Total body irradiation using helical tomotherapy: Set-up experience and in-vivo dosimetric evaluation.

PURPOSE: Helical Tomotherapy (HT) appears as a valuable technique for total body irradiation (TBI) to create highly homogeneous and conformal dose distributions with more precise repositioning than conventional TBI techniques. The aim of this work is to describe the technique implementation, including treatment preparation, planning and dosimetric monitoring of TBI delivered in our institution from October 2016 to March 2019. MATERIAL AND METHOD: Prior to patient care, irradiation protocol was set up using physical phantoms. Gafchromic films were used to assess dose distribution homogeneity and evaluate imprecise patient positioning impact. Sixteen patients' irradiations with a prescribed dose of 12Gy were delivered in 6 fractions of 2Gy over 3 days. Pre-treatment quality assurance (QA) was performed for the verification of dose distributions at selected positions. In addition, in-vivo dosimetry was carried out using optically stimulated luminescence dosimeters (OSLD). RESULTS: Planning evaluation, as well as results of pre-treatment verifications, are presented. In-vivo dosimetry showed the strong consistency of OSLD measured doses. OSLD mean relative dose differences between measurement and calculation were respectively +0,96% and -2% for armpit and hands locations, suggesting better reliability for armpit OSLD positioning. Repercussion of both longitudinal and transversal positioning inaccuracies on phantoms is depicted up to 2cm shifts. CONCLUSION: The full methodology to set up TBI protocol, as well as dosimetric evaluation and pre-treatment QA, were presented. Our investigations reveal strong correspondence between planned and delivered doses shedding light on the dose reliability of OSLD for HT based TBI in-vivo dosimetry.

Complement mediates human immunodeficiency virus type 1 infection of a human T cell line in a CD4- and antibody-independent fashion.

Incubation of the human T cell lymphotropic virus (HTLV)-IIIB and HTLV-RF strains of human immunodeficiency virus type 1 (HIV-1) with normal seronegative human serum under conditions that allow complement activation resulted in enhancement of infection of the MT2 human T cell line cultured in the presence of low amounts of virus. Infection of MT2 cells was assessed by measuring reverse transcriptase activity in supernatants at day 9 of culture. Complement activation by viral suspensions occurred through the alternative pathway. Opsonization of HTLV-RF viral particles with complement was sufficient to allow a productive infection to occur in cells exposed to suboptimal amounts of virus. Infection of MT2 cells with suboptimal amounts of serum-opsonized HIV-1 was suppressed by blocking the C3dg receptor (CR2, CD21) on MT2 cells with monoclonal anti-CR2 antibody and rabbit F(ab')2 anti-mouse immunoglobulin antibodies. Blocking of the gp120-binding site on CD4 under similar experimental conditions had no inhibitory effect on infection of MT2 cells with opsonized virus. Opsonization of HIV-1 with seronegative serum also resulted in a CR2-mediated enhancement of the infection of normal peripheral blood mononuclear cells and T lymphocytes. These results indicate that complement in the absence of antibody may enhance infection of C3 receptor-bearing T cells with HIV-1, and that the interaction of opsonized virus with the CR2 receptor may result by itself in the infection of target T cells in a CD4- and antibody-independent fashion.

Herpes simplex virus glycoprotein D: human monoclonal antibody produced by bone marrow cell line.

Normal bone marrow cells from a donor positive for herpes simplex virus were transformed with Epstein-Barr virus. The resulting lymphoblastoid cell line has secreted immunoglobulin G1 of the kappa type continuously for 2 years. This immunoglobulin, detected both on the cell surface and in the cytoplasm, reacts with cells infected with herpes simplex virus. It defines an antigen that comigrates with the 55-kilodalton glycoprotein D of herpes simplex virus type 1 and neutralizes the infectivity of herpes simplex viruses 1 and 2.

T lymphocyte cloning from rejected human kidney allografts. Growth frequency and functional/phenotypic analysis.

Mechanically harvested lymphocytes invading an irreversibly rejected human kidney allograft were seeded at limiting dilution to calculate the frequency of growing precursors. Optimal growth frequency (1/13) was obtained when Epstein-Barr virus (EBV)-transformed donor B lymphocytes were used as stimulators (D-BLCL) in the presence of interleukin 2 (IL-2). The 55 clones analyzed were all T11+ and T3+, and all expressed DR antigens (45% were T8+ and 55% T4+). Only one clone had a double-labeled (T4+ T8+) surface. All cells proliferated significantly against D-BLCL, although T4+ clones had a significantly shorter average doubling time than T8+ clones. Nearly all T8+ clones were specifically cytotoxic for D-BLCL, while both T4 and T8 did not react against K562, autologous EBV-BLCL, and third-party EBV-BLCL. Detectable IL-2 was found in the culture supernatants of only a minority of clones (all T4+).

