Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc.

The use of symptoms to predict colorectal cancer site. Can we reduce the pressure on our endoscopy services?

OBJECTIVE: Our objective was to determine the value of gastrointestinal symptoms and signs in predicting the site of colorectal cancer (CRC). These symptoms can subsequently be used in determining first-line investigation with either sigmoidoscopy or colonoscopy. METHOD: We interrogated the endoscopic and CRC databases ('Infoflex'), for patients diagnosed with CRC between April 2005 and March 2006 inclusive. These patients were cross-referenced with the pathology database and patient records. Information gathered from these databases include: age, gender, symptoms, site of cancer, histology, Duke's grading, blood parameters, diagnostic tool and treatment. RESULTS: One hundred fifty-three patients were diagnosed with CRC between April 2005 and March 2006. One hundred twenty-six were initially seen in the out-patient department, of whom 38 (29%) were right-sided (proximal to the splenic flexure), and 88 (70%) were left-sided (splenic flexure and beyond). Change in bowel habit (diarrhoea and constipation) and rectal bleeding were significantly associated with left-sided cancers (P < 0.0024 and P < 0.0001, respectively). Haemoglobin (P < 0.0001) and mean corpuscular volume (P < 0.0001) were significantly lower in right-sided cancers. Weight loss, pain and obstruction were not associated with cancer site. C-reactive protein, albumin and carcinoembryonic antigen are not predictive of cancer site, Duke's stage or influenced by patient age or gender. DISCUSSION: Symptoms can accurately predict site of cancer, allowing investigations to be tailored accordingly. We would recommend that patients with altered bowel habit and/or rectal bleeding, and no other symptoms, risk factors or anaemia, can be investigated with a flexible sigmoidoscopy to confirm or refute a diagnosis of colorectal cancer.

Cell-based cardiovascular repair--the hurdles and the opportunities.

Cardiovascular cell therapy offers the first real potential to treat the underlying injuries associated with cardiac and vascular disease. By delivering appropriate exogenous cells to an injury site, the potential exists to mitigate injury or even to begin to reverse damage. Based on their inordinate pre-clinical promise as myogenic or angiogenic precursors, skeletal myoblasts and bone marrow or blood-derived mesenchymal and hematopoietic progenitor cells have all rapidly moved from bench to early clinical studies. From these parallel paths we are learning a number of useful lessons and have begun to visualize the hurdles to be overcome as we move these therapies forward. It is now obvious that cell-based cardiac and vascular repair are feasible-both early and later in the disease process. In fact, cell therapy may offer an unparalleled opportunity for improvement to millions of individuals living with cardiovascular disease. However, many questions about the technology remain. The mechanisms associated with cardiovascular repair remain unclear. Whether a best cell type, delivery method, or route of administration exists is unknown. And, whether cellbased disease prevention is feasible is still unanswerable. Now is the time to delve deeply into the questions of cell-based myocardial and vascular repair-even as we cautiously proceed clinically. Only by understanding these issues will we be able to decrease unanticipated clinical effects and to fulfill the potential promise of the most exciting opportunity yet to treat CVD. As we do so, we must prevent uncontrolled, poorly planned studies and until we understand cell therapy's potential, we must limit "too good to be true" promises. Only by addressing unanswered questions, carefully limiting our promises, and rigorously performing pre-clinical and clinical studies can we provide the surest opportunity for safely moving the field forward.

The reliability of lymph-node staging in rectal cancer after preoperative chemoradiotherapy.

