Toll-Like Receptor 4 Agonistic Antibody Promotes Innate Immunity against Severe Pneumonia Induced by Coinfection with Influenza Virus and Streptococcus pneumoniae

Coinfection with bacteria is a major cause of mortality during influenza epidemics. Recently, Toll-like receptor (TLR) agonists were shown to have immunomodulatory functions. In the present study, we investigated the effectiveness and mechanisms of the new TLR4 agonistic monoclonal antibody UT12 against secondary pneumococcal pneumonia induced by coinfection with influenza virus in a mouse model. Mice were intranasally inoculated with Streptococcus pneumoniae 2 days after influenza virus inoculation. UT12 was intraperitoneally administered 2 h before each inoculation. Survival rates were significantly increased and body weight loss was significantly decreased by UT12 administration. Additionally, the production of inflammatory mediators was significantly suppressed by the administration of UT12. In a histopathological study, pneumonia in UT12-treated mice was very mild compared to that in control mice. UT12 increased antimicrobial defense through the acceleration of macrophage recruitment into the lower respiratory tract induced by c-Jun N-terminal kinase (JNK) and nuclear factor kappaB (NF-κB) pathway-dependent monocyte chemoattractant protein 1 (MCP-1) production. Collectively, these findings indicate that UT12 promoted pulmonary innate immunity and may reduce the severity of severe pneumonia induced by coinfection with influenza virus and S. pneumoniae. This immunomodulatory effect of UT12 improves the prognosis of secondary pneumococcal pneumonia and makes UT12 an attractive candidate for treating severe infectious diseases.     

A case of spinal dural arteriovenous fistula draining to the ventral coronal venous plexus]

Here we report a case of spinal dural areteriovenous fistula (AVF) draining to the anterior spinal vein. An 80-year-old female presented with progressive weakness of lower extremities. MRI showed spinal enlargement at the Th10 to L1 with high intensity signals on T2-weighted image and multiple flow voids on the dorsal and ventral surface of the spinal cord. Angiogram of the left L2 lumbar artery demonstrated a hairpin-shaped vessel with ascending and descending limbs, mimicking radiculomedullary artery. Oblique view angiogram of the left L2 lumbar artery showed that radiculomedullary vein drained to the dilated anterior spinal vein, which then drained cranially and caudally on the anterior and posterior surface of the spinal cord. The patient underwent T9-L2 laminectomy. Several large tortuous dilated veins in the subarachnoid space were found. Examination of the inner surface of the dura revealed an arterialized vein that began at the level of L2 and coursed superiorly. The arterialized vein was coagulated and interrupted. The postoperative angiogram demonstrated the obliteration of the fistula. Postoperative MRI returned to normal with complete disappearance of T2 high signal, cord enlargement. In most spinal dural AVF, the venous drainage is predominantly upward on the posterior surface of the spinal cord. The spinal dural AVF draining to the anterior spinal vein is atypical, and cause difficulty in differentiating the anterior spinal artery from the anterior spinal vein. Oblique view angiogram may be helpful to differentiate the anterior spinal vein from anterior spinal artery.     

Current status of diagnosis and treatment of invasive fungal infections in Japan: the influence of the new Japanese guidelines

One year after the release of the Japanese “Guidelines for the Diagnosis and Treatment of Deep-Seated Mycosis” we conducted a survey of clinicians to determine the extent to which the new guidelines had penetrated clinical practice, and compared these results with those of a previous survey. The responses to the current survey regarding the diagnosis and treatment had changed from those of the previous survey to reflect the new recommendations, showing that the guidelines have had an effect on clinical practice. However, the current survey highlights a need to provide more practical information in the guidelines for use in the clinical settings. In particular, physicians expect the guidelines to proactively provide more information about the features of both current and new drugs. In addition, an effective drug against the genus Aspergillus is eagerly awaited. However, because it is difficult to differentiate among filamentous fungi, there is a need for a drug with broad-spectrum coverage against filamentous fungi. Investigation of combination therapy consequently becomes necessary. Definitive diagnoses of invasive fungal infection are too scarce at the national level. The cooperation of clinicians for organizing more definitive diagnoses would be appreciated when the guidelines are revised.     

