Mechanical relaxation in poly(2.6-dimethyl-1.4-phenylene oxide) in the glassy state

Dynamic mechanical properties (sound velocity, v, and damping factor, Q^?1) have been determined in poly(2.6-dimethyl-1.4-phenylene oxide) over a wide range of temperature (from 80 to 500°K) at acoustic frequencies. The examined polymer exhibits two mechanical relaxation effects, one, α, at temperatures above 480°K, characterized by a sudden strong drop of the elastic modulus and by a rapid increase of the damping factor with increasing temperature, and another, β, below the glass transition point, T_g, characterized by a small drop of the elastic modulus, between 290 and 370°K, and by a damping maximum at about 370^K (f_m = frequency corresponding to the maximum ? 7000 Hz). The α relaxation effect has been attributed to the thermal excitation of cooperative motions in the chain, while the secondary β relaxation has been interpreted as due to oscillation of aromatic rings around C? O? C bond. The damping maximum, for the lateer, is shifted toward higher temperatures with increasing frequency, following an ARRHENIUS -type equation with an apparent activation energy of about 20 kcal/mole.     

Role of heparan sulphate proteoglycans as potential receptors for non-piliated Pseudomonas aeruginosa adherence to non-polarised airway epithelial cells

Tight junctions seal polarised surface epithelial respiratory cells so as to prevent the passage of bacteria and toxins through the epithelial sheet. Disruption of tight junctions, which may occur during injury and repair processes of airway epithelium, favours potential bacterial interaction with receptors from cell basolateral membranes. Earlier studies reported that non-polarised and untight epithelial respiratory cells are highly susceptible to Pseudomonas aeruginosa adherence and internalisation. As heparan sulphate proteoglycans (HSP) from cell basolateral membranes in epithelial cells without tight junctions may become accessible to bacterial ligands, the present study investigated their role as potential receptors for non-piliate P. aeruginosa ligands. Treatment of cells with heparitinase I and II significantly reduced (51.2% and 51.7%, respectively) P. aeruginosa adherence to epithelial respiratory cells without tight junctions. The internalisation of bacteria was not affected by treatment with heparitinases. Treatment of the bacteria with heparin and heparan sulphate also significantly reduced their adherence to respiratory cells (34.3% and 43.7%, respectively). Treatment of cells with other enzymes (trypsin, lipase and chondroitinase ABC) or treatment of bacteria with chondroitin-4-sulphate did not modify the adherence to respiratory cells significantly. Both affinity chromatography and Western blotting assays showed the interaction of different P. aeruginosa outer-membrane proteins (OMPs) with heparin. Several bacterial strains showed differences in their profile of heparin-binding OMPs, but all exhibited low mol. wt (< 30 kDa) reactive proteins. Reactivity of whole bacterial cells with heparin was also observed by transmission electron microscopy. These results suggest that HSP are potential receptors for P. aeruginosa adherence to non-polarised and untight epithelial respiratory cells.     

Assessment of the interaction of human complement regulatory proteins with group A Streptococcus. Identification of a high-affinity group A Streptococcus binding site in FHL-1

Group A Streptococcus (GAS), the most frequent bacterial cause of suppurative infections in humans, expresses on the cell surface M proteins with capacity to bind factor H, FHL-1 and C4b binding protein (C4BP). This has been interpreted as a mechanism developed by this pathogen to decrease phagocytosis by macrophages and polymorphonuclear cells. We report the analysis of the capacity to bind factor H, FHL-1 and C4BP of 69 clinical isolates from 19 different serotypes. We show that strains binding complement regulators (30/69) belong to specific M serotypes. Of these, M18 strains are relatively frequent and interact with all three complement regulators simultaneously. However, the most virulent M1 and M3 strains did not bind complement regulators in our assays. The relevance of the interaction between complement regulators and S. pyogenes was analyzed using different approaches with the conclusion that under physiological conditions only FHL-1 and C4BP bind to streptococci. We show that FHL-1 presents a higher binding affinity for S. pyogenes than factor H because it carries a hydrophobic, high-affinity, GAS binding site in addition to the heparin binding site in SCR7. Using synthetic peptides we provide evidence that the high-affinity GAS binding site in FHL-1 involves the hydrophobic tail (Ser-Phe-Thr-Leu) that distinguishes FHL-1 from factor H.     

RNA interference screening in ticks for identification of protective antigens

Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important arthropod vector of pathogens in North America. Development of vaccines directed against tick proteins may effect reduction of tick infestations and transmission of tick-borne pathogens. The limiting step for the development of tick vaccines has been the identification of tick protective antigens. Reverse vaccinology approaches aimed at reducing animal experimentation while allowing for the rapid screening of pools of potential tick vaccine candidates would greatly facilitate progress towards the development of tick vaccines. Herein, we describe the screening of Ixodes scapularis cDNAs for identification of tick protective antigens using RNA interference (RNAi). The results of the RNAi screening were similar to those obtained previously using expression library immunization and demonstrated that RNAi could serve as a more rapid and cost-effective tool for vaccine antigen discovery in ticks and in other nonmodel organisms.     

