Foscarnet Research Articles

Combined Effect of Basic Antiherpetic Drugs with a New Inhibitor of the Terminase Complex of Herpes Simplex Virus Type 1 in Vero Cell Cultures.

Carriers of herpes simplex virus type 1 (HSV-1) account for more than 90% of the global population. Infection manifests itself in the formation of blisters and ulcers on the face or genitals and can cause blindness, encephalitis, and generalized infection. All first- and second-line modern antiherpetic drugs selectively inhibit viral DNA polymerase. The purine-benzoxazine conjugate LAS-131 ((S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine), which we have described earlier, uses the large subunit of the HSV-1 terminase complex as a biotarget and selectively inhibits HSV-1 reproduction in vitro. Basically new results were for the first time obtained to characterize the combined effect on human herpesvirus infection for LAS-131 used in combination with practically significant antiviral compounds, including the nucleoside analogs acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), brivudine (BVdU), iododeoxyuridine (IdU), and adenine arabinoside (Ara-A); the nucleoside phosphonate analog cidofovir (CDV); and the pyrophosphate analog foscarnet (FOS). A cytopathic effect (CPE) inhibition assay showed that the drug concentration that inhibited the virus-induced CPE by 50% decreased by a factor of 2 (an additive effect, FOS) or more (a synergistic effect; ACV, PCV, GCV, IdU, BVdU, Ara-A, and CDV) when the drugs were used in combination with LAS-131. Nonpermissive conditions for HSV-1 reproduction were thus created at lower drug concentrations, opening up new real possibilities to control human herpesvirus infection.

Hypoxia-Inducible Factor-1α Regulates High Phosphate-Induced Vascular Calcification via Type III Sodium-Dependent Phosphate Cotransporter 1.

Vascular calcification (VC) has a high incidence in patients with chronic kidney disease, which is a worldwide public health problem and presents a heavy burden to society. Hypoxia-inducible factor (HIF)-1α, the active subunit of HIF-1, has been reported to play a vital role in high phosphate-induced VC. However, the underlying mechanism is still undetermined, and effective treatment is unavailable. In the present study, human aortic smooth muscle cells (HASMCs) were cultured under normal or high phosphate media conditions. HIF-1α small interfering RNA and overexpression plasmids were employed to regulate HIF-1α expression. Phosphonoformic acid was employed to restrain the function of type III sodium-dependent phosphate cotransporter 1 (Pit-1). The expression levels of HIF-1α, Pit-1, runt-related transcription factor 2 (Runx2), and smooth muscle 22 alpha (SM22α) were evaluated, and the calcium contents were also examined. Cell growth was assessed using an MTT assay. High phosphate stimulation caused an upregulation in HIF-1α and Pit-1 expression levels and induced calcium depositions in HASMCs. Upregulation of Runx2 expression accompanied by downregulation of SM22α expression was observed in the high phosphate group. Following the suppression of HIF-1α expression, there was a concomitant attenuation in Pit-1 expression, calcium deposition, the alteration of phenotypic transition marker genes, and vice versa. The most serious calcium deposition was noted in HASMCs cultured under high phosphate conditions which were pretreated with a HIF-1α overexpression plasmid. However, when the biological functions of Pit-1 were restrained, the putative serious calcium deposition was not formed even in HASMCs transfected with a HIF-1α overexpression plasmid. The findings confirmed that HIF-1α regulated Pit-1 expression and exerted its pro-calcifying effect through Pit-1, which identified HIF-1α and Pit-1 as therapeutic targets for high phosphate-induced VC.

Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

Inorganic phosphate (Pi) is a critical determinant of calcification, and its concentration is regulated by alkaline phosphatase (ALP) and Pit1. ALP is a key regulator of osteogenic calcification and acts by modulating local inorganic phosphate (Pi) concentrations through hydrolyzing pyrophosphate in the extracellular matrix (ECM). Pit1, a sodium-dependent phosphate transporter, regulates calcification via facilitating phosphate uptake within the cells. To investigate whether zinc differentially regulates osteoblastic and vascular calcifications, we examined ALP activity and Pit1 in osteoblastic and vascular smooth muscle cells (VSMCs). Our findings demonstrate that calcification in osteoblastic MC3T3-E1 cells is decreased via diminished ALP action under zinc deficiency. In contrast, zinc-deficiency-induced calcification in VSMCs is independent of ALP action, as demonstrated by very weak ALP activity and expression in calcified VSMCs. In zinc-deficient A7r5 VSMC, P accumulation increased with increasing Na phosphate concentration (3-7 mM) but not with β-GP treatment, which requires ALP activity to generate Pi. Ca deposition also increased with Na phosphate in a dose-dependent manner; in contrast, β-GP did not affect Ca deposition. In osteoblastic cells, Pit1 expression was not affected by zinc treatments. In contrast, Pit1 expression is highly upregulated in A7r5 VSMC under zinc deficiency. Using phosphonoformic acid, a competitive inhibitor of Pit1, we showed that calcification is inhibited in both A7r5 and MC3T3-E1 cells, indicating a requirement for Pit1 in both calcifications. Moreover, the downregulation of VSMC markers under zinc deficiency was restored by blocking Pit1. Taken together, our results imply that zinc-deficiency-induced calcification in VSMC is independent of ALP action in contrast to osteoblastic calcification. Moreover, Pit1 expression in VSMCs is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency.

The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification.

For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 μM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 μM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 μM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 μM Dynasore. Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.

Phosphonoformic acid reduces hyperphosphatemia-induced vascular calcification via Pit-1.

This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC). Primary human aortic smooth muscle cells and rat aortic rings were cultured in Dulbecco's modified Eagle's medium supplemented with 0.9 mM or 2.5 mM phosphorus concentrations. Type III sodium-dependent phosphate cotransporter-1 (Pit-1) small interfering RNA and phosphonoformic acid (PFA), a Pit-1 inhibitor, were used to investigate the effects and mechanisms of Pit-1 on HPVC. Calcium content shown by Alizarin red staining, expression levels of Pit-1, and characteristic molecules for phenotypic transition of vascular smooth muscle cells were examined. Hyperphosphatemia induced the upregulation of Pit-1 expression, facilitated phenotypic transition of vascular smooth muscle cells, and led to HPVC in cellular and organ models. Treatment with Pit-1 small interfering RNA or PFA significantly inhibited Pit-1 expression, suppressed phenotypic transition, and attenuated HPVC. Our findings suggest that Pit-1 plays a pivotal role in the development of HPVC. The use of PFA as a Pit-1 inhibitor has the potential for therapeutic intervention in patients with HPVC. However, further rigorous clinical investigations are required to ensure the safety and efficacy of PFA before it can be considered for widespread implementation in clinical practice.

At-Home Foscarnet Administration in Patients with Cytomegalovirus Infection Post-Allogeneic Stem Cell Transplantation: A Unicentric, Safe, and Feasible Program.

Cytomegalovirus (CMV) infection is a relevant cause of morbimortality in patients receiving allogeneic stem cell transplantation (allo-HCT). Foscarnet (FCN) is an effective drug against CMV administered intravenously and usually on an inpatient basis. The Home Care Unit (HCU) for hematologic patients at our hospital designed an at-home FCN administration model to avoid the hospitalization of patients requiring FCN treatment. This study analyzes whether the at-home administration of FCN is as safe and effective as its hospital administration. We collected and compared demographic, clinical, analytical, and economic data of patients with CMV infection post-allo-HCT who received FCN in the hospital (n = 16, 17 episodes) vs. at-home (n = 67, 88 episodes). The proportions of patients with cured CMV infections were comparable between the two groups (65.9% vs. 76.5%, p = 0.395). The median duration of FCN treatment was 15 (interquartile range [IQR] 9-23) and 14 (IQR 11-19) days in the HCU and inpatient cohorts, respectively (p = 0.692). There were no significant differences in the FCN toxicities between groups except for hypocalcemia (26.1% vs. 58.8%, p = 0.007), which was more prevalent in the inpatient cohort. A significant cost-effectiveness was found in the HCU cohort, with a median savings per episode of EUR 5270. It may be concluded that home administration of FCN is a safe, effective, and cost-efficient therapeutic option for patients with CMV infection and disease.

