An unusually long-lasting outbreak of community-acquired Legionnaires' disease, 2005-2008, Italy.

An unusually long-lasting community-acquired outbreak of Legionnaires' disease (LD) occurred in the inhabitants of a town in northern Italy from 2005 to 2008. Overall, 43 cases were diagnosed including five deaths. Hundreds of water samples were collected for Legionella isolation but only two clinical samples were obtained. Clinical strains were ST23 as were environmental isolates detected in most Legionella-positive patients' homes and those from a public fountain. Although no Legionella was found in the municipal water mains, a continuous chlorination was applied in 2008. This action resulted in a halving of cases, although incidence remained tenfold higher than the Italian average incidence until the end of 2013, when it dropped to the expected rate. Retrospective analyses of prevalent wind direction suggested that a hidden cooling tower could have been the main cause of this uncommon outbreak, highlighting the importance of implementation of cooling tower registers in supporting LD investigations.

Allopurinol hypersensitivity is primarily mediated by dose-dependent oxypurinol-specific T cell response.

BACKGROUND: Allopurinol is a main cause of severe cutaneous adverse reactions (SCAR). How allopurinol induces hypersensitivity remains unknown. Pre-disposing factors are the presence of the HLA-B*58:01 allele, renal failure and possibly the dose taken. OBJECTIVE: Using an in vitro model, we sought to decipher the relationship among allopurinol metabolism, HLA-B*58:01 phenotype and drug concentrations in stimulating drug-specific T cells. METHODS: Lymphocyte transformation test (LTT) results of patients who had developed allopurinol hypersensitivity were analysed. We generated allopurinol or oxypurinol-specific T cell lines (ALP/OXP-TCLs) from allopurinol naïve HLA-B*58:01(+) and HLA-B*58:01(-) individuals using various drug concentrations. Their reactivity patterns were analysed by flow cytometry and (51) Cr release assay. RESULTS: Allopurinol allergic patients are primarily sensitized to oxypurinol in a dose-dependent manner. TCL induction data show that both the presence of HLA-B*58:01 allele and high concentration of drug are important for the generation of drug-specific T cells. The predominance of oxypurinol-specific lymphocyte response in allopurinol allergic patients can be explained by the rapid conversion of allopurinol to oxypurinol in vivo rather than to its intrinsic immunogenicity. OXP-TCLs do not recognize allopurinol and vice versa. Finally, functional avidity of ALP/OXP-TCL is dependent on both the induction dose and HLA-B*58:01 status. CONCLUSIONS AND CLINICAL RELEVANCE: This study establishes the important synergistic role of drug concentration and HLA-B*58:01 allele in the allopurinol or oxypurinol-specific T cell responses. Despite the prevailing dogma that Type B adverse drug reactions are dose independent, allopurinol hypersensitivity is primarily driven by oxypurinol-specific T cell response in a dose-dependent manner, particular in the presence of HLA-B*58:01 allele.

Anti-leucocyte antibodies in platelet apheresis donors with and without prior immunizing events: implications for TRALI prevention.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) prevention strategies in platelet (PLT) apheresis donors focus on identifying antileucocyte antibody-positive donors. The use of microbead based assays for screening purposes is hampered by the lack of a consensus cut-off for TRALI prevention and the undefined role of anti-leucocyte antibodies in never-alloexposed donors. This study evaluated anti-leucocyte antibody assays in PLT apheresis donors with and without prior immunizing events with special focus on microbead assay cut-offs, antibody specificities and their potential significance in never-alloexposed donors. MATERIAL AND METHODS: Blood samples of male and female PLT apheresis donors with and without history of prior immunization were tested for anti-leucocyte antibodies. RESULTS: Of 262 female and 118 male PLT apheresis donors, 37·4% had prior immunizing events. Fifty-eight of 238 (24·4%) donors without prior immunizing event had anti-HLA antibodies confirmed in microbead single antigen assay (mean fluorescence intensity (MFI) >500). Even with a cut-off MFI >3000, anti-HLA antibodies were detected in 10·6% of female and 4·3% of male donors without history of immunization. Of the antibody specificities found, 6 of 17 (35·3%) anti-HLA-A, 4 of 8 (50·0%) anti-HLA-B and 4 of 6 (66·6%) anti-HLA class II antibodies have been detected in donors associated with TRALI cases in the literature. CONCLUSION: Platelet apheresis donors without history of immunization have anti-leucocyte antibodies that potentially can cause TRALI. In our opinion, this cohort should be included in screening strategies for TRALI prevention. As references and consensus cut-offs have not yet been established, it is premature to use microbead assays as standard for donor screening.

