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Objective: To develop a new immunobead binding test (IBT) procedure that will detect sperm antibody in cervical mucus (CM), especially in very small samples of mucus., Design: After the interaction of donor sperm with bromelin-dissolved CM, the motile sperm cells were separated from other cells and debris and then tested with a standard immunobead method., Setting: The CM and serum samples were obtained from a sequence of referred patients who were sent to this testing laboratory., Patients: There were 60 women who provided 64 samples of CM and 41 samples of blood serum. They were partners in infertile couples., Interventions: None., Main Outcome Measure: A useful distinction was made between positive and negative results for the mucus samples by this CM-IBT procedure. The usable samples of mucus could be as little as 0.05 g (wet weight)., Results: From the 60 women, seven mucus samples were positive; in the repeated testing, the same results were obtained with five of the six positive samples and seven of seven negative samples., Conclusion: This method (CM-IBT) can be clinically useful for detecting sperm antibody in CM, especially because it is effective for tiny samples. In this method, the motile sperm cells are better separated from the debris after the incubation step. When the IBT was applied to the serum samples from the same women, there was no correlation between serum-IBT and CM-IBT, showing that both materials must be tested.

Objective: To determine if female sexual practices are associated with the development of antisperm antibodies., Design: Anonymous questionnaire., Setting: Tertiary care; private practice., Patients: Thirty-nine consecutive patients undergoing antisperm antibody testing; 32 responded., Interventions: Cervical mucus and serum obtained at midcycle., Main Outcome: Female sexual practices were found not to be related to the development of antisperm antibodies., Results: The percent (44.4) of women with antisperm antibodies who practice anal intercourse was not different from the percent (35.7) without these antibodies who engage in the same practice, and the percent (94.4) of women with antisperm antibodies who practice oral intercourse was not different from the percent (92.8) without these antibodies who engage in this practice., Conclusion: Female sexual practices do not appear to be related to the development of antisperm antibodies.

A group of 216 vasovasostomized men were tested with the mixed antiglobulin reaction for immunoglobulin (Ig)G, IgA, and secretory IgA antisperm antibodies bound to the sperm membrane. Free antisperm antibodies in serum and seminal plasma were detected with the gelatin agglutination test and the tray agglutination test. The results were related to the conception rates. In a subgroup with a pure IgG response, the conception rate reached 85.7%, whereas only 42.9% of the men who also had IgA on the sperm induced pregnancy. When 100% of the spermatozoa were covered with IgA, the conception rate was reduced to 21.7%. The combination of IgA on all sperm and a strong immune response (titer in serum greater than or equal to 256) was associated with a conception rate of zero.

To explore possible relationships between genital tract infections, sperm antibodies, and infertility, seminal fluid from 100 men was analyzed. Sperm antibodies, measured by an enzyme-linked immunosorbent assay, were detected in 14 of 29 men (48%) with culture-positive asymptomatic infections, 8 of 17 men (47%) with a history of prostatitis or urethritis, 11 of 33 men (33%) with either a varicocele or an abnormal semen analysis, and only 1 of 21 men (5%) with no infection and a normal semen analysis. The incidence of sperm antibodies in the men with infections or urethritis or prostatitis was significantly higher than in the control subjects (P less than 0.005). The sperm antibody isotype in men with infections was mainly IgA, although IgG or IgM were also sometimes present. Of the men with asymptomatic infections, urethritis or prostatitis, significantly (P less than 0.025) more men without sperm antibodies (12 of 24, 50%) were fertile than were those with sperm antibodies (3 of 22, 13%). Men with genital tract infections have a high incidence of antibodies, reactive with spermatozoa, which is associated with reduced fertility.

This study documents the types and incidence of antisperm antibody, detectable by indirect immunofluorescence, in 114 patients before vasectomy, 112 at 2 months and 71 patients at 6-9 months after vasectomy. Indirect immunofluorescence techniques revealed antibodies to seven distinct sperm antigens. Five of these antigens were readily accessible to antibody in vitro, and the remaining two were accessible only after treatment of spermatozoa with dithiothreitol and trypsin. Antisperm antibodies were detected in 61% of patients before vasectomy. The incidence rose to 77% at 2 months and 90% at 6-9 months after vasectomy. These antibodies were distinguishable into two groups based on their incidence before vasectomy. The first group included antibodies to antigens in the acrosome with a diffuse distribution, the equatorial region, the postacrosomal region and the midpiece of the tail. Its incidence was 61% before vasectomy; increased to 73% at 2 months and 80% at 6-9 months after vasectomy. The second group included antibodies to the sperm nucleus, the tail and to discrete antigens over the acrosome. They were found rarely (3%) in patients before vasectomy; increased in incidence to 25% at 2 months and 55% at 6-9 months after vasectomy. Antisperm antibodies of both groups existed as IgG and IgM classes; an exception being antibodies to sperm nucleus which were almost exclusively IgG. Of the antibodies, 14% were found to fix complement in vitro. Other autoantibodies, including antinuclear, antimitochondrial and antismooth muscle antibodies, did not develop following vasectomy.

