Pragmatic rules for comparability of biological medicinal products

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Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches ’bottle-necks’ are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected ’rate-limiting’ steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates

Biopsy confirmation of metastatic sites in breast cancer patients:clinical impact and future perspectives

Determination of hormone receptor (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2 status in the primary tumor is clinically relevant to define breast cancer subtypes, clinical outcome,and the choice of therapy. Retrospective and prospective studies suggest that there is substantial discordance in receptor status between primary and recurrent breast cancer. Despite this evidence and current recommendations,the acquisition of tissue from metastatic deposits is not routine practice. As a consequence, therapeutic decisions for treatment in the metastatic setting are based on the features of the primary tumor. Reasons for this attitude include the invasiveness of the procedure and the unreliable outcome of biopsy, in particular for biopsies of lesions at complex visceral sites. Improvements in interventional radiology techniques mean that most metastatic sites are now accessible by minimally invasive methods, including surgery. In our opinion, since biopsies are diagnostic and changes in biological features between the primary and secondary tumors can occur, the routine biopsy of metastatic disease needs to be performed. In this review, we discuss the rationale for biopsy of suspected breast cancer metastases, review issues and caveats surrounding discordance of biomarker status between primary and metastatic tumors, and provide insights for deciding when to perform biopsy of suspected metastases and which one (s) to biopsy. We also speculate on the future translational implications for biopsy of suspected metastatic lesions in the context of clinical trials and the establishment of bio-banks of biopsy material taken from metastatic sites. We believe that such bio-banks will be important for exploring mechanisms of metastasis. In the future,advances in targeted therapy will depend on the availability of metastatic tissue

Allergen manufacturing and quality aspects for allergen immunotherapy in Europe and the United States:An analysis from the EAACI AIT Guidelines Project

Adequate quality is essential for any medicinal product to be eligible for marketing. Quality includes verification of the identity, content and purity of a medicinal product in combination with a specified production process and its control. Allergen products derived from natural sources require particular considerations to ensure adequate quality. Here, we describe key aspects of the documentation on manufacturing and quality aspects for allergen immunotherapy products in the European Union and the United States. In some key parts, requirements in these areas are harmonized while other fields are regulated separately between both regions. Essential differences are found in the use of Reference Preparations, or the requirement to apply standardized assays for potency determination. Since the types of products available are different in specific regions, regulatory guidance for such products may also be available in one specific region only, such as for allergoids in the European Union. Region-specific issues and priorities are a result of this. As allergen products derived from natural sources are inherently variable in their qualitative and quantitative composition, these products present special challenges to balance the variability and ensuring batch-to-batch consistency. Advancements in scientific knowledge on specific allergens and their role in allergic disease will consequentially find representation in future regulatory guidelines

Impact of non-axillary sentinel node biopsy on staging and treatment of breast cancer patients

The purpose of this study was to evaluate the occurrence of lymphatic drainage to non-axillary sentinel nodes and to determine the implications of this phenomenon. A total of 549 breast cancer patients underwent lymphoscintigraphy after intratumoural injection of 99mTc-nanocolloid. The sentinel node was intraoperatively identified with the aid of intratumoural administered patent blue dye and a gamma-ray detection probe. Histopathological examination of sentinel nodes included step-sectioning at six levels and immunohistochemical staining. A sentinel node outside level I or II of the axilla was found in 149 patients (27%): internal mammary sentinel nodes in 86 patients, other non-axillary sentinel nodes in 44 and both internal mammary and other non-axillary sentinel nodes in nineteen patients. The intra-operative identification rate was 80%. Internal mammary metastases were found in seventeen patients and metastases in other non-axillary sentinel nodes in ten patients. Staging improved in 13% of patients with non-axillary sentinel lymph nodes and their treatment strategy was changed in 17%. A small proportion of clinically node negative breast cancer patients can be staged more precisely by biopsy of sentinel nodes outside level I and II of the axilla, resulting in additional decision criteria for postoperative regional or systemic therapy

KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA

Host signal-transduction pathways are intimately involved in the switch between latency and productive infection of herpes viruses. As with other herpes viruses, infection by Kaposi's sarcoma herpesvirus (KSHV) displays these two phases. During latency only few viral genes are expressed, while in the productive infection the virus is reactivated with initiation of extensive viral DNA replication and gene expression, resulting in production of new viral particles. Viral reactivation is crucial for KSHV pathogenesis and contributes to the progression of KS. We have recently identified Pim-1 as a kinase reactivating KSHV upon over-expression. Here we show that another Pim family kinase, Pim-3, also induces viral reactivation. We demonstrate that expression of both Pim-1 and Pim-3 is induced in response to physiological and chemical reactivation in naturally KSHV-infected cells, and we show that they are required for KSHV reactivation under these conditions. Furthermore, our data indicate that Pim-1 and Pim-3 contribute to viral reactivation by phosphorylating the KSHV latency-associated nuclear antigen (LANA) on serine residues 205 and 206. This counteracts the LANA–mediated repression of the KSHV lytic gene transcription. The identification of Pim family kinases as novel cellular regulators of the gammaherpesvirus life cycle facilitates a deeper understanding of virus–host interactions during reactivation and may represent potential novel targets for therapeutic intervention

Genome Sequence and Comparative Genome Analysis of Lactobacillus casei: Insights into Their Niche-Associated Evolution

Lactobacillus casei is remarkably adaptable to diverse habitats and widely used in the food industry. To reveal the genomic features that contribute to its broad ecological adaptability and examine the evolution of the species, the genome sequence of L. casei ATCC 334 is analyzed and compared with other sequenced lactobacilli. This analysis reveals that ATCC 334 contains a high number of coding sequences involved in carbohydrate utilization and transcriptional regulation, reflecting its requirement for dealing with diverse environmental conditions. A comparison of the genome sequences of ATCC 334 to L. casei BL23 reveals 12 and 19 genomic islands, respectively. For a broader assessment of the genetic variability within L. casei, gene content of 21 L. casei strains isolated from various habitats (cheeses, n = 7; plant materials, n = 8; and human sources, n = 6) was examined by comparative genome hybridization with an ATCC 334-based microarray. This analysis resulted in identification of 25 hypervariable regions. One of these regions contains an overrepresentation of genes involved in carbohydrate utilization and transcriptional regulation and was thus proposed as a lifestyle adaptation island. Differences in L. casei genome inventory reveal both gene gain and gene decay. Gene gain, via acquisition of genomic islands, likely confers a fitness benefit in specific habitats. Gene decay, that is, loss of unnecessary ancestral traits, is observed in the cheese isolates and likely results in enhanced fitness in the dairy niche. This study gives the first picture of the stable versus variable regions in L. casei and provides valuable insights into evolution, lifestyle adaptation, and metabolic diversity of L. casei