Zinc-binding metallophores protect Pseudomonas aeruginosa from calprotectin-mediated metal starvation.

Pseudomonas aeruginosa is known to exhibit considerable resistance to the antimicrobial activity of the metal-sequestering protein calprotectin (CP). In this study, we demonstrate that although CP induces zinc deficiency in P. aeruginosa, a strain unable to import zinc through the two most important metal acquisition systems, namely ZnuABC and ZrmABCD, maintains significant growth capacity in the presence of high concentrations of CP. Furthermore, we have shown that nicotianamine, a molecule structurally similar to the metallophore pseudopaline, can favor the acquisition of the metal even in the presence of CP. To gain insights into the mechanisms through which metallophores can promote zinc acquisition, we analyzed the effect of nicotianamine on the activity of the metallo-ß-lactamase VIM-1. Our data suggest that metallophores released by bacteria in response to zinc deficiency can extract the protein-bound metal. The ability to interfere with the binding of metals to proteins, as well as favoring the acquisition of zinc, may contribute to increasing the resistance of P. aeruginosa to the antimicrobial action of CP.

Efficient zinc uptake is critical for the ability of Pseudomonas aeruginosa to express virulence traits and colonize the human lung.

We have recently shown that Pseudomonas aeruginosa, an opportunistic pathogen that chronically infects the lungs of patients with cystic fibrosis (CF) and other forms of lung disease, is extremely efficient in recruiting zinc from the environment and that this capability is required for its ability to cause acute lung infections in mice. To verify that P. aeruginosa faces zinc shortage when colonizing the lungs of human patients, we analyzed the expression of three genes that are highly induced under conditions of zinc deficiency (zrmA, dksA2 and rpmE2), in bacteria in the sputum of patients with inflammatory lung disease. All three genes were expressed in all the analyzed sputum samples to a level much higher than that of bacteria grown in zinc-containing laboratory medium, supporting the hypothesis that P. aeruginosa is under zinc starvation during lung infections. We also found that the expression of several virulence traits that play a central role in the ability of P. aeruginosa to colonize the lung is affected by disruption of the most important zinc importing systems. Virulence features dependent on zinc intake include swarming and swimming motility and the ability to form biofilms. Furthermore, alterations in zinc assimilation interfere with the synthesis of the siderophore pyoverdine, suggesting that zinc recruitment could modulate iron uptake and affect siderophore-mediated cell signaling. Our results reveal that zinc uptake is likely to play a key role in the ability of P. aeruginosa to cause chronic lung infections and strongly modulates critical virulence traits of the pathogen. Taking into account the recent discovery that zinc uptake in P. aeruginosa is promoted by the release of a small molecular weight molecule showing high affinity for zinc, our data suggest novel and effective possibilities to control lung infections by these bacteria.

Prognostic performance of MR-pro-adrenomedullin in patients with community acquired pneumonia in the Emergency Department compared to clinical severity scores PSI and CURB.