Classical and quantum many-body effects on the critical properties and thermodynamic regularities of silicon.

Using molecular simulation, we determine the critical properties of Si as well as the loci for several remarkable thermodynamic contours spanning the supercritical region of the phase diagram. We consider a classical three-body potential as well as a quantum (tight-binding) many-body model, and determine the loci for the ideality contours, including the Zeno line and the H line of ideal enthalpy. The two strategies (classical or quantum) lead to strongly asymmetric binodals and to critical properties in good agreement with each other. The Zeno and H lines are found to remain linear over a wide temperature interval, despite the changes in electronic structure undergone by the fluid along these contours. We also show that the classical and quantum model yield markedly different results for the parameters defining the H line, the exponents for the power-laws underlying the line of minima for the isothermal enthalpy and for the density required to achieve ideal behavior, most notably for the enthalpy.

AP-1 is involved in UTP-induced osteopontin expression in arterial smooth muscle cells.

Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The -96 to +1 region mediated UTP-induced OPN overexpression (+276+/-60%). Sequence analysis of this region revealed a potential site for AP-1 located at -76. When this AP-1 site was deleted, UTP-induced activation of the -96 to +1 region was totally inhibited. Thus, this AP-1 (-76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation.

Extracellular nucleotides induce arterial smooth muscle cell migration via osteopontin.

Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y(2) ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors alpha(v)beta(3)/beta(5) by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.

Cytomegalovirus isolations from cell cultures derived from Epstein-Barr virus-associated nasopharyngeal carcinoma.

Cytomegalovirus (CMV) was isolated in cell cultures derived from 2 of 11 nasopharyngeal carcinoma (NPC) biopsy specimens from North African patients. All these cases were Epstein-Barr virus (EBV)-associated NPC. Morphologic cytopathic changes and viral replication not associated with EBV were observed after 2 months in culture. Virus identification was achieved by immunofluorescence studies, and cell culture antigens were tested by the use of complement fixation and indirect hemagglutination. All these NPC patients had been infected by herpes simplex virus, varicella-zoster virus, and CMV, but the antibody titers determined by complement fixation and immunofluorescence were normal. CMV, which is not associated with this cancer, could nevertheless favor carcinogenesis in facilitating fusion between epithelial cells and EBV-positive lymphocytes.

Effect of (E)-5-(2-bromovinyl)uracil on the catabolism and antitumor activity of 5-fluorouracil in rats and leukemic mice.

In contrast to thymine and 5-fluorouracil (FUra) which were cleared from the bloodstream within 2-4 h after their i.p. administration (200 mumol/kg) to rat, (E)-5-(2-bromovinyl)uracil (BVUra) maintained a concentration of 50-70 microM for at least 6 h and was still present in the plasma 24 h after its administration. In vitro experiments with rat liver extracts indicated that BVUra was not a substrate but an inhibitor for the reductive step in pyrimidine degradation catalyzed by dihydrothymine dehydrogenase. Kinetic and dialysis experiments suggested that BVUra was an irreversible inhibitor of this enzyme. The binding of BVUra to the enzyme depended on the presence of reduced nicotinamide adenine dinucleotide phosphate in the reaction mixture. Dihydrothymine dehydrogenase activity was also inhibited in the dialysed 105,000 X g supernatant fraction of livers from rats that had previously been treated with BVUra. Such inhibitory effects also occurred in vivo; previous administration of BVUra increased the plasma half-lives of thymine and FUra by 10- and 5-fold and their area under the curve by 9- and 8-fold, respectively. The effect of BVUra on the antitumor activity of FUra was evaluated in DBA/2 mice inoculated with 10(6) P388 leukemia cells. The mean survival times for the control and FUra-treated mice (5 mg/kg at 1, 3, 5, and 7 days after tumor cell inoculation) were 9.7 and 12.4 days, respectively. When BVUra (200 mumol/kg) was administered 1 h before each injection of FUra, the mean survival time was extended to 17.1 days. BVUra alone did not affect the mean survival time. When the dose of FUra was increased to 20 mg/kg, the mean survival time was 15.3 days; upon a preceding injection of BVUra the mean survival time decreased to 9.2 days. The latter effect probably resulted from an increased toxicity of FUra. Similar results were obtained if FUra was replaced by 5-fluoro-2'-deoxyuridine and BVUra by (E)-5-(2-bromovinyl)-2'-deoxyuridine. The enhancement of both the antitumor and toxic effects of FUra by BVUra were most probably due to an inhibition of FUra degradation, since, like in rats, BVUra increased the plasma half-life of FUra in DBA/2 mice. Hence BVUra appears to be an interesting compound, increasing the potency of FUra by decreasing its degradation.