Sera (vbs, n = 25) and seminal plasma (vsp, n = 21) from vasectomized men (n = 25) were analyzed for cross-reaction with lithium diiodosalicylate (LIS)-solubilized human sperm extract, protamine, and fertilization antigen (FA-1) with an enzyme-linked immunosorbent assay (ELISA). Among the vbs tested, 44% reacted with human sperm extract, 28% reacted with protamine, and 44% reacted with FA-1 for at least one class of antibodies (IgG, IgA, or IgM). In contrast to the sera, the seminal plasma showed minimal reactions. Neither the vbs nor vsp were found to contain immune complexes, indicating that the antibodies were present in free form. Vasectomized sera that reacted with FA-1 showed a significant (p less than 0.0001) inhibition of human sperm penetration of zona-free hamster ova. The immunoabsorption of FA-1-positive sera with purified FA-1 significantly increased the penetration rates. Affinity-purified human immunoglobulins reactive with FA-1 and not those reactive with protamine reduced sperm penetration rates. Thus, antibodies in vbs reactive with FA-1 are relevant to infertility, causing an inhibition of fertilization. These data will have clinical relevance for diagnosis and treatment of infertility after successful vasovasostomy.

Recent studies have demonstrated that many patients with SLE have elevated plasma levels of the minor oestrogen metabolite 16 alpha-hydroxyestrone (16 alpha OHE). This oestrogen is unique in its ability to react with lysine residues and form stable, covalent Heyns products with proteins. Increased levels of 16 alpha OHE-modified proteins have been found to occur on the membranes of red cells and lymphocytes in patients with SLE. In the present study, patient and control sera were analysed for the presence of circulating immunoglobulins which react with an oestrogen hapten. Anti-oestrogen antibodies were detected in 26% (9/34) of male and female SLE patients, and were found to correlate both with levels of plasma 16 alpha OHE (P less than 0.001) and with the presence of active disease (P less than 0.005). Surprisingly, this antibody activity was also observed in 25% (13/52) of normal, disease-free women who had a history of oral contraceptive use. No detectable activity was observed in normal men, women who had not taken oral contraceptives, or patients with a variety of other immunological diseases. The possible role of anti-oestrogen antibodies in both the hormonal exacerbation of SLE and in the long-term sequelae of oral contraceptive usage is discussed.

Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer cells was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17 alpha position. Elution with 50 micro M [3H]estradiol in 10% (vol/vol) dimethyl formamide/0.5 M sodium thiocyanate gave 40% recovery of [3H]estradiol-estrophilin showing 14% of the specific radioactivity expected for the pure complex. Serum from a Lewis rat immunized with this partially purified estradiol-receptor complex contained antiestrophilin antibodies that reacted not only with nuclear and extranuclear estradiol-receptor complexes from MCF-7 cells but also with estrophilin from rat, calf, and monkey uterus, hen oviduct, and human breast cancers. Splenic lymphocytes from the immunized rat were fused with cells of two different mouse myeloma lines (P3-X63-Ag8 and Sp2/0-Ag14) to yield hybridoma cultures, 2% of which produced antibodies to estrophilin. After cloning by limiting dilution, three hybridoma lines secreting antiestrophilin were expanded in suspension culture and as ascites tumors in athymic mice to provide substantial quantities of monoclonal antibodies that recognize mammalian but not avian estrophilin and that show different degrees of reactivity with receptor from nonprimate sources. By growing the clone from Sp2/0 in the presence of [35S]methionine, radiolabeled monoclonal IgG has been prepared. These monoclonal antibodies should prove useful in the study of estrogen receptors of human reproductive tissues, in particular for the radioimmunochemical assay and immunocytochemical localization of receptors in breast cancers.

A metabolic inhibition assay has been employed to investigate the distribution of serum heterocytotoxicity mediated by natural antibody activating the classical complement pathway and by antibody-independent activation of the alternative complement pathway in human serum samples derived from various groups of individuals. Levels in female cancer patients and in women taking oral contraceptives were significantly elevated over normal controls. Newborn infants exhibited approximately half of the average adult levels. Levels were maximally elevated in patients with visceral cancer, particularly in individuals with adenocarcinomas, while patients with sarcomas exhibited reduced levels. These and other data derived from the literature suggest that the metabolic inhibition assay may provide a useful measure of natural antibody and activation of the alternate complement pathway representative of mechanisms of natural immunity vs tumours.

