Isolation of alkane-degrading bacteria from deep-sea Mediterranean sediments.

AIMS: To isolate and identify alkane-degrading bacteria from deep-sea superficial sediments sampled at a north-western Mediterranean station. METHODS AND RESULTS: Sediments from the water/sediment interface at a 2400 m depth were sampled with a multicorer at the ANTARES site off the French Mediterranean coast and were promptly enriched with Maya crude oil as the sole source of carbon and energy. Alkane-degrading bacteria belonging to the genera Alcanivorax, Pseudomonas, Marinobacter, Rhodococcus and Clavibacter-like were isolated, indicating that the same groups were potentially involved in hydrocarbon biodegradation in deep sea as in coastal waters. CONCLUSIONS: These results confirm that members of Alcanivorax are important obligate alkane degraders in deep-sea environments and coexist with other degrading bacteria inhabiting the deep-subsurface sediment of the Mediterranean. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the isolates obtained have potential applications in bioremediation strategies in deep-sea environments and highlight the need to identify specific piezophilic hydrocarbon-degrading bacteria (HCB) from these environments.

Effects of ritonavir-boosted darunavir vs. ritonavir-boosted atazanavir on lipid and glucose parameters in HIV-negative, healthy volunteers.

BACKGROUND: Darunavir (TMC114) is a new HIV protease inhibitor (PI). DESIGN: This Phase I, randomized, open-label trial compared the effects of darunavir plus low-dose ritonavir (RTV) (darunavir/RTV) with those of atazanavir/RTV on lipid and glucose parameters. METHODS: Forty-nine HIV-negative, healthy male volunteers received RTV 100 mg once a day (qd) for 7 days, followed by either darunavir/RTV 800/100 mg qd (n=25) or atazanavir/RTV 300/100 mg qd (n=24) for 21 days. Mean changes in fasting lipid and glucose parameters at day 28 were calculated using post-RTV alone (day 7) and baseline (day -1) values as references. Short-term safety, tolerability and RTV pharmacokinetic parameters were evaluated. RESULTS: After 7 days of RTV treatment, the mean triglyceride concentration increased by approximately 30 mg/dL in both groups, changes in other lipid and glucose parameters were relatively small. Mean concentrations of lipids and glucose over the treatment period were mostly similar between the treatment groups. Mean changes from day 7 to day 28 for the darunavir/RTV and atazanavir/RTV groups, respectively, were -3.6 and -0.5 mg/dL for high-density lipoprotein cholesterol; 5.0 and 5.3 mg/dL for low-density lipoprotein cholesterol; 4.9 and 1.2 mg/dL for total cholesterol; 6.4 and 14.0 mg/dL for triglycerides; -1.7 and -2.4 mg/dL for glucose; and -1.4 and 0.3 mg/dL for insulin. No grade 3 or 4 lipid or glucose laboratory abnormalities were reported. Treatment-emergent hyperbilirubinaemia was reported for all volunteers (including five grade 4 cases) during atazanavir/RTV treatment. CONCLUSIONS: Co-administration of darunavir or atazanavir with low-dose RTV resulted in minor and similar changes in lipid and glucose parameters in HIV-negative healthy volunteers.

Efficacy of once-daily darunavir/ritonavir 800/100 mg in HIV-infected, treatment-experienced patients with no baseline resistance-associated mutations to darunavir.

