ABSTRACT
BACKGROUND: We wished to examine treatment and outcome patterns in older diffuse large B-cell lymphoma (DLBCL) patients, with a focus on the effect of route-to-diagnosis to outcome. METHODS: Data were extracted from Public Health England's National Cancer Registration and Analysis Service between 2013 and 2015 included route-to-diagnosis, disease characteristics and survival for 9186 patients ≥65 years. Systemic Anti-Cancer Therapy data identified front-line regimens, cycles and doses. RESULTS: Route-to-diagnosis were emergency (34%), NHS urgent cancer pathway (rapid haemato-oncologist review <2 weeks), (29%) and standard GP referral (25%). The most common regimen was R-CHOP (n = 4392). 313 patients received R-miniCHOP (7% of R-CHOP). For all patients, 3-year overall survival (OS) for 65-79 years was 57% and for ≥80 years was 32%. Three-year OS for R-CHOP-treated patients diagnosed via emergency presentation was 54% (adjusted hazard ratio (HR) 1.63, p < 0.01) and 75% (adjusted HR 0.81, p < 0.01) on the NHS urgent cancer pathway (reference HR:1.00: GP referrals). 3-year OS was 54% for both R-miniCHOP and R-CHOP in ≥80 years. CONCLUSIONS: Our comprehensive population analysis is the first to show that the NHS urgent cancer pathway is associated with a superior survival after adjusting for multiple confounders. Equivalent survival for R-CHOP and R-mini-CHOP was demonstrated in those ≥80 years.
Subject(s)
Ambulatory Care/methods , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Databases, Factual/statistics & numerical data , Lymphoma, Large B-Cell, Diffuse/drug therapy , Practice Patterns, Physicians'/statistics & numerical data , Aged , Aged, 80 and over , Cyclophosphamide/therapeutic use , Doxorubicin/therapeutic use , England/epidemiology , Humans , Lymphoma, Large B-Cell, Diffuse/epidemiology , Lymphoma, Large B-Cell, Diffuse/pathology , Prednisone/therapeutic use , Retrospective Studies , Rituximab/therapeutic use , Survival Rate , Vincristine/therapeutic useABSTRACT
BACKGROUND: There is no prognostic index for primary cutaneous T-cell lymphomas such as mycosis fungoides (MF) and Sezary syndrome (SS). METHOD: Two prognostic indices were developed for early (IA-IIA) and late stage (IIB-IVB) disease based on multivariate data from 1502 patients. End-points included overall survival (OS) and progression free survival (PFS). External validation included 1221 patients. FINDINGS: Significant adverse prognostic factors at diagnosis consisted of male gender, age >60, plaques, folliculotropic disease and stage N1/Nx for early stage, and male gender, age >60, stages B1/B2, N2/3 and visceral involvement for late stage disease. Using these variables we constructed two separate models each defined using 3 distinct groups for early and late stage patients: 0-1 (low risk), 2 (intermediate risk), and 3-5 factors (high risk). 10 year OS in the early stage model was 90.3% (low), 76.2% (intermediate) and 48.9% (high) and for the late stage model 53.2% (low), 19.8% (intermediate) and 15.0% (high). For the validation set significant differences in OS and PFS in early stage patients (both p<0.001) were also noted. In late stage patients, only OS differed between the groups (p=0.002). INTERPRETATION: This proposed cutaneous lymphoma prognostic index provides a model for prediction of OS in early and late stage MF/SS enabling rational therapeutic choices and patient stratification in clinical trials.
