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Longitudinal studies are needed to evaluate the SARS-CoV-2 mRNA vaccine antibody response under real-world conditions. This longitudinal study investigated the quantity and quality of SARS-CoV-2 antibody response in 846 specimens from 350 patients, comparing BNT162b2-vaccinated individuals (19 previously diagnosed with COVID-19, termed RecoVax; and 49 never diagnosed, termed NaiveVax) with 122 hospitalized unvaccinated (HospNoVax) and 160 outpatient unvaccinated (OutPtNoVax) COVID-19 patients. NaiveVax experienced delay in generating SARS-CoV-2 total antibodies (TAb) and surrogate neutralizing antibodies (SNAb) after the first vaccine dose (D1) but rapid increase in antibody levels after the second dose (D2). However, these never reached RecoVax's robust levels. In fact, NaiveVax TAb and SNAb levels decreased 4 weeks after D2. For the most part, RecoVax TAb persisted, after reaching maximal levels 2 weeks after D2, but SNAb decreased significantly about 6 months after D1. Although NaiveVax avidity lagged behind that of RecoVax for most of the follow-up periods, NaiveVax did reach similar avidity by about 6 months after D1. These data suggest that 1 vaccine dose elicits maximal antibody response in RecoVax and may be sufficient. Also, despite decreasing levels in TAb and SNAb over time, long-term avidity may be a measure worth evaluating and possibly correlating to vaccine efficacy. more...

Longitudinal studies are needed to evaluate the SARS-CoV-2 mRNA vaccine antibody response under “real-world” conditions. This longitudinal study investigated the quantity and quality of SARS-CoV-2 antibody response in 846 specimens from 350 subjects: comparing BNT162b2-vaccinated individuals (19 previously diagnosed with COVID-19 [RecoVax]; 49 never been diagnosed [NaïveVax]) to 122 hospitalized unvaccinated (HospNoVax) and 160 outpatient unvaccinated (OutPtNoVax) COVID-19 patients.NaïveVax experienced a delay in generating SARS-CoV-2 total antibody levels (TAb) and neutralizing antibodies (SNAb) after the 1st vaccine dose (D1), but a rapid increase in antibody levels was observed after the 2nddose (D2). However, these never reached the robust levels observed in RecoVax. In fact, NaïveVax TAb and SNAb levels decreased 4-weeks post-D2 (p=0.003;p more...

The response of the immune system to COVID-19 in end stage kidney disease patients who undergo kidney transplantation has yet to be described. We report data on 72 patients who underwent SARS-CoV-2 antibody testing both before and after kidney transplantation and were followed for a median of 186 days (range 83, 277). Of the 25 patients with a positive antibody test at the time of transplant, 17 (68%) remained positive after transplantation. Patients were significantly more likely to have a persistently positive test if they reported a symptomatic COVID-19 infection prior to transplant (p=0.01). SARS-CoV-2 IgG index values were measured in a subset of kidney transplant recipients and compared to wait -listed dialysis patients. These assays demonstrated a more significant decline in IgG (58% versus 14% p = 0.008) in transplant recipients when compared to dialysis patients tested during the same time period. Additional analysis of the quality of the immune response measuring the binding of SARS-CoV-2 antibodies to the receptor-binding domain (RBD binding), the antibody neutralizing capability, and the antibody avidity demonstrated a more pronounced effect when comparing pre-transplant values to post-induction therapy/post transplant values. The attenuated IgG response seen in transplant patients compared to dialysis patients after induction therapy requires further study. These data have important implications for post-transplant management of vaccinated dialysis patients. more...

Pregnant women were excluded from initial clinical trials for coronavirus disease 2019 (COVID-19) vaccines1,2; thus, the understanding of the immunologic response to vaccination in pregnancy and the transplacental transfer of maternal antibodies is limited.3,4 more...

SummaryPregnant women were excluded from initial clinical trials for COVID-19 vaccines1–2, thus the immunologic response to vaccination in pregnancy and the transplacental transfer of maternal antibodies are just beginning to be studied4–5. more...

Key Points Question Are the quantity and quality of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) different among children, adolescents, and young adults? Findings In this cross-sectional study evaluating 31 426 SARS-CoV-2 antibody tests performed between April 9 and August 31, 2020, immunoglobin G levels were found to vary in different age groups, despite similar seroprevalence in the pediatric and adult patient populations. SARS-CoV-2 immunoglobin G and total antibody levels, neutralizing activity, and avidity exhibited negative correlations with age in patients aged 1 to 24 years. Meaning This analysis revealed distinct antibody responses in different age groups, suggesting that age-targeted strategies for disease screening and management as well as vaccine development may be warranted., This cross-sectional study investigates the association of age with the quantity and quality of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody responses., Importance Accumulating evidence suggests that children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to manifest mild symptoms and are at a lower risk of developing severe respiratory disease compared with adults. It remains unknown how the immune response in children differs from that of adolescents and adults. Objective To investigate the association of age with the quantity and quality of SARS-CoV-2 antibody responses. Design, Setting, and Participants This cross-sectional study used 31 426 SARS-CoV-2 antibody test results from pediatric and adult patients. Data were collected from a New York City hospital from April 9 to August 31, 2020. The semiquantitative immunoglobin (Ig) G levels were compared between 85 pediatric and 3648 adult patients. Further analysis of SARS-CoV-2 antibody profiles was performed on sera from 126 patients aged 1 to 24 years. Main Outcomes and Measures SARS-CoV-2 antibody positivity rates and IgG levels were evaluated in patients from a wide range of age groups (1-102 years). SARS-CoV-2 IgG level, total antibody (TAb) level, surrogate neutralizing antibody (SNAb) activity, and antibody binding avidity were compared between children (aged 1-10 years), adolescents (aged 11-18 years), and young adults (aged 19-24 years). Results Among 31 426 antibody test results (19 797 [63.0%] female patients), with 1194 pediatric patients (mean [SD] age, 11.0 [5.3] years) and 30 232 adult patients (mean [SD] age, 49.2 [17.1] years), the seroprevalence in the pediatric (197 [16.5%; 95% CI, 14.4%-18.7%]) and adult (5630 [18.6%; 95% CI, 18.2%-19.1%]) patient populations was similar. The SARS-CoV-2 IgG level showed a negative correlation with age in the pediatric population (r = −0.45, P more...

