Ethical Acceptability of Postrandomization Consent in Pragmatic Clinical Trials.

Importance: Pragmatic clinical trials that seek informed consent after randomization (ie, postrandomization consent) are increasingly used, but debate on ethics persists because control arm patients are not specifically informed about the trials and randomization occurs before consent for the trials. The public's attitude toward postrandomization consent trials is unknown, but the way the trials are described could bias people's views. Objectives: To assess the attitudes of the US general public toward postrandomization informed consent for pragmatic trials and to measure potential framing and other factors associated with those attitudes. Design, Setting, and Participants: An online, 2 × 2 experimental survey (fielded between February 23 and April 3, 2018) portraying 4 scenarios of postrandomization informed consent (with prior broad consent for medical record use) was conducted. These scenarios included traditional randomized clinical trial language framing vs alternative framing in a high-stakes trial (ie, survival in leukemia) or low-stakes trial (ie, blood glucose level in diabetes). A total of 3793 individuals invited to participate were part of an existing panel representative of the US general public (GfK KnowledgePanel). Main Outcomes and Measures: The proportion of participants who would recommend that an ethics review board approve a postrandomization consent pragmatic trial. Results: A total of 2042 of 3739 invitees (54.6%) responded; after exclusion of 38 incomplete surveys, 2004 participants were included in the analysis. Of these, 997 (49.8%) were women, 1440 (71.9%) were white non-Hispanic, 199 (9.9%) were black non-Hispanic, and 233 (11.6%) were Hispanic. Mean (SD) age was 47.5 (17.4) years. Across scenarios, weighted data showed that 75.4% of the participants would recommend approval of the postrandomization consent pragmatic trial, 20.4% would probably not recommend approval, and 4.2% would definitely not recommend approval. Approval was not sensitive to framing language (traditional vs new framing in high-stakes scenario, 74.3% vs 76.8%, P = .40; in low-stakes scenario, 77.7% vs 72.9%, P = .10) or to the stakes (low vs high stakes in traditional framing, 77.7% vs 74.3%, P = .25; in new framing, 72.9% vs 76.8%, P = .18). Better understanding of the postrandomization consent design was associated with higher rate of approval (78.1% vs 65.0%, P = .002 for high-stakes scenario; 77.2% vs 64.9%, P = .004 for low-stakes scenario), especially among those with less education. However, opinions about personal involvement in the control arm were more cautious (range depending on scenario, 45.6%-59.7%) and sensitive to stakes but not to framing. Conclusions and Relevance: The public's generally high rate of approval of the ethics of postrandomization informed consent for pragmatic trial designs does not appear to be affected by whether postrandomization consent design is framed using traditional randomized clinical trial terminology, regardless of the stakes of the trial. Promoting better understanding of the design may increase its acceptance by the public.

Gait abnormalities and progressive myelin degeneration in a new murine model of Pelizaeus-Merzbacher disease with tandem genomic duplication.

Pelizaeus-Merzbacher disease (PMD) is a hypomyelinating leukodystrophy caused by mutations of the proteolipid protein 1 gene (PLP1), which is located on the X chromosome and encodes the most abundant protein of myelin in the central nervous sytem. Approximately 60% of PMD cases result from genomic duplications of a region of the X chromosome that includes the entire PLP1 gene. The duplications are typically in a head-to-tail arrangement, and they vary in size and gene content. Although rodent models with extra copies of Plp1 have been developed, none contains an actual genomic rearrangement that resembles those found in PMD patients. We used mutagenic insertion chromosome engineering resources to generate the Plp1dup mouse model by introducing an X chromosome duplication in the mouse genome that contains Plp1 and five neighboring genes that are also commonly duplicated in PMD patients. The Plp1dup mice display progressive gait abnormalities compared with wild-type littermates. The single duplication leads to increased transcript levels of Plp1 and four of the five other duplicated genes over wild-type levels in the brain beginning the second postnatal week. The Plp1dup mice also display altered transcript levels of other important myelin proteins leading to a progressive degeneration of myelin. Our results show that a single duplication of the Plp1 gene leads to a phenotype similar to the pattern seen in human PMD patients with duplications.

A germline-competent embryonic stem cell line from NOD.Cg-Prkdc ( scid ) Il2rg ( tm1Wjl )/SzJ (NSG) mice.

