Does the placebo effect on hot flashes depend on the placebo dose?

PURPOSE: To investigate the presence of a placebo dose-response effect in four randomized, double-blind, placebo-controlled, multi-dose hot flash clinical trials conducted at Mayo Clinic. METHODS: Hot flash score, frequency, and hot flash-related distress for each placebo dose level were summarized at each time point by mean and standard deviation and changes from baseline were plotted to visualize a possible placebo dose-effect response. Furthermore, a meta-analysis was conducted for each endpoint in the highest and lowest dosage arms across the four trials. RESULTS: Longitudinal plots of mean hot flash scores, frequencies, and hot flash-related distress scores in patients taking placebo in each study showed a decline in hot flash scores over time without any clinically meaningful differences between the lowest and highest dosage arms in each study. The meta-analysis for each endpoint in the highest and lowest dosage arms across the four trials revealed no clinically important differences either. CONCLUSION: While the current study cannot rule out the existence of a placebo dose-response effect in multi-dose placebo-controlled trials in patients with hot flashes or other conditions, it suggests, along with the available data in the placebo literature, that, at least in well-conducted multi-dose clinical trials in which the placebo was used as control, such an effect, if it exists at all, should be very small. Therefore, pooling data from different placebo subgroups is unlikely to compromise the validity of comparisons between the combined placebo arms and each treatment arm.

[Epidemiology, treatment and mortality in infection by carbapenemase-producing Enterobacteriaceae: retrospective study]. / Epidemiología, tratamiento y mortalidad en pacientes infectados por enterobacterias productoras de carbapenemasas: estudio retrospectivo.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) has become a significant problem in terms of public health and clinical outcome. OBJECTIVE: To assess the epidemiology, treatment and mortality in patients with infection due to CPE. MATERIAL AND METHODS: A retrospective analysis of 163 patients with CPE infection was carried out in a university hospital from July 2013 to October 2015. RESULTS: A total of 163 patients were included over the study period. Klebsiella pneumoniae was isolated in 95.1% of cases, and most of carbapenemases belonged to the OXA-48 group (93.0%). Acquisition was nosocomial in 124 cases (77.0%), healthcare-associated in 30 (18.6%), and 7 cases (4.3%) were community-acquired. The most frequent infections identified in this study were urinary tract (48.4%) and respiratory (19.5%) infections. Approximately half of the patients received antibiotic monotherapy. The 30-day mortality rate was 23.3%. Multivariate analysis revealed that the presence of septic shock at diagnosis (OR 4.2; IC 95% 1.5-11) was independently associated with an increase in death during the first month, unable to identify association with inappropriate antibiotic treatment. DISCUSSION: Further studies are needed to clarify whether antibiotic treatment of EPC infections should be combined or if monotherapy might be sufficient in mild infections.

Epidemiología, tratamiento y mortalidad en pacientes infectados por enterobacterias productoras de carbapenemasas: estudio retrospectivo / Epidemiology, treatment and mortality in infection by carbapenemase-producing Enterobacteriaceae: retrospective study

Resumen Introducción: Las enterobacterias productoras de carbapenemasas (EPC) suponen un reto para la salud pública y la práctica clínica. Objetivo: Analizar la epidemiología, el tratamiento y la mortalidad en pacientes infectados por EPC. Material y Métodos: Análisis retrospectivo de 163 pacientes infectados por EPC en un hospital universitario desde julio de 2013 a octubre de 2015. Resultados: Klebsiella pneumoniae fue aislada en 95,1% de los casos, y la mayoría de las carbapenemasas pertenecían al grupo OXA-48 (93%). La adquisición fue nosocomial en 124 casos (77%), asociada a cuidados sanitarios en 30 (18,6%), y 7 (4,3%) fueron de adquisición comunitaria. Las infecciones más frecuentes fueron las del tracto urinario (48,4%) y las respiratorias (19,5%). Aproximadamente, la mitad de los pacientes recibieron monoterapia antimicrobiana. La tasa de mortalidad a los 30 días fue de 23,3%. El análisis multivariante identificó que la presencia de shock séptico al diagnóstico (OR 4,2; IC 95% 1,5-11) estaba asociada de manera independiente con mayor mortalidad en el primer mes, sin lograr identificar asociación con el tratamiento antimicrobiano inapropiado. Discusión: Son necesarios más estudios para aclarar si el tratamiento antimicrobiano de las infecciones por EPC debe ser combinado o si podría ser suficiente la monoterapia en infecciones leves.