Impact of Early Anti-Cytomegalovirus Therapy on the Incidence of Chronic Graft-Versus-Host Disease

Introduction: Cytomegalovirus (CMV) infections are associated with poor outcomes in recipients of allogeneic hematopoietic stem cell transplants (HSCTs). Therefore, preventive therapy or preemptive treatment have been developed. Ganciclovir (GCV) and foscarnet (FCN) are essential intravenous anti-CMV agents. A previous regional multicenter study revealed a significantly lower risk of chronic graft-versus-host disease (GVHD) in patients who received preemptive FCN therapy. To validate this observation and to investigate the mechanism or target, we executed a comprehensive nationwide study. Patients and Methods: We defined the early anti-CMV therapy as administration within 180 days after transplantation without evidence of target therapy for CMV infections. We retrospectively analyzed patients who received FCN or GCV as early anti-CMV therapy, and who are not received any anti-CMV agents (no anti-CMV) between June 2005 and December 2019. We excluded patients who received FCN, GCV, valganciclovir, or letermovir as prophylactic therapy; those who began anti-CMV therapy before the day of transplantation; those who received both FCN and GCV; those who underwent T cell-depleting treatment. Results: A total of 19536 adults with hematological malignancies who received their first allogeneic HSCT were included. We compared patients who received FCN (n = 1555) or GCV (n = 7335) as early anti-CMV therapy, and no anti-CMV recipients (n = 10646). FCN or GCV recipients showed higher incidence of acute GVHD than no anti-CMV recipients. The cumulative incidence of acute GVHD 200 days after transplantation were 46% (95% confidence interval [CI], 41%-51%), 47% (95% CI, 45%-50%), and 26% (95% CI, 25%-28%) for FCN, GCV, and no anti-CMV recipients, respectively (P < 0.001). The cumulative incidence of chronic GVHD three years after transplantation were 28% (95% CI, 26%-31%), 37% (95% CI, 36%-38%), and 29% (95% CI, 28%-30%) for FCN, GCV, and no anti-CMV recipients, respectively (P < 0.001). The cumulative incidence of extensive chronic GVHD three years after transplantation were 17% (95% CI, 15%-19%), 22% (95% CI, 22%-23%), and 16% (95% CI, 15%-26%) for FCN, GCV, and no anti-CMV recipients, respectively (P < 0.001) (Figure A). In multivariate analyses adjusted for acute GVHD or other important variables, the risks of extensive chronic GVHD among GCV recipients was higher than FCN recipients (hazard ratio [HR], 1.17; 95% CI, 1.01-1.34; P = 0.031), whereas no-anti CMV recipients and FCN did not showed statistically significance. In further investigations, we performed stratified analyses and revealed that the incidence of extensive chronic GVHD was significantly lower among the FCN recipients than among the GCV recipients only in stratifications that involved male recipients of female donor HSCT (according to sex compatibility). Comparing with GCV, the incidence of extensive chronic GVHD was significantly reduced among male recipients of female donor HSCTs who were taking FCN (HR, 1.63; 95% CI, 1.19-2.23; P = 0.002), but FCN did not affect the incidence of extensive chronic GVHD for other sex compatibility-based stratifications (HR, 1.05; 95% CI, 0.90-1.23; P = 0.51). Among male recipients of female donor transplants, the incidence of extensive chronic GVHD three years after transplantation were 13% (95% CI, 10%-17%), 27% (95% CI, 25%-29%), and 20% (95% CI, 18%-21%) for FCN, GCV, and no anti-CMV recipients, respectively (P < 0.001; Figure B). FCN and no anti-CMV recipients showed similar risk of extensive chronic GVHD (HR, 1.22; 95% CI, 0.89-1.66; P = 0.21). The incidence of extensive chronic GVHD was significantly reduced among FCN recipients who received HSCT from female donors, regardless of stem cell source, GVHD prophylaxis, or previous acute GVHD incidence, in male recipients. Conclusions: In this study, patients requiring early anti-CMV therapy were at a high risk of chronic GVHD, primary due to their increased incidence of acute GVHD. Remarkably, FCN could potentially mitigate the incidence of chronic GVHD for them. Especially, FCN would be beneficial for male recipients of female donor transplants who are typically at high risk for chronic GVHD. These findings pave the way towards a deeper understanding of the mechanism underlying chronic GVHD and overcome the unmet needs of allogeneic HSCT recipients.

A Pharmacovigilance Study of Drug-Induced Glaucoma Utilizing the Japanese Adverse Event Reporting System.