Background Low initial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers dropping to undetectable levels within months after infection have raised concerns about long-term immunity. Both the antibody levels and the avidity of the antibody–antigen interaction should be examined to understand the quality of the antibody response. Methods A testing-on-a-probe “plus” panel (TOP-Plus) was developed to include a newly developed avidity assay built into the previously described SARS-CoV-2 TOP assays that measured total antibody (TAb), surrogate neutralizing antibody (SNAb), IgM, and IgG on a versatile biosensor platform. TAb and SNAb levels were compared with avidity in previously infected individuals at 1.3 and 6.2 months after infection in paired samples from 80 patients with coronavirus disease 2019 (COVID-19). Sera from individuals vaccinated for SARS-CoV-2 were also evaluated for antibody avidity. Results The newly designed avidity assay in this TOP panel correlated well with a reference Bio-Layer Interferometry avidity assay (r = 0.88). The imprecision of the TOP avidity assay was Conclusions This highly precise and versatile TOP-Plus panel with the ability to measure SARS-CoV-2 TAb, SNAb, IgG, and IgM antibody levels and avidity of individual sera on one sensor can become a valuable asset in monitoring not only patients infected with SARS-CoV-2 but also the status of individuals’ COVID-19 vaccination response. more...

Background Pregnant women and their neonates represent 2 vulnerable populations with an interdependent immune system that are highly susceptible to viral infections. The immune response of pregnant women to severe acute respiratory syndrome coronavirus 2 and the interplay of how the maternal immune response affects the neonatal passive immunity have not been studied systematically. Objective We characterized the serologic response in pregnant women and studied how this serologic response correlates with the maternal clinical presentation and with the rate and level of passive immunity that the neonate received from the mother. Study Design Women who gave birth and who tested positive for immunoglobulin M or immunoglobulin G against severe acute respiratory syndrome coronavirus 2 using semiquantitative detection in a New York City hospital between March 22, 2020, and May 31, 2020, were included in this study. A retrospective chart review of the cases that met the inclusion criteria was conducted to determine the presence of coronavirus disease 2019 symptoms and the use of oxygen support. Serology levels were compared between the symptomatic and asymptomatic patients using a Welch 2 sample t test. Further chart review of the same patient cohort was conducted to identify the dates of self-reported onset of coronavirus disease 2019 symptoms and the timing of the peak immunoglobulin M and immunoglobulin G antibody levels after symptom onset was visualized using local polynomial regression smoothing on log2-scaled serologic values. To study the neonatal serology response, umbilical cord blood samples of the neonates born to the subset of serology positive pregnant women were tested for serologic antibody responses. The maternal antibody levels of serology positive vs the maternal antibody levels of serology negative neonates were compared using the Welch 2 sample t test. The relationship between the quantitative maternal and quantitative neonatal serologic data was studied using a Pearson correlation and linear regression. A multiple linear regression analysis was conducted using maternal symptoms, maternal serology levels, and maternal use of oxygen support to determine the predictors of neonatal immunoglobulin G levels. Results A total of 88 serology positive pregnant women were included in this study. The antibody levels were higher in symptomatic pregnant women than in asymptomatic pregnant women. Serology studies in 34 women with symptom onset data revealed that the maternal immunoglobulin M and immunoglobulin G levels peak around 15 and 30 days after the onset of coronavirus disease 2019 symptoms, respectively. Furthermore, studies of 50 neonates born to this subset of serology positive women showed that passive immunity in the form of immunoglobulin G is conferred in 78% of all neonates. The presence of passive immunity is dependent on the maternal antibody levels, and the levels of neonatal immunoglobulin G correlate with maternal immunoglobulin G levels. The maternal immunoglobulin G levels and maternal use of oxygen support were predictive of the neonatal immunoglobulin G levels. Conclusion We demonstrated that maternal serologies correlate with symptomatic maternal infection, and higher levels of maternal antibodies are associated with passive neonatal immunity. The maternal immunoglobulin G levels and maternal use of oxygen support, a marker of disease severity, predicted the neonatal immunoglobulin G levels. These data will further guide the screening for this uniquely linked population of mothers and their neonates and can aid in developing maternal vaccination strategies. more...

