The production of anti-PF4 antibodies in anti-phospholipid antibody-positive patients is not affected by COVID-19 vaccination

Antibodies against cationic platelet chemokine, platelet factor 4 (PF4/CXCL4) have been described in heparin-induced thrombocytopenia (HIT) but also in patients positive for anti-phospholipid antibodies (aPL) even in the absence of heparin treatment and HIT-related clinical manifestations. Anti-PF4 antibodies have been recently described also in subjects who developed thrombosis with thrombocytopenia syndrome (TTS) in association with adenoviral vector-based, but not with mRNA-based COVID-19 vaccines. We investigated whether COVID-19 vaccination affects the production of anti-PF4 immunoglobulins detectable by solid phase assay in aPL-positive patients and their ability to induce in vitro platelet activation. Anti-PF4 were found in 9/126 aPL-positive patients, 4/50 COVID-19, 9/49 other infections and 1/50 aPL-negative systemic lupus erythematosus patients. Clinical manifestations of TTS were not observed in any aPL patient positive for anti-PF4, whose sera failed to cause platelet aggregations. The administration of COVID-19 vaccines did not affect the production of anti-PF4 immunoglobulins or their ability to cause platelet aggregation in 44 aPL-positive patients tested before and after vaccination. In conclusion, heparin treatment-independent anti-PF4 antibodies can be found in aPL-positive patients and asymptomatic carriers, but their presence, titer as well as in vitro effect on platelet activation are not affected by COVID-19 vaccination.

Inflammatory biomarkers in pregnant women with COVID-19: a retrospective cohort study

Coronavirns disease 2019 is a pandemic viral disease affecting also obstetric patients and uncertainties exist about the prognostic role of inflammatory biomarkers and hemocytometry values in patients with this infection. To clarify that, we assessed the values of several inflammatory biomarkers and hemocytometry variables in a cohort of obstetric patients hospitalized with coronavirus disease 2019 and we correlated the values at admission with the need of oxygen supplementation during the hospitalization. Overall, among 27 (61%) pregnant women and 17 (39%) post-partum women, 6 (14%) patients received oxygen supplementation and 2 (4%) required admission to intensive care unit but none died. During hospitalization neutrophils (p=0.002), neutrophils to lymphocytes ratio (p=0.037) and C reactive protein (p<0.001) decreased significantly, whereas lymphocytes (p<0.001) and platelets (p<0.001) increased. Leukocytes and lymphocytes values at admission were correlated with oxygen need, with respectively a 1% and 5% higher risk of oxygen supplementation for each 1,000 cells decrease. Overall, in obstetric patients hospitalized with coronavirus disease 2019, C reactive protein is the inflammatory biomarker that better mirrors the course of the disease whereas D-dimer or ferritin are not reliable predictors of poor outcome. Care to the need of oxygen supplementation should be reserved to patients with reduced leukocytes or lymphocytes values at admission.

Characteristics of 1,573 healthcare workers who underwent nasopharyngeal swab for SARS-CoV-2 in Milano, Lombardy, Italy

