Brincidofovir treatment of acyclovir-resistant disseminated varicella zoster virus infection in an immunocompromised host.

Brincidofovir (BCV) is a broad-spectrum antiviral agent active in vitro against double-stranded DNA viruses including herpesviruses, adenoviruses, polyomaviruses, and poxviruses. We report successful BCV use in management of disseminated acyclovir- and cidofovir-resistant varicella zoster virus in an immunocompromised hematopoietic stem cell transplant patient with chronic graft-versus-host disease who was intolerant to foscarnet.

Clinical outcomes, safety, and pharmacokinetics of OPT-80 in a phase 2 trial with patients with Clostridium difficile infection.

OPT-80, a novel, minimally absorbed macrocycle, is a candidate treatment option for Clostridium difficile infection (CDI) based on cure without recurrence of CDI in the hamster challenge model, good in vitro activity against C. difficile, and relative sparing of commensal gram-negative anaerobes. In this open-label, dose-ranging clinical trial, 48 evaluable subjects were randomized to receive either 50, 100, or 200 mg of OPT-80 orally every 12 h for 10 days as treatment for mild to moderately severe CDI. OPT-80 was well tolerated by all subjects. Plasma concentrations were below the lower limit of quantitation in almost one-half of patients and typically

Posaconazole salvage therapy allows successful allogeneic hematopoietic stem cell transplantation in patients with refractory invasive mold infections.

We describe the clinical courses of 3 patients with hematologic malignancies (2 with acute myelogenous leukemia and 1 with multiple myeloma) who developed invasive fungal infections due to uncommon molds (Alternaria spp., Paecilomyces lilacinus, and Zygomycetes). Breakthrough invasive fungal infections of the sinus (n=1), lung (n=3), and pericardium (n=1) developed despite fluconazole prophylaxis and failed to respond to treatment with other licensed antifungal therapies, including amphotericin B (n=3), caspofungin (n=2), and voriconazole (n=3), and surgical intervention (n=2). Salvage therapy with posaconazole oral suspension resulted in successful outcomes in all 3 patients, who subsequently underwent allogeneic hematopoietic stem cell transplantation (HSCT) while on continued posaconazole therapy. The median duration of posaconazole treatment before HSCT was 5 months (range: 1.5-6 months). Posaconazole salvage therapy allowed successful allogeneic HSCT in 3 patients with refractory invasive mold infections.

Breakthrough zygomycosis after voriconazole administration among patients with hematologic malignancies who receive hematopoietic stem-cell transplants or intensive chemotherapy.

Zygomycosis is increasingly reported as a cause of life-threatening fungal infections. A higher proportion of cases reported over the last decades have been in cancer patients, with or without hematopoietic stem cell transplantation (HSCT). The new anti-fungal agent voriconazole is a recently identified risk factor for developing zygomycosis. We reviewed the clinical characteristics and outcomes of a large cohort of cancer patients who developed zygomycosis after exposure to voriconazole. Health care professionals at 13 large cancer centers provided clinical information on cancer patients with zygomycosis and prior exposure to voriconazole. Criteria for inclusion were 5 days or more of voriconazole use and diagnostic confirmation with tissue or histology. Fifty-eight cases were identified among patients with hematologic malignancies, 62% including patients who underwent a HSCT procedure. Fifty-six patients received voriconazole for primary or secondary prophylaxis against fungal infection. In addition to prior exposure to voriconazole, patients also had several of the previously established risk factors for zygomycosis. Amphotericin B was the most commonly prescribed anti-fungal therapy. Overall mortality was 73%. We conclude that zygomycosis after exposure to voriconazole is a recently described entity that is frequently fatal, despite treatment with currently available anti-fungal agents and surgery.

Posaconazole as salvage therapy for zygomycosis.