Data are presented on antibody titers generated in 88 women immunized with three formulations of antihuman chorionic gonadotropin (hCG) vaccine, namely, beta-hCG (formulation B); beta-hCG associated with alpha-subunit of ovine luteinizing hormone (LH) (formulation A) and beta-hCG + beta-ovine LH (formulation M), each linked to tetanus toxoid and cholera toxin chain B as carriers. Each formulation was tested at two dose levels (100 and 500 micrograms). All women without exception developed anti-hCG antibodies having hCG-binding capacity above 20 ng mL-1 (0.5 nM), a level considered to be the threshold for prevention of pregnancy. Formulations A and B gave relatively better immunogenic response in human subjects than M. In each case, the antibody response was reversible. The mean duration of response above 20 ng was 35 to 37 weeks for formulation A, 34 weeks for B, and 17 to 20 weeks for M. Antibodies induced by three formulations of the vaccine had high-affinity (Ka 10(9)-10(10)M-1) for binding with hCG. They were devoid of cross-reaction with human follicle-stimulating hormone and thyroid-stimulating hormone but, as expected, cross-reacted with human LH. Antibodies were competent to block the hCG induced ovarian hyperemia.

Fallopian tube fluid and sera from 33 fertile and 48 infertile patients were investigated for spermatozoal antibodies by gelatin agglutination, immobilization, and indirect immunofluorescence tests. Oviductal fluids were obtained as washings after tubal catheterization. The incidences of antisperm activity in oviductal washings ranged from 9% to 58.3% depending on the tests used. These incidences were higher in infertile patients. Only immunoglobulin (Ig) A antisperm antibody was significantly more common in infertile patients than in fertile patients (P less than 0.01). Gelatin agglutination and antisperm IgG activity in the oviductal washings could be correlated to that in the sera. No relationship was seen between antisperm activity in the oviductal washings and that in the sera for sperm immobilization antibodies and IgA antisperm antibodies.

The umbilical cord blood of 47 out of 54 normal human infants contains hemagglutinating activity directed toward cells coated with a type-L Bence-Jones protein. This hemagglutinating material, in some instances, is present in cord blood in the absence of such activity in the maternal blood. Ultracentrifugation of cord serum in a sucrose gradient shows that the activity is associated with the macroglobulin fractions.

Plaque-forming cells elaborating antibody specific for Salmonella enteritidis somatic antigen have been demonstrated in the thymus of rabbits 5 days after a single systemic injection of a minute amount of this bacterial polysaccharide. A marked decrease in organ weight and loss of thymic cellularity was also seen, and was most pronounced at the time when antibody-forming cells made their appearance.

Selective ion channel modulators play a critical role in physiology in defining the contribution of specific ion channels to physiological function and as proof of concept for novel therapeutic strategies. Antibodies are valuable research tools that have broad uses including defining the expression and localization of ion channels in native tissue, and capturing ion channel proteins for subsequent analyses. In this review, we detail how renewable and recombinant antibodies can be used to control ion channel function. We describe the different forms of renewable and recombinant antibodies that have been used and the mechanisms by which they modulate ion channel function. We highlight the use of recombinant antibodies that are expressed intracellularly (intrabodies) as genetically encoded tools to control ion channel function. We also offer perspectives of avenues of future research that may be opened by the application of emerging technologies for engineering recombinant antibodies for enhanced utility in ion channel research. Overall, this review provides insights that may help stimulate and guide interested researchers to develop and incorporate renewable and recombinant antibodies as valuable tools to control ion channel function.

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P

Influenza remains a major cause of death and disability with limited treatment options. Studies of acute lung injury have identified angiopoietin-2 (Ang-2) as a key prognostic marker and a potential mediator of Acute respiratory distress syndrome. However, the role of Ang-2 in viral pneumonia remains poorly defined. This study characterized the time course of lung Ang-2 expression in severe influenza pneumonia and tested the therapeutic potential of Ang-2 inhibition. We inoculated adult mice with influenza A (PR8 strain) and measured angiopoietin-1 (Ang-1), Ang-2, and Tie2 expressions during the evolution of inflammatory lung injury over the first 7 days post-infection (dpi). We tested a peptide-antibody inhibitor of Ang-2, L1-7, administered at 2, 4, and 6 dpi and measured arterial oxygen saturation, survival, pulmonary edema, inflammatory cytokines, and viral load. Finally, we infected primary human alveolar type II epithelial (AT2) cells grown in air-liquid interface culture with influenza and measured Ang-2 RNA expression. Influenza caused severe lung injury between 5 and 7 dpi in association with increased Ang-2 lung RNA and a dramatic increase in Ang-2 protein in bronchoalveolar lavage. Inhibition of Ang-2 improved oxygenation and survival and reduced pulmonary edema and alveolar-capillary barrier permeability to protein without major effects on inflammation or viral load. Finally, influenza increased the expression of Ang-2 RNA in human AT2 cells. The increased Ang-2 levels in the airspaces during severe influenza pneumonia and the improvement in clinically relevant outcomes after Ang-2 antagonism suggest that the Ang-1/Ang-2 Tie-2 signaling axis is a promising therapeutic target in influenza and potentially other causes of viral pneumonia.