Sugarcane is a highly beneficial crop grown worldwide and is greatly influenced by numerous insect pests. These insect pests have adapted to the diversity of conditions under which sugarcane is grown thus causing a great degree of damage to the crop. These insect pests feed on different parts of the sugarcane. For instance, the early shoot borer attacks the cane before internode formation, while the internode borer feeds after internode formation, the foliar pest feeds on the leaves and some would feed on the rootstalks.Guyana has large areas of land which are free of agricultural chemicals and thus are suitable for organic farming. Organic farming is practiced in Guyana on a small scale. However, crops such as cocoa, pineapple and heart of palm have already been grown organically for export. Similarly, like organic farming, biological control of pests and diseases is also encouraged worldwide. Biological control in agriculture is a method of controlling pests such as insects, weeds and diseases by using natural enemies such as predators, parasites, herbivores, or any other natural mechanisms. In addition, like organic farming biological controls are also ecologically friendly and are very effective. Both approaches (organic farming and biological control) are significant since the use of pesticides and chemical fertilizers would be eliminated, thus reducing health issues in the environment and of consumers.               This research work is on the pathogenicity of Beauveria bassiana on the insect pest Diatraea centrella larva of sugarcane in Guyana. The entomopathogenic fungus, Beauveria bassiana, is a promising control agent against many insect pests [1]. Beauveria bassiana is commonly found in soils worldwide and has a host range of over 700 insect species, including insects from the orders Lepidoptera, Coleoptera and Hymenoptera.  The research carried out focuses on the effect of Beauveria bassiana spores on Diatraea centrella larva and the rate at which these spores are effective, as well as the target site of infection of these spores on the larva.              The study conclusively proves that a higher number of Beauveria bassiana spores are more effective on Diatraea centrella larva in a short period of time and the dorsal, ventral and intestinal tissues of the Diatraea centrella larva are more susceptible to the spores of Beauveria bassiana. more...

A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells. more...

We present a detailed characterization of a single-cycle infection of the bocavirus minute virus of canines (MVC) in canine WRD cells. This has allowed identification of an additional smaller NS protein that derives from an mRNA spliced within the NS gene that had not been previously reported. In addition, we have identified a role for the viral NP1 protein during infection. NP1 is required for read-through of the MVC internal polyadenylation site and, thus, access of the capsid gene by MVC mRNAs. Although the mechanism of NP1's action has not yet been fully elucidated, it represents the first parvovirus protein to be implicated directly in viral RNA processing. more...

Background: Little is known about the mechanisms and treatment of radiation-induced inhibition of craniofacial bone growth. In an earlier study, the radio-protector amifostine (WR-2721) administered to rabbits before irradiation radioprotected cultured orbitozygomatic complex periosteal osteoblast-like cells. This study assessed the effects of amifostine and its active metabolite on the radiation survival, function, and phenotype of mouse calvarial osteoblast-like cells in a cell culture model. Methods: MC3T3-E1 newborn mouse calvarial osteoblast-like cells underwent γ-radiation (0 to 10 Gy) in the presence or absence of either WR-2721 or WR-1065, its active metabolite (10 -7 to 10 -3 M). The effects of radiation with and without drugs were assessed using endpoints of colony-forming ability, cell viability, alkaline phosphatase activity, and expression of osteoblastic phenotype genes (alkaline phosphatase, collagen type I, osteocalcin, and osteopontin). All experiments were replicated at least in triplicate. Results: Irradiation resulted in a dose-dependent inhibition of clonogenic cell survival. Pretreatment with WR-1065, but not WR-2721, resulted in a significant improvement of osteoblast-like cell survival. Specifically, maximum radioprotection was observed with 10 -4 M WR-1065 at a clinically relevant 2-Gy dose of irradiation. No significant radioprotection was observed at the lower (5 X 10 -7 M) concentration of WR-1065. Furthermore, radiation seemed to suppress the expression of ostecblastic phenotype-related genes in a dose-dependent manner. Conclusions: This study reveals improved survival after irradiation in osteoblast-like cells treated with WR-1065 in vitro and corroborates previous findings from animal models. Further studies using this agent and similar drugs are important for devising strategies to prevent radiation-induced inhibition of craniofacial bone growth. more...

Golgi beta1,6-N-acetylglucosaminyltransferase V (Mgat5) produces beta1,6GlcNAc-branched N-glycans on glycoproteins, which increases their affinity for galectins and opposes loss from the cell surface to constitutive endocytosis. Oncogenic transformation increases Mgat5 expression, increases beta1,6GlcNAc-branched N-glycans on epidermal growth factor and transforming growth factor-beta receptors, and enhances sensitivities to ligands, cell motility, and tumor metastasis. Here, we demonstrate that Mgat5(-/-) mouse embryonic fibroblasts (MEFs) display reduced sensitivity to anabolic cytokines and reduced glucose uptake and proliferation. Mgat5(-/-) mice are also hypoglycemic, resistant to weight gain on a calorie-enriched diet, hypersensitive to fasting, and display increased oxidative respiration and reduced fecundity. Serum-dependent activation of the extracellular response kinase (growth) and Smad2/3 (arrest) pathways in Mgat5(-/-) MEFs and bone marrow cells reveals an imbalance favoring arrest. Mgat5(-/-) mice have fewer muscle satellite cells, less osteogenic activity in bone marrow, and accelerated loss of muscle and bone mass with aging. Our results suggest that beta1,6GlcNAc-branched N-glycans promote sensitivity to anabolic cytokines, and increase fat stores, tissue renewal, and longevity. more...

