In Vivo Treatment with Insulin-like Growth Factor 1 Reduces CCR5 Expression on Vaccine-Induced Activated CD4+ T-Cells.

At the heart of the DNA/ALVAC/gp120/alum vaccine's efficacy in the absence of neutralizing antibodies is a delicate balance of pro- and anti-inflammatory immune responses that effectively decreases the risk of SIVmac251 acquisition in macaques. Vaccine efficacy is linked to antibodies recognizing the V2 helical conformation, DC-10 tolerogenic dendritic cells eliciting the clearance of apoptotic cells via efferocytosis, and CCR5 downregulation on vaccine-induced gut homing CD4+ cells. RAS activation is also linked to vaccine efficacy, which prompted the testing of IGF-1, a potent inducer of RAS activation with vaccination. We found that IGF-1 changed the hierarchy of V1/V2 epitope recognition and decreased both ADCC specific for helical V2 and efferocytosis. Remarkably, IGF-1 also reduced the expression of CCR5 on vaccine-induced CD4+ gut-homing T-cells, compensating for its negative effect on ADCC and efferocytosis and resulting in equivalent vaccine efficacy (71% with IGF-1 and 69% without).

Long-acting lenacapavir protects macaques against intravenous challenge with simian-tropic HIV.

BACKGROUND: Long-acting subcutaneous lenacapavir (LEN), a first-in-class HIV capsid inhibitor approved by the US FDA for the treatment of multidrug-resistant HIV-1 with twice yearly dosing, is under investigation for HIV-1 pre-exposure prophylaxis (PrEP). We previously derived a simian-tropic HIV-1 clone (stHIV-A19) that encodes an HIV-1 capsid and replicates to high titres in pigtail macaques (PTM), resulting in a nonhuman primate model well-suited for evaluating LEN PrEP in vivo. METHODS: Lenacapavir potency against stHIV-A19 in PTM peripheral blood mononuclear cells in vitro was determined and subcutaneous LEN pharmacokinetics were evaluated in naïve PTMs in vivo. To evaluate the protective efficacy of LEN PrEP, naïve PTMs received either a single subcutaneous injection of LEN (25 mg/kg, N = 3) or vehicle (N = 4) 30 days before a high-dose intravenous challenge with stHIV-A19, or 7 daily subcutaneous injections of a 3-drug control PrEP regimen starting 3 days before stHIV-A19 challenge (N = 3). FINDINGS: In vitro, LEN showed potent antiviral activity against stHIV-A19, comparable to its potency against HIV-1. In vivo, subcutaneous LEN displayed sustained plasma drug exposures in PTMs. Following stHIV-A19 challenge, while all vehicle control animals became productively infected, all LEN and 3-drug control PrEP animals were protected from infection. INTERPRETATION: These findings highlight the utility of the stHIV-A19/PTM model and support the clinical development of long-acting LEN for PrEP in humans. FUNDING: Gilead Sciences as part of a Cooperative Research and Development Agreement between Gilead Sciences and Frederick National Lab; federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024/HHSN261201500003I; NIH grant R01AI078788.

Bacterial Genotype, Carrier Risk Factors, and an Antimicrobial Stewardship Approach Relevant to Methicillin-resistant Staphylococcus aureus Prevalence in a Population of Macaques Housed in a Research Facility.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a significant problem for human and animal health and can negatively affect the health status of macaques and other nonhuman primates (NHP) in research colonies. However, few publications provide guidance on the prevalence, genotype, or risk factors for macaques with MRSA and even fewer on how to effectively respond to MRSA once identified in a population. After having a clinical case of MRSA in a rhesus macaque, we sought to determine the MRSA carrier prevalence, risk factors, and genotypes of MRSA in a population of research NHPs. Over a 6-wk period in 2015, we collected nasal swabs from 298 NHPs. MRSA was isolated from 28% (n = 83). We then reviewed each macaque's medical record for a variety of variables including animal housing room, sex, age, number of antibiotic courses, number of surgical interventions, and SIV status. Analysis of these data suggests that MRSA carriage is associated with the room location, age of the animal, SIV status, and the number of antibiotic courses. We used multilocus sequence typing and spa typing on a subset of MRSA and MSSA isolates to determine whether the MRSA present in NHPs was comparable with common human strains. Two MRSA sequence types were predominant: ST188 and a novel MRSA genotype, neither of which is a common human isolate in the United States. We subsequently implemented antimicrobial stewardship practices (significantly reducing antimicrobial use) and then resampled the colony in 2018 and found that MRSA carriage had fallen to 9% (26/285). These data suggest that, as in humans, macaques may have a high carrier status of MRSA despite low clinically apparent disease. Implementing strategic antimicrobial stewardship practices resulted in a marked reduction in MRSA carriage in the NHP colony, highlighting the importance of limiting antimicrobial use when possible.