The NOD.Cg-Prkdc ( scid ) Il2rg ( tm1Wjl )/SzJ mouse strain, commonly known as NSG (for NOD SCID Gamma) is severely immunodeficient and thus is an excellent recipient for xenografts, and in particular for engrafting human tumor cells and human hematopoietic stem cells. In the latter case, these cells give rise to many human hematopoetic lineages in their NSG hosts, resulting in recapitulation of many of the features of a human immune system. However, the immune system of these "humanized mice" (huMice) is not completely functional, in part because of a lack of expression of necessary human cytokines and HLA molecules by NSG host tissues. In order to facilitate the genetic modification of this strain in order to improve the huMouse model, we have created germline competent ES cells of this strain in which such modifications can be carried out.

Genetic polymorphisms among C57BL/6 mouse inbred strains.

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina(®) Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyr ( c-2j )/J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.

Cryopreservation of mouse gametes and embryos.

As the use of genetically engineered mouse models continues to expand, the need to cryopreserve strains of mice increases in parallel in order to preserve these unique research resources and provide a low-cost alternative to maintaining the large inventory of strains. This chapter discusses methods for the cryopreservation of mouse embryos, sperm, and oocytes, and briefly discusses other requirements for implementing a successful cryopreservation program.

Transgenic rat model of neurodegeneration caused by mutation in the TDP gene.

TDP-43 proteinopathies have been observed in a wide range of neurodegenerative diseases. Mutations in the gene encoding TDP-43 (i.e., TDP) have been identified in amyotrophic lateral sclerosis (ALS) and in frontotemporal lobe degeneration associated with motor neuron disease. To study the consequences of TDP mutation in an intact system, we created transgenic rats expressing normal human TDP or a mutant form of human TDP with a M337V substitution. Overexpression of mutant, but not normal, TDP caused widespread neurodegeneration that predominantly affected the motor system. TDP mutation reproduced ALS phenotypes in transgenic rats, as seen by progressive degeneration of motor neurons and denervation atrophy of skeletal muscles. This robust rat model also recapitulated features of TDP-43 proteinopathies including the formation of TDP-43 inclusions, cytoplasmic localization of phosphorylated TDP-43, and fragmentation of TDP-43 protein. TDP transgenic rats will be useful for deciphering the mechanisms underlying TDP-43-related neurodegenerative diseases.

The AIM2 inflammasome is critical for innate immunity to Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis. AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.

Developing tTA transgenic rats for inducible and reversible gene expression.

To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 microg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 microg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases.

Targeted disruption of tyrosylprotein sulfotransferase-2, an enzyme that catalyzes post-translational protein tyrosine O-sulfation, causes male infertility.

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2(+/-) mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2(-/-) mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1(-/-) males that have normal fertility, Tpst2(-/-) males are infertile. Tpst2(-/-) sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2(-/-) sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility.

Archiving mouse strains by cryopreservation.

A great deal of time and energy goes into the creation of each new line of transgenic mice; established lines are expensive and labor-intensive to maintain. Archiving of mice by cryopreservation of germ cells or embryos represents a means to free up facility space, while protecting the line from loss due to environmental disasters, genetic drift, or infectious disease. The author reviews the available cryopreservation techniques and presents considerations for setting up a cryopreservation facility.

Diagnosing pediatric asthma: validating the Easy Breathing Survey.

OBJECTIVE: To determine the sensitivity, specificity, and predictive value of a simple, self-administered questionnaire for the diagnosis of asthma in children. STUDY DESIGN: A questionnaire specifically designed to assist primary care providers in making a diagnosis of asthma in children was developed and administered in 4 different primary care and subspecialty clinics, validated, and then used as part of an asthma management program called Easy Breathing. Asthma diagnoses were made according to recommended National Asthma Expert Panel Guidelines. RESULTS: Four questions on the survey were shown to be sensitive and specific for asthma. The sensitivity was greater for all levels (mild, moderate, and severe) of persistent asthma than for mild, intermittent asthma. A positive response to any 1 of the 4 questions was over 94% sensitive for asthma; a negative response to all 4 questions was 55% specific for ruling out asthma. CONCLUSIONS: Patient responses to 4 specific respiratory symptom questions can assist primary care providers in diagnosing asthma in children. Primary care providers serving pediatric populations at high risk for asthma should consider asking patients or their parents these 4 questions regarding asthma symptoms on a regular basis.

Determination and validation of a predictive model for Clostridium difficile diarrhea in hospitalized oncology patients.

