Berberine enhances the function of intestinal stem cells in healthy and radiation-injured mice.

Intestinal stem cells (ISCs) are pivotal for the maintenance and regeneration of the intestinal epithelium. Berberine (BBR) exhibits diverse biological activities, but it remains unclear whether BBR can modulate ISCs' function. Therefore, we investigated the effects of BBR on ISCs in healthy and radiation-injured mice and explored the potential underlying mechanisms involved. The results showed that BBR significantly increased the length of the small intestines, the height of the villi, and the depth and density of the crypts, promoted the proliferation of cryptal epithelial cells and increased the number of OLFM4+ ISCs and goblet cells. Crypts from the BBR-treated mice were more capable of growing into enteroids than those from untreated mice. BBR alleviated WAI-induced intestinal injury. BBR suppressed the apoptosis of crypt epithelial cells, increased the quantity of goblet cells, and increased the quantity of OLFM4+ ISCs and tdTomato+ progenies of ISCs after 8 Gy WAI-induced injury. Mechanistically, BBR treatment caused a significant increase in the quantity of p-S6, p-STAT3 and p-ERK1/2 positive cryptal epithelial cells under physiological conditions and after WAI-induced injury. In conclusion, BBR is capable of enhancing the function of ISCs either physiologically or after radiation-induced injury, indicating that BBR has potential value in the treatment of radiation-induced intestinal injury.

Modeling Indirect Greenhouse Gas Emissions Sources from Urban Wastewater Treatment Plants: Integrating Machine Learning Models to Compensate for Sparse Parameters with Abundant Observations.

Electricity consumption and sludge yield (SY) are important indirect greenhouse gas (GHG) emission sources in wastewater treatment plants (WWTPs). Predicting these byproducts is crucial for tailoring technology-related policy decisions. However, it challenges balancing mass balance models and mechanistic models that respectively have limited intervariable nexus representation and excessive requirements on operational parameters. Herein, we propose integrating two machine learning models, namely, gradient boosting tree (GBT) and deep learning (DL), to precisely pointwise model electricity consumption intensity (ECI) and SY for WWTPs in China. Results indicate that GBT and DL are capable of mining massive data to compensate for the lack of available parameters, providing a comprehensive modeling focusing on operation conditions and designed parameters, respectively. The proposed model reveals that lower ECI and SY were associated with higher treated wastewater volumes, more lenient effluent standards, and newer equipment. Moreover, ECI and SY showed different patterns when influent biochemical oxygen demand is above or below 100 mg/L in the anaerobic-anoxic-oxic process. Therefore, managing ECI and SY requires quantifying the coupling relationships between biochemical reactions instead of isolating each variable. Furthermore, the proposed models demonstrate potential economic-related inequalities resulting from synergizing water pollution and GHG emissions management.

Contrast-enhanced US to Improve Diagnostic Performance of O-RADS US Risk Stratification System for Malignancy.

Background The Ovarian-Adnexal Reporting and Data System (O-RADS) has limited specificity for malignancy. Contrast-enhanced US can help distinguish malignant from benign lesions, but its added value to O-RADS has not yet been assessed. Purpose To establish a diagnostic model combining O-RADS and contrast-enhanced US and to validate whether O-RADS plus contrast-enhanced US has a better diagnostic performance than O-RADS alone. Materials and Methods This prospective study included participants from May 2018 to March 2021 who underwent contrast-enhanced US before surgery and had lesions categorized as O-RADS 3, 4, or 5 by US, with a histopathologic reference standard. From April 2021 to July 2022, participants with pathologically confirmed ovarian-adnexal lesions were recruited for the validation group. In the pilot group, the initial enhancement time and enhancement intensity in comparison with the uterine myometrium, contrast agent distribution pattern, and dynamic changes in enhancement of lesions were assessed. Contrast-enhanced US features were used to calculate contrast-enhanced US scores for benign (score ≤2) and malignant (score ≥4) lesions. Lesions were then re-rated according to O-RADS category plus contrast-enhanced US scores. Receiver operating characteristic curves were constructed and compared using the DeLong method. The combined system was validated in an independent group. Results The pilot group included 76 women (mean age, 44 years ± 13 [SD]), and the validation group included 46 women (mean age, 42 years ± 14). Differences in initial enhancement time (P < .001), enhancement intensity (P < .001), and dynamic changes in enhancement (P < .001) between benign and malignant lesions were observed in the pilot group. Contrast-enhanced US scores were calculated using these features. The O-RADS risk stratification was upgraded one level for contrast-enhanced US scores of 4 or more and downgraded one level for contrast-enhanced US scores of 2 or less. In the validation group, the diagnostic performance of O-RADS plus contrast-enhanced US score was higher (area under the receiver operating characteristic curve [AUC] = 0.93) than O-RADS (AUC = 0.71, P < .001). Conclusion Contrast-enhanced US improved the diagnostic performance for malignancy of the O-RADS categories 3-5. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Grant in this issue.