OBJECTIVE: The objective of this study was to examine the potential of once-daily dosing with darunavir/ritonavir 800/100 mg in a HIV-infected, treatment-experienced patient population with no baseline darunavir resistance-associated mutations (RAMs). METHODS: Patients in the randomized controlled POWER 1 and 2 trials were treatment experienced, with > or =1 International AIDS Society-USA primary protease inhibitor (PI) mutation. The virological and immunological responses in patients with no baseline darunavir RAMs receiving darunavir/r 800/100 mg once daily (n = 23), darunavir/r 600/100 mg twice daily (n = 29), or currently available PI(s) (n = 28) plus an optimized background regimen were compared. RESULTS: The proportion of patients achieving HIV RNA <50 copies per milliliter at week 24 was 67% for the group receiving darunavir/r 800/100 mg once daily and 62% for the group receiving darunavir/r 600/100 mg twice daily (P = 0.774); both were superior to control PI(s) (11%; P < 0.0001). Mean HIV RNA change from baseline was 22.39 and 22.35 log10 copies per milliliter for the group receiving darunavir/r 800/100 mg once daily and for the group receiving 600/100 mg twice daily, respectively (P = 0.895); mean CD4 increases were 88 and 111 cells per milliliter, respectively (P = 0.526). CONCLUSIONS: Treatment-experienced, HIV-infected patients with no baseline darunavir RAMs achieved similar high responses with darunavir/r 800/100 mg once daily and 600/100 mg twice daily. This suggests that once-daily darunavir/r 800/100 mg therapy, which has been shown effective in treatment-naive patients and is currently being studied in treatment-experienced patients, shows potential in patients with no darunavir RAMs.

Satisfaction with and psychological impact of immediate and deferred breast reconstruction.

BACKGROUND: The present work assesses the effect of immediate breast reconstruction (IBR), deferred breast reconstruction (DBR), and no breast reconstruction on the psychological impact. PATIENTS AND METHODS: Standard questionnaires were used to determine the psychological impact suffered by patients who underwent IBR, DBR and no reconstruction, their degree of satisfaction with the results achieved, and their postprocedure opinions regarding reconstruction options. RESULTS: A total of 526 women underwent mastectomy. The response rate to the questionnaires was 71.67%. A significantly greater proportion of the women who underwent no reconstruction suffered psychological problems than those who underwent reconstruction of some type (P = 0.01). Some 94.77% of the women who underwent IBR maintained a postprocedure preference for this option; in contrast, some 87.27% of the DBR and 56.14% of the no-reconstruction patients declared a postprocedure preference for IBR. In all, 63.49% of the women who underwent reconstruction were moderately very satisfied with the aesthetic results achieved, while only 22.80% of the no-reconstruction patients declared such satisfaction (P = 0.0001). CONCLUSIONS: The women who underwent no breast reconstruction suffered more emotional problems than those who underwent a reconstruction procedure. In general, all groups reported a postprocedure preference for IBR in their questionnaire answers. The aesthetic results achieved by IBR seem to be those best accepted.

[Complications and sequelae after nasal trauma]. / Complicaciones y secuela postraumatismo nasal.

Septal haematoma after nasal trauma is a complication that can lead to septal abscess if unrecognized or early intervention is not performed. It can cause compression and thereby necrosis that evolve to a septal abscess in which cultures reveal saprophyte bacteria. Cartilage necrosis and destruction can produce impaired breathing and aesthetic deformities with collapse of the dorsum and the tip of the nose. We present a 10 year old masculine infant, that suffered a nasal fracture with a septal haematoma that remained undiagnosed. The patient developed a septal abscess that required drainage and resulted in nasal sequelae with collapse of dorsum and cranial displacement of tip and columella. Functional and aesthetic reconstruction was performed using rib cartilage grafts. No complications occurred. Functional and aesthetic improvement was observed. Result after 2 months of follow-up is considered favourable.

Reconstrucción del complejo areola-pezón: revisión de 60 casos / Nipple-areola complex reconstruction: revision of 60 cases

