Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay.

We investigated the combined effect of the initial cell density (12,500, 35,000, 75,000, and 100,000 cells cm(-2)) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing time-dependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation between the rate of cell death and the initial cell seeding density was found at 2.5 µM doxorubicin concentration, whereas this was not observed at 5 or 100 µM. By sensing the changes in the cell-substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 µM doxorubicin, whereas the impedance showed at this time point cell viability that was below 25%. These results indicate that impedance detection reveals cytotoxic events undetectable when using the MTS assay, highlighting the importance of combining impedance detection with traditional drug toxicity assays towards a more in depth understanding of the effect of anti-cancer drugs on in vitro assays. Moreover, the detection of doxorubicin induced toxicity determined with impedance under static conditions proved to be 6 times faster than in perfusion culture.

Mechanical and electrophysiological properties of the sarcolemma of muscle fibers in two murine models of muscle dystrophy: col6a1-/- and mdx.

This study aimed to analyse the sarcolemma of Col6a1-/- fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1-/- and increased in mdx fibers. In the membrane of Col6a1-/- fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane conductance and capacitance. In contrast, in mdx intact FDB muscle resting conductance was increased while resting potential and ap depolarization rate were reduced, likely indicating the presence of a consistent population of severely altered fibers which disappear during the culture preparation.

Effect of the anisotropic permeability in the frequency dependent properties of the superficial layer of articular cartilage.

Articular cartilage is a tissue of fundamental importance for the mechanics of joints, since it provides a smooth and lubricated surface for the proper transfer of loads. From a mechanical point of view, this tissue is an anisotropic poroviscoelastic material: its characteristics at the macroscopic level depend on the complex microscopic architecture. With the ability to probe the local microscopic features, dynamic nanoindentation test is a powerful tool to investigate cartilage mechanics. In this work we focus on a length scale where the time dependent behaviour is regulated by poroelasticity more than viscoelasticity and we aim to understand the effect of the anisotropic permeability on the mechanics of the superficial layer of the articular cartilage. In a previous work, a finite element model for the dynamic nanoindentation test has been presented. In this work, we improve the model by considering the presence of an anisotropic permeability tensor that depends on the collagen fibers distribution. Our sensitivity analysis highlights that the permeability decreases with increasing indentation, thus making the tissue stiffer than the case of isotropic permeability, when solicited at the same frequency. With this improved model, a revised identification of the mechanical and physical parameters for articular cartilage is provided. To this purpose the model was used to simulate experimental data from tests performed on bovine tissue, giving a better estimation of the anisotropy in the elastic properties. A relation between the identified macroscopic anisotropic permeability properties and the microscopic rearrangement of the fiber/matrix structure during indentation is also provided.

Topographical Features of Graphene-Oxide-Functionalized Substrates Modulate Cancer and Healthy Cell Adhesion Based on the Cell Tissue of Origin.

Graphene-derived materials, such as graphene oxide (GO), have been widely explored for biomedical and biological applications, including cancer research. Despite some recent works proving that GO inhibits the migration and invasion of different cancer cells, so far most of these in vitro studies have been conducted using GO sheets dispersed in solution or as a planar film. On the contrary, little is known about cellular activities, such as cell viability, adhesion, and spreading, when cancer cells interface with GO functionalized hydrogel-based surfaces, biomechanically and structurally more similar to the tumor environment. Here, we evaluate the interactions of human breast cancer cells (MDA-MB-231) with alginate (Alg)/GO hydrogel-based substrates, and compare them with a cancer cell line from human osteosarcoma (HOS) and healthy murine fibroblasts (3T3). We observed that GO addition selectively inhibits malignant breast cancer cell adhesion efficiency and spreading area, while promotes HOS and 3T3 adhesive processes. Furthermore, we did not observe the same results over Alg substrates with GO nanosheets dispersed in the medium, without embedment into the Alg. This suggests that cancer (MDA-MB-231 and HOS) and healthy (3T3) cell adhesion efficacy does not depend on the cellular tumoral nature and it is driven by the topographical cues provided by the GO-based substrates, whose physical-mechanical characteristics better mimic those of the cell native tissue. We envision that this study can provide a rational for future design and use of graphene-based nanomaterials for cancer research by deepening the knowledge of graphene-cancer cell specific interactions.

Multiwell micromechanical cantilever array reader for biotechnology.

