Calcium-Stimulated Adenylyl Cyclases are Critical Modulators of Fear Learning and Experience-Dependent Plasticity

Stress can exacerbate psychiatric disease, often resulting in cognitive deficits. Consequently, a better understanding of what modulates stress-facilitated memory processing will help identify new targets for possible therapeutic intervention. Recent evidence suggests a role of the Ca2+-stimulated adenylyl cyclases: AC), AC1 and AC8, in modulating fear memory. Ca2+-stimulated AC activity couples neuronal activity and intracellular Ca2+ increases to the production of cAMP, and therefore, can very tightly regulate signal transduction after learning; yet, the details by which this occurs are not well understood. In this dissertation, I first investigated the temporal and regional importance of Ca2+-stimulated AC activity during different stages of memory processing using the tetracycline-off system, which allowed me to produce AC8 Rescue mice with forebrain-specific inducible expression of AC8 on an AC1 and AC8 double knockout: DKO) background. The results showed that forebrain Ca2+-stimulated AC activity was necessary to modulate long-term memory on several learning paradigms, and more specifically, that it was necessary during memory consolidation and retention. This finding is further supported by an overall decrease in transcriptional changes in DKO mice across several time points after conditioned fear: CF) learning, but most strikingly, at periods when memory consolidation and retention should be occurring. Since transcriptional changes are often dictated by synaptic activity and AC1 and AC8 are both localized at the synapse, I examined synaptic activity in DKO mice. Initial analysis of synaptic protein abundance in hippocampal cell cultures revealed decreased SV2 levels in DKO mice, but this can be rescued by infection with an AC8 lentivirus. Moreover, DKO mice also display synaptic deficits after learning as measured by p-synapsin. The CA1 LTP results coincide with the above data as DKO mice, but not AC8 Rescue mice, show impaired LTP. Finally, WT mice show changes in CF memory strength that is dependent on prior environmental exposure, but DKO mice do not, suggesting that Ca2+-stimulated AC activity modulates plasticity at the behavioral level as well. From these studies, I have observed a critical role for Ca2+-stimulated AC activity in modulating the consolidation and retention of fear memory and experience-dependent plasticity

Registration of supine MR mammography with breast ultrasound for surgical planning of breast conserving surgery: a feasibility study

Purpose To report the feasibility, accuracy and initial clinical experience of the use of real-time magnetic resonance navigated ultrasound (rtMRnUS) in the surgical planning of breast-conserving surgery (BCS) via guide wire insertion. Materials and Methods 29 participants were recruited into this prospective ethics committee approved study. The first 4 cases were utilized as a training set. Participants underwent a supine contrast-enhanced breast MR examination with external fiducials and corresponding ink marks placed on the skin of the affected breast to act as co-registration pairs. MR examinations included both functional and morphological images. A LOGIQ E9 ultrasound system (GE Healthcare, Milwaukee, WI, USA) equipped with a 6 - 15 MHz transducer was utilized for rtMRnUS. To facilitate point co-registration of the previously acquired MR dataset with the real-time ultrasound, co-registration pairs were identified on both imaging modalities. The following co-registration quality metrics were recorded: root mean square deviation (RMSD), lesion and global accuracies. Post co-registration guide wire insertion was performed. Results Co-registration was successfully undertaken in all participants. Results from 25 participants are presented. The median (min, max) RMSD was 3.3 mm (0.6 mm, 8.8 mm). The global accuracy was assessed as very good (8), good (12), moderate (3) and poor (2) while the median (min, max) lesion accuracy was recorded at 8.9 mm (2.1 mm, 33.2 mm). Conclusion The use of rtMRnUS to facilitate guide wire insertion is a feasible technique. Generally, very good or good global registration can be expected. Lesion accuracy results indicate that a median difference, in 3 D space, of 9 mm can be expected between imaging modalities

Expression of Human CD4 and chemokine receptors in cotton rat cells confers permissiveness for productive HIV infection

