Multiple Smith-degradations of carcinoembryonic antigen (CEA) and of asialo CEA

Carcinoembryonic antigen (CEA) and asialo CEA were subjected to multiple Smith-degradation (i.e., for each degradation, application in sequence of periodate oxidation, borohydride reduction, and mild hydrolysis with acid; borohydride-t was substituted for unlabelled borohydride). High yields of modified glycoproteins were obtained at each stage. After three complete degradations and a further periodate-borohydride-t treatment, the carbohydrate content of CEA and of asialo CEA had decreased from 45–50% to 11–12% (i.e., 90% removal of carbohydrate). Glycerol was always one of the products obtained after each degradation, but threitol and erythritol were not detected. The second degradation caused a substantial loss of 2-acetamido-2-deoxyglucose, which is consistent with the location of some of this monosaccharide towards the terminal (non-reducing) end of the oligosaccharides. The “core” region of the oligosaccharides is composed of galactose, mannose, and 2-acetamido-2-deoxyglucose. After the fourth oxidation, 2-acetamido-2-deoxyglucose was 50–60% of the total content of residual carbohydrate. After the first degradation, there was a progressive loss in antigenic activity, but this was associated with a small amount of hydrolysis of the protein moiety of CEA.     

CYFRA21-1 and CEA are useful markers for predicting the sensitivity to chemoradiotherapy of esophageal squamous cell carcinoma

Background: Chemoradiotherapy (CRT) is currently performed for patients with advanced esophageal carcinoma. Sensitivity of tumours to CRT differs from one case to another and may be influenced by the expression of biological molecules. The aim of this study was to identify biological markers which could predict sensitivities of esophageal squamous cell carcinoma (ESCC) to CRT.

Methods: A total of 84 patients with stage I–IV ESCC were evaluated. The cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) and carcinoembryonic antigen (CEA) levels were measured before CRT by enzyme-linked immunosorbent assays in patients with primary ESCCs using 3.4?ng ml^?1 and 3.3?ng ml^?1, respectively, as cut-off values. The relationships between pretreatment expression of CYFRA 21-1 and CEA and the effectiveness of CRT were analysed.

Results: The complete response (CR) rates of the primary tumours estimated by computed tomography in patients with high levels of CYFRA21-1 and CEA were 10% (3/30) and 4.2% (1/24), while in cases with low CYFRA21-1 and CEA the CR rates were 50% (27/54) and 48.3% (29/60), respectively (p?=?0.002 and 0.003). The effective rates (CR+PR) in CYFRA21-1 high and low groups were 60% (18/30) and 96.3% (52/54), while in CEA high and low groups they were 58.3% (14/24) and 93.3% (56/60), respectively (p?=?0.013 and 0.013).

Conclusion: CYFRA21-1 and CEA may be helpful in predicting the responsiveness in ESCC of primary lesions to CRT, although the results should be confirmed in larger, more homogeneous studies.     

Phase I trial of a recombinant yeast-CEA vaccine (GI-6207) in adults with metastatic CEA-expressing carcinoma

Yeast-CEA (GI-6207) is a therapeutic cancer vaccine genetically modified to express recombinant carcinoembryonic antigen (CEA) protein, using heat-killed yeast (Saccharomyces cerevisiae) as a vector. In preclinical studies, yeast-CEA induced a strong immune response to CEA and antitumor responses. Patients received subcutaneous vaccines every 2 weeks for 3 months and then monthly. Patients were enrolled at 3 sequential dose levels: 4, 16, and 40 yeast units (10⁷ yeast particles/unit). Eligible patients were required to have serum CEA > 5 ng/mL or > 20 % CEA⁺ tumor block, ECOG PS 0–2, and no history of autoimmunity. Restaging scans were performed at 3 months and then bimonthly. Peripheral blood was collected for the analysis of immune response (e.g., by ELISPOT assay). Twenty-five patients with metastatic CEA-expressing carcinomas were enrolled. Median patient age was 52 (range 39–81). A total of 135 vaccines were administered. The vaccine was well tolerated, and the most common adverse event was grade 1/2 injection-site reaction. Five patients had stable disease beyond 3 months (range 3.5–18 months), and each had CEA stabilization while on-study. Some patients showed evidence post-vaccination of increases in antigen-specific CD8⁺ T cells and CD4⁺ T lymphocytes and decreases in regulatory T cells. Of note, a patient with medullary thyroid cancer had substantial T cell responses and a vigorous inflammatory reaction at sites of metastatic disease. Yeast-CEA vaccination had minimal toxicity and induced some antigen-specific T cell responses and CEA stabilization in a heterogeneous, heavily pre-treated patient population. Further studies are required to determine the clinical benefit of yeast-CEA vaccination.     

