Enhancing the correlation between in vitro and in vivo experiments in dental implant osseointegration: investigating the role of Ca ions.

This study delves into the osteogenic potential of a calcium-ion modified titanium implant surface, unicCa, employing state-of-the-art proteomics techniques both in vitro (utilizing osteoblasts and macrophage cell cultures) and in vivo (in a rabbit condyle model). When human osteoblasts (Hobs) were cultured on unicCa surfaces, they displayed a marked improvement in cell adhesion and differentiation compared to their unmodified counterparts. The proteomic analysis also revealed enrichment in functions associated with cell migration, adhesion, extracellular matrix organization, and proliferation. The analysis also underscored the involvement of key signalling pathways such as PI3K-Akt and mTOR. In the presence of macrophages, unicCa initially exhibited improvement in immune-related functions and calcium channel activities at the outset (1 day), gradually tapering off over time (3 days). Following a 5-day implantation in rabbits, unicCa demonstrated distinctive protein expression profiles compared to unmodified surfaces. The proteomic analysis highlighted shifts in adhesion, immune response, and bone healing-related proteins. unicCa appeared to influence the coagulation cascade and immune regulatory proteins within the implant site. In summary, this study provides a comprehensive proteomic analysis of the unicCa surface, drawing correlations between in vitro and in vivo results. It emphasizes the considerable potential of unicCa surfaces in enhancing osteogenic behavior and immunomodulation. These findings significantly contribute to our understanding of the intricate molecular mechanisms governing the interplay between biomaterials and bone cells, thereby facilitating the development of improved implant surfaces for applications in bone tissue engineering.

Using osteogenic medium in the in vitro evaluation of bone biomaterials: Artefacts due to a synergistic effect.

In vitro tests using bone cells to evaluate the osteogenic potential of biomaterials usually employ the osteogenic medium (OM). The lack of correlation frequently reported between in vitro and in vivo studies in bone biomaterials, makes necessary the evaluation of the impact of osteogenic supplements on these results. This study analysed the proteomic profiles of human osteoblasts (HOb) cultured in the media with and without osteogenic agents (ascorbic acid and ß-glycerol phosphate). The cells were incubated for 1 and 7 days, on their own or in contact with Ti. The comparative Perseus analysis identified 2544 proteins whose expression was affected by osteogenic agents. We observed that the OM strongly alters protein expression profiles with a complex impact on multiple pathways associated with adhesion, immunity, oxidative stress, coagulation, angiogenesis and osteogenesis. OM-triggered changes in the HOb intracellular energy production mechanisms, with key roles in osteoblast maturation. HOb cultured with and without Ti showed enrichment in the skeletal system development function due to the OM. However, differentially expressed proteins with key regenerative functions were associated with a synergistic effect of OM and Ti. This synergy, caused by the Ti-OM interaction, could complicate the interpretation of in vitro results, highlighting the need to analyse this phenomenon in biomaterial testing.

Comparison of patient-centered outcomes measures between low-speed drilling without irrigation and high-speed drilling with irrigation: A randomized clinical trial.

OBJECTIVE: To compare patient satisfaction during surgery, postoperative pain and inflammation and quality of life between high-speed drilling with irrigation and low-speed drilling without irrigation for implant bed preparation. MATERIALS AND METHODS: Sixty-six posterior single edentulous patients were included in a randomized controlled clinical trial. Implant beds were created using high-speed drilling with irrigation (control group) or low-speed drilling without irrigation (test group). Patient satisfaction during surgery (in relation to drilling-time perception, vibration, pressure, noise, comfort, and drowning sensation) and postoperative pain and inflammation were evaluated using a 100-mm visual analogue scale (VAS)-based questionnaire. Quality of life was analyzed with a Likert scale (in relation to mouth opening, chewing, speaking, sleeping, daily routine, and job). The follow-up period was 7 days. RESULTS: Patient satisfaction in relation to drilling-time perception, vibration, pressure, and noise did not show statistically significant differences (p > .05). The highest scores of drowning sensation (p < .05) were correlated (moderate correlation (r = .57)) with lowest scores of comfort (p < .005). Both postoperative pain and inflammation means were significantly higher in the control group than in the test group. No significant differences in quality of life were observed during the postoperative period (p > .05). CONCLUSION: Low-speed drilling without irrigation for single implant site preparation was more comfortable for patients than high-speed drilling with irrigation, due to the correlation between important drowning sensation and low perceived comfort. Postoperative pain and inflammation were lower for low-speed drilling without irrigation. Further studies are needed to validate or refute these results.

