Stereotactic ablative radiotherapy before resection to avoid delay for early-stage lung cancer or oligometastases during the COVID-19 pandemic: Pathologic outcomes from the SABR-BRIDGE protocol.

BACKGROUND: During coronavirus disease 2019 (COVID-19)-related operating room closures, some multidisciplinary thoracic oncology teams adopted a paradigm of stereotactic ablative radiotherapy (SABR) as a bridge to surgery, an approach called SABR-BRIDGE. This study presents the preliminary surgical and pathological results. METHODS: Eligible participants from four institutions (three in Canada and one in the United States) had early-stage presumed or biopsy-proven lung malignancy that would normally be surgically resected. SABR was delivered using standard institutional guidelines, with surgery >3 months following SABR with standardized pathologic assessment. Pathological complete response (pCR) was defined as absence of viable cancer. Major pathologic response (MPR) was defined as ≤10% viable tissue. RESULTS: Seventy-two patients underwent SABR. Most common SABR regimens were 34 Gy/1 (29%, n = 21), 48 Gy/3-4 (26%, n = 19), and 50/55 Gy/5 (22%, n = 16). SABR was well-tolerated, with one grade 5 toxicity (death 10 days after SABR with COVID-19) and five grade 2-3 toxicities. Following SABR, 26 patients underwent resection thus far (13 pending surgery). Median time-to-surgery was 4.5 months post-SABR (range, 2-17.5 months). Surgery was reported as being more difficult because of SABR in 38% (n = 10) of cases. Thirteen patients (50%) had pCR and 19 (73%) had MPR. Rates of pCR trended higher in patients operated on at earlier time points (75% if within 3 months, 50% if 3-6 months, and 33% if ≥6 months; p = .069). In the exploratory best-case scenario analysis, pCR rate does not exceed 82%. CONCLUSIONS: The SABR-BRIDGE approach allowed for delivery of treatment during a period of operating room closure and was well-tolerated. Even in the best-case scenario, pCR rate does not exceed 82%.

Data-driven identification of plasma metabolite clusters and metabolites of interest for potential detection of early-stage non-small cell lung cancer cases versus cancer-free controls.

BACKGROUND: Metabolomics is a potential means for biofluid-based lung cancer detection. We conducted a non-targeted, data-driven assessment of plasma from early-stage non-small cell lung cancer (ES-NSCLC) cases versus cancer-free controls (CFC) to explore and identify the classes of metabolites for further targeted metabolomics biomarker development. METHODS: Plasma from 250 ES-NSCLC cases and 250 CFCs underwent ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) in positive and negative electrospray ionization (ESI) modes. Molecular feature extraction, formula generation, and find-by-ion tools annotated metabolic entities. Analysis was restricted to endogenous metabolites present in ≥ 80% of samples. Unsupervised hierarchical cluster analysis identified clusters of metabolites. The metabolites with the strongest correlation with the principal component of each cluster were included in logistic regression modeling to assess discriminatory performance with and without adjustment for clinical covariates. RESULTS: A total of 1900 UHPLC-QTOF-MS assessments identified 1667 and 2032 endogenous metabolites in the ESI-positive and ESI-negative modes, respectively. After data filtration, 676 metabolites remained, and 12 clusters of metabolites were identified from each ESI mode. Multivariable logistic regression using the representative metabolite from each cluster revealed effective classification of cases from controls with overall diagnostic accuracy of 91% (ESI positive) and 94% (ESI negative). Metabolites of interest identified for further targeted analysis include the following: 1b, 3a, 12a-trihydroxy-5b-cholanoic acid, pyridoxamine 5'-phosphate, sphinganine 1-phosphate, gamma-CEHC, 20-carboxy-leukotriene B4, isodesmosine, and 18-hydroxycortisol. CONCLUSIONS: Plasma-based metabolomic detection of early-stage NSCLC appears feasible. Further metabolomics studies targeting phospholipid, steroid, and fatty acid metabolism are warranted to further develop noninvasive metabolomics-based detection of early-stage NSCLC.