We use a multiwell micromechanical cantilever sensor (MCS) device to measure surface stress changes induced by specific adsorption of molecules. A multiplexed assay format facilitates the monitoring of the bending of 16 MCSs in parallel. The 16 MCSs are grouped within four separate wells. Each well can be addressed independently by different analyte liquids. This enables functionalization of MCS separately by flowing different solutions through each well. In addition, each well contains a fixed reference mirror which allows measuring the absolute bending of MCS. In addition, the mirror can be used to follow refractive index changes upon mixing of different solutions. The effect of the flow rate on the MCS bending change was found to be dependent on the absolute bending value of MCS. Experiments and finite element simulations of solution exchange in wells were performed. Both revealed that one solution can be exchanged by another one after 200 microl volume has flown through. Using this device, the adsorption of thiolated DNA molecules and 6-mercapto-1-hexanol on gold surfaces was performed to test the nanomechanical response of MCS.

Cytokine network and acute primary HIV-1 infection.

OBJECTIVE: To investigate the relationship between cytokine serum levels, peripheral blood lymphocyte subsets and clinical picture in acute primary HIV-1 infection. PATIENTS AND METHODS: Absolute number/microliters total lymphocytes, CD4+, CD8+ and natural killer (NK) cells, as well as serum levels of soluble CD8 receptor, interleukin (IL)-1 beta, IL-2, IL-4, IL-6, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, beta 2-microglobulin and 5'-neopterin were determined in 15 patients with acute primary HIV-1 infection, 16 asymptomatic HIV-1-seropositive individuals and 18 HIV-1-seronegative individuals at risk for HIV-1 infection. RESULTS: Acute primary HIV-1 infection was characterized by significant CD4+ lymphocytopenia with low IL-2 serum concentrations, and by high absolute number of circulating CD8+ and NK cells, with elevated serum levels of soluble CD8 receptor, IL-1 beta, IFN-gamma and 5'-neopterin. Follow-up of acute seroconverters showed a significant decrease in NK cell counts and IL-1 beta levels, with an increase of IL-6. CONCLUSIONS: In acute primary HIV-1 infection, significant alteration of cytokine release, possibly induced by viral antigens, could be responsible for both clinical picture and activation of cytotoxic cells through abnormal mechanisms.

High serum level of the soluble form of CD30 molecule in the early phase of HIV-1 infection as an independent predictor of progression to AIDS.

OBJECTIVE: To determine the serum levels of the soluble form of the CD30 (sCD30) activation molecule in the early phase of HIV-1 infection, and to investigate the possible correlation with evolution to AIDS. METHODS: sCD30 values were determined by an enzyme-linked immunosorbent assay (ELISA) on serum samples collected at the time of the first evidence of HIV-1 infection in 110 individuals with a median follow-up of 56 months (range, 12-88 months), at the A1 (74 cases) or A2 (36 cases) stages of the 1993 revised Centers for Disease Control and Prevention classification. The data were evaluated using established clinical and immunological parameters, including circulating CD4+ T-cell count. The controls were 110 blood donors and 51 HIV-1-negative subjects belonging to groups at risk for HIV-1 infection. RESULTS: Elevated sCD30 levels (> 20 U/ml) were found in 83.6% of HIV-1-infected cases and in 47% of at-risk seronegatives. Data analysis revealed that HIV-1-infected patients with higher sCD30 levels (> 35 U/ml) experienced faster disease progression (P = 0.0002). This was also the case in patients at the earliest stage (A1) of HIV infection (P = 0.0027). In these latter cases the predictive value of sCD30 was independent of the initial absolute number of circulating CD4+ lymphocytes. CONCLUSIONS: Serum levels of sCD30 are increased in the large majority of patients in the early phase of HIV-1 infection and represent an indicator of progression to AIDS independent of other prognostic parameters.

Longitudinal analysis of serum chemokine levels in the course of HIV-1 infection.