Efficacy of Combination Antifungal Therapy with Intraperitoneally Administered Micafungin and Aerosolized Liposomal Amphotericin B against Murine Invasive Pulmonary Aspergillosis

Targeted intrapulmonary delivery of drugs may reduce systemic toxicity and improve treatment efficacy. In the current study, we evaluated the effects of a combination treatment consisting of inhalation of aerosolized liposomal amphotericin B (L-AMB) with intraperitoneal administration of micafungin (MCFG) against murine invasive pulmonary aspergillosis. The combination of aerosolized L-AMB with intraperitoneal MCFG significantly improved the survival rate, and the fungal burdens and histopathology findings after this treatment were superior to those of the control and both monotherapy groups.Invasive pulmonary aspergillosis (IPA) results in significant morbidity and mortality in severely immunocompromised patients (6). Targeted intrapulmonary delivery of antifungals has the potential to reduce systemic toxicity and improve treatment efficacy as well as prophylaxis (1, 8) and may be used as an optional route in combination with other systemic antifungals. In the current study, we evaluated the efficacy of aerosolized liposomal amphotericin B (L-AMB) both singly and in combination with intraperitoneally administered micafungin (MCFG) in a murine model of IPA.Aspergillus fumigatus MF13 was clinically obtained from a patient admitted to the Nagasaki University Hospital. The minimum effective concentration of MCFG (Astellas Pharmaceuticals Inc., Tokyo, Japan) and the MIC of AMB (Sigma, St. Louis, MO) were determined using the microdilution method in accordance with Clinical Laboratory Standards Institute document M38-A2 (2). Drug interactions were assessed using the checkerboard titration broth microdilution-based method (3), and the fractional inhibitory concentration index was determined as previously described (5).Six-week-old female ICR mice (Charles River Breeding Laboratories, Shiga, Japan) were immunosuppressed and then challenged on day 0 with 5 × 10⁶ conidia of A. fumigatus MF13 intratracheally for monitoring of survival, as previously described (7, 11). Eight-week-old female ICR mice were used to determine fungal burdens and for histopathological examination. Mice were immunosuppressed by subcutaneous injection of cortisone acetate (Sigma, Tokyo, Japan) at 250 mg/kg of body weight and intraperitoneally administered cyclophosphamide (Sigma) at 200 mg/kg on days −2 and 0 for the survival study. Only cortisone acetate (200 mg/kg) was used on days −1, 0, and 1 for fungal-burden analysis and histopathological examination. Mice were assigned into the following groups: (i) control mice, (ii) mice receiving MCFG intraperitoneally, (iii) mice receiving aerosolized L-AMB, and (iv) mice receiving a combination treatment of intraperitoneally administered MCFG and aerosolized L-AMB. Each group consisted of 11 and 10 mice for survival and fungal-burden analyses, respectively. MCFG was administered intraperitoneally once daily at 1 mg/kg/day. L-AMB was administered once daily in an 8-ml suspension (at 1.2 mg/ml) per inhalation. Antifungals were initiated 16 h after inoculation and continued for 5 and 3 days for survival and fungal-burden analyses, respectively. The L-AMB solution was aerosolized using a nebulizer (Muromachi Kikai Co., Ltd., Tokyo, Japan), and mice were exposed to aerosol treatment for 60 min as previously described (9). Control mice were treated with sterile saline. Survival was observed until 11 days following the challenge. For fungal-burden and histopathological examinations, mice were sacrificed 4 h after the treatment on day 3. Numbers of CFU per lung tissue were calculated, and removed lungs were fixed and stained with Grocott''s methenamine silver nitrate and hematoxylin-eosin as previously described (11). Survival and fungal burden data are presented from a combination of two sets of experiments. The concentration in blood and the pharmacokinetics of aerosolized L-AMB were evaluated. Uninfected mice were also exposed to several concentrations of aerosolized L-AMB for 5 days, and blood samples and lungs were collected. AMB concentration was quantified as previously described (10). Survival curves were generated using the Kaplan and Meier method, and statistical differences were evaluated by the log rank test. To assess fungal burden in lung tissue, geometric means of numbers of CFU per organ were compared by Student''s t test. Statistical significance was defined as a P of <0.05.The MIC of AMB against A. fumigatus MF-13 was 1.0 μg/ml, and the minimum effective concentration of MCFG was 0.0315 μg/ml. The fractional inhibitory concentration index of AMB and MCFG was 1.5, and drug interaction was classified as indifferent (5).Survival periods of monotherapy groups, in which mice either were treated with