[Research progress on the cyclic GMP-AMP synthase stimulator of interferon genes pathway in respiratory diseases].

Cyclic GMP-AMP synthase (cGAS) is a DNA receptor that produces the second messenger cyclic GMP-AMP (cGAMP). cGAMP activates stimulator of interferon genes (STING), which initiates a signaling cascade leading to immune and inflammatory responses. This intricate molecular pathway plays a pivotal role in the pathogenesis and progression of diverse respiratory ailments, including respiratory infection, lung cancer, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, and acute lung injury. Consequently, the cGAS-STING signaling pathway has emerged as a promising novel therapeutic target, opening up new avenues for the diagnosis and treatment of respiratory disorders. This review focuses on recent advances in our understanding of the cGAS-STING signaling pathway and its intricate involvement in respiratory system diseases.

[Epidemiological investigation on a visceral leishmaniasis case in Zhengzhou City of Henan Province].

OBJECTIVE: To perform an epidemiological investigation on a case with visceral leishmaniasis in Zhengzhou City, Henan Province, and to identify the source of infection, so as to illustrate the transmission chain and assess the risk of local leishmaniasis transmission. METHODS: The medical data were collected from a case with visceral leishmaniasis in Zhengzhou City, and the patient's bone marrow smears were detected by microscopy. Serum anti-Leishmania antibody test and PCR assay were performed among high-risk residents and all dogs in the village where the patient lived. Sandflies were captured using light traps and artificial traps, and the captured female Phlebotomus chinensis was subjected to PCR assay. The internal transcribed spacer 1 (ITS1) gene was amplified with a nested PCR assay using the genomic DNA extracted from visceral leishmaniasis patients, positive dogs and sandflies, and the sequences were aligned with those download from NCBI. In addition, a phylogenetic tree was created based on the ITS1 gene. RESULTS: The visceral leishmaniasis patient had recurrent irregular fever, reduced complete blood counts, low hemoglobin, and a large number of Leishmania amastigotes in bone marrow smears, and was therefore diagnosed as visceral leishmaniasis. Both rk39 rapid diagnostic test and PCR assay tested negative among 324 residents living neighboring the patient's residence, while 21.39% (43/201) dogs were positive for rk39 rapid diagnostic test and 13.93% (28/201) positive for PCR assay. There were 17 female Ph. chinensis tested positive for Leishmania (0.82%) by PCR assay, and the ITS gene sequences from visceral leishmaniasis patients, positive dogs and sandflies shared a 100% homology with L. infantum. The Leishmania species was therefore characterized as L. infantum. CONCLUSIONS: L. infantum infection occurs in visceral leishmaniasis patients, dogs and sandflies in Zhengzhou City, indicating a complete transmission chain and a high transmission risk of visceral leishmaniasis by L. infantum. Intensified control measures are required to prevent local transmission of leishmaniasis in Zhengzhou City.

[Association between puberty with thyroid morphology and function in women].

Objective: To explore the effects of different puberty development stages on thyroid morphology and function in women. Methods: From October to November 2017, a multi-stage cluster sampling method was used to select one junior high school in Minhang district of Shanghai, Haimen city of Jiangsu province, Yuhuan city of Zhejiang province and Deqing county of Zhejiang province respectively. A total of 491 girls in the first grade in 4 schools were included in the study. The subjects were examined with thyroid B-ultrasound and physical examination, and their morning random urine samples and fasting blood samples were collected to detect urinary iodine and thyroid function indexes. Puberty Development Self-rating Scale (PDS) was used to evaluate the stages of puberty; multiple linear regression models and logistic regression models were used to investigate the effects of different puberty stages on thyroid morphology and function. Results: There were differences in thyroid status among women at different stages of puberty. Thyroid volume, the rate of nodules and the level of FT4 were lowest in prepubertal period, followed by pubertal period and postpubertal period (P<0.05). TT3 and FT3 levels were highest in prepubertal period, followed by pubertal period and postpubertal period (P<0.001), and there was an opposite trend on the abnormal rate. TSH and TT4 levels were not affected by the stage of puberty (P>0.05). Multiple linear regression analysis and multivariate logistic regression analysis showed consistent results. There was a negative correlation between puberty development and TT3 and FT3 levels. For each 1 point increase in PDS, TT3 and FT3 levels decreased by 0.067 nmol/L and 0.170 nmol/L in Model 1, respectively, and decreased by 0.065 nmol/L and 0.162 nmol/L in Model 2, respectively. Compared with the prepubertal period, the TT3 and FT3 levels were lower in postpubertal period (Model 1: OR=0.337, 95%CI: 0.173-0.658; OR=0.283, 95%CI: 0.144-0.557; Model 2: OR=0.306, 95%CI: 0.155-0.605; OR=0.263, 95%CI: 0.132-0.524). Conclusions: The process of puberty is related to the thyroid status in women. The better matured during the puberty, the larger volume the thyroid was, more likely the thyroid nodules appeared, and the levels of TT3 and FT3 was much lower.

