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Much has been written about the patient-physician relationship over the years. This relationship is essential in maintaining trust in the complex arena of modern diagnostic techniques, treatment and prevention, including vaccines and vaccine safety. However, a great deal of this material was written from the viewpoint of clinicians and academics. The patient voice may be positive or negative, fragmented or complex. Information sources are weighed and treated differently, according to the value system and risk perceptions of the individual. In post-trust societies, when people have less confidence in health authorities, communication needs to be more than a paternalistic top-down process. Notions of empowerment and individual patient choice are becoming crucial in medical care. The 'voice of the patient', which includes healthy individuals receiving vaccines, needs to be heard, considered and addressed. With respect to childhood immunizations, this will be the voice of the parent or caregiver. The key to addressing any concerns could be to listen more and to develop a communication style that is trust-based and science-informed. Regulatory agencies are encouraging clinical and patient-reported outcomes research under the umbrella of personalized medicine, and this is an important step forward. This paper attempts to reflect the paradigm shift towards increasing attention to the patient voice in vaccination and vaccine safety., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.) more...

Not only the general public, but also those studying to become health professionals, are struggling to keep up with a growing body of evidence and increasingly complex information about the many different types of vaccines available to date. At the same time, a number of increasingly complex subjects of study are competing for their attention during undergraduate and graduate education. In many medical school curricula in German-speaking countries, the subject of vaccines has been entirely omitted, or is regarded a minor subtopic. During the studies, most medical school curricula in German-speaking countries do not offer obligatory courses and/ or hands-on training vaccinology in vaccination. In Germany, private pediatricians administer the majority of immunizations. Even during postgraduate training programs in pediatrics, which are largely hospital-based, vaccinations are rarely a topic, and vaccinology remains a "hobby" and a "field without lobby" lacking specific certification requirements. Studies of acceptance of vaccines among health professionals and medical students have shown that many may still have their own doubts and uncertainties about vaccines revealing a number of unanswered questions during their studies and postgraduate training. more...

The highly complex and controversial topic of vaccine safety communication warrants innovative, user-centered solutions that would start with gaining mutual respect while taking into account the needs, concerns and underlying motives of patients, parents and physicians. To this end, a non-profit collaborative project was conducted by The Vienna Vaccine Safety Initiative, an international think tank aiming to promote vaccine safety research and communication, and the School of Design Thinking in Potsdam, Germany, the first school for innovation in Europe. The revolutionary concept of the Design Thinking approach is to group students in small multi-disciplinary teams. As a result they can generate ground-breaking ideas by combining their expertise and different points of view. The team agreed to address the following design challenge question: "How might we enable physicians to encourage parents and children to prevent infectious diseases?" The current article describes, step-by step, the ideation and innovation process as well as first tangible outcomes of the project. more...

Objectives: Vaccine safety surveillance is highly dependent on accurate reporting of adverse events following immunization (AEFI). An online survey was conducted to assess the utilization of AEFI reporting standards and pathways among pediatricians in Germany, and in Russia where pediatric specialization begins in medical school., Methods: In May 2011, a 31-item online questionnaire was sent to members of the German Professional Association for Pediatricians (BVKJ) and the Union of Pediatricians of Russia (UPR), capturing information on vaccine safety training, awareness of AEFI reporting pathways, and use of standardized case definitions for the ascertainment of AEFI. A convenience sample of 1,632 completed online surveys was analyzed., Results: Participating pediatricians reported spending approximately 50 min per 8-hour workday on vaccine safety consultations, but only 42 % (56 % UPR, 26 % BVKJ) have ever received any formal vaccine safety training. Two-thirds reported having observed AEFI in their practice, but only one-third utilized standardized case definitions for case ascertainment. Only 35 % of participants named accurate AEFI reporting pathways. Every second pediatrician would report AEFI to institutions that are not primarily in charge of vaccine safety surveillance; the remaining reports would either be lost or delayed. Pediatricians who had received formal vaccine safety training were significantly more likely to apply international safety standards and to report adequately, both at the p < 0.05 level., Conclusion: Pediatricians play a key role in the post-marketing surveillance of vaccine safety. The lack of training represents a missed opportunity. There may be a role for professional societies to improve vaccine safety training. more...

Background: The Kids ITP Tools (KIT) is a disease-specific measure of health-related quality of life for children with immune thrombocytopenia (ITP). To facilitate use in international trials it has been cross-culturally adapted for France, Germany, the United Kingdom and Uruguay. This study assessed the validity and reliability of the translated KIT in comparison to generic quality of life measures., Methods: Children 2-18 years of age with ITP and their parents were recruited in France, Germany, the United Kingdom and Uruguay. Participants completed the KIT, PedsQL and KINDL. We examined the Pearson's correlation between these measures for our pooled sample and estimated the reliability over a 2-week time period. Findings were further explored by country., Results: A total of 127 families (81 children self-reported) participated. Mean child-reported scores were: KIT 74.3 (SD = 15.3), PedsQL 81.3 (SD = 13.0), and KINDL 70.5 (SD = 14.3). Corresponding mean parent proxy-reported scores were: 70.6 (SD = 18.1), 75.7 (SD = 16.8) and 72.3 (SD = 12.7), respectively. Correlation between KIT and the generic measures was consistent with our a priori hypothesis (PedsQL r = 0.54, KINDL r = 0.48, both P < 0.0001). Child KIT scores for newly diagnosed ITP patients were significantly lower than for chronic ITP patients (67.3 vs. 77.3; P = 0.005). There was a significant correlation (P < 0.001) between the child and parent proxy KIT scores (ICC = 0.52). Child KIT test-retest reliability was acceptable at 0.71., Conclusions: The cross-culturally translated KIT is valid and reliable with acceptable correlation to the PedsQL and KINDL. There is a significant difference in child self-reported KIT scores between newly diagnosed and chronic ITP., (Copyright © 2012 Wiley Periodicals, Inc.) more...

Background: Thrombocytopenia with absent radii syndrome is defined by bilateral radius aplasia and thrombocytopenia. Due to impaired thrombopoietin signaling there are only few bone marrow megakaryocytes and these are immature; the resulting platelet production defect improves somewhat over time. A microdeletion on chromosome 1q21 is present in all patients but is not sufficient to form thrombocytopenia with absent radii syndrome. We aimed to refine the signaling defect in this syndrome., Design and Methods: We report an extended study of 23 pediatric and adult patients suffering from thrombocytopenia with absent radii syndrome in order to scrutinize thrombopoietin signal transduction by immunoblotting and gel electrophoretic shift assays. In addition, platelet immunotyping and reactivity were analyzed by flow cytometry. Results were correlated with clinical data including age and platelet counts., Results: Two distinct signaling patterns were identified. Juvenile patients showed abrogated thrombopoietin signaling (pattern #1), which is restored in adults (pattern #2). Phosphorylated Jak2 was indicative of activation of STAT1, 3 and 5, Tyk2, ERK, and Akt, showing its pivotal role in distinct thrombopoietin-dependent pathways. Jak2 cDNA was not mutated and the thrombopoietin receptor was present on platelets. All platelets of patients expressed normal levels of CD41/61, CD49b, and CD49f receptors, while CD42a/b and CD29 were slightly reduced and the fibronectin receptor CD49e markedly reduced. Lysosomal granule release in response to thrombin receptor activating peptide was diminished., Conclusions: We show a combined defect of platelet production and function in thrombocytopenia with absent radii syndrome. The rise in platelets that most patients have during the first years of life preceded the restored thrombopoietin signaling detected at a much later age, implying that these events are uncoupled and that an unknown factor mediates the improvement of platelet production. more...

