Animal tissue-based quantitative comparison of dual-energy CT to SPR conversion methods using high-resolution gel dosimetry.

Dual-energy computed tomography (DECT) has been shown to allow for more accurate ion therapy treatment planning by improving the estimation of tissue stopping power ratio (SPR) relative to water, among other tissue properties. In this study, we measured and compared the accuracy of SPR values derived using both dual- and single-energy CT (SECT) based on different published conversion algorithms. For this purpose, a phantom setup containing either fresh animal soft tissue samples (beef, pork) and a water reference or tissue equivalent plastic materials was designed and irradiated in a clinical proton therapy facility. Dosimetric polymer gel was positioned downstream of the samples to obtain a three-dimensional proton range distribution with high spatial resolution. The mean proton range in gel for each tissue relative to the water sample was converted to a SPR value. Additionally, the homogeneous samples were probed with a variable water column encompassed by two ionization chambers to benchmark the SPR accuracy of the gel dosimetry. The SPR values measured with both methods were consistent with a mean deviation of 0.2%, but the gel dosimetry captured range variations up to 5 mm within individual samples.Across all fresh tissue samples the SECT approach yielded significantly greater mean absolute deviations from the SPR deduced using gel range measurements, with an average difference of 1.2%, compared to just 0.3% for the most accurate DECT-based algorithm. These results show a significant advantage of DECT over SECT for stopping power prediction in a realistic setting, and for the first time allow to compare a large set of methods under the same conditions.

Induction of a cross-reactive idiotype dextran-positive antibody response in two IgH-Cb mouse strains treated with anti-J558 cross-reactive idiotype antibodies.

The effect of IdX-specific rabbit and allogeneic antiidiotype antibodies (Ab2) was investigated in vivo in Igh-Cb mouse strains with respect to the induction of a cross-reactive idiotype (IdX)-positive anti-alpha (1-3) Dextran (Dex) response. These C.B20 and C57Bl/6 mice have an allotype-linked incapacity to respond with IdX-positive anti-alpha (1-3) Dex antibodies upon conventional immunization with Dex B1355. 7 d after the rabbit Ab2 injections, IdX-positive Ig (Ab3) and IdX-positive anti-alpha (1-3) Dex antibodies (Ab1') were detected in the sera of each tested mouse. The affinity-purified Ab1' were idiotypically indistinguishable from reference BALB/c IdX-positive myeloma proteins and BALB/c anti-alpha (1-3) Dex antibodies (Ab1) in a competitive inhibition radioimmunoassay, while Ab3 Ig appeared idiotypically deficient and did not bind to Dex. The response to the alpha (1-6) linkage of Dex was not affected in these mice. A large fraction of the Ab1' and Ab3 responses of both mouse strains were of the IgG1 class. The Ab1' antibodies differed from BALB/c Ab1 by lower relative binding to five of eight tested Dex, and by expressing the Igh4b allotype determinants on the IgG1 antibodies. This study identifies the products of a VHDex gene that appears to be under regulatory control in the Ighb mice. Its association with the b haplotype suggests that this gene may differ structurally from the BALB/c VHDex gene.

Kinetics of the plasmacellular reaction in the draining lymph node of mice bearing the BP-8 fibrosarcoma.

The kinetics of the plasmacellular response in the draining lymph node was quantitatively assessed in specific-pathogen-free C3H/He mice grafted S.C. with the syngeneic BP-8 fibrosarcoma. The percentage of plasma cells was estimated on dissociated, cytocentrifuged, and fixed cell preparations by immunoenzymatic staining with either of two B-cell-specific antibody markers. Mice either grafted with liver tumor or given a single implant of tumor homogenate gave a maximal plasma cell response of about 4%. However, this peak was reached at 14 days in the live grafts and at 7 days in the homogenate situations. Moreover, in animals grafted with live tumor an elevated 3% plateau persisted until death, wheras in homogenate-implanted animals a sharp drop to a 0.5% plateau was observed at about Day 20. Homogenate-treated animals displayed a significant enhancement of tumor growth when challenged with a live graft 7 days after homogenate implantation, i.e., during the peak 4% plasmacellular response. Mice given a single implant of X-irradiated (3500 R) tumor responded with a peak of 1.7% plasma cells at 7 days. Despite subsequent boosting with the same material (Days 14 and 21), this value declined and remained at a plateau of about 0.2% until death. Contrary to homogenate-treated mice, these animals were immune to subsequent challenge with a live tumor graft. In the present host-tumor system, there appears to be an inverse relationship between elevated numbers of plasma cells and antitumor immunity.

Immunotoxins containing glucose oxidase and lactoperoxidase with tumoricidal properties: in vitro killing effectiveness in a mouse plasmacytoma cell model.

