Environmental eustress promotes liver regeneration through the sympathetic regulation of type 1 innate lymphoid cells to increase IL-22 in mice.

BACKGROUND AND AIMS: The liver has the unique ability of regeneration, which is extremely important for restoring homeostasis after liver injury. Although clinical observations have revealed an association between psychological stress and the liver, whether stress has a causal influence on the liver regeneration remains markedly less defined. APPROACH AND RESULTS: Rearing rodents in an enriched environment (EE) can induce eustress or positive psychological stress. Herein, EE-induced eustress was found to significantly enhance the ability of liver regeneration after partial hepatectomy or carbon tetrachloride-induced liver injury based on the more rapid restoration of liver/body weight ratio and the significantly increased number of proliferating hepatocytes in EE mice. Mechanistically, the cytokine array revealed that IL-22 was markedly increased in the regenerating liver in response to EE. Blockade of IL-22 signaling abrogated the enhanced liver regeneration induced by EE. Group 1 innate lymphoid cells (ILCs), including type 1 ILCs (ILC1s), have been identified as the major sources of IL-22 in the regenerating liver. EE housing led to a rapid accumulation of hepatic ILC1s after partial hepatectomy and the EE-induced enhancement of liver regeneration and elevation of IL-22 was nearly eliminated in ILC1-deficient Tbx21-/- mice. Chemical sympathectomy or blockade of ß-adrenergic signaling also abolished the effect of EE on ILC1s and attenuated the enhanced liver regeneration of EE-housed mice. CONCLUSION: The study findings support the brain-liver axis and suggest that environment-induced eustress promotes liver regeneration through the sympathetic nerve/ILC1/IL-22 axis.

C-C motif chemokine ligand 5 confines liver regeneration by down-regulating reparative macrophage-derived hepatocyte growth factor in a forkhead box O 3a-dependent manner.

BACKGROUND AND AIMS: Liver regeneration (LR) is vital for the recovery of liver function after hepatectomy. Limited regeneration capacity, together with insufficient remnant liver volume, is a risk factor for posthepatectomy liver failure (PHLF) resulting from small-for-size syndrome. Although inflammation plays an important role in controlling LR, the underlying mechanisms still remain obscure. APPROACH AND RESULTS: We identified C-C motif chemokine ligand (CCL) 5 as an important negative regulator for LR. CCL5 levels were elevated after partial hepatectomy (PHx), both in healthy donors of living donor liver transplantation (LT) and PHx mouse models. Ccl5 knockout mice displayed improved survival after 90% PHx and enhanced LR 36 h after 70% PHx. However, primary hepatocytes from Ccl5-/- mice exposed to growth factors in vitro showed no proliferation advantage compared to those from wild-type (WT) mice. Flow cytometry analysis showed that proportions of Ly6Clo macrophages were significantly increased in Ccl5-/- mice after 70% PHx. RNA-sequencing analysis revealed that sorted macrophages (CD11b+ Ly6Clo&hi ) manifested enhanced expression of reparative genes in Ccl5-/- mice compared to WT mice. Mechanistically, CCL5 induced macrophages toward proinflammatory Ly6Chi phenotype, thereby inhibiting the production of hepatocyte growth factor (HGF) through the C-C motif chemokine receptor (CCR) 1- and CCR5-mediated forkhead box O (FoxO) 3a pathways. Finally, blockade of CCL5 greatly optimized survival and boosted LR in the mouse PHx model. CONCLUSIONS: Our findings suggest that inhibition of CCL5 is a promising strategy to improve regeneration restoration by enhancing HGF secretion from reparative macrophages through the FoxO3a pathway, which may potentially reduce the mortality of PHLF.

Incremental value of radiomics-based heterogeneity to the existing risk criteria in predicting recurrence of hepatocellular carcinoma after liver transplantation.

