Understanding structural/functional properties of immunoconjugates for cancer therapy by computational approaches.

Monoclonal antibodies coupled to highly toxic molecules (immunoconjugates) are currently being developed for cancer therapy. We have used an in silico procedure for evaluating some physicochemical properties of two tumor-targeting anti-HER2 immunoconjugates: (a) the single-chain antibody scFv(FRP5) linked to a bacterial toxin, that has been recently progressed to phase I clinical trial in human cancer; (b) the putative molecule formed by the intrinsically stable scFv(800E6), which has been proposed as toxin carrier to cancer cells in human therapy, joined to the same toxin of (a). Structural models of the immunoconjugates have been built by homology modeling and assessed by molecular dynamics simulations. The trajectories have been analyzed to extract some biochemical properties and to assess the potential effects of the toxin on the structure and dynamics of the anti-HER2 antibodies. The results of the computational approach indicate that the antibodies maintain their correct folding even in presence of the toxin, whereas a certain stiffness in correspondence of some structural regions is observed. Furthermore, the toxin does not seem to affect the antibody solubility, whereas it enhances the structural stability. The proposed computational approach represent a promising tool for analyzing some physicochemical properties of immunoconjugates and for predicting the effects of the linked toxin on structure, dynamics, and functionality of the antibodies.

Percentage of free serum prostate-specific antigen as a predictor of pathologic features of prostate cancer in a screening population.

OBJECTIVES: Measurement of the percentage of free prostate-specific antigen (%FPSA) in serum can improve the specificity of prostate cancer screening. We evaluated the ability of %FPSA to predict pathologic features of screen-detected clinically localized prostate cancer. METHODS: We evaluated the correlation between %FPSA in serum before cancer diagnosis and the pathologic features of the cancers detected in 108 men with clinically localized prostate cancer who were treated with radical prostatectomy and for whom complete embedding of the radical prostatectomy specimen was performed. Ninety-seven men (90%) had a previous negative screening evaluation before prostate cancer was detected. RESULTS: There was a negative correlation of %FPSA with penetration of cancer through the prostatic capsule, cancerous surgical margins, Gleason score, percentage of cancer in the gland, and tumor volume (r = -0.2 to -0.4). After controlling for other preoperative predictors, %FPSA predicted capsular penetration (adjusted odds ratio [OR] 1.6, 95% confidence interval [CI] 1.1 to 2.4, for each 5% decrease in %FPSA) and cancer volume 0.5 cc or greater (adjusted OR 1.6, 95% CI 1.1 to 2.3). Preoperative %FPSA also predicted possibly harmless cancer (OR 1.5, 95% CI 1.1 to 2.2, for each 5% increase in %FPSA). CONCLUSIONS: In a select group of men for whom cancer was detected early via screening, a lower %FPSA in serum suggests a potentially more threatening cancer. This information may aid patients and clinicians in making more informed decisions about the management of prostate cancer, such as selecting patients for watchful waiting. However, more research is needed to determine the performance characteristics of %FPSA in clinical practice.

Prostate-specific antigen as a screening test for prostate cancer. The United States experience.

Serum PSA-based early detection for prostate cancer has been studied fairly extensively for the past several years. It appears that we can state fairly categorically what the relative performances of total serum PSA, DRE, and TRUS are in detecting early-stage prostate cancer; that initial screening is effective in detecting histologically significant and pathologically organ-confined prostate cancer; that annual, serial, repetitive screening, at least over a 4- to 5-year horizon, does not overdetect prostate cancer, and that the results of early detection will improve as our ability to use certain PSA transformations such as PSA density, PSA slope, age-specific PSA adjustment, and knowledge of free versus total serum PSA is better characterized. These advances in our ability to diagnose early-stage prostate cancer likely will be coupled with an increased ability to predict the behavior, curability, and significance of individual tumors. It is hoped that information soon will be available to allow physicians to categorize an individual tumor as insignificant, significant and surgically curable, or significant and incurable by standard approaches. This ability, coupled with the demonstrated ability to detect prostate cancer, will make an even more compelling argument for widespread PSA-based screening. At present, annual DRE and total serum PSA measurements are recommended for men older than 50 and among younger men at high risk for prostate cancer. All suspicious DRE findings should be evaluated with prostatic biopsy. Among younger men, PSA levels over 2.5 ng/mL should be considered worrisome and further evaluated. For men older than 65, serum PSA levels above 4 ng/mL should be considered abnormal and warrant biopsy. Men with persistent serum PSA elevation and a negative biopsy should undergo repeat biopsy at least once, and perhaps more often if PSA slope exceeds 0.75 per year, if density is greater than 0.10, or if f-PSA is less than 20%.