Catabolism of thymidine in human blood platelets: purification and properties of thymidine phosphorylase.

A pyrimidine nucleoside phosphorylase was partially purified from human blood platelets. The purified enzyme, as well as crude enzyme preparations, catalyses the phosphorolysis of thymidine and deoxyuridine, but not of uridine, and is able to catalyse direct pentosyl transfer from these deoxyribonucleosides to uracil or thymine; this enzyme has the properties of a thymidine phosphorylase. It has a molecular weight of about 110,000 and is composed of two identical subunits; it is phosphate dependent, has a maximal activity at a pH value of 5.7, and an isoelectric point of 4.4. This enzyme was mainly of cytoplasmic origin. Although platelet thymidine phosphorylase could promote the degradation or synthesis of thymidine, intact platelets degraded thymidine but were not able to synthesize thymidine from thymine. Blood platelets may play an important role in the degradation of plasma thymidine.

Transient adult T-cell leukemia/lymphoma picture during varicella infection in an HTLV-1 carrier.

HTLV-1 (human T-lymphotropic virus type 1) is associated with tropical spastic paraparesis, adult T-cell lymphoma (ATL), and also with opportunistic infections. The risk for developing ATL in HTLV-1 healthy carriers is low, between 1 and 4%. Nothing is known about the events promoting the evolution from the healthy carrier state to symptomatic ATL. We describe the case of a 44-year-old French Caribbean man with a chronic and recurrent strongyloidiasis in which the occurrence of a hemorrhagic and necrotic varicella led to the discovery of an infection by HTLV-1 and an acute form of ATL. All hematological data were normal before the onset of varicella. ATL completely disappeared at the same time as the varicella healed. This leads us to hypothesize that acute infections such as the reactivation of varicella-zoster may act as a promoting factor for the development of ATL in healthy HTLV-1 carriers.

Effects of hypercholesterolaemic serum on aortic explants from normal rats.

Aortic explants obtained from normal adult rats and composed of all 3 tunics were cultured in a semi-synthetic gelosed medium supplemented by 10% serum. Explants cultured with normocholesterolaemic serum kept their in vivo characteristics for more than 12 days at electron microscope and histometabolic levels. However, explants showed considerable differences when cultured in a medium with hypercholesterolaemic serum. Cell proliferation in the tunica media and enhanced synthesis of macromolecular components of the extracellular matrix were observed. These laterations occurring inside the explants, which had kept all their tissular relationships, suggest that this culture system may be valuable for further studies on atherosclerotic processes.

Toxicity and activity of purified trichosanthin.

Trichosanthin was purified from fresh Chinese root tubers of Trichosanthes kirilowii and evaluated for anti-HIV activity. Trichosanthin inhibited syncytium formation between infected H9 cells and uninfected Sup-T1 cells from 0.5 to 4 micrograms/ml. Trichosanthin also inhibited HIV replication in H9 and CEM-SS cells at 1 microgram/ml, but was toxic for MT-4 cells (HTLV-I-positive), at doses greater than 0.25 microgram/ml. This new purification procedure confirms the anti-HIV activity of trichosanthin on some cell lines in different biological assays.

In vitro HIV-1 entry and replication in Langerhans cells may clarify the HIV-1 genome detection by PCR in epidermis of seropositive patients.