Clinically, glaucoma is a serious problem because it is asymptomatic until a relatively late stage in most cases, which can lead to delays in the diagnosis and treatment of the disease. The purpose of this study was to clarify the rank-order of the association of glaucoma with the causative drugs using a spontaneous reporting system database. Data were extracted from the Japanese Adverse Drug Event Report database of the Pharmaceuticals and Medical Devices Agency (Japan). Based on reports of glaucoma caused by all drugs, we calculated the reporting odds ratio (ROR) and 95% confidence interval (CI) for glaucoma. Among 609 reports of adverse events corresponding to glaucoma (46%, women), the most frequently implicated drug were steroids (prednisolone, betamethasone sodium phosphate, triamcinolone acetonide, and fluorometholone), pregabalin, ranibizumab, crizotinib, tacrolimus hydrate, darbepoetin alfa, and foscarnet sodium hydrate. Among 207 reports involved in angle-closure glaucoma (86%, women), anticholinergic drug and antidepressants ranked high and showed signals. Signals were also detected in bromazepam (ROR, 69.7; 95% CI, 30.9-157.5), oral brotizolam (ROR, 16.6; 95% CI, 6.18-44.8), and oral milnacipran hydrochloride (ROR, 22.8; 95% CI, 8.46-61.4) for angle-closure glaucoma. A national pharmacovigilance database enabled us to identify the drugs that frequently induce glaucoma. The likelihood of the reporting of glaucoma varied among the drugs, which should be used carefully in clinical practice to avoid it.

Abstract 3 Efficacy of Foscarnet in Preventing HHV6 Encephalitis in Adult Single Cord Blood Transplantation

Abstract Introduction The risk of HHV-6 encephalitis is high in cord blood transplantation (CBT) and the prognosis after encephalitis is poor, but there is no clarity regarding effective prevention methods for HHV-6 encephalitis. Objectives We retrospectively evaluated the effect of foscarnet (FCN) in preventing HHV-6 encephalitis in CBT. Methods We included 184 patients who underwent single CBT at Kyoto University Hospital from 2005 to 2022. For those who developed HHV-6 encephalitis, we calculated cumulative steroid doses from transplantation to the date of encephalitis onset, and for those who did not develop encephalitis, we calculated them to 25 days (the median encephalitis onset). Results Median (range) age was 49 years (18-70 years); 82% used tacrolimus and MMF for GVHD prophylaxis; FCN was used prophylactically or preemptively in 141 patients (77%) to prevent HHV6 encephalitis. FCN was used in 35% of patients from day 7 post-transplant, 20% from the time of HHV-6 reactivation, and 8% after the start of steroid administration. HHV-6 encephalitis occurred in 18 (10%) of 184 patients. The median cumulative steroid dose for those receiving steroids was 4.0 mg/kg. Steroid dose (mg/kg) had a significant effect on the development of encephalitis (HR 1.09, P=0.003); pre-engraftment immune reaction had no significant effect. The ROC curve showed that the cutoff for cumulative steroid dose that best predicted the development of encephalitis was 6.5 mg/kg. FCN prophylaxis showed no significant effect in preventing HHV6 encephalitis in the high-steroid group. However, FCN prophylaxis reduced the incidence of HHV6 encephalitis in the low-steroid group with marginal significance (HR 0.22, P=0.070). Patients who developed encephalitis without HHV6 prophylaxis had a significantly lower survival rate than those who did not develop encephalitis (1yr-OS, 25% vs. 69%). However, those who developed encephalitis during HHV6 prophylaxis did not differ significantly from those who did not develop encephalitis (1yr-OS, 59% vs. 69%) (Figure). Discussion FCN prophylaxis significantly reduced the incidence of encephalitis in the low-dose steroid group, whereas prophylaxis was not effective in the high-dose steroid group. However, prophylactic administration may improve the prognosis after the onset of encephalitis.

Novel UL23 and UL30 substitutions in HSV1 and HSV2 viruses related to polymorphism or drug resistance

Data on herpes simplex virus (HSV) polymorphism as well as acyclovir (ACV) and foscarnet (FOS) resistance mutations are not exhaustive and may hinder accurate diagnosis by next-generation sequencing (NGS). Here, we report novel UL23 and UL30 substitutions for HSV1 and HSV2 identified in immunocompromised patients treated for hematological malignancies during the last 6 years of HSV resistance surveillance at the University Hospital of Lyon. For HSV1, 35 novel UL23 substitutions and 52 novel UL30 substitutions were identified. For HSV2, 2 novel UL23 substitutions and 12 novel UL30 substitutions were identified. These results allow to complete the database of HSV1 and HSV2 substitutions, related either to polymorphism or to ACV and FOS resistance.