Proteomics as a tool to study the osteoimmunomodulatory role of metallic ions in a sol-gel coating.

The success of bone implants depends on the osteoimmunomodulatory (OIM) activity of the biomaterials in the interactions with the periimplantary tissues. Many in vitro tests have been conducted to evaluate the osteoimmunology effects of biomaterials. However, results of these tests have often been inconclusive. This study examines the properties of newly developed sol-gel coatings doped with two metal ions associated with bone regeneration, Ca and Zn. The study uses both proteomic methods and traditional in vitro assays. The results demonstrate that proteomics is an effective tool to scrutinize the OIM properties of the materials. Moreover, sol-gel coatings offer excellent base materials to evaluate the effects of metal ions on these properties. The obtained data highlight the highly tunable nature of sol-gel materials; studying the materials with different doping levels supplies valuable information on the interactions between the immune and bone-forming processes.

Influence of abutment shape on peri-implant tissue conditions: A randomized clinical trial.

OBJECTIVE: To analyze the influence of 3-mm high abutments with different shapes (cylindrical abutment vs. wide abutment) on marginal bone-level changes (bone loss and bone remodeling). The influence of abutment shape on implant success, probing pocket depth (PPD), and bleeding on probing (BoP) was studied as secondary objectives. MATERIALS AND METHODS: Patients with a partially edentulous area requiring fixed dental prostheses by two implants in the posterior mandible or maxilla were included. The implants were 1 mm subcrestally placed, and osseointegration healing was submerged. Three-mm high abutments with two different shapes were randomly placed in second-stage surgery: cylindrical abutments (cylindrical group) and wide abutments (wide group). Marginal bone-level changes were measured using parallelized periapical radiographs at abutment placement, at definitive prosthesis placement, and at 1, 3, 6, and 12 months after loading. PPD and BoP were likewise measured at the control visits. RESULTS: Sixty-four dental implants in 25 patients were included. Statistically significant differences were found in bone-level changes. The cylindrical group exhibited less mean marginal bone remodeling (MBR) and marginal bone loss (MBL) than the wide group (p < .05). Moreover, the cylindrical group showed significantly less BoP (p < .05). CONCLUSION: Abutment shape had a significant influence upon marginal bone-level changes during the first 12 months. Cylindrical abutments caused less MBR and MBL than wide abutments. More clinical studies involving longer follow-ups and analyzing other abutment modifications are needed to improve our understanding of how abutments can affect peri-implant tissue stability.

Proteomic Analysis of Mesenchymal Stem Cells and Monocyte Co-Cultures Exposed to a Bioactive Silica-Based Sol-Gel Coating.

New methodologies capable of extensively analyzing the cell-material interactions are necessary to improve current in vitro characterization methods, and proteomics is a viable alternative. Also, many studies are focused on monocultures, even though co-cultures model better the natural tissue. For instance, human mesenchymal stem cells (MSCs) modulate immune responses and promote bone repair through interaction with other cell types. Here, label-free liquid chromatography tandem mass spectroscopy proteomic methods were applied for the first time to characterize HUCPV (MSC) and CD14+ monocytes co-cultures exposed to a bioactive sol-gel coating (MT). PANTHER, DAVID, and STRING were employed for data integration. Fluorescence microscopy, enzyme-linked immunosorbent assay, and ALP activity were measured for further characterization. Regarding the HUCPV response, MT mainly affected cell adhesion by decreasing integrins, RHOC, and CAD13 expression. In contrast, MT augmented CD14+ cell areas and integrins, Rho family GTPases, actins, myosins, and 14-3-3 expression. Also, anti-inflammatory (APOE, LEG9, LEG3, and LEG1) and antioxidant (peroxiredoxins, GSTO1, GPX1, GSHR, CATA, and SODM) proteins were overexpressed. On co-cultures, collagens (CO5A1, CO3A1, CO6A1, CO6A2, CO1A2, CO1A1, and CO6A3), cell adhesion, and pro-inflammatory proteins were downregulated. Thus, cell adhesion appears to be mainly regulated by the material, while inflammation is impacted by both cellular cross-talk and the material. Altogether, we conclude that applied proteomic approaches show its potential in biomaterial characterization, even in complex systems.