AIMS: To determine the prognostic significance of the nodal stage and number of nodes recovered in the surgical specimen after preoperative synchronous chemoradiation (SCRT) and surgery for locally advanced or unresectable rectal cancer. MATERIALS AND METHODS: One hundred and eighty-two consecutive patients with locally advanced or unresectable (T3/T4) rectal carcinomas were entered on a prospective database and treated in this department with preoperative chemoradiation, followed 6-12 weeks later by surgical resection. Most patients received chemotherapy in the form of low-dose folinic acid and 5-fluorouracil (5-FU) 350 mg/m2 via a 60-min infusion on days 1-5 and 29-33 of a course of pelvic radiotherapy delivered at a dose of 45 Gy in 25 fractions over 33 days to a planned volume. After resection, patients with a positive circumferential margin (< or = 1 mm), extranodal deposits or Dukes' C histology received adjuvant 5-FU-based-chemotherapy (n = 40). RESULTS: After SCRT, 161 patients underwent resection. Twenty-one patients remained unresectable or refused an exenterative operation. Median follow-up is 36 months. Down-staging was achieved in most patients, with 19 having a complete pathological response (pT0). The median number of lymph nodes recovered for all patients was five (range 0-21). The 3-year survival rate for node-positive patients is 47%, for node-negative patients with less than three lymph nodes recovered is 62% and for node-negative patients with three or more lymph nodes recovered is 70%. Compared with node-positive patients, simple regression models revealed a reduced hazard ratio (HR) of 0.72 (0.36-1.43) for node-negative patients with less than three nodes recovered and 0.48 (0.26-0.89) for node-negative patients with three or more lymph nodes recovered. In a multivariate model, including nodal status, excision status, age and sex only positive excision margins significantly predicted a poor outcome: HR = 3.05 (1.55-5.97). CONCLUSIONS: The number of nodes found after preoperative chemoradiation is a significant prognostic factor by univariate analysis. In this study, patients with node-negative histology, and at least three nodes recovered, had better long-term survival than patients in whom two or less nodes were recovered or with positive nodes. This effect was attenuated by the inclusion of excision status in multivariate models.

Pretransplant cachexia and morbid obesity are predictors of increased mortality after heart transplantation.

BACKGROUND: Extremes in body weight are a relative contraindication to cardiac transplantation. METHODS: We retrospectively reviewed 474 consecutive adult patients (377 male, 97 female, mean age 50.3+/-12.2 years), who received 444 primary and 30 heart retransplants between January of 1992 and January of 1999. Of these, 68 cachectic (body mass index [BMI]<20 kg/m2), 113 overweight (BMI=>27-30 kg/m2), and 55 morbidly obese (BMI>30 kg/m2) patients were compared with 238 normal-weight recipients (BMI=20-27 kg/m2). We evaluated the influence of pretransplant BMI on morbidity and mortality after cardiac transplantation. Kaplan-Meier survival distribution and Cox proportional hazards model were used for statistical analyses. RESULTS: Morbidly obese as well as cachectic recipients demonstrated nearly twice the 5-year mortality of normal-weight or overweight recipients (53% vs. 27%, respectively, P=0.001). An increase in mortality was seen at 30 days for morbidly obese and cachectic recipients (12.7% and 17.7%, respectively) versus a 30-day mortality rate of 7.6% in normal-weight recipients. Morbidly obese recipients experienced a shorter time to high-grade acute rejection (P=0.004) as well as an increased annual high-grade rejection frequency when compared with normal-weight recipients (P=0.001). By multivariable analysis, the incidence of transplant-related coronary artery disease (TCAD) was not increased in morbidly obese patients but cachectic patients had a significantly lower incidence of TCAD (P=0.05). Cachectic patients receiving oversized donor hearts had a significantly higher postoperative mortality (P=0.02). CONCLUSIONS: The risks of cardiac transplantation are increased in both morbidly obese and cachectic patients compared with normal-weight recipients. However, the results of cardiac transplantation in overweight patients is comparable to that in normal-weight patients. Recipient size should be kept in mind while selecting patients and the use of oversized donors in cachectic recipients should be avoided.

Mechanisms of altered transcription by cigarette smoke.

The article highlighted in this issue is "The Activity of NF-kappaB in Swiss 3T3 Cells Exposed to Aqueous Extracts of Cigarette Smoke Is Dependent on Thioredoxin," by Stephan Gebel and Thomas Müller (pp. 75-81).

