Clonality of HIV-1- and HTLV-1-Infected Cells in Naturally Coinfected Individuals.

BACKGROUND: Coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) diminishes the value of the CD4+ T-cell count in diagnosing AIDS, and increases the rate of HTLV-1-associated myelopathy. It remains elusive how HIV-1/HTLV-1 coinfection is related to such characteristics. We investigated the mutual effect of HIV-1/HTLV-1 coinfection on their integration sites (ISs) and clonal expansion. METHODS: We extracted DNA from longitudinal peripheral blood samples from 7 HIV-1/HTLV-1 coinfected, and 12 HIV-1 and 13 HTLV-1 monoinfected individuals. Proviral loads (PVL) were quantified using real-time polymerase chain reaction (PCR). Viral ISs and clonality were quantified by ligation-mediated PCR followed by high-throughput sequencing. RESULTS: PVL of both HIV-1 and HTLV-1 in coinfected individuals was significantly higher than that of the respective virus in monoinfected individuals. The degree of oligoclonality of both HIV-1- and HTLV-1-infected cells in coinfected individuals was also greater than in monoinfected subjects. ISs of HIV-1 in cases of coinfection were more frequently located in intergenic regions and transcriptionally silent regions, compared with HIV-1 monoinfected individuals. CONCLUSIONS: HIV-1/HTLV-1 coinfection makes an impact on the distribution of viral ISs and clonality of virus-infected cells and thus may alter the risks of both HTLV-1- and HIV-1-associated disease.

A Conformational Escape Reaction of HIV-1 against an Allosteric Integrase Inhibitor.

HIV-1 often acquires drug-resistant mutations in spite of the benefits of antiretroviral therapy (ART). HIV-1 integrase (IN) is essential for the concerted integration of HIV-1 DNA into the host genome. IN further contributes to HIV-1 RNA binding, which is required for HIV-1 maturation. Non-catalytic-site integrase inhibitors (NCINIs) have been developed as allosteric IN inhibitors, which perform anti-HIV-1 activity by a multimodal mode of action such as inhibition of the IN-lens epithelium-derived growth factor (LEDGF)/p75 interaction in the early stage and disruption of functional IN multimerization in the late stage of HIV-1 replication. Here, we show that IN undergoes an adaptable conformational change to escape from NCINIs. We observed that NCINI-resistant HIV-1 variants have accumulated 4 amino acid mutations by passage 26 (P26) in the IN-encoding region. We employed high-performance liquid chromatography (HPLC), thermal stability assays, and X-ray crystallographic analysis to show that some amino acid mutations affect the stability and/or dimerization interface of the IN catalytic core domains (CCDs), potentially resulting in the severely decreased multimerization of full-length IN proteins (IN undermultimerization). This undermultimerized IN via NCINI-related mutations was stabilized by HIV-1 RNA and restored to the same level as that of wild-type HIV-1 in viral particles. Recombinant HIV-1 clones with IN undermultimerization propagated similarly to wild-type HIV-1. Our study revealed that HIV-1 can eventually counteract NCINI-induced IN overmultimerization by IN undermultimerization as one of the escape mechanisms. Our findings provide information on the understanding of IN multimerization with or without HIV-1 RNA and may influence the development of anti-HIV-1 strategies.IMPORTANCE Understanding the mechanism of HIV-1 resistance to anti-HIV-1 drugs could lead to the development of novel drugs with increased efficiency, resulting in more effective ART. ART composed of more potent and long-acting anti-HIV-1 drugs can greatly improve drug adherence and also provide HIV-1 prevention such as preexposure prophylaxis. NCINIs with a multimodal mode of action exert potent anti-HIV-1 effects through IN overmultimerization during HIV-1 maturation. However, HIV-1 can acquire some mutations that cause IN undermultimerization to alleviate NCINI-induced IN overmultimerization. This undermultimerized IN was efficiently stabilized by HIV-1 RNA and restored to the same level as that of wild-type HIV-1. Our findings revealed that HIV-1 eventually acquires such a conformational escape reaction to overcome the unique NCINI actions. The investigation into drug-resistant mutations associated with HIV-1 protein multimerization may facilitate the elucidation of its molecular mechanism and functional multimerization, allowing us to develop more potent anti-HIV-1 drugs and unique treatment strategies.

