The Added Value of Patient Engagement in Early Dialogue at EMA: Scientific Advice as a Case Study.

The European Medicines Agency provides Scientific Advice to medicines developers and patient input has been an integral part of this process for many years. As end users of medicines, patients bring their perspectives to many different processes along EMA's regulatory pathway, complementing the scientific expertise. While the value of including patients has been well-demonstrated over the years, requests for evidence of their impact continue. Using Scientific Advice as a case study, data was collected over a four-year period to assess the number of patients involved, where they contributed, as well as the impact and added value of their input. In this paper, we show that patients' contributions have a tangible impact on the recommendations provided to developers and in over half of the cases, this led to further discussion on relevant patient perspectives. These data provide quantitative evidence of the value of patient input in medicines development and supports EMA's continued inclusion of their voice throughout the medicine's lifecycle.

Individual Trade-Offs Between Possible Benefits and Risks of Cancer Treatments: Results from a Stated Preference Study with Patients with Multiple Myeloma.

BACKGROUND: The objectives of this study were to elicit the preferences of patients with multiple myeloma regarding the possible benefits and risks of cancer treatments and to illustrate how such data may be used to estimate patients' acceptance of new treatments. PATIENTS AND METHODS: Patients with multiple myeloma from the cancer charity Myeloma UK were invited to participate in an online survey based on multicriteria decision analysis and swing weighting to elicit individual stated preferences for the following attributes: (a) 1-year progression-free survival (PFS, ranging from 50% to 90%), (b) mild or moderate toxicity for 2 months or longer (ranging from 85% to 45%), and (c) severe or life-threatening toxicity (ranging from 80% to 20%). RESULTS: A total of 560 participants completed the survey. The average weight given to PFS was 0.54, followed by 0.32 for severe or life-threatening toxicity and 0.14 for mild or moderate chronic toxicity. Participants who ranked severe or life-threatening toxicity above mild or moderate chronic toxicity (56%) were more frequently younger, working, and looking after dependent family members and had more frequently experienced severe or life-threatening side effects. The amount of weight given to PFS did not depend on any of the collected covariates. The feasibility of using the collected preference data to estimate the patients' acceptance of specific multiple myeloma treatments was demonstrated in a subsequent decision analysis example. CONCLUSION: Stated preference studies provide a systematic approach to gain knowledge about the distribution of preferences in the population and about what this implies for patients' acceptance of specific treatments. IMPLICATIONS FOR PRACTICE: This study demonstrated how quantitative preference statements from a large group of participants can be collected through an online survey and how such information may be used to explore the acceptability of specific treatments based on the attributes studied. Results from such studies have the potential to become an important new tool for gathering patient views and studying heterogeneity in preferences in a systematic way, along with other methods, such as focus groups and expert opinions.

Marketing authorisation of orphan medicines in Europe from 2000 to 2013.

An analysis was performed on a data set of 157 orphan designated medicines with an outcome for marketing authorisation application (MAA) between 2000 and 2013. The intention was to understand the factors associated with marketing authorisation success, the challenges developers face regarding orphan medicine development, and how scientific advice (SA) is used during development. The results demonstrated that orphan medicines have a lower success rate compared with non-orphan medicines and that determinants for marketing authorisation success were company size and compliance with SA. Compliance with SA could help orphan medicine developers overcome clinical development challenges.

Animal models for metabolic, neuromuscular and ophthalmological rare diseases.

Animal models are important tools in the discovery and development of treatments for rare diseases, particularly given the small populations of patients in which to evaluate therapeutic candidates. Here, we provide a compilation of mammalian animal models for metabolic, neuromuscular and ophthalmological orphan-designated conditions based on information gathered by the European Medicines Agency's Committee for Orphan Medicinal Products (COMP) since its establishment in 2000, as well as from a review of the literature. We discuss the predictive value of the models and their advantages and limitations with the aim of highlighting those that are appropriate for the preclinical evaluation of novel therapies, thereby facilitating further drug development for rare diseases.

European regulation on orphan medicinal products: 10 years of experience and future perspectives.

