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Free heme, the protein-unbound form of heme, has both toxic and regulatory effects on cells. To detect free heme at low concentrations, we developed a heme sensor using fluorescently labeled heme oxygenase-1 (HO-1), an enzyme that catalyzes oxidative heme degradation and has a high affinity for heme. The response of the heme sensor is based on the fluorescence quenching that occurs when heme binds to the enzyme. Each of the three fluorescently labeled HO-1s exhibits a 1:1 binding stoichiometry and an absorption spectrum similar to that of the heme complex of the wild-type HO-1. Titration of the labeled proteins with hemin resulted in fluorescence quenching in a hemin concentration-dependent manner, presumably due to an energy transfer from the fluorophore to the heme bound to HO-1. The sensor showed a potent affinity for heme with a dissociation constant in the low nanomolar range and a high selectivity for heme. Based on the linear response of the sensor to heme, we performed a fluorometric microplate assay. The sensor was able to selectively detect free heme but did not respond to heme bound to native hemoglobin. This assay will be a useful tool for determination of free heme in biological samples containing protein-bound heme.

Disseminated penicilliosis-an AIDS-indicator disease in Southeast Asian countries -but not Japan- is a systemic fungal infection caused by Penicillium marneffei. A 30-year-old HIV-positive Japanese man visiting Southeast Asia three months before admission and reporting fever, general fatigue, and enlarged lymph nodes lasting over one month was admitted for detailed tests. Blood culture and fine-needle aspiration lymph node biopsy a led to a diagnosis of disseminated penicillioisis, later cured by several anti-fungal agents. Caution is thus recommended regarding the possibility of this disease, given the large number of travelers visiting overseas, geographical proximity to Southeast Asia, and increasing numbers of HIV patients in Japan.

We treated three cases of fungemia in HIV-infected patients. These cases were caused by Candida albicans, Cryptococcus neoformans, and Penicillium marneffei, respectively, and all were diagnosed through the use of mycobacterial blood culture bottles. Although the detection of the etiologic agents of fungal infection is difficult, it has been shown that blood culture media for mycobacteria are more effective for the detection of fungemia than media for aerobes and anaerobes. Some reports have shown that Bactec Myco/F lytic bottles were useful for the diagnosis of fungemia in clinical samples. Here, we report the successful use of BacT MB bottles.

Idiopathic pleuroparenchymal fibroelastosis (IPPFE) is a recently reported rare disease entity characterized by fibrotic thickening of the pleural and subpleural parenchyma predominantly in the upper lobes in idiopathic interstitial pneumonias (IIPs). Because the clinical features of this rare disease are not fully elucidated, we examined the clinical characteristics of IPPFE, especially for serum interstitial biomarkers, surfactant protein-D (SP-D), and Krebs von den Lungen-6 (KL-6). Four consecutive cases of IPPFE who fulfilled the diagnostic criteria were studied. All cases were more than 60 years of age, and were classified as underweight by body mass index. A severe restrictive ventilatory defect was found in all cases on admission. High-resolution computed tomography showed intense pleural thickening associated with fibrosis predominant in upper lobes. Histopathological findings were also confirmed in three out of four cases. Interestingly, the serum level of SP-D was markedly elevated in all cases, while KL-6 was within normal range in three out of four cases. As compared with major IIPs such as idiopathic pulmonary fibrosis and fibrotic nonspecific interstitial pneumonia, IPPFE significantly showed higher frequency of cases with a unique pattern of serum biomarkers, which is characterized by an elevated level of SP-D with a normal range of KL-6. In IPPFE, SP-D might tend to be elevated, while KL-6 was within a normal range. Further study is required to determine the pathogenesis and clinical significance of the elevated SP-D in IPPFE.

Recently, 2 monoclonal antibodies that specifically inhibit mitochondrial creatine kinase (MtCK) activity have been developed. In this study, we measured the serum MtCK activity in HIV-1-infected individuals (n = 100) by employing a novel method using these antibodies. The mean serum MtCK activity in 44 patients treated with highly active antiretroviral therapy (HAART) including tenofovir disoproxil fumarate (TDF) was 16.0 IU/L. The MtCK activity was significantly higher in patients receiving TDF than in those receiving HAART without TDF (3.4 IU/L) or in naive patients (6.9 IU/L) (Tukey–Kramer test, p < 0.0001 and p = 0.0029, respectively). The serum MtCK activity reached a plateau at 1 month after the initiation of TDF administration and decreased upon discontinuation. It showed no significant correlation with the trough plasma TDF concentration, serum creatinine level, or red blood cell count. The activity was elevated in 75% of the patients receiving TDF, and this elevation was specific to TDF; it was not observed with other anti-HIV drugs. In addition, our report emphasizes the careful interpretation of creatine kinase-MB (CK-MB) test results in patients receiving TDF because MtCK in serum could cause false-positive results on a conventional CK-MB test, which does not include MtCK-specific inhibitory antibodies.

