Alpha-1-antitrypsin-deficiency is associated with lower cardiovascular risk: an approach based on federated learning.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory multisystemic disease caused by environmental exposures and/or genetic factors. Inherited alpha-1-antitrypsin deficiency (AATD) is one of the best recognized genetic factors increasing the risk for an early onset COPD with emphysema. The aim of this study was to gain a better understanding of the associations between comorbidities and specific biomarkers in COPD patients with and without AATD to enable future investigations aimed, for example, at identifying risk factors or improving care. METHODS: We focused on cardiovascular comorbidities, blood high sensitivity troponin (hs-troponin) and lipid profiles in COPD patients with and without AATD. We used clinical data from six German University Medical Centres of the MIRACUM (Medical Informatics Initiative in Research and Medicine) consortium. The codes for the international classification of diseases (ICD) were used for COPD as a main diagnosis and for comorbidities and blood laboratory data were obtained. Data analyses were based on the DataSHIELD framework. RESULTS: Out of 112,852 visits complete information was available for 43,057 COPD patients. According to our findings, 746 patients with AATD (1.73%) showed significantly lower total blood cholesterol levels and less cardiovascular comorbidities than non-AATD COPD patients. Moreover, after adjusting for the confounder factors, such as age, gender, and nicotine abuse, we confirmed that hs-troponin is a suitable predictor of overall mortality in COPD patients. The comorbidities associated with AATD in the current study differ from other studies, which may reflect geographic and population-based differences as well as the heterogeneous characteristics of AATD. CONCLUSION: The concept of MIRACUM is suitable for the analysis of a large healthcare database. This study provided evidence that COPD patients with AATD have a lower cardiovascular risk and revealed that hs-troponin is a predictor for hospital mortality in individuals with COPD.

Human Bronchial Epithelial Cell Transcriptome Changes in Response to Serum from Patients with Different Status of Inflammation.

PURPOSE: To investigate the transcriptome of human bronchial epithelial cells (HBEC) in response to serum from patients with different degrees of inflammation. METHODS: Serum from 19 COVID-19 patients obtained from the Hannover Unified Biobank was used. At the time of sampling, 5 patients had a WHO Clinical Progression Scale (WHO-CPS) score of 9 (severe illness). The remaining 14 patients had a WHO-CPS of below 9 (range 1-7), and lower illness. Multiplex immunoassay was used to assess serum inflammatory markers. The culture medium of HBEC was supplemented with 2% of the patient's serum, and the cells were cultured at 37 °C, 5% CO2 for 18 h. Subsequently, cellular RNA was used for RNA-Seq. RESULTS: Patients with scores below 9 had significantly lower albumin and serum levels of E-selectin, IL-8, and MCP-1 than patients with scores of 9. Principal component analysis based on 500 "core genes" of RNA-seq segregated cells into two subsets: exposed to serum from 4 (I) and 15 (II) patients. Cells from a subset (I) treated with serum from 4 patients with a score of 9 showed 5566 differentially expressed genes of which 2793 were up- and 2773 downregulated in comparison with cells of subset II treated with serum from 14 patients with scores between 1 and 7 and one with score = 9. In subset I cells, a higher expression of TLR4 and CXCL8 but a lower CDH1, ACE2, and HMOX1, and greater effects on genes involved in metabolic regulation, cytoskeletal organization, and kinase activity pathways were observed. CONCLUSION: This simple model could be useful to characterize patient serum and epithelial cell properties.

An Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Circulating Pi*Z Alpha1-Antitrypsin Polymers.

Enzyme-linked immunosorbent assay (ELISA) is a sensitive immunoassay based on specific antigen-antibody reaction that is used for quantitative/qualitative analysis of various analytes in serum, plasma, saliva, cell and tissue lysates, and urine. ELISAs are typically performed in multi-well plates and depending on the design require coating antibody/antigen, analyte, detection antibodies, buffer, wash solution, and substrate/chromogen. Here we describe highly specific monoclonal antibody-based ELISA that detects circulating polymers formed by Pi*Z variant of human alpha-1-antitrypsin (Z-AAT). The circulating Z-AAT polymers are present in all individuals with inherited Pi*Z AAT deficiency. Thus, our assay provides a useful tool to examine the clinical significance and utility of Z-AAT polymers.

Diagnostic and therapeutic value of human serpin family proteins.