BACKGROUND: Clostridium difficile colitis in the cancer patient receiving chemotherapy is a frequent cause of morbidity which may prolong hospitalization. Techniques for identifying infection often delay the initiation of therapy. PATIENTS AND METHODS: In this retrospective case-control analysis, we identified predictors for C. difficile-associated diarrhea in 29 patients hospitalized from 1988 to 1993 on a hematologic malignancy/bone marrow transplant unit (hospital A). We then validated our model with 58 C. difficile cases and 74 controls admitted to an oncology unit from a different institution (hospital B). RESULTS: We found that low intensity of chemotherapy (P < 0.001), lack of parenteral vancomycin use (P = 0.03) and hospitalization within the past two months (P = 0.05) were independently predictive of C. difficile colitis by multivariate analysis. These variables were weighted for predictive capability using a receiver operator characteristic score; low intensity chemotherapy was assigned two points, lack of parenteral vancomycin received one point and prior hospitalization one point (P < 0.001 by chi 2 for trend). The receiver operating characteristic (ROC) curve areas were 0.78 for patients at hospital A and 0.70 at hospital B indicating moderate drop off in discrimination. Compared to hospital A patients, hospital B patients hospitalized between 1989 and 1994 were more often women (P = 0.04), received less systemic vancomycin (P = 0.01), were less frequently neutropenic (P < 0.05), and received less intense chemotherapy regimens (P < 0.05). Despite these differences in demographics in patients between these institutions, our predictive model was validated in hospital B patients (P = 0.02 by chi 2 for trend). CONCLUSIONS: The results of this study may help clinicians predict the risk of C. difficile disease in the hospitalized immunocompromised oncology patient and may help guide empiric therapy while awaiting results of stool toxin assays.

Open randomized study of cefepime versus piperacillin-gentamicin for treatment of febrile neutropenic cancer patients.

An open-label randomized trial comparing the efficacy and safety of cefepime versus piperacillin plus gentamicin (P+G) given intravenously for the treatment of febrile episodes in neutropenic patients with underlying malignancy was conducted at two oncology centers. Over a 30-month period 111 patients were enrolled and 99 patients were found to be suitable for evaluation. At the 72-h time of evaluation, cefepime monotherapy and P+G combination therapy produced comparable clinical response rates (78% for both). P+G and cefepime produced comparable response rates in microbiologically documented (78 versus 71%), clinically documented (100 versus 100%), and possible (75 versus 79%) infections. The P+G and cefepime treatments achieved comparable microbiological eradication of gram-negative (100 versus 71%) (P = 0.09) and gram-positive (44 versus 70%) (P = 0.37) organisms. There were no statistically significant differences in the rates of superinfection between the groups; however, more superinfections of fungal origin were noted in the P+G group. Cefepime was demonstrated to be an effective and safe treatment for febrile episodes in neutropenic patients with malignancies, and its lack of nephrotoxicity compared to P+G was noteworthy. Cefepime appears to be a candidate for monotherapy in febrile neutropenic cancer patients.

Paecilomyces sinusitis in an immunocompromised adult patient: case report and review.

A case of fungal sinusitis caused by Paecilomyces lilacinus that was unresponsive to amphotericin B and involved a patient with acute myeloid leukemia is described. Histologic examination of sinus tissue and periorbital bone demonstrated invasion by a fungus with septate hyphae, which was identified in culture as P. lilacinus. The isolate was resistant to amphotericin B but susceptible to itraconazole. The patient responded clinically when itraconazole was added to the treatment regimen. Invasive aspergilar infections are frequently diagnosed by histology. Other fungi such as Fusarium, Pseudallescheria, and Paecilomyces species also produce hyphae in tissue and can be confused with Aspergillus species. However, these pathogens may be resistant to amphotericin B. Since alternative therapy is now available for infections with some of the amphotericin B-resistant fungi, such as P. lilacinus, every effort should be made to recover the fungal pathogen so that effective treatment can be administered.

Cefoperazone/sulbactam versus cefoperazone plus mezlocillin: empiric therapy for febrile, neutropenic bone marrow transplant patients.