China's enhanced urban wastewater treatment increases greenhouse gas emissions and regional inequality.

Sustainable water pollution control requires understanding of historical trajectories and spatial characteristics of greenhouse gas (GHG) emissions from wastewater treatment plants (WWTPs), which remains inadequately studied. Here, we establish plant-level monthly operational emissions inventories of China's WWTPs in 2009-2019. We show that urban wastewater treatment has been enhanced with 80% more chemical oxygen demand being removed annually. However, this progress is associated with 180% more GHG emissions at the national level, up to 58.3 Mt CO2 eq in 2019. We found significant seasonality in GHG emissions. Increasing sludge yield and electricity intensity became primary drivers after 2015 because of stricter standards, causing GHG emissions increase 12.9 and 8.3% until 2019. GHG emissions from urban wastewater treatment show high spatial difference at province, city and plant levels, with different sludge disposal and energy mix approaches combined with different influent and effluent conditions in WWTPs across China. Stricter effluent standard resulted in similar GHG emissions growth pattern in cities. We argue WWTPs focus on resource recovery in developed areas and higher operational efficiency in developing areas.

Assessment of the safety and probiotic properties of Roseburia intestinalis: A potential "Next Generation Probiotic".

Roseburia intestinalis is an anaerobic bacterium that produces butyric acid and belongs to the phylum Firmicutes. There is increasing evidence that this bacterium has positive effects on several diseases, including inflammatory bowel disease, atherosclerosis, alcoholic fatty liver, colorectal cancer, and metabolic syndrome, making it a potential "Next Generation Probiotic." We investigated the genomic characteristics, probiotic properties, cytotoxicity, oral toxicity, colonization characteristics of the bacterium, and its effect on the gut microbiota. The genome contains few genes encoding virulence factors, three clustered regularly interspaced short palindromic repeat (CRISPR) sequences, two Cas genes, no toxic biogenic amine synthesis genes, and several essential amino acid and vitamin synthesis genes. Seven prophages and 41 genomic islands were predicted. In addition to a bacteriocin (Zoocin A), the bacterium encodes four metabolic gene clusters that synthesize short-chain fatty acids and 222 carbohydrate-active enzyme modules. This bacterium is sensitive to antibiotics specified by the European Food Safety Authority, does not exhibit hemolytic or gelatinase activity, and exhibits some acid resistance. R. intestinalis adheres to intestinal epithelial cells and inhibits the invasion of certain pathogens. In vitro experiments showed that the bacterium was not cytotoxic. R. intestinalis did not affect the diversity or abundance of the gut flora. Using the fluorescent labelling method, we discovered that R. intestinalis colonizes the cecum and mucus of the colon. An oral toxicity study did not reveal any obvious adverse effects. The lethal dose (LD)50 of R. intestinalis exceeded 1.9 × 109 colony forming units (CFU)/kg, whereas the no observed adverse effect level (NOAEL) derived from this study was 1.32 × 109 CFU/kg/day for 28 days. The current research shows that, R. intestinalis is a suitable next-generation probiotic considering its probiotic properties and safety.

