Comparative performance of scFv-based anti-BCMA CAR formats for improved T cell therapy in multiple myeloma.

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

Antibody-blocking of a tick transporter impairs Anaplasma phagocytophilum colonization in Haemaphysalis longicornis ticks.

The invasive Asian longhorned tick Haemaphysalis longicornis that vectors and transmits several animal pathogens is significantly expanding in the United States. Recent studies report that these ticks also harbor human pathogens including Borrelia burgdorferi sensu lato, Babesia microti, and Anaplasma phagocytophilum. Therefore, studies that address the interactions of these ticks with human pathogens are important. In this study, we report the characterization of H. longicornis organic anion-transporting polypeptides (OATPs) in interactions of these ticks with A. phagocytophilum. Using OATP-signature sequence, we identified six OATPs in the H. longicornis genome. Bioinformatic analysis revealed that H. longicornis OATPs are closer to other tick orthologs rather than to mammalian counterparts. Quantitative real-time PCR analysis revealed that OATPs are highly expressed in immature stages when compared to mature stages of these ticks. In addition, we noted that the presence of A. phagocytophilum upregulates a specific OATP in these ticks. We also noted that exogenous treatment of H. longicornis with xanthurenic acid, a tryptophan metabolite, influenced OATP expression in these ticks. Immunoblotting analysis revealed that antibody generated against Ixodes scapularis OATP cross-reacted with H. longicornis OATP. Furthermore, treatment of H. longicornis with OATP antibody impaired colonization of A. phagocytophilum in these ticks. These results not only provide evidence that the OATP-tryptophan pathway is important for A. phagocytophilum survival in H. longicornis ticks but also indicate OATP as a promising candidate for the development of a universal anti-tick vaccine to target this bacterium and perhaps other rickettsial pathogens of medical importance.

Gn protein expressed in plants for diagnosis of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes the highly fatal disease in humans. To facilitate diagnosis, the native form of subunit glycoprotein (Gn), a prime target for potential vaccines and therapies, was produced in Nicotiana benthamiana using a Bamboo mosaic virus-based vector system. By fusion with secretory signal tags, SSExt, derived from the extension protein, and the (SP)10 motif, the yield of the recombinant Gn (rGn) was remarkably increased to approximately 7 mg/kg infiltrated leaves. Ultimately, an rGn-based ELISA was successfully established for the detection of SFTSV-specific antibodies in serum samples from naturally infected monkeys. As validated with the reference method, the specificity and sensitivity of rGn-ELISA were 94% and 96%, respectively. In conclusion, utilizing well-suited fusion tags facilitates rGn production and purification in substantial quantities while preserving its antigenic properties. The rGn-ELISA, characterized by its commendable sensitivity and specificity could serve as a viable alternative diagnostic method for assessing SFTSV seroprevalence. KEY POINTS: • SFTSV Gn, fused with secretory signal tags, was expressed by the BaMV-based vector. • The plant fusion tags increased expression levels and eased the purification of rGn. • The rGn-ELISA was established and validated; its specificity and sensitivity > 94%.

Noncovalently particle-anchored cytokines with prolonged tumor retention safely elicit potent antitumor immunity.

Preclinical studies have shown that immunostimulatory cytokines elicit antitumor immune responses but their clinical use is limited by severe immune-related adverse events upon systemic administration. Here, we report a facile and versatile strategy for noncovalently anchoring potent Fc-fused cytokine molecules to the surface of size-discrete particles decorated with Fc-binding peptide for local administration. Following intratumoral injection, particle-anchored Fc cytokines exhibit size-dependent intratumoral retention. The 1-micrometer particle prolongs intratumoral retention of Fc cytokine for over a week and has minimal systemic exposure, thereby eliciting antitumor immunity while eliminating systemic toxicity caused by circulating cytokines. In addition, the combination of these particle-anchored cytokines with immune checkpoint blockade antibodies safely promotes tumor regression in various syngeneic tumor models and genetically engineered murine tumor models and elicits systemic antitumor immunity against tumor rechallenge. Our formulation strategy renders a safe and tumor-agnostic approach that uncouples cytokines' immunostimulatory properties from their systemic toxicities for potential clinical application.

