Scoping review on the efficacy of filter and germicidal technologies for capture and inactivation of micro-organisms and viruses.

The COVID-19 (SARS-CoV-2) pandemic increased the focus on preventing contamination with airborne pathogens (e.g. viruses, bacteria, and fungi) by reducing their concentration. Filtration, UV or ionization technologies could contribute to air purification of the indoor environment and inactivation of micro-organisms. The aim of this study was to identify the relevant literature and review the scientific evidence presented on the efficacy of filter and germicidal technologies (e.g. non-physical technologies) in air purification applications used to capture and inactivate micro-organisms and airborne viruses (e.g. SARS-CoV-2, rhinovirus, influenzavirus) in practice. A scoping review was performed to collect literature. Adopting exclusion criteria resulted in a final number of 75 studies to be included in this research. Discussion is presented on inactivation efficiencies of ultraviolet germicidal irradiation (UVGI) and ionization applications in laboratory studies and in practice. Specific attention is given to studies relating the use of UVGI and ionization to inactivation of the SARS-CoV-2 virus. Based on the consulted literature, no unambiguous conclusions can be drawn regarding the effectiveness of air purification technologies in practice. The documented and well-controlled laboratory studies do not adequately represent the practical situation in which the purifier systems are used.

Establishment and application of test methodology demonstrating the functionality of air purification systems in reducing virus-loaded aerosol in indoor air.

BACKGROUND: The global COVID-19 pandemic has resulted in a greater interest in improving the ventilation of indoor environments in order to remove aerosolized virus and thus reduce transmission. Air purification systems have been proposed as a solution to improve aerosol removal. AIM: The aim was to determine the efficacy of air purification systems in reducing the viral load in the environmental air of a room. METHODS: A containment room equipped with HEPA filter on air intake and exhaust was constructed. It was connected via an inlet with the BSL-2 facility. From the BSL-2, Feline Coronavirus (FCoV)-loaded aerosols were released into the containment room. After nebulization, air sampling was performed to determine the viral load in air prior to assessing the clean air delivery rate of the air purification systems. The infectivity of the captured viruses was also examined. FINDINGS: The air purification systems realized a 97-99% reduction in viral load in air in 1 h. Captured infectious FCoV was reduced by 99.9%-99.99% by use of an ESP technology. CONCLUSIONS: The air purification systems, using ESP technology or HEPA filter, reduce the viral load in air. The ESP purifiers inactivate captured FCoV viruses. Therefore, air purification systems can be used as an adjunctive infection control measure.

Effective ultraviolet C light disinfection of respirators demonstrated in challenges with Geobacillus stearothermophilus spores and SARS-CoV-2 virus.

BACKGROUND: The global COVID-19 pandemic, accompanied by spikes in the number of patients in hospitals, required substantial amounts of respiratory protective devices (respirators), thereby causing shortages. Disinfection of used respirators by applying ultraviolet C (UVC) light may enable safe reuse, reducing shortages. AIM: To determine whether UVC disinfection is applicable to enable repeated safe reuse of respirators. METHODS: The UVC chamber, equipped with low-pressure mercury discharge lamps emitting at 254 nm, was used to determine the sporicidal and virucidal effects. Respirators challenged with spores and viruses were exposed to various UVC energy levels. Deactivation of the biological agents was studied as well as UVC effects on particle filtration properties and respirator fit. FINDINGS: A 5 log10 reduction of G. thermophilus spore viability by a UVC dose of 1.1 J/cm2 was observed. By simulating spores present in the middle of the respirators, a 5 log10 reduction was achieved at a UVC dose of 10 J/cm2. SARS-CoV-2 viruses were inactivated by 4 log10 upon exposure to 19.5 mJ/cm2 UVC. In case UVC must be transmitted through all layers of the respirators to reach the spores and virus, a reduction of >5 log10 was achieved using a UVC dose of 10 J/cm2. Exposure to a six-times higher UVC dose did not significantly affect the integrity of the fit nor aerosol filtering capacity of the respirator. CONCLUSION: UVC was shown to be a mild and effective way of respirator disinfection allowing for reuse of the UVC-treated respirators.

An Alternative Bioassay for Synchytrium endobioticum Demonstrates the Expression of Potato Wart Resistance in Aboveground Plant Parts.

The obligate biotrophic chytrid species Synchytrium endobioticum is the causal agent of potato wart disease. Currently, 39 pathotypes have been described based on their interaction with a differential set of potato varieties. Wart resistance and pathotyping is performed using bioassays in which etiolated tuber sprouts are inoculated. Here, we describe an alternative method in which aboveground plant parts are inoculated. Susceptible plants produced typical wart symptoms in developing but not in fully expanded aboveground organs. Colonization of the host by S. endobioticum was verified by screening for resting spores by microscopy and by molecular techniques using TaqMan polymerase chain reaction and RNAseq analysis. When applied to resistant plants, none of these symptoms were detectable. Recognition of S. endobioticum pathotypes by differentially resistant potato varieties was identical in axillary buds and the tuber-based bioassays. This suggests that S. endobioticum resistance genes are expressed in both etiolated "belowground" sprouts and green aboveground organs. RNAseq analysis demonstrated that the symptomatic aboveground materials contain less contaminants compared with resting spores extracted from tuber-based assays. This reduced microbial contamination in the aboveground bioassay could be an important advantage to study this obligate biotrophic plant-pathogen interaction. Because wart resistance is active in both below- and aboveground organs, the aboveground bioassay can potentially speed up screening for S. endobioticum resistance in potato breeding programs because it omits the requirement for tuber formation. In addition, possibilities arise to express S. endobioticum effectors in potato leaves through agroinfiltration, thereby providing additional phenotyping tools for research and breeding. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Two different R gene loci co-evolved with Avr2 of Phytophthora infestans and confer distinct resistance specificities in potato.