Elevated CEA and CA19-9 serum levels independently predict advanced pancreatic cancer at diagnosis

Abstract

Purpose: It is suggested that tumour markers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) could be used to predict the stage of pancreatic cancer. However, optimal cut-off values for CEA and CA19-9 are disputable. This study aimed to assess the value of CEA and CA19-9 serum levels at diagnosis of pancreatic ductal adenocarcinoma (PDAC) as predictors for the advanced stage of PDAC in patients discussed at pancreatic multidisciplinary team (MDT) meetings.

Methods: Patients with suspected PDAC discussed at MDT meetings from 2013 to 2017 were reviewed, in order to determine optimal cut-off values of both CEA and CA19-9.

Results: In total, 375 patients were included. Optimal cut-off values for predicting advanced PDAC were 7.0?ng/ml for CEA and 305.0?U/ml for CA19-9, resulting in positive predictive values of 83.3%, 73.6%, and 91.4% for CEA, CA19-9 and combined, respectively. Both tumour markers were independent predictors of advanced PDAC, demonstrated by an odds ratio of 4.21 (95% CI:1.85–9.56; p?=?0.001) for CEA and 2.58 for CA19-9 (95% CI:1.30–5.14; p?=?0.007).

Conclusions: CEA appears to be a more robust predictor of advanced PDAC than CA19-9. Implementing CEA and CA19-9 serum levels during MDT meetings as an additional tool for establishing tumour resectability is worthwhile for tailored diagnostics.     

Cell Lysis of Cyanobacteria and Its Implications for Nutrient Dynamics

The dynamics of nutrients, such as phosphorus, nitrogen, and carbohydrates, during cyanobacteria cell lysis was investigated under darkness incubation in the laboratory. The cell lysis rate of cyanobacteria sampled from Lake Taihu was measured using an esterase assay. Based on particulate esterase activity, the calculated cyanobacteria lysis rate was 0.094 d^–1. During 30 days of darkness incubation, Chlorophyll a concentration decreased from 56 μg L^–1 to 2.0 μg L^–1. Parallel to this, total particulate carbohydrate concentration decreased rapidly. The fluctuation of dissolved organic carbon concentration was a function of the production of non‐carbohydrate by cyanobacteria and the decomposition of carbohydrate by bacteria. Total dissolved carbohydrates and dissolved polysaccharides concentrations showed a similar pattern, declining at the beginning of the experiment and keeping relatively stable, thereafter. In contrast, the concentration of dissolved monosaccharides remained constant during the entire process. The concentrations of NH₄⁺ and PO₄^3– increased at the early stage, and then decreased afterwards. A gradual decrease in NO₃^– concentration after day 8 indicated that anaerobic conditions might be produced during the cell lysis process. The present results demonstrated cyanobacteria cell lysis has a big influence on the nutrient status of the surrounding water. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Glycomic profiling of carcinoembryonic antigen isolated from human tumor tissue

Background

Carcinoembryonic antigen (CEA) is a protein commonly found in human serum, with elevated CEA levels being linked to the progression of a wide range of tumors. It is currently used as a biomarker for malign tumors such as lung cancer and colorectal cancer [Urol Oncol: Semin Orig Invest 352: 644–648, 2013 and Lung Cancer 80: 45-49, 2013]. However, due to its low specificity in clinical applications, CEA can be used for monitoring only, rather than tumor diagnosis. The function of many glycoproteins is critically dependent on their glycosylation pattern, which in turn has the potential to serve in tumor detection. However, little is known about the detailed glycan patterns of CEA.

Methods

To determine these patterns, we isolated and purified CEA proteins from human tumor tissues using immunoaffinity chromatography. The glycan patterns of CEA were then analyzed using a Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry³ (MALDI-TOF-MS³) approach.

Results

We identified 61 glycoforms in tumor tissue, where CEA is upregulated. These glycosylation entities were identified as bi-antennary, tri-antennary and tetra-antennary structures carrying sialic acid and fucose residues, and include a multitude of glycans previously not reported for CEA.