Efficient EVs separation and detection by an alumina-nanochannel-array-membrane integrated microfluidic chip and an antibody barcode biochip.

BACKGROUND: Small endosome-derived lipid nanovesicles (30-200 nm) are actively secreted by living cells and serve as pivotal biomarkers for early cancer diagnosis. However, the study of extracellular vesicles (EVs) requires isolation and purification from various body fluids. Although traditional EVs isolation and detection technologies are mature, they usually require large amount of sample, consumes long-time, and have relatively low-throughput. How to efficiently isolate, purify and detect these structurally specific EVs from body fluids with high-throughput remains a great challenge in in vitro diagnostics and clinical research. RESULTS: Herein, we suggest a nanosized microfluidic device for efficient and economical EVs filtration based on an alumina nanochannel array membrane. We evaluated the filtration device performance of alumina membranes with different diameters and found that an optimized chamber array with a hydrophilic-treated channel diameter of 90 nm could realize a filtration efficiency of up to 82% without any assistance from chemical or physical separation methods. Importantly, by integrating meticulously designed multichannel microfluidic biochips, EVs can be captured in-situ and monitored by antibody barcode biochip. The proposed filtration chip together with the high-throughput detection chip were capable of filtration of a few tens of µL samples and recognition of different phonotypes. The practical filtration and detection of EVs from clinical samples demonstrated the high performance of the device. SIGNIFICANT: Overall, this work provides a cost-effective, highly efficient and automated EVs filtration chip and detection dual-function integrated chip platform, which can directly separate EVs from serum or cerebrospinal fluid with an efficiency of 82% and conduct in-situ detection. This small fluidic device can provide a powerful tool for highly efficient identifying and analyzing EVs, presenting great application potential in clinical detection.

Machine learning evaluation for identification of M-proteins in human serum.

Serum electrophoresis (SPEP) is a method used to analyze the distribution of the most important proteins in the blood. The major clinical question is the presence of monoclonal fraction(s) of antibodies (M-protein/paraprotein), which is essential for the diagnosis and follow-up of hematological diseases, such as multiple myeloma. Recent studies have shown that machine learning can be used to assess protein electrophoresis by, for example, examining protein glycan patterns to follow up tumor surgery. In this study we compared 26 different decision tree algorithms to identify the presence of M-proteins in human serum by using numerical data from serum protein capillary electrophoresis. For the automated detection and clustering of data, we used an anonymized data set consisting of 67,073 samples. We found five methods with superior ability to detect M-proteins: Extra Trees (ET), Random Forest (RF), Histogram Grading Boosting Regressor (HGBR), Light Gradient Boosting Method (LGBM), and Extreme Gradient Boosting (XGB). Additionally, we implemented a game theoretic approach to disclose which features in the data set that were indicative of the resulting M-protein diagnosis. The results verified the gamma globulin fraction and part of the beta globulin fraction as the most important features of the electrophoresis analysis, thereby further strengthening the reliability of our approach. Finally, we tested the algorithms for classifying the M-protein isotypes, where ET and XGB showed the best performance out of the five algorithms tested. Our results show that serum capillary electrophoresis combined with decision tree algorithms have great potential in the application of rapid and accurate identification of M-proteins. Moreover, these methods would be applicable for a variety of blood analyses, such as hemoglobinopathies, indicating a wide-range diagnostic use. However, for M-protein isotype classification, combining machine learning solutions for numerical data from capillary electrophoresis with gel electrophoresis image data would be most advantageous.

Pathological findings with vacuoles in anti-mitochondrial antibody-positive inflammatory myopathy.

