Risk factors and titers of COVID-19 infection in a longitudinal statewide seroepidemiology cohort.

BACKGROUND: Virginia is a large state in the USA, yet it remains unclear what percentage of the population has had natural COVID-19 infection and whether risk factors for infection have changed over time. METHODS: Using a longitudinal cohort, from December 2021-July 2022 we performed follow up serology and a questionnaire on 784 individuals from across Virginia who had previously participated in a statewide COVID-19 seroepidemiology study in 2020. Children were also invited to participate and an additional 62 children also completed the study. Serology was performed using Roche nucleocapsid and spike serological assays. RESULTS: The majority of participants were white (78.6%), over 50 years old (60.9%), and reported having received COVID-19 vaccine (93.4%). 28.6% had evidence of prior COVID-19 infection (nucleocapsid positive). Reweighted by region, age, and sex to match the Virginia census data, the seroprevalence of nucleocapsid antibodies was estimated to be 30.6% (95% CI: 24.7, 36.6). We estimated that 25-53% of COVID-19 infections were asymptomatic. Infection rates were lower in individuals > 60 years old and were higher in Blacks and Hispanics. Infection rates were also higher in those without health insurance, in those with greater numbers of household children, and in those that reported a close contact or having undergone quarantine for COVID-19. Participants from Southwest Virginia had lower seropositivity (16.2%, 95% CI 6.5, 26.0) than other geographic regions. Boosted vaccinees had lower infection rates than non-boosted vaccinees. Frequenting indoor bars was a risk factor for infection, while frequently wearing an N95 mask was protective, though the estimates of association were imprecise. Infection rates were higher in children than adults (56.5% vs. 28.6%). Infection in the parent was a risk factor for child infection. Spike antibody levels declined with time since last vaccination, particularly in those that were vaccinated but not previously infected. Neutralizing antibody positivity was high (97-99%) for wild type, alpha, beta, gamma, delta, and omicron variants. Neutralizing antibody levels were higher in the follow-up survey compared to the first survey in 2020 and among individuals with evidence of natural infection compared to those without. CONCLUSIONS: In this longitudinal statewide cohort we observed a lower-than-expected COVID-19 infection rate as of August 2022. Boosted vaccinees had lower infection rates. Children had higher infection rates and infections tracked within households. Previously identified demographic risk factors for infection tended to persist. Even after the omicron peak, a large number of Virginians remain uninfected with COVID-19, underscoring the need for ongoing vaccination strategies.

Assessment of Seroprevalence of SARS-CoV-2 and Risk Factors Associated With COVID-19 Infection Among Outpatients in Virginia.

Importance: Data from seroepidemiologic surveys measuring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure in diverse communities and ascertaining risk factors associated with infection are important to guide future prevention strategies. Objective: To assess the prevalence of previous SARS-CoV-2 infection across Virginia and the risk factors associated with infection after the first wave of coronavirus disease 2019 (COVID-19) infections in the US. Design, Setting, and Participants: In this statewide cross-sectional surveillance study, 4675 adult outpatients presenting for health care not associated with COVID-19 in Virginia between June 1 and August 14, 2020, were recruited to participate in a questionnaire and receive venipuncture to assess SARS-CoV-2 serology. Eligibility was stratified to meet age, race, and ethnicity quotas that matched regional demographic profiles. Main Outcomes and Measures: The main outcome was SARS-CoV-2 seropositivity, as measured by the Abbott SARS-CoV-2 immunoglobulin G assay. Results: Among 4675 adult outpatients (mean [SD] age, 48.8 [16.9] years; 3119 women [66.7%]; 3098 White [66.3%] and 4279 non-Hispanic [91.5%] individuals) presenting for non-COVID-19-associated health care across Virginia, the weighted seroprevalence was 2.4% (95% CI, 1.8%-3.1%) and ranged from 0% to 20% by zip code. Seroprevalence was notably higher among participants who were Hispanic (10.2%; 95% CI, 6.1%-14.3%), residing in the northern region (4.4%; 95% CI, 2.8%-6.1%), aged 40 to 49 years (4.4%; 95% CI, 1.8%-7.1%), and uninsured (5.9%; 95% CI, 1.5%-10.3%). Higher seroprevalence was associated with Hispanic ethnicity (adjusted odds ratio [aOR], 3.56; 95% CI, 1.76-7.21), residence in a multifamily unit (aOR, 2.55; 95% CI, 1.25-5.22), and contact with an individual with confirmed COVID-19 infection (aOR, 4.33; 95% CI, 1.77-10.58). The sensitivity of serology results was 94% (95% CI, 70%-100%) among those who reported receiving a previous polymerase chain reaction test for COVID-19 infection. Among 101 participants with seropositive results, 67 individuals (66.3%) were estimated to have asymptomatic infection. These data suggested a total estimated COVID-19 burden that was 2.8-fold higher than that ascertained by PCR-positive case counts. Conclusions and Relevance: This large statewide serologic study estimated that 2.4% of adults in Virginia had exposure to SARS-CoV-2, which was 2.8-fold higher than confirmed case counts. Hispanic ethnicity, residence in a multifamily unit, and contact with an individual with confirmed COVID-19 infection were significant risk factors associated with exposure. Most infections were asymptomatic. As of August 2020, the population in Virginia remained largely immunologically naive to the virus.