Long-term molecular dynamics simulation of copper azurin: structure, dynamics and functionality.

A long-term molecular dynamics simulation (1.1 ns), at 300 K, of fully hydrated azurin has been performed to put into relationship the protein dynamics to functional properties with particular attention to those structural elements involved in the electron transfer process. A detailed analysis of the root mean square deviations and fluctuations and of the intraprotein H-bonding pattern has allowed us to demonstrate that a rigid arrangement of the beta-stranded protein skeleton is maintained during the simulation run, while a large mobility is registered in the solvent-exposed connecting regions (turns) and in the alpha-helix. Moreover, the structural elements, likely involved in the electron transfer path, show a stable H-bonding arrangement and low fluctuations. Analysis of the dynamical cross-correlation map has revealed the existence of correlated motions among residues connected by hydrogen bonds and of correlated and anti-correlated motions between regions which are supposed to be involved in the functional process, namely the hydrophobic patch and the regions close to the copper reaction center. The results are briefly discussed also in connection to the current through-bond tunneling model for the electron transfer process. Finally, a comparison with the structural and the dynamical behaviour of plastocyanin, whose structure and functional role are very similar to those of azurin, has been performed.

A molecular dynamics simulation study of the solvent isotope effect on copper plastocyanin.

The effect of heavy water on the structure and dynamics of copper plastocyanin as well as on some aspects of the solvent dynamics at the protein-solvent interfacial region have been investigated by molecular dynamics simulation. The simulated system has been analyzed in terms of the atomic root mean square deviation and fluctuations, intraprotein H-bond pattern, dynamical cross-correlation map and the results have been compared with those previously obtained for plastocyanin in H2O (Ciocchetti et al. Biophys. Chem. 69 (1997), 185-198). The simulated plastocyanin structure in the two solvents, averaging 1 ns, is very similar along the beta-structure regions, while the most significant differences are registered, analogous to the turns and the regions likely involved in the electron transfer pathway. Moreover, plastocyanin in D2O shows an increase in the number of both the intraprotein H-bonds and the residues involved in correlated motions. An analysis of the protein-solvent coupling evidenced that D2O makes the H-bond formation more difficult with the solvent molecules for positively charged and polar residues, while an opposite trend is observed for negatively charged residues. On the other hand, the frequency of exchange of the solvent molecules involved in the protein-solvent H-bond formation is significantly depressed in D2O. The results are discussed also in connection with protein functionality and briefly with some experimental results connected with the thermostability of proteins in D2O.

Concerted motions in copper plastocyanin and azurin: an essential dynamics study.

Essential dynamics analysis of molecular dynamics simulation trajectories (1.1 ns) of two copper containing electron transfer proteins, plastocyanin and azurin, has been performed. The protein essential modes have been analysed in order to identify large concerted motions which could be relevant for the electron transfer function exerted by these proteins. The analysis, conducted for temporal windows of different lengths along the protein trajectories, shows a rapid convergence and indicates that for both the proteins the predominant internal motions occur in a subspace of only a few degrees of freedom. Moreover, it is found that for both the proteins the likely binding sites (i.e. the hydrophobic and negative patches) with the reaction partners move in a concerted fashion with a few structural regions far from the active site. Such results are discussed in connection with the possible involvement of large concerted motions in the recognition and binding interaction with physiological electron transfer partners.

Molecular dynamics simulation and essential dynamics study of mutated plastocyanin: structural, dynamical and functional effects of a disulfide bridge insertion at the protein surface.

A molecular dynamics simulation (1.1 ns) at 300 K, of fully hydrated Ile21Cys, Glu25Cys plastocyanin mutant has been performed to investigate the structural, dynamical and functional effects of a disulfide bridge insertion at the surface of the protein. A detailed analysis of the root mean square fluctuations, H-bonding pattern and dynamical cross-correlation map has been performed. An essential dynamics method has also been applied as complementary analysis to identify concerted motions (essential modes), that could be relevant to the electron transfer function. The results have been compared with those previously obtained for wild-type plastocyanin and have revealed that the mutant shows a different pattern of H-bonds, with several interactions lost and a higher flexibility, especially around the electron transfer copper site. The analysis of dynamical cross-correlation map and of essential modes, has shown that the mutant performs different functional concerted motions, which might be related to the binding recognition with its electron transfer partners in comparison with the wild-type protein.

