Clinical and economic impacts of a modified-observational screening approach to well-appearing infants born to mothers with chorioamnionitis.

OBJECTIVE: Term infants born to mothers with chorioamnionitis are at risk for early-onset sepsis (EOS). We aimed to measure the impact of changing from a categorical to a modified-observational EOS screening approach on NICU admission, antibiotic utilization, and hospitalization costs. STUDY DESIGN: Single-center retrospective pre-post cohort study of full-term infants born to mothers with chorioamnionitis. Primary outcomes included NICU admission, antibiotic utilization, and hospitalization costs. Outcomes were adjusted for demographic variables. Budget-impact analysis was performed using bootstrapping with replication. RESULTS: 380 term infants were included (197 categorical; 183 modified-observational). There was a significant decrease in NICU admission and antibiotic utilization (p < 0.05) in the modified-observational cohort but no significant difference in per-patient total hospitalization costs. Budget-impact analysis suggested a high probability of cost savings. CONCLUSION: A modified-observational approach to evaluating term infants of mothers with chorioamnionitis can reduce NICU admission and unnecessary antibiotic therapy, and may lead to cost-savings.

Neonatal Golden Hour: a review of current best practices and available evidence.

PURPOSE OF REVIEW: Recommendations made by several scientific bodies advocate for adoption of evidence-based interventions during the first 60 min of postnatal life, also known as the 'Golden Hour', to better support the fetal-to-neonatal transition. Implementation of a Golden Hour protocol leads to improved short-term and long-term outcomes, especially in extremely premature and extreme low-birth-weight (ELBW) neonates. Unfortunately, several recent surveys have highlighted persistent variability in the care provided to this vulnerable population in the first hour of life. RECENT FINDINGS: Since its first adoption in the neonatal ICU (NICU) in 2009, published literature shows a consistent benefit in establishing a Golden Hour protocol. Improved short-term outcomes are reported, including reductions in hypothermia and hypoglycemia, efficiency in establishing intravenous access, and timely initiation of fluids and medications. Additionally, long-term outcomes report decreased risk for bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH) and retinopathy of prematurity (ROP). SUMMARY: Critical to the success and sustainability of any Golden Hour initiative is recognition of the continuous educational process involving multidisciplinary team collaboration to ensure coordination between providers in the delivery room and beyond. Standardization of practices in the care of extremely premature neonates during the first hour of life leads to improved outcomes. VIDEO ABSTRACT: http://links.lww.com/MOP/A68 .

Six years of activity of the Italian vaccine portal "VaccinarSì": a web traffic evaluation using Google Analytics.

BACKGROUND: Despite the scientific consensus about vaccines safety and effectiveness, there is still a discrepancy between scientific evidence and perception by the general population. The "VaccinarSì" portal was created in 2013 by Italian specialists in Public Health to provide evidence-based information regarding vaccination. STUDY DESIGN: The purpose of this study was to analyse the web traffic on "VaccinarSì" platform during a six-year period (May 8, 2013-May 8, 2019). Moreover, we compared the first six-month with the last six-month period of the website activity, to identify potential areas of improvements. METHODS: This is a descriptive study using Google Analytics data. We collected data regarding the following: total number of sessions to the portal, total number of pages viewed, total number of users and the number of new visitors, geographical locations and demography of users as well as user access mode (type of device used and way of access). We also collected some data that were informative about the possibility to infer the level of visitors' engagement with the portal, such as thee number of returning users, bounce rate, number of pages visited per session and mean session duration. RESULTS: Throughout the relevant period, the portal has consistently increased its popularity, with a remarkable increment of monthly connections (ending up to more than 80,000/month) from all over Italy. Visitors were mainly female (71.1%), aged between 25 and 44 years (64.7%). Healthcare professionals were responsible for a considerable proportion of accesses (50.6%). The mobile has become the dominant device used to access the portal, accounting for 77.8% of total connection in the last six months. Similarly, in the last period, organic search accounted for 92% of all connections. Measles and MPR vaccine, as well as chickenpox and hexavalent vaccine, have remained the most appealing topics of interest among visitors over the years. The page that attracted more visitors over the six years was "real risks and benefits of vaccination", accounting for 5.67% of total sessions with a high mean duration spent of 05:08 minutes. CONCLUSION: During the six years of activity, overall, the level of users' engagement with the portal has dropped with an increased bounce rate and a lower average number of pages visited per session and a lower mean duration of each connection. The lowest engagement involved connections accessed through mobile devices. Results helped "Vaccinarsi" developers to speculate about future strategies to further increase the platform popularity and optimize visitors' engagement.