PurposeThe swell of new and diverse radiotracers to predict or monitor tumor response to cancer immunotherapies invites the opportunity for comparative studies to identify optimal platforms. To probe the significance of antibody format on image quality for PD-L1 imaging, we developed and studied the biodistribution of a library of antibodies based on the anti-PD-L1 IgG1 clone C4.ProcedureA C4 minibody and scFv were cloned, expressed, and characterized. The antibodies were functionalized with desferrioxamine and radiolabeled with Zr-89 to enable a rigorous comparison with prior data collected using 89Zr-labeled C4 IgG1. The biodistribution of the radiotracers was evaluated in C57Bl6/J or nu/nu mice bearing B16F10 or H1975 tumors, respectively, which are models that represent high and low tumor autonomous PD-L1 expression.ResultsThe tumor uptake of the 89Zr-C4 minibody was higher than 89Zr-C4 scFv and equivalent to previous data collected using 89Zr-C4 IgG1. However, the peak tumors to normal tissue ratios were generally higher for 89Zr-C4 scFv compared with 89Zr-C4 minibody and 89Zr-IgG1. Moreover, an exploratory study showed that the rapid clearance of 89Zr-C4 scFv enabled detection of endogenous PD-L1 on a genetically engineered and orthotopic model of hepatocellular carcinoma.ConclusionIn summary, these data support the use of low molecular weight constructs for PD-L1 imaging, especially for tumor types that manifest in abdominal organs that are obstructed by the clearance of high molecular weight radioligands.

Multiple myeloma (MM) is the second most common hematological disorder. Although several drugs have been developed to treat MM, their efficacy is uncertain. In addition, how normal physical activities can decrease inflammatory responses and clinical biomarkers in MM patients needs to be better defined. Therefore, this study evaluated possible clinical and inflammatory markers to determine the early diagnosis of MM during physical activity. This study selected 30 MM patients with normal or no physical activity with ages of >50 years. This study did not require any specific exercise protocols other than noting patients’ daily life activities and considering them as physical activity for 17 days. Then, blood samples were collected to assess clinical and inflammatory markers. Regarding clinical markers, daily life activities in MM patients decreased their LDH, calcium, and β2-microglobulin levels significantly compared to other clinical biomarkers such as creatine and total protein. Further, this study observed no significant differences between daily life activities of MM patients and normal MM patients regarding levels of immunoglobulins except IgM. Furthermore, IL-6 level was significantly increased with the daily life activities of MM patients, suggesting the role of physical activities in increasing anti-inflammatory response along with altering the biochemical profiles including LDH, calcium and β2-microglobulin in MM patients.

PurposeTo determine the maximum tolerated dose (MTD) of [131I]tositumomab in patients with refractory/recurrent Hodgkin lymphoma (HL) and to preliminarily determine if [131I]tositumomab has activity against HL and if positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]DG) performed 6 weeks post-therapy predicted 12-week response.ProceduresSeparate dose-finding studies were performed for patients with and without prior transplant. A single therapeutic total body radiation dose (TBD) of [131I]tositumomab was administered. TBD was escalated/de-escalated based on dose-limiting hematologic toxicity (DLT) using a modified continual reassessment method. [18F]DG-PET/CT scans were performed at baseline and 6 and 12 weeks post therapy.ResultsTwelve patients (nine classical HL, three lymphocyte-predominant [LP] HL) completed two dosing levels (n = 3 each) in the post-transplant (55 cGy, 79 cGy) and no transplant (75 cGy, 87 cGy) groups. Hematologic toxicities were common and transient. Twelve weeks after [131I]tositumomab, 10 patients progressed and two with LPHL achieved complete response. [18F]DG-PET/CT at 6 weeks post therapy appeared more predictive than CT at 6 weeks of a response at 12 weeks.ConclusionsTositumomab and [131I]tositumomab was well-tolerated in patients with relapsed/refractory HL. Complete responses in LPHL support a therapeutic effect in this subtype. Early metabolic response assessments by [18F]DG-PET in HL after radioimmunotherapy appear to be more predictive than purely anatomic assessments.

Giant cell arteritis (GCA) is the most common vasculitis of older age. It usually affects the branches of carotid arteries, especially temporal and ophthalmic artery. In addition to genetic predisposition, environmental factors also contribute to the development of the disease. Interleukin 6 (IL-6), whose level correlates with inflammatory activity, is one of the key cytokines in the pathogenesis of the disease. Sterile inflammation of the artery wall with intimal hyperplasia and the development of occlusion leads to ischemia which is responsible for the onset of symptoms. The clinical picture is dominated by headache together with general symptoms. Sudden loss of vision may occur due to anterior ischemic optic neuropathy (AION). Frequently, signs of rheumatic polymyalgia are also present. Markedly high erythrocyte sedimentation rate (ESR) and elevated C reactive protein (CRP) are typical. The diagnosis is based on clinical and laboratory findings, temporal artery biopsy, and imaging examinations. Prompt diagnosis is crucial for timely and adequate treatment as well as prevention of early and late complications (blindness, arterial aneurysm or dissection). Until recently, treatment has been limited to the use of glucocorticoids and conventional immunomodulating drugs. This review paper outlines new therapeutic approaches arising from a better knowledge of pathophysiology. Insights from recent studies led to corrections of GCA treatment guidelines (as did the Croatian Society for Rheumatology in 2018) by recommending IL-6 inhibitor tocilizumab for patients with refractory or relapsing disease, and initially in patients at increased risk for complications. Clinical trials are underway that could deliver other therapeutic options in the near future.