Short-term additional enfuvirtide therapy is associated with a greater immunological recovery in HIV very late presenters: a controlled pilot study.

OBJECTIVES: To evaluate whether the addition of enfuvirtide to standard highly active antiretroviral therapy (HAART) could confer immunovirological benefits in human immunodeficiency virus (HIV)-infected very late presenters. The current study is an open comparative therapeutic trial of standard protease inhibitor (PI)-based HAART ± additional enfuvirtide in treatment-naïve deeply immunologically impaired HIV-positive patients. METHODS: Very late presenters (CD4 <50/mm(3)), without tuberculosis and neoplasms, were alternatively allocated to two nucleoside reverse transcriptase inhibitors (NRTIs) and lopinavir/ritonavir without (control arm, CO) or with (ENF arm) enfuvirtide 90 mg bid. Enfuvirtide was administered until the achievement of viral load <50 copies/ml and for at least 24 weeks. The primary objective was the magnitude of CD4+ cell recovery at 6 months. HIV RNA was intensively monitored in the first month, and, thereafter, monthly, as for CD4+ cell count and percentage, clinical data, and plasma drug concentrations. RESULTS: Of 22 enrolled patients (11 per arm), 19 completed the study (10 in the ENF arm). Baseline CD4+ cell counts and % were comparable, with 20 CD4+/mm(3) (12-37) and a percentage of 3.3 (1.7-7.1) in the ENF arm, and 16 CD4+/mm(3) (9-29) and a percentage of 3.1 (2.3-3.8) in the CO arm, respectively. The baseline viral load was also comparable between the two arms, with 5.77 log10 (5.42-6) and 5.39 log10 (5.06-6) in the ENF and CO arms, respectively. Enfuvirtide recipients had higher CD4+ percentage at week 8 (7.6 vs. 3.6%, p = 0.02) and at week 24 (10.7 vs. 5.9%, p = 0.02), and a greater CD4+ increase at week 24 (207 vs. 134 cells/mm(3), p = 0.04), with 70% of enfuvirtide intakers versus 12.5% of controls who achieved a CD4+ cell count >200/mm(3) (p = 0.01). At 48 weeks, patients in the ENF arm had CD4+ cell counts higher than controls (251 vs. 153cells/mm(3), p = 0.04) and were also found to be faster in reaching a CD4 cell count over 200/mm(3): 18 (8-24) versus 48 (36-108) weeks (p = 0.01). Viral load decay at week 4 was greater in the ENF arm (-3 vs. -2.2 log, p = 0.04), while the proportion of patients with viral load <50 copies/ml at week 24 was comparable. CONCLUSIONS: In this pilot study, the addition of enfuvirtide to a lopinavir-based HAART was shown to be associated with a significantly faster and greater immunological recovery in newly discovered HIV-positive patients with very low CD4+ cell counts. Induction strategies using an enfuvirtide-based approach in such subjects warrant further investigation.

An international trial of quantitative PCR for monitoring Legionella in artificial water systems.

AIMS: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. METHODS AND RESULTS: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l(-1) ) and culture (CFU l(-1) ) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l(-1) always exceeded CFU l(-1) , and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. CONCLUSIONS: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.

Cluster of travel-associated Legionnaires disease in Lazise, Italy, July to August 2011.

Since 18 August 2011, 17 cases of travel-associated Legionnaires' disease have been reported. They were tourists from five European countries who had stayed in five accommodation sites in Lazise, Italy. The dates of symptom onset ranged from 18 July to 25 August 2011. Control measures were implemented and no further cases associated with stays at the sites have been reported after disinfection. Timely notification of any further cases potentially associated with stay in Lazise is recommended.

A new mechanism of NK cell cytotoxicity activation: the CD40-CD40 ligand interaction.

NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2-activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBL-derived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2-activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.

Molecular basis of the Rh antigen RH48 (JAL).

