Laparoscopic Uterine Preservation and Vaginal Reconstruction in a 13-Year-Old Girl with Uterus Didelphys, Bilateral Vesicovaginal Fistulas, and Transverse Vaginal Septum.

BACKGROUND: Complex female genital tract malformations account for 1.2% of all female genitourinary malformations. Although exceedingly rare, they can cause severe gynecologic symptoms in young women and lead to fertility problems. CASE: We present the case of a 13-year-old girl with primary amenorrhea referred for cyclic abdominal lower pain and menouria. Detailed diagnostics revealed uterus didelphys, transverse vaginal septum, and bilateral vesicovaginal fistulas. Laparoscopic left hemi-hysterectomy and salpingectomy were performed. The vesicovaginal fistula on the right side was excised, and the proximal vagina was anastomosed with the distal dimple. Since the operation, the patient has been pain-free and menstruating regularly from the right uterus. SUMMARY AND CONCLUSION: Preservation of the uterus should be considered in any case of complex female genital tract malformation and, as successful laparoscopic treatment advocates, a minimally invasive approach is feasible.

Modeling Asthma in Mice Using Common Aeroallergens.

Aeroallergens are common inducers of asthma in humans and are widely used in experimental research to generate animal models of this disease. In this chapter, we describe four mouse models of aeroallergen-induced asthma. These models differ in type and number of allergens used, route and duration of allergen exposure, and utilization of an adjuvant, representing different mechanistic variants of asthma. In addition, we describe several basic methods that are commonly used in mechanistic studies of asthma in mice. These methods include tracheotomy and bronchoalveolar lavage, cytospin and morphologic analysis of bronchoalveolar lavage cells, and lung harvest and digestion for generation of single-cell suspension.

Association of Appendicular Skeletal Muscle to Trunk Fat Ratio with Type 2 Diabetes Mellitus in Older Adults.

INTRODUCTION: Mounting evidence has demonstrated that skeletal muscle and visceral adiposity play crucial roles in glucose metabolism. The purpose of this study was to investigate whether the appendicular skeletal muscle mass index (ASMI) to trunk fat mass (TFM) ratio (ASMI/TFM) is a more specific and identifiable factor for type 2 diabetes mellitus (T2DM) in older adults than conventional anthropometric measures. METHODS: This cross-sectional study included 1,370 older adults from the Tianjin Chronic Low-Grade Systemic Inflammation and Health (TCLSIH) cohort. ASMI and TFM were measured by using a bioelectrical impedance analyzer, and T2DM was defined with the criteria of the American Diabetes Association. Odds ratios (ORs) were evaluated using multivariable logistic analysis. RESULTS: The prevalence of T2DM is 20.0% in this study. The multivariable-adjusted ORs (95% confidence interval) of T2DM for increasing categories of ASMI/TFM, BMI, and waist circumference (WC) were 1.00 (reference), 0.70 (0.49, 1.02), 0.61 (0.42, 0.89), and 0.45 (0.30, 0.67; p for trend <0.0001); 1.00 (reference), 1.15 (0.83, 1.60), and 1.37 (0.94, 2.01; p for trend = 0.10); and 1.00 (reference) and 1.78 (1.19, 2.74; p < 0.01), respectively. CONCLUSIONS: Higher ASMI/TFM was associated with a lower prevalence of T2DM in this study of older adults. The T2DM predictive value of ASMI/TFM may be stronger than BMI and WC in this population.

Relationship Between Dietary Patterns and Carotid Atherosclerosis Among People Aged 50 Years or Older: A Population-Based Study in China.

