Foscarnet Research Articles

Incidence, Severity, Day 100 Treatment Efficacy and Therapy Toxicity of Cytomegalovirus (CMV) Infections with Early Pre-Emptive Therapy in Adult Cord Blood (CB) Transplant Recipients

Introduction: Unrelated donor CB transplantation (CBT) is effective therapy for patients (pts) with high-risk hematologic malignancies without suitable adult donors. CBT, however, can be complicated by CMV infection in seropositive recipients with viremia rates as high as 100%. Moreover, CMV infection is associated with increased transplant-related mortality (TRM) in some reports. Therefore, our center is investigating: 1) the efficacy of pre-transplant ganciclovir (GAN) prophylaxis, 2) the efficacy of early pre-emptive therapy based on frequent serial qPCR monitoring, & 3) anti-viral therapy toxicities. The aim of this strategy is to rapidly eradicate viremia & thereby prevent disease without prohibitive toxicity.Methods: Eligible pts were adult CMV seropositive 1st allograft CB recipients transplanted 3/2013-2/2016. Pts received Cy 50/Flu 150/Thio 5-10/TBI 400 cGy myeloablative conditioning + CSA/MMF (no ATG). GAN prophylaxis (5 mg/kg IV daily) was given days -7 to -2. Viremia was evaluated by qPCR (lower detection limit 137 copies/ml) twice weekly from day +14 or earlier if indicated. Treatment for viremia was usually initiated at 1st or 2nd qPCR positivity. Foscarnet (FOS), GAN or valganciclovir (VALGAN) dosing (maintenance vs induction) was selected based on viremia level & assessment of severe infection vs toxicity risk. CMV disease was treated with induction dosing. CMV response was defined as 3 consecutive negative qPCR & no evidence of disease.Results: 42 CBT recipients [median 51 years (range 23-66), 33 acute leukemias, 6 MDS/MPD, 3 lymphomas] received double unit grafts [median donor-recipient HLA-allele match 5/8 (range 2-7/8) & infused CD34+ cell dose 1.3 (range 0.2-3.2) x 105/kg/unit]. 98% of pts engrafted (1 graft failure unrelated to CMV) & 86% (26 gr. II, 7 gr. III, 3 gr. IV) had gr. II-IV aGVHD by day 100 (median onset 29 days, range 19-35). Of the 42 pts, 35 reactivated CMV in the first 100 days [median viremia onset 33 days (range 5-74) & median viral load at 1st detection < 137 copies/mL (range < 137-245)] for a day 100 cumulative incidence of 83% (95%CI 67-92). Of 31 pts with both CMV & aGVHD, 15 developed viremia prior to & 16 after aGVHD onset. The median time from CMV detection to therapy initiation was 3 days (range 0-39). Treatment dosing, viremia vs disease & responses are shown (Figure). Induction dosing was given to one third of pts (n = 11). These pts started therapy later in their transplant course & predominantly received VALGAN. By day 100, just over half cleared viremia but none developed disease. Two-thirds of pts (n = 24) were started on maintenance dosing. They required therapy earlier post-CBT & most received FOS. 10 of these pts cleared viremia by day 100 although GI CMV disease developed in 1 pt after early therapy cessation. This pt was then successfully treated. Notably, half of the maintenance pts required escalation to induction due to persistent viremia in 10 or development of CMV pneumonia in 2 pts. The 2 pneumonia pts developed disease 26 & 12 days post-therapy initiation, respectively, & their peak viremias were 1,330 & 613 IU/ml, respectively. Both pts died of CMV & constituted 2/4 TRM deaths by day 100 in the study (other deaths were 1 DAH & 1 grade IV aGVHD). We then examined therapy toxicity in the first 100 days (Table). 4 pts received only FOS, 10 only GAN/ VALGAN, & 21 had courses of each. Therapy switches were due to toxicity, inadequate response, or convenience of oral VALGAN & therapy duration was highly variable according to infection severity, concurrent GVHD therapy, speed of viremia clearance & tolerance of drug toxicities. One-third of FOS pts & over half of GAN/ VALGAN pts had clinically significant toxicities.Conclusion: CMV infection is very frequent in adult seropositive CBT recipients & pre-CBT GAN is not effective prophylaxis. Early intensive monitoring permits early pre-emptive therapy which is effective in most pts. While maintenance dosing is effective in some, the development of lethal CMV pneumonia in 2 maintenance pts suggests dose escalation may be appropriate if rapid viremia eradication is not achieved. However, FOS/GAN/VALGAN toxicities are also significant. Ultimately, the optimal drug dosing to enhance efficacy but mitigate toxicity, how to predict pts at greatest risk of disease, appropriate therapy duration, & how to predict recurrence risk are not known. Improved CMV prophylaxis & therapy for allograft recipients including CBT pts are needed. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression.

Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I-related chain (MIC) A/B and UL16-binding proteins (ULBP)1-6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell-mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter via its binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response.

Calcium Overload Accelerates Phosphate-Induced Vascular Calcification Via Pit-1, but not the Calcium-Sensing Receptor.

Aim: Vascular calcification (VC) is a risk factor of cardiovascular and all-cause mortality in patients with chronic kidney disease (CKD). CKD–mineral and bone metabolism disorder is an important problem in patients with renal failure. Abnormal levels of serum phosphate and calcium affect CKD–mineral and bone metabolism disorder and contribute to bone disease, VC, and cardiovascular disease. Hypercalcemia is a contributing factor in progression of VC in patients with CKD. However, the mechanisms of how calcium promotes intracellular calcification are still unclear. This study aimed to examine the mechanisms underlying calcium-induced calcification in a rat aortic tissue culture model.Methods: Aortic segments from 7-week-old male Sprague–Dawley rats were cultured in serum-supplemented medium for 10 days. We added high calcium (HiCa; calcium 3.0 mM) to high phosphate (HPi; phosphate 3.8 mM) medium to accelerate phosphate and calcium-induced VC. We used phosphonoformic acid and the calcimimetic R-568 to determine whether the mechanism of calcification involves Pit-1 or the calcium-sensing receptor.Results: Medial VC was significantly augmented by HPi+HiCa medium compared with HPi alone (300%, p < 0.05), and was associated with upregulation of Pit-1 protein. Pit-1 protein concentrations in HPi+HiCa medium were greater than those in HPi medium. Phosphonoformic acid completely negated the augmentation of medial VC induced by HPi+HiCa. R-568 had no additive direct effect on medial VC.Conclusion: These results indicated that exposure to HPi+HiCa accelerates medial VC, and this is mediated through Pit-1, not the calcium-sensing receptor.

Safety of Foscarnet (FOS) Treatment for Human Herpesviruses (HHV) in a Contemporary Cohort of Immunocompromised Patients

Safety of Foscarnet (FOS) Treatment for Human Herpesviruses (HHV) in a Contemporary Cohort of Immunocompromised Patients

Retrospective analysis of foscarnet sodium combined with valacyclovir in the treatment of acute pain in herpes zoster

