Extremely late-onset chronic graft-versus-host disease presenting with bronchiolitis obliterans and pleuroparenchymal fibroelastosis.

A 35-year-old woman experienced left back pain after a 2-h flight. She reported coughing and left back pain 1 day later when she presented to our hospital. Chest computed tomography showed pneumothorax of the left lung, bronchiectasis, thickening of the bronchial wall, nodules, and cavity lesions in both lungs. A pulmonary function test revealed obstructive ventilation disorder with normal lung diffusing capacity. She had a history of haematopoietic stem cell transplantation (HSCT) at 2 years and 3 months of age during the second disease remission of acute myeloid leukaemia. She was diagnosed with chronic graft-versus-host disease (cGVHD) presenting with bronchiolitis obliterans (BO) and pleuroparenchymal fibroelastosis (PPFE). To our knowledge, this is the first reported case of BO and PPFE diagnosed more than 30 years after HSCT.

IgG4 related lung disease mimicking multiple pleural disseminated lung cancer.

IgG4-related lung disease (IgG4-RLD) can present with various types of radiological findings such as nodule, ground-glass opacity, alveolar interstitial, and bronchovascular involvement. IgG4-RLD generally manifests as mild clinical symptoms, despite evidence from the image findings. Herein we report an asymptomatic patient with IgG4-RLD mimicking multiple pleural disseminated lung cancer.

Different abilities of escape mutant-specific cytotoxic T cells to suppress replication of escape mutant and wild-type human immunodeficiency virus type 1 in new hosts.

There is much evidence that in human immunodeficiency virus type 1 (HIV-1)-infected individuals, strong cytotoxic T lymphocyte (CTL)-mediated immune pressure results in the selection of HIV-1 mutants that have escaped from wild-type-specific CTLs. If escape mutant-specific CTLs are not elicited in new hosts sharing donor HLA molecules, the transmission of these mutants results in the accumulation of escape mutants in the population. However, whether escape mutant-specific CTLs are definitively not elicited in new hosts sharing donor HLA molecules still remains unclear. A previous study showed that a Y-to-F substitution at the second position (2F) of the Nef138-10 epitope is significantly detected in HLA-A*2402(+) hemophilic donors. Presently, we confirmed that this 2F mutant was an escape mutant by demonstrating strong and weak abilities of Nef138-10-specific CTL clones to suppress replication of the wild-type and 2F mutant viruses, respectively. We demonstrated the existence of the 2F-specific CTLs in three new hosts who had been primarily infected with the 2F mutant. The 2F-specific CTL clones suppressed the replication of both wild-type and mutant viruses. However, the abilities of these clones to suppress replication of the 2F virus were much weaker than those of wild-type-specific and the 2F-specific ones to suppress replication of the wild-type virus. These findings indicate that the 2F mutant is conserved in HIV-1-infected donors having HLA-A*2402, because the 2F-specific CTLs failed to completely suppress the 2F mutant replication and effectively prevented viral reversion in new hosts carrying HLA-A*2402.

Novel mutation of human DNA polymerase gamma associated with mitochondrial toxicity induced by anti-HIV treatment.

Mitochondrial toxicity is a major adverse effect of the nucleoside reverse-transcriptase inhibitors (NRTIs) used for treatment of human immunodeficiency virus type 1 (HIV-1) infection and can result in life-threatening lactic acidosis. The toxicity is due to inhibition of polymerase gamma (Pol gamma), which is required for replication of mitochondrial DNA (mtDNA). Genetic factors could be involved in this process, given that not all NRTI-treated patients experience the toxicity. In 1 patient with lactic acidosis, a novel homozygous Pol gamma mutation (arginine to cysteine at codon 964 [R964C]) was identified at a site close to polymerase motif B, which is highly conserved among family A polymerases. Recombinant R964C Pol gamma showed only 14% activity, compared with that of wild-type Pol gamma. Culture with stavudine significantly reduced mtDNA levels in patient-derived lymphoblastoid cell lines (LCLs) harboring R964C Pol gamma, compared with those in LCLs harboring wild-type Pol gamma. The novel Pol gamma mutation could be associated with the severe lactic acidosis induced by long-term NRTI use.