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-β (TGF-β) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-β induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-β pathway in the lung is unknown. The αvβ6 integrin is an important in vivo activator of TGF-β activation in the lung. Immunohistochemical analysis of αvβ6 protein expression and bronchoalveolar analysis of TGF-β pathway signaling indicates activation of the αvβ6/TGF-β pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvβ6/TGF-β pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvβ6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvβ6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the β6 integrin, TGF-α transgenic mice were mated with β6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the β6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the β6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvβ6/TGF-β pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

A family of 40 mammalian voltage-gated potassium (Kv) channels control membrane excitability in electrically excitable cells. The contribution of individual Kv channel types to electrophysiological signaling has been difficult to assign, as few selective inhibitors exist for individual Kv subunits. Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors. Here, functionally benign anti-Kv channel monoclonal antibodies (mAbs) were chemically modified to facilitate photoablation of K currents. Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage. Anti-Kv4.2 mAb-porphyrin conjugates facilitated photoablation of Kv4.2 currents. The degree of K current ablation was dependent on photon dose and conjugate concentration. Kv channel photoablation was selective for Kv4.2 over Kv4.3 or Kv2.1, yielding specificity not present in existing neurotoxins or other Kv channel inhibitors. We conclude that antibody-porphyrin conjugates are capable of selective photoablation of Kv currents. These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets.

Cancer therapy has progressed remarkably in recent years. In no area has this been more apparent than in the development of "targeted therapies", particularly those using drugs that inhibit the activity of certain tyrosine kinases, activating mutations or amplifications of which are causal, or strongly contributory, to tumorigenesis. However, some of these therapies have been associated with toxicity to the heart. Here we summarize what is known about the cardiotoxicity of cancer drugs that target tyrosine kinases. We focus on basic mechanisms through which interruption of specific signalling pathways leads to cardiomyocyte dysfunction and/or death, and contrast this with therapeutic responses in cancer cells.

Alterations in the extracellular matrix occur during the cardiac hypertrophic process. Because integrins mediate cell-matrix adhesion and beta(1D)-integrin (beta1D) is expressed exclusively in cardiac and skeletal muscle, we hypothesized that beta1D and focal adhesion kinase (FAK), a proximal integrin-signaling molecule, are involved in cardiac growth. With the use of cultured ventricular myocytes and myocardial tissue, we found the following: 1) beta1D protein expression was upregulated perinatally; 2) alpha(1)-adrenergic stimulation of cardiac myocytes increased beta1D protein levels 350% and altered its cellular distribution; 3) adenovirally mediated overexpression of beta1D stimulated cellular reorganization, increased cell size by 250%, and induced molecular markers of the hypertrophic response; and 4) overexpression of free beta1D cytoplasmic domains inhibited alpha(1)-adrenergic cellular organization and atrial natriuretic factor (ANF) expression. Additionally, FAK was linked to the hypertrophic response as follows: 1) coimmunoprecipitation of beta1D and FAK was detected; 2) FAK overexpression induced ANF-luciferase; 3) rapid and sustained phosphorylation of FAK was induced by alpha(1)-adrenergic stimulation; and 4) blunting of the alpha(1)-adrenergically modulated hypertrophic response was caused by FAK mutants, which alter Grb2 or Src binding, as well as by FAK-related nonkinase, a dominant interfering FAK mutant. We conclude that beta1D and FAK are both components of the hypertrophic response pathway of cardiac myocytes.

Recently, we observed a 15% decrease in the incidence of type 1 diabetes (T1D) in Australian 0-4-year-old children following the introduction of RV vaccination [[2], [3]], suggesting that RV vaccination could contribute to the primary prevention of this autoimmune disease. Australian surveillance data [[11]] show that the prevalence of RV G3 strains increased slightly along with an increase in strain diversity in the post-RV vaccine era, but G3 remains a minor component of disease-causing RV strains. RV was prevalent in nurseries, and the change to rooming-in would have altered the timing of exposure to RV, delaying it until later in the first year of life when, based on NOD mouse studies [[17]-[19]], RV might promote rather than retard development of diabetes. [Extracted from the article]