A case of recovery from aphasia following dose reduction of cefepime by bayesian prediction-based therapeutic drug monitoring.

Cefepime is known to exert bactericidal activity against Pseudomonas aeruginosa. Cefepime-induced neurotoxicity, most likely caused by increased exposure, has recently become a major concern in clinical practice; therefore, appropriate dose reduction of cefepime should be applied with respect to patients with low cefepime clearance (mostly eliminated by the kidneys). Here, we report a case in which Bayesian prediction-based therapeutic drug monitoring (Bayes-TDM) was effectively used to reduce the dose of cefepime in a patient with pneumonia to prevent neurotoxic complications. A woman (age: 59 years, body weight: 32.5 kg, serum creatinine concentration: 1.02 mg/dL) developed pneumonia caused by P. aeruginosa while receiving treatment for scleroderma and systemic lupus erythematosus. She started treatment with a dosing regimen of 1.0 g of cefepime every 8 h (day X). On day X+5, aphasia developed, and the serum cefepime concentration was 71.3 mg/L at trough. This concentration was twice or thrice higher than the reported safe concentration of cefepime (22 or 35 mg/L at trough). Therefore, we reduced the dose of cefepime to 0.5 g every 12 h using Bayes-TDM from day X+7. As a result, the severity of aphasia decreased by day X+10, and this dose was successfully continued up to day X+13 without further adjustment. In conclusion, individualizing doses by Bayes-TDM may be useful in preventing adverse effects associated with cefepime treatment.

Combined use of bortezomib, cyclophosphamide, and dexamethasone induces favorable hematological and organ responses in Japanese patients with amyloid light-chain amyloidosis: a single-institution retrospective study.

Amyloid light-chain amyloidosis (ALA) is a rare disease with poor prognosis and is often associated with monoclonal gammopathy of undetermined significance, multiple myeloma, or Waldenström macroglobulinemia. Only high-dose melphalan with auto-peripheral blood stem cell transplantation (PBSCT) has shown high long-term hematological response rates, but combinations with novel agents, including bortezomib or lenalidomide, have recently shown high hematological response rates for AL amyloidosis patients. In the present study, we treated eight Japanese patients with AL amyloidosis using bortezomib, cyclophosphamide, and dexamethasone (CyBorD). Overall response rate was 100 %; four patients (50 %) had complete remissions (CR), two (25 %) had very good partial responses, and two (25 %) had partial responses. Five of six patients (83 %) had organ responses in the heart and/or kidney. A relapsed patient repeatedly achieved CR with the CyBorD treatment. One patient died of sudden cardiac arrest a month after normalization of his serum free light chain level, which may be attributable to his spending the previous 6 months undergoing PBSCT collection and high-dose melphalan with auto-PBSCT. Altogether, the CyBorD regimen achieved high levels of hematological responses relatively quickly (within 2-3 months). The CyBorD regimen, rather than high-dose melphalan treatment, could serve as a first-line therapy for Japanese patients with ALA.

Therapeutic impact of erythropoietin-encapsulated liposomes targeted to bone marrow on renal anemia.

Bone marrow is a key element in the diagnosis of disorders of erythropoiesis, including anemia, and a potential target in their treatment. However, because efficient delivery of diagnostic and therapeutic agents to bone marrow is difficult, such delivery is achieved by administering drugs in large quantities that often have adverse effects. Here, we achieved selective delivery of recombinant human erythropoietin (rHuEPO) to bone marrow, via its encapsulation in liposomes with l-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA) (liposome-EPO). The result, in a rabbit model of renal anemia, was a beneficial effect on hematopoiesis, better than with rHuEPO alone. Also, we determined that liposome-EPO delivery to bone marrow depended on specific uptake by bone marrow macrophages because of the presence of SA. These results indicate both that liposome-EPO is a new, promising erythropoietin-stimulating agent and that liposomes with SA have potential for diagnostic and therapeutic applications in diseases originating from bone marrow.