La creación del complejo areola-pezón, CAP, constituye el último tiempo de la reconstrucción mamaria, transformando la reconstrucción del montículo mamario en una mama. Debemos considerar pues la reconstrucción de la areola y el pezón, como la culminación de la reconstrucción de la mama. Existen numerosas técnicas descritas para la reconstrucción del CAP. El objetivo de este estudio es determinar el grado de satisfacción psicológica de las pacientes tras la misma. El presente estudio fue diseñado como una revisión clínica retrospectiva de 60 pacientes. Tras la revisión de las historias clínicas, las pacientes fueron entrevistadas procediéndose a la realización del cuestionario. El aspecto a cambiar más deseado fue la falta/pérdida de proyección del pezón. No obstante, el 22% de las pacientes respondieron que no cambiarían nada acerca de su reconstrucción. La satisfacción acerca de la reconstrucción del montículo mamario fue excelente o buena para el 68%, normal para el 23% y pobre para el 9%. En cambio, para la reconstrucción del complejo areola-pezón fue excelente o buena para el 50%, normal para el 45% y pobre para el 5%. No se encontraron diferencias significativas entre las diferentes técnicas en función del tiempo transcurrido entre el momento de la mastectomía y el tercer tiempo de la reconstrucción (p=0,06). La técnica de la donación contralateral de pezón fue la que ofreció una mayor satisfacción (2,67 puntos) y proyección (7,23 puntos). A pesar de las diferencias en sus medias, no se demostró ninguna diferencia estadísticamente significativa. Teniéndo en cuenta la técnica usada para la reconstrucción de la areola, la técnica de la donación-injerto de piel inguinal fue la que ofreció mayor satisfacción (3 puntos) y coloración (8,57 puntos). El estudio estadístico objetivó diferencias estadísticamente significativas (p=0,01). Entre las distintas técnicas de reconstrucción del pezón no hay ninguna cuyos resultados se sobrepongan a las demás, pero sí una vez que se reconstruye la areola siendo la técnica que más satisface la del injerto inguinal. A pesar de ello, el aspecto más notable a mejorar está en la reconstrucción del pezón, dada la frecuente disconformidad de las pacientes con la proyección conseguida a lo largo del tiempo por la posible reabsorción del mismo (AU)

The creation of the nipple-areola complex is the latest time in breast reconstruction, transforming the reconstruction of the breast mound into a real breast. We have to consider the reconstruction of the areola and the nipple as the culmination of breast reconstruction. There are a lot of documented techniques for nipple-areola complex reconstruction. The aim of this study is to determine the grade of psychological satisfaction of patients after this reconstruction This study was designed as a retrospective clinic review of 60 patients. After reviewing medical histories, the patients were interviewed and asked to complete a questionnaire. The most common desired aspect was to correct the absence/lost of nipple projection. However, 22% of patients answered they would not change anything regarding their reconstruction. The satisfaction with the mammary mound was excellent or good for 68%, normal for 23 % and poor for 9 %. On the other hand, satisfaction for the nippleareola complex reconstruction was excellent or good for 50%, normal for 45% and poor for 5%. There were no statistical differences among the different techniques depending on the time between the mastectomy intervention and the third reconstruction (p=0,06). For nipple reconstruction, the contralateral nipple donation technique offered more satisfaction (2.67 points) and projection (7.23 points). In spite of the differences in their means, there were no statistically significant differences. Taking into account the technique used for the areola reconstruction, the donation- graft of inguinal skin was the one that offered more satisfaction (3 points) and better coloration (8.57 points). With the statistical study there were obtained significant statistical differences (p=0,01). Among the different techniques for nipple reconstruction there wasn’t anyone whose results overlapped the others, but when the areola was reconstructed there were some, being the inguinal grafting the technique which satisfies more. In spite of this, the aspect which most needed to improve is nipple reconstruction because many patients were unhuppy with the obtained projection and its reabsorption over time (AU)

Complicaciones y secuela postraumatismo nasal / Complications and sequelae after nasal trauma