Conclusion

Our findings should facilitate a more precise tumor prediction than currently possible, ultimately resulting in improved tumor diagnosis and treatment.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9088-3) contains supplementary material, which is available to authorized users.     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

胃癌根治术后腹腔热灌注化疗对患者血清CEA、CA19-9水平及免疫功能的影响

目的:探讨胃癌根治术后腹腔灌注化疗对患者血清CEA(血清癌胚抗原,carcinoembryonic antigen)、CA19-9(糖链抗原19-9,carbohydrate antigen19-9)水平及免疫功能的影响。方法:回顾性2015年2月至2017年4月我院收治的胃癌患者临床资料,依据接受治疗方案不同分为全身静脉化疗组(对照组)和全身静脉化疗联合腹腔热灌注化疗组(观察组),每组各41例。检测和比较两组患者化疗前(治疗前)与化疗1个月后(治疗后)血清肿瘤标志物CEA、CA19-9与免疫功能指标水平的变化,治疗后毒副作用发生情况及治疗前后生活质量的改善情况。结果:治疗前,两组间血清CEA、CA19-9、CD3~+、CD4~+、CD8~+、CD4+/CD8~+水平比较差异均无统计学意义(P0.05);观察组治疗后血清CEA、CA19-9及CD8~+水平显著低于对照组,CD3~+、CD4~+、CD4~+/CD8~+水平显著高于对照组,差异有统计学意义(P0.05);两组骨髓抑制、恶心呕吐、腹痛腹泻及肠梗发生率比较差异均不显著无统计学意义(P=0.478,0.668,0.315,0.552);观察组生活质量改善总有效率为85.37%,显著高于对照组(70.73%,P=0.017)。结论:与单纯全身化疗相比,胃癌根治术后腹腔灌注化疗可更有效降低患者血清CEA、CA19-9水平,改善患者免疫功能,提高其生活质量,且安全性较高。     

Prognostic Significance of Molecular Analysis of Peritoneal Fluid for Patients with Gastric Cancer: A Meta-Analysis

Background

Accurately distinguishing serosal invasion in patients with gastric cancer (GC) prior to surgery can be difficult. Molecular analysis of peritoneal fluid (MAPF) for free cancer cells with higher sensitivity than other methods; however, its prognostic value for GC remains controversial, precluding its application in clinical practice.

Methods

PubMed, EMBASE and other databases were systematically searched. Thirty-one studies were eligible for the meta-analysis. Hazard ratios (HRs) and 95% confidence intervals (CIs) were pooled for overall survival (OS), disease-free survival (DFS) and peritoneal recurrence-free survival (PRF).

Results

The current meta-analysis focused on patients with GC and negative cytological diagnoses. The results showed that positive MAPF status (MAPF⁺) led to poorer prognoses for OS (HR 2.59, 95% CI 1.99–3.37), DFS (HR 4.92, 95% CI 3.28–7.37) and PRF (HR 2.81, 95% CI 2.12–3.72) compared with negative MAPF status (MAPF^-). Moreover, among the patients with GC who received curative treatment, the MAPF⁺ patients had poorer prognoses for OS (HR 3.27, 95% CI 2.49–4.29), DFS (HR 3.90, 95% CI 2.74–5.57) and PRF (HR 5.45, 95% CI 3.70–8.03). A meta-analysis of multivariate-adjusted HRs demonstrated that MAPF⁺ status was an independent prognostic factor for patients with GC who underwent curative treatment (OS: HR 2.19, 95% CI 1.47–3.28; PRF: HR 3.44, 95% CI 2.01–5.87). Using the identical target genes (CEA, CEA/CK20) as molecular markers, the patients with GC who were MAPF⁺ had significantly worse prognoses for OS (CEA: HR 3.03, 95% CI 2.29–4.01; CEA/CK20: HR 4.24, 95% CI 2.42–7.40), DFS (CEA: HR 3.99, 95% CI 2.24–7.12; CEA/CK20: HR 4.31, 95% CI 1.49–2.48) and PRF (CEA: HR 4.45, 95% CI 2.72–7.31; CEA/CK20: HR 6.46, 95% CI 3.62–11.55) than the patients who were MAPF^-.

