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Pyroptosis is a recently identified form of programmed cell death (PCD) that exerts a vital influence on the antitumor immune response. GA, a xanthone structure isolated from gamboge resin, is a naturally occurring bioactive ingredient with several anticancer activities, such as activities that affect cell cycle arrest, apoptosis, and autophagy. Here, we found that GA decreased the viability of the CRC cell lines, HCT116 and CT26, in a dose- and time-dependent manner, and multiple pores and large bubbles in the membranes, which are morphological characteristics of pyroptosis, were observed by light microscopy and transmission electron microscopy (TEM). Furthermore, the cleavage of gasdermin E (GSDME) was observed after exposure to GA, along with concomitant activation of caspase-3. Additionally, GSDME-dependent pyroptosis triggered by GA could be attenuated by siRNA-mediated knockdown of GSDME and treatment with the caspase-3 inhibitor. Moreover, we found that GA induced pyroptosis and significantly inhibited tumor growth in CT26 tumor-bearing mice. Strikingly, significantly increased proportions of CD3+ T cells, cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) were observed in the tumor microenvironment in the GA-treated groups. Moreover, significantly increased proportions of CTLs and effector memory T cells (TEM) (CD8+ CD44+ CD62L−) were also detected in the spleens of the GA-treated groups, suggesting that the pyroptosis-induced immune response generated a robust memory response that mediated protective immunity. In this study, we revealed a previously unrecognized mechanism by which GA induces GSDME-dependent pyroptosis and enhances the anticancer immune response. Based on this mechanism, GA is a promising antitumor drug for CRC treatment that induces caspase-3-GSDME-dependent pyroptosis. This study provides novel insight into cancer chemoimmunotherapy.

Background: Immune checkpoint blockade (ICB) is considered as a promising approach for cancer treatment. However, the potency of ICB therapy in yolk sac tumors (YSTs) has not been confirmed, and the comprehensive analysis of tumor immune microenvironment and the expression of PD-1/PD-L1 and CTLA4 were also not thoroughly evaluated.Methods: Immunohistochemistry was performed in formalin-fixed, paraffin-embedded tumor specimens from 23 YSTs patients to detect the density and distribution of tumor-infiltrating T cells, tertiary lymphoid structures (TLSs), as well as the expression of PD-1/PD-L1 and CTLA4. Results: Overall, more than half (61%) of all patients exhibited an immune-desert phenotype based on CD3+ T cells. PD-1 expression was identified in five tumor samples (21.7%), and PD-L1 expression exhibited a different positive rate in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) (39.1% and 17.4%). Noteworthily, the rate of positive CTLA4 expression in both TCs and TILs was markedly higher (69.6% and 56.5%) than those of PD-1 and PD-L1 expression. Furthermore, TLSs were observed in 21.74% of all tissues, and samples with TLSs exhibited significantly higher densities of TILs and higher expression of immune checkpoint molecules, particularly PD-1/PD-L1. In addition, tumors located in testes also exhibited a higher density of TILs and higher expression of immune checkpoint molecules.Conclusion: Although a high frequency of CTLA4 expression was found, PD-1/PD-L1 expression, the immune-inflamed phenotype, and TLSs were low frequency in YSTs, suggesting that only a few YSTs patients may be benefit form ICB therapy. Remarkably, patients with tumors located in testes are more likely to benefit from ICB therapy.

