[Elevated Fas expression is related to increased apoptosis of circulating CD8(+)T cell in patients with hepatocellular carcinoma].

Objective: To investigate the mechanism of apoptosis of CD8(+)T lymphocyte in peripheral blood of patients with hepatocellular carcinoma (HCC). Methods: The proportion and apoptosis of peripheral blood CD8(+)T lymphocytes in 30 healthy controls, 30 patients with cirrhosis and 60 HCC patients were detected by Flow cytometry, and the expression of Fas on the surface of CD8(+)T lymphocytes was reported. The differences between groups were compared using independent sample t-test, and data of variance were tested with Mann-Whitney U non-parametric test, P < 0.05 was considered statistically significant. Results: The proportion of CD8(+)T lymphocytes in peripheral blood of patients with HCC was 26.4% ± 9.2%, higher than that of 24.5% ± 7.1% in cirrhosis (t = 0.783, P = 0.489), and and healthy control 19.7% ± 4.7% (t = 2.920, P = 0.004). The proportion of apoptotic CD8(+)T lymphocytes in peripheral blood of HCC patients was 25.3% ± 6.5%, of the total CD8(+)T lymphocytes, which was significantly higher than that of healthy controls 12.1%±6.5% (t = 7.555, P < 0.001) and cirrhotic 13.6% ± 5.8% (t = 5.213, P < 0.001), the differences were statistically significant. The proportion of Fas(+)CD8(+)T lymphocytes in the HCC group was 62.2% ± 18.5%, higher than that in the healthy control group 42.6%±16.5% (t = 4.127, P < 0.001) and 46.1% ± 14.5% (t = 2.561, P < 0.01)of the cirrhosis group, the differences were statistically significant. Fas expression was positively correlated with the apoptosis of CD8(+)T lymphocytes (r (2) = 0.113, P < 0.05). Conclusion: The proportion of CD8(+)T lymphocytes in peripheral blood of patients with HCC is higher than that of healthy controls, but the proportion of CD8(+)T lymphocyte apoptosis based on Fas/FasL pathway increased, which may be an important mechanism for tumor cell immune escape.

[Correlation analysis between glycolipids metabolism and clinicopathologic features in patients with gastric cancer].

OBJECTIVE: To analyze the correlation between glycolipids metabolism and clinicopathologic features in patients with gastric cancer. METHODS: Glycolipids metabolism and clinicopathologic features of 443 gastric cancer patients were collected, and their correlation was analyzed. RESULTS: Compared to gastric cancer patients with normal levels of glycolipids metabolism, there were less male patients who were with low level of total cholesterol (TCH)(χ(2)=7.676, P<0.05), and the number of male patients with low level of high-density lipoprotein (HDL) (χ(2)=7.520) and apoA1 (χ(2)=6.253) was higher (both P<0.05). Serum TCH level showed a negative correlation with age of patients (r=-0.116), tumor size (r=-0.117) and TNM stage (r=-0.111) (P<0.05); serum HDL level was negatively correlated with tumor diameter (r=-0.094), the number of metastatic lymph nodes (r=-0.106), primary tumor invasion depth (r=-0.112), metastatic lymph nodes stage (r=-0.102) and TNM stage (r=-0.107) (P<0.05); serum LDL was negatively correlated with age of patients (r=-0.116) (P<0.05); serum LPa was positively correlated with tumor size (r=0.170), the number of metastatic lymph nodes (r=0.151), primary tumor invasion depth (r=0.160), metastatic lymph nodes stage (r=0.153) and TNM stage (r=0.115) (P<0.05); apoA1 was negatively correlated with distant metastasis (r=-0.168) and TNM stage (r=-0.120) (P<0.05); and apoB was negatively correlated with distant metastases (r=-0.132, P<0.05). Levels of blood glucose and TG had no significant association with clinicopathological features of gastric cancer patients (P>0.05). CONCLUSIONS: Low lipid metabolism but high level of LPa may be the metabolic characteristics of gastric cancer progression. Monitoring the changes of serum lipids levels could be valuable for the prognosis of patients with gastric cancer.