Middle East respiratory syndrome-coronavirus (MERS-CoV) is an emerging virus that causes severe disease with fatal outcomes; however, there are currently no approved vaccines or specific treatments against MERS-CoV. Here, we developed a novel bivalent vaccine against MERS-CoV and rabies virus (RV) using the replication-incompetent P-gene-deficient RV (RVΔP), which has been previously established as a promising and safe viral vector. MERS-CoV spike glycoprotein comprises S1 and S2 subunits, with the S1 subunit being a primary target of neutralizing antibodies. Recombinant RVΔP, which expresses S1 fused with transmembrane and cytoplasmic domains together with 14 amino acids from the ectodomains of the RV-glycoprotein (RV-G), was developed using a reverse genetics method and named RVΔP-MERS/S1. Following generation of RVΔP-MERS/S1 and RVΔP, our analysis revealed that they shared similar growth properties, with the expression of S1 in RVΔP-MERS/S1-infected cells confirmed by immunofluorescence and western blot, and the immunogenicity and pathogenicity evaluated using mouse infection experiments. We observed no rabies-associated signs or symptoms in mice inoculated with RVΔP-MERS/S1. Moreover, virus-specific neutralizing antibodies against both MERS-CoV and RV were induced in mice inoculated intraperitoneally with RVΔP-MERS/S1. These findings indicate that RVΔP-MERS/S1 is a promising and safe bivalent-vaccine candidate against both MERS-CoV and RV. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) play a critical role in host defense against influenza virus infection, and the mechanism of influenza virus to evade type I IFNs responses remains to be fully understood. Here, we found that progranulin (PGRN) was significantly increased both in vitro and in vivo during influenza virus infection. Using a PGRN knockdown assay and PGRN-deficient mice model, we demonstrated that influenza virus-inducing PGRN negatively regulated type I IFNs production by inhibiting the activation of NF-κB and IRF3 signaling. Furthermore, we showed that PGRN directly interacted with NF-κB essential modulator (NEMO) via its Grn CDE domains. We also verified that PGRN recruited A20 to deubiquitinate K63-linked polyubiquitin chains on NEMO at K264. In addition, we found that macrophage played a major source of PGRN during influenza virus infection, and PGRN neutralizing antibodies could protect against influenza virus-induced lethality in mice. Our data identify a PGRN-mediated IFN evasion pathway exploited by influenza virus with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of PGRN in innate immunity. [ABSTRACT FROM AUTHOR]

Oral herpes is a highly prevalent infection caused by herpes simplex virus 1 (HSV-1). After an initial infection of the oral cavity, HSV-1 remains latent in sensory neurons of the trigeminal ganglia. Episodic reactivation of the virus leads to the formation of mucocutaneous lesions (cold sores), but asymptomatic reactivation accompanied by viral shedding is more frequent and allows virus spread to new hosts. HSV-1 DNA has been detected in many oral tissues. In particular, HSV-1 can be found in periodontal lesions and several studies associated its presence with more severe periodontitis pathologies. Since gingival fibroblasts may become exposed to salivary components in periodontitis lesions, we analyzed the effect of saliva on HSV-1 and -2 infection of these cells. We observed that human gingival fibroblasts can be infected by HSV-1. However, pre-treatment of these cells with saliva extracts from some but not all individuals led to an increased susceptibility to infection. Furthermore, the active saliva could expand HSV-1 tropism to cells that are normally resistant to infection due to the absence of HSV entry receptors. The active factor in saliva was partially purified and comprised high molecular weight complexes of glycoproteins that included secretory Immunoglobulin A. Interestingly, we observed a broad variation in the activity of saliva between donors suggesting that this activity is selectively present in the population. The active saliva factor, has not been isolated, but may lead to the identification of a relevant biomarker for susceptibility to oral herpes. The presence of a salivary factor that enhances HSV-1 infection may influence the risk of oral herpes and/or the severity of associated oral pathologies. [ABSTRACT FROM AUTHOR]

Evolving systems, be it an antibody repertoire in the face of mutating pathogens or a microbial population exposed to varied antibiotics, constantly search for adaptive solutions in time-varying fitness landscapes. Generalists refer to genotypes that remain fit across diverse selective pressures; while multi-drug resistant microbes are undesired yet prevalent, broadly-neutralizing antibodies are much wanted but rare. However, little is known about under what conditions such generalists with a high capacity to adapt can be efficiently discovered by evolution. In addition, can epistasis—the source of landscape ruggedness and path constraints—play a different role, if the environment varies in a non-random way? We present a generative model to estimate the propensity of evolving generalists in rugged landscapes that are tunably related and alternating relatively slowly. We find that environmental cycling can substantially facilitate the search for fit generalists by dynamically enlarging their effective basins of attraction. Importantly, these high performers are most likely to emerge at intermediate levels of ruggedness and environmental relatedness. Our approach allows one to estimate correlations across environments from the topography of experimental fitness landscapes. Our work provides a conceptual framework to study evolution in time-correlated complex environments, and offers statistical understanding that suggests general strategies for eliciting broadly neutralizing antibodies or preventing microbes from evolving multi-drug resistance. [ABSTRACT FROM AUTHOR]

