Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Cost-effectiveness of salpingotomy and salpingectomy in women with tubal pregnancy (a randomized controlled trial).

STUDY QUESTION: Is salpingotomy cost effective compared with salpingectomy in women with tubal pregnancy and a healthy contralateral tube? SUMMARY ANSWER: Salpingotomy is not cost effective over salpingectomy as a surgical procedure for tubal pregnancy, as its costs are higher without a better ongoing pregnancy rate while risks of persistent trophoblast are higher. WHAT IS KNOWN ALREADY: Women with a tubal pregnancy treated by salpingotomy or salpingectomy in the presence of a healthy contralateral tube have comparable ongoing pregnancy rates by natural conception. Salpingotomy bears the risk of persistent trophoblast necessitating additional medical or surgical treatment. Repeat ectopic pregnancy occurs slightly more often after salpingotomy compared with salpingectomy. Both consequences imply potentially higher costs after salpingotomy. STUDY DESIGN, SIZE, DURATION: We performed an economic evaluation of salpingotomy compared with salpingectomy in an international multicentre randomized controlled trial in women with a tubal pregnancy and a healthy contralateral tube. Between 24 September 2004 and 29 November 2011, women were allocated to salpingotomy (n = 215) or salpingectomy (n = 231). Fertility follow-up was done up to 36 months post-operatively. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: We performed a cost-effectiveness analysis from a hospital perspective. We compared the direct medical costs of salpingotomy and salpingectomy until an ongoing pregnancy occurred by natural conception within a time horizon of 36 months. Direct medical costs included the surgical treatment of the initial tubal pregnancy, readmissions including reinterventions, treatment for persistent trophoblast and interventions for repeat ectopic pregnancy. The analysis was performed according to the intention-to-treat principle. MAIN RESULTS AND THE ROLE OF CHANCE: Mean direct medical costs per woman in the salpingotomy group and in the salpingectomy group were €3319 versus €2958, respectively, with a mean difference of €361 (95% confidence interval €217 to €515). Salpingotomy resulted in a marginally higher ongoing pregnancy rate by natural conception compared with salpingectomy leading to an incremental cost-effectiveness ratio €40 982 (95% confidence interval -€130 319 to €145 491) per ongoing pregnancy. Since salpingotomy resulted in more additional treatments for persistent trophoblast and interventions for repeat ectopic pregnancy, the incremental cost-effectiveness ratio was not informative. LIMITATIONS, REASONS FOR CAUTION: Costs of any subsequent IVF cycles were not included in this analysis. The analysis was limited to the perspective of the hospital. WIDER IMPLICATIONS OF THE FINDINGS: However, a small treatment benefit of salpingotomy might be enough to cover the costs of subsequent IVF. This uncertainty should be incorporated in shared decision-making. Whether salpingotomy should be offered depends on society's willingness to pay for an additional child. STUDY FUNDING/COMPETING INTERESTS: Netherlands Organisation for Health Research and Development, Region Västra Götaland Health & Medical Care Committee. TRIAL REGISTRATION NUMBER: ISRCTN37002267.

Ectopic pregnancy prediction in women with a pregnancy of unknown location: data beyond 48 h are necessary.

