Fluticasone in mild to moderate atopic dermatitis relapse: A randomized controlled trial.

BACKGROUND: The long-term efficacy of corticosteroids to prevent atopic dermatitis (AD) relapses has partially been addressed in children. This study compared an intermittent dosing regimen of fluticasone propionate (FP) cream 0.05% with its vehicle base in reducing the risk of relapse in children with stabilized AD. METHODS: A randomized controlled, multicentric, double-blind trial was conducted. Children (2-10 years) with mild/moderate AD (exclusion criteria: >30% affected body surface area and/or head) were enrolled into an Open-label Stabilization Phase (OSP) of up to 2 weeks on twice daily FP. Those who achieved treatment success entered the Double-blind Maintenance Phase (DMP). They were randomly allocated to receive FP or vehicle twice-weekly on consecutive days for 16 weeks. The primary study endpoint was relapse rate; time to relapse and severity of disease were also studied. Kaplan-Meier estimates were calculated. RESULTS: Fifty-four patients (29 girls) entered the OSP (23 mild AD) and 49 (26 girls) continued into the DMP. Mean age was 5.5 (SD: 2.8) and 5.1 (SD: 2.3) yrs for FP and vehicle groups, respectively. Four patients withdrew from the DMP (two in every group). Patients treated with FP twice weekly had a 2.7 fold lower risk of experiencing a relapse than patients treated with vehicle (relative risk 2.72, SD: 1.28; p=0.034). FP was also superior to vehicle for delaying time to relapse. Both treatment therapies were well tolerated. CONCLUSION: This long-term study shows that twice weekly FP provides an effective maintenance treatment to control the risk of relapse in children with AD.

High spatial resolution, low-noise Brillouin dynamic gratings reflectometry based on digital pulse compression.

Digital pulse compression was used to enhance the performance of optical time-domain reflectometry, employing Brillouin dynamic gratings (BDGs) in polarization-maintaining fibers. The fundamental and unique issues in BDG field-reflection are addressed, and rules for proper selection of the coding and detection techniques are formulated. While coding in BDG applications generally requires coherent processing of the reflection, conditions are established for use of direct detection. A 256-bit Golay complementary unipolar probe code is used to demonstrate an eightfold signal-to-noise ratio enhancement in the measurement of the Brillouin gain spectrum (BGS), with a spatial resolution of 2 cm and a full-BGS acquisition rate of 133⅓ kHz, resulting in an equivalent reduction in the estimation error of small Brillouin frequency shifts.

Enhanced spontaneous backscattering in Brillouin dynamic gratings.

Spontaneous Brillouin backscattering, which accompanies the operation of Brillouin dynamic gratings (BDGs) setups, is investigated both theoretically and experimentally. It is shown that this noisy emission, which cannot be separated from the signal of interest, contains not only the probe spontaneous Brillouin backscattering but also a significant contribution from the spontaneous/stimulated acoustic field, originating from the high-frequency writing pump. In the absence of the low-frequency writing pump and for a strong enough high-frequency writing pump, the observed Stokes noise can exhibit an average backscattered power much higher than that from the probe alone.

Recombinant, ETA'-based CD64 immunotoxins: improved efficacy by increased valency, both in vitro and in vivo in a chronic cutaneous inflammation model in human CD64 transgenic mice.

BACKGROUND: Dysregulated, activated macrophages play a pivotal role in chronic inflammatory diseases such as arthritis and atopic dermatitis. These cells display increased expression of the high-affinity Fcgamma receptor (CD64), making them ideal targets for CD64-specific immunotoxins. We previously showed that a chemically linked immunotoxin, the monoclonal H22-RicinA, specifically eliminated infiltrating activated macrophages and resolved chronic cutaneous inflammation. However, several disadvantages are associated with classic immunotoxins, and we therefore followed a fusion protein strategy to express the antigen-binding site alone (scFv H22) fused to a derivative of Pseudomonas exotoxin A (ETA'). OBJECTIVES: To assess the potential effect of increased valency on efficacy, we produced monovalent [H22(scFv)-ETA'] and bivalent [H22(scFv)(2)-ETA'] versions and evaluated their potential for eliminating activated macrophages both in vitro and in vivo. METHODS: Both immunotoxins were produced by bacterial fermentation. Binding was assessed by flow cytometry on the monocytic CD64+ cell line U937. Toxicity was analysed by XTT and apoptosis induction by annexin V bioassay. The in vivo effect was tested in a human CD64 transgenic mouse model for cutaneous inflammation. RESULTS: The cytotoxic effects of both immunotoxins were clearly due to apoptosis with an IC(50) of 140 pmol L(-1) for monovalent and only 14 pmol L(-1) for the divalent version. In vivo treatment with H22(scFv)-ETA' reduced CD64+ activated macrophages to 21% of their initial numbers whereas H22(scFv)(2)-ETA' treatment reduced these cells to 4.8% (P < 0.001). CONCLUSIONS: These data clearly show increased efficacy due to increased valency of the anti-CD64 immunotoxin. Both recombinant immunotoxins have a low IC(50), making them suitable for the treatment of diseases involving dysregulated, activated macrophages.