[A 22-year-follow-up cohort study on primary liver cancer in Haimen city of Jiangsu province].

Objective: A prospective cohort study was carried out to assess the mortality and potential risk factors for primary liver cancer (PLC) in Haimen city of Jiangsu province. Methods: The cohort involved 89 789 adult residents aged 25-69 years. Upon the entry of this project, each subject was asked to complete a questionnaire and to provide a blood sample of 10 ml. Surface antigen of hepatitis B virus (HBsAg) was tested by radioimmunoassay. All the subjects were followed-up every year for vital statistics and death certificate information until 2014. Cox proportional hazard model was used to estimate the hazard ratio (HR) and 95% confidence interval (95%CI) for PLC mortality associated with HBsAg status and other risk factors. Results: During the 1 299 611 person-years of follow-up, a total of 2 583 PLC cases were identified, including 2 149 men and 434 women. Mortality of the PLC for men and women were 247.80/100 000 person-years and 100.38/100 000 person-years, respectively. Among those who died of PLC, 73.87% had been tested HBsAg positive. HBV infection seemed the predominant risk factor for PLC and the HRs were 15.97 for men (95%CI: 14.29-17.85) and 21.63 for women (95%CI: 16.16-28.96) respectively. Ageing, cigarette smoking, previous history of hepatitis, and family history of HCC were factors associated with the increased risk for PLC. Conclusion: People living in Haimen city had a high risk on PLC. HBV infection appeared the most important risk factor for HCC mortality in this area.

[Prevalence of loss of activities of daily living and influencing factors in elderly population in China].

Objective: To investigate the prevalence of the loss of basic activities of daily living (ADL) and instrumental ADL (IADL) influencing factors in the elderly population in China and provide evidence for the effective keeping and improvement of the elderly daily living. Methods: The information about demographic characteristics and activities of daily living of elderly Chinese aged ≥60 years were collected by using the data of 2013 China Health and Retirement Longitudinal Study. The elderly's ADL of taking bath, eating, getting in and out of bed, dressing, toilet use, and defecating and the IADL of doing housework, cooking, making phone call, taking medicine, shopping and money managing were evaluated. The differences in loss of ADL among different populations were compared by χ2 test and logistic regression analysis was conducted to identify influencing factors for the loss of ADL and IADL. Results: The overall ADL loss rate was 23.8% and the overall IADL loss rate was 35.4% in elderly Chinese. The proportion of having trouble in toilet use was highest among all ADL items, followed by bathing and getting in and out of bed. The proportion of having trouble in making phone call was highest among all IADL items, followed by doing housework and money managing. Female, older age, low educational level, living in central and western China, chronic diseases and disability were factors associated with ADL loss and IADL loss; the divorced or separated, widowed were more likely to have ADL loss; people living in rural area were more likely to have IADL loss. Conclusion: It is necessary to take comprehensive measures to delay and reduce the ADL and IADL loss in elderly Chinese and provide timely and appropriate health care for the elderly with ADL or IADL loss.

Coexistent gestational choriocarcinoma and mixed adenocarcinoma of the uterus.