TMTP1 is a polypeptide independently screened in our laboratory, which can target tumors in situ and metastases. In previous work, we have successfully developed a near-infrared (NIR) probe TMTP1-PEG4-ICG for tumor imaging. However, the limited ability to target tumor micrometastases hinders its further clinical application. Multimerization of peptides has been extensively demonstrated as an effective strategy to increase receptor binding affinity due to “multivalent effect” or “apparent cooperative affinity”. In this study, a novel TMTP1 homodimer-directed NIR probe (TMTP1-PEG4)2-ICG was successfully constructed and synthesized. The cyclic TMTP1 peptides were bridged by two PEG4 linkers and then labeled with ICG-NHS for tumor imaging and photothermal therapy. In vivo biodistribution were assessed in normal BALB/c mice, and tumor targeting abilities of (TMTP1-PEG4)2-ICG and its monomer were evaluated and compared in 4T1-bearing subcutaneous tumor and lymph node metastasis model mice. Biodistribution analysis in vivo revealed that (TMTP1-PEG4)2-ICG was cleared mainly in both liver and kidney dependent way. Comparing with free ICG dye or TMTP1-PEG4-ICG probe, this improved (TMTP1-PEG4)2-ICG dimer showed more sensitive tumor imaging and could clearly identify tumors at a minimum volume of 10 mm3. Additionally, when compared to its monomer, lymph node (LN) metastases could also be apparently visualized and easily distinguished from normal LN by the novel dimer at 24 h post-injection. The blocking study revealed that the tumor accumulation of this probe was specifically medicated by receptor-ligand interaction. Furthermore, with the increase in stability and tumor targeting ability of ICG in vivo, the probe could also be an attractive photothermal agent to significantly inhibit tumor growth under 808 nm NIR laser irradiation. In conclusion, our work revealed that the novel (TMTP1-PEG4)2-ICG dimer could be a promising theranostic agent for sensitive tumor imaging and imaging-guided photothermal therapy, indicating its broad prospects for further clinical transformation.

Guiying Jiang,1,* Xueqian Wang,1,* Ying Zhou,1 Chenming Zou,2,3 Ling Wang,1 Wei Wang,1 Danya Zhang,1 Hanjie Xu,1 Jie Li,1 Fei Li,1 Danfeng Luo,1 Xiangyi Ma,1 Ding Ma,1 Songwei Tan,2 Rui Wei,1 Ling Xi1 1Department of Obstetrics & Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei Province, People’s Republic of China; 2Tongji School of Pharmacy, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei Province, People’s Republic of China; 3School of Pharmacy, Shanghai Jiao Tong University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ling Xi; Rui WeiDepartment of Obstetrics & Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei, People’s Republic of ChinaTel/Fax +86 27 83662688Email lxi@tjh.tjmu.edu.cn; weirui2018@tjh.tjmu.edu.cnBackground and Purpose: Cisplatin–paclitaxel (TP) combination chemotherapy as the first-line therapy for numerous cancers is hindered by its inadequate accumulation in tumors and severe side effects resulting from non-specific distribution. The aim of this study is to explore whether TMTP1-modified, cisplatin and paclitaxel prodrugs co-loaded nanodrug could improve cervical cancer chemotherapy and relieve its side effects through active and passive tumor targeting accumulation and controlled drug release.Methods: TDNP, with capacities of active targeting for tumors and controlled drug release, was prepared to co-deliver cisplatin and paclitaxel prodrugs. The characteristics were investigated, including the diameter, surface zeta potential, stability and tumor microenvironment (TME) dependent drug release profiles. Cellular uptake, cytotoxicity, drug accumulation in tumors, antitumor effects and safety analysis were evaluated in vitro and in vivo.Results: The oxidized cisplatin and the paclitaxel linked to the polymer achieved a high loading effciency of over 80% and TME-dependent sustained drug release. Moreover, TMTP1 modification enhanced cellular uptake of TDNP and further improved the cytotoxicity of TDNP in vitro. In vivo, TDNP showed an extended blood circulation and increased accumulation in SiHa xenograft models with the aid of TMTP1. More importantly, TDNP controlled tumor growth without life-threatening side effects.Conclusion: Our study provided a novel TP co-delivery platform for targeted chemotherapy of cervical cancer, which was promising to improve the therapeutic effcacy of TP and may also have application in other tumors.Keywords: TME-responsive, targeted co-delivery, combined chemotherapy, cervical cancer

