Intracranial Applications of MR Imaging-Guided Focused Ultrasound.

Initially used in the treatment of prostate cancer and uterine fibroids, the role of focused ultrasound has expanded as transcranial acoustic wave distortion and other limitations have been overcome. Its utility relies on focal energy deposition via acoustic wave propagation. The duty cycle and intensity of focused ultrasound influence the rate of energy deposition and result in unique physiologic and biomechanical effects. Thermal ablation via high-intensity continuous exposure generates coagulative necrosis of tissues. High-intensity, pulsed application reduces temporally averaged energy deposition, resulting in mechanical effects, including reversible, localized BBB disruption, which enhances neurotherapeutic agent delivery. While the precise mechanisms remain unclear, low-intensity, pulsed exposures can influence neuronal activity with preservation of cytoarchitecture. Its noninvasive nature, high-resolution, radiation-free features allow focused ultrasound to compare favorably with other modalities. We discuss the physical characteristics of focused ultrasound devices, the biophysical mechanisms at the tissue level, and current and emerging applications.

Alterations of the immunosuppressive IL4I1 enzyme activity induced by naturally occurring SNP/mutations.

The immunosuppressive phenylalanine oxidase interleukin 4-induced gene 1 (IL4I1), primarily produced by antigen-presenting cells, inhibits T-cell proliferation and promotes the generation of Foxp3(+) regulatory T cells in vitro. Highly expressed by tumour-associated macrophages from human cancers, IL4I1 has a potential role in immune evasion from the anti-tumour immune response. We have reviewed single-nucleotide polymorphisms (SNPs) and mutations described for the exon 4 of the IL4I1 isoform 1, which is expressed in lymphoid tissue. Two of them were expressed in an exogenous system to analyse their effect on the enzymatic activity. The N92D SNP leads to a hyperactive enzyme, while the R102G mutation is hypomorphic. Moreover, we show that IL4I1 activity is not only directed against phenylalanine, as initially described, but also at a lower level against arginine. These data pave the way to more extensive analyses of the mutational state of IL4I1 in pathological conditions such as cancer, where its participation in immune system dysfunctions may have therapeutic implications.

Two atypical cases of cutaneous gamma/delta T-cell lymphomas.

Cutaneous γ/δ T-cell lymphoma (CGD-TCL) is a recent entity described in the newly revised World Health Organization-European Organization for Research and Treatment of Cancer classification of cutaneous lymphomas, and is characterized by the γ/δ T-cell receptor expression on atypical lymphocytes. Only a few cases of primary CGD-TCL have been reported, with an extremely aggressive course (median survival time of 15 months). We describe 2 atypical cases of CGD-TCL. The first case was initially misdiagnosed as an inflammatory panniculitis due to the granulomatous infiltrate on the skin biopsy specimen. Diagnosis was confirmed using δ PCR that revealed γ/δ T-cell clonal expansion. The evolution was marked by predominant γ/δ T-cell infiltrate with diffuse body fat involvement as seen on positron emission tomography-computed tomography. The second case is the first described Epstein-Barr virus (EBV)-associated CGD-TCL with a rapidly fatal evolution. CGD-TCL is also a heterogeneous entity and δ PCR and EBV-encoded RNA probe to detect an EBV latent infection may help diagnose and characterize these cutaneous lymphomas.

The novel immunosuppressive enzyme IL4I1 is expressed by neoplastic cells of several B-cell lymphomas and by tumor-associated macrophages.

We previously reported a strong IL4I1 gene expression in primary mediastinal B-cell lymphoma (PMBL) and recently identified the protein as a secreted L-phenylalanine oxidase, physiologically expressed by myeloid cells, which inhibits T-cell proliferation in vitro. Here, we analyzed the pattern of IL4I1 protein expression in 315 human lymphoid and non-lymphoid malignancies. Besides PMBL, IL4I1 expression in tumors was very frequent. IL4I1 was detected in tumor-associated macrophages from most of the tumors and in neoplastic cells from follicular lymphoma, classic and nodular lymphocyte predominant Hodgkin lymphomas and small lymphocytic lymphoma, three of which are germinal center derived. IL4I1-positive tumor cells were also detected in rare cases of solid cancers, mainly mesothelioma. The enzymatic activity paralleled protein expression, suggesting that IL4I1 is functional in vivo. Depending on the tumor type, IL4I1 may impact on different infiltrating lymphocyte populations with consequences on tumor evolution. In the particular case of follicular lymphoma cells, which are susceptible to antitumor cytotoxic T cells killing but depend on interactions with local T helper cells for survival, a high level of IL4I1 expression seems associated with the absence of bone marrow involvement and a better outcome. These findings plead for an evaluation of IL4I1 as a prognosis factor.

