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Abstract Background In response to the increase in vaccine-derived poliovirus type 2 in Côte d’Ivoire, Mali, and many other African countries from 2017 to 2019, concentrated efforts are needed to improve the effectiveness of vaccination campaigns. Frontline polio health campaign worker engagement and job retention are critical to successful campaign implementation, as well as timely, in-full payment to these workers via an electronic system. Methods The Global Polio Eradication Initiative and its partners designed a road map to implement the World Health Organization Mobile Money digital payment system for health campaign workers across designated African Region countries and country-specific areas. The road map included: (1) strategy communication about Mobile Money to key stakeholders; (2) prioritization of Mobile Money pilot countries; (3) establishment of a digital finance team to support Mobile Money rollout for polio campaigns; (4) implementation of Mobile Money in select pilot areas; and (5) documentation by the digital finance team of Mobile Money implementation across pilot areas. At the country-specific level, and as described in the first pilot campaign in Côte d’Ivoire, implementation of Mobile Money occurred in 3 phases: precampaign, campaign, and postcampaign. Results Mobile Money was piloted in Côte d’Ivoire, Democratic Republic of the Congo, Ghana, Mali, and Republic of the Congo. Although program reach varied by country, the percentages of payments successfully made via Mobile Money in pilot countries were high: In campaign round 1, 99% of campaign workers in 2 regions in Mali, and 99% of campaign workers in 5 districts in Ghana were paid successfully. In Cote d’Ivoire, Mobile Money was piloted in all 113 districts for campaign rounds 1, 2 and 3, and in 4 districts in Abidjan for campaign round 3. In rounds 1, 2 and 3, 99.6%, 99.6%, and 99.9% of payments to polio health campaign workers, respectively, were made successfully. Conclusion Implementation of the Mobile Money pilot program, particularly across Côte d’Ivoire, demonstrates the value of an electronic payment system in addressing frontline polio health campaign worker need for timely, in-full payment. The World Health Organization-led Mobile Money pilot program can serve as a model for agencies committed to delivering greater efficiencies and improved health campaigns in resource-challenged settings.

Waning of neutralizing titres along with decline of protection efficacy after the second dose of COVID-19 vaccines was observed, including China-made inactivated vaccines. Efficacy of a heterologous boosting using one dose of a recombinant SARS-CoV-2 fusion protein vaccine (V-01) in inactivated vaccine-primed population was studied, aimed to restore the immunity. A randomized, double-blind and placebo-controlled phase III trial was conducted in healthy people aged 18 years or older in Pakistan and Malaysia. Each eligible participant received one dose of the V-01 vaccine developed by Livzon Mabpharm Inc. or placebo within the 3-6 months after the two-dose primary regimen, and was monitored for safety and efficacy. The primary endpoint was protection against confirmed symptomatic SARS-CoV-2 infection. A total of 10,218 participants were randomly assigned to receive a vaccine or placebo. Virus-neutralizing antibodies were assessed in 419 participants. A dramatic increase (11.3-fold; 128.3–1452.8) of neutralizing titres was measured in the V-01 group at 14 days after the booster. Over two months of surveillance, vaccine efficacy was 47.8% (95%CI: 22.6–64.7) according to the intention-to-treat principle. The most common adverse events were transient, mild-to-moderate pain at the injection site, fever, headache, and fatigue. Serious adverse events occurred almost equally in V-01 (0.12%) and placebo (0.16%) groups. The heterologous boosting with the V-01 vaccine was safe and efficacious, which could elicit robust humoral immunity under the epidemic of the Omicron variant.Trial registration: ClinicalTrials.gov identifier: NCT05096832.

Supracondylar fracture of the humerus is one of the most common fractures in children. The purpose of our study was to analyze the temporal variations and mechanisms of paediatric supracondylar fractures presented to our hospital during the COVID-19 pandemic lockdown period. As the schools and parks were closed and children were confined to their homes, they were thus more prone to injuries while playing inside. We claim that this would be the first study in Pakistan to provide unique information about these fractures. We conducted this descriptive study in the Accident and Emergency Department at Lady Reading Hospital in Peshawar, Pakistan from 18th March 2020 to 18th June 2020. In the enrolled children data regarding day and time of fracture occurrence, mechanism of fracture, height of furniture or play equipment from which the children fall and types of landing surfaces were all noted. Important variables were compared and a Chi-square test was applied to calculate P value (P