Abstract Background: Carbapenemase-producing Enterobacteriaceae (CPE) has become a significant problem in terms of public health and clinical outcome. Objective: To assess the epidemiology, treatment and mortality in patients with infection due to CPE. Material and Methods: A retrospective analysis of 163 patients with CPE infection was carried out in a university hospital from July 2013 to October 2015. Results: A total of 163 patients were included over the study period. Klebsiella pneumoniae was isolated in 95.1% of cases, and most of carbapenemases belonged to the OXA-48 group (93.0%). Acquisition was nosocomial in 124 cases (77.0%), healthcare-associated in 30 (18.6%), and 7 cases (4.3%) were community-acquired. The most frequent infections identified in this study were urinary tract (48.4%) and respiratory (19.5%) infections. Approximately half of the patients received antibiotic monotherapy. The 30-day mortality rate was 23.3%. Multivariate analysis revealed that the presence of septic shock at diagnosis (OR 4.2; IC 95% 1.5-11) was independently associated with an increase in death during the first month, unable to identify association with inappropriate antibiotic treatment. Discussion: Further studies are needed to clarify whether antibiotic treatment of EPC infections should be combined or if monotherapy might be sufficient in mild infections.

Effect of sorting boar spermatozoa by sex chromosomes on oviduct cell binding.

This study examined the hypothesis that flow sorting sperm by sex chromosomes affects oviduct cell binding which would influence formation of the sperm reservoir in the oviduct. The sperm-rich fraction from boars (n = 5) was collected, sperm were stained with Hoechst 33342 and sorted. Sperm were sorted based on the presence of either an X or Y chromosome and placed into the following treatments: 1) sperm selected for the Y chromosome, 2) selected for the X, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were tested for oviduct cell binding within 12 h of sorting. Additionally, sperm were analyzed for motility characteristics, acrosome status, and binding to the two oviduct glycan motifs that bind porcine sperm, biantennary 6-sialylated N-acetyllactosamine on a mannose core (bi-SiaLN) and sulfated LeX trisaccharide (suLeX). The disaccharide found within both glycan motifs, N-acetyllactosamine (LacNAc), was used as a control. Sperm binding to oviduct cells was reduced by more than half in the three sorted samples when compared to the control sperm that were not sorted. The percentage of sperm that were motile 24 h after sorting was also decreased significantly in each of the sorted sample groups when compared to the unsorted control. In contrast, sorting did not decrease the percentage of sperm that bound purified soluble glycans or the location on sperm to which they bound. There was also no difference in sperm acrosome status among the four groups. In summary, sorting reduced sperm binding to the complex matrix around oviductal cell aggregates but sperm binding to purified soluble oviduct glycans was not affected. The requirement for higher affinity and motility to bind glycans immobilized on oviduct cells may explain this difference. The reduction in sperm fertility observed following sex-sorting may be explained partially by a reduced or altered ability to bind to the oviduct epithelium.

Biolayer interferometry predicts ELISA performance of monoclonal antibody pairs for Plasmodium falciparum histidine-rich protein 2.

Predicting antibody pair performance in a sandwich format streamlines development of antibody-based diagnostics and laboratory research tools, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFAs). We have evaluated panels of monoclonal antibodies against the malarial parasite biomarker Plasmodium falciparum histidine rich protein 2 (HRP2), including 9 new monoclonal antibodies, using biolayer interferometry (BLI) and screened antibody pairs in a checkerboard ELISA. This study showed BLI predicts antibody pair ELISA performance for HRP2. Pairs that included capture antibodies with low off-rate constants and detection antibodies with high on-rate constants performed best in an ELISA format.

Phase II study of temozolomide (TMZ) and everolimus (RAD001) therapy for metastatic melanoma: a North Central Cancer Treatment Group study, N0675.

