Safety, tumor trafficking and immunogenicity of chimeric antigen receptor (CAR)-T cells specific for TAG-72 in colorectal cancer.

BackgroundT cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s.MethodsPatients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 1010 total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72.ResultsFourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had 111Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG-72 binding domain of humanized CC49, reflecting an anti-CAR immune response. No radiologic tumor responses were observed.ConclusionThese findings demonstrate the relative safety of CART72 cells. The limited persistence supports the incorporation of co-stimulatory domains in the CAR design and the use of fully human CAR constructs to mitigate immunogenicity

Design and implementation of GRIP: a computerized glucose control system at a surgical intensive care unit

BACKGROUND: Tight glucose control by intensive insulin therapy has become a key part of critical care and is an important field of study in acute coronary care. A balance has to be found between frequency of measurements and the risk of hypoglycemia. Current nurse-driven protocols are paper-based and, therefore, rely on simple rules. For safety and efficiency a computer decision support system that employs complex logic may be superior to paper protocols. METHODS: We designed and implemented GRIP, a stand-alone Java computer program. Our implementation of GRIP will be released as free software. Blood glucose values measured by a point-of-care analyzer were automatically retrieved from the central laboratory database. Additional clinical information was asked from the nurse and the program subsequently advised a new insulin pump rate and glucose sampling interval. RESULTS: Implementation of the computer program was uneventful and successful. GRIP treated 179 patients for a total of 957 patient-days. Severe hypoglycemia (< 2.2 mmol/L) only occurred once due to human error. With a median (IQR) of 4.9 (4.2 – 6.2) glucose measurements per day the median percentage of time in which glucose fell in the target range was 78%. Nurses rated the program as easy to work with and as an improvement over the preceding paper protocol. They reported no increase in time spent on glucose control. CONCLUSION: A computer driven protocol is a safe and effective means of glucose control at a surgical ICU. Future improvements in the recommendation algorithm may further improve safety and efficiency

A systematic review assessing non-pharmacological conservative treatment studies for people with non-inflammatory multi-joint pain: clinical outcomes and research design considerations

To systematically review the evidence to determine the clinical outcomes and the important methodological quality features of interventional studies on adults with non-inflammatory multi-joint pain (MJP). Systematic search of published and unpublished literature using the databases: AMED, CINAHL, MEDLINE, EMBASE, psycINFO, SPORTDiscus, PEDro, OpenGrey, the EU Clinical Trials Register, World Health Organization International Clinical Trial Registry Platform, ClinicalTrials.gov and the ISRCTN registry (search: inception to 19th October 2017). All papers reporting the clinical outcomes of non-pharmacological interventions for people with non-inflammatory MJP were included. Studies were critically appraised using the Downs and Black Critical Appraisal and the TIDieR reporting checklists. Data were analysed using a Best Evidence Synthesis approach. From 3824 citations, four papers satisfied the eligibility criteria. Three studies reported outcomes from multidisciplinary rehabilitation programmes and one study reported the findings of a spa therapy intervention. All interventions significantly improved pain, function and quality of life in the short-term. There was limited reporting of measures for absenteeism, presenteeism and psychosocial outcomes. The evidence was ‘weak’, and due to a lack of controlled trials, there is limited evidence to ascertain treatment effectiveness. Design consideration for future trials surround improved reporting of participant characteristics, interventions and the standardisation of core outcome measures. There is insufficient high-quality trial data to determine the effectiveness of treatments for non-inflammatory MJP. Given the significant health burden which this condition presents on both individuals and wider society, developing and testing interventions and accurately reporting these, should be a research priority

Cystatin C: current position and future prospects.