Subject(s)
Mycosis Fungoides/diagnosis , Sezary Syndrome/diagnosis , Skin Neoplasms/diagnosis , Biomarkers, Tumor/blood , Disease-Free Survival , Female , Humans , Kaplan-Meier Estimate , L-Lactate Dehydrogenase/blood , Male , Middle Aged , Multivariate Analysis , Mycosis Fungoides/blood , Mycosis Fungoides/mortality , Mycosis Fungoides/pathology , Mycosis Fungoides/therapy , Neoplasm Staging , Proportional Hazards Models , Risk Factors , Sezary Syndrome/blood , Sezary Syndrome/mortality , Sezary Syndrome/pathology , Sezary Syndrome/therapy , Skin Neoplasms/blood , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Skin Neoplasms/therapy , Time FactorsABSTRACT
BACKGROUND: The current management of thyroid lymphomas (TL) includes the combined use of chemotherapy and radiotherapy, with surgery mainly confined to diagnosis through an open biopsy following ultrasound-guided fine-needle aspiration cytology (US-FNAC). AIMS: To analyse the clinical presentation and methods of diagnosis of TL, its pitfalls and the management of these tumours presenting with compression symptoms and airway obstruction. METHODS: A retrospective review of nine patients diagnosed with TL at Guy's and St Thomas Hospital NHS Foundation Trust in London over the past 5 years. RESULTS: Nine consecutive patients were identified with the diagnosis of TL, and seven (78%) of them being women and with a mean age of 65 years. All patients presented with an anterior neck mass while four (44.4%) presented with stridor and vocal cord palsy. Two (22.2%) presented with a hoarse voice, dysphagia, and only one patient had a B symptom of weight loss. FNAC was diagnostic in three patients (33.3%) and a report of multi-nodular goitre in one patient. There was clinical suspicion of TL in three patients (33.3%). Of the three patients presenting with stridor, two had an open biopsy followed by the initiation of dexamethasone therapy and resolution of symptoms within 48 h. One patient had a partial thyroidectomy following a suspected diagnosis of multi-nodular goitre from US-FNAC. One patient required tracheostomy for airway management. CONCLUSION: Diagnosis of TL may be difficult. However, US-FNAC is useful in raising the suspicion of a TL. Open biopsy is still the definitive diagnostic tool of choice. In the emergency setting of airway obstruction, once definitive diagnosis is achieved, dexamethasone therapy and endotracheal intubation for airway management are all that is required for optimal management strategy. Surgical intervention has no role except for providing tissue for diagnosis.
Subject(s)
Airway Obstruction/surgery , Lymphoma/surgery , Thyroid Neoplasms/surgery , Adult , Aged , Aged, 80 and over , Airway Obstruction/etiology , Biopsy, Fine-Needle , Female , Humans , Lymphoma/complications , Lymphoma/diagnosis , Male , Middle Aged , Retrospective Studies , Thyroid Neoplasms/complications , Thyroid Neoplasms/diagnosis , Thyroidectomy , Tomography, X-Ray Computed , TracheostomyABSTRACT
One in every thousand pregnancies is complicated by a concurrent diagnosis of cancer. Lymphoma is currently the fourth most common malignancy diagnosed during pregnancy and its incidence is rising. The diagnosis and management of any malignancy during pregnancy is clearly a clinical and emotional minefield for both patients and health-care professionals. The major challenge is to optimize medical treatment offered to the mother, while limiting the impact on the fetus. Given the relative rarity of the situation, current practice is guided by case reports and personal experience of management of similar patients. Our centre has a large and busy lymphoma practice, and has cared for several women diagnosed with a variety of subtypes of lymphoma over the years. This review aims to summarize current opinion about best practice regarding these patients and discusses options available from the current literature.
