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Background: The prescription of ceftriaxone should be limited to patients with biliary tract infections and those lacking intravenous access. A specific training session for prescribers about the clinical relevance and economic value of prescribing cefotaxime instead of ceftriaxone might decrease the use of the latter in geriatric medicine., Objectives: To determine the clinical and economic impact of a training session for prescribers on the subsequent prescription of third-generation cephalosporin, that is, the percentage of appropriate prescriptions and the cost of third-generation cephalosporin administration before and after the training session., Methods: In a preliminary observational quasi-experimental, open-label study, appropriateness of cefotaxime and ceftriaxone prescription over a 4-week period immediately before the training session were compared with those during a 4-week period immediately afterwards., Key Findings: For 46 patients (29 before the training session and 17 afterwards), the proportion of cefotaxime prescriptions increased (from 3% to 35%; P = 0.007), and the proportion of appropriate prescriptions increased at the start of treatment (from 45% to 76%; P = 0.064) and at the end (from 76% to 88%; P = 0.450). The daily per-patient cost of treatment was €8 for cefotaxime and from €1.63 to €3.42 for ceftriaxone, depending on the administration route., Conclusions: A training session for prescribers was associated with a decrease in ceftriaxone prescriptions encouraging further studies to improve the training session and then evaluate medico-economic impact through randomized clinical trials., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Research on deductive reasoning in adolescents and adults has shown that errors in deductive logic are not necessarily due to a lack of logical ability but can stem from an executive failure to inhibit biases. Few studies have examined this dissociation in children. Here, we used a negative priming paradigm with 64 children (8-10 years old) to test the role of cognitive inhibition in syllogisms with belief-bias effects. On trials where negative priming was predicted, results were as follows: For the first syllogism (A), the strategy 'unbelievable-equals-invalid' had to be inhibited. The logic of the syllogism led to affirming a conclusion inconsistent with one's knowledge of the world, such as 'All elephants are light.' For the second syllogism (B), one's real-world knowledge and the syllogism's logic were congruent but the latter required affirming exactly what had been inhibited for A (i.e. that elephants are heavy). A negative priming effect on the A-B sequence was reflected in a significant drop in reasoning performance on B. This supports the idea that during cognitive development, inhibitory control is required for success on syllogisms where beliefs and logic interfere.

The localization of antigen and the nature of the host antigen-presenting cells (APC) that present it to T cells are two major determinants of antigen immunogenicity. While lymph nodes appear to be the major site for T cell priming, recently the spleen was shown to provide an optimal microenvironment for direct CD8+ cytotoxic T cell (CTL) priming by tumor cells even in the absence of known costimulatory molecules on tumor cells. We analyzed whether the splenic microenvironment would support T cell priming also when host APC are involved (cross-priming) which is probably the major pathway during the generation of anti-tumor immunity. We performed immunization/challenge experiments using different tumor cells (B7.1+, B7.1- and/or beta-gal+, beta-gal-) known to induce CTL to a variable extent either exclusively by cross-priming (B7-) or at least partially by direct priming (B7+). Our results demonstrate that tumor take in the spleen required much less cells than at a subcutaneous injection site. Additionally, intrasplenic immunization was invariably ineffective compared to subcutaneous immunization. We further showed that B cells were not responsible for the inefficient intrasplenic immunization. Therefore delivering the tumor cell antigens inside the spleen by intrasplenic immunization did not improve but rather decreased the efficacy of tumor cell vaccines.

The mechanisms by which dendritic cell (DC) vaccines prime host T cells in vivo was analyzed. Mice were immunized with syngeneic bone marrow-derived DC and as surrogate antigen beta-galactosidase (beta-gal) was used. DC either pulsed with peptide, loaded with beta-gal antigen or gene-modified induced beta-gal-specific cytotoxic T lymphocytes (CTL) and moderate rejection of an in vivo challenge with beta-gal expressing tumors. In addition, beta-gal-specific CTL lysed the syngeneic DC that were used as vaccines. Using SCID mice reconstituted with F1 lymphocytes, direct priming by gene-modified DC vaccines was demonstrated by the presence of beta-gal-specific CTL of the haplotype exclusively expressed by DC while indirect priming by host antigen-presenting cells (APC) was shown by the detection of CTL of the haplotype exclusively present on host APC and absent on DC vaccines. Since DC immunization in syngeneic mice was associated with an increase in NK1.1+/Ly49C- cells and detectable lysis of DC in vitro by lymphokine-activated killer cells, DC vaccines appear to interact with host natural killer cells as well as with antigen-specific T cells. These effector cells in turn may lyse DC vaccines thereby leading to the release of antigens that can be taken up by host APC.