Dietary titanium and infant growth.

Dietary titanium as TiO2+ improved animal growth during infancy while inhibiting the metabolism of intestinal bacteria. TiO2+ was also found capable of inhibiting human cytomegalovirus in tissue culture. These and other findings indicate TiO2+ improves infant growth by acting as an antibacterial and antiviral agent. The behavior of TiO2+ stands in contrast to that of TiO2, which is inert.

Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.

This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine alpha-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca(2+) sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pH-induced shifts in the Ca(2+) dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca(2+) from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.

Exposure to cigarette tar inhibits ribonucleotide reductase and blocks lymphocyte proliferation.

Cigarette smoking causes profound suppression of pulmonary T cell responses, which has been associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. Exposure of human T cells to cigarette tar or its major phenolic components, hydroquinone and catechol, causes an immediate cessation of DNA synthesis without cytotoxicity. However, little is known of the mechanisms by which this phenomenon occurs. In this report we demonstrate that hydroquinone and catechol inhibit lymphocyte proliferation by quenching the essential tyrosyl radical in the M2 subunit of ribonucleotide reductase.

Complicated UTI. Effective treatment in the long-term care setting.

Diagnosis and treatment of complicated urinary tract infection (UTI) in older persons in the long-term care setting presents practitioners with special clinical challenges and is a more complex proposition than management of UTIs that commonly arise in younger persons. Effective care is a function of consideration and understanding of several key issues relating to urinalysis, antibiotic therapy, duration of therapy, and the route of drug administration. Typical diagnostic and management hurdles include collecting a clean specimen; dealing effectively with asymptomatic bacteriuria, a benign condition that often precipitates unnecessary treatment; and appreciation that diagnosis should not be made based solely on a positive culture result.

Biological roles of titanium.

Ti4+ in soil is a natural antibiotic mobilized by bacteria-generated H+. When added to the diet of young mice, Ti4+ enhanced their growth. These and observations of others indicate that Ti4+ has a variety of biological roles.

Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1.

Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and oxidants. They may also contribute to the regulation of cellular proliferation, apoptosis, and malignant progression. We reported previously that the human (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The present study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are transcriptionally activated by hypoxia through metal response elements (MREs) in their proximal promoter regions. These elements bind metal transcription factor-1 (MTF-1). Deletion and mutational analyses of the hMT-IIA promoter indicated that the hMRE-a element is essential for basal promoter activity and for induction by hypoxia, but that other elements contribute to the full transcriptional response. Functional studies of the mMT-I promoter demonstrated that at least two other MREs (mMRE-d and mMRE-c) are responsive to hypoxia. Multiple copies of either hMRE-a or mMRE-d conferred hypoxia responsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibroblasts with targeted deletions of both MTF-1 alleles (MTF-1(-/-); dko7 cells) were not induced by zinc and showed low responsiveness to hypoxia. A transiently transfected MT promoter was unresponsive to hypoxia or zinc in dko7 cells, but inductions were restored by cotransfecting a mouse MTF-1 expression vector. Electrophoretic mobility shift assays detected a specific protein-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Together, these results demonstrate that hypoxia activates MT gene expression through MREs and that this activation involves MTF-1.

Antibiotic use in the elderly: issues and nonissues.

Despite the well-recognized increase in mortality and morbidity due to infections in the elderly, antibiotics may, in most cases, be used in a manner similar to that in younger patients. The decreased lean body weight and reduced renal function typical of elderly patients, however, require consideration of reduced doses and longer dosing intervals, especially for renally excreted antibiotics. Length of therapy should be conservative because underlying anatomic or functional predispositions to infections tend to complicate treatment. Oral antibiotics are equally well absorbed in the elderly and younger patients and may be used for the same indications as for younger patients. A notable, important difference in the choice of antibiotics for serious infections in older versus younger patients is that empirical therapy should be broader in spectrum for elderly patients, and especially for elderly long-term residents, since the variety of infecting bacteria tends to be greater and polymicrobial infections tend to be common.

