Performance of 1,3‐β‐D‐glucan for diagnosing invasive fungal diseases in children

Plasma 1,3‐β‐D‐glucan (BDG) is indicated as a tool for early diagnosis of invasive fungal diseases (IFD). However, data on its diagnostic value are scarce in children. Therefore, definition of BDG test performance in paediatrics is needed. BDG was evaluated in children admitted to “Istituto Giannina Gaslini,” Genoa, Italy, who developed clinical conditions at risk for IFD. Results were analysed for sensitivity, specificity, predictive values, likelihood ratios, accuracy, informedness and probability of missing one case by a negative test. A total of 1577 BDG determinations were performed on 255 patients (49% males, median age 5.4 years). Overall 46 IFD were diagnosed, 72% proven/probable. The test performance was evaluated for 80 pg/mL, 120 pg/mL, 200 pg/mL, 350 pg/mL, 400 pg/mL cut offs. Sensitivity was always <0.80 and specificity > 0.90 only for cut offs ≥200 pg/mL. Negative predictive value was ≥0.90 for all the cut offs evaluated, while positive predictive value resulted barely 0.50 (8% IFD prevalence). Accuracy was never >0.90, and informedness was at best 0.50. The risk of missing one IFD by a negative result was < 10%. Analyses in haemato‐oncological or newborn patients did not show major differences. Detection of serum BDG does not appear a valuable adjunctive diagnostic tool for IFD in paediatrics.     

Detection of (1→3)‐β‐D‐glucan in same‐day urine and serum samples obtained from patients with haematological malignancies

Serum 1,3‐beta‐d ‐glucan (BDG) testing is an established diagnostic marker for invasive fungal infections (IFI) among patients with haematological malignancies. In contrast limited data exist regarding the application of urine BDG testing. Same‐day midstream urine and serum screening samples were collected in adult patients with underlying haematological malignancies. A total of 80 urine samples from 46 patients were investigated: Twenty‐six had positive corresponding serum BDG >120 pg ml^?1, 27 intermediate (60–80 pg ml^?1), and 27 negative serum BDG (<25 pg ml^?1). A significant positive correlation between BDG in serum and urine samples was observed (P = 0.025; r = 0.252). Sensitivity, specificity, positive predictive value and negative predictive value (compared with same‐day serum results) were: 42%, 76%, 46%, 73% when using an 80 pg ml^?1 urine cut‐off, and 35%, 96%, 82%, 75% for a 250 pg ml^?1 cut‐off. Urine BDG seemed to be higher in samples obtained from patients with probable IFI (n = 13, median 145, IQR 22–253) compared to those from patients without IFI (n = 56, median 24, IQR 15–88) but the difference was not significant (P = 0.069). Overall correlation of same‐day urine BDG and serum BDG was moderate. However, urine BDG testing may warrant further investigation in larger studies, as high‐positive urine results correlated with high‐positive corresponding serum levels and clinical performance was comparable to serum BDG.     

Comparative evaluation of pan‐fungal real‐time PCR,galactomannan and (1‐3)‐β‐D‐glucan assay for invasive fungal infection in paediatric cancer patients

Limited specific data and investigations are available for the diagnosis of Invasive Fungal Infection (IFI) in paediatrics cancer patients. Three non‐invasive tests; Platelia Aspergillus EIA for galactomannan (GM), β‐D‐glucan (BDG) assay and pan‐fungal real‐time PCR for fungal DNA in blood were evaluated. One hundred twenty‐five paediatrics cancer patients at the high risk of IFI were enrolled. Single blood and serum samples were evaluated by all the three methods. Patients were classified into 10 proven, 52 probable and 63 no IFI cases in accordance with EORTC MSG 2008 revised guidelines. The sensitivity, specificity, PPV and NPV of all the three tests in proven, probable and no IFIs cases were analysed singly and in combination. The sensitivity, specificity, PPV and NPV of GM, BDG and pan‐fungal real‐time PCR were: 87%, 61%, 81%, 69.5% for GM, 88%, 59.5%, 81%, 71.4% for BDG and 89%, 69.2%, 85%, 67.5% for PCR (95% CI). Among different combinations, best combination was found to be GM and PCR with sensitivity, specificity, PPV and NPV of 98.2%, 89.3%, 97.1% and 90% respectively. Single samples must be evaluated by combination of tests.     