We conducted a prospective, randomized trial in 132 patients undergoing bone marrow transplantation comparing cefoperazone in combination with sulbactam (S), N = 66, vs. cefoperazone plus mezlocillin (CM), N = 66, as empiric antibiotic therapy for fever and neutropenia. Overall duration of neutropenia was 3-55 (median, 13) days. Forty-one patients had positive initial cultures (S = 22 and CM = 19). Twelve of these 41 patients responded to initial study antibacterial agent treatment (S = 6 and CM = 6). Twenty-nine of 41 patients were withdrawn from study because of clinical deterioration, continued fever, or persistently positive cultures (S = 16 and CM = 13). Of the 90 patients who had culture-negative fever (S = 44 and CM = 46), 44 subjects responded with or without the addition of amphotericin B (S = 21 and CM = 23). Thirty-seven of 90 patients were withdrawn from study due to continued fever or clinical deterioration (S = 17 and CM = 20). Nine patients were withdrawn as a result of rash or diarrhea (S = 6 and CM = 3). We conclude that in patients undergoing bone marrow transplantation, there was no difference in efficacy between cefoperazone/sulbactam and the combination of cefoperazone plus mezlocillin in the empiric treatment of the febrile neutropenic patient. Since the majority of initial infections were due to gram positive bacteria, consideration should be given to broadening initial empiric antibacterial agent therapy with drugs that possess potent activity against these organisms.

Nosocomial infections in neutropenic cancer patients.

OBJECTIVE: Identification of overall and site-specific rates of nosocomial infection in neutropenic patients with cancer and associated pathogens. DESIGN: Cumulative continuous prospective surveillance over a 42-month period. Criteria and definitions of infection in neutropenic patients (absolute neutrophil counts < 1,000/mm3) were developed, and surveillance was carried out by a certified infection control nurse and a senior oncology research fellow. SETTING: A cancer research center with two designated oncology nursing units. PATIENTS: Neutropenic patients with hematological and solid malignancies undergoing high-dose chemotherapy with and without autologous bone marrow transplantation. All patients admitted to both of the units during the study period were surveyed. Those who developed neutropenia are included in this report. RESULTS: A total of 444 nosocomial infections were identified in 920 neutropenic patients during 9,582 days of neutropenia for an overall rate of 48.3 per 100 neutropenic patients, or 46.3 per 1,000 days at risk. The rate of bloodstream infection per 100 neutropenic patients was 13.5 (gram-positive, 9.2; gram-negative, 4.8; and Candida 1.2). Other site-specific rates were: urinary tract, 5.7; respiratory tract, 5.5; thrush, 6.6; skin, 3.4; and gastrointestinal tract, 3.4. Among 392 pathogens identified, there were 137 (35%) gram-positive cocci, 105 (27%) gram-negative rods, 70 (18%) Candida, 37 (9%) gram-positive rods, 22 (6%) viruses, and 15 (4%) Aspergillus. CONCLUSIONS: The rate of both overall and site-specific nosocomial infections in neutropenic patients is high. Neutropenia is a significant intrinsic risk factor that should be addressed in surveillance programs. Infection control and infectious diseases practitioners may need to modify techniques for surveillance, control, and management of infection in this population.

Clostridium cadaveris bacteremia in the immunocompromised host.

Clostridium cadaveris, usually considered a non-pathogen, was isolated from blood cultures of two febrile patients with cancer. The bacteremias appeared to have originated from the abdomen. This organism has not been previously reported as the etiological agent in this setting.

Low frequency and low level of elevation of serum CA 72-4 in human carcinomas in comparison with established tumor markers.

We evaluated a new circulating tumor marker, CA 72-4, by comparing its frequency of appearance and level of elevation with other established tumor markers in serial serum specimens from patients with various carcinomas. We found that CA 72-4, though highly expressed and widely found in various tumor tissues, is present at low concentration and frequency in the serum. In breast, colon, ovarian, and pancreatic carcinomas, only 21%, 30.9%, 16%, and 26.5% of specimens, respectively, showed elevated CA 72-4. When elevated, the level of elevation was also low, much lower than that of the dominant markers. Poor response of CA 72-4 to therapy was especially noticeable in serial specimens. In most cases, the CA 72-4 remained normal for the entire series while other markers remained at elevated levels. However, changes of the level of CA 72-4 usually paralleled those of other markers but at a much lower concentration. Simultaneous measurement of CA 72-4 and CA 19-9 appears useful to differentiate colorectal from pancreatic carcinomas when they all contained elevated levels of CA 19-9. There was a much higher ratio of CA 72-4 to CA 19-9 with colon than with pancreatic and other carcinomas (247 +/- 524 vs. 4.7 +/- 6.8).

Disseminated aspergillosis after fungemia in a patient with extragonadal germ cell tumor undergoing autologous bone marrow transplantation.

In a 40-year-old man with extragonadal germ cell tumor, Aspergillus fumigatus disseminated hematogenously from a skin infection at a Hickman catheter exit site after autologous bone marrow transplantation. A notable aspect of this case was the absence of neutropenia.