The Influence of Human IgG Subclass and Allotype on Complement Activation.

Complement activation via the classical pathway is initiated when oligomeric Igs on target surfaces are recognized by C1 of the complement cascade. The strength of this interaction and activation of the complement system are influenced by structural variation of the Ab, including Ab isotype, subclass, and glycosylation profile. Polymorphic variants of IgG have also been described to influence Fc-dependent effector functions. Therefore, we assessed complement binding, deposition, and complement-dependent cytotoxicity (CDC) of 27 known IgG allotypes with anti-trinitrophenyl specificity. Differences between allotypes within subclasses were minor for IgG1, IgG3, and IgG4 allotypes, and more substantial for IgG2. Allelic variant IGHG2*06, containing a unique serine at position 378 in the CH3 domain, showed less efficient complement activation and CDC compared with other IgG2 polymorphisms. We also observed variable cell lysis between IgG1 and IgG3, with IgG3 being superior in lysis of human RBCs and Ramos cells, and IgG1 being superior in lysis of Raji and Wien133 cells, demonstrating that a long-standing conundrum in the literature depends on cellular context. Furthermore, we compared IgG1 and IgG3 under different circumstances, showing that Ag density and Ab hinge length, but not complement regulators, define the context dependency of Ab-mediated CDC activity. Our results point toward a variation in the capacity of IgG subclasses to activate complement due to single amino acid changes and hinge length differences of allotypes to activate complement, which might give new insights on susceptibility to infectious, alloimmune, or autoimmune diseases and aid the design of Ab-based therapeutics.

Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice.

Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency.

Ephrin-Eph signaling usage by a variety of viruses.

Ephrin-Eph signaling is a receptor tyrosine kinase signaling pathway involved in a variety of cellular mechanisms, of which many are related to the adhesion or migration of cells. Both the Eph receptor and ephrin ligand are abundantly present on a wide variety of cell types, and strongly evolutionary conserved. This review provides an overview of how 18 genetically diverse viruses utilize the Eph receptor (Eph), ephrin ligand (ephrin) or ephrin-Eph signaling to their advantage in their viral life cycle. Both Ephs and ephrins have been shown to serve as entry receptors for a variety of viruses, via both membrane fusion and endocytosis. Ephs and ephrins are also involved in viral transmission by vectors, associated with viral replication or persistence and lastly to neurological damage caused by viral infection. Although therapeutic opportunities targeting Ephs or ephrins do not seem feasible yet, the current research does propose two models for the viral usage of ephrin-Eph signaling. Firstly, the viral entry model, in which membrane molecules are used for viral entry, leading to cells being used for replication or as a transporter. Secondly, the advantageous expression ephrin-Eph signaling model, where viruses adapt the expression of Ephs or ephrins to change cell-cell interaction to their advantage. These models can guide future research questions on the usage of Ephs or ephrins by viruses and therapeutic opportunities.

Oncologic Procedures Amenable to Fluorescence-guided Surgery.

OBJECTIVE: Although fluorescence imaging is being applied to a wide range of cancers, it remains unclear which disease populations will benefit greatest. Therefore, we review the potential of this technology to improve outcomes in surgical oncology with attention to the various surgical procedures while exploring trial endpoints that may be optimal for each tumor type. BACKGROUND: For many tumors, primary treatment is surgical resection with negative margins, which corresponds to improved survival and a reduction in subsequent adjuvant therapies. Despite unfavorable effect on patient outcomes, margin positivity rate has not changed significantly over the years. Thus, patients often experience high rates of re-excision, radical resections, and overtreatment. However, fluorescence-guided surgery (FGS) has brought forth new light by allowing detection of subclinical disease not readily visible with the naked eye. METHODS: We performed a systematic review of clinicatrials.gov using search terms "fluorescence," "image-guided surgery," and "near-infrared imaging" to identify trials utilizing FGS for those received on or before May 2016. INCLUSION CRITERIA: fluorescence surgery for tumor debulking, wide local excision, whole-organ resection, and peritoneal metastases. EXCLUSION CRITERIA: fluorescence in situ hybridization, fluorescence imaging for lymph node mapping, nonmalignant lesions, nonsurgical purposes, or image guidance without fluorescence. RESULTS: Initial search produced 844 entries, which was narrowed down to 68 trials. Review of literature and clinical trials identified 3 primary resection methods for utilizing FGS: (1) debulking, (2) wide local excision, and (3) whole organ excision. CONCLUSIONS: The use of FGS as a surgical guide enhancement has the potential to improve survival and quality of life outcomes for patients. And, as the number of clinical trials rise each year, it is apparent that FGS has great potential for a broad range of clinical applications.