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC.

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

NK cells and monocytes modulate primary HTLV-1 infection.

We investigated the impact of monocytes, NK cells, and CD8+ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1WT) or to the HTLV-1p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8+ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8+ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1WT. In contrast, HTLV-1p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1WT and HTLV-1p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 "don't-eat-me" signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.

Impact of Early Introduction to the Neurosciences on West Virginia High School Students via the Brain Bee.

BACKGROUND: The science, technology, engineering, and math (STEM) fields are often underrepresented due to lack of interest or exposure. The Brain Bee is a neuroscience competition for high school students meant to inspire future leaders in the neurosciences. A regional Brain Bee competition hosted in West Virginia was led by medical students and neurology residents with the goal of increasing high school student interest in the neurosciences. ACTIVITY: The West Virginia Brain Bee competition consisted of a neuroanatomy practical, a written exam, and a clinical assessment. Other fun neuroscience educational activities were also offered to students throughout the competition day. Students were surveyed before and after the competition day. RESULTS: Student interest in pursuing a neuroscience career increased (3.24 to 3.58, p = 0.043), and confidence in neuroscience knowledge increased (2.88 to 3.12, p = 0.036). Qualitative assessment revealed the importance of role models and a supportive environment for student learning. CONCLUSION: The West Virginia Brain Bee increased high school student interest in and confidence of neuroscience knowledge. This competition may be a meaningful way to connect high school students with potential mentors in the neurosciences and may also help foster an interest in pursuing a career in the neurosciences in the future.

Dpp/TGFß-superfamily play a dual conserved role in mediating the damage response in the retina.

Retinal homeostasis relies on intricate coordination of cell death and survival in response to stress and damage. Signaling mechanisms that coordinate this process in the adult retina remain poorly understood. Here we identify Decapentaplegic (Dpp) signaling in Drosophila and its mammalian homologue Transforming Growth Factor-beta (TGFß) superfamily, that includes TGFß and Bone Morphogenetic Protein (BMP) signaling arms, as central mediators of retinal neuronal death and tissue survival following acute damage. Using a Drosophila model for UV-induced retinal damage, we show that Dpp released from immune cells promotes tissue loss after UV-induced retinal damage. Interestingly, we find a dynamic response of retinal cells to this signal: in an early phase, Dpp-mediated stimulation of Saxophone/Smox signaling promotes apoptosis, while at a later stage, stimulation of the Thickveins/Mad axis promotes tissue repair and survival. This dual role is conserved in the mammalian retina through the TGFß/BMP signaling, as supplementation of BMP4 or inhibition of TGFß using small molecules promotes retinal cell survival, while inhibition of BMP negatively affects cell survival after light-induced photoreceptor damage and NMDA induced inner retinal neuronal damage. Our data identify key evolutionarily conserved mechanisms by which retinal homeostasis is maintained.

Acute transverse myelitis with Dysautonomia following SARS-CoV-2 infection: A case report and review of literature.

OBJECTIVE: To report a unique case and literature review of post COVID-19 associated transverse myelitis and dysautonomia with abnormal MRI and CSF findings. BACKGROUND: Coronavirus disease have been reported to be associated with several neurological manifestations such as stroke, Guillain-Barré syndrome, meningoencephalitis amongst others. There are only few reported cases of transverse myelitis with the novel coronavirus (n-CoV-2) and only one reported case identifying dysautonomia in COVID-19 patient. Here, we identify a COVID-19 patient diagnosed with acute transverse myelitis in addition to dysautonomia following with complete resolution of symptoms. METHOD: A retrospective chart review of a patient diagnosed with post SARS-CoV-2 infection acute transverse myelitis and dysautonomia, and a review of literature of all the reported cases of transverse myelitis and COVID-19, from December 1st, 2019 till December 25th, 2020, was performed. CONCLUSION: To our knowledge, this is the first reported case of transverse myelitis and dysautonomia in a patient with SARS-CoV-2 infection, who responded to intravenous methyl prednisone and bromocriptine. Follow-up imaging of the spine showed complete resolution of the lesion. Further studies would be recommended to identify the underlying correlation between COVID-19 and transverse myelitis.

