Study of inelastic nuclear interactions of 400 GeV/c protons in bent silicon crystals for beam steering purposes.

Inelastic nuclear interaction probability of 400 GeV/c protons interacting with bent silicon crystals was investigated, in particular for both types of crystals installed at the CERN Large Hadron Collider for beam collimation purposes. In comparison to amorphous scattering interaction, in planar channeling this probability is ∼ 36 % for the quasi-mosaic type (planes (111)), and ∼ 27 % for the strip type (planes (110)). Moreover, the absolute inelastic nuclear interaction probability in the axial channeling orientation, along the ⟨ 110 ⟩ axis, was estimated for the first time, finding a value of 0.6 % for a crystal 2 mm long along the beam direction, with a bending angle of 55 µ rad. This value is more than two times lower with respect to the planar channeling orientation of the same crystal, and increases with the vertical angular misalignment. Finally, the correlation between the inelastic nuclear interaction probability in the planar channeling and the silicon crystal curvature is reported.

The antibody response to myoglobin--I. Systematic synthesis of myoglobin peptides reveals location and substructure of species-dependent continuous antigenic determinants.

Sets of peptides representing all the possible hepta-, octa-, nona- and decapeptides of sperm whale myoglobin were synthesized. An ELISA method was used to detect the ability of antibodies, present in antisera raised against native sperm whale myoglobin, to bind to these peptides. Antisera made in two species were compared. It was found that the peptides recognized by the antibodies were a function of the species in which the antiserum was prepared and of the individual outbred member of that species. Peptides corresponding to surface epitopes of the native antigen were identified by reacting the antisera with native antigen prior to ELISA testing on peptides. More detailed analysis of one epitope revealed that, for some sera, a leucine residue which is facing inwards in the crystal structure is critical for the binding of antibody to the peptide. This suggests that binding between native antigen and antibody can require a restructuring of the native antigen.

Strategies for epitope analysis using peptide synthesis.

A recently developed approach to the synthesis and ELISA screening of large numbers of peptides is described. The method has created the opportunity to tackle questions about the sites and specificity of antigenic determinants which were formerly thought to be too difficult to answer. The various strategies for application of this method are described along with examples of their successful use. They include a procedure for locating all the continuous antigenic peptides of a protein antigen, and the identification of non-replaceable amino acid residues within an antigenic peptide. An approach to the determination of amino acid residues involved in the epitope for any monoclonal antibody is also described. These strategies open up the prospect of rapid mapping of the antigenic properties of hitherto poorly understood antigens.

Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Indications for differential mechanisms of bioactivation.

1,1-Dichloro-2,2-difluoroethylene (DCDFE) produced marked nephrotoxicity in rats upon an i.p. dose of 150 mumole/kg. At doses higher than 375 mumole/kg, DCDFE also produced hepatotoxicity. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, appeared to be slightly nephrotoxic in Wistar rats. Nevertheless it exerted an inhibitory effect on the nephrotoxicity of DCDFE. The N-acetylcysteine conjugate of DCDFE was identified as a major urinary metabolite of DCDFE. When administered as such, this conjugate appeared to be a potent nephrotoxin, without any effect on the liver, indicating that glutathione conjugation of DCDFE is most likely a bioactivation step for nephrotoxicity. The appearance of traces of chlorodifluoroacetic acid in urine of rats treated with higher doses of DCDFE indicates the existence of an oxidative pathway of metabolism of DCDFE, probably involving epoxidation by hepatic mixed-function oxidases. It is speculated that the latter route might account for the hepatotoxicity at higher doses of DCDFE. The nephro- and hepatotoxicity of DCDFE, therefore, most likely are the result of two different mechanisms of bioactivation.

High level production of hybrid potyvirus-like particles carrying repetitive copies of foreign antigens in Escherichia coli.

Synthesis in E. coli of native coat protein of Johnsongrass mosaic virus, and hybrid protein molecules containing foreign antigens, resulted in the intracellular formation of potyvirus-like particles (PVLPs). The foreign antigens used were an octapeptide epitope from Plasmodium falciparum and a decapeptide hormone (luteinizing hormone releasing hormone) at the N- or at both N- and C-terminal regions of the coat protein molecule, and a full length protein antigen (Sj26-glutathione S-transferase of 26 kD from Schistosoma japonicum) replacing the N-terminal 62 amino acids of the coat protein. Electron microscopy of ultrathin sections of E. coli revealed that PVLPs resulting from coat protein molecules containing peptide fusions appeared in vast arrays of parallel strands within the cytoplasm sometimes extending the length of the cell and at times the cells were strung together, with "threads" of PVLPs appearing to connect individual bacterial cells. PVLPs resulting from the fusion of the 26 kD antigen Sj26 to coat protein were shorter and wider. The physical form of the high molecular weight PVLPs enabled purification by simple size exclusion column chromatography. The Sj26-PVLPs administered to mice without adjuvant elicited antibody responses comparable to monomeric Sj26 administered with Freund's Complete Adjuvant.