Zygomycosis, an infection that is associated with significant morbidity and mortality, is becoming common in immunocompromised patients. Posaconazole is a new extended-spectrum azole antifungal that has demonstrated in vitro and in vivo activity against zygomycetes. This report provides the results from the first 24 patients with active zygomycosis who were enrolled in two open-label, nonrandomized, multicentered compassionate trials that evaluated oral posaconazole as salvage therapy for invasive fungal infections. Posaconazole was usually given as an oral suspension of 200 mg four times a day or 400 mg twice a day. Eleven (46%) of the infections were rhinocerebral. Duration of posaconazole therapy ranged from 8 to 1,004 days (mean, 292 days; median, 182 days). Rates of successful treatment (complete cure and partial response) were 79% in 19 subjects with zygomycosis refractory to standard therapy and 80% in 5 subjects with intolerance to standard therapy. Overall, 19 of 24 subjects (79%) survived infection. Survival was also associated with surgical resection of affected tissue and stabilization or improvement of the subjects' underlying illnesses. Failures either had worsening of underlying illnesses or requested all therapy withdrawn; none of the failures received more than 31 days of posaconazole. Posaconazole oral solution was well tolerated and was discontinued in only one subject due to a drug rash. Posaconazole appears promising as an oral therapy for zygomycosis in patients who receive required surgery and control their underlying illness.

International, open-label, noncomparative, clinical trial of micafungin alone and in combination for treatment of newly diagnosed and refractory candidemia.

Candida spp. are the fourth leading cause of bloodstream infections, and non-albicans species are increasing in importance. Micafungin is a new echinocandin antifungal agent with excellent in vitro activity against Candida spp. Pediatric, neonatal, and adult patients with new or refractory candidemia were enrolled into this open-label, noncomparative, international study. The initial dose of micafungin was 50 mg/d (1 mg/kg for patients <40 kg) for infections due to C. albicans and 100 mg/d (2 mg/kg for patients <40 kg) for infections due to other species. Dose escalation was allowed. Maximum length of therapy was 42 days. A total of 126 patients were evaluable (received at least five doses of micafungin). Success (complete or partial response) was seen in 83.3% patients overall. Success rates for treatment of infections caused by the most common Candida spp. were as follows: C. albicans 85.1%, C. glabrata 93.8%, C. parapsilosis 86.4%, and C. tropicalis 83.3%. Serious adverse events related to micafungin were uncommon. Micafungin shows promise as a safe and effective agent for the treatment of newly diagnosed and refractory cases of candidemia. Large-scale, randomized, controlled trials are warranted.

Clinical and magnetic resonance imaging regression of progressive multifocal leukoencephalopathy in an AIDS patient after intensive antiretroviral therapy.

A 36-year-old homosexual man with 6 months of visual symptoms and headaches had right homonymous hemianopia, mild new learning impairment, and alexia with agraphia. The initial brain magnetic resonance imaging (MRI) scan was reported consistent with left occipital infarction. Subsequent MRI demonstrated abnormal demyelination in subcortical white matter and deep parieto-occipital white matter bilaterally, but primarily left. Human immunodeficiency virus testing and cerebrospinal fluid polymerase chain reaction for JC virus DNA were both positive, consistent with progressive multifocal leukoencephalopathy (PML) with AIDS. His clinical status steadily deteriorated, and MRI white matter abnormalities worsened despite high-dose antiretroviral therapy. After the antiretroviral regimen was intensified by the addition of a protease inhibitor, rapid clinical and radiographic improvement occurred with subsequent MRI studies revealing only residual left parieto-occipital encephalomalacia. PML in AIDS patients has been associated with a nearly uniformly poor prognosis until recent reports of improved outcomes after highly active antiretroviral therapy. This patient with PML and AIDS similarly showed a robust clinical and MRI response to intensive antiretroviral combination therapy, which has been maintained for more than 3 years.

Verbal working memory in HIV-seropositive drug users.