ABSTRACT
BACKGROUND: Long-term survival from Hodgkin lymphoma (HL) is 80-90%, but the treatment has serious late adverse effects. Modern risk-adapted treatment requires accurate assessment of the patient's prognosis. This investigation assessed the value of positron emission tomography (PET) with 2-[18F]fluoro-2-deoxy-D-glucose (FDG-PET) after two or three cycles of chemotherapy for prediction of progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: A total of 85 patients with HL underwent FDG-PET after two or three cycles of chemotherapy. Median follow-up was 3.3 years. FDG-PET results were related to PFS and OS using Kaplan-Meier analysis. Regression analyses were employed to test for independence of established pretreatment prognostic factors. RESULTS: After two or three cycles of chemotherapy, 63 patients had negative FDG-PET scans, nine patients had minimal residual uptake (MRU) and 13 patients had positive scans. Three PET-negative patients and one patient from the MRU group relapsed. In the PET-positive group, nine patients progressed and two died. Survival analyses showed highly significant associations between early interim FDG-PET and PFS (P <0.0001) and OS (P <0.03). All advanced-stage patients with positive interim FDG-PET relapsed within 2 years. CONCLUSION: Early interim FDG-PET is an accurate and independent predictor of PFS and OS in HL. A positive interim FDG-PET is highly predictive of relapse in advanced-stage disease.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Hodgkin Disease/diagnostic imaging , Hodgkin Disease/drug therapy , Adolescent , Adult , Aged , Female , Fluorodeoxyglucose F18 , Humans , Male , Middle Aged , Positron-Emission Tomography , Prognosis , Survival AnalysisABSTRACT
BACKGROUND: Less than 50% of all high-grade non-Hodgkin lymphoma (NHL) patients experience lasting disease-free survival. Risk-adapted treatment strategies require better tools for prediction of outcome. This investigation aimed to assess the value of positron emission tomography with 2-[18F]fluoro-2-deoxy-D-glucose (FDG-PET) after two to three cycles of chemotherapy for prediction of progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: One hundred and twenty-one patients with high-grade NHL underwent FDG-PET. The therapy response on FDG-PET was correlated to PFS and OS using Kaplan-Meier survival analysis. Cox regression analyses were employed to test for independence of known pretreatment prognostic factors. RESULTS: Fifty FDG-PET scans were negative, 19 scans showed minimal residual uptake (MRU), and 52 scans were positive. The estimated 5 year PFS was 88.8% for the PET-negative group, 59.3% for the MRU group, and 16.2% for the PET-positive group. Kaplan-Meier analyses showed strong associations between FDG-PET results and PFS (P <0.0001) and OS (P <0.01). Early interim FDG-PET was independent of the other prognostic factors. CONCLUSIONS: Early interim FDG-PET is an accurate and independent predictor of PFS and OS. An early assessment of chemotherapy response with FDG-PET could provide the basis for selection of patients for alternative therapeutic strategies.
Subject(s)
Antineoplastic Agents/therapeutic use , Fluorodeoxyglucose F18/administration & dosage , Lymphoma, Non-Hodgkin/drug therapy , Survival Analysis , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Female , Humans , Lymphoma, Non-Hodgkin/diagnostic imaging , Lymphoma, Non-Hodgkin/pathology , Male , Middle Aged , Positron-Emission TomographyABSTRACT
In the United States, both medical and nonmedical factors have driven the cesarean section rate to over 26% of all deliveries. In addition to questions of increased cost associated with operative delivery, some have questioned the ethics of performing cesarean section for nonmedical reasons. Reduction of both the duration and the pain associated with vaginal delivery would likely bring about a decline in the rate of both medical and nonmedical cesarean sections. This chapter summarizes recent findings that support the premise that through its growth-promoting and softening effects on the cervix, short-term subcutaneous administration of pharmacologic amounts of relaxin to women at term holds promise as a means of reducing the duration and discomfort associated with delivery. Two recent studies conducted in pregnant rats demonstrated that the cervix is highly responsive to relaxin during the antepartum period and that short-term subcutaneous administration of the hormone to relaxin-deficient animals not only promotes growth and softening of the cervix, but also reduces the duration of labor and delivery. Moreover, recent human clinical trials examining the influence of 24 weeks of continuous subcutaneous administration of recombinant human relaxin for the treatment of scleroderma provided evidence not only that the human reproductive tract is responsive to relaxin, but also that the administration of the hormone does not cause serious adverse side effects. It is concluded that recent findings provide an impetus for an investigation into relaxin's potential for cervical remodeling and facilitating birth in women.
Subject(s)
Parturition/drug effects , Relaxin/pharmacology , Animals , Cervix Uteri/drug effects , Cervix Uteri/physiology , Cesarean Section/statistics & numerical data , Female , Humans , Mifepristone/pharmacology , Organ Size/drug effects , Pregnancy , Relaxin/administration & dosage , Relaxin/adverse effects , Relaxin/bloodABSTRACT
Relaxin promotes marked growth of the cervix during the second half of rat pregnancy, and this growth is accompanied by an increase in both epithelial and stromal cells. The objective of this study was to test the hypothesis that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical cells is greatest during late pregnancy in rats. The influence of neutralization of circulating relaxin by iv injection of 5 mg monoclonal antibody against rat relaxin (MCA1) was examined at 3-d intervals throughout the second half of pregnancy. Controls were injected with either 5 mg monoclonal antibody against fluorescein or 0.5 ml PBS vehicle. To evaluate cell proliferation, 5'-bromo-2-deoxyuridine was injected sc 8 h before cervixes were collected. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling and electron microscopy were used to detect apoptotic cells. Neutralization of relaxin with MCA1 decreased the rate of proliferation and increased the rate of apoptosis of cervical cells by d 13. However, the extent to which relaxin influenced these processes was greatest and dramatic by late pregnancy. In MCA1-treated rats on d 22 of pregnancy, the rates of proliferation of both epithelial and stromal cells were less than 20% those in controls, and the rates of apoptosis in epithelial cells and stromal cells were more than 10- and 3-fold, respectively, greater than those in controls. In conclusion, this study provides evidence that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy.