Objective To investigate the clinical curative effect and impact factors of valaciclovir and foscarnet sodium combination therapy on zoster patients who suffered from acute neuralgia. Methods The hospitalization information of 139 herpes zoster patients suffered from acute neuralgia was collected who enrolled in Department of Dermatology, the Second Xiangya Hospital of Central South University between Jan. 1st, 2015 to Dec. 31st, 2015. Their general condition, symptoms, signs and parameters of curative effect evaluation were recorded, including the time days for control rash, rash relief, rash diminish, the pain began to relief and the pain obvious relief. A retrospective cohort study was carried out to analyze the above information. Results The ratio of men to women who were hospitalized because of herpes zoster was 1.07∶1. The rash mostly occurred on head, face and trunk. A percentage (92.8%) of the patients was suffered from basic disease. The curative effect evaluation: the average days for control rash were (2.32 ± 1.10) d. The average days for rash relief were (5.80 ± 2.27) d. The average days for pain began to relief were (3.14 ± 1.32) d. The average days for pain obvious relief were (6.86 ± 2.30) d. The curative effect of patient under the age of 60 was significantly better than the older (P<0.01). The patient with hypoproteinemia showed poorer curative effect compared to patients without hypoproteinemia (P<0.05). The curative effect among patients with more than 3 kinds of diseases was significantly lower than patients with less than 3 kinds of disease (P<0.05). Conclusions The valaciclovir and foscarnet sodium combination therapy displayed definite curative effect on the herpes zoster patients who suffered from acute neuralgia. There are no significant differences among genders in terms of pain relief. The age, serum albumin levels and accompanied diseases were the impact factors for pain relief. Key words: Acyclovir/AA/AD; Foscarnet/AD; Neuralgia, postherpetic/DT

1-Methyl-2-undecyl-4(1H)-quinolone, a derivative of quinolone alkaloid evocarpine, attenuates high phosphate-induced calcification of human aortic valve interstitial cells by inhibiting phosphate cotransporter PiT-1

An abnormally high serum phosphate level induces calcific aortic stenosis (CAS), which is characterized by ectopic valve calcification and stenosis of the orifice area. Inhibition of ectopic calcification is a critical function of any internal medical therapy for CAS disease. The aim of the present study was to investigate the inhibitory effects of several derivatives of evocarpine, methanolic extracts from the fruits of Evodia rutaecarpa Bentham (Japanese name: Go-Shu-Yu) on the high phosphate-induced calcification of human aortic valve interstitial cells (HAVICs) obtained from patients with CAS. High phosphate (3.2 mM) concentrations significantly increased the calcification of HAVICs after 7 days of culture. This calcification was completely inhibited in the presence of sodium phosphonoformate (PFA), a selective inhibitor of the type III sodium-dependent phosphate cotransporter (PiT-1). PiT-1 contributes to phosphate uptake, resulting in calcification. 1-Methyl-2-undecyl-4(1H)-quinolone (MUQ; 30–300 nM), but not evocarpine or its derivatives dihydroevocarpine and 1-methyl-2-nonyl-4(1H)-quinolone, inhibited the high phosphate-induced HAVICs calcification in a concentration-dependent manner. Although all of the evocarpine derivatives attenuated alkaline phosphatase activity, only MUQ also decreased PiT-1 gene expression with cellular PiT-1 protein diminution. These results suggest that MUQ mitigated high phosphate-induced HAVICs calcification by inhibiting PiT-1 gene expression.

Indoxyl sulfate promotes vascular smooth muscle cell calcification via the JNK/Pit-1 pathway

We determined the effect of indoxyl sulfate (IS) on Pit-1 expression and the role of Pit-1 in IS-induced osteoblastic differentiation and calcification of vascular smooth muscle cells (VSMCs). To assess osteoblastic differentiation and Pit-1 expression, VSMCs were incubated with various concentrations of IS for different durations. Phosphonoformic acid (PFA), a competitive inhibitor of Pit-1, was used to verify the role of Pit-1. Western blot analysis and quantitative real-time polymerase chain reaction (PCR) were performed to assess Pit-1 protein and mRNA levels, respectively. To evaluate calcification, calcium content was measured. After IS treatment, we observed osteoblastic differentiation and calcification of VSMCs and up-regulation of Pit-1 expression. Moreover, the effect of IS on osteoblastic differentiation and Pit-1 expression was partly dose- and time-dependent. PFA abrogated the IS-induced osteoblastic differentiation and calcification of VSMCs to a certain extent. The c-Jun N-terminal kinase (JNK) pathway was activated after treatment with IS, whereas inhibition of the JNK pathway partially attenuated the effect of IS on both the stimulation of Pit-1 expression and calcium deposition. Our study is the first to demonstrate that IS promotes Pit-1 expression in part by activation of the JNK pathway that is involved in the mechanism of IS-induced osteoblastic differentiation and matrix mineralization.