Insight into the antibacterial mechanism of Cu-enriched sol-gel coatings employing proteomics.

Advanced antibacterial biomaterials can help reduce the severe consequences of infections. Using copper compounds is an excellent option to achieve this goal; they offer a combination of regenerative and antimicrobial functions. In this study, new CuCl2-doped sol-gel coatings were developed and physicochemically characterised. Their osteogenic and inflammatory responses were tested in vitro using human osteoblasts and THP-1 macrophages. Their antibacterial effect was evaluated using Escherichia coli and Staphylococcus aureus. The Cu influence on the adsorption of human serum proteins was analysed employing proteomics. The materials released Cu2+ and were not cytotoxic. The osteoblasts in contact with these materials showed an increased ALP, BMP2 and OCN gene expression. THP-1 showed an increase in pro-inflammatory markers related to M1 polarization. Moreover, Cu-doped coatings displayed a potent antibacterial behaviour against E. coli and S. aureus. The copper ions affected the adsorption of proteins related to immunity, coagulation, angiogenesis, fibrinolysis, and osteogenesis. Interestingly, the coatings had increased affinity to proteins with antibacterial functions and proteins linked to the complement system activation that can lead to direct bacterial killing via large pore-forming complexes. These results contribute to our understanding of the antibacterial mechanisms of Cu-biomaterials and their interaction with biological systems.

Proteomic evaluation of human osteoblast responses to titanium implants over time.

Titanium is widely used in bone prostheses due to its excellent biocompatibility and osseointegration capacity. To understand the effect of sandblasted acid-etched (SAE) Ti implants on the biological responses of human osteoblast (HOb), their proteomic profiles were analyzed using nLC-MS/MS. The cells were cultured with the implant materials, and 2544 distinct proteins were detected in samples taken after 1, 3, and 7 days. Comparative analyses of proteomic data were performed using Perseus software. The expression of proteins related to EIF2, mTOR, insulin-secretion and IGF pathways showed marked differences in cells grown with SAE-Ti in comparison with cells cultured without Ti. Moreover, the proteomic profiles obtained with SAE-Ti were compared over time. The affected proteins were related to adhesion, immunity, oxidative stress, coagulation, angiogenesis, osteogenesis, and extracellular matrix formation functions. The proliferation, mineralization and osteogenic gene expression in HObs cultured with SAE-Ti were characterized in vitro. The results showed that the osteoblasts exposed to this material increase their mineralization rate and expression of COLI, RUNX2, SP7, CTNNB1, CAD13, IGF2, MAPK2, and mTOR. Overall, the observed proteomic profiles can explain the SAE-Ti osteogenic properties, widening our knowledge of key signaling pathways taking part in the early stages of the osseointegration process in this type of implantations.

The effect of calcium-magnesium mixtures in sol-gel coatings on bone tissue regeneration.