Nonmyeloablative stem cell transplantation is an effective therapy for refractory or relapsed hodgkin lymphoma: results of a spanish prospective cooperative protocol.

We report the results of reduced-intensity conditioning allogeneic stem cell transplantation (allo-RIC) in patients with advanced Hodgkin lymphoma (HL). Forty patients with relapsed or refractory HL were homogeneously treated with an RIC protocol (fludarabine 150 mg/m(2) intravenously plus melphalan 140 mg/m(2) intravenously) and cyclosporin A and methotrexate as graft-versus-host disease (GVHD) prophylaxis. Twenty-one patients (53%) had received >2 lines of chemotherapy, 23 patients (58%) had received radiotherapy, and 29 patients (73%) had experienced treatment failure with a previous autologous stem cell transplantation. Twenty patients (50%) were allografted in resistant relapse, and 38 patients received hematopoietic cells from an HLA-identical sibling. Five patients (12%) died from early transplant-related mortality (before day +100 after allo-RIC). One-year transplant-related mortality was 25%. Acute GVHD developed in 18 patients (45%). Chronic GVHD developed in 17 (45%) of the 31 evaluable patients. The response rate 3 months after the allo-RIC was 67% (21 [52%] complete remissions and 6 [15%] partial remissions). Eleven patients received donor lymphocyte infusions (DLIs) for disease relapse. The response rate after DLI was 54% (3 complete remissions and 3 partial remissions). Overall survival (OS) and progression-free survival (PFS) were 48% +/- 10% and 32% +/- 10% at 2 years, respectively. Refractoriness to chemotherapy was the only adverse prognostic factor for both OS (63% +/- 12% versus 35% +/- 13%; P = .05) and PFS (55% +/- 16% versus 10% +/- 9%; P = .006). For patients with failure of a prior autologous hematopoietic stem cell transplantation, results were especially good for those who experienced late relapses (>/=12 months: 2-year OS and PFS were 75% +/- 16% and 70% +/- 18%, respectively). These data suggest that allo-RIC is feasible in heavily pretreated HL patients and has an acceptable early transplant-related mortality. Results are better in patients allografted in sensitive disease. Both responses observed after the development of GVHD and DLI may suggest a graft-versus-HL effect. Allo-RIC has to be considered an effective therapeutic approach for patients who have had treatment failure with a previous autologous hematopoietic stem cell transplantation.     

Presence of a very small population of Thy-1+, L3T4+ cells producing large amounts of IL-3 in young athymic nude mice.

A mixture of phorbol myristate acetate (PMA) and ionomycin was found to stimulate spleen and lymph node cells (LNC) from 6 to 8 week-old-athymic BALB/c nude mice, as well as from control +/+ mice, to secrete interleukin-3 (IL-3) in vitro. The specificity of the IL-3 bioassay was attested to by the use of rabbit anti-IL-3 antibodies, and by the detection of an accumulation of IL-3 mRNA. Cytotoxic treatment with relevant antibodies showed that the cells responsible for the IL-3 production in athymic nude mice was Thy-1+, L3T4+, Ly2-, while both L3T4+ and Ly 2+ cells produced IL-3 in control +/+ mice. Although the levels of IL-3 secreted by nude LNC varied among experiments, nude LNC were able to produce IL-3 at a level comparable to or higher than +/+ LNC. In addition, nude LNC consistently secreted two to three times more granulocyte-macrophage colony-stimulating factor (GM-CSF) than +/+ LNC, and the majority of GM-CSF secretion was dependent on the presence of L3T4+ cells. In contrast, IL-2 production by nude LNC was markedly limited. Since the flow microfluorometry analysis failed to demonstrate the presence of L3T4+ cells (less than 1%) in nude LNC, compared with 40-50% L3T4+ cells in +/+ LNC, our results suggest that athymic nude mice have a small population of Thy-1+, L3T4+ cells characterized by its ability to secrete IL-3 and GM-CSF at a very high rate.     

Detection of rubella antibodies by hemagglutination inhibition, indirect fluorescent-antibody test, and enzyme-linked immunosorbent assay.