Adenosine Signaling Increases Proinflammatory and Profibrotic Mediators through Activation of a Functional Adenosine 2B Receptor in Renal Fibroblasts.

Interstitial renal fibrosis is a major pathophysiological manifestation of patients diagnosed with Chronic Kidney Disease (CKD), Diabetic Nephropathy (DN) and other inflammatory diseases. Adenosine signaling is an innate autocrine and paracrine cellular signaling pathway involving several key mediators that are elevated in the blood and kidneys of patients with DN. In these studies, we hypothesized that extracellular adenosine signals through one or more functional adenosine GPCRs on renal fibroblasts which increases profibrotic and proinflammatory mediators by inducing an activated fibroblast phenotype. Utilizing the renal fibroblast cell line NRK-49F, the presence and relative abundance of adenosine receptors (AR) A1, A2A, A2B, and A3 were quantified by RT-PCR. Under normal homeostatic conditions, only AR1 and AR2B were detected. The functionality of each receptor was then assessed by receptor specific pharmacological agonism and antagonism and assessed for modulation of the GPCR associated secondary messenger molecule, cyclic adenosine monophosphate (cAMP). Agonism of the AR2B receptor resulted in increased intracellular cAMP while agonism of the AR1 receptor inhibited cAMP modulation. Upon direct agonism of the AR2B receptor, transcripts for profibrotic and inflammatory mediators including SMA-α, IL-6, TGF-ß, CTGF, and fibronectin were elevated between 2-4 fold. These data indicate that renal fibroblasts express a functional AR1 receptor that inhibits cAMP upon stimulation, leading to a functional AR2B receptor that increases cAMP upon stimulation and also induces an activated fibroblast phenotype resulting in increased fibrotic and inflammatory mediators.

Serum Inflammatory and Immune Mediators Are Elevated in Early Stage Diabetic Nephropathy.

BACKGROUND: Diabetes is the leading cause of end stage renal disease (ESRD) in the United States, representing 44% of incident cases [1]. In this study, serum and peripheral blood collected from diabetic patients in five stages of chronic kidney disease (CKD), as defined by glomerular filtration rate (GFR), were compared to healthy (non-CKD) subjects. METHODS: Serum samples were analyzed for 39 inflammatory or immune mediator protein levels and peripheral blood samples were analyzed for expression of 35 gene transcripts. RESULTS: In serum, MCP-1, FGF-2, VEGF, and EGF levels were elevated above controls at all stages of DN. Five mediator levels, GM-CSF, IL-1α, IL-1RA, IL-6, and MIP1ß increased with disease progression until stage 4-5, at which point a decrease was observed paralleling a loss of functional renal mass that occurs in late stage CKD. Five mediator levels: GRO, IFNγ, MDC, Eotaxin, and G-CSF significantly differed from controls at one or more stages without apparent correlation with disease stage. Only a single mediator, sIL2RA, exhibited a linear increase with disease severity consistent with declining GFR. In peripheral blood, the transcript level of seven mediators, ICAM1, TNF-α, TGF-ß, IL-8, IL17RA, IFNγ, and MYD88 were significantly elevated at all disease stages as compared to control. CONCLUSION: Statistically significant differences in protein and transcripts levels between diseased and control can be detected in serum and peripheral blood utilizing high content profiling. These changes occur as early as stage 1-2 before a significant decline in renal function.