Meta-analysis of the association between the HNF1B rs4430796 (A>G) polymorphism and risk of prostate cancer based on case-control studies.

Genome-wide studies have reported an association between the HNF1B rs4430796 (A>G) polymorphism and prostate cancer risk, but results have been inconsistent and recent meta-analyses have been inadequate. This study aimed to integrate previous results and explore the validity of this association. Electronic searches for all relevant publications through May 18, 2014, were conducted across several databases. Additional studies were identified manually, and only the most recent or complete were used in this meta-analysis. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of the association. Seven eligible case-control studies were identified, incorporating a total of 14,049 patients and 12,674 controls. Overall, we found that the rs4430796 (A>G) polymorphism had a decreased risk of prostate cancer (GG vs AA: OR = 0.661, 95%CI = 0.615-0.710, P = 0.304; AG vs AA: OR = 0.782, 95%CI = 0.739-0.828, P = 0.435; dominant model: OR = 0.743, 95%CI = 0.704-0.784, P = 0.912; recessive model: OR = 0.764, 95%CI = 0.718-0.813, P = 0.01). Furthermore, in the stratified analysis, there were significantly decreased risks among studies with population- and hospital-based controls. In the subgroup analysis by ethnicity, significantly decreased risks were also found among Caucasians, Americans, and Asians. Our results suggested that the HNF1B rs4430796 (A>G) polymorphism decreased the risk of prostate cancer. In the future, additional and larger studies on patients from across of the world might be required to validate our findings.

Use of Percutaneous Sonographically Guided Microwave Ablation Therapy to Treat Inoperable Malignant Liver Tumours.

AIMS: Microwave ablation (MWA) is a technique which is used to destroy tumours and soft tissues by using microwave energy to create coagulation and localized tissue necrosis. It is used to treat the tumours which are considered to be inoperable and used to treat those patients who are ineligible for surgery due to some factors. The main objective of this study was to evaluate the use of MWA in the treatment of liver cancer. METHODS: The data were collected from Department of Ultrasound, Harbin Medical University Cancer Hospital. From July 2010 to August 2011, a total of 123 patients with liver tumours was referred to Harbin Medical University Cancer Hospital. One hundred patients were selected for this study and treated with MWA. The study group contained 64 (64%) males and 36 (36%) females with an average age (± SD) of 52 (± 5.1) years. RESULTS: One month after therapy, complete ablation was obtained in nodules. The complete ablation rate in tumors ≤ 3 cm and those > 3 cm was 98% and 94%, respectively. Microwave ablation success was higher with nodules ≤ 3 cm (57/58; 98.3%) in comparison to nodules > 3 cm. CONCLUSION: Sonographically guided percutaneous microwave ablation proved to be safe, fast and effective for treatment of hepatocellular carcinoma.

New roles of filopodia and podosomes in the differentiation and fusion process of osteoclasts.

The cytoskeleton mediates various cellular processes such as differentiation and fusion, including in the filopodia and podosomes. However, apart from cell migration and formation of the sealing zone, little is known regarding the changes and related regulatory mechanisms of the cytoskeleton and additional roles of the filopodia and podosomes during the differentiation and fusion of osteoclasts. The cytomorphology and cytoskeleton of osteoclasts in the differentiation process were evaluated using tartrate-resistant acid phosphatase staining and immunofluorescence staining. Moreover, the expression levels of Rho GTPases and enzymes related to osteoclast differentiation and bone resorption were detected by quantitative reverse transcription-polymerase chain reaction. We detected 3 types of filopodia in osteoclast precursors and only 1 type of filopodia in undifferentiated cells. Mature osteoclasts were completely devoid of filopodia. Interestingly, cell fusion was highly specific, and the fusion initially occurred to the filopodia. Confocal images revealed that F-actin and microtubules significantly differed among fused cells. These results suggest that filopodia and podosomes not only play important roles in cell migration and the formation of sealing zones but also in the pre-fusion selectivity of 2 cells and the movement direction of the cell nucleus and cytoplasm during the fusion process. In addition, cdc42v1, RhoU, and RhoF regulate the formation of 3 types of filopodia during various stages of differentiation, while Rac1, Rac2, and filament A may be associated with cell selectivity during the fusion process.