El hematoma septal después de un traumatismo nasal es una complicación que de no ser diagnosticada y tratada precozmente puede evolucionar a absceso septal. El hematoma septal crea necrosis por compresión del cartílago cuadrangular subyacente y posteriormente puede complicarse con un absceso septal en el que se aisla flora saprófita. La necrosis y destrucción del septo nasal puede afectar a la función respiratoria y estéticamente se manifiesta en hundimiento del dorso y punta nasal. Presentamos el caso de un varón de 10 años que sufrió fractura de huesos propios de la nariz con hematoma septal que pasó desapercibido. El cuadro evolucionó a un absceso septal que fue drenado y que originó como secuelas hundimiento del dorso nasal y retracción craneal de la punta y columela. Se realizó corrección funcional y estética nasal mediante injerto osteocartilaginoso de parrilla costal en dorso y columela. No se observaron complicaciones. Se observó mejoría estética y funcional. El resultado tras 2 meses de seguimiento ha sido favorable (AU)

Septal haematoma after nasal trauma is a complication that can lead to septal abscess if unrecognized or early intervention is not performed. It can cause compression and thereby necrosis that evolve to a septal abscess in which cultures reveal saprophyte bacteria. Cartilage necrosis and destruction can produce impaired breathing and aesthetic deformities with collapse of the dorsum and the tip of the nose. We present a 10 year old masculine infant, that suffered a nasal fracture with a septal haematoma that remained undiagnosed. The patient developed a septal abscess that required drainage and resulted in nasal sequelae with collapse of dorsum and cranial displacement of tip and columella. Functional and aesthetic reconstruction was performed using rib cartilage grafts. No complications occurred. Functional and aesthetic improvement was observed. Result after 2 months of follow-up is considered favourable (AU)

Evolution of genotypic and phenotypic resistance during chronic treatment with the fusion inhibitor T-1249.

T-1249 is a peptide HIV fusion inhibitor (FI) previously under development for use in FI-naive and experienced patients. Here we present prospectively planned longitudinal analyses of FI resistance during 48 weeks of T-1249 dosing in patients with extensive prior FI exposure. T1249-105 was a single-arm rollover study in patients with prior resistance to enfuvirtide (ENF) and 10 days of T-1249 functional monotherapy exposure. The phenotype and genotype of plasma virus envelopes were analyzed at baseline and at study weeks 8, 16, and 48. At study entry, viruses had a geometric mean decrease in susceptibility to ENF of 51.8-fold but to T-1249 of 1.8-fold; extensive genotypic resistance to ENF was observed. A median viral load response of - 1.5 log(10) copies/ml was observed at week 2 that was partially sustained (- 0.5 log(10) copies/ml) through 48 weeks. Resistance to T-1249 gradually increased to a geometric mean 92.7-fold decrease from FI-naive baseline; this occurred concomitant with further evolution of gp41 amino acids 36-45, most commonly the G36D (n = 6, 16%) or N43K (n = 9, 24%) substitutions. A novel substitution, A50V (n = 12, 32%), was also common, as were the N126K and S138A substitutions in heptad-repeat 2 (HR-2). These data point toward a primary role for the gp41 36-45 locus in modulating FI binding and suggest that residues in HR-2 may contribute in a more limited manner to development of peptide FI resistance. These data also point toward a substantial genetic barrier and fitness cost to development of resistance to next-generation fusion inhibitors.

Pharmacokinetics, pharmacodynamics and safety of once-daily versus twice-daily dosing with enfuvirtide in HIV-infected subjects.

