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OBJECTIVE: To assess the safety and efficacy of intensive immunosuppression followed by T cell-depleted autologous hematopoietic stem cell transplantation (ASCT) for induction of disease remission in children with refractory progressive juvenile idiopathic arthritis (JIA). METHODS: Twenty-two patients with progressive refractory JIA were followed up over a median period of 80 months after pretreatment with intensive immunosuppression followed by ASCT in a multicenter, prospective, phase II clinical trial. Hematopoietic stem cells were harvested from the patients' bone marrow, depleted of T cells, and kept frozen until used for ASCT. Pretreatment of patients consisted of a combination of antithymocyte globulin, cyclophosphamide, and low-dose total body irradiation. Patients were followed up for ASCT-related complications, recovery of hematologic and immune system parameters, and disease outcomes. RESULTS: Reconstitution of hematologic values to normal range was rapid. Recovery of immune system parameters, especially normalization of CD4+, CD45RA+ naive T cells, was delayed, occurring at >/=6 months after ASCT. The prolonged period of immune deficiency resulted in a large number of viral infections and may have contributed to the development of macrophage activation syndrome (MAS), leading to death, in 2 patients. After ASCT, 8 of the 20 evaluable patients reached complete clinical remission of their JIA, 7 were partial responders, and 5 experienced a relapse of their disease (occurring 7 years after ASCT in 1 patient). Later during followup, 2 of the patients whose disease relapsed died from infections that developed after restarting immunosuppressive medication. CONCLUSION: Intensive immunosuppression followed by ASCT resulted in sustained complete remission or marked improvement in 15 of 22 patients with progressive refractory JIA. The procedure, however, is associated with significant morbidity and risk of mortality due to prolonged and severe depression of T cell immunity. After fatal complications due to MAS were observed in some patients, the protocol was amended in 1999, to ensure less profound depletion of T cells, better control of systemic disease before transplantation, antiviral prophylaxis after transplantation, and slow tapering of corticosteroids. Following these protocol modifications, no additional ASCT-related deaths were observed among the 11 patients who received the modified treatment. [ABSTRACT FROM AUTHOR]

The major disadvantage of the current gold standard for detection of the food pathogen Campylobacter, i.e. culturing, is the lengthy procedure. In this study we assessed the use of real-time PCR for detection of Campylobacter. To this end, 926 poultry samples, taken from transport containers and broiler caeca in The Netherlands in 2007, were subjected to three different real-time PCR detection methods: one targeting the Campylobacter jejuni hipO gene, one targeting the Campylobacter coli glyA gene, and one generically targeting Campylobacter spp. 16S rDNA sequence. The PCR results from the three different PCR protocols were compared to the work of Nauta et al. (2009) who analyzed the same set of samples collected from 62 broiler flocks by means of enrichment culturing. The results indicate that the generic 16S campylobacter PCR detection is equally reliable but much faster (4 h instead of ≥2 days) than detection by means of culturing. Moreover, PCR detection targeting the hipO and the glyA gene provide the possibility of C. jejuni and C. coli species discrimination. The generic Campylobacter spp. PCR analysis also confirmed the high incidence of Campylobacter spp. in poultry samples (∼90%) and the species specific PCR showed the simultaneous presence of C. jejuni and C. coli in ∼24% of the samples. Furthermore, the results from the three PCR analyses suggested the occurrence of alternative Campylobacter species in almost 10% of the samples. The campylobacter PCR detection methods reported here can replace traditional culturing because of being quicker and more reliable., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The hypothesis that elimination of facultative and strict anaerobic microorganisms from the gastro-intestinal tract by antimicrobial drugs in the period of time around allogeneic bone marrow transplantation (BMT) prevents acute graft-versus-host disease (GVHD), was examined in a cohort of 112 children grafted between 1989 and 2002 for hematological malignancies. All patients received T-cell replete marrow from human leukocyte antigens (HLA) matched sibling donors under identical transplantation conditions. To eliminate microorganisms from the gastro-intestinal tract, total gastro-intestinal decontamination (GID) was applied by high doses of non-absorbable antimicrobial drugs while the graft recipient was maintained in strict protective isolation. About half of the children (51%) proved to be successfully decontaminated, and about half (49%) unsuccessfully. One recipient got acute GVHD in the first group and 8 in the second group (p = 0.013). The degree of success of total GID was decisive for the occurrence of acute GVHD, irrespective of the presence of other risk factors such as higher age of recipient and/or donor, female donor for male recipient and carriership or reactivation of herpesviruses. Our results demonstrate that successful total GID of the graft recipient prevents moderate to severe acute GVHD. We suppose that substantial translocation of gastro-intestinal microorganisms or parts of these, functioning as microbial-associated molecular patterns (MAMP's), triggering macrophages/dendritic cells via pattern recognizing receptors (PRR's) is prohibited. As a consequence the initiation and progression of an inflammatory process leading to acute GVHD is inhibited.

