Genome Dynamics and Temperature Adaptation During Experimental Evolution of Obligate Intracellular Bacteria.

Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt. Yet there is a paucity of such research focusing on strictly host-associated bacteria, even though they are widespread in nature. Here, we used the Acanthamoeba symbiont Protochlamydia amoebophila, a distant relative of the human pathogen Chlamydia trachomatis and representative of a large group of protist-associated environmental chlamydiae, as a model to study how obligate intracellular symbionts evolve and adapt to elevated temperature, a prerequisite for the pivotal evolutionary leap from protist to endothermic animal hosts. We established 12 replicate populations under two temperatures (20 °C, 30 °C) for 510 bacterial generations (38 months). We then used infectivity assays and pooled whole-genome resequencing to identify any evolved phenotypes and the molecular basis of adaptation in these bacteria. We observed an overall reduction in infectivity of the symbionts evolved at 30 °C, and we identified numerous nonsynonymous mutations and small indels in these symbiont populations, with several variants persisting throughout multiple time points and reaching high frequencies. This suggests that many mutations may have been beneficial and played an adaptive role. Mutated genes within the same temperature regime were more similar than those between temperature regimes. Our results provide insights into the molecular evolution of intracellular bacteria under the constraints of strict host dependance and highly structured populations and suggest that for chlamydial symbionts of protists, temperature adaptation was facilitated through attenuation of symbiont infectivity as a tradeoff to reduce host cell burden.

Micronutritional supplementation with a holoBLG-based FSMP (food for special medical purposes)-lozenge alleviates allergic symptoms in BALB/c mice: Imitating the protective farm effect.

BACKGROUND: Previously, the protective farm effect was imitated using the whey protein beta-lactoglobulin (BLG) that is spiked with iron-flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin-iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model. METHODS: Binding of iron-catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic or tolerating milk was assessed to loaded (holo-) versus empty (apo-) BLG and for human mast cell degranulation. BLG and Bet v 1 double-sensitized mice were orally treated with the holoBLG or placebo lozenge, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with apoBLG or holoBLG using catechin-iron complexes as ligands. RESULTS: One major IgE and T cell epitope were masked by catechin-iron complexes, which impaired IgE binding of milk-allergic children and degranulation of mast cells. In mice, only supplementation with the holoBLG lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. CONCLUSION: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen-non-specific manner, thereby conferring immune resilience against allergic symptoms.

Binding to Iron Quercetin Complexes Increases the Antioxidant Capacity of the Major Birch Pollen Allergen Bet v 1 and Reduces Its Allergenicity.

Bet v 1 is the major allergen in birch pollen to which up to 95% of patients sensitized to birch respond. As a member of the pathogenesis-related PR 10 family, its natural function is implicated in plant defense, with a member of the PR10 family being reported to be upregulated under iron deficiency. As such, we assessed the function of Bet v 1 to sequester iron and its immunomodulatory properties on human immune cells. Binding of Bet v 1 to iron quercetin complexes FeQ2 was determined in docking calculations and by spectroscopy. Serum IgE-binding to Bet v 1 with (holoBet v1) and without ligands (apoBet v 1) were assessed by ELISA, blocking experiments and Western Blot. Crosslinking-capacity of apo/holoBet v 1 were assessed on human mast cells and Arylhydrocarbon receptor (AhR) activation with the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for labile iron and phenotypic changes by flow cytometry. Bet v 1 bound to FeQ2 strongly with calculated Kd values of 1 nm surpassing affinities to quercetin alone nearly by a factor of 1000. Binding to FeQ2 masked IgE epitopes and decreased IgE binding up to 80% and impaired degranulation of sensitized human mast cells. Bet v 1 facilitated the shuttling of quercetin, which activated the anti-inflammatory AhR pathway and increased the labile iron pool of human monocytic cells. The increase of labile iron was associated with an anti-inflammatory phenotype in CD14+monocytes and downregulation of HLADR. To summarize, we reveal for the first time that FeQ2 binding reduces the allergenicity of Bet v 1 due to ligand masking, but also actively contributes anti-inflammatory stimuli to human monocytes, thereby fostering tolerance. Nourishing immune cells with complex iron may thus represent a promising antigen-independent immunotherapeutic approach to improve efficacy in allergen immunotherapy.