#4000 ROLE OF ENDOTHELIAL CELLS IN VASCULAR CALCIFICATION

Abstract Background and Aims Vascular calcification (VC) represents a common pathological feature of cardiovascular disease and VC caused by hyperphosphatemia is one of the hallmarks of chronic kidney disease (CKD). Therefore, elucidating the pathogenesis of VC may have significant clinical benefits for CKD patients. Vascular smooth muscle cells challenged with high-phosphate (Pi) actively participate to VC by trans-differentiating into simil-osteoblastic cells that acquire the capability to deposit hydroxyapatite crystals in the extracellular matrix of tunica media in arteries. The role of endothelial cells (ECs) in high-Pi calcification has been poorly investigated until now. Nonetheless, following particular stimuli ECs can lose their endothelial characteristics and acquire several different phenotypes transdifferentiating with a process known as endothelial-to-mesenchymal transition (EndMT). Given the central role of VC for CKD patients wellbeing we decided to investigate whether ECs may have a role in VC process. Method We set up an in vitro calcification model with human aortic ECs (HAECs) challenged with 2, 2.5, 3, 3.5 mM high-Pi for 7 days. Calcium deposition, cell viability and cell trans-differentiation were evaluated. Results In our model we observed deposition of calcium phosphate crystals induced by high-Pi that resulted significant from 3 up to 3.5 mM Pi (Ctr 0.43 ± 0.01; 3 mM Pi 6.72 ± 1.15; 3.5 mM Pi 17.31 ± 1.88; micromol Ca++/mg protein; P < .01; Fig 1A-B). We found that calcification is mediated by Pi-influx, as demonstrated by the inhibition of calcium deposition by Phosphonoformic acid (PFA), an inhibitor of Na/Pi cotransporter Pit-1 (3.5 mM Pi 15.52 ± 1.97; 1 mM PFA 0.6 ± 0.05; micromol Ca++/mg protein; P < .0001; Fig 1A, C). In addition, in our model high-Pi did not show any significant toxic effect at every concentration tested compared to control cells. In Pi-treated ECs we observed a clear change in cell-morphology from cobblestone to spindle shaped, a recognized sign of EndMT. Moreover, we observed a change in extracellular matrix composition more similar to osteoblastic matrix. In facts, ECs treated with Pi were Alcian Blue positive, a staining that binds glycosaminoglycans contained in bone matrix, with blue granules of calcium-phosphate in the cytoplasm of treated cells. Starting from day 5 up to day 7 the transition was confirmed by an up-regulation of SNAIL and N-cadherin mRNA expressions, one of the master genes and one the markers of EndMT process, respectively. A modification of endothelial phenotype was also supported by a progressive decrease in protein expression of Von Willebrand factor and VE-Cadherin observed at day 9 of calcification (−53.7% and −32.6% respectively). Moreover, from day 5 till day 7 we found some osteoblastic differentiation signs such as a progressive increase of RUNX2 and BMP2 mRNA expression and an increase of protective MGP mRNA levels. Conclusion Our data demonstrate that HAECs are able to calcify. High-Pi induces trans-differentiation in simil-osteoblastic cells and a progressive loss of endothelial characteristics through the EndMT process. More studies are needed to better elucidate the role for ECs in CKD VC process.

A case report of long-delayed diagnosis of pseudorabies virus encephalitis with endophthalmitis: lessons from metagenomic next generation sequencing

BackgroundPseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human.Case presentationWe report a case of pseudorabies virus encephalitis and endophthalmitis, diagnosed 89 days after onset, confirmed with intraocular fluid metagenomic next generation sequencing (mNGS) after the result of two cerebrospinal fluid (CSF) mNGS tests were negative. Although treatment with intravenous acyclovir, foscarnet sodium, and methylprednisolone improved the symptoms of encephalitis, significant diagnostic delay resulted in permanent visual loss.ConclusionsThis case suggests that pseudorabies virus (PRV) DNA in the intraocular fluid may have a higher positivity than that in the CSF. PRV may persist in the intraocular fluid for an extended period and may thus require extended antiviral therapy. Patients with severe encephalitis and PRV should be examined with the focus on pupil reactivity and light reflex. A fundus examination should be performed in patients with a central nervous system infection, specifically, those in a comatose state, to help reduce eye disability.