The dynamics of the rec-system in variable environments: haploid selection in a cyclical two-state environment.

The dynamics of a 3-locus infinite population with non-overlapping generations and panmixia was studied. Loci are di-allelic: two loci affect fitness under cyclical symmetric haploid selection while the third one is a modifier of recombination (rec-modifier). Selection favors alternatively haplotypes AB and ab or Ab and aB. It has been proven that under alternating selection (when period of selection consists of two generations) a dominant suppressor of recombination is displaced and the allele for non-zero recombination becomes fixed within the population. For populations with inversion heterozygosity within the selective system (i.e. with zero recombination in heterozygote for rec-modifier and non-zero for homozygotes) fixation of one of the alleles (depending on the initial point) at the rec-modifier locus is predicted. For other values of recombination parameters, the behavior of the system was studied numerically. A full bifurcation picture of parameters was obtained. Many of the results related to the case of a two-generation period hold also in the case of longer period lengths.

Preliminary investigations of ultrasound induced acoustic streaming using particle image velocimetry.

Particle image velocimetry was used to investigate ultrasound-induced acoustic streaming in a system for the enhanced uptake of substances from the aquatic medium into fish. Four distinct regions of the induced streaming in the system were observed and measured. One of the regions was identified as an preferential site for substance uptake, where the highest velocities in proximity to the fish surface were measured. A positive linear relationship was found between the ultrasound intensity and the maximum streaming velocity, where a unitless geometric factor, specific to the system, was calculated for correcting the numerical relationship between the two parameters. The results are part of a comprehensive study aimed at improving mass transdermal administrations of substances (e.g. vaccines, hormones) into fish from the aquatic medium.

Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.

Longitudinal follow-up of cellular and humoral immunity induced by recombinant adenovirus-mediated gene therapy in cancer patients.

Replication-defective adenoviruses are arousing growing interest as both gene therapy and vaccine vectors. In a phase I clinical trial designed to evaluate the feasibility and tolerance of recombinant adenovirus (rAd)mediated gene transfer, we previously demonstrated that a single intratumoral injection of 10(9) PFU of rAd encoding the beta-galactosidase protein (Ad-beta-Gal) induced strong short-term (1-3 months) humoral, helper (Th1 type) and cytotoxic T cell responses specific for the transgene product in patients with advanced lung cancer. The purpose of the present study was to evaluate the persistence of long-lasting immunity to the transgene protein and in parallel, to assess patient immunocompetence revealed by responses to recall antigens (tetanus toxoid, purified protein derivative), viral pathogens (Epstein-Barr virus, influenza virus), and allogeneic antigens in mixed lymphocytic reactions. The beta-Gal-specific proliferative response declined rapidly in patients with progressive disease, as did responses to the other antigens. In contrast, a long-lasting proliferative response to beta-gal was maintained in an immunocompetent patient in complete remission 2 years after an injection of 108 PFU of Ad-beta-Gal. Anti-beta-Gal humoral (IgG and IgA) responses persisted notably, as did responses to TT and poliomyelytic antigens. While T cell effector cytotoxic responses specific for the viral peptides plummeted, the frequency of anti-beta-Gal CTL precursors remained particularly high, thus attesting to major immunization. Despite the impact of both advanced disease and chemotherapy on immunocompetence, we show the long-term persistence of immunity to the transgene protein vectorized by rAd.

Ultrasound-facilitated transport of silver chloride (AgCl) particles in fish skin.

Electron-dense nano-particles in aqueous suspension were administered by immersion into the epidermis of fish using ultrasound in the therapeutic range. Enhanced permeability of the tissues to the particles was achieved by acoustic cavitation, which induced a controlled level of necrosis in the outer cell layers, and by non-cavitational exposures, which widened intercellular spaces of non-necrosed tissue in deeper regions of the epidermis. Both particle concentration and penetration depth were quantified using transmission electron microscopy. While cavitation-induced perforation was necessary for particles to penetrate into the tissues, non-cavitational exposures during immersions increased the particle flux towards the skin surface, as well as the diffusion rate of the particles within the epidermis and their depth of penetration. The technique described above may potentially be applied for non-stressful, mass-administration of substances into aquatic animals, as well as the relatively new field of ultrasound-facilitated delivery in moist epithelial tissues in humans.