Being dendritic antigen-presenting cells in skin and mucous membrane, Langerhans cells (LC) occur in areas at risk for inoculation by human immunodeficiency virus (HIV), and the question whether LC act as a target, reservoir, or vector for transmission of HIV has given rise to much controversy. To address this question, we first analyzed the epidermal compartment of skin from patients seropositive for HIV DNA. Second, we tested the susceptibility of each cell type normally found in this compartment to in vitro infection by HIV-1. A non-denatured DNA was obtained from epidermal sheets after a thermochemical treatment of biopsies (0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.5 at 60 degrees C for 90 seconds). Optimization of amplification of viral genome was performed with three primer pairs derived from gag, env, and pol sequences. Polymerase chain reaction (PCR) products were analyzed by Southern blot. Viral genome was found in five of 11 HIV-seropositive patients. To control the permissivity of epidermal cell population for HIV, cells isolated from the epidermal sheet of normal skin by trypsinization were co-cultured with HIV-1-carrying promonocytic cells (U937) and observed by electron microscopy. After 3-6 h of co-culture, numerous virions were either tightly bound or apparently engaged in the process of internalization through receptor-mediated endocytosis. At day 4 of co-culture, some infected LC appeared to release mature viral particles through bud formation. The in vitro HIV-1 entry and replication in LC may confirm the presence of the HIV-1 genome by PCR in epidermis of seropositive patients. The consequences of the permissivity of LC for HIV on the antigen-presenting function remain to be determined.

Retroviral infection (HTLV-I) induces cytokine-regulated immunomodulation and cytotoxicity of medulloblastoma cells.

Primitive neuroectodermal tumors are thought to result from disturbed differentiation of neuroepithelial stem cells. These tumor cells retain the capacity to differentiate toward the neuron or glia phenotype under extrinsic stimuli. Previously, we have developed a model for the differentiation of a medulloblastoma cell line (Dev cells) induced by infection with the human retrovirus HTLV-I. This virus delivers signals which trigger the Dev cells to differentiate toward an astrocytic lineage. The aim of this study was to characterize the time course of viral infection, to identify the soluble factors released and to analyze their effects on Dev cells. The early phase of viral replication is followed by latent infection. Viral infection induces glial differentiation in a subpopulation of cells and results in the death of others. The inflammatory cytokines TNF alpha, IL1 alpha and IL6 were detected in medium conditioned by infected Dev cells. TNF alpha was cytotoxic and cytostatic for subpopulations of Dev cells. Furthermore, TNF alpha treatment reproduced the modulation of expression of the major histocompatibility complex antigens (MHC class I) observed in infected Dev cells. These observations support the view that HTLV-I infection, which triggers glial differentiation of medulloblastoma Dev cells, also causes the release of soluble factors capable of downregulating proliferation of dividing tumor cells and of modifying their recognition by cellular immune effectors.

A 2-year follow-up of an anti-HIV immune reaction in HIV-1 gp160-immunized healthy seronegative humans: evidence for persistent cell-mediated immunity.

The first trial of an anti-HIV immunization, using a recombinant vaccinia virus expressing gp160 (rV) for priming and paraformaldehyde-fixed rV-infected PBLs and soluble gp 160 for boosting, clearly showed an in vitro HIV-protective immune reaction. This result led us to carry out an additional 2 year Phase I clinical trial in 25 HIV-seronegative volunteers, using HIV gp 160 antigens for immunization in four different protocols. The 2 year trial showed (a) the safety of the preparations, (b) a transient humoral immunity following each boost, and (c) a long-lasting memory T-cell response. Memory cytotoxic T-lymphocytes (CTLs) induced by gp 160 antigen with or without vaccinia vector lysed HLA class I restricted target cells expressing HIV-1 env antigens. These results are consistent with CTLs being an effective component of an AIDS vaccine to control cell-to-cell viral replication, dissemination in the organism, and subsequent evolution toward AIDS.

Zidovudine treatment is not associated with HTLV-1 reverse transcriptase gene mutations in HTLV-I/HIV-1 co-infected patients.

Zidovudine treatment of human immunodeficiency virus (HIV) infection induces drug-resistant viral strains harbouring specific amino acid substitutions in the reverse transcriptase (RT). To investigate whether this phenomenon could be observed in the case of human T lymphotropic virus type I (HTLV-I) infection, we analysed the HTLV-I RT proviral gene sequence in five HTLV-I/HIV-1 co-infected patients treated with zidovudine for HIV-1 infection and in one untreated co-infected subject. In the 816 bp of HTLV-I pol gene sequence determined, no particular nucleotide mutation associated with zidovudine therapy could be identified in the treated subjects. Moreover, the dominant HTLV-1 deduced amino acid sequences determined in treated subjects were identical to that from the untreated subject. Our data show that in the co-infected patients already presenting well-defined mutations associated with zidovudine resistance in HIV-1, no mutations were observed in a part of the pol gene coding for the RT activity of HTLV-I.

