The complement cascade in lung injury and disease.

BACKGROUND: The complement system is an important arm of immune defense bringing innate and adaptive immunity. Although originally regarded as a major complementary defense mechanism against pathogens, continuously emerging evidence has uncovered a central role of this complex system in several diseases including lung pathologies. MAIN BODY: Complement factors such as anaphylatoxins C3a and C5a, their receptors C3aR, C5aR and C5aR2 as well as complement inhibitory proteins CD55, CD46 and CD59 have been implicated in pathologies such as the acute respiratory distress syndrome, pneumonia, chronic obstructive pulmonary disease, asthma, interstitial lung diseases, and lung cancer. However, the exact mechanisms by which complement factors induce these diseases remain unclear. Several complement-targeting monoclonal antibodies are reported to treat lung diseases. CONCLUSIONS: The complement system contributes to the progression of the acute and chronic lung diseases. Better understanding of the underlying mechanisms will provide groundwork to develop new strategy to target complement factors for treatment of lung diseases.

Ghrelin alterations during experimental and human sepsis.

BACKGROUND: Ghrelin is a hormone mainly produced by cells of the gastric mucosa, which has been shown to possess anti-inflammatory and immunomodulatory properties. The objective of the study was to investigate ghrelin levels during sepsis, as well as in an experimental sepsis model. METHODS: All consecutive admissions to the ICU of a tertiary hospital in Athens, Greece were screened for eligibility during the study. Thirty four non-septic patients upon ICU admission who subsequently developed sepsis were enrolled. Clinical data and scores were recorded, and blood samples were obtained at baseline (upon ICU admission), and at sepsis development. Total and active ghrelin, leptin, and cytokines were measured. Moreover, lipopolysaccharide (LPS) was administered to mice in order to induce endotoxemia and at specified time points, blood and tissue samples were collected. RESULTS: In patients, serum total and active ghrelin concentrations were significantly elevated in sepsis compared to baseline (553.8 ±â€¯213.4 vs 193.5 ±â€¯123.2, p < 0.001; 254.3 ±â€¯70.6 vs 56.49 ±â€¯16.3, p < 0.001). Active ghrelin levels at the sepsis stage were inversely correlated with SOFA score and length of stay in the ICU (p = 0.023 and p = 0.027 respectively). In the mouse endotoxemia model ghrelin levels were elevated following LPS treatment, and the same trend was observed for leptin, TNFα and IL-6. Ghrelin administration managed to reduce IL-6 levels in mouse serum and in BALF. Pulmonary expression of ghrelin and its receptor GHSR1a was found decreased in LPS-treated mice. CONCLUSIONS: In a well-defined cohort of ICU patients, we have demonstrated that active and total ghrelin increase in sepsis. The same is true for the experimental sepsis model used in the study. The inverse correlation of active ghrelin levels with SOFA score and length of ICU stay among septic patients is indicative of a potential protective role of active ghrelin during the septic process.

In a sweet mood? Effects of experimental modulation of blood glucose levels on mood-induction during fMRI.

Glucose is the primary source of energy for the human brain. Previous literature has shown that varying blood glucose levels may have a strong impact on behaviour, subjective mood, and the intensity of the BOLD signal measured in fMRI. Therefore, blood glucose levels varying even within the normal range may interact with cognitive and emotional processing as well as BOLD signal. Here, in a placebo-controlled, double-blind crossover study on 20 healthy women, we show that overnight fasting, compared to an elevated glucose condition, influences brain activation and the affective state during mood induction. Results indicate that our brain may compensate for low glucose levels during fasting by stronger recruitment of the brain areas relevant to the task at hand. Additionally, we systematically tested the effect of prior cognitive effort on behavioural and neural patterns and found that elevated activation is only associated with maintained performance as long as no prior cognitively challenging task is administered. Prior cognitive effort leads to deteriorated performance and a further increase in emotion-associated brain activation in the pregenual anterior and posterior cingulate, the superior frontal gyrus, and the pre-SMA. These results are in line with the strength model of self-regulation. Our results corroborate the strength model of self-regulation and extend it to affect regulation processes. Additionally, our observations suggest that experimentally controlling for fasting state or glucose levels may be beneficial, especially when studying processes that involve self-regulation.

