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.

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.

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.

Cefoxitin resistance in community-acquired gram-negative bacillary bacteremia. Associated clinical risk factors.

Among 185 patients with nonneutropenic, community-acquired gram-negative bacillary bacteremias, clinical risk factors for cefoxitin resistance included any antibiotic taken within the last three weeks (25.6% cefoxitin resistance), long-term bladder catheterization or surgical urinary diversion (23.3%), hospitalization within the last 30 days (22.9%), and nursing home residence before admission (20.8%). Patients with none of these risk factors were less likely to have cefoxitin-resistant bacteremias (0.9%). When these risk factors were examined in the subgroups of urinary tract and non-urinary tract sources of community-acquired gram-negative bacillary bacteremia, they were also helpful in predicting sensitivity to trimethoprim-sulfamethoxazole and gentamicin. The presence of one or more of the risk factors identified may be a useful adjunct in determining initial empiric antimicrobial therapy for community-acquired gram-negative bacillary bacteremia.

Improved mortality in gram-negative bacillary bacteremia.

From 1979 to 1982, the four years of this study, episodes of gram-negative bacillary bacteremia occurred in a 489-bed community teaching hospital--an increase of 15.9%. Mortality related to bacteremia was 19.4% overall and only 3.2% for the 158 episodes involving nonfatal underlying illnesses, lower figures than those reported in the past. The severity of underlying illnesses in bacteremic patients dominated all other clinical variables that were studied as prognostic factors for the outcome of the episode. The same bacteremia-related mortality was seen in patients who had empirically received (1) multiple-antibiotic regimens in which one or more drugs were active against the pathogenic organism(s), (2) either an appropriate aminoglycoside or beta-lactam antibiotic alone, or (3) both an aminoglycoside antibiotic and a beta-lactam antibiotic active against the pathogenic organism(s).

Evaluation and management of vaginitis. An update for primary care practitioners.

There are three major causes of vaginitis symptoms that primary care practitioners should be able to diagnose and treat expertly. Recent investigations have shown that bacterial vaginosis, the most common cause of vaginitis symptoms in patients not seen in sexually transmitted disease clinics, has a multifactorial etiology: Gardnerella vaginalis is universally present but is not, per se, the etiologic agent. Diagnosis and treatment are based, therefore, on evidence of a disturbed bacterial ecology as well as the presence of "clue" cells that indicate the presence of Gardnerella. Trichomonas vaginitis is usually easy to diagnose, but treatment failures occasionally occur. Some strains of Trichomonas vaginalis may be relatively resistant to metronidazole, and short-course therapy may lead to reinfection from sexual partners. Candida vulvovaginitis, the third major type of vaginitis, is not a sexually transmitted disease and should be viewed as vaginal "thrush." Earlier treatment regimens have been simplified by the introduction of more potent antifungals.

A stress management workshop improves residents' coping skills.

We describe the effectiveness of a stress management workshop designed for physicians. Of the 64 medicine, pediatrics, and medicine-pediatrics residents who agreed to participate in the workshop, the 43 who could be freed from clinical responsibilities constituted the intervention group; the 21 residents who could not be freed from clinical responsibilities were asked to be the nonintervention group. The ESSI Stress Systems Instrument and Maslach Burnout Inventory were administered to control subjects and workshop participants 2 weeks before and 6 weeks after the workshop. The half-day workshops taught management of the stresses of medical practice through: (1) learning and practicing interpersonal skills that increase the availability of social support; (2) prioritization of personal, work, and educational demands; (3) techniques to increase stamina and attend to self-care needs; (4) recognition and avoidance of maladaptive responses; and (5) positive outlook skills. Overall, the ESSI Stress Systems Instrument test scores for the workshop participants improved (+1.27), while the nonintervention group's mean scores declined (-0.65). All 21 individual ESSI Stress Systems Instrument scale items improved for the workshop, compared with eight of 21 items for the nonintervention group. The workshop group improved in the Maslach Burnout Inventory emotional exhaustion scale and deteriorated less than the nonintervention group in the depersonalization scale. We conclude that a modest, inexpensive stress management workshop was received positively, and can lead to significant short-term improvement in stress and burnout test scores for medicine and pediatrics residents.

The residency-practice training mismatch. A primary care education dilemma.

Primary care practice requires clinical skills and knowledge that differ greatly from those required for successful completion of residency training. Discrepant clinical settings and physician responsibilities have thus created a mismatch between the educational content of residency training and the content of clinical practice, which may result in suboptimal preparation of internists, family practitioners, and pediatricians for patient care. Of equal concern, the psychosocial environment of residency does not prepare physicians for their future community and personal adult roles. Barriers to correcting this worsening mismatch include the following: (1) economic pressures to use house staff to meet service needs of hospitals, (2) changes in patient demographics and the focus of hospital-based medicine that are making hospitals progressively more unsuitable as the principal training site for primary care physicians, (3) the deemphasis of practicing physicians as role models and teachers in postgraduate training, and (4) the often heated disagreement among medical educators regarding the purpose and content of residency training. Efforts to resolve this mismatch should include the following: reexamining the educational objectives of the current system of postgraduate training, better counseling of physicians in training regarding career goals, and emphasizing the primary care physician as role models and faculty.

