Momoyo Azuma

Role of Platelet-Derived Growth Factor/Platelet-Derived Growth Factor Receptor Axis in the Trafficking of Circulating Fibrocytes in Pulmonary Fibrosis

Circulating fibrocytes have been reported to migrate into the injured lungs, and contribute to fibrogenesis via CXCL12-CXCR4 axis. In contrast, we report that imatinib mesylate prevented bleomycin (BLM)-induced pulmonary fibrosis in mice by inhibiting platelet-derived growth factor receptor (PDGFR), even when it was administered only in the early phase. The goal of this study was to test the hypothesis that platelet-derived growth factor (PDGF) might directly contribute to the migration of fibrocytes to the injured lungs. PDGFR expression in fibrocytes was examined by flow cytometry and RT-PCR. The migration of fibrocytes was evaluated by using a chemotaxis assay for human fibrocytes isolated from peripheral blood. The numbers of fibrocytes triple-stained for CD45, collagen-1, and CXCR4 were also examined in lung digests of BLM-treated mice. PDGFR mRNA levels in fibrocytes isolated from patients with idiopathic pulmonary fibrosis were investigated by real-time PCR. Fibrocytes expressed both PDGFR-α and -β, and migrated in response to PDGFs. PDGFR inhibitors (imatinib, PDGFR-blocking antibodies) suppressed fibrocyte migration in vitro, and reduced the number of fibrocytes in the lungs of BLM-treated mice. PDGF-BB was a stronger chemoattractant than the other PDGFs in vitro, and anti-PDGFR-β-blocking antibody decreased the numbers of fibrocytes in the lungs compared with anti-PDGFR-α antibody in vivo. Marked expression of PDGFR-β was observed in fibrocytes from patients with idiopathic pulmonary fibrosis compared with healthy subjects. These results suggest that PDGF directly functions as a strong chemoattractant for fibrocytes. In particular, the PDGF-BB-PDGFR-β biological axis might play a critical role in fibrocyte migration into the fibrotic lungs.

Expression of Soluble Vascular Endothelial Growth Factor Receptor-1 in Human Monocyte-Derived Mature Dendritic Cells Contributes to Their Antiangiogenic Property

The soluble form of vascular endothelial growth factor receptor-1 (sVEGFR-1) is produced from endothelial cells by alternative splicing of VEGFR-1 mRNA, and can inhibit angiogenesis by blocking the biological effects of VEGF. In this study, we show the expression of a large amount of sVEGFR-1 in human monocyte-derived mature dendritic cells (mDCs). As compared with monocytes and immature DCs, mDCs generated by TNF-α or soluble CD40L with IFN-γ, but not LPS or other stimuli, preferentially produce sVEGFR-1. We also detected the mRNA of sVEGFR-1 generated by alternative splicing of VEGFR-1 mRNA in mDCs induced by TNF-α. The production of sVEGFR-1 showed a distinct contrast to those of VEGF in each DC matured with various stimuli. The supernatant of DCs matured with TNF-α or soluble CD40L with IFN-γ showed inhibition of the tube formation of HUVECs, which was neutralized by anti-VEGFR-1 Ab, indicating that sVEGFR-1 secreted from mDCs was biologically active. Interestingly, the supernatant of mDCs generated with LPS increased HUVEC capillary-like formation in vitro. The ratio of sVEGFR-1 to VEGF clearly reflected the net angiogenic property of mDCs. Administration of mDCs induced by TNF-α into the s.c. tumor of PC-14 cells implanted in SCID mice demonstrated the inhibition of tumor growth via reduction of the number of CD31-positive vessels, indicating their in vivo antiangiogenic potential. These results suggest that sVEGFR-1 produced by mDCs contribute to their antiangiogenic property, and the ratio of sVEGFR-1 to VEGF might be a useful tool for evaluating their ability to regulate angiogenesis mediated by VEGF.

Antifibrotic Effects of Focal Adhesion Kinase Inhibitor in Bleomycin-Induced Pulmonary Fibrosis in Mice

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in various biological functions, including cell survival, proliferation, migration, and adhesion. FAK is an essential factor for transforming growth factor β to induce myofibroblast differentiation. In the present study, we investigated whether the targeted inhibition of FAK by using a specific inhibitor, TAE226, has the potential to regulate pulmonary fibrosis. TAE226 showed inhibitory activity of autophosphorylation of FAK at tyrosine 397 in lung fibroblasts. The addition of TAE226 inhibited the proliferation of lung fibroblasts in response to various growth factors, including platelet-derived growth factor and insulin-like growth factor I, in vitro. TAE226 strongly suppressed the production of type I collagen by lung fibroblasts. Furthermore, treatment of fibroblasts with TAE226 reduced the expression of α-smooth muscle actin induced by transforming growth factor β, indicating the inhibition of differentiation of fibroblasts to myofibroblasts. Administration of TAE226 ameliorated the pulmonary fibrosis induced by bleomycin in mice even when used late in the treatment. The number of proliferating mesenchymal cells was reduced in the lungs of TAE226-treated mice. These data suggest that FAK signal plays a significant role in the progression of pulmonary fibrosis and that it can become a promising target for therapeutic approaches to pulmonary fibrosis.

Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice

Sarcoidosis is a granulomatous disease of unknown aetiology. We identified immunological targets for the treatment of pulmonary granulomatosis using a murine model generated with Propionibacterium acnes. Sensitisation and challenge using heat-killed P. acnes and dendritic cells (DCs) were performed to produce pulmonary granulomatosis in C57BL/6 mice. Immunological analyses using ELISA as well as cDNA microarray analysis were used to search for cytokines or chemokines associated with the formation of granulomas in the lungs. Co-administration of P. acnes and DCs reproducibly induced the formation of pulmonary granulomas, which resembled sarcoid granulomas. The cDNA microarray assay demonstrated that the gene expression of CXCL9 and CXCL10, ligands for CXCR3, and of CCL4, a ligand for CCR5, was strongly upregulated during granulomatosis. ELISA confirmed that levels of CXCL9 and CXCL10 as well as T-helper (Th)1 cytokines and chemokines including tumour necrosis factor-&agr; and interferon-&ggr; were elevated in bronchoalveolar lavage fluid (BALF). The blockade of Th1 chemokine receptors using TAK-779, a dual blocker for CXCR3 and CCR5, led to reduced numbers of CXCR3+CD4+ and CCR5+CD4+ T-cells in BALF. Furthermore, administration of TAK-779 ameliorated the granulomatosis. The targeted inhibition of Th1 chemokines might be useful for inhibiting Th1-biased granulomatous diseases, including sarcoidosis.

Murine typhus from Vietnam, imported into Japan.

To the Editor: In Vietnam, many febrile diseases such as malaria, dengue fever, Japanese encephalitis, scrub typhus, and more recently, severe acute respiratory syndrome (SARS) and avian influenza have been reported. Murine typhus cases were also reported during and before the 1960s but not thereafter (1–5). On May 3, 2003, a 54-year-old male resident of Tokushima, Japan, had onset of fever in the suburban town of Cu Chi, ≈60 km northwest of Ho Chi Minh City, Vietnam. Exanthema appeared on his trunk and limbs on May 7. He returned to Japan on May 9 and was admitted to Tokushima University Hospital on May 10. His body temperature was 39.0°C, and serum, C-reactive protein level was high (17.06 mg/dL) on admission (day 8 of illness). Unfortunately, the blood sample taken on that day was discarded. We then collected blood on days 10, 11, 12, 14, 17, and 24 of illness for diagnosis. Minocycline was administered on day 8 and resulted in a gradual decrease in fever and rash. Weil-Felix tests on day 12 showed the serum to be positive for Proteus vulgaris OX19 (titer 160); tests for P. vulgaris OX2 and OXK were negative (titer of 10 for both). We examined blood samples for possible diseases such as malaria, dengue fever, SARS, and rickettsioses. Giemsa-stained peripheral blood samples obtained on day 11 showed no malarial parasites. Results of immunoglobulin M (IgM)-capture ELISA of serum on days 10, 11, and 17 of illness were negative for dengue antibodies. Reverse transcription (RT)–PCR of the serum on day 11 was also negative. RT-PCRs of a pharyngeal swab and urine collected on day 11 were both negative for the SARS coronavirus. These specimens were also injected into Vero cells, and no cytopathic effects were generated. RT-PCR of these cultures was also negative for SARS coronavirus. Moreover, SARS antibodies were not found in serum samples on days 11 and 14 of illness. Serum was also tested for Orientia tsutsugamushi and Coxiella burnttii on day 12 to exclude scrub typhus and Q fever as diagnoses. Indirect immunofluorescence tests for etiologic agents of spotted fever, murine typhus, and epidemic typhus were then performed with serum samples collected on days 10, 14, and 24. We used Rickettsia typhi and R. prowazekii as typhus group (TG) rickettsial antigens and R. japonica and R. conorii as spotted fever group (SFG) rickettsiae. IgM antibody was detected for these antigens, indicating that the disease was a primary infection of rickettsiae (Table). When TG and SFG rickettsioses were compared, TG rickettsiae represented markedly higher elevated titers than SFG rickettsiae, which excluded a diagnosis of SFG rickettsiosis. PCR for the TG rickettsial genome in the convalescent-phase serum on day 10 was negative. Table IFA titers of the patient sera and the cross-absorption test* To demonstrate more detailed antigenic reactivity, Western immunoblotting was performed with serum on day 14 (6). The serum reacted similarly to the ladderlike lipopolysaccharide (LPS) of R. typhi and R. prowazekii. As expected from the group-specific nature of rickettsial LPS, no reaction was demonstrated to LPS of SFG rickettsiae, R. japonica and R. conorii, although weak reactivity, mainly to the major outer member protein of SFG rickettiae, rOmpB, and molecules of smaller sizes was shown (6,7). As described previously, rOmpB has cross-reactive antigenicity between TG and SFG rickettsiae (6). Compared with the trace reaction to rOmpB of SFG rickettsiae, an extremely high level of reaction was demonstrated to rOmpB of TG rickettsiae. These results confirmed the disease to be a TG rickettsiosis. To elucidate whether the disease was murine typhus or epidemic typhus, we conducted cross-absorption tests as described previously (8,9). Serum absorbed by R. typhi showed complete absorption, demonstrating no reaction to R. typhi or R. prowazekii (Table). However, the serum absorbed by R. prowazekii resulted in incomplete absorption, demonstrating no reactivity to R. prowazekii but some reactivity to R. typhi, which was left unabsorbed. Western immunoblotting with the serum absorbed by R. prowazekii showed reactivity only to the rOmpB of R. typhi but not to that of R. prowazekii. These results confirmed the diagnosis of murine typhus. This is the first serodiagnosis of murine typhus in Vietnam since the 1960s (1–5). Since rats inhabit the area where the patient acquired the illness, murine typhus seems to have occurred sporadically or endemically but to have been undiagnosed since the 1960s, maybe because it was thought to have been eradicated and thus widely forgotten. This case was the first imported into Japan since the 1940s, when many Japanese soldiers and residents who returned from abroad had the disease.

