Étude de faisabilité du ganglion sentinelle dans le cancer de la prostate par cœlioscopie : premiers résultats

Objectives

The standard lymphadenectomy is currently a challenge in the management of prostate cancer. The aim of this prospective study was to evaluate the performance of the sentinel lymph node (SLN) by laparoscopy in patients with localized prostate cancer, candidates for local treatment.

Patients and methods

Patients were injected transrectal ultrasound-guided with 0.3 mL/100MBq 99^mTc-Sulfur rhenium colloid in each prostatic lobe, the day before surgery. Lymphoscintigraphy was performed after 2 hours. The detection was realized intraoperatively with a laparoscopic probe (Gamma Sup Clerad^®) followed by extensive dissection. Counts of SLN were performed in vivo and confirmed ex vivo. The histological analysis was performed by HPS staining and followed by immunochemistry if SLN was free.

Results

Seventy patients with carcinoma of the prostate at intermediate or high risk of lymph node metastases (D’Amico), PSA median 9.5 ng/mL [6–130], were included in the study. The lymphoscintigraphic detection rate was 94.2% (66/70) and intraoperative of 97.0% (68/70). Fourteen patients had lymph node metastases, six only in SLN. The false negative rate was 2/14 (14.0%). The internal iliac region is the first metastatic site (40.9%). Limited or standard lymph node dissection would have ignored respectively 72.7% and 59.0% of lymph node metastases.

Conclusion

The laparoscopy is adapted to a broad identification of SLN and targeted dissection of these lymph nodes significantly limits the risk of surgical extended dissection while maintaining the accuracy of the information.     

Staphylococcus aureus Inhibits IL-8 Responses Induced by Pseudomonas aeruginosa in Airway Epithelial Cells

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam₃CysSK₄ but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.     

Characterization by Raman microspectroscopy of the strain-induced conformational transition in fibroin fibers from the silkworm Samia cynthia ricini

Raman microspectroscopy has been used to quantitatively study the effect of a mechanical deformation on the conformation and orientation of Samia cynthia ricini (S. c. ricini) silk fibroin. Samples were obtained from the aqueous solution stored in the silk gland and stretched at draw ratios (lambda) ranging from 0 to 11. Using an appropriate band decomposition procedure, polarized and orientation-insensitive spectra have been analyzed to determine order parameters and the content of secondary structures, respectively. The data unambiguously show that, in response to mechanical deformation, S. c. ricini fibroin undergoes a cooperative alpha-helix to beta-sheet conformational transition above a critical draw ratio of 4. The alpha-helix content decreases from 33 to 13% when lambda increases from 0 to 11, while the amount of beta-sheets increases from 15 to 37%. In comparison, cocoon silk is devoid of alpha-helical structure and always contains a larger amount of beta-sheets. Although the presence of isosbestic points in different spectral regions reveals that the conformational change induced by mechanical deformation is a two-state process, our results suggest that part of the glycine residues might be incorporated into beta-poly(alanine) structures. The beta-sheets are initially isotropically distributed and orient along the fiber axis as lambda increases, but do not reach the high level of orientation found in the cocoon fiber. The increase in the orientation level of the beta-sheets is found to be concomitant with the alpha --> beta conformational conversion, whereas alpha-helices do not orient under the applied strain but are rather readily converted into beta-sheets. The components assigned to turns exhibit a small orientation perpendicular to the fiber axis in stretched samples, showing that, overall, the polypeptide chains are aligned along the stretching direction. Our results suggest that, in nature, factors other than stretching contribute to the optimization of the amount of beta-sheets and the high degree of orientation found in natural cocoon silk.     

Multiple conductance states of the purified calcium release channel complex from skeletal sarcoplasmic reticulum.

The CHAPS-solubilized and purified 30S ryanodine receptor protein complex from skeletal sarcoplasmic reticulum (SR) was incorporated into planar lipid bilayers. The resulting electrical activity displayed similar responses to agents such as Ca2+, ATP, ryanodine, or caffeine as the native Ca2+ release channel, confirming the identification of the 30S complex as the Ca2+ release channel. The purified channel was permeable to monovalent ions such as Na+, with the permeability ratio PCa/PNa approximately 5, and was highly selective for cations over anions. The purified channel also showed at least four distinct conductance levels for both Na+ and Ca2+ conducting ions, with the major subconducting level in NaCl buffers possessing half the conductance value of the main conductance state. These levels may be produced by intrinsic subconductances present within the channel oligomer. Several of these conductances may be cooperatively coupled to produce the characteristic 100 +/- 10 pS unitary Ca2+ conductance of the native channel.     