Objective: To compare long working length distal femoral locking plates with short working length for treating extra-articular distal femur fractures in terms of union and implant failure. Method: The randomised controlled trial was conducted at the Lady Reading Hospital, Peshawar, Pakistan, from April 28, 2018, to March 10, 2021, and comprised all adult patients of either gender with extra-articular distal femur fractures who were randomised into two groups. Group A was exposed to long working length, while group B had short working length. Patients in both the groups were followed up regularly for one year for the assessment of fracture union and implant failure. Data was analysed using SPSS 22. Results: Group Of the 61 patients, 30(49.2%) were in group A; 24(80%) males and 6(20%) females with overall mean age 37.9±9.6 years. The remaining 31(50.8%) were in group B; 26(83.8%) males and 5(16.1%) females with overall mean age 37.2±1 years. The mean working length in group A was 75±5mm and in group B it was 35±9mm. In group A 28(93.3%) fractures healed, while in group B 19(61.2%) fractures achieved union (p=0.01). Non-union was noted in 2(6.6%) patients in group A and 7(22.5%) in group B (p=0.08). Plate breakage was noted in 3(9.6%) patients and screw breakage in 2(6.4%) patients in group B and none in group A (p=0.0001). Conclusion: Long working length titanium locking plates were found to be better than short working length in achieving fracture union and avoiding implant failure. Clinical Trial Number: ACTRN12619001023145 http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=377657&isReview=true Key Words: Bone plate, Bony callus, Femoral fractures, Fracture healings, Internal fixation device, Titanium.

Objective: To determine the epidemiology of nasal polyps at an Army Hospital in Pakistan. Study Design: Cross-sectional study. Place and Duration of Study: This study was conducted at the department of ENT, Combined Military Hospital, Peshawar over 2 years period from Jan 2000 to Dec 2002. Patients and Methods: A total of 80 patients who were diagnosed of nasal polyps during this period were included for analysis after taking written informed consent. A predesigned proforma was used to record patient’s demographic details along with the history, physical examination, hematological and biochemical tests as well as routine radiograph of nose and paranasal sinuses. Results: The age of the patients ranged from 15 years to 40 years with a mean of 26.13 ± 2.5 years. Majority (n=27, 33.75%) of the patients were aged between 26-30 years. There were 49 (61.25%) male and 31 (38.75%) female patients in the study group giving a male to female ratio of 1.6:1. All these patients had bilateral disease. Asthma was seen in 12 (15%) patients, drug hypersensitivity was found in 10 (12.5%) patients while 7 (8.75%) patients had aspirin hypersensitivity. Cervical lymphadenopathy was seen in 8 (10.0%) subjects. Bilateral nasal obstruction was the most frequent presenting symptom seen in all the subjects (100%) followed by postnasal drip (92.5%), excessive sneezing (72.0%), rhino rhea (67.5%) and loss of sense of smell (63.7%). Upon clinical examination bilateral nasal polypi were confirmed in all the subjects. Nasal discharge was seen in 63 (78.75%) patients. It was mucoids in 32 (40.0%) and purulent in 31 (38.75%) patients. Inferior turbinate hypertrophy was seen in 46 (57.5%) patients. It was bilateral in 38 (47.5%) and unilateral in 8 (10.0%) subjects; 4 (5.0%) on each side. Deflected nasal septum was seen in 49 (61.25%) patients. It was on the left in majority of the patients (36.25%). Hypertellurism was seen in 18 (22.5%) patients. Conclusion: With a male predominance of 1.6:1 nasal polypi were seen in adults with a mean age of 29.2 ± 15.93 years. These were bilateral in all the patients and the most common presenting symptoms were nasal obstruction (100%), postnasal drip (92.5%), excessive sneezing (72.0%), rhinorrhea (67.5%) and loss of sense of smell (63.7%) in decreasing order of frequency. Asthma was seen in 12 (15%) patients, drug hypersensitivity was found in 10 (12.5%) patients while 7 (8.75%) patients had aspirin hypersensitivity. Upon clinical examination nasal discharge (78.75%), inferior turbinate hypertrophy (57.5%), deflected nasal septum (61.25%) and Hypertellurism (22.5%) were frequent findings.

Background: Treatment of displaced fractures of the distal end of radius has changed over the course of time. This study was conducted to determine the functional and radiological outcome of percutaneous pinning followed by plaster cast for unstable fractures of distal aspect of radius. Material & Methods: This descriptive study was conducted at DHQ Teaching Hospital D.I.Khan, from April 2009 to September 2012. A total of 32 patients with mean age of 58 years having unstable fractures of distal radius were included in the study. They were all operated within 48 hours by closed reduction and percutaneous K-wire fixation and POP cast. Mean follow-up was 28 weeks (range 22-32 weeks) during which patients were assessed both radiologically and functionally using criteria of Jakim et al. Results: On radiological assessment, mean volar tilt was corrected to 3° (normal 11°). Mean radial height was 10 mm (normal 12 mm) and mean radial inclination was 22o (normal 23°). On functional assessment, 16 (50%) patients showed excellent, 12 (38%) showed good results while 4 (12%) gave fair outcome. Poor result was not encountered in any patient. Conclusion: Percutaneous K-wire fixation and plaster cast immobilization can restore radiographic parameters to nearly normal values and provide good functional results without much complication.