OBJECTIVES: The aim of the study was to evaluate the association between the radiomics-based intratumoral heterogeneity (ITH) and the recurrence risk in hepatocellular carcinoma (HCC) patients after liver transplantation (LT), and to assess its incremental to the Milan, University of California San Francisco (UCSF), Metro-Ticket 2.0, and Hangzhou criteria. METHODS: A multicenter cohort of 196 HCC patients were investigated. The endpoint was recurrence-free survival (RFS) after LT. A CT-based radiomics signature (RS) was constructed and assessed in the whole cohort and in the subgroups stratified by the Milan, UCSF, Metro-Ticket 2.0, and Hangzhou criteria. The R-Milan, R-UCSF, R-Metro-Ticket 2.0, and R-Hangzhou nomograms which combined RS and the four existing risk criteria were developed respectively. The incremental value of RS to the four existing risk criteria in RFS prediction was evaluated. RESULTS: RS was significantly associated with RFS in the training and test cohorts as well as in the subgroups stratified by the existing risk criteria. The four combined nomograms showed better predictive capability than the existing risk criteria did with higher C-indices (R-Milan [training/test] vs. Milan, 0.745/0.765 vs. 0.677; R-USCF vs. USCF, 0.748/0.767 vs. 0.675; R-Metro-Ticket 2.0 vs. Metro-Ticket 2.0, 0.756/0.783 vs. 0.670; R-Hangzhou vs. Hangzhou, 0.751/0.760 vs. 0.691) and higher clinical net benefit. CONCLUSIONS: The radiomics-based ITH can predict outcomes and provide incremental value to the existing risk criteria in HCC patients after LT. Incorporating radiomics-based ITH in HCC risk criteria may facilitate candidate selection, surveillance, and adjuvant trial design. KEY POINTS: • Milan, USCF, Metro-Ticket 2.0, and Hangzhou criteria may be insufficient for outcome prediction in HCC after LT. • Radiomics allows for the characterization of tumor heterogeneity. • Radiomics adds incremental value to the existing criteria in outcome prediction.

Identification and characterization of a 25-lncRNA prognostic signature for early recurrence in hepatocellular carcinoma.

BACKGROUND: Early recurrence is the major cause of poor prognosis in hepatocellular carcinoma (HCC). Long non-coding RNAs (lncRNAs) are deeply involved in HCC prognosis. In this study, we aimed to establish a prognostic lncRNA signature for HCC early recurrence. METHODS: The lncRNA expression profile and corresponding clinical data were retrieved from total 299 HCC patients in TCGA database. LncRNA candidates correlated to early recurrence were selected by differentially expressed gene (DEG), univariate Cox regression and least absolute shrinkage and selection operator (LASSO) regression analyses. A 25-lncRNA prognostic signature was constructed according to receiver operating characteristic curve (ROC). Kaplan-Meier and multivariate Cox regression analyses were used to evaluate the performance of this signature. ROC and nomogram were used to evaluate the integrated models based on this signature with other independent clinical risk factors. Gene set enrichment analysis (GSEA) was used to reveal enriched gene sets in the high-risk group. Tumor infiltrating lymphocytes (TILs) levels were analyzed with single sample Gene Set Enrichment Analysis (ssGSEA). Immune therapy response prediction was performed with TIDE and SubMap. Chemotherapeutic response prediction was conducted by using Genomics of Drug Sensitivity in Cancer (GDSC) pharmacogenomics database. RESULTS: Compared to low-risk group, patients in high-risk group showed reduced disease-free survival (DFS) in the training (p < 0.0001) and validation cohort (p = 0.0132). The 25-lncRNA signature, AFP, TNM and vascular invasion could serve as independent risk factors for HCC early recurrence. Among them, the 25-lncRNA signature had the best predictive performance, and combination of those four risk factors further improves the prognostic potential. Moreover, GSEA showed significant enrichment of "E2F TARGETS", "G2M CHECKPOINT", "MYC TARGETS V1" and "DNA REPAIR" pathways in the high-risk group. In addition, increased TILs were observed in the low-risk group compared to the high-risk group. The 25-lncRNA signature negatively associates with the levels of some types of antitumor immune cells. Immunotherapies and chemotherapies prediction revealed differential responses to PD-1 inhibitor and several chemotherapeutic drugs in the low- and high-risk group. CONCLUSIONS: Our study proposed a 25-lncRNA prognostic signature for predicting HCC early recurrence, which may guide postoperative treatment and recurrence surveillance in HCC patients.

Characterization of hepatitis B virus DNA integration patterns in intrahepatic cholangiocarcinoma.