[Family impact of rotavirus gastroenteritis in children under two years]. / Impacto familiar de la gastroenteritis por rotavirus en menores de dos años.

INTRODUCTION: Acute gastroenteritis (AGE) in infants has a significant impact on the quality of life of their parents. MATERIAL AND METHODS: Cross-sectional study on the sociological family impact related to rotavirus AGE in children under 2 years. The study was carried out in 25 hospitals and 5 primary care centres in Spain. Sociodemographic, epidemiological and clinical data were recorded, as well as the symptomatology of AGE and its severity measured by the Clark scale. Stool samples were tested to determine rotavirus positive (RV+) or negative (RV-). The parents were asked to complete a a family impact questionnaire. RESULTS: Stool specimens were tested in 1087 AGE cases (584 RV+ vs 503 RV-). The 99.5 % of parents whose children were RV+ reported more worries vs. the 97.7 % of RV-, and RV+ had a higher importance score (p < 0.05). A higher percentage of RV+ parents and those with a high importance score reported more time dedicated to dehydration treatment (p < 0.05). The 82.5 % vs. 73.9 % had disruption of their household tasks, with more importance scores (p < 0.05). RV+ had a higher percentage and importance score than RV- ones in all aspects of their child's AGE symptoms, except loss of appetite. CONCLUSION: AGE produces important dysfunctional experiences in daily family life. According to parental perceptions, RV+ produces greater worries and dysfunctions in child behaviour.

[NFLUENCE OF SPONTANEOUS PARTIAL SENSORY DEPRIVATION ON MALE C57BL/6N MICE BEHAVIOR].

Vibrissae loss associated with the peculiarities of the intragroup social interaction may be an important factor affecting the animals' performance in various behavioral tests. To evaluate the influence of spontaneous partial sensory deprivation as a consequence of the barbering activity of a cage mate, the battery of tests was conducted in male C57Bl/6N mice. The results indicate that the behavior of mice without vibrissae significantly differs from control animals in the tube, open field, social interaction and forced swim tests. Thus, the present findings suggest that vibrissae conditions have to be assessed before the inclusion of animals into experimental groups and/or further considered in data analysis.

Human angiogenin fused to human CD30 ligand (Ang-CD30L) exhibits specific cytotoxicity against CD30-positive lymphoma.

A number of different immunotoxins composed of cell-specific targeting structures coupled to plant or bacterial toxins have increasingly been evaluated for immunotherapy. Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based fusion toxin (Ang-CD30L) using the human RNase angiogenin. The completely human fusion gene was inserted into a pET-based expression plasmid. Transformed Escherichia coli BL21(DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta-D-thiogalactoside induction, the M(r) 37,000 His(10)-tagged Ang-CD30L was directed into the periplasmic space and functionally purified by a combination of metal ion affinity followed by enterokinase cleavage of the His(10)-Tag and molecular size chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays showing specific activity against CD30(+) Hodgkin-derived cells. Specific binding activity of Ang-CD30L was verified by competition with anti-CD30 monoclonal antibody Ki-4 and commercially available CD30L-CD8 chimeric protein. Ang-CD30L showed RNase activity in vitro. The human recombinant immunotoxin showed significant toxicity toward several CD30-positive cell lines (HDLM-2, L1236, KM-H2, and L540Cy) and exhibited highest cytotoxicity against L540 cells (IC(50) = 8 ng/ml) as determined by cell proliferation assays. CD30 specificity was confirmed by competitive toxicity assays. This is the first report on the specific cytotoxicity of a recombinant completely human fusion toxin with possibly largely reduced immunogenicity for the treatment of CD30-positive malignancies.

Combining phage display and screening of cDNA expression libraries: a new approach for identifying the target antigen of an scFv preselected by phage display.

