Mean Pneumocystis burden, as determined using the probe method, was 10,119 (1–299,697; median 120) GpA/MSG copies/ng human DNA. Pneumocystis burden determinations using SYBR Green method, reported in a previous study on these same samples [2], click here were concordant with the probe method determinations in this study. Analysis of protein extracts documented a significant

increase in normalized expression levels of hCLCA1 in Pneumocystis-positive samples compared to Pneumocystis-negative samples (P=0.0280) ( Fig. 1), suggesting that Pneumocystis is associated with airway epithelium stimulation including up-regulation of mucus-related responses. The contribution of Pneumocystis burden to the expression of hCLCA1, as analyzed by correlation protein expression

graphics, detected a significant positive correlation between increasing levels of hCLCA1 and Pneumocystis burden suggesting induction by Pneumocystis (Spearman r=0.3479; P=0.0171) ( Fig. 2). Common respiratory viruses were studied in Pneumocystis-positive and Pneumocystis-negative samples to assess their contribution to hCLCA1 expression levels. Respiratory Syncytial Virus was diagnosed Protease Inhibitor Library in three and Adenovirus in one of the Pneumocystis-positive samples. No viruses were detected in the Pneumocystis-negative samples ( Fig. 3). Protein expression levels of hCLCA1 were no different in virus-positive compared to virus-negative samples indicating isothipendyl that, in these samples, viruses do not explain the Pneumocystis-associated increased levels of this protein. Moreover, virus positive samples were grouped for this comparative analysis, and no significant difference in hCLCA1 expression was detected between virus-positive and virus-negative samples (P=0.7648) ( Fig. 3). The increased hCLCA1 protein levels associated with Pneumocystis in infant lungs documented in

this study provide additional evidence that Pneumocystis infection is associated with stimulation of the respiratory epithelium mucus-secretion-system in non-immunocompromised humans ( Fig. 1). Furthermore, all infants in this study were mostly asymptomatic prior to death revealing the mild nature of this Pneumocystis infection, which is consistent with a state of symptomless “colonization”. An association between increased levels of the goblet cell mucin MUC5AC and Pneumocystis was reported in a previous study [2], and the association between hCLCA1 and MUC5AC, which is well-documented in animal models, has been recently reported in patients diagnosed with COPD [21]. Results also document that an increasing burden of Pneumocystis is correlated with increasing levels of hCLCA1 protein expression, suggesting a Pneumocystis-related, stimulatory effect on hCLCA1 induction ( Fig. 2) [6] and [8]. Overexpression of hCLCA1 may explain the increase in mucus proteins, such as MUC5AC, associated with Pneumocystis through stimulation of the STAT6–hCLCA1–MUC5AC proposed pathway [7].