Background: The relationship between dietary patterns and atherosclerosis is inconclusive. Usually, diets vary greatly among different regions due to cultural differences and lifestyles. Few studies to date based on a Chinese population have investigated the relationship between dietary patterns and the formation of atherosclerosis in carotid arteries. We aimed to investigate whether dietary patterns were related to carotid atherosclerosis among an adult population in Tianjin, China. Methods: This cross-sectional study included a total of 2,346 participants aged 50 years or older (mean: 59.7 ± 6.29 years). Dietary intakes were assessed using a validated 81-item semiquantitative food frequency questionnaire, and factor analysis was used to identify dietary patterns. Carotid atherosclerosis was defined as a common carotid artery intima-media thickness ≥1.0 mm or plaques, or a carotid bifurcation intima-media thickness ≥1.2 mm. Multiple logistic regression models were used to explore the relationship between dietary patterns and carotid atherosclerosis. Results: Three factors were determined: "health" dietary pattern (factor 1), "traditional Tianjin" dietary pattern (factor 2), and "sweets" dietary pattern (factor 3). The multivariable-adjusted odds ratios (95% CI) of carotid atherosclerosis for the increasing quartiles of the sweets dietary pattern scores in women were as follows: 1.00 (reference), 1.33 (0.91, 1.97), 1.21 (0.82, 1.79), 1.64 (1.08, 2.51) (p for trend <0.05). No significant difference was found between any dietary pattern and carotid atherosclerosis in men. Conclusion: Greater adherence to "sweets" dietary patterns was positively related to a higher prevalence of carotid atherosclerosis in women aged 50 or older. No relationship was found between any dietary pattern and carotid atherosclerosis in men. Further prospective studies are warranted to test this finding in other populations.

Dietary patterns and risk of non-alcoholic fatty liver disease in adults: A prospective cohort study.

BACKGROUND AND AIMS: Prospective cohort studies linking dietary patterns and non-alcoholic fatty liver disease (NAFLD) are limited, especially in Asian populations. This study aimed to prospectively investigate the association between dietary patterns and risk of NAFLD in a general Chinese adult population. METHODS: This study included a total of 17,360 participants free from NAFLD at baseline. Dietary patterns at baseline were identified with factor analysis based on responses to a validated 100-item food frequency questionnaire. NAFLD was diagnosed by abdominal ultrasound after excluding other causes related to chronic liver disease. Cox proportional regression models were used to assess the association between dietary patterns and risk of NAFLD. RESULTS: During a median follow-up of 4.2 years, 4034 NAFLD cases were documented. Three main dietary patterns were extracted: sugar rich dietary pattern, vegetable rich dietary pattern, and animal food dietary pattern. After adjusting for age, sex, body mass index, smoking, alcohol, education, occupation, income, physical activity, total energy intake, personal and family history of disease, depressive symptoms, dietary supplement use, inflammation markers, and each other dietary pattern score, comparing the highest with the lowest quartiles of dietary pattern scores, the multivariable hazard ratios (95% confidence interval) of NAFLD were 1.11 (1.01, 1.23) for sugar rich dietary pattern, 0.96 (0.86, 1.07) for vegetable rich dietary pattern, and 1.22 (1.10, 1.36) for animal food dietary pattern. Further adjustment for waist circumference instead of body mass index provided similar results. CONCLUSION: Dietary patterns rich in animal foods or sugar were associated with a higher risk of NAFLD among Chinese adults, whereas a vegetable rich dietary pattern was not associated.

The molecular and epigenetic mechanisms of innate lymphoid cell (ILC) memory and its relevance for asthma.

Repetitive exposure of Rag1-/- mice to the Alternaria allergen extract generated a form of memory that elicited an asthma-like response upon a subthreshold recall challenge 3-15 wk later. This memory was associated with lung ICOS+ST2+ ILC2s. Genetic, pharmacologic, and antibody-mediated inhibition and adoptive transfer established an essential role for ILC2s in memory-driven asthma. ATAC-seq demonstrated a distinct epigenetic landscape of memory ILC2s and identified Bach2 and AP1 (JunD and Fosl2) motifs as major drivers of altered gene accessibility. scRNA-seq, gene knockout, and signaling studies suggest that repetitive allergenic stress induces a gene repression program involving Nr4a2, Zeb1, Bach2, and JunD and a preparedness program involving Fhl2, FosB, Stat6, Srebf2, and MPP7 in memory ILC2s. A mutually regulated balance between these two programs establishes and maintains memory. The preparedness program (e.g., Fhl2) can be activated with a subthreshold cognate stimulation, which down-regulates repressors and activates effector pathways to elicit the memory-driven phenotype.