Metabolic Alterations in Sputum and Exhaled Breath Condensate of Early Stage Non-Small Cell Lung Cancer Patients After Surgical Resection: A Pilot Study.

Every year, close to two million people world-wide are diagnosed with and die of lung cancer. Most patients present with advanced-stage cancer with limited curative options and poor prognosis. Diagnosis of lung cancer at an early stage provides the best chance for a cure. Low- dose CT screening of the chest in the high-risk population is the current standard of care for early detection of lung cancer. However, CT screening is invasive due to radiation exposure and carries the risk of unnecessary biopsies in non-cancerous tumors. In this pilot study, we present metabolic alterations observed in sputum and breath condensate of the same population of early- stage non-small cell lung cancer (NSCLC) patients cancer before and after surgical resection (SR), which could serve as noninvasive diagnostic tool. Exhaled breath condensate (EBC) (n=35) and sputum (n=15) were collected from early-stage non-small cell lung cancer (NSCLC) patients before and after SR. Median number of days for EBC and sputum collection before and after SR were 7 and 42; and 7 and 36 respectively Nuclear magnetic resonance (NMR) and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) were used to analyze the metabolic profile of the collected samples. A total of 26 metabolites with significant alteration post SR were identified, of which 14 (54%) were lipids and 12 constituted nine different chemical metabolite classes. Eighteen metabolites (69%) were significantly upregulated and 8 (31%) were downregulated. Median fold change for all the up- and downregulated metabolites (LC-QTOF-MS) were 10 and 8, respectively. Median fold change (MFC) in concentration of all the up- and downregulated metabolites (NMR) were 0.04 and 0.27, respectively. Furthermore, glucose (median fold change, 0.01, p=0.037), adenosine monophosphate (13 log fold, p=0.0037) and N1, N12- diacetylspermine (8 log fold p=0.011) sputum levels were significantly increased post-SR. These identified sputa and EBC indices of altered metabolism could serve as basis for further exploration of biomarkers for early detection of lung cancer, treatment response, and targets for drug discovery. Validation of these promising results by larger clinical studies is warranted.

Canadian ROS proto-oncogene 1 study (CROS) for multi-institutional implementation of ROS1 testing in non-small cell lung cancer.

Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories. Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor. Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases. This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.

Metabolic Changes in Early-Stage Non-Small Cell Lung Cancer Patients after Surgical Resection.

Metabolic alterations in malignant cells play a vital role in tumor initiation, proliferation, and metastasis. Biofluids from patients with non-small cell lung cancer (NSCLC) harbor metabolic biomarkers with potential clinical applications. In this study, we assessed the changes in the metabolic profile of patients with early-stage NSCLC using mass spectrometry and nuclear magnetic resonance spectroscopy before and after surgical resection. A single cohort of 35 patients provided a total of 29 and 32 pairs of urine and serum samples, respectively, pre-and post-surgery. We identified a profile of 48 metabolites that were significantly different pre- and post-surgery: 17 in urine and 31 in serum. A higher proportion of metabolites were upregulated than downregulated post-surgery (p < 0.01); however, the median fold change (FC) was higher for downregulated than upregulated metabolites (p < 0.05). Purines/pyrimidines and proteins had a larger dysregulation than other classes of metabolites (p < 0.05 for each class). Several of the dysregulated metabolites have been previously associated with cancer, including leucyl proline, asymmetric dimethylarginine, isopentenyladenine, fumaric acid (all downregulated post-surgery), as well as N6-methyladenosine and several deoxycholic acid moieties, which were upregulated post-surgery. This study establishes metabolomic analysis of biofluids as a path to non-invasive diagnostics, screening, and monitoring in NSCLC.

Comparative metabolomics studies of blood collected in streck and heparin tubes from lung cancer patients.