Association of autophagy-related 16-like 1 (ATG16L1) gene polymorphism with sepsis severity in patients with sepsis and ventilator-associated pneumonia.

Autophagy is a highly conserved mechanism of eukaryotic cells implicated in cell homeostasis and elimination of intracellular pathogens. Functional polymorphisms in genes encoding for autophagy have been associated with susceptibility to inflammatory and infectious diseases, but data on severe infections are missing. The aim of the present study was to assess whether polymorphisms in genes encoding proteins involved in autophagy influence susceptibility to ventilator-associated pneumonia (VAP). Mechanically ventilated patients with VAP were studied. Genotyping for autophagy-related 16-like 1 (ATG16L1, rs2241880) functional polymorphism was performed using the TaqMan single-nucleotide assay. Monocytes were isolated from patients and stimulated with lipopolysaccharide (LPS). Tumor necrosis factor-α (TNF-α) was measured in the supernatants of monocytes using an enzyme-linked immunosorbent assay. Procalcitonin (PCT) was also measured in the serum of patients by an immuno-time-resolved amplified cryptate technology assay. A total of 155 patients with VAP were enrolled in the study. Carriage of the minor A allele of ATG16L1 was associated with septic shock with at least one organ failure (odds ratio (OR): 2.40, p: 0.036). TNF-α production was significantly greater among the carriers of the polymorphism presenting with at least one organ failure (p: 0.040). PCT was increased upon worsening to septic shock and organ failure only among carriers of the minor frequency A alleles. In a homogeneous cohort of septic patients with VAP, the carriage of autophagy polymorphisms predisposes to VAP severity and septic shock development. This may be related with predisposition to immunoparalysis.

Serum of patients with septic shock stimulates the expression of Trem-1 on U937 monocytes.

OBJECTIVES: To describe the concentrations of sTREM-1 in patients with sepsis and to explore the effects of their serum on the expression of TREM-1 on U937 monocytes. METHODS: Blood was sampled at regular time intervals in 56 patients with sepsis. Concentrations of tumour necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1alpha), IL-6, IL-8, IL-10 and IL-12p70 and sTREM-1 were measured. U937 monocytes were incubated in the presence of serum at sepsis onset. RESULTS: Median sTREM-1 concentration on day 1 for patients with septic shock was 915 pg/ml and 228.5 pg/ml for those without shock (p = 0.002). TNFalpha, IL-1alpha, IL-6, IL-8 and IL-10 did not differ between them. A positive correlation was found between changes of sTREM-1 and SOFA scores from day 1 to 7. Sera of patients with septic shock evoked a significant increase of the expression of TREM-1. The concentrations of TNFalpha and IL-8 in supernatants increased only after stimulating with sera of patients without shock, but not after stimulating with sera of patients with shock. CONCLUSIONS: Levels of sTREM-1 correlated with sepsis severity. sTREM-1 is considerably higher in patients with shock compared to patients without shock. The serum of shocked patients could stimulate the expression of TREM-1 on U937 monocytes.

A case of disseminated tuberculosis mimicking metastatic cancer.

BACKGROUND: Disseminated tuberculosis (TB) or miliary TB is defined as lymphohematogenous dissemination of Mycobacterium tuberculosis bacilli, which may then affect virtually any organ system. The multiple organ involvement in disseminated TB can mimic metastatic cancer and can make the diagnosis challenging. False negatives are common therefore repeating microbiologic and histologic samples is essential. CASE REPORT: We report the case of a young immunocompetent patient presenting with multiple atypical extra-pulmonary TB involvement. The patient presented with pulmonary, pleural, bilateral testicular and multiple bone involvement including calcanerium abscesses. These lesions were initially described as metastasis by the radiologist. Therefore lymphoma and metastatic testicular cancer as well as TB were high on the differential in this young foreign-born male. Pleural, broncho-alveolar lavage, bone marrow and cerebrospinal fluid acid-fast bacilli smear and microbiologic culture were all negative. However the histologic examination of the trans-bronchial biopsy and pleural biopsy showed necrotizing granuloma and helped to narrow down the diagnosis. The patient improved with RIPE therapy. CONCLUSION: This case illustrates the diagnostic difficulty of disseminated TB with atypical organ involvement. Culture is the gold standard for diagnosing TB but is a long process and with 23% of culture negative TB in the United-States, the diagnosis sometimes relies on thoroughly ruling-out differential diagnosis and histologic examination.