Development of three vitrification-based cryopreservation procedures for shoot tips of China's potato.

China is now the largest potato producing country worldwide. The establishment of efficient conservation techniques for potato germplasm is a prerequisite for breeding of elite cultivars. Potato viral diseases have been a great threat for sustainable potato production in China. Use of virus-free seed tubers is an effective and practical means to control viral diseases. In the present study, three vitrification-based cryopreservation techniques, i.e. droplet-vitrification, encapsulation-vitrification and vitrification were successfully developed for the first time for China's potato. Cultivar 'Zihuabai' was used to optimize key parameters involved in the three vitrification-based procedures. With the optimized parameters, shoot regrowth percentages of 71%, 76% and 43% were obtained for droplet-vitrification, encapsulation-vitrification and vitrification, respectively. The three protocols developed were further tested with eight China's major cultivars, with average shoot regrowth of 61%, 38% and 28% for droplet-vitrification, encapsulation-vitrification and vitrification, respectively. Successful development of the three cryopreservation procedures using a single cultivar will facilitate a number of comparative studies such as cryo-injury, regrowth pattern, genetic stability and efficiency of virus elimination. Testing these three cryogenic procedures for potato major cultivars representing a wide range of genetic background, will help the establishment of potato cryobanking in China and the production of virus-free plants.

Benchmark comparison of ab initio microRNA identification methods and software.

MicroRNAs (miRNAs) are short, non-coding RNA molecules that play an important role in the world of genes, especially in regulating the gene expression of target messenger RNAs through cleavage or translational repression of messenger RNA. Ab initio methods have become popular in computational miRNA detection. Most software tools are designed to distinguish miRNA precursors from pseudo-hairpins, but a few can mine miRNA from genome or expressed sequence tag sequences. We prepared novel testing datasets to measure and compare the performance of various software tools. Furthermore, we summarized the miRNA mining methods that study next-generation sequencing data for bioinformatics researchers who are analyzing these data. Because secondary structure is an important feature in the identification of miRNA, we analyzed the influence of various secondary structure prediction software tools on miRNA identification. MiPred was the most effective for classifying real-/pseudo-pre-miRNA sequences, and miRAbela performed relatively better for mining miRNA precursors from genome or expressed sequence tag sequences. RNA-fold performed better than m-fold for extracting secondary structure features of miRNA precursors.

Long noncoding RNA SNHG14 promotes breast cancer cell proliferation and invasion via sponging miR-193a-3p.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Long noncoding RNA SNHG14 promotes breast cancer cell proliferation and invasion via sponging miR-193a-3p, by S.-D. Xie, C. Qin, L.-D. Jin, Q.-C. Wang, J. Shen, J.-C. Zhou, Y.-X. Chen, A.-H. Huang, W.-H. Zhao, L.-B. Wang, published in Eur Rev Med Pharmacol Sci 2019; 23 (6): 2461-2468. DOI: 10.26355/eurrev_201903_17393. PMID: 30964172" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/17393.

Relationship between the MAPK/ERK pathway and neurocyte apoptosis after cerebral infarction in rats.

OBJECTIVE: The aim of this study was to explore the relationship between the mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinase (ERK) pathway and neurocyte apoptosis after cerebral infarction in rats. MATERIALS AND METHODS: Neural stem cells were isolated from rats by establishing the cerebral infarction model and sham model. Isolated cells were cultured in complete culture medium in vitro. Real-time quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect the messenger ribonucleic acid (mRNA) expression of ERK1 and ERK2 in the MARK pathway. Western blotting was applied to examine the activation of the MAPK/ERK pathway and neuron-specific markers. The expression of neuron-specific enolase (NSE) was detected via immunofluorescence. Cell activity and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. RESULTS: The mRNA expressions of ERK1 and ERK2 in neural stem cells increased in a time-dependent manner after cerebral infarction in rats. The expressions of ERK1, ERK2, cyclin D1, Nestin, NSE and glial fibrillary acidic-protein (GFAP) in neural stem cells were significantly decreased after being treated with SCH772984. Cell activity, proliferation and differentiation were markedly inhibited. However, cleaved-caspase 3 protein and apoptosis rate were remarkably increased. CONCLUSIONS: The MAPK/ERK pathway seriously affects neurocyte apoptosis after cerebral infarction in rats. When the MAPK/ERK pathway is inhibited, neurocyte apoptosis is remarkably increased after cerebral infarction in rats.

