Effect of mRNA Vaccine Boosters against SARS-CoV-2 Omicron Infection in Qatar.

BACKGROUND: Waning of vaccine protection against coronavirus disease 2019 (Covid-19) and the emergence of the omicron (or B.1.1.529) variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have led to expedited efforts to scale up booster vaccination. Protection conferred by booster doses of the BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines in Qatar, as compared with protection conferred by the two-dose primary series, is unclear. METHODS: We conducted two matched retrospective cohort studies to assess the effectiveness of booster vaccination, as compared with that of a two-dose primary series alone, against symptomatic SARS-CoV-2 infection and Covid-19-related hospitalization and death during a large wave of omicron infections from December 19, 2021, through January 26, 2022. The association of booster status with infection was estimated with the use of Cox proportional-hazards regression models. RESULTS: In a population of 2,239,193 persons who had received at least two doses of BNT162b2 or mRNA-1273 vaccine, those who had also received a booster were matched with persons who had not received a booster. Among the BNT162b2-vaccinated persons, the cumulative incidence of symptomatic omicron infection was 2.4% (95% confidence interval [CI], 2.3 to 2.5) in the booster cohort and 4.5% (95% CI, 4.3 to 4.6) in the nonbooster cohort after 35 days of follow-up. Booster effectiveness against symptomatic omicron infection, as compared with that of the primary series, was 49.4% (95% CI, 47.1 to 51.6). Booster effectiveness against Covid-19-related hospitalization and death due to omicron infection, as compared with the primary series, was 76.5% (95% CI, 55.9 to 87.5). BNT162b2 booster effectiveness against symptomatic infection with the delta (or B.1.617.2) variant, as compared with the primary series, was 86.1% (95% CI, 67.3 to 94.1). Among the mRNA-1273-vaccinated persons, the cumulative incidence of symptomatic omicron infection was 1.0% (95% CI, 0.9 to 1.2) in the booster cohort and 1.9% (95% CI, 1.8 to 2.1) in the nonbooster cohort after 35 days; booster effectiveness against symptomatic omicron infection, as compared with the primary series, was 47.3% (95% CI, 40.7 to 53.3). Few severe Covid-19 cases were noted in the mRNA-1273-vaccinated cohorts. CONCLUSIONS: The messenger RNA (mRNA) boosters were highly effective against symptomatic delta infection, but they were less effective against symptomatic omicron infection. However, with both variants, mRNA boosters led to strong protection against Covid-19-related hospitalization and death. (Funded by Weill Cornell Medicine-Qatar and others.).

Effects of Previous Infection and Vaccination on Symptomatic Omicron Infections.

BACKGROUND: The protection conferred by natural immunity, vaccination, and both against symptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with the BA.1 or BA.2 sublineages of the omicron (B.1.1.529) variant is unclear. METHODS: We conducted a national, matched, test-negative, case-control study in Qatar from December 23, 2021, through February 21, 2022, to evaluate the effectiveness of vaccination with BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna), natural immunity due to previous infection with variants other than omicron, and hybrid immunity (previous infection and vaccination) against symptomatic omicron infection and against severe, critical, or fatal coronavirus disease 2019 (Covid-19). RESULTS: The effectiveness of previous infection alone against symptomatic BA.2 infection was 46.1% (95% confidence interval [CI], 39.5 to 51.9). The effectiveness of vaccination with two doses of BNT162b2 and no previous infection was negligible (-1.1%; 95% CI, -7.1 to 4.6), but nearly all persons had received their second dose more than 6 months earlier. The effectiveness of three doses of BNT162b2 and no previous infection was 52.2% (95% CI, 48.1 to 55.9). The effectiveness of previous infection and two doses of BNT162b2 was 55.1% (95% CI, 50.9 to 58.9), and the effectiveness of previous infection and three doses of BNT162b2 was 77.3% (95% CI, 72.4 to 81.4). Previous infection alone, BNT162b2 vaccination alone, and hybrid immunity all showed strong effectiveness (>70%) against severe, critical, or fatal Covid-19 due to BA.2 infection. Similar results were observed in analyses of effectiveness against BA.1 infection and of vaccination with mRNA-1273. CONCLUSIONS: No discernable differences in protection against symptomatic BA.1 and BA.2 infection were seen with previous infection, vaccination, and hybrid immunity. Vaccination enhanced protection among persons who had had a previous infection. Hybrid immunity resulting from previous infection and recent booster vaccination conferred the strongest protection. (Funded by Weill Cornell Medicine-Qatar and others.).