[Health care providers' knowledge and abilities to use inhalation devices and spacers]. / Évaluation des connaissances en aérosolthérapie du personnel hospitalier.

INTRODUCTION: Inhaled therapy is the mainstay of asthma treatment due to its local and rapid action. However, its efficiency relies on the teaching of a good inhalation technique by health care providers. We assessed health care providers' knowledge and practical skills in the use of inhalation devices. METHODS: An observational multicenter study was conducted in the pulmonology and paediatric wards in Marseille. The departments' common practices, theoretical knowledge and practical skills were assessed through a questionnaire and a demonstration using a spacer device. RESULTS: Forty health care providers were interviewed (9 attending physicians, 14 residents, 16 nurses and 1 physiotherapist), in 8 different pulmonology and paediatric wards. A total of 42.5% reported previous training in inhalation device technique. When evaluating theoretical knowledge, we found a mean of 54% correct answers. Attending physicians did significantly better than residents and nurses. With regard to practical skills, we found a mean of 1.12 failed steps out of 7. Here again attending physicians did significantly better than residents and nurses. CONCLUSION: Based on the results of our study, we recommend that attending physicians provide training of inhalation technique to nurses and residents, as they did significantly better theoretically and practically.

Validation of the new Sepsis-3 definitions: proposal for improvement in early risk identification.

OBJECTIVES: Sepsis-3 definitions generated controversies regarding their general applicability. The Sepsis-3 Task Force outlined the need for validation with emphasis on the quick Sequential Organ Failure Assessment (qSOFA) score. This was done in a prospective cohort from a different healthcare setting. METHODS: Patients with infections and at least two signs of systemic inflammatory response syndrome (SIRS) were analysed. Sepsis was defined as total SOFA ≥2 outside the intensive care unit (ICU) or as an increase of ICU admission SOFA ≥2. The primary endpoints were the sensitivity of qSOFA outside the ICU and sepsis definition both outside and within the ICU to predict mortality. RESULTS: In all, 3346 infections outside the ICU and 1058 infections in the ICU were analysed. Outside the ICU, respective mortality with ≥2 SIRS and qSOFA ≥2 was 25.3% and 41.2% (p <0.0001); the sensitivities of qSOFA and of sepsis definition to predict death were 60.8% and 87.2%, respectively. This was 95.9% for sepsis definition in the ICU. The sensitivity of qSOFA and of ≥3 SIRS criteria for organ dysfunction outside the ICU was 48.7% and 72.5%, respectively (p <0.0001). Misclassification outside the ICU with the 1991 and Sepsis-3 definitions into stages of lower severity was 21.4% and 3.7%, respectively (p <0.0001) and 14.9% and 3.7%, respectively, in the ICU (p <0.0001). Adding arterial pH ≤7.30 to qSOFA increased sensitivity for prediction of death to 67.5% (p 0.004). CONCLUSIONS: Our analysis positively validated the use of SOFA score to predict unfavourable outcome and to limit misclassification into lower severity. However, qSOFA score had inadequate sensitivity for early risk assessment.

Leptin alterations in the course of sepsis in humans.

Neuroendocrine response to sepsis may be divided into acute and prolonged phase. As leptin is implicated in the stress response, leptin's profile during both phases, and the possible relationships between leptin and the neuroendocrine response to sepsis were investigated. Thirty adult patients with sepsis in an intensive care unit were studied. Blood samples were collected at the acute and the prolonged phases. In acute sepsis, leptin levels were higher in patients than in controls (10.2 +/- 2.5 vs. 4.1 +/- 1.2 ng/ml, p =0.01) and correlated positively with insulin levels and insulin resistance. A decline in leptin levels was found during prolonged sepsis (from 10.2 +/- 2.5 to 6.2 +/- 1.7 ng/ml, p=0.001), which was not related to survival (p=0.913). At the onset of sepsis, leptin levels increased in correlation with insulin and insulin resistance, possibly indicating a cause-effect relationship. However, the decline in leptin levels during the prolonged phase of sepsis was not related either to survival or to metabolic and hormonal changes.

The relationship between experiential deficits of negative symptoms and subjective quality of life in schizophrenia.