Background: Platelet counts below normal values define thrombocytopenia. However, platelet counts alone do not reveal the underlying pathomechanism. New blood cell counters provide additional information on platelet size and volume, and enable the distinction of sub-populations. In this preliminary study, we evaluate whether one of these markers can be used for diagnosis of isolated thrombocytopenia in children., Procedure: We provide normal values for mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), platelet large cell ratio (P-LCR), platelet mean-frequent volume (P-MFV), relative immature platelet fraction (IPF%), and absolute IPF (IPF#) for 100 healthy children and analyzed 87 children with thrombocytopenia., Results: In children with platelet production defects, IPF% was low, while in acute immune thrombocytopenia (ITP), IPF% was markedly increased (median 25.2%, P < 0.01), representing accelerated platelet turnover. Interestingly, children diagnosed with acute lymphocytic leukemia (ALL) also had elevated IPF% (median 10%, P < 0.01), suggesting that thrombopoiesis is stimulated despite virtual absence of bone marrow progenitors. Low IPF# was only found in patients with acute ITP., Conclusions: IPF% is a marker for thrombocytopenia due to defective platelet production while IPF#, representing the immature platelet count, might become a practical parameter to distinguish acute ITP from thrombocytopenia in children with newly diagnosed ALL (P < 0.01)., (Copyright © 2010 Wiley-Liss, Inc.) more...

Objective: To investigate the applicability, efficacy, and safety of single-pass albumin dialysis in children., Design: Retrospective data review of uncontrolled clinical data., Setting: University-based pediatric intensive care unit collaborating with a local center for liver transplantation., Patients: Nine children, aged 2 to 15 yrs, who were treated with single-pass albumin dialysis for acute liver failure of various origins under a compassionate-use protocol between 2000 and 2006. All patients met high-urgency liver transplantation criteria., Interventions: Single-pass albumin dialysis was performed as rescue therapy for children with acute liver failure., Measurements and Main Results: The decrease in hepatic encephalopathy (grades 1-4) and the serum levels of bilirubin, bile acids, and ammonium were measured to assess the efficacy of detoxification. As a measure of liver synthesis function, thromboplastin time and fibrinogen were analyzed. The safety of the procedure was assessed by documenting adverse effects on mean arterial blood pressure, platelet count, and clinical course. Seven out of nine patients were bridged successfully to either native organ recovery (n = 1) or liver transplantation (n = 6), one of them twice. Six out of nine patients undergoing single-pass albumin dialysis (ten treatments) survived. In six patients, hepatic encephalopathy could be reduced at least by one degree. Ammonium, bilirubin, and bile acid levels decreased in all patients. One patient had an allergic reaction to albumin., Conclusions: In childhood acute liver failure, treatment with single-pass albumin dialysis was generally well tolerated and seems to be effective in detoxification and in improving blood pressure, thus stabilizing the critical condition of children before liver transplantation and facilitating bridging to liver transplantation. It may be beneficial in avoiding severe neurologic sequelae after acute liver failure and thereby improve survival. Single-pass albumin dialysis is an inexpensive albumin-based detoxification system that is easy to set up and requires little training. Whether and to what extent single-pass albumin dialysis can support children with acute liver failure until native liver recovery remains unclear. more...

The likelihood of identifying peptides of sufficient quality for the development of effective cancer vaccines by screening of phage display libraries is low. Here, we introduce the sequential application of systematic amino acid substitution by SPOT synthesis. After the substitution of two amino acids within the sequence of a phage display-derived mimotope of disialoganglioside GD2 (mimotope MA), the novel mimotope C3 showed improved GD2 mimicry in vitro. Peptide vaccination with the C3 mimotope induced an 18-fold increased anti-GD2 serum response associated with reduction of primary tumour growth and spontaneous metastasis in contrast to MA mimotope controls in a syngeneic neuroblastoma model. In summary, SPOT provides an ideal optimisation tool for the development of phage display-derived cancer vaccines. more...

Children with relapsed and refractory acute lymphoblastic leukemia (ALL) still face a critical prognosis. We tested the hypothesis that targeted calicheamicin theta (θ) using an anti-CD19-immunoconjugate may provide an effective treatment strategy for CD19(+) ALL. Calicheamicin θ is a rationally designed prodrug of the natural enediyene calicheamicin γ, obtained by total synthesis. It offers the advantage of increased in vivo stability and 1000-fold higher antitumor potency over calicheamicin γ. First, we demonstrate efficacy of calicheamicin θ against primary pre-B leukemic cells and multidrug-resistant leukemia cell lines (IC(50) = 10(-9) to 10(-12) M). Second, conjugation of calicheamicin θ to an internalizing murine anti-CD19 monoclonal antibody was demonstrated to affect neither calicheamicin θ mediated cytotoxicity nor binding of the antibody to the target molecule. Third, anti-CD19-calicheamicin θ immunoconjugate revealed a maximum tolerated dose of 10 μg/kg and CD19-specific and long-lasting eradication of established leukemia was demonstrated in a xenograft model. Finally, we show that the antileukemic effect of anti-CD19-calicheamicin θ is mediated by induction of apoptosis proceeding through the caspase-mediated mitochondrial pathway. On the basis of these results, we conclude that anti-CD19-calicheamicin θ immunoconjugates may offer a novel and effective approach for the treatment of relapsed CD19(+) ALL. more...

Neuroblastoma (NB) is a challenging malignancy of the sympathetic nervous tissue characterized by a very poor prognosis. One important marker for NB is the expression of tyrosine hydroxylase (TH), the first-step enzyme of catecholamine biosynthesis. We could show stable and high TH gene expression in 67 NB samples independent of the clinical stage. Based on this observation, we addressed the question of whether xenogeneic TH DNA vaccination is effective in inducing an anti-NB immune response. For this purpose, we generated three DNA vaccines based on pCMV-F3Ub and pBUD-CE4.1 plasmids encoding for human (h)THcDNA (A), hTH minigene (B), and hTHcDNA in combination with the proinflammatory cytokine interleukin 12 (C), and tested prophylactic and therapeutic efficacy to suppress primary tumor growth and spontaneous metastasis. Here we report that xenogeneic TH DNA vaccination was effective in eradicating established primary tumors and inhibiting metastasis. Interestingly, this effect could not be enhanced by adding the Th1 cytokine interleukin 12. However, increased IFN-gamma production and NB cytotoxicity of effector cells harvested from vaccinated mice suggested the participation of tumor-specific CTLs in the immune response. The depletion of CD8(+)T cells completely abrogated the hTH vaccine-mediated anti-NB immune response. Furthermore, rechallenging of surviving mice resulted in reduced primary tumor growth, indicating the induction of a memory immune response. In conclusion, xenogeneic immunization with TH-derived DNA vaccines is effective against NB, and may open a new venue for a novel and effective immunotherapeutic strategy against this challenging childhood tumor. more...