OBJECTIVES: To investigate the correlation between the serum levels of the CC-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, and the progression of HIV-1 disease. DESIGN: Retrospective analysis of serial serum samples from HIV-1 seroconverters selected according to clinical outcome. METHODS: Twenty-one patients, derived from a cohort recruited between 1985 and 1996 for a prospective study of the natural history of HIV infection, were analysed. All patients had at least one HIV-1-seronegative sample within 1 year prior to the first seropositive test and were followed longitudinally throughout the course of HIV-1 infection (mean follow-up, 73.5 months). Nine were rapid progressors (RP; patients who developed AIDS within 60 months of antibody seroconversion), seven were slow progressors (SP; patients who developed AIDS after 60 months), and five were long-term asymptomatic (LTA; patients with circulating CD4+ cells higher than 400 x 10(6)/l, no signs of HIV disease, no antiretroviral therapy for more than 96 months). A total of 339 serum samples was studied (mean, 16.1 per patient). The levels of RANTES, MIP-1alpha and MIP-1beta were measured by enzyme-linked immunosorbent assay and correlated with different immunological and clinical parameters. RESULTS: Over the entire follow-up period, the geometric mean of serum RANTES was significantly higher in RP [68.6 ng/ml; 95% confidence interval (CI), 56.9-82.7] than in SP (23.7 ng/ml; 95% CI, 20.0-28.2; P < 0.001) and LTA (19.5 ng/ml; 95% CI, 15.5-24.5; P < 0.001). This difference was already significant during the early clinical stages, when patients had peripheral blood CD4+ cell counts still greater than 400 x 10(6)/l (P < 0.001). By contrast, the mean serum levels of MIP-1alpha and MIP-1beta did not differ significantly between the three study groups. Multivariate analysis using the Cox proportional hazard model demonstrated that the mean serum concentration of RANTES before the development of AIDS was independently associated with the time to AIDS (relative risk, 4.5; 95% CI, 1.1-18.2; P = 0.035). In patients with low versus high mean serum RANTES before the fall of CD4+ cells below 400 x 10(6)/l, the median AIDS-free time was 117.5 and 42.7 months, respectively (P = 0.037). CONCLUSION: These data suggest that an elevation of serum RANTES predicts a rapid progression of the disease since the early stages of HIV-1 infection.

Characterisation of an antibody coated microcantilever as a potential immuno-based biosensor.

In this study, we investigated the activity, stability, lifetime and re-usability of monoclonal antibodies to myoglobin covalently immobilised onto microfabricated cantilever surfaces. These sensing surfaces are of interest to us in the development of novel cantilever-based immunosensors. For such sensors the antibody layer represents the sensing element while the microcantilever acts as a mechanical transducer. A procedure for producing re-usable biological coatings has been tested with different independent techniques. An Enzyme Linked Immunosorbent Assay (ELISA) was used to determine the presence of an active antibody coating, and to monitor the lifetime and stability of the immobilised antibody. Through this analysis, the activity of the immobilised antibody layer was found to be more stable with the introduction of sucrose, as a stabilising agent. Sucrose was applied to the immobilised antibody layer after each regeneration step. The immobilised antibody was found to have a stable active lifetime for up to 7 weeks. Fluorescence microscopy was used to give information on the distribution of the coating on the gold and silicon nitride sides of the cantilever. Atomic Force Microscopy was used to determine the presence of the biological coating on the cantilever and to obtain information on the surface morphology of the biological element of the sensor. The combined results provide valuable information on the development of an optimised sensing element and demonstrate a set of methods to use for future sensor-to-sensor characterisation. Preliminary experimental results showing the antibody activity against myoglobin, detected with a microcantilever based sensor prototype confirmed the motivations and potentialities of the proposed immunosensing technique.

Impact of antibiotic changes in empirical therapy on antimicrobial resistance in intensive care unit-acquired infections.

We conducted a one-year prospective study on intensive care unit (ICU)-acquired infections and antimicrobial resistance patterns in an 18-bed medical-surgical ICU of a tertiary-care university hospital. We divided the study into two six-month periods in order to evaluate the impact of antibiotic changes in empirical therapy on antimicrobial resistance profiles of the principal isolated micro-organisms. In the first period no changes were made to the previously applied empirical antibiotic protocol; at the end of this period we found high rates of methicillin resistance (MR) among staphylococci, 93% for Staphylococcus aureus (69 isolates) and 79% for coagulase-negative staphylococci (CNS) (48 isolates), and of multiple drug resistance for Pseudomonas aeruginosa (57 isolates), in particular 67% resistance to piperacillin/tazobactam (PIP/TZ). We therefore decided to substitute PIP/TZ with imipenem in nosocomial pneumonia and with cefepime plus metronidazole in peritonitis. We also considered the previous use of amoxicillin/clavulanate (AM/CL) at admission in critically ill patients inadequate; we therefore advised that no antibiotics should be given unless fever developed and eventually to replace AM/CL with trimethoprim/sulfamethoxazole (TMP/SMX). At the end of this intervention period, we observed a significant decrease of S. aureus MR (93 vs. 73%, P = 0.003) and of P. aeruginosa resistance to PIP/TZ (67 vs. 29%, P < 0.001). A reduction in MR was also seen in CNS (79 vs. 64%, P = 0.09). Other resistance patterns also improved among staphylococci; in contrast P. aeruginosa resistance to imipenem increased in the second period (24 vs. 41%, P = 0.06). A non-premeditated change of antibiotics in empirical therapy, on the basis of detected resistance patterns, provided promising results in reducing some antimicrobial resistance rates. We believe, however, that antibiotic changes must be tailored to local microbiological situation monitoring, and that a repeated rotation is crucial to limit the emergence of new resistance profiles. Furthermore the adoption of this policy should be accompanied by other infection control practices aimed at reducing antimicrobial resistance and nosocomial infection rates.