Subject(s)
Apoptosis/physiology , Cervix Uteri/cytology , Cervix Uteri/physiology , Pregnancy, Animal/physiology , Relaxin/physiology , Stromal Cells/cytology , Stromal Cells/physiology , Animals , Cell Proliferation , Cervix Uteri/ultrastructure , Epithelial Cells/physiology , Epithelial Cells/ultrastructure , Female , Gestational Age , Microscopy, Electron , Pregnancy , Rats , Rats, Sprague-Dawley , Stromal Cells/ultrastructureABSTRACT
There are two pathways for inorganic iron uptake in the intestine, the ferric pathway, mediated by the key protein mobilferrin, and the ferrous pathway, mediated by DMT-1. Previous studies reported that the amount of DMT-1 increased in the intestinal mucosa in iron deficiency and the increase was seen in the apical portion of the villus of the duodenal mucosa. Mobilferrin did not quantitatively increase but became localized at the cell membrane. However, studies on fresh tissue have not previously been performed and the localization to the microvillae has not been demonstrated. In order to more definitively localize these proteins immunofluorescent and electron microscopic studies were undertaken. Samples were also subjected to biochemical analysis and Western analysis. In iron-deficient animals both DMT-1 and Mobilferrin were concentrated in the apical surface of the villae. Electron microscopy revealed that the majority of this increase in the amount of these proteins near the luminal surface was due to increased binding of the proteins to mucin in vesicles near the surface. A significant portion of the iron transport proteins was localized in the goblet cells and outside the cell in the luminal mucin, as demonstrated by immunofluorescence, electron microscopy, and isolation of the mucin by cesium chloride gradient centrifugation and Western analysis. A new model for the transport of metal ions was suggested. The metal transport proteins travel from vesicles inside the cell out to the lumen mucin. This increases the surface area and allows a greater portion of the lumen contents to be exposed to the binding proteins. Once the metal is bound to the externalized protein it is internalized into the cell. This explains many of the unique properties of the iron-binding proteins and suggests that it may be a more general model for the absorption of other nutrients.
Subject(s)
Cation Transport Proteins/metabolism , Duodenum/metabolism , Iron-Binding Proteins/metabolism , Mucins/physiology , Animals , Biological Transport/physiology , Duodenum/ultrastructure , Extracellular Space/metabolism , Fluorescent Antibody Technique , Glycosylation , Male , Microscopy, Electron , Microscopy, Fluorescence , Microvilli/metabolism , Mucins/isolation & purification , Mucins/metabolism , Rats , Rats, Wistar , Staining and Labeling , Tissue DistributionABSTRACT
Every patient in every part of the world has the right to expect the best possible quality of care from health care providers. Non-Hodgkin's lymphomas (NHL) are an extremely heterogeneous group of conditions which require important decisions to be taken at many points along the treatment pathway. To get this right every time requires that high-quality standards are instituted and adhered to, so that the best possible outcome is achieved. In the past this has not always been the case because of the failure of clinicians sometimes to adhere to an optimal management plan. In 1995, the UK government commissioned an inquiry into the running of cancer services in the United Kingdom, which culminated in a series of recommendations to improve them. Subsequently, these recommendations were implemented as objectives of the NHS Cancer Plan which is the framework by which the UK government wishes to improve cancer services. Concurrently another general concept has emerged which is designed to ensure that the highest quality standards may be achieved for all patients across the whole National Health Service (NHS). This concept, termed 'clinical governance', brings together a corporate responsibility of all health care workers to deliver high quality standards, in the hope that this will translate into better long-term survival of patients with malignant disease. This chapter focuses on the issues surrounding clinical governance and how the principles of this concept relate to non-Hodgkin's lymphomas.