STUDY QUESTION: Are there improvements in the accuracy of prediction of ectopic pregnancy (EP) in women with early symptomatic pregnancy using human chorionic gonadotrophin (hCG) curves when clinicians consider visits beyond the first 48 h after initial presentation? SUMMARY ANSWER: Two hCG values, measured 48 h (2 days) apart, are often not sufficient to accurately predict the outcome of a woman with a pregnancy of unknown location (PUL), but adding a third visit on Day 4 or 7 significantly improved the prediction for 1 in 15 women. WHAT IS KNOWN ALREADY: The use of serial hCG values is commonly used to aid in the prediction of the final diagnosis in women with a PUL. Initial outcome predictions based on two hCG values may often be incorrect. STUDY DESIGN, SIZE, DURATION: This retrospective multicenter cohort study included 646 women with a PUL, recruited over 2 years. Of these women, 146 were ultimately diagnosed with EP. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women presenting to the emergency room with first trimester pain or bleeding, with a PUL, at least 2 hCG values and a definitive final diagnosis from the University of Pennsylvania, University of Miami and University of Southern California, were recruited from 2007 to 2009. MAIN RESULTS AND THE ROLE OF CHANCE: Using currently recommended prediction rules, adding a third hCG evaluation on Day 4 after initial presentation significantly improved the accuracy of initial prediction from the first two values (48 h apart, or Day 2) by 9.3% (P = 0.015). Adding a third value on Day 7 improved prediction significantly by 6.7% (P = 0.031), compared with prediction based on first two values. The improvement in prediction by assessing four hCG values (Days 0, 2, 4 and 7) compared with three values (Days 0, 2 and 4) was 1.3% and not statistically significant. LIMITATIONS, REASONS FOR CAUTION: Missing data imputation likely biased results toward the null; predicted outcomes may not match those made by clinicians; and the study does not predict intrauterine pregnancy and spontaneous miscarriage separately. WIDER IMPLICATIONS OF THE FINDINGS: This study provides useful information for the prediction of outcomes for women with a symptomatic first trimester pregnancy of unknown location, but may not be generalizable to all pregnant women. STUDY FUNDING/COMPETING INTEREST(S): Supported by NIH grant numbers R01-HD036455 to Dr Barnhart and Dr Sammel, K24HD060687 to Dr Barnhart, and 5T32MH065218 to Ms. Zee. The authors have no conflicts of interest to declare.

Spontaneous high-grade glial intramedullary tumor of the spine in a rhesus macaque (Macaca mulatta).

BACKGROUND: A 4-year-old rhesus macaque presented with acute, progressive paresis of the extremities. METHODS: A complete blood count, serum biochemical analysis, neurologic exam and necropsy were performed. RESULTS: The clinical, histopathological, and immunohistochemical findings confirmed a high-grade intramedullary glial tumor of the spinal cord that was most consistent with an ependymoma. CONCLUSIONS: We describe a case of a naturally occurring spontaneous spinal cord neoplasia in a non-human primate.

The prognostic profile of subfertile couples and treatment outcome after expectant management, intrauterine insemination and in vitro fertilisation: a study protocol for the meta-analysis of individual patient data.

OBJECTIVE: The current evidence concerning the best treatment option for couples with unexplained and male subfertility is inconclusive. Most studies that have evaluated the effectiveness of treatment options, such as expectant management (EM), intrauterine insemination (IUI), with or without controlled ovarian stimulation (COS), and in vitro fertilisation (IVF), have not taken the couples' prognosis into account. It is very likely that the individual prognosis of the couple influences the effect of treatment. Individual patient data analyses allow us to take these prognostic factors into account, and to evaluate their effect on treatment outcome. This study aims to use anonymised data from relevant published trials to perform an individual patient data meta-analysis, evaluating the effect of couples' prognosis on the effectiveness of EM, IUI, with or without COS, and IVF. METHODS: Based on earlier systematic reviews and an updated search, randomised controlled trials will be considered for inclusion. Untreated subfertile couples with unexplained or male subfertility included in trials comparing EM, IUI, with or without COS, and IVF are included. Authors of the included studies will be invited to share their original anonymised data. The data will be assessed on validity, quality and completeness. The prognosis of the individual couple will be calculated with existing prognostic models. The effect of the prognosis on treatment outcome will be analysed with marker-by-treatment predictiveness curves, illustrating the effect of prognosis on treatment outcome. This study is registered in PROSPERO (registration number CRD42011001832). CONCLUSION: Ultimately, this study may help to select the appropriate fertility treatment, tailored to the needs of an individual couple.

Does a prediction model for pregnancy of unknown location developed in the UK validate on a US population?

