Display of functional alphabeta single-chain T-cell receptor molecules on the surface of bacteriophage

The ability to display functional T-cell receptors (TCR) on the surface of bacteriophage could have numerous applications. For instance, TCR phage-display could be used to develop new strategies for isolating TCRs with unique specificity or it could be used to carry out mutagenesis studies on TCR molecules for analyzing their structure-function. We initially selected a TCR from the murine T-cell hybridoma, DO11.10, as our model system, and genetically engineered a three domain single-chain TCR (scTCR) linked to the gene p8 protein of the Escherichia coli bacteriophage fd. Immunoblotting studies revealed that (1) E. coli produced a soluble scTCR/p8 fusion protein and (2) the fusion protein was packaged by the phage. Cellular competition assays were performed to evaluate the functionality of the TCR and showed the DO11.10 TCR-bearing phage could significantly inhibit stimulation of DO11.10 T hybridoma cells by competing for binding to immobilized MHC/peptide IA(d)/OVA(323-339). Flow cytometric analysis was carried out to evaluate direct binding of DO11.10 TCR-bearing phage onto the surface of cells displaying either IAd containing irrelevant peptide or OVA peptide. The results revealed binding of DO11.10 TCR-bearing phage only on cells expressing IA(d) loaded with OVA peptide showing TCR fine specificity for peptide. To illustrate the generality of TCR phage-display, we also cloned and displayed on phage a second TCR which recognizes a peptide fragment from human tumor suppressor protein p53 restricted by HLA-A2. These findings demonstrate functional TCR can be displayed on bacteriophage potentially leading to the development of novel applications involving TCR phage-display.     

The simian T-lymphotropic virus STLV-PP1664 from Pan paniscus is distinctly related to HTLV-2 but differs in genomic organization

We have isolated a highly divergent simian T-lymphotropic virus, STLV-PP1664, from a wild-caught bonobo (Pan paniscus). Previous phylogenetic analysis suggested that this virus represents an additional type of STLV but this has now become a matter of discussion. We have now obtained and analyzed the entire genome of STLV-PP1664. All major genes and their corresponding viral messengers were identified. Sequence comparison and phylogenetic analysis indicated that this virus, together with the closely related panp isolate, belongs to an early lineage within the PTLV-2 clade, differing from HTLV-2 by about 25%. In contrast to the HTLV-1 and HTLV-2 LTR, only two 21-bp repeats instead of three were found in the STLV-PP1664 LTR. Additional messengers, resulting from alternative splicing, potentially encode five different accessory proteins from open reading frames in the pX region: prorfI, porfII, ptorfV', and two isoforms of Rex. The amino acid sequences of these proteins are only distinctly related to the accessory proteins from HTLV-2. These data suggest a different genomic organization of the STLV-PP1664 pX region than that of HTLV-2. We conclude that STLV-PP1664, although related to HTLV-2, has some distinct features in the LTR and the pX regions, the impact of which needs further investigation. Although arguments pro and contra a distinct classification are nearly equally balanced, we propose to classify this virus as an STLV-2, designated STLV-2PP1664.     

Clear lens extraction and implantation of negative-power posterior chamber intraocular lenses to correct extreme myopia

PURPOSE: To evaluate the effectiveness, predictability, and safety of clear lens extraction to correct extreme myopia. SETTING: Clinica de Nuestra Se?ora de la Concepción, Fundación Jiménez Díaz, Madrid, Spain. METHODS: This retrospective study comprised 26 eyes of 17 highly myopic patients who had clear lens extraction and implantation of a negative-power posterior chamber intraocular lens (IOL). The IOL power was calculated using the SRK/T formula. Analyzed were visual and refractive results and intraoperative and postoperative complications. Follow-up was at least 12 months in all cases. RESULTS: Uncorrected visual acuity improved in all cases, with 80.77% of eyes achieving 20/100 or better and 42.30%, 20/40 or better. Best spectacle-corrected visual acuity (BSCVA) improved in 23 eyes (88.46%). The percentage of eyes achieving a BSCVA of 20/100 or better increased from 73.07% preoperatively to 92.30% postoperatively and the percentage achieving 20/40 or better, from 23.07 to 73.07%. Of the 26 eyes, 76.91% were within 1.00 diopter (D) of refractive error and 96.16% were within 2.00 D. No intraoperative complications occurred. Although postoperatively 3 eyes (11.53%) developed choroidal detachment and 5 (19.23%) had an intraocular pressure greater than 25 mm Hg, all had a favorable outcome. Four eyes (15.38%) developed posterior capsule opacification and had a neodymium:YAG laser posterior capsulotomy 6 months postoperatively. No retinal detachments were observed. CONCLUSION: Clear lens extraction with negative-power IOL implantation using the SRK/T formula had good effectiveness, acceptable predictability, and a low morbidity in eyes with extreme myopia over a short follow-up. A longer follow-up with more cases is needed to assess the safety of the procedure.     

