Search

The primary structure of recombinant human (h) insulin-like growth factor-I (IGF-I) epitopes recognized by a panel of 28 monoclonal antibodies (mAbs) is characterized. Pairwise mAb epitope mapping defines eight 'epitopic clusters' (I–VIII) which cover nearly the entire solvent-exposed IGF-I surface. Monoclonal antibody reactivity with 32 overlapping synthetic peptides and with IGF-I mutants is used to associate these epitopic clusters with the probable primary IGF-I sequences recognized. Epitopic cluster I involves residues in the C-domain and the first α-helix of the A-domain; clusters II, V and VII involve principally the B-domain; clusters III and IV map to amino acid sequences (55–70) and (1–13) respectively; cluster VI includes the A- and B-domains; and cluster VIII involves mainly the C-terminal part of the B-domain. Data indicate that this mAb panel defines 14 distinct IGF-I epitopes. The specific inhibition of HEL 92.1.7 IGF-I-promoted proliferation by these mAbs was explored. Direct correlation between mAb affinity and inhibitory activity was observed except in the case of clusters III- and VII-specific mAbs. Finally, the combination of epitopic cluster I and II mAbs detect 0·5–10 ng/ml hIGF-I in a sandwich immunoassay, with no IGF-II crossreactivity. These anti-IGF-I mAbs are, therefore, useful for both the inhibition of IGF-I mitogenic activity and for the quantification of this growth factor. The potential use of this mAb panel in tumor cell growth control is discussed. Journal of Endocrinology (1997) 154, 293–302

Om de kwaliteit van zorg daadwerkelijk te kunnen verbeteren, moeten richtlijnen aan bepaalde kwaliteitseisen voldoen. De ‘Richtlijn voor richtlijnen’ formuleert twintig criteria waaraan Nederlandse richtlijnen zouden moeten voldoen. Aanvullend op de ‘Richtlijn voor richtlijnen’ is een toetsingskader ontwikkeld dat de ontwikkeling en het onderhoud van kwaliteitsstandaarden in Nederland zou moeten stimuleren. Richtlijnen die aan de eisen voldoen, kunnen worden opgenomen in een landelijk register dat een overzicht biedt van alle richtlijnen en zorgstandaarden die optimale zorg beschrijven.

IL-18, a recently identified cytokine synthesized by different cell types, including Kupffer cells, activated macrophages, and keratinocytes, induces IFN-γ production by T cells and NK cells. The cDNA encoding IL-18 with its natural signal peptide was cloned under control of the CMV promoter and injected into the skin of mice. A single intradermal injection of this construction led to efficient in vivo expression of IL-18 in cutaneous dermal cells and induced IFN-γ mRNA production, indicating that it was produced in a biologically active form. In addition, a massive cellular infiltrate was observed in the skin 2 days after injection. When the mice were subsequently infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG), they produced lower levels of anti-BCG Abs than control animals. However, in contrast to their lowered humoral immune response, the mice produced higher amounts of Ag-specific IFN-γ after in vitro restimulation, as compared with the controls. Therefore, injection of DNA encoding IL-18 into the skin modulates both Ag-specific humoral and T cell responses upon mycobacterial infection. It increases the Th1 type response, which may be particularly useful for the development of new immunotherapeutic or immunoprotective approaches against infections by intracellular parasites, such as mycobacteria.

Chemokine receptor-like 1 (CKR-L1) was described recently as a putative seven-transmembrane human receptor with many of the structural features of chemokine receptors. To identify the ligand of CKR-L1, we have studied chemokine-induced calcium mobilization in 293 cells transfected with CKR-L1. Of 20 different chemokines tested, only I-309 was able to elicit a significant calcium mobilization. In addition, I-309 induced the transfectants to migrate in vitro. As expected for chemokine receptor-mediated effects, pertussis toxin, but not cholera toxin, inhibited both the calcium flux and migration of the CKR-L1 transfectants in response to I-309. All of these data support the conclusion that I-309 is a functional ligand for CKR-L1. According to the current chemokine receptor nomenclature, we have designated this gene as CCR8. The murine CCR8 (mCCR8) gene was cloned, and its predicted amino acid sequence showed a 71% identity with that of human CCR8. As human CCR8, mCCR8 is expressed in thymus. Both I-309 and its murine homologue TCA-3 were able to induce calcium mobilization in transiently transfected 293-EBNA cells expressing mCCR8. The affinity of the binding of 125I-labeled TCA-3 to mCCR8 was high (Kd ≈ 2 nM); the binding was prevented completely by an excess of cold TCA-3, and only partially competed (40%) by I-309. The identification of I-309 and TCA-3 as the functional ligands for CCR8 receptors will help to unravel the role of these proteins in physiologic and pathologic situations.