The stimulation of a specific immune response is an attractive goal in cancer therapy. Gene transfer of co-stimulatory molecules and/or cytokine genes into tumor cells and the injection of these genetically modified cells leads to tumor rejection by syngeneic hosts and the induction of tumor immunity. However, the development of host immune response could be either due to the introduced immunomodulatory genes or due to vector components. In this study, human renal cell carcinoma cell lines were modified by a retrovirus to express the co-stimulatory molecule B7-1 together with the hygromycin/thymidine kinase fusion protein (HygTk) as positive and negative selection markers. These B7-1-transduced renal cell carcinoma cell lines were able significantly to activate allogeneic T cell proliferation. The cytolytic activity of these T cells was determined by employing several transduced and nontransduced renal cell carcinoma cell lines as targets. Evidence for a strong vector-specific T cell reactivity induced by the Hyg/Tk protein was obtained in autologous renal cell carcinoma systems. Antibody blocking experiments as well as peptide binding assays demonstrated an HLA-B7-restricted T cell response directed against both the Hyg and the Tk genes. Thus, the vector itself may mask the generation of immune reactivity against tumor antigens and may even detract from it. Vectors with immunogenic potential may be useful for tumor vaccination via cross priming in vivo, whereas antivector reactivities would be detrimental in situations where gene defects are being corrected and where long term expression of a therapeutic protein is required.

Genetically modifying tumor cells to express a variety of cytokines such as interleukin-2 (IL-2) or the co-stimulatory molecule B7.1 leads to increased immunogenicity and reduced tumorigenicity of tumors in several models with T cells involved in the process. We have previously reported decreased tumorigenicity of the murine plasmacytoma J558L [major histocompatibility complex (MHC) class I+ and class II-] expressing IL-2 or B7.1. When systemic immunity was analyzed, immunization with either J558-IL2 or J558-B7.1 cells generated moderate protection against unmodified J558L tumor cells, comparable to immunization with a tumor cells/adjuvant Corynebacterium parvum mixture. In this study, we asked whether the co-expression of IL-2 and B7.1 in tumor cells would augment vaccine potency, cytotoxic T lymphocyte (CTL) activity and protective immunity. Rejection of single IL-2 or B7.1 or co-transfected IL-2/B7.1 cells occurred in most syngeneic animals but not in T cell-deficient nude mice, thus confirming that T cells were required for tumor rejection. We knew from previous experiments that CD8+ T cells were responsible for rejection. Surprisingly, immunization with J558-IL2/B7.1 cells followed by challenge with parental J558L caused a reduction in systemic protection as compared to J558-B7.1 or J558-IL2 alone. We examined the mechanism underlying this unexpected result: 6 days after injection of J558-IL2/B7.1 cells, tumor were nearly completely destroyed and were almost devoid of CD8+ cells, while CD8+ cells were increased in both IL-2- and B7.1-transfected tumors. In addition, immunization with J558-IL2/B7.1 tumors had an adverse effect on the generation of CTL. Mice immunized with J558-B7.1 and to a lesser extent J558-IL2 cells mounted a CTL response against J558L cells while, in contrast, no CTL activity could be detected in mice immunized with J558-IL2/B7.1, thus showing a correlation between the absence of CTL activity and the lack of in vivo protection. We demonstrate that "hyperstimulation" of the immune response by genetically modified cancer vaccines can have adverse effects on tumor immunity, even though the mechanism is not yet completely understood.