Anti-CD3 and anti-CD2-induced T-cell activation in primary Sjögren's syndrome.

Because T-cell dysfunctions have been reported in patients with primary Sjögren's syndrome (SS), peripheral blood mononuclear cell (PBMC) proliferation obtained with anti-CD3 and anti-CD2 monoclonal antibodies was evaluated in these patients. Anti-CD3-induced mitogenesis, which varied widely among the patients, was lower in subjects with evidence of anti-SSA and anti-SSB antibodies than in controls. Moreover, the anti-CD2-induced response was depressed in about half the patients and the nonresponders were mainly those with anti-SSA and anti-SSB antibodies. Phorbol myristate acetate, a protein kinase C activator, used alone or added to anti-CD3, induced greater proliferation in patients than in control PBMC. In contrast, exogenous recombinant interleukin 2 (rIL-2) did not significantly enhance the anti-CD2-induced response of patients' PBMC, as it did in normal PBMC. Peripheral blood and parotid T cells from a patient with well-defined primary SS and parotid enlargement also responded poorly to anti-CD2 stimulation. Exogenous rIL-2 restored T-cell proliferation only in the salivary gland cultures of this patient. The present findings suggest that there is a T-cell activation defect in subjects with primary SS, particularly in those with circulating anti-SSA and anti-SSB antibodies. In addition, the difference in the response to IL-2 of peripheral blood and parotid-infiltrating T cells would seem to indicate that T-cell subsets are differently distributed in the blood and inflammation site.

Interferon-gamma (r-IFN-gamma) induced activation of alveolar macrophages (AM) from anergic patients with chronic obstructive pulmonary disease (COPD).

Forty-six anergic patients (37 males and 9 females, age range 55-79 yr) were selected from ninety-one patients suffering from COPD due to frequent exacerbations and impaired delayed cutaneous reactivity (43.9%). The phenotype of circulating lymphocytes, their proliferative response to a panel of polyclonal T-cell activators and the candidacidal activity (CA) of circulating PMNs (polymorphonuclear cells) were measured. In 13 patients presenting a defective CA of circulating PMNs, the in vitro response of alveolar macrophage CA to r-IFN-gamma was also determined. We found: 1) a significant reduction in the CL response to PHA in COPD patients vs controls; 2) a low PMN-CA in 23 (57%) COPD patients; 3) a non-significant difference in phenotype analysis in patients and controls; 4) lower CA of AMs in COPD patients than in controls; 5) restoration in vitro of CA by r-IFN-gamma in the group of anergic COPD patients presenting depressed CA. We conclude that a defective cell-mediated immunity could be the basis of the enhanced susceptibility to infectious exacerbations in many COPD patients and that, in vitro, it could be reversed by r-IFN-gamma treatment.

Structural and optical properties of silicon nanocrystals grown by plasma-enhanced chemical vapor deposition.

Silicon nanocrystals (Si-nc) embedded in SiO2 matrix have been prepared by high temperature thermal annealing (1000-1250 degrees C) of substoichiometric SiOx films deposited by plasma-enhanced chemical vapor deposition (PECVD). Different techniques have been used to examine the optical and structural properties of Si-nc. Transmission electron microscopy analysis shows the formation of nanocrystals whose sizes are dependent on annealing conditions and deposition parameters. The spectral positions of room temperature photoluminescence are systematically blue shifted with reduction in the size of Si-nc obtained by decreasing the annealing temperature or the Si content during the PECVD deposition. A similar trend has been found in optical absorption measurements. X-ray absorption fine structure measurements indicate the presence of an intermediate region between the Si-nc and the SiO2 matrix that participates in the light emission process. Theoretical observations reported here support these findings. All these efforts allow us to study the link between dimensionality, optical properties, and the local environment of Si-nc and the surrounding SiO2 matrix.

Renal masses: urologic management.