Macrophage-derived tumor necrosis factor-α mediates diabetic renal injury.

Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-α (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α-dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2(Akita) mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment, and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage-specific TNF-α-deficient mice (CD11b(Cre)/TNF-α(Flox/Flox)). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathologic changes, and kidney macrophage recruitment compared to diabetic TNF-α(Flox/Flox) control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.

Proinflammatory/profibrotic effects of interleukin-17A on human proximal tubule epithelium.

BACKGROUND/AIMS: Interleukin-17A (IL-17A) is a T cell-derived inflammatory cytokine that is upregulated during renal allograft rejection. The present study sought to further describe the IL-17A-mediated proinflammatory/profibrotic activity of proximal tubule epithelium that may contribute to allograft rejection. METHODS: Immortalized (HK-2) and primary (HRPTEpiC) human proximal tubule epithelial cells were utilized for this study. Profibrotic gene alterations were examined by real-time quantitative PCR. Inflammatory mediator secretion was examined by multiplex bead-based detection of secreted proteins. Immunofluorescence microscopy and immunoblotting were utilized to examine alterations in junctional protein expression and cell morphology. RESULTS: In HK-2 cells IL-17A significantly downregulated the expression of the proepithelial gene CDH1 (E-cadherin) while the proinflammatory/profibrotic genes CTGF, CD44 and TGFBR1 were significantly increased. IL-17A also increased the secretion of fractalkine, G-CSF, GM-CSF, VEGF, IL-6 and IL-8. In HRPTEpiC 100 ng/ml IL-17A upregulated the proinflammatory/profibrotic genes ACTA2, CCL2, CHMP1A, CTGF, FN1, IL6, FSP1, SMAD1, SMAD5, TGFB1 and TGFBR2 while treatment with a reduced concentration of IL-17A (0.1 ng/ml) decreased SMAD5, TGFB1 and PDGFRB expression. Changes in ZO-1 and E-cadherin protein expression and cell morphology were examined following IL-17A treatment as indicators of epithelial-to-mesenchymal transition. IL-17A decreased ZO-1 expression in HK-2 and HRPTEpiC; however, E-cadherin was only reduced in HK-2 cells. Neither HK-2 nor HRPTEpiC assumed an elongated, fibroblast-like morphology following IL-17A treatment. CONCLUSIONS: IL-17A directly mediates proximal tubule epithelial cell proinflammatory/profibrotic activity as demonstrated by the alteration in genes associated with extracellular matrix remodeling and cell-cell interaction, and stimulation of inflammatory mediator and immune cell chemoattractant secretion. Additionally, IL-17A may have a negative impact on barrier integrity as indicated by ZO-1 downregulation.

Development of cell-based immunoassays to measure type I collagen in cultured fibroblasts.

Excessive deposition of type I collagen by activated fibroblasts is a hallmark of scarring and fibrotic pathologies. Quantitation of collagen I at the protein level is paramount to measure functionally relevant changes during pathological remodeling of the extracellular matrix. We describe two new cell-based assays to directly quantify the amount of collagen I incorporated into the extracellular matrix of primary human lung fibroblasts. Utilizing a monoclonal antibody specific to native human collagen I, we optimized conditions and parameters including incubation time, specificity and cell density to demonstrate dose-dependent induction of collagen I by transforming growth factor beta, as measured by in-cell enzyme linked immunosorbent assay. The results obtained by this assay were mimicked by an "In situ Quantitative Western Blot" on cultured cells using the same antibody. Results from these assays were comparable to those obtained with a commercial assay for collagen I N-propeptide, which is an index of collagen formation. These assays have been optimized for a 96-well format and provide a novel and useful approach for screening of anti-fibrotic agents in vitro. The assays described here also offer a significant improvement in throughput and specificity over conventional methods that primarily measure soluble collagen.

Generation of bleomycin-induced lung fibrosis is independent of IL-16.