AIM: Hepatitis B virus (HBV) integration is one of the mechanisms contributing to hepatocellular carcinoma (HCC) development. However, the status of HBV integration in intrahepatic cholangiocarcinoma (ICC) is poorly understood. This study aims to characterize the viral integration in HBV-related ICC. METHODS: The presence of HBV S and C gene in ICCs and the paratumor tissue was determined by polymerase chain reaction direct sequencing. Hepatitis B virus integration was detected by a high-throughput capture sequencing method. The expression analysis of the genes targeted by HBV in ICC was undertaken in The Cancer Genome Atlas dataset. RESULTS: Hepatitis B virus S and/or C gene fragments were detected in 71.43% (10/14) ICCs and 57.14% (8/14) paratumor tissues. Using the high-throughput capture sequencing approach, 139 and 183 HBV integration breakpoints were identified from seven ICC and seven paired paratumor tissues, respectively. Seven genes (TERT, CEACAM20, SPATA18, TRERF1, ZNF23, LINC01449, and LINC00486) were recurrently targeted by HBV-DNA in different ICC tissues or different cell populations of the same tissue. TERT, which is the most preferential HBV target gene in HCC, was found to be repeatedly interrupted by HBV-DNA in three different ICC tissues. Based on The Cancer Genome Atlas dataset, TERT, as well as three other HBV recurrently targeted genes (SPATA18, TRERF1, and ZNF23), showed differential expression levels between ICC and para-ICC tissues. CONCLUSIONS: Taken together, HBV integration is a common event in HBV-related ICC. The HBV recurrent integration genes identified from this study, such as TERT, provide new clues for further research on the causative link between HBV infection and ICC.

Characterization of hepatitis B virus infection and viral DNA integration in non-Hodgkin lymphoma.

Hepatitis B virus (HBV) infection has been reported to be associated with non-Hodgkin lymphoma (NHL). However, the evidence is limited to the seroepidemiological study. There is a lack of evidence showing the HBV infection and integration in NHL cells. Here, we reported that in the Shanghai area, the positive rates of serum HBsAg (OR: 3.11; 95% CI: 2.20-4.41) and HBeAg (OR: 3.99; 95% CI: 1.73-9.91) were significantly higher in patients with NHL. HBsAg, HBcAg and HBV DNA were detected in 34.4%, 45.2% and 47.0% of the NHL tissues, respectively. Furthermore, by using a high-throughput viral integration detection approach (HIVID), integrated HBV DNA was identified from 50% (6/12) HBV-related NHL tissues. There were a total of 313 HBV integration sites isolated from the NHL tissues, among which four protein-coding genes (FAT2, SETX, ITGA10 and CD63) were interrupted by HBV DNA in their exons. Seven HBV preferential target genes (ANKS1B, HDAC4, EGFLAM, MAN1C1, XKR6, ZBTB38 and CCDC91) showed significantly altered expression levels in NHL, suggesting a potential role of these genes in NHL development. Taken together, HBV integration is a common phenomenon in NHL. This finding opens up a new direction of research into the mechanistic link between HBV infection and NHL.

ID3 Promotes Stem Cell Features and Predicts Chemotherapeutic Response of Intrahepatic Cholangiocarcinoma.

Cancer stem cells contribute to a high rate of recurrence and chemotherapeutic resistance in many types of cancer, including intrahepatic cholangiocarcinoma (ICC). Inhibitor of differentiation 3 (ID3) has been reported to promote cancer stem cells, but its role in ICC is obscure. In this study, we identified that ID3 is highly expressed in human ICC tissues compared with matched normal tissues and correlates with poor prognosis. Functional studies demonstrate that ID3 is required for stemness maintenance in cholangiocarcinoma both in vitro and in vivo. Consistent with the regulation of cancer stem cell features by ID3, transgenic expression of ID3 enhances chemoresistance of cholangiocarcinoma cells. Moreover, we found that ICC patients with low ID3 levels benefited from postoperative transarterial chemoembolization, whereas patients with high ID3 levels did not, indicating the significance of ID3 in individualized ICC therapy. Mechanistically, ID3 could interact with E47 and block E47 recruitment to the promoter of ß-catenin, which leads to activation of Wnt/ß-catenin signaling. Conclusion: Our results show that ID3 could promote the stemness of ICC by increasing the transcriptional activity of ß-catenin and could serve as a biomarker in predicting ICC patients' response to adjuvant chemotherapeutics.

Risk stratification system to predict recurrence of intrahepatic cholangiocarcinoma after hepatic resection.

