endogenous pulmonary vasoconstrictor substances, as evidenced by the effect of calcium-channel blockers on these vasoconstrictor responses and the measurement of changes in Ca2+ flux or intracellular Ca2+ concentrations in isolated cells. The more vasoselective the calcium-channel blocker, the greater its effect on pulmonary vasoconstriction. However, these drugs are not selective for the pulmonary vascular bed and are not as potent as pulmonary vasodilators when compared with other vasodilator drugs, including prostaglandin E1, isoproterenol, prostacyclin, or nitroglycerin. Moreover, the primary effect of vasoselective calcium-channel blockers on pulmonary vascular resistance is secondary to the effects of these agents on systemic vascular resistance and cardiac output. Although there is improvement in oxygen delivery, exercise tolerance, and survival in patients with primary pulmonary hypertension who respond to calcium-channel blockers, the response of individual patients to these drugs is difficult to predict because the extent of reversible versus irreversible changes in the pulmonary vasculature is not known. The use of these drugs in patients with chronic hypoxia-induced pulmonary vasoconstriction may be associated with a worsening of ventilation-perfusion mismatching secondary to inhibition of HPV.     

Solitary fibrous tumor of the adrenal gland

Solitary fibrous tumors are rare neoplasms, most commonly involving the pleura, recently described in various other locations. We report a solitary fibrous tumor of the right adrenal gland in a 42-year-old woman, discovered incidentally during abdominopelvic ultrasonographic examination. Pathologic and immunohistologic features of the tumor were identical to those of other solitary fibrous tumors. Three-quarters of this unencapsulated infiltrating tumoral mass presented foci of hemorrhage and were made of small, round, epithelioid-like cells that expressed the CD34 antigen more weakly than do the typical spindle cells usually observed in solitary fibrous tumors. Despite hemorrhage and poor limitation, the tumor behaved in a innocuous manner; the mass remaining unchanged for more than 5 years before the patient agreed to surgical intervention, which was recommended because of a sudden enlargement of the mass.     

Epidermal growth factor receptor and transforming growth factor alpha expression in papillary and nonpapillary renal cell carcinoma: correlation with metastatic behavior and prognosis

Papillary renal carcinomas are a cytogenetically unique subset of renal carcinomas that have been reported to be clinically less aggressive. We have examined 19 papillary tumors for immunohistochemical expression of the epidermal growth factor receptor (EGF-R) and its ligand, transforming growth factor alpha (TGF-alpha). EGF-R and TGF-alpha expression was also studied in 149 nonpapillary tumors and 7 mixed papillary/solid tumors. EGF-R and TGF-alpha expression were compared to histology, stage, metastatic behavior, and survival. Formalin-fixed, paraffin-embedded nephrectomy specimens collected between 1977 and 1986 were stained with antibodies to EGF-R and TGF-alpha. Patients with papillary tumors were found to present with earlier stage disease and had significantly longer survival. Papillary tumors had a significantly lower rate of EGF-R positivity than solid pattern tumors (21% versus 73%, P < 0.001). Intermediate or strong cell membrane immunoreactivity for EGF-R was associated with high tumor grade and poor disease-specific survival. EGF-R positivity in the primary tumor was associated with the presence of metastatic disease and with metastatic spread to lung versus bone. Tumor parenchymal TGF-alpha staining was present in 50% of the cases and was not associated with stage or grade. Unrelated to tumor parenchymal TGF staining, tumor vessels stained for TGF-alpha in 56% of the cases. Vessel TGF-alpha staining was absent in papillary tumors (P < 0.001). The improved clinical behavior of papillary tumors as compared to nonpapillary renal tumors may be related, in part, to their relatively lower levels of EGF-R expression.     

Sequence analysis of the CCG polymorphic region adjacent to the CAG triplet repeat of the HD gene in normal and HD chromosomes

The CAG expansion responsible for Huntington's disease (HD) is followed by an adjacent polymorphic CCG repeat region which may interfere with a PCR based diagnosis. We have sequenced this region in 52 unrelated HD patients, from both normal and HD chromosomes. Fifty percent of the normal alleles were (CCG)7(CCT)2, 48% (CCG)10(CCT)2, and 2% (CCG)7(CCT)3. In contrast (CCG)7(CCT)2 was found in 85% of the HD alleles which represents significant linkage disequilibrium with the HD mutation.