Given that CD4+ cells are found in the lungs of patients with fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) we hypothesized that IL-16, a potent chemoattractant for CD4+ cells, may be involved in the pathogenesis of this disease. We found that baseline IL-16 gene expression is greater in fibroblasts isolated from IPF patients compared to non-fibrotic fibroblasts. Furthermore, IL-16 gene expression increased in IPF fibroblasts following stimulation with either of the pro-fibrotic growth factors TGFb1 or PDGF. In contrast, PDGF had no effect on IL-16 gene expression in non-fibrotic lung fibroblasts, whereas TGFb1 down-regulated IL-16 gene expression in non-fibrotic fibroblasts. To gain a better understanding of an association of IL-16 with fibrosis, we used the bleomycin-induced mouse model of fibrosis to examine IL-16 gene expression. Our current study demonstrates that IL-16, and its activator caspase 3, are highly expressed at the mRNA level in the lungs of mice prior to the deposition of collagen following intratracheal bleomycin administration. We then sought to determine the role of IL-16 in the generation of fibrosis in the mouse by using IL-16KO mice. There were no differences observed between IL-16WT and IL-16KO mice (cellular infiltrate, collagen deposition, total lung collagen generation and cytokine expression) following bleomycin instillation. These results indicate that IL-16 is prominently expressed in both murine and human fibrosis however as complete loss of this cytokine did not modulate pulmonary fibrosis, IL-16 is a candidate biomarker for IPF.

BMP-7 fails to attenuate TGF-beta1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells.

BACKGROUND: In rodent models of chronic renal disease bone morphogenetic protein-7 (BMP-7) has been shown to halt disease progression and promote recovery. Subsequent studies utilizing immortalized rodent renal cell lines showed that BMP-7 was renoprotective by antagonizing TGF-beta1-stimulated epithelial-to-mesenchymal transition (EMT). The present study sought to determine if BMP-7 prevents TGF-beta1-induced EMT in primary (RPTEC) and immortalized (HK-2) human proximal tubule epithelial cells. METHODS: EMT was determined by quantitative real-time PCR analysis of e-cadherin, vimentin, CTGF and TGF-beta1 transcript expression and immunocytochemical analysis of ZO-1 and alpha-smooth muscle actin (alpha-SMA) protein expression following TGF-beta1 treatment in RPTEC and HK-2 cells. RESULTS: In RPTEC and HK-2 cells, TGF-beta1 significantly reduced e-cadherin expression and significantly increased vimentin, CTGF and TGF-beta1 expression. TGF-beta1 also diminished ZO-1 immunoreactivity and increased alpha-SMA expression in confluent cell monolayers. Co-incubation of TGF-beta1 with an anti-TGF-beta1 neutralizing antibody substantially reduced the cytokine's effects, which indicated EMT in these cells was inhibitable. Co-administration of BMP-7 over a broad concentration range (0.01-100 microg/ml) with TGF-beta1 failed to attenuate EMT in RPTEC or HK-2 cells, as demonstrated by no inhibition of altered e-cadherin, vimentin, CTGF and TGF-beta1 expression and no restoration of ZO-1 immunoreactivity. Furthermore, when BMP-7 was applied to proximal tubule cells alone, it also decreased e-cadherin expression and increased vimentin, CTGF and TGF-beta1 expression. Additionally, BMP-7 failed to induce the mesenchymal-to-epithelial transition (MET) in NRK-49F rat renal fibroblasts. BMP-7 did however prevent TGF-beta1-mediated e-cadherin downregulation in TCMK-1 mouse renal tubular epithelial cells. BMP-7 activity was routinely confirmed by examining BMP-7-induced phosphorylation of SMADs 1/5/8, BMP-7 regulation of BMPR-IA, BMP-7-mediated reduction of IL-6 transcript expression and BMP-7-mediated reduction of secreted IL-6 and IL-8 proteins. CONCLUSIONS: In the present study, despite confirming BMP-7 regulation of receptor expression and induction of downstream signalling events, we were unable to demonstrate BMP-7 inhibition of EMT in either primary or immortalized human proximal tubule cells. Moreover, we were unable to demonstrate BMP-7-stimulated MET in rat renal fibroblasts. A protective effect was however observed at an elevated BMP-7 concentration in mouse renal tubular epithelial cells.

BMP-7 does not protect against bleomycin-induced lung or skin fibrosis.

