Preclinical trials in autosomal dominant AD: implementation of the DIAN-TU trial.

The Dominantly Inherited Alzheimer's Network Trials Unit (DIAN-TU) was formed to direct the design and management of interventional therapeutic trials of international DIAN and autosomal dominant Alzheimer's disease (ADAD) participants. The goal of the DIAN-TU is to implement safe trials that have the highest likelihood of success while advancing scientific understanding of these diseases and clinical effects of proposed therapies. The DIAN-TU has launched a trial design that leverages the existing infrastructure of the ongoing DIAN observational study, takes advantage of a variety of drug targets, incorporates the latest results of biomarker and cognitive data collected during the observational study, and implements biomarkers measuring Alzheimer's disease (AD) biological processes to improve the efficiency of trial design. The DIAN-TU trial design is unique due to the sophisticated design of multiple drugs, multiple pharmaceutical partners, academics servings as sponsor, geographic distribution of a rare population and intensive safety and biomarker assessments. The implementation of the operational aspects such as home health research delivery, safety magnetic resonance imagings (MRIs) at remote locations, monitoring clinical and cognitive measures, and regulatory management involving multiple pharmaceutical sponsors of the complex DIAN-TU trial are described.

Attenuation of massive cytokine response to the staphylococcal enterotoxin B superantigen by the innate immunomodulatory protein lactoferrin.

Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome.

[Allergic alveolitis after influenza vaccination]. / Allergische Alveolitis nach Grippeschutzimpfung.

Allergic alveolitis as a side effect of vaccination is very rare. We report a life-threatening complication in a female patient after influenza vaccination. The causative antigen was the influenza virus itself. Our Patient has suffered from exogen-allergic alveolitis for 12 years. Because of the guidelines of regular administration of influenza vaccination in patients with chronic pulmonary disease further research in patients with known exogen-allergic alveolitis is vitally important for the pharmaceutical drug safety.

The fate of organs refused locally and transplanted elsewhere.

UNLABELLED: The number of kidney allografts procured from deceased donors has been fairly constant in the past few years, while organs from living donors steadily increase. In our program, existing protocols refused some kidneys which were subsequently accepted and transplanted at other hospitals. Thus, a review of our criteria to accept kidneys became necessary. METHODS: We studied the outcome of all kidneys refused by us but transplanted in other programs between 2002 and 2004. The data analyzed included ID no. donor, transplant center, procurement date, donor age, ischemic times, recipient alive or dead, creatinine level (when it was offered), initial function, hypertension, diabetes mellitus, biopsy, reason why the kidney was not accepted in our program, kidney functioning or lost, and cause of graft failure. The chi-square, Fisher, and t tests were used to analyze our data; P values of <.05 were regarded as significant. RESULTS: Originally 137, we excluded kidneys exported due to mandatory sharing (26 of 137 = 18.97%) and multiorgan placement (10 of 137 = 7.3%). Thus, 101 kidneys were not accepted by us because they did not meet the existing criteria of our program, but were accepted elsewhere. Reasons for nonacceptance were divided into donor quality, donor social history, donor age, donor size/weight, positive serological test, as well as organ preservation time, organ anatomical damage, elevated creatinine, abnormal urinalysis, abnormal biopsy, and decreased urine output. Donor issues were 66 of 101 (65.3%) with a graft loss of 13.6%, and organ issues were 35 of 101 (34.7%) with a graft loss of 66.6%. Donor quality totaled 24 of 66 (36.4%) and donor social history totaled 20 of 66 (30.3%); these were the most common causes for kidney nonacceptance related to donor issues. Reasons related to organ quality included elevated creatinine (15 of 35 = 42.9%; graft loss, 46.6%), and abnormal biopsy (9 of 35 = 25.7%; graft loss, 11.1%) and organ anatomical damage (4 of 35 = 11.4%; graft loss, 75%) (P = .42). Graft loss was more frequent with creatinine levels above 2.4 mg/dL (P < .001, RR gf = 1.5). Long-term fate of these 101 kidneys transplanted elsewhere: 82 (81.2%) were still working while 19 (18.8%) were lost. The causes of graft loss were renal artery thrombosis (42.1%), renal venous thrombosis (26.3%), death for other reasons (15.8%), graft never worked (10.5%), and ESRD (5.7%). The results suggest that the criteria for refusal related to donor issues, including hypertension, diabetes mellitus, donor age and donor size, should be revised owing to the low percentage of graft loss. Other donor issues such as positive serological test and donor social history (drug use, alcoholism) represent a serious potential risk for the health of recipients; for this reason, considering these persons as possible donors is very difficult irrespective of the graft outcome. Kidney refusals related to organ issues (especially elevated creatinine and anatomical damage) due to the very high percentage of graft loss should be considered high risk and probably be excluded. The increase in the demand of kidneys to be transplanted is a very important reason for a continuous and systematic review of donor exclusion criteria in every transplant program. The results presented here have helped us to improve both our outcomes and utilizations based on scientific evidence.

