Efficacy of T cell assays for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis.

Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder associated with autosomal recessive variants in genes required for perforin-mediated lymphocyte cytotoxicity. A rapid diagnosis is crucial for successful treatment. Although defective cytotoxic T lymphocyte (CTL) function causes pathogenesis, quantification of natural killer (NK) cell exocytosis triggered by K562 target cells currently represents a standard diagnostic procedure for primary HLH. We have prospectively evaluated different lymphocyte exocytosis assays in 213 patients referred for evaluation for suspected HLH and related hyperinflammatory syndromes. A total of 138 patients received a molecular diagnosis consistent with primary HLH. Compared to routine K562 cell-based assays, assessment of Fc receptor-triggered NK-cell and T cell receptor-triggered CTL exocytosis displayed higher sensitivity and improved specificity for the diagnosis of primary HLH, with these assays combined providing a sensitivity of 100% and specificity of 98.3%. By comparison, NK-cell exocytosis following K562 target cell stimulation displayed a higher inter-individual variability, in part explained by differences in NK-cell differentiation or large functional reductions following shipment. We thus recommend combined analysis of T cell receptor-triggered CTL and Fc receptor-triggered NK-cell exocytosis for the diagnosis of patients with suspected familial HLH or atypical manifestations of congenital defects in lymphocyte exocytosis.

The impact of metabolic syndrome in breast reconstruction decision-making and postoperative outcomes: A nationwide analysis.

BACKGROUND: Metabolic syndrome (MetS) is a cluster of cardiometabolic abnormalities including hypertension, obesity, insulin resistance, and dyslipidemia. The safety profiles of patients with MetS undergoing breast reconstruction remain underreported. This study aims to evaluate the impact of MetS on the BR decision-making process and postoperative complication rates. METHODS: The ACS-NSQIP database was utilized to identify women who underwent BR between 2012 and 2021. Baseline characteristics were compared based on the presence of MetS, defined as patients receiving medical treatment for diabetes mellitus and hypertension, with a body mass index greater than 30 kg/m2. Group differences were assessed using t tests and Fisher's exact tests. Multivariate logistic regression models evaluated postoperative complications between the groups. RESULTS: A total of 160,115 patients underwent BR. A total of 4570 had a diagnosis of MetS compared to 155,545 without MetS. No statistically significant differences were observed in the type of BR patients received across cohorts. Logistic regression models demonstrated a higher likelihood of postoperative wound complications (OR 2.21; 95% CI 1.399, 3.478; p = 0.001), and readmission rates (OR 2.045; 95% CI 1.337, 3.128; p = 0.001) in the MetS group compared to the non-MetS patients. No significant differences were identified in other postoperative complications between groups. CONCLUSIONS: Patients with MetS appear to have an increased risk of postoperative wound complications and readmission after breast reconstruction. The synergistic effects of these comorbidities on postoperative outcomes underscore the importance of addressing MetS as a holistic condition and considering choosing Delayed breast reconstruction over Immediate Breast Reconstruction in this population. Thus, integrating MetS management and patient counseling at various stages of BR may improve outcomes and facilitate patient decision-making.

Knockdown of chimeric glucocerebrosidase by green fluorescent protein-directed small interfering RNA

Gaucher disease, the most common type of lysosomal storage disorder, is characterized by an inherited deficiency of the membrane-associated hydrolase, glucocerebrosidase. Glucocerebrosidase catalyzes the hydrolysis of glucocerebroside to ceramide and glucose, a crucial step in the recycling of membrane sphingolipids. The exorbitant cost of the current treatment standard for Gaucher disease, enzyme replacement therapy, prevents many from receiving treatment. This limitation has led to a wide-spread search for more efficient and cost-effective methods of protein production and alternate therapies, resulting in a closer examination of glucocerebrosidase biosynthesis and current treatment techniques. The use of specific small interfering RNAs (siRNAs) to knock down target genes is an attractive option for studying such processes, though a glucocerebrosidase-specific siRNA has yet to be reported. We note, however, that green fluorescent protein (GFP)-directed siRNAs can not only provide a positive control to test siRNA delivery and system integrity, but also serve as a means to knock down a fusion partner without having to design siRNAs specific to the partner. After effectively co-transfecting COS-1 cells with enhanced GFP (EGFP)-tagged glucocerebrosidase constructs and GFP-directed siRNAs, we report successful knockdown of all EGFP-containing constructs at both the RNA and protein levels. This provides a method of examining enzyme biosynthesis and treatment options. Furthermore, this technique is applicable to other systems, since we have demonstrated the usefulness of GFP as a siRNA target in mammalian cells when fused to another gene of interest.