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The primary therapeutic goal for the treatment of diabetes is maintenance of a long-term, near-normoglycemic condition and prevention of the onset or progression of the complications associated with the disease. Although several analogs of human insulin have been developed, the currently prescribed long-acting insulin analogs do not provide a stable basal glycemia for more than a few hours. Here, we report the development of Albulin, a long-acting insulin analog obtained by direct gene fusion of a single-chain human insulin to human serum albumin. Albulin showed an elimination [t.sub.1/2] of ~7 h in normoglycemic mice. In vitro pharmacodynamic profiles for Albulin characterized by receptor binding, inhibition of gluconeogenesis, induction of glucose uptake, and global regulation of gene expression in relevant cell types showed that Albulin produced similar activity profiles compared with that of recombinant human insulin. A single Albulin administration in vivo normalized blood glucose level in diabetic mice in a relatively peakless and sustained (24-h) fashion. A further reduction in glucose levels was achieved by administering a recombinant human insulin a few hours after Albulin injection in mice, indicating the potential for Albulin therapy in combination with available fast-acting insulin derivatives. In summary, Albulin displays characteristics of a potent long-acting insulin analog that can be evaluated for use as a novel insulin therapy for patients with insulin-dependent diabetes., Management of type 1 and type 2 diabetes with insulin therapy typically includes a long-acting basal component for between-meal and nocturnal glycemic control, together with preprandial bolus injections of a [...]

Objective:To evaluate the effect of an employer-sponsored T2D management virtual program (Onduo) on blood glucose changes, via an independent data source, within the first year of participation.Methods:This retrospective pre-post study included participants with T2D who engaged in Onduo Dec 2020-Sept 2021. Participants had to have completed an employer-sponsored wellness program (independent of Onduo) with year-end biometric screening in 2019, 2020 and 2021, which included HbA1c, fasting glucose (FG), lipids, and other biometric measurements (Table). Year-to-year changes pre-intervention (2019 vs 2020 wellness screening, before Onduo) and post-intervention (2020 vs 2021) were compared using paired t-tests (significant at P≤.05). We also assessed year-to-year changes based on use of specific Onduo telehealth tools (mobile app, blood glucose meter [BGM], health coach consultations, continuous glucose monitor [CGM], and telemedicine visits).Results:All 578 participants included (Table) used a mobile app, 84% used personalized coaching, 69% BGM, 13% telehealth visits, and 10% CGM. Seventy (12.1%) participants had HbA1c ≥9% in 2019, 79 (13.7%) in 2020, and 57 (9.9%) in 2021 (2019 vs 2020: P=.3; 2020 vs 2021: P=.01; Χ2tests). Within the first year of intervention, year-to-year changes in HbA1c, FG, HDL, and LDL significantly improved compared to pre-intervention changes (Table). The largest glycemic improvements post-intervention were in participants with baseline HbA1c ≥9% (P<.001; ANOVA) or baseline FG ≥126 mg/dL (P<.001; 2-sample t-test). Participants who used a CGM or telemedicine visits (n=81) had greater post-intervention drops (2020 vs 2021) in HbA1c than did others (-0.7 vs -0.2%, P<.01; 2-sample t-test).Conclusions:Participation in Onduo improved year-to-year trends in HbA1c, FG, HDL, and LDL. Improvements in glycemia were greater in participants with the highest baseline glycemic indices and those who used a CGM or telemedicine visits.

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4–500 ng/ml), tissue factor pathway inhibitor (TFPI) (42–1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2–250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430–1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.

We analyzed expression of candidate genes encoding cell surface or secreted proteins in normal kidney and kidney cancer. This screen identified a bone morphogenetic protein (BMP) antagonist, SOSTDC1 (sclerostin domain–containing-1) as down-regulated in kidney tumors. To confirm screening results, we probed cDNA dot blots with SOSTDC1. The SOSTDC1 message was decreased in 20/20 kidney tumors compared with normal kidney tissue. Immunohistochemistry confirmed significant decrease of SOSTDC1 protein in clear cell renal carcinomas relative to normal proximal renal tubule cells (p < 0.001). Expression of SOSTDC1 was not decreased in papillary and chromophobe kidney tumors. SOSTDC1 was abundantly expressed in podocytes, distal tubules, and transitional epithelia of the normal kidney. Transfection experiments demonstrated that SOSTDC1 is secreted and binds to neighboring cells and/or the extracellular matrix. SOSTDC1 suppresses both BMP-7–induced phosphorylation of R-Smads-1, -5, and -8 and Wnt-3a signaling. Restoration of SOSTDC1 in renal clear carcinoma cells profoundly suppresses proliferation. Collectively, these results demonstrate that SOSTDC1 is expressed in the human kidney and decreased in renal clear cell carcinoma. Because SOSTDC1 suppresses proliferation of renal carcinoma cells, restoration of SOSTDC1 signaling may represent a novel target in treatment of renal clear cell carcinoma.

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21low B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21low B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21low B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.

During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2α, HE2β1 and HE2β2 and found that the full-length proteins (10 μg/ml or ~1 μM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human β-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25–100 μg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2α, HEβ1 and HEβ2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human β-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human β-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon™ (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-β (IFN-β ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-β induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 μg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 μg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-β, and the terminal half-life was 36-40 h compared with 8 h for IFN-β. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2′,5′ mRNA expression. At a molar dose equivalent to one-half the dose of IFN-β, Albuferon beta elicited comparable neopterin responses and significantly higher 2′,5′-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-β when fused to serum albumin suggest a clinical opportunity for improved IFN-β therapy.

Background:Behavioral counseling has been shown to improve modifiable cardiovascular risk factors. We asked whether the 10-year risk for ASCVD events could be reduced within the first year of a digital (on-line) counseling program.Methods:Individuals were included in the study if they (i) participated in an annual employer-sponsored wellness program with year-end biometric screening for 3 years (2016, 2017, and 2018), (ii) had metabolic syndrome at the end of 2017, and (iii) completed at least 9 lessons in a digital behavioral counseling program initiated at the beginning of 2018. Paired t-Tests and McNemar tests were used to assess changes in ASCVD risk before and after intervention.Results:The baseline characteristics of the 144 participants are shown in the table. Participants engaged in the counseling program for a mean of 8.9 months (SD, 1.3) and completed a mean of 24 lessons (SD, 12). In the year before participation in counseling (2016-2017), mean 10-year ASCVD risk increased from 3.5% to 4.4%, P=0.0002 for difference in the annual ASCVD risk change before and after the program. In the year after participation in the counseling program (2017-2018), mean 10-year ASCVD risk decreased from 4.4% to 3.6%, and the fraction of participants with 10-year ASCVD risk ?5% decreased from 30% to 22%: P=0.04. Metabolic syndrome was substantially reduced (p<0.0001), and CVD risk factors were shifted in a favorable direction (Table).Conclusions:In participants with metabolic syndrome digital behavioral counseling reduced 10-year ASCVD risk after the first year on the program.