Combination IETA Ultrasonographic Characteristics Simple Scoring Method With Tumor Biomarkers Effectively Improves the Differentiation Ability of Benign and Malignant Lesions in Endometrium and Uterine Cavity.

OBJECTIVES: To evaluate International Endometrial Tumor Analysis (IETA) ultrasonographic characteristics simple scoring method and tumor biomarkers for the diagnosis of uterine cavity and endometrial lesions. METHODS: We classified and scored the normalized description of IETA ultrasonic characteristics, according to IETA expert consensus literature, previous IETA-related research articles, and the previous research experience of this project group. We conducted a retrospective analysis of the ultrasound images of 594 patients enrolled from January 2017 to June 2020, scored them item by item, and finally calculated the total score of each case. Meanwhile, we combined the results of seven tumor biomarkers. The objective was to evaluate the sensitivity, specificity, coincidence rate, and the area under receiver operating characteristic (ROC) curve of IETA ultrasonographic characteristics simple scoring method and tumor biomarkers for benign and malignant uterine cavity or endometrial lesions. The diagnostic efficiency between the combined method and the single method was compared. RESULTS: A total of 594 cases were confirmed by postoperative pathology or surgery records, including 475 benign lesions and 119 malignant lesions. In the simple ultrasound scoring method, the average score of benign lesions was 3.879 ± 1.279 and that of malignant lesions was 9.676 ± 4.491. If ≥6.5 points was taken as the cutoff value for the judgment of malignant lesions, the sensitivity, specificity, coincidence rate, and the area under receiver operating characteristic (ROC) curve (AUC) were 76.5%, 96.0%, 92.1%, and 0.935, respectively. The difference in tumor antigen 19-9 (CA19-9) and human epididymal protein 4 (HE4) between benign and malignant lesions was statistically significant (all p ≤ 0.01). The other five tumor biomarkers (CA125, CA15-3, SCC-Ag, AFP, and CEA) showed no statistically significant difference in benign and malignant lesions. If the value of CA19-9 ≥13.96 U/ml was taken as cutoff value, the sensitivity, specificity, and coincidence rate of the diagnosis of endometrial benign and malignant lesions were 54.8%, 74.7%, and 70.7%, respectively, and the AUC was 0.620. If the value of HE4 ≥ 39.075 pmol/L was taken as cutoff point, the sensitivity, specificity, coincidence rate, and AUC were 77.4%, 67.9%, 69.8%, and 0.796, respectively. The sensitivity was increased to 97.6% and the AUC was 0.939 when IETA ultrasound characteristics simple scoring method combined CA19-9 and HE4 in parallel test. CONCLUSIONS: In IETA ultrasound characteristics simple scoring method, with ≥6.5 points as the cutoff value, it could quickly and accurately assess the benign and malignant in uterine cavity and endometrial lesions, with high diagnostic value. The diagnostic efficacy of seven tumor biomarkers was all mediocre. Combining with these two methods, the comprehensive diagnosis could improve sensitivity and accuracy and reduce the risk of missed diagnosis.

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials ("Phase II-like mouse clinical trials"); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 µm cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 µM Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

The effect of Lactobacillus fermentum DALI02 in reducing the oxidative stress and inflammatory response induced by high-fat diet of rats.

A long-term high-fat diet (HFD) leads to significant oxidative stress in the body and induces inflammation. A preliminary evidence suggests a potential therapeutic utility of probiotics for this condition. To evaluate the potential effect of Lactobacillus fermentum DALI02 on the oxidative stress and inflammatory damage induced by HFD, we used a hyperlipidemic rat as a model fed with HFD. Results revealed that HFD induced a significant oxidative stress and inflammation. However, results reveal that L. fermentum DALI02, manifested a significant decrease in levels of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and resistin, while the catalase (CAT), total antioxidant capability (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and adiponectin (ADPN) levels significantly increased. And it was dose-dependent that the effect of high dose groups with high viable count was particularly notable. The results suggest that L. fermentum DALI02 could alleviate oxidative stress and inflammation as it appeared to reduce lipid peroxidation and improved the lipid metabolism in vivo.

