Macrophage-Derived Factors with the Potential to Contribute to Pathogenicity of HIV-1 and HIV-2: Role of CCL-2/MCP-1.

Human immunodeficiency virus type 2 (HIV-2) is known to be less pathogenic than HIV-1. However, the mechanism(s) underlying the decreased HIV-2 pathogenicity is not fully understood. Herein, we report that ß-chemokine CCL2 expression was increased in HIV-1-infected human monocyte-derived macrophages (MDM) but decreased in HIV-2-infected MDM when compared to uninfected MDM. Inhibition of CCL2 expression following HIV-2 infection occurred at both protein and mRNA levels. By microarray analysis, quantitative PCR, and Western blotting, we identified that Signal Transducer and Activator of Transcription 1 (STAT1), a critical transcription factor for inducing CCL2 gene expression, was also reduced in HIV-2-infected MDM. Blockade of STAT1 in HIV-infected MDM using a STAT1 inhibitor significantly reduced the production of CCL2. In contrast, transduction of STAT1-expressing pseudo-retrovirus restored CCL2 production in HIV-2-infected MDM. These findings support the concept that CCL2 inhibition in HIV-2-infected MDM is meditated by reduction of STAT1. Furthermore, we showed that STAT1 reduction in HIV-2-infected MDM was regulated by the CUL2/RBX1 ubiquitin E3 ligase complex-dependent proteasome pathway. Knockdown of CUL2 or RBX1 restored the expression of STAT1 and CCL2 in HIV-2-infected MDM. Taken together, our findings suggest that differential regulation of the STAT1-CCL2 axis may be one of the mechanisms underlying the different pathogenicity observed for HIV-1 and HIV-2.

FDA Approval Summary: Pembrolizumab for Treatment of Metastatic Non-Small Cell Lung Cancer: First-Line Therapy and Beyond.

On October 24, 2016, the U.S. Food and Drug Administration (FDA) approved pembrolizumab (Keytruda; Merck & Co., Inc., https://www.merck.com) for treatment of patients with metastatic non-small cell lung cancer (mNSCLC) whose tumors express programmed death-ligand 1 (PD-L1) as determined by an FDA-approved test, as follows: (a) first-line treatment of patients with mNSCLC whose tumors have high PD-L1 expression (tumor proportion score [TPS] ≥50%), with no epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) genomic tumor aberrations, and (b) treatment of patients with mNSCLC whose tumors express PD-L1 (TPS ≥1%), with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving pembrolizumab.Approval was based on two randomized, open-label, active-controlled trials demonstrating statistically significant improvements in progression-free survival (PFS) and overall survival (OS) for patients randomized to pembrolizumab compared with chemotherapy. In KEYNOTE-024, patients with previously untreated mNSCLC who received pembrolizumab (200 mg intravenously [IV] every 3 weeks) had a statistically significant improvement in OS (hazard ratio [HR] 0.60; 95% confidence interval [CI]: 0.41-0.89; p = .005), and significant improvement in PFS (HR 0.50; 95% CI: 0.37-0.68; p < .001). In KEYNOTE-010, patients with disease progression on or after platinum-containing chemotherapy received pembrolizumab IV 2 mg/kg, 10 mg/kg, or docetaxel 75 mg/m2 every 3 weeks. The HR and p value for OS was 0.71 (95% CI: 0.58-0.88), p < .001 comparing pembrolizumab 2 mg/kg with chemotherapy and the HR and p value for OS was 0.61 (95% CI: 0.49-0.75), p < .001 comparing pembrolizumab 10 mg/kg with chemotherapy. IMPLICATIONS FOR PRACTICE: This is the first U.S. Food and Drug Administration approval of a checkpoint inhibitor for first-line treatment of lung cancer. This approval expands the pembrolizumab indication in second-line treatment of lung cancer to include all patients with programmed death-ligand 1-expressing non-small cell lung cancer.

FDA Approval Summary: Accelerated Approval of Pembrolizumab for Second-Line Treatment of Metastatic Melanoma.