OBJECTIVE To examine physician and patient adherence to prevention guidelines in non-insulin-dependent diabetes mellitus (NIDDM) with attention to possible differences in adherence by patient race or ethnicity. RESEARCH DESIGN AND METHODS We performed a cross-sectional chart review study of prevention practices, complications, and risk factors in 378 NIDDM patients (at least 5 years known duration) who were Permanente Medical Care Program, Oakland, CA. There were 232 blacks, 81 whites, 29 Hispanics, and 36 members of other races/ethnicities. RESULTS Age- and duration-adjusted prevalence of 14 complications was low and, with the exception of diabetic retinopathy, did not differ significantly by race. An eight-point prevention score that reflected recommended use of glucose, HbA1c, high-density lipoprotein/total cholesterol, blood pressure and proteinuria testing, ophthalmology a and foot examinations, and diabetes education during a 2-year period did not differ by race/ethnicity (P = 0.36). The mean score (4.9 ± 1.6) indicated that on average, five of eight guidelines were performed appropriately. Compliance was poorest for annual proteinuria checks (32.2%). No differences were noted by race/ethnicity in referrals to ophthalmology, weight reduction, or smoking cessation or in missed appointments or failure to keep referral visits. CONCLUSIONS The absence of racial differences in either prevention practices or complication rates in this insured prepaid setting suggests that improved access to preventive services may be effective in reducing such differences nationally. However, adherence to several guidelines was ≤ 50%, indicating that physicians are not sufficiently convinced of the necessity for these prevention measures.

OBJECTIVE To determine the predictors of lower extremity amputation (LEA) in a cohort of persons with diabetes (primarily non-insulin-dependent). RESEARCH DESIGN AND METHODS We conducted a nested case-control study among 10,068 patients from a large health maintenance organization who reported a diagnosis of diabetes at a multiphasic health checkup (MHC) (baseline) between 1964 and 1984. Average length of follow-up after baseline was 13.2 years. Case patients were 150 cohort members with a first, nontraumatic LEA after baseline. Control subjects were 278 cohort members who did not experience an LEA during follow-up, matched to patients on age, sex, and year of baseline MHC. The presence of diabetes at baseline was verified by chart review for both patients and control subjects. Cardiovascular disease risk factors were obtained at the baseline MHC; glucose control, other diabetes-related variables, preventive services, and other complications were obtained from chart review. RESULTS Level of glucose control (P < 0.0001), duration of diabetes (P = 0.04), and baseline systolic blood pressure (P = 0.004) were independent predictors of amputation, as were microvascular complications (retinopathy, neuropathy, and nephropathy). History of stroke, but not myocardial infarction, was also independently predictive; type of diabetes, cigarette smoking, and total cholesterol level were not. Being African-American was unrelated to amputation risk in univariate or multivariate analyses in this insured population. CONCLUSIONS LEA shares a risk factor profile with other microvascular complication of diabetes. Thus, control of blood glucose and blood pressure should reduce risk for amputation. African-Americans do not appear to be at increased risk for diabetes-related amputation when access to medical care is comparable.

BACKGROUND Low-density lipoprotein (LDL) subclass phenotype B, characterized by predominance of small, dense LDL particles, is associated with elevated plasma triglycerides and apolipoprotein B and with lower high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I. Because these abnormalities resemble the dyslipidemia of insulin resistance, we examined associations of LDL subclass phenotype with plasma insulin levels and with other aspects of the insulin resistance syndrome. METHODS AND RESULTS LDL subclass phenotypes were determined by gradient gel electrophoresis in 682 female twins aged 30 to 91 years who participated in the second examination of the Kaiser Permanente Women Twins Study. Prevalence of phenotype B and the intermediate phenotype (I) increased strongly with age, obesity, and non-insulin-dependent diabetes. In multivariate analysis of nondiabetic women, phenotype B or I was independently associated with each aspect of the insulin resistance syndrome, including higher plasma triglycerides, waist-hip ratio, fasting and postload insulin levels, and systolic blood pressure and lower HDL cholesterol levels after adjustment for age and body mass index. The prevalence of phenotype B or I rose progressively from 5.6% in women with no manifestations of the insulin resistance syndrome to 100% in women with four syndrome components. In 25 nondiabetic, monozygotic twin pairs discordant for subclass phenotype, the twins with phenotype B (or I) had significantly higher levels of body mass index, waist-hip ratio, and systolic blood pressure than their twins with phenotype A. Thus, nongenetic variation in these risk factors is important in explaining their associations with LDL subclass phenotype. CONCLUSIONS Small, dense LDL is an integral feature of the insulin resistance syndrome. Nongenetic (ie, behavioral or environmental) factors are important for the expression of the phenotype and for its association with other heart disease risk factors.

