Citations

Abstract Stimulation of NK cell-mediated cytotoxicity involves the coupling of proximal Src and Syk family protein tyrosine kinases to downstream effectors. However, the mechanisms linking these second messenger pathways are incompletely understood. Here, we describe a key role for the LAT (p36) adaptor protein in human NK cell activation. LAT is tyrosine phosphorylated upon stimulation of NK cells through FcγRIII receptors and following direct contact with NK-sensitive target cells. This NK stimulation induces the association of LAT with several phosphotyrosine-containing proteins. In addition to the biochemical evidence showing LAT involvement in NK cell activation, a genetic model shows that LAT is required for FcR-dependent phosphorylation of phospholipase C-γ. Furthermore, overexpression of LAT in NK cells leads to increased Ab-dependent cell-mediated cytotoxicity and “natural cytotoxicity,” thus demonstrating a functional role for LAT in NK cells. These data suggest that LAT is an important adaptor protein for the regulation of human NK cell-mediated cytotoxicity.

AbstractSTUDY QUESTIONAre chromosome abnormalities detected at Day 3 post-fertilization predominantly retained in structures of the blastocyst other than the inner cell mass (ICM), where chromosomally normal cells are preferentially retained?SUMMARY ANSWERIn human embryos, aneuploid cells are sequestered away from the ICM, partly to the trophectoderm (TE) but more significantly to the blastocoel fluid within the blastocoel cavity (Bc) and to peripheral cells (PCs) surrounding the blastocyst during Day 3 to Day 5 progression.WHAT IS KNOWN ALREADYA commonly held dogma in all diploid eukaryotes is that two gametes, each with ‘n’ chromosomes (23 in humans), fuse to form a ‘2n’ zygote (46 in humans); a state that remains in perpetuity for all somatic cell divisions. Human embryos, however, display high levels of chromosomal aneuploidy in early stages that reportedly declines from Day 3 (cleavage stage) to Day 5 (blastocyst) post-fertilization. While this observation may be partly because of aneuploid embryonic arrest before blastulation, it could also be due to embryo ‘normalization’ to a euploid state during blastulation. If and how this normalization occurs requires further investigation.STUDY DESIGN, SIZE, DURATIONA total of 964 cleavage-stage (Day 3) embryos underwent single-cell biopsy and diagnosis for chromosome constitution. All were maintained in culture, assessing blastulation rate, both for those assessed euploid and aneuploid. Pregnancy rate was assessed for those determined euploid, blastulated and subsequently transferred. For those determined aneuploid and blastulated (174 embryos), ICM (all 174 embryos), TE (all 174), Bc (47 embryos) and PC (38 embryos) were analyzed for chromosome constitution. Specifically, concordance with the original Day 3 diagnosis and determination if any ‘normalized’ to euploid karyotypes within all four structures was assessed.PARTICIPANTS/MATERIALS, SETTING, METHODSAll patients (144 couples) were undergoing routine preimplantation genetic testing for aneuploidy in three IVF clinical settings. Cleavage-stage biopsy preceded chromosome analysis by next-generation sequencing. All patients provided informed consent. Additional molecular testing was carried out on blastocyst embryos and was analyzed for up to four embryonic structures (ICM, TE, Bc and PC).MAIN RESULTS AND THE ROLE OF CHANCEOf 463/964 embryos (48%) diagnosed as euploid at Day 3, 70% blastulated (leading to a 59% pregnancy rate) and 30% degenerated. Conversely, of the 501 (52%) diagnosed as aneuploid, 65% degenerated and 35% (174) blastulated, a highly significant difference (P < 0.0001). Of the 174 that blastulated, the ratio of ‘(semi)concordant-aneuploid’ versus ‘normalized-euploid’ versus ‘other-aneuploid’ embryos was, respectively, 39%/57%/3% in the ICM; 49%/48%/3% in the TE; 78%/21%/0% in the PC; and 83%/10%/5% in the Bc. The TE karyotype therefore has a positive predictive value of 86.7% in determining that of the ICM, albeit with marginally higher aneuploid rates of abnormalities (P = .071). Levels of abnormality in Bc/PC were significantly higher (P < 0.0001) versus the ploidy of the ICM and TE and nearly all chromosome abnormalities were (at least partially) concordant with Day 3 diagnoses.LIMITATIONS, REASONS FOR CAUTIONThe results only pertain to human IVF embryos so extrapolation to the in vivo situation and to other species is not certain. We acknowledge (rather than lineage-specific survival, as we suggest here) the possibility of other mechanisms, such as lineage-specific movement of cells, during blastulation. Ethical considerations, however, make investigating this mechanism difficult on human embryos.WIDER IMPLICATIONS OF THE FINDINGSMosaic human cleavage-stage embryos can differentiate into a euploid ICM where euploid cell populations predominate. Sequestering of aneuploid cells/nuclei to structures no longer involved in fetal development has important implications for preimplantation and prenatal genetic testing. These results also challenge previous fundamental understandings of mitotic fidelity in early human development and indicate a complex and fluid nature of the human embryonic genome.STUDY FUNDING/COMPETING INTEREST(S)This research was funded by Organon Pharmaceuticals and Merck Serono by grants to W.G.K. W.G.K. is also an employee of AdvaGenix, who could, potentially, indirectly benefit financially from publication of this manuscript. R.C.M. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133747. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.K.G. provides paid consultancy services for Care Fertility.TRIAL REGISTRATION NUMBERN/A.