[Fam-] trastuzumab deruxtecan (DS-8201a) is a HER2 (ERBB2)-targeting antibody-drug conjugate, composed of a HER2-targeting antibody and a topoisomerase I inhibitor, exatecan derivative, that has antitumor effects in preclinical xenograft models and clinical trials. Recently, [fam-] trastuzumab deruxtecan was reported to enhance antitumor immunity and was beneficial in combination with an anti–PD-1 antibody in a mouse model. In this study, the antitumor effect of [fam-] trastuzumab deruxtecan in combination with an anti–CTLA-4 antibody was evaluated. [Fam-] trastuzumab deruxtecan monotherapy had antitumor activity in an immunocompetent mouse model with EMT6 human HER2-expressing mouse breast cancer cells (EMT6-hHER2). [Fam-] trastuzumab deruxtecan in combination with the anti–CTLA-4 antibody induced more potent antitumor activity than that by monotherapy with either agent. The combination therapy increased tumor-infiltrating CD4+ and CD8+ T cells in vivo. Mechanistically, cured mice with treatment of [fam-] trastuzumab deruxtecan and an anti–CTLA-4 antibody completely rejected EMT6-mock cells similar to EMT6-hHER2 cells, and splenocytes from the cured mice responded to both EMT6-hHER2 and EMT6-mock cells as measured by interferon-gamma release. Taken together, these results indicate that antitumor immunity is induced by [fam-] trastuzumab deruxtecan and is facilitated in combination with anti–CTLA-4 antibody. [ABSTRACT FROM AUTHOR]

RNA contains various chemical modifications, among which N6-methyladenosine (m6A) is the most prevalent modified nucleotide in eukaryotic mRNA. Emerging evidence suggests that m6A plays an important role in regulating a variety of cellular functions by controlling mRNA processing, translation and degradation. Because m6A is not detectable by standard chemical modification-based approaches, immunological methods, such as ELISA, immunoblotting, immunohistochemistry, m6A RNA immunoprecipitation sequencing and m6A individual-nucleotide resolution cross-linking and immunoprecipitation, have been employed to detect m6A in RNA. Although the most important factor determining the success of these methods is the integrity of highly specific antibodies against m6A, the development of m6A-specific monoclonal antibodies has been challenging. We developed anti-m6A monoclonal antibodies using our recently developed single cell-based monoclonal antibody production system. The binding of one selected antibody, #B1-3, to RNA oligoribonucleotide containing a single m6A had an equilibrium dissociation constant of 6.5 nM, and this antibody exhibited negligible binding to oligoribonucleotides containing a single N1-methyladenosine and unmodified adenosine. The binding was competed by the addition of increasing concentrations of N6-methyl-ATP but not N1-methyl-ATP or ATP. Furthermore, this mAb specifically crosslinked m6A-containing oligoribonucleotide by ultraviolet light, resulting in the induction of cDNA truncation at m6A position. These results show the feasibility of using the validated m6A monoclonal antibody for the specific detection of m6A in RNA. [ABSTRACT FROM AUTHOR]

In this study, we analyzed PRRS virus (PRRSv) specific lymphocyte function in piglets vaccinated with Ingelvac PRRSFLEX EU® at two and three weeks of age in the presence of homologous maternal immunity. Complete analysis of maternal immunity to PRRSv was evaluated postpartum, as well as passive transfer of antibodies and T cells to the piglet through colostrum intake and before and after challenge with a heterologous PRRSv at ten weeks of age. Maternal-derived antibodies were detected in piglets but declined quickly after weaning. However, vaccinated animals restored PRRSv-specific antibody levels by anamnestic response to vaccination. Cell analysis in colostrum and milk revealed presence of PRRSv-specific immune cells at suckling with higher concentrations found in colostrum than in milk. In addition, colostrum and milk contained PRRSv-specific IgA and IgG that may contribute to protection of newborn piglets. Despite the presence of PRRSv-specific Peripheral Blood Mononuclear cells (PBMCs) in colostrum and milk, no PRRSv-specific cells could be detected from blood of the piglets at one or two weeks of life. Nevertheless, cellular immunity was detectable in pre-challenged piglets up to 7 weeks after vaccination while the non-vaccinated control group showed no interferon (IFN) γ response to PRRSv stimulation. After challenge, all piglets developed a PRRSv-specific IFNγ-response, which was more robust at significantly higher levels in vaccinated animals compared to the primary response to PRRSv in non-vaccinated animals. Cytokine analysis in the lung lumen showed a reduction of pro-inflammatory responses to PRRSv challenge in vaccinated animals, especially reduced interferon (IFN) α levels. In conclusion, vaccination of maternally positive piglets at 2 and 3 weeks of age with Ingelvac PRRSFLEX EU induced a humoral and cellular immune response to PRRSv and provided protection against virulent, heterologous PRRSv challenge. [ABSTRACT FROM AUTHOR]