The inhibitor of apoptosis protein survivin is highly expressed in neuroblastoma (NB) and survivin-specific T cells were identified in Stage 4 patients. Therefore, we generated a novel survivin minigene DNA vaccine (pUS-high) encoding exclusively for survivin-derived peptides with superior MHC class I (H2-K(k)) binding affinities and tested its efficacy to suppress tumor growth and metastases in a syngeneic NB mouse model. Vaccination was performed by oral gavage of attenuated Salmonella typhimurium SL7207 carrying pUS-high. Mice receiving the pUS-high in the prophylactic setting presented a 48-52% reduction in s.c. tumor volume, weight and liver metastasis level in contrast to empty vector controls. This response was as effective as a survivin full-length vaccine and was associated with an increased target cell lysis, increased presence of CD8(+) T-cells at the primary tumor site and enhanced production of proinflammatory cytokines by systemic CD8(+) T cells. Furthermore, depletion of CD8(+) but not CD4(+) T-cells completely abrogated the pUS-high mediated primary tumor growth suppression, demonstrating a CD8(+) T-cell mediated effect. Therapeutic vaccination with pUS-high led to complete NB eradication in over 50% of immunized mice and surviving mice showed an over 80% reduction in primary tumor growth upon rechallenge in contrast to controls. In summary, survivin-based DNA vaccination is effective against NB and the rational minigene design provides a promising approach to circumvent potentially hazardous effects of using full length antiapoptotic genes as DNA vaccines. more...

Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I antigen H2-K(k) according to the prediction program SYFPEITHI and computer modeling of epitopes into the MHC class I antigen binding groove. Subsequently, a DNA minigene vaccine was generated based on the expression vector pCMV-F3Ub encoding mutated ubiquitin (Gly(76) to Ala(76)) and mTH3. Prophylactic and therapeutic efficacies of this vaccine were established following oral delivery with attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependent on ubiquitin and high affinity of the mTH epitopes to MHC class I antigens. Specifically, we showed a crucial role for minigene expression as a stable ubiquitin-Ala(76) fusion peptide for vaccine efficacy. The immune response following the mTH3 DNA minigene vaccination was mediated by CD8(+) T cells as indicated by infiltration of primary tumors and TH-specific cytolytic activity in vitro. Importantly, no cell infiltration was detectable in TH-expressing adrenal medulla, indicating the absence of autoimmunity. In summary, we show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy. more...

Background: The replacement of established evidence-based cancer therapy protocols (mainstream therapy) by unevaluated complementary and alternative medicine (CAM) is a challenge in pediatric oncology. We tested the hypothesis that oral application of L-lysine and ascorbic acid (Lysin C Drink) in combination with epigallocatechin-gallate (EGCG) and amino-acids (Epican forte) is effective in a preclinical model of neuroblastoma., Methods: Primary tumors and spontaneous metastases were induced in A/J mice by injection of NXS2 neuroblastoma cells. Mice were treated by daily oral gavage with L-lysine and ascorbic acid (Lysin C Drink) (equivalent to 150 mg ascorbic acid/day/mouse) (treatment A) or with EGCG plus ascorbic- and amino-acids (Epican forte) (9.2 mg/mouse) (treatment B). Treatment A was started in the prophylactic setting (7 days before tumor cell injection) as well as in the therapeutic setting (1 day after tumor cell inoculation). Finally, treatment B was evaluated alone and in combination with treatment A in the therapeutic setting. The effect on primary tumor growth and the development of spontaneous liver metastases was evaluated., Results: L-lysine and ascorbic acid (Lysin C Drink) and EGCG plus ascorbic- and amino-acids (Epican forte) are ineffective in reduction of primary tumor growth and prevention of spontaneous liver metastases in this model., Conclusions: Neither a formal clinical development nor the use of these substances can be recommended for neuroblastoma., ((c) 2007 Wiley-Liss, Inc.) more...

Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by perturbed or absent beating of motile cilia, which is referred to as Kartagener syndrome (KS) when associated with situs inversus. We present a German family in which five individuals have PCD and one has KS. PCD was confirmed by analysis of native and cultured respiratory ciliated epithelia with high-speed video microscopy. Respiratory ciliated cells from the affected individuals showed an abnormal nonflexible beating pattern with a reduced cilium bending capacity and a hyperkinetic beat. Interestingly, the axonemal ultrastructure of these respiratory cilia was normal and outer dynein arms were intact, as shown by electron microscopy and immunohistochemistry. Microsatellite analysis indicated genetic linkage to the dynein heavy chain DNAH11 on chromosome 7p21. All affected individuals carried the compound heterozygous DNAH11 mutations c.12384C>G and c.13552&#95;13608del. Both mutations are located in the C-terminal domain and predict a truncated DNAH11 protein (p.Y4128X, p.A4518&#95;A4523delinsQ). The mutations described here were not present in a cohort of 96 PCD patients. In conclusion, our findings support the view that DNAH11 mutations indeed cause PCD and KS, and that the reported DNAH11 nonsense mutations are associated with a normal axonemal ultrastructure and are compatible with normal male fertility., ((c) 2007 Wiley-Liss, Inc.) more...

We report the uncommon clinical course of tetanus in a completely immunized 14-year-old boy. His initial symptoms, which included a flaccid paralysis, supported a diagnosis of botulism. Preliminary mouse-test results with combined botulinum antitoxins A, B, and E, obtained from tetanus-immunized horses, backed this diagnosis. The change in his clinical course from paralysis to rigor and the negative, more specific, botulinum mouse test with isolated botulinum antitoxins A, B, and E, obtained from nonvaccinated rabbits, disproved the diagnosis of botulism. Tetanus was suspected despite complete vaccination. The final results of a positive mouse test performed with isolated tetanus antitoxin confirmed the diagnosis. Adequate treatment was begun, and the boy recovered completely. more...

Fractalkine (FKN) is a unique CX3C chemokine (CX3CL1) known to induce both adhesion and migration of leukocytes mediated by a membrane-bound and a soluble form, respectively. Its function is mediated through CX3C receptor (CX3CR), which is expressed by T(H)1 immune cells including T cells and natural killer (NK) cells. FKN was shown to be expressed in >90% of 68 neuroblastoma samples as determined by cDNA microarray analysis. Here, we characterized the effect of FKN in the neuroblastoma microenvironment using a syngeneic model genetically engineered to secrete FKN. We show FKN-mediated migration, adhesion, and IFN-gamma secretion of immune effector cells, but limited antineuroblastoma activity, in vitro and in vivo. Therefore, we tested the hypothesis that a combined increase of FKN and interleukin-2 (IL-2) in the neuroblastoma microenvironment induces an effective antitumor immune response. For this purpose, IL-2 was targeted to ganglioside GD2, which is highly expressed on neuroblastoma tissue, using an anti-GD2 antibody IL-2 immunocytokine (ch14.18-IL-2). Only mice bearing FKN- and IL-2-enriched neuroblastoma tumors exhibited a reduction in primary tumor growth and a complete eradication of experimental liver metastases. The depletion of T cells and NK cells in vivo abrogated the effect, and these effector cells showed the highest cytolytic activity in vitro. Finally, only the FKN- and IL-2-enriched neuroblastoma microenvironment resulted in T-cell activation and the release of proinflammatory cytokines. In summary, we showed for the first time the immunologic mechanisms by which targeted IL-2 treatment of neuroblastoma with an FKN-rich microenvironment induces an effective antitumor response. more...

