Nonimmune Serum Research Articles

Detection and Level Evaluation of Antibodies Specific to Environmental Bacteriophage I11mO19 and Related Coliphages in Non-Immunized Human Sera

Bacteriophages (phages) are viruses infecting bacteria. They are widely present in the environment, food, and normal microflora. The human microbiome is a mutually interdependent network of bacteria, bacteriophages, and human cells. The stability of these tri-kingdom interactions may be essential for maintaining immunologic and metabolic health. Phages, as with each other's antigens, may evoke an immune response during a human's lifetime and induce specific antibody generation. In this manuscript, we labeled these antibodies as naturally generated. Naturally generated antibodies may be one of the most important factors limiting the efficacy of phage therapy. Herein, we attempted to determine the physiological level of these antibodies specific to a population bacteriophage named I11mO19 in human sera, using an ELISA-based assay. First, we purified the phage particles and assessed the immunoreactivity of phage proteins. Then, affinity chromatography was performed on columns with immobilized phage proteins to obtain a fraction of human polyclonal anti-phage antibodies. These antibodies were used as a reference to elaborate an immunoenzymatic test that was used to determine the level of natural anti-phage antibodies. We estimated the average level of anti-I11mO19 phage antibodies at 190 µg per one milliliter of human serum. However, immunoblotting revealed that cross-reactivity occurs between some proteins of I11mO19 and two other coliphages: T4 and ΦK1E. The antigens probably share common epitopes, suggesting that the determined level of anti-I11mO19 phage might be overestimated and reflects a group of antibodies reactive to a broad range of other E. coli phages. Anti-I11mO19 antibodies did not react with Pseudomonas bacteriophage F8, confirming specificity to the coliphage group. In this work, we wanted to show whether it is possible to determine the presence and level of anti-phage antibodies in nontargeted-immunized sera, using an immunoenzymatic assay. The conclusion is that it is possible, and specific antibodies can be determined. However, the specificity refers to a broader coliphage group of phages, not only the single phage strain.

Immunochemical activity of <i>Yersinia pseudotuberculosis</i> ompF and ompC porins evaluated by optical trapping

Introduction. Study of features for interacting antigen-antibody system is of great importance for developing new modern tools for diagnostics and therapy of infectious diseases. In this regard, it is of great interest to study the rupture force between bacterial antigens and antibodies using modern biophysical methods including optical trapping. The importance of surface antigens in the immunochemical activity of Yersinia pseudotuberculosis assessed by such method has not been evaluated yet. In this work we examined an opportunity to evaluate the interaction of hydrophobic Y. pseudotuberculosis porins OmpF and OmpC with specific antibodies using optical trapping method. Materials and methods. Polystyrene microspheres (d = 1 m) were coated by passive adhesion with purified preparations of OmpF and OmpC porins; microsphere sensitization was verified by enzyme immunoassay. Antibodies from mouse sera were adsorbed onto the glass surface by chemical linking. The rupture force in the porins-antibodies system was determined using a laser trap according to the previously developed algorithm. Results. Using a model system including polystyrene microspheres sensitized with the proteins and aminated glass substrate coated with immune or nonimmune serum, significant differences in binding strength of OmpF and OmpC porins to homologous immune versus nonimmune sera were detected. The average forces of interaction with immune sera was 60 pN for OmpF microspheres (control 40 pN) and 69 pN for OmpC microspheres (control 49 pN). The proportion of irreversible substrate binding of the microspheres coated by the antigens to the treated with immune vs. non-immune sera was significantly higher. The results of assessing the average interaction force, as well as the predominance of the proportion of irreversible binding of antigen-coated microspheres with sera-treated substrates, indicates that specific interactions contribute significantly to the force of interaction. The aforementioned method can be used to evaluate the forces of intermolecular interaction in similar model systems using other microbial antigens.