Long noncoding RNA SNHG14 promotes breast cancer cell proliferation and invasion via sponging miR-193a-3p.

OBJECTIVE: Breast cancer (BC) is one of the most ordinary fatal cancers. Recent studies have identified the vital role of long noncoding RNAs (lncRNAs) in the development and progression of BC. In this research, lncRNA SNHG14 was studied to identify how it functioned in the development and metastasis of BC. PATIENTS AND METHODS: SNHG14 expression of tissues was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) in 50 paired patients with BC. And cell proliferation assay, colony formation assay, and transwell assay were enrolled to observe the biological behavior changes of BC cells through gain or loss of SNHG14. In addition, luciferase assays and RNA immunoprecipitation assay (RIP) were performed to discover the potential targets of SNHG14 in BC cells. RESULTS: SNHG14 expression level of BC samples was higher than that of adjacent ones. Besides, cell growth ability and cell invaded ability of BC cells were inhibited after SNHG14 was silenced, while cell growth ability and cell invaded ability of BC cells were promoted after SNHG14 was overexpressed. In addition, miR-193a-3p was upregulated after silence of SNHG14 in BC cells, while miR-193a-3p was downregulated after overexpression of SNHG14 in BC cells. Furthermore, luciferase assays and RNA immunoprecipitation assay (RIP) showed that miR-193a-3p was a direct target of SNHG14 in BC. CONCLUSIONS: Our study uncovers a new oncogene in BC and suggests that SNHG14 could enhance BC cell proliferation and invasion via sponging miR-193a-3p, which provided a novel therapeutic target for BC patients.

Elimination of two viruses which interact synergistically from sweetpotato by shoot tip culture and cryotherapy.

Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae) and Sweet potato feathery mottle virus (SPFMV; Potyviridae) interact synergistically and cause severe diseases in co-infected sweetpotato plants (Ipomoea batatas). Sweetpotato is propagated vegetatively and virus-free planting materials are pivotal for sustainable production. Using cryotherapy, SPCSV and SPCSV were eliminated from all treated single-virus-infected and co-infected shoot tips irrespective of size (0.5-1.5mm including 2-4 leaf primordia). While shoot tip culture also eliminated SPCSV, elimination of SPFMV failed in 90-93% of the largest shoot tips (1.5mm) using this technique. Virus distribution to different leaf primordia and tissues within leaf primordia in the shoot apex and petioles was not altered by co-infection of the viruses in the fully virus-susceptible sweetpotato genotype used. SPFMV was immunolocalized to all types of tissues and up to the fourth-youngest leaf primordium. In contrast, SPCSV was detected only in the phloem and up to the fifth leaf primordium. Because only cells in the apical dome of the meristem and the two first leaf primordia survived cryotherapy, all data taken together could explain the results of virus elimination. The simple and efficient cryotherapy protocol developed for virus elimination can also be used for preparation of sweetpotato materials for long-term preservation.

Clostridium butyricum promotes intestinal motility by regulation of TLR2 in interstitial cells of Cajal.