Ornithine is a key mediator in hyperphosphatemia-mediated human umbilical vein endothelial cell apoptosis: Insights gained from metabolomics

AimsHyperphosphatemia is associated with accelerated vascular endothelial dysfunction in patients with chronic kidney disease (CKD). The purpose of this study is to investigate the molecular mechanisms underlying hyperphosphatemia-caused endothelial dysfunction. Main methodsThe metabolic fingerprinting of human umbilical vein endothelial cells (HUVECs) subjected to hyperphosphatemia was characterized using an integrated metabolomics approach. HUVECs cultured in physiologically simulated hyperphosphatemia with or without phosphonoformic acid, a sodium-dependent phosphate transporter inhibitor (N=6) were collected for metabolomics analysis. Multivariate principle component analysis and partial least squares discriminant analysis were applied to analyze the metabolic data. The key metabolites were confirmed by quantitative analysis using liquid chromatography coupled with tandem mass spectrometer (LC–MS/MS). Key findings36 metabolites were significantly altered in HUVECs following the challenges of hyperphosphatemia mimic, involving several metabolic pathways (all P<0.05). Among them, ornithine increased significantly in the HUVECs mediated by hyperphosphatemia mimic, and its levels positively correlated with cell apoptosis rate (r=0.674, P=0.002), and several additional metabolites in multiple metabolic pathways. The changes in the levels of ornithine and other several metabolites were supported by subsequent quantitative analyses using LC–MS/MS. Further study demonstrated that the increase in ornithine level may result from the increased expression of arginase 2 in HUVECs, which mediates the hydrolysis of arginine to form ornithine. SignificanceThis is the first study demonstrating ornithine a key molecule mediating hyperphosphatemia-induced apoptosis of ECs. Arginase 2 may be a therapeutic target for hyperphosphatemia-associated cardiovascular events.

The Bisphosphonates Clodronate and Etidronate Exert Analgesic Effects by Acting on Glutamate- and/or ATP-Related Pain Transmission Pathways

Bisphosphonates (BPs) are typical anti-bone-resorptive drugs, with nitrogen-containing BPs (N-BPs) being stronger than non-nitrogen-containing BPs (non-N-BPs). However, N-BPs have inflammatory/necrotic effects, while the non-N-BPs clodronate and etidronate lack such side effects. Pharmacological studies have suggested that clodronate and etidronate can (i) prevent the side effects of N-BPs in mice via inhibition of the phosphate transporter families SLC20 and/or SLC34, through which N-BPs enter soft-tissue cells, and (ii) also inhibit the phosphate transporter family SLC17. Vesicular transporters for the pain transmitters glutamate and ATP belong to the SLC17 family. Here, we examined the hypothesis that clodronate and etidronate may enter neurons through SLC20/34, then inhibit SLC17-mediated transport of glutamate and/or ATP, resulting in their decrease, and thereby produce analgesic effects. We analyzed in mice the effects of various agents [namely, intrathecally injected clodronate, etidronate, phosphonoformic acid (PFA; an inhibitor of SLC20/34), and agonists of glutamate and ATP receptors] on the nociceptive responses to intraplantar injection of capsaicin. Clodronate and etidronate produced analgesic effects, and these effects were abolished by PFA. The analgesic effects were reduced by N-methyl-D-aspartate (agonist of the NMDA receptor, a glutamate receptor) and α,β-methylene ATP (agonist of the P2X-receptor, an ATP receptor). SLC20A1, SLC20A2, and SLC34A1 were detected within the mouse lumbar spinal cord. Although we need direct evidence, these results support the above hypothesis. Clodronate and etidronate may be representatives of a new type of analgesic drug. Such drugs, with both anti-bone-resorptive and unique analgesic effects without the adverse effects associated with N-BPs, might be useful for osteoporosis.

