Pilot study on the use of basophil activation tests and skin tests for the prevention of allergic transfusion reactions.

Background and objectives: Management of severe allergic transfusion reactions (ATR) is challenging. In this study, we investigate the usefulness of skin tests and basophil activation tests (BAT) in chronically transfused patients for the prevention of future ATR. Materials and methods: BAT and skin tests were carried with the supernatant of red blood cell (RBC) units for a sickle-cell disease patient under chronic exchange transfusion who has presented a severe ATR, in order to prevent potential future ATR. If the results for both BAT and skin tests were negative, the RBC units could be transfused to the patient. If either one of the results was positive, the tested RBC unit was discarded for the patient. Results: 192 RBC units were tested with both tests. The level of results concordance between the two tests was 95%. Out of the 169 negative units with both tests, 118 units were transfused to the patient for which he presented no ATR. Conclusion: In our study, combining both BAT and skin tests was associated with a good negative predictive value since we were able to safely transfuse our patient. Further studies are still necessary to confirm this result but this pilot study indicates that skin tests and BAT might help prevent ATR. When BAT is not available, skin tests may also be useful in preventing ATR.

Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective.

As the global burden of disease caused by multidrug resistant bacteria is a major source of concern, credible clinical alternatives to antibiotic therapy, such as personalized phage therapy, are actively explored. Although phage therapy has been used for more than a century, the issue of an easy to implement diagnostic tool for determining phage susceptibility that meets current routine clinical needs is still open. In this Review, we summarize the existing methods used for determining phage activity on bacteria, including the three reference methods: the spot test, the double agar overlay plaque assay, and the Appelmans method. The first two methods rely on the principle of challenging the overnight growth of a lawn of bacteria in an agar matrix to a known relative phage to bacteria concentration and represent good screening tools to determine if the tested phage can be used for a "passive" and or "active" treatment. Beside these methods, several techniques, based on "real-time" growth kinetics assays (GKA) have been developed or are under development. They all monitor the growth of clinical isolates in the presence of phages, but use various detection methods, from classical optical density to more sophisticated techniques such as computer-assisted imagery, flow-cytometry, quantitative real-time polymerase chain reaction (qPCR) or metabolic indicators. Practical considerations as well as information provided about phage activity are reviewed for each technique. Finally, we also discuss the analytical and interpretative requirements for the implementation of a phage susceptibility testing tool in routine clinical microbiology.

Evaluation and Modelling of the Performance of an Automated SARS-CoV-2 Antigen Assay According to Sample Type, Target Population and Epidemic Trends.

The Lumipulse® G SARS-CoV-2 Ag assay performance was evaluated on prospectively collected saliva and nasopharyngeal swabs (NPS) of recently ill in- and outpatients and according to the estimated viral load. Performances were calculated using RT-PCR positive NPS from patients with symptoms ≤ 7 days and RT-PCR negative NPS as gold standard. In addition, non-selected positive NPS were analyzed to assess the performances on various viral loads. This assay yielded a sensitivity of 93.1% on NPS and 71.4% on saliva for recently ill patients. For NPS with a viral load > 103 RNA copies/mL, sensitivity was 96.4%. A model established on our daily routine showed fluctuations of the performances depending on the epidemic trends but an overall good negative predictive value. Lumipulse® G SARS-CoV-2 assay yielded good performance for an automated antigen detection assay on NPS. Using it for the detection of recently ill patients or to screen high-risk patients could be an interesting alternative to the more expensive RT-PCR.

Leveraging of SARS-CoV-2 PCR Cycle Thresholds Values to Forecast COVID-19 Trends.

Introduction: We assessed the usefulness of SARS-CoV-2 RT-PCR cycle thresholds (Ct) values trends produced by the LHUB-ULB (a consolidated microbiology laboratory located in Brussels, Belgium) for monitoring the epidemic's dynamics at local and national levels and for improving forecasting models. Methods: SARS-CoV-2 RT-PCR Ct values produced from April 1, 2020, to May 15, 2021, were compared with national COVID-19 confirmed cases notifications according to their geographical and time distribution. These Ct values were evaluated against both a phase diagram predicting the number of COVID-19 patients requiring intensive care and an age-structured model estimating COVID-19 prevalence in Belgium. Results: Over 155,811 RT-PCR performed, 12,799 were positive and 7,910 Ct values were available for analysis. The 14-day median Ct values were negatively correlated with the 14-day mean daily positive tests with a lag of 17 days. In addition, the 14-day mean daily positive tests in LHUB-ULB were strongly correlated with the 14-day mean confirmed cases in the Brussels-Capital and in Belgium with coinciding start, peak, and end of the different waves of the epidemic. Ct values decreased concurrently with the forecasted phase-shifts of the diagram. Similarly, the evolution of 14-day median Ct values was negatively correlated with daily estimated prevalence for all age-classes. Conclusion: We provide preliminary evidence that trends of Ct values can help to both follow and predict the epidemic's trajectory at local and national levels, underlining that consolidated microbiology laboratories can act as epidemic sensors as they gather data that are representative of the geographical area they serve.