Metabolomics analysis of blood from patients (n = 42) undergoing surgery for suspected lung cancer was performed in this study. Venous and arterial blood was collected in both Streck and Heparin tubes. A total of 96 metabolites were detected, affected by sex (n = 56), collection tube (n = 33), and blood location (n = 8). These metabolites belonged to a wide array of compound classes including lipids, acids, pharmaceutical agents, signalling molecules, vitamins, among others. Phospholipids and carboxylic acids accounted for 28% of all detected compounds. Out of the 33 compounds significantly affected by collection tube, 18 compounds were higher in the Streck tubes, including allantoin and ketoleucine, and 15 were higher in the Heparin tubes, including LysoPC(P-16:0), PS 40:6, and chenodeoxycholic acid glycine conjugate. Based on our results, it is recommended that replicate blood samples from each patient should be collected in different types of blood collection tubes for a broader range of the metabolome. Several metabolites were found at higher concentrations in cancer patients such as lactic acid in Squamous Cell Carcinoma, and lysoPCs in Adenocarcinoma and Acinar Cell Carcinoma, which may be used to detect early onset and/or to monitor the progress of the cancer patients.

Outcome of Locally Advanced Esophageal Cancer Patients Treated With Perioperative Chemotherapy and Chemoradiotherapy Followed by Surgery.

OBJECTIVES: Perioperative chemotherapy (P-CT) or neoadjuvant chemoradiation (C-RT) followed by surgical resection is the standard of care for locally advanced esophageal cancer (LAEC). We present an institutional review and outcome of patients with LAEC treated with neoadjuvant C-RT or P-CT followed by surgery. METHODS: Patients were identified through the Manitoba Cancer Registry. Overall survival (OS), recurrence-free survival (RFS), and time to recurrence (TTR) were compared using proportion hazard regression analysis. Metabolic and pathologic response rates were compared by the Fisher exact test. RESULTS: Sixty-seven patients were treated with C-RT and 32 with P-CT. Fifty-two percent of the patients had pretreatment and posttreatment positron emission tomography scans before surgery. Ninety-five percent of the patients in C-RT and 91% in P-CT had a partial metabolic response or stable disease. Sixty-one percent of C-RT and 34% of P-CT patients had tumor regression grade (TRG) 0 to 1; 39% of C-RT and 66% of P-CT had TRG 2 to 3 (P=0.018). Median OS was 37 and 18 months for patients with TRG 0 to 1 and 2 to 3, respectively (P=0.013, hazard ratio [HR]=1.96). Three-year OS was 43% versus 37% (P=0.37, HR=1.30), RFS was 34% versus 26% (P=0.87, HR=0.96), and median TTR was 30 versus 13 months (P=0.07, HR=0.59) for C-RT and P-CT, respectively. CONCLUSIONS: C-RT was associated with a higher degree of pathologically tumor regression. Patients with major tumor regression had a better outcome than those with minimal to poor response. There was a trend toward improved TTR with C-RT but no difference in OS or RFS.

Immunotherapy Benefit in a Patient With Non-Small Cell Lung Cancer and a Rare BRAF Mutation.

Immunotherapy is less effective in non-small cell lung cancer (NSCLC) with driver mutations in epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) and some may extrapolate this trend to other driver mutations. Up to 4% of NSCLC cases contain a BRAF mutation. Most BRAF mutations are V600E, and little is known about the impact of treatment in rare BRAF G469A mutations. We present a case of a patient found to have BRAF G469A mutated NSCLC. She was diagnosed with Stage IIIB NSCLC and treated with concurrent chemotherapy and radiation. Post-treatment imaging demonstrated disease progression and she was started on nivolumab, resulting in a dramatic and prolonged response which is ongoing after 76 cycles. Her substantial response and prolonged benefit suggest that BRAF-mutated NSCLC may respond better than EGFR- or ALK-driven disease to immunotherapy. Due to the rarity of specific mutations, this case adds to the limited current published literature on NSCLC harbouring a BRAF G469A mutation and suggests that immunotherapy is a reasonable treatment option.