BACKGROUND: A logistic regression model (M4) was developed in the UK to predict the outcome for women with a pregnancy of unknown location (PUL) based on the initial two human chorionic gonadotrophin (hCG) values, 48 h apart. The purpose of this paper was to assess the utility of this model to predict the outcome for a woman (PUL) in a US population. METHODS: Diagnostic variables included log-transformed serum hCG average of two measurements, and linear and quadratic hCG ratios. Outcomes modeled were failing PUL, intrauterine pregnancy (IUP) and ectopic pregnancy (EP). This model was applied to a US cohort of 604 women presenting with symptomatic first-trimester pregnancies, who were followed until a definitive diagnosis was made. The model was applied before and after correcting for differences in terminology and diagnostic criteria. RESULTS: When retrospectively applied to the adjusted US population, the M4 model demonstrated lower areas under the curve compared with the UK population, 0.898 versus 0.988 for failing PUL/spontaneous miscarriage, 0.915 versus 0.981 for IUP and 0.831 versus 0.904 for EP. Whereas the model had 80% sensitivity for EP using UK data, this decreased to 49% for the US data, with similar specificities. Performance only improved slightly (55% sensitivity) when the US population was adjusted to better match the UK diagnostic criteria. CONCLUSIONS: A logistic regression model based on two hCG values performed with modest decreases in predictive ability in a US cohort for women at risk for EP compared with the original UK population. However, the sensitivity for EP was too low for the model to be used in clinical practice in its present form. Our data illustrate the difficulties of applying algorithms from one center to another, where the definitions of pathology may differ.

Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Continuous dosing of a novel contraceptive vaginal ring releasing Nestorone® and estradiol: pharmacokinetics from a dose-finding study.

BACKGROUND: As part of a program to develop a novel estradiol-releasing contraceptive vaginal ring (CVR), we evaluated the pharmacokinetic (PK) profile of CVRs releasing segesterone acetate (Nestorone® (NES)) combined with one of three different estradiol (E2) doses. STUDY DESIGN: A prospective, double-blind, randomized, multi-centered study to evaluate a 90-day CVR releasing NES [200mcg/day] plus E2, either 10mcg/day, 20mcg/day, or 40mcg/day in healthy reproductive-age women with regular cycles. Participants provided blood samples twice weekly for NES and E2 levels during the first 60 days (ring 1) and the last 30 days (ring 2) of use. A subset underwent formal PK assessments at ring initiation, ring exchange (limited PK), and study completion. RESULTS: The main study enrolled 197 women; 22 participated in the PK substudy. Baseline characteristics between the main and PK participants were comparable, with an average BMI of 25.8 kg/m2 (SD 4.3). In the PK substudy, all three rings showed similar NES PK: mean area under the curve (AUC(0-72)) 34,181 pg*day/mL; concentration maximum (Cmax) 918 pg/mL; time to maximum concentration (Tmax) 3.5 h. For E2, the Cmax occurred at 2 h, and was significantly higher with the 20 mcg/day ring (mean 390 pg/mL); 10 mcg/day, 189 pg/mL, p=.003; 40 mcg/day, 189 pg/mL, p<.001), and declined rapidly to≤50 pg/mL for all doses by 24 h. For all subjects, the median E2 levels remained under 35 pg/mL during treatment. CONCLUSION: PK parameters of NES were not affected when paired with different doses of E2, but E2 levels from all three doses were lower than anticipated and no dose response was observed. IMPLICATIONS: While these novel estradiol-releasing combination contraceptive vaginal rings provided sustained release of contraceptive levels of Nestorone over 90 days, the E2 levels achieved were not consistent with bone protection, and a dose-response was not observed.

Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene.