Prevalence of cytomegalovirus antiviral drug resistance in transplant recipients

Cytomegalovirus (CMV) is a significant human pathogen, especially for immunocompromised patients, often treated with one or more antiviral drugs. Although the prevalence of resistance is low, the impact of drug resistant CMV infections on patient outcomes is high and genotypic testing is recommended when resistance is suspected. To assess the prevalence of CMV drug resistance mutations among samples submitted for genotypic testing, 2750 patient sample results were analyzed. Testing was performed by sequencing for ganciclovir (GCV), cidofovir (CDV), foscarnet (FOS), maribavir (MBV) and/or letermovir (LMV) resistance conferring mutations. Of the 2750 patient samples, 826 (30.04%) had resistance to one or more anti-CMV drug. Resistance mutations were most common in UL97, with 27.64% and 9.96% of samples having GCV and MBV mutations, respectively. Resistance mutations in UL54 were less common, with 6.11%, 5.98% and 1.76% of samples having GCV, CDV and FOS mutations, respectively. For LMV, resistance mutations in UL56 were present in 7.17% of samples, with mutations at codon 325 representing 80.95% of the observed LMV resistance mutations. Resistance to two drugs was identified in 215 samples and to 3 or more drugs in 35 samples. While a high prevalence of CMV resistance mutations was identified, this must be taken in the context of healthcare providers submitting samples from patients with suspected resistant CMV strains. For these patients, rapid monitoring for resistance allows treatment modifications based on objective results rather than empiric drug selection, which is particularly relevant given the presence of mutations conferring resistance to more than one drug.

Pharmacology of Mammalian Na+-Dependent Transporters of Inorganic Phosphate.

Inorganic phosphate (Pi) is an essential component of many biologically important molecules such as DNA, RNA, ATP, phospholipids, or apatite. It is required for intracellular phosphorylation signaling events and acts as pH buffer in intra- and extracellular compartments. Intestinal absorption, uptake into cells, and renal reabsorption depend on a set of different phosphate transporters from the SLC20 (PiT transporters) and SLC34 (NaPi transporters) gene families. The physiological relevance of these transporters is evident from rare monogenic disorders in humans affecting SLC20A2 (Fahr's disease, basal ganglia calcification), SLC34A1 (idiopathic infantile hypercalcemia), SLC34A2 (pulmonary alveolar microlithiasis), and SLC34A3 (hereditary hypophosphatemic rickets with hypercalciuria). SLC34 transporters are inhibited by millimolar concentrations of phosphonoformic acid or arsenate while SLC20 are relatively resistant to these compounds. More recently, a series of more specific and potent drugs have been developed to target SLC34A2 to reduce intestinal Pi absorption and to inhibit SLC34A1 and/or SLC34A3 to increase renal Pi excretion in patients with renal disease and incipient hyperphosphatemia. Also, SLC20 inhibitors have been developed with the same intention. Some of these substances are currently undergoing preclinical and clinical testing. Tenapanor, a non-absorbable Na+/H+-exchanger isoform 3 inhibitor, reduces intestinal Pi absorption likely by indirectly acting on the paracellular pathway for Pi and has been tested in several phase III trials for reducing Pi overload in patients with renal insufficiency and dialysis.

Changes and Treatment Prognosis of Aqueous Humor Cytokine Concentrations of Patients with Acquired Immune Deficiency Syndrome Complicated by Cytomegalovirus Retinitis.

Purpose: The purposes of this study were to investigate cytokine changes in the aqueous humor after treatment of acquired immune deficiency syndrome (AIDS) complicated with cytomegalovirus retinitis (CMVR) and to determine whether these changes are useful prognostic indicators. Methods: This study included 12 patients (15 eyes) undergoing treatment for AIDS and CMVR. The patients received intravitreal injections and systemic intravenous treatment with ganciclovir and foscarnet sodium. The aqueous humor of each eye was sampled before treatment and before the third and fifth injections. The samples were tested to determine the concentrations of each of 27 cytokines using the Luminex 200™ liquid phase chip. Results: The concentrations of cytokines interleukin (IL)-1rα (P = 0.002), IL-1b (P = 0.001), IL-8 (P = 0.001), basic fibroblast growth factor (bFGF) (P < 0.001), interferon γ-induced protein 10 (IP-10) (P = 0.001), and tumor necrosis factor (TNF)-α (P = 0.004) in the aqueous humor before the third and fifth injections were significantly lower after than before treatment. The reductions in TNF-α (P = 0.028) and IL-1b (P = 0.028) concentrations after treatment were statistically significant compared with the postoperative visual acuity improvement (≥3 lines and <3 lines). The difference in TNF-α (P = 0.018) level before and after treatment (the difference between before treatment and before the fifth intravitreal injection) was also statistically significant compared with the number of injections (≥6 times and <6 times). Conclusion: The cytokines IL-1rα, IL-1b, IL-8, bFGF, IP-10, and TNF-α may offer new avenues for evaluation of therapeutic effect, and TNF-α and IL-1b may be important cytokines for prognostic evaluation (based on visual acuity and the number of injections) in patients suffering from AIDS and CMVR. Clinical Trial Registration: Number: ChiCTR2200056955.