OBJECTIVE: Mammalian target of rapamycin (mTOR) pathway is activated in malignant melanoma and in situ lesions as opposed to benign nevi. Inhibition of PI3K-Akt-mTOR signaling is implicated in sensitization of melanoma cells to alkylating agents (temozolomide [TMZ]) and inhibition of tumor angiogenesis. METHODS: We conducted a single-arm phase II multi-institution cooperative group study to assess the antitumor activity and safety profile of the combination of TMZ and the rapamycin derivative everolimus in patients with metastatic unresectable malignant melanoma. Patients received 10 mg/d of RAD001 for 5 of 7 days (ie, 50 mg/wk) and 200 mg/m/d of TMZ for 5 days each cycle. RESULTS: Of the first 39 eligible patients, 17 were PFS-9 successes, for a predetermined threshold of 18/39 patients for a positive trial. Overall, 21 of 48 patients were progression free at 9 weeks, for an event-free survival rate of 44% (95% confidence interval, 29%-59%). The median progression-free survival was 2.4 months and the median overall survival was 8.6 months. Four patients achieved a partial response; the median duration of response was 15.1 months. No complete remissions were observed. Treatment was in general well tolerated with only 1 patient discontinuing therapy due to toxicity (hyperlipidemia). CONCLUSIONS: The combination of TMZ and RAD001 was well tolerated but failed to meet/exceed our study threshold for promising clinical activity in patients with metastatic melanoma.

A randomized phase 2 study of temozolomide and bevacizumab or nab-paclitaxel, carboplatin, and bevacizumab in patients with unresectable stage IV melanoma : a North Central Cancer Treatment Group study, N0775.

BACKGROUND: Increasing evidence shows chemotherapy in combination with vascular endothelial growth factor (VEGF) inhibition is a clinically active therapy for patients with metastatic melanoma (MM). METHODS: A phase 2 trial was conducted in chemotherapy-naive patients with unresectable stage IV MM who were randomized to temozolomide (200 mg/m(2) on days 1 through 5) and bevacizumab (10 mg/kg intravenously on days 1 and 15) every 28 days (Regimen TB) or nab-paclitaxel (100 mg/m(2) , or 80 mg/m(2) post-addendum 5 secondary to toxicity, on days 1, 8, and 15), bevacizumab (10 mg/kg on days 1 and 15), and carboplatin (area under the curve [AUC] 6 on day 1, or AUC 5 post-addendum 5) every 28 days (Regimen ABC). Accrual goal was 41 patients per regimen. The primary aim of this study was to estimate progression-free survival rate at 6 months (PFS6) in each regimen. A regimen would be considered promising if its PFS6 rate was > 60%. RESULTS: Ninety-three eligible patients (42 TB and 51 ABC) were enrolled. The majority of patients had M1c disease (20 TB and 26 ABC). The median PFS and overall survival times with ABC were 6.7 months and 13.9 months, respectively. Median PFS time and median overall survival with TB were 3.8 months and 12.3 months, respectively. The most common severe toxicities (≥ grade 3) in both regimens were cytopenias, fatigue, and thrombosis. Among the first 41 patients enrolled onto each regimen, PFS6 rate was 32.8% (95% confidence interval: 21.1%-51.2%) for TB and 56.1% (90% confidence interval: 44.7%-70.4%) for ABC. CONCLUSIONS: The addition of bevacizumab to nab-paclitaxel and carboplatin shows promising activity despite tolerability issues.

Epratuzumab with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy in patients with previously untreated diffuse large B-cell lymphoma.

Approximately 60% of patients with diffuse large B-cell non-Hodgkin lymphoma (DLBCL) are curable with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemoimmunotherapy. Epratuzumab (E) is an unlabeled anti-CD22 monoclonal antibody with efficacy in relapsed DLBCL. This phase 2 trial tested the safety and efficacy of combining E with R-CHOP (ER-CHOP) in untreated DLBCL. A secondary aim was to assess the efficacy of interim positron emission tomography (PET) to predict outcome in DLBCL. Standard R-CHOP with the addition of E 360 mg/m(2) intravenously was administered for 6 cycles. A total of 107 patients were enrolled in the study. Toxicity was similar to standard R-CHOP. Overall response rate in the 81 eligible patients was 96% (74% CR/CRu) by computed tomography scan and 88% by PET. By intention to treat analysis, at a median follow-up of 43 months, the event-free survival (EFS) and overall survival (OS) at 3 years in all 107 patients were 70% and 80%, respectively. Interim PET was not associated with EFS or OS. Comparison with a cohort of 215 patients who were treated with R-CHOP showed an improved EFS in the ER-CHOP patients. ER-CHOP is well tolerated and results appear promising as a combination therapy. This study was registered at www.clinicaltrials.gov as #NCT00301821.