Subject(s)
Lymphoma, Non-Hodgkin/therapy , Quality of Health Care/organization & administration , Humans , Interdisciplinary Communication , Lymphoma, Non-Hodgkin/diagnosis , National Health Programs , Quality of Health Care/ethics , Quality of Health Care/legislation & jurisprudence , United KingdomABSTRACT
A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.
Subject(s)
Dependovirus/genetics , Hemophilia B/genetics , Muscle, Skeletal/metabolism , Spermatozoa/virology , Animals , DNA Primers/chemistry , DNA, Viral/analysis , Dogs , Factor IX/genetics , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Hemophilia B/pathology , Hemophilia B/therapy , In Situ Hybridization, Fluorescence , Injections, Intramuscular , Male , Mice , Polymerase Chain Reaction , Rabbits , Rats , Recombinant Proteins/genetics , Semen/virology , Testis/virologyABSTRACT
The safety of several gene therapy approaches for treatment of the severe, X-linked bleeding disorder hemophilia is currently being evaluated in early phase clinical trials. One strategy seeks to correct deficiency of functional coagulation factor IX (hemophilia B) by intramuscular (IM) administration of an adeno-associated viral (AAV) vector. A potentially serious complication of any treatment for hemophilia is formation of inhibitory antibodies against the coagulation factor protein, a risk that increases in the setting of null mutations in the factor IX gene (F9). Here, we describe hemophilia B mice with a large F9 deletion that form inhibitors within 1 to 2 months after IM administration of an AAV vector expressing mouse F9 or after repeated intravenous infusion of mouse F9 concentrate. In both cases, inhibitors are primarily IgG1 immunoglobulins representing a Th2-driven humoral immune response. We further demonstrate that anti-mouse F9 antibody formation in the gene-based approach can be reduced by transient immune modulation at the time of vector administration. Moreover, this maneuver resulted in complete absence of anti-mouse F9 and sustained expression of functional mouse F9 in some hemophilia B mice, particularly in those animals treated with the immunosuppressive drug cyclophosphamide. These data have direct relevance for design of clinical trials and strategies aimed at avoiding immune responses against a secreted transgene product.
Subject(s)
Dependovirus/genetics , Factor IX/genetics , Factor IX/immunology , Gene Deletion , Genetic Therapy/methods , Hemophilia B/genetics , Hemophilia B/immunology , Animals , Antibodies/immunology , CHO Cells , Cricetinae , Cyclophosphamide/pharmacology , Factor IX/administration & dosage , Factor IX/therapeutic use , Gene Expression/drug effects , Genetic Vectors/genetics , Hemophilia B/drug therapy , Hemophilia B/therapy , Immunosuppressive Agents/pharmacology , Injections, Intramuscular , Injections, Intravenous , Mice , Mice, Inbred C57BL , Partial Thromboplastin Time , RNA, Messenger/genetics , RNA, Messenger/metabolism , Risk Factors , Time Factors , Transgenes/geneticsABSTRACT
We examined the effects of temperature and stabilizing solutes on A4-lactate dehydrogenase (A4-LDH) from warm- and cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate-limiting for A4-LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A4-LDH from a warm-temperate goby (Gillichthys mirabilis), which has lower values for kcat and the Michaelis constant for pyruvate ( K m PYR), was intrinsically more stable than the orthologs of the cold-adapted Antarctic notothenioids Parachaenichthys charcoti and Chionodraco rastrospinosus, as shown by a higher apparent transition ('melting') temperature (Tm(APP)). We used four solutes, glycerol, sucrose, trimethylamine-N-oxide and poly(ethylene glycol) 8000, which stabilize proteins through different modes of preferential exclusion, to study temperature-solute interactions of the three orthologs. Changes in Tm(APP) were similar for all orthologs in each solute tested, but the catalytic rate of G. mirabilis A4-LDH was decreased most by solutes and increased most by temperature. In contrast, the K m PYR values of the Antarctic orthologs were more affected than that of the goby by both solutes and temperature. We conclude that (a) preferential exclusion of solutes functions within the native state of A4-LDH to favor conformational microstates with minimal surface area; (b) the varied effects of the different solutes on the kinetic properties are due to the interaction between this nonspecific stabilization and the differing intrinsic stabilities of the orthologs; (c) the catalytic rates of A4-LDH orthologs are equally affected by stabilizing solutes, if measurements are made at physiologically appropriate temperatures; and (d) global stability and localized flexibility of these A4-LDH orthologs may evolve independently.