BACKGROUND: A few patients with inflammatory myopathy showed anti-mitochondrial antibody (AMA) positivity. This study aimed to report the clinical and pathological findings with vacuoles in 3 cases of such patients. METHODS: Three cases with myositis from the Myositis Clinical Database of Peking University First Hospital were identified with AMA positivity. Their clinical records were retrospectively reviewed and the data was extracted. All the 3 cases underwent muscle biopsy. RESULTS: Three middle-aged patients presented with chronic-onset weakness of proximal limbs, marked elevation of creatine kinase, and AMA-positivity. Two of the 3 cases meet the criteria of primary biliary cholangitis. All the 3 cases presented with cardiac involvement and proteinuria. Two cases developed type 2 respiratory failure. MRI of the thigh muscle showed multiple patches of edema bilaterally in both cases, mostly in the adductor magnus. Pathological findings include degeneration of muscle fibers, diffused MHC-I positivity, and complement deposits on cell membranes. Vacuoles without rims of different sizes were discovered under the membrane of the muscle fibers. A few RBFs were discovered in case 1, while a diffused proliferation of endomysium and perimysium was shown in case 2. CONCLUSIONS: AMA-positive inflammatory myopathy is a disease that could affect multiple systems. Apart from inflammatory changes, the pathological findings of muscle can also present vacuoles.

SERS biosensor with plastic antibodies for detection of a cancer biomarker protein.

Surface-enhanced Raman scattering (SERS) is a powerful method for detecting breast cancer-specific biomarkers due to its extraordinary enhancement effects obtained by localized surface plasmon resonance (LSPR) in metallic nanostructures at hotspots. In this research, gold nanostars (AuNSs) were used as SERS probes to detect a cancer biomarker at very low concentrations. To this end, we combined molecularly imprinted polymers (MIPs) as a detection layer with SERS for the detection of the biomarker CA 15-3 in point-of-care (PoC) analysis. This required two main steps: (i) the deposition of MIPs on a gold electrode, followed by a second step (ii) antibody binding with AuNSs containing a suitable Raman reporter to enhance Raman signaling (SERS). The MPan sensor was prepared by electropolymerization of the monomer aniline in the presence of CA 15-3. The template molecule was then extracted from the polymer using sodium dodecyl sulfate (SDS). In parallel, a control material was prepared in the absence of the protein (NPan). Surface modification for the control was performed using electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The performance of the sensor was evaluated using the SERS technique, in which the MPan sensor is first incubated with the protein and then exposed to the SERS probe. Under optimized conditions, the device showed a linear response to CA 15-3 concentrations from 0.016 to 248.51 U mL-1 in a PBS buffer at pH 7.4 in 1000-fold diluted serum. Overall, this approach demonstrates the potential of SERS as an optical reader and opens a new avenue for biosensing applications.

VISTA checkpoint inhibition by pH-selective antibody SNS-101 with optimized safety and pharmacokinetic profiles enhances PD-1 response.

VISTA, an inhibitory myeloid-T-cell checkpoint, holds promise as a target for cancer immunotherapy. However, its effective targeting has been impeded by issues such as rapid clearance and cytokine release syndrome observed with previous VISTA antibodies. Here we demonstrate that SNS-101, a newly developed pH-selective VISTA antibody, addresses these challenges. Structural and biochemical analyses confirmed the pH-selectivity and unique epitope targeted by SNS-101. These properties confer favorable pharmacokinetic and safety profiles on SNS-101. In syngeneic tumor models utilizing human VISTA knock-in mice, SNS-101 shows in vivo efficacy when combined with a PD-1 inhibitor, modulates cytokine and chemokine signaling, and alters the tumor microenvironment. In summary, SNS-101, currently in Phase I clinical trials, emerges as a promising therapeutic biologic for a wide range of patients whose cancer is refractory to current immunotherapy regimens.

Expression of Collagen XIII in Tissues of the Thyroid and Orbit With Relevance to Thyroid-Associated Ophthalmopathy.

Purpose: Antibodies against collagen XIII have previously been identified in patients with active thyroid-associated ophthalmopathy (TAO). Although collagen XIII expression has been described in extraocular muscles and orbital fat, its detailed localization in extraocular and thyroid tissues and the connection to autoimmunity for collagen XIII remain unclear. Our objective was to map the potential targets for these antibodies in the tissues of the orbit and thyroid. Methods: We evaluated the expression of collagen XIII in human patient and mouse orbital and thyroid tissues with immunostainings and RT-qPCR using Col13a1-/- mice as negative controls. COL13A1 expression in Graves' disease and goiter thyroid samples was compared with TGF-ß1 and TNF, and these were also studied in human thyroid epithelial cells and fibroblasts. Results: Collagen XIII expression was found in the neuromuscular and myotendinous junctions of extraocular muscles, blood vessels of orbital connective tissue and fat and the thyroid, and in the thyroid epithelium. Thyroid expression was also seen in germinal centers in Graves' disease and in neoplastic epithelium. The expression of COL13A1 in goiter samples correlated with levels of TGF-B1. Upregulation of COL13A1 was reproduced in thyroid epithelial cells treated with TGF-ß1. Conclusions: We mapped the expression of collagen XIII to various locations in the orbit, demonstrated its expression in the pathologies of the Graves' disease thyroid and confirmed the relationship between collagen XIII and TGF-ß1. Altogether, these data add to our understanding of the targets of anti-collagen XIII autoantibodies in TAO.

