Guy B. Faguet

Radiation-induced clastogenic plasma factors

Ionizing irradiation induces chromosomal aberrations in directly exposed cells and is known to have mutagenic and carcinogenic potential for the exposed host. Under controlled conditions, we examined whether such clastogenic effects of irradiation might be due in part to radiation-induced plasma factors. Irradiated cells and sera from CF-Nelson rats were used at 15 min, and 1, 7, 14, and 56-70 days after total body irradiation (250 R, n = 67 or 400 R, n = 39). Control rats (n = 44) served as donors of nonirradiated sera and cells. In addition, sera from six rats were irradiated (250 R or 400 R) in vitro. On the average, 298 metaphases from six rats were studied at each time-point. Cytogenetic abnormalities observed included chromatid- and chromosome-type lesions and hyperdiploidy. The frequency of abnormalities was comparable at both radiation doses. Nonirradiated cells exposed in vitro to irradiated serum (15 min postirradiation) exhibited a 36- to 48-fold increment in hyperdiploidy (p = 0.0001) and a 2.- to 2.2-fold rise in chromatid gaps and breaks (p less than 0.01), but none of the chromosome-type aberrations seen in cells exposed to radiation. The clastogenic activity of irradiated plasma persisted in circulation for the 10-wk duration of the study and was not abrogated by dilution with nonirradiated serum. Serum irradiated in vitro was not clastogenic. This study shows that irradiation of rats results in the prompt appearance of clastogenic activity in their plasma. This activity is not due to radiation-induced depletion of protective factors nor to chemical-physical changes of normal plasma components, but results from circulating factors released by irradiated cells.

Immunologically diagnosed malignancy in Sjögren's pseudolymphoma

Studies of lymphocyte markers in a patient with Sjögrens syndrome who exhibited histologically benign lymphoproliferation in the lung revealed a malignant cell clone. T and B cells were quantitated according to their ability to form spontaneous rosettes with sheep erythrocytes and to fluoresce with fluorescein-conjugated antiserums, respectively. Circulating lymphocytes were 66 percent T cells (N = 58 +/- 2 per cent) and 14 percent B cells (N = 22+/- 1 percent), the latter exhibiting normal polyclonal distribution of membrane immunoglobulins. However, lymphocyte suspensions obtained from fresh lymph node and from biopsy specimens from a lymphoid lung nodule revealed 95 percent and 88 percent B cells, with 1 percent and 2 percent T cells, respectively. Moreover, when cryostat-frozen sections from both tissues were reacted with each of the heavy and light chain-specific antiserums, most cells demonstrated the presence of intracytoplasmic mu kappa immunoglobulin exclusively. Twenty-two months later, a clinically and histologically classic lymphoma developed. Repeat marker studies performed on cells freshly isolated and on frozen sections from the histologically malignant lymph node revealed persistence of the monoclonal marker on most cells.

Clone Emergence and Evolution in Chronic Lymphocytic Leukemia: Characterization of Clinical, Laboratory and Immunophenotypic Profiles of 25 Patients

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease usually involving B-cells. Characteristically, B-CLL cells express CD5, CD19, CD20, CD23, cCLLa, Fc and mouse RBC receptors, and low density monoclonal surface immunoglobulins (Moslgs). In order to shed light into the presentation and natural history of B-CLL, patients with a sustained, mild relative (>45%) or absolute lymphocytosis (<104/μL) and a non-diagnostic marrow (waived if lymphocyte count <5 × 103μL) were phenotyped in search of a B-cell clone. Immunophenotyping, initially done by fluorescence microscopy was confirmed by one and two color flow cytometry. Positive and negative controls included 95 patients with overt B-CLL and 45 healthy volunteers, respectively. While patients with overt B-CLL exhibited a cCLLa+ (100%), CD19+ (92%), MosIgs+ (92%) and CD5+ (80%) clone, healthy volunteers did not. Out of 39 patients with lymphocytosis of unknown significance (LUS), 15 had relatively normal immunophenotypes. However, 24 exhibited a ...