Transition metals as protease inhibitors.

An alternative approach to the development of clinically useful protease inhibitors was investigated. The approach utilized coordination chemistry of transition metal ions rather than substrate analogs to block active sites of these enzymes. In the case of serine proteases it was found that aqueous Ti(IV) is a potent inhibitor of the trypsin subclass, but not the chymotrypsin subclass. The direct binding of Ti(IV) to trypsin was made possible by the presence of a free carboxyl group at the bottom of the substrate binding pocket of the enzyme, and the five-coordinate geometry of TiO(SO4)(H2O). Although initial binding of Ti(IV) was reversible, it was followed in time by irreversible inhibition. Direct binding of octahedral or tetrahedral metal ion complexes was prevented by the inability of the enzyme active sites to promote formation of a five-coordinate transition state of the metal ion required for reaction. These studies demonstrate the ability of direct metal ion binding as a way to enhance blocking of enzyme active sites as compared with that of traditional organic inhibitors. Application of these findings was investigated by measuring the affect Ti(IV) had on growth of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa. Five-coordinate titanyl sulfate completely inhibited the growth of these organisms. This suggests that five-coordinate titanyl sulfate, which is easier and less expensive to manufacture than conventional antibiotics, may be useful in controlling endemic infections of E. coli and S. typhimurium.

Cloning and characterization of cDNAs for a bovine (Bos taurus) Mx protein.

Mx proteins are GTPases that are stringently induced in cells from many vertebrates on exposure to type I interferons (IFNs), and expression of some Mx proteins potently inhibits replication of specific viruses. Two cDNAs encoding bovine Mx proteins were isolated from an endometrial phage library. The open reading frames (ORFs) of these two clones predict proteins of 654 (Mxl) and 648 (Mxl-a) residues. Both possess the tripartite GTPase domains, dynamin signature, and leucine zipper motifs conserved in all other Mx proteins identified. The bovine protein sequences show highest identity to ovine Mx (93%) and are substantially similar to human MxA (73%) and mouse Mx1 (63%). Based on differences between the two bovine clones in the coding and 3'-untranslated regions, it was concluded that they represent two alleles of one gene, and heterozygous and homozygous cattle were identified. Expression of Mx mRNA was rapidly induced in cultured bovine cells by treatment with IFN.

A proposal for minimum detectable compartment in MIRD dosimetry modelling.

The accuracy of radiation dose estimates from radiopharmaceutical administrations has recently become more important for three main reasons: (i) clinical providers are demanding more information on diagnostic procedures; (ii) regulatory groups are scrutinizing dosimetry for research subjects; and (iii) accurate organ doses are crucial in therapeutic administrations. These dose estimates are a sensitive function of the residence times. Because most clinical data acquisition protocols are limited to the first 24 h after dose administration, the area under the remainder of the time-activity curve (TAC) must be estimated. Estimation methods range from assuming physical decay only (overly conservative) to extrapolating end point physiological kinetics (overly liberal). This study demonstrates how much the results from these two methods vary and develops an alternative method which more accurately estimates this remainder term. A method, called the minimum detectable compartment (MDC), is constructed so that an accurate dose estimate can be made with a realistic measure of the remainder term. The method for determining MDC uses standard hypothesis testing. Using an analogue of the traditional minimal detectable activity calculation, a model with and without constant compartments is fitted to the TAC. The size of the constant compartment is varied until the relative likelihood of the two models meets the desired measure of power and sensitivity. Computer simulations of a simple mono-exponential are used to demonstrate the MDC as a function of the model, the number of data points, the range of the data and the noise in the data. The MDC is a very sensitive function of the data range. It falls by more than 50% when the data range is increased from two to three half-lives. In addition, the MDC is moderately sensitive to the noise in the data and relatively insensitive to the number of data points. These findings suggest that the MDC method can also be uses a priori to indicate what type of data collection regimen is necessary to achieve a certain accuracy.