[Escalating management of progressive neuropathic cancer pain]. / Therapieeskalation bei progredienten neuropathischen Tumorschmerzen.

The management of tumor-related pain, particularly that which is neuropathic in origin, is both difficult and challenging. According to WHO guidelines, opioids are the first-line analgesics in case of severe tumor-related pain syndromes. Some patients receiving continuous opioid therapy rapidly require increase of the opioid dose. In this case it can be necessary to extend the analgesic regime and to either apply interventional procedures or change the opioid or the route of administration. The case presented is remarkable regarding the severity and persistent nature of the patient's pain despite a variety of commonly employed treatments.

[Uncommon course of radiation-induced pneumonitis with subsequent bronchiolitis obliterans with organizing pneumonia (BOOP) in a female with breast cancer]. / Ungewöhnlicher Verlauf einer Strahlenpneumonitis mit nachfolgender Bronchiolitis obliterans mit organisierender Pneumonie (BOOP) bei einer Patientin mit Mammakarzinom.

Radiation-induced pneumonitis is a familiar complication of breast cancer radiotherapy, whereas bronchiolitis obliterans with organizing pneumonia occurs extremely rarely after this treatment. Consequently, testing for BOOP is rarely included in differential diagnosis. This results in delayed diagnosis, so that treatment is ultimately commenced too late. The infiltrations are often located in the unirradiated lung, impeding diagnosis. The radiotherapy-induced BOOP-reaction that can also occur after typical radiation pneumonitis is illustrated by reference to a characteristic case report.

Identification and characterization of fhuD1 and fhuD2, two genes involved in iron-hydroxamate uptake in Staphylococcus aureus.

Staphylococcus aureus can utilize several hydroxamate siderophores for growth under iron-restricted conditions. Previous findings have shown that S. aureus possesses a cytoplasmic membrane-associated traffic ATPase that is involved in the specific transport of iron(III)-hydroxamate complexes. In this study, we have identified two additional genes, termed fhuD1 and fhuD2, whose products are involved in this transport process in S. aureus. We have shown that fhuD2 codes for a posttranslationally modified lipoprotein that is anchored in the cytoplasmic membrane, while the deduced amino acid sequence predicts the same for fhuD1. The predicted FhuD1 and FhuD2 proteins share 41.0% identity and 56.4% total similarity with each other, 45.9 and 49.1% total similarity with the FhuD homolog in Bacillus subtilis, and 29.3 and 24.6% total similarity with the periplasmic FhuD protein from Escherichia coli. Insertional inactivation and gene replacement of both genes showed that while FhuD2 is involved in the transport of iron(III) in complex with ferrichrome, ferrioxamine B, aerobactin, and coprogen, FhuD1 shows a more limited substrate range, capable of only iron(III)-ferrichrome and iron(III)-ferrioxamine B transport in S. aureus. Nucleotide sequences present upstream of both fhuD1 and fhuD2 predict the presence of consensus Fur binding sequences. In agreement, transcription of both genes was negatively regulated by exogenous iron levels through the activity of the S. aureus Fur protein.