Proteomic changes and molecular effects associated with Cr(III) and Cr(VI) treatments on germinating kiwifruit pollen.

The present study is aimed at identifying molecular changes elicited by Cr(III) and Cr(VI) on germinating kiwifruit pollen. To address this question, comparative proteomic and DNA laddering analyses were performed. While no genotoxic effect was detected, a number of proteins whose accumulation levels were altered by treatments were identified. In particular, the upregulation of some proteins involved in the scavenging response, cell redox homeostasis and lipid synthesis could be interpreted as an oxidative stress response induced by Cr treatment. The strong reduction of two proteins involved in mitochondrial oxidative phosphorylation and a decline in ATP levels were also observed. The decrease of pollen energy availability could be one of the causes of the severe inhibition of the pollen germination observed upon exposure to both Cr(III) and Cr(VI). Finally, proteomic and biochemical data indicate proteasome impairment: the consequential accumulation of misfolded/damaged proteins could be an important molecular mechanism of Cr(III) toxicity in pollen.

Phase 2 trial of carboplatin, weekly paclitaxel, and biweekly bevacizumab in patients with unresectable stage IV melanoma: a North Central Cancer Treatment Group study, N047A.

BACKGROUND: Vascular endothelial growth factor (VEGF) plays an important role in the growth and metastatic progression of melanoma. Exposure of melanoma cells to chemotherapy induces VEGF overproduction, which in turn may allow melanoma cells to evade cell death and become chemotherapy resistant. Therefore, in patients with metastatic melanoma, the combination of chemotherapy with an agent that specifically targets VEGF might be able to control tumor growth and progression more effectively than chemotherapy alone. METHODS: A 2-stage phase 2 clinical trial was conducted in patients with unresectable stage IV (metastatic) melanoma to assess antitumor activity and the toxicity profile of the combination of carboplatin (area under the curve 6 iv on Day 1 of a 28-day cycle), paclitaxel (80 mg/m2 iv on Days 1, 8, and 15), and bevacizumab (10 mg/kg iv on Days 1 and 15). Treatment was continued until progression or intolerable toxicity. RESULTS: Fifty-three patients (62.3% male) were enrolled. Nine (17%) patients achieved partial remission, and another 30 (57%) achieved stable disease for at least 8 weeks. Median progression-free survival and median overall survival were 6 months and 12 months, respectively. One patient died after 8 treatment cycles from intracranial hemorrhage into undiagnosed brain metastases. The most common severe (grade>or=3) toxicities were neutropenia (53%), thrombocytopenia (11%), hypertension (9%), and anemia (8%). CONCLUSIONS: This combination of carboplatin, paclitaxel, and bevacizumab appears to be moderately well tolerated and clinically beneficial in patients with metastatic melanoma. Further study of this combination is warranted.

Tamoxifen-associated hot flashes in women.

PURPOSE: The most prominent toxicity from the antiestrogenic agent tamoxifen is hot flashes. The objective of this project was to better describe the incidence and severity of hot flashes in younger women receiving tamoxifen. PATIENTS AND METHODS: This article reports the vasomotor effects associated with the initiation of tamoxifen in 27 women aged < 50 years, most of whom were premenopausal and had received chemotherapy. It then compares the results from these women to results from women aged > 50 years, who participated in a previous similar study. Hot flash data in both studies were obtained by utilizing a validated hot flash diary. RESULTS: Fifty-nine percent of the women aged < 50 years experienced significant hot flashes; women who were postmenopausal reported more severe trouble with hot flashes, compared with premenopausal women. There was a trend toward more severe hot flashes in women who had received chemotherapy. When data from both studies were combined and analyzed by menopausal status and whether hot flashes had occurred during menopause, both factors were related to more hot flash trouble. Hot flashes were more prominent in postmenopausal women who had significant previous history of hot flashes, compared with those without a significant history of hot flashes. CONCLUSION: Premenopausal women, and postmenopausal women without a previous significant history of hot flashes, appear to have less trouble with tamoxifen-associated hot flashes, compared with postmenopausal women with a previous history of moderate or worse hot flashes.