AIM: (i) evaluate the performance of MR-pro-ADM in reflecting the outcome and risk for CAP patients in the emergency department, and (ii) compare the prognostic performance of MR-pro-ADM with that of clinical scores PSI and CURB65. METHODS: Observational prospective, single-center study in patients with suspected community acquired pneumonia (CAP). Eighty one patients underwent full clinical and laboratory assessment as by protocol, and were followed up a 28 days. Primary endpoints measured were: death, death at 14 days, non-invasive mechanical ventilation (NIMV), endotracheal intubation (EI), ICU admission, overall hospital stay >10 days, emergency department stay >4 days. The discriminative performance of MR-pro-ADM and clinical scores was assessed by AUROC analysis. RESULTS: The distribution for MR-pro-ADM followed an upward trend, increasing with the increase of both PSI (p<0.001) and CURB65 (p<0.001) classes. However, the difference between MRproADM values and score classes was significant only in the case of CURB65 classes 0 and 1 (p = 0.046), 2 (p = 0.013), and 3 (p = 0.011); and with PSI classes 5, 3 (p = 0.044), and 1 (p = 0.020). As to the differences among variables for the six end-points, MR-pro-ADM values in the two groups selected for each considered end-point differed in a statistically significant manner for all endpoints. Both PSI and CURB65 differed significantly for all end-points, except for stay in the ED longer than 4 days and the hospital stay longer than 10 days and endotracheal intubation (only PSI classes differed with statistical significance). ROC analyses evidenced that MR-pro-ADM values gave the greatest AUC for the prediction of death, endotracheal intubation, hospital stay >10 days and DE stay >4 days, compared to the PSI and CURB (though difference not statistically significant). For each endpoint measured, the best thresholds values for Mr-pro-ADM were: 1.6 (specificity 76.5%; sensitivity 77.8%) for death; 2.5 (specificity 88.9%; sensitivity 80.0%) for death at 14 days; 1.5 (specificity 77.0%; sensitivity 87.5%) for NIMV; 2.4 (specificity 88.7%; sensitivity 83.3%) for endotracheal intubation; 0.9 (specificity 53.5%; sensitivity 70.6%) for DE stay greater than 4 days; 1.9 (specificity 82.1%; sensitivity 55.3%) for hospital stay greater than 10 days. The AUC for the combination of MR-pro-ADM and PSI was 81.29% [63.41%-99.17%], but not in a statistically significant manner compared to the AUCs of the single predictors. Conversely, the AUC for the combination of MR-pro-ADM and CURB65 was 87.58% [75.54%-99.62%], which was significantly greater than the AUC of CURB65 (p = 0.047) or PSI (p = 0.017) alone. CONCLUSIONS: The present study confirms that assessment of MR-pro-ADM levels in CAP patients in addition to CURB scores increases the prognostic accuracy of CURB alone and may help rule out discrepancies arising from flawed clinical severity classification. With particular reference to patients scoring in the upper classes of CURB and PSI, MR-pro-ADM values provided additional information towards a better risk stratification of those patients. In particular, our results pointed towards two MR-pro-ADM threshold values that appear to predict with a good degree of accuracy the patient's need for non-invasive mechanical ventilation, endotracheal intubation, or intensive care. This aspect, however, deserves further investigation.

Growth of Pseudomonas aeruginosa in zinc poor environments is promoted by a nicotianamine-related metallophore.

Previous studies have suggested that P. aeruginosa possesses redundant zinc uptake systems. To identify uncharacterized zinc transporters, we analyzed the genome-wide transcriptional responses of P. aeruginosa PA14 to zinc restriction. This approach led to the identification of an operon (zrmABCD) regulated by the zinc uptake regulator Zur, that encodes for a metallophore-mediated zinc import system. This operon includes the genes for an uncharacterized TonB-dependent Outer Membrane Protein (ZrmA) and for a putative nicotianamine synthase (ZrmB). The simultaneous inactivation of the ZnuABC transporter and of one of these two genes markedly decreases the ability of P. aeruginosa to grow in zinc-poor media and compromises intracellular zinc accumulation. Our data demonstrate that ZrmB is involved in the synthesis of a metallophore which is released outside the cell and mediates zinc uptake through the ZrmA receptor. We also show that alterations in zinc homeostasis severely affect the ability of P. aeruginosa to cause acute lung and systemic infections in C57BL/6 mice, likely due to the involvement of zinc in the expression of several virulence traits. These findings disclose a hitherto unappreciated role of zinc in P. aeruginosa pathogenicity and reveal that this microorganism can obtain zinc through a strategy resembling siderophore-mediated iron uptake.

The capability of Pseudomonas aeruginosa to recruit zinc under conditions of limited metal availability is affected by inactivation of the ZnuABC transporter.