Antibody-based imaging strategies for cancer.

Although mainly developed for preclinical research and therapeutic use, antibodies have high antigen specificity, which can be used as a courier to selectively deliver a diagnostic probe or therapeutic agent to cancer. It is generally accepted that the optimal antigen for imaging will depend on both the expression in the tumor relative to normal tissue and the homogeneity of expression throughout the tumor mass and between patients. For the purpose of diagnostic imaging, novel antibodies can be developed to target antigens for disease detection, or current FDA-approved antibodies can be repurposed with the covalent addition of an imaging probe. Reuse of therapeutic antibodies for diagnostic purposes reduces translational costs since the safety profile of the antibody is well defined and the agent is already available under conditions suitable for human use. In this review, we will explore a wide range of antibodies and imaging modalities that are being translated to the clinic for cancer identification and surgical treatment.

A ratiometric threshold for determining presence of cancer during fluorescence-guided surgery.

BACKGROUND AND OBJECTIVE: Fluorescence-guided imaging to assist in identification of malignant margins has the potential to dramatically improve oncologic surgery. However, a standardized method for quantitative assessment of disease-specific fluorescence has not been investigated. Introduced here is a ratiometric threshold derived from mean fluorescent tissue intensity that can be used to semi-quantitatively delineate tumor from normal tissue. METHODS: Open-field and a closed-field imaging devices were used to quantify fluorescence in punch biopsy tissues sampled from primary tumors collected during a phase 1 trial evaluating the safety of cetuximab-IRDye800 in patients (n = 11) undergoing surgical intervention for head and neck cancer. Fluorescence ratios were calculated using mean fluorescence intensity (MFI) from punch biopsy normalized by MFI of patient-matched tissues. Ratios were compared to pathological assessment and a ratiometric threshold was established to predict presence of cancer. RESULTS: During open-field imaging using an intraoperative device, the threshold for muscle normalized tumor fluorescence was found to be 2.7, which produced a sensitivity of 90.5% and specificity of 78.6% for delineating disease tissue. The skin-normalized threshold generated greater sensitivity (92.9%) and specificity (81.0%). CONCLUSION: Successful implementation of a semi-quantitative threshold can provide a scientific methodology for delineating disease from normal tissue during fluorescence-guided resection of cancer.

Laparoscopic Fluorescent Visualization of the Ureter With Intravenous IRDye800CW.

OBJECTIVES: Ureter injury is a serious complication of laparoscopic surgery. Current strategies to identify the ureters, such as placement of a ureteral stent, carry additional risks for patients. We hypothesize that the systemically injected near-infrared (NIR) dye IRDye800CW-CA can be used to visualize ureters intraoperatively. METHODS: Adult female mixed-breed pigs weighing 24 to 41 kg (n = 2 per dose) were given a 30, 60, or 120 µg/kg systemic injection of IRDye800CW-CA. Using the Food and Drug Administration-cleared Pinpoint laparoscopic NIR system, images of the ureter and bladder were captured every 10 minutes for 60 minutes after injection. To determine the biodistribution of the dye, tissues were collected for ex vivo analysis with the Pearl Impulse system. ImageJ software was used to quantify fluorescence signal and signal-to-background ratio (SBR) for the intraoperative images. RESULTS: The ureter was identified in all pigs at each dose, with peak intensity reached by 30 minutes and remaining elevated throughout the duration of imaging (60 minutes). The 60 µg/kg dose was determined to be optimal for differentiating ureters according to absolute fluorescence (>60 counts/pixel) and SBR (3.1). Urine fluorescence was inversely related to plasma fluorescence (R(2) = -0.82). Ex vivo imaging of kidney, ureter, bladder, and abdominal wall tissues revealed low fluorescence. CONCLUSION: Systemic administration of IRDye800CW-CA shows promise in providing ureteral identification with high specificity during laparoscopic surgery. The low dose required, rapid time to visualization, and absence of invasive ureteral instrumentation inherent to this technique may reduce complications related to pelvic surgery.

Fluorescence-guided resection of experimental malignant glioma using cetuximab-IRDye 800CW.

