Analyzing Fcγ-Receptor Interactions on Monocytes with the Proximity Ligation Assay (PLA).

Proximity ligation assays (PLA) enable the detection and characterization of protein interactions independent of protein abundance or genetic modifications. This technique exploits both antibody and DNA-binding features, providing high selectivity and sensitivity for protein recognition and visualization of single-protein molecules with high spatial accuracy. Here, we describe the general procedure for a direct PLA on splenic monocytes to analyze FcγRIIb homodimerization. However, this method can be applied to other cells and receptors of interest.

Role of lipid nanodomains for inhibitory FcγRIIb function.

The inhibitory Fcγ receptor FcγRIIb is involved in immune regulation and is known to localize to specific regions of the plasma membrane called lipid rafts. Previous studies suggested a link between the altered lateral receptor localization within the plasma membrane and the functional impairment of the FcγRIIb-I232T variant that is associated with systemic lupus erythematosus. Here, we conducted microsecond all-atom molecular dynamics simulations and IgG binding assays to investigate the lipid nano-environment of FcγRIIb monomers and of the FcγRIIb-I232T mutant within a plasma membrane model, the orientation of the FcγRIIb ectodomain, and its accessibility to IgG ligands. In contrast to previously proposed models, our simulations indicated that FcγRIIb does not favor a cholesterol- or a sphingolipid-enriched lipid environment. Interestingly, cholesterol was depleted for all studied FcγRIIb variants within a 2-3 nm environment of the receptor, counteracting the usage of raft terminology for models on receptor functionality. Instead, the receptor interacts with lipids that have poly-unsaturated fatty acyl chains and with (poly-) anionic lipids within the cytosolic membrane leaflet. We also found that FcγRIIb monomers adopt a conformation that is not suitable for binding to its IgG ligand, consistent with a lack of detectable binding of monomeric IgG in experiments on primary immune cells. However, our results propose that multivalent IgG complexes might stabilize FcγRIIb in a binding-competent conformation. We suggest differences in receptor complex formation within the membrane as a plausible cause of the altered membrane localization or clustering and the altered suppressive function of the FcγRIIb-I232T variant.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Side effects and antibody response of an inactive severe acute respiratory syndrome coronavirus 2 vaccine among health care workers.

OBJECTIVE: This study aims to investigate the antibody response and the side effects of the two-dose inactive SARS-CoV-2 vaccine (CoronaVac, Sinovac, China) among a health care worker population in Turkey. METHODS: This study was a prospective, cross-sectional, single-center study conducted between December 16, 2020, and March 15, 2021. We evaluated the side effects from a questionnaire, and anti-spike immunoglobulin G response to the vaccine (0- and 28-day schedule) using an enzyme-linked immunosorbent assay. RESULTS: A total of 94 of 184 health care workers completed this study. The percentages of participants who were seronegative at baseline and achieved to the seropositivity were 21.3 and 97.9%, respectively, on day 21 after vaccinations. The seropositivity was predominantly detected in 31-45 years of the age group (55.4%, p=0.636), normal body mass index (47.8%, p=0.999), nonsmokers (64.1%, p=0.999), those without any comorbidities (73.9%, p=0.463), and those without any side effects (70.2%, p=0.256). The frequencies of overall side effects within seven days after the first and second doses of CoronaVac were 37.2 and 28.7%, respectively. The most common side effects was localized pain at the injection site (15.7 and 11.6%, respectively). CONCLUSIONS: We found that vaccination by two-dose CoronaVac could elicit a specific humoral response, and it was well tolerated in health care workers. The high seropositivity developed after the second dose attracted attention. Our study will be useful in terms of showing short-term immunity and side effects.

Change of respiratory functions, the STOP-Bang questionnaire, and Epworth sleepiness scale after bariatric surgery.

INTRODUCTION AND OBJECTIVE: Obesity is a highly morbid and fatal syndrome that reduces respiratory function. Obstructive sleep apnea syndrome (OSAS) is a common sleep disorder in morbid obesity. Herein, we aimed to determine how respiratory function tests changed over time after bariatric surgery and to assess non-PSG (polysomnography) tests, namely STOP-Bang questionnaire and Epworth sleepiness tests, for predicting OSAS risk. METHOD: This retrospectively conducted study enrolled 35 patients who underwent bariatric surgery. Patients were divided into three groups formed on the basis of time passed after surgery (1, 2, or 3 years). Preoperative and postoperative respiratory function test parameters assessed by spirometry, body mass index (BMI), STOP-Bang questionnaire, and Epworth sleepiness test scores were recorded. RESULTS: Twenty-four (68.6%) patients were female, 11 (31.4%) male. The mean age was 36.5±10.5 years. Postoperative weight loss of the study groups was 26% p=0.001, 23.6% p=0.002 and 25.9% p=0.005. Reductions in BMI were 32 kg/m2 p=0.001, 34.5 kg/m2 p=0.002, 35.8 kg/m2 p=0.005 respectively. Postoperative FVC (440 ml, 390 ml, 430 ml p = 0.005) and FEV1 (220 ml p = 0.005, 250 ml p = 0.004, 214 ml p = 0.005) increased in all three groups. STOP-Bang questionnaire and Epworth sleepiness scale scores significantly decreased after weight loss compared to preoperative period in all the study groups. CONCLUSION: We showed that FVC and FEV1 increased in the short and long term after weight loss by bariatric surgery; we also found that STOP-Bang questionnaire and Epworth sleepiness scale scores decreased postoperatively. These tests may be helpful to assess OSAS risk before and after surgery. KEY WORDS: Bariatric surgery, Obesity, Respiratory functions, STOP-Bang questionnaire and Epworth sleepiness test.