Differential chromosome sensitivity to 5-azacytidine in Alzheimer''s disease

The chemiluminescence (CL) technique with scavengers for superoxide anion (superoxide dismutase) and hydrogen peroxide (catalase) was used to characterize the generation of reactive oxygen species (ROS) inside and outside the human neutrophil after stimulation with both soluble (formyl-methionyl-leucyl-phenylalanine, FMLP) and particulate (urate crystals, zymosan, oxidized LDL) stimuli. Depending on the stimulus used, ROS generation differed in composition and absolute amounts. The ratio between extracellularly and intracellularly produced ROS ranged from 0.3 (zymosan) to 4.2 (FMLP). While enhancing substantially FMLP-stimulated CL, horseradish peroxidase inhibited CL induced by particulate stimuli by 40-80%. Furthermore, an azide-insensitive and therefore peroxidase-independent part of CL was found in FMLP-, LDL- and zymosan-stimulated cells. The results indicate that different agonists may lead through distinct chemical pathways to neutrophil luminol-amplified light generation.     

Morphologic comparison of atherosclerotic lesions in native coronary arteries and saphenous vein graphs with intracoronary angioscopy in patients with unstable angina

The purpose of this study was to investigate platelet effects on postischemic heart function in conjunction with adenosine effects on intracoronary platelet adhesion. Homologous platelets were infused into the coronaries of isolated guinea pig hearts, either during low-flow ischemia or during reperfusion, and external heart work (EHW) and intracoronary platelet adhesion were determined. In most experiments, thrombin was added to the perfusate. The influence of endogenous adenosine was studied by use of the uptake blocker dipyridamole and the unspecific adenosine-receptor blocker theophylline, the A1-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and the A2-receptor blocker 3,7-dimethyl-1-propargylxanthine (DMPX). The importance of nitric oxide and prostaglandin I2 (PGI2) was tested by using nitro-L-arginine (NOLAG) and indomethacin, respectively. When platelets were applied with thrombin during low-flow ischemia, EHW recovered to only 63 +/- 4% of the preischemic value, as compared with 89 +/- 3% without platelets (p < 0.05). Despite thrombin, platelets incurred no significant functional loss when applied in the first minute of reperfusion (but again in the fifth minute); however, when theophylline was also present, recovery of EHW amounted to only 42 +/- 12%. Intracoronary adhesion of platelets was negligible without thrombin, and highest during low-flow ischemia with thrombin (35 +/- 3% of the applied number). No adhesion occurred during the first minute of reperfusion, whereas in the fifth minute, adhesion was again 20.8 +/- 4%. Dipyridamole increased adenosine release and attenuated adhesion at this time. Theophylline increased adhesion in the first minute of reperfusion (33 +/- 6.4%), whereas NOLAG and indomethacin proved to be ineffective. DPCPX and DMPX each increased platelet retention during the first minute of reperfusion, their effects being additive. Intracoronary adhesion of platelets induced by thrombin in isolated hearts can reduce postischemic recovery of heart function. During reperfusion, but not during low-flow, endogenous adenosine can prevent platelet adhesion and loss of myocardial function, an action mediated both by A1- and A2-receptor-dependent mechanisms.     

Overview of otolaryngic allergy management. An eclectic and cost-effective approach

In this paper, we studied three species of prosimian primate (Propithecus diadema edwardsi, Eulemur fulvus rufus, and Eulemur rubriventer) from June-July 1995 at the Ranomafana National Park to answer three questions: 1) how they handle and process seeds, 2) how the physical properties of seeds influence seed handling and seed fate, and 3) whether handling and processing patterns influence seed dispersal. Seeds from five plant species were collected from feces and examined for external damage (punctures and scrapes), weighed, measured, and checked daily for germination. P. d. edwardsi masticated seeds into two or more pieces while feeding. Seed fragments were either dropped under the parent tree or chewed and swallowed; seeds never emerged as recognizable units in feces. In contrast, both Eulemur species either dropped or swallowed seeds whole while feeding. E. rubriventer passed seeds that were longer, wider, and heavier than seeds passed by E.f. rufus. Although seeds emerged as separate units when passed by both Eulemur species, 65 Protorhus sp. seeds were scraped and/or punctured prior to being swallowed. Based on physical property tests, Protorhus seeds were more susceptible to mastication than undamaged seeds from Eugenia sp., Cissus sp., and Chrysophyllum madagascariensis. H. madagascariensis seeds also were undamaged but had physical properties comparable to Protorhus and may avoid being masticated due to their small size (2-3 mm). All damaged seeds were moldy or rotten within 6 days, and only 15% of the undamaged seeds passed by E. rubriventer germinated. None of the seeds passed by E.f. rufus germinated. Eulemur species also rested in the same tree after feeding and defecated prior to a new feeding bout or before moving, so seeds were most likely to be dispersed under the parent tree. Consequently, we concluded that these primate species do not appear to serve as effective seed dispersers for these plant species at this time of year.     