Detection of cytomegalovirus by polymerase chain reaction in aqueous samples from patients with acquired immune deficiency syndrome

Objective To analyze the sensitivity and specificity of polymerase chain reaction (PCR) tests in the detection of cytomegalovirus (CMV) in the diagnosis of patients with acquired immune deficiency syndrome (AIDS), using aqueous humor samples. Methods 25 AIDS patients (including 21 men and 4 women) were studied. The age of the patients varied from 24 to 59 years, with an average of (39.2±9.3) years. The CD4+ T cell count was from 1 to 523 cells/μl, with a medium of 40 cells/μl. They were infected with human immunodeficiency virus(HIV)for a period from 15 days to 9 years with a median of 10 months. They were divided into three groups according to the fundus and treatment, including untreated cytomegalovirus retinitis (CMVR), treated CMVR and control group. There were 10 patients without anti-CMV treatment and 7 patients treated previously with foscarnet or ganciclovir whose eyes were diagnosed CMVR. Control group has 8 patients who had normal fundus or minor retinopathy excluded from CMVR. Approximately 100 μl of aqueous humor was obtained by anterior-chamber paracentesis and PCR was performed in all cases. Results There were CMV DNA in 9 of 10 eyes with untreated CMVR (90.0% sensitivity). Of 7 specimens from eyes with treated CMVR, 3 were CMV PCR positive (42.9% sensitivity). All 8 samples of the control group were negative for CMV DNA, indicating the clinical specificity of our PCR was greater than 99.9% for CMVR. The anterior chamber paracentesis did not cause any complications in our patients except for a patient with subconjunctival hemorrhage. Conclusions The assay had an estimated sensitivity of 90.0% in detecting untreated CMVR and a sensitivity of 42.9% in detecting CMVR that had been treated. The specificity of this assay was greater than 99.9%. Key words: Acquired immunodeficiency syndrome/complications; Cytomegalovirus retinitis/diagnosis; Aqueous humor; Polymerase chain reaction

Cellular immunotherapy for cytomegalovirus infection after hematopoietic stem cell transplantation

The incidence of cytomegalovirus (CMV) infection was significantly increased due to the impaired immune function of the patients after hematopoietic stem cell transplantation (HSCT). Anti CMV drugs mainly include ganciclovir and foscarnet, which will decline the immune function of the patients and easily lead to a further virus resistance, and the late-onset CMV disease may actually increase. Therefore, cellular immunotherapy has become a new prevention and therapy of CMV, and the phase Ⅱ and phase Ⅲ clinical trials are being carried out in many countries. It is confirmed that cellular immunotherapy is effective on CMV infection and it is rarely caused the risk of graft versus host disease(GVHD). This article reviews literatures on the cellular immunotherapy of CMV infection. Key words: Cytomegalovirus; Immunotherapy; Hematopoietic stem cell transplantation

A salmon DNA scaffold promotes osteogenesis through activation of sodium-dependent phosphate cotransporters