Sprouty2 positively regulates T cell function and airway inflammation through regulation of CSK and LCK kinases.

The function of Sprouty2 (Spry2) in T cells is unknown. Using 2 different (inducible and T cell-targeted) knockout mouse strains, we found that Spry2 positively regulated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling by modulating the activity of LCK. Spry2-/- CD4+ T cells were unable to activate LCK, proliferate, differentiate into T helper cells, or produce cytokines. Spry2 deficiency abrogated type 2 inflammation and airway hyperreactivity in a murine model of asthma. Spry2 expression was higher in blood and airway CD4+ T cells from patients with asthma, and Spry2 knockdown impaired human T cell proliferation and cytokine production. Spry2 deficiency up-regulated the lipid raft protein caveolin-1, enhanced its interaction with CSK, and increased CSK interaction with LCK, culminating in augmented inhibitory phosphorylation of LCK. Knockdown of CSK or dislodgment of caveolin-1-bound CSK restored ERK1/2 activation in Spry2-/- T cells, suggesting an essential role for Spry2 in LCK activation and T cell function.

JNK pathway and heat shock response mediate the survival of C26 colon carcinoma bearing mice fed with the mushroom Pleurotus eryngii var. eryngii without affecting tumor growth or cachexia.

In the last few years, there has been emerging interest in developing treatments against human diseases using natural bioactive content. Here, the powder of the edible mushroom Pleurotus eryngii var. eryngii was mixed with the normal diet of mice bearing C26 colon carcinoma. Interestingly, it was evidenced by a significant increase in the survival rate of C26 tumor-bearing mice accompanied by a significant increase in Hsp90 and Hsp27 protein levels in the tumors. These data were paralleled by a decrease in Hsp60 levels. The mushroom introduced in the diet induced the inhibition of the transcription of the pro-inflammatory cytokines IL-6 and IL-1 exerting an anti-inflammatory action. The effects of the mushroom were mediated by the activation of c-Jun NH2-terminal kinases as a result of metabolic stress induced by the micronutrients introduced in the diet. In the tumors of C26 bearing mice fed with Pleurotus eryngii there was also a decreased expression of the mitotic regulator survivin and the anti-apoptotic factor Bcl-xL as well as an increase in the expression levels of Atg7, a protein that drives autophagy. In our hypothesis the interplay of these molecules favored the survival of the mice fed with the mushroom. These data are promising for the introduction of Pleurotus eryngii as a dietary supplement or as an adjuvant in anti-cancer therapy.

Effect of the freeze-drying process on the phenotypic diversity of Pseudomonas putida strains isolated from the interior of healthy roots of Sida hermaphrodita: Phenotype microarrays (PMs).

The objective of this study was to research the effect of the freeze-drying process on the metabolic changes of Pseudomonas putida strains (E41, E42, R85) isolated from the interior of Sida hermaphrodita roots with the use of the phenotypic microarrays (PM) technology. The proposed method of the freeze-drying process with inulin as component lycoprotectant demonstrated a high bacterial survival ratio (BSR) immediately after freeze-drying and storage after 12 months. While, after 360 days of freeze-drying BSR decreased to value of 74.38. Pseudomonas putida strains were assayed on microplates PM1-PM5, and PM9-PM13 testing 664 different substrates. However, no significant differences in the use of C substrates were observed either before or after the freeze drying process. An insignificant negative effect of the freeze-drying on the use of these substrates was observed. The utilization of N, P and S sources was low or showed no metabolic activity for most of the compounds after freeze-drying. The freeze-drying process increased the sensitivity of the bacteria to antibiotics and selected chemicals. In this study, the freeze-drying process decreased the metabolic activities of the tested strains and their resistance to antibiotics and chemicals.

Hsp60 Post-translational Modifications: Functional and Pathological Consequences.