The ability of a large number of bacterial pathogens to multiply in the infected host and cause disease is dependent on their ability to express high affinity zinc importers. In many bacteria, ZnuABC, a transporter of the ABC family, plays a central role in the process of zinc uptake in zinc poor environments, including the tissues of the infected host. To initiate an investigation into the relevance of the zinc uptake apparatus for Pseudomonas aeruginosa pathogenicity, we have generated a znuA mutant in the PA14 strain. We have found that this mutant strain displays a limited growth defect in zinc depleted media. The znuA mutant strain is more sensitive than the wild type strain to calprotectin-mediated growth inhibition, but both the strains are highly resistant to this zinc sequestering antimicrobial protein. Moreover, intracellular zinc content is not evidently affected by inactivation of the ZnuABC transporter. These findings suggest that P. aeruginosa is equipped with redundant mechanisms for the acquisition of zinc that might favor P. aeruginosa colonization of environments containing low levels of this metal. Nonetheless, deletion of znuA affects alginate production, reduces the activity of extracellular zinc-containing proteases, including LasA, LasB and protease IV, and decreases the ability of P. aeruginosa to disseminate during systemic infections. These results indicate that efficient zinc acquisition is critical for the expression of various virulence features typical of P. aeruginosa and that ZnuABC also plays an important role in zinc homeostasis in this microorganism.

Hepatic nodules in liver transplantation candidates: MR imaging and underlying hepatic disease.

OBJECTIVE: To assess by MR imaging the frequency of hepatic nodules in patients waiting on the liver transplant list and to determine whether certain underlying hepatic diseases were more often associated with the development of such hepatic nodules. MATERIAL AND METHODS: We reviewed the MR and clinical records in all patients seen by the liver transplant service at our center since its inception in January 1998 until September 2002. A total of 371 patients (207 men and 164 women, age range 18-68 years, mean 45 years) were included in the study. The presence of hepatic nodules, size, number and underlying hepatic diseases were determined in all patients. Magnetic resonance imaging was performed on a 1.5-T MR imager using T1-weighted, T2-weighted and multi-phase gadolinium-enhanced sequences. Odds ratio (OR) and 95% confidence intervals (CIs) were computed to evaluate the association between the underlying hepatic disease and the development of hepatic nodule. RESULTS: Among 371 liver transplantation candidates, the most common underlying hepatic disease was hepatitis C virus (HCV) infection, either alone (n=93; 25%) or associated with other hepatic diseases (n=40; 10.8%). Of all patients, 33 (8.9%) had regenerative nodules (RNs), 40 (10.7%) dysplastic nodules (DNs) and 57 (15.3%) hepatocellular carcinomas (HCCs). Hepatocellular carcinoma was observed in 35.3% of patients with HCV infection and alcohol abuse combined, 24.5% with cryptogenic cirrhosis, 25% with hemochromatosis and 19% with alcohol abuse. Patients who had either DNs or HCC were 2.5 times more likely to have either alcohol abuse or HCV, alone or combined, as the substrate of their liver disease (OR 2.54, 95% CI 1.56-4.13). Our data suggest a supra-additive interaction between HCV infection and ethanol in their association with MR imaging detected lesions. CONCLUSION: Patients with cryptogenic cirrhosis, alcohol abuse, HCV infection (alone or combined) and hemochromatosis had the greatest likelihood of having HCC, with the combination of HCV infection and alcohol abuse having the highest of all.

Angiogenesis in hepatocellular nodules: correlation of MR imaging and vascular endothelial growth factor.

PURPOSE: To assess the correlation between magnetic resonance (MR) imaging findings and angiogenetic activity in hepatocellular nodules evaluated by immunohistochemical staining with antibody of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: We searched the pathologic records of our institution from December 1999 to April 2002, and included 16 patients with hepatocellular carcinoma (N = 14), large regenerative nodule (N = 1), and dysplastic nodule (N = 1) who underwent orthotopic liver transplantation (10 patients) or partial hepatectomy (six patients) and MR imaging within an interval of two weeks. The MR images were retrospectively assessed qualitatively and quantitatively. Angiogenetic activity of the hepatic nodules was evaluated by means of immunohistochemical study for VEGF. Analysis of variance and the Scheffé criterion were used for statistical evaluation. RESULTS: Hepatic nodules with moderate to strong immunoreactivity for VEGF showed higher signal intensity on T1-weighted images (P < 0.05) and those with intense immunoreactivity for VEGF showed higher signal intensity on T2-weighted images (P < 0.05). No correlation was found between the immunoreactivity for VEGF and tumor vascularity on postcontrast early- and late-phase images. CONCLUSION: Our current results suggest that signal intensity on unenhanced T1- and T2-weighted MR images may correlate with immunoreactivity for VEGF. Correlation was not found between immunoreactivity for VEGF and signal intensity on gadolinium-enhanced MR images.