A potential method for identifying new tumor-specific antibody structures as well as tumor-associated antigens is by selecting scFv phage libraries on tumor cells. This phage display technique involves multiple rounds of phage binding to target cells, washing to remove non-specific phage and elution to retrieve specific binding phage. Although the binding properties of an isolated tumor-specific scFv can be evaluated by ELISA, FACS and immunohistochemistry, it still remains a challenge to define the corresponding antigen. Here, we provide evidence that the target antigen of a given scFv displayed on phages can be detected in an immobilized lambda phage cDNA expression library containing thousands of irrelevant clones. The library contained CD30-negative breast-cancer specific cDNA as well as human CD30 receptor cDNA. The interaction of anti-CD30 scFv phages and their target antigen after blotting onto nitrocellulose filters was documented under defined conditions. Screening of different ratios between CD30 receptor and breast cancer specific clones (1:1 and 1:200) revealed that the CD30 antigen could be detected by anti-CD30 scFv phages using at least 5x10(12) plaque forming units of filamentous phages per blot. These investigations demonstrate that it is possible to detect the target antigen of a preselected scFv displayed on filamentous phages in lambda phage cDNA expression libraries.

Persistence of spectral variations in DGD statistics.

We observe distinct variations between the DGD temporal statistics for different channels in a field installed system. This phenomenon is confirmed with statistical analysis, using a model in which DGD dynamics are due only to a finite number of active points along the link.

[Morphine-induced Straub tail reaction as a model of spasticity in mice: effects of serotonergic compounds]. / Vyzvannaia morfinom reaktsiia Shtrauba kak model' spastichnosti u myshei: éffekty serotoninergicheskikh soedinenii.

AIM: To adopt and validate the Straub tail reaction (SR) for comparative assessment of spastic effects of serotonergic compounds. MATERIAL AND METHODS: To measure the muscle relaxant activity, the morphine-induced Straub-tail assay was used. SR was graded according to modified intensity-score basis in a scale decribed by Kameyama et al. (1978). Subcutaneous injections of different doses of morphine (10-60 mg/kg) induced a dose-dependent SR with maximum response obtained 15-30 min after the morphine administration. RESULTS AND CONCLUSION: The centrally acting muscle relaxant baclofen (3-10 mg/kg) reduced SR induced by morphine (40 mg/kg) at all used doses; tizanidine decreased the intensity of SR at highest doses tested (0.6 and 1 mg/kg). Dantrolene (20-100 mg/kg), a peripherally acting muscle relaxant, did not affect SR. Effects of serotonergic agents depended on the specific mechanism of action. SR appears to be available for rapid evaluation of the effect of antispasticity drugs.

Basal perfusion of the cutaneous microcirculation: measurements as a function of anatomic position.

Noninvasive optical techniques of photopulse plethysmography (PPG) and laser Doppler velocimetry (LDV) have been used to identify regional variations in the basal skin blood flow of humans. The procedures assess either the volume (PPG) or the volume-velocity product (LDV) of cutaneous blood vessel perfusion. Fifty-two anatomic positions have been studied in 10 normal subjects resting horizontally. The mean perfusion levels were ranked to reveal the variations in cutaneous blood flow as a function of body site. Groups of data were collected into cohorts and average perfusion values for the subjects within each cohort were compared by the Newman-Keuls multiple comparison test. Most transparently, the results reveal a collection of regions (fingers, palms, face, ears) for which cutaneous perfusion is much higher than all other positions. More subtle differences and some unexpected similarities, however, are also apparent and, in some cases, agree or, in others, conflict, with previously published information. With some exceptions, good general agreement between the two techniques was observed.

Noninvasive assessment of local nicotinate pharmacodynamics by photoplethysmography.

The local pharmacodynamics of a topical vasodilator (methyl nicotinate) has been followed noninvasively using photopulse plethysmography. This technique is sensitive to changes in blood flow through the cutaneous microcirculation and responds to the pharmacologic stimulus of the vasoactive agent employed. Five different application sites for the drug were studied and the time course of the local effect (i.e., onset, duration, and decay) was recorded. The applied amount of drug elicited, within a short period, a response which was saturable such that the observed increase in blood flow reached a plateau level. The decay of the elevated perfusion required approximately 1 h, suggesting a half-life for elimination of the drug from the skin of about 10 min. This result agrees closely with other reported values and suggests that the pharmacodynamic measurements of this study may prove useful in elucidating aspects of dermal pharmacokinetics.

Selection of scFv phages on intact cells under low pH conditions leads to a significant loss of insert-free phages.

Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens. However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking antibody fragments. Moderate adhesive binding activities and production advantages of these "empty" phages results in their subsequent enrichment during selection on target cells. To date, very limited effort has been made to develop strategies removing nonspecific binding phages during the selection processes. To efficiently reduce insert-free phages when panning on intact cells, we increased the washing stringency by lowering the pH of the buffer with citric acid. Under standard washing procedures (pH 7.4), only approximately 73% of recovered phages were insert-free after three rounds of selection. Using stringent washing procedures (pH 5.0), approximately 12% of recovered phages contained no scFv. Using this protocol, we have cloned an antibody fragment from a mouse/human hybridoma cell line directed against the disialoganglioside GD2. This study confirms that selection of phage antibodies on cells is efficiently enhanced by assays augmenting the stringency to remove nonspecific binding phages.