Organ procurement organization (OPO), best practices.

There are currently 59 organ procurement organizations (OPOs) in the United States which serve their assigned geographic areas with variable productivity. Knowledge of organizational characteristics, programs and practices of more successful OPOs may be useful to increase the productivity of less successful OPOs. A preliminary survey of all OPO executive directors in the United States ascertained the most important beneficial and detrimental factors affecting their success. Site visits were then conducted at OPOs based on a selection process utilizing population size, geographic location, minority population, donors per million population and donors per thousand deaths among potential donors. All OPOs were categorized and the highest ranking OPOs in each of seven categories, based on 4 years of national data, were selected for the site visits. Regression analysis and correlation analysis using Pearson's product-moment correlation were performed. The survey to identify the important factors was returned by 47 (77%) of 61 OPOs existent in 1999. The most important beneficial factors identified by responding OPOs were adequate staffing and experience, allocation of responsibilities, hospital development and leadership. The most important detrimental factors were inadequate staffing and experience, poor donor hospital/transplant center/ OPO relationships and failure in the consent process. Site visits of the highest-ranking OPOs demonstrated all had respected, experienced leadership focused on the donation process; efficient mechanisms for resolving allocation or transplant center conflicts; systems for monitoring activity and tracking outcomes; excellent communication between OPO and transplant centers; open internal communication at all levels of the OPO; immediate, on-site response to vascular donor referrals; and volunteer support of public and/or professional education. Regression and correlation analysis demonstrated that as minority population increases, OPO performance declines (P < 0.03). Moreover, independent OPOs were associated with poorer performance regardless of minority population (P < 0.05). All of the successful OPOs visited had strong leadership, excellent donor hospital and transplant center relationships, well-developed communication and innovative methods to deal with their minority populations. Application of these practices within all OPOs could significantly enhance organ donation.

Quality of life and retrospective perception of the effect of growth hormone treatment in adult patients with childhood growth hormone deficiency.

Divergent findings on the quality of life (QoL) and the psychosocial functioning of adults treated during childhood with growth hormone (GH) because of GH deficiency (GHD) have been reported. In the present study we evaluated the QoL and the perception of the effect of former GH treatment in Belgian young adults with childhood GHD. Thirty-six patients (22 males) were included in the study. They all were treated during childhood with GH for GHD. QoL was evaluated with a standardised questionnaire: the Quality of Life Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDA). Psychosocial functioning, sexual experience and schooling were evaluated by semi-structured interviews and questionnaires. The influence of gender, type of hormone deficiency (isolated GHD vs multiple pituitary hormone deficiencies [MPHD]), age at the start of GH therapy (before 12 yr vs after 12 yr) and the height deficit at the start of GH therapy (< -3 SDS vs > -3 SDS) were studied. In addition, the patients' and parents' perception of height and of the effect of GH treatment was retrospectively evaluated by semi-structured interviews. Age (mean +/- SD) at the time of evaluation was 20.0 +/- 1.3 yr and final height was -0.5 +/- 0.9 SDS, comparable to mid-parental height (-0.6 +/- 0.8 SDS). The QoL-AGHDA score was 9 +/- 6. About half of the patients, especially those in whom GH treatment was started after the age of 12 years, complained of retrospective difficulties with self-confidence and social contact, and about one-quarter of the patients had current difficulties with self-confidence, social contact, contact with the opposite sex and with emotional life. Only 44% of the patients had had sexual intercourse--none of those with MPHD. According to the parents, the patients had and still have more difficulties with self-confidence and social contact than their siblings and/or peers, and they needed and still need more emotional support. In one out of four patients the parents expected difficulties in finding a job, in one out of three patients parents expected difficulties in leaving home or in having a stable relationship. The educational level of patients with a height deficit < -3 SDS at start of GH therapy was lower than in patients with a height deficit > -3 SDS. According to the parents, about half of the patients, especially those with MPHD, had more study problems compared to siblings. In all patients, satisfaction with final height and GH therapy was obvious. In conclusion, the psychosocial outcome of young adults with childhood GHD was more satisfying than in previous studies. This could be due to a more adequate GH treatment with better final height results. Nevertheless, more difficulties with respect to psychosocial functioning were observed in patients with MPHD, in patients in whom GH treatment was started after 12 years of age and in patients with a height deficit < -3 SDS at the start of GH therapy, underlining the need for early diagnosis and treatment of childhood GHD, and of continuing medical follow-up and psychosocial counselling, particularly in these subgroups of patients with GHD.

Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus.

Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An S. aureus mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn917-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis. DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG, all of which show significant similarities to ferric hydroxamate uptake (fhu) genes in B. subtilis. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the S. aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S. aureus. This regulation is attributable to Fur in S. aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.

Distribution of core oligosaccharide types in lipopolysaccharides from Escherichia coli.

In the lipopolysaccharides of Escherichia coli there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the waa (core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of E. coli. This system was applied to the 72 isolates in the E. coli ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69. 4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic E. coli isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal E. coli isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic E. coli (VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic E. coli serogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.

Characterization of dTDP-4-dehydrorhamnose 3,5-epimerase and dTDP-4-dehydrorhamnose reductase, required for dTDP-L-rhamnose biosynthesis in Salmonella enterica serovar Typhimurium LT2.

The thymidine diphosphate-L-rhamnose biosynthesis pathway is required for assembly of surface glycoconjugates in a growing list of bacterial pathogens, making this pathway a potential therapeutic target. However, the terminal reactions have not been characterized. To complete assignment of the reactions, the four enzymes (RmlABCD) that constitute the pathway in Salmonella enterica serovar Typhimurium LT2 were overexpressed. The purified RmlC and D enzymes together catalyze the terminal two steps involving NAD(P)H-dependent formation of dTDP-L-rhamnose from dTDP-6-deoxy-D-xylo-4-hexulose. RmlC was assigned as the thymidine diphosphate-4-dehydrorhamnose 3,5-epimerase by showing its activity to be NAD(P)H-independent. Spectrofluorometric and radiolabeling experiments were used to demonstrate the ability of RmlC to catalyze the formation of dTDP-6-deoxy-L-lyxo-4-hexulose from dTDP-6-deoxy-D-xylo-4-hexulose. Under reaction conditions, RmlC converted approximately 3% of its substrate to product. RmlD was unequivocally identified as the thymidine diphosphate-4-dehydrorhamnose reductase. The reductase property of RmlD was shown by equilibrium analysis and its ability to enable efficient biosynthesis of dTDP-L-rhamnose, even in the presence of low amounts of dTDP-6-deoxy-L-lyxo-4-hexulose. Comparison of 23 known and predicted RmlD sequences identified several conserved amino acid residues, especially the serine-tyrosine-lysine catalytic triad, characteristic for members of the reductase/epimerase/dehydrogenase protein superfamily. In conclusion, RmlD is a novel member of this protein superfamily.

The LifeLink Foundation and cadaver kidney transplantation in Tampa.