The development of pulmonary arterial hypertension (PAH) is the leading cause of mortality in patients with thalassemia and sickle cell anemia and was reported to occur in hemolytic anemias such as hereditary stomatocytosis, and paroxysmal nocturnal hemoglobinuria. Here, we report for the first time on the development of PAH in a patient with Hb-Mainz hemolytic anemia. Hb-Mainz is an unstable hemoglobin variant resulting from mutations at codon 98 of the beta chain gene (Val>Glu) characterized by severe chronic hemolytic anemia. The development of PAH in this patient further supports the contention that there is a clinical syndrome of hemolysis-associated development of PAH. more...

Disialoganglioside GD2 is an established target for immunotherapy in neuroblastoma. We tested the hypothesis that active immunization against the glycolipid GD2 using DNA vaccines encoding for cyclic GD2-mimicking decapeptides (i.e., GD2 mimotopes) is effective against neuroblastoma. For this purpose, two GD2 peptide mimotopes (MA and MD) were selected based on docking experiments to anti-GD2 antibody ch14.18 (binding free energy: -41.23 kJ/mol for MA and -48.06 kJ/mol for MD) and Biacore analysis (K(d) = 12.3 x 10(-5) mol/L for MA and 5.3 x 10(-5) mol/L for MD), showing a higher affinity of MD over MA. These sequences were selected for DNA vaccine design based on pSecTag2-A (pSA) also including a T-cell helper epitope. GD2 mimicry was shown following transfection of CHO-1 cells with pSA-MA and pSA-MD DNA vaccines, with twice-higher signal intensity for cells expressing MD over MA. Finally, these DNA vaccines were tested for induction of tumor protective immunity in a syngeneic neuroblastoma model following oral DNA vaccine delivery with attenuated Salmonella typhimurium (SL 7207). Only mice receiving the DNA vaccines revealed a reduction of spontaneous liver metastases. The highest anti-GD2 humoral immune response and natural killer cell activation was observed in mice immunized with the pSA-MD, a finding consistent with superior calculated binding free energy, dissociation constant, and GD2 mimicry potential for GD2 mimotope MD over MA. In summary, we show that DNA immunization with pSA-MD may provide a useful strategy for active immunization against neuroblastoma. more...

The cause of idiopathic thrombocytopenia (ITP) is largely unknown, although the underlying pathophysology is an autoimmune process. Anti-idiotypic antibodies and their role on regulatory T-cells might play an important role in the switch from acute to chronic ITP. The exact interaction remains to be elucidated. The effects of the dysregulated immune system and of the autoimmune process on thrombocytopoiesis and megakaryopiesis are ill defined. Therapy of acute and chronic ITP is directed to the risk of bleeding., (Copyright (c) 2006 Wiley-Liss, Inc.) more...

Background: Intracellular antibodies (intrabodies) have been used for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. Intrabodies present an alternative to methods of gene inactivation that target genomic DNA or m-RNA, such as RNA interference. Several studies suggest that the VEGF receptor pathway and the Tie-2 pathway are independent and essential mediators of angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects in tumor growth., Methods: In order to generate a precise tool for the simultaneous silencing of two independent signaling pathways essential for angiogenesis, we developed a bispecific, tetravalent endoplasmatic reticulum (ER)-targeted intradiabody, against Tie-2 and VEGF-R2., Results: Using an adenovirus mediated gene delivery system, we achieved the simultaneous downregulation of the two cell surface receptors and demonstrate that the intradiabody is significantly more powerful with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2 when compared to scFv intrabodies. In a human melanoma xenograft mouse model, we could show that blockade of both VEGF-R2 and Tie-2 pathways or the VEGF receptor pathway alone resulted in a significant inhibition of tumor growth and tumor angiogenesis (92.2 % and 74.4 %)., Conclusion: We demonstrate for the first time that simultaneous inhibition of the VEGF and the Tie-2 receptor pathways result in additive antiangiogenic effects in vitro and in vivo as compared to single VEGF receptor pathway blockade, strengthening the potential of simultaneous targeting of multiple pathways as a therapeutic strategy. more...

Background: A trivalent measles-mumps-rubella (MMR) vaccine (MMR Berna) has been developed with a new mumps component, BBM-18, to replace a previously licensed MMR vaccine containing the Rubini mumps strain. Previous studies showed Rubini to confer insufficient long term protection against mumps infection. This study compared the immunogenicity and safety of MMR Berna, which is produced entirely in human diploid cells, with those of the licensed vaccine M-M-RVax (Merck & Co.)., Methods: We vaccinated 467 subjects, 12-24 months of age, in an open, randomized (1:1), phase II, multicenter study. Antibody titers were determined for each vaccine component with a plaque neutralization test (PNT) and a commercial enzyme-linked immunosorbent assay. Solicited local and systemic reactions were recorded in subject diaries for 6 weeks after vaccination., Results: Seroconversion rates 6 to 8 weeks after vaccination for measles and rubella were statistically comparable for the 2 vaccines. However, mumps seroconversion rates were highly assay dependent, with significant differences being measured with the enzyme-linked immunosorbent assay (Berna, 77.4%; Merck, 91.3%; P < 0.001) but not the PNT (Berna, 84.8%; Merck, 87.6%; P = 0.42). The overall rate of systemic reactions was lower in the MMR Berna group (36.8% versus 45.9%; P < 0.05), including a significantly lower rate of fever of >38 degrees C (37.2% versus 51.8%; P < 0.01)., Conclusions: MMR Berna was statistically noninferior to M-M-RVax with respect to seroconversion rates, and the BBM-18 strain elicited a level of functional antimumps antibodies comparable to the Jeryl Lynn strain, as measured with the PNT. Overall, MMR Berna was better tolerated than the comparison vaccine, particularly with respect to the frequency of fever. more...

The induction of tumor protective immunity against neuroblastoma remains a major challenge for active immunotherapy. Fractalkine is a unique Th1 CX3C chemokine known to induce adhesion and migration of leukocytes mediated by both, a membrane-bound and soluble form, respectively. Here, we tested the hypothesis that chemokine gene therapy with fractalkine (FKN) induces an effective anti-neuroblastoma immune response amplified by targeted IL-2 using the anti-GD2 antibody ch14.18 fused with IL-2 (ch14.18-IL-2). For this purpose, NXS2 cells were genetically engineered to stably produce murine FKN (NXS2-FKN). Transcription and expression of the mFKN gene in tumor tissue of mice inoculated with NXS2-FKN cells were demonstrated in vivo. Importantly, mFKN exhibited a reduction in primary tumor growth and spontaneous liver metastases in syngenic A/J mice. This effect was boosted by targeted IL-2 using small non-curative doses of ch14-18-IL-2. The amplification of the FKN induced immune response was specific, since a non-specific antibody-IL-2 fusion protein ch225-IL-2 was ineffective. In summary, we demonstrated for the first time that chemokine gene therapy is amplified by targeted IL-2 suggesting a combination of both strategies as an adjuvant therapy for neuroblastoma. more...