Canadian Multicenter Project on Standardization of Programmed Death-Ligand 1 Immunohistochemistry 22C3 Laboratory-Developed Tests for Pembrolizumab Therapy in NSCLC.

INTRODUCTION: The programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) assay is used to select patients for first or second-line pembrolizumab monotherapy in NSCLC. The PD-L1 IHC 22C3 pharmDx assay requires an Autostainer Link 48 instrument. Laboratories without this stainer have the option to develop a highly accurate 22C3 IHC laboratory-developed test (LDT) on other instruments. The Canadian 22C3 IHC LDT validation project was initiated to harmonize the quality of PD-L1 22C3 IHC LDT protocols across 20 Canadian pathology laboratories. METHODS: Centrally optimized 22C3 LDT protocols were distributed to participating laboratories. The LDT results were assessed against results using reference PD-L1 IHC 22C3 pharmDx. Analytical sensitivity and specificity were assessed using cell lines with varying PD-L1 expression levels (phase 1) and IHC critical assay performance controls (phase 2B). Diagnostic sensitivity and specificity were assessed using whole sections of 50 NSCLC cases (phase 2A) and tissue microarrays with an additional 50 NSCLC cases (phase 2C). RESULTS: In phase 1, 80% of participants reached acceptance criteria for analytical performance in the first attempt with disseminated protocols. However, in phase 2A, only 40% of participants reached the desired diagnostic accuracy for both 1% and 50% tumor proportion score cutoff. In phase 2B, further protocol modifications were conducted, which increased the number of successful laboratories to 75% in phase 2C. CONCLUSIONS: It is possible to harmonize highly accurate 22C3 LDTs for both 1% and 50% tumor proportion score in NSCLC across many laboratories with different platforms. However, despite a centralized approach, diagnostic validation of predictive IHC LDTs can be challenging and not always successful.

Non-invasive exploration of metabolic profile of lung cancer with Magnetic Resonance Spectroscopy and Mass Spectrometry.

BACKGROUND: Lung cancer is a major cause of global morbidity and mortality. Current low dose CT screening is invasive and its role remains contentious. There are no known biomarkers to monitor treatment response, detect disease recurrence and patient selection for adjuvant treatment after curative surgical resection. Hence there is an urgent need to explore non-conventional and non-invasive tools to develop novel biomarkers to improve the outcome of this lethal cancer. METHODS: This is an ongoing exploratory and translational study involving collection of bio fluids from 50 patients with early stage non-small cell lung cancer before and after surgical resection. The primary objective is to identify cancer specific metabolome in body fluids - sputum, exhaled breath condensate, blood and urine of the patients with early stage non-small cell lung cancer using Magnetic Resonance Spectroscopy and Mass Spectroscopy. CONCLUSION: The trajectory of change in metabolic profile of body fluids before and after surgical resection may have potential clinical applications in lung cancer screening, as biomarkers for disease recurrence and exploration of novel targets for therapeutic intervention.

Sustained suppression of IL-18 by employing a vaccine ameliorates intestinal inflammation in TNBS-induced murine colitis.

AIM: To develop IL-18 peptide-based virus-like particle vaccines that elicit autoantibodies against IL-18 and to evaluate the in vivo effects of the vaccines in murine colitis. METHODS: Recombinant IL-18 vaccines were constructed, and the effects of the vaccines were evaluated in trinitrobenzene sulfonic acid-induced acute and chronic colitis in mice. RESULTS: Two murine IL-18 peptide-based vaccines (A and D) were developed, which induced relative long-lasting specific antibodies against IL-18. Vaccine-immunized mouse antisera could partially block IL-18-induced IFN-γ production in vitro. Mice receiving vaccine D, not vaccine A, had a significant decrease in intestinal inflammation, collagen deposition and pro-inflammatory cytokine levels in colon tissue. CONCLUSION: IL-18 vaccine may provide a potential therapeutic approach in the treatment of Crohn's disease.

