The effect of coughing at extubation on oxygenation in the post-anaesthesia care unit.

We prospectively studied 84 patients to investigate whether there is a relationship between coughing during emergence and tracheal extubation, and impaired oxygenation in the post-anaesthesia care unit. Our primary outcome measure was a change in the alveolar-arterial oxygen partial pressure gradient ((A-a)DO2 ) between time A (during general anaesthesia) and time B (1 h after extubation). Patients demonstrated a worsening of oxygenation with mean (SD) (A-a)DO2 increasing from 7.5 (5.2) kPa at time A to 13.9 (4.2) kPa at time B (p < 0.01). An overall linear regression model was not predictive for the observed change (adjusted R(2) = 0.01, p = 0.31) and nor were any of the individual predictors studied, including subjective cough score (p = 0.33), number of coughs (p = 0.95) and duration of coughing (p = 0.39). Despite the abnormal cough that occurs while tracheally intubated, we have been unable to demonstrate that coughing at extubation is associated with impaired oxygenation in the immediate postoperative period.

Outpatient Follow Up and Reconstructive Surgery Rates in Massive Burn Survivors: Investigating the Social Determinants.

Burn care continues to improve and larger total body surface area (TBSA) burn survival is increasing. These survivors require more extensive care than smaller burns and are at higher risk for wound/scar related complications. Prior work has shown low rates of follow up for burn survivors linked to socioeconomic factors such as housing insecurity and substance use. There are limited studies that evaluate socioeconomic factors that contribute to follow up and reconstructive surgery rates in massively burned patients. Patients that survived to discharge with >50% TBSA burns and planned return to treating institution were included in the study. Univariate and multivariate analyses were performed on the data collected. Sixty-Five patients were included with an average TBSA of 63.1%. Fifty-three patients (81.5%) attended at least one follow up appointment with median of four follow-up appointments. Younger patients (33±9 vs 44±11; p=0.0006), patients with larger TBSA burns (65±13 vs 55±5%; p=0.02), those with private insurance and those without housing insecurity (1.8% vs 45.4%; p=0.003) were more likely to follow up. On multivariate regression analysis, patients with housing insecurity were independently associated with lack of follow up (OR: 0.009 CI: 0.00001-0.57). Thirty-five patients had at least one reconstructive surgery and 31 patients had reconstructive surgery after discharge. No patients with housing insecurity underwent reconstructive surgery. Follow up rates in massive burns were higher than reported for smaller TBSA burns and more than half received reconstructive surgery. Housing insecure patients should be targeted for improved follow up and access to reconstructive surgery.

Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts.

Genetic defects of lysosomal hydrolases result in severe storage diseases and treatments based on enzyme replacement have been proposed. In mice lacking beta-glucuronidase, which develop a disease homologous to human mucopolysaccharidosis type VII (Sly syndrome), we have used autologous implants of genetically-modified skin fibroblasts for the continuous in vivo production of the enzyme. The human beta-glucuronidase cDNA was introduced with a retroviral vector into mutant mice skin fibroblasts grown in primary culture. Fourteen mutant mice were implanted intraperitoneally with these modified cells embedded into collagen lattices. All animals expressed beta-glucuronidase from the vascularized neo-organs that developed after implantation and accumulated the enzyme in their tissues. A complete disappearance of the lysosomal storage lesions was observed in their liver and spleen.

Massive Burn Injuries: Characteristics and Outcomes From a Single Institution.