Effects of Leptin antagonist treatments on testosterone and testis histological characteristics of immature male mice

The present study aimed to ascertain how leptin antagonist injection affected testis weights, testis morphology and testosterone levels in immature male Swiss mice. Animals were administered with anti-leptin antibody subcutaneously, with or without equine chorionic gonadotropin (eCG). Control animals were treated with non-immune serum. Blood and testis were collected. The Androgen profile was analyzed in serum and tissue homogenates, and testes were histologically examined. Compared to controls, mice treated with an anti-leptin antibody with or without gonadotropins had a significant (p<0.05) increase in testis weight. Testosterone concentrations in the testis were significantly (p<0.05) higher in mice administered with anti-leptin antibody compared to control, but testosterone concentrations in blood were not affected. The diameter of seminiferous tubules, the diameter of the lumen and the width of spermatogenic cells were significantly (p<0.05) higher in mice in treatment groups compared to controls. We conclude that anti-leptin antibody administration in immature male mice increased testosterone concentrations in the testis and improved testis histological characteristics. Keywords: leptin; mouse; histology; testis; testosterone; immature male

Development of a biosafe ELISA-based platform for assessing immunogenicity in the production of an inactivated whole-virion coronavirus vaccine

BACKGROUND: SARS-CoV-2 vaccine immunogenicity is evaluated in neutralization test with live virus. It is performed in a biosafety level 3 zone because requires live virus stage. Therefore, control laboratories should be certified for this class of work. The development of technology based on enzyme-linked immunosorbent assay as an analogue of the neutralization reaction makes it possible to create an immunobiological product in a shorter time and in conditions without special requirements for control laboratories.
 AIM: Development of an enzyme-linked immunosorbent assay for assessing SARS-CoV-2 vaccine immunogenicity by measuring neutralizing antibodies production in immunized animals.
 MATERIALS AND METHODS: Recombinant receptor-binding domain fused to a С-terminal hexahistidine sequence was produced in Escherichia coli cells and purified via metal-affinity chromatography on WorkBeads NiMAC (Bio-Works). Purified protein was used in enzyme-linked immunosorbent assay as an antigen for sorption. The sera of mice immunized with the vaccine preparation were tested for neutralizing activity against the SARS-CoV-2, as well as in the developed enzyme-linked immunosorbent assay.
 RESULTS: Sera with high neutralizing titers showed a high degree of binding to recombinant receptor-binding domain fused to a С-terminal hexahistidine sequence in enzyme-linked immunosorbent assay, while sera from non-immunized animals or sera with neutralization titers less than 1:8 were not reactive in enzyme-linked immunosorbent assay. The Spearman and Pearson correlation coefficients for neutralization test titers and optical density in enzyme-linked immunosorbent assay were 0.759 and 0.76, respectively. The developed assay can be used as a semi-quantitative method for assessing the immunogenicity of a vaccine against coronavirus infection.
 CONCLUSIONS: The developed platform makes it possible to reliably assess the immunogenicity of an inactivated coronavirus vaccine under conditions that do not require a high biosafety conditions.

Optimization of a protocol for the evaluation of antibody responses to human papillomavirus (HPV) vaccination in low-resource settings

IntroductionAvailable human papillomavirus (HPV) vaccines could have an important primary role in cervical cancer prevention once their long-term immunogenicity and safety are evaluated at the population level. The aim of this study was to optimize an assay to be used in evaluating the long-term durability of HPV vaccine response following a pilot vaccination of adolescent girls in Ghana.MethodsA rapid, high-throughput, indirect enzyme-linked immunosorbent assay (ELISA) was optimized for the detection and quantitation of anti-HPV L1 (late expression protein: types 6, 11, 16 and 18) immunoglobulin G (IgG) in human serum (n = 89). The utility of the assay was demonstrated using serum collected from a cohort of pre-adolescent girls (n = 49) previously vaccinated with a quadrivalent vaccine and non-immune serum obtained from age-matched controls (n = 40).ResultsThe assay showed good discrimination of antibody levels between cases and control sera: seroprevalence of anti-HPV IgG antibodies was significantly higher among vaccinated than unvaccinated girls for both HPV-16 (63.3% vs. 12.5%; p < 0.001) and HPV-18 (34.7% vs. 20.0%; p = 0.042), respectively. Thirty-six months after receiving the third dose of vaccine, significantly higher mean anti-HPV-16 (0.618 vs. 0.145), anti-HPV-18 (0.323 vs. 0.309), and anti-HPV-6 (1.371 vs. 0.981) antibody levels were measured, compared to unvaccinated girls (all p < 0.05). A correlation between optical density and antibody activity indicated assay sensitivity to increasing levels of antibody activity.ConclusionWe have successfully optimized and implemented a robust and sensitive assay for the evaluation of antibody responses among immunized adolescent girls for monitoring future large-scale HPV vaccination studies in low-income settings. Our results demonstrated greater immunoglobulin G antibody activity within serum drawn from adolescent girls immunized 36 months prior.