Hsp60 is a chaperone belonging to the Chaperonins of Group I and typically functions inside mitochondria in which, together with the co-chaperonin Hsp10, maintains protein homeostasis. In addition to this canonical role, Hsp60 plays many others beyond the mitochondria, for instance in the cytosol, plasma-cell membrane, extracellular space, and body fluids. These non-canonical functions include participation in inflammation, autoimmunity, carcinogenesis, cell replication, and other cellular events in health and disease. Thus, Hsp60 is a multifaceted molecule with a wide range of cellular and tissue locations and functions, which is noteworthy because there is only one hsp60 gene. The question is by what mechanism this protein can become multifaceted. Likely, one factor contributing to this diversity is post-translational modification (PTM). The amino acid sequence of Hsp60 contains many potential phosphorylation sites, and other PTMs are possible such as O-GlcNAcylation, nitration, acetylation, S-nitrosylation, citrullination, oxidation, and ubiquitination. The effect of some of these PTMs on Hsp60 functions have been examined, for instance phosphorylation has been implicated in sperm capacitation, docking of H2B and microtubule-associated proteins, mitochondrial dysfunction, tumor invasiveness, and delay or facilitation of apoptosis. Nitration was found to affect the stability of the mitochondrial permeability transition pore, to inhibit folding ability, and to perturb insulin secretion. Hyperacetylation was associated with mitochondrial failure; S-nitrosylation has an impact on mitochondrial stability and endothelial integrity; citrullination can be pro-apoptotic; oxidation has a role in the response to cellular injury and in cell migration; and ubiquitination regulates interaction with the ubiquitin-proteasome system. Future research ought to determine which PTM causes which variations in the Hsp60 molecular properties and functions, and which of them are pathogenic, causing chaperonopathies. This is an important topic considering the number of acquired Hsp60 chaperonopathies already cataloged, many of which are serious diseases without efficacious treatment.

Maternal diesel particle exposure promotes offspring asthma through NK cell-derived granzyme B.

Mothers living near high-traffic roads before or during pregnancy are more likely to have children with asthma. Mechanisms are unknown. Using a mouse model, here we showed that maternal exposure to diesel exhaust particles (DEP) predisposed offspring to allergic airway disease (AAD, murine counterpart of human asthma) through programming of their NK cells; predisposition to AAD did not develop in DEP pups that lacked NK cells and was induced in normal pups receiving NK cells from WT DEP pups. DEP NK cells expressed GATA3 and cosecreted IL-13 and the killer protease granzyme B in response to allergen challenge. Extracellular granzyme B did not kill, but instead stimulated protease-activated receptor 2 (PAR2) to cooperate with IL-13 in the induction of IL-25 in airway epithelial cells. Through loss-of-function and reconstitution experiments in pups, we showed that NK cells and granzyme B were required for IL-25 induction and activation of the type 2 immune response and that IL-25 mediated NK cell effects on type 2 response and AAD. Finally, experiments using human cord blood and airway epithelial cells suggested that DEP might induce an identical pathway in humans. Collectively, we describe an NK cell-dependent endotype of AAD that emerged in early life as a result of maternal exposure to DEP.

Optimal identification of human conventional and nonconventional (CRTH2-IL7Rα-) ILC2s using additional surface markers.

