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.

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.

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.

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.

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.

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.

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.

Retro-orbital late relapse in a child with leukaemia after allogeneic bone marrow transplantation.

Relapse after allogeneic bone marrow transplantation (BMT) usually occurs in the bone marrow and is often associated with a poor prognosis. Isolated extramedullary relapse following BMT is exceedingly rare. We observed an isolated relapse in the left retro-orbital region of a 13-year-old girl, 3 years after BMT performed for acute lymphoblastic leukaemia in third complete remission. Computerized tomography revealed a tumor at the inferomedial part of the orbit infiltrating the maxillary and ethmoid sinuses and nasal cavities and also involving the rectus muscles. Histology demonstrated a monomorphic leukaemia infiltrate. Complete disappearance of the retro-orbital mass was obtained with chemotherapy and local irradiation.

Pulmonary infections in lung cancer patients at diagnosis.

We carried out a prospective study involving 96 consecutive lung cancer patients at diagnosis, in order to determine through quantitative cultures of the bronchoalveolar lavage (BAL) fluid, the prevalence of pulmonary infections; we also evaluated the relationship between a patient's performance status, immunocompetence, lung cancer stage, histotype and the occurrence of respiratory infections. The patients (81 males, 15 females) had a mean age of 64 +/- 9 years. Of these, 62 were smokers, 30 were ex-smokers and four had never smoked. Sixty-seven patients had a prior history of chronic bronchitis. A total of 42 micro-organisms were cultured from the BAL fluids of 33 patients (34.3%). Fifty percent of these micro-organisms were gram-negative, 33.3% were gram-positive and the remaining 16.7% were other micro-organisms. The bacilli most often isolated were the Haemophilus species, accounting for 38.8% of all gram-negative bacilli. The most frequently isolated gram-positive pathogen was the Staphylococcus aureus. We have not found a significant relationship between the presence of a respiratory infection and the different cell types separately analyzed, nor with SCLC and NSCLC patient groups, nor with the stage of the disease. The performance status, the immunoregulatory ratio and the lymphocyte subsets were not significantly different in patients with or without a pulmonary infection. We think that the identification of a definite etiologic agent is of great importance for a rational anti-microbial treatment of pulmonary infections.

Microgranular variant of acute promyelocytic leukemia in children.

PURPOSE: The microgranular variant (M3v) of acute promyelocytic leukemia (APL) rarely has been reported in a pediatric series of acute nonlymphoblastic leukemia (AnLL). We reviewed the clinical and biologic features of childhood M3v cases in our AnLL series. PATIENTS AND METHODS: From January 1970 to January 1991, 11 children with M3v were admitted and treated at our center. A diagnosis was made according to French-American-British (FAB) criteria. Morphologic examination, cytochemical analysis, and immunophenotyping were performed by a single pathologist. From January 1984, the diagnosis was confirmed by cytogenetic and, subsequently, by molecular analysis on frozen material. RESULTS: In our series, the overall incidence of children with APL was unusually high, 31.2% of the AnLL and M3v constituted one case in every four cases of APL. Even restriction of the analysis to the time when either cytogenetic and DNA studies confirmed the diagnosis, the incidence did not change. The immunophenotype of M3v cases was identical to that described for the hypergranular type, but an unexpected association of CD2 with M3v was shown. The onset was characterized by marked hyperleukocytosis (median WBC count, 87 x 10(9)/L) unlike classic APL. Disseminated intravascular coagulation (DIC) was always present and severe. Hyperleukocytosis and DIC were responsible for the high incidence of deaths for hemorrhagic events in the first days after onset (eight of 11 patients). CONCLUSIONS: In our experience, for unknown reasons, M3v may occur in childhood more than generally was considered. The clinical course and prognosis seem worse in M3v than in typical APL cases.

Bone marrow transplantation for childhood leukemia: five years' experience in a pediatric hematology center.

BACKGROUND: Fifty-three children (39 male, 14 female, median age 9 years 3 months) with different forms of leukemia underwent bone marrow transplantation (BMT) at our center. Various conditioning regimens were used according to the disease and time of BMT. In this paper we evaluate the impact of the experience of a pediatric hematology center on BMT-related problems in children. METHODS: We analyzed disease-free survival (DFS), early BMT-related effects, hepatic, cardiac and respiratory function and late endocrine effects as shown by standard instrumental and laboratory tests. RESULTS AND CONCLUSIONS: Outcome (overall median follow-up 34 months) was satisfactory. Three years DFS was 50.1% in all patients, 58.8% in lymphoblastic leukemia in 2nd complete remission (CR), and 50.0% in acute myeloid leukemia (some in 2nd or subsequent CR). Three of four patients with chronic myeloid leukemia were alive at 38 months. Management of the problems causing early post-BMT toxicity contributed to a progressive fall in early morbidity and mortality (14.3% in the last 3 years). Pre-BMT hepatitis in most patients was not associated with increased post-BMT hepatotoxicity. Cardiac function, even in patients given aggressive anthracycline treatment before BMT, remained normal 3 years after transplantation, as did respiratory function, although 8 cases presented subclinical restrictive and/or obstructive alterations. Compensated hypothyroidism was observed in 9 patients. Six boys received replacement treatment for hypogonadism. Severe height impairment was seen in 2 patients. Post-BMT endocrine and growth effects require a longer follow-up for definitive conclusions to be drawn.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated testicular relapse in a boy with acute lymphoblastic leukemia following allogeneic bone marrow transplantation.

About 5-10% of boys with acute lymphoblastic leukemia (ALL) present with isolated testicular relapse. Very frequently these relapses occur during treatment or in the following six months, and in these cases the prognosis is very severe. The patients are usually treated with radiotherapy and chemotherapy or bone marrow transplantation (BMT). Testicular relapses after BMT are relatively rare. We report the case of a child with ALL who presented testicular relapse during therapy and was treated with local radiotherapy (2000 cGy), chemotherapy and allogeneic matched BMT. The preparative regimen consisted of Cyclophosphamide (60 mg/kg/day x 2 days) and total body irradiation (200 cGy x 2/day x 3 days). Engraftment was documented at day + 14. The patient presented again with testicular relapse at day + 146, and was therefore treated with orchiectomy, local radiotherapy and systemic chemotherapy. A marrow relapse followed, however, at day + 284 and the patient died of progressive disease.