Molecular impact of selective NFKB1 and NFKB2 signaling on DLBCL phenotype.

Diffuse large B-cell lymphoma (DLBCL) has been categorized into two molecular subtypes that have prognostic significance, namely germinal center B-cell like (GCB) and activated B-cell like (ABC). Although ABC-DLBCL has been associated with NF-κB activation, the relationships between activation of specific NF-κB signals and DLBCL phenotype remain unclear. Application of novel gene expression classifiers identified two new DLBCL categories characterized by selective p100 (NF-κB2) and p105 (NF-κB1) signaling. Interestingly, our molecular studies showed that p105 signaling is predominantly associated with GCB subtype and histone mutations. Conversely, most tumors with p100 signaling displayed ABC phenotype and harbored ABC-associated mutations in genes such as MYD88 and PIM1. In vitro, MYD88 L265P mutation promoted p100 signaling through TAK1/IKKα and GSK3/Fbxw7a pathways, suggesting a novel role for this protein as an upstream regulator of p100. p100 signaling was engaged during activation of normal B cells, suggesting p100's role in ABC phenotype development. Additionally, silencing p100 in ABC-DLBCL cells resulted in a GCB-like phenotype, with suppression of Blimp, IRF4 and XBP1 and upregulation of BCL6, whereas introduction of p52 or p100 into GC cells resulted in differentiation toward an ABC-like phenotype. Together, these findings identify specific roles for p100 and p105 signaling in defining DLBCL molecular subtypes and posit MYD88/p100 signaling as a regulator for B-cell activation.

Listeria Monocytogenes: an uncommon pathogen of cervical necrotizing fasciitis.

The aim of this paper is to present a unique case of neck-necrotizing fasciitis caused by Listeria Monocytogenes in a young woman, successfully treated by surgery and IV antibiotic therapy. Necrotizing fasciitis is a rare, rapidly progressing and potentially life-threatening infection that infrequently occurs in the head and neck region. Pathogens involved in necrotizing fasciitis are heterogeneous and include aerobic and anaerobic bacteria. To the best of our knowledge, this is the only case of neck necrotizing fasciitis caused by Listeria Monocytogenes studied in literature so far.

Neonatal bacterial meningitis.

Neonatal bacterial meningitis (NM) continues to be a serious disease with an unchanging rate of adverse outcome of 20-60%, despite a worldwide decline in mortality. The 3 major pathogens in developed countries are: Group B streptococcus, gram negative rods and Lysteria monocytogenes. Signs and symptoms of NM may be subtle, unspecific, vague, atypical or absent. In order to exclude NM, all infants with proven or suspected sepsis should undergo lumbar puncture. Positive culture of cerebrospinal fluid may be the only way to diagnose NM and to identify the pathogen, as CSF parameters Smay be normal at early stages and NM may occur frequently (up to 30% of cases) in the absence of bacteraemia. When NM is suspected, treatment must be aggressive, as the goal is to achieve bactericidal concentration of antibiotics and to sterilize CSF as soon as possible. Antibiotics should be administered intravenously, at the highest clinically validated doses. Empiric antibiotic treatment should include agents active against all main pathogens; currently the recommended empiric treatment of NM is ampicillin, plus an aminoglycoside and a third-generation cephalosporn. Therapy should be reassessed after cultures and antibiotic susceptibility is available. Prevention of neonatal sepsis, early recognition of infants at risk, prompt treatment and future adjunctive therapies will improve prognosis. Finally, we present the first preliminary Italian data on GBS meningitis. Data are obtained from an area-based study conducted In Emilia-Romagna during 2003 to 2009.

Evaluation of a new rapid immunoassay for the detection of Helicobacter pylori in faeces: a prospective pilot study.

BACKGROUND: Detection of Helicobacter pylori antigen in faeces is a valid method to diagnose H. pylori infection. Presently available stool tests are performed in the laboratory, and diagnostic report is delayed. AIM: To evaluate a new rapid stool test in a pre-treatment setting and to compare it with a validated laboratory stool test. METHODS: A total of 105 patients underwent gastroscopy with brush cytology, and biopsies for histology and rapid urease test, to assess H. pylori presence. Helicobacter pylori-status was considered positive if at least two tests were positive; negative if all tests were negative; indeterminate if one test was positive and two negative. Stool specimens were tested using either a rapid immunoassay kit (ImmunoCard STAT) or a laboratory enzyme immunoassay kit (Hp StAR). RESULTS: Sixty patients were infected with H. pylori, 44 non-infected, one indeterminate. The sensitivity and specificity of ImmunoCard STAT were 85 and 93%; those of Hp StAR were 88 and 100% (not significant). CONCLUSIONS: ImmunoCard STAT seems a reliable method for detecting H. pylori in untreated patients. It could replace laboratory stool tests, as it is easy and can be performed quickly. These characteristics might be a breakthrough for diagnosing H. pylori in the doctor's office.

