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.

Estimation of peptide concentration by a modified bicinchoninic acid assay.

Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 degrees C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 degrees C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.

The European searchable tumour line database.

The European Searchable Tumour line Database (ESTDAB) ( http://www.ebi.ac.uk/ipd/estdab ) is a freely available and fully searchable database of melanoma-derived cell lines, which have been characterised for over 250 immunologically relevant markers by a consortium of European scientists. The database is linked to a cell bank, which can provide melanoma cell lines to non-profit investigators for a nominal handling charge. All cells are fully HLA typed at the genomic and surface expression levels. The expression of a number of surface antigens, apoptotic markers, tumour-associated antigens and extracellular matrix proteins has also been determined. Cytokine secretion has been tested and polymorphisms in cytokine genes have been identified. Glycans at the cell surface were identified and glycosyltransferase activity quantified. Cell lines with a particular constellation of these parameters can be sought online via the ESTDAB interface, which is included as part of the Immuno-Polymorphism Database (IPD) section of the European Bioinformatics Institute's (EBI) website.

High purity and yield of natural Tregs from cord blood using a single step selection method.

Natural regulatory T cells (Tregs), characterized as CD4 CD25high Foxp3+, have been described as paramount contributors in immuno-regulation and self-tolerance. CD4 and CD25 have been the main markers used for their isolation, resulting in cells with potent suppressive properties. Nevertheless, low purity and yield continue to be an issue when attempting thorough characterizations and/or up scaling to bigger models and for clinical trials. Here we present a single-step methodology optimized for cord blood CD25+ isolation, using magnetic microbeads that achieves a reproducible purity of 89% for CD4 CD25high CD127low. These cells showed a more consistent suppressive effect in mixed lymphocyte cultures. In addition, the proportion of contaminating effector T cells was < 9% whilst the yield of Tregs was doubled compared to the standard protocol. Gating on CD4 CD25high CD127low populations post isolation showed better correlation with suppressive efficacy compared to CD4 CD25+ gate. These data should facilitate the clinical scale-up of this procedure to obtain consistent Tregs for clinical application and research.

Layer guided-acoustic plate mode biosensors for monitoring MHC-peptide interactions.

The transduction signals from the immobilisation of a class I heavy chain, HLA-A2, on a layer guided acoustic plate mode device, followed by binding of beta(2)-microglobulin and subsequent selective binding of a target peptide are reported.

High frequency of homozygosity of the HLA region in melanoma cell lines reveals a pattern compatible with extensive loss of heterozygosity.

Malignant transformation of cells is frequently associated with abnormalities in human leukocyte antigen (HLA) expression. MHC class I loss or down-regulation in cancer cells is a major immune escape route used by a large variety of human tumours to evade antitumour immune responses mediated by cytotoxic T lymphocytes. The goal of our study was to explore HLA genotyping and phenotyping in a variety of melanoma tumour cell lines. A total of 91 melanoma cell lines were characterised for HLA class I and II genotype. In addition, 61 out of the 91 cell lines were also analysed for HLA class I and II cell surface molecule expression by flow cytometry. Unexpectedly, we found that 19.7% of the melanoma cell lines were homozygous for HLA class I genotypes, sometimes associated with HLA class II homozygosity (8.79%) and sometimes not (10.98%). The frequency of homozygosity was significantly higher compared with the control groups (1.6%). To identify the reasons underlying the high frequency of HLA homozygosity we searched for genomic deletions using eight pairs of highly polymorphic microsatellite markers covering the entire extended HLA complex on the short arm of chromosome 6. Our results were compatible with hemizygous deletions and suggest that loss of heterozygosity on chromosome arm 6p is a common feature in melanoma cell lines. In fact, although autologous normal DNA from the patients was not available and could not be tested, the retention in some cases of heterozygosity for a number of microsatellite markers would indicate a hemizygous deletion. In the rest of the cases, markers at 6p and 6q showed a single allele pattern indicating the probable loss of part or the whole of chromosome 6. These results led us to conclude that loss of heterozygosity in chromosome 6 is nonrandom and is possibly an immunologically relevant event in human malignant melanoma. Other well-established altered HLA class I phenotypes were also detected by flow cytometry that correspond to HLA class I total loss and HLA-ABC and/or specific HLA-B locus down-regulation.

