Detection of drug-responsive B lymphocytes and antidrug IgG in patients with ß-lactam hypersensitivity.

BACKGROUND: Delayed-type ß-lactam hypersensitivity develops in subset of patients. The cellular immunological processes that underlie the drug-specific response have been described; however, little is known about involvement of the humoral immune system. Thus, the aim of this study was to utilize piperacillin hypersensitivity as an exemplar to (i) develop cell culture methods for the detection of drug-specific B-cell responses, (ii) characterize drug-specific IgG subtypes and (iii) assess reactivity of IgG antibodies against proteins modified to different levels with piperacillin haptens. METHODS: IgG secretion and CD19+ CD27+ expression on B cells were measured using ELISPOT and flow cytometry, respectively. A piperacillin-BSA adduct was used as an antigen in ELISA antibody binding studies. Adducts generated using different ratios of drug to protein were used to determine the degree of conjugation required to detect IgG binding. RESULTS: B cells from hypersensitive patients, but not controls, were stimulated to secrete IgG and increase CD27 expression when cultured with soluble piperacillin. A piperacillin-BSA adduct with cyclized and hydrolysed forms of the hapten bound to eight lysine residues was used to detect hapten-specific IgG 1-4 subclasses in patient plasma. Hapten inhibition and the use of structurally unrelated hapten-BSA adducts confirmed antigen specificity. Antibody binding was detected with antigens generated at piperacillin/BSA ratios of 10:1 and above, which corresponded to a minimum epitope density of 1 for antibody binding. CONCLUSION: These data show that antigen-specific B lymphocytes and T lymphocytes are activated in piperacillin-hypersensitive patients. Further work is needed to define the role different IgG subtypes play in regulating the iatrogenic disease.

The chemical, genetic and immunological basis of idiosyncratic drug-induced liver injury.

Idiosyncratic drug reactions can be extremely severe and are not accounted for by the regular pharmacology of a drug. Thus, the mechanism of idiosyncratic drug-induced liver injury (iDILI), a phenomenon that occurs with many drugs including ß-lactams, anti-tuberculosis drugs and non-steroidal anti-inflammatories, has been difficult to determine and remains a pressing issue for patients and drug companies. Evidence has shown that iDILI is multifactorial and multifaceted, which suggests that multiple cellular mechanisms may be involved. However, a common initiating event has been proposed to be the formation of reactive drug metabolites and covalently bound adducts. Although the fate of these metabolites are unclear, recent evidence has shown a possible link between iDILI and the adaptive immune system. This review highlights the role of reactive metabolites, the recent genetic innovations which have provided molecular targets for iDILI, and the current literature which suggests an immunological basis for iDILI.

HLA-DQ allele-restricted activation of nitroso sulfamethoxazole-specific CD4-positive T lymphocytes from patients with cystic fibrosis.

BACKGROUND: For certain HLA allele-associated drug hypersensitivity reactions, the parent drug has been shown to associate directly with the risk allele. In other forms of hypersensitivity, HLA risk alleles have not been identified and T cells are activated in an allele unrestricted manner. Chemically reactive drug metabolites bind to multiple proteins; thus, it is assumed that the derived peptide antigens interact with a number of HLA molecules to activate T cells; however, HLA restriction of the drug metabolite-specific T-cell response has not been studied. OBJECTIVE: To utilize T cells from sulfamethoxazole (SMX) hypersensitive patients with cystic fibrosis to examine the HLA molecules that interact with nitroso SMX (SMX-NO)-derived antigens. METHODS: T-cell clones were generated from 4 hypersensitive patients. Drug-specific proliferative responses and cytokine secretion were measured. Anti-human class I and class II antibodies were used to analyse HLA restriction. Antigen-presenting cells expressing different HLA molecules were used to determine the alleles involved in the presentation of SMX-NO-derived antigens to T cells. RESULTS: A total of 976 clones were tested for SMX-NO reactivity. Thirty-nine CD4+ clones were activated with SMX-NO and found to proliferate and secrete cytokines. The SMX-NO-specific response was blocked with an antibody against HLA-DQ. SMX-NO-specific responses were detected with antigen-presenting cells expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. CONCLUSION AND CLINICAL RELEVANCE: HLA-DQ plays an important role in the activation of SMX-NO-specific CD4+ T cells. Detection of HLA-DQ allele-restricted responses suggests that T cells are activated by a limited repertoire of SMX-NO-modified peptides.

Trends in American Board of Psychiatry and Neurology specialties and neurologic subspecialties.