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteobacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intracellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characterization through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.

Murine Cytomegalovirus-induced Complement-fixing Antibodies Deposit in Murine Renal Allografts During Acute Rejection.

Human cytomegalovirus (CMV) infection is associated with renal allograft dysfunction and loss, particularly in combination with acute rejection. Emerging literature suggests that non-HLA antibodies may contribute to antibody-mediated rejection, but pathogen-induced antibodies have not been investigated in this context. This study examines the presence of CMV-induced antibodies in murine CMV (MCMV)-infected renal allografts during acute rejection. Intragraft immunoglobulin G (IgG) and complement C3 immunostaining were compared among allogeneic MCMV D-/R-, D+/R-, and D+/R+ renal transplants. Intragraft antibody deposition was examined in B cell-deficient recipients treated with MCMV immune sera. Antibody binding and complement-dependent cytotoxicity (CDC) of D-/R- and D+/R+ sera against infected renal tubular epithelial cells (TECs) were measured in vitro. IgG immunostaining was performed in D+/R+ allografts and native kidneys and in D+/R- allografts treated with ganciclovir to inhibit viral replication. D+/R- and D+/R+ transplants had more abundant IgG and C3 deposition compared with D-/R- recipients. Greater IgG deposition was associated with more severe allograft injury in B cell-deficient recipients treated with MCMV immune sera compared with nonimmune sera. D+/R+ sera induced greater CDC of infected TECs compared with D-/R- sera. Native kidneys had lower IgG deposition compared with allografts, despite similar organ viral loads. Ganciclovir-treated allografts had reduced IgG deposition compared with untreated allografts. In this murine model, complement-fixing antibodies can deposit into MCMV-infected renal allografts, are associated with allograft damage, and can induce CDC of MCMV-infected renal TECs. The allogeneic response and viral replication may also contribute to intragraft antibody deposition.

CD39 and CD73 Activity Are Protective in a Mouse Model of Antiphospholipid Antibody-Induced Miscarriage

Antiphospholipid antibodies (aPL-ab) including lupus anticoagulant, anticardiolipin (aCL) antibodies and anti-β2-glycoprotein I (β2GP1) antibodies initiate inflammation, tissue factor-activated coagulation, oxidative stress and complement activation. These pathways contribute to the pathophysiology of thrombotic and obstetric manifestations in antiphospholipid syndrome (APS). Not all patients with aPL-ab develop disease phenotype and hence a ‘second hit’ is postulated to be necessary. Extracellular purines (ATP, ADP, and adenosine) are important signalling molecules with the general acceptance that ATP promotes inflammation, ADP is pro platelet-aggregatory, while adenosine has vasodilatory, antithrombotic and anti-inflammatory effects. Extracellular ATP/ADP levels are tightly regulated by CD39 and CD73. CD39 hydrolyses ATP and ADP to AMP, which is then hydrolysed to adenosine by the ecto-5'-nucleotidase CD73.We hypothesised that CD39 and CD73 mitigate both vascular thrombosis as well as inflammatory responses in APS. We developed a murine model of APS-miscarriages by passive infusion of aPL-ab harvested from patients into pregnant mice. Comparison made with infusion of non-immune IgG. We applied this model to wild type (WT), CD39-transgenic mice as well as CD39-null and CD73-null mice. Non-immune IgG administration to WT or CD39-/- C57BL/6 mice did not cause miscarriages, whereas aPL-ab-treated WT mice displayed a mean resorption frequency of 3.67%. In comparison, aPL-ab-treated CD39-/- mice showed a significantly higher mean resorption frequency of 20.1% (p<0.001 vs aPL-ab-treated WT; Fig. 1a). aPL-ab-treated CD73-/- mice also had a significantly increased mean resorption frequency of 10.5% (p<0.05 vs aPL-ab-treated WT).We have previously demonstrated that CD39 over expression in BALB/c transgenic mice is associated with enhanced adenosine generation under prothrombotic conditions. WT BALB/c mice injected with non-immune IgG had a mean resorption frequency of 21.2%, which doubled with aPL-ab injection (39.7%; p<0.05 vs non-immune IgG-treated WT). In transgenic BALB/c mice over-expressing CD39, mean resorption frequencies were significantly lower in the aPL-ab-treated group (11.4% vs 39.7% in aPL-ab-treated WT, p<0.01).QRT-PCR showed 1.6-fold and 2.4-fold increase in TF expression in aPL-ab administered CD39-/- and CD73-/- mice relative to both WT C57BL/6 mice and littermates injected with non-immune sera and. CD39 over expression suppressed TF expression compared with WT BALB/c mice after both non-immune IgG and aPL-ab challenge particularly with 6.3 fold lower TF mRNA levels after aPL-ab infusion. Decidua from aPL-ab injected CD39-/- and CD73-/- C57BL/6 mice showed increased TF immunoreactivity compared to aPL-ab-treated or nonimmune IgG-treated WT mice. C3d deposition was increased by aPL-ab treatment relative to non-immune IgG treatment in all groups except the CD39 transgenic mice (Fig. 3). C3d deposition was most marked in aPL-ab-treated CD39-/- and CD73-/- mice.Placental expression of inflammatory cytokines C57BL/6was measured at the mRNA level by qRT-PCR. TNF-α expression was doubled in CD39-/- and CD73-/- mice relative to non-immune IgG controls. CD39 Tg mice showed suppression of TNF- α after aPL-ab challenge. Consistent with the previously documented role for IL-10 in exacerbating lupus/auto-immune diseases, we noted a significant reduction in IL-10 mRNA in aPL-ab-treated CD39 Tg mice compared to aPL-ab-treated WT mice.Therefore, we demonstrate a protective role for CD39 in APS and provide rationale for the development of soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease DisclosuresDwyer:Kyowa Kirin Pharmaceutical Development, Inc.: Employment.