Progress made towards the development of a CMV peptide vaccine.

Cytomegalovirus disease still remains a major cause of morbidity and mortality in hematopoietic stem cell transplantation recipients. The cell-mediated immune response is essential in the maintenance of latency and the resolution of primary infections. The identification of immunodominant cytomegalovirus antigens has enabled researchers to determine the best candidate antigens to be included in a cytomegalovirus vaccine. Such a vaccine would have to stimulate both a cell-mediated and humoral immune response. Recent advances have enabled the rapid identification of minimal cytotoxic epitopes required to trigger such responses. Epitope mapping to date has mainly focused on the pp65 antigen but other antigens such as IE1 are starting to be mapped. A human leukocyte antigen allele hierarchy is starting to emerge that is dependent on the alleles present in an individual; this is relevant when considering what peptides should be included in a vaccine. This review looks at the current methods available for epitope mapping and the progress that has been made to date.

Heat-shock protein 60-reactive CD4+CD28null T cells in patients with acute coronary syndromes.

BACKGROUND: CD4+CD28null T cells are present in increased numbers in the peripheral blood of patients with acute coronary syndrome (ACS) compared with patients with chronic stable angina (CSA). The triggers of activation and expansion of these cells to date remain unclear. METHODS AND RESULTS: Twenty-one patients with ACS and 12 CSA patients with angiographically confirmed coronary artery disease and 9 healthy volunteers were investigated. Peripheral blood leukocytes were stimulated with human cytomegalovirus (HCMV), Chlamydia pneumoniae, human heat-shock protein 60 (hHSP60), or oxidized LDL (ox-LDL). CD4+CD28null cells were separated by flow cytometry and assessed for antigen recognition using upregulation of interferon-gamma and perforin mRNA transcription as criteria for activation. CD4+CD28null cells from 12 of 21 patients with ACS reacted with hHSP60. No response was detected to HCMV, C pneumoniae, or ox-LDL. Incubation of the cells with anti-MHC class II and anti-CD4 antibodies but not anti-class I antibodies blocked antigen presentation, confirming recognition of the hHSP60 to be via the MHC class II pathway. Patients with CSA had low numbers of CD4+CD28null cells. These cells were nonreactive to any of the antigens used. Circulating CD4+CD28null cells were present in 5 of the 9 healthy controls. None reacted with hHSP60. CONCLUSIONS: We have shown that hHSP60 is an antigen recognized by CD4+CD28null T cells of ACS patients. Endothelial cells express hHSP60 either constitutively or under stress conditions. Circulating hHSP60-specific CD4+CD28null cells may, along other inflammatory mechanisms, contribute to vascular damage in these patients.

CD4(+) bias in T cells cloned from a CML patient with active graft versus leukemia effect.

BACKGROUND: The ability to generate a GvL response by infusion of donor leukocytes (DL) in patients following relapse after BMT is now well documented and has been demonstrated to be particularly effective in patients with CML. METHODS: We generated T-cell lines from a patient who was undergoing an active GvL response following withdrawal of immunosuppression for cytogenetic relapse of CML. Cryopreserved pre-transplant leukemic cells were used as stimulators, to generate T-cell lines and oligoclonal lines from the lymphocytes. In total 38 sub-lines were generated from different bulk cultures. The lines were tested for their proliferative and cytotoxic capability to patient pre-transplant leukemic cells, PHA-transformed lymphoblasts, allogeneic CML cells, and autologous and allogeneic B-LCL. RESULTS: Four of the cloned lines tested recognized the patient's pre-transplant leukemic cells. Specifically, two were both cytotoxic and proliferative in response to patient leukemic cells and two were cytotoxic only. Six clonal lines recognized PHA blasts only and were proliferative; one was specific for PHA blasts and CML cells. The sub-lines were phenotyped for cell-surface markers and all were CD4(+) CD8(-) CD 16/56(-). The proliferative response of the leukemia-specific clonal lines could be blocked with anti-MHC Class II MAbs. DISCUSSION: These data suggest that CD4(+) cells play a crucial role in mediating the GvL effect in CML patients. Our observations can be used to delineate strategies for enhancing and investigating the GvL effect in CML.