Isolated amyloidosis presenting with lumbosacral radiculoplexopathy: description of two cases and pathogenic review.

In this study, we present two cases of infiltrative, localized amyloidosis involving lumbosacral root and plexus, e.g., isolated amyloidomas. Rare and poorly understood amyloidomas may occur in both neurologic and non-neurologic tissues. The described cases emphasize potential for localized peripheral amyloidomas: (1) potential for associated lambda light chain lymphoplasmacytic lymphoma association; (2) e isolated amyloidosis without evidence for systemic plasma cell dyscrasia; (3) features suggestive of potential pathogenesis; and (4) discussion of treatment options including immunotherapy and resection. The limited literature and experience among other cases is described.

Pilot evaluation of bupropion for the treatment of hot flashes.

Bupropion is commonly used in the treatment of nicotine dependence and depression, and in most people, does not cause sexual dysfunction, weight gain, or sedation. Given its attractive side effect profile, the efficacy of other newer antidepressants against hot flashes and anecdotal observations of resolution of hot flashes in some patients taking bupropion for nicotine dependence, it was decided to explore its clinical activity as a hot flash remedy in a pilot study. Between January 1999 and October 2004, 21 patients (7 men and 14 women) were enrolled in the study. Self-completed daily hot flash diaries were used to document the frequency and severity of hot flashes at baseline (week 1) and during the treatment period (weeks 2 through 5). Participants received bupropion 150 mg every morning for the first 3 days and then 150 mg twice per day for a total of 4 weeks. One woman did not provide any hot flash information and was excluded from the analysis. Five women could not complete the study because of side effects. The study did not show a reduction in hot flash frequency and/or severity significantly higher than what would be expected with a placebo. Even though the sample size was small, these results are consistent with bupropion's mechanism of action (norepinephrine reuptake inhibition without serotonergic effects) and what it is now hypothesized about the pathophysiology of hot flashes (increased noradrenergic activity and decreased serotonergic activity). These data suggest that bupropion should not be further investigated as a remedy for hot flashes.

High variability in CYP21A2 mutated alleles in Spanish 21-hydroxylase deficiency patients, six novel mutations and a founder effect.

OBJECTIVE: To detect common as well as rare and novel CYP21A mutations in 21-hydroxylase deficiency patients. To estimate the distribution of mutations and compare them with other European studies. To construct haplotypes linked to a recurrent novel mutation. DESIGN: Genetic analysis by sequencing the entire CYP21A2 gene plus Southern blot. PATIENTS: A total of 138 unrelated Spanish patients: 122 nonclassical forms (NCF) and 16 classical forms (CF) were studied. RESULTS: Among the 266 nonrelated mutated alleles; CYP21A2 deletions/conversions and a spectrum of 27 different mutated alleles were found: 15 different single point mutations, 8 nucleotide deletions in exon 3, 3 mutation clusters in exon 6, 9 alleles with more than one mutation, one 21-nucleotide duplication in exon 10, and one allele with CYP21A2 duplicated and both copies mutated. The most frequent mutation in NCF alleles is V281L (71.8%). Among CFs, the most common is I2 g (20%) and Q318X (16%) and rare alleles (21.9%). Six novel causative mutations were found, four associated with CF: I46+1nt, R444X, P463L and M473_R479dup and two associated with NCF: W302 and D322G. The R444X mutation was found in seven unrelated patients and it appeared only once in an ancestral haplotype. In addition, we found a novel single nucleotide polymorphism with a 31.5% frequency for the rare allele. CONCLUSION: A great diversity of haplotypes with a large spectrum of mutated alleles was found. The frequency of the V281L mutation was the highest reported and the relatively high frequency of R444X was the result of a founder effect.

Newer antidepressants and other nonhormonal agents for the treatment of hot flashes.

The reluctance to use estrogen in breast cancer survivors with hot flashes has extended to its use in healthy women since the 2002 publication of the Women's Health Initiative study. This article reviews the clinical development of nonhormonal agents as alternatives to hormonal therapy for the management of hot flashes.

Pilot evaluation of mirtazapine for the treatment of hot flashes.