The disruption of self-tolerance against neuroblastoma is the ultimate goal of an effective DNA-vaccine. We demonstrate the induction of protective immunity against syngeneic murine NXS2 neuroblastoma in A/J mice following vaccination with tyrosine hydroxylase (TH)-derived antigens. Oral gene delivery was accomplished using an attenuated strain of Salmonella typhimurium as a carrier harboring vectors encoding for mouse tyrosine hydroxylase (mTH) antigens. Vaccination was effective in protecting animals from a lethal challenge with wild-type NXS2 tumor cells. These findings were extended by comparing efficacy of mTH minigene vaccines with a minigene vaccine comprising three novel epitopes isolated fom NXS2 neuroblastoma cells. For this purpose, MHC class I was immunoprecipitated from NXS2 cell lysates, and peptides were eluted and examined in tandem-mass spectrometry analysis. This led to the identification of three novel natural MHC class I peptide ligands: TEALPVKLI, from ribonucleotide reductase M2; NEYIMSLI, from Ser/Thr protein phosphatase 2A; and FEMVSTLI, of unknown origin. Two minigenes were constructed, one encoding for the three novel epitopes and the second for three known mTH-derived epitopes with high predicted binding affinity to MHC class I, by cloning them into the mammalian expression vector pCMV-3FUB. Immunized mice showed a reduction in primary tumor growth and the absence of spontaneous liver metastasis in the majority of animals. Importantly, there was no significant difference between the two minigenes, suggesting that, compared with tumor peptide isolation, mTH epitope prediction is similarly effective for designing efficient DNA-minigene vaccines. In summary, these findings establish proof of the concept that disruption of self-tolerance against neuroblastoma-associated epitopes may be an effective adjuvant therapeutic strategy. more...

The diagnosis of Kawasaki disease is based on 6 clinical criteria, 5 of which must be fulfilled. The presence of uncommon symptoms in addition to the classic criteria can be as misleading as the lack of common ones. Here we report 2 infants with marked pulmonary symptoms in the course of Kawasaki disease who were initially diagnosed with pneumonia. more...

Objective: To compare the immunogenicity and safety of a virosome-adjuvanted influenza vaccine (Inflexal V; Berna Biotech, Berne, Switzerland) and a split influenza vaccine (Fluarix; GlaxoSmithKline Biologicals, Rixensart, Belgium) in children., Subjects and Methods: The subjects, 453 children ages 6 to 71 months, were stratified into primed and unprimed and age groups (6 to 35 and 36 to 71 months) and then randomized 1:1 to receive virosome-adjuvanted (n = 224) or split influenza vaccine (n = 229), a half or full dose was given intramuscularly according to age. Unprimed children received a second dose after 4 weeks. Blood samples (n = 326) collected pre-and 28 days postvaccination were analyzed by hemagglutination inhibition test. Safety assessments were made at baseline and follow-up visits by the investigators and by parents for the 4 days after vaccinations., Results: Both vaccines induced an effective immune response. Seroconversion rates (>4-fold titer rise) against the WHO recommended strains A/New Caledonia (H3N2), A/Moscow (H1N1) and B/Hongkong (B) were 80.1, 66.0 and 90.4% for the virosome-adjuvanted and 75.9, 62.9 and 89.4% for the split influenza vaccine, respectively. Unprimed children's seroconversion rates for H3N2 were significantly higher (P = 0.02) for the virosome-adjuvanted (88.8%) than for split influenza vaccine (77.5%). Seroprotection rates (titer of > 40) for H3N2, H1N1 and B, respectively, were 87.8, 80.1 and 90.4% after vaccination with the virosome-adjuvanted vaccine and 82.9, 78.2 and 89.4% after the split influenza vaccine. Unprimed children's seroprotection rate was significantly higher (P = 0.03) for H3N2 after the virosome-adjuvanted (88.8%) than those for the split influenza vaccine (78.3%). Equivalent geometric mean titer fold increases were evident for both vaccines. No serious adverse events were seen. Pain/ tenderness, redness and swelling/induration was found in 25.4, 11.2 and 8.9% for the virosome-adjuvanted vaccine and in 24.0, 9.2 and 6.1% for the split influenza vaccine, respectively. The rates of fever, malaise/irritability and shivering was 6.3, 11.6 and 2.7% for the virosome-adjuvanted vaccine and 8.3, 11.8 and 2.6% for the split influenza vaccine, respectively., Conclusions: The virosome-adjuvanted influenza vaccine showed greater immunogenicity over the split influenza vaccine in unprimed children and showed a trend toward better immunogenicity in the rest of the study population. Both vaccines were well-tolerated. more...

In December 2000 and February 2001, two children with suspected meningococcal disease were admitted to our pediatric intensive unit. Their Glasgow Meningococcal Septicaemia Prognostic score was 12 points. General treatment including antibiotics, steroids in case of meningitis, and fluid replacement, was performed. Despite appropriate volume replacement, intubation and ventilation, noradrenaline and adrenaline continuous infusions < or =1.0 microg/kg/min, and additional bolus infusions, cardiac output deteriorated within minutes in both children. Calcium and bicarbonate were given without sustained effect. Echocardiography demonstrated no pericardial effusion and shortening fraction was <10%. External cardiac massage had to be performed immediately in one case for electromechanical uncoupling. Both patients received a bolus of enoximone 2 mg/kg and 5 mg/kg body weight, respectively, followed by a continuous infusion of 20-23 microg/kg/min. Thereafter, both children had an adequate blood pressure and their shortening fraction increased to >30%. Within minutes, the catecholamine infusion could be reduced in both patients. The children completely recovered from their life-threatening situations. In patients with severe prolonged catecholamine and volume refractory endotoxin shock in Waterhouse-Friderichsen syndrome, even with electromechanical uncoupling and complete myocardial arrest, enoximone can immediately restore myocardial contractility. more...

Acute gastroenteritis is a major source of morbidity and mortality among young children in developed and developing countries. Enteropathogenic viruses are regarded as particularly relevant causative agents. Between February 2001 and January 2002, fecal specimens were obtained from German children admitted to hospital with acute gastroenteritis and examined for rotaviruses, Noroviruses, enteric adenoviruses, and astroviruses using (RT-)PCR methods. Of the 59% (129/217) samples positive for > or =1 viral agent, 79% (102/129) carried rotavirus, whereas Norovirus was detected in 35% (45/129), enteric adenovirus in 14% (18/129), and astrovirus in 4% (5/129). Thirty-eight specimens contained at least two enteropathogenic viruses, with the majority of coinfections attributable to rotavirus/Norovirus dual infections. Sequence analysis revealed a cocirculation of G1, G3, G4, and G9 type rotavirus with G1 being the most common and G9 the second most common rotavirus G-type. Emergence of G9 rotaviruses in Germany may have implications for future vaccine development. A variety of Norovirus genotypes, most belonging to GGII, were found. Apart from subgenus F, adenovirus related genetically to subgenera A-C were detected. All astroviruses belonged to genotype 1. This is the first study concerning German children admitted to hospital that assesses the relative importance of these viruses by nested (RT-) PCR methods., (Copyright 2003 Wiley-Liss, Inc.) more...