Recent evidence suggests that HIV-seropositive drug users are impaired on tasks of visuospatial working memory compared with drug users seronegative for HIV. In the current study we evaluated the performance of 30 HIV-seropositive male drug users and 30 risk-matched seronegative controls on two measures of verbal working memory, the Listening Span and the verbal Self Ordered Pointing Task. Impaired working memory performance was significantly more common among HIV-seropositive persons compared to controls, with the highest incidence of deficit among symptomatic participants. These findings indicate that working memory deficits in persons with HIV are not domain-specific and can be demonstrated reliably in drug users.

Reaction times are faster in HIV-seropositive patients on antiretroviral therapy: A preliminary report.

We evaluated subclinical mental and motor slowing in 142 HIV-seropositive patients without dementia, using computerized simple and choice reaction time tasks and self-report measures of psychological distress. Patients on antiretroviral therapy at the time of testing (n = 79) had significantly faster choice reaction times (p < 0.05), indicating faster mental processing speed, than untreated patients (n = 63). These faster RTs could not be attributed to differences in age, education, risk factors, degree of immunosuppression, substance abuse history, peripheral neuropathy, or psychological distress. Reaction time tasks should be investigated further as potential outcome measures in clinical trials, particularly for subjects with few or no overt cognitive deficits.

Signaling from polyomavirus middle T and small T defines different roles for protein phosphatase 2A.

Polyomavirus causes a broad spectrum of tumors as the result of the action of its early proteins. This work compares signaling from middle T antigen (MT), the major transforming protein, to that from small T antigen (ST). The abilities of MT mutants to promote cell cycle progression in serum-starved NIH 3T3 cells were compared. Transformation-defective mutants lacking association with SHC or with phosphatidylinositol 3-kinase (PI3-K) retained the ability to induce DNA synthesis as measured by bromodeoxyuridine incorporation. Only when both interactions were lost in the Y250F/Y315F double mutant was MT inactive. ST promoted cell cycle progression in a manner dependent on its binding of protein phosphatase 2A (PP2A). Since the Y250F/Y315F MT mutant was wild type for PP2A binding yet unable to promote cell cycle progression, while ST was capable of promoting cell cycle progression, these experiments revealed a functional difference in MT and ST signaling via PP2A. Assays testing the abilities of MT and ST to induce the c-fos promoter and to activate c-jun kinase led to the same conclusion. ST, but not Y250F/Y315F MT, was able to activate the c-fos promoter through its interaction with PP2A. In contrast, MT, but not ST, was able to activate c-jun kinase by virtue of its interaction with PP2A.

Impaired phagocyte oxidative capacity in patients with human immunodeficiency virus infection.

Bacterial infections that may be related to impaired phagocyte function often develop in patients infected with human immunodeficiency virus (HIV). We examined the oxidative capacity of circulating phagocytes in 78 HIV+ patients and 31 control subjects by measuring chemiluminescence with a whole blood assay. Phagocytes were stimulated with zymosan opsonized with human complement (hC-OPZ) or immunoglobulin (hI-OPZ) with or without exogenous primers. Patients with CD4+ < 500/microL showed reduced whole blood chemiluminescence at maximal opsonin receptor (MOR) activity after priming in response to hC-OPZ relative to control subjects, and the difference was significant for patients with CD4+ < 100/microL. Patients had lower absolute phagocyte counts; however, the chemiluminescence activity calculated per phagocyte count was significantly depressed in advanced HIV infection, indicating the impairment of phagocytic cell function and a reduction in number. Data were similar when hI-OPZ was used as a stimulus. The chemiluminescence of unprimed phagocytes at circulating opsonin receptor (COR) activity relative to maximally primed phagocytes (COR/MOR ratio) was significantly higher for HIV+ patients as compared with control subjects and indicates a defect in phagocyte priming. Alternatively, the phagocytes do not increase chemiluminescence with priming because they have already been primed or activated in vivo. In late-stage disease, decreased opsonin receptor-dependent respiratory burst activity contributes to the risk of secondary bacterial infections.

Information processing and antiretroviral therapy in HIV-1 infection.

