THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

Lipid remodeling in Rhodopseudomonas palustris TIE‐1 upon loss of hopanoids and hopanoid methylation

The sedimentary record of molecular fossils (biomarkers) can potentially provide important insights into the composition of ancient organisms; however, it only captures a small portion of their original lipid content. To interpret what remains, it is important to consider the potential for functional overlap between different lipids in living cells, and how the presence of one type might impact the abundance of another. Hopanoids are a diverse class of steroid analogs made by bacteria and found in soils, sediments, and sedimentary rocks. Here, we examine the trade‐off between hopanoid production and that of other membrane lipids. We compare lipidomes of the metabolically versatile α‐proteobacterium Rhodopseudomonas palustris TIE‐1 and two hopanoid mutants, detecting native hopanoids simultaneously with other types of polar lipids by electrospray ionization mass spectrometry. In all strains, the phospholipids contain high levels of unsaturated fatty acids (often >80 %). The degree to which unsaturated fatty acids are modified to cyclopropyl fatty acids varies by phospholipid class. Deletion of the capacity for hopanoid production is accompanied by substantive changes to the lipidome, including a several‐fold rise of cardiolipins. Deletion of the ability to make methylated hopanoids has a more subtle effect; however, under photoautotrophic growth conditions, tetrahymanols are upregulated twofold. Together, these results illustrate that the ‘lipid fingerprint’ produced by a micro‐organism can vary depending on the growth condition or loss of single genes, reminding us that the absence of a biomarker does not necessarily imply the absence of a particular source organism.     

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.     

TIGIT Marks Exhausted T Cells,Correlates with Disease Progression,and Serves as a Target for Immune Restoration in HIV and SIV Infection

HIV infection induces phenotypic and functional changes to CD8⁺ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8⁺ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT⁺ and TIGIT⁺ PD-1⁺ CD8⁺ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8⁺ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8⁺ T cell effector responses. The frequency of TIGIT⁺ CD4⁺ T cells correlated with the CD4⁺ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.     

Non-Classical Monocytes and Monocyte Chemoattractant Protein-1 (MCP-1) Correlate with Coronary Artery Calcium Progression in Chronically HIV-1 Infected Adults on Stable Antiretroviral Therapy

Background

Persistent inflammation and immune activation has been hypothesized to contribute to increased prevalence of subclinical atherosclerosis and cardiovascular disease (CVD) risk in patients with chronic HIV infection. In this study, we examined the correlation of peripheral monocyte subsets and soluble biomarkers of inflammation to coronary artery calcium (CAC) progression, as measured by cardiac computed tomography scan.

Methods

We conducted a longitudinal analysis utilizing baseline data of 78 participants with HIV infection on stable antiretroviral therapy (ART) in the Hawaii Aging with HIV-Cardiovascular study who had available baseline monocyte subset analysis as well as CAC measurement at baseline and at 2-year follow up. Monocyte phenotypes were assessed from cryopreserved blood by flow cytometry and plasma was assayed for soluble biomarkers using antibody-coated beads in a high sensitivity Milliplex Luminex platform. Change in CAC over 2 years was analyzed as the primary outcome variable.

Results

Of all monocyte subsets and biomarkers tested, higher non-classical monocyte percentage (ρ = 0.259, p = 0.022), interleukin (IL)-6 (ρ = 0.311, p = 0.012), and monocyte chemoattractant protein (MCP)-1 (ρ = 0.524, p = <0.001) were significantly correlated to higher 2-year CAC progression in unadjusted Spearman’s correlation. Non-classical monocyte percentage (ρ = 0.247, p = 0.039), and MCP-1 (ρ = 0.487, p = <0.001), remained significantly correlated to 2-year CAC progression, while IL-6 was not (ρ = 0.209, p = 0.120) after adjustment for age, hypertension, diabetes mellitus, total/HDL cholesterol ratio, smoking history, and BMI.

Conclusion

The percentage of non-classical monocytes and plasma MCP-1 levels were independently associated with CAC progression and may be related to the progression of atherosclerosis and increased CVD risk associated with chronic HIV infection on stable ART.     