Understanding the link between quality of life and symptoms in schizophrenia is important in enhancing the prospect of patient recovery. Only weak associations have been found between subjective quality of life (SQOL) and negative symptoms. However, this may be because many existing symptom assessment scales inadequately assess the experiential deficits of negative symptoms. This study aimed to re-evaluate these findings using the Clinical Assessment Interview for Negative Symptoms (CAINS), which as been designed to capture both the expressive and experiential subdomains of negative symptoms as separate constructs. In this observational study 275 participants with at least moderate negative symptoms were assessed three times over nine months using the CAINS, the Positive and Negative Syndrome Scale (PANSS), and the Manchester Short Assessment of Quality of Life (MANSA). A significant negative association between SQOL and the CAINS experiential subscale was found in the cross-sectional analysis (adj. B=-0.28, 95% CI=-0.44 to -0.12, P=0.001), and in the change scores (adj. B=-0.13, 95% CI=-0.26 to -0.01, P=0.032). No associations between SQOL and expressive symptoms, or negative symptoms measured using the PANSS were detected in the multivariable models. These findings suggest that the association between negative symptoms and SQOL is related primarily to experiential deficits, and highlights the importance of measuring the separate subdomains of negative symptoms as distinct constructs. The findings also highlight the impact of negative symptoms and experiential deficits in particular on social outcomes, further emphasising the need to develop new treatments for these symptoms.

Exhaled nitric oxide and carbon monoxide in mechanically ventilated brain-injured patients.

The inflammatory influence and biological markers of prolonged mechanical-ventilation in uninjured human lungs remains controversial. We investigated exhaled nitric oxide (NO) and carbon monoxide (CO) in mechanically-ventilated, brain-injured patients in the absence of lung injury or sepsis at two different levels of positive end-expiratory pressure (PEEP). Exhaled NO and CO were assessed in 27 patients, without lung injury or sepsis, who were ventilated with 8 ml kg(-1) tidal volumes under zero end-expiratory pressure (ZEEP group, n = 12) or 8 cm H2O PEEP (PEEP group, n = 15). Exhaled NO and CO was analysed on days 1, 3 and 5 of mechanical ventilation and correlated with previously reported markers of inflammation and gas exchange. Exhaled NO was higher on day 3 and 5 in both patient groups compared to day 1: (PEEP group: 5.8 (4.4-9.7) versus 11.7 (6.9-13.9) versus 10.7 (5.6-16.6) ppb (p < 0.05); ZEEP group: 5.3 (3.8-8.8) versus 9.8 (5.3-12.4) versus 9.6 (6.2-13.5) ppb NO peak levels for days 1, 3 and 5, respectively, p < 0.05). Exhaled CO remained stable on day 3 but significantly decreased by day 5 in the ZEEP group only (6.3 (4.3-9.0) versus 8.1 (5.8-12.1) ppm CO peak levels for day 5 versus 1, p < 0.05). The change scores for peak exhaled CO over day 1 and 5 showed significant correlations with arterial blood pH and plasma TNF levels (r s = 0.49, p = 0.02 and r s = -0.51 p = 0.02, respectively). Exhaled NO correlated with blood pH in the ZEEP group and with plasma levels of IL-6 in the PEEP group. We observed differential changes in exhaled NO and CO in mechanically-ventilated patients even in the absence of manifest lung injury or sepsis. These may suggest subtle pulmonary inflammation and support application of real time breath analysis for molecular monitoring in critically ill patients.

Prolonged fasting impairs neural reactivity to visual stimulation.

Previous literature has shown that hypoglycemia influences the intensity of the BOLD signal. A similar but smaller effect may also be elicited by low normal blood glucose levels in healthy individuals. This may not only confound the BOLD signal measured in fMRI, but also more generally interact with cognitive processing, and thus indirectly influence fMRI results. Here we show in a placebo-controlled, crossover, double-blind study on 40 healthy subjects, that overnight fasting and low normal levels of glucose contrasted to an activated, elevated glucose condition have an impact on brain activation during basal visual stimulation. Additionally, functional connectivity of the visual cortex shows a strengthened association with higher-order attention-related brain areas in an elevated blood glucose condition compared to the fasting condition. In a fasting state visual brain areas show stronger coupling to the inferior temporal gyrus. Results demonstrate that prolonged overnight fasting leads to a diminished BOLD signal in higher-order occipital processing areas when compared to an elevated blood glucose condition. Additionally, functional connectivity patterns underscore the modulatory influence of fasting on visual brain networks. Patterns of brain activation and functional connectivity associated with a broad range of attentional processes are affected by maturation and aging and associated with psychiatric disease and intoxication. Thus, we conclude that prolonged fasting may decrease fMRI design sensitivity in any task involving attentional processes when fasting status or blood glucose is not controlled.