Objectives: ACL reconstruction is one of the most common knee procedures performed annually in the United States. Athletes participating in cutting and pivoting sports are at high risk for ACL injury. In the general population, most graft choices including allograft, bone-patellar tendon-bone (BTB) autograft, hamstring autograft and quadriceps autograft have all been shown to excellent results following reconstruction. However, in the high-level cutting and pivoting athlete, the optimal graft for reconstruction remains controversial. Most consider BTB autograft to be the gold standard for such athletes. However, BTB autografts have drawbacks including anterior knee pain, difficulty kneeling and possible patellar fracture and patellar tendon rupture. Quadriceps autograft has increased in popularity since it offers a thicker graft with more favorable tensile properties compared to BTB and hamstring autografts. The quadriceps autograft has nearly twice the cross sectional area, higher load to failure and greater stiffness than the BTB autograft. Studies have shown equivalent outcomes when directly comparing BTB autograft vs. soft tissue quadriceps autograft in the general population. No studies have directly compared these two grafts in athletes participating in cutting and pivoting sports. We hypothesized that the quadriceps autograft would lead to similar patient outcomes, re-tear rates, return to sport and complications as BTB autografts in the cutting and pivoting athlete. Methods: A retrospective review of cutting and pivoting athletes with ACL tears treated with BTB autograft or soft tissue quadriceps autograft with at least 2 years of follow up was performed. Only athletes participating in cutting and pivoting sports were included in the study. Four sports were considered to be cutting and pivoting including soccer, football, lacrosse and basketball. The decision on which graft to use was based on the athlete’s choice after discussing the pros and cons of each graft. Exclusion criteria included those athletes with recurrent ACL tears, multiligamentous injuries, previous meniscal surgery and those requiring osteotomies. International Knee Documentation Committee Subjective Knee Evaluation (IKDC) and Lysholm knee scoring scale were used to evaluate patient reported outcomes. Through chart review, ability to return to sport, time to return to sport and complications were identified. The postoperative rehabilitation protocol for both grafts was identical. Results: There were 32 athletes in the soft tissue quadriceps autograft group and 36 in the BTB autograft group. The average age was 18.6 years for the quad group and 19.7 years for the BTB autograft group (p=0.63). Females made up 62.5% of the quadriceps group and 44.4% of the BTB group (p=0.14). The quad group was made up of 56.3% high school and 43.7% college athletes compared to 61.1% high school and 38.9% college athletes in the BTB autograft group (p=0.53). Soccer was the most common sport with 16 in the quad group and 14 in the BTB group. The rest of the quad group included 8 football players, 4 lacrosse and 4 basketball players. The remaining BTB group consisted of 12 football players, 8 lacrosse and 2 basketball players. Meniscal surgery was performed in combination with the ACL reconstruction in 17 (53.1%) of the quad group and 22 (61.1%) of the BTB autograft group (p=0.37). Average graft size was 9.5mm for the quad autograft group and 10 for the BTB autograft group. The 2-year IKDC score was 93.6 for the quad group and 95.1 for the BTB group (p=0.45). The 2 year Lysholm scores were 95.7 and 96.1 for the quad and BTB groups respectively. Return to play at the same or higher level was 90.6% in the quad group and 86.1% in the BTB autograft group (p=0.82). Time to return was also similar between the groups with 7.1 months for the quad group and 7.6 months for the BTB autograft group. There was 1 re-tear which required revision in the BTB group and no re-tears in the quad group (p=0.34). Arthrofibrosis requiring MUA and lysis of adhesions occurred in 2 quad autografts and 4 BTB autografts (p=0.49). One contralateral ACL rupture occurred in the quad autograft group and 4 in the BTB autograft group (p=0.21). Conclusions: The optimal ACL graft in high level athletes participating in cutting and pivoting sports remains in question. In our comparison of quadriceps autograft compared to BTB autograft in this athletic population, no difference in patient reported outcomes, return to sport or re-tear rates was identified. Based on these findings, quadriceps autograft is as effective as BTB autografts in cutting and pivoting athletes and should be part of the graft choice discussion with the athlete.

AbstractBackgroundHeterozygote carriers of potentially pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have increased asthma risk. However, the frequency and impact of CFTR variation among individuals with asthma is unknown.ObjectiveTo determine whether potentially pathogenic CFTR variants associate with disease severity and whether individuals with two potentially pathogenic variants exist in a severe asthma‐enriched cohort.MethodsWe analyzed sequencing data spanning a 190.5Kb region of CFTR in participants from the Severe Asthma Research Program (SARP1‐3). Potentially pathogenic, rare CFTR variants (frequency < 0.05) were classified as CF‐causing or of varying clinical consequences (VVCC) (CFTR2. org). Regression‐based models tested for association between CFTR genotypes (0‐2 potentially pathogenic variants) and severity outcomes.ResultsOf 1401 participants, 9.5% (134) had one potentially pathogenic variant, occurring more frequently in non‐Hispanic white (NHW, 10.1% [84 of 831]) compared to African American individuals (AA, 5.2% [22 of 426]). We found ≥2 potentially pathogenic CFTR variants in 1.4% (19); 0.5% (4) of NHW and 2.8% (12) of AA. Potentially pathogenic CFTR variant genotypes (≥1 or ≥2 variants) were not cumulatively associated with lung function or exacerbations. In NHW, we found three F508del compound heterozygotes with F508del and a VVCC (two 5 T; TG12[c.1210‐11 T > G] and one Arg1070Trp) and a homozygote for the VVCC, 5 T; TG12.ConclusionsWe found potentially pathogenic CFTR variants within a severe asthma‐enriched cohort, including three compound heterozygote genotypes variably associated with CF in NHW individuals. These findings provide the rationale for CFTR sequencing and phenotyping of CF‐related traits in individuals with severe asthma.