Clinical Evaluation of Pharmacist Interventions in Patients Treated with Anti-methicillin-Resistant Staphylococcus aureus Agents in a Hematological Ward

The therapeutic effects of anti-methicillin-resistant Staphylococcus aureus (MRSA) agents, vancomycin (VCM), teicoplanin (TEIC), and arbekacin (ABK), depend on their concentrations in blood. Therefore, therapeutic drug monitoring (TDM) is important when these antibiotics are used. In the hematological ward at Tokushima University Hospital, pharmacists have ordered the measurement of blood VCM, TEIC, and ABK concentrations to promote the use of TDM in accordance with an agreed protocol since 2013. Moreover, the infection control team includes several medical disciplines and has advised on the optimal treatment using VCM, TEIC, and ABK since 2013. This study aimed to investigate the clinical effectiveness of these pharmacist interventions. We retrospectively studied 145 cases in which patients were treated with VCM, TEIC, or ABK between January 2012 and December 2013 in the hematological ward at Tokushima University Hospital. The patients were divided into a control group (71 cases) and an intervention group (74 cases), and their clinical outcomes were compared. The rate of achievement of effective drug concentrations significantly increased in the intervention group (74%), compared to the rate in the control group (55%). Moreover, univariate and multivariate Cox proportional hazard regression revealed that pharmacist intervention and appropriate concentrations of anti-MRSA agents were independent factors associated with reduced hospitalization periods in patients with lymphoma. Our study revealed that proactive pharmacist intervention may improve the therapeutic effect of anti-MRSA agents in hematology ward patients.

Potential Usefulness of Early Potassium Supplementation for Preventing Severe Hypokalemia Induced by Liposomal Amphotericin B in Hematologic Patients: A Retrospective Study

PURPOSE Liposomal amphotericin B (L-AMB) is an essential antifungal agent for patients with hematologic diseases; however, the drug causes severe hypokalemia at a high frequency. Meanwhile, there is little evidence regarding the risk factors for L-AMB-induced severe hypokalemia, and the prevention protocol has not been established. The goal of this study was to identify the risk factors related to severe hypokalemia induced by L-AMB in hematologic patients. METHODS Seventy-eight hematologic patients with a first administration of L-AMB were enrolled in the study. Eleven patients who had serum potassium levels <3.0 mmol/L before L-AMB administration and 12 patients who received L-AMB administration within 3 days were excluded. Patients who had a serum potassium level <3.0 mmol/L during L-AMB administration were classified into a hypokalemia group (n = 26), and those who had a serum potassium level ≥3.0 mmol/L were classified into a non-hypokalemia group (n = 29). The patient characteristics were analyzed retrospectively. In addition, the usefulness of potassium supplementation was analyzed for those patients who received potassium formulations (non-hypokalemia group, n = 15; hypokalemia group, n = 24). FINDINGS Twenty-six patients had hypolalemia after L-AMB administration. Hypokalemia with serum potassium levels <3.0 mmol/L was observed ~7 days after starting L-AMB administration. The patient characteristics, L-AMB dose, and L-AMB administration period did not differ between the 2 groups. In the patients who received potassium formulations, the period between starting L-AMB administration and starting potassium supplementation was significantly shorter in the non-hypokalemia group than in the hypokalemia group (median, 0 vs 4 days, respectively; P < 0.01); the potassium dose was not different between the 2 groups. A receiver-operating characteristic curve revealed that the cutoff time for the start of potassium supplementation to reduce the incidence of L-AMB-induced hypokalemia was 3 days. Multivariate logistic regression analysis revealed that beginning potassium supplementation within 2 days from the start of L-AMB administration was an independent factor reducing the risk of L-AMB-induced hypokalemia (odds ratio, 0.094 [95% CI, 0.019-0.47]). IMPLICATIONS This study showed that starting administration of a potassium formulation within 2 days from the start of L-AMB administration was a risk reduction factor for L-AMB-induced hypokalemia. This finding indicates that early potassium supplementation should be incorporated into the regimen of hypokalemia management when L-AMB is used.