BTLA interaction with HVEM expressed on CD8(+) T cells promotes survival and memory generation in response to a bacterial infection.

The B and T lymphocyte attenuator (BTLA) is an Ig super family member that binds to the herpes virus entry mediator (HVEM), a TNF receptor super family (TNFRSF) member. Engagement of BTLA by HVEM triggers inhibitory signals, although recent evidence indicates that BTLA also may act as an activating ligand for HVEM. In this study, we reveal a novel role for the BTLA-HVEM pathway in promoting the survival of activated CD8(+) T cells in the response to an oral microbial infection. Our data show that both BTLA- and HVEM-deficient mice infected with Listeria monocytogenes had significantly reduced numbers of primary effector and memory CD8(+) T cells, despite normal proliferation and expansion compared to controls. In addition, blockade of the BTLA-HVEM interaction early in the response led to significantly reduced numbers of antigen-specific CD8(+) T cells. HVEM expression on the CD8(+) T cells as well as BTLA expression on a cell type other than CD8(+) T lymphocytes, was required. Collectively, our data demonstrate that the function of the BTLA-HVEM pathway is not limited to inhibitory signaling in T lymphocytes, and instead, that BTLA can provide crucial, HVEM-dependent signals that promote survival of antigen activated CD8(+) T cell during bacterial infection.

Crosstalk between adaptive and innate immune cells leads to high quality immune protection at the mucosal borders.

Mucosal effector memory CD8 T cells are located at the epithelium and have a heightened and immediate effector function. By contrast, central memory T cells reside within lymphoid tissues and require proliferation and differentiation to become effector cells that migrate to epithelial surfaces. The accumulation of effector memory T cells at the pathogen entry site(s) is essential for protective immunity, but the mechanisms that drive the differentiation of memory cell subsets are poorly understood. We recently showed that CD8αα, induced selectively on the most highly activated primary CD8αß T cells, together with its ligand, the thymic leukemia (TL) antigen, induced on mucosal antigen-presenting cells and constitutively expressed on intestinal epithelial cells (IEC), serve as key components to mediate the selective accumulation of the fittest effector cells to form mucosal effector memory T cells. Therefore, the generation of mucosal effector memory is controlled by an innate-adaptive crosstalk that provides for host defense at the body's largest interface.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid suppresses the pro-oncogenic effects induced by hepatitis B virus pre-S2 mutant oncoprotein and represents a potential chemopreventive agent in high-risk chronic HBV patients.

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S(2) mutant large HBV surface antigen (LHBS) in type II ground glass hepatocytes (GGHs) has been recognized as an emerging viral oncoprotein; it directly interacts with the c-Jun activation domain-binding protein 1 (JAB1) and subsequently causes hyperphosphorylation of the tumor-suppressor retinoblastoma and, consequently, leads to disturbed cell cycle progression. The interaction of the pre-S(2) mutant LHBS with JAB1 could provide a potential target for chemoprevention. In this study, we found that the preneoplastic type II GGHs showed a significant decrease of the cyclin-dependent kinase inhibitor p27(Kip1), which serves as a marker for pre-S(2) mutant-JAB1 complex formation. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) elevated expression of the tumor-suppressor thioredoxin-binding protein 2 (TBP2), which subsequently enhanced the JAB1-TBP2 interaction and abolished the pre-S(2) mutant LHBS-induced degradation of p27(Kip1), which, in turn, recovered the normal cell cycle checkpoint. The pre-S(2) mutant LHBS-induced pro-oncogenic effects: increased cell proliferation, nuclear/cytoplasmic ratio and proliferating cell nuclear antigen expression, were all greatly ameliorated after SAHA treatments, which suggested SAHA as a promising chemopreventive agent for the pre-S(2) mutant oncoprotein-induced HCC. In conclusion, this study provides the mechanism of histone deacetylase (HDAC) inhibitor in preventing the pre-S(2) mutant-induced oncogenic phenotype. The HDAC inhibitor SAHA is therefore a potential chemopreventive agent for high-risk chronic HBV patients who may develop HCC.