Diagnostic value of serum galactomannan, (1,3)‐β‐d‐glucan,and Aspergillus fumigatus‐specific IgA and IgG assays for invasive pulmonary aspergillosis in non‐neutropenic patients

Detection of serum galactomannan (GM) and (1,3)‐β‐d ‐glucan (BG) is considered useful for non‐culture diagnosis of invasive pulmonary aspergillosis (IPA) in neutropenic patients. Only few studies evaluated these seromarkers in non‐neutropenic patients suspected of having IPA. The aim of this study was to evaluate both tests together with the Aspergillus fumigatus‐specific serum IgG and IgA (IgAG) test for serological IPA diagnosis in non‐neutropenic patients. Sera from 87 patients suspected of having IPA were retrospectively analysed. Patients were categorised into groups of proven IPA (n = 10), putative IPA (n = 31) and non‐IPA colonisation (n = 46). When the GM, BG and IgAG assays were used for patients included in the study, the sensitivity/specificity/positive predictive value (PPV)/negative predictive value (NPV) were 48.8%/91.3%/83.3%/66.7%, 82.9%/73.9%/73.9%/82.9% and 75.6%/95.7%/93.9%/81.5%, respectively. Thus, the highest specificity and PPV were confirmed for the IgAG assay. Improvements in the sensitivity and NPV were achieved by “at least one positive” analysis with the GM and BG assays, with the sensitivity/specificity/PPV/NPV values being 85.0%/69.6%/71.4%/84.2%. Nevertheless, the highest sensitivity and NPV were achieved by the “at least one positive” analysis combining the GM, BG and IgAG tests (97.6% and 96.8%, respectively). The involvement of the IgAG assay could improve IPA diagnosis in non‐neutropenic patients by increasing the sensitivity and NPV when combined with the GM or BG assays. Furthermore, improvement was achieved by combining the GM, BG and IgAG assays using the “at least one positive test” strategy, especially if doubt exists.     

Reliability of serum 1,3‐beta‐d‐glucan assay in patients undergoing renal replacement therapy: a review of the literature

The serum 1,3‐beta‐d ‐glucan (BDG) test is a pan‐fungal serum marker considered to detect the majority of pathogenic fungi, including Aspergillus spp. and Candida spp. For this review we searched for publications dealing with serum BDG levels in patients undergoing renal replacement therapy (RRT). The influence of various different membrane materials used for RRTs in these publications on serum BDG has been reviewed. We found that unmodified cellulose containing membranes increased the serum BDG levels highly, whereas conflicting results have been observed for modified cellulose containing materials. Synthetic materials (e.g. polysuflone) had no influence on serum BDG levels in the majority of the reviewed publications.     

Use of (1→3)‐β‐d‐glucan for diagnosis and management of invasive mycoses in HIV‐infected patients

People living with HIV (PLHIV) are highly vulnerable to invasive fungal infections (IFIs) due to their immune dysfunction. Diagnosis and treatment of IFIs remain challenging due to the requirement of deep tissue sampling to visualise and culture fungi before initiating treatment. Such techniques are less practical in resource‐limited settings due to their cost and requirement of relatively invasive procedures. Hence, identification of surrogate markers for the early diagnosis and therapeutic monitoring of IFIs is required. Recent studies have shown that (1→3)‐β‐d ‐glucan (BDG), a major fungal cell wall antigen, represents a promising soluble marker for the presumptive diagnosis and therapeutic monitoring of IFIs in HIV‐infected patients. Herein, we review findings on the merits of BDG assays in the diagnosis of IFIs and monitoring of antifungal therapies for PLHIV. Conversely to other types of immunocompromised patients, HIV infection is associated with gut damage and subsequent bacterial and fungal translocation leading to elevated BDG plasma levels.     