Effective chemotherapy in neuroblastoma is limited by poor anti-tumor efficacy, systemic toxicity and the induction of drug resistance. Here, we provide further evidence that a hydrolytic activated prodrug design may overcome these problems. For this purpose, VP-16 was functionally blocked by a carbonate linker to generate two novel chemically stable prodrugs of VP-16, ProVP-16 I and II. We demonstrate profoundly different biological effects in vitro and in vivo of the prodrugs compared to parental VP-16. First, we established an up to >2 log higher in vitro toxicity of the two prodrugs compared to VP-16 on a panel of neuroblastoma cell lines. The highest increase of prodrug mediated cytotoxicity was observed in multi drug resistant cell lines. Second, in vivo studies showed a maximum tolerated dose (MTD) of ProVP-16 II (60 mg/kg), which was at least threefold higher than that of VP-16 (20 mg/kg). Tests of ProVP-16 II in a syngeneic NXS2 neuroblastoma model indicated that mice treated with this prodrug at 1/3 of the MTD was as effective as VP-16 parental compound used at the MTD in suppression of tumor growth. In summary, the etoposide prodrugs proved effective and less toxic and are therefore highly promising new anti-neuroblastoma compounds. more...

The induction of T-cell mediated immunity against neuroblastoma is a challenge due to poor immunogenicity of this malignancy. Here, we demonstrate the induction of protective immunity in a syngeneic murine neuroblastoma model following vaccination with minigenes comprising of three novel natural MHC class I ligands. First, after immunoprecipitation of MHC class I from NXS2 cells, peptides were eluted and examined in tandem-MS analysis which lead to the identification of three novel natural MHC class I peptide ligands, TEALPVKLI from ribonucleotide reductase M2, NEYIMSLI from Ser/Thr protein phosphatase 2A and FEMVSTLI with unknown origin. Second, we constructed two different minigenes, one encoding for the three novel epitopes and the second for three known mTH derived epitopes with high predicted binding affinity to MHC class I by cloning them into the mammalian expression vector pCMV-3FUB. This lead to constructs with an ubiquitin-tag upstream the inserted epitopes in order to facilitate proteasomal degradation. Furthermore the epitopes were separated by a spacer peptide (AAY), which proved to be a preferential proteasome cleavage site. Third, we demonstrate the induction of protective immunity against neuroblastoma using an attenuated strain of Salmonella typhimurium as a carrier harboring pCMV 3FUb vectors encoding for the two minigenes. These findings establish proof of concept that disruption of self tolerance against neuroblastoma associated epitopes may be an effective adjuvant therapeutic strategy. more...

Tumor directed cytotoxic therapy is one of the major challenges for the success of chemotherapy. In order to accomplish this goal in neuroblastoma, we rationally designed a prodrug of etoposide as substrate for tyrosine hydroxylase, a well established neuroblastoma associated enzyme. Here, we report synthesis and characterization of a 3,4 dihydroxy-phenyl carbamate derivative of etoposide. In order to demonstrate activation by tyrosine hydroxylase, the coding sequence of murine tyrosine hydroxylase was generated by reverse transcriptase-polymerase chain reaction from NXS2 neuroblastoma cells and cloned into the pRSET-A bacterial expression vector. The enzyme was expressed in Escherichia coli, characterized by Western blot and enzymatic activity was demonstrated by conversion of tyrosine into DOPA in the presence of cofactors using reversed phase high-performance liquid chromatography. Under these enzymatic conditions, we demonstrate conversion of 3,4 dihydroxy-phenyl carbamate prodrug into free etoposide. This effect was clearly mediated by the enzyme since bacteria transformed with the empty vector were ineffective of prodrug activation. Furthermore, tyrosine hydroxylase positive cells exposed to the etoposide prodrug were effectively killed in contrast to tyrosine hydroxylase negative controls. These findings demonstrate that etoposide can be designed as a prodrug substrate for tyrosine hydroxylase and thereby establish proof of concept for neuroblastoma directed enzyme prodrug therapy. more...

Effective therapy of high-risk leukemia with established cytotoxic drugs may be limited by poor antitumor efficacy, systemic toxicity, and the induction of drug resistance. Here, we provide the first evidence that hydrolytically activated prodrugs may overcome these problems. For this purpose, VP16 was functionally blocked by hydrolytically cleavable carbonate linkers with unique characteristics to generate 2 novel prodrugs of VP16. First, we established a more than 3-log higher efficacy of the 2 prodrugs compared with VP16 on a panel of naturally drug-resistant tumor cell lines. Second, the prodrugs did overcome VP16-induced multidrug resistance-1 gene (MDR-1)-mediated multidrug resistance in vitro in a newly established VP16-resistant T-cell leukemia cell line MOVP-3 by functionally blocking MDR-1-mediated efflux. Third, in vivo studies showed a maximum tolerated dose of ProVP16-II (> 45mg/kg), which was at least 3-fold higher than that of VP16 (15 mg/kg). Finally, tests of ProVP16-II in a multidrug-resistant xenograft model of T-cell leukemia expressing MDR-1 indicated that only the mice treated with this prodrug revealed a complete and long-lasting regression of established, drug-resistant leukemia. In summary, the hydrolytically activated etoposide prodrugs proved effective against multidrug-resistant T-cell leukemia in vitro and in vivo and provide proof of concept for a highly promising new strategy for the treatment of MDR-1 drug-resistant malignancies. more...

The development of novel strategies for the treatment of malignancies by successful intervention in advanced stage disease is a major challenge in oncology. We tested the hypothesis that this can be achieved by the rational design of taxoid onium salts modified at C-7 and C-2' positions. The characterization of these molecules revealed a dramatically improved water solubility and prodrug behavior in plasma. Specifically, all compounds released parental paclitaxel with half-lives ranging from 0.9 to 180 min. In the absence of plasma, only the 2'-(N-methylpyridinium acetate) derivative of paclitaxel (2'-MPA-paclitaxel) revealed a complete abrogation of paclitaxel specific microtubule assembly disassembly dynamics and a 3 log reduction in cellular binding, indicating that reversible blockage of the C-2' position by methylpyridinium acetate yields a true paclitaxel prodrug. Structure/activity profiles of all compounds in tissue culture revealed cytotoxicity effective at picomolar concentrations with a panel of 16 cancer cell lines in contrast to 4 nonmalignant cell lines. Importantly, the decisive cytotoxic potential observed in vitro for all compounds correlated only with in vivo findings for 2'-MPA-paclitaxel. Specifically, the 2'-MPA-paclitaxel prodrug induced regression of primary tumors in three xenograft models of nonsmall cell lung carcinoma, ovarian carcinoma and prostate cancer, in contrast to ineffective C-7 derivatives and parental paclitaxel. At the same time, a reduced systemic toxicity of 2'-MPA-paclitaxel was observed in contrast to a far more toxic parental paclitaxel. Taken together, these findings demonstrate that the 2'-MPA-paclitaxel prodrug is a promising new candidate for cancer therapy. more...

Two 4'-propylcarbonoxy derivatives (2,3) of etoposide (1), a topoisomerase II inhibitor, were synthesized and evaluated as potential prodrugs for anticancer therapy. Their activation via hydrolysis mechanisms was determined as a function of pH in buffer solutions, in human serum and in the presence of carboxyl ester hydrolase. Cytotoxicity was determined on various tumor cell lines and compared to the parent compound. On cell lines exhibiting resistance to etoposide we observed an enhanced cytotoxicity of the prodrugs of up to three orders of magnitude. more...