A simple technique for the rapid enrichment of class and subclass hybridoma switch variants: A 1000-fold enrichment in half the time, for half the cost

Switching parental hybrids in vitro to downstream switch variant clones producing more desirable monoclonal antibodies (MoAbs) requires either labor intensive and time consuming subcloning techniques, or fluorescence activated sorting of the desired clones. We tested the hypothesis that enrichment of downstream switch variant clones might be achieved by selective lysis of upstream hybridoma cells followed by expansion of the enriched downstream clone. Using a parental hybridoma with surface and secretory IgM, we attempted to enrich downstream switch variant clones producing class (IgG) and subclass (IgG1 or IgG2a) MoAbs. Enrichment of downstream IgG, IgG1 and IgG2a MoAb-producing switch variants was achieved by single or repeated antibody-dependent, complement-mediated lysis of the upstream IgM-bearing parental hybridoma cells followed by limited subcloning. Two exposures of parental hybridoma cells to lysis followed by plating at 100 cells/well enriched the frequency of switch variants up to 1235-fold, enabling the development of IgG1 or IgG2a-producing subclones exhibiting high yield antibody production. Using this protocol, production time and costs were reduced by > 50% when compared to the standard technique. This novel technique for the rapid isolation and expansion of switch variant clones should be ideal for most laboratories, particularly those without access to cell sorting capabilities.

Structural characterization of γ1-heavy chain disease protein BAZ: Heterogeneity of the constant region

Abstract γ 1 -heavy chain disease protein (HCD) BAZ has a molecular weight of 60,000 daltons and contains a substantial quantity of carbohydrate (14%) in comparison to normal γ-chains which is distributed as follows: sialic acid (5%), fucose (1.7%), hexoses (3.5%), glucosamine (1.7%), and galactosamine (2.0%). Dansylation of the protein indicates that it has a blocked N -terminal amino acid, whereas treatment of the protein with hydrazine liberates glycine as the C-terminal amino acid. Thus, both amino and carboxy terminal residues appeared to be homologous to normal γ-chains. The amino acid composition of the protein also shows a markedly lower half-cystine content in comparison to other γ 1 -chains, indicating that the protein lacks interchain disulfide bridges connecting the heavy chains due to a deletion encompassing the hinge region. Amino acid sequence analysis of the C-terminal octadecapeptide of protein BAZ demonstrated a previously unrecognized amino acid substitution in the constant region. It contains a substitution of glycine for alanine at position 431. This exchange may conceivably represent another allotypic variant of IgGl proteins.

A brief history of cancer: age-old milestones underlying our current knowledge database.

This mini‐review chronicles the history of cancer ranging from cancerous growths discovered in dinosaur fossils, suggestions of cancer in Ancient Egyptian papyri written in 1500–1600 BC, and the first documented case of human cancer 2,700 years ago, to contributions by pioneers beginning with Hippocrates and ending with the originators of radiation and medical oncology. Fanciful notions that soon fell into oblivion are mentioned such as Paracelsus and van Helmont substituting Galens black bile by mysterious ens or archeus systems. Likewise, unfortunate episodes such as Virchow claiming Remaks hypotheses as his own remind us that human shortcomings can affect otherwise excellent scientists. However, age‐old benchmark observations, hypotheses, and practices of historic and scientific interest are underscored, excerpts included, as precursors of recent discoveries that shaped modern medicine. Examples include: Petits total mastectomy with excision of axillary glands for breast cancer; a now routine practice, Peyrilhes ichorous matter a cancer‐causing factor he tested for transmissibility one century before Rous confirmed the virus‐cancer link, Hills warning of the dangers of tobacco snuff; heralding todays cancer pandemic caused by smoking, Pott reporting scrotum cancer in chimney sweepers; the first proven occupational cancer, Velpeaus remarkable foresight that a yet unknown subcellular element would have to be discovered in order to define the nature of cancer; a view confirmed by cancer genetics two centuries later, ending with Röntgen and the Curies, and Gilman et al. ushering radiation (1896, 1919) and medical oncology (1942), respectively.

Hodgkin's Disease: Basing Treatment Decisions on Prognostic Factors

The purpose of this study was to review the current status of risk factor assessment in Hodgkins disease (HD) clinically useful for managing this disease. Regarding database retrieval and selection a literature search restricted to English-language articles, abstracts, book chapters and reports published between 1980 and 1993 was conducted both electronically using MEDLINE and CANCERLIT and manually using the bibliographies of the retrieved database. Out of approximately 500 publications identified for analysis, 34 were selected as illustrative. Results showed that most patients with Hodgkins disease are curable at the outset with standard radio- or chemo-therapy, depending on stage and other risk factors. However, up to 40% of patients will either fail at initial induction, or experience early or late relapses. Risk factors analysis at these various times provide a solid base for selecting the therapy best suited to optimize outcome for each individual patient. Patients with truly refractory disease pose a serious challenge to clinicians and are best managed in specialized centers conducting controlled clinical trials. In conclusion it appears that although approximately 75% of newly diagnosed patients with HD can expect long-term, disease-free survival, refractory patients exhibit a dismal survival. Improving their outcome will require innovative approaches.