TNF‐α enhances TGF‐β‐induced endothelial‐to‐mesenchymal transition via TGF‐β signal augmentation

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer‐associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF‐β, which induces endothelial‐to‐mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor‐infiltrating inflammatory cells secrete various cytokines, including TNF‐α. However, the role of TNF‐α in TGF‐β‐induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF‐α on TGF‐β‐induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF‐β and TNF‐α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF‐β and TNF‐α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF‐β type I receptor, TGF‐β2, activin A, and integrin αv, suggesting that TNF‐α enhanced TGF‐β‐induced EndMT by augmenting TGF‐β family signals. Furthermore, oral squamous cell carcinoma‐derived cells underwent epithelial‐to‐mesenchymal transition (EMT) in response to humoral factors produced by TGF‐β and TNF‐α‐cultured ECs. This EndMT‐driven EMT was blocked by inhibiting the action of TGF‐βs. Collectively, our findings suggest that TNF‐α enhances TGF‐β‐dependent EndMT, which contributes to tumor progression.     

Modulation of TGF‐β activity by latent TGF‐β‐binding protein 1 in human malignant glioma cells

High biological activity of the transforming growth factor (TGF)‐β‐Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF‐β has become a novel target for the experimental treatment of these tumors. TGF‐β is processed by furin‐like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency‐associated peptide (LAP). Latent TGF‐β‐binding protein 1 (LTBP‐1) covalently binds to this small latent TGF‐β complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP‐1 protein in vivo increase with the grade of malignancy in gliomas. LTBP‐1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP‐1 into the medium is decreased by the inhibition of FLP activity. Gene‐transfer mediated overexpression of LTBP‐1 in glioma cell lines results in an increase inTGF‐β activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF‐β activity is enhanced. Conversely, LTBP‐1 gene silencing reduces TGF‐β activity and Smad2 phosphorylation without affecting TGF‐β protein levels. Collectively, we identify LTBP‐1 as an important modulator of TGF‐β activation in glioma cells, which may contribute to the malignant phenotype of these tumors. © 2009 UICC     

Clinicopathological analysis of 17 primary cutaneous T‐cell lymphoma of the γδ phenotype from Japan

Primary cutaneous γδ T‐cell lymphoma (PCGD‐TCL) is an aggressive lymphoma consisting of clonal proliferation of mature activated γδ T‐cells of a cytotoxic phenotype. Because primary cutaneous γδ T‐cell lymphoma is a rare disease, there are few clinicopathological studies. In addition, T‐cell receptor (TCR) γδ cells are typically immunostained in frozen sections or determined by TCRβ negativity. We retrospectively analyzed 17 primary cutaneous T‐cell lymphomas of the γδ phenotype (CTCL‐γδ) in a clinicopathological and molecular study using paraffin‐embedded sections. Among 17 patients, 11 had CTCL‐γδ without subcutaneous panniculitis‐like T‐cell lymphoma (SPTCL) features and six had CTCL‐γδ with SPTCL features. Immunophenotypically, some significant differences were found in CD8 and CD56 positivity between our patient series of CTCL‐γδ patients with SPTCL features and SPTCL‐γδ patients described in the previous literature. A univariate analysis of 17 CTCL‐γδ patients showed that being more than 60 years old, presence of visceral organ involvement, and small‐to‐medium cell size were poor prognostic factors. In addition, the 5‐year overall survival rate was 42.4% for the CTCL‐γδ patients without SPTCL features and 80.0% for those with SPTCL features. Consequently, there was a strikingly significant difference in overall survival among SPTCL, CTCL‐γδ with SPTCL features and CTCL‐γδ without SPTCL features (P = 0.0005). Our data suggests that an indolent subgroup may exist in CTCL‐γδ. Studies on more cases, including those from other countries, are warranted to delineate the clinicopathological features and the significance in these rare lymphomas.     

