A comprehensive model for the biochemistry of ageing, senescence and longevity.

Various models for ageing, each focussing on different biochemical and/or cellular pathways have been proposed. This has resulted in a complex and non-coherent portrayal of ageing. Here, we describe a concise and comprehensive model for the biochemistry of ageing consisting of three interacting signalling hubs. These are the nuclear factor kappa B complex (NFκB), controlling the innate immune system, the mammalian target for rapamycin complex, controlling cell growth, and the integrated stress responses, controlling homeostasis. This model provides a framework for most other, more detailed, biochemical pathways involved in ageing, and explains why ageing involves chronic inflammation, cellular senescence, and vulnerability to environmental stress, while starting with the spontaneous formation of advanced glycation end products. The totality of data underlying this model suggest that the gradual inhibition of the AMPK-ISR probably determines the maximal lifespan. Based on this model, anti-ageing drugs in general, are expected to show hormetic dose response curves. This complicates the process of dose-optimization. Due to its specific mechanism of action, the anti-aging drug alkaline phosphatase is an exception to this rule, because it probably exhibits saturation kinetics.

An AMP Kinase-pathway dependent integrated stress response regulates ageing and longevity.

The purpose of this article is to investigate the role of the AMP-kinase pathway (AMPK pathway) in the induction of a concomitant set of health benefits by exercise, numerous drugs, and health ingredients, all of which are adversely affected by ageing. Despite the AMPK pathway being frequently mentioned in relation to both these health effects and ageing, it appears challenging to understand how the activation of a single biochemical pathway by various treatments can produce such a diverse range of concurrent health benefits, involving so many organs. We discovered that the AMPK pathway functions as an integrated stress response system because of the presence of a feedback loop in it. This evolutionary conserved stress response system detects changes in AMP/ATP and NAD/NADH ratios, as well as the presence of potential toxins, and responds by activating a common protective transcriptional response that protects against aging and promotes longevity. The inactivation of the AMPK pathway with age most likely explains why ageing has a negative impact on the above-mentioned set of health benefits. We conclude that the presence of a feedback loop in the AMP-kinase pathway positions this pathway as an AMPK-ISR (AMP Kinase-dependent integrated stress response) system that responds to almost any type of (moderate) environmental stress by inducing various age-related health benefits and longevity.

Osteoporosis therapy in patients with inflammatory rheumatic diseases and osteonecrosis of the jaw.

BACKGROUND AND OBJECTIVES: The aim of the present study was to assess the prevalence of medication-related osteonecrosis of the jaw (MRONJ) in osteoporosis patients suffering from inflammatory rheumatic diseases, as well as to assess the prevalence of relevant dental, behavioral, and medical risk factors for MRONJ. MATERIALS AND METHODS: A total of 198 patients with inflammatory rheumatic diseases and osteoporosis therapy were recruited from a tertiary rheumatological/immunological referral center between June 2015 and September 2016. They were assessed using a structured interview. A maxillofacial surgeon later examined patients complaining of possible symptoms of osteonecrosis. In cases of osteonecrosis, dental records were obtained and evaluated. Preventive measures taken and dental as well as other clinical risk factors were evaluated. RESULTS: Of the 198 patients, three suffered from osteonecrosis of the jaw, none of whom had any history of malignant disease or radiation therapy, resulting in a prevalence of 1.5%. Of these three patients, only one was given bisphosphonates intravenously (i.v.), whereas all three had been treated orally. All three diagnoses of MRONJ had been previously known to the patients and their maxillofacial surgeons. Two of the patients had rheumatoid arthritis, and one patient suffered from large vessel vasculitis. Long anti-osteoporotic treatment duration, low functional status, and low bone density of the femur were significantly associated with MRONJ development. CONCLUSION: Inflammatory rheumatic diseases constitute a risk factor for MRONJ in patients treated with bisphosphonates for osteoporosis. Patients should be counseled accordingly and should be offered dental screening and regular dental check-ups.