BACKGROUND: Human type 2 innate lymphoid cells (ILC2s) are identified by coupled detection of CRTH2 and IL7Rα on lineage negative (Lin-) cells. Type 2 cytokine production by CRTH2-IL7Rα- innate lymphoid cells (ILCs) is unknown. OBJECTIVE: We sought to identify CRTH2-IL7Rα- type 2 cytokine-producing ILCs and their disease relevance. METHODS: We studied human blood and lung ILCs from asthmatic and control subjects by flow cytometry, ELISA, RNA sequencing, quantitative PCR, adoptive transfer to mice, and measurement of airway hyperreactivity by Flexivent. RESULTS: We found that IL-5 and IL-13 were expressed not only by CRTH2+ but also by CRTH2-IL7Rα+ and CRTH2-IL7Rα- (double-negative [DN]) human blood and lung cells. All 3 ILC populations expressed type 2 genes and induced airway hyperreactivity when adoptively transferred to mice. The frequency of type 2 cytokine-positive IL7Rα and DN ILCs were similar to that of CRTH2 ILCs in the blood and lung. Their frequency was higher in asthmatic patients than in disease controls. Transcriptomic analysis of CRTH2, IL7Rα, and DN ILCs confirmed the expression of mRNA for type 2 transcription factors in all 3 populations. Unexpectedly, the mRNA for GATA3 and IL-5 correlated better with mRNA for CD30, TNFR2, ICOS, CCR4, and CD200R1 than for CRTH2. By using a combination of these surface markers, especially CD30/TNFR2, we identified a previously unrecognized ILC2 population. CONCLUSIONS: The commonly used surface markers for human ILC2s leave a majority of type 2 cytokine-producing ILC2s unaccounted for. We identified top GATA3-correlated cell surface-expressed genes in human ILCs by RNA sequencing. These new surface markers, such as CD30 and TNFR2, identified a previously unrecognized human ILC2 population. This ILC2 population is likely to contribute to asthma.

Mouse Models of Asthma.

Mouse models are critical for delineating the mechanisms that underlie asthma pathogenesis and developing new treatments. In this chapter we describe four different asthma models that offer unique benefits and allow investigators to answer distinct research questions. We also describe key surgical procedures that are necessary for assessing experimental asthma.

Experimental asthma persists in IL-33 receptor knockout mice because of the emergence of thymic stromal lymphopoietin-driven IL-9+ and IL-13+ type 2 innate lymphoid cell subpopulations.

BACKGROUND: IL-33 plays an important role in the development of experimental asthma. OBJECTIVE: We sought to study the role of the IL-33 receptor suppressor of tumorigenicity 2 (ST2) in the persistence of asthma in a mouse model. METHODS: We studied allergen-induced experimental asthma in ST2 knockout (KO) and wild-type control mice. We measured airway hyperresponsiveness by using flexiVent; inflammatory indices by using ELISA, histology, and real-time PCR; and type 2 innate lymphoid cells (ILC2s) in lung single-cell preparations by using flow cytometry. RESULTS: Airway hyperresponsiveness was increased in allergen-treated ST2 KO mice and comparable with that in allergen-treated wild-type control mice. Peribronchial and perivascular inflammation and mucus production were largely similar in both groups. Persistence of experimental asthma in ST2 KO mice was associated with an increase in levels of thymic stromal lymphopoietin (TSLP), IL-9, and IL-13, but not IL-5, in bronchoalveolar lavage fluid. Expectedly, ST2 deletion caused a reduction in IL-13+ CD4 T cells, forkhead box P3-positive regulatory T cells, and IL-5+ ILC2s. Unexpectedly, ST2 deletion led to an overall increase in innate lymphoid cells (CD45+lin-CD25+ cells) and IL-13+ ILC2s, emergence of a TSLP receptor-positive IL-9+ ILC2 population, and an increase in intraepithelial mast cell numbers in the lung. An anti-TSLP antibody abrogated airway hyperresponsiveness, inflammation, and mucus production in allergen-treated ST2 KO mice. It also caused a reduction in innate lymphoid cell, ILC2, and IL-9+ and IL-13+ ILC2 numbers in the lung. CONCLUSIONS: Genetic deletion of the IL-33 receptor paradoxically increases TSLP production, which stimulates the emergence of IL-9+ and IL-13+ ILC2s and mast cells and leads to development of chronic experimental asthma. An anti-TSLP antibody abrogates all pathologic features of asthma in this model.

Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: The role of thymic stromal lymphopoietin.