Covid-19 Vaccine Protection among Children and Adolescents in Qatar.

BACKGROUND: The BNT162b2 vaccine against coronavirus disease 2019 (Covid-19) has been authorized for use in children 5 to 11 years of age and adolescents 12 to 17 years of age but in different antigen doses. METHODS: We assessed the real-world effectiveness of the BNT162b2 vaccine against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among children and adolescents in Qatar. To compare the incidence of SARS-CoV-2 infection in the national cohort of vaccinated participants with the incidence in the national cohort of unvaccinated participants, we conducted three matched, retrospective, target-trial, cohort studies - one assessing data obtained from children 5 to 11 years of age after the B.1.1.529 (omicron) variant became prevalent and two assessing data from adolescents 12 to 17 years of age before the emergence of the omicron variant (pre-omicron study) and after the omicron variant became prevalent. Associations were estimated with the use of Cox proportional-hazards regression models. RESULTS: Among children, the overall effectiveness of the 10-µg primary vaccine series against infection with the omicron variant was 25.7% (95% confidence interval [CI], 10.0 to 38.6). Effectiveness was highest (49.6%; 95% CI, 28.5 to 64.5) right after receipt of the second dose but waned rapidly thereafter and was negligible after 3 months. Effectiveness was 46.3% (95% CI, 21.5 to 63.3) among children 5 to 7 years of age and 16.6% (95% CI, -4.2 to 33.2) among those 8 to 11 years of age. Among adolescents, the overall effectiveness of the 30-µg primary vaccine series against infection with the omicron variant was 30.6% (95% CI, 26.9 to 34.1), but many adolescents had been vaccinated months earlier. Effectiveness waned over time since receipt of the second dose. Effectiveness was 35.6% (95% CI, 31.2 to 39.6) among adolescents 12 to 14 years of age and 20.9% (95% CI, 13.8 to 27.4) among those 15 to 17 years of age. In the pre-omicron study, the overall effectiveness of the 30-µg primary vaccine series against SARS-CoV-2 infection among adolescents was 87.6% (95% CI, 84.0 to 90.4) and waned relatively slowly after receipt of the second dose. CONCLUSIONS: Vaccination in children was associated with modest, rapidly waning protection against omicron infection. Vaccination in adolescents was associated with stronger, more durable protection, perhaps because of the larger antigen dose. (Funded by Weill Cornell Medicine-Qatar and others.).

Waning of BNT162b2 Vaccine Protection against SARS-CoV-2 Infection in Qatar.

BACKGROUND: Waning of vaccine protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or coronavirus disease 2019 (Covid-19) is a concern. The persistence of BNT162b2 (Pfizer-BioNTech) vaccine effectiveness against infection and disease in Qatar, where the B.1.351 (or beta) and B.1.617.2 (or delta) variants have dominated incidence and polymerase-chain-reaction testing is done on a mass scale, is unclear. METHODS: We used a matched test-negative, case-control study design to estimate vaccine effectiveness against any SARS-CoV-2 infection and against any severe, critical, or fatal case of Covid-19, from January 1 to September 5, 2021. RESULTS: Estimated BNT162b2 effectiveness against any SARS-CoV-2 infection was negligible in the first 2 weeks after the first dose. It increased to 36.8% (95% confidence interval [CI], 33.2 to 40.2) in the third week after the first dose and reached its peak at 77.5% (95% CI, 76.4 to 78.6) in the first month after the second dose. Effectiveness declined gradually thereafter, with the decline accelerating after the fourth month to reach approximately 20% in months 5 through 7 after the second dose. Effectiveness against symptomatic infection was higher than effectiveness against asymptomatic infection but waned similarly. Variant-specific effectiveness waned in the same pattern. Effectiveness against any severe, critical, or fatal case of Covid-19 increased rapidly to 66.1% (95% CI, 56.8 to 73.5) by the third week after the first dose and reached 96% or higher in the first 2 months after the second dose; effectiveness persisted at approximately this level for 6 months. CONCLUSIONS: BNT162b2-induced protection against SARS-CoV-2 infection appeared to wane rapidly following its peak after the second dose, but protection against hospitalization and death persisted at a robust level for 6 months after the second dose. (Funded by Weill Cornell Medicine-Qatar and others.).