A major goal of current vaccine development is the induction of strong immune responses against protective antigens delivered by mucosal routes. One of the most promising approaches in that respect relies on the use of live recombinant vaccine carriers. In this study, Mycobacterium bovis BCG was engineered to produce an intracellular glutathione S-transferase from Schistosoma haematobium (Sh28GST). The gene encoding Sh28GST was placed under the control of the mycobacterial hsp60 promoter on a replicative shuttle plasmid containing a mercury resistance operon as the only selectable marker. The recombinant Sh28GST produced in BCG bound glutathione and expressed enzymatic activity, indicating that its active site was properly folded. Both intraperitoneal and intranasal immunizations of BALB/c mice with the recombinant BCG resulted in strong anti-Sh28GST antibody responses, which were enhanced by a boost. Mice immunized intranasally produced a mixed response with the production of Sh28GST-specific immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgA in the serum. In addition, high levels of anti-Sh28GST IgA were also found in the bronchoalveolar lavage fluids, demonstrating that intranasal delivery of the recombinant BCG was able to induce long-lasting secretory and systemic immune responses to antigens expressed intracellularly. Surprisingly, intranasal immunization with the BCG producing the Sh28GST induced a much stronger specific humoral response than intranasal immunization with BCG producing the glutathione S-transferase from Schistosoma mansoni, although the two antigens have over 90% identity. This difference was not observed after intraperitoneal administration.

BACKGROUND Evidence based guidelines are needed to guide effective long term follow up (LTFU) of childhood cancer survivors (CCS) at risk of late adverse effects (LAEs). We aimed to ascertain the use of LTFU guidelines throughout Europe and seek views on the need for pan European LTFU guidelines. PROCEDURES One expert clinician from each of 44 European countries was invited to participate in an online survey. Information was sought regarding the use and content of LTFU guidelines in the respondent's centre and country and their views about developing pan European LTFU guidelines. RESULTS Thirty one countries (70) responded including 24 of 26 full EU countries (92). LTFU guidelines were implemented nationally in 17 countries (55). All guidelines included recommendations about physical LAEs specific risk groups and frequency of surveillance and the majority about psychosocial LAEs (70) and healthy lifestyle promotion (65). A minority of guidelines described recommendations about transition to age appropriate LTFU services (22) where LTFU should be performed (22) and by whom (30). Most respondents (94) agreed on the need for pan European LTFU guidelines specifically including recommendations about surveillance for specific physical LAEs (97) action to be taken if a specific LAE is detected (90) minimum requirements for LTFU (93) transition and health promotion (both 87). CONCLUSIONS Guidelines are not universally used throughout Europe. However there is strong support for developing pan European LTFU guidelines for CCS. PanCareSurFup (www.pancare.eu) will collaborate with partners to develop such guidelines including recommendations for hitherto relatively neglected topics such as minimum LTFU requirements transition and health promotion. Pediatr Blood Cancer © 2014 Wiley Periodicals Inc.

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.

Schistosomiasis is a group of severe parasitic diseases, in humans and domestic animals, that are especially of importance in the developing world. No efficacious vaccine is currently available. However, Ab-mediated immune responses against the 28-kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST) appear to be involved in protection. This Ag was produced in recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG). The recombinant protein bound glutathione and expressed enzymatic activity, indicating that the active site of Sm28GST was folded properly. Single i.v., i.p., s.c., or intranasal immunizations with rBCG in BALB/c mice resulted in significant anti-SM28GST Ab responses, which were enhanced by a booster dose. The Ab responses remained high for at least 1 yr after immunization. Analyses of the isotype profiles indicated that i.v. immunized mice produced high titers of anti-Sm28GST IgG2a, and less IgG2b and IgG1. Mice immunized by the s.c. route initially also produced high levels of IgG2a and low titers of IgG1 and IgG2b, but the titers of the latter two isotypes increased gradually thereafter, tending toward a mixed profile. Intraperitoneal immunization provided a mixed profile directly after the first administration. High titers of anti-Sm28GST Abs also corresponded to high levels of neutralization of the enzymatic activity. These results indicate that rBCG induces strong IgG1, IgG2a, and IgG2b, and neutralizing Ab responses against Sm28GST, which has been found to correlate with protection against S. mansoni in humans.

5-year overall survival rates have surpassed 90% for childhood acute lymphocytic leukaemia, but survivors are at risk for permanent health sequelae. Although event-free survival appropriately represents the outcome for cancers with poor overall survival, this metric is inadequate when cure rates are high but challenged by serious, persistent complications. Accordingly, a group of experts in paediatric haematology-oncology, representative of 17 international acute lymphocytic leukaemia study groups, launched an initiative to construct a measure, designated severe toxicity-free survival (STFS), to quantify the occurrence of physician-prioritised toxicities to be integrated with standard cancer outcome reporting. Five generic inclusion criteria (not present before cancer diagnosis, symptomatic, objectifiable, of unacceptable severity, permanent, or requiring unacceptable treatments) were used to assess 855 health conditions, which resulted in inclusion of 21 severe toxicities. Consensus definitions were reached through a modified Delphi process supplemented by two additional plenary meetings. The 21 severe toxicities include severe adverse health conditions that substantially affect activities of daily living and are refractory to therapy (eg, refractory seizures), are without therapeutic options (eg, blindness), or require substantially invasive treatment (eg, cardiac transplantation). Incorporation of STFS assessment into clinical trials has the potential to improve and diversify treatment strategies, focusing not only on traditional outcome events and overall survival but also the frequencies of the most severe toxicities. The two major aims of this Review were to: prioritise and define unacceptable long-term toxicity for patients with childhood acute lymphocytic leukaemia, and define how these toxicities should be combined into a composite quantity to be integrated with other reported outcomes. Although STFS quantifies the clinically unacceptable health tradeoff for cure using childhood acute lymphocytic leukaemia as a model disease, the prioritised severe toxicities are based on generic considerations of relevance to any other cancer diagnosis and age group.