Congenital hypothyroidism

Congenital hypothyroidism (CH) occurs in 1/3 500 newborn infants. Interest in newborn screening for CH has been widely demonstrated in most countries. It was introduced in France in the last 70's; by the measurement of serum TSH level from an eluate of whole blood collected on filter paper on 3 days of life. It allows earlier diagnosis and treatment preventing most of the serious sequelae of the disease such as postnatal neurological damage. Their neuropsychological evaluation has shown normal mental development in most cases. Although CH with eutopic gland (15% of the cases) has recently been recognized as associated with different molecular defect, the cause of thyroid agenesis (85% of the cases with athyreosis or ectopic gland) remains unknown. These latter forms are usually sporadic but familial cases are described.     

Therapeutic approach in blast injuries of the ear

Otic blast injury is caused by arrhythmic air blast wave. The perforation of the tympanic membrane is the commonet finding associated with lacerations of mucosa in the middle ear. Makki [6] published 34 cases of myringoplasty after war blast injuries. However, healing of such perforations is common; Kerr [7] noted a healing rate of 83% after the blast injury. The aim of the study was to evaluate different therapic procedures of otic blast injuries. There were 74 patients with bilateral otic blast injuries, who underwent otomycroscopic examination. The following parameters were noted: Integrity of tympanic membrane and size of perforation, presence of haemorrhagic exudate in the middle ear and median value of conductive deafness (Table 2). Main symptoms, presented in Table 1 were: pain, deafness and otohaematorrhoea. The first group of 19 patients received antibiotics by parenteral way during 7 days according to the bacterial finding in ear exudate. Healing rate was dependent on the size of tympanic membrane perforation, and rated from 71% in perforation of one third of tympanic membrane to 25% in total perforation of tympanic membrane. Data are presented in Table 3. The second group of 24 patients received the same therapy as the first treatment, plus otomycroscopic removal of haemorrhagic exudate, lacerated middle ear mucosa, and repair of tympanic membrane lacerations. Healing rate was significantly better than the one obtained in the first group (Table 4). Persistent tympanic perforation, as indicator of failed therapy, was present in 5 (35%) of all examined ears with two thirds of tympanic membrane perforation in the first group, while in the second group the rate was 3 (12%). The third group received the same therapy as the second treatment, including administration of amicacyn into the external auditory canal. The results were statistically compared by chi 2 test, and we found that the second therapy protocol was significantly better. There was no significant difference between the second and the third therapy protocols. We found blast ruptures of tympanic membrane and auditory ossicles chain discontinuity in 88% of examined ears. In our material the high incidence of total tympanic membrane ruptures and subtotal ruptures (48%) is quite different in comparison to other authors [5-7]. We consider it as the effect of high power blast wave. Loss of conductive hearing was present in 91% of blast injured patients, while 7.4% of patients had mixed, predominantly senzoneural deafness. Consequently, in addition to mechanic blast injuries acoustic trauma could profoundly damage the inner ear. Spontaneous healing of tympanic membrane occurred in 71% of injured persons and this was a better result than the results obtained by other authors [3, 4, 6]. The spontaneous healing of tympanic membrane failed if infection of the middle ear occurred or blast caused the total tympanic membrane perforation. In the second and third therapy protocols significantly higher healing of tympanic membrane ruptures was evident; it rated from 88% to 91%. Better results could be explained by the effect of optimal healing conditions, based on removal of exudate from the middle ear and necrotic parts of tympanic membrane, completed by fitting of lacerated parts of tympanic membrane and antibiotic prophylaxis. Healing of tympanic membrane without scars and adhesions was more frequent than in patients treated only with antibiotics. Incidence of undesired outcome of persistent perforation of tympanic membrane was reduced. To prevent posttraumatic complications in the middle ear, we recommend early cleaning of margins, reposition of lacerated fragments of tympanic membrane, and removal of haemorrhagic exudate. Myringoplasty should be performed if spontaneous healing of tympanic membrane did not occur after 6 months. CONCLUSION: Otic blast injury was frequently found in war induced trauma. (ABSTRACT TRUNCATED)