1. LifeLink Foundation, a not-for-profit organization, has been the driving force and absolutely essential entity for kidney and liver transplantation in Tampa providing all the components (patient, organs and clinicians) save for inpatient hospitalization. It also plays a big role in the heart transplant program. LifeLink has increased the kidney transplant rate from the first 1,000 done in 17 years to the second 1,000 in 7 years and is on a pace for the third 1,000 in 5 1/2 years. 2. Because of its innovative programs, cadaver donor procurement by the Tampa LifeLink OPO has been roughly double the national average for the past 10 years. Because of cadaver kidney availability the median wait time from activation on the wait list to transplantation over the past 5 years was 159 days. The recent transplant rate is 14.7-22.7% higher than the national average, dependent upon the parameter measured. Similar results are seen for Tampa patients awaiting heart and liver transplantation. 3. The overall outcome of 1,184 cadaver kidney transplants performed in the decade 1989-98 was similar to that reported from the UNOS database in this series of publications. a) One- and 2-year graft survival increased 2% per year over the decade with a recent one-year graft survival rate of 96%. The overall T1/2 was 10 years. b) Our disastrous 1994 results were quickly reversed by a more intense pretransplant medical evaluation, the introduction of mycophenolate mofetil, more aggressive and earlier treatment of rejection episodes, and mandatory T- and B-cell flow cytometry crossmatching for all transplants. The incidence of rejection episodes decreased from 40 to 20%, and the first year immunological graft loss decreased from 5%, to 1.9%, to 0.8%, to 1.4% and 0% over the succeeding 4 years. 4. Individual factors affecting allograft survival were strikingly similar to national data, although all did not react statistical significance probably due to the smaller numbers. a) Primary and second grafts had similar survival rates (p = 0.97) whereas the third or subsequent graft survival was 7-32% poorer (p = 0.02). b) Black recipients had survival rates 10-13% lower than Caucasians and other races (p = 0.003). c) Patients with a peak PRA > 50 had survival values 4-13% poorer than those with < 50 PRA (p = 0.14). d) Patients with 2-4 HLA mismatches had graft survival rates 4-10% poorer than those with 0-1 mismatch (p = 0.12), whereas those with 5-6 mismatches had rates 6-17% poorer (p = 0.04). e) Although 22% of our transplants were to patients > 60 years of age, there was no difference (p = 0.81 to 0.90) in graft survival for the age groups 0-40, 41-60 and > 61. However, the proportion of grafts lost due to patient death compared with all allografts lost, was very different at 21% in the youngest group, 43% in those 41-60 years of age, and 63% in recipients > 61 years. 5. The rate of delayed graft function with imported kidneys was higher (27 vs. 16%, p = 0.006) but essentially the same as local kidneys with the same ischemia times. However, 41% of local kidneys were transplanted within 12 hours of procurement. Totally, 78% of local kidneys were transplanted within 18 hours (11% DGF rate) versus 79% of imports being transplanted at > 18 hours (32% DGF rate). Ischemia time, not the kidney source is the key issue since: a) There was no difference in overall graft survival of imported versus local kidneys (p = 0.95) nor in comparing local versus import kidneys with (p = 0.66) or without (p = 0.69) DGF. b) There was, however, a 11-17% overall poorer graft survival over 3 years in kidneys with DGF (p < 0.001) seen with both local (9-18% poorer, p = 0.0002) and imported (12-19% poorer, p = 0.008) kidneys. c) Kidneys displaying DGF came from older donors (40 vs. 34 years, p = 0.023) and had longer ischemia times (21 vs. 15 hours, p < 0.0005). 6. Dual kidney transplants were started in late 1996 with older or marginal donors to provide a better chance of success fo

Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica.

Bacterial lipopolysaccharides (LPS) are unique and complex glycolipids that provide characteristic components of the outer membranes of Gram-negative bacteria. In LPS of the Enterobacteriaceae, the core oligosaccharide links a highly conserved lipid A to the antigenic O-polysaccharide. Structural diversity in the core oligosaccharide is limited by the constraints imposed by its essential role in outer membrane stability and provides a contrast to the hypervariable O-antigen. The genetics of core oligosaccharide biosynthesis in Salmonella and Escherichia coli K-12 have served as prototypes for studies on the LPS and lipo-oligosaccharides from a growing range of bacteria. However, despite the wealth of knowledge, there remains a number of unanswered questions, and direct experimental data are not yet available to define the precise mechanism of action of many gene products. Here we present a comparative analysis of the recently completed sequences of the major core oligosaccharide biosynthesis gene clusters from the five known core types in E. coli and the Ra core type of Salmonella enterica serovar Typhimurium and discuss advances in the understanding of the related biosynthetic pathways. Differences in these clusters reflect important structural variations in the outer core oligosaccharides and provide a basis for ascribing functions to the genes in these model clusters, whereas highly conserved regions within these clusters suggest a critical and unalterable function for the inner region of the core.

Involvement of waaY, waaQ, and waaP in the modification of Escherichia coli lipopolysaccharide and their role in the formation of a stable outer membrane.