Antibody-mediated depletion of viral reservoirs is limited in SIV-infected macaques treated early with antiretroviral therapy.

The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4+ T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection. During cART, 5 animals received 5 to 6 anti-CD4 antibody administrations and CD4+ T cell populations were then allowed 1 year on cART to recover. Despite profound CD4+ T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not significantly delayed in anti-CD4-treated animals compared with controls. Viral reactivation rates, determined based on rebounding SIVmac239M clonotype proportions, also were not significantly different in CD4-depleted animals. Notably, antibody-mediated depletion was limited in rectal tissue and negligible in lymphoid follicles. These results suggest that, even if robust viral reactivation can be achieved, antibody-mediated viral reservoir depletion may be limited in key tissue sites.

Abnormal cerebellar volume in somatic vs. non-somatic delusional disorders.

BACKGROUND: There is abundant evidence for cerebellar involvement in schizophrenia, where the cerebellum has been suggested to contribute to cognitive, affective and motor dysfunction. More recently, specific cerebellar regions have also been associated with psychotic symptoms, particularly with auditory verbal hallucinations. In contrast, little is known about cerebellar contributions to delusions, and even less is known about whether cerebellar involvement differs by delusional content. METHODS: Using structural magnetic resonance imaging at 1.0 T together with cerebellum-optimized segmentation techniques, we investigated gray matter volume (GMV) in 14 patients with somatic-type delusional disorder (S-DD), 18 patients with non-somatic delusional disorder (NS-DD) and 18 patients with schizophrenia (SZ) with persistent non-somatic delusions. A total of 32 healthy controls (HC) were included. Between-group comparisons were adjusted for age, gender, chlorpromazine equivalents and illness duration. RESULTS: Compared to HC, S-DD patients showed decreased GMV in left lobule VIIIa. In addition, S-DD patients showed decreased GMV in lobule V and increased GMV in bilateral lobule VIIa/crus II compared to NS-DD. Patients with SZ showed increased GMV in right lobule VI and VIIa/crus I compared to HC. Significant differences between HC and NS-DD were not found. CONCLUSIONS: The data support the notion of cerebellar dysfunction in psychotic disorders. Distinct cerebellar deficits, predominantly linked to sensorimotor processing, may be detected in delusional disorders presenting with predominantly somatic content.

Immunological Considerations for Retinal Stem Cell Therapy.

There is an increasing effort toward generating replacement cells for neuronal application due to the nonregenerative nature of these tissues. While much progress has been made toward developing methodologies to generate these cells, there have been limited improvements in functional restoration. Some of these are linked to the degenerative and often nonreceptive microenvironment that the new cells need to integrate into. In this chapter, we will focus on the status and role of the immune microenvironment of the retina during homeostasis and disease states. We will review changes in both innate and adaptive immunity as well as the role of immune rejection in stem cell replacement therapies. The chapter will end with a discussion of immune-modulatory strategies that have helped to ameliorate these effects and could potentially improve functional outcome for cell replacement therapies for the eye.

Preferential Small Intestine Homing and Persistence of CD8 T Cells in Rhesus Macaques Achieved by Molecularly Engineered Expression of CCR9 and Reduced Ex Vivo Manipulation.

Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer.IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.

TLR7 agonist administration to SIV-infected macaques receiving early initiated cART does not induce plasma viremia.

Reduction/elimination of HIV-1 reservoirs that persist despite combination antiretroviral therapy (cART) will likely require induction of viral expression by residual infected cells and enhanced clearance of these cells. TLR7 agonists have potential to mediate these activities. We evaluated immunologic and virologic effects of repeated doses of the TLR7 agonist GS-9620 in SIV-infected rhesus macaques receiving cART, which was initiated at 13 days after infection and was continued for 75 weeks prior to GS-9620 administration. During cART, GS-9620 induced transient upregulation of IFN-stimulated genes in blood and tissues, increases in plasma cytokines, and changes in immune cell population activation and phenotypes but did not result in measurable increases in plasma viremia or viral RNA-to-viral DNA ratio in PBMCs or tissues nor decreases in viral DNA in PBMC or tissues. SIV-specific CD8+ T cell responses, negligible prior to GS-9620 treatment, were not measurably boosted by treatment; a second course of GS-9620 administration overlapping with later cART discontinuation was associated with increased CD8+ T cell responses during viral recrudescence. These results confirm and extend evidence for GS-9620-mediated enhancement of antiviral immune responses in SIV-infected macaques but suggest that GS-9620-mediated viral induction may depend critically on the timing of initiation and duration of cART and resulting characteristics of viral reservoirs.