The feasibility of establishing probiotic bacteria in the intestine of broiler chickens by in ovo inoculation was investigated, followed by verifying possible subsequent protection against Salmonella Enteriditis infection. In a first study, 7 commercially available probiotics were screened for compatibility with in ovo inoculation. Two of these probiotics, one being a Enterococcus faecium and the other a Bacillus subtilis, were selected for colonizing the chick gut without compromising hatchability. In a second study, these 2 products were administered in ovo and in the feed to chicks reared until 18 d in comparison with noninoculated chicks and with chicks fed an antibiotic. All chicks were orally challenged with Salmonella Enteritidis at 4 d of age. Results showed reduced performance of Salmonella Enteritidis challenged chicks fed no additives compared with challenged chicks fed antibiotic, but no significant differences in mortality was observed. Probiotics offered in ovo or through the diet could only partially recover performance compared with antibiotic-fed chicks. A significant reduction in the number of Salmonella Enteritidis positive chicks was observed when chicks were in ovo inoculated with E. faecium and continued receiving it in the diet. This work establishes standards for future in ovo colonization research and emphasizes its value as a promising method to deliver individual precise dose of probiotics to poultry in mass scale at the earliest possible age based on the competitive exclusion concept. In ovo colonization with probiotic can therefore become an important ally in combination with other approaches to combat Salmonella and other intestinal bacterial infections in poultry.

Microbiota plays a role in the release and absorption of nutrients from feed components, thereby affecting digesta composition and moisture content of the excreta. The objective of the current study was to determine the effects of 5 different diets varying in ingredients (medium-chain fatty acids, nonstarch polysaccharides, and starch) on the microbiota composition of ileal digesta of broiler chickens and excreta DM content. Each treatment was repeated 6 times in cages each containing 18 Ross 308 broilers, with growth performance measured from 0 to 34 d of age and excreta DM and ileal microbiota composition analyzed at 34 d of age. Microbiota composition was evaluated using a novel ribosomal RNA microarray technology containing 370 different probes covering various genera, groups of microbial species, and individual species of the chicken gut microbiota, of which 321 had a signal above the background threshold. Replacing part of the animal fat and soybean oil in the wheat-based diet with medium-chain fatty acids (MCFA; 0.3% C10 and 2.7% C12) improved feed efficiency compared with the other dietary treatments. This coincided with a suppression of gram-positive bacteria belonging to the phylum of the Firmicutes, including Lactobacillus species, and species belonging to the family of the Enterococcaceae and Micrococcaceae, whereas the gram-negative bacteria belonging to the family of the Enterobacteriaceae were promoted. None of the other diets used in the present study notably changed the ileal digesta bacteria composition. Excreta DM content was not affected by dietary treatment. The variation between individual birds per dietary treatment was more pronounced than variation caused by feed composition, with the exception of the digesta microbiota of the birds fed the MCFA diet. It is concluded that a diet with MCFA significantly changes the ileal microbiota composition, whereas the effect of the other diets on the composition of the microbiota and excreta DM content is small in broiler chickens.

In 2006/2007, two vaccines were licensed against two of the most common HPV types that cause about 70% of cervical cancers. Clinical trials show that vaccinated individuals develop high levels of neutralizing antibodies. Although these data suggest that serum antibodies are the mode of action against HPV infection, it is uncertain whether immune responses generated by vaccination are similar to those induced by a natural infection. In this review, the current knowledge of humoral immune responses after natural infection and vaccination is described. Serosurveillance can be used as a monitoring tool to study vaccine uptake, the impact of HPV16/18 vaccination on other HPV types, dynamics of HPV infection and herd-immunity. In addition, factors that contribute to a higher seroresponse after a natural infection, which are summarized in this article (a persistent DNA infection, increased viral load, immunosuppression and high sexual risk behavior), can help to interpret these indirect effects better., (Copyright © 2013 Wiley Periodicals, Inc.)