Early heterogeneous enhancement of the liver: magnetic resonance imaging findings and clinical significance.

PURPOSE: To investigate and assess the radiologic, serological, and histopathologic findings in patients who presented with early heterogeneous enhancement (EHE) on gadolinium-enhanced early-phase magnetic resonance imaging (MRI) of the liver. MATERIALS AND METHODS: We searched our radiologic records of MRI of the liver from July 1999 to April 2002 to identify patients with EHE. Three investigators retrospectively evaluated in consensus the MR images in each patient for intensity and characteristic of EHE blinded to clinical information. Serological laboratory values and clinical information were obtained in all patients, and histologic findings were available in 19. RESULTS: We identified 67 patients with EHE. Of them, 62 patients (93%) had underlying chronic liver disease. Twenty-seven patients had viral hepatitis, 13 had alcohol abuse, 6 had primary sclerosing cholangitis, and the others had miscellaneous etiologies. The five patients without chronic liver disease had the following clinical histories: concurrent chemotherapy for extrahepatic malignancy (two patients), concurrent intraabdominal infection (one), and no known associated disease (two). Intensity of EHE was intense in 6 (9%), moderate in 22 (33%), and mild in 39 (58%). Pattern of EHE was geographic in 15 patients (22%), patchy in 37 (55%), and miliary in 15 (22%). All EHE showed rapid fading on postcontrast late-phase images. EHE showed mild to moderate hyperintensity on T2-weighted images in 30 patients (45%). In the 19 patients with histological correlation, 19 (100%) had hepatocellular necrosis, 19 (100%) had fibrosis, 18 (95%) had inflammatory cell infiltration, 17 (89%) had capillary-size vessels within fibrous septa, and 16 (84%) had ductal proliferation. No statistical correlation was found between the intensity or pattern of EHE on MR images and the extent of elevation of serological laboratory values or severity of histologic findings. CONCLUSION: Several different types of underlying chronic liver disease were observed in most of the patients with EHE. Hepatocyte necrosis, fibrosis, or inflammatory processes were found in all patients with EHE who had histopathological correlation.

Cavernous hemangiomas in patients with chronic liver disease: MR imaging findings.

The purpose of our study was to assess the difference in magnetic resonance imaging (MRI) features of cavernous hemangiomas in patients with chronic liver disease compared them with hemangiomas in normal livers. We retrospectively searched our records of MRI of the liver between October 1998 and June 2002, and identified 76 hemangiomas in 49 patients (18 men and 31 women; age range 29-81 years [mean, 57 years]). Hemangiomas were classified into 3 groups: patients with cirrhosis [group 1, 8 lesions in 8 patients], patients with chronic hepatitis [group 2, 6 lesions in 5 patients], and patients without underlying liver disease [group 3, 62 lesions in 36 patients]. Four radiologists, blinded to clinical information, retrospectively reviewed in consensus the MRI findings of hemangiomas for number, size, signal intensities on T1- and T2-weighted images, and enhancement patterns on early- and late-phase postcontrast images. The mean lesion numbers and sizes were 1.0 and 16.2 +/- 9.6 mm, 1.2 and 15.3 +/- 7.1 mm, and 1.7 and 26.1 +/- 24.7 mm in groups 1-3, respectively. There was a correlation (p < 0.05, coefficient: 0.35) between lesion number and severity of liver disease. Although there was no significant difference in lesion size among the 3 groups, all of 11 lesions larger than 4 cm in diameter belonged to group 3. Almost all lesions appeared moderately hypointense on T1-weighted images and moderately hyperintense on T2-weighted images. Twenty-seven lesions showed immediate homogeneous enhancement (pattern 1), and 49 showed peripheral nodular enhancement with centripetal enhancement progression (pattern 2). There was no difference in frequency of enhancement patterns among the 3 groups. Hemangiomas were more often solitary in livers with chronic liver disease, large lesions were exclusively seen in livers without chronic liver disease, and there was a trend for small lesions in patients with chronic liver disease.