intraperitoneally administered MCFG or inhaled aerosolized L-AMB were significantly longer than that of the control group (MCFG alone versus the control, P = 0.006; L-AMB versus the control, P < 0.001) (Fig. ​(Fig.1).1). The combination treatment group showed significantly longer survival than the intraperitoneal-MCFG (P < 0.001), aerosolized-L-AMB (P = 0.037), and control (P < 0.001) groups. Numbers of CFU in the lungs of mice in the combination treatment group were significantly reduced compared to those in each of the intraperitoneal-MCFG (P < 0.001), aerosolized-L-AMB (P = 0.027), and control (P < 0.001) groups (Fig. ​(Fig.2).2). The lungs of aerosolized-L-AMB-administered and combination treatment mice showed obviously smaller numbers of hyphae and fewer foci of inflammation than the intraperitoneal-MCFG and control groups (Fig. ​(Fig.3).3). The mean AMB concentrations in the lung tissue following L-AMB inhalation at 1.2, 2.6, and 4.0 mg/ml were 35.5, 73.2, and 94.2 μg/g, respectively. Recorded levels in sera were 0.02, 0.06, and 0.06 μg/ml when inhaled-L-AMB suspensions were administered at 1.2, 2.6, and 4.0 mg/ml, respectively.Open in a separate windowFIG. 1.Survival curves for mice with IPA (Kaplan-Meier plot). Groups of 11 mice were treated with a combination of intraperitoneal administration of MCFG (1 mg/kg/day) and inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml [open squares]), inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml [filled triangles]), intraperitoneal administration of MCFG (1 mg/kg/day [open triangles]), and no therapy (control [filled circles]). *, P < 0.05 versus the control; **, P < 0.05 versus the control group, intraperitoneal-MCFG group, or aerosolized-L-AMB group (log rank test). The survival times for all treatment groups were longer than that for controls (P < 0.05). The survival time for the combination treatment group was significantly longer than those of the intraperitoneal-MCFG group and the aerosolized-L-AMB group (P < 0.05).Open in a separate windowFIG. 2.Numbers of CFU from homogenized lung tissues of mice with IPA. Groups of 10 mice were treated once per day with a combination of intraperitoneally administered MCFG (1 mg/kg/day) and inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml), aerosolized L-AMB (8 ml at 1.2 mg/ml), intraperitoneal MCFG (1 mg/kg/day), and saline (control). CFU counts, as a parameter of A. fumigatus burden in the lungs of IPA mice at 4 h after day 3 of treatment, are shown. *, P < 0.05 (Student''s t test).Open in a separate windowFIG. 3.Histopathology of lung tissues. Both lungs were obtained from IPA mice 4 h after 3 days of treatment with a combination of intraperitoneally administered MCFG and inhalation of aerosolized L-AMB, aerosolized L-AMB, intraperitoneal MCFG, and saline alone as a control. The lungs obtained from aerosolized-L-AMB-treated and combination treatment mice showed obviously smaller numbers of hyphae and fewer foci of inflammation than intraperitoneal-MCFG and control mice. HE, hematoxylin-eosin; GMS, Grocott''s methenamine silver nitrate stain.The current study demonstrated the efficacy of monotherapy of aerosolized L-AMB in a murine IPA model. The AMB concentrations in lung tissue in our study were relatively higher but extremely lower in serum than those from another report of a murine model of intravenously administered L-AMB, although experimental conditions were not the same (10). These results suggested that systemic toxicity generally caused by AMB treatment may be reduced by L-AMB inhalation therapy.The effect of combined intraperitoneal-MCFG and aerosolized-L-AMB treatment was an enhanced survival rate, even though this drug interaction was classified as indifferent in vitro. Since 78% of all control mice died in first 3 days in a survival analysis, we changed the experimental conditions for analysis of fungal burden and histopathological examination. In this model, no mice died before euthanasia, a prerequisite for the organ CFU assay. Both fungal-burden data and histopathological findings supported the survival data in our study.Unlike in our study, Graybill et al. previously reported that combination therapy demonstrated a lack of synergistic effects following intravenous-L-AMB and intraperitoneal-MCFG treatment in a model of murine IPA (4). These discrepancies are likely due to differences between our model and Graybill et al.''s model, including (i) the route of infection, (ii) the status of immunosuppression, and (iii) the administration route of antifungal drugs. These differences also suggest that targeted intrapulmonary delivery of drugs by inhalation raises the drug concentration at the active site of infection in the lungs, thus contributing to the efficacy of combination therapy. Further comparative efficacy studies in a clinical setting are warranted.     