Huntington's disease (HD) is an inherited neurodegenerative disease characterized by neuropsychiatric symptoms including chorea, dementia, and depression. It is inherited in an autosomal dominant fashion and exhibits anticipation leading to earlier and more severe symptoms in the affected offspring of a patient. With a much lower prevalence in Asia than in Europe and other parts of the world, its diagnosis can be missed easily in the early stages due to mildness of the symptoms and significant overlap between its symptoms and those of other diseases. We present the case of a 38-year-old male of Pashtun ethnicity presenting with mild cognitive impairment and clumsiness who was eventually diagnosed with HD, but his mild clinical features, no documented history of HD in his parents, and his relatively young age coupled with the relatively low prevalence of HD in his geographical location presented a significant challenge in our diagnosis of his condition. This case underscores the importance of keeping a high clinical suspicion for HD in patients with chorea despite a negative parental history, especially in resource-limited areas where the parents may have gone undiagnosed and highlights the need for further research on HD's prevalence in different parts of the world as well as the barriers to its diagnosis., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Alphaherpesviruses infect humans and most animals. They can cause severe morbidity and mortality. The pseudorabies virus (PRV) is a neurotropic alphaherpesvirus that can infect most mammals. The PRV persists in the host by establishing a latent infection, and stressful stimuli can induce the latent viruses to reactivate and cause recurrent diseases. The current strategies of antiviral drug therapy and vaccine immunization are ineffective in eliminating these viruses from the infected host. Moreover, overspecialized and complex models are also a major obstacle to the elucidation of the mechanisms involved in the latency and reactivation of the PRV. Here, we present a streamlined model of the latent infection and reactivation of the PRV. A latent infection established in N2a cells infected with the PRV at a low multiplicity of infection (MOI) and maintained at 42 °C. The latent PRV was reactivated when the infected cells were transferred to 37 °C for 12 to 72 h. When the above process was repeated with a UL54 -deleted PRV mutant, it was observed that the UL54 deletion did not affect viral latency. However, viral reactivation was limited and delayed. This study establishes a powerful and streamlined model to simulate PRV latency and reveals the potential role of temperature in PRV reactivation and disease. Meanwhile, the key role of the early gene UL54 in the latency and reactivation of PRV was initially elucidated.

Background: Today, healthcare is more complex than just ensuring clients receive quality care; it also involves consistently delivering excellent client experience. A non-profit community support services agency conducted an extensive diagnostic journey to determine root causes of inconsistent care delivery between regular and relief frontline staff., Local Problem: Clients and family caregivers noted lower satisfaction in care delivery when a relief staff (ie, internal staff or an external agency that is covering a shift) provided service in comparison with their regular staff. The diagnostic journey discovered that the shift exchange process-when outgoing staff transfers critical knowledge to incoming staff for continuing care-varied significantly between the 11 service locations, leading to a lack of consistent service delivery, thereby impacting client experience., Methods: A working group consisting of Supervisors of Client Services, Personal Support Workers (PSW) and management were tasked with process mapping the current state, highlighting gaps and outlining the ideal state of the shift exchange process., Interventions: Using best practices from the aviation industry, a checklist was developed that encapsulated all the critical steps needed to be undertaken for a successful, consistent shift exchange. The theory was that the utilisation of the checklist would enable consistency and improve client satisfaction with care delivery, especially when care is delivered by a staff unfamiliar with clients., Results: Prior to the checklist implementation, 74% of clients were satisfied or very satisfied with their relief staff, and post checklist implementation client satisfaction improved to 90%. Staff self-assessments also indicated that PSWs agreed that the checklist helped provide consistent care., Conclusion: The use of checklists can transform the way care is delivered in the community support sector and other service delivery agencies alike to bring greater standardisation of care between providers, thus significantly improving client experience across the healthcare sector., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: To determine the level of awareness of outpatients, their preferences regarding the appropriate time for discussions regarding do-not-resuscitate (DNR) order  in Saudi Arabia. Methods: This cross-sectional, self-administered survey was conducted at King Fahd Medical City, a tertiary care hospital in Riyadh, Saudi Arabia between December 2012 and January 2013. Demographic parameters of the participants were analyzed by frequency distribution, and the data on their responses by percentage analysis. Results: The survey participants constituted 307 randomly selected outpatients/caregivers presenting for outpatient care at primary and tertiary care centers, 70% were female. Three-fourths of the participants had heard of DNR order, of which 50% defined it accurately. Ninety percent preferred a discussion while ill, and 10% while healthy. More than 70% expressed willingness to share the decision with their spouses/family members. Almost one-third believed DNR orders were consistent with Islamic beliefs, almost as many believed they were inconsistent, and almost a third did not take either position. Almost all the participants showed a willingness to learn more about the order. Conclusion: A divided opinion exists regarding religious and ethical aspects of the issue among the participants. However, almost all the participants showed a willingness to learn more about the DNR order.