<p>Abstract</p> <p>Background</p> <p>Current small animal models for studying HIV-1 infection are very limited, and this continues to be a major obstacle for studying HIV-1 infection and pathogenesis, as well as for the urgent development and evaluation of effective anti-HIV-1 therapies and vaccines. Previously, it was shown that HIV-1 can infect cotton rats as indicated by development of antibodies against all major proteins of the virus, the detection of viral cDNA in spleen and brain of challenged animals, the transmission of infectious virus, albeit with low efficiency, from animal to animal by blood, and an additional increase in the mortality in the infected groups.</p> <p>Results</p> <p>Using <it>in vitro </it>experiments, we now show that cotton rat cell lines engineered to express human receptor complexes for HIV-1 (hCD4 along with hCXCR4 or hCCR5) support virus entry, viral cDNA integration, and the production of infectious virus.</p> <p>Conclusion</p> <p>These results further suggest that the development of transgenic cotton rats expressing human HIV-1 receptors may prove to be useful small animal model for HIV infection.</p

Human Erythrocytes Selectively Bind and Enrich Infectious HIV-1 Virions

Although CD4(+) cells represent the major target for HIV infection in blood, claims of complement-independent binding of HIV-1 to erythrocytes and the possible role of Duffy blood group antigen, have generated controversy. To examine the question of binding to erythrocytes, HIV-1 was incubated in vitro with erythrocytes from 30 healthy leukapheresis donors, and binding was determined by p24 analysis and adsorption of HIV-1 with reduction of infectivity for CD4(+) target cells. All of the cells, regardless of blood group type, bound HIV-1 p24. A typical preparation of erythrocytes bound <2.4% of the added p24, but erythrocytes selectively removed essentially all of the viral infectivity as determined by decreased infection of CD4(+) target cells; however, cell-associated HIV-1 was approximately 100-fold more efficient, via trans infection, than unadsorbed virus for infection of CD4(+) cells. All of the bound HIV-1 p24 was released by treatment of the cells with EDTA, and binding was optimized by adding Ca2+ and Mg2+ during the washing of erythrocytes containing bound HIV-1. Although the small number of contaminating leukocytes in the erythrocyte preparation also bound HIV-1 p24, there was no significant binding to CD4, and it thus appears that the binding occurred on leukocytes at non-CD4 sites. Furthermore, binding occurred to erythrocyte ghosts from which contaminating leukocytes had been previously removed. The results demonstrate that erythrocytes incubated in vitro with HIV-1 differentially adsorb all of the infectious HIV-1 virions (as opposed to non-infectious or degraded virions) in the absence of complement and independent of blood group, and binding is dependent on divalent cations. By analogy with HIV-1 bound to DC-SIGN on dendritic cells, erythrocyte-bound HIV-1 might comprise an important surface reservoir for trans infection of permissive cells

Hypothalamic-pituitary-adrenal axis and behavioral dysfunction following early binge-like prenatal alcohol exposure in mice

The range of defects that fall within fetal alcohol spectrum disorder (FASD) includes persistent behavioral problems, with anxiety and depression being two of the more commonly reported issues. Previous studies of rodent FASD models suggest that interference with hypothalamic-pituitary-adrenal (HPA) axis structure and/or function may be the basis for some of the prenatal alcohol (ethanol) exposure (PAE)-induced behavioral abnormalities. Included among the previous investigations are those illustrating that maternal alcohol treatment limited to very early stages of pregnancy (i.e., gestational day [GD]7 in mice; equivalent to the third week post-fertilization in humans) can cause structural abnormalities in areas such as the hypothalamus, pituitary gland, and other forebrain regions integral to controlling stress and behavioral responses. The current investigation was designed to further examine the sequelae of prenatal alcohol insult at this early time period, with particular attention to HPA axis-associated functional changes in adult mice. The results of this study reveal that GD7 PAE in mice causes HPA axis dysfunction, with males and females showing elevated corticosterone (CORT) and adrenocorticotropic hormone (ACTH) levels, respectively, following a 15-min restraint stress exposure. Males also showed elevated CORT levels following an acute alcohol injection of 2.0 g/kg, while females displayed blunted ACTH levels. Furthermore, analysis showed that anxiety-like behavior was decreased after GD7 PAE in female mice, but was increased in male mice. Collectively, the results of this study show that early gestational alcohol exposure in mice alters long-term HPA axis activity and behavior in a sexually dimorphic manner

Innate immune cell activation after HIV-1 vaccine administration is associated with increased antibody production

