Background: Pandemic type A (H1N1) influenza arose in early 2009, probably in Mexico and the United States, and reappeared in North America in September for seven more months. An amino acid substitution in the hemagglutinin (HA), D222G, has been reported in a significant proportion of patients with a severe and fatal outcome. We studied the prevalence of HA222 substitutions in patients in Mexico during the second wave and its association with clinical outcome and pathogenicity in a mouse model. Methods: The nucleotide sequences of hemagglutinin (HA) from viruses collected from 77 patients were determined including 50 severe and fatal cases and 27 ambulatory cases. Deep sequencing was done on 5 samples from severe or fatal cases in order to determine the quasispecies proportion. Weight loss and mortality due to infection with cultured influenza viruses were analyzed in a mouse model. Results: Viruses from 14 out of 50 hospitalized patients (28%) had a non aspartic acid residue at the HA 222 position (nD222), while all 27 ambulatory patients had D222 (p = 0.0014). G222 was detected as sole species or in coexistence with N222 and D222 in 12 patients with severe disease including 8 who died. N222 in coexistence with D222 was detected in 1 patient who died and co-occurrence of A222 and V222, together with D222, was detected in another patient who died. The patients with a nD222 residue had higher mortality (71.4%), compared to the group with only D222 (22.2%, p = 0.0008). Four of the 14 viruses from hospitalized patients were cultured and intranasally infected into mice. Two viruses with G222 were lethal while a third virus, with G222, caused only mild illness in mice similar to the fourth virus that contained D222. Conclusions: We confirm the elevated incidence of HA222 (H1N1)pdm09 variants in severe disease and mortality. Both clinical and mouse infection data support the idea that nD222 mutations contribute to increased severity of disease but additional determinants in disease outcome may be present.

Background: CD4⁺ T cell activation indicators have been reported to be a common phenomenon underlying diverse manifestations of immune reconstitution inflammatory syndrome (IRIS). However, we have found that a high frequency of circulating CD8⁺ T cells is a specific risk factor for mycobacterial IRIS. Therefore, we investigated whether CD8⁺ T cells from patients who develop TB IRIS were specifically activated. Methods: We obtained PBMCs from HIV+ patients prior to and 4, 8, 12, 24, 52 and 104 weeks after initiating antiretroviral therapy. CD38 and HLADR expression on naive, central memory and effector memory CD8⁺ and CD4⁺ T cells were determined by flow cytometry. Absolute counts and frequencies of CD8⁺ T cell subsets were compared between patients who developed TB IRIS, who developed other IRIS forms and who remained IRIS-free. Results: TB IRIS patients showed significantly higher counts of naive CD8⁺ T cells than the other groups at most time points, with a contraction of the effector memory subpopulation occurring later in the follow-up period. Activated (CD38⁺ HLADR⁺) CD8⁺ T cells from all groups decreased with treatment but transiently peaked in TB IRIS patients. This increase was due to an increase in activated naive CD8⁺ T cell counts during IRIS. Additionally, the CD8⁺ T cell subpopulations of TB IRIS patients expressed HLADR without CD38 more frequently and expressed CD38 without HLADR less frequently than cells from other groups. Conclusions: CD8⁺ T cell activation is specifically relevant to TB IRIS. Different IRIS forms may involve different alterations in T cell subsets, suggesting different underlying inflammatory processes.

How T cells integrate environmental cues into signals that limit the magnitude and length of immune responses is poorly understood. Here, we provide data that demonstrate that B55β, a regulatory subunit of protein phosphatase 2A, represents a molecular link between cytokine concentration and apoptosis in activated CD8⁺ T cells. Through the modulation of AKT, B55β induced the expression of the proapoptotic molecule Hrk in response to cytokine withdrawal. Accordingly, B55β and Hrk were both required for in vivo and in vitro contraction of activated CD8⁺ lymphocytes. We show that this process plays a role during clonal contraction, establishment of immune memory, and preservation of peripheral tolerance. This regulatory pathway may represent an unexplored opportunity to end unwanted immune responses or to promote immune memory.

Immune reconstitution inflammatory syndrome (IRIS) is an exacerbated immune response that can occur to HIV+ patients after initiating antiretroviral therapy (ART). IRIS pathogenesis is unclear, but dysfunctional and exhausted cells have been reported in IRIS patients, and the TIM-3/Gal-9 axis has been associated with chronic phases of viral infection. This study aimed to evaluate the soluble levels of TIM-3 and Gal-9 and their relationship with IRIS development. TIM-3, Gal-9, TNF-α, IFN-γ, IL-6, TNFR1, TNFR2, E-cadherin, ADAM10, and ADAM17 were measured to search for IRIS-associated biomarkers in plasma samples from 0-, 4-, 8-, 12-, and 24-weeks after ART initiation of 61 HIV+ patients (15 patients developed IRIS, and 46 did not). We found that patients who developed IRIS had higher levels of TIM-3 [median 4806, IQR: 3206–6182] at the time of the IRIS events, compared to any other follow-up time evaluated in these patients or compared with a control group of patients who did not develop IRIS. Similarly, IRIS patients had a higher TNF-α level [median 10.89, IQR: 8.36–12.34] at IRIS events than any other follow-up time evaluated. Other molecules related to the TIM-3 and TNF-α pathway (Gal-9, IL-6, IFN-γ, TNFR1, TNFR2, ADAM-10, and ADAM-17) did not change during the IRIS events. In conclusion, our data suggest that a high level of soluble TIM-3 and TNF-α could be used as an IRIS biomarker.

Idiopathic pulmonary fibrosis (IPF) is a chronic, deathly disease with no recognized effective cure as yet. Furthermore, its diagnosis and differentiation from other diffuse interstitial diseases remain a challenge. Circulating miRNAs have been measured in IPF and have proven to be an adequate option as biomarkers for this disease. These miRNAs, released into the circulation outside the cell through exosomes and proteins, play a crucial role in the pathogenic pathways and mechanisms involved in IPF development. This review focuses on the serum/plasma miRNAs reported in IPF that have been validated by real-time PCR and the published evidence regarding the fibrotic process. First, we describe the mechanisms by which miRNAs travel through the circulation (contained in exosomes and bound to proteins), as well as the mechanism by which miRNAs perform their function within the cell. Subsequently, we summarize the evidence concerning miRNAs reported in serum/plasma, where we find contradictory functions in some miRNAs (dual functions in IPF) when comparing the findings in vitro vs. in vivo. The most relevant finding, for instance, the levels of miRNAs let-7d and miR-21 reported in the serum/plasma in IPF, correspond to those found in studies in lung fibroblasts and the murine bleomycin model, reinforcing the usefulness of these miRNAs as future biomarkers in IPF.

Idiopathic pulmonary fibrosis, a chronic and fatal condition with no effective cure, is a challenging disease within the group of interstitial lung diseases. The pathogenic mechanisms of this disease are still not fully understood. In this context, the research on miRNAs, which are small non-coding RNAs that regulate messenger RNAs, is of utmost importance. These miRNAs, released into the circulation outside the cell through exosomes, play a crucial role in the pathogenic pathways and mechanisms involved in IPF development. The analysis of miRNAs in the serum/plasma of IPF patients has opened up new possibilities in the search for biomarkers. This review focuses on circulating miRNAs validated by real-time PCR in IPF and the evidence reported about the pulmonary fibrotic process.