Smoking and infection with Gram-negative bacterial pathogens are risk factors for alveolar bone loss. The aims of this study were: 1) to examine the combined effects of an aryl hydrocarbon, benzo[a]pyrene (BaP), that is concentrated in cigarette smoke, and lipopolysaccharide (LPS) extracted from Porphyromonas gingivalis on osteogenesis in a rat bone marrow cell (RBMC) model of osteogenesis; and 2) to determine whether resveratrol (Res), an aryl hydrocarbon receptor antagonist, could reverse the putative inhibitory effects of BaP + LPS on osteogenesis.LPS of P. gingivalis strain 2561 was introduced in various concentrations to the RBMC in 96-well plates and kept in culture for 8 to 12 days. The same protocol was used for studying BaP and LPS + BaP combinations. Following the incubation periods, parameters of osteogenesis were measured, including formation of mineralized bone nodules, alkaline phosphatase activity, and total cell protein. Transcription of the pro-inflammatory cytokine interleukin (IL)-1beta in the cultures was determined by reverse transcriptase polymerase chain reaction (RT-PCR).Bone nodule formation generally decreased significantly with increasing LPS concentrations (P0.05), whereas total cell protein decreased only slightly (P0.05). BaP in previously high concentrations alone also caused a significant dose-dependent decrease in bone nodule formation (P0.05) but when half maximal doses were used, significant decreases were most often seen when LPS was added. Hence, in combination, the inhibitory effects of LPS + BaP on osteogenesis were additive, inhibiting bone nodule formation up to 9-fold. Resveratrol partially reversed the inhibitory effects of low concentrations of LPS alone, and completely reversed the inhibition of nodule formation when low concentrations of LPS were combined with BaP. IL-1beta expression generally fluctuated inversely to the inhibitory activity of LPS, LPS + BaP, and LPS + BaP + Res combinations.Smoke-derived aryl hydrocarbons and bacterial LPS may act additively to inhibit bone formation. The findings may explain, in part, why net periodontal bone loss is greater and bone healing is less successful in smokers than non-smokers with periodontal infections. Reversal of the inhibitory effects in vitro by resveratrol suggests that this phytoalexin should be studied further for its potential therapeutic value, given its aryl hydrocarbon receptor antagonism and apparent anti-inflammatory activity. more...

Acquired haemophilia A due to the development of auto-antibodies directed against factor VIII (FVIII) is a rare disorder that may result in serious haemorrhagic episodes. Although in many cases no associated underlying disorders are apparent, the condition has been reported in association with autoimmune disorders, haematological malignancies, treatment with certain drugs and pregnancy. The reaction kinetics of auto-antibodies to FVIII differ from those observed with allo-antibodies in congenital haemophilia. Previous studies in congenital haemophilia have raised the possibility that inhibitory antibodies vary in their reactivity with the factor VIII molecules in different concentrates used for treatment. However, the interaction of FVIII in concentrates and inhibitors in acquired haemophilia has never been previously studied. In this study, the effect of different FVIII concentrates was studied on neutralization in vitro by performing inhibitor titres using the New Oxford inhibitor assay method. The inhibitor titre in eight patients with acquired haemophilia A was assayed against five commercially available FVIII concentrates of varying purity. The intermediate purity concentrate 8Y and the high purity concentrate that contains normal amounts of von Willebrand's Factor (vWF) (Alphanate) gave lower titres than the high purity concentrates with low (Monoclate-P) or no (Kogenate) von Willebrand content. All but one antibody had very low reactivity with porcine FVIII. Further work will be required to establish whether concentrates manifesting a low level of in vitro reactivity with the inhibitor have a better haemostatic effect in vivo. more...

Treatment of patients with haemophilia A with coagulation factor concentrates may result in the development of inhibitory antibodies directed against factor VIII (FVIII). In this study, a previously unpublished ELISA test for FVIII inhibitor screening (Genetic Testing Institute [GTI] FVIII inhibitor, Brookfield, WI, USA) was evaluated in 131 blood samples (124 samples from patients with haemophilia A, and seven serial samples from one patient with an acquired FVIII inhibitor). Comparisons were made with the routine screening assay (based on recovery of FVIII) and confirmed where positive (< 90% recovery) with the New Oxford assay. The ELISA kit had a sensitivity of 97.7% and specificity of 78.4%. The high negative predictive value of this new test (98.6%) suggests it may be useful as a reliable, rapid (< 2 h) and flexible (microwell strip format) tool for inhibitor screening of samples from both patients with haemophilia A and those with suspected acquired FVIII inhibitors. more...

Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene. more...

The serum glycoprotein fetuin is expressed during embryogenesis in multiple tissues including limb buds and has been shown to promote bone remodeling and stimulate cell proliferation in vitro. In this report, we demonstrate that fetuin antagonizes the antiproliferative action of transforming growth factor-beta1 (TGF-beta1) in cell cultures. Surface plasmon resonance measurements show that fetuin binds directly to TGF-beta1 and TGF-beta2 and with greater affinity to the TGF-beta-related bone morphogenetic proteins (BMP-2, BMP-4, and BMP-6). In a competitive enzyme-linked immunosorbent assay, fetuin blocked binding of TGF-beta1 to the extracellular domain of TGF-beta receptor type II (TbetaRII), one of the primary TGF-beta-binding receptors. A comparison of fetuin and TbetaRII shows homology in an 18-19-amino acid sequence, which we have designated TGF-beta receptor II homology 1 domain (TRH1). Since the TRH1 sequence is known to form a disulfide loop in fetuin, cyclized TRH1 peptides from both fetuin and TbetaRII were chemically synthesized and tested for cytokine binding activity. Cyclized TRH1 peptide from TbetaRII bound to TGF-beta1 with greater affinity than to BMP-2, while the cyclized TRH1 peptide from fetuin bound preferentially to BMP-2. Finally, fetuin or neutralizing anti-TGF-beta antibodies blocked osteogenesis and deposition of calcium-containing matrix in cultures of dexamethasone-treated rat bone marrow cells. In summary, these experiments define the TRH1 peptide loop as a cytokine-binding domain in both TbetaRII and fetuin and suggest that fetuin is a natural antagonist of TGF-beta and BMP activities. more...

