Imaging the dome of Santa Maria del Fiore using cosmic rays.

The dome of Santa Maria del Fiore, Florence Cathedral, was built between 1420 and 1436 by architect Filippo Brunelleschi and it is now cracking under its own weight. Engineering efforts are under way to model the dome's structure and reinforce it against further deterioration. According to some scholars, Brunelleschi might have built reinforcement structures into the dome itself; however, the only known reinforcement is a wood chain 7.75 m above the springing of the Cupola. Multiple scattering muon radiography is a non-destructive imaging method that can be used to image the interior of the dome's wall and therefore ascertain the layout and status of any iron substructure in it. A demonstration measurement was performed at the Los Alamos National Laboratory on a mock-up wall to show the feasibility of the work proposed, and a lightweight and modular imaging system is currently under construction. We will discuss here the results of the demonstration measurement and the potential of the proposed technique, describe the imaging system under construction and outline the plans for the measurement.This article is part of the Theo Murphy meeting issue 'Cosmic-ray muography'.

The presence of anti-carcinoembryonic antigen (CEA) antibodies in the sera of patients with gastrointestinal malignancies.

Using an enzyme-linked immunoassay we tested the sera of 71 patients with digestive system cancer, 35 patients with various nonmalignant disorders, and 28 normal individuals for anti-CEA activity. Antibodies were found in the sera of 51% of the patients. Most of the patients positive for the antibodies (70%) had no evidence of metastatic disease. Fewer than 10% of the sera from control groups had anti-CEA activity. The authors concluded that the patients suffering from cancer of the GI system are capable of producing tumor-specific antibodies. These antibodies could be used as a tumor marker and/or as a possible index for the function of the immune system. The presence of a large tumor mass could lead to the removal of these antibodies from the circulation.

Clonogenic potential of myeloid leukaemia cells in vitro is restricted to leukaemia cells expressing the CD34 antigen.

Cells from patients with acute myeloid leukaemia (AML) or chronic myeloid leukaemia (CML) were separated into CD34-enriched and CD34-depleted subpopulations. The clonogenic capacities of these two subpopulations were then compared to each other and to the original unseparated cell population. In every study, the CD34-enriched subpopulation demonstrated a substantial increase in clonogenicity in vitro in comparison with the original cell population, while the reverse was the case for the CD34-depleted subpopulations. For reasons not clear at present, the enrichment for clonogenic cells far exceeded the enrichment for cells expressing the CD34 antigen. Additionally, the clonogenic potential was found to be unrelated to the level of myc expression in the various cell populations.

Nucleotide sequence of messenger RNA encoding VHDJH and VKJK of a highly conserved idiotype-defined primary response anti-hapten antibody.

The primary humoral immune response of mice to the hapten phthalate (Xmp) is focused upon two adjacent immunodominant negatively charged carboxyl groups on a benzene ring that are in positions meta and para to the azolinkage (i.e., Xmp) to the protein carrier keyhole limpet hemocyanin. A significant fraction of the anti-Xmp antibodies raised in several different inbred mouse strains (BALB/c, DBA/2, A/HeHa; C3H, and SM/J), and many wild mouse populations express a cross-reactive Id, CRIXmp-1. This CRIXmp-1 is conspicuously absent in C57BL/6 mice. In order to obtain a better understanding of the events and parameters that influence the selection and regulation of the primary response B cell repertoire, and to explore the structural basis of Ag binding, we have determined the nucleotide sequence of the entire V region gene complexes, which encode the H and L chains of these highly conserved and dominant CRIXmp-1+ antibodies. Our data establish that the H chain gene complex consists of a single VH germ-line gene that is identical to VH Oxazolone-1, encoding the H chain of another highly conserved and dominant cross-reactive Id family associated with the primary response to Oxazolone. In CRIXmp-1+ Xmp-specific hybridomas this gene is joined to a limited set of D region sequences that express a conserved amino acid motif-GLR. At least three of the five D regions examined are coded for by DFL16.2. This VHD complex can be utilized with one of three different JH region genes (JH1, JH2, and JH4) without any significant effect upon antibody fine specificity or Id. In spite of this lack of JH fidelity all of the CRIXmp-1+ hybridomas have precisely maintained the same length in the H chain CDR3 and FRW4 by altering either the length of the D segment or the length of JH. Nucleotide sequence analysis of the VL gene complex of CRIXmp-1+ anti-Xmp antibodies indicates that the L chain V region is also encoded by a single germ-line gene. The amino acid sequence predicted from the nucleotide sequence of the VKJK from Xmp-specific CRIXmp-1+ hybridomas is identical to the sequence of the anti-arsonate antibody 1210.7, which is the prototype of another Id family (CRI) that is conserved and dominant in BALB/c mice.

Risk factors for epidemic Xanthomonas maltophilia infection/colonization in intensive care unit patients.