Abstract Cystatin C is a low-molecular-weight protein which has been proposed as a marker of renal function that could replace creatinine. Indeed, the concentration of cystatin C is mainly determined by glomerular filtration and is particularly of interest in clinical settings where the relationship between creatinine production and muscle mass impairs the clinical performance of creatinine. Since the last decade, numerous studies have evaluated its potential use in measuring renal function in various populations. More recently, other potential developments for its clinical use have emerged. This review summarises current knowledge about the physiology of cystatin C and about its use as a renal marker, either alone or in equations developed to estimate the glomerular filtration rate. This paper also reviews recent data about the other applications of cystatin C, particularly in cardiology, oncology and clinical pharmacology. Clin Chem Lab Med 2008;46:1664-86

Avaliação do efeito do eucaliptol nas convulsões induzidas por pentilenotetrazol em camundongos

The developmental process of epilepsies involves diverse mechanisms that culminate in the hyperactivity of a population of neurons, resulting in a pattern of repeated and rhythmic depolarizations. Antiepileptic drugs act by increasing GABAergic neurotransmission, reducing the effects of glutamate, or blocking ion channels, and are endowed with serious adverse effects that make it difficult for patients to adhere to treatment. This fact has encouraged the search for compounds of natural origin with potential anticonvulsant effect. Thus, the present study aimed to evaluate the effect of eucalyptol in seizures induced by pentylenetetrazole (PTZ). For this, male Swiss mice, orally treated with monotrepene, were used. The first protocol evaluated the toxicity and the estimated LD50 of the compound. Based on the value of LD50, the doses of terpene used in the behavioral and neurochemical tests were selected. For the behavioral tests, groups of mice were pretreated with saline (10 mL/kg, vol), diazepam (2 mg/kg, ip) and eucalyptol (100, 200 and 400 mg/kg, vol) and then with pentylenetetrazole 80 mg/kg, ip) and evaluated for the following parameters: seizure intensity, latency for first seizure and time of death. For neurochemical tests, groups of mice were pretreated with saline (10 mL/kg, v.o.) and eucalyptol (400 mg/kg, i.p.) and subsequently with pentylenetetrazole (80 mg/kg, i.p.); The determination of the concentration of neurotransmitters (monoamines - dopamine, noradrenaline and serotonin) and oxidative stress markers (nitrite and thiobarbituric acid reactive substances - TBARs) were the parameters evaluated. The results were analyzed by ANOVA or Kruskal-Wallis, followed by Student-Newman-Keuls, and Dunns, respectively. Values of p <0.05 were considered significant. The results showed that oral administration of eucalyptol had low toxicity and the estimated LD50 was greater than 2000 mg / kg. In the PTZ-induced seizure test, only the higher dose of monoterpene (400 mg/kg) significantly reduced seizure intensity by 60%, increased latency for onset of the first seizure by 85% and time of death of the animals in 75% in relation to the control. Similarly, treatment with eucalyptol (400 mg/kg) significantly reduced the concentration of noradrenaline, dopamine and serotonin by 50%, 33% and 70%, respectively, in relation to the PTZ-treated group (80 mg/kg). In addition, treatment with eucalyptol (400 mg/kg) significantly reduced the concentration of TBARs by 33%, but not nitrite, relative to the PTZ treated group (80 mg/kg). Taken together, the results show that the monoterpene studied has low oral toxicity and an important anticonvulsant effect, since its administration is capable of attenuating the convulsions chemically induced by pentylenetetrazol with consequent reduction of the concentration of monoamines and the reactive substances of thiobarbituric acid, elements whose increase is associated with the epileptogenesis phenomenon.O processo de desenvolvimento das epilepsias envolve mecanismos diversos que culminam na hiperatividade de uma população de neurônios, resultando em um padrão de despolarizações repetidas e rítmicas. Os fármacos antiepilépticos agem através do aumento da neurotransmissão GABAérgica, da redução dos efeitos do glutamato, ou do bloqueio de canais iônicos, sendo dotados de efeitos adversos sérios que dificultam a adesão do paciente ao tratamento. Este fato tem incentivado a busca por compostos de origem natural com potencial efeito anticonvulsivante. Desta forma, o presente trabalho teve como objetivo avaliar o efeito do eucaliptol nas convulsões induzidas por pentilenotetrazol (PTZ). Para tanto, foram utilizados camundongos Swiss machos, tratados oralmente com o monotrepeno. O primeiro protocolo realizado avaliou a toxicidade e a DL50 estimada do composto. Com base no valor da DL50, foram selecionadas as doses do terpeno utilizadas nos testes comportamentais e neuroqímicos. Para os testes comportamentais, grupos de camundongos foram previamente tratados com salina (10 mL/kg, v.o.), diazepam (2 mg/kg, i.p.) e eucaliptol (100, 200 e 400 mg/kg, v.o.) e posteriormente com pentilenotetrazol (80 mg/kg, i.p.) e avaliados quanto aos seguintes parâmetros: intensidade das convulsões, latência para primeira convulsão e tempo de morte. Para os testes neuroquímicos, grupos de camundongos foram previamente tratados com salina (10 mL/kg, v.o.) e eucaliptol (400 mg/kg, i.p.) e posteriormente com pentilenotetrazol (80 mg/kg, i.p.); a determinação da concentração de neurotransmissores (monoaminas – dopamina, noradrenalina e serotonina) e dos marcadores de estresse oxidativo (nitrito e substâncias reativas do ácido tiobarbitúrico – TBARs) foram os parâmetros avaliados. Os resultados foram analisados por ANOVA ou Kruskal-Wallis, seguido dos testes de Student-Newman-Keuls, e Dunns, respectivamente. Foram considerados significativos os valores de p < 0,05. Os resultados mostraram que a administração oral do eucaliptol apresentou baixa toxicidade e a DL50 estimada foi superior a 2000 mg/kg. No teste das convulsões induzidas por PTZ apenas a dose maior do monoterpeno (400 mg/kg) reduziu de forma significativa a intensidade das convulsões em 60%, aumentou a latência para aparecimento da primeira convulsão em 85% e o tempo de morte dos animais em 75% em relação ao controle. De forma semelhante, o tratamento com eucaliptol (400 mg/kg) reduziu de forma significativa a concentração de noradrenalina, dopamina e serotonina, em 50%, 33% e 70%, respectivamente, em relação ao grupo tratado com PTZ (80 mg/kg). Além disso, o tratamento com eucaliptol (400 mg/kg) reduziu de forma significativa a concentração de TBARs em 33%, mas não de nitrito, em relação ao grupo tratado com PTZ (80 mg/kg). Tomados em conjunto, os resultados mostram que o monoterpeno estudado apresenta baixa toxicidade oral e importante efeito anticonvulsivante, visto que sua administração é capaz de atenuar as convulsões quimicamente induzidas por pentilenotetrazol com consequente redução da concentração de monoaminas e das substâncias reativas do ácido tiobarbitúrico, elementos cujo aumento está associado ao fenômeno da epileptogênese