In this study we have analyzed the feasibility of gene transfer in human dendritic cells (DCs). DCs were generated from T and B cell-depleted peripheral blood mononuclear cells cultured for 7 days in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The cells showed morphologic and immunophenotypical features typical of DCs, including expression of major histocompatibility complex (MHC) class I and II molecules, CD1a, CD80, CD86, CD13, CD33, CD40, and CD54. The cells showed high stimulatory activity in both allogeneic and autologous mixed lymphocyte reaction (MLR). The bacterial reporter gene lacZ coding for beta-galactosidase (beta-gal) was introduced in DCs by three sequential cycles of infection using a MFG retroviral vector system. After 7 days of culture 35-67% of the cells showed high expression of beta-gal activity, proving successful gene transfer. Stable integration of the lacZ gene was demonstrated by genomic DNA-polymerase chain reaction (PCR) up to 20 days after gene transfer. The percentage of transduction was similar when DCs were further purified by immunomagnetic separation according to CD1a-expression. We conclude that human DCs can be efficiently gene modified, further broadening the spectrum of possible DC-based clinical applications.

To improve the vaccine potency of gene-modified tumor cells, using retroviruses, we have expressed the B7.1 gene in J558L cells and a subline previously transfected with the gene for interleukin-4 (IL-4). Complete longterm tumor eradication occurred in only 73-82% of syngeneic BALB/c mice injected with IL-4 or B7.1 transfectants or tumor cells mixed with the adjuvant Corynebacterium parvum. In contrast, none of the mice injected with J558-IL4/B7.1 cells developed a tumor, thus demonstrating that IL-4 and B7.1 together induced a more potent antitumor immune response compared to either molecule alone. Immunization/challenge experiments demonstrated that IL-4/B7.1 co-transfected cells possessed improved and tumor-specific vaccine potency when compared to single gene transfectants and, more importantly, to a tumor cell/C. parvum mixture. Furthermore, irradiation of vaccine cells almost completely abrogated the vaccine effect. Together, our results mean a step toward an improved tumor cell vaccine that acquires efficacy by the concerted action of IL-4 and B7.1 and the use of viable cells.

The herpes simplex virus thymidine kinase (HSV-TK) converts ganciclovir (GCV) into a toxic product and allows selective elimination of TK+ cells in vitro and in vivo. It is currently being used in clinical gene therapy trials as a therapeutic gene or as a safety marker. We have analyzed the susceptibility of different tumor cell lines to the TK/GCV-mediated "suicide" effect. Therefore, tumor cells TSA, J558L, EB, and ESB and, as a control, NIH-3T3 cells were infected with a retrovirus containing a hygromycin/TK fusion gene. All cell lines were sensitive to GCV in vitro; however, the concentration of GCV and the time needed to eliminate tumor cells completely considerably varied between different tumor cell lines. TSA-TK cells were completely eliminated within 10 days in 1 microg/ml GCV, whereas ESB-TK cells required 22 days in 10 microg/ml GCV. When two cell lines were examined, the differing sensitivity to GCV in vitro correlated with the ability to eradicate TK+ tumors in vivo. TSA-TK tumors could be eliminated in almost all animals by systemic GCV administration, whereas ESB-TK tumors were completely resistant. Different sensitivity to GCV was not due to different TK expression levels because the cells were similarly resistant to hygromycin, and Western blot analysis with an anti-TK antiserum revealed similar protein amounts in TSA/TK and ESB-TK cells. Together, the results demonstrate that tumor cells are highly different concerning the susceptibility to the TK/GCV effect, which, however, may be tested for in vitro.