Laparoscopic diagnosis of mesenteric venous thrombosis.

Diagnosis of mesenteric venous thrombosis is almost always delayed, due to the aspecificity of the complaints and the clinical findings, as well as the laboratory investigations. Earlier diagnosis is essential to improve the present grim mortality rate. We report a case of mesenteric venous thrombosis in a 25-year-old female. Early diagnosis was made by gynecological laparoscopy. After resection and anastomosis, the outcome was uneventful.

Preterm labor: the role of amnionitis.

This prospective study represents an attempt to find a link between subclinical intra-amniotic infection and preterm singleton labor in 27 asymptomatic patients with intact membranes. Liquor amnii was obtained by transabdominal amniocentesis and cultured for anaerobic and aerobic bacteria and mycoplasmas. None of these cultures was positive. The value of gas-liquid chromatographic determination of volatile and non-volatile acids in liquor amnii could not be determined because amniotic infections did not occur in the population under study.

Simple and sensitive quantification of anthracyclines in mouse atrial tissue using high-performance liquid chromatography and fluorescence detection.

Anthracyclines are very effective against soft tissue sarcomas, with cardiotoxicity being an important side effect after repeated administration. To estimate the relative cardiotoxicity of various anthracyclines and their metabolites, we developed an isolated mouse left atrium model. To relate an effect of doxorubicin, 4'-epidoxorubicin and their four main metabolites (doxorubicinol, epidoxorubicinol and the aglycons 7-deoxydoxorubicinon and 7-deoxydoxorubicinolon) to concentrations in the tissue instead of the incubation bath, a method of quantifying the anthracyclines in small tissue samples was developed. Atria were homogenized by sonication followed by extraction of the anthracyclines with methanol. The extract was directly analyzed by high-performance liquid chromatography with fluorescence detection. Recoveries for the six compounds tested ranged from 67.5% for 4'-epidoxorubicin to 100.6% for 7-deoxydoxorubinol aglycon with coefficients of variation of 2-3% at two spiked concentrations (0.1 and 1 nmol/mg of tissue). The calibration plots were linear (r2 greater than 0.996) over the concentration range tested (0.05-1 nmol/mg wet weight). The limits of detection (4-10 pmol/mg of tissue) were low enough to allow the determination of the anthracyclines at all relevant tissue concentrations.

The role of biotransformation in anthracycline-induced cardiotoxicity in mice.

Anthracyclines are highly efficacious antineoplastic agents but they become cardiotoxic after repeated dosing. For the major anthracycline, doxorubicin (Dox), this toxicity is thought to be associated with the formation of the 13-dihydro metabolite. Paced mouse left atria were used to assess the cardiotoxicity of Dox, 4'-epidoxorubicin (Epi), daunorubicin (Dauno) and their major metabolites. Apart from the aglycons, all compounds (1-500 microM) reduced the contractile force. To correct for differences in cellular uptake, anthracycline concentrations were determined in the atria after 1 h of incubation. IC50 values ranged from 0.33 mumol/g for 13-dihydro-Dox to 3.5 mumol/g for Dauno. The toxicities relative to Dox, i.e., the ratio of IC50,Dox/IC50,anthracycline, ranged from 0.19 for Dauno to 2.1 for 13-dihydro-Dox (the most toxic). For Dox, Epi and Dauno, the 13-dihydro metabolite had greater toxicity than the corresponding parent compound. The pharmacokinetics of Dox and Epi in the murine heart are comparable and, thus, cannot explain the reduced cardiotoxicity of Epi. However, when pharmacokinetic data of Dox and Epi in murine heart tissue were interpreted using the relative toxicity factors, Epi would be expected to be threefold less cardiotoxic than Dox, thus providing a better correlation with in vivo data. This simple pharmacological model in combination with preclinical pharmacokinetics may contribute to the prediction of the cardiotoxic potency of new anthracyclines relative to Dox.

