High dimensional profiling identifies specific immune types along the recovery trajectories of critically ill COVID19 patients.

The COVID-19 pandemic poses a major burden on healthcare and economic systems across the globe. Even though a majority of the population develops only minor symptoms upon SARS-CoV-2 infection, a significant number are hospitalized at intensive care units (ICU) requiring critical care. While insights into the early stages of the disease are rapidly expanding, the dynamic immunological processes occurring in critically ill patients throughout their recovery at ICU are far less understood. Here, we have analysed whole blood samples serially collected from 40 surviving COVID-19 patients throughout their recovery in ICU using high-dimensional cytometry by time-of-flight (CyTOF) and cytokine multiplexing. Based on the neutrophil-to-lymphocyte ratio (NLR), we defined four sequential immunotypes during recovery that correlated to various clinical parameters, including the level of respiratory support at concomitant sampling times. We identified classical monocytes as the first immune cell type to recover by restoration of HLA-DR-positivity and the reduction of immunosuppressive CD163 + monocytes, followed by the recovery of CD8 + and CD4 + T cell and non-classical monocyte populations. The identified immunotypes also correlated to aberrant cytokine and acute-phase reactant levels. Finally, integrative analysis of cytokines and immune cell profiles showed a shift from an initially dysregulated immune response to a more coordinated immunogenic interplay, highlighting the importance of longitudinal sampling to understand the pathophysiology underlying recovery from severe COVID-19.

Community-acquired pneumonia (CAP) hospitalizations and deaths: is there a role for quality improvement through inter-hospital comparisons?

OBJECTIVE: To assess between-hospital variations in standardized in-hospital mortality ratios of community-acquired pneumonia (CAP), and identify possible leads for quality improvement. DESIGN: We used an administrative database to estimate standardized in-hospital mortality ratios for 111 Belgian hospitals, by carrying out a set of hierarchical logistic regression models, intended to disentangle therapeutic attitudes and biases. To facilitate the detection of false-negative/positive results, we added an inconclusive zone to the funnel plots, derived from the results of the study. Data quality was validated by comparison with (i) alternative data from the largest Belgian Sickness Fund, (ii) published German hospital data and (iii) the results of an on-site audit. SETTING: All Belgian hospital discharge records from 2004 to 2007. STUDY PARTICIPANTS: A total of 111 776 adult patients were admitted for CAP. MAIN OUTCOME MEASURE: Risk-adjusted standardized in-hospital mortality ratios. RESULTS: Out of the 111 hospitals, we identified five and six outlying hospitals, with standardized mortality ratios of CAP consistently on the extremes of the distribution, as providing possibly better or worse care, respectively, and 18 other hospitals as having possible quality weaknesses/strengths. At the individuals' level of the analysis, adjusted odds ratios showed the paramount importance of old age, comorbidity and mechanical ventilation. The data compared well with the different validation sources. CONCLUSIONS: Despite the limitations inherent to administrative data, it seemed possible to establish inter-hospital differences in standardized in-hospital mortality ratios of CAP and to identify leads for quality improvement. Monitoring is needed to assess progress in quality.

Cellular and molecular features related to exceptional therapy response and extreme long-term survival in glioblastoma.

Glioblastoma Multiforme (GBM) remains the most common malignant primary brain tumor with a dismal prognosis that rarely exceeds beyond 2 years despite extensive therapy, which consists of maximal safe surgical resection, radiotherapy, and/or chemotherapy. Recently, it has become clear that GBM is not one homogeneous entity and that both intra-and intertumoral heterogeneity contributes significantly to differences in tumoral behavior which may consequently be responsible for differences in survival. Strikingly and in spite of its dismal prognosis, small fractions of GBM patients seem to display extremely long survival, defined as surviving over 10 years after diagnosis, compared to the large majority of patients. Although the underlying mechanisms for this peculiarity remain largely unknown, emerging data suggest that still poorly characterized both cellular and molecular factors of the tumor microenvironment and their interplay probably play an important role. We hereby give an extensive overview of what is yet known about these cellular and molecular features shaping extreme long survival in GBM.