Computerized reaction time (RT) tasks are sensitive measures of subclinical HIV-related mental slowing. We previously reported that nondemented HIV-seropositive patients on antiretroviral therapy at the time of testing had faster choice RTs compared to matched untreated seropositive participants. In the present study, we evaluated the performance of 163 nondemented HIV-seropositive participants on a reaction time version of the Stroop task as a function of antiretroviral status. Persons on antiretroviral therapy at the time of testing had significantly faster reaction times than untreated individuals, although treated asymptomatic participants showed significantly less Stroop interference than treated symptomatic participants. These effects could not be attributed to differences in demographic variables, disease status, substance abuse, or psychological distress. These data indicate that central information processing is faster for patients treated with antiretroviral compounds compared to untreated patients, and suggest that reaction time tasks may have significant potential utility in clinical trials of neuroprotective compounds.

Serine 257 phosphorylation regulates association of polyomavirus middle T antigen with 14-3-3 proteins.

Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.

Working memory deficits in HIV-seropositive drug users.

We studied the integrity of working memory operations in 38 HIV-seropositive and 20 seronegative drug users, using a modified version of the Tower of London task. This new task, the Tower of London-Working Memory version (TOL-WM), includes a delayed-response component in addition to the planning required for successful performance of the standard TOL. Symptomatic HIV-seropositive participants solved significantly fewer TOL-WM problems compared to matched seronegative controls. However, seropositive and seronegative subjects showed similar overall levels of planning efficiency, suggesting that the TOL-WM deficit may be associated primarily with failure to encode or maintain an adequate online memory representation. The results of this study confirm our previous report of a possible working memory deficit in HIV-1 infection and suggest that measures of working memory have particular utility in the evaluation of HIV-related cognitive deficits.

DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication.

The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage lambda in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.

Cardioprotection with a novel adenosine regulating agent mediated by intravascular adenosine.

Adenosine is cardioprotective in models of myocardial stunning and infarction, but the precise compartment within the heart in which adenosine elicits its cardioprotective effects has not been determined. The goals of the present study were to (i) investigate the effects of a novel adenosine regulating agent, GP531 (5-amino-1-beta-n-(5-benzylamino-5-deoxyribofuranosyl) imidazole-4-carboxamide), on post-ischemic myocardial function, and (ii) examine the contribution of endogenous adenosine in the intravascular and interstitial compartments in mediating the beneficial effects. Pigs were instrumented for measurement of myocardial segment shortening, and for sampling of coronary venous blood and myocardial interstitial fluid for determination of adenosine concentration. Myocardial dysfunction was induced by 4 x 8 min coronary occlusions, and recovery of regional function was monitored for 2 h. In control pigs, function recovered to 24 +/- 2% of baseline after 2 h. Treatment with GP531 improved functional recovery to 55 +/- 3%. GP531-mediated cardioprotection was prevented by adenosine receptor blockade with 8-sulfophenyltheophylline (23 +/- 2%). GP531 did not affect basal adenosine levels, but caused a 2-fold greater increase in vascular adenosine concentration with ischemia (54.6 +/- 10.6 vs. 28.1 +/- 8.0 microM in controls. P < 0.05). In contrast, the interstitial adenosine concentration was not significantly different in treated vs. untreated control pigs (9.4 +/- 3.9 vs. 15.0 +/- 1.8 microM in controls). These data indicate that (1) GP531 improves recovery of myocardial function following ischemia reperfusion injury via an adenosine receptor-dependent mechanism, and (2) the cardioprotection is associated with increased intravascular, but not interstitial, adenosine concentration during ischemia. Therefore, we conclude that cardioprotection elicited by GP531-enhanced endogenous adenosine is dependent on an intravascular site of action.

Dual activation of adenosine A1 and A3 receptors mediates preconditioning of isolated cardiac myocytes.