Oral administration of soluble β‐glucans extracted from Grifola frondosa induces systemic antitumor immune response and decreases immunosuppression in tumor‐bearing mice

Maitake D (MD)‐Fraction is a highly purified soluble β‐glucan derived from Grifola frondosa (an oriental edible mushroom). Intraperitoneal (i.p.) injection of MD‐Fraction has been reported to inhibit tumor growth via enhancement of the host immune system. In this study, we demonstrated that oral administration of MD‐Fraction as well as i.p. injection significantly inhibited tumor growth in murine tumor models. After oral administration, MD‐Fraction was not transferred to the blood in its free form but was captured by antigen‐presenting cells such as macrophages and dendritic cells (DCs) present in the Peyer's patch. The captured MD‐Fraction was then transported to the spleen, thereby inducing the systemic immune response. Our study showed that MD‐Fraction directly induced DC maturation via a C‐type lectin receptor dectin‐1 pathway. The therapeutic response of orally administered MD‐Fraction was associated with (i) induced systemic tumor‐antigen specific T cell response via dectin‐1‐dependent activation of DCs, (ii) increased infiltration of the activated T cells into the tumor and (iii) decreased number of tumor‐caused immunosuppressive cells such as regulatory T cells and myeloid‐derived suppressor cells. Our preclinical study suggests that MD‐Fraction is a useful oral therapeutic agent in the management of patients with cancer.     

1,3‐ß‐D‐glucan concentrations in blood products predict false positive post‐transfusion results

1,3‐ß‐D‐glucan (BDG) is increasingly used to diagnose invasive fungal infections (IFI), although false positive results are a concern. To evaluate the potential interaction of blood products with the BDG assay, human albumin (HA), fresh frozen plasma (FFP), undiluted platelet transfusion (UPT) and packed red blood cells (PRBC) were tested for their BDG content using two different b‐D‐glucan tests. UPTs tested negative, FFP, PBRC and HA tested positive for BDG. In serial dilution, BDG concentration correlated with blood product concentration. To investigate the clinical impact of blood product transfusions, we measured BDG levels before and after the transfusion in three patients (2 PRBC, 1 HA). In the patients receiving PRBC transfusions, BDG values increased from 13 and 17 pg ml^?1 to 183 and 361 pg ml^?1, the HA transfusion increased the serum level from 42 to 58 pg ml^?1. BDG concentrations measured in blood products can be used to predict false positive BDG results.     

Endoplasmic reticulum stress‐mediated autophagy protects against β,β‐dimethylacrylshikonin‐induced apoptosis in lung adenocarcinoma cells

β,β‐Dimethylacrylshikonin (DMAS) is an anti‐cancer compound extracted from the roots of Lithospermum erythrorhizon. The present study aims to investigate the effects of DMAS on human lung adenocarcinoma cells in vitro and explore the mechanisms of its anti‐cancer action. We showed that DMAS markedly inhibited cell viability in a dose‐ and time‐dependent way, and induced apoptosis as well as autophagy in human lung adenocarcinoma cells. Furthermore, we found that DMAS stimulated endoplasmic reticulum stress and mediated autophagy through the PERK‐eIF2α‐ATF4‐CHOP and IRE1‐TRAF2‐JNK axes of the unfolded protein response in human lung adenocarcinoma cells. We also showed that the autophagy induced by DMAS played a prosurvival role in human lung adenocarcinoma cells and attenuated the apoptotic cascade. Collectively, combined treatment of DMAS and pharmacological autophagy inhibitors could offer an effective therapeutic strategy for lung adenocarcinoma treatment.     