The RV144 Thai phase III clinical trial’s canarypox–protein HIV vaccine regimen showed modest efficacy in reducing infection. We therefore sought to determine the effects of vaccine administration on innate cell activation and subsequent associations with vaccine-induced immune responses. RV306 was a randomized, double-blind clinical trial in HIV-uninfected Thai adults that tested delayed boosting following the RV144 regimen. PBMC collected from RV306 participants prior to and 3 days after the last boost were used to investigate innate immune cell activation. Our analysis showed an increase in CD38+ mucosal associated invariant T (MAIT) cells, CD38+ invariant natural killer T (iNKT) cells, CD38+ γδ T cells, CD38+, CD69+ and HLA-DR+ NK cells 3 days after vaccine administration. An increase in CD14-CD16+ non-classical monocytes and CD14+CD16+ intermediate monocytes accompanied by a decrease in CD14+CD16- classical monocytes was also associated with vaccine administration. Inclusion of ALVAC-HIV in the boost did not further increase MAIT, iNKT, γδ T, and NK cell activation or increase the proportion of non-classical monocytes. Additionally, NK cell activation 3 days after vaccination was positively associated with antibody titers of HIV Env-specific total IgG and IgG1. Vδ1 T cell activation 3 days after vaccine administration was associated with HIV Env-specific IgG3 titers. Finally, we observed trending associations between MAIT cell activation and Env-specific IgG3 titers and between NK cell activation and TH023 pseudovirus neutralization titers. Our study identifies a potential role for innate cells, specifically NK, MAIT, and γδ T cells, in promoting antibody responses following HIV-1 vaccine administration

Convalescent human IgG, but not IgM, from COVID-19 survivors confers dose-dependent protection against SARS-CoV-2 replication and disease in hamsters

IntroductionAntibody therapeutic strategies have served an important role during the COVID-19 pandemic, even as their effectiveness has waned with the emergence of escape variants. Here we sought to determine the concentration of convalescent immunoglobulin required to protect against disease from SARS-CoV-2 in a Syrian golden hamster model.MethodsTotal IgG and IgM were isolated from plasma of SARS-CoV-2 convalescent donors. Dose titrations of IgG and IgM were infused into hamsters 1 day prior to challenge with SARS-CoV-2 Wuhan-1.ResultsThe IgM preparation was found to have ~25-fold greater neutralization potency than IgG. IgG infusion protected hamsters from disease in a dose-dependent manner, with detectable serum neutralizing titers correlating with protection. Despite a higher in vitro neutralizing potency, IgM failed to protect against disease when transferred into hamsters.DiscussionThis study adds to the growing body of literature that demonstrates neutralizing IgG antibodies are important for protection from SARS-CoV-2 disease, and confirms that polyclonal IgG in sera can be an effective preventative strategy if the neutralizing titers are sufficiently high. In the context of new variants, against which existing vaccines or monoclonal antibodies have reduced efficacy, sera from individuals who have recovered from infection with the emerging variant may potentially remain an efficacious tool

Phase I Study of Safety and Immunogenicity of an Escherichia coli-Derived Recombinant Protective Antigen (rPA) Vaccine to Prevent Anthrax in Adults

The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA).A total of 73 healthy adults ages 18-40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 µg Alhydrogel®-formulated rPA.The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses.ClinicalTrials.gov NCT00057525

The Role of Natural Killer (NK) Cells and NK Cell Receptor Polymorphisms in the Assessment of HIV-1 Neutralization

The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses

The Role of Regulatory T Cells in Cancer

There has been an explosion of literature focusing on the role of regulatory T (Treg) cells in cancer immunity. It is becoming increasingly clear that Treg cells play an active and significant role in the progression of cancer, and have an important role in suppressing tumor-specific immunity. Thus, there is a clear rationale for developing clinical strategies to diminish their regulatory influences, with the ultimate goal of augmenting antitimor immunity. Therefore, manipulation of Treg cells represent new strategies for cancer treatment. In this Review, I will summarize and review the explosive recent studies demonstrating that Treg cells are increased in patients with malignancies and restoration of antitumor immunity in mice and humans by depletion or reduction of Treg cells. In addition, I will discuss both the prognostic value of Treg cells in tumor progression in tumor-bearing hosts and the rationale for strategies for therapeutic vaccination and immunotherapeutic targeting of Treg cells with drugs and microRNA