Improved quantification of immune-gold labeling and its use to compare the distribution of cellular factors among sub-chloroplast compartments

Quantification of immuno-gold labeling can provide valuable information on the quantity and localization of a target within a region of interest (ROI). Background subtraction usually requires preparation of material with a deliberately reduced amount of target component often by gene knockout/knockdown. This paper reports a modified method without the need for gene knockout/knockdown, by using a region outside the ROI as a background and non-immune serum to verify the reliability of the data. An optimized parameter for use in image processing was also developed to improve semi-automatic segmentation of gold particles, by using the standard deviation of pixel intensity together with default parameters (size and intensity) to improve specificity. The modified methods were used to quantify the gold labeling of various components within chloroplasts and their 3 sub-organelle compartments (thylakoid, stroma and starch). Rubisco, actin, myosin, β-tubulin, Endoplasmic reticulum-retention signal HDEL, Sterol methyltransferase 1, and double stranded RNA were all effectively and consistently quantified at the level of the different sub-chloroplast compartments. The approach should be applicable more widely for high resolution labelling of samples in which a background requiring gene knockout/knockdown is not a realistic option.

A Protective and Pathogenic Role for Complement During Acute Toxoplasma gondii Infection.

The infection competence of the protozoan pathogen Toxoplasma gondii is critically dependent on the parasite’s ability to inactivate the host complement system. Toxoplasma actively resists complement-mediated killing in non-immune serum by recruiting host-derived complement regulatory proteins C4BP and Factor H (FH) to the parasite surface to inactivate surface-bound C3 and limit formation of the C5b-9 membrane attack complex (MAC). While decreased complement activation on the parasite surface certainly protects Toxoplasma from immediate lysis, the biological effector functions of C3 split products C3b and C3a are maintained, which includes opsonization of the parasite for phagocytosis and potent immunomodulatory effects that promote pro-inflammatory responses and alters mucosal defenses during infection, respectively. In this review, we discuss how complement regulation by Toxoplasma controls parasite burden systemically but drives exacerbated immune responses locally in the gut of genetically susceptible C57BL/6J mice. In effect, Toxoplasma has evolved to strike a balance with the complement system, by inactivating complement to protect the parasite from immediate serum killing, it generates sufficient C3 catabolites that signal through their cognate receptors to stimulate protective immunity. This regulation ultimately controls tachyzoite proliferation and promotes host survival, parasite persistence, and transmissibility to new hosts.