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level >or=1 x 10(-4) were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n=1-4). The intervention was associated with graft versus host disease >or=grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0-30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells.

Chemokine receptor/ligand interactions orchestrate the migration of cells to peripheral tissues such as the skin. We analysed chemokine receptor expression by acute myeloid leukaemic (AML) cells present in peripheral blood (n = 7), bone marrow (n = 6), or skin (n = 11) obtained from 15 paediatric AML patients with skin involvement and in 10 AML patients without skin involvement. High percentages of circulating CCR2(pos) AML cells were only detected in patients with extramedullary disease. Skin-residing AML cells displayed a different set of receptors in situ, namely: CCR5, CXCR4, CXCR7 and CX3CR1. These results suggest the involvement of different chemokine/chemokine receptor interactions in homing and retention of AML blasts in the skin., ((c) 2010 Wiley-Liss, Inc.)

CsA is commonly used after haematological SCT (HSCT) as GVHD prophylaxis. In solid organ transplantation, area under the blood concentration vs time curve (AUC) correlates with clinical outcome. However, in HSCT, it has not been determined whether the AUC is superior to trough level monitoring to optimize clinical efficacy of CsA therapy. Therefore, the aim of this study was to investigate the relationships between CsA trough levels and/or AUC early after HSCT with clinical outcome. A total of 91 children (1.1-17.3 years) were treated consecutively with HSCT for a haematological malignancy. CsA trough levels were obtained and were used to estimate the AUC, retrospectively, with a NONMEM (Non-Linear Mixed Effects Modelling) method. Subsequently, these exposure parameters were correlated to the occurrence of acute GVHD, relapse risk (RR) and OS. Low CsA trough levels were found to correlate with the occurrence of acute GVHD. In addition, a CsA AUC over 3000 mcg h/l in AML patients was associated with a higher RR and a reduced OS. This was not the case for ALL patients. Thus, monitoring CsA exposure early after HSCT and adjusting the CsA dose to a predefined target trough level and AUC may provide a tool to influence GVHD/GVL balance.

The transplant policy for unrelated donor (UD) BMT at Leiden Paediatrics' SCT-Centre consisted of the use of (1) fully HLA-matched donors or, if not available, HLA-class I matched and/or cytotoxic T-lymphocyte precursor (CTLp)-negative donors and (2) protective isolation of the recipient and antimicrobial suppression of his/her gut microflora to prevent infections and acute GVHD. Engraftment, GVHD, relapse in the case of malignancy and survival were studied retrospectively in 126 evaluable children, transplanted between 1988 and 2005. In addition to the effect of HLA-matching, that of other transplant-relevant variables on the outcome was also studied. Actuarial OS was 65% and the EFS was 59%, 13% graft failures occurred and 7.5% > or =grade II acute GVHD. HLA-class II mismatches combined with HLA-class I matches resulted in a superior OS of 92%, as did a negative vs positive CTLp test, that is, 65 vs 33%. Analysis of other variables showed a poorer OS in patients > or =10 yrs vs <10 yrs, that is, 54 vs 73%, and in male recipients of a female donor graft, that is, 53 vs 69% for other combinations. UD-BMT can be optimized by permitting HLA-class I-matched and/or CTLp-negative donors, and probably by choosing male donors for male recipients.

Background: Recent evidence indicates that bone marrow-derived cells contribute to endothelial and epithelial cell renewal in recipients of an allogeneic stem-cell transplantation (SCT). Controversy remains on the biological significance of these donor-derived cells., Methods: This study investigated the occurrence of endothelial and epithelial cell chimerism in relation to the conditioning regimen, time interval after SCT, and development of acute and chronic graft-versus-host disease (GVHD). Fifty-five skin biopsy samples from 35 female patients transplanted with a male donor were screened for donor-derived endothelial and epithelial cells using in situ hybridization for Y chromosomes in combination with immunohistochemical cell-marking techniques., Results: Endothelial cell chimerism was found in 25% of the biopsies and increased in time after SCT. Its appearance was increased in patients with acute GVHD more than 2 weeks before biopsy. Epithelial cell chimerism was found in 85% of the biopsies. Appearance of epithelial cell chimerism was not correlated with the time interval after SCT or with tissue damage caused by GVHD., Conclusion: From these results, we conclude that donor-derived endothelial cell chimerism results from repair of damaged endothelium and maintenance of vascular homeostasis. In contrast, epithelial cell chimerism follows a more uniform pattern of engraftment, not influenced by tissue damage.