BACKGROUND: Type 2 innate lymphoid cells (ILC2s) represent an important type 2 immune cell. Glucocorticoid regulation of human ILC2s is largely unknown. OBJECTIVE: We sought to assess steroid resistance of human blood and airway ILC2s from asthmatic patients and to examine its mechanism of induction. METHODS: We studied human blood and lung ILC2s from asthmatic patients and control subjects using flow cytometry and ELISA. RESULTS: Dexamethasone inhibited (P = .04) chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes and type 2 cytokine expression by blood ILC2s stimulated with IL-25 and IL-33. However, it did not do so when ILC2s were stimulated with IL-7 and thymic stromal lymphopoietin (TSLP), 2 ligands of IL-7 receptor α. Unlike blood ILC2s, bronchoalveolar lavage (BAL) fluid ILC2s from asthmatic patients were resistant to dexamethasone. BAL fluid from asthmatic patients had increased TSLP but not IL-7 levels. BAL fluid TSLP levels correlated (r = 0.74) with steroid resistance of ILC2s. TSLP was synergistically induced in epithelial cells by IL-13 and human rhinovirus. Mechanistically, dexamethasone upregulated ILC2 expression of IL-7 receptor α, which augmented and sustained signal transducer and activator of transcription (STAT) 5 signaling by TSLP. TSLP induced mitogen-activated protein kinase kinase (MEK), c-Fos, inhibitor of DNA binding 3, phosphorylated signal transducer and activator of transcription (pSTAT) 3, and pSTAT5, molecules linked to steroid resistance. Dexamethasone inhibited c-Fos, inhibitor of DNA binding 3, and pSTAT3 but not pSTAT5 and MEK. The MEK inhibitor trametinib, the Janus kinase-STAT inhibitor tofacitinib, and the STAT5 inhibitor pimozide reversed steroid resistance of BAL ILC2s. CONCLUSIONS: Dexamethasone inhibited type 2 cytokine production by blood ILC2s. IL-7 and TSLP abrogated this inhibition and induced steroid resistance of ILC2s in a MEK- and STAT5-dependent manner. BAL fluid ILC2s from asthmatic patients with increased TSLP levels were steroid resistant, which was reversed by clinically available inhibitors of MEK and STAT5.

Natural killer cells in asthma.

PURPOSE OF REVIEW: This review article discusses current knowledge on natural killer (NK) cells in asthma. RECENT FINDINGS: It is now well accepted that NK cell activities go beyond cancer immune surveillance and antiviral defense. Recent reports indicate that NK cells are activated in response to allergens in vivo. NK cells promote allergic sensitization, type-2 immune response, development of eosinophilic inflammation, and airway hyperresponsiveness. NK cells are activated by respiratory syncytial virus and other respiratory viruses. When infection occurs in the setting of active allergic inflammation, NK cells augment its magnitude and contribute to asthma exacerbations. Proasthma activities of NK cells can be programmed during embryogenesis through maternal exposure to environmental pollutants. Prenatally programmed NK cells produce type-2 and type-3 cytokines and mediate asthma predisposition. NK cells can also act as asthma antagonists. NK cells contribute to the resolution of inflammation through suppression of antigen-specific CD4+ T cells and type-3 immunity. When viral infection occurs in naïve mice prior to allergic sensitization, NK cells antagonize type-2 immunity and prevent development of asthma. SUMMARY: NK cells are nonredundant participants of allergic inflammation. The environmental context determines whether NK cells act as protagonists or antagonists.

Mechanism of TH2/TH17-predominant and neutrophilic TH2/TH17-low subtypes of asthma.