Three erythroid cell factors that bind the murine alpha-globin promoter were enriched more than 1,000-fold by conventional and DNA sequence affinity chromatography. Visualization of enriched polypeptides revealed simple patterns suggesting that each binding activity was purified. Two of the purified proteins, alpha-CP1 and alpha-CP2, have been shown previously to interact with distinct binding sites that overlap in the alpha-globin CCAAT box. Affinity purification of alpha-CP1 revealed seven polypeptides with Mrs raging from 27,000 to 38,000. In contrast, purified alpha-CP2 was made up of a polypeptide doublet with Mrs of 64,000 and 66,000. The third purified binding activity, alpha-IRP, interacted with sequences that formed an inverted repeat (IR) between the alpha-globin CCAAT and TATAA boxes. Affinity-purified alpha-IRP was made up of a single polypeptide with an Mr of 85,000. We confirmed that the purified polypeptides corresponded to alpha-CP1-, alpha-CP2-, and alpha-IRP-binding activities by UV cross-linking experiments (alpha-CP2 and alpha-IRP) or by renaturation of binding activity after elution of polypeptides from sodium dodecyl sulfate-polyacrylamide gels (alpha-CP1 and alpha-CP2). The apparent complexity of the polypeptides accounting for alpha-CP1 binding activity prompted a further physical characterization of this factor. Sedimentation of affinity-purified alpha-CP1 in glycerol gradients containing 100 mM KCl showed that all seven polypeptides migrated as a complex that cosedimented with alpha-CP1-binding activity. In contrast, when sedimented in glycerol gradients containing 500 mM KCl, alpha-CP1 dissociated into at least two components. Under these conditions, alpha-CP1-binding activity was reduced or lost. Activity was reconstituted, however, by combining fractions that were enriched in the two components. These results were confirmed by experiments in which we showed that alpha-CP1-binding activity can be recovered only by combining distinct sets of polypeptides that were isolated and renatured from sodium dodecyl sulfate-polyacrylamide gels. Our results suggest that the seven polypeptides visualized after affinity purification of alpha-CP1 interact to form a heterotypic complex (or set of complexes) required for alpha-CP1-binding activity.

Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.

Tissue-specific gene expression in the pituitary: the glycoprotein hormone alpha-subunit gene is regulated by a gonadotrope-specific protein.

The molecular mechanisms for the development of multiple distinct endocrine cell types in the anterior pituitary have been an area of intensive investigation. Though the homeodomain protein Pit-1/GHF-1 is known to be involved in differentiation of the somatotrope and lactotrope lineages, which produce growth hormone and prolactin, respectively, little is known of the transcriptional regulators important for the gonadotrope cell lineage, which produces the glycoprotein hormones luteinizing hormone and follicle-stimulating hormone. Using transgenic mice and transfection into a novel gonadotrope lineage cell line, we have identified a regulatory element that confers gonadotrope-specific expression to the glycoprotein hormone alpha-subunit gene. A tissue-specific factor that binds to this element is purified and characterized as a 54-kDa protein which is present uniquely in cells of the gonadotrope lineage and is not Pit-1/GHF-1. The human and equine alpha-subunit genes are also expressed in placental cells. However, the previously characterized placental transcription factors designated TSEB and alpha-ACT are not found in the pituitary gonadotrope cells, indicating that independent mechanisms confer expression of these genes in the two different tissues.

Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene.

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

Promoter elements and erythroid cell nuclear factors that regulate alpha-globin gene transcription in vitro.

We have previously purified four factors (alpha-IRP, alpha-CP1, alpha-CP2, and NF-E1) that interact with the promoter of the alpha-globin gene. One of these (NF-E1) is a tissue-restricted factor that has recently been cloned. The binding sites of these factors identify DNA sequence elements that might mediate the tissue-specific and inducible transcription of the alpha-globin gene. This possibility was tested in a series of in vitro transcription experiments. An examination of 5' truncated templates and synthetic promoters constituted from individual factor-binding sites apposed to the alpha-TATAA box showed that the binding elements of three factors (alpha-CP1, alpha-IRP, and NF-E1) mediate four- to sixfold activation of transcription in vitro. In contrast, one element (alpha-CP2) stimulated transcription less than twofold. The 5- to 10-fold stimulation of these latter templates upon addition of a DNA sequence affinity-purified factor suggests that alpha-CP2 is functionally limiting in nuclear extracts. Additional experiments further tested the effect of supplementing extracts with factors purified from erythroid cell nuclear extracts or, in the case of NF-E1, enriched from a bacterial cDNA expression system. Each factor tested stimulated transcription in vitro in a binding-site-dependent manner. Our results provide a comprehensive functional view of the murine alpha-globin promoter and suggest possible mechanisms for activation of alpha-globin gene transcription during induced differentiation of murine erythroleukemia cells.