A case of human immunodeficiency virus-related heart failure resembling dilated cardiomyopathy but accompanied by high cardiac output.

A 34-year-old man presented with heart failure (HF). He suffered opportunistic infections and was shown to be human immunodeficiency virus (HIV)-positive (viral load: 156,013 copies/mL) and have low CD4 lymphocytes (3/mm3), so he was initially treated for the opportunistic infections. Initial investigations showed high elevation of brain natriuretic peptide (BNP: 969 pg/mL). Transthoracic echocardiography showed an enlarged left ventricle (LV: 70 mm), a reduced LV ejection fraction (EF: 19%), but no LV hypertrophy or significant valvular diseases. After treatments for the infections, we started standard HF medications. Cardiac catheterization, after recovery from the opportunistic infections with negative inflammatory markers, showed no significant coronary stenosis, and endomyocardial biopsy did not show findings of myocarditis, without HIV structural protein on immunohistochemistry. Despite reduced EF, the cardiac output was elevated at 7.1 l/min [cardiac index (CI): 4.3 l/min/m2] and the systemic vascular resistance index was decreased at 1358 dynes s/cm5 m2. Hematologists began anti-retroviral therapy; the viral load was gradually reduced to negative, and the CD4 count was increased to 50/mm3 at Day 182. EF was accordingly improved up to 54%, but the cardiac output decreased to a normal level at 3.9 l/min (CI: 2.4 l/min/m2), leading to normalization of plasma BNP (<5 pg/mL). This case indicates that high cardiac output might be involved in the pathogenic mechanisms of HIV-related HF. .

Switching tenofovir/emtricitabine plus lopinavir/r to raltegravir plus Darunavir/r in patients with suppressed viral load did not result in improvement of renal function but could sustain viral suppression: a randomized multicenter trial.

BACKGROUND: Whether tenofovir nephrotoxicity is reversible after its withdrawal is unknown. Furthermore, there are no data on the viral efficacy of raltegravir (RAL) plus ritonavir-boosted Darunavir (DRV/r) in patients with suppressed viral load. METHODS: This multicenter, randomized trial compared renal function and viral efficacy in patients with suppressed viral load treated with RAL+DRV/r and ritonavir-boosted lopinavir (LPV/r) plus tenofovir/emtricitabine (TVD), who had been previously on LPV/r+TVD. The primary endpoint was the proportion of patients with >10% improvement in estimated glomerular filtration rate (eGFR) at 48 weeks calculated with Cockcroft-Gault equation. RESULTS: 58 randomized and treatment-exposed patients were analyzed (28 on RAL+DRV/r and 30 on LPV/r+TVD). Greater than 10% improvement in eGFR was noted in 6 (25%) out of 24 with RAL+DRV/r and 3 (11%) of 28 with LPV/r+TVD, and the difference was not statistically significant (p=0.272, 95% CI -0.067 to 0.354). Sensitivity analyses using three other equations for eGFR showed the same results. Urinary ß2 microglobulin, a sensitive marker of tenofovir tubulopathy, significantly improved with RAL+DRV/r than with LPV/r+TVD (-271 versus -64 µg/gCr, p=0.026). Per protocol analysis showed that the HIV-RNA was <50 copies/mL at week 48 in all patients of both arms (24 in RAL+DRV and 29 in LPV/r+TVD). CONCLUSIONS: Switching LPV/r+TVD to RAL+DRV/r did not significantly increase the proportion of patients who showed >10% improvement in renal function among those with relatively preserved eGFR. However, the switch improved urinary ß2 microglobulin, suggesting that discontinuation of TDF might be beneficial in the long-term. RAL+DRV/r showed favorable viral efficacy in patients with suppressed viral load. TRIAL REGISTRATION: ClinicalTrials.gov NCT01294761 http://clinicaltrials.gov/ct2/show/NCT01294761?term=SPARE&rank=2, Umin Clinical Trials Registry UMIN000005116 http://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type=summary&recptno=R000006083&language=J).