Active TB screening among homeless people attending soup kitchens in Verona (Italy).

Background: The hard-to-reach populations, including the homeless, are particularly vulnerable to the development of active tuberculosis. According to the World Health Organization, tuberculosis rates among the homeless in industrialized Countries are up to 20 times higher if compared with the general popula-tion, representing a relevant public health problem. The aim of our study was to describe the results of an active tuberculosis screening applied in order to find out suspected active TB cases among the homeless in Verona. Methods: As part of a partnership between the non-profit association Medici per la Pace and one of the Local Health Units of Veneto Region (ULSS 9 Scaligera) in 2018, a tuberculosis screening, based on thoracic radiographs, was offered to the homeless guests of two Verona's soup kitchens. Results: The studied population included 139 people, and three cases of suspected active tuberculosis, all in males, were observed. Among these, two received a diagnostic confirmation of active tuberculosis (a prevalence of 1.44% - CI: 0,17 - 5,1). Moreover, radiographic patterns of tuberculosis aftermaths were found in six additional subjects. Conclusions: Interventions specifically dedicated to hard-to-reach populations, can be useful in identifying tuberculosis active cases and controlling the disease in low tuberculosis burden countries. In particular, the active research of subjects, the screening carried out with mobile X-ray, and also the constant caring of the patients with active disease, could be the right method to keep under control this relevant public health problem.

A Quality Improvement Approach to Reduce Unplanned Extubation in the NICU While Avoiding Sedation and Restraints.

The unplanned extubation (UE), a common adverse event in the neonatal intensive care unit (NICU), may result in airway trauma, cardiopulmonary resuscitation, and, in extreme cases, death. As part of the Nationwide Children's Hospital NICU's effort to optimize NICU graduates' neurodevelopmental outcomes, skin-to-skin care of intubated infants is encouraged, while sedation and restraints to prevent UE are strongly discouraged. This project aimed to decrease the UE rate from 1.85 to 1.5 per 100 endotracheal tube (ETT) days. METHODS: The project occurred in a 114-bed, level-IV NICU with approximately 850 admissions per year and 100% outborn infants. A multidisciplinary team began biweekly meetings to review all UE events, later separating these into preventable and nonpreventable. Important ongoing tests of change included assigning a single process owner for UE reporting, ensuring proper ETT securement, and using 2 clinical staff during patient and/or ETT manipulation. RESULTS: Early in the project, enhanced detection led to an increased rate from 1.85 to 3.26 per 100 ETT days. However, identifying preventable events empowered staff to decrease the frequency to 2.03 per 100 ETT days. In August 2017, an ETT taping method change produced an increase in special causes due to decreased compliance. However, when securement methods were enhanced, noncompliance reversed and is now trending favorably. CONCLUSIONS: Decreasing UE in a neurodevelopmentally friendly unit, which avoids sedation and restraints, is challenging. Using a multidisciplinary quality improvement approach and after appropriately capturing events, we reduced UE, with the highest impact of intervention being ETT securement standardization.

Clinical Decision Support to Improve Dosing Weight Use in Infants with Neonatal Abstinence Syndrome.