Calcium and magnesium are two elements essential for bone structure and metabolism. However, their synergistic or competitive effects on bone regeneration are often overlooked during biomaterial development. We examined the interactions between Ca and Mg in sol-gel coatings doped with mixtures of CaCl2 (0.5%) and MgCl2 (0.5, 1, and 1.5%). After physicochemical characterisation, the materials were incubated in vitro with MC3T3-E1 osteoblastic cells and RAW264.7 macrophages, and the protein adsorption was analysed using nLC-MS/MS. The incorporation of the ions did not lead to the formation of crystalline structures and did not affect the sol-gel network cross-linking. The release of the ions did not cause cytotoxic effects at any tested concentration. The proteomic analysis showed that adding the Ca and Mg ions elevated the adsorption of proteins associated with inflammatory response regulation (e.g., ALBU, CLUS, HPT, HPTR, A1AG1 and A1AG2) but decreased the adsorption of immunoglobulins. The CaMg coatings had reduced affinity to proteins associated with coagulation (e.g., FA9, FA10, FA11, FA12) but increased the adsorption of proteins involved in cell adhesion (DSG1, DESP, FBLN1, ZA2G). In vitro assays revealed that the cellular response was affected by changing the concentration of Mg. Moreover, our results show that these differences reflect the changes in the concentrations of both ions in the mix but are not a simple additive effect.

Complex effects of Mg-biomaterials on the osteoblast cell machinery: A proteomic study.

The cell-biomaterial interface is highly complex; thousands of molecules and many processes participate in its formation. Growing demand for improved biomaterials has highlighted the need to understand the structure and functions of this interface. Proteomic methods offer a viable alternative to the traditional in vitro techniques for analyzing such systems. Magnesium is a promoter of cell adhesion and osteogenesis. Here, we used the LC-MS/MS to compare the protein expression profiles of human osteoblasts (HOb) exposed to sol-gel coatings without (MT) and with Mg (MT1.5Mg) for 1, 3, and 7 days. PANTHER, DAVID, and IPA databases were employed for protein identification and data analysis. Confocal microscopy and gene expression analysis were used for further characterization. Exposure to MT1.5Mg increased the HOb cell area and the expression of SP7, RUNX2, IBP3, COL3A1, MXRA8, and FBN1 genes. Proteomic analysis showed that MT1.5Mg affected the early osteoblast maturation (PI3/AKT, mTOR, ERK/MAPK), insulin metabolism, cell adhesion (integrin, FAK, actin cytoskeleton regulation) and oxidative stress pathways. Thus, the effects of Mg on cell adhesion and osteogenesis are rather complex, affecting several pathways rather than single processes. Our analysis also confirms the potential of proteomics in biomaterial characterization, showing a good correlation with in vitro results.

Protein adsorption/desorption dynamics on Ca-enriched titanium surfaces: biological implications.

Calcium ions are used in the development of biomaterials for the promotion of coagulation, bone regeneration, and implant osseointegration. Upon implantation, the time-dependent release of calcium ions from titanium implant surfaces modifies the physicochemical characteristics at the implant-tissue interface and thus, the biological responses. The aim of this study is to examine how the dynamics of protein adsorption on these surfaces change over time. Titanium discs with and without Ca were incubated with human serum for 2 min, 180 min, and 960 min. The layer of proteins attached to the surface was characterised using nLC-MS/MS. The adsorption kinetics was different between materials, revealing an increased adsorption of proteins associated with coagulation and immune responses prior to Ca release. Implant-blood contact experiments confirmed the strong coagulatory effect for Ca surfaces. We employed primary human alveolar osteoblasts and THP-1 monocytes to study the osteogenic and inflammatory responses. In agreement with the proteomic results, Ca-enriched surfaces showed a significant initial inflammation that disappeared once the calcium was released. The distinct protein adsorption/desorption dynamics found in this work demonstrated to be useful to explain the differential biological responses between the titanium and Ca-ion modified implant surfaces.

Characterization of magnesium doped sol-gel biomaterial for bone tissue regeneration: The effect of Mg ion in protein adsorption.