Estimating protection afforded by prior infection in preventing reinfection: Applying the test-negative study design.

The COVID-19 pandemic has highlighted the need to use infection testing databases to rapidly estimate effectiveness of prior infection in preventing reinfection ($P{E}_S$) by novel SARS-CoV-2 variants. Mathematical modeling was used to demonstrate a theoretical foundation for applicability of the test-negative, case-control study design to derive $P{E}_S$. Apart from the very early phase of an epidemic, the difference between the test-negative estimate for $P{E}_S$ and true value of $P{E}_S$ was minimal and became negligible as the epidemic progressed. The test-negative design provided robust estimation of $P{E}_S$ and its waning. Assuming that only 25% of prior infections are documented, misclassification of prior infection status underestimated $P{E}_S$, but the underestimate was considerable only when >50% of the population was ever infected. Misclassification of latent infection, misclassification of current active infection, and scale-up of vaccination all resulted in negligible bias in estimated $P{E}_S$. The test-negative design was applied to national-level testing data in Qatar to estimate $P{E}_S$ for SARS-CoV-2. $P{E}_S$ against SARS-CoV-2 Alpha and Beta variants was estimated at 97.0% (95% CI: 93.6-98.6) and 85.5% (95% CI: 82.4-88.1), respectively. These estimates were validated using a cohort study design. The test-negative design offers a feasible, robust method to estimate protection from prior infection in preventing reinfection.

Coronavirus Disease 2019 Disease Severity in Children Infected With the Omicron Variant.

SHORT SUMMARY: Severe acute respiratory syndrome coronavirus 2 infection from the Omicron variant in children/adolescents is less severe than infection from the Delta variant. Those 6 to <18 years also have less severe disease than those <6 years old. BACKGROUND: There are limited data assessing coronavirus 2019 (COVID-19) disease severity in children/adolescents infected with the Omicron variant. METHODS: We identified children and adolescents <18 years of age with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with Delta and propensity score-matched controls with Omicron variant infection from the National COVID-19 Database in Qatar. Primary outcome was disease severity, determined by hospital admission, admission to the intensive care unit (ICU), or mechanical ventilation within 14 days of diagnosis, or death within 28 days. RESULTS: Among 1735 cases with Delta variant infection between 1 June and 6 November 2021, and 32 635 cases with Omicron variant infection between 1 January and 15 January 2022, who did not have prior infection and were not vaccinated, we identified 985 propensity score-matched pairs. Among those who were Delta infected, 84.2% had mild, 15.7% had moderate, and 0.1% had severe/critical disease. Among those who were Omicron infected, 97.8% had mild, 2.2% had moderate, and none had severe/critical disease (P < .001). Omicron variant infection (vs Delta) was associated with significantly lower odds of moderate or severe/critical disease (adjusted odds ratio [AOR], 0.12; 95% confidence interval [CI], .07-.18). Those aged 6-11 and 12 to <18 years had lower odds of developing moderate or severe/critical disease compared with those younger than age 6 years (aOR, 0.47; 95% CI, .33-.66 for 6-11 year olds; aOR, 0.45; 95% CI, .21-.94 for 12 to <18 year olds). CONCLUSIONS: Omicron variant infection in children/adolescents is associated with less severe disease than Delta variant infection as measured by hospitalization rates and need for ICU care or mechanical ventilation. Those 6 to <18 years of age also have less severe disease than those <6 years old.