SERPIN (serine proteinase inhibitors) is an acronym for the superfamily of structurally similar proteins found in animals, plants, bacteria, viruses, and archaea. Over 1500 SERPINs are known in nature, while only 37 SERPINs are found in humans, which participate in inflammation, coagulation, angiogenesis, cell viability, and other pathophysiological processes. Both qualitative or quantitative deficiencies or overexpression and/or abnormal accumulation of SERPIN can lead to diseases commonly referred to as "serpinopathies". Hence, strategies involving SERPIN supplementation, elimination, or correction are utilized and/or under consideration. In this review, we discuss relationships between certain SERPINs and diseases as well as putative strategies for the clinical explorations of SERPINs.

Ex vivo study on the human blood neutrophil circadian features and effects of alpha1-antitrypsin and lipopolysaccharide.

AIMS: Neutrophils perform various functions in a circadian-dependent manner; therefore, we investigated here whether the effect of alpha1-antitrypsin (AAT), used as augmentation therapy, is dependent on the neutrophil circadian clock. AAT is a vital regulator of neutrophil functions, and its qualitative and/or quantitative defects have significant implications for the development of respiratory diseases. METHODS: Whole blood from 12 healthy women age years, mean (SD) 29.92 (5.48) was collected twice daily, 8 h apart, and incubated for 30 min at 37 °C alone or with additions of 2 mg/ml AAT (Respreeza) and/or 5 µg/ml lipopolysaccharide (LPS) from Escherichia coli. Neutrophils were then isolated to examine gene expression, migration and phagocytosis. RESULTS: The expression of CD14, CD16, CXCR2 and SELL (encoding CD62L) genes was significantly higher while CDKN1A lower in the afternoon than in the morning neutrophils from untreated blood. Neutrophils isolated in the afternoon had higher migratory and phagocytic activity. Morning neutrophils isolated from AAT-pretreated blood showed higher expression of CXCR2 and SELL than those from untreated morning blood. Pretreatment of blood with AAT enhanced migratory properties of morning but not afternoon neutrophils. Of all genes analysed, only CXCL8 expression was strongly upregulated in morning and afternoon neutrophils isolated from LPS-pretreated blood, whereas CXCR2 expression was downregulated in afternoon neutrophils. The addition of AAT did not reverse the effects of LPS. SIGNIFICANCE: The circadian clock of myeloid cells may affect the effectiveness of various therapies, including AAT therapy used to treat patients with AAT deficiency, and needs further investigation.

An association between plasma levels of α2-macroglobulin and α1-antitrypsin in PiMM and PiZZ individuals differing in COPD presentation.

INTRODUCTION: Compared to normal PiMM, individuals with severe α1-antitrypsin (AAT) PiZZ (Glu342Lys) genotype deficiency are at higher risk of developing early-onset chronic obstructive pulmonary disease (COPD)/emphysema associated with Z-AAT polymers and neutrophilic inflammation. We aimed to investigate putative differences in plasma levels of acute phase proteins (APP) between PiMM and PiZZ subjects and to determine plasma Z-AAT polymer levels in PiZZ subjects. MATERIALS AND METHODS: Nephelometric analysis of seven plasma APPs was performed in 67 PiMM and 44 PiZZ subjects, of whom 43 and 42, respectively, had stable COPD. Of the PiZZ-COPD patients, 21 received and 23 did not receive intravenous therapy with human AAT preparations (IV-AAT). Plasma levels of Z-AAT polymers were determined by Western blotting using specific mouse monoclonal antibodies (2C1 and LG96). RESULTS: In addition to lower plasma AAT, PiZZ patients had higher α2-macroglobulin (A2MG) levels than PiMM patients. In contrast, PiZZ who received IV-AAT had higher AAT values but lower A2MG values than PiZZ without IV-AAT. Regardless of the AAT genotype, AAT levels were inversely correlated with A2MG, and the AAT/A2MG ratio was correlated with lung diffusion capacity (DCLO%). All PiZZ patients had circulating Z-AAT polymer levels that correlated directly with A2MG. In PiZZ without IV-AAT therapy polymer levels correlated inversely with the ratio of forced expiratory volume in 1 s to forced vital capacity (FEV1/FVC). CONCLUSION: Combined measurement of plasma AAT and A2MG levels may be of clinical value in assessing the progression of COPD and requires further attention.