Clinical indicators for success of misoprostol treatment after early pregnancy failure.

OBJECTIVE: To identify clinical indicators for success of misoprostol treatment after early pregnancy failure. METHODS: A total of 473 women with early pregnancy failure received 800 microg of vaginal misoprostol on treatment day 1. At the follow-up visit on day 3, a second dose was given if expulsion was incomplete. On day 8, vacuum aspiration was offered if expulsion had not occurred. Ultrasonography was used as gold standard for success. A Classification and Regression Tree analysis was undertaken to derive two decision trees for the success of misoprostol treatment on study days 3 and 8. RESULTS: Heavy bleeding after the first dose and an open cervical os were identified as clinical indicators of treatment success on day 3. Treatment success occurred in 84% of women with either or both indicators. Reporting passage of tissue after a second misoprostol dose and old blood in the vagina were potential indicators of treatment success or failure on day 8. A woman with either of these indicators has a 65% chance of treatment success after the second dose. Conversely, a woman with neither indicator on day 8 has a 94% chance of treatment failure. CONCLUSION: Standard clinical findings may be useful as indicators for success or failure of medical management of early pregnancy failure in settings with limited or no access to ultrasonography. More research to identify even better indicators is warranted.

An atypical case of Trypanosoma cruzi infection in a young English Mastiff.

A case of Trypanosoma cruzi infection in a young English Mastiff from Texas is presented. Clinical signs and laboratory findings included subcutaneous edema, lymphadenopathy, weight loss, and hypoalbuminemia. Cytology of a lymph node revealed numerous amastigotes. No trypomastigotes were observed in buffy coat preparation of peripheral blood, and on histologic evaluation, most organs contained numerous interstitial pseudocysts. Initial serology was positive for both T. cruzi and Leishmania, and immunohistochemistry supported a diagnosis of Leishmania. However, additional serology supported a T. cruzi infection, and cultivation of organisms isolated from a lymph node revealed morphology consistent with T. cruzi. In addition, PCR analysis resulted in a 504 bp fragment with 99% homology to a flagellar protein of T. cruzi. Although uncommon, autochthonous cases of both T. cruzi and Leishmania have been reported in the United States. Clinical signs observed with both diseases can show many similarities, cytology may be indistinguishable, as in this case, and serological cross-reactivity is common. This case demonstrates an unusual presentation of T. cruzi and the use of multiple testing strategies to support its diagnosis.

Antitumor effects of interferon-omega: in vivo therapy of human tumor xenografts in nude mice.

The antitumor effect of the type I IFN, IFN-omega, was evaluated in both in vitro and in vivo studies of human cancer. For these studies, the cDNA for human IFN-omega was cloned into a eukaryotic expression plasmid DNA (pDNA) driven by the cytomegalovirus promoter. Supernatants from UM449 cells transfected in vitro with IFN-omega pDNA had antiproliferative effects on 11 of 13 human tumor cell lines. For in vivo studies, nude mice were implanted s.c. with one of the following human tumors: NIH: OVCAR-3 ovarian carcinoma, A375 melanoma, or A431 epidermoid carcinoma. Direct intratumoral injection of 100 microg of a IFN-omega pDNA DMRIE/DOPE complex (1:1 DNA:DMRIE mass ratio) for 6 consecutive days resulted in a significant reduction in the tumor volume of NIH: OVCAR-3 ovarian carcinoma or A375 melanoma (P = 0.02). IFN-omega pDNA delivered by i.m. injection also had an antitumor effect. Nude mice bearing s.c. A431 epidermoid carcinoma and injected i.m. with 100 microg of IFN-omega pDNA, twice per week for 3 weeks, had a significant reduction in tumor volume (P = 0.009). These results demonstrate for the first time that IFN-omega can have in vivo antitumor effects in several models of human cancer.