OBJECTIVE: To determine risk factors for and modes of transmission of Xanthomonas maltophilia infection/colonization. DESIGN: Surveillance and cohort study. SETTING: A 470-bed tertiary trauma-referral community hospital. PATIENTS: From January 1, 1988 to March 17, 1989, 106 intensive care unit patients developed X maltophilia infection/colonization. We defined a case as any intensive care unit patient who, from July 15, 1988, through March 17, 1989 (epidemic period), had X maltophilia infection/colonization greater than or equal to 48 hours after intensive care unit admission. We identified 45 case patients and 103 control patients (persons in the shock-trauma intensive care unit for greater than or equal to 72 hours during the epidemic period who had no X maltophilia-positive culture). RESULTS: Cases were significantly more likely to occur in the shock-trauma intensive care unit than in all other intensive care units combined. Mechanical ventilation, tracheostomy, being transported to the hospital by airplane, and receipt of a higher mean number of antimicrobials were risk factors for X maltophilia infection/colonization. Risk of X maltophilia infection/colonization was significantly greater among cases exposed to a patient with a X maltophilia surgical wound infection than among those without such exposure (relative risk = 1.3, p = .03). Animate and inanimate cultures revealed X maltophilia contamination of the hospital room of a patient with an X maltophilia surgical wound infection, of respiratory therapy equipment in this patient's room, of respirometers shared between patients, and of shock-trauma intensive care unit personnel's hands. Related environmental and clinical isolates were serotype 10. CONCLUSIONS: Mechanically ventilated patients receiving antimicrobials in the shock-trauma intensive care unit were at increased risk of X maltophilia infection/colonization. Patients with draining X maltophilia surgical wound infections served as reservoirs for X maltophilia, and contamination of the respirometers and the hands of shock-trauma intensive care unit personnel resulted in patient-to-patient transmission of X maltophilia.

Detection of single-stranded DNA damage using monoclonal anti-thymidine antibody.

A method to detect single-stranded DNA damage from individual cells has been developed using a monoclonal anti-thymidine antibody (MoAb20B7). Initially, HL-60 cells were incubated with daunomycin at different concentrations, and processed by MoAb20B7. While 73.5% of the cells incubated with 5 micrograms/ml of daunomycin for 24 h reacted positively with MoAb20B7, 83.5% cells at 10 micrograms/ml daunomycin dose were positive. Next, this method was combined with unscheduled DNA synthesis to simultaneously measure repair and damage from individual cells. Finally, patients with acute myeloid leukemias were studied before and 24 h after therapy with a daunomycin containing regimen. In vivo damage could be determined in a prompt fashion.

Proto-oncogene expression in differentiating and non-differentiating chronic myelogenous leukaemia cells.

Despite the profound differences between the chronic and blastic phases of chronic myelogenous leukaemia, no differences between chronic and blastic phase cells have been described at the molecular level. Differences have been found in the levels of expression of c-myc, c-myb and p53, which fell when chronic phase cells were cultured, while the levels of expression of the genes were stable when blastic crisis cells were cultured. In contrast c-fms expression increased and MRS expression decreased after culture of chronic or blastic phase cells. The data suggest that the regulation of expression of some genes in blastic crisis cells is unaltered while that of others is disrupted. It is not known whether the failure of c-myc, c-myb and p53 expression to fall during the culture of blastic phase cells is the cause of or a reflection of the failure of these cells to differentiate.

DNA damage/repair studies using a combination of monoclonal thymidine antibody and unscheduled DNA synthesis simultaneously.

The ability of a new monoclonal antibody against thymidine (MoAb 20B7) to detect chemotherapy induced single stranded DNA damage is described. HL-60 cells were used for these experiments. Damage to DNA was caused by incubation of cells with alkylating agents. Portions of DNA which is damaged are cleaved by cellular endonucleases, thus exposing thymidine on the opposite DNA strand. Processing of these samples by MoAb 20B7 showed that such damaged segments could be detected consistently. Furthermore, this method was combined with autoradiographic detection of unscheduled DNA synthesis thereby allowing for assessment of DNA repair simultaneously from the same cell.

Selective elimination of idiotype-binding cells in vivo by a drug-idiotype conjugate demonstrates the functional significance of these cells in immune regulation.

A receptor-specific cytotoxic drug delivery system has been used to eliminate idiotype-binding cells in vivo to ascertain the possible functional significance of these cells in regulating the humoral immune response to dextran. Protein M104E, a mouse myeloma protein that binds dextran, expresses a private idiotope that is present on a significant proportion of the normal dextran-specific antibody repertoire. Immunocompetent cells that bind and internalize M104E idiotype-bearing molecules were eliminated by the intravenous administration of a single dose of cytosine arabinonucleoside conjugated to purified M104E protein. The administration of this cytotoxic drug-idiotype conjugate had a profound effect upon the expression of the M104E idiotype in euthymic but not in athymic BALB/c mice following immunization with dextran. In euthymic mice, the depletion of the idiotype-binding cells resulted in a marked elevation in the level of M104E idiotype present in the immune sera. Moreover, treated but not control mice developed idiotype-positive molecules that did not bind dextran. These results demonstrate the functional significance of idiotype-binding cells in the regulation of individual clonotypes during an immune response.