Subject(s)
Isoenzymes/chemistry , L-Lactate Dehydrogenase/chemistry , Animals , Enzyme Stability , Fishes , Fluorescence , Isoenzymes/metabolism , L-Lactate Dehydrogenase/metabolism , Lactate Dehydrogenase 5 , Protein Conformation , TemperatureABSTRACT
No organism can survive across the entire temperature range found in the biosphere, and a given species can rarely support active metabolism across more than a few tens of degrees C. Nevertheless, life can be maintained at surprisingly extreme temperatures, from below -50 to over 110 degrees C. That proteins, which are assembled with the same 20 amino acids in all species, can function well at both extremes of this range illustrates the plasticity available in the construction of these macromolecules. In studying proteins from extremophiles, researchers have found no new amino acids, covalent modifications or structural motifs that explain the ability of these molecules to function in such harsh environments. Rather, subtle redistributions of the same intramolecular interactions required for protein stabilization at moderate temperatures are sufficient to maintain structural integrity at hot or cold extremes. The key to protein function, whether in polar seas or hot springs, is the maintenance of an appropriate balance between molecular stability on the one hand and structural flexibility on the other. Stability is needed to ensure the appropriate geometry for ligand binding, as well as to avoid denaturation, while flexibility is necessary to allow catalysis at a metabolically appropriate rate. Comparisons of homologous proteins from organisms spanning a wide range of thermal habitats show that adaptive mutations, as well as stabilizing solutes, maintain a balance between these two attributes, regardless of the temperature at which the protein functions.
Subject(s)
Enzyme Stability , Proteins/chemistry , Proteins/physiology , Temperature , Amino Acid Sequence , Amino Acid Substitution , Amino Acids/chemistry , Animals , Enzymes/chemistry , Enzymes/physiology , Molecular Sequence DataABSTRACT
The growth of the cervix and vagina that occurs during the second half of rat pregnancy is accompanied by an increase in both epithelial and stromal cells. Neither the mechanism(s) that regulates this accumulation of cells nor its hormonal control is known. To test the hypothesis that the rate of apoptosis declines during the second half of pregnancy, cervices and vaginas were collected on days 5, 10, 15, 18, and 21 of pregnancy. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling was used to detect apoptotic cells. The rate of apoptosis declined (P < 0.05) in epithelial and stromal cells in both the cervix and vagina during the second half of pregnancy, when blood levels of relaxin are increasing. To test the hypothesis that relaxin inhibits apoptosis, cervices and vaginas were collected 6, 12, 24, 48, and 72 h after the neutralization of endogenous relaxin, on days 19-21 of pregnancy, with a monoclonal antibody for rat relaxin. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling method and electron microscopy were used to detect apoptotic cells. Withdrawal of relaxin caused an increase in the rate of apoptosis in both the cervix and the vagina (P < 0.05). It is concluded that the rate of apoptosis declines in the cervix and the vagina during the second half of rat pregnancy, and that relaxin likely contributes to this process.
Subject(s)
Apoptosis/drug effects , Cervix Uteri/cytology , Relaxin/pharmacology , Vagina/cytology , Animals , Female , Gestational Age , In Situ Nick-End Labeling , Microscopy, Electron , Pregnancy , Rats , Rats, Sprague-DawleyABSTRACT
Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.
Subject(s)
Adenoviridae/genetics , Factor IX/immunology , Genetic Vectors , Immune Tolerance , Adenovirus E1 Proteins/genetics , Adenovirus E3 Proteins/genetics , Animals , Cell Division/immunology , Haplotypes , Injections, Intravenous , Interleukin-10/metabolism , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , T-Lymphocytes/immunologyABSTRACT
Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical gamma-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and gamma-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.