Medical-grade polycaprolactone scaffolds made by melt electrospinning writing for oral bone regeneration - a pilot study in vitro.

BACKGROUND: The spectrum of indications for the use of membranes and scaffolds in the field of oral and maxillofacial surgery includes, amongst others, guided bone regeneration (GBR). Currently available membrane systems face certain disadvantages such as difficult clinical handling, inconsistent degradation, undirected cell growth and a lack of stability that often complicate their application. Therefore, new membranes which can overcome these issues are of great interest in this field. METHODS: In this pilot study, we investigated polycaprolactone (PCL) scaffolds intended to enhance oral wound healing by means of melt electrospinning writing (MEW), which allowed for three-dimensional (3D) printing of micron scale fibers and very exact fiber placement. A singular set of box-shaped scaffolds of different sizes consisting of medical-grade PCL was examined and the scaffolds' morphology was evaluated via scanning electron microscopy (SEM). Each prototype sample with box sizes of 225 µm, 300 µm, 375 µm, 450 µm and 500 µm was assessed for cytotoxicity and cell growth by seeding each scaffold with human osteoblast-like cell line MG63. RESULTS: All scaffolds demonstrated good cytocompatibility according to cell viability, protein concentration, and cell number. SEM analysis revealed an exact fiber placement of the MEW scaffolds and the growth of viable MG63 cells on them. For the examined box-shaped scaffolds with pore sizes between 225 µm and 500 µm, a preferred box size for initial osteoblast attachment could not be found. CONCLUSIONS: These well-defined 3D scaffolds consisting of medical-grade materials optimized for cell attachment and cell growth hold the key to a promising new approach in GBR in oral and maxillofacial surgery.

Nutrition management for head and neck cancer patients improves clinical outcome and survival.

Up to 80% of patients with head and neck cancers are malnourished because of their lifestyle and the risk factors associated with this disease. Unfortunately, nutrition management systems are not implemented in most head and neck cancer clinics. Even worse, many head and neck surgeons as well as hospital management authorities disregard the importance of nutrition management in head and neck cancer patients. In addition, the often extensive resection and reconstruction required for tumors in the upper aerodigestive tract pose special challenges for swallowing and sufficient food intake, placing special demands on nutrition management. This article presents the basics of perioperative metabolism and nutrition management of head and neck cancer patients and makes recommendations for clinical practice. Implementing a nutrition management system in head and neck cancer clinics will improve the clinical outcome and the survival of the patients.

Evaluation of miRNA-expression and clinical tumour parameters in oral squamous cell carcinoma (OSCC).

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common malignancy worldwide. The past decades have not led to substantial improvement in diagnosis and therapy. Analysis of miRNA-expression may help to determine the progression profiles and outcomes of many different diseases, including HNSCC. Therefore, in this investigation, 43 formalin-fixed, paraffin-embedded (FFPE) samples of oral squamous cell carcinoma were micro-dissected, analysed for expression of 30 miRNAs and were compared with non-tumorous tissue. Furthermore, correlation analysis was performed, investigating possible correlations of miRNA-expression and patient or tumour-linked data, such as age, sex, tumour stage and size. miRNA extraction from FFPE samples functioned well for OSCC, and several miRNAs were differently expressed in tumours compared with non-tumorous tissue (i.e., miR-99*; miR-224; miR-205*), indicating their possible utility as biomarkers. Moreover, some miRNAs showed significant correlations with clinical and pathological data (e.g. tumour size: miR-3156, P = 0.033; T-stage: miR-212, P = 0.0009).

Expression of MAGE-A1-A12 subgroups in the invasive tumor front and tumor center in oral squamous cell carcinoma.