OBJECTIVE: To investigate the pharmacokinetics, safety/tolerability and antiviral activity of enfuvirtide administered once-daily (QD) versus twice-daily (BID). DESIGN: An open-label, randomized, multiple dose, two-period crossover study comparing 180 mg enfuvirtide, two injections QD versus 90 mg enfuvirtide, two injections, BID. METHODS: Steady-state intensive pharmacokinetic samples were obtained on days 7 and 14. RESULTS: Thirty-seven subjects received at least one dose of enfuvirtide. Thirty-three subjects completed both dosing periods. The regimens were bioequivalent based on the ratio of geometric mean area under the curve (AUC)0-tau [112 +/- 6.2 microg x h/ml QD; 115 +/- 6.4 microg x h/ml 2 x BID; QD/BID 0.98; 90% confidence interval (CI) 0.89,1.07]. The maximum observed plasma concentration within a dosing interval (Cmax) was 49% higher for QD (9.5 +/- 2.7 microg/ml) versus BID (6.3 +/- 1.7 microg/ml) and the pre-dose plasma concentration (Ctrough) was 57% lower for QD (1.6 +/- 1.1 microg/ml) versus BID (3.8 +/- 1.3 microg/ml). The LSM decrease in viral load from baseline to day 7 was 1.0 +/- 0.14 log10 (n = 18) for QD and 1.4 +/- 0.2 log10 (n = 17) for BID (LSM difference 0.385; P = 0.07). Linear regression analysis suggested that decline in viral load up to day 7 was associated with Ctrough but not Cmax or AUC. There were no significant differences in adverse events between the two dosing regimens. CONCLUSIONS: Administration of enfuvirtide 180 mg QD results in bioequivalence compared with 90 mg BID based on AUC with a similar short-term safety profile, but a trend towards a weaker antiretroviral effect. Larger and longer-term studies are needed to determine if 180 mg once daily is an effective dosing alternative for enfuvirtide.

T-1249 retains potent antiretroviral activity in patients who had experienced virological failure while on an enfuvirtide-containing treatment regimen.

BACKGROUND: T-1249 is a 39-amino acid synthetic peptide fusion inhibitor (FI) shown to preserve antiretroviral activity in vitro against human immunodeficiency virus (HIV) isolates that have decreased susceptibility to enfuvirtide (ENF). METHODS: A 10-day phase 1/2 study of the safety and antiretroviral activity of T-1249 was conducted in 53 HIV-1-infected adults with detectable viremia while on an ENF-containing treatment regimen. RESULTS: From FI-naive baseline levels, the geometric mean (GM) decrease in susceptibility to ENF was 116.3-fold, and the GM decrease in susceptibility to T-1249 was 2.0-fold. Patients continued to administer their failing treatment regimen but replaced ENF with T-1249 at a dose of 192 mg/day. T-1249 was generally well tolerated; injection site reactions, which were generally mild, were the most commonly reported adverse event (64% of patients). The median change from levels of HIV-1 RNA at baseline to levels on day 11 was -1.26 log(10) copies/mL (95% confidence interval, -1.40 to -1.09 log(10) copies/mL); on day 11, a decrease from baseline HIV-1 RNA levels of >/=1.0 log(10) copies/mL was seen in 73% of patients. Antiretroviral activity, as measured by levels of HIV-1 RNA, was not predicted by baseline susceptibility to T-1249 or to ENF; genotypic substitutions that emerged during T-1249 treatment were identified in virus from some patients. CONCLUSIONS: These results indicate that FIs constitute an expanding class of antiretroviral agents with the potential to be sequenced.

Short-term safety and antiretroviral activity of T-1249, a second-generation fusion inhibitor of HIV.

T-1249 is a 39-aa synthetic peptide that inhibits fusion of human immunodeficiency virus (HIV) to the host target cell. A 14-day open-label, phase 1/2 dose-escalation monotherapy study of the safety and antiretroviral activity of T-1249 was performed on 115 HIV-1-infected adults. At baseline, the majority of the patients had advanced HIV disease (baseline median CD4(+) cell count, 57 cells/microL) and had extensive pretreatment (i.e., pre-T-1249) experience with antiretroviral medications (median, 11 antiretroviral drugs). Patients received T-1249 monotherapy by subcutaneous injection, for 14 days, at doses ranging from 6.25 to 192 mg/day. T-1249 was generally well tolerated, and no dose-limiting toxicity was identified. Injection-site reactions were the most commonly reported adverse event (57%). Dose-dependent decreases in plasma HIV-1 RNA load were observed; the median maximum change from baseline across dose groups ranged from -0.29 log(10) copies/mL (95% confidence interval [CI], -0.43 to -0.05 log(10) copies/mL) for the lowest dose to -1.96 log(10) copies/mL (95% CI, -2.02 to -1.37 copies/mL) for the highest dose. These results indicate that T-1249 is a potent new therapeutic agent for HIV-1 infection.