Severity, Criticality, and Fatality of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Beta Variant.

Beta (B.1.351)-variant coronavirus disease 2019 (COVID-19) disease was investigated in Qatar. Compared with the Alpha (B.1.1.7) variant, odds (95% confidence interval) of progressing to severe disease, critical disease, and COVID-19-related death were 1.24-fold (1.11-1.39), 1.49-fold (1.13-1.97), and 1.57-fold (1.03-2.43) higher, respectively, for the Beta variant.

Association of Prior SARS-CoV-2 Infection With Risk of Breakthrough Infection Following mRNA Vaccination in Qatar.

Importance: The effect of prior SARS-CoV-2 infection on vaccine protection remains poorly understood. Objective: To assess protection from SARS-CoV-2 breakthrough infection after mRNA vaccination among persons with vs without prior SARS-CoV-2 infection. Design, Setting, and Participants: Matched-cohort studies in Qatar for the BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines. A total of 1 531 736 individuals vaccinated with either vaccine between December 21, 2020, and September 19, 2021, were followed up beginning 14 days after receiving the second dose until September 19, 2021. Exposures: Prior SARS-CoV-2 infection and COVID-19 vaccination. Main Outcomes and Measures: Incident SARS-CoV-2 infection, defined as a polymerase chain reaction (PCR)-positive nasopharyngeal swab regardless of reason for PCR testing or presence of symptoms. Cumulative incidence was calculated using the Kaplan-Meier estimator method. Results: The BNT162b2-vaccinated cohort comprised 99 226 individuals with and 290 432 matched individuals without prior PCR-confirmed infection (median age, 37 years; 68% male). The mRNA-1273-vaccinated cohort comprised 58 096 individuals with and 169 514 matched individuals without prior PCR-confirmed infection (median age, 36 years; 73% male). Among BNT162b2-vaccinated persons, 159 reinfections occurred in those with and 2509 in those without prior infection 14 days or more after dose 2. Among mRNA-1273-vaccinated persons, 43 reinfections occurred in those with and 368 infections in those without prior infection. Cumulative infection incidence among BNT162b2-vaccinated individuals was an estimated 0.15% (95% CI, 0.12%-0.18%) in those with and 0.83% (95% CI, 0.79%-0.87%) in those without prior infection at 120 days of follow-up (adjusted hazard ratio for breakthrough infection with prior infection, 0.18 [95% CI, 0.15-0.21]; P < .001). Cumulative infection incidence among mRNA-1273-vaccinated individuals was an estimated 0.11% (95% CI, 0.08%-0.15%) in those with and 0.35% (95% CI, 0.32%-0.40%) in those without prior infection at 120 days of follow-up (adjusted hazard ratio, 0.35 [95% CI, 0.25-0.48]; P < .001). Vaccinated individuals with prior infection 6 months or more before dose 1 had statistically significantly lower risk for breakthrough infection than those vaccinated less than 6 months before dose 1 (adjusted hazard ratio, 0.62 [95% CI, 0.42-0.92]; P = .02 for BNT162b2 and 0.40 [95% CI, 0.18-0.91]; P = .03 for mRNA-1273 vaccination). Conclusions and Relevance: Prior SARS-CoV-2 infection was associated with a statistically significantly lower risk for breakthrough infection among individuals receiving the BNT162b2 or mRNA-1273 vaccines in Qatar between December 21, 2020, and September 19, 2021. The observational study design precludes direct comparisons of infection risk between the 2 vaccines.

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing.

Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%;P< 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P< 0.05) and improved the sensitivity of pathogen detection (P< 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

Innate immune control of EBV-infected B cells by invariant natural killer T cells.