Interleukin-7 (IL-7) and the membrane molecule B7 are both able to provide proliferation and activation signals for T cells. However, tumor cells transfected to express either molecule alone are not reliably rejected in syngeneic hosts or are not sufficiently immunogenic to serve as potent tumor vaccines. Since IL-7 and B7 have shown synergistically to induce activation and proliferation of T cells in vitro, we have expressed B7.1 by means of a retrovirus in the mammary adenocarcinoma TS/A which arose spontaneously in a BALB/c mouse and in the plasmacytoma J558L and their IL-7-transfected sublines to improve vaccine efficacy. Expression of IL-7 or B7.1 alone in tumor cells decreased tumorigenicity, but nevertheless tumors grew in a substantial number of mice. In contrast, IL-7/B7.1 cotransfected cells did not grow as tumor in a single case. This inhibition of tumor growth was completely T cell dependent, because TS/A-IL-7/B7.1 cells retained their full tumorigenic potential in T cell-deficient mice. Analysis of tumor-infiltrating T lymphocytes revealed increased numbers of T cells in B7, IL-7 and IL-7/B7 transfected compared to parental tumors. In IL-7/B7 transfected tumors, T cell numbers were not further increased compared to that in single-gene-transfected tumors. However, T cells in B7 and IL-7 transfected tumors differed phenotypically with respect to activation markers. In B7 transfected tumors, T cells were predominantly CD28+ and CD25-, while in IL-7 transfected tumors, T cells were mainly CD28- and CD25+. In IL-7/B7 cotransfected tumors, the majority of T cells was CD28+ and CD25+. Thus, IL-7 and B7 induced an anti-tumor immune response by complementary T cell directed pathways in a cooperative fashion. Importantly, immunization of mice with the transfected cells and subsequent contralateral challenge with parental tumor cells showed that IL-7/B7 co-expressing cells induced the most strongly protective immunity, which is superior to that induced by single-gene transfectants and to the adjuvant Corynebacterium parvum. Vaccine efficacy was abrogated when irradiated cells were used for vaccination. Together, our results show that IL-7 and B7.1 transfected tumor cells induce strong T cell activation and tumor immunity.

In the present study, plasma prostaglandin E2 (PGE2) levels were quantitated serially in 14 patients during the period preceding and following autologous bone marrow transplantation (ABMT) or autologous stem cell transplantation (ABSCT). In eight ABMT recipients, the pre-transplant conditioning regimen consisted of combined supralethal doses of chemo- and radiotherapy which was followed by ABMT using autografts purged in vitro either with mafosfamide or monoclonal antibodies (moAbs) plus immunomagnetic beads (IB). A further two ABMT recipients received only high-dose chemotherapy without irradiation as conditioning, followed by unpurged autologous BM. The four ABSCT recipients were also treated solely with chemotherapy followed by untreated autologous stem cell reinfusion. Elevation of plasma PGE2 levels occurred in all 14 patients, despite the difference in the pre-transplant conditioning. The peak levels of PGE2 ranged from about three times the mean value obtained with ten control individuals (29.8 +/- 3.7 pg/ml) to ten times this level. In all cases plasma PGE2 normalised with recovery of the peripheral leucocyte count.

Intracellular cyclic adenosine monophosphate (cAMP) levels were measured in the peripheral mononuclear cells of 25 recipients of ABMT after stimulation with forskolin (10(-4) M), cholera toxin (1 microgram/ml) and prostaglandin E2 (PGE2, 10(-6) M). Significantly increased post-stimulatory cAMP levels were found in cells derived from patients' cells less than 30 days after ABMT (after forskolin stimulation: mean 89.7 +/- 45.4 pmol per 5 x 10(6) cells, p < 0.001; after cholera toxin stimulation: mean 78.6 +/- 37.9 pmol per 5 x 10(6) cells, p = 0.003; after PGE2 stimulation: mean 93.6 +/- 36.6 pmol per 5 x 10(6) cells, p < 0.001). However, at a later time after ABMT (7-60 months), cAMP levels had returned to normal and no significant differences in intracellular cAMP content between cells from patients and normal individuals could be detected. In addition, in six patients cAMP levels after PGE2 stimulation were measured serially pre- or peri-transplantation up to a time when haematologic recovery had occurred: the significantly elevated inducible cAMP levels found early post-ABMT returned to normal with peripheral blood reconstitution. This observation could be of interest in the post-transplantation period, because cAMP, as a 'second messenger', is a modulator of the immune system.