A Fast Scoring of Human Primary Respiratory Epithelia Grown at Air-Liquid Interface (ALI) to Assess Epithelial Morphology in Research and Personalized Medicine Settings.

BACKGROUND: In recent years, increasingly complex ALI protocols involving specialized, albeit laboratory-specific media have been established, while at the same time, many studies compile the data of only a few ALI donors in spite of site-, protocol- and donor-specific differentiation. METHODS: We describe a simple morphology scoring protocol using histology material derived from epithelia grown on ALI inserts in parallel to other, more complex readouts. RESULTS: Among more than 100 ALI inserts derived from different donors, significant differences in layer score (p = 0.001) and goblet cell score (p = 0.002) were observed when ALI epithelia derived from explanted lung material were compared to trachea-derived ALI cultures. Cortisol withdrawal for the final 2 days of ALI cultures influenced goblet cell density (p = 0.001). CONCLUSIONS: While the histology score provides less resolution than FACS- or OMICs- based single cell analyses, the use of a subportion of the ALI epithelia grown on inserts makes it feasible to combine morphology assessment and other readouts of the same insert. This allows us to control for basic ALI morphology in research and personalized medicine settings in order to assess and, if desired, control for the impact of ALI culture protocols, site- and donor-specific influences on outcome of studies of ALI-derived epithelia.

Alpha1-antitrypsin improves survival in murine abdominal sepsis model by decreasing inflammation and sequestration of free heme.

Background: Excessive inflammation, hemolysis, and accumulation of labile heme play an essential role in the pathophysiology of multi-organ dysfunction syndrome (MODS) in sepsis. Alpha1-antitrypsin (AAT), an acute phase protein with heme binding capacity, is one of the essential modulators of host responses to inflammation. In this study, we evaluate the putative protective effect of AAT against MODS and mortality in a mouse model of polymicrobial abdominal sepsis. Methods: Polymicrobial abdominal sepsis was induced in C57BL/6N mice by cecal ligation and puncture (CLP). Immediately after CLP surgery, mice were treated intraperitoneally with three different forms of human AAT-plasma-derived native (nAAT), oxidized nAAT (oxAAT), or recombinant AAT (recAAT)-or were injected with vehicle. Sham-operated mice served as controls. Mouse survival, bacterial load, kidney and liver function, immune cell profiles, cytokines/chemokines, and free (labile) heme levels were assessed. In parallel, in vitro experiments were carried out with resident peritoneal macrophages (MPMΦ) and mouse peritoneal mesothelial cells (MPMC). Results: All AAT preparations used reduced mortality in septic mice. Treatment with AAT significantly reduced plasma lactate dehydrogenase and s-creatinine levels, vascular leakage, and systemic inflammation. Specifically, AAT reduced intraperitoneal accumulation of free heme, production of cytokines/chemokines, and neutrophil infiltration into the peritoneal cavity compared to septic mice not treated with AAT. In vitro experiments performed using MPMC and primary MPMΦ confirmed that AAT not only significantly decreases lipopolysaccharide (LPS)-induced pro-inflammatory cell activation but also prevents the enhancement of cellular responses to LPS by free heme. In addition, AAT inhibits cell death caused by free heme in vitro. Conclusion: Data from the septic CLP mouse model suggest that intraperitoneal AAT treatment alone is sufficient to improve sepsis-associated organ dysfunctions, preserve endothelial barrier function, and reduce mortality, likely by preventing hyper-inflammatory responses and by neutralizing free heme.

Distinct metabolic responses to heme in inflammatory human and mouse macrophages - Role of nitric oxide.

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Serum Levels of Alpha1-antitrypsin and Their Relationship With COPD in the General Spanish Population / Niveles séricos de alfa-1 antitripsina y su relación con la EPOC en la población española