BACKGROUND AND OBJECTIVES: RH48 (JAL) is a low-incidence Rh antigen of unknown molecular background associated with weakened expression of RhCE antigens. The objective of this study was to establish the molecular basis of JAL. MATERIALS AND METHODS: Seventeen JAL+ samples, from seven black (one of them a Brazilian of mixed race: black/Caucasian), nine European Caucasians and one Asian individuals, were typed with anti-D, -C, -c, -E and -e. Some samples were also tested for V/VS and ce (f). Titration studies and flow cytometry were used to analyse the expression of the JAL antigen and genomic DNA sequencing of all RHCE exons was conducted on all samples. Routine genotyping for RHCE was carried out on all samples. Screening of RHD exons 1-10, which included detection of the DAU allele, was carried out on all except one of the black samples. The Caucasian samples and remaining black sample were screened for the DAU mutation 1136C>T (T379M). RESULTS: Six black individuals had the Dce haplotype with RHCE mutations 340C>T (R114W) and 733C>G (L245V) [V/VS] and the RHD mutation T379M [DAU]. One mixed race individual had the Dce haplotype with the RHCE mutation 340C>T (R114W) but without the V/VS or DAU mutation. Eight Caucasians had the DCe haplotype with the 340C>T mutation. One Caucasian and one Asian had the Dce haplotype with a different mutation in an adjacent nucleotide, 341G>A (R114Q). All Caucasian individuals were negative for the DAU mutation 1136C>T (T379M). Previously described weakness of CE-related Rh antigens when present in single dose on JAL+ samples of DCe and Dce haplotypes was observed. Weak expression of V/VS was observed in the three black samples tested and weakness of JAL was observed in the black samples compared to the Caucasian samples. CONCLUSION: The same mutation (340C>T, R114W) in two different haplotypes (DCe and Dce) and another mutation (341G>A, R114Q) in one of these haplotypes (Dce) are associated with expression of the JAL antigen. One of the RHCE mutations detected in our samples (340C>T) has been previously described but not in association with the JAL antigen. Our results indicate that the previously described RhCeMA and ce(s)(340) alleles encode the JAL antigen. Expression of V/VS antigen is weakened in the presence of JAL and expression of JAL is usually weaker when associated with the Dce haplotype compared to DCe.

Production of an egg yolk antibody against Parietaria judaica 2 allergen.

Specific antibodies are essential tools for studying proteins as well as for diagnostic research in biomedicine. The egg yolk of immunized chicken is an inexpensive source of high-quality polyclonal antibodies. The 12-kDa Parietaria judaica 2 allergen was expressed as a fusion protein and was used to immunize Leghorn chickens. In this paper, we show, using 2-dimensional gel electrophoresis and immunoblotting, that chicken antibodies raised against a recombinant allergen can be used to recognize similar proteins from a pollen raw extract. Allergen identity was confirmed by nanoLC-nanospray-tandem mass spectrometry analysis. Our data demonstrate for the first time that a synergistic combination of molecular biology, 2-dimensional PAGE, and use of nonmammalian antibodies represents a powerful tool for reliable identification of allergens.

Long-term evaluation of the efficacy on the podalic support and postural control of a new elastic functional orthopaedic device for the correction of Class III malocclusion.

AIM: Correlations between occlusion and posture are open to new perspectives, which include treatment of functional alterations traditionally approached separately. The aim of this study is to evaluate whether the treatment of Class III malocclusion, through an innovative elastic functional orthopaedic device, allows an overall improvement of the podalic support. MATERIALS AND METHODS: A 5½-year-old patient with Angle Class III malocclusion and c anterior ross bite in deciduous dentition has been treated for 7 years with a functional orthopaedic device (MSB Class III). Assessment of frontal and lateral postural plumb line was performed with stabilo-baro-podometric platform analysis, in order to record the podalic support discrepancy between feet, both in static phase and in dynamic phase. The patient has been posturally re-evaluated at nine and twelve years old. RESULTS: The functional device allowed the restoration of the correct intermaxillary relationship, favourably conditioning also the posture. In particular, the correction of the valgus flat foot and a significative reduction of the podalic support discrepancy between feet has been obtained. CONCLUSIONS: A global approach to the patient can successfully address both malocclusion and postural alterations.

[What would you do in front of a patient with a Horner syndrome?] / ¿Qué haría usted ante un paciente con síndrome de Horner?

Horner's syndrome (HS) occurs when there is disruption to the oculosympathetic pathway. Its features include eyelid ptosis, miosis and anhidrosis. The aetiology of this syndrome is varied and includes tumours, trauma, vascular disease and iatrogenic. Different pharmacologic tests are used for diagnosis, such as cocaine, hydroxyamphetamine and apraclonidine; while neuroimaging helps elucidating the aetiology. We present a case of a 63-year-old female referred to our service with a 4-month history of right eyelid ptosis. During examination right miosis was noted. The patient reported a history of multinodular goiter. Pharmacologic tests and neuroimaging confirmed the diagnosis of HS secondary to thyroid disease.