Bone morphogenic protein (BMP)-7 is a member of the BMP family which are structurally and functionally related, and part of the TGFbeta super family of growth factors. BMP-7 has been reported to inhibit renal fibrosis and TGFbeta1-induced epithelial-mesenchymal transition (EMT), in part through negative interactions with TGFbeta1 induced Smad 2/3 activation. We utilized in vivo bleomycin-induced fibrosis models in the skin and lung to determine the potential therapeutic effect of BMP-7. We then determined the effect of BMP-7 on TGFbeta1-induced EMT in lung epithelial cells and collagen production by human lung fibroblasts. We show that BMP-7 did not affect bleomycin-induced fibrosis in either the lung or skin in vivo; had no effect on expression of pro-fibrotic genes by human lung fibroblasts, either at rest or following exposure to TGFbeta1; and did not modulate TGFbeta1-induced EMT in human lung epithelial cells. Taken together our data indicates that BMP-7 has no anti-fibrotic effect in lung or skin fibrosis either in vivo or in vitro. This suggests that the therapeutic options for BMP-7 may be confined to the renal compartment.

Hyper-responsiveness of IPF/UIP fibroblasts: interplay between TGFbeta1, IL-13 and CCL2.

One of the hallmarks of idiopathic pulmonary fibrosis with a usual interstitial pneumonia histological pathology (IPF/UIP) is excess collagen deposition, due to enhanced fibroblast extracellular matrix synthetic activity. Studies using murine models of lung fibrosis have elucidated a pro-fibrotic pathway involving IL-13 driving CCL2, which in turn drives TGFbeta1 in lung fibroblasts. Therefore, we sought to determine whether this pathway exists in the human fibrotic setting by evaluating human IPF/UIP fibroblasts. IPF/UIP fibroblasts have an increased baseline fibrotic phenotype compared to non-fibrotic fibroblasts. Interestingly, non-fibrotic fibroblasts responded in a pro-fibrotic manner to TGFbeta1 but were relatively non-responsive to IL-13 or CCL2, whereas, IPF/UIP cells were hyper-responsive to TGFbeta1, IL-13 and CCL2. Interestingly, TGFbeta1, CCL2 and IL-13 all upregulated TGFbeta receptor and IL-13 receptor expression, suggesting an ability of the mediators to modulate the function of each other. Furthermore, in vivo, neutralization of both JE and MCP5, the two functional orthologs of CCL2, during bleomycin-induced pulmonary fibrosis significantly reduced collagen deposition as well as JE and CCR2 expression. Also in the bleomycin model, CTGF, which is highly induced following TGFbeta stimulation, was attenuated with anti-JE/anti-MCP5 treatment. Overall this study demonstrates an interplay between TGFbeta1, IL-13 and CCL2 in IPF/UIP, where these three mediators feedback on each other, promoting the fibrotic response.

Expression and characterization of a human BMP-7 variant with improved biochemical properties.

Bone morphogenetic protein-7 (BMP-7, OP-1) is a secreted growth factor that is predominantly known for its osteoinductive properties, though it has also been implicated as having a role in mammalian kidney development. Clinical efficacy of recombinant BMP-7 has been demonstrated in the treatment of orthopedic injuries through topical application. However, the pharmaceutical development of recombinant BMP-7 for systemic delivery has presented many challenges. Specifically, the expression level of recombinant mature BMP-7 protein in mammalian cells is very low, the molecule has poor solubility at neutral pH, and intracellular proteolytic processing events result in a secreted BMP-7 having multiple amino-termini, creating a heterogeneous mixture of proteins. Utilizing structural information, we have designed and generated a number of rational BMP-7 mutations that improved both expression levels in mammalian cells and solubility at neutral pH, while limiting the amino-terminal heterogeneity of the mature protein. Introduction of these mutations did not compromise BMP-7 in vitro bioactivity. This improved BMP-7 molecule is better suited for pharmaceutical development and clinical advancement for indications where systemic delivery may be required.

An in vivo model to assess factors that may stimulate the generation of an immune reaction to erythropoietin.