INTRODUCTION: Opioid abuse in the United States is a public health emergency. From 2000 to 2009, prenatal maternal opiate use increased from 1.19 to 5.63 per 1,000 births, with up to 80% of in utero opioid-exposed infants requiring pharmacotherapy. This study aimed to increase the percentage of neonatal abstinence syndrome (NAS) medication orders based on birth weight (BW) in neonates admitted to a neonatal intensive care unit with a principal diagnosis of NAS from 29% to 90%, within 4 months of project initiation, and to sustain this for 6 months. METHODS: This project occurred at an academic medical center with 5,000 deliveries per year and a 49-bed Level III neonatal intensive care unit. We used the Institute for Healthcare Improvement methodology, largely focusing interventions on clinical decision support (CDS) tools. We plotted all measures on Shewhart charts, and Nelson rules differentiated special versus common cause variation. RESULTS: The percent of orders based on BW increased from 29% to 78% after implementing multiple interventions focused primarily on CDS. However, this later decreased to 48% as workarounds began. There was also a significant decrease in the length of stay variability, which persisted throughout the project. DISCUSSION: CDS is a helpful tool to guide prescribing behavior; however, workarounds can negate its usefulness. Standardized use of BW for weight-based NAS medication prescribing can decrease the length of stay variability. Further studies are needed using a human factors approach to minimize workarounds in CDS and potentially decrease the length of stay in neonates with NAS.

[Psychological processes of stress management and neuroendocrine regulation in incarcerated adolescent offenders: A pilot study]. / Processus psychologiques de gestion du stress et régulation neuroendocrinienne chez les adolescents délinquants en institution fermée : une étude pilote.

BACKGROUND: Adolescence is a stressful period where important biological, psychological and social changes occur. Adolescents are particularly vulnerable during this developmental period and can use various strategies to deal with daily stress, such as substance use or externalizing behaviors. In previous studies, stress in adolescents with externalizing behaviors was often linked to ineffective cognitive coping strategies (i.e., constructive thinking) and overlooking the biological aspects involved in stress management such as neuroendocrine regulation. Indeed, repeated activation of the hypothalamic-pituitary-adrenal (HPA) axis in chronic stress situations may have long-term effects on subsequent cortisol regulation and lead to psychological difficulties. It was also shown that basal cortisol levels are lower in adolescents with externalizing behaviors. This study aims to assess the links between constructive thinking and neuroendocrine regulation in adolescent offenders and their association with externalizing symptoms (e.g., aggression, delinquency, psychopathic traits, substance use). Identifying particular biopsychological patterns can help to better understand stress management in youth with externalizing behaviors and to improve clinical treatments. METHOD: Sixteen adolescent males aged from 12 to 18 years were recruited in an institution for juvenile offenders. Exclusion criteria were insufficient reasoning abilities assessed using the Raven Matrices Test. Regarding psychological dimensions, constructive thinking was assessed through the Constructive thinking inventory (CTI), psychopathic traits through the Youth psychopathic traits inventory (YPI), externalizing behaviors through 30 items (out of 113) and 2 subscales (aggressive behavior and delinquency problems) from the Child behavior checklist-youth self-report (CBCL), and substance use through the Dep-ado. Regarding biological dimensions, cortisol daily secretion and regulation were assessed through saliva samples that were collected during 3 consecutive days (4 samples per day: directly after awakening, at 10 a.m., at 4 a.m., and before going to bed). RESULTS: Adolescent offenders presented maladaptative thinking styles and a particular neuroendocrine regulation in their daily management with stress. In particular, their level of cortisol in the morning was higher than those expected in a general population (20.34 nmol/L while the norm is around 10 nmol/L). They also showed more agressive and delinquent behaviors (CBCL) as well as more psychopathic traits (YPI) than the general population. Moreover, constructive thinking style was associated with personality and behavioral dimensions. Indeed, results indicated positive and significant correlations between categorical thinking style (CTI), psychopathic traits (YPI) (r=0.57, P=0.021) and externalizing behaviors (CBCL) (r=0.55, P=0.028). In other words, the more adolescent offenders used categorical thinking, the more they presented psychopathic traits and externalizing behaviors. With respect to the association between psychological and biological dimensions in stress management, we observed a significant and positive correlation between cortisol regulation and esoteric thinking (r=0.57, P=0.028) and a trend with superstitious thinking (r=0.47, P=0.075). The more adolescent offenders used esoteric and superstitious thinking, the poorer was their cortisol regulation. We also observed a trend between the life style scale of the YPI (i.e., impulsive, irresponsible) and the daily secretion of cortisol (r=0.51; P=0.052) as well as cortisol regulation (r=0.49, P=0.065). The more adolescent offenders presented psychopathic traits, the higher tended to be their daily secretion of cortisol and the poorer their cortisol regulation. Finally, cortisol regulation (r=0.54, P=0.038) and secretion (r=0.73, P=0.002) were significantly correlated with the DEP-Ado score. In other words, a poor cortisol regulation and a high secretion of cortisol seem to be associated with substance use. CONCLUSIONS: Adolescent offenders face an important amount of daily stress and do not always have the appropriate skills to deal with it. Indeed, we know from clinical experience that they often report a sense of hopelessness toward their lack of professional perspectives as well as familial conflicts which can be important stressors in addition to the incarceration in itself. Therefore, treatment aiming to improve psychic elaboration can help these adolescents to make their thinking styles more flexible and use more appropriate ways of coping with stress instead of externalizing behaviors and substance use. Moreover, considering the complex cases of these adolescents and the many changes of caregivers and institutions where they have lived, which can be important stressors as well, professionals working with these youth should be aware of their emotional reactions toward them and try to encourage continuity of care.