Development of a Simple Method for the Specific Detection of Anthrax Antibody in Sera by Colored Slide Agglutination Test

This study was aimed to identify humoral immune response against anthrax vaccine in mice model by using colored slide agglutination test and detection of field infectivity of anthrax. The field isolates of B. anthracis (n=05) and F34 stern strain vaccine was isolated on agar plates in order to carry out the slide agglutination test. The field isolates of B. anthracis and vaccine bacteria grew on PLET agar medium produced roughly circular and creamy white colonies with ground glass appearance. The bacteria on sheep blood agar media produced rough, sticky, white-gray non hemolytic colonies. Colony polymerase chain reaction (PCR) protocol was adapted to detect fragments of pX01 (596bp) and pX02 (777bp) plasmid of virulent field isolates of B. anthracis. The fragment of pX01 plasmid was only detected in vaccine bacteria. Growth of a field isolate and a vaccine bacteria were colored with crystal violet and used in slide agglutination test to detect anthrax antibodies. The anti-anthrax antibody was prepared by immunizing female mice with 100ül anthrax vaccine through subcutaneous route. Tail bleed were collected on day 0, 30, 60, 120 and 180 of immunization. Cardiac bleed was collected on day 180 of immunization for extensive study. 25μl of diluted (1:10, 1:20, 1:50 and 1:100) antisera and 25μl colored antigen was mixed together onto a clean slide at room temperature and the results was red following 5min, 10mins, 15mins and 20mins of reaction. Unstained antigens and non-immunized sera from the mice were used as control. Results of slide agglutination test showed that the colored vaccine bacteria and field isolates clumped the mice anti-antisera (day 30, 60, and 120) at 1:20 dilution as seen in naked eye but the reaction was seen only at 1:10 dilution while colorless antigens were used. Under microscopic investigation of slide agglutination test, the reaction was read up to 1:100 dilutions with the sera collected at day 30, 60, 120 and 180 of immunization. The Anthrax Sterne strain vaccine induced anti-anthrax immunity in mice that was detected until day 180 of immunization. The clumping reaction was distinct while colored anthrax antigen was used in slide agglutination tests. The colored slide agglutination tests protocol developed in this study can be used to detect anti-anthrax immune response and anthrax bacteria in the field condition with minimum laboratory facilities.&#x0D; Res. Agric., Livest. Fish.7(3): 497-506, December 2020

Immunotherapy targeting the Streptococcus pyogenes M protein or streptolysin O to treat or prevent influenza A superinfection.

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.

Toxoplasma gondii Recruits Factor H and C4b-Binding Protein to Mediate Resistance to Serum Killing and Promote Parasite Persistence in vivo.

Regulating complement is an important step in the establishment of infection by microbial pathogens. Toxoplasma gondii actively resists complement-mediated killing in non-immune human serum (NHS) by inactivating C3b, however the precise molecular basis is unknown. Here, a flow cytometry-based C3b binding assay demonstrated that Type II strains had significantly higher levels of surface-bound C3b than Type I strains. However, both strains efficiently inactivated C3b and were equally resistant to serum killing, suggesting that resistance is not strain-dependent. Toxoplasma activated both the lectin (LP) and alternative (AP) pathways, and the deposition of C3b was both strain and lectin-dependent. A flow cytometry-based lectin binding assay identified strain-specific differences in the level and heterogeneity of surface glycans detected. Specifically, increased lectin-binding by Type II strains correlated with higher levels of the LP recognition receptor mannose binding lectin (MBL). Western blot analyses demonstrated that Toxoplasma recruits both classical pathway (CP) and LP regulator C4b-binding proteins (C4BP) and AP regulator Factor H (FH) to the parasite surface to inactivate bound C3b–iC3b and C3dg and limit formation of the C5b-9 attack complex. Blocking FH and C4BP contributed to increased C5b-9 formation in vitro. However, parasite susceptibility in vitro was only impacted when FH was blocked, indicating that down regulation of the alternative pathway by FH may be more critical for parasite resistance. Infection of C3 deficient mice led to uncontrolled parasite growth, acute mortality, and reduced antibody production, indicating that both the presence of C3, and the ability of the parasite to inactivate C3, was protective. Taken together, our results establish that Toxoplasma regulation of the complement system renders mice resistant to acute infection by limiting parasite proliferation in vivo, but susceptible to chronic infection, with all mice developing transmissible cysts to maintain its life cycle.