We previously reported the promotion of bone regeneration in calvarial defects of both normal and ovariectomy-induced osteoporotic rats, with the use of biodegradable DNA/protamine scaffold. However, the method by which this DNA-containing scaffold promotes bone formation is still not understood. We hypothesize that the salmon DNA, from which this scaffold is derived, has an osteoinductive effect on pre-osteoblasts and osteoblasts. We examined the effects of salmon DNA on osteoblastic differentiation and calcification in MC3T3-E1 cells, mouse osteoblasts, in vitro and bone regeneration in a calvarial defect model of aged mouse in vivo. The salmon DNA fragments (300 bps) upregulated the expression of the osteogenic markers, such as alkaline phosphatase, Runx2, and osterix (Osx) in MC3T3E1 cells compared with incubation with osteogenic induction medium alone. Measurement of phosphate ion concentrations in cultures showed that the DNA scaffold degraded phosphate ions were released to the cell cultures. Interestingly, we found that the inclusion of DNA in osteoblastic cell cultures upregulated the expression of sodium-dependent phosphate (NaPi) cotransporters, SLC20A1 and SLC34A2, in MC3T3-E1 cells in a time dependent manner. Furthermore, the inclusion of DNA in cell cultures increased the transcellular permeability of phosphate. Conversely, the incubation of phosphonoformic acid, an inhibitor of NaPi cotransporters, attenuated the DNA-induced expression and activation of SLC20A1 and SLC34A2 in MC3T3-E1 cells, resulting in suppression of the osteogenic markers. The implantation of a salmon DNA scaffold disk promoted bone regeneration using calvarial defect models in 30-week-old mice. Our results indicate that the phosphate released from salmon DNA upregulated the expression and activation of NaPi cotransporters, resulting in the promotion of bone regeneration.

Resistance testing of clinical herpes simplex virus type 2 isolates collected over 4 decades

There is only little information about the role of mutations of the thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type 2 (HSV-2) for the development of antiviral resistance. In this study, the polymorphism of TK and DNA pol genes was examined in 82 clinical isolates collected routinely between 1973 and 2013. If novel, presently unclear or resistance-related mutations were found, the resistance phenotype against acyclovir (ACV) and foscarnet (FOS) was analyzed. The four novel amino acid changes G150D, A157T, R248W, L342W and the hitherto phenotypically unclear substitution T131M within the TK gene were identified as natural polymorphisms. Within the DNA pol gene, 17 novel substitutions and the to-date unclear change R628C were characterized as part of natural gene polymorphism. Two novel DNA pol mutations were linked to resistance (M910T) and weak susceptibility to ACV (684 insertion ED), respectively. In one isolate, the genomic cause of ACV resistance could not be identified. Phylogenetic analysis including sequences of this study and of the GenBank revealed a hierarchy of mutation clusters in TK displaying G39E as first common mutation step, followed by N78D and L140F. In conclusion, the present findings allow a deeper insight in the variability of HSV-2 TK and DNA pol genes. The most common substitution G39E can be excluded as unique cause of HSV-2 resistance. This study supports once more the importance of phenotypic adjustment of genotypic results to enhance the clinical utility of genotypic findings.

Artesunate demonstrates in vitro synergism with several antiviral agents against human cytomegalovirus.

Human cytomegalovirus (HCMV) infections remain a major problem in immunocompromised patients. Three antiviral agents, ganciclovir (GCV), foscarnet (FOS) and cidofovir (CDV), are currently approved for the treatment of HCMV infections. They all target the viral DNA polymerase and are associated with significant side effects. Combinations of novel antiviral compounds acting on different targets such as artesunate (ART) with currently approved drugs or eventually letermovir or maribavir (MBV) may result in synergistic effects. Here, we evaluated the in vitro activity of a series of two-drug combinations against a wild-type recombinant HCMV strain by the Gaussia luciferase (GLuc) reporter assay. The in vitro activity of each drug was first tested individually against HCMV by using the GLuc reporter assay. The activity of two-drug combinations consisting of ART and currently approved drugs, as well as letermovir or MBV, was then analysed by the Chou-Talalay method. The concentrations of GCV, FOS, CDV and ART that reduced the GLuc activity by 50% (EC50 values) were 3.92 ±1.64 µM, 62.45 ±8.39 µM, 0.68 ±0.19 µM and 3.86 ±1.25 µM, respectively, whereas those of MBV and letermovir were 64 ±22 nM and 2.50 ±0.83 nM, respectively. The combination of ART with GCV, CDV or MBV was associated with synergism, whereas combination of ART with FOS or letermovir resulted in moderate synergism. As expected, the combination of MBV with GCV was antagonistic. These results suggest that the combination of ART with the antiviral agents tested in this study could be an interesting strategy for the treatment of HCMV infections to reduce toxicity and drug-resistance development.