The incidence of pure red cell aplasia (PRCA) in patients with chronic kidney disease associated with the subcutaneous (s.c.) administration of epoetin alfa (EPREX) began to increase in 1998. As part of an intensive investigation into the reasons for this increase, in vivo models were developed to assess the ability of potential causative factors to stimulate an immune response to recombinant human erythropoietin (rHuEPO). It was difficult to generate anti-EPO antibodies in mice. In animals injected with rHuEPO alone, anti-EPO antibodies were either absent or present at very low levels. The addition of an adjuvant to the immunization protocol was able to increase both the frequency of occurrence and titer of the immune response and resulted in the generation of anti-EPO antibodies that, in most cases, recognized both human and mouse EPO. Some mice exhibited a reduction in hematocrit, suggesting neutralization of endogenous EPO by anti-EPO antibodies. To evaluate the primary lead identified in the technical investigation, leachates from the uncoated syringe stoppers of EPREX syringes, a surrogate antigen (chicken egg albumin, OVA) was used to avoid possible interferences that could arise from the use of an endogenous protein like EPO. These leachates yielded a positive, concentration-dependent antibody response in the OVA animal model, demonstrating their adjuvant properties and providing support for the hypothesis generated through the technical investigation that leachates were capable of enhancing the immune response to rHuEPO.

Recombinant epoetins do not stimulate tumor growth in erythropoietin receptor-positive breast carcinoma models.

We investigated the significance of erythropoietin receptor (EPOR) expression following treatment with recombinant human erythropoietin (rHuEPO; epoetin alpha) and the effect of recombinant epoetins (epoetin alpha, epoetin beta, and darbepoetin alpha) alone or in combination with anticancer therapy on tumor growth in two well-established preclinical models of breast carcinoma (MDA-MB-231 and MCF-7 cell lines). Expression and localization of EPOR under hypoxic and normoxic conditions in MDA-MB-231 and MCF-7 cells were evaluated by immunoblotting, flow cytometry, and immunohistochemistry. EPOR binding was evaluated using [125I]rHuEPO. Proliferation, migration, and signaling in MDA-MB-231 and MCF-7 cells following treatment with rHuEPO were evaluated. Tumor growth was assessed following administration of recombinant epoetins alone and in combination with paclitaxel (anticancer therapy) in orthotopically implanted MDA-MB-231 and MCF-7 breast carcinoma xenograft models in athymic mice. EPOR expression was detected in both tumor cell lines. EPOR localization was found to be exclusively cytosolic and no specific [125I]rHuEPO binding was observed. There was no stimulated migration, proliferation, or activation of mitogen-activated protein kinase and AKT following rHuEPO treatment. In mice, treatment with recombinant epoetins alone and in combination with paclitaxel resulted in equivalent tumor burdens compared with vehicle-treated controls. Results from our study suggest that although EPOR expression was observed in two well-established breast carcinoma cell lines, it was localized to a cytosolic distribution and did not transduce a signaling cascade in tumors that leads to tumor growth. The addition of recombinant epoetins to paclitaxel did not affect the outcome of paclitaxel therapy in breast carcinoma xenograft models. These results show that recombinant epoetins do not evoke a physiologic response on EPOR-bearing tumor cells as assessed by numerous variables, including growth, migration, and cytotoxic challenge in preclinical in vivo tumor models.

Potential involvement of BMP receptor type IB activation in a synergistic effect of chondrogenic promotion between rhTGFbeta3 and rhGDF5 or rhBMP7 in human mesenchymal stem cells.

Chondrogenic promotion by rhGDF5 with or without rhTGFbeta3 was studied in pellet culture of human mesenchymal stem cells (HMSCs). A synergy between rhGDF5 and rhTGFbeta3 was observed in promoting chondrogenesis. rhBMP2, rhBMP6, rhBMP7 and rhTGFbeta1 were further tested and showed the same effect. To explore the mechanism, the expression of TGFbetatype I and II receptors, ALK5, ALK2, ALK3, ALK6, TGFbetaRII, BMPRII, ActRII was studied. ALK6 showed increase by the rhTGFbeta1 or rhTGFbeta3 treatment. ALK6 protein expression also showed increase by rhTGFbeta3. rhTGFbeta1/rhTGFbeta3 induced ALK6 up-regulation was inhibited by SD-208, a TGFbeta type I receptor inhibitor. Chondrogenesis by rhTGFbetal/rhTGFbeta3 or the combination between rhTGFbetal/rhTGFbeta3 and rhGDF5 also was diminished by SD-208. SMAD1/5/8 phosphorylation in nascent human mesenchymal stem cells (HMSCs) was stimulated weakly by rhGDF5 but strongly by rhBMP7. The rhGDF5 stimulated SMAD1/5/8 phosphorylation was enhanced by rhTGFbetal/rhTGFbeta3 but inhibited by SD-208. The rhBMP7 stimulated SMAD1/5/8 phosphorylation did not show influence by rhTGFbeta3 and SD-208. Our results indicated the potential involvement of ALK6 activation by rhTGFbetas in the synergy between rhTGFbetas and rhBMPs.