MAGE-A proteins are highly expressed in oral squamous cell carcinoma (OSCC) and are promising targets for cancer immunotherapy. This study examined the presence of MAGE-A expression within the tumor center (TC) and tumor invasive front (TIF) and evaluated its relationship to poor prognosis. The expression rate of each MAGE-A subtype, A1-A12, was examined in 68 OSCCs at the TIF and TC. Slides (1-µm) of tissue microarrays (diameter =0.6 mm) were immunohistochemically stained, and the findings were correlated to clinical data. Approximately 95% of the tumors had MAGE-A expression. Higher expression in the TC was shown significantly for MAGE-A1, -A5, -A6, -A9 and -A12 (P<0.05). MAGE-A2 and -A3 exhibited the opposite behavior (not significant, P>0.05). Age, tumor size, grade and survival time were not associated with the expression of certain MAGE-A subgroups. When expression in the whole tumor tissue was considered, only MAGE-A1 was expressed at a significantly higher rate in male patients (P=0.034). At the TIF, MAGE-A9 and the UICC disease stage were significantly correlated (P=0.0263), and MAGE-A6 and the UICC disease stage exhibited a strong trend (P=0.0596). The expression of MAGE-A3, -A4, -A5, -A9 and -A11 was significantly associated with lymph node metastasis, while MAGE-A4 was expressed in all regions of the tumors (TIF and TC). This study showed that higher expression of most MAGE-A antigens occurred at the TC rather than at the TIF. MAGE­A1, -A3, -A4, -A5, -A9 and -A11 were significantly associated with clinically advanced stages of disease and seem to be of particular interest.

Phosphorylated epidermal growth factor receptor expression and KRAS mutation status in salivary gland carcinomas.

OBJECTIVES: Salivary gland carcinomas (e.g., adenoidcystic carcinoma or mucoepidermoid carcinoma) are rare and often unresectable head and neck tumors. They are also weakly affected by most chemotherapeutic drugs, which emphasize the need for further studies on this topic. In clinical practice, various drugs target the well-characterized EGFR pathway in many epithelial tumors. There is limited reliable data on phophorylated EGFR expression, such as activated conformation, in salivary gland tumors. MATERIALS AND METHODS: This study investigates the pEGFR expression in salivary gland carcinomas (n = 43). Three different carcinoma varieties, that represent >50 % of all salivary gland tumors, were included: adenoidcystic carcinoma (n = 23), mucoepidermoid carcinoma (n = 17), and adenocarcinoma NOS (not otherwise specified) (n = 3). The specimens were investigated by immunohistochemistry. Additionally, mutations of KRAS oncogene were screened with gene sequencing. The findings were correlated with clinical data by using SPSS. RESULTS: In 34 out of 43 specimens (79 %), a positive staining for pEGFR was found. Sex, tumor entity, tumor site, and grading had no significant correlation with pEGFR expression. A weak correlation was found for tumor size and pEGFR expression. Significant correlations were found for pEGFR expression with patient's age and lymph node metastasis (pN). No specimen showed a KRAS mutation in codon 12 or 13. CONCLUSION: Salivary gland carcinomas show a high expression of pEGFR. This high expression correlates with lymph node metastasis, which supports the hypothesis that a high pEGFR expression facilitates lymphogenous metastasis. Due to this pEGFR expression, status may be a negative predictive factor in salivary gland carcinoma diagnostics. Patients with pN-positive salivary gland cancer may benefit from EGFR-inhibiting drugs. CLINICAL RELEVANCE: The EGFR pathway may be a potential target for chemotherapy of advanced unresectable salivary gland carcinomas.

Microdosing of a Carbon-14 Labeled Protein in Healthy Volunteers Accurately Predicts Its Pharmacokinetics at Therapeutic Dosages.

Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 µg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs.

Recombinant human tissue non-specific alkaline phosphatase successfully counteracts lipopolysaccharide induced sepsis in mice.