The health and economic burden of chickenpox and herpes zoster in Belgium.

Varicella-zoster virus causes chickenpox (CP) and after reactivation herpes zoster (HZ). Vaccines are available against both diseases warranting an assessment of the pre-vaccination burden of disease. We collected data from relevant Belgian databases and performed five surveys of CP and HZ patients. The rates at which a general practitioner is visited at least once for CP and HZ are 346 and 378/100 000 person-years, respectively. The average CP and HZ hospitalization rates are 5·3 and 14·2/100 000 person-years respectively. The direct medical cost for HZ is about twice as large as the direct medical cost for CP. The quality-adjusted life years lost for ambulatory CP patients consulting a physician is more than double that of those not consulting a physician (0·010 vs. 0·004). In conclusion, both diseases cause a substantial burden in Belgium.

Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity.

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

Non-invasive diagnosis of endometriosis based on a combined analysis of six plasma biomarkers.

BACKGROUND: Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. The aim of this study was to evaluate the combined performance of six potential plasma biomarkers in the diagnosis of endometriosis. METHODS: This case-control study was conducted in 294 infertile women, consisting of 93 women with a normal pelvis and 201 women with endometriosis. We measured plasma concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha, high-sensitivity C-reactive protein (hsCRP), and cancer antigens CA-125 and CA-19-9. Analyses were done using the Kruskal-Wallis test, Mann-Whitney test, receiver operator characteristic, stepwise logistic regression and least squares support vector machines (LSSVM). RESULTS: Plasma levels of IL-6, IL-8 and CA-125 were increased in all women with endometriosis and in those with minimal-mild endometriosis, compared with controls. In women with moderate-severe endometriosis, plasma levels of IL-6, IL-8 and CA-125, but also of hsCRP, were significantly higher than in controls. Using stepwise logistic regression, moderate-severe endometriosis was diagnosed with a sensitivity of 100% (specificity 84%) and minimal-mild endometriosis was detected with a sensitivity of 87% (specificity 71%) during the secretory phase. Using LSSVM analysis, minimal-mild endometriosis was diagnosed with a sensitivity of 94% (specificity 61%) during the secretory phase and with a sensitivity of 92% (specificity 63%) during the menstrual phase. CONCLUSIONS: Advanced statistical analysis of a panel of six selected plasma biomarkers on samples obtained during the secretory phase or during menstruation allows the diagnosis of both minimal-mild and moderate-severe endometriosis with high sensitivity and clinically acceptable specificity.

Stereoselective pharmacokinetics of atenolol in the rat: influence of aging and of renal failure.

The pharmacokinetics of R- and S-atenolol after intravenous administration of racemic atenolol were studied in 3-, 12- and 24-month-old rats and in 3-month-old rats with renal failure induced by uranyl nitrate. In all age groups, the area under the plasma concentration-time curves is higher for R- than for S-atenolol; volume of distribution, total clearance and renal clearance are lower for R-atenolol than for S-atenolol, but the differences are small. In function of age there is for both enantiomers a significant increase in AUC, due, at least in part, to a decreased renal clearance; the effect of aging is not stereoselective. In rats with renal failure, the AUC of both enantiomers increases, due mainly to a decrease in renal clearance, but to a lesser degree also to a decrease in nonrenal clearance. For both enantiomers, the volume of distribution decreases and the half-life increases in the uraemic rats. The total amount of both enantiomers excreted in the urine is decreased in the rats with renal failure. There are no stereoselective effects of treatment of the rats with uranyl nitrate.

Influence of age on stereoselective pharmacokinetics and metabolism of hexobarbital in the rat.