Magnesium is the fourth most abundant element in the human body with a wide battery of functions in the maintenance of normal cell homeostasis. In the bone, this element incorporates in the hydroxyapatite structure and it takes part in mineral metabolism and regulates osteoclast functions. In this study, sol-gel materials with increasing concentrations of MgCl2 (0.5, 1, and 1.5%) were synthesized and applied onto Ti surfaces as coatings. The materials were first physicochemically characterized. In vitro responses were examined using the MC3T3-E1 osteoblastic cells and RAW264.7 macrophages. Human serum protein adsorption was evaluated employing nLC-MS/MS. The incorporation of Mg did not affect the crosslinking of the sol-gel network, and a controlled release of Mg was observed; it was not cytotoxic at any of the tested concentrations. The cytoskeleton arrangement of MC3T3-E1 cells cultured on the Mg-doped materials changed in comparison with controls; the cells became more elongated, with protruded lamellipodia and increased cell surface. The expression of integrins (ITGA5 and ITGB1) was boosted by Mg-coatings. The ALP activity and expression of TGF-ß, OSX and RUNX2 genes were also increased. In RAW264.7 cells, TNF-α secretion was reduced, while TGF-ß and IL-4 expression rose. These changes correlated with the altered protein adsorption patterns. The Mg-doped coatings showed increased adsorption of anti-inflammatory (CLUS, IC1, CFAH, and VTNC), cell adhesion (DSG1, FILA2, and DESP) and tissue regeneration (VTNC and CYTA) proteins. This integrated approach to biomaterial characterization revealed the potential of Mg in bone tissue regeneration.

Influence of calcium ion-modified implant surfaces in protein adsorption and implant integration.

BACKGROUND: Calcium (Ca) is a well-known element in bone metabolism and blood coagulation. Here, we investigate the link between the protein adsorption pattern and the in vivo responses of surfaces modified with calcium ions (Ca-ion) as compared to standard titanium implant surfaces (control). We used LC-MS/MS to identify the proteins adhered to the surfaces after incubation with human serum and performed bilateral surgeries in the medial section of the femoral condyles of 18 New Zealand white rabbits to test osseointegration at 2 and 8 weeks post-implantation (n=9). RESULTS: Ca-ion surfaces adsorbed 181.42 times more FA10 and 3.85 times less FA12 (p<0.001), which are factors of the common and the intrinsic coagulation pathways respectively. We also detected differences in A1AT, PLMN, FA12, KNG1, HEP2, LYSC, PIP, SAMP, VTNC, SAA4, and CFAH (p<0.01). At 2 and 8 weeks post-implantation, the mean bone implant contact (BIC) with Ca-ion surfaces was respectively 1.52 and 1.25 times higher, and the mean bone volume density (BVD) was respectively 1.35 and 1.13 times higher. Differences were statistically significant for BIC at 2 and 8 weeks and for BVD at 2 weeks (p<0.05). CONCLUSIONS: The strong thrombogenic protein adsorption pattern at Ca-ion surfaces correlated with significantly higher levels of implant osseointegration. More effective implant surfaces combined with smaller implants enable less invasive surgeries, shorter healing times, and overall lower intervention costs, especially in cases of low quantity or quality of bone.

Evaluation of the inflammatory responses to sol-gel coatings with distinct biocompatibility levels.

The immune system plays a crucial role in determining the implantation outcome, and macrophages are in the frontline of the inflammatory processes. Further, cellular oxidative stress resulting from the material recognition can influence how cell responses develop. Considering this, the aim of this study was to study oxidative stress and macrophages phenotypes in response to sol-gel materials with distinct in vivo outcomes. Four materials were selected (70M30T and 35M35G30T, with high biocompatibility, and 50M50G and 50V50G, with low biocompatibility). Gene expression, immunocytochemistry and cytokine secretion profiles for M1 and M2 markers were determined. Moreover, oxidative stress markers were studied. Immunocytochemistry and ELISA showed that 50M50G and 50V50G lead to a higher differentiation to M1 phenotype, while 70M30T and 35M35G30T promoted M2 differentiation. In oxidative stress, no differences were found. These results show that the balance between M1 and M2, more than individual quantification of each phenotype, determines a biomaterial outcome.

A possible use of melatonin in the dental field: Protein adsorption and in vitro cell response on coated titanium.