We have tested the tumoricidal potency of enzyme immunotoxins constructed of antibodies conjugated to glucose oxidase and to lactoperoxidase. Murine plasmacytoma cells were targeted in vitro with the use of affinity-purified rabbit anti-plasmacytoma membrane antibodies (conjugated to glucose oxidase or lactoperoxidase) or rabbit serum raised against plasmacytoma microsome membranes followed by goat anti-rabbit immunoglobulin conjugates (to glucose oxidase or lactoperoxidase). Cytotoxicity was generated subsequently by incubation of the washed cells in a medium supplemented with glucose and sodium iodide, which were the substrates of these enzymes. This resulted in the presumed metabolic release of highly toxic reduced oxygen species and iodinated derivatives. Targeting of tumor cells with both conjugates, as opposed to one of them alone, produced a synergistic killing effect. The gain of specific versus unspecific cytotoxicity was upwards of 10,000-fold. The killing rates were elevated (t10 values less than 30 min) and linear over time. The resultant reduction in tumor cell viability was in the order of 5 to 6 logs after only 20 to 90 min of incubation in the glucose/NaI medium. Cytotoxicity was enhanced by the gamma-glutamyl cysteine synthetase inhibitor buthionine-S,R-sulfoximine and by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, while catalase was inhibitory. The results suggest that these enzyme immunotoxins may be suitable for the ex vivo purging of autologous bone marrow grafts.

Regulation of the response to alpha(1-3) dextran: an anti-dextran associated idiotope of BALB/c mice is also expressed on A/J anti-NIP antibodies.

The functional properties of a mAb reactive to the B5 idiotope were analyzed in BALB/c, A/J and C.B20 mice. the B5 idiotope was specifically associated with the alpha(1-3) DEX response in a strain-specific manner in BALB/c mice (responder strain). It was serologically distinct from the previously described IdX idiotype and overlapped on part of the IdX+ and IdX- BALB/c anti-alpha(1-3) DEX response. While treatment of adult BALB/c mice with mAb B5 enhanced the serum anti-alpha(1-3) DEX antibody synthesis and induced de novo antibody synthesis in non-responder C.B20 mice, A/J non-responder mice remained unresponsive, despite elevated levels of serum B5+ dextran non-binding Ab3 immunoglobulins. However, in some of these A/J mice, we detected an enhancement of anti-NIP antibody synthesis. This anti-NIP antibody component was (1) specifically induced by mAb B5, (2) found in A/J, but not in BALB/c or C.B20 mice, and (3) specific towards the NIP hapten. These A/J anti-NIP antibodies bore either the lambda 1 or the kappa light chain and some of them also expressed the B5 idiotope. The B5 id thus appears as a typical id determinant shared by antibodies having different binding specificities, and it may have a role in the regulation of these responses.

Immunoenzymatic methods for the study of surface bound immunoglobulins in mice.

Methods are described for the study of lymphocyte surface bound immunoglobulins (Ig) for light microscopy experimentation. a) Live peripheral lymph node cells (LNC) or spleen cells were reacted in suspension under capping conditions with rabbit anti-mouse Ig antibodies. After washing the cells were cytocentrifuged, fixed and relabeled with either peroxidase-(PO) of fluorescein-(Fl) conjugated anti-rabbit IgG antibodies (Method SC). The staining was heterogeneous and three main categories of lymphocytes were distinguished on the basis of a characteristic surface label distribution (1) all label within distinct caps (caps), (2) all label evenly distributed over the cell surface (rings), and (3) an intermediate pattern where only part of the label was concentrated over one pole (polarized). b) LNC or spleen cells were treated for the simultaneous detection of lymphocyte surface Ig and cytoplasmic Ig of plasma cells. For this, live cells were cytocentrifuged, and after fixation were labeled with PO-conjugated antibodies. Lymphocytes stained in a continuous ring and were easily distinguished on morphological grounds from the intense cytoplasmic stain of plasma cells (Method D). Positive lymphocyte counts done by this method compared favorably with those obtained by Method SC. Comparison of Fl- and PO-conjugated reagents were done by either Method SC or D, by scoring total positive cells. Although either reagent gave closely similar percentage values, the lymphocytes stained by the immunoenzymatic technique contrasted sharply from negative cells and were as a result more easily counted. Other practical advantages of immunoenzymatic staining over immunofluorescence are (1) the simultaneous visualization of cell morphology and type of label distribution over the cell surface, (2) cell scoring can be done rapidly on a large population sample, and (3) stained slides can be reexamined on subsequent days. A common advantages of Method SC for both immunofluorescence and immoenzymology is that antibody labeled slides can be stored before the final staining step.

Establishment and biological activity of a proliferative anti-idiotype-activated T cell line.