The influence of age on stereoselective pharmacokinetics and in vitro metabolism of R- and S-hexobarbital was studied in the rat. After intravenous administration of the racemate, the plasma concentrations of S-hexobarbital are markedly lower than those of R-hexobarbital. For S-hexobarbital the half-life is somewhat shorter and the volume of distribution and plasma clearance is higher than for its antipode. For both enantiomers an increase in AUC and half-life, and a decrease in clearance are observed with aging. These changes occur mainly between the 3rd and the 12th month and are slightly more pronounced for R- than for S-hexobarbital, as appears from the S/R ratios. The volume of distribution shows no changes with aging. In vitro disappearance rate in 3-month-old rats is significantly higher for S- than for R-hexobarbital. There is for both enantiomers an increase in disappearance rate in 12-month-old rats as compared to younger or older rats, but this is significant only for the R-enantiomer. There are pronounced differences in the kinetics and metabolism of both hexobarbital enantiomers; changes with aging occur, but are only slightly and not always significantly more important for R- than for S-hexobarbital.

The presence of dopamine beta-hydroxylase in the cerebrospinal fluid of rabbits and its increased concentration after stimulation of peripheral nerves and cold stress.

The presence of dopamine beta-hydroxylase activity in the cerebrospinal fluid of rabbits has been shown using a sensitive radiochemical assay; the identity of the reaction product was confirmed by using thin layer chromatographic techniques. The enzyme found in this fluid has some properties (sedimentation and electrophoretic migration) in common with the best characterized preparation of dopamine beta-hydroxylase, that prepared from bovine adrenal chromaffin granules. It also has these properties in common with the enzyme present in the high-speed supernatants obtained from osmotically disrupted synaptosomes prepared from rabbit brain. When the sciatic nerves of rabbits under urethane anaesthesia were stimulated, or when shaved rabbits were subjected to cold stress, the level of dopamine beta-hydroxylase in the cerebrospinal fluid increased. The increase in response to nerve stimulation was gradual, starting within 90 min of stimulation and remained high for at least 3 h after the stimulation had ended, at which time it was 280% of the normal value. There was no equivalent increase in the protein concentration of the cerebrospinal fluid nor was there a change in the enzyme activity when sciatic nerves were exposed but not stimulated. The enzyme present in the cerebrospinal fluid during this period of high activity is identical in its sedimentation and electrophoretic properties to that present in normal fluid. It is suggested that the dopamine beta-hydroxylase activity in cerebrospinal fluid may be derived from noradrenergic neurons within the brain and that the enzyme is released together with noradrenaline.

Influence of human recombinant interleukin-1 beta on the enantioselective disposition of propranolol in rats.

The influence of i.v. administration of 10 micrograms/kg recombinant human interleukin-1 beta (rhIL-1 beta), a putative mediator of inflammation, on the pharmacokinetics and metabolism of the propranolol enantiomers was studied in rats aged 3, 12 and 24 months. After oral administration of rac-propranolol to control rats of the three age groups, the plasma concentrations of (R)-propranolol were higher than those of (S)-propranolol. Administration of IL-1 beta increased the plasma concentrations of the (R)-enantiomer markedly and significantly, those of the (S)-enantiomer only to a lesser degree. For both enantiomers an important increase in plasma binding was found in the IL-1 beta-treated rats, which was linked to the increase in alpha 1-acid glycoprotein levels. The in vitro clearance, measured in 3-month-old rats using the 9000 g liver fraction, was for neither of the propranolol enantiomers influenced by IL-1 beta treatment, which is in keeping with the unchanged cytochrome P450 content. The enantioselective influence of IL-1 beta treatment on the pharmacokinetics of propranolol was also present in 12- and 24-month-old rats, although somewhat less pronounced in the latter group. Our results show an enantioselective influence of IL-1 beta treatment on the pharmacokinetics of propranolol in the rat, favouring the (R)-enantiomer.

The effect of colchicine treatment on the buoyant density of noradrenaline storage particles in the rabbit superior cervical ganglion.