Background Tissue non-specific alkaline phosphatase (TN-AP) is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic activity. This enzyme is distributed virtually in all mammalian tissues during embryonic development (it can be demonstrated as early as the 2-cell stage) where its expression is stage specific. The expression of TN-AP is frequently associated with cell differentiation and as such it has been used as a marker for this process. By employing a stable gene transfer and forced gene expression technique, previous findings suggested that TN-AP expression might influence cellular proliferation and morphological differentiation. The focus of this study was to determine whether this was a cell-specific effect or not. Methods The effects of TN-AP on various aspects of cellular activity were assessed by transferring and expressing the gene for this enzyme into three target populations; 1) CHO, 2) R1610, and 3) Rat-2. The parameters of cellular activity studied included cellular proliferation, cell cycle, cell migration, and tumorigenic potential (in the nude mouse). Cell cycle and cell proliferation analyses were accomplished, in part, through the use of fluorescence activated cell sorting (FACS) as were determinations of cell-associated TN-AP activity and amount. Northern and southern blot analyses were used to estimate gene copy number and to evaluate gene expression respectively in transfected cell lines. Results Our data indicate that the TN-AP gene under control of various gene promoters was stably integrated into three fibroblast-like cell lines (CHO, R1610, and Rat-2). TN-AP activity and TN-AP protein levels were correlated to the strength of the various gene promoters, but not to inserted gene copy numbers. The expression of the TN-AP gene in these three cell types further suggests cell-specific effects as demonstrated by the following findings. The expression of TN-AP under control of a weak gene promoter in CHO and Rat-2 cells clearly decreased cell proliferation and cell migration. However, the expression of TN-AP under control of either a weak or even a strong gene promoter in R1610 cells did not induce any changes in that cell line's behaviour (apart from expression of TN-AP). Such changes in CHO cells were also associated with a 2.2-fold increase in tubulin transcription as well as suppressed tumorigenic potential. Conclusion Taken together, these findings suggest that the inhibitory effects of TN-AP expression on proliferation and cell migration are not non-specific and that high expression of TN-AP may induce changes in cell behaviour which may be consistent with or at least related to induction of terminal differentiation. © 1996 Wiley-Liss, Inc. more...

Glucocorticoids and sex-steroids can modulate osteogenesis in vivo and in vitro. Although the effects of glucocorticoids on bone cells in vitro have been described in detail, the role of sex-steroids is not as well defined. We examined whether sex-steroids influence bone metabolism indirectly by regulating glucocorticoid effects on bone. Interactions of the sex-steroid progesterone or its analog RU38486 with the glucocorticoid dexamethasone (dex) were studied in functional assays of osteogenesis. Three osteoblastic models were evaluated: (1) the rat bone marrow stromal cell (RBMC) nodule system; (2) the chick periosteal osteogenesis (CPO) model; and (3) ROS 17/2.8 cells. RU38486, progesterone, and unlabelled dex competitively inhibited 3H-dex uptake by ROS 17/2.8 cells as well as its (3H-dex) binding to cytosol preps. Both RU38486 and progesterone inhibited dex-induced increases in alkaline phosphatase in CPO cultures, in RBMC cultures, and in ROS 17/2.8 cells. Dex-induced decreases in cell proliferation in ROS 17/2.8 cells were reversed by RU38486 but dex-induced increases in proliferation in the CPO model were not affected. In CPO cultures, dex-induced increases in collagen synthesis were inhibited completely by RU38486 and progesterone. Dex-dependent nodule formation in the RBMC was blocked by RU38486. Both RU38486 and dex mediated reduction of calcium uptake in the CPO model but did not affect mineralized tissue area. The data indicate that RU38486 and progesterone competitively inhibit dex-mediated stimulation of osteogenesis in vitro; this inhibition is exerted on early but not late stage differentiation events of osteoprogenitor cells. more...

The amount and function of von Willebrand factor (VWF), measured against a panel of laboratory tests, is the normal basis for the diagnosis of von Willebrand's disease (VWD). The normal range for each test is usually obtained by assaying samples from a cross section of the local population or from a manufacturer's kit insert. While collecting normal controls for another study, population from Durban in South Africa, with its distinct ethnic mix of Africans, Indians and Caucasians were also studied. Previously, Indians from their subcontinent have not been looked at separately and compared with Africans and Caucasians. It is confirmed in a previous study (Miller, Dilley, Richardson, Craig, Evatt. (2001) American Journal of Hematology 67, 125) that African Americans had significantly higher VWF:Ag and FVIII levels when compared with Caucasians. In addition, it was found that there was a significant difference in VWF:Ag levels between Indians and Africans, and between Caucasians and Africans, whereas no significant difference between Indians and Caucasians. Africans, Indians and Caucasians with blood group O showed significantly lower VWF:Ag and FVIII than the other ABO blood groups. Normal ranges of VWF for different blood groups are well established and this information should be utilized while considering a diagnosis of VWD. It is proposed here that the influence of racial origin should also be considered in the clinical and laboratory evaluation of VWD. more...

It is well documented that oral microorganisms play a significant role in the initiation and progression of periodontal disease. By using various in vitro models, it has been shown that some bacteria considered periodontal pathogens or their products can stimulate bone resorption and some other parameters of osteoblast-like cell activity. However, the effects of these organisms and their products on osteogenesis itself are not known. This study was undertaken to determine the direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis on bone formation in the chick periosteal osteogenesis model. Cultures of P. gingivalis 2561 were grown under standard anaerobic culture conditions. The spent medium was collected, and following centrifugation, sonicated bacterial extracts were prepared from the bacterial pellet. These were added in various proportions to the chick periosteal osteogenesis cultures. Sonicated extracts were further fractionated into five molecular-size ranges and similarly tested. Parameters of osteogenesis, including alkaline phosphatase activity, calcium and Pi accumulation, and collagen synthesis, were measured on 6-day-old cultures. Compared with controls devoid of bacterial products, osteogenesis was inhibited significantly in cultures treated with either conditioned medium or extracts obtained from P. gingivalis. Various amounts of inhibitory activity were observed in the different ultrafiltration molecular-size fractions, with very profound inhibitory effects observed in the < 5-kDa range. Histological observations indicated the presence of cells, some bone, and/or new fibrous connective tissue at all concentrations, indicating that toxicity was not a factor. These results suggest that periodontal pathogens such as P. gingivalis might contribute to the bone loss in periodontal diseases not only by stimulating resorption but, possibly, by inhibiting bone formation directly. more...