Antisera were raised in rabbits against a monoclonal antibody (McAb 103) of C3H.SW origin which is specific to the synthetic polypeptide (T,G)-A-L and was shown to express the major idiotypic determinants of conventional anti-(T,G)-A-L antibodies. Antibodies were purified and were shown in a binding assay to recognize McAb 103 as well as C3H.SW anti-(T,G)-A-L antibodies. C57BL/6 mice were immunized with the purified rabbit anti-McAb 103 (Ra 103) and their lymph nodes were studied in a proliferation assay. Proliferation was observed in the presence of both Ra 103 and (T,G)-A-L, although the latter stimulated the cells to a lesser extent, suggesting the induction in vivo of (T,G)-A-L-specific clones in low frequency. A T cell line was established from these lymph node cells. The line is kept in continuous growth in the presence of IL-2 and periodic triggering with Ra 103. A significant proliferative response was obtained with Ra 103 only. This proliferation could be almost completely inhibited by either McAb 103 or by conventional anti-(T,G)-A-L antibodies of C3H.SW origin, indicating the cross reaction between the idiotypes expressed on the T cell line and the (T,G)-A-L-specific antibodies. No proliferation could be detected in the presence of either normal rabbit IgG or rabbit anti-mouse IgG. Thus, the T cell line TId 103 allows the analysis of the role of idiotype in T cell recognition and regulation.

Enhanced in vitro cytotoxicity of 1,3-bis-(2-chloroethyl)-1-nitrosourea or buthionine sulfoximine combined with a reactive oxygen-generating enzyme immunotoxin.

The glutathione inhibitor drugs, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and buthionine sulfoximine (BSO), were tested in vitro in order to assess their cytotoxic effectiveness when combined with an enzyme immunotoxin (eIT) composed of a T-cell reactive monoclonal antibody (mAb) 097 coupled to the reactive oxygen-generating enzyme, glucose oxidase (GO) (EC 1.1.3.4). As targets of this eIT we used mature human T-cells or leukemia cells that expressed the 097 epitope. We found that treatment of the cells with subtoxic amounts of mixtures of both a drug and the 097 eIT markedly potentiated cytotoxicity compared to either drug or eIT alone.

T cell depletion of human bone marrow using an oxidase-peroxidase enzyme immunotoxin.

We have shown in a previous paper that an antibody-enzyme immunotoxin (eIT) constructed by chemically coupling the 097 monoclonal antibody to glucose oxidase and to lactoperoxidase (097 eIT) effectively eliminated T cells of human peripheral blood origin and killed cultured human thymoma cells. Here we tested its effectiveness against T cells present in human bone marrow cell suspensions contaminated by large numbers of erythrocytes. The T cell-depleting capacity of the 097 eIT was assessed by means of four different assay methods, three of which gave concordant results and indicated an effective depletion comparable in efficiency to published work. For example, by limiting dilution analysis assay of IL-2-producing T cells we found approximately 3 logs of T cell depletion. The growth of bone marrow stem cells (CFU-GM, CFU-E and BFU-E) was not affected by treatment with the 097 conjugates.

An immunotoxin system intended for bone marrow purging composed of glucose oxidase and lactoperoxidase coupled to monoclonal antibody 097.

The effectiveness of an antibody-enzyme immunotoxin (eIT) was investigated on human T cells. This enzyme immunotoxin contained glucose oxidase (GO) and lactoperoxidase (LPO) chemically coupled to the pan-leukocyte-specific mouse monoclonal antibody (MoAb) 097 (097-GO and 097-LPO). Human peripheral blood mononuclear cells or tumor cells were suspended in a mixture of 097-GO and 097-LPO for 30 min, and then for 2 h with glucose and NaI. The effectiveness of this eIT system was indicated by the almost complete reduction of T cell viability, as estimated by a phytohemagglutinin induced proliferation assay (99.4% +/- 0.31 depletion, mean +/- SEM of 15 experiments). The specificity of the cytotoxicity reaction was indicated by the lack of cytotoxicity of control irrelevant MoAb conjugates to T cells (1.9% +/- 4.17 of T cell depletion, eight experiments). The growth of human bone marrow myeloid progenitors (CFU-GM) was not affected by the conjugates even by increasing 100-fold the optimal cytotoxic dose. T cells were susceptible to the conjugates in the presence of up to 90% of erythrocytes. This eIT system may thus represent a new alternative immunospecific procedure for allograft and/or autograft purging, and appears to effectively replace complement-mediated methods of T cell depletion.

Release of hydrogen peroxide from human T cell lines and normal lymphocytes co-infected with HIV-1 and mycoplasma.