Effects of α‐tocopherol and β‐carotene supplementation on cancer incidence and mortality: 18‐Year postintervention follow‐up of the Alpha‐Tocopherol,Beta‐Carotene Cancer Prevention Study

In the Alpha‐Tocopherol, Beta‐Carotene Cancer Prevention Study among 29,133 Finnish male smokers aged 50–69 years, daily α‐tocopherol (50 mg) for a median of 6.1 years decreased the risk of prostate cancer, whereas β‐carotene (20 mg) increased risk of lung cancer and overall mortality. To determine the postintervention effects of α‐tocopherol and β‐carotene, 25,563 men were followed 18 years for cancer incidence and all causes of mortality through national registers. Neither supplement had significant effects on post‐trial cancer incidence. Relative risk (RR) for lung cancer (n = 2,881) was 1.04 (95% confidence interval [CI], 0.96–1.11) among β‐carotene recipients compared with nonrecipients. For prostate cancer (n = 2,321), RR was 0.97 (95% CI, 0.89–1.05) among α‐tocopherol recipients compared with nonrecipients with the preventive effect of α‐tocopherol continuing ～8 years postintervention. Body mass index significantly modified the effect of α‐tocopherol on prostate cancer (p for interaction = 0.01) RR 1.00 (95% CI, 0.88–1.14) in normal‐weight men, 0.87 (95% CI, 0.77–0.98) in overweight men, and 1.25 (95% CI, 1.01–1.55) in obese men. The post‐trial relative mortality (based on 16,686 deaths) was 1.02 (95% CI, 0.98–1.05) for α‐tocopherol recipients compared with nonrecipients and 1.02 (95% CI, 0.99–1.05) for β‐carotene recipients compared with nonrecipients. α‐Tocopherol decreased post‐trial prostate cancer mortality (RR, 0.84; 95% CI, 0.70–0.99), whereas β‐carotene increased it (RR, 1.20; 95% CI, 1.01–1.42). In conclusion, supplementation with α‐tocopherol and β‐carotene appeared to have no late effects on cancer incidence. The preventive effect of moderate‐dose α‐tocopherol on prostate cancer continued several years post‐trial and resulted in lower prostate cancer mortality.     

Curcumin β‐D‐glucuronide exhibits anti–tumor effects on oxaliplatin‐resistant colon cancer with less toxicity in vivo

The NF‐kappa B (NF‐κB) pathway plays a pivotal role in tumor progression and chemoresistance, and its inhibition has been shown to suppress tumor growth in a variety of preclinical models. Recently, we succeeded in synthesizing a water‐soluble injectable type of curcumin β‐D‐glucuronide (CMG), which is converted into a free‐form of curcumin by β‐glucuronidase in vivo. Herein, we aimed to clarify the efficacy, safety and pharmacokinetics of CMG in a xenograft mouse model. First, we confirmed that the presence of KRAS/TP53 mutations significantly increased the IC₅₀ of oxaliplatin (L‐OHP) and NF‐κB activity in HCT116 cells in vitro. Then, we tested the efficacy of CMG in an HCT116 colon cancer xenograft mice model. CMG demonstrated superior anticancer effects compared to L‐OHP in an L‐OHP‐resistant xenograft model. With regard to safety, significant bodyweight loss, severe myelosuppression and AST/ALT elevation were observed in L‐OHP‐treated mice, whereas none of these toxicity was noted in CMG‐treated mice. The combination of CMG and L‐OHP exhibited additive effects in these xenograft models without increasing toxicity. Pharmacokinetic analysis revealed that high levels of free‐form curcumin were maintained in the tumor tissue after 48 hours following CMG administration, but it was not detected in other major organs, such as the heart, liver and spleen. Immunohistochemistry revealed reduced NF‐κB activity in the tumor tissue extracted from CMG‐treated mice compared with that from control mice. These results indicated that CMG could be a promising anticancer prodrug for treating colon cancer with minimal toxicity.     

