RESUMO
BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare, fatal, premature aging disease caused by a toxic protein called progerin. Circulating progerin has not been previously detected, precluding research using readily available biological samples. This study aimed to develop a plasma progerin assay to evaluate progerin's quantity, response to progerin-targeted therapy, and relationship to patient survival. METHODS: Biological samples were collected by The Progeria Research Foundation Cell and Tissue Bank from a non-HGPS cohort cross-sectionally and a HGPS cohort longitudinally. HGPS donations occurred at baseline and intermittently while treated with farnesylation inhibitors lonafarnib±pravastatin and zoledronate, within 3 sequential open-label clinical trials at Boston Children's Hospital totaling >10 years of treatment. An ultrasensitive single-molecule counting progerin immunoassay was developed with prespecified performance parameters. Intra- and interpatient group statistics were descriptive. The relationship between progerin and survival was assessed by using joint modeling with time-dependent slopes parameterization. RESULTS: The assay's dynamic detection range was 59 to 30 000 pg/mL (R2=0.9987). There was no lamin A cross-reactivity. Mean plasma progerin in non-HGPS participants (n=69; 39 male, 30 female; age, 0.2-71.3 years) was 351±251 pg/mL, and in drug-naive participants with HGPS (n=74; 37 female, 37 male; age, 2.1-17.5 years) was 33 261±12 346 pg/mL, reflecting a 95-fold increase in affected children (P<0.0001). Progerin levels did not differ by sex (P=0.99). Lonafarnib treatment resulted in an average per-visit progerin decrease from baseline of between 35% to 62% (all P<0.005); effects were not augmented by adding pravastatin and zoledronate. Progerin levels fell within 4 months of therapy and remained lower for up to 10 years. The magnitude of progerin decrease positively associated with patient survival (P<0.0001; ie, 15 000 pg/mL decrease yields a 63.9% decreased risk of death). For any given decrease in progerin, life expectancy incrementally increased with longer treatment duration. CONCLUSIONS: A sensitive, quantitative immunoassay for progerin was developed and used to demonstrate high progerin levels in HGPS plasma that decreased with lonafarnib therapy. The extent of improved survival was associated with both the magnitude of progerin decrease and duration at lower levels. Thus, plasma progerin is a biomarker for HGPS whose reduction enables short- and long-term assessment of progerin-targeted treatment efficacy. REGISTRATION: URL: https://www. CLINICALTRIALS: gov. Unique identifiers: NCT00879034 and NCT00916747.
Assuntos
Progéria , Criança , Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Progéria/diagnóstico , Progéria/tratamento farmacológico , Progéria/metabolismo , Ácido Zoledrônico/uso terapêutico , Pravastatina/uso terapêutico , Piperidinas/uso terapêutico , Lamina Tipo A/metabolismoRESUMO
Cardiac troponins (cTns) are released and cleared slowly after myocardial injury. Cardiac myosin-binding protein C (cMyC) is a similar cardiac-restricted protein that has more rapid release and clearance kinetics. Direct comparisons are hampered by the lack of an assay for cMyC that matches the sensitivity of the contemporary assays for cTnI and cTnT. Using a novel pair of monoclonal antibodies, we generated a sensitive assay for MyC on the Erenna platform (Singulex) and compared serum concentrations with those of cTnI (Abbott) and cTnT (Roche) in stable ambulatory cardiac patients without evidence of acute cardiac injury or significant coronary artery stenoses. The assay for cMyC had a lower limit of detection of 0.4 ng/L, a lower limit of quantification (LLoQ) of 1.2 ng/L (LLoQ at 20% coefficient of variation [CV]) and reasonable recovery (107.1 ± 3.7%; mean ± standard deviation), dilutional linearity (101.0 ± 7.7%), and intraseries precision (CV, 11 ± 3%) and interseries precision (CV, 13 ± 3%). In 360 stable patients, cMyC was quantifiable in 359 patients and compared with cTnT and cTnI measured using contemporary high-sensitivity assays. cMyC concentration (median, 12.2 ng/L; interquartile range [IQR], 7.9-21.2 ng/L) was linearly correlated with those for cTnT (median, <3.0 ng/L; IQR, <3.0-4.9 ng/L; R = 0.56, P < 0.01) and cTnI (median, 2.10 ng/L; IQR, 1.3-4.2 ng/L; R = 0.77, P < 0.01) and showed similar dependencies on age, renal function, and left ventricular function. We have developed a high-sensitivity assay for cMyC. Concentrations of cMyC in clinically stable patients are highly correlated with those of cTnT and cTnI. This high correlation may enable ratiometric comparisons between biomarkers to distinguish clinical instability.