The immortalized UROtsa cell line as a potential cell culture model of human urothelium.

The UROtsa cell line was isolated from a primary culture of normal human urothelium through immortalization with a construct containing the SV40 large T antigen. It proliferates in serum-containing growth medium as a cell monolayer with little evidence of uroepithelial differentiation. The working hypothesis in the present study was that this cell line could be induced to differentiate and express known features of in situ urothelium if the original serum-containing growth medium was changed to a serum-free formulation. We demonstrated that the UROtsa cells could be successfully placed into a serum-free growth medium consisting of a 1:1 mixture of Dulbeco's modified Eagle's medium and Ham's F-12 supplemented with selenium (5 ng/mL), insulin (5 microg/mL), transferrin (5 microg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). Under serum-free growth conditions, confluent UROtsa cells were shown by light microscopy to produce raised, three-dimensional structures. Routine ultrastructural examination disclosed these three-dimensional areas to consist of a stratified layer of cells that strongly resembled in situ urothelium. The cells displayed numerous desmosomal connections, complex interactions of the lateral membranes, and abundant intermediate filaments within the cytoplasm. Freeze fracture analysis demonstrated that the cells possessed tight-junction sealing strands and gap junctions. The overall morphology was most consistent with that found in the intermediate layers of in situ urothelium. The basal expression patterns of the metallothionein (MT) and heat shock proteins 27, 60, and 70 were determined in these cells, and expression was in agreement with that known to occur for in situ urothelium. The cells were also successfully tested for their ability to be stably transfected using expression vectors containing the MT-3 or MT-2A genes. The findings suggest that the UROtsa cells grown with a serum-free medium could be a valuable adjunct for studying environmental insult to the human urothelium in general and for the stress response in particular.

[Current epidemiological and clinical features meningitis in a northern Italian area] / Attuali aspetti epidemiologici e clinici delle meningiti in un'area del nord Italia.

Objectives: To study etiological, epidemiological and clinical features of 97 cases of acute meningitis. Methods: Ninety-seven cases of acute meningitis were examined in adult HIV-negative patients admitted to the Infectious Diseases Unit of the Azienda Ospedale-Universita S. Anna in Ferrara. Demographic, etiological, epidemiological and clinical data were analyzed. Results: All cases were divided into two groups according to the macroscopic aspect of cerebrospinal fluid (CSF): purulent CSF (50 cases) or non-purulent CSF (47 cases). Purulent CSF meningitis more frequently affected male patients (64% vs 47%) and older patients (average 52 vs 44 years). The main epidemiological features in both groups were underlying bacterial diseases (i.e. otomastoiditis and/or sinusitis in 50% of pneumococcal meningitis) and iatrogenic immunodeficiency. From a clinical point of view the following alterations in the state of consciousness (stupor, confusion and coma) were most frequently found in purulent meningitis. The following non purulent forms of meningitis were diagnosed: 5 tubercular, 3 viral infections, 2 by Listeria monocytogenes, 1 by Entoameba histolytica, 1 by Cryptococcus neoformans and 35 (74,4%) unknown causes. Purulent meningitis were: 20 (40%) Streptococcus pneumoniae, 10 Neisseria meningitidis, 3 Staphylococcus aureus, 2 Escherichia coli, 1 Haemophilus influenzae and 1 Pseudomonas aeruginosa; 13 cases were unidentified. From 1989 to 1993 and from 1994-98 both groups of meningitis increased; respectively from 17 to 30 cases for non-purulent meningitis and from 18 to 32 cases for purulent meningitis. Meningitis due to Streptococcus pneumoniae increased from 27.7% to 46.8% during the period 1994-98. Conclusions: The study shows the high incidence of pneumococcal meningitis, during 1994-98, because a large number of patients with sinusitis and otomastoiditis were observed. The incidence of meningococcal meningitis appears stable. These data confirm the importance of timely diagnosis and correct therapy for such infections with reserved prognosis.

Tissue culture of human renal epithelial cells using a defined serum-free growth formulation.