Improvement of SCD morbimortality in children: experience in a remote area of an African country.

BACKGROUND: Sickle cell disease (SCD) is a public health problem in the Democratic Republic of Congo. While reference sickle cell centers have been implemented in capital cities of African countries and have proven to be beneficial for SCD patients. In the Democratic Republic of Congo, they have never been set up in remote areas for families with low or very low sources of income. METHOD: A cohort of 143 children with SCD aged 10 years old (IQR (interquartile range): 6-15 years) (sex ratio male/female = 1.3) were clinically followed for 12 months without any specific intervention aside from the management of acute events, and then for 12 months with a monthly medical visit, biological follow-up, and chemoprophylaxis (folic acid/penicillin), adequate fluids and malaria prevention. RESULTS: The median age of patients at the diagnosis of SCD was 2 years (IQR: 1-5). The implementation of standardized and regular follow-ups in a new sickle cell reference center in a remote city showed an increase in the annual mean hemoglobin level from 50 to 70 g/L (p = 0.001), and a decrease in the lymphocyte count and spleen size (p < 0.001). A significant decrease (p < 0.001) in the average annual number of hospitalizations and episodes of vaso-occlusive crises, blood transfusions, infections, and acute chest syndromes were also observed. CONCLUSIONS: The creation of a sickle cell reference center and the regular follow-up of children with sickle cell disease are possible and applicable in the context of a remote city of an African country and represent simple and accessible measures that can reduce the morbimortality of children with sickle cell disease.

Covid-19 and blood groups: ABO antibody levels may also matter.

BACKGROUND: Susceptibility to Covid-19 has been found to be associated with the ABO blood group, with O type individuals being at a lower risk. However, the underlying mechanism has not been elucidated. Here, we aimed to test the hypothesis that Covid-19 patients might have lower levels of ABO antibodies than non-infected individuals as they could offer some degree of protection. METHODS: After showing that the viral spike protein harbors the ABO glycan epitopes when produced by cells expressing the relevant glycosyltransferases, like upper respiratory tract epithelial cells, we enrolled 290 patients with Covid-19 and 276 asymptomatic controls to compare their levels of natural ABO blood group antibodies. RESULTS: We found significantly lower IgM anti-A + anti-B agglutination scores in blood group O patients (76.93 vs 88.29, P-value = 0.034) and lower levels of anti-B (24.93 vs 30.40, P-value = 0.028) and anti-A antibodies (28.56 vs 36.50, P-value = 0.048) in blood group A and blood group B patients, respectively, compared to controls. CONCLUSION: In this study, we showed that ABO antibody levels are significantly lower in Covid-19 patients compared to controls. These findings could indicate that patients with low levels of ABO antibodies are at higher risk of being infected.

Neural progenitors derived from human induced pluripotent stem cells survive and differentiate upon transplantation into a rat model of amyotrophic lateral sclerosis.

Human induced pluripotent stem cells (iPSCs) offer hope for personalized regenerative cell therapy in amyotrophic lateral sclerosis (ALS). We analyzed the fate of human iPSC-derived neural progenitors transplanted into the spinal cord of wild-type and transgenic rats carrying a human mutated SOD1(G93A) gene. The aim was to follow survival and differentiation of human neural progenitors until day 60 post-transplantation in two different in vivo environments, one being ALS-like. iPSC-derived neural progenitors efficiently engrafted in the adult spinal cord and survived at high numbers. Different neural progenitor, astroglial, and neuronal markers indicated that, over time, the transplanted nestin-positive cells differentiated into cells displaying a neuronal phenotype in both wild-type and transgenic SOD1 rats. Although a transient microglial phenotype was detected at day 15, astroglial staining was negative in engrafted cells from day 1 to day 60. At day 30, differentiation toward a neuronal phenotype was identified, which was further established at day 60 by the expression of the neuronal marker MAP2. A specification process into motoneuron-like structures was evidenced in the ventral horns in both wild-type and SOD1 rats. Our results demonstrate proof-of-principle of survival and differentiation of human iPSC-derived neural progenitors in in vivo ALS environment, offering perspectives for the use of iPSC-based therapy in ALS.

Bimodal modulation of tau protein phosphorylation and conformation by extracellular Zn2+ in human-tau transfected cells.

Abnormal homeostasis of heavy metals is a well-documented physiopathological mechanism in Alzheimer's disease. An exacerbation of these abnormalities is best illustrated in the amyloid plaques in Alzheimer's disease brain tissue, in which zinc reaches the enormous concentration of 1000 microM. Zinc in the plaques is thought to originate from impaired glutamatergic neurons distributed in the associative cortex and limbic structures of normal brain. Although the characteristics of zinc binding to Abeta and its role in promotion of Abeta aggregation have been intensively studied, the contribution of zinc to the development of tau pathology remains elusive. To further document the effect of zinc we have investigated the modifications of tau phosphorylation, conformation and association to microtubules induced by zinc in clonal cell lines expressing a human tau isoform. A bimodal dose dependent effect of zinc was observed. At 100 microM zinc induced a tau dephosphorylation on the PHF-1 epitope, and at higher zinc concentrations induced the appearance of the abnormal tau conformational epitope MC1 and reduced the electrophoretic mobility of tau, known to be associated to increased tau phosphorylation. High zinc concentrations also increased glycogen synthase kinase-3beta (GSK-3beta) phosphorylation on tyrosine 216, a phosphorylation associated with increased activity of this tau kinase. Live imaging of tau-EGFP expressing cells demonstrated that high zinc concentrations induced a release of tau from microtubules. These results suggest that zinc plays a significant role in the development of tau pathology associated to Alzheimer's disease.