Massive burn injuries are a unique patient population with unique treatment paradigms. Data from 155 adult patients, admitted from 2009 to 2019, with >50% total body surface area burns (TBSA) were collected and analyzed. Average burn size was 70% TBSA and 63% had a concomitant inhalation injury. Approximately 30% of patients (46/155) transitioned to comfort care-only measures within 24 hours of admission. Standard treatment patients were younger (37 ± 13 vs 60 ± 19 years; p < .00001), male (94% vs 28%; p = .001) and had smaller TBSA (66 ± 13 vs 80 ± 16; p < .00001). Of the standard treatment group, 72 (66%) survived to discharge. Survivors had smaller TBSA (64 ± 13 vs 71 ± 13; p = .003), less third-degree TBSA (48 ± 25 vs 71 ± 13; p = .003) and lower incidence of renal failure requiring dialysis (22% vs 73%, p < .00001). Multivariate regression analysis showed that age (OR 1.05; p = .025), total TBSA (OR 1.07; p = .005), and renal failure (OR 10.2; p = .00005) were independently associated with mortality. Inhalation injury was not significantly associated with mortality. About 23% (35/155) of patients had a psychiatric condition on admission and 19% (30/155) of patients were burned attempting suicide. Patients with psychiatric conditions spent more time in the hospital (62 vs 30 days; p = .004), more time on ventilator (31 vs 12 days; p = .046), underwent more surgery (4 vs 2 operations, p = .03), and were less likely to die (34% vs 59%; p = .02). In summary, age, burn size, and renal failure were independently associated with mortality, with renal failure being the strongest factor. Psychiatric conditions are prevalent pre-injury and tend to require more inpatient care.

Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts.

We investigated tetracycline regulation of gene expression in an experimental model relevant to gene therapy. Mouse primary myogenic cells were engineered for doxycycline-inducible and skeletal muscle-specific expression of the mouse erythropoietin (Epo) cDNA by using two retrovirus vectors. Gene expression increased 200 fold in response to both myogenic cell differentiation and doxycycline stimulation. After transplantation of transduced cells into mouse skeletal muscles, Epo secretion could be iteratively switched on and off over a five-month period, depending on the presence or the absence of doxycycline in the drinking water. We conclude that tetracycline regulation provides a suitable control system for adjusting the delivery of therapeutic proteins from engineered tissues over long periods of time.

Long-term delivery of a lysosomal enzyme by genetically modified fibroblasts in dogs.

We have evaluated the feasibility and efficacy of intraperitoneal implants (neo-organs) for protein delivery in large animals. Skin biopsies were taken from four healthy dogs. Primary fibroblast cultures were transduced with a retroviral vector coding for the human beta-glucuronidase. One to six lattices each containing 10(9) skin fibroblasts were implanted into the omentum of the donor animal. Laparotomies performed at regular intervals showed vascularized neo-organs without local inflammation. Human beta-glucuronidase levels equivalent to 0.8 to 3.1% of the endogenous canine activity were detected for up to 340 days on liver biopsy samples. These results indicate that neo-organs can be considered for the long-term delivery of therapeutic proteins or enzymes in humans.

Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.

Like other pathogenic viruses, HIV-1 down-modulates surface expression of major histocompatibility complex class I (MHC-I) molecules in infected cells, thus impairing lysis by cytotoxic T lymphocytes. We have observed that this phenomenon depends on the expression of Nef. nef is an early gene of primate lentiviruses, which is necessary for maintaining high virus loads and inducing AIDS. Nef is not necessary for viral replication in vitro and stimulates the endocytosis of CD4. We show that the expression of MHC-I at the surface of lymphoid, monocytic and epithelial cells was reduced in the presence of Nef protein from various HIV-1 strains. Whereas MHC-I protein synthesis and transport through the endoplasmic reticulum and cis Golgi apparatus occurred normally in Nef(+) cells, surface MHC-I molecules were rapidly internalized, accumulated in endosomal vesicles and were degraded. The stimulation of MHC-I endocytosis by Nef represents a previously undocumented viral mechanism for evading the immune response.

MHC-I-restricted presentation of HIV-1 virion antigens without viral replication.

Dendritic cells and macrophages can process extracellular antigens for presentation by MHC-I molecules. This exogenous pathway may have a crucial role in the activation of CD8+ cytotoxic T lymphocytes during human viral infections. We show here that HIV-1 epitopes derived from incoming virions are presented through the exogenous MHC-I pathway in primary human dendritic cells, and to a lower extent in macrophages, leading to cytotoxic T-lymphocyte activation in the absence of viral protein synthesis. Exogenous antigen presentation required adequate virus-receptor interactions and fusion of viral and cellular membranes. These results provide new insights into how anti-HIV cytotoxic T lymphocytes can be activated and have implications for anti-HIV vaccine design.