Construction of phage display libraries from reactive lymph nodes of breast carcinoma patients and selection for specifically binding human single chain Fv on cell lines.

The display of recombinant antibody fragments on the surface of filamentous phage mimicks B cells and is therefore a technology ideal to generate antibodies against any potential target antigen in vitro. In order to obtain tumor specific, high-affinity single chain antibody fragments (scFv), it has been speculated that lymph node tissue from cancer patients infiltrated with activated B cells must be a valuable source of antibody V-genes. The aim of this study was to generate a human scFv-phage library from lymph nodes of patients with breast cancer and to develop a stringent depletion and selection protocol in order to isolate specific single chain antibodies recognizing potentially new antigens in breast cancer. The amplification of the V-genes cloned from regional lymph node tissue and their assembly to single chain variable fragments was optimized in terms of library size and diversity. A large set of degenerated primers, annealing to all known V-gene families, was designed and used under optimized PCR conditions. The amplified V-genes were genetically fused in all possible combinations and cloned into a phagemid vector. Depletion and selection on mammary epithelial and primary breast carcinoma cell lines, respectively led to the isolation of a breast cancer cell line specific scFv (BCK-1 scFv) from this patient-derived scFv-phage display library as demonstrated in polyclonal and monoclonal ELISA, using immobilized cell membrane fractions of the indicated cell lines. A new recombinant breast cancer cell line specific antibody based on V-genes derived from reactive B-lymphocyte-infiltrated lymph nodes of patients with breast cancer was isolated via phage display, performing stringent depletion and selection protocols. We believe that this combination of antibody V-gene source and elaborated phage display depletion and selection strategy will be successful for the retrieval of numerous other recombinant, tumor specific antibody fragments.

An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines.

Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.

[Delayed diagnosis of homocystinuria by major deficiency in cystathionine beta synthase]. / Diagnostic tardif d'homocystinurie par déficit majeur en cystathionine beta synthase.

INTRODUCTION: Homocystinuria due to cystathionine beta synthase (CBS) deficiency is a special type of hyperhomocysteinemia because of its clinical expression (thrombotic events, ectopic lens and mental retardation). It's a rare, hereditary recessive autosomic disease generally diagnosed during childhood. EXEGESIS: Thrombophilia examination in a 50-year-old man found a dramatically increase homocysteinemia. Homocystinuria, profile of plasmatic amino acids and reduced CBS activity, (0.05 microkat/kg protein; N = 1.5 +/- 0.8) confirmed homocystinuria's diagnosis. Family study demonstrates that three siblings suffer from homocystinuria. Vitamin enriched diet with pyridoxin, vitamin B12 and folates induced reducing hyperhomocysteinemia and homocystinuria. CONCLUSION: This case report, original because of the diagnosis age, suggests a hyperhomocysteinemia's screening in patients with recurrent thrombotic events.

[The intra-arterial chemotherapy of tumorous lesions of the stomach, rectum and liver]. / Vnutrishn'oarterial'na khimioterapiia pukhlynnykh urazhen' shlunka, priamoï kyshky ta pechinky.

Roentgenosurgical treatment of malignant tumor of the stomach, straight intestine, liver is based on a directed bringing of cytostatics to the tumor by way of catheterization of afferent vessels together with the use of the modifier of drug resistance verapamil. A higher therapeutic effect can be achieved by securing an easier access of the medicinal substances to the membrane of the tumor cells. There can be noted regression and destruction of the tumor, enhancement of ablasticity of surgical intervention, with metastatic spreading and local relapses developing at a lower rate, which facts taken together contribute to a better outlook for the patients' survival and quality of their life.

Generation of recombinant antibody fragments that target canine dendritic cells by phage display technology.

One of the main goals in cancer immunotherapy is the efficient activation of the host immune system against tumour cells. Dendritic cells (DCs) can induce specific anti-tumour immune responses in both experimental animal models and humans. However, most preclinical studies using small animal models show only limited correlation with studies carried out in clinical settings, whereas laboratory dogs naturally develop tumours that are biologically and histopathologically similar to their human counterparts. Here, we describe the generation and characterization of recombinant antibodies against canine DCs, isolated using the Tomlinson phage display system. We successfully isolated highly specific single-chain variable fragment (scFv) antibodies in a sequential three-step panning strategy involving depletion on canine peripheral blood mononuclear cells followed by positive selection on native canine DCs. This provides the basis for an antibody-based method for the immunological detection and manipulation of DCs and for monitoring antigen-specific immune responses.