Residents' views of the value of moonlighting.

The opportunity for residents to moonlight is threatened by legal liability concerns and legislation designed to limit the duration of workdays and workweeks. We sought the opinion of all 40 second- or third-year residents and fellows in a hybrid university/community hospital internal medicine residency program regarding their motivation to moonlight and the value of their experiences. Sixty-five percent were moonlighters; moonlighters had a higher average debt ($41 644) than nonmoonlighters ($32 917). Residents viewed moonlighting as a positive educational experience that helped them with career decisions. They believed they acquired important skills and knowledge not learned elsewhere, and that moonlighting did not interfere with their job and educational responsibilities. A program in operation for 10 years that was designed to control, monitor, and facilitate moonlighting experiences is described. We believe our residents' positive views may be in part a result of the supervision and integration of moonlighting in a residency training program with a controlled workload.

Amrinone-induced thrombocytopenia.

The frequency and characteristics of thrombocytopenia resulting from administration of amrinone, a new inotropic and vasodilator agent, was evaluated in 43 patients. Thrombocytopenia attributable to amrinone developed in eight patients (18.6%). The thrombocytopenia was due to accelerated peripheral loss of platelets. There appeared to be a dose relationship with regard to the rapidity of onset and degree of thrombocytopenia. Although platelet-associated IgG levels were elevated when measured in patients with thrombocytopenia, the clinical features were suggestive of a direct, perhaps nonimmunologic effect of amrinone on platelets. Thrombocytopenia was mild in most cases and bleeding attributable to thrombocytopenia did not occur. Several patients continued amrinone therapy over long periods despite low platelet counts, showing that mild to moderate thrombocytopenia is not necessarily an indication that therapy should be discontinued, but that platelet counts should be observed closely.

Do clinical and formal assessments of the capacity of patients in the intensive care unit to make decisions agree?

BACKGROUND: The complex environment and technology of intensive care unit (ICU) care may impair the ability of patients to participate in medical decision making or give informed consent. We studied the agreement of the intuitive assessments of residents and nurses of ICU patients' cognition, judgment, and decision-making capacity, and whether those assessments agreed with abbreviated formal mental status testing. METHODS: Using a prospective survey case study, we assessed 200 English-speaking patients within 24 hours of their ICU admission. Formal assessment of cognition, judgment, and insight was performed by a research assistant. We obtained independent intuitive ratings by nurses and residents of patient cognition, judgment, and ability to participate in medical decision making or give informed consent. RESULTS: Residents' and nurses' assessment of cognition and judgment showed a high degree of agreement with weighted ks of greater than 0.76. Assessments of cognition by residents and nurses agreed with Folstein Mini-Mental State Examination in 70% and 73.6% of cases, respectively. Forty percent of the population had an unimpaired Mini-Mental State Examination score of greater than 23, and an additional 12% of the subjects were mildly impaired with scores of 20 to 23. When asked whether they would approach patient or family for consent for an invasive procedure, nurses and physicians said they would request informed consent from 66% and 62% of the patients, respectively. CONCLUSIONS: Residents and nurses caring for patients newly admitted to the ICU agree in their assessment of cognition, judgment, and capacity to participate in medical decision making, and are not unduly influenced by ventilator status. Their assessments correlate highly with abbreviated formal mental status testing.

Dialysis discontinuation. A 'good' death?

BACKGROUND: Approximately 10% of the deaths of patients receiving long-term dialysis for end-stage renal disease are preceded by discontinuation of dialysis. We prospectively studied the decision to discontinue dialysis and whether, as is often stated, these patients have a prompt, predictable, and comfortable death. METHODS: All patients receiving hemodialysis in a hospital-based and a freestanding unit whose long-term dialysis was discontinued in 1990 were included in the study. Patients, providers, and families of prospectively enrolled cases were interviewed to determine the reasons for discontinuation; the patients' terminal courses were reviewed daily to collect information describing their quality of death. Retrospectively enrolled cases were studied by chart review and interviews of providers. The reasons for discontinuation of dialysis and a rating of the quality of their deaths (for prospectively studied patients only) were determined by interdisciplinary team consensus. Quality of death was rated on scales of 1 (worst) to 5 (best) according to duration of dying, discomfort, and psychosocial circumstances. RESULTS: Eighteen patients discontinued dialysis after a mean duration of 43.6 months of hemodialysis, and they lived a mean of 9.6 days after termination. The quality of death of the 11 patients who were enrolled prospectively was subjectively assessed as "good" (> 10 of a possible 15 points) for seven patients and "poor" for four patients. A good quality of death was more likely if dialysis was discontinued because of medical deterioration from progressive chronic disease (P = .009); none of the three patients whose dialysis was discontinued for other reasons had a good death (P = .024). CONCLUSIONS: A majority of the prospective cohort of patients who discontinued dialysis experienced a good death by our largely subjective criteria. Improved palliative therapy for some of these dying patients, however, could have ameliorated prolonged suffering, delirium, and inadequately treated pain that led to a poor quality of death.