HLA tetramers and anti-CMV immune responses: from epitope to immunotherapy.

BACKGROUND: Identification of HLA class I-restricted CMV epitopes, and the subsequent synthesis of HLA class I-peptide tetrameric complexes, have provided investigators with an important tool for visualising and quantifying the precise in vivo CTL response to CMV reactivation following stem cell transplantation. In conjunction with PCR-monitoring of the viral load, the magnitude and dynamic of the host's specific CD8(+) T cell response to viral replication can be studied. METHODS: CMV peptide epitopes can be identified be searching the CMV-pp65 antigen for HLA class I allele binding motifs, by testing their binding affinity and ability to generate CTLs, and by screening for CTL responses in as many individuals as possible to assess their general applicability for monitoring large number of patients. HLA tetramers are synthesized by refolding recombinant class I heavy chains and beta(2)m with CMV-pp65(495-503) peptide. After biotinylation and tetramerisation to PE-conjugated streptavidin, they are used to stain CD8(+) T cells taken from patients at different time points after SCT. RESULTS: The T-cell mediated immune response is mainly directed against epitopes derived from the CMV tegument protein pp65. CMV-specific CTL's confer protection against CMV reactivation above a threshold level of 10(7) to 2 x 10(7)/L. CMV reactivation is required to stimulate CTL responses. Transfer of CMV immunity from seropositive donors is associated with better outcome and steroids suppress the Ag-specific immune response. DISCUSSION: Initial studies with CMV-specific HLA class I tetramers have helped to define the nature of anti-CMV T cell response in SCT patients and to determine a threshold CTL level required for controlling CMV reactivation. Monitoring patients with HLA-tetramers should therefore allow clinicians to predict and assess the risk of reactivation and to balance the risks and benefits of early anti-viral treatment, thereby avoiding the hazards of anti-viral prophylaxis. HLA-tetramers can also be used to isolate antigen-specific cells for further in vitro expansion and transfer to patients for antiviral immunotherapy. The threshold level determined from patient monitoring can be used as a guide for estimating an effective target cell dose.

Direct evidence that leukemic cells present HLA-associated immunogenic peptides derived from the BCR-ABL b3a2 fusion protein.

The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3-positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.

Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants.

The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

[Vertical transmission of the hepatitis C virus]. / La trasmissione verticale del virus dell'epatite C.

The incidence of hepatitis C in childhood is approximately 0.4%. The mode of transmission can be parenteral, sexual, occupational and also vertical. The latter has an incidence that varies widely and it increases in the case of human immuno-deficiency virus (HIV) coinfection and high titers of HCV in the mother. The vertical transmission is not influenced by breast feeding, however, data are discrepant with regard to child delivery (cesarean section vs vaginal delivery). Ninety-seven babies born from mothers with hepatitis C from 1996 to 1999, were evaluated prospectively in the Day Hospital of the Pediatric Department of Parma. The protocol of observation established a blood sampling for titers of antibodies anti-HCV and HCV-RNA at the 3rd trimester of pregnancy and subsequent clinical and biochemical controls at 3-6-9-12-15 and 18 months. Thirty (31.2%) out of the 96 mothers evaluated were positive for antibodies anti-HCV and 66 (68.8%) were positive for antibodies anti-HCV and HCV-RNA. Five (5.15%) out of the 97 babies evaluated were infected by HCV. Of these 4 were delivered vaginally and 1 by cesarean section. Of the 3 babies born to mothers with HIV coinfection, none was infected by HIV, but 1 was infected by HCV. Vertical transmission is increased by HCV viral load or HIV coinfection in the mother. The vaginal delivery and breastfeeding do not represent an additional risk factor.