Output of selenium in milk, urine, and feces is proportional to selenium intake in dairy cows fed a total mixed ration supplemented with selenium yeast.

Fifteen rumen fistulated Holstein cows in late lactation and fed a total mixed ration offered ad libitum were supplemented with Se yeast to provide 0, 11, 20, 30, or 42 mg of supplemental Se/day to test the hypothesis that amounts of Se secreted in milk, excreted in urine and feces, and apparently retained in tissues would increase in direct proportion to Se intake. One-half of the yeast supplement was placed directly into the rumen through the fistula of each cow just before milking in the morning and again in the evening, and estimates of average daily excretion of Se were made using total collections of urine and feces from 25 to 31 d after treatments commenced. Amounts of Se secreted daily in milk and apparently retained in tissues increased linearly with average daily intake of Se. The amount of Se excreted in feces and total excretion of Se in urine plus feces increased curvilinearly with Se intake, such that proportionately less Se was excreted as the amount of Se fed increased. On average, total Se excretion accounted for 66%, Se secretion in milk accounted for 17%, and Se apparently retained in tissues accounted for 17% of total Se intake by cows. Thus, in herds fed large amounts of Se yeast, most of the Se will be excreted and retained on-farm. High concentrations of Se will be found where urine and feces accumulate (e.g., yards and effluent ponds), and effluent management practices must be tailored to avoid environmental issues.

The impact of COVID-19 on rare metabolic patients and healthcare providers: results from two MetabERN surveys.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has caused disruption in all aspects of daily life, including the management and treatment of rare inherited metabolic disorders (IMDs). To perform a preliminary assessment of the incidence of COVID-19 in IMD patients and the impact of the coronavirus emergency on the rare metabolic community between March and April 2020, the European Reference Network for Hereditary Metabolic Diseases (MetabERN) has performed two surveys: one directed to patients' organizations (PO) and one directed to healthcare providers (HCPs). The COVID-19 incidence in the population of rare metabolic patients was lower than that of the general European population (72.9 × 100,000 vs. 117 × 100,000). However, patients experienced extensive disruption of care, with the majority of appointments and treatments cancelled, reduced, or postponed. Almost all HCPs (90%) were able to substitute face-to-face visits with telemedicine, about half of patients facing treatment changes switched from hospital to home therapy, and a quarter reported difficulties in getting their medicines. During the first weeks of emergency, when patients and families lacked relevant information, most HCPs contacted their patients to provide them with support and information. Since IMD patients require constant follow-up and treatment adjustments to control their disease and avoid degradation of their condition, the results of our surveys are relevant for national health systems in order to ensure appropriate care for IMD patients. They highlight strong links in an interconnected community of HCPs and PO, who are able to work quickly and effectively together to support and protect fragile persons during crisis. However, additional studies are needed to better appreciate the actual impact of COVID-19 on IMD patients' health and the mid- and long-term effects of the pandemic on their wellbeing.

Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes.

The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants. Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.

Increasing amounts of crushed wheat fed with pasture hay reduced dietary fiber digestibility in lactating dairy cows.