Comparative sequence analysis and radiation hybrid mapping of two epidermal type II keratin genes in the dog: keratin 1 and keratin 2e.

In order to extend knowledge of the process of cornification across species and to be better able to recognize inborn errors in keratin synthesis in the dog, we describe the organization and chromosome mapping of canine KRT1 and KRT2E and compare these results to human and murine sequence data. The coding regions of KRT1 and KRT2E are 1,860 bp and 1,902 bp respectively, distributed over nine exons. Both genes are localized on the canine radiation hybrid map to chromosome 27 in the type II keratin gene cluster close to polymorphic markers. These genes are highly conserved across species and based on both genomic and amino acid sequences, canine KRT1 and KRT2E share greater homology with humans than with mice.

The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone alpha-subunit gene in pituitary gonadotropes.

Tissue-specific expression of the glycoprotein hormone alpha-subunit gene in pituitary gonadotropes relies on a gonadotrope-specific element (GSE), which binds an approximately 54-kilodalton protein termed GSE-binding protein 1 (GSEB1). We report here that GSEB1 is the orphan nuclear receptor steroidogenic factor-1 (SF-1), which has been shown to be a primary regulator of steroidogenic enzymes in the adrenal gland and gonadal tissues. GSEB1 from alpha T3-1 pituitary gonadotrope cells and SF-1 from Y1 adrenocortical cells and R2C testicular Leydig cells display identical binding properties with both the GSE and SF-1 elements. Antiserum specific to the SF-1 DNA-binding domain abolishes the binding of both GSEB1 and SF-1 to both elements. SF-1 mRNA is found in the mouse pituitary and in the alpha T3-1 cell line but not in other pituitary cell lines, consistent with the pattern of GSEB 1-binding activity. The GSE element specifically enhances transcription in SF-1-containing cells. The discovery that an orphan nuclear receptor regulates the expression of both the gonadotropin hormones in the pituitary and the steroidogenic enzymes in the gonad provides a potential molecular mechanism for coordinate control in reproductive function, perhaps through an as yet unidentified endocrine ligand for SF-1.

Comparative sequence analysis and radiation hybrid mapping of the canine keratin 10 gene.

The type I keratin, K10, is expressed in epidermal keratinocytes undergoing terminal differentiation to form the stratum corneum, a barrier essential for life. In order to facilitate the study of keratinization disorders in the dog, the sequence and mapping of KRT10 is reported. The coding region of KRT10 is 1707 bp and is comprised of eight exons. Although the length of KRT10 has been reported to be polymorphic in humans, this was not observed in the eight domestic dog breeds studied, although one wild canid displayed a size difference. The structure and sequence of this gene is highly conserved across mammalian species. Canine K10 had an 86% amino acid identity with the human gene. KRT10 was localized to the on-going canine radiation hybrid map to chromosome 9 in the type I keratin gene cluster.

A snapshot of biologic drug development: Challenges and opportunities.

Since the approval of insulin as the first recombinant therapeutic protein, the prominence of biologic therapies in drug development has grown significantly. Many modalities beyond traditional biologics are now being developed or explored for various indications with significant unmet medical needs. From early traditional replacement proteins to more recent, highly engineered antibodies, oligonucleotides, fusion proteins, and gene constructs, biologic agents have delivered life-changing therapies, despite often having scientifically and technically challenging development programs. This brief review outlines some of the major biotherapeutic classes and identifies the advantages and challenges with the development of these products.