OBJECTIVE: To review the current status and recent trends in the American Board of Psychiatry and Neurology (ABPN) specialties and neurologic subspecialties and discuss the implications of those trends for subspecialty viability. METHODS: Data on numbers of residency and fellowship programs and graduates and ABPN certification candidates and diplomates were drawn from several sources, including ABPN records, Web sites of the Accreditation Council for Graduate Medical Education and the American Medical Association, and the annual medical education issues of the Journal of the American Medical Association. RESULTS: About four-fifths of neurology graduates pursue fellowship training. While most recent neurology and child neurology graduates attempt to become certified by the ABPN, many clinical neurophysiologists elect not to do so. There appears to have been little interest in establishing fellowships in neurodevelopmental disabilities. The pass rate for fellowship graduates is equivalent to that for the "grandfathers" in clinical neurophysiology. Lower percentages of clinical neurophysiologists than specialists participate in maintenance of certification, and maintenance of certification pass rates are high. CONCLUSION: The initial enthusiastic interest in training and certification in some of the ABPN neurologic subspecialties appears to have slowed, and the long-term viability of those subspecialties will depend upon the answers to a number of complicated social, economic, and political questions in the new health care era.

Detection of GD2-positive cells in bone marrow samples and survival of patients with localised neuroblastoma.

The impact of bone marrow (BM) GD2-positive cells on survival has been evaluated in 145 Italian children with localised neuroblastoma (NB) evaluated at diagnosis by anti-GD2 immunocytochemistry. Nineteen of these (13.1%) were found to be BM GD2-positive, with the number of positive cells ranging between 1 and 155 out of 1 x 10(6) total cells analysed. Seven/19 (38.8%) GD2-positive vs 12/126 (9.5%) GD2-negative patients relapsed. The 5-year event-free survival (EFS) and overall survival of the GD2-positive patients was significantly worse than that of the GD2-negative ones (62.2 vs 89.9%, P<0.001; and 74.9 vs 95.9%, P=0.005, respectively). GD2 positivity was not associated to other known risk factors, and in particular to Myc-N amplification and 1p deletion. Among Myc-N-negative patients, the EFS of those negative for both GD2 and 1p deletion was significantly better than in children positive for either one of these two markers (EFS=96.9 vs 66.0%, P<0.001). In conclusion, GD2 positivity may represent a prognostic marker for patients with non-metastatic NB without Myc-N amplification, and its combination with genetic alterations might help identifying patients that require a more careful follow-up.

Temperature influences whole-animal rates of metabolism but not protein synthesis in a temperate intertidal isopod.

The effects of temperature on two important biological rate processes, whole-body rates of oxygen uptake (M dot o2) and protein synthesis (k(s)), were investigated in the temperate intertidal isopod Ligia oceanica at two different times of the year. Animals were collected in January (winter) and June (summer) and either subjected to an acute temperature change after 24 h (acclimatized) or acclimated to various temperatures for 4 wk. In both cases, M dot o2 increased with temperature, with a Q(10) of 2.2 between 5 degrees and 20 degrees C, but increased in thermal sensitivity at 25 degrees C. Winter isopods were characterized by significantly higher M dot o2 levels, greater thermal sensitivities, and lower thermal tolerances than summer animals. Seasonal differences in M dot o2 persisted after acclimation, indicating that temperature alone was not responsible for the changes. In sharp contrast, whole-body k(s) showed no variation with temperature, although overall rates decreased upon acclimation. In acclimatized animals, k(s) was higher in the summer than in the winter. After acclimation, a compensatory increase in RNA capacity in winter animals reversed this situation. The temperature independence of whole-body k(s) in L. oceanica could ensure survival in a highly liable thermal environment, as thermal tolerances of intertidal invertebrates are thought to be more closely related to protein than to energy metabolism.

Characterisation of attaching-effacing Escherichia coli isolated from animals at slaughter in England and Wales.

Escherichia coli isolates were recovered from faecal samples taken from cattle, sheep and pigs at slaughter in England and Wales. Isolates (n = 1227) selected at random from this collection were each hybridised in colony dot-blot experiments with an eae gene probe that presumptively identified attaching-effacing E. coli (AEEC). Of the 99 (8.1%) eae positive isolates 72 were of ovine origin, 24 were of bovine origin and three of porcine origin. None were typed as O157:H7 whereas 78 were assigned to 23 serogroups and 21 were untypable. The most frequently isolated eae positive serogroups were O156 (10), O26 (8), O103 (8), O108 (7) O56 (6) and O168 (6) of which serogroups O103 and O156 only were recovered from all three animal species. In tissue culture adherence assays, 36 representatives of eae positive isolates of all serogroups and host of origin tested induced intimate attachment with varying degrees of actin accumulation and pedestal formation in the HEp-2 cells. The identity of the eae type for these 36 was determined by specific PCR and the most prevalent intimin types were eaebeta (15), eaegamma (12) and eae (4). Isolates were examined by PCR for the presence of other virulence determinants and five possessed stx1 but none possessed stx2. One O115 eae isolate possessed cnf1 and 2, hlyA, etpD and katP genes which is a novel combination of virulence determinants.