A reduced linezolid dosage maintains favorable efficacy with minimal hematologic toxicity in a methicillin-resistant Staphylococcus aureus-infected patient with renal insufficiency.

The optimal dosage of linezolid to avoid hematologic toxicity is unknown. We report the case of an 87-y-old woman with renal insufficiency who developed a surgical site infection with refractory methicillin-resistant Staphylococcus aureus. The standard dosage of linezolid (1200 mg daily) was not initially tolerated by the patient due to severe thrombocytopenia, but she was successfully treated when the dose was reduced by half (600 mg daily) based on a population pharmacokinetic-pharmacodynamic model. Appropriate dose adjustments can be made to optimize linezolid therapy especially in cases with preexisting renal dysfunction.

Whole brain radiation alone produces favourable outcomes for AIDS-related primary central nervous system lymphoma in the HAART era.

Primary central nervous system lymphoma (PCNSL) related to acquired immunodeficiency syndrome (AIDS) is a lethal disorder, but the recent application of highly active antiretroviral therapy (HAART) has significantly improved prognosis. This retrospective cohort study of AIDS-related PCNSL examined the actual clinical outcomes and prognostic variables affecting overall survival (OS) in the HAART era. Twenty-three newly diagnosed AIDS-related PCNSL at 12 regional centre hospitals for HIV/AIDS in Japan between 2002 and 2008 were consecutively enrolled. The estimated 3-yr OS rate of the entire cohort was 64% (95%CI, 41.0-80.3%). Whole brain radiation therapy (WBRT) had an independent positive impact on survival (WBRT >or=30 Gy vs. others, P = 0.02). Nine of 10 patients with a good performance status (PS) (0-2) remained alive with complete response, whereas 10 (77%) of 13 of those with a poor PS (3-4) died mostly after a short period. The estimated 3-yr OS rate of the groups with a good and poor PS was 100% and 38% (95%CI, 14-63%), respectively (P = 0.01). Leukoencephalopathy (grade >or= 2) developed in 21% of those that survived more than 12 months after radiation. The patients receiving a curative intent radiation dose (>or=30 Gy) of WBRT achieved prolonged survival while maintaining a good quality of life in the HAART era, especially among patients with a favourable PS.

Non-cleavage site gag mutations in amprenavir-resistant human immunodeficiency virus type 1 (HIV-1) predispose HIV-1 to rapid acquisition of amprenavir resistance but delay development of resistance to other protease inhibitors.

In an attempt to determine whether mutations in Gag in human immunodeficiency virus type 1 (HIV-1) variants selected with a protease inhibitor (PI) affect the development of resistance to the same or a different PI(s), we generated multiple infectious HIV-1 clones carrying mutated Gag and/or mutated protease proteins that were identified in amprenavir (APV)-selected HIV-1 variants and examined their virological characteristics. In an HIV-1 preparation selected with APV (33 passages, yielding HIV(APVp33)), we identified six mutations in protease and six apparently critical mutations at cleavage and non-cleavage sites in Gag. An infectious recombinant clone carrying the six protease mutations but no Gag mutations failed to replicate, indicating that the Gag mutations were required for the replication of HIV(APVp33). An infectious recombinant clone that carried wild-type protease and a set of five Gag mutations (rHIV(WTpro)(12/75/219/390/409gag)) replicated comparably to wild-type HIV-1; however, when exposed to APV, rHIV(WTpro)(12/75/219/390/409gag) rapidly acquired APV resistance. In contrast, the five Gag mutations significantly delayed the acquisition of HIV-1 resistance to ritonavir and nelfinavir (NFV). Recombinant HIV-1 clones containing NFV resistance-associated mutations, such as D30N and N88S, had increased susceptibilities to APV, suggesting that antiretroviral regimens including both APV and NFV may bring about favorable antiviral efficacy. The present data suggest that the preexistence of certain Gag mutations related to PI resistance can accelerate the emergence of resistance to the PI and delay the acquisition of HIV resistance to other PIs, and these findings should have clinical relevance in the therapy of HIV-1 infection with PI-including regimens.

