Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening.

CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.

Acute Hospital Outcomes for Renal Transplantation in Patients With Moderate or Severe Congenital Heart Disease.

Children and adults with congenital heart disease (CHD) are increasingly recognized to be at risk for acute and chronic renal injury. Some of these may progress to the need for renal transplantation. We hypothesized that patients with underlying moderate or severe CHD who undergo renal transplantation will have worse acute hospital outcomes. Using a national administrative database, we queried for admissions aged 0 to 50 years with moderate or severe CHD and renal transplantation and compared these to admissions without CHD. There were 56 admissions for renal transplantation in the CHD group (0.04%) and 26,285 admissions in the group without CHD (0.21%, p<0.001). The CHD group were younger, had a higher proportion of Whites, longer length of stay, higher complication rates, higher in-hospital mortality, and higher costs. In conclusion, although renal transplantation is still relatively uncommon in the CHD population, there is an increasing recognition of severe chronic renal disease in the setting of CHD, making it important to understand the potential implications of these findings.

Novel use of the double kissing crush technique to stent complex pulmonary artery stenosis in a child with Alagille syndrome.

Coronary bifurcation lesions and treatment with two-stent techniques have been developed, including the double kissing (DK) crush technique. The use of this technique in children or noncoronary vessels, including pulmonary arteries, has not been described. We present a 12-year-old girl with Alagille syndrome, a ventricular septal defect (VSD), and complex bilateral pulmonary artery (PA) stenoses who is status post six catheterizations for PA angioplasty and stenting to improve her marked right ventricular hypertension. With collaboration between the congenital and structural teams, she successfully underwent the DK crush technique for a complex lesion in her PA. This improved pulmonary flow and allowed for successful surgical VSD closure.

Direct capture of neutralized RBD enables rapid point-of-care assessment of SARS-CoV-2 neutralizing antibody titer.

Neutralizing antibody (NAb) titer is a key biomarker of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but point-of-care methods for assessing NAb titer are not widely available. Here, we present a lateral flow assay that captures SARS-CoV-2 receptor-binding domain (RBD) that has been neutralized from binding angiotensin-converting enzyme 2 (ACE2). Quantification of neutralized RBD in this assay correlates with NAb titer from vaccinated and convalescent patients. This methodology demonstrated superior performance in assessing NAb titer compared with either measurement of total anti-spike immunoglobulin G titer or quantification of the absolute reduction in binding between ACE2 and RBD. Our testing platform has the potential for mass deployment to aid in determining at population scale the degree of protective immunity individuals may have following SARS-CoV-2 vaccination or infection and can enable simple at-home assessment of NAb titer.

Novel dithiocarbamate derivatives are effective copper-dependent antimicrobials against Streptococcal species.

Despite the availability of several vaccines against multiple disease-causing strains of Streptococcus pneumoniae, the rise of antimicrobial resistance and pneumococcal disease caused by strains not covered by the vaccine creates a need for developing novel antimicrobial strategies. N,N-dimethyldithiocarbamate (DMDC) was found to be a potent copper-dependent antimicrobial against several pathogens, including S. pneumoniae. Here, DMDCs efficacy against Streptococcal pathogens Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus anginosus was tested using bactericidal and inductively coupled plasma - optical emission spectrometry. After confirming DMDC as broad-spectrum streptococcal antimicrobial, DMDC was derivatized into five compounds. The derivatives' effectiveness as copper chelators using DsRed2 and as copper-dependent antimicrobials against S. pneumoniae TIGR4 and tested in bactericidal and animal models. Two compounds, sodium N-benzyl-N-methyldithiocarbamate and sodium N-allyl-N-methyldithiocarbamate (herein "Compound 3" and "Compound 4"), were effective against TIGR4 and further, D39 and ATCC® 6303™ _(a type 3 capsular strain). Both Compound 3 and 4 increased the pneumococcal internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. However, in an in vivo murine pneumonia model, Compound 3, but not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of S. pneumoniae-infected mice. These derivatives also had detrimental effects on the other streptococcal species. Collectively, derivatizing DMDC holds promise as potent bactericidal antibiotics against relevant streptococcal pathogens.