BACKGROUND: Low plasma level of alpha1-antitrypsin (AAT) is an established risk factor for early-onset chronic obstructive lung disease (COPD). However, less attention is given to the levels of AAT in the general population. METHODS: This is a part of a multicentre, population-based study conducted at 11 sites throughout Spain. Plasma levels of AAT were available for 837 persons with a mean (SD) age of 58.05 (11.3) years: 328-smokers, 272-ex-smokers and 237 non-smokers. Out of 837, 303 (36.2%) had a diagnosis of COPD, 222 (26.5%) had respiratory symptoms but no COPD, and 312 (37.3%) were healthy controls. RESULTS: In the whole cohort, the mean level of plasma AAT was 1.51 (0.47) g/L. Levels were higher in COPD patients [1.55 (0.45) g/L] and individuals with respiratory symptoms [1.57 (0.47) g/L] than in controls [1.43 (0.47) g/L], p < 0.001, a finding which persisted after correction for age and CRP. Plasma AAT levels were negatively associated with FEV1/FVC ratio, after adjustment for age, sex, smoking status, CRP, TNFα, fibrinogen and albumin. The risk for COPD was significantly associated with higher AAT levels in univariate and multivariate models, with odds ratios of 1.8 and 1.5, respectively. In the univariate and multivariate models smoking status, gender, and CRP levels were also associated with COPD probability, demonstrating that they act independently. CONCLUSION: Increased circulating levels of AAT, similarly to CRP and other markers of systemic inflammation, is an important feature of COPD. Our results highlight a complex interrelationship between levels of AAT and health of respiratory system

INTRODUCCIÓN: Los niveles plasmáticos bajos de alfa-1 antitripsina (AAT) constituyen un factor de riesgo para el desarrollo temprano de la enfermedad pulmonar obstructiva crónica (EPOC). Sin embargo, se ha prestado una menor atención a los niveles de AAT en la población general. MÉTODOS: Este trabajo forma parte de un estudio poblacional multicéntrico llevado a cabo en 11 centros españoles. Se incluyeron niveles de AAT de 837 personas con una edad media (DE) de 58,05 (11,3) años: 328 fumadores, 272 exfumadores y 237 no fumadores. De los 837, a 303 (36,2%) se les diagnosticó EPOC, 222 (36,5%) presentaron síntomas respiratorios pero no EPOC y 312 (37,3%) eran controles sanos. RESULTADOS: En la cohorte total, los niveles plasmáticos medios de AAT fueron 1,51 (0,47) g/l. Los pacientes con EPOC y los individuos con síntomas respiratorios presentaron niveles más elevados (1.55 [0.45] g/l y 1.57 [0.47] g/l respectivamente) que los sujetos control (1.43 [0.47] g/l, p < 0.001). Este resultado se mantuvo tras la corrección por edad y niveles de proteína C reactiva (PCR). Los niveles plasmáticos de AAT se asociaron negativamente con la relación FEV1/FVC tras el ajuste por edad, sexo, hábito tabáquico, niveles de PCR, TNFα, fibrinógeno y albúmina. El riesgo de EPOC se asoció significativamente con niveles más elevados de AAT tanto en el modelo univariante como el multivariante, con odds ratios de 1,8 y 1.5 respectivamente. En los modelos univariantes y multivariantes, el hábito tabáquico, el sexo, y los niveles de PCR también se asociaron con la probabilidad de sufrir EPOC, lo cual demostró que se trataba de variables independientes. CONCLUSIÓN: Un aumento de los niveles circulantes de AAT, al igual que los niveles de PCR y otros marcadores de inflamación sistémica, son características importantes de la EPOC. Nuestros resultados evidencian la compleja interrelación existente entre los niveles de AAT y la salud del aparato respiratorio

?_1-antichymotrypsin interaction with Alzheimer’s peptide A?_(1-42) in affecting expression of transcription factors in human neuroblastoma cells / 中华神经科杂志

Objective To study the interaction between ?_1-antichymotrypsin (ACT) and amyloid peptide A?_~1-42 in vitro, and the effects of their complex on expression of transcription factors PPAR? and NF?B in human neuroblastoma (Kelly) cells. Methods A?_~1-42 and ACT were mixed at a 10∶1 molar ratio at room temperature, the complex formed in 2 hours and 24 hours were expressed as A?_~1-42 /ACT (2 hours), A?_~1-42 /ACT (24 hours), respectively. The complexes were studied by using agrose electrophoresis, and their effects on PPAR? and NF?B expressions were assessed by electrophoresis mobility shift assay (EMSA). Results At conditions used, ACT interacted with A?_~1-42 in vitro, the complexes formed and functioned differently according to their interaction period. Compared with unstimulated control cells, A?_~1-42 /ACT (2 hours) strongly increased PPAR? and NF?B expressions by Kelly cells by 158% (P