An optimized permeabilization step for flow cytometry analysis of nuclear proteins in myeloid differentiation of blood cells into neutrophils

Polymorphonuclear leukocytes (PMNLs) or neutrophils play an important role in the innate immune response. Working with human neutrophils is challenging because these cells are sensitive to changes in the surrounding media and quickly become apoptotic. Meanwhile the experiments with mature neutrophils may be very important for studies of blood function. In this paper we propose an improved technique of flow cytometry nuclear protein analysis with double antibody labeling, which allows direct comparison of protein quantity (overlay histograms) in the primary cells (neutrophils) and progenitor cell lines (line HL-60), to study differentiation process and for other research purposes. We suggest improved technique to analyze and compare nuclear proteins levels in the myeloid differentiation model system (HL-60 cell line) and / or primary human neutrophils. This method was justified with measurement of GFI1 protein expression level, as well-known transcription factor, typical and essential for mature neutrophils.The key protocol features are as follows:•Suggested protocol allows simply, direct and correct visual comparison of flow cytometry data in overlay diagrams for myeloid blood cells on various stages of differentiation.•70% ethanol permeabilization of neutrophils and HL-60 cells results in lower background fluorescence and better peak resolution than MeOH and Saponin permeabilization.•Non-specific antibody binding in neutrophils can be efficiently blocked by using 1% BSA and non-immune goat serum.

Maternal immunization downregulates offspring TCD4 regulatory cells (Tregs) thymic maturation without implications for allergy inhibition.

The regulation of offspring allergy development mediated by maternal immunization was evidenced by several groups, and this mechanism seems to involve the induction of regulatory T cells (Tregs) on offspring. Here, we aimed to evaluate whether the effect of maternal immunization on offspring Tregs occurs as a result of peripheral or central modulation. Briefly, C57BL/6 female mice were immunized with OVA in Alum or Alum alone and boosted with OVA in saline or saline only after 10 and 20days. Non-immunized offspring serum, thymus and spleen were evaluated at 3 or 20days old, and some groups of pups were submitted to neonatal OVA-immunization protocol for the subsequent evaluation of antibody production and allergic response. Our experimental protocol could be validated because maternal OVA-immunization inhibited offspring allergic response as evidenced by the suppression of offspring IgE production and allergic lung inflammation. Interestingly, maternal immunization reduced the frequency of offspring thymic Tregs with an opposite effect on spleen Tregs. Furthermore, after neonatal immunization, the frequency of lung-infiltrated Tregs was also augmented on offspring from immunized mothers. In conclusion, maternal OVA-immunization can inhibit the thymic maturation of offspring Tregs without implications on peripheral Tregs induction and allergy inhibition.

Acinetobacter baumannii maintains its virulence after long-time starvation.

Acinetobacter baumannii is a cause of healthcare-associated infections. Although A. baumannii is an opportunistic pathogen, its infections are notoriously difficult to treat due to intrinsic and acquired antimicrobial resistance, often limiting effective therapeutic options. A. baumannii can survive for long periods in the hospital environment, particularly on inanimate surfaces. Such environments may act as a reservoir for cross-colonization and infection outbreaks and should be considered a substantial factor in infection control practices. Moreover, clothing of healthcare personnel and gadgets may play a role in the spread of nosocomial bacteria. A link between contamination of hospital surfaces and A. baumannii infections or between its persistence in the environment and its virulence has not yet been established. Bacteria under stress (i.e., long-term desiccation in hospital setting) could conserve factors that favor infection. To investigate whether desiccation and/or starvation may be involved in the ability of certain strains of A. baumannii to retain virulence factors, we have studied five well-characterized clinical isolates of A. baumannii for which survival times were determined under simulated hospital conditions. Despite a considerable reduction in the culturability over time (up to 88% depending on strain and the condition tested), some A. baumannii strains were able to maintain their ability to form biofilms after rehydration, addition of nutrients, and changing temperature. Also, after long-term desiccation, several clinical strains were able to grow in the presence of non-immune human serum as fine as their non-stressed homologs. Furthermore, we also show that the ability of bacterial strains to kill Galleria mellonella larvae does not change although A. baumannii cells were stressed by long-term starvation (up to 60 days). This means that A. baumannii can undergo a rapid adaptation to both the temperature shift and nutrients availability, conditions that can be easily found by bacteria in a new patient in the hospital setting.