This prospective, single-arm, pilot clinical trial, developed to evaluate the efficacy and tolerability of mirtazapine for alleviating hot flashes, was conducted between May 2001 and January 2002. Patients' baseline characteristics were collected during the first week of the study. At the beginning of the second week, patients were started on mirtazapine at a dose of 7.5 mg at bedtime. The dose of mirtazapine was then increased to 15 mg at week 3 and to 30 mg at week 4. For week 5, patients could choose whether to take 15 mg/d or 30 mg/d. Data were obtained primarily from patient-completed questionnaires. Data from 22 evaluable women were available. For the 16 patients who completed the study, the median reductions in total daily hot flashes and weekly hot-flash scores from their baselines were 52.5% and 59.5%, respectively. Patients reported improvements in tension, trouble sleeping, abnormal sweating, distress from hot flashes, satisfaction with hot-flash control, overall quality of life, and impact of hot flashes on quality of life. Patients also reported increases in appetite and dry mouth. Although data from a double-blind, placebo-controlled clinical trial would be necessary to more definitively elucidate the efficacy and toxicity of mirtazapine in patients with hot flashes, the available data suggest that mirtazapine is a reasonable treatment to consider in patients with hot flashes, particularly in those with anxiety and sleep disturbances.

[Evaluating the simulation of memory problems within the legal and forensic fields]. / Evaluacion de la simulacion de problemas de memoria dentro del ambito legal y forense.

The present article is a theoretic review of the techniques used to assess the malingering of memory problems in the forensic and legal context. The necessity of instruments to facilitate the detection of malingerers is confirmed. This phenomenon is more relevant in the memory pathology context because their exaggeration or false performance is very common. The reason of this preference is the ease of the simulation of these very well known symptoms. The consequences, economics primarily, of the confirmation of brain damage sequels have produced that malingerers are well informed about the characteristics of this syndrome. This review is focus on, both tests assessing different components of memory, such as the Wechsler Memory Scale III and the California Verbal Learning Test, and tests designed specifically to detect malingering. We also differentiate tests assessing explicit and implicit memory. Likewise, future lines of research are proposed in this important legal context.

Hot flashes in breast cancer survivors.

Hot flashes can be a major problem for patients with a history of breast cancer. The precipitation of menopause in premenopausal women who undergo chemotherapy for breast cancer can lead to the rapid onset of hot flash symptoms that are more frequent and more severe than those associated with natural menopause. In addition, tamoxifen, historically the most commonly prescribed pharmacologic agent for the treatment of breast cancer, is associated with hot flashes in more than 50% of its users. Although estrogen relieves hot flashes in 80-90% of women who initiate treatment, its use in women with a history of breast cancer is controversial, and most physicians in the community will not use this treatment modality. In addition, the results of the long-awaited Women's Health Initiative study and other recent studies suggest that long-term estrogen therapy should not be recommended for most women for a variety of reasons. However, hot flashes in breast cancer survivors should no longer be considered untreatable, as there are many pharmacologic and nonpharmacologic treatments that can help alleviate this problem. This article reviews the current strategies for the management of hot flashes in breast cancer survivors and the evidence supporting their use.

Multiple display of peptides and proteins on a macromolecular scaffold derived from a multienzyme complex.

The acyltransferase components (E2) from the family of 2-oxo acid dehydrogenase multienzyme complexes form large protein scaffolds, to which multiple copies of peripheral enzymes bind tightly but non-covalently. Sixty copies of the E2 polypeptide from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus assemble to form a pentagonal dodecahedral scaffold with icosahedral symmetry. This protein scaffold can be modified to present foreign peptides and proteins on its surface. We show that it is possible to display two epitopes (MAL1 and MAL2) from the circumsporozoite CS proteins of Plasmodium falciparum and Plasmodium berghei, respectively, and a green fluorescent protein (EGFP), on the E2 surface. Immunization with an E2 scaffold displaying the MAL1 epitope elicited MAL1-specific antibodies in rabbits. EGFP (25 kDa) displayed as an N-terminal fusion in each of the 60 copies of the E2 chain folded into its active form, as judged by its fluorescence and detection in localized foci in Escherichia coli cells in vivo. Simultaneous display of a hexahistidine affinity tag, the MAL1 epitope and the green fluorescent protein, all on the same E2 scaffold, could be achieved by reversible denaturation and reassembly of mixtures of appropriately modified E2 chains. This new methodology offers several important advantages over other current display technologies, not least in the size of insert that can be accommodated and the multiplicity of display that can be achieved.