On September 4, 2014, the FDA approved pembrolizumab (KEYTRUDA; Merck Sharp & Dohme Corp.) with a recommended dose of 2 mg/kg every 3 weeks by intravenous infusion for the treatment of patients with unresectable or metastatic melanoma who have progressed following treatment with ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. Approval was based on demonstration of objective tumor responses with prolonged response durations in 89 patients enrolled in a randomized, multicenter, open-label, dose-finding, and activity-estimating phase 1 trial. The overall response rate (ORR) by blinded independent central review per RECIST v1.1 was 24% (95% confidence interval, 15-34); with 6 months of follow-up, 86% of responses were ongoing. The most common (≥20%) adverse reactions were fatigue, cough, nausea, pruritus, rash, decreased appetite, constipation, arthralgia, and diarrhea. Immune-mediated adverse reactions included pneumonitis, colitis, hepatitis, hypophysitis, and thyroid disorders. The benefits of the observed ORR with prolonged duration of responses outweighed the risks of immune-mediated adverse reactions in this life-threatening disease and represented an improvement over available therapy. Important regulatory issues in this application were role of durability of response in the evaluation of ORR for accelerated approval, reliance on data from a first-in-human trial, and strategies for dose selection. Clin Cancer Res; 23(19); 5666-70. ©2017 AACR.

Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

BACKGROUND: The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. RESULTS: We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. CONCLUSIONS: Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.

Tuberoinfundibular Peptide of 39 residues is required for germ cell development.

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as a PTH 2 receptor ligand. We report that mice with deletion of Tifp39, the gene encoding TIP39, are sterile. Testes contained Leydig and Sertoli cells and spermatogonia but no spermatids. Labeling chromosome spreads with antibodies to proteins involved in recombination showed that spermatogonia do not complete prophase of meiosis I. Chromosomes were observed at different stages of recombination in single nuclei, a defect not previously described with mutations in genes known to be specifically involved in DNA replication and recombination. TIP39 was previously shown to be expressed in neurons projecting to the hypothalamus and within the testes. LH and FSH were slightly elevated in Tifp39(-/-) mice, suggesting intact hypothalamic function. We found using in situ hybridization that the genes encoding TIP39 and the PTH 2 receptor are expressed in a stage-specific manner within seminiferous tubules. Using immunohistochemistry and quantitative RT-PCR, TIP39 expression is greatest in mature testes, and appears most abundant in postmeiotic spermatids, but TIP39 protein and mRNA can be detected before any cells have completed meiosis. We used mice that express Cre recombinase under control of a spermatid-specific promoter to express selectively a cDNA encoding TIP39 in the testes of Tifp39(-/-) mice. Spermatid production and fertility were rescued, demonstrating that the defect in Tifp39(-/-) mice was due to the loss of TIP39. These results show that TIP39 is essential for germ cell development and suggest that it may act as an autocrine or paracrine agent within the gonads.

Characterization of big stanniocalcin variants in mammalian adipocytes and adrenocortical cells.

The hormone stanniocalcin (STC) is widely distributed, and in rodents the highest levels of expression are in the ovaries. In both cows and rodents, ovarian STC consists of three high-molecular-weight variants collectively known as big STC. In the ovary, big STC is made by theca cells and interstitial cells and is targeted to lipid storage droplets of nearby luteal cells to inhibit progesterone release. An endocrine pathway is operative during pregnancy and lactation. Whether or not big STC is made by tissues other than ovary has never been addressed. Therefore, the purpose of this study was to determine via a detailed characterization of adrenal glands and adipocytes whether big STC is present in other cells that store and release lipids. The results showed that STC was made in bovine and mouse adrenals, mainly in steroidogenic, adrenocortical cells. The majority of ligand and receptor were likewise confined to cortical zone cells. As in luteal cells, high levels of ligand and receptor were found in the adrenocortical cell lipid droplet fraction. However, adrenals made only the largest (135 kDa) of the three big STC variants. Nonetheless, adrenal STC had much greater receptor affinity than a mixture of the three big STC variants. Adipocytes contained all three big STC variants, and both ligand and receptor were heavily concentrated on the lipid droplets. Moreover, adipocyte lipid storage droplets had 50-fold more receptors than those in steroidogenic cells, indicating that big STC is heavily targeted to adipose cells. The findings collectively support the hypothesis that big STC is not unique to ovarian steroidogenic cells but is in fact common to cells with a role in lipid storage and release.

Evidence for cross-talk between stanniocalcins.

There are 2 forms of stanniocalcin (STC) produced by the STC-1 gene; a 50 kDa polypeptide known as STC50 and a recently discovered group of higher molecular weight variants that are collectively referred to as big STC. Both have different tissue patterns of expression and different intracellular targeting pathways. STC50 functions locally in tissues such as muscle, liver, and kidney and is targeted to mitochondria. Big STC, on the other hand, is made by the ovaries. It signals both locally on nearby corpus luteal cells and systemically. Interestingly, however, receptor binding assays employing STC50 as the tracer have shown that the smaller ligand can bind equally to tissue receptors targeted by either form of the hormone. This suggests there may be cross-talk between ligands. The present study provides credence to this notion by demonstrating how the 2 hormones can compete for tissue receptors normally targeted by 1 form of the hormone (big STC). The results also reveal how STC50 can completely block the inhibitory effects of big STC on luteal cell progesterone release when added simultaneously. The findings therefore add credence to the possibility that there may be circumstances during which the 2 ligands functionally antagonize each other's actions.