BACKGROUND: Previous nomograms for intrahepatic cholangiocarcinoma (ICC) were conducted to predict overall survival, which could be influenced by various factors. Herein, we conducted our nomogram to predict recurrence of the tumor only after hepatic resection. METHODS: The nomogram was established with prognostic factors for the relapse-free survival (RFS) analyzed from our single center cohort and was evaluated by comparing with the American Joint Committee on Cancer (AJCC) staging system for the predictive accuracy. RESULTS: Seropositivity of hepatitis B surface antigen (hazard ratio [HR], 0.505; 95% confidence interval [CI], 0.279 to 0.914; P = 0.024), tumor size of larger than 5 cm (HR, 1.947; 95% CI, 1.177 to 3.219; P = 0.009), Child-Pugh score of B (HR, 3.067; 95% CI, 1.293 to 7.275; P = 0.011), and lymph node metastasis (HR, 2.790; 95% CI, 1.628 to 4.781; P < 0.001) were found to be independent prognostic factors that significantly affected RFS. The calibration curve for the prediction revealed excellent agreement between estimation by our stratification system and actual RFS. The concordance C index of the nomogram (0.71; 95% CI, 0.65 to 0.77) revealed to be significantly higher than the AJCC staging system (0.66; 95% CI, 0.60 to 0.72). In the validation cohort, our risk stratification system (C-index 0.65; 95% CI, 0.59 to 0.71) also revealed more precise prediction than the AJCC staging system (C-index, 0.57; 95% CI, 0.50 to 0.64). CONCLUSIONS: Our nomogram could more accurately predict recurrence of ICC after hepatic resection than the AJCC staging system.

ZBP1-mediated apoptosis and inflammation exacerbate steatotic liver ischemia/reperfusion injury.

Steatotic donor livers are becoming more and more common in liver transplantation. However, the current use of steatotic grafts is less acceptable than normal grafts due to their higher susceptibility to ischemia/reperfusion (I/R) injury. To investigate the mechanism underlying the susceptibility of steatotic liver to I/R injury, we detected cell death markers and inflammation in clinical donor livers and animal models. We found that caspase-8-mediated hepatic apoptosis is activated in steatotic liver I/R injury. However, ablation of caspase-8 only slightly mitigated steatotic liver I/R injury without affecting inflammation. We further demonstrated that RIPK1 kinase induces both caspase-8-mediated apoptosis and cell death-independent inflammation. Inhibition of RIPK1 kinase significantly protects against steatotic liver I/R injury by alleviating both hepatic apoptosis and inflammation. Additionally, we found that RIPK1 activation is induced by Z-DNA binding protein 1 (ZBP1) but not the canonical TNF-α pathway during steatotic liver I/R injury. Deletion of ZBP1 substantially decreases the steatotic liver I/R injury. Mechanistically, ZBP1 is amplified by palmitic acid-activated JNK pathway in steatotic livers. Upon I/R injury, excessive reactive oxygen species trigger ZBP1 activation by inducing its aggregation independent of the Z-nucleic acids sensing action in steatotic livers, leading to the kinase activation of RIPK1 and the subsequent aggravation of liver injury. Thus, ZBP1-mediated RIPK1-driven apoptosis and inflammation exacerbate steatotic liver I/R injury, which could be targeted to protect steatotic donor livers during transplantation.

Necroptosis-Mediated Synergistic Photodynamic and Glutamine-Metabolic Therapy Enabled by a Biomimetic Targeting Nanosystem for Cholangiocarcinoma.

Targeted delivery of glutamine metabolism inhibitors holds promise for cholangiocarcinoma therapy, yet effective delivery vehicles remain a challenge. This study reports the development of a biomimetic nanosystem, termed R-CM@MSN@BC, integrating mesoporous organosilicon nanoparticles with reactive oxygen species-responsive diselenide bonds for controlled release of the glutamine metabolism inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and the photosensitizer Ce6. Erythrocyte membrane coating, engineered with Arg-Gly-Asp (RGD) peptides, not only enhanced biocompatibility but also improved tumor targeting and tissue penetration. Upon laser irradiation, R-CM@MSN@BC executed both photodynamic and glutamine-metabolic therapies, inducing necroptosis in tumor cells and triggering significant immunogenic cell death. Time-of-flight mass cytometry analysis revealed that R-CM@MSN@BC can remodel the immunosuppressive tumor microenvironment by polarizing M1-type macrophages, reducing infiltration of M2-type and CX3CR1+ macrophages, and decreasing T cell exhaustion, thereby increasing the effectiveness of anti-programmed cell death ligand 1 immunotherapy. This strategy proposed in this study presents a viable and promising approach for the treatment of cholangiocarcinoma.