Multiple factors may determine insulin resistance and the insulin resistance syndrome. The contributions of genes and environment to the distribution of fasting insulin levels and to the associations of fasting insulin with elements of the syndrome were evaluated in the second examination of the Kaiser Permanente Women Twins Study (Oakland, California, 1989-1990). Subjects included 556 white women (165 monozygous twin pairs, 113 dizygous pairs; 455 women with normal glucose tolerance, 75 with impaired glucose tolerance, and 26 with non-insulin-dependent diabetes by World Health Organization criteria). The intraclass correlation coefficients for log fasting insulin for monozygous and dizygous twin pairs were 0.64 and 0.40, respectively. After adjustment for age, behavioral factors, and body mass index, the estimated classic heritability was 0.53 (p = 0.003). Commingling analysis of fasting insulin indicated the presence of four distributions (p < 0.001), consistent with at least one, and perhaps two, genes influencing this trait. In an unmatched multiple regression model among women from monozygous twin pairs only, log fasting insulin was independently associated with body mass index (p < 0.0001), waist/hip ratio (p = 0.02), and glucose intolerance (p = 0.04), but not with triglycerides, high density lipoprotein cholesterol, or hypertension. After removal of genetic influences by analysis of monozygous intrapair differences, only body mass index (p < 0.0001) remained independently related to fasting insulin. The authors conclude that, in addition to significant genetic influences on fasting insulin, environmental or behavioral factors (particularly nongenetic variation in obesity) are important determinants of fasting insulin and the insulin resistance syndrome.

Background. Genetic factors are firmly established as determinants of plasma lipoprotein(a) [Lp(a)] concentration. This study focused on behavioral or environmental factors that might also explain some of the variation in levels of this cardiovascular disease risk factor. Methods. The study considers the 644 women twins (597 whites, 47 blacks; ages 30-91 years) who participated in the second examination of the Kaiser Permanente Women Twins Study. Cross-sectional associations of behaviors and environmental factors with Lp(a) concentration were studied before and after removing genetic influences on Lp(a) levels. Results. Lp(a) levels were substantially higher among blacks than whites (P < 0.0001). The distribution of apo(a) size phenotypes also differed between blacks and whites, but this variation did not explain the difference in Lp(a) levels. A positive association of Lp(a) concentration with age was noted among blacks (P = 0.06) but not among whites (P = 0.86). No evidence was found for associations of Lp(a) with menopausal status, cigarette smoking, alcohol consumption, total or heavy recreational physical activity, 11-year weight gain, use of several antihypertensive medications, or diabetes status in either race. Among postmenopausal women, however, estrogen replacement therapy was associated with lower Lp(a) levels among whites (7.9 vs 9.9 mg/dl, P = 0.05). Removing genetic variation in Lp(a) concentration by matching 171 monozygotic (MZ) twins to their genetically identical co-twins did not alter these findings. Conclusion. The plasma concentration of Lp(a), unlike other lipoprotein risk factors for heart disease, has few behavioral or environmental correlates, at least among white women. Neither behavioral or environmental factors nor variation in the apo(a) size phenotype appeared to explain the higher mean Lp(a) levels among black compared with white women; further study seems warranted in larger samples of black women.

We evaluated the long-term gynecologic risks of postmenopausal estrogen therapy in conjunction with cyclic, monthly progestin (progestin-estrogen replacement therapy, or PERT). Our medical record review showed that incidence of abnormal vaginal bleeding necessitating gynecologic procedures for evaluation was significantly higher (RR, 3.1; 95% CI, 2.1-4.5), as was the rate of endometrial biopsy (RR, 3.4; 95% CI, 2.3-5.1), among women receiving PERT than among women not receiving hormone therapy. We also identified a non-significant trend toward a higher rate of dilation and curettage (RR, 1.5; CI, 0.7-3.3) among women receiving PERT. However, rates of endometrial hyperplasia and hysterectomy were similarly low in both groups. PERT apparently protects women against these serious gynecologic consequences previously seen in women taking unopposed estrogen.