Cooperative contribution of gag substitutions to nelfinavir-dependent enhancement of precursor cleavage and replication of human immunodeficiency virus type-1.

We previously described a clinical human immunodeficiency virus type-1 (HIV-1) isolate, CL-4, which showed nelfinavir (NFV)-dependent enhancement of replication (Matsuoka-Aizawa, S., Sato, H., Hachiya, A., Tsuchiya, K., Takebe, Y., Gatanaga, H., Kimura, S., Oka, S, 2003. Isolation and molecular characterization of a nelfinavir (NFV)-resistant human immunodeficiency virus type 1 that exhibits NFV-dependent enhancement of replication. J. Virol. 77, 318-327.). To identify the responsible region(s) of HIV-1 proteins for such replication enhancement, we constructed a panel of recombinant HIV-1 clones harboring portions of the Gag and protease of CL-4 and analyzed their replication capabilities and Gag processing patterns. Our data suggested that the substitutions in the matrix and N-terminal half of capsid of CL-4 were indispensable for the NFV-dependent enhancement of replication and that NFV facilitated the cleavage between the matrix and capsid of the Gag precursor harboring these substitutions. The substitutions in C-terminal half of capsid rather decreased the cleavability of Gag precursor and NFV counteracted such negative impact. Efficient replication enhancement with NFV can be observed only in the presence of the substitutions in entire Gag and protease of CL-4.

Emergence of protease inhibitor resistance-associated mutations in plasma HIV-1 precedes that in proviruses of peripheral blood mononuclear cells by more than a year.

HIV-1 genotype assay using plasma viruses has been widely applied for detection of resistant viruses in infected individuals, whereas there are only a few reports about proviral genotype in peripheral blood mononuclear cells (PBMCs). To determine which sample, plasma or PBMC, should be used for early detection of drug-resistant viruses during antiretroviral treatment, we analyzed 275 plasma-derived and 211 PBMC-derived HIV-1 protease sequences obtained from HIV-1-infected patients during protease inhibitor (PI) therapy. In 70 of 107 pairs (65.4%) of plasma and PBMC samples taken from the same blood draws, the numbers of PI resistance-associated mutations in the plasma-derived genotype were different from those in the PBMC-derived genotype. Plasma viruses had more PI resistance-associated mutations than PBMC proviruses (P = 0.0004). Analysis of serial samples showed that plasma-derived genotype assay could detect primary mutations about 425 days earlier than PBMC-derived genotype when the plasma viral load was less than 10(4 )copies/mL. Our data suggest that genetic turnover of PBMC proviruses is slower than that of plasma viruses and that time lag between emergence of mutations in plasma-derived and PBMC-derived genotypes correlates inversely with viral load. Plasma viruses should be the material of choice for early detection of drug resistance during antiretroviral treatment.

Primary nelfinavir (NFV)-associated resistance mutations during a follow-up period of 108 weeks in protease inhibitor naïve patients treated with NFV-containing regimens in an HIV clinic cohort.