BACKGROUND: Development of the culture of renal epithelial cells in a serum-free growth medium was driven by the need to examine the effects of hormones and other effector molecules on differentiated cell function without interference from the complex mixture of substances in serum. The present report details this laboratory's cumulative experience in the use of a defined growth medium for the propagation of epithelial cells from adult, fetal, and malignant human renal tissue. METHODS: Routine cell culture technology was used to determine the capability of a defined growth medium to support the growth of renal epithelial cells isolated by collagenase dissociation of tissue from adult and fetal kidneys, renal cell carcinoma, and Wilms' tumors. RESULTS: The defined growth medium formulation consistently allows the isolation and growth of transporting renal epithelial cells from both normal adult and fetal kidneys. This growth medium only rarely supports the growth of epithelial cells from renal cell carcinomas and Wilms' tumors. CONCLUSIONS: The method developed for the culture of human proximal tubule cells requires minimal cell culture expertise and equipment, and results in the repeatable isolation of transporting epithelial cell cultures that retain features of differentiated proximal tubule cells.

Helicobacter pylori stool antigen test: clinical evaluation and cost analysis of a new enzyme immunoassay.

Noninvasive tests for Helicobacter pylori are increasingly used. Recently, an enzyme immunoassay for H. pylori detection in feces has been put on the market. Aim of this multicenter study was to evaluate the usefulness of this novel test as a predictor of H. pylori status in the pretreatment setting. Three hundred consecutive patients were enrolled. None of the patients had received any eradicating treatment in the last 12 months, and all underwent gastroscopy with biopsies of the antrum and body for histology (H) and rapid urease test (RUT). H. pylori status was defined positive (or negative) if both H and RUT were positive (or negative). When H and RUT gave conflicting results, the patients were classified as H. pylori-indeterminate. A stool specimen was collected for each patient and tested by using a novel enzyme immunoassay for H. pylori detection (HpSAT). Sensitivity, specificity, and diagnostic accuracy of the test were calculated, as was the cost of each assay. H. pylori status was positive in 159 patients, negative in 131, and indeterminate in 10. HpSAT gave evaluable results (positive or negative) in 293 patients, and doubtful results in 7 (2.3%). Sensitivity, specificity, and diagnostic accuracy of HpSAT were 96.8%, 89.7%, and 93.6% respectively. Considering the H. pylori-indeterminate patients as positive, the percentages were 95.8%, 98.7%, and 93.2% respectively. The cost for each assay was about US $27. These results suggest that HpSAT is a noninvasive, simple, reliable, fast, and cheap method for evaluating H. pylori status in the pretreatment setting.

Evaluation of a new enzyme immunoassay for detecting Helicobacter pylori in feces: a prospective pilot study.

OBJECTIVE: There is an increasing interest in noninvasive tests for detecting Helicobacter pylori (H. pylori) infection. Unlike serological and urea breath tests, the possibility of searching for H. pylori in feces has been scarcely investigated. The aim of this prospective pilot study was to evaluate the usefulness of a new enzyme immunoassay for detecting H. pylori antigens in feces, as a predictor of H. pylori status in the pre- and posttreatment settings. METHODS: One hundred and fifty-four symptomatic, anti-H. pylori untreated patients (Group A) and 116 anti-H. pylori treated patients (Group B) underwent gastroscopy with biopsies of the antrum and corpus for histology (H) and rapid urease test (RUT). In the anti-H. pylori treated group, a 13C-urea breath test (UBT) was also performed. In Group A, H. pylori status was defined as positive or negative when both H and RUT gave concordant positive or negative results. In Group B, the patients were considered eradicated if all three tests were negative. A stool specimen was collected from all patients the day after gastroscopy, and tested by using an enzyme immunoassay commercial kit for detecting H. pylori antigens in feces (HpSAT). RESULTS: Eighty-five patients in Group A (55%) and 44 in Group B (38%) were H. pylori infected. On the whole, HpSAT showed a sensitivity of 94% and specificity of 86%. In Group A and Group B, sensitivity and specificity were 94% versus 93%, and 90% versus 82%, respectively (p < 0.05). CONCLUSIONS: HpSAT seems to be a reliable method for predicting H. pylori status in anti-H. pylori untreated patients. Conversely, the test appears less suitable to evaluate the outcome of the eradicating treatment. Consequently, it is likely to be accepted for the primary diagnosis of H. pylori status, particularly in dyspeptic young patients.