2010 Use it but still lose it: Exploring age-related changes in skeletal stem cell location and activation in response to physical stimulation

OBJECTIVES/SPECIFIC AIMS: Our goal is to assess age-related changes in osteogenic stem cell populations of bone tissue. We hypothesize that aging mice have reduced osteogenic capacity in response to physical stimulation due to aging-associated decline in osteoprogenitor cell number and their proliferative capacity. METHODS/STUDY POPULATION: Mechanical loading: The NYU School of Medicine Institutional Animal Care and Use Committee approved all procedures. The response of tibial periosteal cells to physical stimulation or mechanical loading was assessed in 16-week-old adult (n=6) and aged 78-week-old female (n=4) mice subjected to 4 consecutive days of strain-matched axial compressive loading (1400 μm, 120 cycles, 2 Hz). Whole Mount Staining: Baseline periosteal cell numbers and nuclear morphology were assessed by whole bone DAPI staining of the antero-medial region of the tibiae in adult and aged mice (n=6). Immunohistochemistry: Tibiae were fixed in 4% PFA, decalcified in 19% EDTA, OCT-embedded, and thickly sectioned (150 μm) at midshaft. Sca1+, Prrx1+, and Ki67+cell numbers were quantified by simultaneous fluorescent immunohistochemical staining from loaded and nonloaded contralateral tibiae. Nonimmune species specific serum served as negative controls. Imaging: 3D image datasets of the periosteum at the antero-medial region of the tibial midshaft were acquired by multi-photon and confocal microscopy. Quantification of Sca1+, Prrx1+, and Ki67+ cells was carried out using Particle Analysis software (ImageJ) and Imaris 7.4.2 Surface Rendering Statistics functions. Cell number was normalized to periosteal area (~0.04 mm2). A Student t-test determined significance at p&lt;0.05. RESULTS/ANTICIPATED RESULTS: At baseline, aged periosteal cell nuclei (DAPI+) area (14% decrease, p&lt;0.0001), nuclei number, and Prrx1+ cell number (22% decrease) was significantly lower compared with adult mice. In loaded adult mice, Prrx1+but not Sca1+cell number increased significantly (35%, p=0.0115). Proliferating Sca1+(top panel) and Prrx1+(top panel) cells also increased with loading, 62%, p=0.0253 and 115%, p=0.0004, respectively, in adult but not aged mice. The percentage of Prrx1+ cells undergoing proliferation (co-expressing Ki67+) in the total Prrx1+ cell population increased significantly with loading (bottom panel). Aged mice did not exhibit significant differences in loaded versus nonloaded controls for all other outcomes. Our data suggest fundamental changes in periosteal cell morphology, number and response to mechanical loading with aging. The significant increase in total Prrx1+ cell number and the number of Prrx1+ cells undergoing proliferation with loading in adult mice, suggest that the Prrx1+ cell population expands through proliferation. In fact, loading resulted in a 2-fold increase in the percentage of Prrx1+ preosteogenic cells undergoing proliferation. Accordingly, the significant age-related decrease in Prrx1+ cells may explain, in part, the attenuation of load-induced bone formation in aged mice. Loading resulted in greater numbers of proliferating Sca1+ cells (the more primitive cell) in adult mice, though this represented only a small percentage (&lt;10%) of the total Sca1+ population. Mechanical loading expands the Prrx1+ pre-osteogenic cell population, but not the more primitive Sca1+ population. However, this load-induced osteogenic effect in the periosteum is not observed in aged mice, which may explain age-related diminishment of load-induced bone formation. DISCUSSION/SIGNIFICANCE OF IMPACT: Mechanical loading presents an inexpensive treatment for increasing bone mass and bone strength, but may be insufficient to prevent or reverse age-related bone loss due to reduced numbers of osteogenic progenitors in the periosteum. Therapeutic approaches targeting the osteogenic capacity of periosteal cells will be required to address declining mechanoresponsiveness with age.