The DNA sequence and comparative analysis of human chromosome 10.

The finished sequence of human chromosome 10 comprises a total of 131,666,441 base pairs. It represents 99.4% of the euchromatic DNA and includes one megabase of heterochromatic sequence within the pericentromeric region of the short and long arm of the chromosome. Sequence annotation revealed 1,357 genes, of which 816 are protein coding, and 430 are pseudogenes. We observed widespread occurrence of overlapping coding genes (either strand) and identified 67 antisense transcripts. Our analysis suggests that both inter- and intrachromosomal segmental duplications have impacted on the gene count on chromosome 10. Multispecies comparative analysis indicated that we can readily annotate the protein-coding genes with current resources. We estimate that over 95% of all coding exons were identified in this study. Assessment of single base changes between the human chromosome 10 and chimpanzee sequence revealed nonsense mutations in only 21 coding genes with respect to the human sequence.

The DNA sequence and analysis of human chromosome 13.

Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.

Acceleration threshold detection during short anterior and posterior perturbations on a translating platform.

Balance control systems have usually been studied under two conditions, during quiet standing or under large postural perturbations of a magnitude that requires a postural adjustment to prevent falling. Between these two extremes lie perturbations that can be repeated and measured while not forcing adaptive strategies from the postural control system. Unlike other studies of postural control, we employed very short translations with varying accelerations at the edge of psychophysical detectability. These perturbations were vibration-free anterior or posterior translations of the platform on which a subject stood. Using a full Latin-square design set of perturbations in the forward or backward direction, with a smooth or jerk acceleration profile, and of length 4 or 20 mm, were presented to five subjects. Perceptual peak acceleration thresholds were determined by an iterative psychophysical method that forced the subjects to choose in which of two sequential intervals that they perceived a stimulus to have been presented. The only factor found that significantly correlated with detection was perturbation length. The 4 mm peak thresholds averaged 14.51 mm/s2 while 20 mm thresholds averaged 8.55 mm/s2. For the short perturbations employed in this study, detection of motion thus was dependent upon the magnitude of the acceleration, but it was independent of the acceleration profile (jerk versus smooth) or movement direction. By understanding the influences on the ability to perceptually detect motion underfoot, we can begin to understand what elements of the postural control system might be involved in the second-to-second control of balance.

Transfer of macrophage-derived mycobacterial antigens to dendritic cells can induce naïve T-cell activation.

Mycobacteria are capable of surviving and replicating in host macrophages, where they can release antigenic material into the environment. However, unlike dendritic cells (DCs), macrophages do not appear to be capable of activating naïve T cells. Therefore, this work investigated antigen transfer between macrophages and DCs. We generated culture supernatants from bacille Calmette-Guérin (BCG)-infected and uninfected macrophages and then determined whether DCs could present these extracellular mycobacterial antigens to T cells. Here, we show that DCs pulsed with antigens released from BCG-infected macrophages can stimulate primed T cells in vitro and initiate naïve T-cell responses in vivo. These results suggest that antigen transfer can occur between macrophages and DCs.

Influenza hemagglutinin peptides fused to interferon gamma and encapsulated in liposomes protects mice against influenza infection.

The immunogenicity of a peptide vaccine may be improved by fusing antigen to a cytokine and administering this chimeric protein in a particulate delivery system. We have investigated this using a vaccine comprising an immunodominant T cell epitope and a B cell epitope from influenza haemagglutinin (HATB) fused to interferon gamma and encapsulated in liposomes (HATB/IFN-gamma/lipo). Controls comprised groups receiving HATB/IFN-gamma mixed with liposomes, HATB incorporated in liposomes or heat inactivated PR8 influenza virus (HI PR8). IFN-gamma production in mice treated with HATB/IFN-gamma/lipo was significantly higher than in mice inoculated with either HATB/IFN-gamma mixed with liposomes or HATB incorporated in liposomes but less than HI PR8. Lung viral titres were significantly lower in mice treated with HATB/IFN-gamma/lipo compared with those treated with HATB/IFN-gamma mixed with liposomes. HI PR8-treated mice recorded a nil viral titre. There was no correlation between the level of antibody production and clearance of virus from the lungs. These data suggest that particulate delivery systems may be useful adjuncts to improve immune responses to chimeric proteins and to induce protection against disease.

A collaborative clerkship with a focus on rural community health.