In 2000, regulation on orphan medicinal products was adopted in the European Union with the aim of benefiting patients who suffer from serious, rare conditions for which there is currently no satisfactory treatment. Since then, more than 850 orphan drug designations have been granted by the European Commission based on a positive opinion from the Committee for Orphan Medicinal Products (COMP), and more than 60 orphan drugs have received marketing authorization in Europe. Here, stimulated by the tenth anniversary of the COMP, we reflect on the outcomes and experience gained in the past decade, and contemplate issues for the future, such as catalysing drug development for the large number of rare diseases that still lack effective treatments.

The Trw type IV secretion system of Bartonella mediates host-specific adhesion to erythrocytes.

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The alpha-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks.

Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.

A secreted anti-activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri.

Bacteria of Shigella spp. are responsible for shigellosis in humans and use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages. Transit of translocator and effector proteins through the TTS apparatus is activated upon contact of bacteria with host cells. Transcription of approximately 15 genes encoding effectors is regulated by the TTS apparatus activity and controlled by MxiE, an AraC family activator, and its coactivator IpgC, the chaperone of IpaB and IpaC translocators. Using a genetic screen, we identified ospD1 as a gene whose product negatively controls expression of genes regulated by secretion activity. OspD1 associates with the chaperone Spa15 and the activator MxiE and acts as an anti-activator until it is secreted. The mechanism regulating transcription in response to secretion activity involves an activator (MxiE), an anti-activator (OspD1), a co-anti-activator (Spa15), a coactivator (IpgC) and two anti-coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the TTS apparatus activity.

Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri.

Proteins directly involved in entry and dissemination of Shigella flexneri into epithelial cells are encoded by a virulence plasmid of 200 kb. A 30-kb region (designated the entry region) of this plasmid encodes components of a type III secretion (TTS) apparatus, substrates of this apparatus and their dedicated chaperones. During growth of bacteria in broth, expression of these genes is induced at 37 degrees C and the TTS apparatus is assembled in the bacterial envelope but is not active. Secretion is activated upon contact of bacteria with host cells and is deregulated in an ipaB mutant. The plasmid encodes four transcriptional regulators, VirF, VirB, MxiE and Orf81. VirF controls transcription of virB, whose product is required for transcription of entry region genes. MxiE, with the chaperone IpgC acting as a co-activator, controls expression of several effectors that are induced under conditions of secretion. Genes under the control of Orf81 are not known. The aim of this study was to define further the repertoires of virulence plasmid genes that are under the control of (i) the growth temperature, (ii) each of the known virulence plasmid-encoded transcriptional regulators (VirF, VirB, MxiE and Orf81) and (iii) the activity of the TTS apparatus. Using a macroarray analysis, the expression profiles of 71 plasmid genes were compared in the wild-type strain grown at 37 and 30 degrees C and in virF, virB, mxiE, ipaB, ipaB mxiE and orf81 mutants grown at 37 degrees C. Many genes were found to be under the control of VirB and indirectly of VirF. No alteration of expression of any gene was detected in the orf81 mutant. Expression of 13 genes was increased in the secretion-deregulated ipaB mutant in an MxiE-dependent manner. On the basis of their expression profile, substrates of the TTS apparatus can be classified into three categories: (i) those that are controlled by VirB, (ii) those that are controlled by MxiE and (iii) those that are controlled by both VirB and MxiE. The differential regulation of expression of TTS effectors in response to the TTS apparatus activity suggests that different effectors might be required at different times following contact of bacteria with host cells.

Topological analysis of GtrA and GtrB proteins encoded by the serotype-converting cassette of Shigella flexneri.