Subject(s)
Factor IX/biosynthesis , Muscle Fibers, Skeletal/cytology , Protein Processing, Post-Translational , 1-Carboxyglutamic Acid/analysis , Adenoviridae/genetics , Carbohydrates/analysis , Cell Culture Techniques , Culture Media, Conditioned/chemistry , Factor IX/chemistry , Factor IX/genetics , Genetic Vectors , Humans , Molecular Sequence Data , Muscle Fibers, Skeletal/metabolism , Partial Thromboplastin Time , Phosphorylation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Analysis, Protein , Serine/metabolism , Sulfates , Transduction, Genetic , Tyrosine/metabolismABSTRACT
Defining immune responses against the secreted transgene product in a gene therapy setting is critical for treatment of genetic diseases such as hemophilia B (coagulation factor IX deficiency). We have previously shown that intramuscular administration of an adeno-associated viral (AAV) vector results in stable expression of therapeutic levels of factor IX (F.IX) and may be associated with humoral immune responses against F.IX. This study demonstrates that intramuscular injection of an AAV vector expressing F.IX fails to activate F.IX-specific cytotoxic T lymphocytes (CTLs) in hemostatically normal or in hemophilia B mice, so that there is an absence of cellular immune responses against F.IX. However, transgene-derived F.IX can cause B cell responses characterized by production of T helper cell-dependent antibodies (predominantly IgG1, but also IgG2 subclasses) resulting from activation of CD4+ T helper cells primarily of the Th2 subset. In contrast, administration of an adenoviral vector efficiently activated F.IX-specific CTLs and T helper cells of both Th1 and Th2 subsets, leading to inflammation and destruction of transduced muscle tissue and activation of B cells as well. Therefore, vector sequences fundamentally influence T cell responses against transgene-encoded F.IX. In conclusion, activation of the immune system in AAV-mediated gene transfer is restricted to pathways mediated by F.IX antigen presentation through MHC class II determinants resulting in T and B cell responses that are more comparable to responses in the setting of protein infusion rather than of viral infection/gene transfer.
Subject(s)
Factor IX/metabolism , Gene Transfer Techniques/adverse effects , Genetic Vectors/immunology , T-Lymphocyte Subsets/immunology , Transgenes , Adenoviridae/genetics , Adoptive Transfer , Animals , Cytokines/metabolism , Factor IX/genetics , Factor IX/immunology , Fluorescent Antibody Technique , Hemophilia B/immunology , Hemophilia B/metabolism , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Mice , Mice, Inbred C57BL , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose-dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the alpha-skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol. (Blood. 2000;95:2536-2542)
Subject(s)
Factor IX/genetics , Gene Transfer Techniques , Genetic Vectors , Actins/genetics , Animals , Cytomegalovirus/genetics , Dogs , Enhancer Elements, Genetic/genetics , Factor IX/biosynthesis , Gene Expression , Humans , Mice , Muscle, SkeletalABSTRACT
We reported that the nucleotide sequence of a cDNA generated from rabbit placental poly(A)(+) RNA using porcine preprorelaxin primers was identical to SQ10, a product of squamous differentiated tracheal epithelial cells. However, these results did not confirm that SQ10 was the biologically active rabbit relaxin that had been isolated previously yet not sequenced. In this study, a 7-kDa protein isolated from rabbit placentas exhibited relaxin bioactivity and cross-reacted with a porcine relaxin antiserum. A partial amino acid sequence of this protein revealed a sequence identical to that of SQ10. Although the amino acid sequence of the putative relaxin receptor-binding domain found in the B chain of relaxin was modified in SQ10 from CGRDYVR to CRNDFVR, the placental protein was bioactive. These results suggest that SQ10 is the rabbit relaxin. In situ hybridization, using an SQ10 riboprobe, indicated radiolabeling in the syncytiotrophoblast cells of the rabbit placenta. The pattern of labeling corresponded with the immunohistochemical staining for relaxin observed with use of a porcine relaxin antiserum. These results indicate that the syncytiotrophoblast cells are a site of synthesis for SQ10 and that the immunostaining is not solely the result of sequestering SQ10 through receptor-mediated endocytosis. A potential role for relaxin in implantation is discussed.