Investigation of ganciclovir resistance in CMV UL54 and UL97 gene regions in immunocompromised patients receiving gancyclovir treatment

Cytomegalovirus (CMV) is a main cause of severe morbidity and mortality in immunocompromised patients. Ganciclovir (GCV) is used for both prophylaxis and treatment of CMV disease with successful results, however GCV resistance has been increasingly reported. The aim of this study was to investigate the GCV resistance in patients whose viral loads did not decline (≥1000 copies/mL) despite of receiving GCV treatment, by using sequence analysis method. A total of 30 patients, 25 of them were bone marrow transplant (BMT) and five who were followed in hematology clinics (non-Hodgkin lymphoma, lung cancer, diffuse large B cell lymphoma, combined immune deficiency, chronic lymphocytic leukemia) were included in the study. CMV-DNA levels were monitored by real-time polymerase chain reaction (QIAsymphony, Artus® CMV QS-RGQ kit, Qiagen, Germany), and DNA sequence analysis (ABI 310 Genetic Analyzer, Applied Biosystems, USA) was performed to detect the mutations leading to CMV antiviral drug resistance in following gene regions: 420-664 codons in UL97 gene region and 261 to 588 and 740 to 987 codons in UL54 gene region. Of 30 patients included, M460V mutation in CMV UL97 gene region was detected in one (3.3%) (1st case) and L802M mutation in UL54 gene region, in addition to P887S and S897L variant sequences in another patient (3.3%) (2nd case). The first patient was a 20-year-old male with acute myeloid leukemia who underwent BMT. The blood sample for the investigation of antiviral drug resistance was taken on the 117th day of transplantation (with simultaneous viral load 4470 copies/mL) and the patient has been using GCV for 70 days when the sample was taken. Valganciclovir (VGCV) and foscarnet (FOS) were used for the therapy of the first patient and monitored. The second patient was a 19-year-old male with acute lymphoblastic leukemia who underwent BMT. The blood sample for the investigation of antiviral drug resistance was taken on the 109th day of transplantation (with simultaneous viral load 4830 copies/mL) and the patient received GCV for 26 days and VGCV for 40 days when the sample was taken. FOS and cidofovir were used for the therapy of the second patient but the patient was lost due to the underlying diseases. In conclusion, mutations responsible for GCV resistance was detected in 6.6% (2/30) of immunocompromised patients receiving GCV, indicating that the determination of CMV antiviral drug resistance may help clinicians for planning the antiviral therapy.

Sequence Analysis of Herpes Simplex Virus 1 Thymidine Kinase and DNA Polymerase Genes from over 300 Clinical Isolates from 1973 to 2014 Finds Novel Mutations That May Be Relevant for Development of Antiviral Resistance.

A total of 302 clinical herpes simplex virus 1 (HSV-1) strains, collected over 4 decades from 1973 to 2014, were characterized retrospectively for drug resistance. All HSV-1 isolates were analyzed genotypically for nonsynonymous mutations in the thymidine kinase (TK) and DNA polymerase (Pol) genes. The resistance phenotype against acyclovir (ACV) and/or foscarnet (FOS) was examined in the case of novel, unclear, or resistance-related mutations. Twenty-six novel natural polymorphisms could be detected in the TK gene and 69 in the DNA Pol gene. Furthermore, three novel resistance-associated mutations (two in the TK gene and one in the DNA Pol gene) were analyzed, and eight known but hitherto unclear amino acid substitutions (two encoded in TK and six in the DNA Pol gene) could be clarified. Between 1973 and 2014, the distribution of amino acid changes related to the natural gene polymorphisms of TK and DNA Pol remained largely stable. Resistance to ACV was confirmed phenotypically for 16 isolates, and resistance to ACV plus FOS was confirmed for 1 isolate. Acyclovir-resistant strains were observed from the year 1995 onwards, predominantly in immunosuppressed patients, especially those with stem cell transplantation, and the number of ACV-resistant strains increased during the last 2 decades. The data confirm the strong genetic variability among HIV-1 isolates, which is more pronounced in the DNA Pol gene than in the TK gene, and will facilitate considerably the rapid genotypic diagnosis of HSV-1 resistance.