Changes in CD4+ T-cell differentiation phenotype during structured treatment interruption in patients with chronic HIV-1 infection.

Markers of maturation and activation were measured on peripheral CD4+ T cells in chronically HIV-1-infected patients in a randomized, controlled pilot study of structured treatment interruption (STI). Eight subjects underwent 2 cycles of 1 month off and 1 month on highly active antiretroviral therapy (HAART), followed by a final 3-month interruption. During STI, CD4+ T-cell percentage remained relatively stable in 4 of 8 subjects. The remaining 4 STI subjects had significant rapid decline in CD4+ T-cell percentage during STI, followed by return to pre-STI baseline while on HAART. Changes in overall CD4+ T-cell percentage corresponded with fluctuations in the CD45RA+CCR7+ naive and CD45RA-CCR7+ central memory subsets. Subjects with variable CD4+ T-cell percentages tended to have higher pre-HAART plasma HIV-1 RNA set-points and experienced higher levels of plasma HIV-1 RNA rebound during STI. These results suggest that interruptions should be avoided whenever possible in patients on HAART with high plasma HIV-1 RNA set-points.

[Spanish adaptation of the Seasonal Pattern Assessment Questionnaire (SPAQ) in the adult and children-adolescent versions]. / Adaptación española del Cuestionario de Evaluación de Perfil Estacional (Seasonal Pattern Assessment Questionnaire, SPAQ) en las versiones de adultos e infanto-juvenil.

INTRODUCTION: In 1984, Rosenthal et al. described the seasonal affective disorder as a cyclic pattern of depressive episodes appearing in autumn and winter, showing atypical symptoms as hypersomnia, overeating, and carbohydrate craving. They also introduced the self-applied Seasonal Pattern Assessment Questionnaire, which includes a seasonality index. A children and adolescent version was also introduced later too. In this paper the test retest reliability and internal consistency of both the adult and the children and adolescent SPQA version are presented. METHODS: 30 adults y 30 adolescents filled out the corresponding questionnaires in an interval of one week. Kappa and intraclass correlation coefficients were applied. Internal consistency was measured with Cronbach alpha. RESULTS: The adult version obtained coefficients between 0.47 y 0.81, and a Cronbach alpha of 0.85 for the seasonality index. The children and adolescent version included several items with low reliability, which were then rewritten. The renewed version was tested again in a new 30 subjects sample. Coefficients ranged from 0.50 to 0.83, with a Cronbach's alpha of 0.69 for the seasonality index. CONCLUSIONS: The Spanish version of the SPAQ (Both Adult and Children-Adolescent) showed good reliability values and also appropriate internal consistency coefficients. Therefore, they are ready to be used in clinical and epidemiological research.

Cartilage repair using new polysaccharidic biomaterials: macroscopic, histological and biochemical approaches in a rat model of cartilage defect.

OBJECTIVE: The present study aims at evaluating, in a rat model of cartilage defect, the potential of various polymers as filling and repair biomaterials. The macroscopic and histological observations are compared to biochemical parameters in order to appreciate the pertinence of the latter as suitable criteria in tissue engineering. METHODS: A hydrogel, consisting of hyaluronic acid (HA), covalently substituted by hydrophobic alkyl chains (HA12, HA18) and an alginate sponge, alone (Asp) or combined with HA (AHAsp) or combined with HA and chondrocytes (HYBsp) were evaluated. Cartilage lesions were drilled in femoral trochlea of rats. The analyses were performed on trochlea as well as on patella and condyles. RESULTS: Repairs achieved with hydrogels had a similar macroscopic appearance than those afforded by AHAsp and HYBsp. Best macroscopic and histological scores were obtained with HA18 and HYBsp in comparison with alginate group (P< 0.01 and P< 0.02 respectively). Biochemical evaluations confirmed the presence of similar amounts of proteoglycans in the repaired zones and in the controls, though with different DeltadiC4S/DeltadiC6S ratios and enhanced HA levels. CONCLUSIONS: Hydrogels or sponges proved to be colonized by cells synthesizing a matrix with a high HA content. The matrix obtained eventually turns hyaline and takes over the scaffold. The addition of HA and/or chondrocytes to Asp significantly improves the macroscopic and histological scores (P< 0.05 and P< 0.02 respectively). However, biochemical parameters are significantly different of those evaluated in native cartilage. The present study shows that only biochemical parameters allow to discriminate between various biomaterials in tissue engineering and are essential informations which should be taken into account in addition to macroscopic and histological observations.