OBJECTIVE: Clostridium butyricum (C. butyricum) as a probiotic has been reported to have an important role in the pathogenesis of gastrointestinal diseases. However, the effects of C. butyricum on regulation of intestinal motility of ulcerative colitis (UC) remain unclear. Our study aimed to explore the cross-regulation effect of C. butyricum and toll-like receptor 2 (TLR-2) on UC. MATERIALS AND METHODS: Interstitial cells of Cajal (ICCs) were treated by C. butyricum for 2 h, the mRNA and protein levels of TLR-2, IL-6, and IL-8 were detected by RT-qPCR and Western blot. Then, TLR2-specific small interfering RNA (si-TLR2) was transfected into ICCs, and the relative expressions of IL-6 and IL-8, SCF, cell viability, ghrelin, SP, and ET were measured by RT-qPCR, Western blot, CCK-8, and ELISA. Besides, the signal pathways of NF-κB and JNK were determined by Western blot. RESULTS: C. butyricum significantly increased TLR2, IL-6, and IL-8 expressions in ICCs. However, TLR2 silence alleviated C. butyricum-induced IL-6 and IL-8 expressions. Moreover, TLR2 silence significantly inhibited C. butyricum-induced cell viability in ICCs. Additionally, C. butyricum significantly increased SCF expression and promoted the secretion of ghrelin and SP. However, a significant reduction in the levels of SCF, ghrelin, and SP was evident in the silence of TLR2 expression. Besides, TLR2 silence reduced C. butyricum-activation NF-κB and JNK signal pathways in ICCs. CONCLUSIONS: These findings revealed that C. butyricum promoted intestinal motility by regulation of TLR2 in ICCs, which contributed to understand the molecular mechanisms of C. butyricum on UC.

[Analysis of sonographic characteristics of thyroid nodules diagnosed as false positive or negative results in shear wave elastography].

Objective:In order to improve diagnostic accuracy, we study the characteristics of two dimensional ultrasound and shear wave elastography in the diagnosis of false negative or false positive thyroid nodules by shear wave elastography.Method:One hundred and eighty-nine nodules in 189 consecutive patients who had been determined by surgical operation and pathology. Conventional ultrasound features and SWE elasticity imaging characteristics and properties of the final postoperative pathology were recorded. A comparative study between true and false results of quantitative SWE elasticity imaging, and the corresponding conventional ultrasound nodule characteristics were compared.Result:Postoperative pathology showed 189 nodules, 74(39.2%) were benign and 115(60.8%) were malignant. The sensitivity, specificity of conventional ultrasound in the diagnosis of thyroid nodules were 56.5% and 81.1% respectively, and those of SWE were 60.9% and 85.1%. The false positive rate of shear wave elastography in diagnosing benign nodules and the false negative rate of malignant nodules were 14.9% and 39.1%, respectively. The false negative rate was higher than the false positive rate. A vertical growth (P< 0.01) and smaller diameter of the masses were significantly associated with false SWE findings (P< 0.01).Conclusion:The SWE imaging has important significance for differentiating benign and malignant thyroid nodules, but false results are inevitable, which requires clinicians conjunction with other test results to prevent errors judgment when reviewing the SWE imaging.

Kinetics of protein synthesis inactivation in human T-lymphocytes by selective monoclonal antibody-ricin conjugates.

Immunotoxins synthesized with the pan-T-cell monoclonal antibody T101 and ricin, acetylricin, or ricin A-chain have been compared. Native ricin was acetylated with N-acetylimidazole to block the galactose-binding site of the toxin B (binding)-chain. In the presence of lactose, both whole-ricin-containing immunotoxins were selectively cytotoxic but the ricin A-chain conjugate was less effective in blocking cellular protein synthesis. Immunotoxin-treated cells cultured in fresh growth medium exhibited no growth, declining viabilities, and no protein synthesis activity. Lymphocytes treated with T101:ricin or ricin did not form clusters or colonies when plated in 0.3% Bacto-agar. Ammonium chloride markedly enhanced the efficacy of T101:ricin and T101:ricin A-chain. Our results suggest that: (a) all immunotoxins were selectively cytotoxic; (b) in the presence of ammonium chloride the effectiveness of the T101:ricin A-chain conjugate approached that of T101:ricin; and (c) the toxin B-chain may facilitate conjugate internalization and/or processing.

Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized Fv fragment.

Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have recently described a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin (PE38KDEL). This disulfide-stabilized immunotoxin is indistinguishable in activity and specificity from its single-chain immunotoxin counterpart (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90: 7538-7542, 1993). We have now constructed and evaluated the stability, pharmacokinetics, and antitumor effect of a very similar disulfide-stabilized immunotoxin B3(dsFv)-PE38. This immunotoxin is specifically cytotoxic to human cancer cell lines such as A431 that express the B3 antigen on their surface. In addition, the dsFv-immunotoxin is more stable at 37 degrees C in human serum than the corresponding single-chain immunotoxin B3(Fv)-PE38. The survival of the disulfide-stabilized immunotoxin in the circulation of mice was determined by a bioassay on cultured A431 cells after administering the immunotoxin i.v. The half-life in blood was 23 min. To determine the therapeutic effects of the disulfide-stabilized immunotoxin, it was given i.v. to immunodeficient mice bearing s.c. human epidermoid carcinomas. The dsFv-immunotoxin caused complete regression of tumors with no toxic effect on mice. The antitumor effect was similar to that reported for the single-chain Fv-immunotoxin. Our data show that dsFv-immunotoxins retain full in vitro as well as in vivo activity when compared to scFv-immunotoxins. Because dsFv-immunotoxins have full activity, are more stable, and can be produced with significantly improved yields compared to scFv-immunotoxins, the dsFv-immunotoxins may be more useful for therapeutic applications than scFv-immunotoxins.

Aminosyn II effectively blocks renal uptake of 18F-labeled anti-tac disulfide-stabilized Fv.

Because intact IgG has limitations as a tumor-imaging agent, radiolabeled Fv fragments are being evaluated. Due to the high renal accumulation of Fv fragments, methods to block renal uptake are being sought. This study evaluated how well Aminosyn II, a Food and Drug Administration-approved 15% amino acid solution, would block the renal accumulation of 18F anti-Tac disulfide-stabilized Fv (dsFv) fragments (small fragments with high renal uptake). The anti-Tac dsFv is directed against the alpha subunit of the interleukin 2 receptor. It was labeled at specific activities of 1.1-2.7 mCi/mg using N-succinimidyl 4-[18F]fluoromethyl benzoate. Four adult baboons were injected i.v. with 0.7-1.9 mCi and 150 microg of dsFv. Each baboon was preinjected with Aminosyn II i.v. and, on a separate occasion, with a control solution. Thirty min before injection of 18F-labeled anti-Tac dsFv, a bolus of either solution was given, followed by a constant infusion of 13.3 ml/kg/h. Quantitative positron emission tomography imaging was performed. The amino acid levels in serum were measured serially. The baseline levels of lysine (and other amino acids) in plasma were not significantly different in either the Aminosyn II or control infusion group and did not change during the control infusion. In the Aminosyn II group, lysine levels in plasma 5 min before anti-Tac dsFv infusion were 5-15 times higher than the baseline value and continued to rise during the infusion. The areas under the curve in blood of the 18F-labeled anti-Tac dsFv, from time of injection to end of imaging, expressed as percentage injected dose (%ID), were 28.94 +/- 4.05%ID x h/liter (mean +/- SD) for the control group and 32.09 +/- 11.15%ID x h/liter for the Aminosyn II group (P = 0.54). The peak concentration of 18F-labeled anti-Tac dsFv in the kidney of the controls was 24.53 +/- 4.34%ID; the value in the Aminosyn II group was 5.39 +/- 1.89%ID, representing a mean decrease of 78.5%. The times to reach 90% of the peak levels of 18F in the kidney were 5.6 +/- 3.0 min for the Aminosyn II group and 33.8 +/- 4.8 min for the control group. The amounts excreted in urine by 90 min were 47.7 +/- 8.55%ID and 78.5 +/- 12.8%ID (P = 0.01) for the controls and Aminosyn II group, respectively. In conclusion, Aminosyn II effectively blocks the renal accumulation of 18F-labeled anti-Tac dsFv. Use of Aminosyn II should allow much higher tracer administration for the same radiation exposure to the target organ (kidney).

