[Efficacy and safety of neoadjuvant chemotherapy combined with PD-1 antibody for esophageal squamous cell carcinoma in the real world].

Objective: To evaluate the efficacy and safety of neoadjuvant chemotherapy combined with programmed death-1 (PD-1) antibody in operable, borderline or potentially resectable locally advanced esophageal squamous cell carcinoma(ESCC) in the real world. Methods: The study retrospectively analyzed 28 patients with operable or potentially resectable locally advanced ESCC patients treated with preoperative chemotherapy combined with PD-1 inhibitor in Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School from April 2020 to March 2021. According to the clinical TNM staging system of the 8th edition of the American Joint Committee on Cancer, there were 1, 15, 10, 1 and 1 case of stage Ⅱ, Ⅲ, ⅣA, ⅣB and unknown stage respectively. The treatment was two cycle of dual drug chemotherapy regimen including taxane plus platinum or fluorouracil combined with PD-1 antibody followed by tumor response assessment and surgery if the patient was eligible for resection. Results: Of the 28 patients, 1, 2, 3 and 4 cycles of chemotherapy combined with PD-1 antibody treatment completed in 1, 21, 5, and 1 patient, respectively. Objective response rate (ORR) was 71.4% (20/28), and disease control rate (DCR) was 100% (28/28). The incidence of adverse events exceeding grade 3 levels was 21.4% (6/28), including 3 neutropenia, 1 leukopenia, 1 thrombocytopenia and 1 immune hepatitis. There was no treatment-related death. Of the 23 patients underwent surgery, R0 resection rate was 87.0% (20/23), 13 patients had down staged to the T1-2N0M0 I stage, the pCR rate was 17.3% (4/23), and the pCR rate of primary tumor was 21.7% (5/23). Four patients received definitive chemoradiotherapy. One patient rejected surgery and other treatment after achieved PR response. Conclusion: Neoadjuvant chemotherapy combined PD-1 inhibitor is safe and has high efficacy in operable, borderline or potentially resectable locally advanced ESCC, and it is a promising regimen.

Structure-activity relationship (SAR) studies of synthetic glycogen synthase kinase-3ß inhibitors: A critical review.

Glycogen Synthase Kinase-3 (GSK-3) is a constitutively dynamic, omnipresent serine/threonine protein kinase regularly called as a "multitasking kinase" due to its pliable function in diverse signaling pathways. It exists in two isoforms i.e., GSK-3α and GSK-3ß. Inhibition of GSK-3 may be useful in curing various diseases such as Alzheimer's disease, type II diabetes, mood disorders, cancers, chronic inflammatory agents, stroke, bipolar disorders and so on, but the approach poses significant challenges. Lithium was the first GSK-3ß inhibitor to be used for therapeutic outcome and has been effectively used for many years. In recent years, a large number of structurally diverse potent GSK-3ß inhibitors are reported. The present review focuses on the recent developments in the area of medicinal chemistry to explore the diverse chemical structures of potent GSK-3ß inhibitors and also describes its structure-activity relationships (SAR) and molecular binding interactions of favorable applicability in various diseases.

PHRF1 promotes genome integrity by modulating non-homologous end-joining.

Methylated histone readers are critical for chromatin dynamics, transcription, and DNA repair. Human PHRF1 contains a plant homeodomain (PHD) that recognizes methylated histones and a RING domain, which ubiquitinates substrates. A recent study reveals that PHRF1 is a tumor suppressor that promotes TGF-ß cytostatic signaling through TGIF ubiquitination. Also, PHRF1 is a putative phosphorylation substrate of ataxia telangiectasia-mutated/ataxia telangiectasia and Rad3-related kinases; however, the role of PHRF1 in DNA damage response is unclear. Here we report a novel function of PHRF1 in modulating non-homologous end-joining (NHEJ). PHRF1 quickly localizes to DNA damage lesions upon genotoxic insults. Ablation of PHRF1 decreases the efficiency of plasmid-based end-joining, whereas PHRF1 overexpression leads to an elevated NHEJ in H1299 reporter cells. Immunoprecipitation and peptide pull-down assays verify that PHRF1 constitutively binds to di- and trimethylated histone H3 lysine 36 (H3K36) (H3K36me2 and H3K36me3) via its PHD domain. Substitution of S915DT917E to ADAE in PHRF1 decreases its affinity for NBS1. Both PHD domain and SDTE motif are required for its NHEJ-promoting activity. Furthermore, PHRF1 mediates PARP1 polyubiquitination for proteasomal degradation. These results suggest that PHRF1 may combine with H3K36 methylation and NBS1 to promote NHEJ and stabilize genomic integrity upon DNA damage insults.

