Adipokines as New Biomarkers of Immune Recovery: Apelin Receptor, RBP4 and ZAG Are Related to CD4+ T-Cell Reconstitution in PLHIV on Suppressive Antiretroviral Therapy.

A significant proportion of people living with HIV (PLHIV) who successfully achieve virological suppression fail to recover CD4+ T-cell counts. Since adipose tissue has been discovered as a key immune organ, this study aimed to assess the role of adipokines in the HIV immunodiscordant response. This is a multicenter prospective study including 221 PLHIV starting the first antiretroviral therapy (ART) and classified according to baseline CD4+ T-cell counts/µL (controls > 200 cells/µL and cases ≤ 200 cells/µL). Immune failure recovery was considered when cases did not reach more than 250 CD4+ T cells/µL at 144 weeks (immunological nonresponders, INR). Circulating adipokine concentrations were longitudinally measured using enzyme-linked immunosorbent assays. At baseline, apelin receptor (APLNR) and zinc-alpha-2-glycoprotein (ZAG) concentrations were significantly lower in INRs than in immunological responders (p = 0.043 and p = 0.034), and they remained lower during all ART follow-up visits (p = 0.044 and p = 0.028 for APLNR, p = 0.038 and p = 0.010 for ZAG, at 48 and 144 weeks, respectively). ZAG levels positively correlated with retinol-binding protein 4 (RBP4) levels (p < 0.01), and low circulating RBP4 concentrations were related to a low CD4+ T-cell gain (p = 0.018 and p = 0.039 at 48 and 144 weeks, respectively). Multiple regression adjusted for clinical variables and adipokine concentrations confirmed both low APLNR and RBP4 as independent predictors for CD4+ T cells at 144 weeks (p < 0.001). In conclusion, low APLNR and RBP4 concentrations were associated with poor immune recovery in treated PLHIV and could be considered predictive biomarkers of a discordant immunological response.

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug.

Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

Development of a mouse IgA monoclonal antibody-based enzyme-linked immunosorbent sandwich assay for the analyses of RBP4.

Elevated circulating Retinol-binding protein 4 (RBP4) has been associated with insulin resistance, dyslipidemia, and hypertension. However, many commonly used RBP4 ELISAs have limited dynamic range. We therefore developed an enzyme-linked immunosorbent sandwich assay (ELISA) employing a novel immunoglobulin A (IgA)-type capture mAb called AG102 instead of IgG subtypes, which was selected for its stability, capture efficiency, and specificity for human RBP 4. These features of RBP4 have hampered the development of quantitative immunological assays. Molecular analysis of AG102 revealed IgA heavy and light chains and a J chain, as expected. AG102 demonstrated notable detection of both bacterial- and HEK293-expressed RBP4 in Western blots. Serial and internal deletion experiments suggested that a putative epitope may be located in the first 35 amino acids of the mature RBP4. Compared with commercial ELISAs, the AG102-based system exhibited more significant recovery of RBP4 from serum or urine at any given dilution factor. To substantiate its quantitation capacity, comparison between RBP4 measurements from quantitative western blots and the AG102-based ELISA demonstrated a significant correlation (R2 = 0.859). After measurement for those analytes, our data suggested that IgA-based ELISA could be adapted for quantitative measurement of those analytes existing as major serum proteins or as multi-protein complexes like RBP4.

The complex P2X7 receptor/inflammasome in perivascular fat tissue of heavy smokers.

OBJECTIVE: Smoking is a recognized cardiovascular risk factor. Perivascular visceral adipose tissue (PVAT) is a source of inflammatory molecules, thus contributing to atherosclerosis progression. The P2X7 receptor (P2X7 R)-inflammasome complex, crucial in determining IL-1ß and IL-18 release, participates in this scenario. We evaluated whether smoking might affect the PVAT inflammatory phenotype and explored the putative role of the axis P2X7 R-inflammasome in this picture. SUBJECTS AND METHODS: TNFα, IL-6, RBP4, MCP-1, as well as P2X7 R and inflammasome components NLRP3, ASC, caspase-1 and IL-1ß and IL-18 expression was determined in adipocytes isolated by PVAT of healthy smokers (Smok) and nonsmokers (No-Smok) subjects. Plasma and culture medium levels of these cytokines were also determined. RESULTS: Perivascular adipose tissue of Smok had a higher expression of P2X7 R and inflammasome components; via P2X7 R activation, it released more IL-1ß and IL-18, whose serum levels were also higher in Smok than in No-Smok. Linear correlations of NLRP3 with P2X7 R and IL-18 expression and release emerged. Smok also had a higher PVAT expression of the chemotactic factor MCP-1. However, no difference was observed in the PVAT expression of genes more strictly related to insulin resistance, like TNFα, RBP4, IL-6; this was coupled with similar plasma levels of TNFα and RBP4 in the two groups. CONCLUSION: Smoking contributes to the pro-inflammatory status of the PVAT by enhancing expression and activity of the P2X7 R-inflammasome complex; the effect on adipocytokines more related to insulin resistance and metabolic abnormalities appears trivial.

[Expression of human retinol-binding protein 4 in insect baculovirus system and preparation of its polyclonal antibody].