The standard treatment for glioblastoma multiforme (GBM) remains maximal safe surgical resection. Here, we evaluated the ability of a systemically administered antibody-dye probe conjugate (cetuximab-IRDye 800CW) to provide sufficient fluorescent contrast for surgical resection of disease in both subcutaneous and orthotopic animal models of GBM. Multiple luciferase-positive GBM cell lines (D-54MG, U-87MG, and U-251MG; n = 5) were implanted in mouse flank and tumors were fluorescently imaged daily using a closed-field near-infrared (NIR) system after cetuximab-IRDye 800CW systemic administration. Orthotopic models were also generated (n = 5), and tumor resection was performed under white light and fluorescence guidance using an FDA-approved wide-field NIR imaging system. Residual tumor was monitored using luciferase imaging. Immunohistochemistry was performed to characterize tumor fluorescence, epidermal growth factor receptor (EGFR) expression, and vessel density. Daily imaging of tumors revealed an average tumor-to-background (TBR) of 4.5 for U-87MG, 4.1 for D-54MG, and 3.7 for U-251MG. Fluorescence intensity within the tumors peaked on day-1 after cetuximab-IRDye 800CW administration, however the TBR increased over time in two of the three cell lines. For the orthotopic model, TBR on surgery day ranged from 19 to 23 during wide-field, intraoperative imaging. Surgical resection under white light on day 3 after cetuximab-IRDye 800CW resulted in an average 41% reduction in luciferase signal while fluorescence-guided resection using wide-field NIR imaging resulted in a significantly (P = 0.001) greater reduction in luciferase signal (87%). Reduction of luciferase signal was found to correlate (R (2) = 0.99) with reduction in fluorescence intensity. Fluorescence intensity was found to correlate (P < 0.05) with EGFR expression in D-54MG and U-251MG tumor types but not U-87MG. However, tumor fluorescence was found to correlate with vessel density for the U-87MG tumors. Here we show systemic administration of cetuximab-IRDye 800CW in combination with wide-field NIR imaging provided robust and specific fluorescence contrast for successful localization of disease in subcutaneous and orthotopic animal models of GBM.

A case of pulmonary tumor embolism syndrome with thrombus in transit.

The incidence of pulmonary tumor embolism in patients with solid tumors is estimated to be between 3% and 26% yet is rarely diagnosed. In this case, a 74-year-old male with sarcomatoid variant of urothelial carcinoma and recently diagnosed left renal vein thrombus treated with low-molecular-weight-heparin, presented to the emergency department with acute syncope and dyspnea. He was found to have CT imaging of segmental and subsegmental arterial filling defects, a right atrial filling defect concerning for thrombus in transit and was diagnosed with pulmonary tumor embolism syndrome. The patient was treated with aspiration thrombectomy, with pathology demonstrating sarcomatoid urothelial carcinoma cells. He was initiated on a combination of gemcitabine plus carboplatin to decrease the tumor burden. While pulmonary tumor embolism syndrome is associated with a poor prognosis, prompt diagnosis and initiation of cancer-specific therapies can significantly improve survival.

C1-inhibitor treatment in patients with severe complement-mediated autoimmune hemolytic anemia.

Complement-mediated (CM) autoimmune hemolytic anemia (AIHA) is characterized by the destruction of red blood cells (RBCs) by autoantibodies that activate the classical complement pathway. These antibodies also reduce transfusion efficacy via the lysis of donor RBCs. Because C1-inhibitor (C1-INH) is an endogenous regulator of the classical complement pathway, we hypothesized that peritransfusional C1-INH in patients with severe CM-AIHA reduces complement activation and hemolysis, and thus enhances RBC transfusion efficacy. We conducted a prospective, single-center, phase 2, open-label trial (EudraCT2012-003710-13). Patients with confirmed CM-AIHA and indication for the transfusion of 2 RBC units were eligible for inclusion. Four IV C1-INH doses (6000, 3000, 2000, and 1000 U) were administered with 12-hour intervals around RBC transfusion. Serial blood samples were analyzed for hemolytic activity, RBC opsonization, complement activation, and inflammation markers. Ten patients were included in the study. C1-INH administration increased plasma C1-INH antigen and activity, peaking at 48 hours after the first dose and accompanied by a significant reduction of RBC C3d deposition. Hemoglobin levels increased briefly after transfusion but returned to baseline within 48 hours. Overall, markers of hemolysis, inflammation, and complement activation remained unchanged. Five grade 3 and 1 grade 4 adverse event occurred but were considered unrelated to the study medication. In conclusion, peritransfusional C1-INH temporarily reduced complement activation. However, C1-INH failed to halt hemolytic activity in severe transfusion-dependent-CM-AIHA. We cannot exclude that posttransfusional hemolytic activity would have been even higher without C1-INH. The potential of complement inhibition on transfusion efficacy in severe CM-AIHA remains to be determined.