Impaired renal function caused by tenofovir disoproxil fumarate (TDF) is considered reversible by discontinuing TDF administration, but there are occasional cases of incomplete recovery. We investigated the recovery of renal function after the discontinuation of TDF. Subjects comprised patients who had been started on TDF but in whom it was later discontinued because of impaired renal function. We investigated renal function until 96 weeks after the discontinuation of TDF, and the duration of TDF administration, up to May 2010. TDF was discontinued because of impaired renal function in 21 of 766 patients (2.7%). Following discontinuation, a significant recovery was seen in eGFR (p = 0.003). The median duration of administration was 28 days (6–941 days) in 9 patients whose eGFR recovered to pre-administration levels, 405 days (250–1,379) in 7 patients in whom mild recovery was seen, and 1,110 days (421–1,470) in 5 patients in whom eGFR was much lower than at the time of discontinuation. A significant correlation was seen between the eGFR recovery rate and the duration of TDF administration. TDF administration was discontinued because of renal impairment in 2.7% of patients. The duration of TDF administration was short in patients whose renal function recovered to pre-administration levels, but patients in whom sufficient recovery was not seen after discontinuation had received TDF over long periods and included many whose renal function gradually declined, even after discontinuation. Recovery of renal function after discontinuation of TDF is likely affected by the duration of TDF administration.

The expression levels of various cytokines increase with the progression of HIV-1 infection. However, the effects of antiretroviral therapy (ART) on serum cytokine levels have not been fully determined. In this study we measured serum cytokine levels of 35 HIV-1-infected Japanese adults. We first performed a cross-sectional study and observed that TNF-α, IL-6, IL-10, IL-18, and IL-7 levels all showed significant increases in those with advanced disease, and that this had a significant negative correlation with the CD4 cell count. However, IFN-γ levels did not show this relationship. A longitudinal study in 18 HIV-1-infected patients with a CD4 cell count350/μL revealed that the introduction of ART reduced cytokine levels. Significant reductions of IL-7, IL-10, IFN-γ, and IL-18 levels were observed on days 30, 60, 90, and 90 after the initiation of ART, respectively. These results indicate a discrepancy between cross-sectional and longitudinal studies of serum levels of IFN-γ. To clarify this, we investigated serum IFN-γ levels in each patient. In 5 of the 15 patients IFN-γ levels did not decrease, even after ART initiation, and remained at 5 pg/mL or higher on day 120 after ART initiation. Higher IFN-γ levels (5 pg/mL) were also observed in 2 of 7 asymptomatic patients, and 2 of 11 patients who underwent ART for 1 year or longer. These data demonstrate that IFN-γ levels in some patients increased and remained high even after the initiation of ART, which was a specific observation different from those of the other cytokines.

Background Viral reservoir size refers to cellular human immunodeficiency virus-1 (HIV-1) DNA levels in CD4+ T lymphocytes of peripheral blood obtained from patients with plasma HIV-1-RNA levels (viral load, VL) maintained below the detection limit by antiretroviral therapy (ART). We measured HIV-1 DNA levels in CD4+ lymphocytes in such patients to investigate their clinical significance. Methods CD4+ T lymphocytes were isolated from the peripheral blood of 61 patients with a VL maintained at less than 50 copies/ml for at least 4 months by ART and total DNA was purified. HIV-1 DNA was quantified by nested PCR to calculate the copy number per 1 million CD4+ lymphocytes (relative amount) and the copy number in 1 ml of blood (absolute amount). For statistical analysis, the Spearman rank or Wilcoxon signed-rank test was used, with a significance level of 5%. Results CD4 cell counts at the time of sampling negatively correlated with the relative amount of HIV-1 DNA (median = 33 copies/million CD4+ lymphocytes; interquartile range [IQR] = 7-123 copies/million CD4+ lymphocytes), but were not correlated with the absolute amounts (median = 17 copies/ml; IQR = 5-67 copies/ml). Both absolute and relative amounts of HIV-1 DNA were significantly lower in six patients in whom ART was initiated before positive seroconversion than in 55 patients in whom ART was initiated in the chronic phase, as shown by Western blotting. CD4 cell counts before ART introduction were also negatively correlated with both the relative and absolute amounts of HIV-1 DNA. Only the relative amounts of HIV-1 DNA negatively correlated with the duration of VL maintenance below the detection limit, while the absolute amounts were not significantly correlated with this period. Conclusions The amounts of cellular HIV-1 DNA in patients with VLs maintained below the detection limit by the introduction of ART correlated with the timing of ART initiation but not with the duration of ART. In addition, CD4+ T lymphocytes, which were newly generated by ART, diluted latently infected cells, indicating that measurements of the relative amounts of cellular HIV-1 DNA might be underestimated.