How to mitigate the risk of inducing transfusion-associated adverse reactions.

Transfusion has become extremely safe but can still be associated with adverse reactions. Some adverse reactions can be mitigated by applying measures to donor selection, the process of separating blood components as well as hospital-based procedures consisting in matching the donor and the recipient; special attention is given to optimizing the best fit between the component and the beneficiary, which is not only an immuno-hematological challenge (fresh versus old blood, testing for certain viruses such as CMV, parvovirus B19, etc.). Considerable progress has also been achieved to strengthen the overall quality and safety of the whole transfusion chain. Guidelines and recommendations have resulted in substantial progress, and the recent revisiting of patients as part of a more holistic approach has enabled blood management programs to be created. Such programs, when wisely applied in a context of optimal blood use, reinforce patient safety; they enhance hospital recognition of transfusion and hemovigilance specialists as useful players acting in the interests of patients in full compliance with hospital budgets. This review considers the step-by-step processes that reinforce transfusion safety and identifies hurdles that cannot yet be properly addressed; it proposes steps for further progress, in light of personalized medicine.

Male breast cancer with mandibular metastasis. A case report.

Female breast cancer is one of the major causes of death among women while male breast cancer is relatively uncommon and accounts for about 1% of all breast cancers in both sexes. Influencing factors are: gynecomasty, familiarity for male breast cancer, Jewish and African-American male population. From the histological point of view, it is not different from the female breast cancer, except for the infiltrant ductal carcinoma, but with a much severe prognosis. Breast cancer metastases to the jaws are rare, only 1%; the most common sites of metastases are: lungs(59-69%), liver (58-65%), bone (44-71%), pleura (23-37%), brain (9-22%) and kidney (4-17%). At present, based on a literature research (May 2006), there have been just two other case reports of male breast cancer metastasis to the maxillofacial region, both to the mandible. The case of a 69-year-old white man who in 2001 underwent a radical mastectomy due to ductal breast cancer is reported. In 2005 the patient was referred to our department by his oncologist for multiple oral fistula. A mandibular TC revealed osteolytic lesions and the patient underwent mandibular surgery to remove the lesions and clean up the area. The histological examination was consistent with that of a metastatic deposit of adenocarcinoma of the breast. In June 2006 the patient died due to worsening of the general clinical conditions, in particular due to ascites and hepatic insufficiency.

Growth of vertically aligned carbon nanofibers from nickel nanodot arrays produced from diblock copolymer thin film templates.

Diblock copolymers of polystyrene and poly(methyl methacrylate) are utilized in a thin film to create a nanoscale template, which is used to deposit a nanoscale array of nickel nanodots. Systematic experiments show that this process must be optimized to successfully transfer the order of the diblock copolymer template to the resultant nickel nanodot array. It is found that the amount of nickel deposited dramatically impacts the fidelity of the final nanodot array, with a thickness ratio for copolymer:metal of 8:1 found to be optimal. The results also indicate that the method by which the surface is neutralized for the diblock domain alignment impacts the amount of nickel nanodots that remain on the surface after template removal. Finally, the nanoscale array of nickel nanodots is utilized to successfully grow vertically aligned carbon nanofibers from 18 nm and 40 nm diameter nickel dots.

Expression of major histocompatibility complex class I antigens in normal and transformed rat thyroid epithelial cell lines.

Recent evidence suggests that the expression of abnormally high amounts of major histocompatibility complex (MHC) class I molecules may be a feature of at least some kinds of transformed cells. To investigate this aspect of neoplastic transformation we studied the expression of MHC class I antigens in an experimental model of normal, tumor-derived, and virus-transformed thyroid epithelial cell lines. The expression of MHC class I antigens has been studied by means of several monoclonal antibodies directed against either monomorphic or polymorphic epitopes and quantified by flow cytometry. Class I specific mRNA transcripts have been also analyzed by Northern blot hybridization, using a mouse genomic H-2 DNA probe. Our results indicate a modulation of MHC class I expression associated with loss of the differentiated phenotype and with transformation of thyroid epithelial cell lines. Undifferentiated cells in fact show a small quantity of these antigens, because acquisition of a fully differentiated phenotype is associated with an increase in their expression. Cell lines derived from thyroid tumors show reduced expression of MHC class I antigens, as compared to differentiated cells. Conversely, cells transformed in vitro by a retrovirus carrying the v-raski oncogene exhibit an increase in these antigens in comparison to their normal differentiated counterparts. Cells infected with a mutant virus able to transform cells only at the permissive temperature of 33 degrees C show a similar increased expression. After a shift to the nonpermissive temperature of 39 degrees C, infected cells, even those losing the transformed phenotype retain the same increased amount of MHC class I antigens. Our data suggest that the modulation of MHC class I antigen expression is strongly associated with transformation in thyroid epithelial cells.