OBJECTIVE: To report the first case of a uterine gestational choriocarcinoma coexisting with an endometrial carcinoma (EC) and to discuss its possible pathogenesis. MATERIALS AND METHODS: All tissues were examined histologically and monoclonal antibodies were used to evaluate the expression of HCG, HPL, P53, PTEN, and ER. Genotyping was performed on DNA extracted from the freshly dissected choriocarcinoma and the paraffin-embedded endometrial carcinoma along with parental blood DNA using multiplex STR-PCR at 16 loci. RESULTS: Histology identified two distinct tumors: a uterine tumor containing cytotrophoblastic and syncytiotrophoblastic cells and a second distinct neoplasm composed of adenocarcinoma resembling endometrioid and mucinous adenocarcinoma. Genotyping of the choriocarcinoma revealed alleles from both the patient and her husband and was classified as biparental in origin. The endometrial adenocarcinoma contained only maternal alleles and was thus classified as maternal in origin. CONCLUSIONS: This is the first description of the simultaneous diagnosis of a uterine gestational choriocarcinoma and an EC within the same patient. DNA genotyping and immunohistochemistry are valuable tools in distinguishing the different origins of coexisting tumors.

Discovery of altered protein profiles in epithelial ovarian carcinogenesis by SELDI mass spectrometry.

OBJECTIVE: Identification of proteomic alterations in epithelial ovarian tumorigenesis may facilitate the understanding of progression of this disease. METHODS: Specific protein peak patterns were identified in 20 microdissected epithelial ovarian tumors (13 epithelial ovarian cancers (EOCs) and 7 low malignant potential (LMP) tumors), as well as in the matched normal cells. Protein profiles were generated by surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) from all the different types of cells. RESULTS: Among seven protein peaks from EOC cells, six were significantly increased while one was decreased compared with normal cells, and three peaks from LMP cells were markedly increased while one was decreased compared with normal cells. CONCLUSIONS: The combination of SELDI and laser capture microdissection (LCM) is effective in finding the key molecules in ovarian tumorigenesis. Further identification of these protein peaks is important and these malignant protein signatures lend themselves to identification of populations at high-risk for EOC and for monitoring response to EOC chemopreventive agents.

Trace element removal from coal ash leachate by a 10-year-old constructed wetland.

This study investigated the ability of a 10-yr-old constructed wetland to treat metal-contaminated leachate emanating from a coal ash pile at the Widows Creek electric utility, Alabama (USA). The two vegetated cells, which were dominated by cattail (Typha latifolia L.) and soft rush (Juncus effusus L.), were very effective at removing Fe and Cd from the wastewater, but less efficient for Zn, S, B, and Mn. The concentrations were decreased by up to 99% for Fe, 91% for Cd, 63% for Zn, 61% for S, 58% for Mn, and 50% for B. Higher pH levels (>6) in standing water substantially improved the removing efficiency of the wetland for Mn only. The belowground tissues of both cattail and soft rush had high concentrations of all elements; only for Mn, however, did the concentration in the shoots exceed those in the belowground tissues. The concentrations of trace elements in fallen litter were higher than in the living shoots, but lower than in the belowground tissues. The trace element accumulation in the plants accounted for less than 2.5% of the annual loading of each trace element into the wetland. The sediments were the primary sinks for the elements removed from the wastewater. Except for Mn, the concentrations of trace elements in the upper layer (0-5 cm) of the sediment profile tended to be higher than the lower layers (5-10 and 10-15 cm). We conclude that constructed wetlands are still able to efficiently remove metals in the long term (i.e.,>10 yr after construction).

Removal and distribution of iron, manganese, cobalt, and nickel within a Pennsylvania constructed wetland treating coal combustion by-product leachate.