Assuntos
Biomarcadores/sangue , Proteínas de Transporte/sangue , Imunoensaio/métodos , Calibragem , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Doenças Vasculares Periféricas/sangue , Doença Pulmonar Obstrutiva Crônica/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Troponina I/sangue , Troponina T/sangueRESUMO
BACKGROUND: There is a paucity of information regarding cardiac troponin (cTn) concentrations in peripheral blood of nonhuman primates (NHP). Even less is known regarding cTn concentrations in monkeys that are restrained for oral or intravenous (iv) dosing. OBJECTIVES: The objectives of these studies were to (1) determine cardiac troponin I (cTnI) concentration in resting Cynomolgus monkeys and investigate biologic variability in cTnI concentration over time, (2) determine cTnI changes in restrained monkeys given sham oral dosing, and (3) determine cTnI changes in restrained NHP given a sham intravenous dosing. METHODS: The Research Use Only Erenna cTnI ultrasensitive immunoassay based on single molecule counting technology was used to determine serum cTnI concentration in longitudinal studies of male Cynomolgus monkeys at rest, and after sham oral and intravenous dosing. Animals were catheterized prestudy, and blood samples were collected by an automated sampling device to limit disturbance of the animals during studies. RESULTS: In resting monkeys cTnI concentrations were relatively low and constant and ranged from 0.2 to 9.6 pg/mL (mean = 2.5 pg/mL), with minimal variability during a 24-hour period. Animals given sham oral dosing also had low cTnI concentration with little variability similar to the resting values. Several animals restrained for intravenous dosing had a small transient increase in cTnI concentration (~5-25 pg/mL) that resolved quickly within one to 3 hours postinjection. CONCLUSIONS: Results of this longitudinal study provide information that may be important in differentiating effects of animal handling from those associated with compound-related effects in preclinical toxicology studies of drugs in development.
Assuntos
Troponina I/sangue , Administração Intravenosa/veterinária , Animais , Imunoensaio/veterinária , Estudos Longitudinais , Macaca fascicularis , Masculino , Miocárdio/metabolismo , Restrição Física/veterinária , Sensibilidade e Especificidade , Albumina Sérica , Troponina I/administração & dosagemRESUMO
Management of chronic abdominal pain can be challenging. Sometimes patients fail to get adequate response from multiple medications and nerve blocks. We present a patient case report of chronic abdominal pain with a history of multiple surgeries managed successfully by neuromodulation of the transverse abdominis plane (TAP). The TAP block is a procedure in which local anesthetic is injected into the abdominal fascial plane that carries sensory nerves to the abdominal wall in order to block pain sensation. It has been shown to reduce postoperative pain and analgesic dependence after abdominal and gynecological surgeries. A 60-year-old woman presented to us for chronic abdominal pain for which medications provided little relief. She had an extensive history of abdominal surgeries and was also treated for lower back pain with surgery and less invasive procedures in the past. Under our care, she underwent 2 TAP blocks with almost complete resolution of her abdominal pain. Her pain, however, came back within a few of weeks of the procedures. Since our patient found pain relief from the TAP blocks, we proceeded with neurostimulation of the TAP for long-term pain relief. We placed a dorsal column stimulator 16 contact lead for lower back and leg pain and 8 contact leads placed in the TAP under ultrasound guidance. She has had multiple follow-ups since her TAP lead placement procedure with continued and near complete resolution of her abdominal pain. The TAP lead stimulation was helping her abdominal pain and the dorsal column lead stimulation was helping her back and leg pain.
Assuntos
Dor Abdominal/terapia , Parede Abdominal/inervação , Terapia por Estimulação Elétrica/métodos , Bloqueio Nervoso/métodos , Manejo da Dor/métodos , Dor Abdominal/tratamento farmacológico , Parede Abdominal/fisiopatologia , Anestésicos Locais/administração & dosagem , Anestésicos Locais/farmacologia , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Bupivacaína/administração & dosagem , Bupivacaína/farmacologia , Combinação de Medicamentos , Feminino , Humanos , Metilprednisolona/administração & dosagem , Metilprednisolona/análogos & derivados , Metilprednisolona/farmacologia , Acetato de Metilprednisolona , Pessoa de Meia-Idade , Manejo da Dor/instrumentação , Resultado do TratamentoRESUMO
On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrP(Sc)), we have been interested in how these peptides interact with PrP(Sc). After screening peptides from the entire human PrP sequence, we found two peptides (PrP(19-30) and PrP(100-111)) capable of binding full-length PrP(Sc) in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrP(C)). The limit of detection for captured PrP(Sc) was calculated to be 8 amol from a approximately 10(5)-fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to PrP(Sc) over PrP(C). Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrP(Sc). Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-PrP(Sc) interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrP(Sc), is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrP(C) may play a role in the recruitment of PrP(C) to PrP(Sc).