The subcellular distribution of noradrenaline (NA) was studied in the rabbit superior cervical ganglion before and after colchicine treatment. One-third of the total NA could be sedimented with the microsomal material in control conditions. Upon colchicine treatment the percentage of the NA which was particle-bound increased only slightly. The absolute NA content of the superior cervical ganglion from colchicine-treated rabbits, however, showed a 3.5-fold increase compared to control animals. Density gradient centrifugation of the microsomal fraction revealed that the distribution of the NA-containing particles changed significantly. Before colchicine treatment the NA was found in a broad band covering the gradient fractions where 'light' and 'heavy' NA vesicles were expected to equilibrate. After colchicine treatment the NA was concentrated in the 'heavy' NA vesicles part of the gradient. The dopamine beta-hydroxylase activity and the NA content in the gradient are increased 3 times after colchicine treatment. These experimental results can be explained by the axoplasmic transport impairing action of colchicine. The neuronal cell body accumulates 'heavy' NA vesicles formed there, unable to transport them towards the periphery.

Neonatal infections with Pseudomonas aeruginosa associated with a water-bath used to thaw fresh frozen plasma.

In our 15-bed neonatal intensive care unit (NICU), four new-borns were found to be colonized or infected with Pseudomonas aeruginosa within a period of one week. To identify the outbreak source, three independent studies were performed: epidemiological investigation, environmental surveillance and genotypic typing of isolates. Although epidemiological investigation by a case-control study revealed no conclusive results, the transfusion of fresh frozen plasma (FFP) and human albumin (HA) appeared to be the factor with highest risk. Environmental surveillance and random amplification of polymorphic DNA (RAPD) of isolates identified a water-bath used to warm FFP and HA as the likely reservoir for the outbreak. Further spread of the organism did not occur after elimination of this water-bath from the NICU. RAPD identified in addition an isolate from an infant hospitalized in the NICU five months before the outbreak with a pattern matching the one of the outbreak cluster.

Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma.

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.

Effect of turpentine-induced inflammation on the disposition kinetics of propranolol, metoprolol, and antipyrine in the rat.

Plasma concentrations after oral administration of the high extraction drug propranolol are increased in patients and animals with inflammation. This could be due to increased serum propranolol binding, but also to decreased first-pass metabolism. We studied the pharmacokinetics of 3 drugs in control rats and in rats with turpentine-induced inflammation: propranolol, which is bound extensively to alpha 1-acid glycoprotein (alpha 1-AGP); metoprolol, another high extraction drug, but which is negligibly bound to alpha 1-AGP; and antipyrine, a low extraction drug, not bound to serum proteins. After IV administration of propranolol in rats with inflammation, systemic clearance, volume of distribution, and free fraction decreased, and the area under the curve (AUC) increased, whereas the half-life did not change. As the systemic clearance of a high extraction drug such as propranolol depends on hepatic blood flow only, a fall in hepatic blood flow or transition to a low extraction situation should be postulated. After oral administration of propranolol, the AUC was increased 20-fold in rats with inflammation; as the decrease in free fraction was only 4-fold, it can be concluded that a considerable decrease in hepatic intrinsic clearance was present. For metoprolol, in contrast to propranolol, after IV administration, no changes in pharmacokinetic parameters as a result of inflammation were observed. After oral administration, the AUC was increased about 4 times in rats with inflammation; as metoprolol is only negligibly bound to serum proteins, the increase in AUC can be attributed to a decrease in hepatic intrinsic clearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of lignocaine on plasma protein binding and pharmacokinetics of verapamil in dogs.