Individuals with X-linked lymphoproliferative disease lack invariant natural killer T (iNKT) cells and are exquisitely susceptible to Epstein-Barr virus (EBV) infection. To determine whether iNKT cells recognize or regulate EBV, resting B cells were infected with EBV in the presence or absence of iNKT cells. The depletion of iNKT cells increased both viral titers and the frequency of EBV-infected B cells. However, EBV-infected B cells rapidly lost expression of the iNKT cell receptor ligand CD1d, abrogating iNKT cell recognition. To determine whether induced CD1d expression could restore iNKT recognition in EBV-infected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-α agonist that upregulates CD1d expression via the nuclear protein, lymphoid enhancer-binding factor 1 (LEF-1). AM580 significantly reduced LEF-1 association at the CD1d promoter region, induced CD1d expression on LCL, and restored iNKT recognition of LCL. CD1d-expressing LCL elicited interferon γ secretion and cytotoxicity by iNKT cells even in the absence of exogenous antigen, suggesting an endogenous iNKT antigen is expressed during EBV infection. These data indicate that iNKT cells may be important for early, innate control of B cell infection by EBV and that downregulation of CD1d may allow EBV to circumvent iNKT cell-mediated immune recognition.

Microbiology of bloodstream infections in Ontario, Canada during COVID-19 pandemic.

Background: Bloodstream infections (BSI) caused by a wide range of bacterial and fungal pathogens are associated with high rates of morbidity and mortality. Based on an estimate in 2017, the number of BSI incidences in Ontario is 150 per 100,000 population. The epidemiology of BSIs may be affected by many factors, including the social and travel restrictions and increased rates of hospitalizations in Ontario during the coronavirus disease 2019 (COVID-19) pandemic. Objectives: This study aimed to assess the changes in the microbiology of BSIs in Ontario during the COVID-19 pandemic compared to the pre-pandemic period. Methods: Retrospective blood culture data (n=189,106) from LifeLabs Ontario (July 2018 to December 2021) were analyzed. Blood culture positivity rates for common bacterial pathogens were compared between pre-COVID-19 (July 2018 to March 2020) and COVID-19 (April 2020 to December 2021) periods in community and hospital settings, using the chi-square test for significance. Results: During the COVID-19 period, blood culture positivity rates in the community remained the same, while hospital rates increased by approximately threefold (p=0.00E-00). In the community, the isolation rates of most bacterial species remained unchanged, except for an increase in Enterococcus spp. and a decrease in Salmonella spp. The rates of antibiotic-resistant organisms (AROs) also significantly decreased in the community. In hospitals, all bacterial species, including AROs, showed significant increases in isolation rates during the COVID-19 period. Conclusion: The study revealed shifts in the microbiology of BSIs and suggests changes in the epidemiology of BSIs during the COVID-19 pandemic in Ontario, both in hospitals and in the community.

Addressing bias in the definition of SARS-CoV-2 reinfection: implications for underestimation.

Introduction: Reinfections are increasingly becoming a feature in the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, accurately defining reinfection poses methodological challenges. Conventionally, reinfection is defined as a positive test occurring at least 90 days after a previous infection diagnosis. Yet, this extended time window may lead to an underestimation of reinfection occurrences. This study investigated the prospect of adopting an alternative, shorter time window for defining reinfection. Methods: A longitudinal study was conducted to assess the incidence of reinfections in the total population of Qatar, from February 28, 2020 to November 20, 2023. The assessment considered a range of time windows for defining reinfection, spanning from 1 day to 180 days. Subgroup analyses comparing first versus repeat reinfections and a sensitivity analysis, focusing exclusively on individuals who underwent frequent testing, were performed. Results: The relationship between the number of reinfections in the population and the duration of the time window used to define reinfection revealed two distinct dynamical domains. Within the initial 15 days post-infection diagnosis, almost all positive tests for SARS-CoV-2 were attributed to the original infection. However, surpassing the 30-day post-infection threshold, nearly all positive tests were attributed to reinfections. A 40-day time window emerged as a sufficiently conservative definition for reinfection. By setting the time window at 40 days, the estimated number of reinfections in the population increased from 84,565 to 88,384, compared to the 90-day time window. The maximum observed reinfections were 6 and 4 for the 40-day and 90-day time windows, respectively. The 40-day time window was appropriate for defining reinfection, irrespective of whether it was the first, second, third, or fourth occurrence. The sensitivity analysis, confined to high testers exclusively, replicated similar patterns and results. Discussion: A 40-day time window is optimal for defining reinfection, providing an informed alternative to the conventional 90-day time window. Reinfections are prevalent, with some individuals experiencing multiple instances since the onset of the pandemic.