Respiratory syncytial virus infection suppresses IFN-gamma production of gammadelta T cells

The immunological mechanisms by which respiratory syncytial virus (RSV) contributes to the development of asthma are poorly understood. gammadelta T cells are important in mucosal defence, and may contribute to the establishment of primary immune responses by producing cytokines early during respiratory infections. Thus, we used flow cytometry and intracellular cytokine staining to investigate the expression of interferon (IFN)-gamma and interleukin (IL)-4 by mitogen-stimulated gammadelta T cells from the peripheral blood of 15 hospitalized infants with RSV bronchiolitis, seven rotavirus-infected infants and eight normal controls. gammadelta T cells from RSV-infected infants had a lower proportion of IFN-gamma-producing cells (median, 4.00%; range, 0.58-6.60%) and a slightly but significantly higher proportion of IL-4-producing cells (median, 0.40%; range, 0.13-2.76%) than rotavirus-infected infants (median, 32.10%; range, 14.43-61.21%; P < 0.01, median, 0.00%; range, 0.00-0.00%; P < 0.05) in the acute phase. By contrast, differences in cytokine production by total CD3+ T cells did not differ significantly between patient groups. Thus, reduced IFN-gamma-production by gammadelta T cells in the peripheral blood of RSV-infected infants is accompanied by increased Th2 cytokine production during the acute phase of disease. At follow-up, eight children had recurrent episodes of wheezing. The frequencies of IFN-gamma-producing gammadelta T cells were significantly lower in patients who developed recurrent wheezing (median, 0.65%; range, 0.02-1.75%) than in patients without recurrent wheezing (median, 6.90%; range, 5.25-10.98%; P < 0.005). Cytokine production by gammadelta T cells may therefore be important in the pathogenesis of acute RSV disease, and play a part in the development of recurrent childhood wheezing after bronchilolitis.     

Prospective clinical evaluation of the serologic tuberculous glycolipid test in combination with the nucleic acid amplification test

We have conducted a prospective controlled multicenter study to evaluate differences in the levels of clinical utility of the tuberculous glycolipid (TBGL) serodiagnostic test and the nucleic acid amplification test in patients with smear-negative active pulmonary tuberculosis (TB). The TBGL test and the PCR test were individually not so useful for the rapid diagnosis of smear-negative active pulmonary TB. However, clinical utility was considerably improved by using the TBGL test and the PCR test in combination, especially in patients with smear-negative and culture-negative active pulmonary TB and in patients with minimally advanced lesions.     