Sixteen cows in mid-lactation (milk yield of 23.8 +/- 2.3 kg/d) were individually fed diets consisting of chopped perennial ryegrass hay, offered at 3 kg of dry matter (DM)/100 kg of body weight (BW), fed either alone or supplemented with amounts of crushed wheat ranging from 0.4 to 1.6 kg of DM/100 kg of BW (increasing at nominal intervals of 0.4 kg of DM/100 kg of BW; 5 nominal treatments in total). Three cows were allocated to each treatment except the mid-range wheat treatment, which had 4 cows. Results were analyzed by regression because the intake of the wheat by cows within treatments varied. The hay was used to reflect the characteristics of summer pastures in southeastern Australia. Feed intake and fecal output were measured to determine digestion coefficients, feeds were incubated in nylon bags in the rumen, and rumen variables were monitored. Estimates of metabolizable energy (ME) of the hay from in vivo or in vitro digestibility were also compared. The digestibility of neutral detergent fiber (NDF) was depressed linearly as the amount of crushed wheat consumed increased to 36% of DM intake. The extent to which negative associative effects on NDF digestion were associated with the hay could not be determined, as it was not possible to distinguish between the NDF from hay and that from wheat. However, acid detergent fiber (ADF) digestion also declined, suggesting that most of the response lay with the hay because ADF was negligible in the wheat. Most data indicated that effects of proportion of wheat in the diet on the utilization of consumed nutrients were small. Despite substitution of wheat for hay reducing the forage intake of cows, there was a positive linear effect on marginal milk responses (1.3 kg of energy-corrected milk/kg of DM wheat). Mean rumen fluid pH declined as the proportion of wheat in the diet increased. The lowest pH for any individual cow during a 24-h period was 5.4, and the amount of time that rumen fluid pH was <6.0 ranged from 0 to 14 h depending on the amount of wheat consumed. It was concluded that these perturbations of the rumen environment were probably sufficient to result in negative associative effects. In addition, estimates of the ME content of the hay were higher when calculated from in vitro compared with in vivo digestibility, which has implications when estimating the amount of feed required for production.

Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.

Multistep virus-induced leukemogenesis in vitro: description of a model specifying three steps within the myeloblastic malignant process.

A helper-independent Friend leukemia virus was used to infect bone marrow cultures. This virus induces myeloblastic leukemia in mice after a long latency period. Infection of the bone marrow cultures resulted in the in vitro production of myeloblastic leukemogenesis after a long latency period. Three steps were observed in the evolution of the infected cultures, and permanent cell lines were derived at each step. This allowed us to individualize three successive events in the course of the myeloblastic transformation: (i) an abnormal responsiveness to the physiological hormone granulo-macrophagic colony-stimulating factor, (ii) the acquisition of growth autonomy, and (iii) the acquisition of in vivo tumorigenicity.

Monoclonal proliferation of Friend murine leukemia virus-transformed myeloblastic cells occurs early in the leukemogenic process.

Integrated Friend murine leukemia virus copies were analyzed by the Southern blotting procedure in myeloblastic cell lines obtained after in vitro infection of long-term mouse bone marrow cultures. Several steps leading to the generation of malignant myeloblastic cells after a long latency period were observed in the evolution of infected cultures. Shortly after infection, a random distribution of integrated provirus copies was observed in the DNA of normally differentiating myeloid cells. In contrast, a distinct pattern of integrated Friend murine leukemia virus copies was evident in the first non-differentiating immature myeloblastic cells appearing in cultures, suggesting a monoclonal origin of these cells. For each cell line, characteristic hybridizing fragments were conserved during the 1-year culture period necessary for the acquisition of tumorigenic properties and were also observed in tumors grafted in vivo. We can conclude that monoclonality is effective very early in the myeloid transformation process, as soon as the precursor cells are blocked in their differentiation.

Increasing selenium concentration in milk: effects of amount of selenium from yeast and cereal grain supplements.