The successful induction of T cell-mediated protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. Here, we demonstrate that the induction of tumor-protective immunity by IL-12 in a murine neuroblastoma model depends entirely on the CXC chemokine IFN-gamma-inducible protein 10 (IP-10). This was established by in vivo depletion of IP-10 with mAbs in mice vaccinated against NXS2 neuroblastoma by gene therapy with a linearized, single-chain (sc) version of the heterodimeric cytokine IL-12 (scIL-12). The efficacy of IP-10 depletion was indicated by the effective abrogation of scIL-12-mediated antiangiogenesis and T cell chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2 cells. These findings were extended by data demonstrating that IP-10 is directly involved in the generation of a tumor-protective CD8+ T cell-mediated immune response during the early immunization phase. Four lines of evidence support this contention: First, A/J mice vaccinated with NXS2 scIL-12 and depleted of IP-10 by two different anti-IP-10 mAbs revealed an abrogation of systemic-protective immunity against disseminated metastases. Second, CD8+ T cell-mediated MHC class I Ag-restricted tumor cell lysis was inhibited in such mice. Third, intracellular IFN-gamma expressed by proliferating CD8+ T cells was substantially inhibited in IP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth, systemic tumor protective immunity was completely abrogated in mice depleted of IP-10 in the early immunization phase, but not if IP-10 was depleted only in the effector phase. These findings suggest that IP-10 plays a crucial role during the early immunization phase in the induction of immunity against neuroblastoma by scIL-12 gene therapy. more...

We present an 11-year-old girl with sensory and autonomic neurological dysfunction, and respiratory insufficiency caused by recurrent aspiration. The diagnosis of familial dysautonomia (FD) was confirmed by a missing axonal flare to histamine, miosis in response to conjunctival methacholine and homozygous polymorphic linked markers DS58(18) and DS159(7) on chromosome 9. Ashkenazi Jewish descent could not be ascertained by history. A variety of positive tests for autoantibodies were initially interpreted as evidence for systemic lupus erythematosus vs. overlap syndrome with pulmonary, cerebral, skin, and ocular involvement. The diagnosis of FD was delayed because of the rarity of this disorder in Germany (second case reported). We discuss possible explanations for the misleading immunological findings, including interference by antibodies binding to milk proteins used as blocking reagents in enzyme-linked immunoassays and circulating immune-complexes due to chronic aspiration pneumonitis. more...

The development of novel chemotherapy strategies based on prodrugs remains a major challenge for effective treatment of malignancies. We tested the hypothesis that this can be achieved by a prodrug of paclitaxel where one biologically active center, represented by the C7 hydroxyl group, was blocked by a dihydroxypropyl side chain which can be hydrolytically cleaved by a pH-dependent, slow-release mechanism. The prodrug was synthesized by condensation of solketal chloroformate with the C7 hydroxyl group of paclitaxel followed by a ring-opening reaction to the dihydroxyl derivative. The cytotoxicity of the prodrug was similar to paclitaxel, when tested in vitro against a variety of human tumor cell lines. In vitro cell cycle analysis indicated that concentrations within the micromolar range of both drug and prodrug are required to induce sufficient G2M arrest. The hydrophilic paclitaxel prodrug proved to be more than 50-fold more water soluble than the parental drug and effectively converted to paclitaxel by pH dependent hydrolysis. Importantly, the prodrug could be used at a 3-fold higher maximum tolerated dose (MTD) and revealed a markedly improved antitumor activity in mice compared to paclitaxel. Taken together, our results demonstrate, that a hydrolytically activated paclitaxel prodrug exhibits greater water solubility and superior antitumor activity than the parental drug. more...

The etiology of Kawasaki disease (KD) is still unknown. Therefore, the diagnosis relies on clinical criteria only. Although a specific therapy for KD is not available, coronary complications can be significantly reduced with the help of intravenous immunoglobulin (IVIG) therapy. It is not clear how IVIG interacts with the immune system. Previously, we selected a large number of IgG and IgM Fab fragments specifically reacting with IVIG molecules by phage display and antiidiotypic panning from three patients with autoimmune thrombocytopenia, a patient with lupus, and a healthy individual. Sequencing revealed that the favored V(H) germline gene segments of these IVIG-bound Fabs were 3-23 or 3-30/3-30.5, the most frequently rearranged V(H) genes among human B cells. The binding pattern suggested a B cell superantigen-like, specific interaction of an IVIG subset with B cells that present B cell receptors derived from these two germline genes. The aim of the current study was to investigate whether treatment with IVIG influences this restricted interaction. Therefore we cloned and selected Fab fragments from a patient with KD before and after IVIG therapy. A favored selection of antibodies derived from both the 3-23 and the 3-30/3-30.5 germline gene segments as before was observed. Importantly, the reactivity with IVIG was significantly higher for clones from the library prepared after the IVIG treatment, providing the first in vivo functional evidence that a subset of IVIG may selectively activate B cells of this germline origin. This mechanism may add to the therapeutic effect of IVIG in the treatment of KD., (Copyright 2001 Academic Press.) more...

Background: The disruption of self-tolerance against neuroblastoma is the ultimate goal of an effective DNA-vaccine., Procedure: Here we demonstrate the induction of a protective immunity against syngeneic murine NXS2 neuroblastoma in A/J mice, following vaccination with tyrosine hydroxylase (TH) derived antigens. Oral gene delivery was accomplished using an attenuated strain of Salmonella typhimurium as a carrier harboring vectors encoding for mTH antigens., Results: Vaccination was effective in protecting animals from a lethal challenge with wild-type NXS2 tumor cells., Conclusions: These results provide the first evidence of the TH self antigen being recognized by T-cells and demonstrate that a TH-based DNA vaccine is a potentially useful immunotherapeutic strategy for neuroblastoma., (Copyright 2000 Wiley-Liss, Inc.) more...

Objective: To perform a comparative analysis of 1) intravenous Ig (IVIG)-bound Fab fragments from a patient with autoimmune thrombocytopenia that had progressed to systemic lupus erythematosus (SLE) and 2) IVIG-selected Fabs from an SLE patient without thrombocytopenia., Methods: IVIG preparations have been successfully used to treat certain cases of autoimmune thrombocytopenia and SLE. Specific interactions of IVIG with the components of the immune system are not well characterized. To investigate these, we had previously cloned a large number of phage-displayed IgG Fab fragments, derived from 3 patients with autoimmune thrombocytopenia, that were specifically bound by IVIG molecules during panning. Many of these Fabs reacted with platelets. Sequencing revealed that the most frequently used VH germline gene segments of all IVIG-bound Fabs were 3-23 and 3-30/3-30.5. One patient's autoimmune thrombocytopenia had progressed to SLE. Using the same cloning and panning procedures, we performed a comparative analysis of this patient's IVIG-bound Fab fragments and the IVIG-selected Fabs from an SLE patient without thrombocytopenia., Results: We observed an exclusive selection of antibodies derived from 3-23 and 3-30/3-30.5 germline segments. In contrast to the Fab fragments from the autoimmune thrombocytopenia patient who developed SLE, none of the IVIG-selected Fabs from the SLE patient without thrombocytopenia bound to thrombocytes., Conclusion: Our results suggest a preferential interaction of a subfraction of IVIG-representative of normal Ig repertoires-with antibodies and B cell receptors derived from these 2 gene segments. Importantly, these are the most frequently rearranged VH germline genes among human B cells. This kind of interaction is characteristic of a B cell superantigen, since light chains, antigen specificity, and the high variation in the third complementarity-determining region 3 showed little influence on the selection of 3-23- or 3-30/3-30.5-derived Fabs by IVIG. However, at least some of the contact residues on Fabs for IVIG appear to be different from those for staphylococcal protein A and human immunodeficiency virus gp 120. The IVIG-selected Fabs may now be used to clone antibodies representative of this IVIG subfraction to study their possible regulatory influence on the B cell repertoire during normal development and disease. more...