The pharmacokinetics of erythropoietin in the cerebrospinal fluid after intravenous administration of recombinant human erythropoietin.

OBJECTIVES: Erythropoietin (EPO) was originally described as a regulator of erythropoiesis. Recently, synthesis of EPO and expression of the EPO receptor (EPO-R) have been reported for the central nervous system (CNS). The potential use of EPO to prevent or reduce CNS injury and the paucity of information regarding its entry into the human CNS led us to examine the pharmacokinetics (PK) of recombinant human EPO (r-HuEPO) in the serum and cerebrospinal fluid (CSF). METHODS: Four patients with Ommaya reservoirs were enrolled to facilitate serial CSF sampling. R-HuEPO was given intravenously (IV) in single doses of 40,000 IU or 1,500 IU/kg and in multiple doses of 40,000 IU daily for 3 days. RESULTS: The EPO concentrations in the CSF increased after a period of slow equilibration. Linear first-order distribution kinetics were observed for serum and CSF. The concentration of EPO in the CSF was proportional to the serum concentration of EPO and the permeability of the blood-brain barrier (BBB), as determined by the albumin quotient (QA=[albumin] CSF/[albumin] serum). A rise in the CSF concentration was seen as early as 3 h after IV administration. Peak levels (C(max)) were reached between 9 h and 24 h. After a single dose of 1,500 IU/kg, the Cmax in the CSF ranged from 11 mIU/ml to 40 mIU/ml, and the ratios of CSF/serum Cmax ranged from 3.6x10-4 to 10.2x10-4. The terminal half-life (t1/2) values of EPO in serum and CSF were similar. The t(1/2) of r-HuEPO in the CSF ranged from 25.6 h to 35.5 h after a single dose of 1,500 IU/l. Using these parameters a PK model was generated that predicts the concentration-time profile of EPO in the CSF. CONCLUSIONS: We report that r-HuEPO can cross the human BBB and describe for the first time the PK of EPO in the CSF after IV administration. Our data suggest that the concentration-time profile of EPO in the CSF can be predicted for individual patients if the serum concentration of EPO and the Q(A) are known. This information may be useful in the design of clinical trials to explore the potential therapeutic effects of EPO during CNS injury.

The erythropoietin receptor and its expression in tumor cells and other tissues.

Erythropoietin (EPO) is the primary regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. The EPO receptor belongs to the cytokine receptor superfamily. Although the primary role of EPO is the regulation of red blood cell production, EPO and its receptor have been localized to several nonhematopoietic tissues and cells, including the central nervous system (CNS), endothelial cells, solid tumors, the liver, and the uterus. The presence of EPO receptors and the possibility of EPO signaling in these tissues and cells have led to numerous studies of the effects of EPO at these sites. In particular, expression of EPO and the EPO receptor in cancer cells has generated much interest because of concern that administration of recombinant human erythropoietin (rHuEPO) to patients with breast and other cancer cells expressing the EPO receptor may promote tumor growth via the induction of cell proliferation or angiogenesis. However, evidence supporting a growth-promoting effect has been inconclusive. Moreover, several preclinical studies have shown a beneficial effect of EPO on delaying tumor growth. Further, it is conceivable that increased expression of EPO could reduce tumor hypoxia and ameliorate the deleterious effects of hypoxia on tumor growth, metastasis, and treatment resistance. On the other hand, EPO has also been shown to produce an angiogenic effect in vascular endothelial cells in vitro. However, there is no evidence that these effects occur in vivo to promote tumor growth. EPO and EPO receptors are expressed in neural tissue, and they are upregulated there by hypoxia. Animal studies have shown that administration of epoetin alfa (an rHuEPO) reduces tissue injury due to ischemic stroke, blunt trauma, and experimental autoimmune encephalomyelitis. These findings suggest that epoetin alfa may provide a therapeutic benefit in patients with stroke, trauma, epilepsy, and other CNS-related disorders. Clearly, further study of EPO and the EPO receptor in nonhematopoietic tissue is warranted to determine the potential therapeutic usefulness of rHuEPO as well as to determine the signaling pathway responsible for its effect in vivo.