Brain Sample Preparation After Performing in Utero Electroporation to Proliferating and Differentiated Cells in the Developing Mouse Neocortex.

In utero electroporation (IUE) enables labeling and manipulating specific types of cells by introducing DNA plasmids with desired promoters. After the surgery, mouse brains are fixed at any stage and analyzed after staining using specific antibodies. Here, we describe the flow of the IUE experiment from the preparation to microscopic observations.

Identification of Inhibitors of the Disease-Associated Protein Phosphatase Scp1 Using Antibody Mimetic Molecules.

Protein phosphorylation is a prevalent translational modification, and its dysregulation has been implicated in various diseases, including cancer. Despite its significance, there is a lack of specific inhibitors of the FCP/SCP-type Ser/Thr protein phosphatase Scp1, characterized by high specificity and affinity. In this study, we focused on adnectin, an antibody-mimetic protein, aiming to identify Scp1-specific binding molecules with a broad binding surface that target the substrate-recognition site of Scp1. Biopanning of Scp1 was performed using an adnectin-presenting phage library with a randomized FG loop. We succeeded in identifying FG-1Adn, which showed high affinity and specificity for Scp1. Ala scanning analysis of the Scp1-binding sequence in relation to the FG-1 peptide revealed that hydrophobic residues, including aromatic amino acids, play important roles in Scp1 recognition. Furthermore, FG-1Adn was found to co-localize with Scp1 in cells, especially on the plasma membrane. In addition, Western blotting analysis showed that FG-1Adn increased the phosphorylation level of the target protein of Scp1 in cells, indicating that FG-1Adn can inhibit the function of Scp1. These results suggest that FG-1Adn can be used as a specific inhibitor of Scp1.

Thermodynamic and molecular dynamic insights into how fusion influences peptide-tag recognition of an antibody.

To understand the effect of protein fusion on the recognition of a peptide-tag by an antibody, we fused a CCR5-derived peptide-tag (pep1) to GFP and investigated its recognition by an anti-pep1 antibody, 4B08. First, to characterize the thermodynamic properties associated with the pep1-4B08 binding, isothermal titration calorimetry experiments were conducted. It was found that pep1 fused to the C-terminus of GFP (GFP-CT) enhanced the enthalpic gain by 2.1 kcal mol-1 and the entropic loss only by 0.9 kcal mol-1, resulting in an 8-fold increase in the binding affinity compared to the unfused pep1. On the other hand, pep1 fused to the N-terminus of GFP (GFP-NT) enhanced the enthalpic gain by 3.0 kcal mol-1 and the entropic loss by 3.2 kcal mol-1, leading to no significant enhancement of the binding affinity. To gain deeper insights, molecular dynamics simulations of GFP-NT, GFP-CT, and pep1 were performed. The results showed that the location of the fusion point sensitively affects the interaction energy, the solvent accessible surface area, and the fluctuation of pep1 in the unbound state, which explains the difference in the experimental thermodynamic properties.

The physiological interactome of TCR-like antibody therapeutics in human tissues.