Mucosal memory CD8⁺ T cells are selected in the periphery by an MHC class I molecule.

The presence of immune memory at pathogen-entry sites is a prerequisite for protection. Nevertheless, the mechanisms that warrant immunity at peripheral interfaces are not understood. Here we show that the nonclassical major histocompatibility complex (MHC) class I molecule thymus leukemia antigen (TL), induced on dendritic cells interacting with CD8αα on activated CD8αß(+) T cells, mediated affinity-based selection of memory precursor cells. Furthermore, constitutive expression of TL on epithelial cells led to continued selection of mature CD8αß(+) memory T cells. The memory process driven by TL and CD8αα was essential for the generation of CD8αß(+) memory T cells in the intestine and the accumulation of highly antigen-sensitive CD8αß(+) memory T cells that form the first line of defense at the largest entry port for pathogens.

[Changes of iron metabolism indices in children with various genotypes of thalassema].

OBJECTIVE: To study the value of iron metabolism indices, serum iron (SI), total iron blinding capacity (TIBC) and transferring (Tf), in thalassema. METHODS: The serum samples from 9 children with silent alpha thalassema, 56 with standard alpha thalassema, 26 with HbH disease, 40 with beta+ thalassema, 56 with beta0 thalassema, 45 with iron deficiency anemia (IDA) and 70 healthy children were detected for SI, TIBC and Tf levels. RESULTS: The SI level increased (p<0.01), while the TIBC level decreased significantly in the beta0 thalassema group compared with those in the other groups (p<0.05 or 0.01), but the Tf level was not different. The Tf level of both the silent alpha thalassema and the standard alpha thalassema groups was statistically lower than that of the healthy group (p<0.01), but the levels of SI and TIBC were similar to the healthy group. Though the SI level of the HbH disease group was similar to the healthy group, the TIBC and Tf levels were statistically lower (p<0.01). CONCLUSIONS: Compared with Tf, SI and TIBC are better indices for monitoring iron loading in children with thalassema. The increased SI level and decreased TIBC level are two indices for the diagnosis of beta(0) thalassema in children with cellule anaemia.

Streptococcal pyrogenic exotoxin B cleaves human S-adenosylhomocysteine hydrolase and induces hypermethioninemia.

Group A Streptococcus is a common pathogen that causes pharyngitis, impetigo, myositis, and lethal streptococcal toxic shock syndrome. Streptococcal pyrogenic exotoxin B (SPE B) is strongly associated with the severity of disease. SPE B is a cysteine protease and matures itself by autocatalysis. We found that SPE B was directly associated with human S-adenosylhomocysteine hydrolase (AdoHcyase), an essential factor for a delayed-type immune response. AdoHcyase protein levels and enzymatic activities were significantly higher in human cells infected with the Streptococcus pyogenes SW510 speB mutant strain than in cells infected with the NZ131 wild-type strain. SPE B also inactivated AdoHcyase, shown by a decrease in homocysteine, the main product of AdoHcyase. We found that in vivo and in vitro, SPE B induced hypermethioninemia, which is caused by an AdoHcyase defect. We also found that AdoHcyase is a substrate of SPE B cysteine protease. SPE B, therefore, potentially causes immunosuppression by cleaving AdoHcyase.

Hepatitis B virus pre-S2 mutant surface antigen induces degradation of cyclin-dependent kinase inhibitor p27Kip1 through c-Jun activation domain-binding protein 1.