Conclusion/Significance

The above results demonstrate that MAPF could be a prognostic indicator for patients with GC who have a negative cytological diagnosis and/or are receiving curative treatment. MAPF could provide clinicians with additional prognostic information that could aid in developing individualized treatment plans prior to surgery. The widely used target genes CEA, CEA/CK20 were confirmed to be valuable MAPF markers for predicting the prognosis of GC.     

四磨汤对宫颈癌化疗患者胃肠功能的保护作用及对血清IL-8、CEA、CA125水平的影响

目的:研究四磨汤对恩度联合力朴素治疗的宫颈癌患者胃肠功能的保护作用及对其血清白细胞介素8(IL-8)、癌胚抗原(CEA)、糖类抗原125(CA125)的影响。方法:选取2015年9月至2016年8月我院收治的88例宫颈癌患者,根据患者入院顺序分为观察组和对照组,每组44例。对照组在放疗基础上加以恩度联合力朴素完成化疗,观察组在对照组治疗基础上加以四磨汤。比较两组患者治疗前后血清IL-8、CEA、CA125水平及外周血CD3~+、CD4~+、CD8~+细胞比例的变化和胃肠道毒副反应、胃肠道放射性损伤的发生情况。结果:治疗后,两组患者血清IL-8、CEA、CA125水平均较治疗前显著降低(P0.05),且观察组的血清IL-8、CEA、CA125水平较对照组明显降低(P0.05);两组患者CD3~+、CD4~+细胞比例较治疗前显著降低(P0.05),CD8~+细胞比例较治疗前显著升高(P0.05),但观察组的CD3~+、CD4~+、CD8~+细胞比例细胞比例显著高于对照组(P0.05)。观察组不良反应发生率、早期胃肠道放射性损伤发生率、Ⅱ、Ⅲ级胃肠道放射性损伤率均显著低于对照组(P0.05)。结论:四磨汤用于恩度联合力朴素治疗的宫颈癌患者能有效保护患者胃肠功能,降低患者血清IL-8、CEA、CA125水平,增强患者免疫力。     

CpG oligonucleotides enhance the tumor antigen-specific immune response of an anti-idiotype antibody-based vaccine strategy in CEA transgenic mice

A murine monoclonal anti-idiotype (Id) antibody, 3H1 has been developed and characterized previously. Anti-Id 3H1 mimics a specific epitope of carcinoembryonic antigen (CEA) and can be used as a surrogate antigen for CEA. 3H1 induced anti-CEA immunity in different species of animals as well as humans and showed promise as a potential vaccine candidate in phase I/II clinical trials for colon cancer patients. One area of interest to us has been the development of new immune adjuvants that may augment the potency of 3H1 as a tumor vaccine. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. In this study, we have evaluated the efficacy of 3H1 as a tumor vaccine when admixed with a select CpG ODN 1826 in transgenic mice that express human CEA. The vaccine potential of 3H1 was also assessed in the presence of another widely used adjuvant, QS-21. 3H1 coupled to keyhole limpet hemocyanin (KLH) and mixed with Freund’s adjuvant (FA) was used as a gold standard in this system. 3H1 vaccination with different adjuvants induced both humoral and cellular anti-3H1, as well as anti-CEA immunity in CEA transgenic mice. The immune sera could lyse CEA-transfected murine colon carcinoma cells, C15 effectively in an antibody-dependent cellular cytotoxicity assay. The anti-CEA antibody responses were somewhat comparable in each adjuvant-treated group of mice, whereas cellular immune responses were significantly greater when CpG was used as an adjuvant. Splenocytes obtained from 3H1–CpG-immunized mice showed an increased proliferative CD4⁺ Th1-type T-cell response when stimulated in vitro with 3H1 or CEA and secreted elevated levels of Th1 cytokines (IL-2, IFN-γ). This vaccine also induced MHC class I antigen-restricted CD8⁺ T-cell responses. In a solid tumor model, C15 tumor growth was significantly inhibited by 3H1 vaccinations. In 3H1–CpG-vaccinated mice, the duration of survival was, however, longer compared to the 3H1–QS21-vaccinated mice. These findings suggest that 3H1-CpG vaccinations can break peripheral tolerance to CEA and induce protective antitumor immunity in this murine model transgenic for human CEA.     