Melatonin (MLT) is widely known for regulating the circadian cycles and has been studied for its role in bone regeneration and inflammation. Its application as a coating for dental implants can condition the local microenvironment, affecting protein deposition on its surface and the cellular and tissue response. Using sol-gel coatings as a release vehicle for MLT, the aim of this work was to assess the potential of this molecule in improving the osseointegration and inflammatory responses of a titanium substrate. The materials obtained were physicochemically characterized (scanning electron microscopy, contact angle, roughness, Fourier-transform infrared spectroscopy, nuclear magnetic resonance, Si release, MLT liberation, and degradation) and studied in vitro with MC3T3-E1 osteoblastic cells and RAW264.7 macrophage cells. Although MLT application led to an increased gene expression of RUNX2 and BMP2 in 10MTL, it did not improve ALP activity. On the other hand, MLT-enriched sol-gel materials presented potential effects in the adsorption of proteins related to inflammation, coagulation and angiogenesis pathways depending on the dosage used. Using LC-MS/MS, protein adsorption patterns were studied after incubation with human serum. Proteins related to the complement systems (CO7, IC1, CO5, CO8A, and CO9) were less adsorbed in materials with MLT; on the other hand, proteins with functions in the coagulation and angiogenesis pathways, such as A2GL and PLMN, showed a significant adsorption pattern.

Silica-gelatin hybrid sol-gel coatings: A proteomic study with biocompatibility implications.

Osseointegration, including the foreign body reaction to biomaterials, is an immune-modulated, multifactorial, and complex healing process in which various cells and mediators are involved. The buildup of the osseointegration process is immunological and inflammation-driven, often triggered by the adsorption of proteins on the surfaces of the biomaterials and complement activation. New strategies for improving osseointegration use coatings as vehicles for osteogenic biomolecules delivery from implants. Natural polymers, such as gelatin, can mimic Collagen I and enhance the biocompatibility of a material. In this experimental study, two different base sol-gel formulations and their combination with gelatin were applied as coatings on sandblasted, acid-etched titanium substrates, and their biological potential as osteogenic biomaterials was tested. We examined the proteins adsorbed onto each surface and their in vitro and in vivo effects. In vitro results showed an improvement in cell proliferation and mineralization in gelatin-containing samples. In vivo testing showed the presence of a looser connective tissue layer in those coatings with substantially more complement activation proteins adsorbed, especially those containing gelatin. Vitronectin and FETUA, proteins associated with mineralization process, were significantly more adsorbed in gelatin coatings.

Characterization of serum proteins attached to distinct sol-gel hybrid surfaces.

The success of a dental implant depends on its osseointegration, an important feature of the implant biocompatibility. In this study, two distinct sol-gel hybrid coating formulations [50% methyltrimethoxysilane: 50% 3-glycidoxypropyl-trimethoxysilane (50M50G) and 70% methyltrimethoxysilane with 30% tetraethyl orthosilicate (70M30T)] were applied onto titanium implants. To evaluate their osseointegration, in vitro and in vivo assays were performed. Cell proliferation and differentiation in vitro did not show any differences between the coatings. However, four and eight weeks after in vivo implantation, the fibrous capsule area surrounding 50M50G-implant was 10 and 4 times, respectively, bigger than the area of connective tissue surrounding the 70M30T treated implant. Thus, the in vitro results gave no prediction or explanation for the 50M50G-implant failure in vivo. We hypothesized that the first protein layer adhered to the surface may have direct implication in implant osseointegration, and perhaps correlate with the in vivo outcome. Human serum was used for adsorption analysis on the biomaterials, the first layer of serum proteins adhered to the implant surface was analyzed by proteomic analysis, using mass spectrometry (LC-MS/MS). From the 171 proteins identified; 30 proteins were significantly enriched on the 50M50G implant surface. This group comprised numerous proteins of the immune complement system, including several subcomponents of the C1 complement, complement factor H, C4b-binding protein alpha chain, complement C5 and C-reactive protein. This result suggests that these proteins enriched in 50M50G surface might trigger the cascade leading to the formation of the fibrous capsule observed. The implications of these results could open up future possibilities to predict the biocompatibility problems in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1477-1485, 2018.