Impact of Plasma Membrane Domains on IgG Fc Receptor Function.

Lipid cell membranes not only represent the physical boundaries of cells. They also actively participate in many cellular processes. This contribution is facilitated by highly complex mixtures of different lipids and incorporation of various membrane proteins. One group of membrane-associated receptors are Fc receptors (FcRs). These cell-surface receptors are crucial for the activity of most immune cells as they bind immunoglobulins such as immunoglobulin G (IgG). Based on distinct mechanisms of IgG binding, two classes of Fc receptors are now recognized: the canonical type I FcγRs and select C-type lectin receptors newly referred to as type II FcRs. Upon IgG immune complex induced cross-linking, these receptors are known to induce a multitude of cellular effector responses in a cell-type dependent manner, including internalization, antigen processing, and presentation as well as production of cytokines. The response is also determined by specific intracellular signaling domains, allowing FcRs to either positively or negatively modulate immune cell activity. Expression of cell-type specific combinations and numbers of receptors therefore ultimately sets a threshold for induction of effector responses. Mechanistically, receptor cross-linking and localization to lipid rafts, i.e., organized membrane microdomains enriched in intracellular signaling proteins, were proposed as major determinants of initial FcR activation. Given that immune cell membranes might also vary in their lipid compositions, it is reasonable to speculate, that the cell membrane and especially lipid rafts serve as an additional regulator of FcR activity. In this article, we aim to summarize the current knowledge on the interplay of lipid rafts and IgG binding FcRs with a focus on the plasma membrane composition and receptor localization in immune cells, the proposed mechanisms underlying this localization and consequences for FcR function with respect to their immunoregulatory capacity.

Evaluation of Subclinical Atherosclerosis with Carotid Intima-Media and Epicardial Fat Thickness in Patients with Sarcoidosis.

OBJECTIVES: Since many similar mechanisms may play a role in the pathophysiology of sarcoidosis and atherosclerosis, the risk of subclinical atherosclerosis may be increased in patients with sarcoidosis. The aim of this study was to evaluate known markers of subclinical atherosclerosis, namely epicardial fat thickness (EFT) and carotid intima-media thickness (CIMT) in patients with sarcoidosis. MATERIALS AND METHODS: This cross-sectional study included a total of 183 subjects, including 94 patients with sarcoidosis (patient group) and a control group of 89 healthy individuals. Measurements of EFT and CIMT were taken from all subjects and recorded. The groups were compared, and differences were analyzed statistically. RESULTS: EFT was higher in patients than in control subjects (6.42±1.12 mm vs 7.13±1.41 mm, p<0.001). CIMT was higher in patients than in control subjects (0.51±0.02 mm vs 0.52±0.02 mm, p=0.003). CONCLUSION: EFT and CIMT were found to be higher in patients with sarcoidosis than in healthy people. These results indicate that the risk of subclinical atherosclerosis might be increased in these patients.

Predicting Pulmonary Complications Following Upper and Lower Abdominal Surgery: ASA vs. ARISCAT Risk Index.

OBJECTIVE: Postoperative pulmonary complications (POPC) account for a substantial proportion of risk related to surgery and anaesthesia. The American Society of Anesthesiologists (ASA) classification and the Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) risk index correlate well with POPC. Here, we compared their accuracy in predicting pulmonary complications following upper and lower abdominal surgery. METHODS: We retrospectively reviewed the medical records of patients undergoing upper and lower abdominal surgery. We collected patients' demographic data, comorbidities, preoperative pulmonary risk score, laboratory results, surgical data, respiratory tract infection history within one month before surgery, surgical urgency, ASA scores and pulmonary complications within one month after the surgery. RESULTS: We evaluated 241 patients [upper abdominal surgery (UAS) n=121; lower abdominal surgery (LAS) n=120; mean age 55.7±3.1 years]. In the UAS, 55.8% of the patients were male. In LAS, all patients were female. In both groups, the most common POPC was pleural effusion with compressive atelectasis (CA). Regarding risk score, in both groups, patients with high-risk developed a higher rate of pulmonary complications [UAS (50%), LAS (40%)]. In patients with low-risk scores, the rate of pulmonary complications was significantly lower than the intermediate and high-risk groups (p<0.001). A positive correlation was observed between preoperative risk score and complications (UAS r=0.34; LAS r=0.35 p<0.05). No association was observed between the ASA scores and POPC (p=0.8). CONCLUSION: The ASA classification was found to be a weaker modality than ARISCAT risk index to predict pulmonary complications after the upper and lower abdominal surgeries.