Italian guidelines for the management of infectious complications in pediatric oncology: empirical antimicrobial therapy of febrile neutropenia.

The Italian Association for Paediatric Haematology and Oncology prepared a guideline document aimed at unifying and rationalising as much as possible the management of febrile neutropenia in children with cancer, because of the potential impact of these procedures on hospital costs and on the development of antibiotic resistance. Before starting anti-infective therapy, at least 2 blood cultures, a throat swab, urine-culture, and cultures from any suspected infected site, should be performed. Routine chest X-rays at onset of febrile neutropenia are probably not necessary, in absence of respiratory signs. At the present time, the safer option probably remains the combination of a beta-lactam and an aminoglycoside, and treating febrile neutropenia outside of hospital should be considered an investigational approach. The choice of the most appropriated regimen for each institution should be based also on the local bacteriological statistics and patterns of bacterial resistance. Antibiotic toxicity and cost should be other important factors. Every subsequent addition or substitution of antibiotics should be based on objective signs of clinical deterioration. The only accepted empirical modification is empirical antifungal therapy, while the empirical addition of a glycopeptide antibiotic cannot be recommended.

Fatal sepsis from Vibrio vulnificus in a hemodialyzed patient.

Vibrio vulnificus, a particularly virulent halophilic vibrio, has been isolated from the blood and skin necrotic lesion of a hemodialyzed patient with sepsis. The patient has had exposure of the skin to seawater. Various chronic conditions including renal failure have a great risk for developing septicemia due to V vulnificus. It is necessary to inform persons with liver diseases or immunocompromising conditions of hazards associated with the consumption of undercooked seafood and seawater exposure.

Febrile complications in the first 100 days after bone marrow transplantation in children: a single center's experience.

One hundred fifty-six episodes of fever occurred in 102 children during the first 100 days after bone marrow transplantation (BMT) performed at a single institution: fever of undetermined origin (FUO), 40.3%; septicemia, 7.1%; pneumonia, 19.2%; other infections, 33.4% of cases. The overall incidence of mortality was 22.6% and of mortality due to infections 17.4%. All FUO episodes resolved. Pneumonia was the major cause of death; 60% of recipients who developed pneumonia died, accounting for 90% of deaths attributable to febrile complications. Interstitial pneumonia, occurred rarely, in 3.9% of all febrile episodes. The Cox model showed that the presence of graft-versus-host disease (GVHD) was related to an approximately ninefold increase in the risk of a first episode of FUO (P value .03). The risk of developing pneumonia was fourfold greater in children who received a transplant from a matched unrelated donor or a mismatched family donor (P value .01). Developments in diagnostic tools are needed to diagnose febrile episodes earlier and more precisely with the aim of reducing early mortality after BMT.

[Respiratory circuits and infections of the airway]. / Circuiti dei respiratori e infezioni delle vie aeree.

OBJECTIVE: To study the effects of ventilator circuit changes on the rate of airway infections and to investigate the relationship between the microorganisms responsible for circuit colonization and those responsible for infection. DESIGN: Prospective study, carried out in two different periods, of one year duration each. SETTING: General Intensive Care Units (ICUs) in a University Hospital. PATIENTS: Seventy-eight patients, requiring mechanical ventilation (VAM) for more than 5 days, were enrolled. They were divided into two groups: in the first (group I, n. 36, 1st period) the ventilator circuit was changed every 5 days; in the second (group II, n. 42, 2nd period) every 10 days. INTERVENTION: Ventilator circuit change every 5 or 10 days. Daily culvert and filling of cascade humidifiers with sterile irrigation water. Daily replacement of mount catheter. MEASUREMENTS: Qualitative cultures of tracheobronchial aspirate and of fluid from the humidifying cascades and the expiratory tubing traps. RESULTS: The two groups were similar. Pneumonia and tracheobronchitis were found in the 25% and 11% of patients of group I and in 26% and 12% of those of group II respectively. The VAM duration was 26.5 +/- 15 days in patients who developed airway infection, and 12.9 +/- 11.6 days (p < 0.001) in patients who did not. The Gram+ organisms were predominant. An identical microorganism was found both in the tracheo-bronchial aspirate and in the circuit in 44 of 78 (56%) patients; no difference was found between infected (16 of 29) and non infected (28 of 49) patients. CONCLUSIONS: Changing the ventilator circuit every 10 days rather than every 5 days, does not increase the incidence of airway infections and result in considerable savings in the expenses of tubing and personnel time. The infection or colonization rates due to the same microorganisms are quite low and it seems not useful to make routine cultures of fluid from humidifying cascades and the expiratory tubing traps in order to characterize in time the microorganism that could be responsible of airway infections.