A good definition of commensal microflora and an understanding of its relation to health are essential in preventing and combating disease. We hypothesized that the species richness of human oral microflora is underestimated. Saliva and supragingival plaque were sampled from 71 and 98 healthy adults, respectively. Amplicons from the V6 hypervariable region of the small-subunit ribosomal RNA gene were generated by PCR, pooled into saliva and plaque pools, and sequenced by means of the Genome Sequencer 20 system at 454 Life Sciences. Data were evaluated by taxonomic and rarefaction analyses. The 197,600 sequences generated yielded about 29,000 unique sequences, representing 22 taxonomic phyla. Grouping the sequences in operational taxonomic units (6%) yielded 3621 and 6888 species-level phylotypes in saliva and plaque, respectively. This work gives a radically new insight into the diversity of human oral microflora, which, with an estimated number of 19,000 phylotypes, is considerably higher than previously reported.

Aims: To develop a limited sampling strategy to determine ciclosporin systemic exposure [area-under-the-curve(AUC)]. This is meant to be the first step in a future study of the relationship between AUC and the biological effects of ciclosporin., Methods: The pharmacokinetics of ciclosporin was investigated prospectively following stem cell transplantation (SCT) in 17 children, aged 1.8-16.1 years. Ciclosporin was given twice daily, intravenously over a short infusion of 2 h duration during the early post-SCT period, or orally later on, when oral medication was well tolerated. Parameter estimation was performed using nonlinear mixed effect modelling as implemented in the NONMEM program. Individual empirical Bayes estimates of clearance and distribution volume were correlated with the demographic variables., Results: Pharmacokinetics was described adequately with a two-compartment model with lag time (population estimates: CL = 11.3 l h(-1); V(c) = 16.5 l; V(p) = 59.9 l; t(1/2) absorption = 0.78 h, t(lag) = 0.6 h). The AUCs, determined for the combination of trough level with one time point between 2 and 3 h after dosing, correlated very well with the reference AUC (r(2) = 0.97). No correlation was found between clearance and distribution volume, and the demographic patient variables length, body weight, age and glomerular filtration rate., Conclusion: A two-point limited sampling strategy, in combination with a Bayesian fitting procedure using the pharmacokinetic population model described, can adequately determine the AUC of ciclosporin. Since no correlation between clearance and body weight was found, dosing ciclosporin per kg bodyweight is not supported by the results of this study. We suggest starting with a fixed dose, followed by AUC determination and dose adjustment.

Purpose: To evaluate results of high-dose total-body irradiation (TBI) regimens for hematopoietic stem cell transplantation., Methods and Materials: A total of 1,032 patients underwent TBI in one or two fractions before autologous or allogeneic hematologic stem cell transplantation for acute leukemia and non-Hodgkin's lymphoma. The TBI regimens were normalized by using the biological effective dose (BED) concept. The BED values were divided into three dose groups. Study end points were relapse incidence (RI), non-relapse mortality (NRM), relapse-free survival (RFS), and overall survival (OS). Multivariate analysis was performed, stratified by disease., Results: In the highest TBI dose group, RI was significantly lower and NRM was higher vs. the lower dose groups. However, a significant influence on RFS and OS was not found. Relapses in the eye region were found only after shielding to very low doses. Age was of significant influence on OS, RFS, and NRM in favor of younger patients. The NRM of patients older than 40 years significantly increased, and OS decreased. There was no influence of age on RI. Men had better OS and RFS and lower NRM. Type of transplantation significantly influenced RI and NRM for patients with acute leukemia and non-Hodgkin's lymphoma. There was no influence on RFS and OS., Conclusions: Both RI and NRM were significantly influenced by the size of the BED of single-dose or two-fraction TBI regimens; OS and RFS were not. Age was of highly significant influence on NRM, but there was no influence of age on RI. Hyperfractionated TBI with a high BED might be useful, assuming NRM can be reduced.

Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated.