The hepatitis B virus (HBV) large surface antigen (LHBS) mutant with deletion at the pre-S(2) region accumulates in endoplasmic reticulum (ER) and is associated with HBV-induced hepatocellular carcinogenesis. In this study, we found that the pre-S(2) LHBS mutant directly interacts with the Jun activation domain-binding protein 1 (JAB1). Association of pre-S(2) LHBS with JAB1 dissociated JAB1 from the JAB1/IRE1 complex in ER. The free (active) JAB1 then translocated into cell nuclei and rendered the Cdk inhibitor p27(Kip1) to cytosolic proteasome for degradation. The pre-S(2) LHBS mutant induced hyperphosphorylation of tumor suppressor retinoblastoma (RB) via cyclin-dependent kinase 2 (Cdk2), a downstream molecule regulated by p27(Kip1). This effect is independent of the ER stress signaling pathway. The transgenic mice carrying the pre-S(2) mutant LHBS gene also exhibited Cdk2 activation, p27(Kip1) degradation, as well as RB hyperphosphorylation. The mouse hepatocytes exhibited morphologic abnormalities such as chromatin condensation, multinucleation, and dysplasia of hepatocytes. In summary, the pre-S(2) LHBS mutant causes p27(Kip1) degradation through direct interaction with JAB1. The pre-S(2) mutant LHBS is suggested to be a potential oncoprotein for HBV-related hepatocellular carcinoma.

Tumor resistance to CD8+ T cell-based therapeutic vaccination.

CD8(+) cytotoxic T lymphocytes (CTLs) play an important role in antitumor immunity. Induction of tumor-specific CTLs is one major strategy for tumor immunotherapy. However, therapeutic vaccinations used to treat firmly established tumors are generally ineffective. A thorough understanding of the mechanisms underlying tumor resistance to CTL-based therapeutic vaccination is very important in the tumor immunology field. There are two main mechanisms by which tumors develop resistance to CTL-based therapeutic vaccinations. One is that tumors induce peripheral tolerance of tumor-specific CD8(+) T cells. The other is that tumor cells themselves develop immune evasion mechanisms to prevent recognition and killing by CTLs. This review focuses on recently reported cellular and molecular mechanisms of CD8(+) T cell tolerance and immune evasion in tumors and discusses about the possibilities to improve tumor immunotherapy.

Induction of mucosal and systemic immune responses against human carcinoembryonic antigen by an oral vaccine.

Carcinoembryonic antigen (CEA) is a tumor-associated antigen targeted for the development of colorectal tumor vaccines. In this study, we developed papillomavirus pseudoviruses encoding the truncated CEA without NH2-terminal signal peptide (PV-CEA) as an oral vaccine to induce CEA-specific CTL responses. In CEA transgenic (CEA-Tg) mice orally immunized with PV-CEA, the immunologic tolerance to CEA as a "self-antigen" was overcome and both mucosal and systemic CEA-specific cytolytic activities were detected by in vitro 51Cr release assays. In a tumor prevention model, the growth rate of CEA+ tumors was significantly delayed in CEA-Tg mice orally immunized with PV-CEA when compared with the control vaccine. Further, the IFN-gamma enzyme-linked ImmunoSPOT and in vitro 51Cr release assay results showed that HLA-A2-restricted, CEA-specific CTL responses were induced in both mucosal and systemic lymphoid tissues in A2 transgenic mice after oral immunization with PV-CEA. Finally, we showed that coadministration of papillomavirus pseudoviruses encoding interleukin-2 with PV-CEA enhanced the generation of A2-restricted, CEA-specific CTLs in aged CEA/A2 double transgenic mice, which were more clinically relevant. Our data suggest that PV-CEA pseudovirus vaccine is a promising oral CEA vaccine for humans to induce CEA-specific CTLs at the site of colorectal tumors (i.e., intestinal mucosa), which might efficiently eliminate CEA+ colorectal tumor cells in the mucosa.

Turning on/off tumor-specific CTL response during progressive tumor growth.