Two experiments were conducted to establish responses in milk Se concentrations in grazing dairy cows to different amounts of dietary Se yeast, and to determine the effects of the Se concentration of the basal diet. The hypothesis tested was that the response in milk, blood, and tissue Se concentrations to supplemental Se would not be affected by whether the Se was from the basal diet or from Se yeast. In addition, by conducting a similar experiment in either early (spring; experiment 1) or late (autumn; experiment 2) lactation, we hypothesized that different Se input-output relationships would result. Both 6-wk experiments involved 60 multiparous Holstein-Friesian cows, all of which had calved in spring. They were allocated to 1 of 10 dietary Se treatments that included 2 types of crushed triticale grain (low Se, approximately 165 microg of Se/kg of DM; or high Se, approximately 580 microg/kg of DM) fed at 4 kg of DM/d, and 1 kg of DM/d of pellets formulated to carry 5 quantities of Se yeast (0, 4, 8, 12, or 16 mg of Se). Daily total Se intakes ranged from <2 to >18 mg/cow in both experiments. Milk Se concentrations plateaued after 15 and 7 d of supplementation in experiments 1 and 2, respectively, and then remained at plateau concentrations. Average milk Se concentrations for the plateau period increased as the amount of Se yeast increased, and low- and high-Se grain treatments were different at all quantities of Se yeast, although there was a tendency for this difference to diminish at the greatest concentrations of yeast. There were significant positive, linear relationships between Se intake and the concentrations of Se in milk, which were not affected by the source of Se, and the relationships were similar for both experiments. Therefore, the output of Se in milk in experiment 1 was greater than that in experiment 2 because the milk yield of the cows in early lactation was greater. The estimated proportions of Se partitioned to destinations other than milk and feces increased with the amount of Se in the diet and were greater in experiment 2 than in experiment 1, a result that was supported by Se concentrations in whole blood and plasma and in semitendinosus muscle tissue. If high-Se products are to be produced for human nutrition, it is important to be able to develop feeding systems that produce milk with consistent and predictable Se concentrations so that products can consistently meet specifications. The results indicate that this objective is achievable.

Role of Food Handlers in Norovirus Outbreaks in London and South East England, 2013 to 2015.

Outbreaks caused by norovirus infection are common and occur throughout the year. Outbreaks can be related to food outlets either through a contaminated food source or an infected food handler. Both asymptomatic and symptomatic food handlers are potentially implicated in outbreaks, but evidence of transmission is limited. To understand potential food handler transmission in outbreak scenarios, epidemiological and microbiological data on possible and confirmed norovirus outbreaks reported in London and South East England in a 2-year period were reviewed. One hundred eighty-six outbreaks were associated with a food outlet or registered caterer in this period. These occurred throughout the year with peaks in quarter 1 of study years. A case series of 17 outbreaks investigated by the local field epidemiological service were evaluated further, representing more than 606 cases. In five outbreaks, symptomatic food handlers were tested and found positive for norovirus. In four outbreaks, symptomatic food handlers were not tested. Asymptomatic food handlers were tested in three outbreaks but positive for norovirus in one only. Environmental sampling did not identify the causative agent conclusively in any of the outbreaks included in this analysis. Food sampling identified norovirus in one outbreak. Recommendations from this study include for outbreak investigations to encourage testing of symptomatic food handlers and for food and environmental samples to be taken as soon as possible. In addition, sampling of asymptomatic food handlers should be considered when possible. However, in light of the complexity in conclusively identifying a source of infection, general measures to improve hand hygiene are recommended, with specific education among food handlers about the potential for foodborne pathogen transmission during asymptomatic infection, as well as reinforcing the importance of self-exclusion from food handling activities when symptomatic.

Comparative effects of polycythemia-inducing Friend leukemia virus and Moloney leukemia virus on hematopoietic progenitors in murine long-term bone marrow cultures.

The specificity of murine leukemia virus-induced myelomonocytic phenotypic changes in long-term bone marrow culture have been examined by comparing the effects of polycythemia-inducing Friend leukemia virus (FVP) and Moloney murine leukemia virus (M-MuLV) known to have in vivo target cells in the erythroid and lymphoid lineage, respectively. Noninfected adn M-MuLV-infected cultures showed no modification in granulocyte macrophage colony-forming cell behavior and failed to generate cell line in WEHI-3-conditioned medium. In contrast, in FVP-infected cultures, granulocyte macrophage colony-forming cells became colony-stimulating factor independent, and the nonadherent cells gave rise to two cell lines in WEHI-3 conditioned medium with monocytic characteristics and no leukemogenic potential in vivo. These results confirm the ability of long-term bone marrow culture to unmask target cells for FVP within myelomonocytic progenitors, and the negative results in M-MuLV-infected cultures underline the specificity of the FVP-induced phenotypic changes. Despite a high level of virus production and the presence of T-cell precursors in the M-MuLV infected culture, T-cell transformation was not observed.