Autologous stem cell transplantation (ASCT) to treat autoimmune diseases (AID) is thought to reset immunological memory directed against autoantigens. This hypothesis can only be studied indirectly because the exact nature of the pathogenetic autoantigens is unknown in most AID. Therefore, 19 children with juvenile idiopathic arthritis (JIA) or systemic lupus erythematodes (SLE) and 10 adults with multiple sclerosis (MS) were vaccinated with the T-cell-dependent neoantigen rabies and the recall antigen tetanus toxoid after, respectively before, bone marrow harvest. Both vaccinations were repeated after ASCT. All except two of the responders mounted a primary antibody response to rabies after revaccination, and 44% of the responders mounted a primary antibody response to tetanus boost after ASCT. These data show that immunological memory to a neoantigen is lost in most patients with AID after immunoablative pretreatment; however, memory to a recall antigen boosted before bone marrow harvest is only lost in part of the patients. Disease progression was arrested in all patients with JIA/SLE except one, but only in a minority of MS patients. Clinical outcome on a per case basis was not associated with the profile of the immune response toward the vaccination antigens after ASCT.

Objective: In January 1997 we introduced a protocol for the treatment with GH of children with impaired growth after unfractionated total body irradiation (TBI). This study is an evaluation of that protocol., Patients and Methods: Between January 1997 and July 2005, 66 patients (48 male) treated for haematological malignancies had at least two years of disease-free survival after TBI-based conditioning for stem cell transplantation (SCT). Stimulated and/or spontaneous GH secretion was decreased in 8 of the 29 patients tested because of impaired growth. Treatment with GH (daily dose 1.3 mg/m2 body surface area) was offered to all 29 patients and initiated in 23 of them (17 male). The main outcome measure was the effect of GH therapy on height standard deviation scores (SDS) after onset of GH therapy, estimated by random-effect modelling with corrections for sex, age at time of SCT and puberty (data analysed on intention-to-treat basis)., Results: At time of analysis, median duration of therapy was 3.2 years; median follow-up after start of GH therapy was 4.2 years. The estimated effect of GH therapy, modelled as nonlinear (logit) curve, was +1.1 SD after 5 years. Response to GH therapy did not correlate to GH secretion status., Conclusion: GH therapy has a positive effect on height SDS after TBI, irrespective of GH secretion status.

To examine relapse, survival and transplant-related complications in relationship to disease- and pre-treatment-related characteristics, we evaluated 132 children, who consecutively received an allogeneic HLA-identical SCT for acute leukaemia in our centre: ALL in first remission (n=24), ALL in second remission (n=53) and AML in first remission (n=55). The source of the stem cells was bone marrow in all but three cases. Most patients (89%) were pre-treated with cyclophosphamide and an age-related dose of TBI. Initially, GVHD prophylaxis consisted of long-course MTX only (n=24), later short-course MTX and CsA (n=102) was given. All patients were nursed in strictly protective isolation and received total gut decontamination to suppress their potentially pathogenic enteric microflora. The 5-year probability of overall survival was 63, 53 and 74% for ALL1, ALL2 and AML1, respectively (median follow-up: 10.6 years). The overall transplant-related mortality was 6%. The incidence of acute GVHD was 17%; 6% was grades II-IV. A higher total biologically effective TBI dose (BED) resulted in a decreased relapse frequency (P=0.034) and increased overall survival. AML patients with acute GVHD got no relapse (P=0.02); this was not the case in ALL patients. Fractionated TBI regimens with higher BED should be evaluated in prospective studies.

The immune system of patients with severe combined immunodeficiency (SCID) reconstitutes to a large extent during the first years after hematopoietic stem cell transplantation (HSCT). It was suggested, however, that accelerated loss of thymus output may cause impaired immune function at the long term. To address this issue, we studied patients with SCID who underwent allogeneic HSCT 5 to 32 years earlier and identified early determinants of long-term T-cell reconstitution. A variety of immune parameters were analyzed both early (1-4 years) and late (5-32 years) after HSCT. Late after HSCT, a clear distinction could be made between a group of 8 patients with impaired T-cell reconstitution and 11 patients with good immune reconstitution. Importantly, in patients with decreased long-term T-cell reconstitution, T-cell recovery was already poor early after HSCT, demonstrating that long-term immune failure was not caused by accelerated loss of thymus output or long-term graft failure, but resulted from poor early grafting. The number of T-cell receptor excision circles (TRECs) early after HSCT was most predictive for long-term T-cell reconstitution. Frequent monitoring of T-cell immunity and TREC numbers early after HSCT may thus serve to timely identify patients who will fail to reconstitute properly and who may need additional treatment.