A flow-through wetland treatment system was constructed to treat coal combustion by-product leachate from an electrical power station at Springdale, Pennsylvania. In a nine-compartment treatment system, four cattail (Typha latifolia L.) wetland cells (designated Cells 1 through 4) successfully removed iron (Fe) and manganese (Mn) from the inlet water; Fe and Mn concentrations were decreased by an average of 91% in the first year (May 1996-May 1997), and by 94 and 98% in the second year (July 1997-June 1998), respectively. Cobalt (Co) and nickel (Ni) were decreased by an average of 39 and 47% in the first year, and 98 and 63% in the second year, respectively. Most of the metal removed by the wetland cells was accumulated in sediments, which constituted the largest sink. Except for Fe, metal concentrations in the sediments tended to be greater in the top 5 cm of sediment than in the 5- to 10- or 10- to 15-cm layers, and in Cell 1 than in Cells 2, 3, and 4. Plants constituted a much smaller sink for metals; only 0.91, 4.18, 0.19, and 0.38% of the Fe, Mn, Co, and Ni were accumulated annually in the aboveground tissues of cattail, respectively. A greater proportion of each metal (except Mn) was accumulated in cattail fallen litter and submerged Chara (a macroalga) tissues, that is, 2.81, 2.75, and 1.05% for Fe, Co, and Ni, respectively. Considerably higher concentrations of metals were associated with cattail roots than shoots, although Mn was a notable exception.

Targeting of anti-tumor responses with bispecific antibodies.

T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such "targeted cytolysis" requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials.

Targeted cytokine production.

It has been well established that bispecific antibodies containing anti-T-cell receptor MAbs crosslinked to anti-tumor MAbs induce T cells to lyse tumor cells, as measured in a 51Cr-release assay. Such lysis requires direct attachment between target and cytotoxic cells and most probably involves the exocytosis of cytolytic substances into the cell:cell interface. In addition, targeted T cells mediate a second activity, the secretion into the medium of factors that can block the growth of bound tumor cells and unbound bystander cells. In order to test how targeted effector cells mediate anti-tumor effects in vivo, we are currently developing a totally syngeneic murine system in which murine T cells are targeted against mouse mammary tumors. The system allows us to treat both primary tumors and tumor transplants, using a mammary-tumor-virus antigen as the entity that is specifically recognized on the tumor cells.

An ERP-based, control-question lie detector analog: algorithms for discriminating effects within individuals' average waveforms.

Two experimental, P3-based analog control question tests were run. In both, guilty subjects were presented with a set of seven phrases describing antisocial acts of which they were innocent, plus one phrase describing a guilty act (the analog relevant question), and one act to which a "yes" response (yes-target stimulus) was required to assure attention. Innocent subjects (run only in Experiment 1) saw all innocent acts plus the yes-target act. Thus nine acts were seen by guilty and innocent subjects. In both experiments, all subjects had to selectively review their guilty acts privately. Also in both experiments, all subjects were especially questioned about four acts of which guilty subjects were known to be innocent of all but one, and of which innocent subjects were known to be innocent of all. (These falsely accused acts were regarded as control question analogs.) In Experiment 1, the private review and rehearsal took place on the same day as the main test. In Experiment 2, one subgroup (delay-only) of guilty subjects was run as in Experiment 1, except that the private review-rehearsal was separated from the main run by 7-14 days. Another subgroup (delay-rehearsal) of guilty subjects was run just as was the subgroup delay-only, except that the delay-rehearsal subgroup additionally received a non-selective additional interrogation/rehearsal on the delayed main run day. Parietally maximal P3 responses were obtained to yes-target items in all groups. In Experiment 1, only in the guilty group was the relevant-minus-control P3 amplitude difference significant. In Experiment 2, the difference was significant only in the delay-rehearsal subgroup. A four-step algorithm (involving relevant-control amplitude differences and relevant target vs. control-target cross-correlations) was used to assess effects within individuals. In Experiment 1, 12 of 13 guilty subjects and 13 of 15 innocent subjects were correctly diagnosed. In Experiment 2, 3 of 8 delay-only subjects and 7 of 8 delay-rehearsal subjects were correctly diagnosed. In Experiment 2, the relevant-minus-control group P3 amplitude difference was significant in the delay-rehearsal but not in the delay-only subgroup. The results suggest that temporally proximal, non-selective rehearsal procedures are sufficient to activate personal knowledge of a salient (oddball), P3-generating stimulus phrase, and that even selective rehearsal of guilty acts is not sufficient without temporal proximity.

Human peripheral blood lymphocytes targeted with bispecific antibodies release cytokines that are essential for inhibiting tumor growth.