Trichosanthin-monoclonal antibody conjugate specifically cytotoxic to human hepatoma cells in vitro.

A plant single-chain ribosome-inactivating protein derived from the root tuber of Trichosanthes kirilowii, termed trichosanthin (TCS), was modified with 2-iminothiolane. It was not like trichokirin, a ribosome-inactivating protein derived from the seeds of the same plant, in that TCS retained full activity when 1.5 sulfhydryl groups were introduced into each TCS molecule by 2-iminothiolane modification. The 2-iminothiolane-TCS was conjugated to Hepama-1, a monoclonal antibody directed against human hepatoma with a cross-linking reagent, N-succinimidyl-3-(2-pyridyl)-dithiopropionate. The hepatoma cytotoxicity of the immunotoxin, TCS-Hepama-1, was 500-fold higher than that of free TCS and only 1 log lower than that of free ricin. However, the immunotoxin was approximately 600-fold less cytotoxic to HeLa cells. The results suggested that the immunotoxin was a potent and quite specific antihepatoma agent and might have considerable potential in hepatoma therapy.

Polyethylene glycol-modified chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin.

Modification of proteins with monomethoxy-polyethylene glycol (mPEG) has been shown to prolong circulation time and to reduce immunogenicity. To make a mPEG-modified recombinant toxin that retained cytotoxic activity but had a longer residence time in circulation, we have constructed an altered form of TGF alpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGF alpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell-binding domain. In the newly designed protein, termed TGF alpha R29-L2-CH2-PE38QQ delta (TCP), there are no lysine residues in the TGF alpha and PE38 portions. Human IgG4 constant region CH2 and a tetradecapeptide linker; L2, are inserted between TGF alpha and PE38. Together, L2 and CH2 contain 13 lysine residues as potential modification sites for mPEG. mPEG conjugates of TCP (PEG-TCP) were generated and the products were resolved by ion exchange chromatography. Two PEG-TCP species termed B4 and B6 retained 15 and 4% of cytotoxicity, respectively, and 26% of their receptor binding activity compared with the unmodified TCP. Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in animals. The mean residence times of B4 and B6 were 37 and 68 min, respectively, compared to 7 min for TCP. When administered i.v. to tumor bearing mice, both B4 and B6 produced marked antitumor effects whereas the unmodified TCP had none. Also, the immunogenicity of PEG-TCP was 5-10 times less than that of TCP. We suggest that the prolonged circulating time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.

Cytotoxic activity of disulfide-stabilized recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) toward fresh malignant cells from patients with B-cell leukemias.

Chemical conjugates of anti-CD22 monoclonal antibodies and toxins have been used to treat CD22+ hematological malignancies. A new anti-CD22 recombinant immunotoxin RFB4(dsFv)-PE38, composed of the Fv portion of the monoclonal antibody RFB4 fused to a truncated form of Pseudomonas exotoxin A, is being developed to target CD22+ tumor cells. To explore the potential clinical utility of this recombinant toxin in treating patients with B-cell malignancies, the fresh cells of patients were incubated ex vivo with RFB4(dsFv)-PE38. Specific cytotoxicity was demonstrated in the malignant cells of 25 of 28 patients with a variety of B-cell malignancies, including acute and chronic lymphocytic leukemias and large cell, mantle cell, and follicular lymphomas. The IC50S, the concentrations necessary for 50% inhibition of protein synthesis, were 3-10 ng/ml in five patients and 10-50 ng/ml in seven patients. Cytotoxicity correlated with cell death upon direct examination of the malignant cells. Significant cytotoxicity was observed with cells containing as few as 350 CD22 sites/cell. A more active derivative of RFB4(dsFv)-PE38, RFB4(dsFv)-PE38KDEL, was produced and was slightly to more than 10-fold more cytotoxic toward patient cells and about twice as toxic to mice. Thus, RFB4(dsFv)-PE38 was specifically cytotoxic toward malignant cells from patients with B-cell leukemias. These data support the testing of RFB4(dsFv)-PE38 in patients with CD22+ leukemias and lymphomas, which is presently under way.