Fluconazole versus amphotericin B as empirical antifungal therapy of unexplained fever in granulocytopenic cancer patients: a pragmatic, multicentre, prospective and randomised clinical trial.

Amphotericin B, despite its intrinsic servere toxicity, is the most commonly used empirical antifungal therapy in cancer patients with unexplained fever not responding to empirical antibacterial therapy. The aim of this study was to show whether fluconazole was as effective as, and less toxic than, amphotericin, with no effort made to compare the antifungal activity of the two drugs. A group of 112 persistently febrile (> 38 degrees C) and granulocytopenic (< 1000 cells/mm3) cancer patients, not receiving any absorbable antifungal antibiotic for prophylaxis, with a mean age of 27 years (range 1-73 years), undergoing chemotherapy for a variety of malignancies and with a diagnosis of unexplained fever after at least 96 h of empirical antibacterial therapy, were randomised to receive either fluconazole (6 mg/kg/day up to 400 mg/day) or amphotericin B (0.8 mg/kg/day) as empirical antifungal treatment. Patients were required to have normal chest X-rays at randomisation, no previous history of aspergillosis and negative surveillance cultures for Aspergillus. The intention-to-treat analysis showed defervescence and survival without treatment modification in 42 of 56 patients (75%) in the fluconazole group and in 37 of 56 (66%) in the amphotericin B group (P = 0.4). Duration of therapy was 6 days (95% CI = 4-8 days) in both groups. Death occurred in 3 patients (5%) in the fluconazole and in 2 (4%) in the amphotericin B group. No fungal death was documented in either group. Adverse events developed in 18 of 56 patients (32%) in the fluconazole group and in 46 of 56 (82%) in the amphotericin B group (P < 0.001). In the amphotericin B group, 5 patients had treatment discontinued because of toxicity, versus none in the fluconazole group, a difference which approached statistical significance (P = 0.06). This study shows that fluconazole is by far less toxic than amphotericin B and suggests that it might be as effective as amphotericin B, in pragmatical terms and for this specific indication. However, numbers are too small to allow definitive conclusions about efficacy, and the use of fluconazole for this indication remains experimental. Future studies should try to identify patients more at risk of fungal infections, with the aim of individualising antifungal approaches.

Pharmacokinetics of HD-MTX in infants, children, and adolescents with non-B acute lymphoblastic leukemia.

A retrospective pharmacokinetic analysis was done of methotrexate serum levels after high-dose treatment (HD-MTX, four cycles at two-week intervals of 5 g/sq.1m. over 24 h i.v.) in children with non-B acute lymphoblastic leukemia (ALL) with the specific aim of seeking differences in patients of different ages, including infants under one year. A total of 122 children (seven infants aged 3 months-1 year, 26 children aged 1-3 years, 68 children aged 3-10 years and 21 adolescents aged 10-15 years) with normal liver and renal function, receiving consolidation therapy at the Pediatric Clinic of Monza between May 1988 and April 1992, were enrolled in this study. MTX was given as an intravenous infusion in 24 h and serum concentrations were measured up to at least 72 h after the start of infusion by an enzyme immunoassay (TDX Abbot, Dallas, TX) in order to modulate folinic acid rescue. Pharmacokinetic analysis of MTX levels according to a two-compartment open model indicated that, compared to all children up to 10 years old, in adolescents older than 10 years the drug reached higher concentrations in serum and was cleared at a lower rate. Steady-state levels and AUC were from 60% higher to more than double and the total clearance of the compound, expressed either per square meter surface area or per kg body weight, in each cycle was significantly lower in adolescents > 10 years of age, sometimes being only one-third of the clearance in infants (0.2 vs. 0.6 1/h/kg and 6.6 vs. 10.7 1/h/sq.m). The relationship between each age and systemic clearance was highly significant as measured by regression analysis. Methotrexate systemic clearance progressively decreased as a function of age. Subsequent treatments did not induce changes in MTX pharmacokinetics. These data suggest that the better tolerance of HD-MTX in children may have a pharmacokinetic basis. The faster elimination of MTX in infants, who usually show the worst prognosis, suggests that full doses could be safely used in order to maximize the antileukemic effect without a high risk of toxicity.