The relationship of modulation of major histocompatibility complex class I antigens to retrovirus transformation in rat cell lines.

The expression of major histocompatibility complex (MHC) Class I antigens has been studied, by means of monoclonal antibodies directed against nonpolymorphic determinants of MHC Class I molecules, in two epithelial differentiated cell lines (FRTL-5 clone 2 and PC clone 3) and in one fibroblast cell line (FRT Fibro) of Fischer rat thyroid origin, before and after infection with various acute retroviruses carrying the v-ras-Ha, v-mos, v-src, polyoma middle T, and c-myc oncogenes. The results obtained indicate that a single virus does not produce identical changes in MHC Class I molecule expression in all tested lines, but a general increase occurs in lines derived from FRTL-5 clone 2 and a decrease occurs in lines derived from PC clone 3 and from FRT Fibro. Thus the modulation of expression seems to proceed always in the same direction in each cell line regardless of the infecting retrovirus and appears to involve posttranscriptional mechanisms, since no modification of expression of mRNA levels has been observed between normal and transformed cells. Only one line of PC clone 3 origin, transformed by the cooperation of two oncogenes (human c-myc and middle T), almost completely lost MHC Class I antigens on the cell surface and presented a significantly reduced synthesis of Class I mRNA.

Sentinel node biopsy after neoadjuvant treatment in breast cancer: Five-year follow-up of patients with clinically node-negative or node-positive disease before treatment.

PURPOSE: It is controversial whether sentinel node biopsy (SNB) without axillary dissection (AD) should be performed in cN1/2 breast cancer patients who become cN0 after neoadjuvant treatment, since the false negative rate (FNR) may be unacceptably high. We assessed outcomes to address this issue. METHODS: We retrospectively assessed 396 cT1-4, cN0/1/2 patients, who became or remained cN0 after neoadjuvant treatment and underwent SNB with at least one sentinel node (SN) found, and AD not performed if the SN was negative. RESULTS: After a median follow-up of 61 months (interquartile range 38-82), five-year overall survival was 90.7% (95% CI, 87.7-93.7) in the whole cohort, 93.3% (95% CI, 90.0-96.6) in those initially cN0, and 86.3% (95% CI, 80.6-92.1) in those initially cN1/2 (P = 0.12). Axillary failure occurred in only 1 (0.7%) initially cN1/2 patient who became cN0. In initially cN0 patients, and also initially cN1/2 patients who responded well to neoadjuvant treatment (ypT0/ypTx), SN-negativity was a significant predictor of good outcome, consistent with the known prognostic significance of axillary status, and suggesting that SN status accurately reflected axillary status. By contrast, in initially cN1/2 patients found to be ypT1/2/3, SN status (and whether or not AD was performed) had no influence on survival. CONCLUSIONS: These findings suggest that SNB is acceptable in cN1/2 patients who become cN0 after neoadjuvant therapy.

Proteomic analysis of protein components in periodontal ligament fibroblasts.

BACKGROUND: Characterization of periodontal ligament (PDL) fibroblast proteome is an important tool for understanding PDL physiology and regulation and for identifying disease-related protein markers. PDL fibroblast protein expression has been studied using immunological methods, although limited to previously identified proteins for which specific antibodies are available. METHODS: We applied proteomic analysis coupled with mass spectrometry and database knowledge to human PDL fibroblasts. RESULTS: We detected 900 spots and identified 117 protein spots originating in 74 different genes. In addition to scaffold cytoskeletal proteins, e.g., actin, tubulin, and vimentin, we identified proteins implicated with cellular motility and membrane trafficking, chaparonine, stress and folding proteins, metabolic enzymes, proteins associated with detoxification and membrane activity, biodegradative metabolism, translation and transduction, extracellular proteins, and cell cycle regulation proteins. CONCLUSIONS: Most of these identified proteins are closely related to the extensive PDL fibroblasts' functions and homeostasis. Our PDL fibroblast proteome map can serve as a reference map for future clinical studies as well as basic research.