The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli. This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule. In the R1 core prototype strain, E. coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function. In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy. All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability. Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII. Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY. Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity. Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations.

The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides.

The major core oligosaccharide biosynthesis operons from prototype Escherichia coli strains displaying R1 and R4 lipopolysaccharide core types were polymerase chain reaction-amplified and analyzed. Comparison of deduced products of the open reading frames between the two regions indicate that all but two share total similarities of 94% or greater. Core oligosaccharide structures resulting from nonpolar insertion mutations in each gene of the core OS biosynthesis operon in the R1 strain allowed assignment of all of the glycosyltransferase enzymes required for outer core assembly. The difference between the R1 and R4 core oligosaccharides results from the specificity of the WaaV protein (a beta1, 3-glucosyltransferase) in R1 and WaaX (a beta1, 4-galactosyltransferase) in R4. Complementation of the waaV mutant of the R1 prototype strain with the waaX gene of the R4 strain converted the core oligosaccharide from an R1- to an R4-type lipopolysaccharide core molecule. Aside from generating core oligosaccharide specificity, the unique beta-linked glucopyranosyl residue of the R1 core plays a crucial role in organization of the lipopolysaccharide. This residue provides a novel attachment site for lipid A-core-linked polysaccharides and distinguishes the R1-type LPS from existing models for enterobacterial lipopolysaccharides.

The assembly system for the lipopolysaccharide R2 core-type of Escherichia coli is a hybrid of those found in Escherichia coli K-12 and Salmonella enterica. Structure and function of the R2 WaaK and WaaL homologs.

In Escherichia coli F632, the 14-kilobase pair chromosomal region located between waaC (formerly rfaC) and waaA (kdtA) contains genes encoding enzymes required for the synthesis of the type R2 core oligosaccharide portion of lipopolysaccharide. Ten of the 13 open reading frames encode predicted products sharing greater than 90% total similarity with homologs in E. coli K-12. However, the products of waaK (rfaK) and waaL (rfaL) each resemble homologs in Salmonella enterica serovar Typhimurium but share little similarity with E. coli K-12. The F632 WaaK and WaaL proteins therefore define differences between the type R2 and K-12 outer core oligosaccharides of E. coli lipopolysaccharides. Based on the chemical structure of the core oligosaccharide of an E. coli F632 waaK::aacC1 mutant and in vitro glycosyltransferase analyses, waaK encodes UDP-N-acetylglucosamine:(glucose) lipopolysaccharide alpha1, 2-N-acetylglucosaminyltransferase. The WaaK enzyme adds a terminal GlcNAc side branch substituent that is crucial for the recognition of core oligosaccharide acceptor by the O-polysaccharide ligase, WaaL. Results of complementation analyses of E. coli K-12 and F632 waaL mutants suggest that structural differences between the WaaL proteins play a role in recognition of, and interaction with, terminal lipopolysaccharide core moieties.

Age-related perception of stature, acceptance of therapy, and psychosocial functioning in human growth hormone-treated girls with Turner's syndrome.

This study evaluated the perception of stature, acceptance of therapy, and psychosocial functioning in relation to age at onset and time on treatment during 2 yr of GH therapy in 31 girls with Turner's syndrome grouped by age (group A: 3.7-5.8 yr, n = 9; group B: 7.2-11.8 yr, n = 13; group C: 12.5-16.4 yr, n = 9). The growth response after 2 yr was significant in the 3 groups when calculated in terms of growth norms for untreated Turner girls (mean increase in height SD score: +1.2, +1.5, and +1.1, respectively). The effect was less marked in terms of growth norms for normal girls, particularly in group B (+0.5 SD score). Height was perceived as a problem by most patients, except in the youngest girls at the start of treatment (group A) and in the majority of the adolescents after 2 yr of GH therapy (group C), without evidence of relation to growth response during therapy. The GH injections were fairly well accepted by all patients, except those younger than 6 yr. In all patients, expected adult height was unrealistic and became more realistic with age, whereas no consistent changes were observed in relation to growth response to GH therapy. The Child Behavior Checklist revealed elevated mean scores at the behavioral subscales of attention problems (group A and B), social problems, withdrawal, and anxiety-depression (most obviously in group B). No significant changes were seen during GH therapy. In group C, an elevated mean social problem score at the Youth Self Report and a low mean social self-esteem score at the Self-Esteem Inventory were observed before therapy and showed a significant improvement during 2 yr of GH treatment. These results, however, might be biased due to an increase in social desirability during therapy. We conclude that the perception of height, acceptance of GH therapy, and psychosocial functioning in girls with Turner's syndrome show important differences between age groups, with only slight changes observed during GH therapy.