Human Anti-CEA Antibodies detected by Radioimmunoelectrophoresis

THE carcinoembryonic antigen (CEA) of the human digestive system was first described in adenocarcinomata of the human digestive system and in human foetal digestive organs during the first two trimesters of gestation^1,2. Subsequently, it was shown that the CEA is glycoprotein which is intimately associated with the surface of the tumour cell^3,4. Recently, there has been considerable interest in the measurement of circulating CEA by a variety of radioimmunoassay techniques^5–7 in the sera of patients with digestive system tumours and related diseases for diagnostic and therapeutic purposes. Relatively little is known, however, about whether the CEA evokes an antibody response in the tumour bearing host⁸.     

Growth and biochemical composition of filamentous microalgae Tribonema sp. as potential biofuel feedstock

Filamentous oleaginous microalgae Tribonema minus have advantages in relatively easy harvesting and grazers resistance in mass cultivation due to its filaments in previous study. To evaluate whether the genus Tribonema is a valuable candidate for use in biofuel production, the morphology, growth, biochemical composition and fatty acid profile of six filamentous microalgae strains Tribonema sp. were investigated. All the strains are unbranched filament in single row of elongated cylinder, attaining 0.5–3 mm in length. The growth rates of tested strains were 0.35–0.42 g L^?1 d^?1. Generally, for all strains, decrease in protein content was followed by a slight increase in lipid and significant increase in carbohydrate in early phase, afterwards, lipid increased constantly inversely to decrease in carbohydrate content. After 15-day cultivation, total lipid contents of tested strains ranged from 38–61 %, of which TAG were the majority and palmitic acid (C16:0) and palmitoleic acid (C16:1) were the dominant components. The study confirmed that the genus Tribonema is the potential for biodiesel and bioethanol production upon culture time.     

Combination Therapy with Local Radiofrequency Ablation and Systemic Vaccine Enhances Antitumor Immunity and Mediates Local and Distal Tumor Regression

Purpose

Radiofrequency ablation (RFA) is a minimally invasive energy delivery technique increasingly used for focal therapy to eradicate localized disease. RFA-induced tumor-cell necrosis generates an immunogenic source of tumor antigens known to induce antitumor immune responses. However, RFA-induced antitumor immunity is insufficient to control metastatic progression. We sought to characterize (a) the role of RFA dose on immunogenic modulation of tumor and generation of immune responses and (b) the potential synergy between vaccine immunotherapy and RFA aimed at local tumor control and decreased systemic progression.

Experimental Design

Murine colon carcinoma cells expressing the tumor-associated (TAA) carcinoembryonic antigen (CEA) (MC38-CEA⁺) were studied to examine the effect of sublethal hyperthermia in vitro on the cells’ phenotype and sensitivity to CTL-mediated killing. The effect of RFA dose was investigated in vivo impacting (a) the phenotype and growth of MC38-CEA⁺ tumors and (b) the induction of tumor-specific immune responses. Finally, the molecular signature was evaluated as well as the potential synergy between RFA and poxviral vaccines expressing CEA and a TRIad of COstimulatory Molecules (CEA/TRICOM).

Results

In vitro, sublethal hyperthermia of MC38-CEA⁺ cells (a) increased cell-surface expression of CEA, Fas, and MHC class I molecules and (b) rendered tumor cells more susceptible to CTL-mediated lysis. In vivo, RFA induced (a) immunogenic modulation on the surface of tumor cells and (b) increased T-cell responses to CEA and additional TAAs. Combination therapy with RFA and vaccine in CEA-transgenic mice induced a synergistic increase in CD4⁺ T-cell immune responses to CEA and eradicated both primary CEA⁺ and distal CEA^– s.c. tumors. Sequential administration of low-dose and high-dose RFA with vaccine decreased tumor recurrence compared to RFA alone. These studies suggest a potential clinical benefit in combining RFA with vaccine in cancer patients, and augment support for this novel translational paradigm.     

Analysis of neuropeptide Y-induced feeding: Dissociation of Y1 and Y2 receptor effects on natural meal patterns