Antigen-specific drug-targeting used to manipulate an immune response in vivo.

The administration of dextran-conjugated cytosine arabinonucleoside (araC) to BALB/c mice at various times prior to but not subsequent to immunization with native dextran renders mice unresponsive to this thymic-independent antigen. These results demonstrate that the primary immune response to an antigen can be selectively and efficiently suppressed or eliminated in vivo by the delivery of a single dose of an appropriate antigen-cytotoxic drug conjugate. Evidence presented here indicates that the dextran-araC conjugate (toxogen) acts directly and selectively upon unprimed dextran-specific antibody-forming cell precursors, presumably by binding to their receptors and subsequent internalization of the resultant receptor-toxogen complexes. The resistance of antigen-primed mice to the cytotoxic effect of the toxogen could result from the failure of dextran-primed cells to reexpress antigen-specific receptors, from an alternative processing of the toxogen, or from the inability of the antigen-primed cells to internalize a second round of receptor-ligand complexes. We also determined that B cells responding to thymic-dependent antigens were not affected by the prior exposure to a toxogen. The inability to eliminate or suppress the primary response to a thymic-dependent antigen via the administration of a cytotoxic drug-antigen conjugate distinguishes the thymic-independent set of B cells from the thymic-dependent B-cell repertoire. The difference between these two B-cell compartments could be due either to differences in the amount of ligand bound to receptors or to differences in the trafficking patterns of receptor-ligand complexes within each cell type.

Proliferative characteristics of acute nonlymphocytic leukemia in vivo.

The proliferative characteristics of myeloid leukemias were defined in vivo following intravenous bromodeoxyuridine (BrdU). Fifteen patients received a 2-hour infusion of BrdU. A monoclonal anti-BrdU antibody was used to detect the in vivo incorporation of BrdU by S-phase cells. The percentage of S-phase cells obtained from the biopsies (mean 17.3%) was significantly higher (p = 0.00001) than the percentage determined from the aspirates (7.8%). It is concluded that the true estimate of S-phase cells can only be obtained from biopsies following in vivo labeling of cells synthesizing DNA. The persistence of BrdU-labeled cells in follow-up studies can be used to recognize 'residual leukemia', and the subsequent fate of these cells can be defined in vivo.

A new method for studying cell cycle characteristics in ANLL using double-labeling with BrdU and 3HTdr.

Ten patients with acute nonlymphocytic leukemia (ANLL) received bromodeoxyuridine (BrdU) at 100 mg/M2 intravenously over 1 h. BrdU is incorporated into the DNA by S-phase cells and was detected by using a monoclonal anti-BrdU antibody in the bone marrow aspirate (BM) and biopsy specimens obtained at the end of the infusion. Additionally, BM was incubated in vitro with tritiated thymidine (3HTdr) and processed by our previously described double-label method. This allowed us to measure the duration of S-phase (Ts) and total cell cycle time (Tc) of myeloblasts. Data revealed a higher number of S-phase cells from biopsies (21%) than BM (5%). The Ts ranged from 9 to 35 h and Tc ranged between 36 and 152 h in different patients. Using this method, data are available within 48 h and if shown to be clinically relevant, may be useful for prospective planning of therapy in individual patients.

Failure of PFC inhibition assays to distinguish idiotypically between clonotypes that are readily distinguishable by RIA analysis.

Two different hemolytic plaque assay protocols that are commonly used to quantitate idiotype-positive antibody-secreting cells have been compared to a standard radioimmunoassay (RIA) to test for their ability to discriminate between related, but idiotypically distinct, clonotypes. The idiotype proband used in this analysis is the individual specific idiotype associated with the dextran-binding myeloma protein M104E (M104E IdI). Antibodies specific for this private idiotype (anti-M104E IdI) were purified by a combination of adsorption and affinity chromatography of the immunoglobulin fraction isolated from the sera of rabbits repeatedly immunized with M104E. The same affinity-purified anti-M104E IdI antibodies were used in the hemolytic plaque assays and in the RIA. In one of the plaque assays, the putative idiotype-positive antibody-forming cells were scored by lysis of target erythrocytes to which the anti-idiotype had been covalently coupled. In the other plaque assay, the idiotype-positive cells were determined indirectly by anti-idiotype inhibition of PFC produced on dextran-coupled target erythrocytes. The fidelity of these two assays to quantitate the M104E private idiotype expression in individual BALB/c mice after a single immunization with dextran B-1355S was determined by comparing the plaque assay data to the data generated by a double-antibody, post-precipitation RIA of either the antibodies in the serum or of monoclonal antibodies produced by hybridomas. Our data indicate that both plaque assay protocols reflect an overestimate of the actual expression of M104E private idiotype. By using a library of dextran-specific hybridomas (that have been characterized in an RIA with respect to their M104E IdI cross-reactivity), we have shown that the PFC overestimate of the M104E expression observed in dextran-immune mice is due to the inability of both plaque assay protocols to distinguish between dextran-specific clonotypes that express idiotypes cross-reactive with, but not identical to, the 104E IdI. We conclude that the plaque assay should be used only in conjunction with an RIA to estimate the idiotype expression. This is especially true in situations where closely related cross-reactive idiotype families exist.