Emergence of drug-resistant HIV-1 variants in patients undergoing structured treatment interruptions.

We report the emergence of drug-resistant viral mutations in chronically HIV-infected individual undergoing structured treatment interruptions (STI). THe protease mutations K101E and K103N were detected at the end of the second or third STI. We concluded that the repeated abrupt termination and resumption of certain antiretroviral drug regimens during STI therapy may lead to the development of drug resistance in chronically HIV-infected individuals.

Addition of cyclophosphamide to antiretroviral therapy does not diminish the cellular reservoir in HIV-infected persons.

The chronically HIV-infected cellular reservoir in lymphoid tissue (LT) represents a formidable obstacle to the long-term success of antiretroviral therapy. Cytoreductive chemotherapy with cyclophosphamide (CTX) reduces cells in LT, and we hypothesized that coadministration of antiretroviral therapy with CTX may diminish the cellular reservoir over time. Ten antiretroviral treatment-naive subjects were recruited, and they received stavudine, lamivudine and nelfinavir (antiretroviral therapy, ART) until 2 consecutive plasma HIV RNA levels measured < 50 copies/ml (step 1). Five subjects then received ART alone, whereas five subjects received ART plus three escalating doses of CTX (step 2). Viral DNA was measured in LT obtained by excisional lymph node biopsy and peripheral blood mononuclear cells (PBMCs), using quantitative polymerase chain reaction at three time points in both groups (before steps 1 and 2, and after CTX). Viral DNA declined in both groups after the initiation of ART alone in step 1. During step 2 both groups experienced a modest decline compared with step 1. However, no significant differences were observed in viral DNA in LT or PBMCs between the ART alone and the ART plus CTX groups. Suppression of plasma HIV RNA levels < 50 copies/ml was not maintained in the ART plus CTX group, perhaps because of inadequate medication adherence. The group receiving ART plus CTX had lower CD4(+) lymphocyte counts and absolute total lymphocytes compared with the ART alone group. We conclude that the addition of CTX to ART did not diminish the cellular reservoir in HIV-infected persons.

Structured antiretroviral treatment interruptions in chronically HIV-1-infected subjects.

The risks and benefits of structured treatment interruption (STI) in HIV-1-infected subjects are not fully understood. A pilot study was performed to compare STI with continuous highly active antiretroviral therapy (HAART) in chronic HIV-1-infected subjects with HIV-1 plasma RNA levels (VL) <400 copies per ml and CD4(+) T cells >400 per microl. CD4(+) T cells, VL, HIV-1-specific neutralizing antibodies, and IFN-gamma-producing HIV-1-specific CD8(+) and CD4(+) T cells were measured in all subjects. STIs of 1-month duration separated by 1 month of HAART, before a final 3-month STI, resulted in augmented CD8(+) T cell responses in all eight STI subjects (P = 0.003), maintained while on HAART up to 22 weeks after STI, and augmented neutralization titers to autologous HIV-1 isolate in one of eight subjects. However, significant decline of CD4(+) T cell count from pre-STI level, and VL rebound to pre-HAART baseline, occurred during STI (P = 0.001 and 0.34, respectively). CD4(+) T cell counts were regained on return to HAART. Control subjects (n = 4) maintained VL <400 copies per ml and stable CD4(+) T cell counts, and showed no enhancement of antiviral CD8(+) T cell responses. Despite increases in antiviral immunity, no control of VL was observed. Future studies of STI should proceed with caution.