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.

Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA.

RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAi-based therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log(10) decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.

Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.

The idiotypic determinant (Id) of the immunoglobulin expressed by a B-cell malignancy can serve as an effective tumor-specific antigen but is only weakly immunogenic. This study demonstrates that the immunogenicity of the tumor Id protein can be dramatically increased by directing it to antigen-presenting cells (APCs). Cytotoxic T-lymphocyte antigen 4 (CTLA-4) present on activated T cells has a strong binding affinity to both B7-1 and B7-2 molecules, which are primarily expressed on APCs. After construction of a fusion protein consisting of Id and CTLA-4 (Id-CTLA4), mice immunized with the fusion protein induced high titers of Id-specific antibody and T-cell proliferative responses without adjuvants and were protected from lethal tumor challenge. The Id-CTLA4 fusion protein was so potent that even low doses (down to 0.1 microg) of the immunogen were able to elicit strong antibody responses. By using an Id-CTLA4 mutant protein, the ability to bind B7 molecules on APCs was shown to be required for the enhanced immunogenicity of Id-CTLA4. These findings demonstrate that fusing CTLA-4 to a potential tumor antigen represents an effective approach to prime antitumor immunities in vivo and may be applicable to the design of vaccines for a variety of other diseases. (Blood. 2000;96:3663-3670)

Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM.

Tumor therapy by the preferential activation of a prodrug at tumor cells targeted with an antibody-enzyme conjugate may allow improved treatment efficacy with reduced side effects. We examined antibody-mediated clearance of poly(ethylene glycol)-modified beta-glucuronidase (betaG-sPEG) as a method to reduce serum concentrations of enzyme and minimize systemic prodrug activation. Enzyme-linked immunosorbent assay and immunoblot analysis of two monoclonal antibodies generated by immunization of BALB/c mice with an antibody-betaG-sPEG conjugate showed that mAb 1E8 (IgG1) bound betaG and betaG-sPEG whereas mAb AGP3 (IgM) bound poly(ethylene glycol). Neither antibody affected the betaG activity. mAb 1E8 and AGP3 were modified with 36 and 208 galactose residues (1E8-36G and AGP3-208G) with retention of 72 and 48% antigen-binding activity, respectively, to target immune complexes to the asialoglycoprotein receptor on liver cells. mAb 1E8 and AGP3 cleared betaG-PEG from the circulation of mice as effectively as 1E8-36G and AGP3-208G, respectively. mAb AGP3, however, cleared betaG-sPEG more completely and rapidly than 1E8, reducing the serum concentration of betaG-sPEG by 38-fold in 8 h. AGP3 also reduced the concentration of an antibody-betaG-sPEG conjugate in blood by 280-fold in 2 h and 940-fold in 24 h. AGP3-mediated clearance did not produce obvious damage to liver, spleen, or kidney tissues. In addition, AGP3 clearance of betaG-sPEG before administration of BHAMG, a glucuronide prodrug of p-hydroxyaniline mustard, prevented toxicity associated with systemic activation of the prodrug based on mouse weight and blood cell numbers. AGP3 should be generally useful for accelerating the clearance of PEG-modified proteins as well as for improving the tumor/blood ratios of antibody-betaG-PEG conjugates for glucuronide prodrug therapy of cancer.

The management of patients with advanced motor neuron disease.

BACKGROUND: There is no specific treatment for motor neuron disease (MND) except hospice or palliative care to improve patients' quality of life and decrease complications. This topic is seldom discussed in Taiwan. METHODS: A retrospective study was conducted of patients with terminal MND who were treated and died at the Veterans General Hospital-Taipei from March 1986 through April 1996. Patients' characteristics, management, length of survival and cause of death were analyzed. RESULTS: Twenty-three patients (M/F, 17/6) were included. The median age of onset was 59 years (range, 24-69). The median interval from onset of symptoms to diagnosis was nine months (range, 2-36). Seventeen patients received mechanical ventilation for an average median of six months. Nineteen patients had dysphagia, 17 received long-term nasogastric tube feeding, one had gastrostomy and one was treated with cricopharyngeal myotomy. Pain over the neck, trunk or limbs was reported by 18 patients; none received narcotics. Only two patients received respiratory exercise training and two had a cervical collar for stabilization. Electronic communication aids were not available. The median survival from onset of symptoms was 36 months (range, 7-99). The causes of death included sepsis (n = 13), respiratory failure (n = 7), heart disease (n = 2) and MND-related cachexia (n = 1). Cardiopulmonary resuscitation was performed for 12 patients. CONCLUSIONS: In Taiwan, management of patients with advanced MND is mainly hospital-based and most of the effort is focused on life-sustaining. More attention needs to be paid to improvement of the quality of life and dignity of the patient.