Dopaminergic modulation of salt sensitivity in patients with essential hypertension

The present study was designed to investigate the possible role of dopaminergic mechanisms in contributing to the pathogenesis of hypertension in salt sensitive patients. Eighteen patients with essential hypertension were studied while under a diet ranging from low salt to high salt, which enabled a classification in "salt-sensitive" (SS) and "nonsalt-sensitive" (NSS) groups based on a tentative criteria of a 10% increase of mean blood pressure with high salt diet. The SS patients showed reduced urinary excretion of sodium as compared with that from NSS patients. Urinary norepinephrine excretion in all patients with salt loading was suppressed, but urinary excretion of epinephrine showed a tendency to increase in SS patients after salt loading. Urinary excretion of dopamine increased in NSS patients with salt loading, but did not change in SS patients. To further evaluate the role of dopaminergic mechanisms in salt-sensitive hypertension, metoclopramide, a dopamine antagonist, was injected intravenously to all patients. With salt loading, plasma aldosterone levels increased after injection of metoclopramide in NSS patients, but did not change in SS patients. These results suggest that salt-sensitive hypertension is modulated by dopaminergic activity, which in turn is attenuated in SS patients. Decreased dopaminergic activity induced sodium retention both by a direct effect on the kidney as well as indirectly via relatively increased aldosterone secretion. Both mechanisms would help to increase intravascular volume and blood pressure in salt-sensitive hypertension.     

Marine biofilms on submerged surfaces are a reservoir for Escherichia coli and Vibrio cholerae

The enteric bacterium and potential human pathogen, Escherichia coli, is known to persist in tropical soils and coastal waters. Vibrio cholerae causes the disease cholera and inhabits marine environments including microbial films on submerged surfaces. The abundances of E. coli and V. cholerae were quantified in biofilm and water-column samples from three harbors in Honolulu, Hawai‘i, which differ in their local and international ship traffic. E. coli and, in some cases V. cholerae, occurred in relatively high abundances in marine biofilms formed on abiotic surfaces, including the exterior hulls of ships. The community fingerprints of the biofilms and the water harboring these pathogens were further analyzed. The community compositions of biofilms from different locations were more similar to each other than to water-column communities from the same locations. These results suggest that biofilms are an overlooked reservoir and a source of dissemination for E. coli and V. cholerae.     

Siglec-9 defines and restrains a natural killer subpopulation highly cytotoxic to HIV-infected cells

Siglec-9 is an MHC-independent inhibitory receptor expressed on a subset of natural killer (NK) cells. Siglec-9 restrains NK cytotoxicity by binding to sialoglycans (sialic acid-containing glycans) on target cells. Despite the importance of Siglec-9 interactions in tumor immune evasion, their role as an immune evasion mechanism during HIV infection has not been investigated. Using in vivo phenotypic analyses, we found that Siglec-9⁺ CD56^dim NK cells, during HIV infection, exhibit an activated phenotype with higher expression of activating receptors and markers (NKp30, CD38, CD16, DNAM-1, perforin) and lower expression of the inhibitory receptor NKG2A, compared to Siglec-9^- CD56^dim NK cells. We also found that levels of Siglec-9⁺ CD56^dim NK cells inversely correlate with viral load during viremic infection and CD4⁺ T cell-associated HIV DNA during suppressed infection. Using in vitro cytotoxicity assays, we confirmed that Siglec-9⁺ NK cells exhibit higher cytotoxicity towards HIV-infected cells compared to Siglec-9^- NK cells. These data are consistent with the notion that Siglec-9⁺ NK cells are highly cytotoxic against HIV-infected cells. However, blocking Siglec-9 enhanced NK cells’ ability to lyse HIV-infected cells, consistent with the known inhibitory function of the Siglec-9 molecule. Together, these data support a model in which the Siglec-9⁺ CD56^dim NK subpopulation is highly cytotoxic against HIV-infected cells even whilst being restrained by the inhibitory effects of Siglec-9. To harness the cytotoxic capacity of the Siglec-9⁺ NK subpopulation, which is dampened by Siglec-9, we developed a proof-of-concept approach to selectively disrupt Siglec/sialoglycan interactions between NK and HIV-infected cells. We achieved this goal by conjugating Sialidase to several HIV broadly neutralizing antibodies. These conjugates selectively desialylated HIV-infected cells and enhanced NK cells’ capacity to kill them. In summary, we identified a novel, glycan-based interaction that may contribute to HIV-infected cells’ ability to evade NK immunosurveillance and developed an approach to break this interaction.