Complement-Dependent Activity of CD20-Specific IgG Correlates With Bivalent Antigen Binding and C1q Binding Strength.

Monoclonal antibodies directed against the CD20 surface antigen on B cells are widely used in the therapy of B cell malignancies. Upon administration, the antibodies bind to CD20 expressing B cells and induce their depletion via cell- and complement-dependent cytotoxicity or by induction of direct cell killing. The three antibodies currently most often used in the clinic are Rituximab (RTX), Ofatumumab (OFA) and Obinutuzumab (OBI). Even though these antibodies are all of the human IgG1 subclass, they have previously been described to vary considerably in the effector functions involved in therapeutic B cell depletion, especially in regards to complement activation. Whereas OFA is known to strongly induce complement-dependent cytotoxicity, OBI is described to be far less efficient. In contrast, the role of complement in RTX-induced B cell depletion is still under debate. Some of this dissent might come from the use of different in vitro systems for characterization of antibody effector functions. We therefore set out to systematically compare antibody as well as C1q binding and complement-activation by RTX, OFA and OBI on human B cell lines that differ in expression levels of CD20 and complement-regulatory proteins as well as human primary B cells. Applying real-time interaction analysis, we show that the overall strength of C1q binding to live target cells coated with antibodies positively correlated with the degree of bivalent binding for the antibodies to CD20. Kinetic analysis revealed that C1q exhibits two binding modes with distinct affinities and binding stabilities, with exact numbers varying both between antibodies and cell lines. Furthermore, complement-dependent cell killing by RTX and OBI was highly cell-line dependent, whereas the superior complement-dependent cytotoxicity by OFA was independent of the target B cells. All three antibodies were able to initiate deposition of C3b on the B cell surface, although to varying extent. This suggests that complement activation occurs but might not necessarily lead to induction of complement-dependent cytotoxicity. This activation could, however, initiate complement-dependent phagocytosis as an alternative mechanism of therapeutic B cell depletion.

Comparative analysis of the patients with community-acquired pneumonia (CAP) and health care-associated pneumonia (HCAP) requiring hospitalization.

INTRODUCTION: The recently introduced concept of health care-associated pneumonia (HCAP), referring to patients with frequent healthcare contacts and at higher risk of contracting resistant pathogens is controversial. MATERIALS AND METHODS: A prospective study comparing patients with HCAP and community-acquired pneumonia (CAP) in the our center. The primary outcome was 30 day mortality. RESULT: A total of the 169 patients HCAP 36 (21.3%); CAP 133 (78.7%) were evaluated. HCAP patients were older than patients with CAP [median age was 72.5 (43-96), 60.0 (18-91) years p<0.05]. The most common Klebsiella pneumoniae (16.6%) and Pseudomonas aeruginosa (8.3%) were gram-negative bacteria in the SBIP group; In the TGP group, gram-positive bacteria were more frequently isolated. Polymicrobial agents (22.2% vs. 3.7% p<0.05) and MDR pathogens (57.1% vs. 24% p<0.05) were more common in patients with HCAP. Mortality rate (22.2% vs. 6% p<0.05) was also higher in HCAP more than CAP. CONCLUSIONS: HCAP was common among patients with pneumonia requiring hospitalization and mortality rate was high. The patients with HCAP were different from CAP in terms of demographic and clinical features, etiology, outcome.

Pneumonia in Renal Transplant Recipients: A Single-Center Study.

OBJECTIVES: Pulmonary infections are a significant cause of morbidity and mortality in solid-organ transplant recipients despite enhanced facilities for perioperative care. The aim of this study was to evaluate the demographic characteristics, clinical course, and outcomes of renal transplant recipients with pneumonia. MATERIALS AND METHODS: The medical records of all renal transplant recipients from January 2010 to December 2014 were retrospectively reviewed, and patients diagnosed with pneumonia according to Centers for Disease Control and Prevention criteria were evaluated. Pneumonia was classified as community acquired or nosocomial. Patient demographics, microbiologic findings, need for intensive care/mechanical ventilation over the course of treatment, and information about clinical follow-up and mortality were all recorded. RESULTS: Eighteen (13.4%) of 134 renal transplant recipients had 25 pneumonia episodes within the study period. More than half (56%) of the pneumonia episodes developed within the first 6 months of transplant, whereas 44% developed after 6 months (all > 1 year). Eight cases (32%) were considered nosocomial pneumonia, and 17 (68%) were considered community-acquired pneumonia. Bacteria were the most common cause of pneumonia (28%), and fungi ranked second (8%). No viral or mycobacterial agents were detected. No patients required prolonged mechanical ventilation. No statistically significant difference was found in the need for intensive care or regarding mortality between patients with nosocomial and community-acquired pneumonia. Two patients (11%) died, and all remaining patients recovered. CONCLUSIONS: The present study confirmed that pneumonia after renal transplant is not a rare complication but a significant cause of morbidity. Long-term and close follow-up for pneumonia is necessary after renal transplant.