Selective binding of TCR-like antibodies that target a single tumour-specific peptide antigen presented by human leukocyte antigens (HLA) is the absolute prerequisite for their therapeutic suitability and patient safety. To date, selectivity assessment has been limited to peptide library screening and predictive modeling. We developed an experimental platform to de novo identify interactomes of TCR-like antibodies directly in human tissues using mass spectrometry. As proof of concept, we confirm the target epitope of a MAGE-A4-specific TCR-like antibody. We further determine cross-reactive peptide sequences for ESK1, a TCR-like antibody with known off-target activity, in human liver tissue. We confirm off-target-induced T cell activation and ESK1-mediated liver spheroid killing. Off-target sequences feature an amino acid motif that allows a structural groove-coordination mimicking that of the target peptide, therefore allowing the interaction with the engager molecule. We conclude that our strategy offers an accurate, scalable route for evaluating the non-clinical safety profile of TCR-like antibody therapeutics prior to first-in-human clinical application.

SIRPB1 regulates inflammatory factor expression in the glioma microenvironment via SYK: functional and bioinformatics insights.

BACKGROUND: SIRPB1 expression is upregulated in various tumor types, including gliomas, and is known to contribute to tumor progression; nevertheless, its function in the immune milieu of gliomas is still mainly unknown. METHODS: This study, we analyzed 1152 normal samples from the GTEx database and 670 glioma samples from the TCGA database to investigate the relationship between the expression of SIRPB1 and clinicopathological features. Moreover, SIRPB1 gene knockout THP-1 cell lines were constructed using CRISPR/Cas9 and were induced into a co-culture of macrophages and glioma cells in vitro to learn more about the role of SIRPB1 in the glioma immune milieu. Lastly, we established a prognostic model to predict the effect of SIRPB1 on prognosis. RESULTS: Significantly higher levels of SIRPB1 expression were found in gliomas, which had an adverse effect on the immune milieu and correlated poorly with patient survival. SIRPB1 activation with certain antibodies results in SYK phosphorylation and the subsequent activation of calcium, MAPK, and NF-κB signaling pathways. This phenomenon is primarily observed in myeloid-derived cells as opposed to glioma cells. In vitro co-culture demonstrated that macrophages with SIRPB1 knockout showed decreased IL1RA, CCL2, and IL-8, which were recovered upon ectopic expression of SIRPB1 but reduced again following treatment with SYK inhibitor GS9973. Critically, a lower overall survival rate was linked to increased SIRPB1 expression. Making use of SIRPB1 expression along with additional clinicopathological variables, we established a nomogram that showed a high degree of prediction accuracy. CONCLUSIONS: Our study demonstrates that glioma cells can be activated by macrophages via SIRPB1, subsequently reprogramming the TME, suggesting that SIRPB1 could serve as a promising therapeutic target for gliomas.

Updated Evaluation of Agalsidase Alfa Enzyme Replacement Therapy for Patients with Fabry Disease: Insights from Real-World Data.

The clinical use of agalsidase alfa as enzyme replacement therapy (ERT) for Fabry disease (FD) has spread since 2001, and a large body of evidence of its effectiveness has been collected. This review presents the clinical and laboratory results achieved with agalsidase alfa, which has been published in the literature. Agalsidase alfa infusion slows down or stops the progression of renal damage, expressed by reduction or stabilization of the annual decline of the glomerular filtration rate; yearly decrease of glomerular filtration rate (slope) sometimes is reduced until its stabilization. ERT prevents or reduces the occurrence of hypertrophic cardiomyopathy or slows the increase over time if it is already present. Moreover, regarding neurological manifestations, ERT improves neuropathic pain and quality of life, and recent data indicated that it may also prevent the burden of cerebrovascular disease. In addition to ERT's clinical benefits, crucial topics like the most appropriate time to start therapy and the role of anti-drug antibodies (ADA) are analyzed. Treatment with agalsidase alfa in patients with FD substantially improves their outcomes and enhances their quality of life in patients with FD.

STING agonist diABZI enhances the cytotoxicity of T cell towards cancer cells.