Antibody response of BALB/c mice to dextran B1355S: alterations in the expression of an idiotype associated with the depletion of idiotype-binding cells.

In this study, we established that BALB/c mice recognize and respond to the idiotype (M104E IdI) of a major dextran-specific clonotype within the BALB/c mouse repertoire. This idiotype recognition is established by demonstrating the presence of idiotype-binding cells and by the production of antibodies specific for the private M104E idiotype. To determine whether or not the idiotype-recognizing cells play a regulatory role during an immune response to dextran, the idiotype-binding cells were selectively removed either by panning or by radiation-induced killing. Two significant effects are observed when the depleted spleen cells are immunized with dextran. First, there is a substantial increase in the proportion of anti-dextran antibodies that are M104E IdI+. The second effect of the idiotype-specific cell depletion is the production of significant amounts of M104E IdI+ immunoglobulin molecules which do not bind dextran. The depletion experiments produced no alteration in the concentration of anti-dextran antibodies found in the serum or in the number of dextran-specific PFC in the spleen. The data indicate that idiotype-reactive cells can play a role in regulating the level of individual clonotype expression (i.e., the M104E clonotype), but that an alternative mechanism must exist for regulating the absolute amount of anti-dextran antibody produced after immunization.

Double labeling of S-phase murine cells with bromodeoxyuridine and a second DNA-specific probe.

A rapid method has been developed which combines immunofluorescence and autoradiography and permits the double labeling of DNA. P388 murine leukemic cells were incubated with bromodeoxyuridine and tritiated thymidine simultaneously. After fixation, the sample was first processed with a monoclonal antibody to bromodeoxyuridine (RPMB I) so that any cell in S-phase was brightly fluorescent (RPMB technique). Next, tritiated thymidine grains were developed by autoradiography, and the result demonstrated fluorescence as well as black grains in each S-phase cell. P388 cells sensitive (P388S) and resistant (P388R) to 1-beta-D-arabinofuranosylcytosine (ara-C) were incubated with bromodeoxyuridine and [3H]ara-C simultaneously. Processing by autoradiography and RPMB techniques revealed that all S-phase cells in the P388S sample demonstrated vivid "double labeling," whereas P388R cells only revealed bright green fluorescence in S-phase cells, but no grains, confirming a lack of ara-C incorporation into the DNA by this line. Finally, a computerized digital analysis system attached to a microphotometer was used to quantitate fluorescence and grains per cell, and the data demonstrated that the number of [3H]ara-C grains in each P388S cell was inversely proportional to the degree of fluorescence in that cell, indicating that DNA synthesis was inhibited by ara-C. In conclusion, a simple, easy-to-use double-labeling method has been introduced which will be useful to a wide variety of researchers, because this technique together with the digital analysis system offers the possibility of measuring drug sensitivities in individual cells.

The use of protein A in solid-phase binding assays: a comparison of four radioiodination techniques.

Preparations of protein A radioiodinated by 4 different methods have been compared in indirect radioimmunoassays. The oxidative methods (chloramine-T and iodogen) for direct iodination of tyrosyl and histidyl residues were applied with high efficiency and gave a suitable product, provided the substitution ratio was kept low (1 iodine atom/molecule of protein A). Higher levels of modification tended to perturb the Fc-binding characteristics of the protein, especially with the use of iodogen. Introduction of the isotope via substitution of lysyl residues (Bolton-Hunter and Wood reagents) was also examined. The Bolton-Hunter modification of protein A gave an unsuitably low labeling efficiency; in contrast, the Wood reagent gave efficiencies approaching 50%. Protein A could be extensively substituted with the latter reagent (greater than 5 diiodinated benzimidate molecules per protein molecule). Thus, the use of the Wood-labeled protein A could raise the sensitivity of the binding assay at least an order of magnitude compared to using protein A iodinated by the oxidative methods. The effects on the biological activity of protein A exerted by the different labeling procedures are rationalized on the basis of the amino acid composition and tertiary structure of the protein.