Assuntos
Fragmentos de Peptídeos/síntese química , Proteínas PrPC/síntese química , Proteínas PrPSc/síntese química , Anticorpos/metabolismo , Humanos , Imunoglobulina G/metabolismo , Microesferas , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/imunologia , Proteínas PrPC/sangue , Proteínas PrPC/classificação , Proteínas PrPC/imunologia , Proteínas PrPSc/sangue , Proteínas PrPSc/classificação , Proteínas PrPSc/imunologia , Doenças Priônicas/diagnóstico , Doenças Priônicas/imunologia , Ligação ProteicaRESUMO
Small cell lung cancer (SCLC) is an aggressive form of lung cancer associated with cigarette smoking and presently accounts for approximately 20% of all lung cancer cases. SCLC cells derive from a neuroendocrine origin and therefore their antigenic profile coincides, to a great extent, with that of neuroendocrine cells. Multiple attempts to generate SCLC-specific MoAbs during the past decade have failed because all SCLC-specific MoAbs isolated also react against neuroendocrine tissues or normal immune cells. Cross-reactivity with normal antigens raises safety concerns due to the inevitable toxicity of such interactions and the dreaded effects. The concept of DIAAD trade mark ( Differential Immunization for Antigen and Antibody Discovery) provides for an immune response that can be effectively focused on cancer antigens. The object is to overcome obstacles resulting from an antigenic hierarchical pattern biased towards a response to dominant antigens in order to induce a robust immune response to cancer antigens. Cancer antigens are weak or nonimmunogenic molecules. Due to the fact that the immune system responds more strongly to immunodominant antigens than to weak immunogenic antigens, cancer cell proliferation is unencumbered. DIAAD employs protocols of induction of tolerance and immunity, conducted in sequential order to "biologically subtract" the immune response of dominant antigens expressed by normal cells. This biological subtraction is achieved in a laboratory animal by first eliminating the immune response to the normal cells or closely related cancer cells, followed by immunization of the same laboratory animal with diseased cells. This procedure directs the immune response exclusively towards antigens expressed by the diseased and not the normal cells. Our objective was to use DIAAD to generate monoclonal antibodies specific to SCLC antigens that are not shared by neuroendocrine cells by contrasting a pool of human SCLC cell lines with a pool of human neuroendocrine cancer cell lines. Four monoclonal antibodies reacted strongly and exclusively with SCLC cells and identified a membrane molecule comprising a single chain glycoprotein. Two of four antibodies were selected for a detailed analysis that revealed a narrow tissue specificity of antigen expressed by colon, lung, and pancreatic cancers (less than 20% staining was found on breast, ovarian and prostate cancer). These antibodies did not bind to various other cancers such as kidney, carcinoid, lymphoma, sarcoma, adrenal, liver, melanoma, seminoma, leiomyoma, basal cell cancer, or undifferentiated cancer. The epitope recognized by the selected MoAbs was destroyed with the removal of carbohydrates from SCLC cells. This result does not exclude the possibility of protein-carbohydrate cooperation in epitope recognition. However, it strongly suggests the pivotal role of carbohydrates in antibody binding to this molecule. Upon binding to the extracellular molecule on SCLC cells, the antibodies were shown to internalize. A low or insignificant level of internalization was recorded following incubation of the antibodies with neuroendocrine-derived tumors. The capacity of these antibodies to internalize upon binding the extracellular receptors renders them potential candidates for prodrug or immunotoxin-targeted therapeutics. In a qualitative experiment involving immunoaffinity purification, the SCLC antigen was shown to be differentially detected in sera of SCLC patients. Plans are being generated to explore the possible utility of this novel SCLC-specific antigen recognized by the above MoAbs as a new biomarker for early diagnosis of the disease, as well as for therapeutic intervention for SCLC.
Assuntos
Antígenos de Neoplasias , Biomarcadores Tumorais , Carcinoma de Células Pequenas/imunologia , Neoplasias Pulmonares/imunologia , Neuroblastoma/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Linfócitos B/citologia , Western Blotting , Membrana Celular/metabolismo , Separação Celular , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Hibridomas/metabolismo , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Pâncreas/citologia , Células Tumorais CultivadasRESUMO
The urokinase receptor is a multi-functional protein that plays a central role in cell surface plasminogen activation, cell migration, and cell adhesion. We previously demonstrated that high affinity peptide ligands for the urokinase receptor, which are urokinase competitors, can be obtained from a 15mer peptide library (Goodson et al., 1994). In order to probe for additional urokinase receptor binding sites we affinity selected the same bacteriophage library on complexes of soluble urokinase receptor (suPAR) and the receptor binding domain of urokinase, residues 1-48 (uPA1-48). Bacteriophage were isolated which bound to suPAR and suPAR:uPA1-48 complexes with high yield. The peptide sequences encoded by these bacteriophage were distinct from those obtained previously on urokinase receptor expressing cells, and comprise two groups based upon effects on su-PAR:1-anilino-8-napthalene sulfonate (ANS) fluorescence, and vitronectin binding competition. Alanine scanning mutagensis of the soluble peptides was used to define minimal regions and key residues for suPAR binding by competition with the parent bacteriophage. A comparison of these results with sequences of domains of both vitronectin and integrin alpha-chains, which have been reported to be important for urokinase receptor binding, suggests that the homology with the peptide sequences selected is functionally significant.