Background: As important as the vertebral ligaments are in maintaining the integrity of the spinal column and protecting the contents of the spinal canal, a single detailed review of their histology and embryology is missing in the literature., Methods: A literature search using online search engines was conducted., Results: Single comprehensive reviews of the histology and embryology of the spinal ligaments are not found in the extant medical literature., Conclusions: This review will be useful to those who study or treat patients with pathology of the spine.

Background: As important as the vertebral ligaments are in maintaining the integrity of the spinal column and protecting the contents of the spinal canal, a single detailed review of their anatomy and function is missing in the literature., Methods: A literature search using online search engines was conducted., Results: Single comprehensive reviews of the spinal ligaments are not found in the extant medical literature., Conclusions: This review will be useful to those who treat patients with pathology of the spine or who interpret imaging or investigate the anatomy of the ligaments of the vertebral column.

Radial access has been increasingly utilized for coronary intervention due to higher safety profile in comparison to femoral access site with lower bleeding rate. Radial artery occlusion is not uncommon with radial access site. This usually does not lead to any harm due to ulnar artery collaterals that are sufficient to prevent hand ischemia and is usually left alone. However, in the case of significant hand ischemia, treatment is often necessary. We are reporting an interesting case of symptomatic radial artery thrombosis leading to arm ischemia that was successfully treated percutaneously using femoral access. Using femoral access for radial artery intervention has not been reported previously. This case is followed by review of the literature., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Meditative movement, such as tai chi, yoga, and qi gong, may benefit people with cystic fibrosis (CF), as a form of gentle exercise incorporating meditation, breathing, and relaxation. Respiratory function is the most common issue in CF. In this systematic review we synthesized the evidence on the effect of meditative movement on respiratory function in patients with CF., Methods: We searched Chinese and English language databases with terms relating to tai chi/yoga/qi gong, and respiratory function/cough/dyspnea. Articles were screened and selected by 2 researchers. We included controlled studies published in English or Chinese after 1980, and extracted data using a specially designed spreadsheet. Two researchers independently evaluated study quality and reporting, using 3 standardized checklists. Meta-analysis was not possible due to heterogeneous methods., Results: We found 1,649 papers, included 43 (30 in English, 13 in Chinese), 23 of which were randomized controlled trials, and 20 were non-randomized trials. No studies were concerned with CF. Eleven studies included patients with respiratory disorders, and 27 included healthy people. Very few studies were high quality. The main problems with the randomized controlled trials was the randomization and non-random and/or poorly reported sampling. The main problems with the non-randomized studies were poor reporting of samples and non-equivalent groups. Although no clinically important changes were found, meditative movement may improve FEV1 in healthy people, compared to no treatment/exercise (the intervention groups showed effect-size changes from 0.07 to 0.83), but meditative movement did not appear to affect FEV1/FVC in subjects with COPD. Key study limitations were: poor reporting of sampling or methods; inadequate sample size; non-randomized design; inadequate description of randomization; randomization by center; no blinding; lack of reporting of important aspects of meditative movement; and short-term follow-up., Conclusions: The available evidence does not support meditative movement for patients with CF, and there is very limited evidence for respiratory function in healthy populations. The available studies had heterogeneous populations and provided inadequate sampling information, so clinically relevant conclusions cannot be drawn. Well powered, randomized studies of meditative movement are needed.