Therapeutic vaccinations used to induce CTLs and treat firmly established tumors are generally ineffective. To understand the mechanisms underlying the failure of therapeutic vaccinations, we investigated the fate of tumor-specific CD8+ T cells in tumor-bearing mice with or without vaccinations. Our data demonstrate that tumor-specific CD8+ T cells are activated at the early stage of tumor growth, tumor-specific CTL response reaches a maximal level during progressive tumor growth, and tumor-specific CD8+ T cells lose cytolytic function at the late stage of tumor growth. The early stage therapeutic vaccination induces efficient antitumor activity by amplifying the CTL response, whereas the late-stage therapeutic vaccination is invalid due to tumor-induced dysfunction of CD8+ T cells. However, at the late stage, tumor-specific CD8+ T cells are still present in the periphery. These tumor-specific CD8+ T cells lose cytolytic activity, but retain IFN-gamma secretion function. In contrast to in vitro cultured tumor cells, in vivo growing tumor cells are more resistant to tumor-specific CTL killing, despite an increase of tumor Ag gene expression. Both tumor-induced CD8+ T cell dysfunction at the late stage and immune evasion developed by in vivo growing tumor cells contribute to an eventual inefficacy of therapeutic vaccinations. Our study suggests that it is important to design a vaccination regimen according to the stages of tumor growth and the functional states of tumor-specific CD8+ T cells.

Oral administration with papillomavirus pseudovirus encoding IL-2 fully restores mucosal and systemic immune responses to vaccinations in aged mice.

Infectious diseases are one of the major threats for the elderly because their immune system is often compromised, and vaccinations to prevent these infections are not effective. A major defect in their immune system seems to be the inability of T cells to produce IL-2. We used papillomavirus (PV) pseudoviruses (PSVs) as a model vaccine and a gene delivery vector to address how to enhance immune responses to vaccinations. We found that oral immunization with PV PSV induced minimal mucosal and systemic Abs and CTLs specific for the PSVs in aged mice compared with young adult mice. In addition, fewer specific Th cells were generated in the aged mice. When aged mice were immunized with PV PSVs encoding human IL-2, specific Th cells were generated, producing murine IL-2, IL-4, and IFN-gamma. Further, specific Abs and CTLs were induced, resulting in protection against mucosal viral challenge. Thus, this study provided a basis for clinical trials using PV PSVs encoding IL-2 for vaccination of the elderly.

Induction of mucosal and systemic neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) by oral immunization with bovine Papillomavirus-HIV-1 gp41 chimeric virus-like particles.

Human immunodeficiency virus type 1 (HIV-1) envelope-specific neutralizing antibodies are generated late after initial infection, and the neutralizing antibody response is weak in the infected individuals. Administration of neutralizing antibodies such as 2F5 to HIV-1-infected individuals resulted in reductions in viral loads. Because HIV-1 is transmitted mainly via mucosa and because HIV-specific neutralizing antibodies reduce HIV-1 in infected individuals, a vaccine that can induce both mucosal and systemic HIV-1-specific neutralizing antibodies may be used to prevent and to treat HIV-1 infection. In this study, we made a bovine papillomavirus (BPV) L1-HIV-1 gp41 fusion protein in which ELDKWA of gp41 was inserted into the N terminus of BPV L1 (amino acids 130 to 136). Expression of the fusion protein in insect cells led to the assembly of chimeric virus-like particles (CVLPs). The CVLPs had sizes similar to those of BPV particles and were able to bind to the cell surface and penetrate the cell membrane. Oral immunization of mice with CVLPs induced gp41-specific serum immunoglobulin G (IgG) and intestinal secretory IgA. However, intramuscular immunization with the CVLPs resulted in similar amounts of gp41-specific IgG but low levels of secretory IgA. The antibodies specifically recognized the fixed HIV-1 gp41 on the cell surface. Importantly, the sera and fecal extracts from mice orally immunized with the CVLPs neutralized HIV-1(MN) in vitro. Thus, BPV-HIV-1 gp41 CVLPs may be used to prevent and to treat HIV-1 infection.