Pulmonary capillary endothelium-bound angiotensin-converting enzyme activity in acute lung injury.

BACKGROUND: Pulmonary capillary endothelium-bound (PCEB) angiotensin-converting ectoenzyme (ACE) activity alteration is an early, sensitive, and quantifiable lung injury index in animal models. We hypothesized that (1) PCEB-ACE alterations can be found in patients with acute lung injury (ALI) and (2) PCEB-ACE activity correlates with the severity of lung injury and may be used as a quantifiable marker of the underlying pulmonary capillary endothelial dysfunction. METHODS AND RESULTS: Applying indicator-dilution techniques, we measured single-pass transpulmonary hydrolysis of the synthetic ACE substrate (3)H-benzoyl-Phe-Ala-Pro (BPAP) in 33 mechanically ventilated, critically ill patients with a lung injury score (LIS) ranging from 0 (no lung injury) to 3.7 (severe lung injury) and calculated the kinetic parameter A(max)/K(m). Both parameters decreased early during the ALI continuum and were inversely related to APACHE II score and LIS. Hydrolysis decreased with increasing cardiac output (CO), whereas 2 different patterns were observed between CO and A(max)/K(m). CONCLUSIONS: PCEB-ACE activity decreases early during ALI, correlates with the clinical severity of both the lung injury and the underlying disease, and may be used as a quantifiable marker of underlying pulmonary capillary endothelial dysfunction.

Single-Incision Laparoscopic Right Hemi-Colectomy: a Systematic Review.

As surgeons became more adept with laparoscopic colon surgery, other less invasive procedures, such as single-incision laparoscopic right hemi-colectomy (SIL-RH), have been applied. The objective of this study was to evaluate the safety of SIL-RH as well as its intraoperative and postoperative outcomes for right-sided colon diseases. A detailed search in PubMed for citations that included SIL-RH from 2000 to 2014 revealed 21 studies fulfilling the criteria of the present review. A total of 684 patients were analyzed. Of the patients, 50.2 % were men. Mean patient age was 64.8 years. Of the patients, 36.1 % had already undergone an abdominal operation before the performance of SIL-RH, while 69 % of the patients underwent SIL-RH for colon cancer. Relatively low rates of overall morbidity (15 %) and mortality (0.75 %) were reported in the included studies. Mean length of postoperative hospital stay (LOS) was 5.5 days. Bowel motility return had a mean value of 2.8 days. Mean number of harvested lymph nodes (LN) was 19.2 LN. All resection margins were tumor-free. SIL-RH was a safe alternative to multiport laparoscopic right hemi-colectomy (ML-RH) in terms of morbidity and mortality, postoperative gastrointestinal function recovery, LOS, as well as oncological radicalness.

Elevated soluble endothelial protein C receptor levels at ICU admission are associated with sepsis development.

BACKGROUND: The endothelial protein C receptor (EPCR) is a protein that regulates the protein C anticoagulant and anti-inflammatory pathways. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits activated protein C (APC) activities. The clinical impact of sEPCR and its involvement in the septic process is under investigation. In this study, we assessed the role of sEPCR levels as an early indicator of sepsis development. METHODS: Plasma sEPCR levels were measured in 59 critically-ill non-septic patients at the time of admission to the intensive care unit (ICU). Multiple logistic regression analysis was performed to identify potential risk factors for sepsis development and Cox-Regression models were fitted for variables to examine their relationship with time to sepsis development. RESULTS: Thirty patients subsequently developed sepsis and 29 did not. At ICU admission, sequential organ failure assessment (SOFA) scores were significantly higher in the subsequent sepsis group as compared to the non sepsis group (mean ± SD: 6.4±2.7 and 5±2.3, respectively, P=0.037). sEPCR levels were also higher in the patients who subsequently developed sepsis compared to the patients who did not (median and interquartile range: 173.4 [104.5-223.5] ng/mL vs. 98.3 [69.8-147.7] ng/mL, respectively; P=0.004). Cox regression analysis identified sEPCR as the only parameter related to sepsis development with time (relative risk: 1.078, 95% confidence interval: 1.016-1.144, by 10 units; P=0.013). CONCLUSION: Upon ICU admission, sEPCR levels in initially non-septic critically-ill patients appear elevated in the subjects who will subsequently become septic.