Skin Rounds: A Quality Improvement Approach to Enhance Skin Care in the Neonatal Intensive Care Unit.

BACKGROUND: Skin injuries are common among neonatal intensive care unit (NICU) patients and may lead to significant complications. Standardized methods of preventing, detecting, and treating skin injuries are needed. PURPOSE: The aim of this project was to standardize the assessment, documentation, and tracking of skin injuries among hospitalized neonatal patients and to determine the incidence of pressure ulcers in this patient population. METHODS: (1) Creation of an interdisciplinary skin team to identify skin injuries through weekly skin rounds. (2) Assessment of all patients at least twice daily for the presence of skin injuries. Interventions were implemented upon identification of a skin injury. Pressure ulcers of Stage II or more were further assessed by wound/ostomy nurses. FINDINGS: A total of 2299 NICU patients were hospitalized and assessed between July 2011 and December 2015. After the initiation of skin rounds, the baseline incidence of pressure ulcers increased from 0.49 per 1000 patient days to 4.6 per 1000 patient days, reflecting an improvement in detection and reporting. The most common skin injuries detected included erythema, skin tears, and ecchymosis; the most common cause of injuries was medical devices. IMPLICATIONS FOR PRACTICE: A dedicated skin team can improve the detection and reporting of skin injuries among NICU patients. Determination of the incidence of pressure ulcers in this population is critical to develop targeted interventions. IMPLICATIONS FOR RESEARCH: Further research is needed to determine the most effective interventions to prevent and treat skin injuries among hospitalized neonates.

Red shift for CdTe nanoparticle thin films and suspensions during heating.

The work that we have conducted shows that temperature affects the wavelength of light emitted from CdTe nanoparticle clusters that are in a suspension or deposited into thin films via a layer-by-layer process. Compared with the stock suspension, the films show an initial photoluminescent shift, of circa 6-8 nm to the red, when the particles are deposited. A shift of circa 6-8 nm is also seen when the suspensions are first heated to 85 degrees C from room temperature (20 degrees C) having been stored in a fridge at 5 degrees C. This shift is non-recoverable. With continual cycling from room temperature to 85 degrees C the suspensions show a slight tendency for the emission to move increasingly to the red; whereas the films show no such tendency. In both cases, the range in emission is ca 10 nm from the room temperature state to 80 degrees C. The intensity of the emission from the film drops abruptly (ca 50% reduction) after one cycle of heating; in the suspension there is an initial increase (ca 3-5% increase) in intensity before it decays. We see that the shift towards the red has been attributed to energy transfer or a rearrangement of the packing of the particles in the thin films. After conducting analysis of the films using scanning probe microscopy we have determined that a change in the morphology is responsible for the permanent shift in emission wavelength associated with prolonged heating. The influence of traps has not been ruled out, but the morphological change in the samples is very large and is likely to be the dominating mechanism affecting change for the red shift at room temperature.

Hydrogen-induced surface metallization of beta-SiC(100)-(3x2) revisited by density functional theory calculations.