Successful simplification of HAART in patients with acute primary HIV infection.

Aggressive treatment has been advocated for the management of primary HIV infection (PHI), but the composition and the optimal duration of therapy are still to be determined. In addition, time to undetectable viral load (VL), rate and duration of VL suppression as well as subsequent therapeutic choices remain issues widely debated. We evaluated the rate and duration of VL suppression in 12 consecutive patients with PHI given triple-drug treatment with zidovudine, lamivudine and indinavir (highly active antiretroviral therapy, HAART) at onset of the acute illness and subsequently switched to a simplified 2-NRTI-based regimen once VL suppression was maintained for at least 6 months. Throughout the study, no patient discontinued treatment because of symptoms attributed to the study medications. In the study population, undetectable VL was achieved after a median of 84 days (range: 67-135) on HAART and was maintained for a median of 194 days (range: 179-205) before simplification. After switching to simplified maintenace, undetectable VL was maintained in all patients for at least 6 months. Only one patient experienced virological failure, plasma HIV-RNA remaining suppressed for a median foliow-up of 33 months (15-54) and T-CD4+ being steadily higher than 500/mL in the remaining patients. Our results suggest that simplification of HAART in patients promptly treated during PHI and maintaining undetectable VL for at least 6 months before simplification may be a valid option capable of controlling viral replication and maintaining an optimal immunological profile for a prolonged time.

Is a 3-month course of treatment with Peg-interferon effective in acute HCV hepatitis?

From February 2002, all the consecutive patients referring to the Department of Infectious Diseases, University of Turin, who were diagnosed as having acute HCV hepatitis were included in a prospective cohort study to evaluate if a 3-month course of Peg-Interferon alpha-2b (1.5 microg/kg once weekly) is effective to decrease the risk of progression to chronic disease. ALT and HCV-RNA measurements were scheduled at week 4 and 12 during treatment, and 24 weeks after the end of therapy. As of April 2003, ten patients were enrolled in the study. As to HCV genotype, seven patients had type 1 and 3 type non-1. At entry, median HCV-RNA level was 129500 (range: 3000-3100000 copies/mL) and six patients were symptomatic. Treatment was given within 20 days (range: 8-30) of the ALT peak. All patients completing 4 weeks (n = 9) and 12 weeks of treatment (n = 7) had undetectable HCV-RNA. Five patients who completed the 24-week follow-up after the end of treatment had a sustained viral response with ALT levels within normal range. Therapy was well tolerated in all patients. Even if our data are not definitive, our results show that once-weekly administration of Peg-interferon alfa-2b in patients with acute HCV infection may be an effective and convenient regimen.

[Pharmaco-radiodiagnosis of the digestive tract]. / La farmaco-radiodiagnostica dell'apparato digerente.

A brief account of the subject from the chronological standpoint is followed by a classification of the drugs most often used in radiological examination of the digestive tract into two groups according to their effects on individual viscera, namely excitomotor and hypotonising drugs. The indications and contraindications for each drug are explained, and the most common methods for the pharmacoradiological investigation of each organ are indicated. It is felt that this form of examination leads to an earlier radiological diagnosis and one more consonant with the real anatomical and pathological situation.

Is the clinical course of HIV-1 changing? Cohort study.