Benefit of crizotinib in a lung cancer patient with discordant ALK testing results.

Crizotinib is a first line treatment for patients with non-small cell lung cancer (NSCLC) harboring translocations in anaplastic lymphoma kinase (ALK). The current gold standard for determining ALK status is fluorescence in-situ hybridisation (FISH), but immunohistochemistry (IHC) is becoming increasingly popular due to lower cost. There are currently few reports on clinical outcomes with crizotinib therapy in patients who have tested negative by FISH and positive by IHC. A 53 year old lifelong non-smoking, physically active male with newly diagnosed Stage IV NSCLC presented with shortness of breath on exertion one month prior to referral. Staging CT scan failed to show a discreet lung lesion, but the left lower lobe was collapsed due to pleural effusion. Pleural fluid showed adenocarcinoma and IHC was positive for an ALK mutation, while FISH was negative. Pre-treatment PET-CT showed hypermetabolic, enlarged lymph nodes in the mediastinum and retroperitoneum. Partially due to patient concerns about cytotoxic chemotherapy toxicity, crizotinib therapy was instituted. Repeat CT conducted two months after crizotinib initiation showed a decrease in lymphadenopathy at all sites compared to the PET-CT. Furthermore, the patient showed clinical improvement, with less drainage through his PleurX catheter and stability of his excellent performance status. After 12 months on crizotinib CT showed ongoing improvement in lymphadenopathy. His bloodwork has been stable, and he denies significant drug toxicity. This case illustrates a sustained response to crizotinib therapy in a patient with an ALK translocation identified by IHC, but with negative FISH testing. The literature suggests that the population with these discordant results could be up to 19% of ALK positive NSCLC. Patients in this subgroup who are receiving crizotinib should be identified and outcome data pooled. However, in the interim, oncologists may wish to consider targeted therapy for these discordant patients.

Androgen Receptor and Ki67 Expression and Survival Outcomes in Non-small Cell Lung Cancer.

Lung cancer is the most common cause of cancer-related deaths worldwide with non-small cell lung cancer (NSCLC) making up most of these cases. Males have poorer overall survival compared to women following a lung cancer diagnosis. Many studies have focused on the effects of estrogen to explain higher survival rates among women, but few have looked at the effects of androgens. We describe the expression of the androgen receptor (AR) and Ki67 in lung cancer specimens in the Manitoba Tumor Bank (MTB) and correlate these factors with patient outcome. Using the MTB, we performed immunohistochemistry on lung cancer tissue to determine expression of the AR and Ki67. These were then correlated with patient outcome. Of the 136 cases, 55% were female and 55% were adenocarcinoma. AR expression was not independently associated with outcome. Ki67 was associated with a significantly higher hazard ratio for death and recurrence (HR 2.19, 95% CI 1.30-3.70; HR 1.92, 95% CI 1.07-3.46, respectively). AR expression modified the effect of Ki67 on outcome, such that when both were expressed, there was no association with recurrence or survival (HR 2.39, 95% CI 1.31-4.36 for AR- Ki67+ vs HR 1.54, 95% CI 0.44-5.37 for AR+ Ki67+). Ki67 was associated with poorer outcomes alone. AR status alone was not associated with outcome. Although the mechanism remains unclear, AR status seems to negate the association of a high Ki67 and poor outcome.

Malignant mesothelioma in situ.