[Risk of materno-fetal transmission of the HIV infection with antiretroviral therapy and cesarean section: experience of the Parma group]. / Rischio di trasmissione materno-fetale dell'infezione da HIV mediante terapia antiretrovirale e taglio cesareo: esperienza del gruppo di Parma.

Vertical transmission of HIV is by far the most important way of infection in pediatric patients. Transmission rate of infection varies between 15-40% in the absence of antiretroviral prophylaxis. Only 2% of infected pregnant women who underwent caesarean section and zidovudine treatment transmitted the infection to their newborns. From January 1995 to September 2000 twenty seropositive pregnant women and their twenty newborns were followed at the Azienda Ospedaliera of Parma. Nine women (45%) were treated with only zidovudine according to the ACTG 076 protocol; eight women (40%) continued the treatment they were assuming before pregnancy with the eventual addition of zidovudine. 3 women (15%) were not treated because HIV infection was only detected after delivery. 15 women underwent caesarean section, in 13 cases in association to antiretroviral prophylaxis: in the remaining 2 cases no intrapartum treatment was started due to the urgency of delivery. The rate of vertical transmission among the 20 women was 5% (1/20), significantly less then that observed (20.5%) among 31 pregnant HIV women followed in Parma from January 1987 to December 1994 and not treated with antiretroviral prophylaxis and/or cesarean section (Magnani G. Personal data). The only infected baby was born by vaginal delivery. No transmission was observed in the group of pregnant women who underwent the combination of antiretroviral prophylaxis and cesarean section.

A high resolution HLA class I and class II matching method for bone marrow donor selection.

HLA matching in bone marrow transplantation has an important role in determining successful outcome. However HLA typing of both potential related and unrelated donors can be both time-consuming and laborious, and does not always resolve accurately the true level of histocompatibility. We have utilised a method, reference strand mediated conformation analysis (RSCA), which is technically simple and allows high resolution matching for all HLA loci, for the typing of 48 patients and their potential 120 donors. The results indicate that RSCA can detect many mismatches that are not routinely identified by conventional HLA typing methods. In addition, RSCA can be applied for the simultaneous analysis of multiple potential BM donor samples in order to quickly identify the best match for each patient.

[Atypical cat-scratch disease: a case report of splenic granulomatosis]. / La malattia da graffio di gatto atipica: descrizione di un caso di granulomatosi splenica.

Generally cat-scratch disease is a benign inflammatory adenopathy. The Authors describe an atypical form of this disease, characterized by persistent fever and splenic granulomatosis requiring a diagnostic and therapeutic prolonged effort. They point out the important role of new immuno-fluorescent techniques to exactly identify the bacterium--Bartonella henselae--causing cat-scratch disease and suggest to include cat-scratch disease among the causes of unknown origin fever.

[Liver damage and obesity in pediatric age]. / Alterazioni epatiche e obesità in età pediatrica.

In adult obese patients both an increase of aminotranspherase values and hepatic steatosis have been frequently showed. Conversely in childhood the existence of a liver's damage is often not investigated. To assess the prevalence of hepatic alterations in obese children, we studied 135 subjects, all affected by simple obesity, showing in a 20% of them the presence of ultrasonographic evidence of hepatic steatosis and/or hyperaminotransferasemia. Our study demonstrates the existence of silent hepatic alterations also in obese children and suggests to improve the treatment of obesity in childhood to prevent the progression of liver's damage.