Recent experiments on the silicon terminated (3 x 2)-SiC(100) surface indicated an unexpected metallic character upon hydrogen adsorption. This effect was attributed to the bonding of hydrogen to a row of Si atoms and to the stabilization of a neighboring dangling bond row. Here, on the basis of density-functional calculations, we show that multiple-layer adsorption of H at the reconstructed surface is compatible with a different geometry: in addition to saturating the topmost Si dangling bonds, H atoms are adsorbed at rather unusual sites, i.e., stable bridge positions above third-layer Si dimers. The results thus suggest an alternative interpretation for the electronic structure of the metallic surface.

Oligonucleotide-mediated gene editing for neuromuscular disorders.

In the last decade, outstanding progress has been made in the development of new approaches to treat neuromuscular disorders and in particular Duchenne muscular dystrophy (DMD). The use of oligonucleotides to induce single base pair alterations in the dystrophin gene and restore gene expression in skeletal muscle has proven to be a feasible alternative approach to dystrophin gene replacement. Oligonucleotide-mediated gene editing for dystrophin has the potential to treat the disorder permanently and effectively. Many hurdles however still need to be overcome before this technology can enter into a clinical setting. Understanding the mechanisms of the repair process is a key for the design of oligonucleotides capable to induce gene repair more efficiently and precisely. The future of this technology will depend, ultimately, on the development of safe delivery systems capable to target a large number of muscles. Furthermore, before using oligonucleotides into a clinical setting, we will need to evaluate issues of toxicity, which will have to be balanced with the severity of the disease and the prognosis of the patient. Finally, the rapid progress that has been made to scale up the production of synthetic oligonucleotides will enable the synthesis from micrograms to milligrams quantities allowing this field to move from the bench to the bedside. This review will describe the basic mechanisms of oligonucleotide-mediated gene editing and will explain the potential, hurdles and substantial results obtained using this technology in the treatment of muscular dystrophies.

Ab initio simulations of homoepitaxial SiC growth.

We present first-principle calculations on the initial stages of SiC homoepitaxial growth on the beta-SiC(111)-(sqrt[3]xsqrt[3]) surface. We show that the nonstoichiometric reconstruction plays a relevant role in favoring the attainment of high-quality films. The motivation is twofold: On one hand, we find that the reconstruction controls the kinetics of adatom incorporation; on the other hand, we observe that the energy gain upon surface stability can induce the reorganization of the deposited material into a crystalline structure, thus revealing that a surface-driven mechanism is able to stabilize defect-free layer deposition on Si-rich surfaces.

BRCA1 mutations and clinicopathological features in a sample of Italian women with early-onset breast cancer.

Breast cancer in young women is uncommon and often presents with unfavourable biopathological features. Although early age at onset could suggest a genetic susceptibility to cancer, the appropriateness of BRCA1 testing for women with early-onset breast cancer and modest family history (FH) is controversial. 40 Women diagnosed with breast cancer at the age of 35 years or less, unselected for FH, were screened for germ line BRCA1 mutations by automated sequencing of exons 2, 5, 6, 11, 13 and 20. Overall, deleterious mutations were evidenced in 6 (15%) patients. With regard to FH, mutations were detected in 14%, 11% and 29% of women with none, weak and strong FH, respectively. Large tumour size, grade 3, lack of oestrogen receptors and high proliferation rate were significantly more common in mutation carriers (MC). Our data support both the appropriateness of testing young breast cancer patients and the frequency of unfavourable features in BRCA1-related breast cancer. It is hypothesised that BRCA1 mutations partially justify the high rate of aggressive breast cancer in young patients and that combining age and breast cancer phenotype could help to identify probable MC.

Comparison between genotype and phenotype identifies a high-risk population carrying BRCA1 mutations.

