Scanning and transmission electron microscopy of various plant and animal tissue

Includes bibliographical references.The focus of the work is to learn basic and advanced techniques in the preparation of plant and animal tissue for both the TEM and SEM. Techniques used would include fixation, critical point drying, heavy metal coating, and photography of the SEM samples as well as negative staining and shadowing for the TEM samples (e.g. collagen, bacteria). Electron microscopy work leads to exciting discovery about plant and animal tissues which would otherwise be beyond our sight. The minute nature of the specimens studied in this work required the powerful application of TEM and SEM microscopes. Electron microscopy is an exciting and rewarding field of research.B.S. (Bachelor of Science

Small airways dysfunction:The importance of utilising Z-scores to define MMEF abnormalities in clinical practice

BACKGROUND: The small airways comprise the largest cross-sectional area of the lungs, however, assessing and reporting abnormalities for this region of the bronchial tree has been practically and scientifically uncertain.METHODS: Using routinely collected spirometry data for patients with either asthma or COPD, the accuracy of % predicted values for defining small airways dysfunction was assessed. A z-score of ≤ -1.645 of the maximal-mid expiratory flow (MMEF) was used as the gold standard for defining abnormality in the small airways.RESULTS: Records of 3396 patients were included in the analysis. The false positive (FP) rates were 24.6 %, 16.1 %, 11.5 %, or 7.9 % when the % predicted value of 80 %, 70 %, 65 %, or 60 % were used, respectively. Sex, age, and BMI were associated with FP rates. Males were more likely to be categorised as FP with odds ratio (OR) between 1.10 and 1.49 across % predicted groups. Age was associated with FP rates with an OR between 1.01 and 1.08. The BMI was also associated with FP rates with an OR of 1.03 across all % predicted groups. Assessing the association of age groups with FP rate showed that those above 60 years old were more likely to be categorised as FP with an OR between 1.23 and 73.2 compared to those less than 30 years old.CONCLUSION: When assessing the small airways in clinical practice or for scientific purposes, the % predicted values overestimate the actual impairment leading to FP interpretation. Utilising z-score values are recommended to assess the small airways using the spirometric index, MMEF.</p

The prevalence of bronchodilator responsiveness of the small airway (using mid-maximal expiratory flow) in COPD - a retrospective study

Abstract Background Bronchodilator responsiveness (BDR) using FEV1 is often utilised to separate COPD patients from asthmatics, although it can be present in some COPD patients. With the advent of treatments with distal airway deposition, BDR in the small airways (SA) may be of value in the management of COPD. We aimed to identify the prevalence of BDR in the SA, utilizing maximal mid-expiratory flow (MMEF) as a measure of SA. We further evaluated the prevalence of BDR in MMEF with and without BDR in FEV1 and its association with baseline demographics, including conventional airflow obstruction severity and smoking history. Methods Lung function data of ever-smoking COPD patients were retrospectively analysed. BDR was evaluated 20 min after administering 2.5 mg of salbutamol via jet nebulizer. Increase in percent change of ≥ 12% and absolute change of ≥ 200 ml was used to define a BDR in FEV1, whereas an increase percent change of MMEF ≥ 30% was used to define a BDR in MMEF. Patients were classified as one of three groups according to BDR levels: group 1 (BDR in MMEF and FEV1), group 2 (BDR in MMEF alone) and group 3 (no BDR in either measure). Result BDR in MMEF was present in 59.2% of the patients. Of note, BDR in MMEF was present in all patients with BDR in FEV1 (group 1) but also in 37.9% of the patients without BDR in FEV1 (group 2). Patients in group 1 were younger than in groups 2 and 3. BMI was higher in group 1 than in group 3. Baseline FEV1% predicted and FVC % predicted were also higher in groups 1 and 2 than in group 3. Conclusion BDR in the SA (evaluated by MMEF) is common in COPD, and it is also feature seen in all patients with BDR in FEV1. Even in the absence of BDR in FEV1, BDR in MMEF is detected in some patients with COPD, potentially identifying a subgroup of patients who may benefit from different treatment strategies