To differentiate NPY receptor subtypes, Y₁ and Y₂, in terms of their impact on feeding behavior, the intact molecule NPY(1–36) and the 3 fragments, NPY(2–36), the Y₁ agonist [Leu³¹,Pro³⁴]NPY, and the Y₂ agonist NPY(13–36), were injected (100 pmol/0.3 μl) into the hypothalamic paraventricular nucleus (PVN) of freely feeding rats. A computer-automated data acquisition system was employed in these experiments to permit a detailed analysis of feeding over the 12-h nocturnal cycle, in animals maintained on pure macronutrient diets. The results demonstrate that: 1) NPY(1–36) potentiates feeding behavior, primarily carbohydrate ingestion, by increasing the size and duration of the first meal after injection, rather than by affecting meal number or feeding rate, suggesting that NPY acts through mechanisms of satiety. The potentiation of carbohydrate intake occurs in association with a suppression of protein intake, which is strongest during the second meal after injection and which further increases the proportion of carbohydrate in the diet. No changes in fat ingestion are seen. 2) NPY(2–36), with the N-terminal tyrosine residue deleted, is equally potent to NPY(1–36) in potentiating carbohydrate intake and increasing meal size; however, it is less selective than NPY(1–36), producing an additional, smaller increase in consumption of protein. 3) The stimulatory effect of these peptides on carbohydrate intake and meal size is similarly observed, with somewhat reduced potency, after PVN injection of the selective Y₁ agonist [Leu³¹,Pro³⁴]NPY which, like NPY(1–36), also reduces protein intake. 4) The Y₂ receptor agonist, NPY(13–36), causes a decrease in the ingestion of carbohydrate, a smaller decline in protein intake, and a reduction in meal size. It is proposed that hypothalamic Y₁ receptors mediate the stimulatory effect of NPY on carbohydrate intake and meal size, while Y₂ receptors have the opposite effect of suppressing carbohydrate intake, possibly by altering presynaptic release of monoamines known to influence nutrient ingestion.     

Phytoplankton ecology in the Southern Benguela current. I. Biochemical composition

Investigations on phytoplankton communities in a nearshore region off the Cape Peninsula revealed three types of upwelled water. During active upwelling temperatures were < 10 °C and concentrations of inorganic nutrients were high (Type 1). Maturing upwelled water was characterized by temperatures > 10°C and nitrate concentrations varying between 2 and 15 μg-at. NO₃-N · 1^?1 (Type 2), while aged upwelled water (Type 3) contained low concentrations of nitrate (<2 μg-at. NO₃-N · 1^?1) at temperatures > 10°C. During the summer of 1978–1979 diatoms dominated the communities from October to January but microflagellates were dominant in February and March. In both types of community, low concentrations of ATP, chlorophyll a, protein and carbohydrate were measured in Type 1 water with protein/carbohydrate ratios being > 1. In Type 2 water concentrations of chlorophyll a, ATP and protein were high and the protein/carbohydrate ratio was > 1. Concentrations of chlorophyll a and ATP remained high in Type 3 water but the protein/carbohydrate ratio decreased to < 1 due to an increase in the concentration of acid-soluble glucan. It was concluded that the communities were in an active phase of growth in Type 1 and Type 2 water when adequate nutrients were available, but were in a slow-growing phase in Type 3 water when nitrate concentrations were low. Correlation coefficients, simple linear regressions and stepwise multiple regressions between biochemical and environmental variables confirmed that nitrate was the nutrient most closely related to the biochemical composition of phytoplankton. Using linear regression equations of biochemical variables on glucan it was estimated that chlorophyll a existed in a ratio of ≈ 1: 1 between living phytoplankton and bacteria/detritus, while the percentage of ATP was high in the phytoplankton component of Type 1 water but low in that of Type 2 water. The percentage of protein in detritus was greater than in living phytoplankton, and the carbohydrate content of living phytoplankton increased as the upwelled water matured from Type 1 and Type 2 to Type 3.     

Pattern recognition of neuron specific enolase and carcinoembryonic antigen in whole blood samples

New tools and methods for pattern recognition of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) were proposed for the screening of whole blood samples. The new tools were based on stochastic sensors designed using nanoporous gold microspheres, graphite, graphene, diamond paste as well as α‐CDs, and 5,10,15,20‐tetraphenyl‐21H,23H‐porphyrin. The best sensor for the assay of CEA was the one based on P/graphite (the limit of determination was 16 fg/ml and sensitivity was 2.32 × 10⁷ s mg^?1 ml), while for the assay of NSE the, best sensor was the one based on P/graphene (the limit of determination was 7.45 pg/ml and sensitivity was 2.49 × 10⁸ s mg^?1 ml). The sensor of choice for simultaneous detection of NSE and CEA is the one based on P/graphene because we need high sensitivity and low limit of determination for NSE. To our knowledge, this is the only one screening test for early detection of lung cancer, by identification of NSE and CEA in whole blood samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Radioimmunoimaging of Liver Metastases with PET Using a 64Cu-Labeled CEA Antibody in Transgenic Mice