The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation.

Objectives: The complement system is an important component of innate immunity. The alternative pathway (AP) amplification loop is considered an essential feed forward mechanism for complement activation. However, the role of the AP in classical pathway (CP) activation has only been studied in ELISA settings. Here, we investigated its contribution on physiologically relevant surfaces of human cells and bacterial pathogens and in antibody-mediated complement activation, including in autoimmune haemolytic anaemia (AIHA) setting with autoantibodies against red blood cells (RBCs). Methods: We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP-depleted sera or antibodies against factor B and factor D. Results: We show that the amplification loop is redundant when efficient CP activation takes place. This is independent of the presence of membrane-bound complement regulators. The role of the AP may become significant when insufficient CP complement activation occurs, but this depends on antibody levels and (sub)class. Our data indicate that therapeutic intervention in the amplification loop will most likely not be effective to treat antibody-mediated diseases. Conclusion: The AP can be bypassed through efficient CP activation. The AP amplification loop has a role in complement activation during conditions of modest activation via the CP, when it can allow for efficient complement-mediated killing.

Signatures of Mitochondrial Dysfunction and Impaired Fatty Acid Metabolism in Plasma of Patients with Post-Acute Sequelae of COVID-19 (PASC).

Exercise intolerance is a major manifestation of post-acute sequelae of severe acute respiratory syndrome coronavirus infection (PASC, or "long-COVID"). Exercise intolerance in PASC is associated with higher arterial blood lactate accumulation and lower fatty acid oxidation rates during graded exercise tests to volitional exertion, suggesting altered metabolism and mitochondrial dysfunction. It remains unclear whether the profound disturbances in metabolism that have been identified in plasma from patients suffering from acute coronavirus disease 2019 (COVID-19) are also present in PASC. To bridge this gap, individuals with a history of previous acute COVID-19 infection that did not require hospitalization were enrolled at National Jewish Health (Denver, CO, USA) and were grouped into those that developed PASC (n = 29) and those that fully recovered (n = 16). Plasma samples from the two groups were analyzed via mass spectrometry-based untargeted metabolomics and compared against plasma metabolic profiles of healthy control individuals (n = 30). Observational demographic and clinical data were retrospectively abstracted from the medical record. Compared to plasma of healthy controls or individuals who recovered from COVID-19, PASC plasma exhibited significantly higher free- and carnitine-conjugated mono-, poly-, and highly unsaturated fatty acids, accompanied by markedly lower levels of mono-, di- and tricarboxylates (pyruvate, lactate, citrate, succinate, and malate), polyamines (spermine) and taurine. Plasma from individuals who fully recovered from COVID-19 exhibited an intermediary metabolic phenotype, with milder disturbances in fatty acid metabolism and higher levels of spermine and taurine. Of note, depletion of tryptophan-a hallmark of disease severity in COVID-19-is not normalized in PASC patients, despite normalization of kynurenine levels-a tryptophan metabolite that predicts mortality in hospitalized COVID-19 patients. In conclusion, PASC plasma metabolites are indicative of altered fatty acid metabolism and dysfunctional mitochondria-dependent lipid catabolism. These metabolic profiles obtained at rest are consistent with previously reported mitochondrial dysfunction during exercise, and may pave the way for therapeutic intervention focused on restoring mitochondrial fat-burning capacity.

Therapeutic Lessons to be Learned From the Role of Complement Regulators as Double-Edged Sword in Health and Disease.

The complement system is an important part of the innate immune system, providing a strong defense against pathogens and removing apoptotic cells and immune complexes. Due to its strength, it is important that healthy human cells are protected against damage induced by the complement system. To be protected from complement, each cell type relies on a specific combination of both soluble and membrane-bound regulators. Their importance is indicated by the amount of pathologies associated with abnormalities in these complement regulators. Here, we will discuss the current knowledge on complement regulatory protein polymorphisms and expression levels together with their link to disease. These diseases often result in red blood cell destruction or occur in the eye, kidney or brain, which are tissues known for aberrant complement activity or regulation. In addition, complement regulators have also been associated with different types of cancer, although their mechanisms here have not been elucidated yet. In most of these pathologies, treatments are limited and do not prevent the complement system from attacking host cells, but rather fight the consequences of the complement-mediated damage, using for example blood transfusions in anemic patients. Currently only few drugs targeting the complement system are used in the clinic. With further demand for therapeutics rising linked to the wide range of complement-mediated disease we should broaden our horizon towards treatments that can actually protect the host cells against complement. Here, we will discuss the latest insights on how complement regulators can benefit therapeutics. Such therapeutics are currently being developed extensively, and can be categorized into full-length complement regulators, engineered complement system regulators and antibodies targeting complement regulators. In conclusion, this review provides an overview of the complement regulatory proteins and their links to disease, together with their potential in the development of novel therapeutics.