Pharmacological management of cardiac cachexia: a review of potential therapy options.

Cardiac cachexia is a syndrome of progressive skeletal muscle and fat loss affecting a significant number of congestive heart failure patients. With the potential detrimental effects of cardiac muscle wasting, greater attention is needed to understanding the prevention and treatment of the condition. Potential therapeutic approaches are aimed at the various mechanisms for the pathogenesis of cardiac cachexia including neurohormonal abnormalities, immune activation and inflammation, metabolic hormonal imbalance, and gastrointestinal abnormalities. While there are no current guideline-recommended treatments for the prevention of cardiac cachexia, targeting an imbalance of the renin-angiotensin-aldosterone system with beta-blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers appears to be the most well-studied therapeutic approaches. Treatment of inflammation with monoclonal antibodies, hormonal imbalance with testosterone, and nutritional deficiencies with appetite stimulants has also been suggested. Proposed therapies may prove beneficial in heart failure patients; however, further studies specifically focusing on the cardiac component of cachexia are needed before definitive therapy options can be established.

Sodium chloride pica causing recurrent nephrolithiasis in a patient with iron deficiency anemia: a case report.

BACKGROUND: Iron deficiency anemia is a common finding in women of child-bearing age. Pica, or the ingestion of non-food or non-nutritive items, is a well-known manifestation of iron deficiency. A high sodium diet increases risk for nephrolithiasis. We describe the case of a 31-year-old woman with recurrent calcium nephrolithiasis and anemia who ate ice chips as well as spoons of salt daily. Treatment of pica may prove effective in preventing recurrent nephrolithiasis. CASE PRESENTATION: A 31-year-old white woman with a past medical history of menorrhagia, anemia, and recurrent calcium nephrolithiasis presented for preoperative evaluation prior to ureterolithotomy. She described a daily pattern of eating continually from a cup of ice chips accompanied by multiple spoons of salt directly out of a salt shaker. These cravings had been present for many years, were bothersome to her, and interfered with her daily life. Laboratory findings revealed hemoglobin of 10.9 g/dL with ferritin of 3 ng/mL. History, physical, and laboratory data were consistent with pica secondary to iron deficiency anemia. She was prescribed orally administered ferrous sulfate 325 mg three times a day with meals. She continues to struggle with the symptoms of pica and orally administered supplementation. CONCLUSIONS: It is important that clinicians consider the possible diagnosis of sodium chloride pica in patients with iron deficiency anemia and recurrent nephrolithiasis. Treatment of anemia and resolution of pica may prove effective in preventing future nephrolithiasis. Specific questioning about pica symptoms in patients with iron deficiency anemia and recurrent nephrolithiasis may be helpful diagnostically and therapeutically.

Polylutidines: Multifunctional Surfaces through Vapor-Based Polymerization of Substituted Pyridinophanes.

We report a new class of functionalized polylutidine polymers that are prepared by chemical vapor deposition polymerization of substituted [2](1,4)benzeno[2](2,5)pyridinophanes. To prepare sufficient amounts of monomer for CVD polymerization, a new synthesis route for ethynylpyridinophane has been developed in three steps with an overall yield of 59 %. Subsequent CVD polymerization yielded well-defined films of poly(2,5-lutidinylene-co-p-xylylene) and poly(4-ethynyl-2,5-lutidinylene-co-p-xylylene). All polymers were characterized by infrared reflection-absorption spectroscopy, ellipsometry, contact angle studies, and X-ray photoelectron spectroscopy. Moreover, ζ-potential measurements revealed that polylutidine films have higher isoelectric points than the corresponding poly-xylylene surfaces owing to the nitrogen atoms in the polymer backbone. The availability of reactive alkyne groups on the surface of poly(4-ethynyl-2,5-lutidinylene-co-p-xylylene) coatings was confirmed by spatially controlled surface modification by means of Huisgen 1,3-dipolar cycloaddition. Compared to the more hydrophobic poly-p-xylylyenes, the presence of the heteroatom in the polymer backbone of polylutidine polymers resulted in surfaces that supported an increased adhesion of primary human umbilical vein endothelial cells (HUVECs). Vapor-based polylutidine coatings are a new class of polymers that feature increased hydrophilicity and increased cell adhesion without limiting the flexibility in selecting appropriate functional side groups.

Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice.

RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1f/fSos2-/- mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.