Sodium alginate sponges with or without sodium hyaluronate: in vitro engineering of cartilage.

Studies are underway to design biosystems containing embedded chondrocytes to fill osteochondral defects and to produce a tissue close to native cartilage. In the present report, a new alginate three-dimensional support for chondrocyte culture is described. A sodium alginate solution, with or without hyaluronic acid (HA), was freeze-dried to obtain large-porosity sponges. This formulation was compared with a hydrogel of the same composition. In the sponge formulation, macroscopic and microscopic studies demonstrated the formation of a macroporous network (average pore size, 174 microm) associated with a microporous one (average pore size, 250 nm). Histological and biochemical studies showed that, when loaded with HA, the sponge provides an adapted environment for proteoglycan and collagen synthesis by chondrocytes. Cytoskeleton organization was studied by three-dimensional fluorescence microscopy (CellScan EPR). Chondrocytes exhibit a marked spherical shape with a nonoriented and sparse actin microfilament network. Type II collagen was detected in both types of sponges (with or without HA) using immunohistochemistry. In conclusion, the sponge formulation affords new perspectives with respect to the in vitro production of "artificial" cartilage. Furthermore, the presence of hyaluronate within the alginate sponge mimics a functional environment, suitable for the production by embedded chondrocytes of an extracellular matrix.

The role of 3D-microscopy in the study of chondrocyte-matrix interaction (alginate bead or sponge, rat femoral head cap, human osteoarthritic cartilage) and pharmacological application.

The potentialities of a new non-invasive optical scanning microscopy technique were evaluated through 3D analysis of chondrocyte-matrix interactions. Five different 2D or 3D culture systems were used: (1) MonoLayer (ML) of human chondrosarcoma cell line; (2) rat or human chondrocytes encapsulated in Alginate Bead (AB); (3) human chondrocytes encapsulated in Alginate Sponge (AS); (4) Rat Femoral Head Cap (RFHC); (5) slices of knee human Osteoarthritic Cartilage (HOAC). Chondrocytes ML, AB, RFHC were incubated for 24 h in vitro in the presence of recombinant human interleukin1-beta (rhIL1-beta) and the effects on cytoskeleton organisation (F-actin filament), Focal Adhesion Kinase (FAK) expression (tyrosine kinase), collagenase B expression (metalloprotease) were studied. Furthermore, the production of intracellular IL1-beta by LPS- or rhIL1-beta-stimulated chondrocytes was shown to be partly suppressed by rhein (active metabolite of diacerhein) in all culture systems. This high resolution light microscopy gave complementary information that could be important for a better understanding of the interaction of chondrocytes with the extracellular matrix in a variety of culture devices.

Evaluation of distinct blood lymphocyte populations in human immunodeficiency virus type 1-infected subjects in the absence or presence of effective therapy.

Virus reservoirs can persist in human immunodeficiency virus type 1 (HIV-1)-infected subjects despite effective plasma virus suppression. To compare viral dynamics in the absence and presence of antiretroviral therapy, blood mononuclear cells from 19 subjects with high plasma RNA levels and 18 subjects following prolonged virus suppression were examined, by use of in situ hybridization, to detect virus RNA expression before and after in vitro T cell activation. This approach reveals circulating lymphocytes expressing HIV-1 RNA before activation and an increase in cells with detectable HIV-1 RNA transcription after in vitro activation. The frequencies of these 2 cell populations are strongly correlated with plasma virus load and appear to be stable once a new steady state is established during therapy. The frequency of viral RNA-positive cells is equivalent to the frequency of cells that produce infectious virus. Thus, in HIV-1-infected subjects there are distinct virus reservoirs comprising both latent and replication-active cells.