Antigen-specific T cell receptor-engineered T cell (TCR-T) based immunotherapy has proven to be an effective method to combat cancer. In recent years, cross-talk between the innate and adaptive immune systems may be requisite to optimize sustained antigen-specific immunity, and the stimulator of interferon genes (STING) is a promising therapeutic target for cancer immunotherapy. The level of expression or presentation of antigen in tumor cells affects the recognition and killing of tumor cells by TCR-T. This study aimed at investigating the potential of innate immune stimulation of T cells and engineered T cells to enhance immunotherapy for low-expression antigen cancer cells. We systematically investigated the function and mechanism of cross-talk between STING agonist diABZI and adaptive immune systems. We established NY-ESO-1 full knockout Mel526 cells for this research and found that diABZI activated STING media and TCR signaling pathways. In addition, the results of flow cytometry showed that antigens presentation from cancer cells induced by STING agonist diABZI also improved the affinity of TCR-T cells function against tumor cells in vitro and in vivo. Our findings revealed that diABZI enhanced the immunotherapy efficacy of TCR-T by activating STING media and TCR signaling pathways, improving interferon-γ expression, and increasing antigens presentation of tumor cells. This indicates that STING agonist could be used as a strategy to promote TCR-T cancer immunotherapy.

Unlocking PD-1 antibody resistance: The MUC1 DNA vaccine augments CD8+ T cell infiltration and attenuates tumour suppression.

In light of increasing resistance to PD1 antibody therapy among certain patient populations, there is a critical need for in-depth research. Our study assesses the synergistic effects of a MUC1 DNA vaccine and PD1 antibody for surmounting PD1 resistance, employing a murine CT26/MUC1 colon carcinoma model for this purpose. When given as a standalone treatment, PD1 antibodies showed no impact on tumour growth. Additionally, there was no change observed in the intra-tumoural T-cell ratios or in the functionality of T-cells. In contrast, the sole administration of a MUC1 DNA vaccine markedly boosted the cytotoxicity of CD8+ T cells by elevating IFN-γ and granzyme B production. Our compelling evidence highlights that combination therapy more effectively inhibited tumour growth and prolonged survival compared to either monotherapy, thus mitigating the limitations intrinsic to single-agent therapies. This enhanced efficacy was driven by a significant alteration in the tumour microenvironment, skewing it towards pro-immunogenic conditions. This assertion is backed by a raised CD8+/CD4+ T-cell ratio and a decrease in immunosuppressive MDSC and Treg cell populations. On the mechanistic front, the synergistic therapy amplified expression levels of CXCL13 in tumours, subsequently facilitating T-cell ingress into the tumour setting. In summary, our findings advocate for integrated therapy as a potent mechanism for surmounting PD1 antibody resistance, capitalizing on improved T-cell functionality and infiltration. This investigation affords critical perspectives on enhancing anti-tumour immunity through the application of innovative therapeutic strategies.

Comparative analysis of antibodies to four major periodontal bacteria in respiratory diseases: a cohort study.

OBJECTIVES: To make a descriptive comparison of antibodies to four major periodontal bacteria and their relation to the respiratory diseases asthma and bronchitis/emphysema, and to cancer incidence. METHODS: The serum of a random sample of men with no history of cancer incidence (n=621) was analysed by the ELISA method for antibody levels of four periodontal bacteria; the anaerobes of the so-called red complex Tannerella forsythia (TF), Porphyromonas gingivalis (PG), and Treponema denticola (TD), and the facultative anaerobe Aggregatibacter actinomycetemcomitans (AA). The antibody readings were divided into quartiles and the distribution of cases of the relevant diseases as compared with the non-cases. Comparisons of the quartile distributions were by the Pearson χ2 test. Data and serum from the Oslo II study of Norwegian men from 2000 were used. The ELISA analyses were performed on thawed frozen serum. Cancer data from 17.5 years of follow-up were provided by the Norwegian Cancer Registry. RESULTS: In all, 52 men had reported asthma and 23 men had bronchitis/emphysema at the health screening. Results on cancer incidence are given for all respiratory cancers, n=23, and bronchi and lung cancers separately, n=18. Stratified analyses were performed for the four endpoints showing significant association with low levels of TD antibodies for bronchitis; p=0.035. Both TF and TD were significant for low levels of antibodies among daily smokers; p=0.030 for TF and p<0.001 for TD in the analysis of the full study sample. For PG and AA, no such associations were observed. An association with respiratory cancers was not observed. CONCLUSION: A low level of TD was associated with bronchitis/emphysema compared with the rest of the cohort. In the total study sample, low levels of antibodies to both TF and TD were associated with daily smoking.

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.