Effectiveness of two and three doses of COVID-19 mRNA vaccines against infection, symptoms, and severity in the pre-omicron era: A time-dependent gradient.

BACKGROUND: Vaccines were developed and deployed to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This study aimed to characterize patterns in the protection provided by the BNT162b2 and mRNA-1273 mRNA vaccines against a spectrum of SARS-CoV-2 infection symptoms and severities. METHODS: A national, matched, test-negative, case-control study was conducted in Qatar between January 1 and December 18, 2021, utilizing a sample of 238,896 PCR-positive tests and 6,533,739 PCR-negative tests. Vaccine effectiveness was estimated against asymptomatic, symptomatic, severe coronavirus disease 2019 (COVID-19), critical COVID-19, and fatal COVID-19 infections. Data sources included Qatar's national databases for COVID-19 laboratory testing, vaccination, hospitalization, and death. RESULTS: Effectiveness of two-dose BNT162b2 vaccination was 75.6% (95% CI: 73.6-77.5) against asymptomatic infection and 76.5% (95% CI: 75.1-77.9) against symptomatic infection. Effectiveness against each of severe, critical, and fatal COVID-19 infections surpassed 90%. Immediately after the second dose, all categories-namely, asymptomatic, symptomatic, severe, critical, and fatal COVID-19-exhibited similarly high effectiveness. However, from 181 to 270 days post-second dose, effectiveness against asymptomatic and symptomatic infections declined to below 40%, while effectiveness against each of severe, critical, and fatal COVID-19 infections remained consistently high. However, estimates against fatal COVID-19 often had wide 95% confidence intervals. Analogous patterns were observed in three-dose BNT162b2 vaccination and two- and three-dose mRNA-1273 vaccination. Sensitivity analyses confirmed the results. CONCLUSION: A gradient in vaccine effectiveness exists and is linked to the symptoms and severity of infection, providing higher protection against more symptomatic and severe cases. This gradient intensifies over time as vaccine immunity wanes after the last vaccine dose. These patterns appear consistent irrespective of the vaccine type or whether the vaccination involves the primary series or a booster.

Vascular endothelial growth factor promotes pericyte coverage of brain capillaries, improves cerebral blood flow during subsequent focal cerebral ischemia, and preserves the metabolic penumbra.

BACKGROUND AND PURPOSE: Therapeutic angiogenesis aims at improving cerebral blood flow by amplification of vascular sprouting, thus promoting tissue survival under conditions of subsequent ischemia. It remains unknown whether induced angiogenesis leads to the formation of functional vessels that indeed result in hemodynamic improvements. Observations of hemodynamic steal phenomena and disturbed neurovascular integrity after vascular endothelial growth factor delivery questioned the concept of therapeutic angiogenesis. METHODS: Mice were treated with recombinant human vascular endothelial growth factor (0.02 µg/d; intracerebroventricular) for 3 to 21 days and subsequently exposed to 90-minute middle cerebral artery occlusion. Angiogenesis, histological brain injury, IgG extravasation, cerebral blood flow, protein synthesis and energy state, and pericyte coverage on brain capillaries were evaluated in a multiparametric approach combining histochemical, autoradiographic, and regional bioluminescence techniques. RESULTS: Vascular endothelial growth factor increased brain capillary density within 10 days and reduced infarct volume and inflammation after subsequent middle cerebral artery occlusion, and, when delivered for prolonged periods of 21 days, enhanced postischemic blood-brain barrier integrity. Increased cerebral blood flow was noted in ischemic brain areas exhibiting enhanced angiogenesis and was associated with preservation of the metabolic penumbra, defined as brain tissue in which protein synthesis has been suppressed but ATP preserved. Vascular endothelial growth factor enhanced pericyte coverage of brain endothelial cells via mechanisms involving increased N-cadherin expression on cerebral microvessels. CONCLUSIONS: That cerebral blood flow is increased during subsequent ischemic episodes, leading to the stabilization of cerebral energy state, fosters hope that by promoting new vessel formation brain tissue survival may be improved.