Utilization of membranous lipid substrates by membranous enzymes: activation of the latent sphingomyelinase of hen erythrocyte membrane

Previous studies demonstrated that hen erythrocytes have an inoperative, latent sphingomyelinase which is activated when the cells are hemolyzed in a hypotonic medium. Within minutes after hemolysis about 60-80% of the sphingomyelin (SPM) of the RBC "ghost" membrane was hydrolyzed. In this paper, expression of sphingomyelinase activity was further investigated. The percentage of total SPM hydrolyzed depended on the volume of the hypotonic hemolyzing buffer. Thus, suspending the erythrocytes in 4 vol of the buffer resulted in clumping of the hemolyzed "ghosts" and no hydrolysis of SPM. In comparison, suspension in 19 vol of the hypotonic buffer showed no clumping and sphingomyelinase activity was fully expressed. But centrifugation of the latter or, alternatively, addition of concanavalin A induced clumping and elimination of sphingomyelinase activity. Hen RBC could also be hemolyzed in an isotonic medium in the presence of Triton X-100, mellitin, halothane, and phospholipase C. Activation of the latent sphingomyelinase occurred at concentrations of these reagents which caused cell lysis. Hen RBC were dispersed in an isotonic medium containing glutaraldehyde (0.1%) or formaldehyde (10%). This rendered the cells resistant to hemolysis, even when subsequently dispersed in a hypotonic medium or water. But incubation of the "fixed" cells in a hypotonic or isotonic medium activated the enzyme, resulting in hydrolysis of 60% of the cellular SPM. In contrast, when glutaraldehyde was included in the hypotonic buffer, hemolysis occurred but sphingomyelinase activity was eliminated.     

Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei

The identity and location of nuclear cyclic nucleotide phosphodiesterases (PDE) has yet to be ascertained. Intact cardiac nuclei and subnuclear fractions from ovine hearts were isolated to determine cAMP-specific PDE activity which was 3-fold greater than that of cGMP PDE, the latter being insensitive to Ca-calmodulin and zaprinast. Specific hydrolytic activities of the cardiac nuclear envelopes (NE) were similar to those measured in the corresponding intact nuclei, thus suggesting that most PDE activity is associated with the nuclear membrane. Moreover, the main hydrolytic activities in cardiac nuclei were attributed to PDE4 (56%) and PDE3 (44%). The pharmacological sensitivity of each isoform in terms of IC(50), K(m) and K(i) values was typical of previously characterized cardiac PDE 3 and 4 isoforms. PDE2 (cGMP-stimulated PDE) represented a minor component (8-9%) of total hydrolytic activity. Solubilization of nuclear envelopes and HPLC separation also yielded rolipram-sensitive PDE activities. Upon 1% Triton X-100 extractions, the presence of PDE3 and PDE4 was revealed in a low speed, nucleopore complex-enriched, P1 pellet. In addition, Western blot analysis demonstrated the presence of PDE4B and PDE4D subtypes in the nuclei as well as enrichment in NE. However, in the same preparations, the presence of PDE4A could not be ascertained. Altogether, these results suggest an intrinsic and predominant association of these nuclear PDEs with the NE and much likely with nucleopore complexes.     

Size Distribution of Air Bubbles Entering the Brain during Cardiac Surgery

Background

Thousands of air bubbles enter the cerebral circulation during cardiac surgery, but whether high numbers of bubbles explain post-operative cognitive decline is currently controversial. This study estimates the size distribution of air bubbles and volume of air entering the cerebral arteries intra-operatively based on analysis of transcranial Doppler ultrasound data.

Methods

Transcranial Doppler ultrasound recordings from ten patients undergoing heart surgery were analysed for the presence of embolic signals. The backscattered intensity of each embolic signal was modelled based on ultrasound scattering theory to provide an estimate of bubble diameter. The impact of showers of bubbles on cerebral blood-flow was then investigated using patient-specific Monte-Carlo simulations to model the accumulation and clearance of bubbles within a model vasculature.

Results

Analysis of Doppler ultrasound recordings revealed a minimum of 371 and maximum of 6476 bubbles entering the middle cerebral artery territories during surgery. This was estimated to correspond to a total volume of air ranging between 0.003 and 0.12 mL. Based on analysis of a total of 18667 embolic signals, the median diameter of bubbles entering the cerebral arteries was 33 μm (IQR: 18 to 69 μm). Although bubble diameters ranged from ~5 μm to 3.5 mm, the majority (85%) were less than 100 μm. Numerous small bubbles detected during cardiopulmonary bypass were estimated by Monte-Carlo simulation to be benign. However, during weaning from bypass, showers containing large macro-bubbles were observed, which were estimated to transiently affect up to 2.2% of arterioles.

Conclusions

Detailed analysis of Doppler ultrasound data can be used to provide an estimate of bubble diameter, total volume of air, and the likely impact of embolic showers on cerebral blood flow. Although bubbles are alarmingly numerous during surgery, our simulations suggest that the majority of bubbles are too small to be harmful.     

Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1

Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.     

Characteristics of unbuffered gel-immobilized urease particles. II. Overall rate of reaction

The overall rates of reaction of unbuffered gel-immobilized urease particles have been investigated with the aid of a packed-bed differential recycle reactor. Both substrate and enzyme concentrations have received attention. Cylindrical gel particles contained within impermeable tubelets were used to provide the physical strength necessary for the packed-bed arrangement and a one dimensional diffusion path to aid understanding of the complex interactions between substrate and product diffusion, and their effect on the reactions taking place. The experimental data have been interpreted with the aid of an enzyme rate equation (ERE) which relates the free solution characteristics of the enzyme to the conditions within a diffusion limited particle. The internal hydrogen ion profiles have been accommodated by a lumped parameter, the apparent pH (pH_app). Two methods have been suggested for the calculation of pH_app and the loss of activity on particle preparation, these methods are based on the use of the ERE in conjunction with experimental data.