for the Prevention of Severe Pneumonia and Endotracheal Colonization Trial (PROSPECT) Investigators and the Canadian Critical Care Trials Group IMPORTANCE Growing interest in microbial dysbiosis during critical illness has raised questions about the therapeutic potential of microbiome modification with probiotics. Prior randomized trials in this population suggest that probiotics reduce infection, particularly ventilator-associated pneumonia (VAP), although probiotic-associated infections have also been reported.OBJECTIVE To evaluate the effect of Lactobacillus rhamnosus GG on preventing VAP, additional infections, and other clinically important outcomes in the intensive care unit (ICU). DESIGN, SETTING, AND PARTICIPANTS Randomized placebo-controlled trial in 44 ICUs in Canada, the United States, and Saudi Arabia enrolling adults predicted to require mechanical ventilation for at least 72 hours. A total of 2653 patients were enrolled from October 2013 to March 2019 (final follow-up, October 2020). INTERVENTIONS Enteral L rhamnosus GG (1 × 10 10 colony-forming units) (n = 1321) or placebo (n = 1332) twice daily in the ICU. MAIN OUTCOMES AND MEASURES The primary outcome was VAP determined by duplicate blinded central adjudication. Secondary outcomes were other ICU-acquired infections including Clostridioides difficile infection, diarrhea, antimicrobial use, ICU and hospital length of stay, and mortality. RESULTS Among 2653 randomized patients (mean age, 59.8 years [SD], 16.5 years), 2650 (99.9%) completed the trial (mean age, 59.8 years [SD], 16.5 years; 1063 women [40.1%.]with a mean Acute Physiology and Chronic Health Evaluation II score of 22.0 (SD, 7.8) and received the study product for a median of 9 days (IQR, 5-15 days). VAP developed among 289 of 1318 patients (21.9%) receiving probiotics vs 284 of 1332 controls (21.3%; hazard ratio [HR], 1.03 (95% CI, 0.87-1.22; P = .73, absolute difference, 0.6%, 95% CI, -2.5% to 3.7%). None of the 20 prespecified secondary outcomes, including other ICU-acquired infections, diarrhea, antimicrobial use, mortality, or length of stay showed a significant difference. Fifteen patients (1.1%) receiving probiotics vs 1 (0.1%) in the control group experienced the adverse event of L rhamnosus in a sterile site or the sole or predominant organism in a nonsterile site (odds ratio, 14.02; 95% CI, 1.79-109.58; P < .001).CONCLUSIONS AND RELEVANCE Among critically ill patients requiring mechanical ventilation, administration of the probiotic L rhamnosus GG compared with placebo, resulted in no significant difference in the development of ventilator-associated pneumonia. These findings do not support the use of L rhamnosus GG in critically ill patients.

Pre-TCR complexes are thought to signal in a ligand-independent manner because they are constitutively targeted to lipid rafts. We report that ligand-independent signaling is not a unique capability of the pre-TCR complex. Indeed, the TCRα subunit restores development of pTα-deficient thymocytes to the CD4+CD8+ stage even in the absence of conventional MHC class I and class II ligands. Moreover, we found that pre-TCR and αβTCR complexes exhibit no appreciable difference in their association with lipid rafts, suggesting that ligand-independence is a function of the CD4−CD8− (DN) thymocytes in which pre-TCR signaling occurs. In agreement, we found that only CD44−CD25+ DN thymocytes (DN3) enabled activation of extracellular signal-regulated kinases by the pre-TCR complex. DN thymocytes also exhibited a lower signaling threshold relative to CD4+CD8+ thymocytes, which was associated with both the markedly elevated lipid raft content of their plasma membranes and more robust capacitative Ca2+ entry. Taken together these data suggest that cell-autonomous, ligand-independent signaling is primarily a property of the thymocytes in which pre-TCR signaling occurs.

The first checkpoint in T cell development occurs between the CD4−CD8− and CD4+CD8+ stages and is associated with formation of the pre-T cell receptor (TCR). The signaling mechanisms that drive this progression remain largely unknown. Here, we show that extracellular signal–regulated kinases (ERKs)-1/2 are activated upon engagement of the pre-TCR. Using a novel experimental system, we demonstrate that expression of the pre-TCR by developing thymocytes induces ERK-1/2 activation within the thymus. In addition, the activation of this pre-TCR signaling cascade is mediated through Lck. These findings directly link pre-TCR complex formation with specific downstream signaling components in vivo.