Nucleophosmin 1, upregulated in adenomas and cancers of the colon, inhibits p53-mediated cellular senescence.

Dysregulation of nucleophosmin 1 (NPM1) has been found in numerous solid and hematological malignancies. Our previous meta-analysis of colorectal cancer (CRC) high throughput gene expression profiling studies identified it as a consistently reported up-regulated gene in the malignant state. Our aims were to compare NPM1 expression in normal colon, adenoma and CRC, to correlate their expressions with clinico-pathological parameters, and to assess the biological role of aberrant NPM1 expression in CRC cells. NPM1 transcript levels were studied in human CRC cell lines, whereas a tissue microarray of 57 normal human colon, 40 adenoma and 185 CRC samples were used to analyze NPM1 protein expression by immunohistochemistry. CRC cell lines were subjected to transient siRNA-mediated knockdown to study NPM1's roles on cell viability and senescence. NPM1 transcript levels were 7-11-folds higher in three different human CRC cell lines compared to normal colon cells. NPM1 protein expression was found to be progressively and significantly upregulated in CRC compared to adenomas and in adenomas compared to normal mucosa. Reducing NPM1 expression by siRNA had caused a significant decrease in cell viability, a concomitant increase in cellular senescence and cell cycle arrest. Cellular senescence induced under conditions of forced NPM1 suppression could be prevented by knocking down p53. The differential expression of NPM1 along the normal colon-adenoma-carcinoma progression and its involvement in resisting p53 related senescent growth arrest in CRC cell lines implicate its role in supporting CRC tumorigenesis.

The novel proteasome inhibitor BSc2118 protects against cerebral ischaemia through HIF1A accumulation and enhanced angioneurogenesis.

Only a minority of stroke patients receive thrombolytic therapy. Therefore, new therapeutic strategies focusing on neuroprotection are under review, among which, inhibition of the proteasome is attractive, as it affects multiple cellular pathways. As proteasome inhibitors like bortezomib have severe side effects, we applied the novel proteasome inhibitor BSc2118, which is putatively better tolerated, and analysed its therapeutic potential in a mouse model of cerebral ischaemia. Stroke was induced in male C57BL/6 mice using the intraluminal middle cerebral artery occlusion model. BSc2118 was intrastriatally injected 12 h post-stroke in mice that had received normal saline or recombinant tissue-plasminogen activator injections during early reperfusion. Brain injury, behavioural tests, western blotting, MMP9 zymography and analysis of angioneurogenesis were performed for up to 3 months post-stroke. Single injections of BSc2118 induced long-term neuroprotection, reduced functional impairment, stabilized blood-brain barrier through decreased MMP9 activity and enhanced angioneurogenesis when given no later than 12 h post-stroke. On the contrary, recombinant tissue-plasminogen activator enhanced brain injury, which was reversed by BSc2118. Protein expression of the transcription factor HIF1A was significantly increased in saline-treated and recombinant tissue-plasminogen activator-treated mice after BSc2118 application. In contrast, knock-down of HIF1A using small interfering RNA constructs or application of the HIF1A inhibitor YC1 (now known as RNA-binding motif, single-stranded-interacting protein 1 (RBMS1)) reversed BSc2118-induced neuroprotection. Noteworthy, loss of neuroprotection after combined treatment with BSc2118 and YC1 in recombinant tissue-plasminogen activator-treated animals was in the same order as in saline-treated mice, i.e. reduction of recombinant tissue-plasminogen activator toxicity through BSc2118 did not solely depend on HIF1A. Thus, the proteasome inhibitor BSc2118 is a promising new candidate for stroke therapy, which may in addition alleviate recombinant tissue-plasminogen activator-induced brain toxicity.