AIMS: The existence of malignant mesothelioma in situ (MIS) is often postulated, but there are no accepted morphological criteria for making such a diagnosis. METHODS AND RESULTS: Here we report two cases that appear to be true MIS on the basis of in-situ genomic analysis. In one case the patient had repeated unexplained pleural unilateral effusions. Two thoracoscopies 9 months apart revealed only visually normal pleura. Biopsies from both thoracoscopies showed only a single layer of mildly reactive mesothelial cells. However, these cells had lost BRCA1-associated protein 1 (BAP1) and showed loss of cyclin-dependent kinase inhibitor 2 (CDKN2A) (p16) by fluorescence in-situ hybridisation (FISH). NF2 was not deleted by FISH but 28% of the mesothelial cells showed hyperploidy. Six months after the second biopsy the patient has persisting effusions but no evidence of pleural malignancy on imaging. The second patient presented with ascites and minimal omental thickening on imaging, but no visual evidence of tumour at laparoscopy. Omental biopsy showed a single layer of minimally atypical mesothelial cells with rare tiny foci of superficial invasion of fat. BAP1 immunostain showed loss of nuclear BAP1 in all the surface mesothelial cells and the invasive cells. There was CDKN2A deletion, but no deletion of NF2 by FISH. CONCLUSIONS: These cases show that morphologically bland single-layered surface mesothelial proliferations with molecular alterations seen previously only in invasive malignant mesotheliomas exist, and presumably represent malignant MIS. More cases are need to understand the frequency of such changes and the time-course over which invasive tumour develops.

Metabolic Signatures of Lung Cancer in Sputum and Exhaled Breath Condensate Detected by 1H Magnetic Resonance Spectroscopy: A Feasibility Study.

OBJECTIVES: Lung cancer is one of the most lethal cancers. Currently, there are no biomarkers for early detection, monitoring treatment response, and detecting recurrent lung cancer. We undertook this study to determine if 1H magnetic resonance spectroscopy (MRS) of sputum and exhaled breath condensate (EBC), as a noninvasive tool, can identify metabolic biomarkers of lung cancer. MATERIALS AND METHODS: Sputum and EBC samples were collected from 20 patients, comprising patients with pathologically confirmed non-small cell lung cancer (n = 10) and patients with benign respiratory conditions (n = 10). Both sputum and EBC samples were collected from 18 patients; 2 patients provided EBC samples only. 1H MR spectra were obtained on a Bruker Avance 400 MHz nuclear magnetic resonance (NMR) spectrometer. Sputum samples were further confirmed cytologically to distinguish between true sputum and saliva. RESULTS: In the EBC samples, median concentrations of propionate, ethanol, acetate, and acetone were higher in lung cancer patients compared to the patients with benign conditions. Median concentration of methanol was lower in lung cancer patients (0.028 mM) than in patients with benign conditions (0.067 mM; P = 0.028). In the combined sputum and saliva and the cytologically confirmed sputum samples, median concentrations of N-acetyl sugars, glycoprotein, propionate, lysine, acetate, and formate were lower in the lung cancer patients than in patients with benign conditions. Glucose was found to be consistently absent in the combined sputum and saliva samples (88%) as well as in the cytologically confirmed sputum samples (86%) of lung cancer patients. CONCLUSION: Absence of glucose in sputum and lower concentrations of methanol in EBC of lung cancer patients discerned by 1H MRS may serve as metabolic biomarkers of lung cancer for early detection, monitoring treatment response, and detecting recurrence.

The potential protective role of caveolin-1 in intestinal inflammation in TNBS-induced murine colitis.