Poly(ethylene glycol) modification of beta-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs.

Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of antibody-betaG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect betaG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified betaG was coupled via a thioether bond to mAb RH1, an IgG2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-betaG-3PEG), 5.2 (RH1-betaG-5PEG) or 9.8 (RH1-betaG-10PEG) PEG molecules per betaG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-betaG. In contrast to the rapid serum clearance of RH1-betaG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-betaG-3PEG and RH1-betaG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-betaG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-betaG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-betaG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective.

Effects of the callipyge phenotype on serum creatinine, total cholesterol, low-density lipoproteins, very-low-density lipoproteins, high-density lipoproteins, and triacylglycerol in growing lambs.

The goals of this study were to investigate the effects of the callipyge (CLPG) phenotype on serum creatinine and lipid profiles of growing lambs. Preliminary studies in our laboratories indicated that creatinine may have utility in distinguishing the CLPG phenotype and that expression of the CLPG gene altered concentrations of serum total cholesterol (TC). As a result, in this study, we examined the influence of the CLPG gene on concentrations of creatinine, TC, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), and triacylglycerol (TG) at varying stages of maturity in lambs. Ten homozygous (c/c) Polypay ewes were crossed with Dorset rams heterozygous for the CLPG gene (C/c). From this cross, 20 lambs (13 females and 7 males) were born, of which 11 were homozygotic (c/c) and 9 were heterozygotic (C/c; CLPG) based on muscle weights and longissimus dorsi (LD) area at slaughter. Blood samples were taken at monthly intervals and serum lipid constituents were assayed. At 1 mo of age, no differences (P > .05) in plasma lipids were detectable between phenotypes. However, at 2 mo age, CLPG lambs had higher (P < .01) concentration of TG, TC, HDL, and VLDL compared to homozygotic (c/c) lambs. Triglycerides and VLDL were elevated (P < .05) in CLPG lambs at 3 mo of age. By slaughter, no differences (P > .05) in serum lipid constituents were detectable between genotypes. Hence, the increase in serum TC is due to elevated levels of HDL and VLDL. These observations indicate that creatinine may be used to distinguish CLPG lambs and that the CLPG gene alters serum lipid profiles during the postnatal period.

[Ultrasonography of hips in neonates].

For evaluation of the development of the hip joints in young infants, between Jun., 1988 and Oct., 1991, 522 hips of the infants under 4 months of age were studied by ultrasonography based on the method well described by Graf. The results revealed only 48% of the hips of neonatal group was type I (stable type). But this percentage increased with age: 90% in the group under 2 months of age; 97% in the group under 4 months of age. Compared with the findings of other studies in Europe, the development and the stability of the hips of our neonatal group seemed poorer. But this condition improved much within 2 months after birth. So we concluded that, except the high risk group, the ultrasonographic screening approach should be performed after 2 months of age, but not in neonatal period under considering the cost-benefit.

Eradication of Helicobacter mustelae from the ferret stomach: an animal model of Helicobacter (Campylobacter) pylori chemotherapy.

Colonization of the ferret stomach by Helicobacter mustelae has been suggested as a possible animal model for Helicobacter pylori-associated gastroduodenal disease of humans. Our study was designed to determine whether antimicrobial chemotherapy could eradicate H. mustelae from ferrets. Triple antimicrobial therapy combining amoxicillin, metronidazole, and bismuth subsalicylate was successful in eradicating the organism from 5 of 7 (71%) adult ferrets. Despite apparent in vitro susceptibility, neither chloramphenicol monotherapy nor a polytherapeutic regimen combining tetracycline, metronidazole, and bismuth subsalicylate proved effective in the eradication of H. mustelae. Several strains isolated after unsuccessful polytherapy showed markedly increased resistance to metronidazole. These preliminary findings are similar to results of H. pylori treatment trials with humans and suggest that the ferret may be a useful model for evaluating and comparing potential antimicrobial modalities for the eradication of H. pylori.