In search for a new therapeutic approach for metastasized renal cell carcinoma (RCC), we evaluated the cytotoxicity of a novel prodrug chemoimmunoconjugate with monoclonal antibody (mAb) 138H11 and the DNA-cleaving enediyne calicheamicin thetaI1 (Camtheta) in vitro and in vivo. Previously, mAb 138H11, produced against human renal gamma-glutamyltransferase, stained over 99% clear cell and papillary RCC on frozen sections, showing a membranous expression of the target antigen. In contrast, in normal kidneys gammaGT was restricted to the brush-border in the lumen of proximal tubules and not accessible to the circulation. Thus, human tumor-bearing kidneys perfused in an extra-corporeal system with 99mTc-138H11 revealed a high, specific uptake into the tumor. In this study, fluorescence-activated cell sorting analysis showed binding of mAb 138H11 to RCC cell lines, whereas squamous cell carcinoma lines, fibroblasts, and the murine RENCA were negative. XTT cell proliferation assays revealed efficient killing of the Caki-1 cell line by the 138H11-Camtheta conjugate using SPDP (EC50 = 5 x 10(-11) M) as a covalent linker. For in vivo testing, five groups of eight nude mice each were injected with 2.5 x 10(6) Caki-1 cells s.c. and treated with the following: (a) PBS; (b) 138H11; (c) Camtheta; (d) a mixture of 138H11 and Camtheta; and (e) 138H11-Camtheta conjugate. Treatment started on day 1 after tumor induction and was repeated three times. The data show a highly significant inhibition of tumor growth with the 138H11-Camtheta conjugate versus PBS (P = 0.004). Only mice treated with 138H11-Camtheta showed a tumor shrinkage to minimal residues. In a second experiment, lower doses of the 138H11-Camtheta conjugate were compared with an antineuroblastoma mAb (ch14.18), confirming targeted killing of RCC by the 138H11-Camtheta conjugate at tolerable toxicity in vivo. In conclusion, these combined results encourage further studies for targeted therapy of metastatic RCC with mAb 138H11 conjugates. more...

Intravenous immunoglobulin preparations (IVIG) have shown positive effects in the treatment of immune defects and autoimmune diseases. It is not clear how IVIG interacts with the components of the immune system. To investigate this, we cloned previously a large number of phage displayed IgG Fab fragments derived from three patients with autoimmune thrombocytopenia (AITP) that were specifically bound by IVIG molecules. Many of these Fabs reacted with platelets. Sequencing revealed that the most frequently used germ-line gene segments of all IVIG-bound Fabs were identical to those observed for many other autoantibodies. Particularly, the loci 3-30 or 3-30/3-30. 5, 3-23 and 3r, 3l, and 2a2 represented the most abundant genes used for the heavy (VH) and light chain V region (VL), respectively. This suggested a specific interaction of IVIG molecules with B cells that present B cell receptors derived from these germ-line genes. In the current study we determined the genetic origin of IVIG-reactive IgG and IgM cloned from a healthy person. A favoured selection of antibodies derived from the same germ-line origins as in AITP was observed. Because 3-30 and 3-23 are the most frequently rearranged VH germ-line gene segments among human B cells, our results suggest that this favoured anti-idiotypic interaction may have an important role for the development and control of the normal B cell repertoire. more...

Beta-thalassaemia is inherited as an autosomal recessive trait in most families. Particular interest has recently been focused on the molecular pathology of the rare forms with a dominant mode of inheritance. The index patient and her mother, who are described in this report, displayed typical clinical and haematological features of beta-thalassaemia intermedia with significant ineffective erythropoiesis and additional peripheral haemolysis. Molecular analysis demonstrated a heterozygous genotype for a novel 6 bp (TGGTCT) deletion of the beta-globin gene involving codons 33-35. This deletion results in the removal of two valine residues from the beta-globin chain at position 33/34 (B15/B16) and the substitution of the tyrosine residue at position 35 (C1) by an aspartic acid (beta 33-35 [B15-C1] Val-Val-Tyr-->0-0-Asp). According to the index patient's place of birth, this abnormal haemoglobin has been termed Hb Dresden. The stability of the variant and the normal beta-globin chains were similar during the incubation period of in vitro globin chain synthesis analysis. However, Hb Dresden is exquisitely unstable and cannot be detected in the peripheral blood by haemoglobin electrophoresis, high-performance liquid chromatography (HPLC) or isoelectric focusing. This instability can be explained by the vital structural role of the three affected amino acids that, in normal haemoglobin, establish a total of nine intermolecular bonds (five hydrophobic and four polar) at both the alpha1beta1 (alpha2beta2) and the alpha1beta2 (alpha2beta1) interface. more...

We present the case of a 16-year-old girl with an extended thrombosis of the femoral and iliac vein and the inferior vena cava during pleuropneumonia; predisposing risk factors for thrombophilia were: use of contraceptives, nicotine abuse and congenital deficiency of antithrombin III (not previously diagnosed). Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA; initial dose: 0.08 mg/kg/h) was started. 2 days later--after diagnosis of an extended hemothorax: 1500 ml blood were obtained after thoracocentesis, transfusion of packed red blood cells was necessary--rt-PA was stopped, with only heparin (400 U/kg/d) being administered. 36 h later--the thrombosis had not yet changed--the thrombolytic therapy with rt-PA was continued in a markedly reduced dose (0.015 mg/kg/d) with no further bleeding complications. 8 days later--after successful thrombolysis--t-PA was stopped, heparin was given for another 10 days, then cumarin was administered orally. more...

Background: Spiral CT of the chest is an imaging technique with unequivocal indications and proven higher sensitivity and specificity than conventional chest X-rays. However, particularly in children, attempts should be made to reduce radiation exposure to a minimum. OBJECTIVE. To evaluate whether a low-dose technique in spiral CT scanning results in adequate diagnostic information., Materials and Methods: In a prospective study, 27 children (range 3 weeks to 14 years, mean 7 years) underwent a low-dose CT examination of the chest for various indications. The tube energy was 12.5 mAs (n = 5), 25 mAs (n = 17), 50 mAs (n = 3), or 75 mAs (n = 2) per slice. Two radiologists evaluated, in consensus, the CT scans with respect to their diagnostic value and comparison was made with 20 standard-dose chest CT examinations of adults (175 mAs per slice, mean age 56 years) with respect to technical image quality (noise and artefacts). In a second part of the study, dose measurements were carried out by means of exposing thermoluminescent dosimeters attached to a water/air phantom simulating a child's chest., Results: All low-dose CT scans were of diagnostic image quality and no additional studies were necessary. The average image noise was significantly higher than in standard-dose CT examinations (SD 39.5 compared with 12.5 for unenhanced soft tissue, P < 0.01), but did not hinder accurate diagnosis. Artefacts were exclusively due to patient motion. Radiation exposure per slice was approx. 4 mGy at 25 mAs and 34 mGy at 250 mAs, regardless of slice thickness., Conclusions: For all indications in paediatric CT scanning of the chest, low-dose technique provides adequate image quality without loss of diagnostic information. The radiation exposure is approximately 5-20 % of a standard-dose CT. more...