Children with acute lymphoblastic leukemia (ALL) diagnosed with resistant phenotypes, and those who relapse, have a dismal prognosis for cure. The antifolate methotrexate (MTX), a universal component of ALL therapies, is metabolized by folylpoly-gamma-glutamate synthetase (FPGS) into long-chain polyglutamates (MTX-PG(3-7)), resulting in enhanced cytotoxicity from prolonged inhibition of dihydrofolate reductase (DHFR) and thymidylate synthetase (TS). Using DNaseI assays, we identified a hypersensitive site upstream from exon-1, suggesting chromatin remodeling could alter FPGS expression. We demonstrated that histone deacetylase-1 (HDAC1) is recruited by NFY and Sp1 transcription factors to the FPGS promoter in ALL cell lines. We examined the effect of histone deacetylase inhibitors (HDACIs) sodium butyrate and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes. HDACIs increased FPGS mRNA expression by 2- to 5-fold, whereas DHFR and TS mRNA expression was decreased. Combination treatment with MTX plus SAHA significantly increased cytotoxicity and apoptosis in B- and T-ALL cell lines as compared with each drug alone (CI

Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-alpha-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways.

The antiviral compound azidothymidine (AZT), alone or in combination with other agents, induces apoptosis in early-passage, Epstein-Barr virus-positive Burkitt lymphoma (EBV+ BL) lines and has clinical activity in EBV+ BL. We report here a mechanism of AZT's antitumor activity. The nuclei of these cells contain activated nuclear factor-kappaB (NF-kappaB) subunits p50, c-Rel, RelB, and p52, but not p65. Treatment of primary EBV+ BL lines with AZT inhibited NF-kappaB within 1 to 2 hours. This was followed by up-regulation of EBV gene expression including viral thymidine kinase (vTK) and apoptosis. Subclones of EBV+ BL cells that demonstrated activated p65 were resistant to AZT. In EBV+ BLs, AZT but not ganciclovir (GCV) was highly phosphorylated to its monophosphate form (AZT-MP). Phosphorylation, as well as apoptosis, was markedly enhanced in the presence of hydroxyurea. AZT inhibits NF-kappaB and up-regulates EBV gene expression in primary EBV+ BLs. AZT with hydroxyurea may represent an inexpensive, targeted regimen for endemic BL.

In an attempt to discover potent anti-HIV agents devoid of the serious toxicity of the current HIV-reverse transcriptase inhibitors, three steroid prodrugs of AZT have been synthesized and their anti-HIV profiles determined with CEM-SS cell line. Two of the prodrugs were active against HIV, though weaker than AZT. The third agent was totally inactive against HIV. However, it demonstrated remarkably high anti-growth activities. Further experiments established that growth inhibition of the third agent was caused by induction of apoptosis rather than general necrosis.

The survival of viral mediated lymphomas depends upon constitutive nuclear factor kappa B (NF-kappaB) activity. AIDS-related human herpesvirus type 8-associated primary effusion lymphoma (PEL) responds poorly to chemotherapy and is almost invariably fatal. We have previously demonstrated that the antiviral combination of interferon alpha (IFN-alpha) and azidothymidine (AZT) induces apoptosis in PEL cell lines. We therefore used these agents as therapy for an AIDS patient with PEL. The patient had a dramatic response, with complete resolution of his malignant effusion in 5 days. In PEL cells, the death receptor ligand known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is markedly up-regulated by IFN-alpha; however, signals transduced by death receptors may also activate an antiapoptotic response mediated by NF-kappaB. In both the primary tumor cells from our patient and PEL cell lines, AZT selectively blocked nuclear entry of the NF-kappaB heterodimer p50 and p65, an effect not seen with other nonthymidine antiviral nucleosides. AZT monophosphate, the principal intracellular metabolite, inhibited phosphorylation and degradation of IkappaB by the IkappaB kinase complex. AZT- and IFN-alpha-mediated apoptosis was blocked by expression and nuclear localization of an IkappaB-resistant form of NF-kappaB (the p50 subunit linked to the transactivation domain of herpes simplex virus VP16). The proapoptotic effect of AZT and IFN-alpha in PEL occurs through the concomitant activation of TRAIL and blockade of NF-kappaB and represents a novel antiviral therapy for a virally mediated tumor.

Mutations in HIV-1 reverse transcriptase (RT) give rise to 3'-azido-3'-deoxythymidine (AZT) resistance by a mechanism that has not been previously reproduced in vitro. We show that mutant RT has increased ability to remove AZTMP from blocked primers through a nucleotide-dependent reaction, producing dinucleoside polyphosphate and extendible primer. In the presence of physiological concentrations of ATP, mutant RT extended 12% to 15% of primers past multiple AZTMP termination sites versus less than 0.5% for wild type. Although mutant RT also unblocked ddAMP-terminated primers more efficiently than wild-type RT, the removal of ddAMP was effectively inhibited by the next complementary dNTP (IC50 approximately equal to 12 microM). In contrast, the removal of AZTMP was not inhibited by dNTPs except at nonphysiological concentrations (IC50 > 200 microM).