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease in potato. For sustainable management of this economically important disease, resistance breeding relies on the availability of resistance (R) genes. Such R genes against P. infestans have evolved in wild tuber-bearing Solanum species from North, Central and South America, upon co-evolution with cognate avirulence (Avr) genes. Here, we report how effectoromics screens with Avr2 of P. infestans revealed defense responses in diverse Solanum species that are native to Mexico and Peru. We found that the response to AVR2 in the Mexican Solanum species is mediated by R genes of the R2 family that resides on a major late blight locus on chromosome IV. In contrast, the response to AVR2 in Peruvian Solanum species is mediated by Rpi-mcq1, which resides on chromosome IX and does not belong to the R2 family. The data indicate that AVR2 recognition has evolved independently on two genetic loci in Mexican and Peruvian Solanum species, respectively. Detached leaf tests on potato cultivar 'Désirée' transformed with R genes from either the R2 or the Rpi-mcq1 locus revealed an overlapping, but distinct resistance profile to a panel of 18 diverse P. infestans isolates. The achieved insights in the molecular R - Avr gene interaction can lead to more educated exploitation of R genes and maximize the potential of generating more broad-spectrum, and potentially more durable control of the late blight disease in potato.

Short communication: Growth of dairy isolates of Geobacillus thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species.

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of ß-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006.

Real-time PCR detection of Campylobacter spp.: A comparison to classic culturing and enrichment.

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.

In ovo inoculation of chicken embryos with probiotic bacteria and its effect on posthatch Salmonella susceptibility.

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.

[Extreme types - a comparison of public health systems in Thuringia and the Warthegau]. / Extreme Typen - Die öffentlichen Gesundheitsdienste in Thüringen und im Warthegau im Vergleich.

Special policing measures in Nazi Germany required centralisation in the public health service which was mainly achieved through the Law for the Unification of Municipal and State Health Administration in 1934. The long-term aim for public health officers was now hereditary and racial welfare. Several following legal regulations contributed to the implementation of a specific national-socialist health and social policy on the basis of racial hygiene and gave immense power to the health officers. Especially with the Law for the Prevention of Hereditarily Sick Offspring, compulsory sterilisation was legalised and resulted in about 400,000 victims up to 1945. The central force in this new system was the public health office. The most extreme variation of public health administration was launched in Thuringia, where professional powers and state authority implemented an extensive institutional machinery which managed to reinforce Nazi population policy almost to perfection. After the invasion of western Poland the Germans built up an inhumane regime in the annexed and occupied regions, persecuted and murdered the Polish population and tried to exterminate the Jewish people. Here the health officers of the Warthegau could promote a rigorously racial dominated population policy of segregation.

Ileal microbiota composition of broilers fed various commercial diet compositions.

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.

Identification of RFLP and NBS/PK profiling markers for disease resistance loci in genetic maps of oats.

Two of the domains most widely shared among R genes are the nucleotide binding site (NBS) and protein kinase (PK) domains. The present study describes and maps a number of new oat resistance gene analogues (RGAs) with two purposes in mind: (1) to identify genetic regions that contain R genes and (2) to determine whether RGAs can be used as molecular markers for qualitative loci and for QTLs affording resistance to Puccinia coronata. Such genes have been mapped in the diploid A. strigosa × A. wiestii (Asw map) and the hexaploid MN841801-1 × Noble-2 (MN map). Genomic and cDNA NBS-RGA probes from oat, barley and wheat were used to produce RFLPs and to obtain markers by motif-directed profiling based on the NBS (NBS profiling) and PK (PK profiling) domains. The efficiency of primers used in NBS/PK profiling to amplify RGA fragments was assessed by sequencing individual marker bands derived from genomic and cDNA fragments. The positions of 184 markers were identified in the Asw map, while those for 99 were identified in the MN map. Large numbers of NBS and PK profiling markers were found in clusters across different linkage groups, with the PK profiling markers more evenly distributed. The location of markers throughout the genetic maps and the composition of marker clusters indicate that NBS- and PK-based markers cover partly complementary regions of oat genomes. Markers of the different classes obtained were found associated with the two resistance loci, PcA and R-284B-2, mapped on Asw, and with five out of eight QTLs for partial resistance in the MN map. 53 RGA-RFLPs and 187 NBS/PK profiling markers were also mapped on the hexaploid map A. byzantina cv. Kanota × A. sativa cv. Ogle. Significant co-localization was seen between the RGA markers in the KO map and other markers closely linked to resistance loci, such as those for P. coronata and barley yellow dwarf virus (Bydv) that were previously mapped in other segregating populations.

Preemptive alloimmune intervention in high-risk pediatric acute lymphoblastic leukemia patients guided by minimal residual disease level before stem cell transplantation.

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.

CsA exposure is associated with acute GVHD and relapse in children after SCT.

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.

Unrelated donor marrow transplantation in children: transplant policy and outcome in Leiden Paediatrics SCT-Centre.

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.

Pyrosequencing analysis of the oral microflora of healthy adults.

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.

Ciclosporin kinetics in children after stem cell transplantation.

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.

Resetting the adaptive immune system after autologous stem cell transplantation: lessons from responses to vaccines.

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.

The characterisation of Bacillus spores occurring in the manufacturing of (low acid) canned products.

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.

HLA-identical haematopoietic stem cell transplantation for acute leukaemia in children: less relapse with higher biologically effective dose of TBI.

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.

Autologous stem cell transplantation in children with severe progressive systemic or polyarticular juvenile idiopathic arthritis: long-term follow-up of a prospective clinical trial.

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.