Individuals and families carrying mutations in BRCA1 and BRCA2 (BRCA1/2) have a markedly elevated risk of developing breast and ovarian cancers. The first-generation of BRCA1/2 mutation analysis targeted only the coding exons and has implicated protein-truncating mutations (indel, nonsense) in BRCA1/2 inactivation. Recently, heritable breast cancers have also been attributed to other exonic mutations (missense, silent) and mutations in introns and untranslated regions. However, analysis of these alterations has been prohibitively laborious and cost intensive, and the proportion of cases carrying mutations in unscreened regions of BRCA1/2 and other predisposition genes is unknown. We have developed and validated a next-generation sequencing (NGS) approach for BRCA1/2 mutation analysis by applying long-range PCR and deep sequencing. Genomic DNA from familial breast cancer patients (N = 12) were screened and NGS successfully identified all 19 distinct (51 total) BRCA1 and 35 distinct (63 total) BRCA2 sequence alterations detectable by the Sanger sequencing, with no false-negative or positive results. In addition, we report the robust detection of variants from introns and untranslated regions. These results illustrate that NGS can provide comprehensive genetic information more quickly, accurately, and at a lower cost than conventional approaches, and we propose NGS to be a more effective method for BRCA1/2 mutational analysis. Advances in NGS will play an important role in enabling molecular diagnostics and personalized treatment of breast and ovarian cancers. more...

Five adenovirus (Ad) gene products are required for efficient replication of co-infecting adeno-associated virus (AAV); however, the combined net enhancement by these factors is composed of both positive and negative effects. Similar to previous results with AAV Rep52, AAV2 large Rep was targeted for ubiquitination and degradation by the Ad E4orf6/E1b 55 kDa, cullin 5-containing, E3-ubiquitin ligase. Additionally, large Rep was targeted for ubiquitination via extension of ubiquitin lysine K48 and K63 both in the presence and absence of E4orf6. more...

The ability of cells to traverse pores in a biocompatible filter provides means for examining cell chemoattraction. Filter-based assays also permit rapid, quantitative assessment of the in vitro migratory and invasive potential of tumor cells. Scoring migration has relied on visual counting of stained cells which appear on the underside of the filter and determining a true percentage score involves arduous counting of cells on both filter surfaces. Visual counting of random fields may be unreliable, and counting all fields is laborious. In the present study we developed and compared two alternative methods for scoring cell numbers in filter-based assays, a colorimetric assay of toluidine blue binding, and a radioassay of cells prelabeled with [ 3 H]thymidine. Each method was evaluated for sensitivity, variability, ease of use and efficiency, and suitability for use in assays of cell migration and invasion. The radiolabeling method proved to be sensitive and reliable and was the most efficient technique. Although less sensitive and specific, the colorimetric dye method offered a rapid and reliable, nonradioactive alternative with the distinct advantage of preserving intact cultures for follow-up visual assessments. We conclude that colorimetric and radiolabel scoring of filter-based assays are reliable and efficient semiantomated methods which provide means to obtain more complete assessments of cell migration and invasion. more...

Microfilaments and microtubules play a part in the extension of neuronal processes but their roles in the formation of glial processes have not yet been determined. The objectives of this study were to determine the organization of microfilaments in differentiating glial progenitors (RB2 cells) and to study the effects of microfilament or microtubule disruption on process extension. Intense F-actin staining (crown of microfilaments) was observed at the leading edge of a small extending conical tip in differentiating RB2 cells, but was absent in process-bearing TE671 rhabdomyosarcoma cells. No significant difference was noted in the mean number of TE671 cells with processes treated with a microfilament disrupter from that of similarly treated controls. In contrast, a significant difference was noted in the mean number of RB2 cells with processes after microfilament disruption treatment from that of similarly treated controls. Microtubule disruption arrested extension and caused process retraction in both cell types. The results of this study demonstrate that microtubules play an equally important part in the extension and stabilization of the RB2 and TE671 processes. Moreover, the crown of microfilaments concentrated in the glial RB2 process (and not in the TE671 process) may be critical to its extension during differentiation. more...

Adeno-associated virus (AAV) type 2 and 5 proteins Rep52 and Rep40 were polyubiquitinated during AAV-adenovirus type 5 (Ad5) coinfection and during transient transfection in either the presence or absence of Ad5 E4orf6 and E1b-55k. Polyubiquitination of small Rep proteins via lysine 48 (K48) linkages, normally associated with targeting of proteins for proteasomal degradation, was detected only in the presence of E4orf6. The small Rep proteins were ubiquitinated via lysine 63 (K63) following transfection in either the presence or absence of E4orf6 or following coinfection with Ad5. E4orf6/E1b-55k-dependent K48-specific polyubiquitination of small Rep proteins could be inhibited using small interfering RNA (siRNA) to cullin 5. more...