Chemical and Redox Speciation of Uranyl with Three Environmentally Relevant Bifunctional Chelates: Multi-Technique Approach Combined with Theoretical Estimations.

Carbon and phosphorous are two primary elements common to the bio-geosphere and are omnipresent in both biotic and abiotic arenas. Phosphonate and carboxylate are considered as building blocks of glyphosate and humic substances and constituents of the cellular wall of bacteria and are the driving functionalities for most of the chemical interactions involving these two elements. Phosphonocarboxylates, a combination of both the functionalities in one moiety, are ideal models to dig deep into for understanding the chemical interactions of the two functional groups with metal ions. Phosphorous and carbon majorly exist as inorganic/organic phosphate and carboxylate, respectively, in the bio-geosphere. Aquatic contamination is a major concern for uranium, and the presence of complexing agents would alter the uranium concentrations in aquifers. Determination of solution thermodynamic parameters, speciation plots, redox patterns, Eh-pH diagrams, coordination structures, and molecular-level understanding by density functional theory calculations was carried out to interpret the uranyl (UO22+) interaction with three environmentally relevant phosphonocarboxylates, namely, phosphono-formic acid (PFA), phosphono-acetic acid (PAA), and phosphono-propanoic acid (PPA). UO22+ forms 1:1 complexes with the three phosphonocarboxylates in the monoprotonated form, having nearly the same stability, and the complexes [UO2(PFAH)], [UO2(PAAH)], and [UO2(PPAH)] involve chelate formation of five, six, and seven membered rings, respectively, through the participation of an oxygen each from the carboxylate and phosphonate, strengthened by an intra-molecular hydrogen bonding through the proton of the phosphonate moiety with uranyl oxygen. The complex formations are favored both enthalpically and entropically, with the latter being more contributive to the overall free energy of formation. The redox speciation showed an aqueous soluble complex formation over a wide pH range of 1-8. Electrospray ionization mass spectrometry and extended X-ray absorption fine structure established the coordination modes, which are further corroborated by density functional calculations. The knowledge gained from the present studies provide potential inputs in framing the cleanup, sequestering, microbial, and bio-remediation strategies for uranyl from aquatic environments.

Artificial stem cells mediated inflammation-tropic delivery of antiviral drugs for pneumonia treatment

BackgroundCytomegalovirus (CMV) pneumonia is a major cause of morbidity and mortality in immunodeficiency individuals, including transplant recipients and Acquired Immune Deficiency Syndrome patients. Antiviral drugs ganciclovir (GCV) and phosphonoformate (PFA) are first-line agents for pneumonia caused by herpesvirus infection. However, the therapy suffers from various limitations such as low efficiency, drug resistance, toxicity, and lack of specificity.MethodsThe antiviral drugs GCV and PFA were loaded into the pH-responsive nanoparticles fabricated by poly(lactic-co-glycolic acid) (PLGA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and further coated with cell membranes derived from bone marrow mesenchymal stem cells to form artificial stem cells, namely MPDGP. We evaluated the viral suppression effects of MPDGP in vitro and in vivo.ResultsMPDGP showed significant inflammation tropism and efficient suppression of viral replication and virus infection-associated inflammation in the CMV-induced pneumonia model. The synergistic effects of the combination of viral DNA elongation inhibitor GCV and viral DNA polymerase inhibitor PFA on suppressing the inflammation efficiently.ConclusionThe present study develops a novel therapeutic intervention using artificial stem cells to deliver antiviral drugs at inflammatory sites, which shows great potential for the targeted treatment of pneumonia. To our best knowledge, we are the first to fabricate this kind of artificial stem cell to deliver antiviral drugs for pneumonia treatment.Graphical

Abstract 2408: Elevated phosphate promotes a pro-migration phenotype in lung cancer