Immunization vectors based on cytomegalovirus (CMV) have attracted a lot of interest in recent years because of their high efficacy in the simian immunodeficiency virus (SIV) macaque model, which has been attributed to their ability to induce strong, unusually broad, and unconventionally restricted CD8+ T cell responses. To evaluate the ability of CMV-based vectors to mediate protection by other immune mechanisms, we evaluated a mouse CMV (MCMV)-based vector encoding Friend virus (FV) envelope (Env), which lacks any known CD8+ T cell epitopes, for its protective efficacy in the FV mouse model. When we immunized highly FV-susceptible mice with the Env-encoding MCMV vector (MCMV.env), we could detect high frequencies of Env-specific CD4+ T cells after a single immunization. While the control of an early FV challenge infection was highly variable, an FV infection applied later after immunization was tightly controlled by almost all immunized mice. Protection of mice correlated with their ability to mount a robust anamnestic neutralizing antibody response upon FV infection, but Env-specific CD4+ T cells also produced appreciable levels of interferon γ. Depletion and transfer experiments underlined the important role of antibodies for control of FV infection but also showed that while no Env-specific CD8+ T cells were induced by the MCMV.env vaccine, the presence of CD8+ T cells at the time of FV challenge was required. The immunity induced by MCMV.env immunization was long-lasting, but was restricted to MCMV naïve animals. Taken together, our results demonstrate a novel mode of action of a CMV-based vaccine for anti-retrovirus immunization that confers strong protection from retrovirus challenge, which is conferred by CD4+ T cells and antibodies. [ABSTRACT FROM AUTHOR]

Open data science and algorithm development competitions offer a unique avenue for rapid discovery of better computational strategies. We highlight three examples in computational biology and bioinformatics research in which the use of competitions has yielded significant performance gains over established algorithms. These include algorithms for antibody clustering, imputing gene expression data, and querying the Connectivity Map (CMap). Performance gains are evaluated quantitatively using realistic, albeit sanitized, data sets. The solutions produced through these competitions are then examined with respect to their utility and the prospects for implementation in the field. We present the decision process and competition design considerations that lead to these successful outcomes as a model for researchers who want to use competitions and non-domain crowds as collaborators to further their research. [ABSTRACT FROM AUTHOR]

Swine influenza virus (SIVs) infections cause a significant economic impact to the pork industry. Moreover, pigs may act as mixing vessel favoring genome reassortment of diverse influenza viruses. Such an example is the pandemic H1N1 (pH1N1) virus that appeared in 2009, harboring a combination of gene segments from avian, pig and human lineages, which rapidly reached pandemic proportions. In order to confront and prevent these possible emergences as well as antigenic drift phenomena, vaccination remains of vital importance. The present work aimed to evaluate a new DNA influenza vaccine based on distinct conserved HA-peptides fused with flagellin and applied together with Diluvac Forte as adjuvant using a needle-free device (IntraDermal Application of Liquids, IDAL®). Two experimental pig studies were performed to test DNA-vaccine efficacy against SIVs in pigs. In the first experiment, SIV-seronegative pigs were vaccinated with VC4-flagellin DNA and intranasally challenged with a pH1N1. In the second study, VC4-flagellin DNA vaccine was employed in SIV-seropositive animals and challenged intranasally with an H3N2 SIV-isolate. Both experiments demonstrated a reduction in the viral shedding after challenge, suggesting vaccine efficacy against both the H1 and H3 influenza virus subtypes. In addition, the results proved that maternally derived antibodies (MDA) did not constitute an obstacle to the vaccine approach used. Moreover, elevated titers in antibodies both against H1 and H3 proteins in serum and in bronchoalveolar lavage fluids (BALFs) was detected in the vaccinated animals along with a markedly increased mucosal IgA response. Additionally, vaccinated animals developed stronger neutralizing antibodies in BALFs and higher inhibiting hemagglutination titers in sera against both the pH1N1 and H3N2 influenza viruses compared to unvaccinated, challenged-pigs. It is proposed that the described DNA-vaccine formulation could potentially be used as a multivalent vaccine against SIV infections. [ABSTRACT FROM AUTHOR]

The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies. [ABSTRACT FROM AUTHOR]

Background: Zika virus (ZIKV) and Dengue virus (DENV) are often co-endemic. The high protein-sequence homology of flaviviruses renders IgG induced by and directed against them highly cross-reactive against their antigen(s), as observed on a large set of sera, leading to poorly reliable sero-diagnosis. Methods: We selected Domain III of the ZIKV Envelope (ZEDIII) sequence, which is virus specific. This recombinant domain was expressed and purified for the specific detection of ZEDIII-induced IgG by ELISA from ZIKV-RT-PCR-positive, ZIKV-IgM-positive, flavivirus-positive but ZIKV-negative, or flavivirus-negative sera. We also assessed the reactivity of ZEDIII-specific human antibodies against EDIII of DENV serotype 4 (D4EDIII) as a specific control. Sera from ZEDIII-immunized mice were also tested. Results: Cross-reactivity of IgG from 5,600 sera against total inactivated DENV or ZIKV was high (71.0% [69.1; 72.2]), whereas the specificity and sensitivity calculated using a representative cohort (242 sera) reached 90% [84.0; 95.8] and 92% [84.5; 99.5], respectively, using a ZEDIII-based ELISA. Moreover, purified human IgG against D2EDIII or D4EDIII did not bind to ZEDIII and we observed no D4EDIII reactivity with ZIKV-induced mouse polyclonal IgGs. Conclusions: We developed a ZEDIII-based ELISA that can discriminate between past or current DENV and ZIKV infections, allowing the detection of a serological scar from other flaviviruses. This could be used to confirm exposure of pregnant women or to follow the spread of an endemic disease. [ABSTRACT FROM AUTHOR]