Primary in vitro immunization with multimeric synthetic peptides of HIV-1 envelope glycoproteins: generation of neutralizing human monoclonal antibodies.

Peripheral blood lymphocytes from healthy HIV-1 seronegative donors were immunized in vitro with the following synthetic peptides: (i) an octameric poly-L-lysine conjugated peptide of the HIV-1MN V3 loop and (ii) a resin bound synthetic peptide aa642-665 of HIV-1 gp41. Lymphoblastoid cell lines (LCL) were obtained by immortalization with Epstein-Barr virus (EBV). We produced four LCL secreting human monoclonal antibodies (HuMoAbs) of the IgM isotype: three were directed against the V3 domain (FC10, FC81 and CF41) and one against aa642-665 (CA45C). Two of these HuMoAbs (FC81 and CA45C) reacted to viral surface antigen on HIV-1-infected cells. All the HuMoAbs inhibited 40-53% of cell fusion induced by HIV-1-infected H9 cells at 5 micrograms/ml. They also neutralized, at lower concentrations, cell-free infection with HIV-1MN, HIV-1IIIB and four primary clinical HIV-1 isolates. No enhancing activity of the HuMoAbs in the presence of complement was observed. The results presented here show the feasibility of generating neutralizing human monoclonal antibodies against HIV-1 by primary in vitro immunization with selected synthetic peptides of HIV-1 envelope glycoproteins. This approach has provided tools for further studies of synergistic neutralization assays, and generated potential immunoglobulin candidates for passive immunotherapy.

The humanized severe combined immunodeficient mouse as a model for primary human humoral response against HIV1 peptides.

Adequate animal models for the study of human immunodeficiency virus (HIV) infection are important for the analysis of specific cellular and humoral immune responses. Humanized severe combined immunodeficiency (SCID) mice can be constructed either by injecting human peripheral blood lymphocytes (hu-PBL-SCID) or by transplanting human fetal tissues--liver, thymus and bone fragments--(SCID-hu) into these mice. Such animals can produce human immunoglobulins and SCID-hu mice exhibit circulating T and B lymphocytes of human origin. These humanized mice were injected with immunogenic HIV peptides and the specific humoral response was studied. A human antibody response was obtained after de novo contact with HIV1 peptides p583 and p642, from gp41. In SCID-hu mice, a primary, then a secondary response were demonstrated to occur with 225 mg/l of human immunoglobulin (Ig)M and 300-1860 mg/l human IgG. When tested in ELISA, these human antibodies recognized specifically both the immunization peptides and the HIV1 antigens. The antibody response was obviously of a primary nature since the human cells derived from naive fetal cells. When SCID mice received intraperitoneal injections of human peripheral blood lymphocytes pre-incubated in vitro with peptide p583 for 1 week, and when the resulting hu-PBL-SCID mice were injected with the same peptide, only IgM anti-HIV antibodies were produced (372-424 mg/l) and the switch to IgG antibodies did not occur. This model may provide a means to produce human monoclonal antibodies to HIV and to check candidate HIV vaccines.

Comparison between direct binding, competition and agglutination assays in the characterization of polyclonal anti-idiotypes against anti-HBs human monoclonal antibodies.

Polyclonal anti-idiotypic antibodies to human monoclonal anti-HBs antibodies (MoAb1) were raised in rabbits and designated Ab2-H1 and Ab2-H2. These Ab2 were characterized using three assays. A direct binding ELISA evaluated specificity towards a panel of human monoclonal antibodies and gamma globulins. Competition radioimmunoassay (CRIA) revealed Ab2 specificities towards Ab1 antigen binding sites by inhibition of HBsAg/Ab1 binding. Ab2-H1 and Ab2-H2 had comparable reactivities in ELISA and CRIA, whereas, using affinity purified Ab2, a fast (10 min) agglutination test (Spherotest) revealed different Ab1/Ab2 binding properties. Ab2-H1 reacted in this Spherotest with the Ab1 against which it was known to be specific (Ab1-H1), whereas in the same assay Ab2-H2 showed no activity towards the variable regions of the Ab1 used for its production (Ab1-H2). When injected into rabbits Ab2-H1 induced anti-HBs Ab3 antibodies but Ab2-H2 did not, thereby confirming the assay results.