Human T-cell lines and normal lymphocytes persistently or acutely co-infected with the human immunodeficiency virus type 1 (HIV-1) and mycoplasmas were found to release hydrogen peroxide (H2O2), a likely cause of oxidative stress in these cells. The spectrofluorometric measurement of H2O2 release from these cells, using the scopoletin fluorescence quenching technique, gave values of 16-84 p moles/10(6) cells/min. In CEM cells, H2O2 was released only when acutely co-infected with HIV-1 and mycoplasmas, and not when infected with either organism alone. Anti-mycoplasmal antibiotics strongly reduced H2O2 release, and improved cell viability without blocking virus replication. These results suggest that the simultaneous infection by HIV-1 and mycoplasma leads to the release of H2O2, a toxic and potentially lethal metabolite, which in vivo may contribute to HIV-1 pathogenicity.

A flexible apparatus for attosecond photoelectron spectroscopy of solids and surfaces.

We describe an apparatus for attosecond photoelectron spectroscopy of solids and surfaces, which combines the generation of isolated attosecond extreme-ultraviolet (XUV) laser pulses by high harmonic generation in gases with time-resolved photoelectron detection and surface science techniques in an ultrahigh vacuum environment. This versatile setup provides isolated attosecond pulses with photon energies of up to 140 eV and few-cycle near infrared pulses for studying ultrafast electron dynamics in a large variety of surfaces and interfaces. The samples can be prepared and characterized on an atomic scale in a dedicated flexible surface science end station. The extensive possibilities offered by this apparatus are demonstrated by applying attosecond XUV pulses with a central photon energy of ∼125 eV in an attosecond streaking experiment of a xenon multilayer grown on a Re(0001) substrate.

Two IgG1 (IgF) subclasses in mice: presence in normal serum and representation within lymphocyte and plasma cell populations.

Monospecific anti-gamma1 sera were prepared in rabbits against each of two IgG1 mouse myeloma globulins isolated from myeloma A2 and MOPC21. Both these sera react with IgG1 of normal mouse serum by immunoelectrophoresis and cannot be distinguished any further. The antibodies contained in the sera were cross-isolated on immunoadsorbents containing either the A2 or MOPC21 antigenic material and tested by immunoelectrophoresis against normal mouse serum. An interesting spur was formed between these two antibodies. The two isotypes thus revealed in normal serum are tentatively designated IgG1a (globulin A2) and Ig G1b (globulin MOPC21). With live peripheral lymph node cells almost all lymphocytes which stain with antikappa antibodies (B lymphocytes) also stain with the specific anti-gamma1a antibodies, but only 5-10% stain with the specific anti-gamma1b antibodies. On fixed spleen cells (C57B16 mice) the anti-gamma1a antibodies stain almost identical percentages of plasma cells as anti-kappa antibodies: 1.8% are gamma1 and 2.2% are kappa. Anti-gamma1b antibodies stain only 5% of kappa-positive plasma cells. The % values estimated on both the normal lymphocyte and plasma cell populations were not significantly affected by inhibtion experiments of the specific anti-gamma1 antibodies performed with isolated myeloma globulins representative of several Ig classes.

A common VH marker relating BALB/c alpha 1-3 dextran-binding and A/J P-azophenylarsonate-binding antibody families.

Antibodies that define the idiotypic marker 104E-MPC11 were purified from rabbit anti-M104E sera. The expression of this marker was searched for in a panel of representative myeloma and hybridoma proteins differing in antigen-binding specificities and idiotype expression. By radioimmune competition assays, the marker was distinguished from the IdX Dex idiotype of BALB/c alpha 1-3 dextran-binding proteins (alpha 1-3 Dex-BP) in that it was expressed on both IdX Dex-positive and IdX Dex-negative proteins on the one hand, and on A/J p-azophenylarsonate-binding proteins (Ar-BP) on the other. Among the later, six of eight of the proteins also expressed the major cross-reactive idiotype (CRI). The marker was expressed weakly or not at all on A/J Ar-BP, which did not express the CRI, as well as on a number myeloma and hybridoma proteins of different antigen-binding specificities, and normal IgG. The marker was localized in VH, but its full expression required the light chain, and was influenced by quaternary structure.

In-vitro cytolysis of myeloma tumor cells with glucose oxidase and lactoperoxidase antibody conjugates.

We have assessed the tumoricidal potential of enzyme-antibody conjugates on murine myeloma cells. Conjugates of glucose oxidase (EC 1.1.3.4) and lactoperoxidase (EC 1.11.1.7) were specifically targeted on the NSO tumor cells. Optimal conditions for tumor cell killing, as assayed by [51Cr] release required the binding of both antibody conjugates to the cell membrane. This is followed by washing and incubation in medium containing glucose and 0.1 mM iodide. Under these conditions 90% of the incorporated [51Cr] labeled is released from the cells, and NSO clonogenicity is reduced by a factor greater than 5 logs by 2 h of incubation.