OBJECTIVE: To assess whether the clinical course of HIV infection has changed from 1985 to 1995. DESIGN: Cohort Study. SETTING: Infectious disease clinic. SUBJECTS: 285 patients recruited from September 1985 to January 1995 with < or = 12 months between the dates of their last seronegative and first seropositive test result and with first follow up visit in the six months after seroconversion and at least 12 months' follow up. Patients were grouped according to the date of seroconversion. MAIN OUTCOME MEASURES: Time to CD4 cell count of < 500, 400, and 200 x 10(6) cells/l, and clinical outcome defining AIDS; variation in cell count per day between consecutive visits, and ratio between this variation and time from estimated date of seroconversion at each visit. RESULTS: The groups were similar in age, number with acute primary HIV infection, CD4 cell count at intake, and cell count at the beginning of antiretroviral treatment; they differed in sex ratio, risk factors for HIV, probability of CD4 cell decline to < 500, 400, and 200 x 10(6) cells/l. and risk of developing AIDS. Acute infection, seroconversion after December 1989, and serum beta 2 microglobulin > 296 nmol/l were independent predictors of poor clinical course. The speed of CD4 cell decline, expressed as cell variation divided by the number of days between consecutive visits, increased with more recent seroconversion (P = 0.02). Ratio between the speed of CD4 cell decline and time from estimated date of seroconversion at each visit was also higher in the patients who seroconverted after December 1989. CONCLUSIONS: The faster disease progression and the higher speed of CD4 cell decline at early stages in the patients with recently acquired HIV infection suggest changes in the clinical course of HIV infection.

[Use of echography for the diagnosis of joint amyloidosis in dialyzed patients]. / Emploi de l'échographie pour le diagnostic de l'amyloïdose articulaire des dialysés.

Amyloid arthropathy is an important complication of long-term hemodialysis. This condition may lead to destructive bone lesions and to severe functional impairment. We studied with sonography of both knees 32 long-term dialysis patients (mean dialytic age: 69.7 months), whose 7 were carrying palpable joint swellings. In 29/32 patients, ultrasound scans were associated with knee X-rays films, patellar views included. In one case, opaque arthrography was performed too. Sonography demonstrated the presence of fluid collections within the articular space and the synovial cavities in 19/32 patients (59%). Their content was transonic or mildly hypoechoic. X-ray films were positive for amyloid bone involvement in 19/29 cases (66%), with intra-osseous cysts and cortical erosions. There was a good general agreement between the results obtained with the two techniques: therefore, in some cases only one examination was positive. Sonography is proposed, in association with standard X-ray films, as a simple and reliable method for the diagnosis of articular amyloidosis in dialysis patients and for the follow-up of this disease.

[Acute necrotizing ulcerative gingivitis (ANUG) in HIV antibody-positive subjects]. / Gengivite ulcero-necrotica acuta (GUNA) in soggetti AbHIV positivi.

The paper reports five cases of acute ulcero-necrotic gingivitis in AbHIV-positive subjects and stresses that the frequency of this pathology in the AbHIV-positive population is 0.67% (3.3% if only the homosexual population is taken into consideration) compared to 0.02-0.08% in the normal population. These findings may therefore suggest an association between AUNG and HIV-1 infection.

Poroelastic response of articular cartilage by nanoindentation creep tests at different characteristic lengths.

Nanoindentation is an experimental technique which is attracting increasing interests for the mechanical characterization of articular cartilage. In particular, time dependent mechanical responses due to fluid flow through the porous matrix can be quantitatively investigated by nanoindentation experiments at different penetration depths and/or by using different probe sizes. The aim of this paper is to provide a framework for the quantitative interpretation of the poroelastic response of articular cartilage subjected to creep nanoindentation tests. To this purpose, multiload creep tests using spherical indenters have been carried out on saturated samples of mature bovine articular cartilage achieving two main quantitative results. First, the dependence of indentation modulus in the drained state (at equilibrium) on the tip radius: a value of 500 kPa has been found using the large tip (400 µm radius) and of 1.7 MPa using the smaller one (25 µm). Secon, the permeability at microscopic scale was estimated at values ranging from 4.5×10(-16) m(4)/N s to 0.1×10(-16) m(4)/N s, from low to high equivalent deformation. Consistently with a poroelastic behavior, the size-dependent response of the indenter displacement disappears when characteristic size and permeability are accounted for. For comparison purposes, the same protocol was applied to intrinsically viscoelastic homogeneous samples of polydimethylsiloxane (PDMS): both indentation modulus and time response have been found size-independent.