We have compared the mechanisms by which human PBL targeted with bispecific antibodies either lyse tumor cells or block their growth in culture or in mice. We found that resting PBL were unable to mediate lysis, but were able to block tumor growth. Moreover, targeted PBL were unable to lyse bystander cells, whereas targeted PBL did block the growth of bystander tumor cells in culture and in nude mice. Supernatants from cultures of targeted PBL, or from PBL grown on anti-CD3-coated flasks, blocked the growth of tumor cells in the absence of added effector cells, and antibodies against TNF-alpha and IFN-gamma reversed the inhibition of tumor growth, but had no effect upon cytolysis mediated by targeted by PBL. Our results show that targeted human PBL mediate two different antitumor activities: lysis, which occurs rapidly and requires the direct attachment of the target cell to the cytotoxic cell, and tumor growth inhibition, which is mediated by cytokines released into the medium as a result of receptor cross-linking. The inhibition of bystander tumor growth in mice by targeted PBL suggests that factor release is sufficient to block tumor growth in vivo. Targeted factor release therefore provides a mechanism by which targeted PBL could block the growth of tumor cells in vivo that were not bound by the effector cells, but which were located in the vicinity of tumor cells that were bound.

Studies on the induction of tolerance to alloantigens. III. Induction of antibodies directed against alloantigen-specific delayed-type hypersensitivity T cells by a single injection of allogeneic lymphocytes via portal venous route.

BALB/c mice were inoculated with normal C3H/He spleen cells via the portal venous (p.v.) route. Intravenous injection of serum from these BALB/c mice into naive syngeneic mice resulted in almost complete abrogation of their ability to generate anti-C3H/He delayed-type hypersensitivity (DTH) responses as induced by s.c. immunization with C3H/He cells. Since a portion of the same serum did not inhibit the development of anti-C57BL/6 DTH responses, the suppressive effect of the transferred serum was alloantigen-specific. Such serum factor(s) was produced in normal but not in nude mice and the suppressive activity was transferred in H-2- or immunoglobulin allotype-incompatible combinations. Immunochemical analyses of this serum suppressive factor have revealed that its m.w. was approximately 150,000, corresponding to the size of immunoglobulin (Ig)G, and that the activity was trapped by protein A or by an anti-immunoglobulin column. Although the absorption of the serum from anti-C3H/He-tolerant BALB/c mice with C3H/He target spleen cells did not abrogate the suppressive activity, the additional absorption with spleen cells from anti-C3H/He hyperimmune BALB/c mice almost completely eliminated the suppressive potential. Moreover, pretreatment of BALB/c anti-C3H/He DTH effector spleen cells with the above serum from tolerant mice induced the inhibition of anti-C3H/He DTH responses. Taken together, these results indicate that a single injection of allogeneic cells via the p.v. route results in the production of antibody capable of inhibiting the capacity of DTH effector cells specific for alloantigens used for the p.v. presensitization.

Targeting of cytotoxic cells against tumors with heterocrosslinked, bispecific antibodies.

Cytotoxic cells express specific receptors on their surfaces by which they distinguish altered or foreign cells from normal autologous cells. Recently, a method has been developed by which the natural recognition system of cytotoxic cells can be artificially manipulated, giving rise to cytotoxic cells of any desired specificity, including specificity against tumor and virally infected cells. The method for retargeting cytotoxic cells employs heterocrosslinked antibodies, in which one antibody is directed against the cytotoxic cell receptor (CCR) involved in lysis, while the second antibody is directed against a target cell structure, for example a tumor or viral antigen. By linking the CCR directly to the target cell, the heterocrosslinked antibodies promote the formation of effector: target conjugates and signal the cytotoxic cell to deliver a lethal hit. T cells can be targeted by heteroconjugates containing antibodies against components of the T cell receptor complex, e.g., Ti or CD3, while several types of antibody-dependent cellular cytotoxicity (ADCC) effector cells, including K/NK cells, macrophages, and neutrophils, are targeted using heteroconjugates containing antibodies against Fc gamma receptors. In peripheral blood from normal donors at least six types of targetable activities have been identified in vitro. In Winn type tumor neutralization assays in nude mice, targeted T and K cells can prevent the establishment of subcutaneous tumor at low effector: tumor ratios. Moreover, targeted human peripheral blood T cells cause the eradication of established intraperitoneal human ovarian carcinoma in nude mouse models. Targeted cytotoxic cells therefore hold great promise as a novel form of cancer immunotherapy in humans.