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution.

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.

Isolated mouse atrium as a model to study anthracycline cardiotoxicity: the role of the beta-adrenoceptor system and reactive oxygen species.

Cancer chemotherapy with anthracyclines, of which doxorubicin (DX2) is the main representative, is limited by cardiomyopathy developing in animals and patients after cumulative dosing. The toxicity is probably related to free radical formation by the anthracycline as well as its metabolites with concomitant O2.- and .OH generation resulting in lipid peroxidation and subsequent membrane damage. An in vitro model is required to investigate the individual contribution of each metabolite to cardiotoxicity. For in vivo studies, the species of choice is the mouse because it lacks the DX-induced nephrotic syndrome seen for instance in rats and rabbits. Thus, isolated mouse heart muscle was chosen as an in vitro model. To characterize the model, we used l-isoprenaline/dl-propranolol and metacholine/atropine to measure the beta-adrenergic and the muscarinic responses of (spontaneously beating) right and (paced) left atrium. Dose response curves (n greater than or equal to 4) were highly reproducible: pD2,iso = 8.0 +/- 0.3 (left) and 8.5 +/- 0.4 (right); pD2,met = 6.7 +/- 0.1 (left) and 6.2 +/- 0.3 (right). Propranolol as well as atropine behaved as competitive antagonists, with pA2-values of 8.4 +/- 0.2/8.5 +/- 0.2 (l/r) and 9.1 +/- 0.1/9.1 +/- 0.2 (l/r), respectively. These values corresponded to those obtained with other organ preparations. We tested the effect of DX in two ways: a) by measuring the direct inotropic and chronotropic effect during 60 minutes of incubation with 10-100 microM DX in the organ bath, and b) by determining the remaining beta-adrenergic response to l-isoprenaline after the incubation period. Both variables turned out to be equally affected. For paced left atria an IC50 (causing 50% depression of contractile force) of 35 microM was determined. Right atria stopped beating at concentrations above 50 microM, thus hampering IC50 determination. The results indicate that anthracyclines exert an effect not related to receptor integrity, but directly to the functionality of heart muscle. To check whether radical stress can be involved in the observed negative inotropic effect, incubations with xanthine/xanthine oxidase (to produce reactive oxygen species) were performed. A pronounced negative effect on mouse atrial contraction was indeed observed. However, initially a positive inotropic effect accompanied by an increased resting tension were seen. It can be concluded that mouse atrium can be used as a model to compare anthracyclines and their metabolites with regard to their acute cardiotoxic effects.

Clinical experience with an ethinyl estradiol-desogestrel oral contraceptive.

Desogestrel (150 micrograms), a potent progestogen virtually devoid of androgenic activity, was used in combination with 30 micrograms ethinyl estradiol as an oral contraceptive preparation (Marvelon). 219 women completed a total of 4074 cycles, and the use-effectiveness was 0.58 Pearl Units. Serious side effects were not observed. The drug-related discontinuation index was 12.8% in six months.

Chimeric potyvirus-like particles as vaccine carriers.

Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses. Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs). The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly. Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs. Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant. Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles. This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier.

Human phase I vaccine trials of 3 recombinant asexual stage malaria antigens with Montanide ISA720 adjuvant.

Two phase I vaccine trials were conducted to test the immunogenicity and safety of a vaccine containing three recombinant malaria antigens from the asexual stage of Plasmodium falciparum. The three antigens are a fragment of MSP1 (190LCS.T3); MSP2 and a portion of RESA and were formulated in Montanide ISA720 adjuvant. These trials investigated the dose response of each antigen for eliciting both antibody and T-cell responses and the immunogenicity of a mixture of the antigens compared with the antigens injected separately. All three antigens elicited both antibody and T-cell responses. Strong T-cell responses were observed with 190LCS.T3 and RESA with stimulation indices exceeding 100 for peripheral blood leucocytes in some individuals. The antibody responses were generally weak. The human antibody responses observed with MSP2 in Montanide ISA720 were not significantly different from those obtained in an earlier trial which used MSP2 with alum as the adjuvant. No antigenic competition was observed: volunteers receiving a mixture of antigens had similar responses to those receiving the three antigens at separate sites. Tenderness and pain at the injection site were common over the first few days following immunization. In some volunteers, especially those receiving the highest doses tested, there was a delayed reaction at the injection site with pain and swelling occurring approximately 10 days after injection.