Renal cell carcinoma in a transplanted kidney: MR imaging findings.

We report the MR findings of a 42-year-old man who developed renal cell carcinoma in an allograft kidney, 10 years after transplantation. The lower pole of the transplant kidney showed a solid lesion which was well shown on the post gadolinium fat suppressed images as a heterogeneously enhancing 2 cm mass lesion.

Hepatocellular carcinoma of diffuse type: MR imaging findings and clinical manifestations.

PURPOSE: To assess MR imaging findings and clinical manifestations of diffuse-type hepatocellular carcinoma (HCC). MATERIALS AND METHODS: We retrospectively reviewed our experience with diffuse HCC from November 1994 to October 2001. MR imaging findings and clinical features were assessed. RESULTS: Twenty-two consecutive patients with diffuse-type HCC (19 men and three women, age range 16-80 years [mean, 52 years]) were identified in a review of liver MR studies. This represented 13% of all patients with HCC imaged during this time period. Diffuse HCC showed a permeative, infiltrative pattern with ill-defined borders and no evidence of convex margination in all cases. At least 50% of the liver volume was involved with tumor. Diffuse-type HCC showed hypointensity in 15 patients, mixed intensity in three, and isointensity in four on T1-weighted images; heterogeneous hyperintensity in 16 patients; and homogeneous hyperintensity in six on T2-weighted MR images. Diffuse-type HCC showed patchy enhancement in 12 patients, miliary enhancement in nine, and minimal enhancement in one on postcontrast early-phase images, and showed heterogeneous wash-out in all patients on postcontrast late-phase images. Proximal portal venous tumor thrombosis was seen in all patients. Serum alpha-fetoprotein (AFP) value was elevated (>10 ng/mL) in 14 of 18 patients, and 13 showed a value greater than 500 ng/mL. The four patients who did not have elevated AFP had tumors which were indistinguishable from those in patients with elevated AFP; they also did not have a distinctive clinical history. CONCLUSION: Diffuse-type HCC was typically seen as an extensive, heterogeneous permeative hepatic tumor, with portal venous tumor thrombosis on MR images in all cases. Early enhancement, observed as patchy in 12 and miliary in nine of 22 patients, was a distinctive imaging feature. Elevated serum AFP value was a common finding; however, 22% had normal values.

Hoffman's syndrome: muscle stiffness, pseudohypertrophy and hypothyroidism.

Primary hypothyroidism is a chronic and insidious disease caused by failure of thyroid hormone production. We observed a 38-year-old woman admitted to our hospital due to progressive proximal weakness, muscle pain and fatigue during mild exercise. Laboratory tests showed features of rhabdomyolysis and hypothyroidism. After examination of the thyroid, we reached a diagnosis of Hashimoto's thyroiditis and hypothyroid myopathy. Hypothyroidism should be considered as a differential diagnosis of creatine kinase elevation; actually, neuromuscular symptoms and signs occur in most newly diagnosed patients with thyroid diseases. Hypothyroidism presenting as muscle stiffness and pseudohypertrophy is called 'Hoffman's syndrome'.

Epithelioid hemangioendothelioma of the liver. MR imaging findings.

We report the MR appearance of epithelioid hemangioendothelioma (EHE) in a 51 year old man, who presented with a 14-month history of fatigue and anemia to his physician. The tumor showed massive replacement of the liver and had an unusual appearance of multiple high fluid content focal lesions with diminished vascularity.