The Deans' Rural Primary Care Clerkship was developed through the collaborative efforts of South Carolina's two medical schools. The clerkship provides students an innovative learning experience in rural community medicine through the unique combination of learning opportunities with community-oriented primary care, continuous quality improvement, interdisciplinary health care teams, and cultural competency. Much of students' learning addresses current directives for population health training. The positive experience students are having in these rural, underserved South Carolina communities will help them better understand the rewards and challenges of rural, community-responsive health care.

IL-2 linked to a peptide from influenza hemagglutinin enhances T cell activation by affecting the antigen-presentation function of bone marrow-derived dendritic cells.

Chimeric proteins containing antigen linked to cytokines have shown some promise as vaccine candidates but little is known of their mechanism of action, particularly at the level of the antigen-presenting cell. We have investigated this using a chimeric protein in which an immunodominant T cell epitope from influenza hemagglutinin peptide (HA), recognized in the context of I-E(d), was fused to IL-2. Immature murine dendritic cells (DC) derived from bone marrow (BMDC) were used to present the chimeric protein to a T cell hybridoma with TCR specific for the HA peptide (A5 cell line). HA-IL-2 was found to induce significantly higher T cell activation than HA alone. Although the inclusion of IL-2 and HA separately did increase the response of A5 cells compared to HA alone, they were not as effective as the HA-IL-2 chimeric protein. When an antibody known to block IL-2 receptor alpha chain (CD25) was included, A5 activation was reduced, suggesting a role for the receptor in this process. Expression of CD25 on A5 cells was low during activation, implying that the effect was mediated by CD25(+) BMDC. Antigen uptake and processing of HA-IL-2 by BMDC was required since fixing BMDC, prior to antigen exposure, greatly reduced their ability to activate A5 cells. The function of CD25 on DC is currently unknown. Our results suggest this receptor may play a role in antigen uptake and subsequent T cell activation by receptor-mediated endocytosis of antigen attached to IL-2. This finding that may have implications for the development of a new generation of vaccines.

Structure of the Maize streak virus geminate particle.

The Geminiviridae is an extensive family of plant viruses responsible for economically devastating diseases in crops worldwide. Geminiviruses package circular, single-stranded DNA (ssDNA) genomes. The characteristic twinned or "geminate" particles, which consist of two joined, incomplete T = 1 icosahedra, are unique among viruses. We have determined the first structure of a geminivirus particle, the Nigerian strain of Maize streak virus (MSV-N), using cryo-electron microscopy and three-dimensional image reconstruction methods. The particle, of dimensions 220 x 380 A, has an overall 52-point-group symmetry, in which each half particle "head" consists of the coat protein (CP) arranged with quasi-icosahedral symmetry. We have modeled the MSV-N CP as an eight-stranded, antiparallel beta-barrel motif (a structural motif common to all known ssDNA viruses) with an N-terminal alpha-helix. This has produced a model of the geminate particle in which 110 copies of the CP nicely fit into the reconstructed density map. The reconstructed density map and MSV-N pseudo-atomic model demonstrate that the geminate particle has a stable, defined structure.

In vivo cytoreduction studies and cell sorting--enhanced tumor-cell detection in high-risk neuroblastoma patients: implications for leukapheresis strategies.

PURPOSE: To improve autologous leukapheresis strategies in high-risk neuroblastoma (NB) patients with extensive bone marrow involvement at diagnosis. PATIENTS AND METHODS: Anti-G(D2) immunocytochemistry (sensitivity, 1 in 10(5) to 10(6) leukocytes) was used to evaluate blood and bone marrow disease at diagnosis and during the recovery phase of the first six chemotherapy cycles in 57 patients with stage 4 NB and bone marrow disease at diagnosis. A total of 42 leukapheresis samples from the same patients were evaluated with immunocytology, and in 24 of these patients, an anti-G(D2) immunomagnetic enrichment step was used to enhance tumor-cell detection. RESULTS: Tumor cytoreduction was much faster in blood compared with bone marrow (3.2 logs after the first cycle and 2.1 logs after the first two cycles, respectively). Bone marrow disease was often detectable throughout induction, with a trend to plateau after the fourth cycle. By direct anti-G(D2) immunocytology, a positive leukapheresis sample was obtained in 7% of patients after either the fifth or sixth cycle; when NB cell immunomagnetic enrichment was applied, 25% of patients had a positive leukapheresis sample (sensitivity, 1 in 10(7) to 10(8) leukocytes). CONCLUSION: Standard chemotherapy seems to deliver most of its in vivo purging effect within the first four cycles. In patients with overt marrow disease at diagnosis, postponing hematopoietic stem-cell collection beyond this point may not be justified. Tumor-cell clearance in blood seems to be quite rapid, and earlier collections via peripheral-blood leukapheresis might be feasible. Immunomagnetically enhanced NB cell detection can be highly sensitive and can indicate whether ex vivo purging should be considered.