[Aspergillosis of masticator space including mandibular bone responding to itraconazole treatment in a diabetic adult: case report].

Aspergillosis of the bone is rare and resistant to treatment. We report a case of Aspergillus infection of the masticator space including mandibular bone in a diabetic adult. After extraction of a posterior tooth, the patient began to suffer from facial pain. The pain worsened in spite of antibiotic treatment. The results of serum tests and biopsy showed an invasive aspergillosis of the left masticator space including the mandibular bone six months after the onset. Although invasive aspergillosis can be fatal, the infection in our case responded to itraconazole treatment. Even in diabetes mellitus, invasive aspergillosis may occur after surgical interventions such as tooth extraction.

Structural and molecular interactions of CCR5 inhibitors with CCR5.

We have characterized the structural and molecular interactions of CC-chemokine receptor 5 (CCR5) with three CCR5 inhibitors active against R5 human immunodeficiency virus type 1 (HIV-1) including the potent in vitro and in vivo CCR5 inhibitor aplaviroc (AVC). The data obtained with saturation binding assays and structural analyses delineated the key interactions responsible for the binding of CCR5 inhibitors with CCR5 and illustrated that their binding site is located in a predominantly lipophilic pocket in the interface of extracellular loops and within the upper transmembrane (TM) domain of CCR5. Mutations in the CCR5 binding sites of AVC decreased gp120 binding to CCR5 and the susceptibility to HIV-1 infection, although mutations in TM4 and TM5 that also decreased gp120 binding and HIV-1 infectivity had less effects on the binding of CC-chemokines, suggesting that CCR5 inhibition targeting appropriate regions might render the inhibition highly HIV-1-specific while preserving the CC chemokine-CCR5 interactions. The present data delineating residue by residue interactions of CCR5 with CCR5 inhibitors should not only help design more potent and more HIV-1-specific CCR5 inhibitors, but also give new insights into the dynamics of CC-chemokine-CCR5 interactions and the mechanisms of CCR5 involvement in the process of cellular entry of HIV-1.

Potent anti-R5 human immunodeficiency virus type 1 effects of a CCR5 antagonist, AK602/ONO4128/GW873140, in a novel human peripheral blood mononuclear cell nonobese diabetic-SCID, interleukin-2 receptor gamma-chain-knocked-out AIDS mouse model.

We established human peripheral blood mononuclear cell (PBMC)-transplanted R5 human immunodeficiency virus type 1 isolate JR-FL (HIV-1(JR-FL))-infected, nonobese diabetic-SCID, interleukin 2 receptor gamma-chain-knocked-out (NOG) mice, in which massive and systemic HIV-1 infection occurred. The susceptibility of the implanted PBMC to the infectivity and cytopathic effect of R5 HIV-1 appeared to stem from hyperactivation of the PBMC, which rapidly proliferated and expressed high levels of CCR5. When a novel spirodiketopiperazine-containing CCR5 inhibitor, AK602/ONO4128/GW873140 (molecular weight, 614), was administered to the NOG mice 1 day after R5 HIV-1 inoculation, the replication and cytopathic effects of R5 HIV-1 were significantly suppressed. In saline-treated mice (n = 7), the mean human CD4(+)/CD8(+) cell ratio was 0.1 on day 16 after inoculation, while levels in mice (n = 8) administered AK602 had a mean value of 0.92, comparable to levels in uninfected mice (n = 7). The mean number of HIV-RNA copies in plasma in saline-treated mice were approximately 10(6)/ml on day 16, while levels in AK602-treated mice were 1.27 x 10(3)/ml (P = 0.001). AK602 also significantly suppressed the number of proviral DNA copies and serum p24 levels (P = 0.001). These data suggest that the present NOG mouse system should serve as a small-animal AIDS model and warrant that AK602 be further developed as a potential therapeutic for HIV-1 infection.