The Chronic Lymphocytic Leukemia Antigen (cCLLa) as Immunotherapy Target: Pharmacokinetics and Biodistribution of Two Divalent, Ricin-Based Immunotoxins in Xenografted Athymic Mice

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is potentially suitable for targeted immunotherapy given its restriction to clonal CLL cells and lack of expression by normal lymphocytes. In order to assess the pharmacokinetics and biodistribution of two potent anti-cCLLa immunotoxins (ITs) were examined in the mouse model. The IgG fraction of anti-cCLLa monoclonal antibody CLL2m was conjugated with 125I-labeled intact (RTA) or deglycosylated (dgA) ricin chain A, injected intravenously into athymic mice engrafted with cCLLa-expressing human tumors, and monitored over 120 hours. Blood concentrations of CLL2m/125I-RTA and CLL2m/125I-dgA were best fit to biexponential equations but the latter exhibited a lower alphaT1/2 and betaT1/2 (4.1 and 102 min vs 5.9 and 126 min), a smaller volume of distribution (5.1 g vs 9.7 g), and a lower blood clearance (2.2 g/hr vs 4.6 g/hr). Both ITs exhibited preferential tumor uptake that followed distinct kinetics: rising tumor uptake for 2 hrs post-injection (while tissue uptake decreased), reaching tumor/non-tumoral tissue uptake ratios up to 16.9; and slower dissociation rates of tumor- vs tissue-bound ITs (>45% vs <20% remaining tissue-bound 6 hrs post-injection, respectively). Non-specific liver uptake was not prominent for either IT. In vivo IT deconjugation reached 50% approximately 12 hours pos-injection. The pharmacokinetics and biodistribution data in the mouse model suggest that ricin-based anti-cCLLa ITs are suitable for use in human trials.

Physicochemical and immunochemical properties of γ1 heavy chain disease protein baz

Abstract The physicochemical and antigenic properties of a γ1 heavy chain disease protein (γl HCD BAZ) are described. Protein BAZ is positive for the Glm(1) determinant and gives a reaction of identity with Fc fragment, whether derived from Cohn Fraction II or from an IgG1 myeloma protein. It also gives a reaction of complete identity with the γHCD proteins CRA and ZUC. The mol. wt of the native protein was determined from (1) sedimentation-diffusion, (2) sedimentation-viscosity, and (3) sedimentation-equilibrium measurements to be approx 60,000 daltons. The mol. wt of the unreduced protein in 6 M guanidine hydrochloride was found to be 30,000 daltons, which was identical to that of the reduced-alkylated form in 6 M guanidine. These results established that γ1 HCD BAZ is a noncovalently linked dimer and suggested the possibility of a missing hinge region.

MECHANISMS OF LYMPHOCYTE ACTIVATION. II. NEGATIVE COOPERATIVITY IN THE BINDING OF PHYTOHEMAGGLUTININ (PHA) TO ITS LYMPHOCYTE RECEPTORS

Publisher Summary Several studies on the mechanism of human lymphocytes has revealed that under optimal experimental conditions, equilibrium interactions between 125I-PHA (phytohemagglutinin) and its specific lymphocyte membrane receptors result in a curvilinear Scatchard plot, suggestive of the heterogeneity of receptor sites with different binding affinity or the existence of site–site interactions. The chapter describes an experiment to demonstrate that PHA-containing sites lower the affinity of adjacent sites for PHA, a phenomenon known as negative cooperativity. The chapter illustrates the Scatchard plot of the binding data obtained under steady state conditions in the experiment conducted. The slope of the line, which reflects the equilibrium constant of the receptor–ligand interactions, shows a progressive change with increasing ligand concentrations. The demonstration of negative cooperativity rests on the displacement of receptor-bound 125I-PHA induced by an excess of PHA, a phenomenon that can easily be measured from the resulting acceleration of the complex dissociation.