The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) has been characterized with respect to inhibition by azidothymidylate (AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using either poly(rA)/poly(dT) or poly(rG)/poly(dC) as model substrate and either Mg2+ or Mn2+ as divalent cation activator. The inhibitory potency of AZTMP and other nucleotide analogues was found to be dependent on both the composition of the substrate and the divalent cation. The enzyme was significantly more sensitive to AZTMP inhibition with poly(rG)/poly(dC) than with poly(rA)/poly(dT) as substrate and in Mn2+ than in Mg2+ with either substrate. Kinetic studies indicated that AZTMP is a competitive inhibitor with respect to the substrate in Mn2+ whereas it behaves as an uncompetitive inhibitor in Mg2+. These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2+ or Mn2+ is the divalent cation activator. Consistent with this suggestion is the alteration in the mode of cleavage of the substrate upon substitution of Mg2+ with Mn2+. In Mg2+, hydrolysis of poly(rA)/poly(dT) appears to be solely endonucleolytic, whereas in Mn2+, hydrolysis is both endonucleolytic and exonucleolytic. With poly(rG)/poly(dC) as substrate, hydrolysis is both endonucleolytic and exonucleolytic in either Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonuclease activity of RNase H is preferentially inhibited by AZTMP. The sensitivity of RNase H to inhibition by N-ethylmaleimide was also found to be markedly influenced by the substrate composition and the divalent cation activator, being most sensitive under conditions in which endonucleolytic activity predominates.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was undertaken to elucidate the mechanism(s) of cross-resistance (4.9-fold) to mitomycin C (MMC) in a multi-drug-resistant cell line, P388/R-84. Intracellular accumulation of MMC by sensitive (P388/S) and P388/R-84 cells was comparable. Despite a 32% reduction in NADPH cytochrome P-450 reductase activity (responsible for MMC activation) in P388/R-84 cells, the rate of MMC bio-reduction by sensitive and resistant cells was similar. These results suggested that MMC resistance in P388/R-84 cell line must depend on factors other than impaired drug accumulation or bio-activation. Recent studies suggest that glutathione transferase (GST) dependent drug detoxification also contributes to cellular resistance of a variety of alkylating agents. Even though overexpression of GST has been noted in some MMC resistant tumor cells, it is not known if its level affects sensitivity to MMC. We have, therefore, determined the effect of ethacrynic acid (an inhibitor of GST activity) treatment on MMC cytotoxicity in P388/R-84 cells, which have about 2-fold higher GST activity than P388/S cells. The IC50 value for the inhibition of GST activity in vitro by ethacrynic acid (EA) was 16.5 microM (5 micrograms/ml). A depletion in intracellular GSH was also observed by treating P388/R-84 cells with EA alone or in combination with MMC. A non-toxic concentration of EA (1 microgram/ml; 3.3 microM) increased MMC cytotoxicity by 36% in P388/R-84 cells. MMC cytotoxicity was increased 2-fold by EA treatment in glutathione (GSH)-depleted P388/R-84 cells. These results suggest that GST mediated drug inactivation may represent another important mechanism of MMC resistance.

Chronic exposure of H9 cells to 25 microM zidovudine (H9-AZT cells) causes a 2- to 3-fold increase in thymidine kinase (TK) activity (Agarwal RP, Int J Purines Pyrimidine Res, in press). The present study compared thymidine (TdR) and AZT anabolism in H9 and H9-AZT cells. After a 3.5-hr incubation with 10 microM TdR or AZT, the total intracellular accumulations of AZT (48.7 microM in H9 cells and 32.8 microM in H9-AZT cells) were 46.4% of TdR accumulation. Other major differences between TdR and AZT anabolism were: (i) the majority of TdR (84-87%) was incorporated into DNA compared to less than 1% of AZT; and (ii) whereas distribution of TdR in the nucleotides was TTP greater than TMP greater than TDP, zidovudine distributed was AZT-MP much greater than AZT-TP much greater than AZT-DP. Because of the poor substrate activity of AZT-MP for thymidylate kinase (TMP-kinase), most of the AZT (95-98%) remained as AZT-MP. TMP-kinase activities with TMP as substrate were 47.6 +/- 20.3 and 91.4 +/- 28.8 pmol/mg protein/min in H9 and H9-AZT cells, respectively. 5'-Nucleotidase activities with TMP as substrate were 428.9 +/- 37.8 and 255.9 +/- 28.7 pmol/mg protein/min in H9 and H9-AZT cells, respectively. Activities of these enzymes with AZT-MP as a substrate were very low. Despite an increase in TK and TMP-kinase, and a decrease in 5'-nucleotidase activities, the total intracellular accumulations of TdR and AZT were reduced significantly (P less than 0.05) to 67.5% in H9-AZT cells. Thymidine transport (0.66 to 0.68 pmol/sec/10(6) cells) was similar in both the cell lines. The severe reductions of TdR salvage caused by chronic exposure of cells to AZT, if it occurs in AIDS patients on AZT chemotherapy, may explain some of the long-term clinical toxicities of the drug.