BackgroundThe management of healthcare workers (HCWs) exposed to confirmed cases of COVID-19 is still a matter of debate. It is unclear whether these subjects should be tested in the absence of symptoms and if those can guide diagnosis. MethodsOccupational and clinical characteristics of all the consecutive HCWs who performed a nasopharyngeal swab for the detection of SARS-CoV-2 in a University Hospital from February 24, 2020, to March 31, 2020, were collected. Frequencies of positive tests were compared according to selected variables. Multivariable logistic regression analyses were then applied. FindingsPositive tests were 138 among 1,573 HCWs (8.8%, 95% confidence interval [CI]: 7.4-10.3), with a marked difference between symptomatic (20.2%, 95% CI: 16.7-24.1) and asymptomatic (3.7%, 95% CI: 2.7-5.1) subjects (p<0.001). Physicians were the group with the highest frequency of positive tests (10.6%, 95% CI: 8.3-13.4) whereas clerical workers and technicians displayed the lowest frequency (2.9%, 95% CI: 0.8-7.3). The likelihood of being positive increased with the number of reported symptoms and the strongest predictors of a positive test were taste and smell alterations (odds ratio [OR] = 29.7) and fever (OR = 7.21). The median time from first positive test to a negative test was 23 days (95% CI: 19-24). InterpretationIn this Italian group of HCWs exposed to confirmed cases of COVID-19 the presence of symptoms, especially taste and smell alterations and fever, was associated with SARS-CoV-2 infection. The median time to clear the virus from nasopharynx was 23 days. Fundingnone related to the content of this manuscript. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed for articles published in English up to April 25, 2020, using the keywords "SARS-CoV-2", "COVID-19", "2019-nCoV", AND "healthcare workers","HCW", AND "testing", "nasopharyngeal swab". We found one article: Roll-out of SARS-CoV-2 testing for healthcare workers at a large NHS Foundation Trust in the United Kingdom, March 2020 published in Euro Surveillance. Reviewing the pre-print website medRxiv with the same keywords we identified two additional studies: SARS-CoV-2 infection in Health Care Workers in a large public hospital in Madrid, Spain, during March 2020, and SARS-CoV-2 infection in 86 healthcare workers in two Dutch hospitals in March. Added value of this studyWe showed that, even if symptomatic healthcare workers had a much higher probability of positive test, almost one third of those infected were asymptomatic. Specific symptoms, namely taste and smell alterations and fever, were strongly associated with the infection. Finally, the median time to clear the virus from nasopharynx was 23 days. Implications of all the available evidenceScreening strategies for healthcare workers exposed to COVID-19 patients should take in account the significant proportion of asymptomatic carriers and the predictive role of specific symptoms. Moreover, healthcare workers coming back to work after a positive test should be aware of the long-time of viral shedding from nasopharynx.

Early phases of COVID-19 are characterized by a reduction of lymphocyte populations and the presence of atypical monocytes

BackgroundSevere acute respiratory syndrome coronavirus 2 is a recently discovered pathogen responsible of coronavirus disease 2019 (COVID-19). The immunological changes associated with this infection are largely unknown. MethodsWe evaluated the peripheral blood mononuclear cells profile of 63 patients with COVID-19 at diagnosis and the presence of association with inflammatory biomarkers and 28-days mortality. ResultsLymphocytopenia was present in 51 of 63 (80.9%) patients. This reduction was mirrored also on CD8+ lymphocytes (128 cells/L), natural killer cells (67 cells/L) and natural killer T cells (31 cells/L). Monocytes were preserved in total number but displayed a subpopulation composed mainly of cells with a reduced expression of both CD14 and HLA-DR. A direct correlation was found between serum values of IL-6 and the frequency of Th2 lymphocytes (R=0.17; p=0.04) but not with the monocytes count (R=0.01; p=0.60). Patients who died in the 28 days from admission (N=10, 15.9%), when compared to those who did not, displayed lower mean values of CD3+ (p=0.028) and CD4+ cells (p=0.042) and higher mean percentages of CD8+/CD38+/HLA-DR+ lymphocytes (p=0.026). ConclusionsThe early phases of COVID-19 are characterized by lymphocytopenia, predominance of Th2 lymphocytes and less immunocompetent monocytes, which include atypical mononuclear cells. eTOC-At diagnosis patients with COVID-19 have lymphocytopenia -Monocytes with both normal or altered scatter properties display a reduced expression of CD14 and HLA-DR in most of COVID-19 patients -Patients who die in the 28 days from admission have lower values of CD3+ and CD4+ cells and higher percentages of activated CTL cells compared to those who survive

Discovery of Entrectinib: A New 3-Aminoindazole As a Potent Anaplastic Lymphoma Kinase (ALK), c-ros Oncogene 1 Kinase (ROS1), and Pan-Tropomyosin Receptor Kinases (Pan-TRKs) inhibitor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.

The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition.

The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.

Crystal structures of anaplastic lymphoma kinase in complex with ATP competitive inhibitors.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the development of several human cancers and, as a result, is a recognized target for the development of small-molecule inhibitors for the treatment of ALK-positive malignancies. Here, we present the crystal structures of the unphosphorylated human ALK kinase domain in complex with the ATP competitive ligands PHA-E429 and NVP-TAE684. Analysis of these structures provides valuable information concerning the specific characteristics of the ALK active site as well as giving indications about how to obtain selective ALK inhibitors. In addition, the ALK-KD-PHA-E429 structure led to the identification of a potential regulatory mechanism involving a link made between a short helical segment immediately following the DFG motif and an N-terminal two-stranded beta-sheet. Finally, mapping of the activating mutations associated with neuroblastoma onto our structures may explain the roles these residues have in the activation process.