Hereditary breast carcinomas constitute about 10% of all malignant mammary tumors, but the selection criteria to identify a high-risk population carrying BRCA1 mutations are not yet well-defined. We have collected 51 pedigrees of familial breast cancer, 16 pedigrees of familial breast and ovarian cancer, and 30 cases of early-onset breast cancer (<35 years of age) without any family history of breast cancer. The index cases of the 97 selected families were further subdivided into three groups based on histopathological parameters: group A (n = 19) was characterized by tumor grade III, negative estrogen and progesterone receptors, and high proliferative rate; group B (n = 20) was characterized by grade I-II tumors, positive hormonal receptors, and low proliferative rate; and group C (n = 58) was not homogeneous for the histopathological criteria. The aim of our study was to evaluate, in patients with a family history of breast cancer or with early diagnosis of breast cancer, the incidence of BRCA1 mutation on the basis of tumor phenotype. We found the highest rate of BRCA1 mutations in group A (53%), and low frequencies in groups B (5%) and C (0%). Our data strongly indicate that an aggressive tumor phenotype in patients with a positive family history or early diagnosis identifies a population with high probability of carrying BRCA1 mutations. Genes Chromosomes Cancer 27:130-135, 2000.

Catabolism of asialo-GM2 in man and mouse. Specificity of human/mouse chimeric GM2 activator proteins.

Tay-Sachs disease is an inborn lysosomal disease characterized by excessive cerebral accumulation of GM2. The catabolism of GM2 to GM3 in man requires beta-hexosaminidase A (HexA) and a protein cofactor, the GM2 activator. Thus, Tay-Sachs disease can be caused by the deficiency of either HexA or the GM2 activator. The same cofactor found in mouse shares 74.1% amino acid identity (67% nucleotide identity) with the human counterpart. Between the two activators, the mouse GM2 activator can effectively stimulate the hydrolysis of both GM2 and asialo-GM2 (GA2) by HexA and, to a lesser extent, also stimulate HexB to hydrolyze GA2, whereas the human activator is ineffective in stimulating the hydrolysis of GA2 (Yuziuk, J. A., Bertoni, C., Beccari, T., Orlacchio, A., Wu, Y.-Y., Li, S.-C., and Li, Y.-T. (1998) J. Biol. Chem. 273, 66-72). To understand the role of these two activators in stimulating the hydrolyses of GM2 and GA2, we have constructed human/mouse chimeric GM2 activators and studied their specificities. We have identified a narrow region (Asn(106)-Tyr(114)) in the mouse cDNA sequence that might be responsible for stimulating the hydrolysis of GA2. Replacement of the corresponding site in the human sequence with the specific mouse sequence converted the ineffective human activator into an effective chimeric protein for stimulating the hydrolysis of GA2. This chimeric activator protein, like the mouse protein, is also able to stimulate the hydrolysis of GA2 by HexB. The mouse model of human type B Tay-Sachs disease recently engineered by the targeted disruption of the Hexa gene showed less severe clinical manifestation than found in human patients. This has been considered to be the result of the catabolism of GM2 via converting it to GA2 and further hydrolysis of GA2 to lactosylceramide by HexB with the assistance of mouse GM2 activator protein. The chimeric activator protein that bears the characteristics of the mouse GM2 activator may therefore be able to induce an alternative catabolic pathway for GM2 in human type B Tay-Sachs patients.

With regard to glycohemoglobin measurement: are we sure that high-performance liquid chromatography currently works in the clinical routine?

The clinical usefulness of glycated hemoglobin (HbA1C) depends crucially on the accuracy and precision of its assay. When we compared an immunological bench-top analyzer (DCA 2000, Bayer Diagnostici, Milan) to the high-performance liquid chromatography (HPLC) reference method used in a routine hospital laboratory (Diamat and Fast Diamat, Bio-Rad Lab., Milan) by assaying multiple control sera, we found so many sources of systematic analytical errors in the routine use of HPLC as to compromise between-assay precision. DCA 2000 showed intra- and interassay coefficients of variation (CV) of 1.1% and 2.3% with the normal standard serum, 1.0% and 4.2% with the pathological one; Diamat yielded CVs of 1.3% and 7.0%, 1.3% and 5.7%, respectively. Although the measurement of 161 blood samples showed that Diamat usually overestimated HbA1C (paired t-test, P<0.001), a great variability of Diamat performance became evident when the relationship Diamat vs DCA was evaluated day by day over 17 days of observation (analysis of variance, ANOVA, P<0.001). Intra- and interassay CVs of Fast Diamat initially (new instrument still on approval) were 0.6% and 2.5% (normal standard serum), 0.3% and 1.9% (high standard serum), yet after 6 months of routine laboratory use, they became 3.1% and 3.2%, 1% and 12.3%, respectively. Main sources of error were: inaccurate autodilution, unsuitable parameter settings, disregard of the maintenance schedule. We conclude that the tendency to overlook major critical aspects in the routine use of HPLC is detrimental to the quality of HbA1C determination and implies the loss of HbA1C value in clinical practice. Both carefully supervising laboratory quality and checking the likelihood of the analytical result with the clinical setting appear even more important.