Automatic Compilation from High-Level Biologically-Oriented Programming Language to Genetic Regulatory Networks

Background The field of synthetic biology promises to revolutionize our ability to engineer biological systems, providing important benefits for a variety of applications. Recent advances in DNA synthesis and automated DNA assembly technologies suggest that it is now possible to construct synthetic systems of significant complexity. However, while a variety of novel genetic devices and small engineered gene networks have been successfully demonstrated, the regulatory complexity of synthetic systems that have been reported recently has somewhat plateaued due to a variety of factors, including the complexity of biology itself and the lag in our ability to design and optimize sophisticated biological circuitry. Methodology/Principal Findings To address the gap between DNA synthesis and circuit design capabilities, we present a platform that enables synthetic biologists to express desired behavior using a convenient high-level biologically-oriented programming language, Proto. The high level specification is compiled, using a regulatory motif based mechanism, to a gene network, optimized, and then converted to a computational simulation for numerical verification. Through several example programs we illustrate the automated process of biological system design with our platform, and show that our compiler optimizations can yield significant reductions in the number of genes () and latency of the optimized engineered gene networks. Conclusions/Significance Our platform provides a convenient and accessible tool for the automated design of sophisticated synthetic biological systems, bridging an important gap between DNA synthesis and circuit design capabilities. Our platform is user-friendly and features biologically relevant compiler optimizations, providing an important foundation for the development of sophisticated biological systems.National Institutes of Health (U.S.) (Grant # 7R01GM74712-5)United States. Defense Advanced Research Projects Agency (contract HR0011-10-C-0168)National Science Foundation (U.S.) (NSF CAREER award 0968682)BBN Technologie