The current status of, and challenges in, the development of CCR5 inhibitors as therapeutics for HIV-1 infection.

The discovery of CCR5 as a HIV-1 co-receptor unfolded the cryptic and complicated process of HIV-1 cellular entry and has provided more than a few entry steps as possible modalities for effective viral intervention. The proof-of-principle has already been established for the use of entry inhibitors against HIV-1 and there is a cautious optimism that several CCR5 inhibitors might soon be added to our armamentarium for therapy of HIV-1 infection.

Spirodiketopiperazine-based CCR5 inhibitor which preserves CC-chemokine/CCR5 interactions and exerts potent activity against R5 human immunodeficiency virus type 1 in vitro.

We identified a novel spirodiketopiperazine (SDP) derivative, AK602/ONO4128/GW873140, which specifically blocked the binding of macrophage inflammatory protein 1alpha (MIP-1alpha) to CCR5 with a high affinity (K(d) of approximately 3 nM), potently blocked human immunodeficiency virus type 1 (HIV-1) gp120/CCR5 binding and exerted potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates, including multidrug-resistant HIV-1 (HIV-1(MDR)) (50% inhibitory concentration values of 0.1 to 0.6 nM) in vitro. AK602 competitively blocked the binding to CCR5 expressed on Chinese hamster ovary cells of two monoclonal antibodies, 45523, directed against multidomain epitopes of CCR5, and 45531, specific against the C-terminal half of the second extracellular loop (ECL2B) of CCR5. AK602, despite its much greater anti-HIV-1 activity than other previously published CCR5 inhibitors, including TAK-779 and SCH-C, preserved RANTES (regulated on activation normal T-cell expressed and secreted) and MIP-1beta binding to CCR5(+) cells and their functions, including CC-chemokine-induced chemotaxis and CCR5 internalization, while TAK-779 and SCH-C fully blocked the CC-chemokine/CCR5 interactions. Pharmacokinetic studies revealed favorable oral bioavailability in rodents. These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection.

Identification of amino acid residues critical for LD78beta, a variant of human macrophage inflammatory protein-1alpha, binding to CCR5 and inhibition of R5 human immunodeficiency virus type 1 replication.

In an attempt to determine which amino acid(s) of LD78beta, a variant of human macrophage inflammatory protein-1alpha, plays a critical role in the interaction with CCR5, we generated six LD78beta variants with an amino acid substituted to Ala at the NH(2) terminus of LD78beta. There was no significant difference in eliciting Ca(2+) flux and chemotaxis among the variants with the exception of LD78beta(T9A) showing a substantially reduced activity. The comparative order for human immunodeficiency virus type 1 (HIV-1) replication inhibition was: LD78beta(P8A) > LD78beta(D6A) > LD78beta(WT), LD78beta(L3A) > LD78beta(T7A), LD78beta(P2A) > LD78beta(T9A). In binding inhibition assays of LD78beta variants using 2D7 monoclonal antibody and (125)I-labeled macrophage inflammatory protein-1alpha, the comparative order was: LD78beta(P8A), LD78beta(D6A) > LD78beta(WT) > LD78beta(L3A) > LD78beta(T7A) > LD78beta(T9A), LD78betaP2A). The order for CCR5 down-regulation induction was comparable to that for binding inhibition. The present data suggest that Pro-2, Asp-6, Pro-8, and Thr-9 are critical for LD78beta binding to CCR5 and HIV-1 replication inhibition, and that LD78beta binding to CCR5, regardless of affinity, is sufficient for the initial signal transduction of LD78beta, whereas the greater anti-HIV-1 activity requires the greater magnitude of binding. The data also suggest that LD78beta variants with appropriate amino acid substitution(s) such as LD78beta(D6A) and LD78beta(P8A) may represent effective chemokine-based anti-HIV-1 therapeutics while preserving LD78beta-CCR5 interactions.