Urinary excretion rates of multiple renal indicators after kidney transplantation: clinical significance for early graft outcome.

Post-reperfusion inflammation as well as anti-allograft response occur following kidney transplantation. We evaluated tissue damage by multiple renal indicators and searched for rejection predictors forewarning serum creatinine upturns. Twenty recipients (43 +/- 9 y; donors' age 35 +/- 16 y) of first renal grafts were studied. All through their hospital stay (35 +/- 18 d, range 17-75 d) we measured serum levels of urea, creatinine and electrolytes along with urinary excretion rates of total protein, albumin, enzymes (GGT, NAG, AAP) and electrolytes. During the period of observation, peaks were seen on the 1st day for serum creatinine, serum K+ and urine albumin output; on the 2nd day for urine Na+, GGT, AAP and protein excretion rates; on the 4th day for urea and creatinine outputs; on the 5th day for NAG output. On the 14th day, serum urea and creatinine as well as urine GGT, NAG, AAP, albumin and total protein were still elevated compared to 20 healthy control subjects. Delayed/slow graft function was observed in six recipients with higher pre-transplantation plasma lipids and lower donor HDL cholesterol. Hospital stay time was correlated with need for post-transplantation dialysis (p = 0.01) and recipient proteinuria by time 0 (TO) to day 3 (p = 0.02). Cold ischemia time was positively associated with 0-3 d serum creatinine, 0-3 d urinary urea and protein outputs (multiple r 0.9, p < 0.001). Multivariate analysis of longitudinal data showed that recipients' serum creatinine was positively correlated (p < 0.001) with urine AAP and negatively correlated with urine albumin, with diuresis volume and urine creatinine (p < 0.01). Serum creatinine elevations were preceded (previous 1-7 d) by increases in urinary indicators, the probability being higher in the presence of multiple simultaneous abnormalities. Useful parameters predictive of favorable graft outcome prior to transplantation included a brief cold ischemia time and a normal donor/recipient serum lipoprotein profile. Following transplantation, useful parameters were a high diuresis volume at time zero along with low urine NAG and high albumin outputs; early (first opst-graft 3 d) polyuria, low urea and GGT, high K, NAG and total protein excretions.

Specificity of mouse GM2 activator protein and beta-N-acetylhexosaminidases A and B. Similarities and differences with their human counterparts in the catabolism of GM2.

Tay-Sachs disease, an inborn lysosomal disease featuring a buildup of GM2 in the brain, is caused by a deficiency of beta-hexosaminidase A (Hex A) or GM2 activator. Of the two human lysosomal Hex isozymes, only Hex A, not Hex B, cleaves GM2 in the presence of GM2 activator. In contrast, mouse Hex B has been reported to be more active than Hex A in cleaving GM2 (Burg, J., Banerjee, A., Conzelmann, E., and Sandhoff, K. (1983) Hoppe Seyler's Z. Physiol. Chem. 364, 821-829). In two independent studies, mice with the targeted disruption of the Hexa gene did not display the severe buildup of brain GM2 or the concomitant abnormal behavioral manifestations seen in human Tay-Sachs patients. The results of these two studies were suggested to be attributed to the reported GM2 degrading activity of mouse Hex B. To clarify the specificity of mouse Hex A and Hex B and to better understand the observed results of the mouse model of Tay-Sachs disease, we have purified mouse liver Hex A and Hex B and also prepared the recombinant mouse GM2 activator. Contrary to the findings of Burg et al., we found that the specificities of mouse Hex A and Hex B toward the catabolism of GM2 were not different from the corresponding human Hex isozymes. Mouse Hex A, but not Hex B, hydrolyzes GM2 in the presence of GM2 activator, whereas GM2 is refractory to mouse Hex B with or without GM2 activator. Importantly, we found that, in contrast to human GM2 activator, mouse GM2 activator could effectively stimulate the hydrolysis of GA2 by mouse Hex A and to a much lesser extent also by Hex B. These results provide clear evidence on the existence of an alternative pathway for GM2 catabolism in mice by converting GM2 to GA2 and subsequently to lactosylceramide. They also provide the explanation for the lack of excessive GM2 accumulation in the Hexa gene-disrupted mice.