Bronchial hyperresponsiveness and airway inflammation in adolescents with asymptomatic childhood asthma

BACKGROUND: About 70% of childhood asthmatics become free of asthma-related symptoms during adolescence. Little is known about bronchial hyperresponsiveness (BHR) and airway inflammation in young adults with "outgrown" childhood asthma. METHODS: We studied 61 nonsmoking medical students (18 intermittent mild asthmatics, 23 students with outgrown childhood asthma but free of asthma-related symptoms for 10 years (asymptomatic asthmatics) and 20 healthy students). BHR and lung function were measured, and induced sputum samples analyzed for eosinophil count, eosinophilic cationic protein (ECP), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). RESULTS: BHR was still present in most asymptomatic asthmatics, but it was milder compared with healthy students. Only three subjects with previous asthma had no BHR and no signs of airway inflammation. Percentages of eosinophil, and ECP, TNF-alpha and GM-CSF concentrations in induced sputum of mild asthmatics and asymptomatic asthma groups were higher than in the healthy group. In asymptomatic asthmatics group, the duration of asthma, sputum eosinophil percentage, and the level of TNF-alpha in sputum correlated significantly with BHR. CONCLUSIONS: Only a few subjects with longstanding asymptomatic asthma could be considered as cured; most asymptomatic asthmatics continued to exhibit BHR and signs of airway inflammation. The outcome of childhood asthma and BHR was associated with the degree of airway inflammation and the duration of childhood asthma.     

Frequent expression of gp46 in adult T-cell leukemia/lymphoma: an immunohistochemical study of 40 cases

Adult T-cell leukemia/lymphoma (ATLL) is a lymphoproliferative disease caused by human T-cell lymphotropic virus type 1 (HTLV-1) infection. HTLV-1 is spread by cell-to-cell transmission via the gp46-197 region, from Asp197 to Leu216, in the envelope protein gp46. A correlation exists between the prevalence and titer of the antibody recognizing the gp46-197 region (anti-gp46-197 antibody) and the severity of ATLL. In the present study, immunohistochemical staining was performed on samples of paraffin embedded lymph nodes of three different histological types of ATLL (anaplastic large cell type, n = 10; pleomorphic type, n = 10; and Hodgkin's-like type, n = 10) from 30 cases and 10 cases of HTLV-associated lymphadenitis. Of the three ATLL subtypes, gp46 expression was highest in the anaplastic large cell type, followed by the pleomorphic type and Hodgkin's-like type (mean: 53.4%, 34.9% and 16.0%, respectively; P= 0.0003). In HTLV-1 associated lymphadenitis cases, gp46 positive cells were rarely seen (4.0%). These results suggest that gp46-197 immunohistochemical staining can be a useful histological indicator for prediction of the aggressiveness of ATLL and prognosis for ATLL patients.     

Solitary intrapulmonary cystic lymphangioma in an infant: a case report with literature review

Lymphangioma rarely presents as a solitary pulmonary lesion. We encountered a case of solitary cystic lymphangioma and present its clinicopathologic and immunohistochemical findings. A 2-month-old boy was referred to the hospital after developing a persistent cough. Chest X-ray showed a large cyst in the right lung. Under the preoperative diagnosis of bronchogenic cyst, he underwent right lower lobectomy at the age of 11 months. The resected specimen contained a 5.5-cm septate cystic lesion. Microscopically, the lesion consisted of a large cystic space and interconnected slit-like spaces surrounding bronchovascular islands. The cyst was lined by a monolayer of flat cells with focal multinucleated giant cells. Immunohistochemically, the cells lining the cystic lesion were positive for D2-40, Prox1, CD34, and CD31, and weakly positive for VEGFR-3, but were negative for AE1/3, HMB45, VEGF-A, VFGF-C, VEGFR-1. Differential diagnoses included lobar or interstitial emphysema, bronchogenic cyst, congenital pulmonary airway malformation and alveolar adenoma. D2-40 and Prox1 were useful in differentiation and in determining the extent of the lesion. A review of the literature found only 15 cases of solitary pulmonary lymphangioma. In younger patients, the lesions tend to occupy more of the lung. Focal giant cell reaction has not been described in the reported papers.