Ischemic preconditioning reduces post-ischemic myocardial injury by activating myocellular adenosine A1 receptors. Adenosine A3 receptors have also been implicated but there is no evidence for A3 receptors in cardiac myocytes. The aim of this study was to develop a model of preconditioning in isolated cardiac myocytes to evaluate the role of the adenosine A1 and A3 receptors in preconditioning-induced protection from ischemic injury. Reverse transcription polymerase chain reaction (PCR) was also employed to establish the presence of adenosine A3 receptors in these cells. In the preconditioning studies, ischemic injury was simulated by exposing isolated rabbit myocytes (placed in the cell chamber and paced at l Hz) to buffer containing (in mM) 2'-deoxyglucose (20), NaCN (1), Na (+)-lactate (20), KCl (10) at pH 6.6 (37 degrees C). Changes of diastolic and systolic cell length were monitored with an optical-video edge imaging system, and hypercontracture was assessed as an index of irreversible cell injury. Preconditioning (2 min brief ischemia and 15 min reperfusion) significantly reduced cell injury resulting from a subsequent prolonged ischemia (10 min) and reperfusion (15 min), as indicated by a reduction in the incidence of cell hypercontracture from 67 +/- 6% to 29 +/- 5% (P < 0.001). Preconditioning-induced cardioprotection was only partially blocked by a maximally effective concentration (100 nM) of the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (cell hypercontracture = 43 +/- 3%, P < 0.05 vs. control) but completely blocked by either the combination of DPCPX (100 nM) with the adenosine A1/A3 receptor antagonist DPCPX +8-(4-carboxyethylphenyl)-1,3-dipropylxanthine (BWA1433; 1 microM) or the non-selective adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (8-SPT; 100 microM) (cell hypercontracture = 64 +/- 4%, 59 +/- 5%, respectively; P = NS vs. control). In non-hypercontractured myocytes, preconditioning also substantially enhanced the recovery of the contractile amplitude and, similarly, this effect was only partially blocked by DPCPX but completely blocked by either the combination of DPCPX with BWA1433, or 8-SPT. These studies suggest that preconditioning protects isolated cardiac myocytes from ischemic injury independent of other cell types, and that maximal preconditioning-induced cardioprotection requires activation of both adenosine A1 and A3 receptors. Reverse transcription-PCR using primers for the rabbit receptor provide evidence for the presence of adenosine A3 receptors in these cells.

Accelerated neutrophil apoptosis in the acquired immunodeficiency syndrome.

Neutrophil (PMNL) function defects occur as a consequence of HIV infection. This study examined PMNL apoptosis in patients with the acquired immunodeficiency syndrome (AIDS) to determine if accelerated apoptosis contributes to impaired function. PMNL were isolated from 10 HIV-infected patients with CD4+ lymphocyte counts < 200/mm3 without signs of active infection and 7 healthy volunteers. PMNL were stained with acridine orange and ethidium bromide after 0, 3, 6, and 18 h in culture, and examined for the morphologic changes of apoptosis and viability by fluorescent microscopy. Apoptosis was also demonstrated by electron microscopy, flow cytometry, and DNA gel electrophoresis. Apoptosis was minimal at 0 h, but PMNL from AIDS patients exhibited significantly greater apoptosis than controls at 3 h (22.5+/-11.5 vs. 8.9+/-6.9%, P = 0.015), 6 h (38.1+/-14.2 vs. 18.1+/-4.5%, P = 0.003), and 18 h (71.3+/-19.0 vs. 38.8+/-16.7%, P = 0.002). Viabilities were > or = 88.0% for both groups from 0-6 h, but by 18 h viability was significantly decreased for the HIV group (58.8+/-12.4 vs. 83.5+/-10.4%, P = 0.001) due to an increase in non-viable apoptotic cells. Incubation with serum from AIDS patients had no effect on control PMNL, and incubation with control serum did not reduce the rate of apoptosis of PMNL from AIDS patients. Incubation with granulocyte colony-stimulating factor (G-CSF) in vitro significantly decreased apoptosis for PMNL from AIDS patients. PMNL from patients with AIDS exhibit markedly accelerated apoptosis ex vivo. In vivo, apoptosis and functional impairment of PMNL may contribute to the risk of secondary infections, and cytokine therapy may be of potential clinical benefit in this circumstance.