Erytropoietin concentrations in cerebrospinal fluid of nonhuman primates and fetal sheep following high-dose recombinant erythropoietin.

Erythropoietin (Epo) decreases neuronal injury and cell death in vitro and in vivo. To lay the groundwork for use of Epo as a potential therapy for brain injury, we tested the hypothesis that systemic dosing of high-dose recombinant Epo (rEpo) would result in neuroprotective rEpo concentrations in the spinal fluid of adult and developing animals. This report characterizes the pharmacokinetics of high-dose rEpo in the blood and spinal fluid of juvenile and adult nonhuman primates (n = 7) and fetal sheep (n = 37) following a single injection. Timed blood and spinal fluid samples were collected following rEpo injection. Epo accumulation in spinal fluid was dependent on peak serum concentration and time following injection. We demonstrate that high-dose rEpo was well tolerated and results in neuroprotective concentrations in spinal fluid of adult and developing animal models by 2-2.5 h after injection.

Human recombinant erythropoietin significantly improves tumor oxygenation independent of its effects on hemoglobin.

Tumor oxygenation is known to be an important predictive/prognostic marker in a variety of tumors, including cervix, head/neck, sarcoma, non-small cell of the lung, and breast. Tumor oxygenation is influenced by many interactions, including oxygen delivery (angiogenesis, permeability, and HgB) and consumption (metabolic and growth rates). This study randomized 30 nonanemic, female Fischer 344 rats into three treatment arms to examine the effects of recombinant human erythropoietin (EPO) on R3230 rodent mammary carcinoma oxygenation. The three treatment arms were: (a) placebo; (b) EPO after tumor implantation (2000 units/kg/SQdose, M/W/F for six doses); and (c) EPO before tumor implantation (2000 units/kg/SQdose, M/W/F for six doses). Tumors were implanted in the hindflank, and in vivo oxygenation was measured at day 22 after implantation using the Oxylite system (Oxford Optronix, Oxford, England). An average of 180 measurements/animal were performed. On day 22, median tumor volume was 399 mm(3) (range: 65-950 mm(3)), and no differences in tumor volume were seen between treatment arms. Mean hematocrit was equal between arms at therapy initiation but were significantly higher for both arms receiving EPO at day 22 (placebo versus Arm B versus Arm C; Wilcoxon P = 0.052). EPO-treated tumors had significantly less hypoxic measurements when compared with either the placebo or those receiving EPO before implantation. These data confirm that tumor oxygenation in nonanemic individuals may be improved through the administration of EPO, and this improvement appears to be independent of HgB effects.

Pharmacokinetic and pharmacodynamic modeling of recombinant human erythropoietin after intravenous and subcutaneous dose administration in cynomolgus monkeys.

The pharmacokinetics (PK) and pharmacodynamics (PD) of recombinant human erythropoietin (rHuEpo) were investigated in monkeys. A two-compartment model with dual input and nonlinear disposition could adequately characterize the PK of rHuEpo upon three intravenous and six s.c. administrations. The kinetic model suggests rapid zero-order absorption of part of the s.c. dose (35%) followed by a slow first-order entry through the lymphatics. The s.c. treatments caused a delayed dose-dependent rise in reticulocyte numbers peaking between 100 and 200 h and returning to baseline by 300 to 400 h. This was followed by steady rises in red blood cell (RBC) and hemoglobin counts. A physiological catenary model based on a life span concept with rHuEpo stimulating the production of two cell populations (progenitor cells and erythroblasts) was applied. The model could adequately describe the reticulocyte responses upon the various s.c. treatments, giving estimates of maturation times for cells in the various stages of differentiation including the early progenitor cells (70.4 h), erythroblasts (15.0 h), and reticulocytes (141.6 h) that are close to the literature reported values. An Smax of 3.13 was estimated indicating a moderate maximum stimulation of erythropoiesis, whereas the SC50 was 842 IU/l. The model was used to effectively predict the increases in RBC and hemoglobin counts as well. In conclusion, the physiological PK/PD model developed could adequately describe the time course of rHuEpo effects, yielding realistic estimates of cell life span parameters.