A single-chain variable fragment selected against a conformational epitope of a recombinantly produced snake toxin using phage display.

Phage display technology is a powerful tool for selecting monoclonal antibodies against a diverse set of antigens. Within toxinology, however, it remains challenging to generate monoclonal antibodies against many animal toxins, as they are difficult to obtain from venom. Recombinant toxins have been proposed as a solution to overcome this challenge, but so far, few have been used as antigens to generate neutralizing antibodies. Here, we describe the recombinant expression of α-cobratoxin in E. coli and its successful application as an antigen in a phage display selection campaign. From this campaign, an scFv (single-chain variable fragment) was isolated with similar binding affinity to a control scFv generated against the native toxin. The selected scFv recognizes a structural epitope, enabling it to inhibit the interaction between the acetylcholine receptor and the native toxin in vitro. This approach represents the first entirely in vitro antibody selection strategy for generating neutralizing monoclonal antibodies against a snake toxin.

Electrophoresis microchip with integrated waveguides for simultaneous native UV fluorescence and absorbance detection.

Simultaneous label-free detection of UV absorbance and native UV-excited fluorescence in an electrophoresis microchip is presented. UV transparent integrated waveguides launch light at a wavelength of 254 nm from a mercury lamp along the length of a 1-mm long detection cell. Transmitted UV light is collected by another waveguide in the opposite end of the detection cell, while visible fluorescence is collected vertically through the lid of the chip. The background of scattered excitation light is suppressed by detection perpendicular to the excitation, the limited UV transparency of the borosilicate lid and by choosing a PMT insensitive to the excitation light. This way, the need for a fluorescence filter is eliminated. Calibration curves were measured for serotonin, tryptophan, propranolol and acetaminophen, and separations of the four compounds were demonstrated by electrophoresis and MEKC. All compounds could be detected in the micromolar range by absorbance detection, but fluorescence detection improved detection limits for compounds displaying native UV fluorescence up to ten times. The simultaneous detection also proved useful for the identification of compounds with similar retention times and even enables accurate quantification of co-eluting compounds.

[Color Doppler ultrasonography in retinitis pigmentosa. Preliminary study]. / Echo-Doppler couleur dans la rétinopathie pigmentaire. Etude préliminaire.

PURPOSE: Retinitis pigmentosa is a bilateral retinal degeneration. The primary disorder is still debated. METHODS: We performed a prospective investigation of the ocular circulation directly by color Doppler imaging (CDI). A total of 28 eyes of 14 patients (8 men and 6 women, affected with retinitis pigmentosa) were recruited for this study. For each case were evaluated protosystolic velocity and the resistive index of the ophthalmic artery, central retinal artery, posterior ciliary arteries and choroid. These values, furthermore, have been compared with a control group. RESULTS: The results of the CDI in the group of RP and in the CG were: in the OA: PSV 31.177 +/- 5.119 cm/sec vs 36.700 +/- 3.152 cm/sec (p < 0.007); RI 0.713 +/- 0.058 vs 0.717 +/- 0.019 (p < 0.0839); in the CRA PSV 7.075 +/- 1.611 cm/sec vs 12.710 +/- 2.795 cm/sec (p < 0.001); RI 0.560 +/- 0.062 vs 0.550 +/- 0.051 (p < 0.234); in the PCA: PSV 8.569 +/- 3.408 cm/sec vs 14.100 +/- 2.571 cm/sec (p < 0.001) with RI 0.634 +/- 0.090 vs 0.681 +/- 0.045 (p < 0.145). In the CHO: PSV 12.312 +/- 2.327 cm/sec vs 16.170 +/- 1.846 cm/sec (p < 0.001) with RI 0.581 +/- 0.072 vs 0.638 +/- 0.050 (p < 0.065). CONCLUSION: Our results suggest that in the affected eyes there is a statistically significant reduction in blood flow in ophthalmic and ciliary arteries. These data offer new views on the retinitis cause of pigmentosa and possible therapeutics to be studied.

[Prognosis of renal vein thrombosis in the adult: influence of treatment]. / Pronostic des thromboses des veines rénales de l'adulte: influence du traitement.

The medical histories of 27 patients with renal vein thrombosis were evaluated retrospectively. Short and long term evolution as well as prognostic factors were analysed. 24 of the patients were suffering from a nephrotic syndrome, 15 from renal failure. In the 20 biopsies performed we observed: 14 cases of extramembranous glomerulonephritis, 2 cases of minimal glomerular lesions, 3 cases of segmental focal glomerulosclerosis and 1 periarteritis nodosa. The renal vein thrombosis was bilateral (18), left (7) or right (2). In 7 patients this was associated with thrombosis of the inferior vena cava. 9 patients were treated with anticoagulants alone, 9 underwent thrombectomy, 7 were treated by thrombolysis and 2 received no treatment. Within the first six months 11 patients died as a result of complications due to hemorrhaging (5), septic infection (2) or embolism (1) and 3 patients died of undetermined causes. The progression of the remaining patients was followed up over a period of 6 months to 19 years. In 12 patients, the nephrotic syndrome regressed and renal function did not deteriorate in any of the cases. The main causes for poor prognosis are the existence of initial renal failure and glomerulonephritis which is not extramembranous. Since the evolution of the glomerulopathy does not appear to be affected by extramembranous glomerulonephritis, renal vein thrombosis associated with this complication should be treated with anticoagulants alone. The risk-benefit ratio of thrombectomy leads us to conclude that this technique should be abandoned. Thrombolysis could be considered in cases where renal vein thrombosis is associated with acute renal failure.

Do-not-resuscitate orders for critically ill patients in the hospital. How are they used and what is their impact?

We studied compliance with do-not-resuscitate (DNR) orders at a university hospital where a DNR protocol has existed since 1979. Documentation of DNR status in patient progress notes and chart orders increased through 1983. During a 12-month period (March 1983 through April 1984), we studied in detail the medical records of 521 patients who had a cardiopulmonary arrest in the hospital. Seventy-five percent (389 of 521) of these patients were designated DNR. Patients who were designated DNR were significantly more likely to be older, to have malignancy or an abnormal mental status, and to be less likely to have acute myocardial infarction, stroke, or chronic obstructive pulmonary disease than patients in whom resuscitation was attempted. Eighty-six percent of families, but only 22% of patients, were involved in the decision to designate a patient DNR. The decision to designate a patient DNR occurred late in the course of a patient's illness, often when the patient was in coma. For 28% of patients, some form of medical care was withdrawn or withheld after they were designated DNR. These data suggest that use of the DNR protocol requires changes if patients are to participate in the decision not to undergo cardiopulmonary resuscitation.

KIE: Records of 521 patients who suffered cardiopulmonary arrest during a 12-month period at the Harvard-affiliated Beth Israel Hospital were examined to determine compliance with the hospital's policy on do-not-resuscitate (DNR) orders. Seventy-five percent of the DNR patients were older and more likely to have a malignancy or an abnormal mental status than those on whom resuscitation was attempted. Families of 86% of the patients, but only 22% of the patients themselves, participated in the DNR decisions, which often were made after the patient was comatose. Some form of medical care was withdrawn or withheld from 28% of the patients after they had been designated DNR. The authors evaluated their data according to the four elements of the hospital's DNR policy--physician record keeping; involvement of patients, health personnel, and families in decision making; selection of patients designated DNR; and effect of a DNR order on subsequent medical care.