Cytological analysis of interference in mouse meiosis.

In most eukaryotes, meiotic crossovers (COs) are non-randomly placed along the bivalents, such that the presence of a CO reduces the probability of additional COs nearby. This phenomenon, named CO interference, was originally defined genetically, but can also be analyzed cytologically by studying the chromosomal positions of protein complexes that are involved in CO formation, or by studying the positions of chiasmata. Here we focus on the cytological analysis of interference among protein complexes involved in meiotic recombination and CO formation in the mouse. During the pachytene stage of meiosis, these protein complexes can be visualized as immunofluorescent foci along synaptonemal complexes (SCs), which are linear protein structures that are formed along homologous chromosome pairs (bivalents) during meiotic prophase. We describe how to make cytological preparations that are suitable for the analysis of interference among these foci, and how to estimate the strength of interference among foci, using the gamma distribution as a mathematical model for focus/CO positioning.

Protocol for Chromatin Immunoprecipitation of Meiotic-Stage-Specific Tomato Anthers.

Interactions occurring between DNA and proteins across the nuclear genome regulate numerous processes, including meiosis. Meiosis ensures genetic variation and balanced segregation of homologous chromosomes. It involves complex DNA-protein interactions across the entire genome to regulate a broad range of processes, including formation and repair of double-strand DNA breaks (DSBs), chromosome compaction, homolog pairing, synapsis, and homologous recombination. The latter meiotic event, meiotic recombination, often occurs at discrete locations in a genome, within a tight time window. The identification of genomic binding sites of meiotic proteins is a major step toward understanding the molecular mechanisms underlying meiotic recombination and provides important information for plant breeding. Collecting meiotic cells from plants is challenging, tedious, and time consuming, since the meiocyte-producing organs, the anthers, are generally small and limited to certain developmental stages of plants. Here we provide a protocol to isolate meiotic-stage-specific anthers and perform ChIP on this material. We have developed a ChIP protocol specifically suited to (1) small amounts of input material and (2) proteins that bind transiently to chromatin and at very low frequency. © 2018 by John Wiley & Sons, Inc.

Sugar-sweetened beverages intake is associated with blood pressure and sympathetic nervous system activation in children.

INTRODUCTION: Research in adults shows high intakes of sugar-sweetened beverages (SSBs) increases blood pressure (BP), however evidence in children is scarce and inconclusive. No studies have investigated autonomic nervous system (ANS) activation due to SSBs in children. Our aim was to investigate the associations between SSBs intake, BP and ANS activation in children. METHODS: Cross-sectional data collected in 2008-2010 at age 5/6 (n = 2519) and in 2015-2016 at age 11/12 (n = 769) years in the Amsterdam Born Children and their Development-study were analyzed in 2017. Systolic blood pressure (SBP) and diastolic blood pressure (DBP), as well as pre-ejection period (PEP; sympathetic activation) and respiratory sinus arrhythmia (RSA; parasympathetic activation) were measured during supine position. SSBs intake was self-reported. RESULTS: After adjustments for covariates, including weight status, no associations between SSBs intake, BP and ANS measures were found at age 5/6. At age 11/12, one serving/day increase in SSBs was associated with 0.8 mmHg increase in SBP (95%CI:0.4; 1.2), 0.3 mmHg increase in DBP (0.0; 0.5) and 0.9 msec decrease in PEP (-1.5;-0.2). Children in the highest SSBs tertile had an increase of 2.3 mmHg in SBP (0.7; 4.0) and a decrease of 3.6 msec in PEP (-6.7;-0.6) compared to children in the lowest tertile. CONCLUSION: In children aged 11/12, high intake of SSBs was associated with increased BP, which might be due to increased sympathetic nervous system activation. Importantly, these associations were independent of weight status.