Management strategies for urinary and vaginal infections.

Detailed history, physical examination, laboratory and follow-up data were obtained from 821 women coming to a primary care clinic over a two-year period with the symptoms of urinary tract (UTI) or vaginal infection. Using all available information, each patient retrospectively was given one of several mutually exclusive diagnoses. Vaginitis without UTI was diagnosed in 70% of patients, UTI without vaginitis in 12%, UTI and vaginitis in 2%. The conditional probability of the several possible diagnoses was calculated, given various combinations of clinical data; a diagnosis of vaginitis was twice as likely as a diagnosis of UTI in a patient with dysuria. On the basis of these calculations we identified efficient clinical strategies for when to perform a pelvic examination, a urinalysis, and a urine culture, and when to diagnose UTI presumptively on the basis of urinalysis.

Interference of new penicillins and cephalosporins with urine glucose monitoring tests.

The possibility that three new penicillins (mezlocillin, pipericillin, and azlocillin) and two new cephalosporins (ceftazidime and cefoperazone) might give false positive urine glucose testing results was studied. High and low concentrations of antibiotics were dissolved in urine and tested with Clinitest (Ames Division, Miles Laboratories, Elkhart, Indiana) and Tes-Tape (Eli Lilly and Company, Indianapolis, Indiana) at four different concentrations of glucose. None of the antibiotics interfered with the accuracy of Tes-Tape readings. All antibiotics interfered with Clinitest readings; the greatest interference was noted at high concentrations of antibiotics and low concentrations of glucose, and with the two cephalosporins. A summary of all reported cephalosporin interactions with Clinitest urine glucose testing is included.

Changes in oxygen radical scavenging by human blood cell lysates concurrent with viral infections.

The higher superoxide dismutase (SOD) levels found in human blood cells when Nitro Blue Tetrazolium (NBT) rather than Cytochrome C was used as the colorimetric detector of superoxide (O-2) was investigated. the NBT was found to react with oxygen radicals other than O-2, thus providing measurement of total oxygen radical scavenging ability. Also investigated was the biomodal distribution of SOD activity in lymphocytes and granulocytes from a randomly selected human populace. Human lymphocytes rapidly increased their SOD activity by two- to four-fold during respiratory viral distress. The response to certain infections was probably responsible for the bimodal distribution of SOD activity in the general populace. It was concluded that the chemical events that prepare lymphocytes and granulocytes for their role in defense against infections begin long before these cells are sequestered from circulation by the infections.

Expression of a murine gonadotropin-releasing hormone receptor-luciferase fusion gene in transgenic mice is diminished by immunoneutralization of gonadotropin-releasing hormone.

A line of transgenic mice harboring a fusion gene consisting of 1900 bp of proximal 5'-flanking region from the murine GnRH receptor gene linked to the complementary DNA encoding luciferase was established to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for identifying the molecular mechanisms underlying hormonal regulation of this gene. Of 10 tissues screened, luciferase was detected predominantly in pituitary gland, but also in brain and testis. To assess hormonal regulation, luciferase activity was measured in intact males and ovariectomized females treated with an anti-GnRH serum alone, and in combination with testosterone or 17beta-estradiol. No effect of steroid treatment on transgene expression was detected. However, immunoneutralization of GnRH resulted in decreased serum LH concentrations and suppressed pituitary expression of luciferase. Furthermore, the effects of GnRH antiserum could be prevented by the administration of a noncross-reactive GnRH agonist. Thus, 1900 bp of 5'-flanking DNA from the murine GnRH receptor gene are sufficient to target luciferase expression in transgenic mice to established sites of GnRH receptor gene expression. Furthermore, we suggest that GnRH regulation of GnRH receptor gene expression is mediated by regulatory elements residing within 1900 bp of the 5'-flanking region.

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.

Influence of medical and premedical education on important personal qualities of physicians.

Three interrelated personal qualities of physicians are believed important for sensitive patient care and optimal individual adjustment to the stresses of medical practice: maturity, social competence, and moderation in aggressive competitive (exaggerated "type A" behavior). Despite widespread recognition of the importance of these qualities by patients and physicians alike, they have commonly been neglected in favor of scientific and scholastic excellence in the selection process for medical schools. In addition, some aspects of premedical and medical education may actually have an adverse influence on these personal qualities of future physicians. More emphasis in premedical and medical education on the importance of physicians' noncognitive abilities, and more individualized feedback to students and residents on the interactions between their personal qualities and their success and happiness as physicians, are needed.

Training internists: insights from private practice.

University internal medicine training programs concentrate on the traditional curriculum designed to produce well-trained academicians and researchers. Increasingly internists are involved in primary patient care with over two-thirds being office based practitioners. Residency training at these institutions must make available to all residents the opportunity to learn the skills taught by primary care programs. Clinical problem solving, skills in patient-physician negotiations and patient comfort, psychiatric techniques, medical ethics, cost effectiveness analysis, and practice management are areas in which the private practitioner frequently needs help. Feedback from training program graduates in private practice could help identify such deficiencies which could be incorporated into the teaching responsibility of a division of general internal medicine.