Regulation of luteal cell big stanniocalcin production and secretion.

In mammals, the ovaries have the highest levels of stanniocalcin (STC) gene expression, most or all of which is confined to androgen-producing thecal-interstitial cells (TICs). Ovarian TICs also synthesize a different STC that consists of three high molecular weight species collectively known as big STC. Upon release in response to LH stimulation, TIC-derived big STC is sequestered locally by target cells, particularly steroidogenic cells of the corpus luteum, via a receptor-mediated process. Although there is little or no STC gene expression in luteal cells in the in vivo setting, this report describes how the gene is turned on, STC mRNA becomes readily detectable, and big STC is secreted when bovine luteal cells are cultured in vitro. STC gene expression and secretion were both positively regulated by activation of the adenylate cyclase/protein kinase A signaling pathway (forskolin and 8-bromo-cAMP). However, prostaglandin E2 was the only natural luteal cell ligand capable of replicating the effects of forskolin and 8-bromo-cAMP (LH had no consistent effect). Sex steroids such as 17beta-estradiol, androstenedione, and progesterone significantly decreased luteal cell STC expression and secretion. However, only androstenedione was capable of reducing STC production and secretion to undetectable levels. This report is the first to show that once removed from their normal context within the ovary, luteal cells are capable of synthesizing and secreting big STC. It is also the first to delineate the regulatory mechanisms involved in STC production and secretion by luteal cells. These results therefore suggest that under certain physiological conditions, the corpus luteum could very well serve as a source of STC production.

Targeting of big stanniocalcin and its receptor to lipid storage droplets of ovarian steroidogenic cells.

Stanniocalcin (STC) is a large polypeptide hormone that is widely distributed in tissues such as kidney, adrenal, and ovary. In most tissues, STC exists as a 50-kDa homodimer (STC50). The ovaries produce a higher molecular weight variant (big STC) in androgen-producing theca cell and interstitial cell compartments. Luteal cells, which do not express the STC gene, nonetheless contain high levels of STC protein, suggesting they are targeted by and sequester big STC through a receptor-mediated process. Recently, an STC.alkaline phosphatase fusion protein was used to characterize mitochondrial targeting and sequestration of STC50 and its receptor in liver and kidney. The main objective of the present study was to characterize big STC and its receptor in mammalian ovary and determine whether the ovarian STC variant was similarly targeted to luteal cell mitochondria. By in situ ligand binding, we identified large numbers of STC receptors on corpus luteal cells. However, a more detailed analysis of sub-cellular fractions revealed that both STC and its receptor were not preferentially targeted to mitochondria but instead to cholesterol/lipid storage droplets, which was more indicative of a role in steroidogenesis. Functional studies revealed that additions of big STC had concentration-dependent inhibitory effects on both basal and stimulated progesterone output by primary cultured luteal cells. Furthermore, STC receptor levels were up-regulated in luteal cells in response to protein kinase A activation. Taken together, these findings indicate that theca cell-derived big STC is targeted to the cholesterol/lipid storage droplets of luteal cells to regulate steroidogenesis. This constitutes the first reported description of polypeptide hormone and receptor targeting to cholesterol/lipid droplets and the first biological role for the big STC variant.

Ovarian stanniocalcin is structurally unique in mammals and its production and release are regulated through the luteinizing hormone receptor.

Stanniocalcin (STC) is a recently discovered mammalian hormone that is widely distributed in many tissues. In rodents the STC gene is most highly expressed in ovary, specifically in androgen-producing thecal and interstitial cells. In addition, ovarian levels of expression rise 15-fold over pregnancy. The objective of this study was to develop a primary culture system for ovarian thecal-interstitial cells (TICs) to identify factors governing STC production and release. We used highly purified primary cultures of rat and bovine TICs, the purity of which was routinely assessed with antigenic and enzymatic markers. The functionality of cells was assured by their responsiveness to LH in the form of progesterone release. We found that forskolin significantly increased STC gene expression and secretion by both rat and bovine TICs, an effect that was only replicated by human (h) chorionic gonadotropin (CG). Coincubation of TICs with hCG and phosphodiesterase inhibitors further increased STC secretion, whereas coincubation of TICs with hCG and protein kinase A inhibitors attenuated hCG-stimulated release. Intriguingly, ovarian STC proved to be substantially larger than the 50-kDa homodimer produced in most other tissues. These results indicate that ovarian STC is physically distinct, a feature that could explain its presence in serum during pregnancy and lactation.