Dual FGFR and VEGFR inhibition synergistically restrain hexokinase 2-dependent lymphangiogenesis and immune escape in intrahepatic cholangiocarcinoma.

BACKGROUND: Therapies for cholangiocarcinoma are largely limited and ineffective. Herein, we examined the role of the FGF and VEGF pathways in regulating lymphangiogenesis and PD-L1 expression in intrahepatic cholangiocarcinoma (iCCA). METHODS: The lymphangiogenic functions of FGF and VEGF were evaluated in lymphatic endothelial cells (LECs) and iCCA xenograft mouse models. The relationship between VEGF and hexokinase 2 (HK2) was validated in LECs by western blot, immunofluorescence, ChIP and luciferase reporter assays. The efficacy of the combination therapy was assessed in LECs and xenograft models. Microarray analysis was used to evaluate the pathological relationships of FGFR1 and VEGFR3 with HK2 in human lymphatic vessels. RESULTS: FGF promoted lymphangiogenesis through c-MYC-dependent modulation of HK2 expression. VEGFC also upregulated HK2 expression. Mechanistically, VEGFC phosphorylated components of the PI3K/Akt/mTOR axis to upregulate HIF-1α expression at the translational level, and HIF-1α then bound to the HK2 promoter region to activate its transcription. More importantly, dual FGFR and VEGFR inhibition with infigratinib and SAR131675 almost completely inhibited lymphangiogenesis, and significantly suppressed iCCA tumor growth and progression by reducing PD-L1 expression in LECs. CONCLUSIONS: Dual FGFR and VEGFR inhibition inhibits lymphangiogenesis through suppression of c-MYC-dependent and HIF-1α-mediated HK2 expression, respectively. HK2 downregulation decreased glycolytic activity and further attenuated PD-L1 expression. Our findings suggest that dual FGFR and VEGFR blockade is an effective novel combination strategy to inhibit lymphangiogenesis and improve immunocompetence in iCCA.

UDP-glucuronate metabolism controls RIPK1-driven liver damage in nonalcoholic steatohepatitis.

Hepatocyte apoptosis plays an essential role in the progression of nonalcoholic steatohepatitis (NASH). However, the molecular mechanisms underlying hepatocyte apoptosis remain unclear. Here, we identify UDP-glucose 6-dehydrogenase (UGDH) as a suppressor of NASH-associated liver damage by inhibiting RIPK1 kinase-dependent hepatocyte apoptosis. UGDH is progressively reduced in proportion to NASH severity. UGDH absence from hepatocytes hastens the development of liver damage in male mice with NASH, which is suppressed by RIPK1 kinase-dead knockin mutation. Mechanistically, UGDH suppresses RIPK1 by converting UDP-glucose to UDP-glucuronate, the latter directly binds to the kinase domain of RIPK1 and inhibits its activation. Recovering UDP-glucuronate levels, even after the onset of NASH, improved liver damage. Our findings reveal a role for UGDH and UDP-glucuronate in NASH pathogenesis and uncover a mechanism by which UDP-glucuronate controls hepatocyte apoptosis by targeting RIPK1 kinase, and suggest UDP-glucuronate metabolism as a feasible target for more specific treatment of NASH-associated liver damage.

Early detection of hepatocellular carcinoma via no end-repair enzymatic methylation sequencing of cell-free DNA and pre-trained neural network.

BACKGROUND: Early detection of hepatocellular carcinoma (HCC) is important in order to improve patient prognosis and survival rate. Methylation sequencing combined with neural networks to identify cell-free DNA (cfDNA) carrying aberrant methylation offers an appealing and non-invasive approach for HCC detection. However, some limitations exist in traditional methylation detection technologies and models, which may impede their performance in the read-level detection of HCC. METHODS: We developed a low DNA damage and high-fidelity methylation detection method called No End-repair Enzymatic Methyl-seq (NEEM-seq). We further developed a read-level neural detection model called DeepTrace that can better identify HCC-derived sequencing reads through a pre-trained and fine-tuned neural network. After pre-training on 11 million reads from NEEM-seq, DeepTrace was fine-tuned using 1.2 million HCC-derived reads from tumor tissue DNA after noise reduction, and 2.7 million non-tumor reads from non-tumor cfDNA. We validated the model using data from 130 individuals with cfDNA whole-genome NEEM-seq at around 1.6X depth. RESULTS: NEEM-seq overcomes the drawbacks of traditional enzymatic methylation sequencing methods by avoiding the introduction of unmethylation errors in cfDNA. DeepTrace outperformed other models in identifying HCC-derived reads and detecting HCC individuals. Based on the whole-genome NEEM-seq data of cfDNA, our model showed high accuracy of 96.2%, sensitivity of 93.6%, and specificity of 98.5% in the validation cohort consisting of 62 HCC patients, 48 liver disease patients, and 20 healthy individuals. In the early stage of HCC (BCLC 0/A and TNM I), the sensitivity of DeepTrace was 89.6 and 89.5% respectively, outperforming Alpha Fetoprotein (AFP) which showed much lower sensitivity in both BCLC 0/A (50.5%) and TNM I (44.7%). CONCLUSIONS: By combining high-fidelity methylation data from NEEM-seq with the DeepTrace model, our method has great potential for HCC early detection with high sensitivity and specificity, making it potentially suitable for clinical applications. DeepTrace: https://github.com/Bamrock/DeepTrace.