Pulmonary endothelium in acute lung injury: from basic science to the critically ill.

BACKGROUND: Pulmonary endothelium is an active organ possessing numerous physiological, immunological, and metabolic functions. These functions may be altered early in acute lung injury (ALI) and further contribute to the development of acute respiratory distress syndrome (ARDS). Pulmonary endothelium is strategically located to filter the entire blood before it enters the systemic circulation; consequently its integrity is essential for the maintenance of adequate homeostasis in both the pulmonary and systemic circulations. Noxious agents that affect pulmonary endothelium induce alterations in hemodynamics and hemofluidity, promote interactions with circulating blood cells, and lead to increased vascular permeability and pulmonary edema formation. OBJECTIVE: We highlight pathogenic mechanisms of pulmonary endothelial injury and their clinical implications in ALI/ARDS patients.

Effect of high blood flow on pulmonary vascular permeability to protein.

The elevated cardiac output associated with exercise increases lung lymph flow and may increase extravascular lung water. However, it is not known if extremely elevated cardiac output alters pulmonary vascular permeability. The hematocrit-protein method was used to determine the solvent drag reflection coefficient, an index of vascular permeability to proteins, in the isolated blood-perfused canine lung lobe. Microvascular pressure was obtained by double vascular occlusion. Lobes filtered fluid during perfusion at normal flow, 0.451 +/- 0.005 l/min (LF; n = 8), or high flow, 2.319 +/- 0.080 l/min (HF; n = 7). In the LF, venous pressure was elevated to 19.0 +/- 0.5 Torr to induce filtration, whereas Pv was 3.3 +/- 0.1 Torr in the HF. In HF vs. LF, respectively, arterial pressure was 61.4 +/- 7.1 vs. 28.0 +/- 1.0 Torr (P < 0.05), microvascular pressure was 31.9 +/- 3.0 vs. 22.2 +/- 0.9 Torr (P < 0.05), and sigma was 0.52 +/- 0.07 vs. 0.51 +/- 0.02 (P > 0.05). The fivefold increase in blood flow did not alter pulmonary vascular permeability to proteins; however, the capillary filtration coefficient was fivefold greater in the HF vs. LF group (0.328 +/- 0.059 vs. 0.067 +/- 0.007; P < 0.002). These data are compatible with enzyme activity measures indicating a direct linear relationship between blood flow rate and perfused pulmonary microvascular surface area. Although the data do not rule out the possibility of increased pulmonary vascular permeability to water during very elevated blood flow rates, the greater filtration rate during elevated flow is more likely related to increases in both microvascular pressure and surface area.

Primary retroperitoneal mucinous cystadenoma. A rare case with two cysts and review of the literature.

BACKGROUND: Primary retroperitoneal mucinous cystadenoma is a rare neoplasm, with benign biological behavior. Delay in diagnosis and treatment of this tumor may be fatal for the patient, because of complications, such as rupture, infection and malignant transformation. CASE PRESENTATION: We present a 23-year-old woman, who was admitted to the hospital because of a palpable abdominal mass and discomfort since 4 months. Computed Tomography and Magnetic Resonance Imaging scans were performed and showed two retroperitoneal cystic masses, which were excised by laparoscopy. Histological and immunohistochemical examination revealed that the inner surfaces of the cysts were lined by epithelium with features of mesothelial cells, in addition to ovarian mucinous cystadenoma. This is the 29(th) case and the second reported case with two contemporary cysts. CONCLUSION: The origin of retroperitoneal mucinous cystadenomas is still unclear. Pathological and immunohistochemical findings proved that these tumors resemble ovarian mucinous cystadenomas but are unattached to the ovary and can arise at any location in the retroperitoneum. Surgical excision of the aforementioned tumors is the treatment of choice. Hippokratia 2014; 18 (3): 278-281.