BACTERICIDAL EFFECT OF HUMAN SERUM ON BORRELIA MIYAMOTOI, CAUSATIVE AGENT OF IXODES TICK-BORNE BORRELIOSIS

Aim. In this paper we investigate the impacts of co-incubation of Borrelia miyamotoi with neutrophils. Materials and methods. Spirochetes B. miyamotoi, strain HT31, were incubated 3 hours at 37°C with neutrophils of healthy donors (5*106 cells/ml) in a 1:1 ratio. The incubation medium contained also non-immune serum of healthy blood donors (SHD) and, in some experiments, high-immune serum of patients recovered from ITBB-BM (S-ITBB-BM). The proportion of neutrophils that bound borrelia, as well as the number and viability (mobility) of free borrelia, was estimated by dark-field microscopy. Results. Free-swimming borrelia remain viable in SHD or heat-inactivated S-ITBB-BM, but about 10% of borrelia are associated with neutrophils. In S-ITBB-BM with neutrophils, the proportion of viable borrelia among free ones decreases by approximately 10% compared to S-ITBB-BM without neutrophils; in addition about 15% of bor-relia become bound by neutrophils. If chemoattractant fMLP was added, the proportion of neutrophils binding borrelia increases to 25%, and the proportion of immobilized non-bound bor-relia reaches 40%. Conclusion. Although neutrophils are able to destroy borrelia with or without direct contact, under model conditions the combined effect of blood neutrophils and high-immune human serum does not provide 100% elimination of B. miyamotoi.

BACTERICIDAL EFFECT OF HUMAN SERUM ON BORRELIA MIYAMOTOI, CAUSATIVE AGENT OF IXODES TICK-BORNE BORRELIOSIS (ITBB-BM)

Aim. Our aim was to study the bactericidal effect of human serum on Borrelia miyamotoi in vitro. Materials and methods. B. miyamotoi spirochetes (strains HT31 and LB-2001) were incubated in non-immune serum of healthy donors (SHD) and in heat inactivated complement-depleted SHD, as well as in serum samples of the patients recovered from ITBB-BM. The viability, that is motility, of borrelia after incubation was investigated by dark-field microscopy. The levels ofserum antibody to B.miyamofoi-specificproteins (GlpQ enzyme and four variable major proteins Vlpl5/16, Vlpl8, Vspl, and Vlp5) were measured by specially designed plane protein microarray. Results. Borrelia fully retain their viability in non-immune SHD, but their motility is partially or completely suppressed by the addition of serum from ITBB-BM convalescents or rabbit antibodies to Д. miyamotoi. The immobilizing effect of the immune serum is substantially inhibited by its heat-inactivation, which indicates that immobilizing effect is mediated by the complement system. Conclusion. Antibody-dependent complement-mediated bactericidal action ofhuman blood serum is probably not the only and 100% effective mechanism for human defense against B. miyamotoi infection, but requires support from cellular immunity.

Prophylactic immunization with human papillomavirus vaccines induces oral immunity in mice.

Although it has been shown that prophylactic vaccination can induce genital immunity, there is inadequate information on human papillomavirus (HPV) vaccine-induced oral immunity, which is of particular interest due to HPV-associated oropharyngeal malignancies and recurrent respiratory papillomatosis. Therefore, we assessed the efficacy of various HPV vaccines against oral HPV pseudovirus (PsV) infection in mice. Preclinical scientific investigation. C57BL/6 mice were vaccinated three times at 2-week intervals with either Gardasil (Merck, Kenilworth, NJ) (50 µL intramuscular injection) or a candidate pan-HPV L2 vaccine with alum adjuvant (25 µg subcutaneous injection). Additional mice were immunized with passive transfer of either Gardasil (Merck) human antisera or nonimmunized sera (100 µL intraperitoneal injection). All vaccinated and naïve control mice were then challenged with HPV16 E6E7 luciferase PsV in the oral mucosa. Visualization of HPV PsV infection was monitored through in vivo luciferase imaging. Oral luciferase-expressing HPV16 PsV infection was not detected in Gardasil (Merck), L2 vaccine, and Gardasil (Merck) antisera-immunized mice, whereas robust luciferase expression was observed in all control mice. An in vitro neutralization assay from sera of Gardasil-vaccinated (Merck) mice confirmed that vaccine efficacy was due to neutralizing antibodies. Oral HPV16 PsV infection in mice was completely prevented with all methods of prophylactic HPV immunization. These findings provide preliminary evidence that human vaccines induce protection against oral HPV infection, which has significant public health implications for HPV-associated oropharyngeal malignancies. NA. Laryngoscope, 128:E16-E20, 2018.