Tolerance induction of alloreactivity by portal venous inoculation with allogeneic cells followed by the injection of cyclophosphamide. II. Cellular and molecular mechanisms underlying tolerance.

BALB/c mice receiving allogeneic C3H/He or C57BL/6 spleen cells via portal venous (p.v.) route or a single administration of cyclophosphamide (Cy) were capable of rejecting the respective allogeneic C3H/He- or C57BL/6-derived tumor cells. In contrast, the combined treatment of p.v. inoculation with allogeneic lymphocytes and Cy administration abrogated the capability of rejecting allogeneic tumor cells. Such abrogation of alloreactivity was alloantigen-specific and associated with the suppression of potentials to generate delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses to alloantigens. This was further substantiated by the inhibition of molecular mechanisms underlying anti-allo-DTH and -CTL responses. Thus, the above combined treatment led to the decreased production of lymphokines such as macrophage-activating factor (MAF) and interleukin 2 (IL2) following the stimulation with the relevant alloantigens. These results demonstrate that p.v. inoculation of allogeneic cells followed by a single administration of Cy results in the effective elimination of alloreactivity as verified by the suppression of cellular and molecular mechanisms of alloreactive responses.

Abrogation of the capacity of delayed-type hypersensitivity responses to alloantigens by intravenous injection of neuraminidase-treated allogeneic cells.

BALB/c or C3H/He mice were inoculated i.v. with allogeneic spleen cells untreated or treated with neuraminidase. Appreciable or potent anti-allo-delayed-type hypersensitivity (DTH) responses were observed when mice were inoculated i.v. with untreated allogeneic cells or inoculated i.v. with those cells followed by s.c. immunization with untreated allogeneic cells. In contrast, i.v. inoculation of neuraminidase-treated allogeneic cells (presensitization) not only failed to induce any significant anti-allo-DTH responses but also abolished the capability of the animals to develop DTH responses after s.c. immunization, indicating the tolerance induction. This tolerance was alloantigen-specific, and rapidly inducible and long lasting. The induction of suppressor cell activity was demonstrated in tolerant mice. However, this activity was associated only with the tolerant state around 4 to 7 days after the i.v. presensitization, but was no longer detected in mice more than 14 days after the presensitization, although these mice exhibited complete tolerant state. When spleen cells from such tolerant mice were transferred i.v. into 600 R x-irradiated syngeneic recipient mice alone or together with normal syngeneic spleen cells, these tolerant spleen cells themselves failed to induce DTH responses but did not exhibit suppressive effect on the generation of DTH responses induced by normal spleen cells co-transferred. These results indicate that i.v. administration of neuraminidase-treated allogeneic cells results in the induction of alloantigen-specific tolerance which is not always associated with the induction of suppressor cell activity but rather with the elimination or functional impairment of alloantigen-specific clones.

Suppressive effect of portal venous inoculation with tumor cells on the anti-tumor immune potential.

The effect of portal venous (pv) administration of syngeneic tumor cells on the potential to generate anti-tumor immune resistance was examined. C3H/He mice were inoculated with syngeneic X5563 tumor cells via the pv or intravenous (iv) route. A single pv administration of 10,000 R X-irradiated X5563 cells into C3H/He mice not only failed to induce anti-X5563 immunity but also abolished the capability of the mice to develop anti-X5563 in vitro cytotoxic and in vivo protective immunity as induced by intradermal (id) inoculation of viable X5563 cells followed by the surgical resection of the tumor (immunization procedure). Such immunosuppression was also obtained by pv inoculation of tumor cells after the above immunization procedure. The immunosuppression induced by the pv injection of tumor cells before or after the immunization procedure was tumor-specific, since the pv sensitizing regimen using X5563 tumor cells did not interfere with the generation of immunity against another syngeneic tumor, MH134. More importantly, the pv route-induced suppression contrasted with the lack of inhibiting effect of iv inoculation with tumor cells on the induction of the tumor immunity. These results indicate that the host's tumor-specific immune capability is markedly suppressed in an anti-tumor sensitizing stage-independent manner when tumor cells enter the pv circulation.