Previously, we demonstrated that through binding a novel intracellular receptor of microM affinity (HIC), histamine mediates, and the HIC antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine. HCl (DPPE) inhibits, platelet aggregation and serotonin granule secretion; the latter response is dependent upon the same processes that mediate histamine release from mast cell granules. We now show that, as for platelet serotonin release, DPPE blocks concanavalin A-stimulated mast cell histamine release with a potency (IC50 = 30 microM) greater than the H1-antagonist, pyrilamine (IC50 = 150 microM) or the H2-antagonist cimetidine (IC50 = 5 mM), correlating with rank order of potency to inhibit 3H-histamine binding in rat brain membranes and liver microsomes. We postulate that histamine release from mast cells is mediated at HIC by second messenger intracellular histamine. However, unlike platelets, mast cells do not appear to rely on newly synthesized histamine. Rather, as for calcium, histamine may be mobilized from bound stores to mediate histamine secretion. more...

Radiation-induced craniofacial bone growth inhibition is a consequence of therapeutic radiation in the survivors of pediatric head and neck cancer. Previously, the infant rabbit orbitozygomatic complex (OZC) was established as a reliable animal model. The purpose of this study was to develop a cell culture model from the rabbit OZC to study the effects of radiation in the craniofacial skeleton. Infant (7-week-old) New Zealand white rabbits were used in this study. Periostea from both OZC were harvested in sterile conditions, introduced into cell culture by way of sequential digestion, and subcultured at confluence. Cultures were analyzed for cellular proliferation (methylthiazoletetrazolium assay), alkaline phosphatase activity, collagen type I expression, and mineralization. Electron microscopy was performed to reveal the in vitro ultrastructure. Subsequently, rabbits were irradiated with sham or 15 Gy radiation, and cell cultures were developed and analyzed for cell numbers. Cell cultures, grown from OZC periostea, expressed osteoblast-like phenotype, with high alkaline phosphatase activity, collagen type 1 expression, and mineralization in an osteogenic environment. Electron microscopy confirmed the characteristic ultrastructural features of osteogenesis in vitro. Finally, significantly (P < 0.01) fewer cells were obtained from animals treated with 15 Gy radiation compared with those from control animals.A primary cell culture with osteoblast-like cellular phenotype was developed from infant rabbit OZC periosteum. This cell culture system responded to in vivo administered radiation by a significant decrease in cell numbers. This in vitro model will be subsequently used to study the cellular mechanisms of radiation and radioprotection in craniofacial osteoblast-like cells. more...

Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1. Furthermore, Vaxfectin had no effect on IFN-γ or IL-4 production by splenocytes re-stimulated with antigen, suggesting that the Th1 type responses typical of intramuscular pDNA immunization were not altered. Studies with IL-6 −/− mice suggest that the antibody enhancement is IL-6 dependent and results in a correlative increase in antigen specific antibody secreting cells. more...

This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhances antibody responses when complexed with an antigen-encoding plasmid DNA (pDNA). In mice, intramuscular injection of Vaxfectin formulated with pDNA encoding influenza nucleoprotein (NP) increased antibody titers up to 20-fold, to levels that could not be reached with pDNA alone. As little as 1 microg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 microg naked pDNA. The antibody titers in animals injected with Vaxfectin-pDNA remained higher than in the naked pDNA controls for at least 9 months. The enhancement in antibody titers was dependent on the Vaxfectin dose and was accomplished without diminishing the strong anti-NP cytolytic T cell response typical of pDNA-based vaccines. In rabbits, complexing pDNA with Vaxfectin enhanced antibody titers up to 50-fold with needle and syringe injections and also augmented humoral responses when combined with a needle-free injection device. Vaxfectin did not facilitate transfection and/or increase synthesis of beta-galactosidase reporter protein in muscle tissue. ELISPOT assays performed on bone marrow cells from vaccinated mice showed that Vaxfectin produced a three- to five-fold increase in the number of NP-specific plasma cells. Thus, Vaxfectin should be a useful adjuvant for enhancing pDNA-based vaccinations. more...

Aryl hydrocarbon receptor (AhR) ligands are environmental contaminants found in cigarette smoke and other sources of air pollution. The prototypical compound is TCDD (2,3,7, 8-tetrachlorodibenzo-p-dioxin), also known as dioxin. There is an increasing body of knowledge linking cigarette smoking to osteoporosis and periodontal disease, but the direct effects of smoke-associated aryl hydrocarbons on bone are not well understood. Through the use of resveratrol (3,5,4'-trihydroxystilbene), a plant antifungal compound that we have recently demonstrated to be a pure AhR antagonist, we have investigated the effects of TCDD on osteogenesis. It was postulated that TCDD would inhibit osteogenesis in bone-forming cultures and that this inhibition would be antagonized by resveratrol. We employed the chicken periosteal osteogenesis (CPO) model, which has been shown to form bone in vitro in a pattern morphologically and biochemically similar to that seen in vivo, as well as a rat stromal cell bone nodule formation model. In the CPO model, alkaline phosphatase (AP) activity was reduced by up to 50% (P more...