[Reduction of bacterial contamination during mechanical autotransfusion by uv irradiation--initial results]. / Reduktion von bakteriellen Kontaminationen während maschineller Autotransfusion durch UV-Bestrahlung--Erste Ergebnisse.

OBJECTIVE: To investigate the effects of UV-radiation of autologous blood on bacteria and red blood cells when using intraoperative sampling of autologous blood using a cell separator--an established method for reducing the need for donor blood during surgery--which is reported to have a bacterial contamination rate of 5-75%, due mainly to coagulase-negative staphylococci (CNS). METHODS: Cell-separator blood was diluted to a haematocrit level permitting transmission of 1% of the UV-radiation used in this study (lambda 254 nm, coat thickness 1 mm). CNS samples were irradiated for 2, 4, 10, 20 and 30 seconds. Free haemoglobin and methaemoglobin levels were measured, and erythrocytes examined microscopically at the end of the procedure. RESULTS: Blood samples had to be diluted to a haematocrit of 1% to permit transmission of 1% of the UV light. The optimal irradiation duration was 4 seconds, when bacteria were completely eliminated. Longer irradiation durations were associated with increasing levels of free haemoglobin and methaemoglobin, the levels of which at 4 seconds exposure were 12.5 mg/L and 15.5%, respectively. CONCLUSIONS: It is possible to prevent CNS contamination of cell-separator blood by irradiation with UV light. Prior to clinical application, however, the method will need to be modified to minimize side effects and increase its decontamination efficacy.

Longitudinal study of behavioral and affective patterns in girls with central precocious puberty during long-acting triptorelin therapy.

The aim of the present study was to evaluate the behavioral and affective characteristics and the changes in psychosocial functioning resulting from precocious puberty in 15 girls with central precocious puberty treated for 2 y using the GnRH agonist long-acting triptorelin, and in 5 untreated girls. After diagnosis of precocious puberty at 6.6-10.4 y of age, height, weight and pubertal development were evaluated at 3-month intervals over 2 y. Semi-structured interviews were carried out with the patient, the parents and the pediatric endocrinologists at 1, 8, 16 and 24 months after diagnosis. Standardized questionnaires (Child Behavior Checklist, Self-esteem Inventory) were administered at 1 and 24 months or 16 and 24 months, respectively. There was a mean 1.5-y delay between the observation of signs of puberty as reported by the parents and the diagnosis of precocious puberty at the first consultation of a pediatric endocrinologist. Before follow-up, all 20 girls were very concerned about physical differences from peers, particularly breast development. During therapy, breast regression to minimal or absent development occurred in 5/15 treated patients, who then no longer felt embarrassed about pubertal development in contrast to the other patients. Fear of sexuality remained obvious throughout the study in most patients. Feelings of loneliness and exemplary behavior were observed and tended to decrease in the treated patients and to increase in the untreated patients. Elevated scores of withdrawal, anxiety/depression and somatic complaints at Child Behavior Checklist were still observed after 2 y. These changes in behavioral and affective characteristics appeared to be related neither to height and weight, nor to development of pubic hair, which progressed in most patients. After 2 y, the physical differences remained a concern for 13 girls and the risk of short adult stature for 6. In summary, some behavioral and affective characteristics and particularities in psychosocial functioning are observed in girls with precocious puberty. During treatment with long acting triptorelin, problematic behavior and functioning decrease slightly, particularly in the few girls showing breast regression to minimal or absent development.