The reliability of urinary albumin assay by immunonephelometry in the clinical practice. The critical role of medical surveillance on laboratory routine.

Measurement of the urinary albumin excretion rate (UAER) is essential for the early diagnosis and monitoring of diabetic nephropathy; immunonephelometry is a procedure used worldwide for routine screening of diabetic patients. Since we have met with occasional inconsistent values of UAER in serial urine collections, we searched for possible sources of analytic error. To assess the best working conditions of the instrument in use, the stability of urine samples during storage and the need for previous urine centrifugation, we assayed repeatedly the six automatically diluted points of the standard curve (55.6 to 1.7 mg/l), four control samples of human albumin in saline (100 to 1 mg/l) and 24-h urine collections from outpatient diabetic subjects. The last were also assayed with and without previous centrifugation, and both immediately after collection as well as after storage at -20 degrees C for 7, 42, 79, 97, 128 and 161 days. We concluded that: (1) pre-analytic centrifugation of urine samples in unnecessary; (2) the intra-assay coefficient of variation (CV) of the standard curve changed from 2.4% to 9.3% when moving from the highest to the lowest concentration; the inter-assay CV changed from 4.1% to 14.4%, respectively; (3) the intra-assay CV of the control samples (manually prepared) changed from 5.7% to 10.2% and the inter-assay CV from 7.7% to 22.9%; there was a constant and significant (P < 0.01) underestimation (from -9% to -30%) of the obtained values compared with the expected concentrations; (4) a progressive decrease in recovered albumin by multiple freezing and thawing of urine samples did occur, which became significant after 161 days of storage. In the BNA workbook (menu 7.1, assay protocols), a 7-day validity of the reference curve is reported. Moreover, to economize, pre-dilution cuvettes were often recycled in our hospital central laboratory. We observed that: the intra-assay CV for urine samples was 79.4% with recycled cuvettes and stored standard curve, 11.3% with new cuvettes and stored standard curve, 4.9% with both new cuvettes and newly performed standard curve; the inter-assay CV was 32.6%, 10.5% and 6.4%, respectively. These data emphasize, from the laboratory viewpoint, the need for both accurate calibration of BNA and use of native urines; in addition, they stress the importance of careful supervision of laboratory routine and interpreting analytic results in the clinical setting.

Structural organization and expression of the gene for the mouse GM2 activator protein.

The GM2 activator protein is an essential component for the degradation of GM2 ganglioside by hexosaminidase A in vivo. Mutations in the human gene coding for the GM2 activator protein cause the AB variant of GM2-gangliosidosis, a condition that is clinically indistinguishable from Tay-Sachs disease. To understand better factors affecting the expression of the GM2 activator protein gene (Gm2a) in mouse tissues, we have determined its exon-intron organization and analyzed its promoter region. Gm2a is about 14 kb, has four exons, and the 5' flanking region contains a CAAT box, Sp1 binding sites, AP-1, AP-2 sites, and a pair of IRE sites. A 1.2-kb fragment upstream from the initiation codon was shown to have promoter activity in NIH 3T3 cells. Similarities between the elements present in Gm2a and Hexa promoters might in part explain their similar expression patterns in mouse tissues. The different levels of GM2 activator protein mRNA in liver, kidney, brain, and testis are not owing to the use of different transcription start sites, because a single start site was found 50 bp upstream from the initiation codon in each these tissues. Northern blot analysis demonstrated variation in the GM2 activator protein mRNA expression during mouse development. Gm2a was mapped to Chromosome (Chr) 11, where it co-segregated with Csfgm.