Osteoporosis is a common problem of aging and results from a failure of homeostatic mechanisms to regulate osteogenesis and mineralization. Bovine and human forms of fetuin glycoprotein bind to the transforming growth factor (TGF)-beta/BMP (bone morphogenic protein) cytokines and block their osteogenic activity in cell culture assays (Demetriou, M., Binkert, C., Sukhu, B., Tenenbaum, H. C., and Dennis, J. W. (1996) J. Biol. Chem. 271, 12755-12761). Fetuin is a prominent serum glycoprotein and a major noncollagenous component of mineralized bone in mammals. In this study, we show that recombinant fetuin and native serum protein have similar potency as inhibitors of osteogenesis in dexamethasone-treated rat bone marrow cell cultures (dex-RBMC). Recombinant bovine fetuin also bound to TGF-beta1 and BMP-2 in vitro with kinetics similar to native fetuin. Although TGF-beta1 is required for osteogenesis in dex-RBMC, the cytokine also inhibited osteogenesis at concentrations >/=10 pM. Titration of fetuin or anti-TGF-beta1 antibodies into the bone marrow cultures in the presence of 10 pM TGF-beta1 restored osteogenesis, whereas titrations of the same reagents into cultures with 0.3 pM added TGF-beta1 were inhibitory, confirming the biphasic nature of the TGF-beta1 response. Suppression of osteogenesis by both TGF-beta1 and the antagonist proteins required their presence within the first 6 days of culture, well before mineralization at 10-12 days. Northern analysis showed that both fetuin and high dose TGF-beta1 suppressed expression of the bone-associated transcripts alkaline phosphatase, osteopontin, collagen type I, and bone sialoprotein. The suppression of osteogenesis by fetuin and by high dose TGF-beta1 was accompanied by the differentiation of an alternate cell lineage with adipocyte characteristics. In summary, the biphasic osteogenic response to TGF-beta1 suggests that overlapping gradients of TGF-beta/BMP cytokines and fetuin regulate osteogenesis in remodeling bone. more...

Tamoxifen is a synthetic estrogen analog which may regulate osteogenesis in vivo by virtue of its antiglucocorticoid properties. We have examined tamoxifen regulation of glucocorticoid-induced osteogenesis in two different in vitro bone systems: the chicken periosteal osteogenesis model (CPO) and rat bone marrow stromal cells (RBMC). Hormone uptake studies were conducted with the osteosarcoma cell line, ROS 17/2.8. In the CPO model, alkaline phosphatase (AP) activity and collagen synthesis were stimulated by the glucocorticoid dexamethasone (Dex; 0.1 microM). These Dex-mediated effects were inhibited by increasing concentrations of tamoxifen (10-100 microM). Similarly, in the RBMC model, Dex-dependent (0.01 microM Dex) mineralized tissue formation and AP activity were blocked by tamoxifen (0.1 microM). Although tamoxifen inhibited Dex-mediated increases of AP activity in ROS 17/2.8 cells, it did not inhibit uptake of 3H-Dex or of 3H-estrogen. Northern analyses showed that tamoxifen did not affect messenger RNAs (mRNAs) for AP. Tamoxifen did seem to reduce mRNA for collagen type I, but not bone sialoprotein, osteopontin, and osteocalcin. Dex-induced increases for all proteins mRNAs in the RBMC model were not reduced by tamoxifen. Similarly, tamoxifen had no effects on cellular proliferation. We conclude that tamoxifen has no direct effect on gene expression of bone-related proteins of osteoblastic cells. Further, in the ROS 17/2.8 cell line, the antiglucocorticoid properties of tamoxifen do not appear to be mediated through either Dex or estrogen receptors. more...

Mineralized bone formation in vitro can be induced by the alkaline phosphatase substrate beta-glycerophosphate (GP). GP may not only be essential for mineralization in vitro, but could also modulate other metabolic activities of bone cells, particularly if GP is presented to these cells during different phases of development. To assess GP modulation of bone cell metabolism, biochemical and autoradiographic analyses of chick periosteal cultures treated with GP were performed. About 50% less (p less than 0.05) Type I collagen was produced in periosteal cultures treated with GP. If the fibrous portion of the periostem was microdissected from the osteogenic layer prior to culture, GP inhibition of Type I collagen synthesis was even more marked (60%: p less than 0.05). To define organic phosphate-sensitive phases of osteogenesis, cultures were exposed to GP for various time periods. Mineralization occurred reproducibly when periosteal cultures were treated with GP from the outset of the incubation period (positive control). However, if GP was added after the third day of incubation, phosphate content was the same as in positive control cultures, whereas calcium content was significantly (20%: p less than 0.05) lower. Moreover, if GP was added on day 6, there was virtually no calcium accumulation by day 12, while massive amounts of phosphate had accumulated. Taken together, these findings indicate that organic phosphates may modulate phenotypic expression of osteogenic cells, and that osteogenic cells traverse an organic phosphate-sensitive phase, after which they may be incapable of normal mineralization. more...

Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta-aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE. more...

Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by the DNA. The preferred vehicle for plasmid DNA injections has been saline (154 mM sodium chloride) or PBS (154 mM NaCl plus 10 mM sodium phosphate). Here, it is shown that injection of luciferase or beta-galactosidase encoding plasmid DNA in a 150 mM sodium phosphate vehicle into murine muscle resulted in a two- to seven-fold increase in transgene expression compared with DNA injected in saline or PBS. When the DNA encoded secreted alkaline phosphatase, preproinsulin or interferon, sodium phosphate vehicle increased their serum levels by two- to four-fold. When the DNA encoded mouse erythropoietin, sodium phosphate vehicle increased hematocrits by two-fold compared with DNA injected in saline. When the DNA encoded influenza nucleoprotein, sodium phosphate increased anti-nucleoprotein antibody titers by two-fold. The expression of luciferase from plasmid DNA instilled into lung was increased five-fold compared with that in vehicle without sodium phosphate. Incubation of plasmid DNA with muscle extract or serum showed that sodium phosphate protected the DNA from degradation. Thus, a change from sodium chloride to sodium phosphate vehicle can enhance the expression of plasmid DNA in a tissue, possibly by inhibiting DNA degradation. Gene Therapy (2000) 7, 1171-1182. more...