The Platelet Function Analyser (PFA)-100 and von Willebrand disease: a story well over 16 years in the making.

The Platelet Function Analyser (PFA)-100 and von Willebrand disease: a story well over 16 years in the making.

Phosphate enhances Fgf23 expression through reactive oxygen species in UMR-106 cells.

Fibroblast growth factor 23 (FGF23) has been shown to work as a phosphotropic hormone. Although FGF23 reduces the serum phosphate level, it has not been established that phosphate directly regulates FGF23 production. In this study, we investigated whether phosphate can enhance Fgf23 expression using the rat osteoblastic cell line UMR-106, which has been shown to express Fgf23 in response to 1,25-dihydroxyvitamin D [1,25(OH)2D]. Phosphate increased Fgf23 expression in a dose- and time-dependent manner in the presence of 1,25(OH)2D. Phosphate also increased Fgf23 promoter activity, but showed no effect on the half-life of Fgf23 messenger RNA. Phosphonoformic acid and PD98059, an inhibitor of MEK, inhibited the effects of phosphate on Fgf23 expression and promoter activity. In addition, phosphate enhanced production of reactive oxygen species (ROS) in UMR-106 cells, and hydrogen peroxide enhanced FGF23 production in a dose- and time-dependent manner. Hydrogen peroxide also enhanced Elk1 reporter activity, a target of the MEK-extracellular-signal-regulated kinase (ERK) pathway. Furthermore, the effect of phosphate on ROS production and Fgf23 expression was inhibited by apocynin, an inhibitor of NADPH oxidase. These results indicate that phosphate directly enhances Fgf23 transcription without affecting the stability of Fgf23 messenger RNA by stimulating NADPH-induced ROS production and the MEK-ERK pathway in UMR-106 cells.

Derivatives of Mesoxalic Acid Block Translocation of HIV-1 Reverse Transcriptase

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors.

A comparison study on the clinical effects of foscarnet sodium injection and interferon on human immunodeficiency virus-infected patients complicated with herpes zoster.

Objective:To compare the clinical effects of foscarnet sodium injection and interferon on human immunodeficiency virus (HIV)-infected patients complicated with herpes zoster.Methods:Ninety HIV-infected patients complicated with herpes zoster were divided into a treatment group and a control group that were both treated routinely first. Then the control group and treatment group were administered with interferon and foscarnet sodium injection respectively for four consecutive weeks.Results:After four weeks, the effective rates of the treatment and control groups were 95.6% and 80.0% respectively, which were significantly different (P < 0.05). The pain scores of the two groups were similar before treatment, but the scores of the treatment group were significantly lower than those of the control group two and four weeks after treatment (P < 0.05) as well as were significantly lower than those before treatment (P < 0.05). The numbers of CD4+ cells and the contents of IL-2 of both groups two and four weeks after treatment significantly exceeded those before treatment (P < 0.05), with significant inter-group differences also (P < 0.05). Two and four weeks after treatment, the treatment group scored significantly higher in physical activity, energy, sleep, social life and emotional reaction than the control group did (P < 0.05).Conclusions:HIV-infected patients are prone to being complicated with herpes zoster. Compared with interferon, foscarnet sodium injection better improves the clinical outcomes by effectively relieving pain and by regulating immune mediated inflammatory diseases, thus boosting the prognostic quality of life.