The antiviral activity, uptake, and metabolism of 2',3'-dideoxyguanosine was investigated in human immunodeficiency virus- (HIV) infected and noninfected human cells. 2',3'-Dideoxyguanosine had anti-HIV activity (effective dose 50%: 0.1-1.0 microM) in H-9 and MT-2 cells. The addition of excess (greater than or equal to 30 microM) guanosine, deoxyguanosine, or 8-aminoguanosine had no effect on the anti-HIV activity of 2',3'-dideoxyguanosine. In [8-3H]2',3'-dideoxyguanosine-exposed cells, the intracellular radioactivity was twofold higher than the extracellular. When guanosine, deoxyguanosine, or 8-aminoguanosine was preincubated or added simultaneously to 2',3'-dideoxyguanosine, uptake of 2',3'-dideoxyguanosine was reduced by 28 to 34%, whereas addition of p-nitrobenzylmercaptopurine riboside (20 microM) had no effect. In metabolism studies using H-9 cells, dideoxyguanosine triphosphate could not be detected despite a 24-h incubation of 2',3'-dideoxyguanosine at effective anti-HIV concentrations. The addition of excess (greater than or equal to 30 microM) guanosine, deoxyguanosine, and 8-aminoguanosine, while inhibiting the catabolism of 2',3'-dideoxyguanosine, did not enhance the anabolic conversion of 2',3'-dideoxyguanosine to dideoxyguanosine triphosphate. Our failure to detect the formation of dideoxyguanosine triphosphate and the lack of reversal of antiviral effects by natural purine nucleosides raises questions on the role of this metabolite in the anti-HIV activity of 2',3'-dideoxyguanosine.

The uptake of 2',3'-dideoxyadenosine was examined in a human immunodeficiency virus (HIV) infected and uninfected T cell line (H9 cells), a B cell line (Namalwa), and in normal peripheral blood mononuclear cells. After a 10-minute incubation at ambient temperature, the intracellular 2',3'-dideoxyadenosine-derived radioactivity was 8- to 16-fold higher than the extracellular radioactivity. In metabolically inactive cells (0 degrees C), the intracellular and extracellular 2',3'-dideoxyadenosine-derived radioactivities were nearly equal. In infected and noninfected H9 cells, a large excess of p-nitrobenzylmercaptopurine riboside or pyrimidine nucleosides weakly inhibited the uptake of 2',3'-dideoxyadenosine (7-30%), whereas deoxycoformycin was a stronger inhibitor (50-80%). Purine nucleosides minimally enhanced the uptake (10-20%). The cellular uptake was not associated with the accumulation of dideoxyadenosine triphosphate. In normal peripheral blood mononuclear cells, the uptake of 2',3'-dideoxyadenosine was inhibited by all agents except 2'-deoxyadenosine (15% enhancement). In contrast to H9 cells, the formation and accumulation of dideoxyadenosine triphosphate paralleled the uptake of dideoxyadenosine. The results of these studies suggest that the major route of transport of 2',3'-dideoxyadenosine into cells is by simple diffusion and that different metabolic patterns exist among cell lines and normal peripheral blood mononuclear cells. An understanding of these cellular differences could aid in the development of therapeutic strategies directed against HIV.

6-Thio-3-deazaguanine (TDG), a relatively new purine antimetabolite, exhibits significant antitumor activity against a variety of experimental animal tumor models including C3H mammary adenocarcinoma, Lewis lung carcinoma, adenocarcinoma 755, and leukemias L1210 and P388. However, the drug was ineffective against 3-deazaguanine-resistant L1210 (both in vitro and in vivo) and CEM cells (in vitro). The resistant cells appear to lack HGPRTase activity because the extracts from these cell lines failed to convert hypoxanthine to IMP. These data indicate that TDG needs to be activated by hypoxanthine guanine phosphoribosyltransferase prior to its growth inhibitory effects. Cytotoxicity of TDG was completely reversed by hypoxanthine and inosine. TDG inhibited the synthesis of DNA and RNA equally and effectively, whereas the inhibition of protein synthesis required a prolonged drug exposure and appears to be a consequence of the inhibition of DNA and RNA synthesis. Data from these studies suggest that TDG is an effective antitumor agent, and its spectrum of antitumor activity and mechanism of action appears to be different from that of 3-deazaguanine.