Abstract Despite advances in treatment, metastasis remains a major cause of cancer-associated death in the US. Thus, identification of intrinsic and extrinsic factors that promote or inhibit tumor migration and invasion are critical. Increasingly, the dysregulation of cellular metabolism is being linked not only to tumor growth but tumor cell migration. Phosphorus (Pi) is an essential micronutrient necessary for energy metabolism, cell signaling via ATP, formation of nucleic acids and the cell membrane, and mineralization of the skeleton. Unsurprisingly, high levels of Pi are observed in tumor tissue compared to normal tissue, consistent with a demand for this metabolite in rapidly growing cells. Further, our lab has reported that high extracellular Pi increases cell growth, alters gene expression and protein translation, and promotes angiogenesis. High dietary Pi levels has also been shown to increase tumorigenesis in both lung and skin cancer models. However, it remains unknown how phosphate metabolism affects tumor migration and invasion. Here we report that high levels of Pi increases gene expression of EMT-related genes such as Twist, Vimentin, CD44, and OPN in the lung cancer cell line, A549. Further, increased Pi promotes tumor cell migration in a transwell assay and decreased Pi levels or inhibition of phosphate transport by phosphonoformic acid (PFA) impaired migration. Suggesting that high Pi can promote a pro-migration phenotype by upregulating EMT in cancer cells and by increasing directed cell migration. Ongoing research will focus on how intracellular Pi flux changes during cell invasion using a novel FRET-based phosphate sensor in vitro and the effect of Pi inhibition on metastasis in vivo. These findings provide further evidence for the role dysregulated Pi metabolism in promoting cancer progression and the potential for therapeutic intervention with broad impacts on patient outcomes. Citation Format: Jamie Arnst, George Beck. Elevated phosphate promotes a pro-migration phenotype in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2408.

Case Report: Unusual Varicella-Zoster Virus Meningoencephalitis With Meningomyelitis Mimicking Central Nervous System Leukemia

BackgroundVaricella-Zoster Virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. In immunocompromised patients, VZV may produce disease of the central nervous system (CNS). Clinical manifestations of CNS infection with VZV are non-specific and can mimic other infectious and non-infectious diseases. Due to similar symptoms, CNS infection with VZV represents a diagnostic challenge. Here, we report a case of a patient who showed laboratory and imaging manifestations mimicking the neoplastic etiology.CaseA 31-year-old man presented with a 3-day history of headache, 5-h of confusion, generalized tonic-clonic seizures, and transient fever. The patient has a history of acute myelogenous leukemia (AML). His cerebrospinal fluid (CSF) studies revealed markedly elevated protein (10.326 g/L) and atypical cells. Meanwhile, the MRI of brain, cervical, and thoracic spine was reported as extensive (frontal, parieto-occipital and temporal pachymeningeal, and falx cerebri) enhancement and irregular thickening. These examinations suggested a suspicion of CNS involvement of AML. However, based on further investigations with metagenomic next-generation sequencing, a final diagnosis of VZV meningoencephalitis with meningomyelitis was made. With acyclovir and foscarnet sodium therapy, repeated CSF studies revealed normal cell count and protein. No atypical cells were found. The repeated brain MRI also revealed obvious resolution of the previous abnormal pachymeningeal enhancement.ConclusionThis case highlights the importance of recognizing the unusual phenomenon of traditional tests in VZV meningoencephalitis with meningomyelitis, and timely using of further precise examinations to detect viral DNA, which is required to prevent missed diagnosis.

Foscarnet-Type Inorganic-Organic Hybrid Nanoparticles for Effective Antiviral Therapy.

[ZrO]2+[(FCN)0.4(OH)0.8]2- and Gd3+[FCN]3- inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a pyrophosphate analogue serves as a prototype of a viral DNA polymerase inhibitor. FCN is used for the treatment of herpesvirus infections, including the drug-resistant cytomegalovirus (CMV) and herpes simplex viruses, HSV-1 and HSV-2. The novel [ZrO]2+[(FCN)0.4(OH)0.8]2- and Gd3+[FCN]3- IOH-NPs are characterized by aqueous synthesis, small size (20-30 nm), low material complexity, high biocompatibility, and high drug load (up to 44 wt % FCN per nanoparticle). The antiviral activity of the FCN-type IOH-NPs is probed for the human cytomegalovirus (HCMV). Moreover, the uptake of FCN-type IOH-NPs into vesicles, cytoplasm, and nuclei of nonphagocytic lung epithelial cells is evaluated. As a result, a promising antiviral activity of the FCN-type IOH-NPs that significantly outperforms freely dissolved FCN at the level of clinical formulations is observed, encouraging a future use of FCN-type IOH-NPs for the delivery of antivirals against respiratory viruses.