Tissue factor: a mini-review.

Tissue factor (TF) is historically known as the trigger of the coagulation cascade. This integral membrane glycoprotein forms a ternary complex with factor VIIa (FVIIa) and zymogen factor (FX), which is then activated to factor Xa (FXa). The latter cleaves prothrombin into thrombin (FIIa), which in turn activates fibrinogen in fibrin monomers. What is less known is its additional non-haemostatic roles in inflammation, tumour growth and angiogenesis. This aspect will be developed here. TF, as a transmembrane protein, has a signalling effect requiring FVIIa. TF-FVIIa complex activates G protein-coupled receptor protease-activated receptor 2 (PAR-2) and therefore modulates various cellular processes, such as cell proliferation and survival, gene transcription and protein translation. In this review we will first highlight, using recent structural data, the 'potentially' active domain able to modulate the triggered intracellular response. We also will focus on the still emerging and promising results deciphering the diverse locations in which TF appears. We conclude with a description of an emerging and atypical use of tissue factor in platelet gel surgery for sinus augmentation.

Osteosarcoma cell-calcium signaling through tissue factor-factor VIIa complex and factor Xa.

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.

Factor Xa and thrombin evoke additive calcium and proinflammatory responses in endothelial cells subjected to coagulation.

Endothelial cells react to factor Xa and thrombin by proinflammatory responses. It is unclear how these cells respond under physiological conditions, where the serine proteases factor VIIa, factor Xa and thrombin are all simultaneously generated, as in tissue factor-driven blood coagulation. We studied the Ca(2+) signaling and downstream release of interleukins (ILs), induced by these proteases in monolayers of human umbilical vein endothelial cells. In single cells, factor Xa, but not factor VIIa, complexed with tissue factor, evoked a greatly delayed, oscillatory Ca(2+) response, which relied on its catalytic activity and resembled that of SLIGRL, a peptide specifically activating the protease-activated receptor 2 (PAR2). Thrombin even at low concentrations evoked a rapid, mostly non-oscillating Ca(2+) response through activation of PAR1, which reinforced the factor Xa response. The additive Ca(2+) signals persisted, when factor X and prothrombin were activated in situ, or in the presence of plasma that was triggered to coagulate with tissue factor. Further, thrombin reinforced the factor Xa-induced production of IL-8, but not of IL-6. Both interleukins were produced in the presence of coagulating plasma. In conclusion, under coagulant conditions, factor Xa and thrombin appear to contribute in different and additive ways to the Ca(2+)-mobilizing and proinflammatory reactions of endothelial cells. These data provide first evidence that these serine proteases trigger distinct signaling modules in endothelium that is activated by plasma coagulation.

Sinus grafting using recombinant human tissue factor, platelet-rich plasma gel, autologous bone, and anorganic bovine bone mineral xenograft: histologic analysis and case reports.

PURPOSE: The purpose of this study was to analyze healthy bone formation by means of histology and immunohistochemistry after grafting with a mixture of autologous ground calvarial bone, inorganic xenograft, platelet-rich plasma (PRP), and recombinant human tissue factor (rhTF). MATERIALS AND METHODS: Maxillary sinus floor augmentation was performed on 3 patients by grafting with 5 to 10 mL of a paste consisting of autologous powder from calvarial bone (diameter < 1 mm), 50% v/v anorganic bovine bone mineral xenograft (PepGen P-15, a new tissue-engineered bone replacement graft material), PRP (1.8 x 10(6) platelets/mm3 plasma), and about 1 microg rhTF. Six and 10 months after grafting, bone cores were extracted for implant fixation and analyzed. RESULTS: Histology demonstrated a high degree of inorganic xenograft integration and natural bone regeneration. Both the xenograft and newly synthesized bone were colonized with osteocytes and surrounded by osteoblasts. Six-month-old bone cores demonstrated a ratio of synthesized bone to xenograft particles ratio of 0.5, whereas 10-month-old cores demonstrated a ratio of 2. A low degree of inflammation could also be observed using S100A8 immunohistochemistry. DISCUSSION: Autologous grafting in edentulous patients is a complex procedure; the successful substitution of synthetic analogs for ground bone is a major challenge. CONCLUSION: In this investigation, it was shown that inorganic xenograft in the harvested bone paste could be safe for patients and had high bone regeneration capacity over time. The sinus graft showed intense bone formation 6 months after grafting and a further increase in bone growth 10 months after grafting.