To prepare recombinant human retinol binding protein 4 (RBP4) by using the baculovirus expression system and to detect its immunogenicity, the fusion DNA fragment of secretory signal peptide SS64 and human RBP4 gene was subcloned into a baculovirus transfer vector pFastBac-dual(pFBd), and the corresponding recombinant transfer plasmid was transformed into E. coli strain DH10bac, after transposition recombinant shuttle bacmid was screened out. The logarithmic phase Sf9 cells were transfected with the recombinant bacmid and then the recombinant baculovirus containing hRBP4 expression box were generated. After amplification of recombinant baculovirus, the recombinant baculovirus seeds were obtained. To express human RBP4, logarithmic phase Sf9 cells were infected with the virus seeds and SDS-PAGE and Western blotting were used to detect and identify the expression. Finally, to prepare a batch of RBP4 protein, logarithmic phase Sf9 cells in suspension culture were infected with recombinant baculovirus seeds and the supernatant was harvested after 120 hours post-infection for purification. Finally for preparation of polyclonal antibody and evaluation of immunogenicity, the recombinant hRBP4 from insect cells and from E. coli were immunized rabbits. Restriction enzyme digestion and sequencing confirmed that the recombinant baculovirus transfer plasmid was constructed correctly, and subsequently recombinant RBP4-bacmid was generated successfully. SDS-PAGE and Western blotting analysis suggested that human RBP4 protein was highly expressed in Sf9 cells with the molecular weight of approximately 23 kDa. The recombinant RBP4 protein could be secreted into the medium efficiently, and the expression level was calculated amount of 100 mg/L. Finally the rabbit antiserum was harvested after recombinant RBP4 immunization, therein the titer of antiserum against baculovirus recombinant RBP4 is 1:100 000 whereas the titer of antiserum against E. coli recombinant RBP4 is only 1:10 000. Overall, human RBP4 was high efficiently expressed successfully with good antigenicity in baculovirus system, and high affinity antiserum was obtained. A solid foundation was laid for the next step of the preparation of human serum RBP4 detection kit.

Prenatally acquired vitamin A deficiency alters innate immune responses to human rotavirus in a gnotobiotic pig model.

We examined how prenatally acquired vitamin A deficiency (VAD) modulates innate immune responses and human rotavirus (HRV) vaccine efficacy in a gnotobiotic (Gn) piglet model of HRV diarrhea. The VAD and vitamin A-sufficient (VAS) Gn pigs were vaccinated with attenuated HRV (AttHRV) with or without concurrent oral vitamin A supplementation (100,000 IU) and challenged with virulent HRV (VirHRV). Regardless of vaccination status, the numbers of conventional and plasmacytoid dendritic cells (cDCs and pDCs) were higher in VAD piglets prechallenge, but decreased substantially postchallenge as compared with VAS pigs. We observed significantly higher frequency of CD103 (integrin αEß7) expressing DCs in VAS versus VAD piglets postchallenge, indicating that VAD may interfere with homing (including intestinal) phenotype acquisition. Post-VirHRV challenge, we observed longer and more pronounced diarrhea and higher VirHRV fecal titers in nonvaccinated VAD piglets. Consistent with higher VirHRV shedding titers, higher IFN-α levels were induced in control VAD versus VAS piglet sera at postchallenge day 2. Ex vivo HRV-stimulated mononuclear cells (MNCs) isolated from spleen and blood of VAD pigs prechallenge also produced more IFN-α. In contrast, at postchallenge day 10, we observed reduced IFN-α levels in VAD pigs that coincided with decreased TLR3(+) MNC frequencies. Numbers of necrotic MNCs were higher in VAD pigs in spleen (coincident with splenomegaly in other VAD animals) prechallenge and intestinal tissues (coincident with higher VirHRV induced intestinal damage) postchallenge. Thus, prenatal VAD caused an imbalance in innate immune responses and exacerbated VirHRV infection, whereas vitamin A supplementation failed to compensate for these VAD effects.

Increased retinol-binding protein (RBP) 4 and anti-RBP4 antibody in alopecia areata.

BACKGROUND: Neither the underlying pathogenesis of alopecia areata (AA) nor the molecular mechanisms leading to hair loss have been fully elucidated. OBJECTIVES: To compare the protein profiles of sera obtained from patients with AA with those from healthy controls. METHODS: Protein profiles of sera obtained from subjects with AA and healthy controls were compared using proteomics techniques. Serum levels of the identified protein were quantified by specific enzyme-linked immunosorbent assay (ELISA). The relative serum reactivities of the recombinant human protein were compared between patients with AA and healthy controls using Western blots and double indirect immunofluorescence. RESULTS: The upregulated expression of retinol-binding protein (RBP) 4 was identified, and RBP4 ELISA demonstrated significantly increased serum levels of RBP4 among subjects with AA when compared with healthy controls. Western blots using recombinant human RBP4 and the sera from both groups presented serum reactivity of antihuman recombinant RBP4 IgG antibodies in 10/15 subjects with AA (67%) and 2/15 healthy controls (13%). Double indirect immunofluorescence demonstrated merged fluorescence signals of serum anti-RBP4 IgG antibodies and monoclonal antibodies to RBP4 in subjects with AA on the outer root sheath and companion layer. CONCLUSIONS: Our data demonstrate that AA is associated with increased serum levels of RBP4 and positive IgG immunoreactivity against recombinant human RBP4. These results suggest that the major components for the retinoic acid biosynthesis pathway may be crucially involved in the pathogenic process of AA.

Development of retinol-binding protein 4 immunocolloidal gold fast test strip using high-sensitivity monoclonal antibodies generated by DNA immunization.

DNA immunization is an efficient method for high-affinity monoclonal antibody generation. Here, we describe the generation of several high-quality monoclonal antibodies (mAbs) against retinol-binding protein 4 (RBP4), an important marker for kidney abnormality and dysfunction, with a combination method of DNA priming and protein boost. The mAbs generated could bind to RBP4 with high sensitivity and using these mAbs, an immunocolloidal gold fast test strip was constructed. The strip can give a result in <5 min and is very sensitive with a detection limit of about 1 ng/ml. A small-scale clinical test revealed that the result of this strip was well in accordance with that of an enzyme-labeled immunosorbent assay kit currently available on the market. Consequently, it could be useful for more convenient and faster RBP4 determination in the clinic.