Sepsis is a life threatening condition that arises when the body's response to an infection injures its own tissues and organs. Sepsis can lead to shock, multiple organ failure and death especially if not recognized early and treated promptly. Molecular mechanisms underlying the systemic inflammatory response syndrome associated with sepsis are still not completely defined and most therapies developed to target the acute inflammatory component of the disease are insufficient. In this study we investigated a possibility of combating sepsis in a mouse model by intravenous treatment with recombinant human tissue non-specific alkaline phosphatase (rhTNAP) derived from transgenic rabbit milk. We induced sepsis in mice by intraperitoneal injection of LPS and three hours later treated experimental group of mice by intravenous injection with rhTNAP derived from transgenic rabbits. Such treatment was proved to be physiologically effective in this model, as administration of recombinant rhTNAP successfully combated the decrease in body temperature and resulted in increased survival of mice (80 % vs. 30 % in a control group). In a control experiment, also the administration of bovine intestinal alkaline phosphatase by intravenous injection proved to be effective in increasing survival of mice treated with LPS. Altogether, present work demonstrates the redeeming effect of the recombinant tissue non-specific AP derived from milk of genetically modified rabbits in combating sepsis induced by LPS.

A rare case of a groin hernia: the Hesselbach's hernia.

A 61-year-old woman presented with pain in the left groin. The pain radiated from the groin to the knee. At physical examination, a non-reducible swelling was found lateral to the femoral vessels. A CT-scan showed herniation of fatty tissue through the lacuna musculorum. A laparoscopic repair was carried out. A large adipose structure was found herniating through the lacuna musculorum and originating from the Bogros area. It ran along the iliopsoas muscle, suppressing the femoral cutaneous nerve. The tissue was reduced and excised, and a polypropylene mesh was placed to cover the defect. Inguinal hernias are categorized as medial, lateral or femoral hernias. We describe a case in which a hernia through the lacuna musculorum was found, which is very rare and referred to as a Hesselbach's hernia.

Correlation of MAGE-A tumor antigens and the efficacy of various chemotherapeutic agents in head and neck carcinoma cells.

OBJECTIVES: The present study examined the relationship between MAGE-A tumor antigens and the efficacy of diamindichloridoplatin (DDP), 5-fluorouracil (5-FU), docetaxel, and paclitaxel for in vitro treatment of head and neck cancer. METHODS: In the present study, five cell lines of human squamous cell carcinomas were treated with DDP (25-400 µM), 5-FU (0.75-12 mM), docetaxel (1.56-25 nM), and paclitaxel (1.56-25 nM) for a period of 24 or 48 h. The efficacy of the agents was observed dynamically using real-time cell analysis. Subsequently, the expression levels of MAGE-A1, MAGE-A5, MAGE-A8, MAGE-A9, MAGE-A11, and MAGE-A12 were determined by quantitative real-time polymerase chain reaction. Chemosensitivity and MAGE-A-expression were correlated by linear regression. RESULTS: The tumor cell lines showed a highly differentiated response to the chemotherapeutic agents. Expression of MAGE-A11 was significantly associated with a poorer response to treatment with DDP, 5-FU, docetaxel, and paclitaxel. Two cell lines, one of which was MAGE-A11-positive, showed a significant and concentration-dependent cisplatin-induced growth spurt during the first 24 h after treatment. MAGE-A5 was connected to a positive effect on treatment with paclitaxel within the first 24 h after application. In association with docetaxel treatment, MAGE-A8 was connected to a poorer susceptibility. CONCLUSIONS: The results describe, for the first time, a correlation between these MAGE-A tumor antigens and the susceptibility of head and neck cancer cells to DDP, 5-FU, docetaxel, and paclitaxel. CLINICAL RELEVANCE: These findings could affect the antineoplastic treatment of patients with MAGE-A11-positive tumors.

High level expression of tissue-nonspecific alkaline phosphatase in the milk of transgenic rabbits.