The effect of lignocaine (lidocaine) on the plasma protein binding of verapamil has been studied in-vitro and in-vivo in dogs. The binding of verapamil was ca 85%. In-vitro addition of lignocaine at therapeutic concentrations displaced verapamil from its plasma binding sites. Lignocaine in this regard was equipotent with tris(2-butoxyethyl)phosphate, suggesting an interaction at the level of alpha 1-acid glycoprotein binding sites. On in-vivo administration of 4 mg kg-1 in a bolus to dogs in which steady state concentrations of verapamil were present, the free fraction of verapamil increased transiently. During the lignocaine maintenance infusion, it then decreased to a level higher than that before administration of the local anaesthetic. The free verapamil concentrations increased suddenly upon the administration of the lignocaine loading dose, and then returned to values slightly higher than those before lignocaine. After a bolus injection of verapamil during a lignocaine infusion, the verapamil total plasma concentrations were lower than during a saline infusion, but the free concentrations were not different. The volume of distribution of verapamil was increased, whereas the blood clearance had not changed; the lignocaine infusion did not change the hepatic blood flow, as measured by indocyanine green clearance. These results show that lignocaine displaces verapamil in-vitro and in-vivo from its plasma protein binding sites, but the ensuing pharmacokinetic changes do not lead to significant changes in free verapamil concentrations.

A study of the heterogeneity of human alpha 1-acid glycoprotein with monoclonal antibodies.

Monoclonal antibodies (MABS) were raised against human alpha 1-acid glycoprotein (AGP) and their reaction with the polymorphic forms of this plasma protein were evaluated. Spleen cells of BALB/c mice, immunized with native or desialylated human AGP, were fused with NSO mouse myeloma cells. The hybridoma products were screened with a direct ELISA test, in which the immunoplates were coated with a mixture of native and desialylated AGP. In this test, 14 anti-AGP antibody producing clones were retained. Coating the wells with either native or desialylated AGP showed that eleven clones reacted with both types of AGP ('Type I' MABS), while three MABS reacted specifically with the desialylated form ('Type II' MABS). Precoating the immunoplates with polyclonal anti-human AGP followed by incubation with native or desialylated AGP before the addition of hybridoma supernatant (indirect ELISA), confirmed the specificities observed in the direct ELISA. The molecular heterogeneity of both native AGP and desialylated AGP, based on Concanavalin A reactivity and isoelectric point, was not reflected by any specificity in the antibody reactions. Thus 'Type I' MABS reacted with all molecular forms present in native and desialylated AGP while 'Type II' MABS reacted with all molecular forms present in desialylated AGP.

Body mass index and HER-2 overexpression in breast cancer patients over 50 years of age.

PURPOSE: In breast cancer, in vitro as well as in vivo experiments have shown an inverse relationship between HER-2 and steroid hormone receptors. It is unknown whether circulating estrogens affect HER-2 expression. We hypothesize that the postmenopausal body mass index (BMI) as a surrogate marker for bio-available estrogens, is inversely associated with HER-2 over-expression. PATIENTS AND METHODS: A total of 535 women over age 50 or with known postmenopausal status, with a unilateral, not previously treated, operable breast cancer were evaluated the evening prior to surgery for body weight, height, abdominal and hip circumference over a 3 years period. Waist-to-hip ratio (WHR) and BMI were calculated. HER-2, estrogen receptor and progesterone receptor staining was done by immunohistochemistry. All tumours with DAKO 2+ staining were submitted for HER-2 detection by FISH analysis. HER-2 was defined as positive if DAKO 3+ or FISH positive. We assessed the frequency of HER-2 positivity in each of 6 quantiles for all parameters of body composition and tested for a trend in HER-2 expression across the 6 quantiles. Furthermore, we investigated whether BMI contributed, together with other known predictors for HER-2, in a standard multivariate logistic regression model that predicts HER-2 over-expression. RESULTS: There is a decrease in HER-2 over-expression per increasing quantile of BMI. In a multivariate model-including both steroid receptors-BMI remains an independent predictor for HER-2 over-expression. CONCLUSION: In women over age 50 or with known postmenopausal status with an operable breast cancer, there is an inverse association between BMI and HER-2 over-expression.