This article reviews the differential diagnoses for solid renal masses and staging for renal cell carcinoma, and also discusses several areas of controversy in regard to the management of solid renal masses, such as the role of nephron-sparing surgery, extended lymphadenectomy, and ipsilateral adrenalectomy. The management of renal cell carcinoma associated with tumor thrombus within the vena cava is discussed, as are issues regarding both surgical and medical management (including chemotherapy and immunotherapy) of metastatic disease. Lastly, the emerging role of laparoscopic nephrectomy is examined.

Occult thyrotoxicosis in patients with chronic and paroxysmal isolated atrial fibrillation.

Thyroid function tests, including thyrotropin releasing hormone administration (TRH), were performed in 40 consecutive patients with isolated atrial fibrillation (IAF) (i.e., without any other evidence of cardiac disease). The arrhythmia was chronic in 5 and paroxysmal in 35 patients. Thyrotoxicosis could not be diagnosed either clinically or by abnormal serum levels of T4, T3, T3 BC, and thyroid stimulating hormone (TSH). Thyroid stimulating hormone response to TRH, which was normal in 35 patients, was absent in 5 (12.5%) who were considered to have occult thyrotoxicosis. One had chronic and the other 4 had paroxysmal IAF. The arrhythmia did not recur after antithyroid treatment in these four patients who were in sinus rhythm after a mean follow-up period of 21 months. Full exploration of the thyroid function therefore seems useful not only in patients with chronic IAF, but also in those affected by the paroxysmal form.

Assessment of sinus node function in asymptomatic subjects with sinus bradycardia and in symptomatic patients with sino-atrial disease.

Sinus node function was evaluated in 18 patients with sinus bradycardia without complaints (Group I), in 16 patients with sinus bradycardia and/or sinoatrial block with complaints (subgroup IIa) and in 14 patients with the bradycardia-tachycardia syndrome (subgroup IIb). Mean values of corrected sinus node recovery time (CSRT), atrial effective refractory period (AERP) and atrial functional refractory period (AFRP) differentiated significatively asymptomatic subjects of group I from the two subgroups of patients with sinoatrial disease, but failed to differentiate each subgroup from the other one. There was no significative difference in mean sinoatrial conduction time (SACT) between group I and each of the two subgroups. Three patients of subgroup IIa and 1 patient of subgroup IIb had a false negative response after both overdrive and premature programmed atrial pacing. Spontaneous cycle length was directly correlated with the sinus node recovery time and the atrial refractoriness in group I, and with the only sinus node recovery time in subgroup IIb. No direct correlations were observed in subgroup IIa. This suggests a less disturbed sinus node automaticity in bradycardia-tachycardia syndrome.

[Association of pulmonary and portal hypertension]. / Associazione tra ipertensione arteriosa polmonare ed ipertensione portale.

The association between portal venous hypertension and pulmonary arterial hypertension has received scarce attention in the italian medical literature. Nevertheless the association is relatively frequent, it needs a multidisciplinary approach and it is a stimulus for the search of causes of so-called primary pulmonary hypertension. The purpose of the article is to review the frequency of the association, the main pathogenetic hypothesis formulated to explain the appearance of pulmonary hypertension, the clinical and the laboratory findings, the evolution of the association and to present briefly a personal series of cases. The pulmonary arterial hypertension has been found in approximately 2% of patients with portal hypertension due to either hepatic cirrhosis or extraepatic lesions. Microembolism from the portocavat system or a number of vasoactive substances which enter the pulmonary circulation without being inactivated by the liver have been held responsible for the appearance of pulmonary hypertension in predisposed patients. Clinical and laboratory findings do not differ from those a patients with primary pulmonary hypertension. Also the prognosis is similar. In conclusion on accurate examination of the pulmonary circulation by noninvasive methods, in particular by echocardiography, appears to be mandatory in patients with chronic hepatic lesions. When pulmonary arterial hypertension is detected the study of the biochemical factors which at present are known to determine pulmonary hypertension may be warranted. The study may enhance our knowledge of the pathogenesis of the so-called primary pulmonary hypertension.

Phenotypic analysis of T cell subsets in the blood of chronically aborting women.

A relative decrease in helper and a parallel increase in suppressor-cytotoxic T lymphocytes was found in the blood of six habitually aborting women, as compared with the T-cell subset distribution in ten normal multiparae and eight multigravid post-partum women. It is suggested that an imbalance between the immunoregulatory T-cell subpopulations may contribute to the rejection of the semiallogenic fetus.