Alkaline phosphatase is a promising therapeutic agent in the Gram-negative bacterial lipopolysaccharide (LPS) mediated acute and chronic diseases. Contrary to other alkaline phosphatase isozymes, purified tissue-nonspecific alkaline phosphatase (TNAP) is not available in large quantities from tissue sources, which would enable to analyse its efficacy in animal sepsis models. Two transgenic rabbit lines were created by pronuclear microinjection with the whey acidic protein promoter-humanTNAP minigene (WAP-hTNAP). Lactating females of both lines produced biologically active human TNAP. As indicated by fractionation of milk samples the recombinant alkaline phosphatase was associated with the membrane of milk fat globules. Alkaline phosphatase enzymatic activity was two orders of magnitude higher compared to normal human serum levels. The demonstration that this TNAP is physiologically active would provide the clue to use transgenic animals as bioreactor for bulk production of the human tissue-nonspecific alkaline phosphatase in milk. This may be a valuable and possibly viable option with important implication in attenuating LPS mediated inflammatory responses.

The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture.

Fetal bovine serum (FBS) is a common supplement to in vitro culture media. A workshop was organized to discuss whether or not fetuses might suffer when blood is withdrawn, and to discuss serum replacement methods. When bovine fetuses are exposed after slaughter of the dam, they can suffer only if they inflate their lungs with air and increase their blood oxygen to levels compatible with awareness. Preventing fetuses from breathing air or killing them by an efficient method, according to clearly defined safeguards, ensures that fetal blood collection is humane. Since serum is a supplement of unknown composition, which could be contaminated with unwanted factors, there are scientific and safety reasons for omitting FBS from culture media. Several media have been developed in which minimal or no animal derived components are present. Also, different cell types have been adapted to serum-free media. As yet, no standard serum free media are present, and each cell type requires its own medium composition. Among other recommendations, the establishment of a public database with information on cell types and their serum-free medium composition is proposed.

Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity.

Local mucosal IgA antibodies play a central role in protection of the respiratory tract against influenza virus infection. Therefore, new-generation influenza vaccines should aim at stimulating not only systemic, but also local antibody responses. Previously, we demonstrated that the recombinant B subunit of the Escherichia coli heat-labile toxin (LTB) is a potent adjuvant towards nasally administered influenza subunit antigen. Here, we investigated the protection conferred by LTB-supplemented influenza subunit antigen given intranasally (i.n.) or intramuscularly (i.m.) to mice. Both i.n. and i.m. immunization with subunit antigen and LTB completely protected the animals against viral infection. Protection upon i.n. immunization was associated with the induction of antigen-specific serum IgG and mucosal IgA, whereas protection upon i.m. immunization correlated with strong serum and mucosal IgG, but not IgA responses. We conclude that LTB-supplemented influenza subunit antigen, given either i.n. or i.m, induces protective antibody-mediated mucosal immunity and thus represents a promising novel flu vaccine candidate.

Generation and characterization of reassortant influenza A viruses propagated in serum-free cultured MDCK-SF1 cells.

The replacement of embryonated chicken eggs by tissue culture cells for the production of influenza vaccines is likely to take place in the near future. Vaccines have already been produced in Madin Darby Canine Kidney (MDCK) cells (Brands et al, in this issue) and extensively tested in phase III trials in humans (Palache et al, in this issue) and it seems a matter of time before such vaccines will become available. For this reason, the generation of high-growth reassortants of influenza A virus strains in MDCK cells has been examined. Influenza A virus reassortants of the field strains A/Taiwan/1/86, A/Johannesburg/82/96 and A/Shenzhen/227/95 (all H1N1) were generated in serum-free cultured MDCK-SF1 cells by dual infection with A/Hong Kong/2/68 (H3N2), a strain selected for its high-growth phenotype. These reassortant viruses all contained at least the matrix gene of A/Hong Kong/2/68 which apparently correlates with an improvement of the viral yield.

Influvac: a safe Madin Darby Canine Kidney (MDCK) cell culture-based influenza vaccine.

Influenza vaccine production technology based on large scale cell culture technology has been developed. From the characterization of the continuous cell line MDCK as well as drug safety studies we conclude that this cell line and the cell culture system are suitable for biological production. The Down Stream Process (DSP) of the virus-containing harvest fluids guarantees sufficient inactivation of influenza viruses and adequate removal or inactivation of putative adventitious or endogenous viruses, mycoplasma or bacteria. Our data indicate that the tissue culture-based production technology is feasible.