The significance of the F variant of alpha-1-antitrypsin and unique case report of a PiFF homozygote

BACKGROUND: Inheritance of the F variant of alpha-1-antitrypsin is associated with normal circulating protein levels, but it is believed to be dysfunctional in its ability to inhibit neutrophil elastase and therefore has been implicated as a susceptibility factor for the development of emphysema. In this study, its functional characteristics were determined following the identification of a unique patient with the PiFF phenotype, and the implications as a susceptibility factor for emphysema are considered both in homozygotes and heterozygotes. METHODS: Second order association rate constants were measured for M, Z, S and F variants of alpha-1-antitrypsin with neutrophil elastase and proteinase 3. Clinical characteristics of the PiFF homozygote and six PiFZ heterozygote subjects were studied. RESULTS: The F variant had a reduced association rate constant with neutrophil elastase (5.60 ± 0.83 × 10(6) M(-1) s(-1)) compared to the normal M variant (1.45 ± 0.02 × 10(7) M(-1) s(-1)), indicating an increased time to inhibition that was comparable to that of the Z variant (7.34 ± 0.03 × 10(6) M(-1) s(-1)). The association rate constant for the F variant and proteinase 3 (1.06 ± 0.22 × 10(6) M(-1) s(-1)) was reduced compared to that with neutrophil elastase, but was similar to that of other alpha-1-antitrypsin variants. Of the six PiFZ heterozygotes, five had airflow obstruction and radiological evidence of emphysema. The PiFF homozygote had airflow obstruction but no emphysema. None of the patients had clinical evidence of liver disease. CONCLUSIONS: The F variant may increase susceptibility to elastase-induced lung damage but not emphysema, whereas co-inheritance with the Z deficiency allele may predispose to emphysema despite reasonable plasma concentrations of alpha-1-antitrypsin

TNF- α Autocrine Feedback Loops in Human Monocytes:The Pro- and Anti-Inflammatory Roles of the TNF- α Receptors Support the Concept of Selective TNFR1 Blockade in Vivo

Selective TNFR1 blockade in inflammatory diseases is emerging as a clinical strategy. We studied the roles of the two TNF-α receptors, TNFR1 and TNFR2, in human monocytes, the principal producer of TNF-α and central to many TNF-α driven diseases. We hypothesised that TNF-α has pro- and anti-inflammatory effects on monocytes, occurring differentially via TNFR1 and TNFR2. Monocytes were isolated from healthy human subjects and exposed to LPS, plus/minus the addition of blocking antibodies to TNF-α or its receptors. Pro- and anti-inflammatory cytokine production was quantified using real-time PCR and ELISAs. Cell surface expression of TNFR1/2 was measured by flow cytometry. We demonstrated that monocytes vary in the expression patterns of TNFR1 and TNFR2. Autocrine binding of TNF-α led to sustained upregulation of proinflammatory cytokines via TNFR1. In contrast, autocrine binding via TNFR2 upregulated the anti-inflammatory cytokine, IL-10, without proinflammatory effect. TNFR2 was responsible for binding soluble TNF-α secreted by monocytes, clearing the cytokine from the pericellular environment. TNFR1 blockade did not change the cell surface expression of TNFR2, leaving this receptor free to upregulate IL-10. These novel results support the concept of selective TNFR1 blockade in vivo in order that positive anti-inflammatory effects of TNF-α can be retained via TNFR2 ligation

Effect of expectoration on inflammation in induced sputum in α-1-antitrypsin deficiency