Cow's milk protein ß-lactoglobulin confers resilience against allergy by targeting complexed iron into immune cells.

BACKGROUND: Beta-lactoglobulin (BLG) is a bovine lipocalin in milk with an innate defense function. The circumstances under which BLG is associated with tolerance of or allergy to milk are not understood. OBJECTIVE: Our aims were to assess the capacity of ligand-free apoBLG versus loaded BLG (holoBLG) to protect mice against allergy by using an iron-quercetin complex as an exemplary ligand and to study the molecular mechanisms of this protection. METHODS: Binding of iron-quercetin to BLG was modeled and confirmed by spectroscopy and docking calculations. Serum IgE binding to apoBLG and holoBLG in children allergic to milk and children tolerant of milk was assessed. Mice were intranasally treated with apoBLG versus holoBLG and analyzed immunologically after systemic challenge. Aryl hydrocarbon receptor (AhR) activation was evaluated with reporter cells and Cyp1A1 expression. Treated human PBMCs and human mast cells were assessed by fluorescence-activated cell sorting and degranulation, respectively. RESULTS: Modeling predicted masking of major IgE and T-cell epitopes of BLG by ligand binding. In line with this modeling, IgE binding in children allergic to milk was reduced toward holoBLG, which also impaired degranulation of mast cells. In mice, only treatments with holoBLG prevented allergic sensitization and anaphylaxis, while sustaining regulatory T cells. BLG facilitated quercetin-dependent AhR activation and, downstream of AhR, lung Cyp1A1 expression. HoloBLG shuttled iron into monocytic cells and impaired their antigen presentation. CONCLUSION: The cargo of holoBLG is decisive in preventing allergy in vivo. BLG without cargo acted as an allergen in vivo and further primed human mast cells for degranulation in an antigen-independent fashion. Our data provide a mechanistic explanation why the same proteins can act either as tolerogens or as allergens.

Facing mortality: a qualitative in-depth interview study on illness perception, lay theories and coping strategies of adult patients with acute leukemia 1 week after diagnosis.

In patients with acute leukemia we investigated the illness perceptions, lay theories and coping strategies 1 week after diagnosis. Semi-structured in-depth interviews were conducted with 12 patients. The transcribed interviews were analyzed by methods of qualitative research. Dramatic narrations of overwhelming threat in younger patients contrast to rationalization in elderly patients. Feelings of helplessness and efforts of normalization become apparent. Intense descriptions of physical injury due to invasive procedures allow verbalizing the fear of the disease. While coping strategies are complex, the overall importance of trust is recognized. Mortal fears are indirectly indicated. The results have consequences for psycho-oncological training and patient education.

[Illness perceptions of patients suffering from acute leukaemia one week after diagnosis]. / Subjektive Krankheitsvorstellungen bei Patienten mit akuter Leukämie eine Woche nach Diagnostellung.

OBJECTIVES: To investigate illness perceptions, treatment expectations, and treatment experiences of patients suffering from acute leukaemia in the initial stage of their disease. METHODS: In the first week of treatment we interviewed twelve patients with acute leukaemia using a detailed semi-structured interview guide. To investigate the transcribed interviews we applied methods of qualitative research. Case analyses were assigned to interindividual comparison tables representing the following subject areas: complaints, diagnostics, causes, controllability, treatment experiences, and prognosis. This allowed us to describe similarities and contrasts. RESULTS: Dramatic narrations of overwhelming threat in younger patients are in contrast to downplaying, rationalizing, and factual descriptions especially in elderly patients. Feelings of helplessness and efforts of normalization become apparent. Experiences of visible and sensible physical injury are perceived as threatening. Prognosis is estimated as too positive. Mortal fears are only indirectly indicated. CONCLUSIONS: Our results have practical consequences for both patient education and psycho-oncological training.