BACKGROUND: The mechanism of TH2/TH17-predominant and TH2/TH17-low asthma is unknown. OBJECTIVE: We sought to study the immune mechanism of TH2/TH17-predominant and TH2/TH17-low asthma. METHODS: In a previously reported cohort of 60 asthmatic patients, 16 patients were immunophenotyped with TH2/TH17-predominant asthma and 22 patients with TH2/TH17-low asthma. We examined bronchoalveolar lavage (BAL) fluid leukocytes, cytokines, mediators, and epithelial cell function for these asthma subgroups. RESULTS: Patients with TH2/TH17-predominant asthma had increased IL-1ß, IL-6, IL-23, C3a, and serum amyloid A levels in BAL fluid, and these correlated with IL-1ß and C3a levels. TH2/TH17 cells expressed higher levels of the IL-1 receptor and phospho-p38 mitogen-activated protein kinase. Anakinra, an IL-1 receptor antagonist protein, inhibited BAL TH2/TH17 cell counts. TH2/TH17-low asthma had 2 distinct subgroups: neutrophilic asthma (45%) and pauci-inflammatory asthma (55%). This contrasted with patients with TH2/TH17-predominant and TH2-predominant asthma, which included neutrophilic asthma in 6% and 0% of patients, respectively. BAL fluid neutrophils strongly correlated with BAL fluid myeloperoxidase, IL-8, IL-1α, IL-6, granulocyte colony-stimulating factor, and GM-CSF levels. Sixty percent of the patients with neutrophilic asthma had a pathogenic microorganism in BAL culture, which suggested a subclinical infection. CONCLUSION: We uncovered a critical role for the IL-1ß pathway in patients with TH2/TH17-predminant asthma. A subgroup of patients with TH2/TH17-low asthma had neutrophilic asthma and increased BAL fluid IL-1α, IL-6, IL-8, granulocyte colony-stimulating factor, and GM-CSF levels. IL-1α was directly involved in IL-8 production and likely contributed to neutrophilic asthma. Sixty percent of neutrophilic patients had a subclinical infection.

Doxorubicin anti-tumor mechanisms include Hsp60 post-translational modifications leading to the Hsp60/p53 complex dissociation and instauration of replicative senescence.

The chaperone Hsp60 is pro-carcinogenic in certain tumor types by interfering with apoptosis and with tumor cell death. In these tumors, it is not yet known whether doxorubicin anti-tumor effects include a blockage of the pro-carcinogenic action of Hsp60. We found a doxorubicin dose-dependent viability reduction in a human lung mucoepidermoid cell line that was paralleled by the appearance of cell senescence markers. Concomitantly, intracellular Hsp60 levels decreased while its acetylation levels increased. The data suggest that Hsp60 acetylation interferes with the formation of the Hsp60/p53 complex and/or promote its dissociation, both causing an increase in the levels of free p53, which can then activate the p53-dependent pathway toward cell senescence. On the other hand, acetylated Hsp60 is ubiquitinated and degraded and, thus, the anti-apoptotic effect of the chaperonin is abolished with subsequent tumor cell death. Our findings could help in the elucidation of the molecular mechanisms by which doxorubicin counteracts carcinogenesis and, consequently, it would open new roads for the development of cancer treatment protocols targeting Hsp60.

Aurintricarboxylic acid structure modifications lead to reduction of inhibitory properties against virulence factor YopH and higher cytotoxicity.

Yersinia sp. bacteria owe their viability and pathogenic virulence to the YopH factor, which is a highly active bacterial protein tyrosine phosphatase. Inhibition of YopH phosphatase results in the lack of Yersinia sp. pathogenicity. We have previously described that aurintricarboxylic acid inhibits the activity of YopH at nanomolar concentrations and represents a unique mechanism of YopH inactivation due to a redox process. This work is a continuation of our previous studies. Here we show that modifications of the structure of aurintricarboxylic acid reduce the ability to inactivate YopH and lead to higher cytotoxicity. In the present paper we examine the inhibitory properties of aurintricarboxylic acid analogues, such as eriochrome cyanine R (ECR) and pararosaniline. Computational docking studies we report here indicate that ATA analogues are not precluded to bind in the YopH active site and in all obtained binding conformations ECR and pararosaniline bind to YopH active site. The free binding energy calculations show that ECR has a stronger binding affinity to YopH than pararosaniline, which was confirmed by experimental YopH enzymatic activity studies. We found that ATA analogues can reversibly reduce the enzymatic activity of YopH, but possess weaker inhibitory properties than ATA. The ATA analogues induced inactivation of YopH is probably due to oxidative mechanism, as pretreatment with catalase prevents from inhibition. We also found that ATA analogues significantly decrease the viability of macrophage cells, especially pararosaniline, while ATA reveals only slight effect on cell viability.