Proteome analysis of human serum proteins adsorbed onto different titanium surfaces used in dental implants.

Titanium dental implants are commonly used due to their biocompatibility and biochemical properties; blasted acid-etched Ti is used more frequently than smooth Ti surfaces. In this study, physico-chemical characterisation revealed important differences in roughness, chemical composition and hydrophilicity, but no differences were found in cellular in vitro studies (proliferation and mineralization). However, the deposition of proteins onto the implant surface might affect in vivo osseointegration. To test that hypothesis, protein layers formed on discs of both surface type after incubation with human serum were analysed. Using mass spectrometry (LC/MS/MS), 218 proteins were identified, 30 of which were associated with bone metabolism. Interestingly, Apo E, antithrombin and protein C adsorbed mostly onto blasted and acid-etched Ti, whereas the proteins of the complement system (C3) were found predominantly on smooth Ti surfaces. These results suggest that physico-chemical characteristics could be responsible for the differences observed in the adsorbed protein layer.

Complement component 4d immunostaining in liver allografts of patients with de novo immune hepatitis.

De novo immune hepatitis (DNIH) is a form of late graft dysfunction after liver transplantation. The fine mechanisms leading to the development of DNIH are not known, and whether this hepatitis is a form of rejection or a result of an auto/alloimmune injury has not been established. In our patients, DNIH was always preceded by the production of donor-specific antibodies against the glutathione S-transferase T1 (GSTT1) enzyme because of a genetic mismatch in which the donors carried the wild-type gene and the recipients displayed the null genotype. Complement component 4d (C4d) immunopositivity in 12 paraffin-embedded liver biopsy samples from 8 patients diagnosed with DNIH associated with anti-GSTT1 antibodies was retrospectively evaluated. Six patients with a diagnosis of chronic rejection (CR) and 7 patients with hepatitis C virus recurrence were included as control groups. Among the patients with DNIH, 7 showed C4d-positive immunostaining localized in the portal tracts, whereas in the tested biopsy samples of the 2 control groups, this staining pattern was absent. Four biopsy samples of the CR group showed C4d-positive sinusoidal staining. This study confirms the activation of the complement pathway in the presence of donor-specific antibodies, which was shown by the deposition of C4d elements in liver biopsy samples of patients with DNIH. The use of C4d as a marker of antibody-mediated rejection in liver allografts in the presence of antidonor antibodies is discussed, and it may contribute to improved differential diagnoses based on biopsy findings.

Glutathione S-transferase T1 mismatch constitutes a risk factor for de novo immune hepatitis after liver transplantation.

A new form of autoimmune hepatitis referred to as de novo, has been reported after liver transplantation during the past 5 years. The features are identical to those of classical autoimmune hepatitis (AIH), but the facts involved in the onset and outcome of this type of graft dysfunction are still unclear. The identification of antibodies directed to glutathione S-transferase T1 (GSTT1) in the sera of patients with de novo immune hepatitis led us to the description of an alloimmune reaction due to a GSTT1 genetic incompatibility between donor and recipient. We analyzed a cohort of 110 liver transplant patients treated in the liver transplant unit of our hospital during a period of 1 year, from September 2002 to October 2003. We found the following distribution of the GSTT1 genotypes (recipient/donor): +/+ = 66, +/- = 23, -/+ = 15, -/- = 6. Six of these patients were diagnosed with de novo immune hepatitis; all of them belong to the group of negative recipients with positive donors, and all produced anti-GSTT1 antibodies. This genetic combination is associated with a statistically significant increased risk of de novo immune hepatitis (IH) in liver transplant patients (P < .0001 by the Fisher exact test). In conclusion, our results clearly establish the importance of the GSTT1 genotype from donor and recipient of a liver transplant as a predictive marker for de novo IH. At the same time, we confirmed our initial results that only this particular donor/recipient combination triggers the anti-GSTT1 antibody production.