Immune response to recombinant adenovirus in humans: capsid components from viral input are targets for vector-specific cytotoxic T lymphocytes.

We previously demonstrated that a single injection of 10(9) PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218-2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8(+) CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses.

Ultrasound-induced intercellular space widening in fish epidermis.

Transmission electron microscopy was employed to determine the effects of therapeutic ultrasound (US) (I(sata) < or =2.2 W cm(-2), 3 MHz), sonicated at different angles and durations, on the external epithelia of fish skin. Sonication at 1.7 W cm(-2) (90 s), where the ultrasonic beam was perpendicular to the skin surface, produced minor intercellular space widening (ICSW), as well as the disruption of desmosomes connecting between the cells. Increasing the intensity to 2.2 W cm(-2) increased ICSW, the extent of which was positively correlated to the duration of exposure (30 to 90 s). Perpendicular sonication produced ICSW, almost exclusively between cells of the two outermost cell layers, parallel to the skin surface. Sonicating at 45 degrees (2.2 W cm(-2), 90 s) produced ICSW in deeper cell layers in the tissues, in which the spaces were at seemingly random orientations. Mucous cells and macrophages were also found to be damaged, as were apoptotic epidermal cells. The suggested mechanism for ICSW is the formation of transverse (shear) waves at the interface between the aquatic medium and the skin surface. The waves, which are damped out within a few cell layers, give rise to shear stresses that, in turn, cause strains that act to separate between cells and damage some of the relatively weaker cells.

Ultrasound-induced cavitation damage to external epithelia of fish skin.

Transmission electron microscopy was used to show the effects of therapeutic ultrasound (< or = 1.0 W/cm2, 1 MHz) on the external epithelia of fish skin. Exposures of up to 90 s produced damage to 5 to 6 of the outermost layers. Negligible temperature elevations and lack of damage observed when using degassed water indicated that the effects were due to cavitation. The minimal intensity was determined for inducing cellular damage, where the extent and depth of damage to the tissues was correlated to the exposure duration. The results may be interpreted as a damage front, advancing slowly from the outer cells inward, presumably in association with the slow replacement of the perforated cell contents with the surrounding water. This study illustrates that a controlled level of microdamage may be induced to the outer layers of the tissues.

Prognostic significance of apoptosis regulators in breast cancer.

Dysregulation of normal programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X(L) has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Though the mechanisms by which Bcl-2 family proteins regulate apoptosis are diverse, ultimately they govern decision steps that determine whether certain caspase family cell death proteases remain quiescent or become active. To date, approximately 17 cellular homologs of Bcl-2 and at least 15 caspases have been identified in mammals. Other types of proteins may also modulate apoptotic responses through effects on apoptosis-regulatory proteins, such as BAG-1-a heat shock protein 70 kDa (Hsp70/Hsc70)-binding protein that can modulate stress responses and alter the functions of a variety of proteins involved in cell death and division. In this report, we summarize our attempts thus far to explore the expression of several Bcl-2 family proteins, caspase-3, and BAG-1 in primary breast cancer specimens and breast cancer cell lines. Moreover, we describe some of our preliminary observations concerning the prognostic significance of these apoptosis regulatory proteins in breast cancer patients, contrasting results derived from women with localized disease (with or without node involvement) and metastatic cancer.

Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein.

Embolic mesenteric infarction: a unique initial manifestation of renal cell carcinoma.

Renal cell carcinoma is an unpredictable tumor that often has already metastasized when first seen. It can affect many organs before the primary tumor is found. We describe a unique example of renal cell carcinoma first seen as repeated episodes of small-bowel infarction caused by tumor emboli from a metastasis in the left ventricle. Although intestinal ischemia caused by emboli is not uncommon, intestinal ischemia resulting from metastatic tumor emboli occurs in only a small percentage of cases. We suggest that surgeons include the possibility of tumor emboli in the differential diagnosis for mesenteric or peripheral ischemia that cannot be attributed to more common causes.