BACKGROUND: Caveolin-1 (Cav-1) is a multifunctional scaffolding protein serving as a platform for the cell's signal-transduction and playing an important role in inflammation. However, its role in inflammatory bowel disease is not clear. A recent study showed that Cav-1 is increased and mediates angiogenesis in dextran sodium sulphate-induced colitis, which are contradictory to our pilot findings in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. In the present study, we further clarified the role of Cav-1 in TNBS-induced colitis. METHODS: In BALB/c mice, acute colitis was induced by intra-rectal administration of one dose TNBS, while chronic colitis was induced by administration of TNBS once a week for 7 weeks. To assess the effects of complete loss of Cav-1, Cav-1 knockout (Cav-1-/-) and control wild-type C57 mice received one TNBS administration. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified through ELISAs. Inflammation was evaluated through histological analysis. RESULTS: Colon Cav-1 levels were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation scores and proinflammatory cytokine levels (IL-17, IFN-γ and TNF) significantly. Furthermore, after administration of TNBS, Cav-1-/- mice showed significantly increased clinical and colon inflammatory scores and body weight loss when compared with control mice. CONCLUSIONS AND SIGNIFICANCE: Cav-1 may play a protective role in the development of TNBS-induced colitis. Our findings raise an important issue in the evaluation of specific molecules in animal models that different models may exhibit opposite results because of the different mechanisms involved.

Employing an IL-23 p19 vaccine to block IL-23 ameliorates chronic murine colitis.

BACKGROUND: Overexpression of IL-23 has been implicated in the pathogenesis of Crohn's disease. Using vaccines to block overexpressed endogenous cytokines has emerged as a new therapeutic strategy for the long-term treatment of the disease. AIM: We sought to develop peptide-based vaccines specific to IL-23 and evaluate their effects in colitis mice. MATERIALS & METHODS: The vaccine was developed by inserting a peptide derived from mouse IL-23 p19 into the carrier protein, hepatitis B core antigen, using molecular engineering methods. One vaccine against IL-23 p19 was obtained that induced high-titered and long-lasting antibodies to IL-23 without the use of adjuvants. The inhibitory effect of vaccine-immunized serum was subsequently evaluated in vitro. To evaluate the in vivo effects, mice were subcutaneously injected with the vaccine, carrier or saline three times. Two weeks after the last injection, chronic colitis was induced in mice by seven weekly administrations with 2,4,6-trinitrobenzene sulfonic acid. RESULTS: In vitro studies revealed that serum IL-23 p19-specific IgG significantly suppressed IL-23-induced IL-17 production by splenocytes. In vivo evaluation of the effect of the vaccine in mice with chronic colitis indicated that vaccine-immunized mice exhibited a decrease in colon inflammation, collagen deposition and levels of IL-23 and IL-12 cytokines, compared with control groups. CONCLUSION: IL-23 p19 vaccine is capable of downregulating inflammatory responses in chronic murine colitis, providing a novel therapeutic approach in Crohn's disease.

The role and potential therapeutic application of myeloid-derived suppressor cells in TNBS-induced colitis.

MDSCs, a heterogeneous population of cells that expand during many pathogenic conditions, have remarkable abilities to suppress T cell responses. Their role in murine colitis, induced by TNBS and therapeutic application, remains unclear. Murine colitis was induced through intrarectally administrating TNBS, twice. MDSCs in spleen and colonic LPMCs were identified using flow cytometric analysis. In adoptive transfer, MDSCs were isolated from spleen after TNBS challenges by using microbeads or generated in vitro by coculturing bone marrow cells with HSCs and then transferred into naïve mice. Two hours later, mice were then challenged with TNBS, once/week for 2 weeks. The mice were killed four days after the second TNBS delivery, and intestinal inflammation and cytokine levels and MDSC percentages were evaluated. The percentages of CD11b+Gr-1+MDSCs and subsets (CD11b+Ly6C+ and CD11b+Ly6G+MDSCs) were increased in spleen and/or colonic LPMCs in colitis mice and also correlated with the severity of intestinal inflammation. MDSCs isolated from colitis mice suppressed the proliferation of splenocytes in vitro. Adoptive transfer of MDSCs, isolated from colitis mice or generated in vitro, decreased intestinal inflammation, levels of IFN-γ, IL-17, and TNF, and percentages of spleen MDSCs when compared with controls. MDSCs that have inhibitory function in vitro and in vivo are increased and correlated with intestinal inflammation, suggesting that they may be used as a biomarker of disease activity and a cell-based biotherapy in IBD.