A series of nucleotide homo- and heterodimers [3'-azido-3'-deoxythymidilyl-(5',5')-2',3'-di-deoxy-5' adenylic acid (AZT-P-ddA), 3'-azido-3'-deoxythymidilyl-(5',5')-2', 3'-dideoxy;-5'-adenylic acid, 2-cyanoethyl ester [AZT-P(CyE)-ddA], 3'-azido-3'-deoxythymidilyl-(5',5')-2',3'-dideoxy-5'-inosinic acid (AZT-P-ddI), and 3'-azido-3'-deoxythymidilyl-(5',5')-3'-azido-3'-deoxy-5'-thymid ilic acid (AZT-P-AZT)] were synthesized and compared with respect to their anti-HIV and cytotoxic properties to their component monomers in vitro. MT-2 cells were infected with HIV (TM) followed by the addition of drug. The dimers and their respective monomers inhibited HIV-induced syncytia formation, reverse transcriptase production, and the expression of HIV p24 antigen. However, on an equimolar basis, greater anti-HIV potency and enhanced cytotherapeutic indices were observed with the heterodimers when compared with their monomers. Nucleotide dimers, such as AZT-P-ddA, should be actively considered for further evaluation as anti-HIV agents.

Synthesis of 2-deoxyribofuranosides of 3-deazaguanine (IX-XII) has been achieved by a base-catalyzed ring closure of appropriate 2-deoxyribofuranosides of methyl 5(4)-(cyanomethyl)imidazole-4(5)-carboxalate (IV-VII). The separation of isomers and anomers were accomplished by column chromatography and HPLC. The site of glycosidic linkage and the anomeric configurations were established on the basis of C-13 and proton magnetic resonance spectroscopy, as well as UV absorption characteristics. Preliminary results of the antitumor activity of these derivatives, in vitro and in vivo, are described.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.

1-beta-D-Arabinofuranosyl-2-amino-1,4(2H)-4-iminopyrimidine (ara-AIPy), a new deaminase-resistant analog of cytarabine, exhibited extremely potent antitumor activity against P388 leukemia [400 mg/kg on Days 1-5; increase in life span (ILS), 211%] and significant inhibition against Lewis lung carcinoma (inhibition of tumor weight, 68%) and mammary adenocarcinoma 755 (inhibition of tumor weight, 82%). Schedule-dependency studies indicate that this drug, unlike cytarabine, was effective irrespective of its treatment schedules. The drug exhibited therapeutic efficacy against established P388 tumor transplants (400 mg/kg on Days 3-7; ILS, 131%) and inhibited the tumor growth effectively even when administered as a single dose on Day 1 by both ip (2000 mg/kg; ILS, 150%) and iv (500 mg/kg; ILS, 68%) routes. Ara-AIPy was most effective when administered on Days 1, 3, 5, 7, and 9 after tumor transplantation (400 mg/kg; ILS, 210%, with 50% of animals 60-day survivors). Ara-AIPy inhibited the growth of L1210 leukemia when both the tumor transplantation and the drug treatment were administered by iv route (500 mg/kg on Days 1, 5, and 9; ILS, 181%). The routes of administration of ara-AIPy experiments showed that the drug was effective by both ip and iv routes of administration; however, better therapeutic values were obtained by ip schedules. These studies demonstrate that ara-AIPy exhibits highly significant and broad-spectrum antitumor activity against a variety of experimental animal tumor models and suggest a possible future role for this drug in the treatment of human neoplasia.

1-beta-D-Arabinofuranosyl-2-amino-1,4(2H)-imino-5-fluoropyrimidine (10), 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-imino-5-fluoropyrimidine 3'-phosphate (9), and 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-imino-5-chloropyrimidine (11) have been synthesized from 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine (5), 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine 3'-phosphate (4), and 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-chlorocytosine (6), respectively. 2,2'-Anhydro-1-beta-D-arabinofuranosylcytosine 3'-phosphate (7), 1-beta-D-arabinofuranosyl-2-amino-1,4-(2H)-iminopyrimidine (13), 1-beta-D-arabinofuranosyl-2-amino-1,3(2H)-iminopyrimidine 3'-phosphate (12), and compounds 4, 5, and 9 showed significant in vitro activity against a number of DNA viruses. Compounds 7 and 12 were also effective in vivo against type 1 herpes simplex virus. Compounds 7, 12, and 13 were extremely effective in the treatment of mice bearing leukemia L1210.