The objective of our study was to evaluate the biologic role of granulocyte colony-stimulating factor (G-CSF) for hematologic reconstitution following autologous bone marrow transplantation (ABSCT). Using a commercially available enzyme-linked immunosorbent assay, serum levels of G-CSF were measured in samples from 48 patients (30 male/18 female) who underwent ABMT or ABSCT. Their median age was 34.5 years (range 16 to 51). Autografting was performed (40 ABMT, 8 ABSCT) in 23 AML, 8 ALL and 17 malignant lymphoma patients. Patients transplanted with blood stem cells had a faster leukocyte and neutrophil recovery compared with the ABMT patients (p < 0.025 and p < 0.05, respectively). During marrow aplasia G-CSF serum levels were elevated in all patients, with a median peak value of 2199 pg/mL (range 453 to 8676 pg/mL). A strong reverse correlation (R = -0.76, p < 0.01) could be demonstrated between G-CSF serum level and white blood count (WBC). An additional increase of G-CSF serum levels on days of fever (> or = 38.5 degrees C) or documented infectious disease was observed. During the early phase of marrow aplasia, the endogenously produced amounts of G-CSF reached concentrations which are used for in vitro stimulation of colony-forming unit granulocyte (CFU-G). The relationship between G-CSF serum level and WBC supports the central role of this circulating hemopoietin following myeloablative treatment and autotransplantation. During periods of higher demand such as fever and infectious complications, endogenous G-CSF production is enhanced.

We investigated the effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) on the pool of circulating hemopoietic progenitor cells in 11 patients with hematological malignancies of nonmyeloid origin and 1 patient with sarcoma. These patients were eligible for autologous blood stem cell transplantation rather than autologous bone marrow transplantation because sufficient marrow aspirates could not be performed due to damage at the usual sites of bone marrow harvest by previous chemo- and/or radiotherapy. Recombinant human GM-CSF was given as continuous i.v. infusion via central venous line for a median time of 11.5 days (range 5-22 days), during which a median number of six aphereses were performed. In comparison to the pretreatment level the median increase in the number of granulocyte-macrophage colony-forming units (CFU-GM)/ml of peripheral blood was 8.5-fold. In all 12 patients a median decrease of the platelet count of 21% (range 7%-67%) was observed during rhuGM-CSF treatment prior to the start of the apheresis procedures. Six patients were treated with a myeloablative conditioning therapy consisting of total body irradiation and/or high-dose polychemotherapy followed by autografting with blood stem cells. Five of them achieved a sustained engraftment. Recombinant human GM-CSF proved to be highly efficient in increasing the number of circulating progenitor cells in these patients with severely compromised hemopoiesis. Blood stem cells harvested under a rhuGM-CSF treatment are capable of restoring hemopoiesis in man after a myeloablative pretransplant therapy.

Cockayne syndrome (CS) is a rare autosomal recessive disease characterized by acute sun sensitivity, cachectic dwarfism, and neurologic and skeletal abnormalities. Cultured skin fibroblasts from patients with this disease are known to be hypersensitive to the lethal effects of 254-nm UV radiation. We have studied the sensitivity of 254-nm UV radiation of lymphoblastoid lines derived from 3 typical CS patients, 1 atypical CS patient who had a very late age of onset of clinical manifestations, 2 patients who had both xeroderma pigmentosum (XP) and typical CS, and 3 heterozygous parents of these patients. Post-UV survival was determined by the trypan-blue dye-exclusion method. The lymphoblastoid lines from the 3 typical CS patients, the atypical CS patient, and the 2 patients with both CS and XP had decreased post-UV viability in comparison with lines from normal donors. Lines from the heterozygous parents had normal post-UV viability. The post-UV viability of the typical CS lines was similar to that of a XP complementation group C line. The relative post-UV viability of lymphoblastoid lines from the typical CS patients was similar to the relative post-UV survival of their fibroblast lines. The lymphoblastoid line from the atypical CS patient had a post-UV viability similar to that of the typical CS patients. Thus, the relative hypersensitivity of CS patients' cells in vitro does not reflect the severity or age of onset of the patients' clinical manifestations. The lymphoblastoid lines from the 2 patients who had both CS and XP were significantly more sensitive to the UV radiation than those from patients with only CS. Our studies demonstrate that lymphoblastoid lines from patients with CS are appropriate and useful cell lines for the study of the inherited hypersensitivity to UV radiation.