BACKGROUND: Nelfinavir (NFV) is a widely prescribed HIV-1 specific protease inhibitor (PI). However, there are only a few reports that have described the long-term effects of NFV-containing regimens, especially with regard to the emergence of drug resistance in inner-city clinics. OBJECTIVES: The aim of this study was to investigate the clinical and virologic responses to treatment with NFV-containing regimens for up to 108 weeks and determine the timing and rate of emergence of primary NFV-resistance associated mutations in daily clinical practice. STUDY DESIGN: A cohort study in an inner-city clinic. Our study included 51 consecutive patients who were PI-nai;ve and commenced therapy in February 1997 through April 1999. RESULTS AND CONCLUSIONS: The proportions of patients who continued the same therapeutic regimen and showed virologic success (viral load <400 copies/ml) up to 108 weeks were 78 and 63%, respectively, based on intent-to-treat analysis. Among patients with a viral load persistently >400 copies/ml at week 12 (n=30), 11 developed primary NFV-resistance associated mutations by 108 weeks (stratified log-rank test; P<0.05). The Cox proportional hazard model showed that prior use of reverse transcriptase inhibitors (n=22) (relative hazard (RH); 2.10, 95% CI; 0.67-6.62), prior AIDS diagnosis (n=6) (RH; 1.70, 95% CI; 0.37-7.77), CD4 < 200/microl at baseline (n=19) (RH; 2.48, 95% CI; 0.78-7.81) and viral load >30,000 copies/ml at baseline (n=21) (RH; 2.10, 95% CI; 0.67-6.62) were not independent predictors of the NFV-resistance, although some tendency was noted. In total, 77% of the patients continued NFV-containing treatment without the NFV-resistance for 108 weeks. The viral load at week 12 could be used as a predictor of treatment success in our cohort study.

"All-in-One Assay", a direct phenotypic anti-human immunodeficiency virus type 1 drug resistance assay for three-drug combination therapies that takes into consideration in vivo drug concentrations.

A novel phenotypic anti-human immunodeficiency virus type 1 (HIV-1) drug resistance assay is described. Three drugs at concentrations equivalent to those determined in in vivo pharmacokinetics, were mixed in a well, serially diluted by 10-folds, and added to incubations of clinical HIV-1 isolates and CCR-5 expressing HeLa/CD4+ cells which was previously reported as the MAGIC-5 cells (Antimicrob. Agents Chemother. 45 (2001) 495) to determine the 95% inhibitory dilution (ID(95)) of the combination regimens. The ID(95) of efavirenz (EFV)-containing regimens was ten-times lower than that of nevirapine (NVP)-containing regimens against HIV-1 isolated from antiviral therapy naive patients. However, the difference was not apparent by the conventional fold resistance measurement based on the 50% inhibitory concentration. Furthermore, the synergistic effects of drug combinations against clinical HIV-1 isolates can be evaluated by our assay. The ID(95)s of EFV- and nelfinavir (NFV)- containing regimens against HIV-1 from naive patients were less than 0.01 whereas those against resistant viruses were over 0.05, although the clinical cut-off values are to be determined in larger clinical studies. Our assay, designated "All-in-One Assay", that can examine resistance to three drugs simultaneously under consideration of in vivo drug concentrations described above might be useful in practice.

Isolation and molecular characterization of a nelfinavir (NFV)-resistant human immunodeficiency virus type 1 that exhibits NFV-dependent enhancement of replication.

During the use of a phenotypic anti-human immunodeficiency virus type 1 (HIV-1) drug resistance assay in a large set of clinical virus isolates, we found a unique variant (CL-4) that exhibited a high level of nelfinavir (NFV) resistance and rather enhanced replication under subinhibitory concentrations of NFV (0.001 to 0.1 micro M). Comparison of gag-pol sequences of the CL-4 variant and its predecessor virus isolates showed a stepwise accumulation of a total of 19 amino acid substitutions in protease (PR) and Gag p17 during 32-month NFV-containing antiretroviral therapy, while other Gag regions including the cleavage sites of the p55 precursor remained highly conserved. To understand the relationship between the genetic and phenotypic changes in CL-4, we constructed chimeric viruses using pNL4-3, replacing the PR, p24PR, or p17PR gene segment of CL-4 or its predecessor. A series of tissue culture infections with the chimeras in the absence or presence of increasing concentrations of NFV demonstrated that only the p17PR segment of CL-4 could confer the NFV-dependent replication enhancement phenotype on NL4-3. Our data suggest a novel adaptation mechanism of HIV-1 to NFV, in which coevolution of Gag and PR genes generates a variant that replicates more efficiently in the cellular environment in the presence of NFV than without the drug.