Liver Transplant and Reexpansion Pulmonary Edema: A Case Report.

Hydrothorax occurs frequently in patients with endstage liver disease and usually requires drainage of pulmonary effusion during the hepatectomy phase of liver transplant. Reexpansion pulmonary edema is a rare but potentially fatal complication seen after rapid reexpansion of the collapsed lung following thoracentesis of pleural fluid or tube drainage of pneumothorax. This condition, which manifests with various degrees of clinical severity, is rarely reported following liver transplantation. Herein, we present a 62-year-old male patient who developed reexpansion pulmonary edema after drainage of massive pleural effusion, which caused a total collapse in the right hemithorax during liver transplant. Six hours after pleural fluid drainage, the patient developed a nonproductive cough, mild tachypnea, shortness of breath, and low oxygen saturation (88%). His chest radiograph showed diffuse heterogeneous opacities in the right hemithorax. Computed tomography of the thorax revealed consolidations containing air bronchograms and ground glass opacities in the parenchyma of the right lung; these findings did not extend to the periphery and were observed less frequently in the inferoposterior left lung. These symptoms and radiologic findings were diagnosed as reexpansion pulmonary edema. Complete clinical and radiologic improvements were achieved within 72 hours of mechanical ventilatory support.

Genome-wide identification and comparative expression analysis of LEA genes in watermelon and melon genomes.

Late embryogenesis abundant (LEA) proteins are large and diverse group of polypeptides which were first identified during seed dehydration and then in vegetative plant tissues during different stress responses. Now, gene family members of LEA proteins have been detected in various organisms. However, there is no report for this protein family in watermelon and melon until this study. A total of 73 LEA genes from watermelon (ClLEA) and 61 LEA genes from melon (CmLEA) were identified in this comprehensive study. They were classified into four and three distinct clusters in watermelon and melon, respectively. There was a correlation between gene structure and motif composition among each LEA groups. Segmental duplication played an important role for LEA gene expansion in watermelon. Maximum gene ontology of LEA genes was observed with poplar LEA genes. For evaluation of tissue specific expression patterns of ClLEA and CmLEA genes, publicly available RNA-seq data were analyzed. The expression analysis of selected LEA genes in root and leaf tissues of drought-stressed watermelon and melon were examined using qRT-PCR. Among them, ClLEA-12-17-46 genes were quickly induced after drought application. Therefore, they might be considered as early response genes for water limitation conditions in watermelon. In addition, CmLEA-42-43 genes were found to be up-regulated in both tissues of melon under drought stress. Our results can open up new frontiers about understanding of functions of these important family members under normal developmental stages and stress conditions by bioinformatics and transcriptomic approaches.

Radiation recall pneumonitis caused by erlotinib after palliative definitive radiotherapy.

BACKGROUND: Radiation recall pneumonitis (RRP) occurs in a previously irradiated field and is triggered by certain cytotoxic drugs, principally chemotherapeutic agents such as erlotinib. Erlotinib is a reversible epidermal growth factor receptor tyrosine kinase inhibitor (TKI) and is an effective second-line treatment for patients with advanced-stage non-squamous-cell lung cancer. Previously, only 2 cases of radiation recall after erlotinib treatment have been reported. Here, we report a case of RRP caused by treatment with erlotinib 4 months after palliative definitive hypofractionated radiation therapy (RT). PATIENT AND METHODS: A 58-year-old male patient with non-small cell lung cancer (adenocarcinoma) was treated with polychemotherapy, palliative RT (30 Gy in 10 fractions), and erlotinib thereafter. RESULTS: Dosimetric analysis obtained from a 3-dimensional conformal RT planning system revealed that the volume of lung receiving at least 20 Gy (V20) was 21.2% and the mean lung dose was 12.7 Gy. These data indicate that systemic administration of a TKI, even after palliative RT, may lead to unexpected toxicity when the radiation field encompasses visceral organs. CONCLUSION: We conclude that the use of a TKI after RT may trigger radiation pneumonitis. Although evidence is limited, we advise clinicians to be cautious of RRP after erlotinib treatment.