Purpose

Colorectal cancer is one of the most common forms of cancer, and the development of novel tools for detection and efficient treatment of metastases is needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (PET) imaging and radioimmunotherapy. Since carcinoembryonic antigen (CEA) is an important target in colorectal cancer, the CEA-specific M5A antibody has been extensively studied in subcutaneous xenograft models; however, the M5A antibody has not yet been tested in advanced models of liver metastases. The aim of this study was to investigate the ⁶⁴Cu-DOTA-labeled M5A antibody using PET in mice bearing CEA-positive liver metastases.

Procedures

Mice were injected intrasplenically with CEA-positive C15A.3 or CEA-negative MC38 cells and underwent micro-computed tomography (micro-CT) to monitor the development of liver metastases. After metastases were detected, PET/MRI scans were performed with ⁶⁴Cu-DOTA-labeled M5A antibodies. H&E staining, immunohistology, and autoradiography were performed to confirm the micro-CT and PET/MRI findings.

Results

PET/MRI showed that M5A uptake was highest in CEA-positive metastases. The %ID/cm³ (16.5%±6.3%) was significantly increased compared to healthy liver tissue (8.6%±0.9%) and to CEA-negative metastases (5.5%±0.6%). The tumor-to-liver ratio of C15A.3 metastases and healthy liver tissue was 1.9±0.7. Autoradiography and immunostaining confirmed the micro-CT and PET/MRI findings.

Conclusion

We show here that the ⁶⁴Cu-DOTA-labeled M5A antibody imaged by PET can detect CEA positive liver metastases and is therefore a potential tool for staging cancer, stratifying the patients or radioimmunotherapy.     

Comparison of multiple anti-CEA immunotoxins active against human adenocarcinoma cells

Summary Anti-carcinoembryonic antigen (CEA) immunotoxins constructed with multiple anti-CEA antibodies (goat and baboon polyclonal, and three murine monoclonal antibodies) by covalently linking them to the A chain of ricin via a disulfide bond all function as potent and specific toxins for CEA-bearing cells, suggesting that the CEA molecule is capable of directing productive internalization of ricin A chain. The high potency of anti-CEA immunotoxins apparently makes addition of ricin B chain unnecessary for high toxic efficiency, as in some other systems, because presence of the B chain reduces target cell specificity. Several characteristics of the immunotoxins which might account for their cytotoxic potency were studied. Equilibrium association constants of the goat, baboon, and murine monoclonal C-19 antibodies with fluid-phase CEA were determined by using Langmuir plots and were found to be 8.79, 6.61, and 8.13×10⁹ M ^–1, respectively, indicating the high and similar affinities of the three antibodies toward CEA. Radioimmunoassay binding studies of the three immunotoxins with ¹²⁵I-CEA showed that the antibody portions of the molecules retained the ability to form complexes with CEA after conjugation to ricin A chain. The maximum number of anti-CEA antibody molecules bound per cell, as demonstrated by ¹¹¹In-labeled C-19 binding assays with CEA-bearing cell lines, varied from 2.65×10⁵ per cell for HT29 to 2.01×10⁶ for LoVo, with an intermediate value of 1.17×10⁶ per cell for WiDr. Cytotoxicity of the immunotoxins was assessed by inhibition of protein synthesis and expressed as a median inhibitory dose (ID₅₀). Comparison of the ID₅₀'s of each immunotoxin on the three cell lines has shown that the immunotoxin made of the monoclonal C-19 antibody is in general 6 to 7 times more cytotoxic than the goat and baboon antibody immunotoxins. The affinity of CEA-antibody binding is probably an important, but not a sole factor in determining the immunotoxin potency. The fact that the antibodies with very similar affinity toward fluid phase CEA make immunotoxins of different potency might indicate that interactions with membrane-bound CEA are more complex and/or the efficiency of internalization of various immunotoxins is different. An important factor in immunotoxin action appears to be the CEA content in target adenocarcinoma cells.Supported in part by the NIH BRSG grant SO7RRO5712, the American Cancer Society, Mass. Div. Research Grant 1543-C-1, and by the Aid for Cancer Research (Boston) award to L. V. L., and by RO1 CA 29160 and RO1 CA 39748 grants to T. W. G.