Using hemagglutination inhibition (HAI) as a reference method, 292 (40 nonimmune, 252 immune) human serum samples were tested by indirect fluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) for immune status and quantitation of rubella antibodies. The overall agreement with HAI for immune status was 99.7% (291/292) with IFA and 98.6% (288/292) with ELISA. Two specimens (0.7%, 2/292), negative by HAI, were equivocal by ELISA. Initially a 6.5% (19/292) overall disagreement was obtained for immune status evaluation between HAI and IFA, which was reduced to 0.3% (1/292) upon repeat testing. All of these samples were near the immune/nonimmune cutoff point (95 samples), reflecting an initial disagreement of 20% (19/95) in this category (HAI titers less than 1:20). Likewise, an initial overall disagreement of 4.5% (13/292) was obtained between HAI and ELISA which was reduced to 0.7% (2/292) upon repeated testing. Eleven of the 13 samples were near the immune/nonimmune cutoff point, reflecting an initial disagreement of 11.6% (11/95) with sera having an HAI antibody titer of less than 1:20. Quantitation of rubella antibodies by IFA showed an overall correlation with HAI of 86.6% within less than twofold titer and 99.3% within less than fourfold titers. In testing the ability of ELISA to quantitate antibody, a correlation coefficient (r) of 0.996 was obtained by plotting the measured average optical density (405 nm) of ELISA against the corresponding log of HAI titer. Both IFA and ELISA showed good correlation with HAI for immune status evaluation and for quantitation of rubella antibodies. Technically the HAI was the most cumbersome to perform, whereas IFA was the least technically demanding. Originally, 308 samples were tested; 16 samples (5.2%) could not be evaluated by IFA because of a high level of nonspecific fluorescence. The strict requirement of controlling the temperature range (23 to 24 degrees C) during substrate hydrolysis proved to be a problem with the ELISA test in our laboratory.     

Protection against Toxoplasma gondii in mice immunized with Toxoplasma cell fractions, RNA and synthetic polyribonucleotides

Mice immunized with fractions obtained by centrifugation of disrupted Toxoplasma gondii trophozoites as well as with 200 μg of Toxoplasma ribonucleic acid (RNA) were resistant (as measured by time to death and total mortality) to challenge with Toxoplasma 30 days later. When mice were challenged at 15 days no protection was noted. A dose of 50 μg of Toxoplasma RNA was effective in protecting mice against lethal challenge only when incorporated into Freund's incomplete adjuvant. In studies performed to determine the specificity of the resistance observed, resistance was also noted in mice immunized with 200 μg of RNA extracted from normal mouse peritoneal macrophages, as well as in mice immunized with 100 μg of the synthetic polyribonucleotide polycytidylic acid. Polyadenylicuridylic acid conferred protection only when incorporated into Freund's incomplete adjuvant and polyinosinic—cytidylic acid had no effect. The protection induced by Toxoplasma RNA was eliminated by prior treatment of the preparation with ribonuclease but not by treatment with pronase, suggesting that the moiety responsible for the protective effect was RNA. In experiments designed to explore the mechanism of resistance in the vaccinated mice, macrophages harvested from mice which had been injected with Toxoplasma RNA 15 days earlier were found to be activated in that they resisted challenge with Listeria monocytogenes.     

The relationship between the family doctor and the hospital

From this paper based on the opinions of hospital patients, a representative group of Dutch family doctors and of specialists, as well as on the results of an investigation in three practices of the Nijmegen University Department of General Practice, two conclusions can be drawn.

(1) The family doctor can fulfil an important role for his patients in hospital. This role arises from his function as a doctor to the family and is complementary to the technical specialist's work in hospital. The family doctor can therefore bridge the gap between hospital and home and between the patient and his family.

(2) A strict referral and hospital admission policy by the family doctor has important consequences and gives him a key position in health care. He can promote the shift from hospital-centred care to domiciliary-centred care which is now generally considered to be necessary.

It seems likely that such a strict referral system means a better quality of medical care for patients.

This is a priority of the first order, greater than that of perfecting hospital medical care, which will require an ever-increasing effort and cost for a decreasing group of patients.

     

DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene. Large rearrangements in the gene are found in about two-thirds of DMD patients, with approximately 60% carrying deletions and 5-10% carrying duplications. Most of the remaining 30-35% of patients are expected to have small nucleotide substitutions, insertions, or deletions. To detect these subtle changes within the coding and splice site determining sequences of the dystrophin gene, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. The DGGE scan covers the dystrophin gene with 95 amplicons, PCRed either individually or in a multiplex setup. PCR and pooling were performed semiautomatically, using a pipetting robot and 384-well plates, enabling concurrent amplification of DNA of four patients in one run. Amplification of individual fragments was performed using one PCR program. The products were pooled just before gel loading; DGGE requires only a single gel condition. Validation was performed using DNA samples harboring 39 known DMD variants, all of which could be readily detected. DGGE mutation scanning was applied to analyze 135 DMD/BMD patients and potential DMD carriers without large deletions or duplications. In DNA from 25 out of 44 DMD patients (57%) and from 5 out of 39 BMD patients (13%), we identified clear pathogenic changes. All mutations were different, with the exception of one DMD mutation, which occurred twice. In DNA from 10 out of 44 potential DMD carriers, including four obligate carriers, we detected causative changes, including one pathogenic change in every obligate carrier. In addition to these pathogenic changes, we detected 15 unique unclassified variants, i.e., changes for which a pathogenic nature is uncertain.