Serotype conversion (O-antigen glucosylation) in Shigella flexneri is mediated by temperate bacteriophages, which encode a three-gene cluster that contains gtrA, gtrB, and gtr([type]). Sequence analysis has revealed that gtrA and gtrB are conserved and readily interchangeable between serotypes. The gtr([type]) is unique in each serotype and responsible for specifically mediating conversion by the addition of a glucosyl group to the O-antigen units. Analysis of the GtrA and GtrB amino acid sequence using computer prediction programs indicated that GtrA and GtrB have four and two transmembrane segments, respectively. The topology model of GtrA was analyzed by constructing consecutive sandwich fusions using a dual reporter PhoA/LacZ at predetermined positions targeting each of the 3 cytoplasmic and 2 periplasmic hypothetical loops. The topology of GtrB was determined by constructing C-terminal truncated fusions of GtrB to full-length PhoA and LacZ by a PCR-mediated method. These approaches revealed that GtrA consists of four transmembrane segments with both the N-terminal and C-terminal ends in the cytoplasm. Accordingly, GtrB consists of two transmembrane segments with both ends also in the cytoplasm. Furthermore, membrane anchorage of the extended N-terminal end of GtrB was found to be important in catalysis. This study completes the topology of all three proteins (GtrA, GtrB, and the gtr([type]): GtrV) involved in the glucosyltransferase activity that results in serotype conversion of S. flexneri. A model is proposed showing how both O-antigen synthesis and modification take place in S. flexneri.

Microbial-gut interactions in health and disease. Epithelial cell responses.

Pathogenic bacteria use many strategies to secure their survival within the host. Enteropathogens exploit intestinal epithelial cells in many ways, including the manipulation of normal cellular functioning, or of cellular structural components, or by the induction of signalling pathways, such as the production of pro-inflammatory cytokines. However, the enterocyte warns the host of impending danger and, in turn, elicits a protective response. Pathogens are detected by epithelial cells owing to their vast array of surface antigens and secreted products. Epithelial cells have developed both extracellular and intracellular sensing proteins that function as a first line of defence against pathogens; this is followed by acquired immunity, namely IgA, which is used as reinforcement. Thus, in a game of constant attack and defence, the pathogen and the enterocyte aim to outsmart each other in an effort to survive.

Identification of the cis-acting site involved in activation of promoters regulated by activity of the type III secretion apparatus in Shigella flexneri.

Bacteria of Shigella spp. use a virulence plasmid-encoded type III secretion (TTS) system to invade the colonic epithelium in humans. The activity of the TTS apparatus is tightly regulated in the wild-type strain and is induced upon contact of bacteria with epithelial cells, whereas it is deregulated, i.e., constitutively active, in some mutants. Under conditions of deregulated secretion, approximately 20 proteins are secreted, including VirA, OspB to OspG, and at least three members of the IpaH family, all of which are encoded by the virulence plasmid. Conditions inducing or deregulating the activity of secretion also induce the transcription of virA and four ipaH genes. The transcription of virA and ipaH9.8 requires both MxiE, a transcriptional activator of the AraC family, and IpgC, the chaperone of IpaB and IpaC, acting as a coactivator. Using reporter plasmids containing lacZ transcriptional fusions, we showed that the ipaH7.8. ipa4.5. ospC1, and ospF promoters are activated under conditions of deregulated secretion and that both MxiE and IpgC are necessary and sufficient for their activation in both Shigella flexneri and Escherichia coli. Promoter mapping and deletion analysis of the ipaH9.8. virA, and ospC1 promoters identified a 17-bp motif, the MxiE box, which overlaps the -35 region and is essential for the activation of these promoters. The presence of eight MxiE boxes on the virulence plasmid suggests that 11 genes encoding secreted proteins may be regulated by the activity of secretion. We also present evidence that at least one ipaH gene that is carried by the chromosome is controlled by MxiE and IpgC.

Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus.

The virulence plasmid-encoded type III secretion system of Shigella flexneri consists of the Mxi-Spa secretion apparatus, secreted proteins IpaA-D and IpgD involved in entry of bacteria into epithelial cells,cytoplasmic chaperones IpgC and IpgE and 15 other secreted proteins of unknown function, including VirA and members of the IpaH family. The activity of the Mxi-Spa apparatus is regulated by external signals, and transcription of virA and IpaH genes is specifically induced in conditions of active secretion. We present genetic evidence that regulation of these genes involves both MxiE, the transcriptional activator of the AraC family encoded by the mxi operon, and IpgC, the chaperone for IpaB and IpaC. We also show that together MxiE and IpgC are sufficient to activatevirA and IpaH 9.8 promoters in Escherichia coli. InS. flexneri, increasing the expression of IpgC led to a concomitant increase in IpaH production in conditions of non-secretion. This suggests that the activity of secretion is sensed by the presence of free IpgC, which acts as a coactivator to allow MxiE to activate transcription at its target promoters.