Regulatory mechanisms mediated by peroxisome proliferator‐activated receptor‐β/δ in skin cancer

Considerable progress has been made during the past 20 years towards elucidating the role of peroxisome proliferator‐activated receptor‐β/δ (PPARβ/δ) in skin cancer. In 1999, the original notion that PPARβ/δ was involved with epithelial cell function was postulated based on a correlation between PPARβ/δ expression and the induction of messenger RNAs encoding proteins that mediate terminal differentiation in keratinocytes. Subsequent studies definitively revealed that PPARβ/δ could induce terminal differentiation and inhibit proliferation of keratinocytes. Molecular mechanisms have since been discovered to explain how this nuclear receptor can be targeted for preventing and treating skin cancer. This includes the regulation of terminal differentiation, mitotic signaling, endoplasmic reticulum stress, and cellular senescence. Interestingly, the effects of activating PPARβ/δ can preferentially target keratinocytes with genetic mutations associated with skin cancer. This review provides the history and current understanding of how PPARβ/δ can be targeted for both nonmelanoma skin cancer and melanoma and postulates how future approaches that modulate PPARβ/δ signaling may be developed for the prevention and treatment of these diseases.     

β‐glucan restores tumor‐educated dendritic cell maturation to enhance antitumor immune responses

Tumors can induce the generation and accumulation of immunosuppressive cells such as myeloid‐derived suppressor cells (MDSCs) in a tumor microenvironment, contributing to tumor escape from immunological attack. Although dendritic cell‐based cancer vaccines can initiate antitumor immune responses, tumor‐educated dendritic cells (TEDCs) involved in the tolerance induction have attracted much attention recently. In this study, we investigated the effect of β‐glucan on TEDCs and found that β‐glucan treatment could promote the maturation and migration of TEDCs and that the suppressive function of TEDCs was significantly decreased. Treatment with β‐glucan drastically decreased the levels of regulatory T (Treg) cells but increased the infiltration of macrophages, granulocytes and DCs in tumor masses, thus elicited Th1 differentiation and cytotoxic T‐lymphocyte responses and led to a delay in tumor progression. These findings reveal that β‐glucan can inhibit the regulatory function of TEDCs, therefore revealing a novel function for β‐glucan in immunotherapy and suggesting its potential clinical benefit. β‐Glucan directly abrogated tumor‐educated dendritic cells‐associated immune suppression, promoted Th1 differentiation and cytotoxic T‐lymphocyte priming and improved antitumor responses.     

Dkk3, downregulated in cervical cancer,functions as a negative regulator of β‐catenin

The Wnt/β‐catenin signaling pathway is activated during the malignant transformation of keratinocytes that originate from the human uterine cervix. Dkk1, 2 and 4 have been shown to modulate the Wnt‐induced stabilization of the β‐catenin signaling pathway. However, the function of Dkk3 in this pathway is unknown. Comparison of the Dkk3 gene expression profiles in cervical cancer and normal cervical tissue by cDNA microarray and subsequent real‐time PCR revealed that the Dkk3 gene is frequently downregulated in the cancer. Methylation studies showed that the promoter of Dkk3 was methylated in cervical cancer cell lines and 22 (31.4%) of 70 cervical cancer tissue specimens. This promoter methylation was associated with reduced expression of Dkk3 mRNA in the paired normal and tumor tissue samples. Further, the reintroduction of Dkk3 into HeLa cervical cancer cells resulted in reduced colony formation and retarded cell growth. The forced expression of Dkk3 markedly attenuated β‐catenin‐responsive luciferase activity in a dose‐dependent manner and decreased the β‐catenin levels. By utilizing a yeast two‐hybrid screen, βTrCP, a negative regulator of β‐catenin was identified as a novel Dkk3‐interacting partner. Coexpression with βTrCP synergistically enhanced the inhibitory function of Dkk3 on β‐catenin. The stable expression of Dkk3 blocks the nuclear translocation of β‐catenin, resulting in downregulation of its downstream targets (VEGF and cylcin D), whereas knockdown of Dkk3 abrogates this blocking. We conclude from our finding that Dkk3 is a negative regulator of β‐catenin and its downregulation contribute to an activation of the β‐catenin signaling pathway. © 2008 Wiley‐Liss, Inc.