SummaryIt is unclear how chronic expectoration influences airway inflammation in patients with chronic lung disease. The aim of this study was to investigate factors influencing inflammation in induced sputum samples, including, in particular, chronic sputum production. Myeloperoxidase, interleukin-8, leukotriene B4 (LTB4), neutrophil elastase, secretory leukoprotease inhibitor (SLPI) and protein leakage were compared in induced sputum samples from 48 patients (36 with chronic expectoration) with COPD (with and without alpha-1-antitrypsin deficiency; AATD), 9 individuals with AATD but without lung disease and 14 healthy controls. There were no differences in inflammation in induced sputum samples from healthy control subjects and from AATD deficient patients with normal lung function but without chronic expectoration (P>0.05). Inflammation in induced sputum from AATD patients with airflow obstruction and chronic sputum expectoration was significantly greater than for similar patients who did not expectorate: Interleukin-8 (P<0.01), elastase activity (P=0.01), and protein leakage (P<0.01). The presence of spontaneous sputum expectoration in AATD patients with airflow obstruction was associated with increased neutrophilic airway inflammation in induced sputum samples. The presence of chronic expectoration in some patients will clearly complicate interpretation of studies employing sputum induction where this feature has not been identified

Small airways disease:time for a revisit?

James A Stockley,1 Brendan G Cooper,1 Robert A Stockley,2 Elizabeth Sapey3 1Department of Lung Function and Sleep, 2Department of Respiratory Medicine, University Hospital Birmingham, 3Institute of Inflammation and Ageing, Centre for Translational Inflammation Research, University of Birmingham, Edgbaston, Birmingham, UK Abstract: It is increasingly acknowledged that delays in the diagnosis of chronic inflammatory lung conditions have hampered our understanding of pathogenesis and thus our ability to design efficacious therapies. This is particularly true for COPD, where most patients are diagnosed with moderate-to-severe airflow obstruction and little is known about the inflammatory processes present in early disease. There is great interest in developing screening tests that can identify those most at risk of developing COPD before airflow obstruction has developed for the purpose of research and clinical care. Landmark pathology studies have suggested that damage to the small airways precedes the development of airflow obstruction and emphysema and, thus, presents an opportunity to identify those at risk of COPD. However, despite a number of physiological tests being available to assess small airways function, none have been adopted into routine care in COPD. The reasons that tests of small airways have not been utilized widely include variability in test results and a lack of validated reference ranges from which to compare results for some methodologies. Furthermore, population studies have not consistently demonstrated their ability to diagnose disease. However, the landscape may be changing. As the equipment that delivers tests of small airways become more widely available, reference ranges are emerging and newer methodologies specifically seek to address variability and difficulty in test performance. Moreover, there is evidence that while tests of small airways may not be helpful across the full range of established disease severity, there may be specific groups (particularly those with early disease) where they might be informative. In this review, commonly utilized tests of small airways are critically appraised to highlight why these tests may be important, how they can be used and what knowledge gaps remain for their use in COPD. Keywords: small airways, COPD, early disease, physiology, emphysema, airflow obstructio

The prevalence of bronchiectasis in patients with alpha-1 antitrypsin deficiency: initial report of EARCO

Alpha-1 antitrypsin deficiency; Emphysema; PrevalenceDeficiència d'alfa-1 antitripsina; Emfisema; PrevalençaDeficiencia de alfa-1 antitripsina; Enfisema; PrevalenciaBackground Although bronchiectasis has been recognised as a feature of some patients with Alpha1-Antitrypsin deficiency the prevalence and characteristics are not widely known. We wished to determine the prevalence of bronchiectasis and patient characteristics. The first cohort of patients recruited to the EARCO (European Alpha1 Research Collaboration) International Registry data base by the end of 2021 was analysed for radiological evidence of both emphysema and bronchiectasis as well as baseline demographic features. Results Of the first 505 patients with the PiZZ genotype entered into the data base 418 (82.8%) had a reported CT scan. There were 77 (18.4%) with a normal scan and 38 (9.1%) with bronchiectasis alone. These 2 groups were predominantly female never smokers and had lung function in the normal range. The remaining 303 (72.5%) ZZ patients all had emphysema on the scan and 113 (27%) had additional evidence of bronchiectasis. Conclusions The data indicates the bronchiectasis alone is a feature of 9.1% of patients with the PiZZ genotype of Alpha1-antitrypsin deficiency but although emphysema is the dominant lung pathology bronchiectasis is also present in 27% of emphysema cases and may require a different treatment strategy.The International EARCO registry is funded by unrestricted grants of Grifols, CSL Behring, Kamada, pH Pharma and Takeda to the European Respiratory Society (ERS)