Efficacy and safety of liver transplantation in patients with cholangiocarcinoma: a systematic review and meta-analysis.

The aim of our study was to evaluate the efficacy and safety of liver transplantation in patients with cholangiocarcinoma. According to the requirements of Cochrane systematic review, a thorough literature search was performed in PubMed/Medline, Embase and Cochrane electronic databases between 1995 and 2009 in terms of the key words "liver transplantation" and "cholangiocarcinoma," "cholangiocellular carcinoma" or "bile duct cancer," with restricted articles for the English language. Data were processed for a meta-analysis by Stata 10 software. Altogether 14 clinical trials containing 605 transplanted patients of bile duct cancers were finally enrolled in our study. The overall 1-, 3- and 5-year pooled survival rates were 0.73 [95% confidence interval (CI) = 0.65-0.80], 0.42 (95% CI = 0.33-0.51) and 0.39 (95% CI = 0.28-0.51), respectively. Of note, preoperative adjuvant therapies [orthotopic liver transplantation (OLT)-PAT group] rendered the transplanted individuals with comparably favorable outcomes with 1-, 3- and 5-year pooled survival rates of 0.83 (95% CI = 0.57-0.98), 0.57 (95% CI = 0.18-0.92) and 0.65 (95% CI = 0.40-0.87). In addition, the overall pooled incidence of complications was 0.62 (95% CI = 0.44-0.78), among which that of OLT-PAT group (0.58; 95% CI = 0.20-0.92) was relatively acceptable compared to those of liver transplantation alone (0.61; 95% CI = 0.33-0.85) and liver transplantation with extended bile duct resection (0.78; 95% CI = 0.55-0.94). In comparison to curative resection of cholangiocarcinoma with the 5-year survival rate reported from 20 to 40%, the role of liver transplantation alone is so limited. In the future, attention will be focused on liver transplantation following neoadjuvant radiochemotherapy, which requires a well-designed, prospective randomized controlled study.

The influence of membrane molecular weight cutoff on a novel bioartificial liver.

Given the xenogeneic immune reaction relevant to the molecular weight cutoff of the membrane of a bioartificial liver (BAL) system, we investigated the influence of membrane molecular weight cutoff in our BAL system in this study. Acute liver failure in beagles was induced by d-galactosamine administration. Eight beagles were divided into two groups by the membrane molecular weight cutoff of the plasma component separator. Group 1 beagles were treated with BAL containing 200 kDa retention rating membrane. Group 2 beagles were treated with BAL containing 1200 kDa retention rating membrane. Each group underwent two 6-h BAL treatments that were performed on day 1 and day 21. The hemodynamic and hematologic response, humoral immune responses, and cytotoxic immune response to BAL therapy were studied before and after treatments. All beagles remained hemodynamically and hematologically stable during BAL treatments. BAL treatment was associated with a significant decline in levels of complement; however, a longer time of level maintenance was observed in Group 2. Group 2 beagles experienced a significant increase in levels of IgG and IgM after two BAL treatments. Significant levels of canine proteins were detected in BAL medium from Group 2; only trace levels of canine proteins were detected in BAL medium from Group 1. The posttreatment viability of co-culture cells in Group 2 was lower compared with Group 1, and the viability of co-culture cells after treatments was associated with deposition of canine proteins on the cells. Xenogeneic immune response was influenced by membrane molecular weight cutoff in the BAL.