Tough gel adhesive is an effective method for meniscal repair in a bovine cadaveric study.

PURPOSE: To test tough gel adhesives to repair meniscus tears under relevant loading conditions and determine if they have adequate biomechanical properties to repair meniscus tears in a bovine cadaveric study. METHODS: Cyclic compression tests on 24 dissected bovine knees were performed. The tough gel adhesive was used either as an adhesive patch or as a coating bonded onto commercially available surgical sutures. Forty-eight menisci were tested in this study; 24 complete radial tears and 24 bucket-handle tears. After preconditioning, the specimens underwent 100 cycles of compression, (800 N/0.5 Hz) on an Instron© machine and the size of the gaps measured. One third of the menisci were repaired with pristine sutures, one third with adhesive patches, and one third with sutures coated in adhesive gel. The size of the gaps was compared after 100 and 500 cycles of compression. RESULTS: The mean gap measured at the tear site without treatment was 6.46 mm (± 1.41 mm) for radial tears and 1.92 mm (± 0.65 mm) for bucket-handle tears. After treatment and 500 cycles of compression, the mean gap was 1.63 mm (± 1.41 mm) for pristine sutures, 1.50 mm (± 1.16 mm) for adhesive sutures and 2.06 mm (± 1.53 mm) for adhesive gel patches. There was no significant difference between treatments regardless of the type of tear. Also, the gaps for radial tears increased significantly with the number of compression cycles applied (p > 0.001). CONCLUSION: From a biomechanical standpoint, the tough adhesive gel patch is as effective as suturing. In addition, it would allow the repair of non-suturable tears and thus broaden the indications for meniscus repair. LEVEL OF EVIDENCE: Controlled laboratory study.

Increased circulating fibronectin, depletion of natural IgM and heightened EBV, HSV-1 reactivation in ME/CFS and long COVID.

Myalgic Encephalomyelitis/ Chronic Fatigue syndrome (ME/CFS) is a complex, debilitating, long-term illness without a diagnostic biomarker. ME/CFS patients share overlapping symptoms with long COVID patients, an observation which has strengthened the infectious origin hypothesis of ME/CFS. However, the exact sequence of events leading to disease development is largely unknown for both clinical conditions. Here we show antibody response to herpesvirus dUTPases, particularly to that of Epstein-Barr virus (EBV) and HSV-1, increased circulating fibronectin (FN1) levels in serum and depletion of natural IgM against fibronectin ((n)IgM-FN1) are common factors for both severe ME/CFS and long COVID. We provide evidence for herpesvirus dUTPases-mediated alterations in host cell cytoskeleton, mitochondrial dysfunction and OXPHOS. Our data show altered active immune complexes, immunoglobulin-mediated mitochondrial fragmentation as well as adaptive IgM production in ME/CFS patients. Our findings provide mechanistic insight into both ME/CFS and long COVID development. Finding of increased circulating FN1 and depletion of (n)IgM-FN1 as a biomarker for the severity of both ME/CFS and long COVID has an immediate implication in diagnostics and development of treatment modalities.

Modification of the properties of a suture thread with a tough gel coating: A baseline ex-vivo study.

The mechanical properties of sutures are important for wound closure and meniscus repair. A tough gel coating technology has been developed to modify and functionalize sutures, but its effects on suture degradation remain unexplored. Our aim is to investigate how a tough gel coating mediates the properties of the suture. The Polyglactin910 (Vicryl) suture was chosen because it is widely used, strong, easy to handle, and degradable. This study compared six pristine Vicryl sutures and six coated Vicryl sutures at 0, 2, 4, and 6 weeks. All the sutures were soaked in phosphate-buffered saline (PBS), to mimic degradation in physiological conditions, and tensile strength was tested at each time point. The pH of the soaking mediums was measured weekly and compared at 4, 5, and 6 weeks. No significant difference (p = 0.059 and p = 0.576) was found between the absolute and normalized breaking force of coated and pristine Vicryl sutures at 0, 2, 4, and 6 weeks. After 4 weeks of immersion, the soaking medium became more acidic for both suture types. The decrease in pH was less significant for coated Vicryl sutures than for pristine ones (p < 0.001) at 4, 5, and 6 weeks of immersion. Although coating does not affect the strength of Vicryl sutures soaked in PBS, it can effectively act as a buffer to the acidic environment caused by suture degradation, which could help reduce inflammation. Hydrogel coating is a promising technology to modify suture characteristics.

A Complete and Versatile Protocol: Decoration of Cell-Derived Matrices with Mass-Encoded Peptides for Multiplexed Protease Activity Detection.

This article provides guidance toward a platform technology for monitoring enzyme activity within the extracellular matrix (ECM) assessed by quantifying reporters secreted into the cell culture supernatant and analyzed by tandem mass spectrometry. The reporters are enzymatically and covalently bound to the ECM by transglutaminases (TG) using the peptide sequence of human insulin-like growth factor I's (IGF-I) D-domain which is known to be bound to the ECM by transglutaminase. The IGF-I D-domain sequence is followed by a peptide sequence cleaved by the intended target protease. This protease-sensitive peptide sequence (PSS) is cleaved off the ECM and can be used to monitor target-enzyme activity by employing a downstream mass tag designed according to isobaric mass encoding strategies, i.e., the combination of isotopically labeled, heavy amino acids. Thereby, cleavage events are linked to the appearance of encoded mass tags, readily allowing multiplexing. This article presents the design and synthesis of these mass reporters. It further aims at detailing the search for peptide sequences responding to target proteases to facilitate future work on enzyme activity measurement for enzymatic activities of hitherto unknown enzymes. In conclusion, the goal of this article is to arm scientists interested in measurements of local enzymatic activities within the ECM with robust protocols and background knowledge.

Development and Application of a Chemical Probe Based on a Neuroprotective Flavonoid Hybrid for Target Identification Using Activity-Based Protein Profiling.

Alzheimer's disease (AD) is the most common form of dementia, and up to now, there are no disease-modifying drugs available. Natural product hybrids based on the flavonoid taxifolin and phenolic acids have shown a promising pleiotropic neuroprotective profile in cell culture assays and even disease-modifying effects in vivo. However, the detailed mechanisms of action remain unclear. To elucidate the distinct intracellular targets of 7-O-esters of taxifolin, we present in this work the development and application of a chemical probe, 7-O-cinnamoyltaxifolin-alkyne, for target identification using activity-based protein profiling. 7-O-Cinnamoyltaxifolin-alkyne remained neuroprotective in all cell culture assays. Western blot analysis showed a comparable influence on the same intracellular pathways as that of the lead compound 7-O-cinnamoyltaxifolin, thereby confirming its suitability as a probe for target identification experiments. Affinity pulldown and MS analysis revealed adenine nucleotide translocase 1 (ANT-1) and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as intracellular interaction partners of 7-O-cinnamoyltaxifolin-alkyne and thus of 7-O-esters of taxifolin.

Human Herpesvirus-6 Reactivation, Mitochondrial Fragmentation, and the Coordination of Antiviral and Metabolic Phenotypes in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multifactorial disorder with many possible triggers. Human herpesvirus (HHV)-6 and HHV-7 are two infectious triggers for which evidence has been growing. To understand possible causative role of HHV-6 in ME/CFS, metabolic and antiviral phenotypes of U2-OS cells were studied with and without chromosomally integrated HHV-6 and with or without virus reactivation using the histone deacetylase inhibitor trichostatin-A. Proteomic analysis was conducted by pulsed stable isotope labeling by amino acids in cell culture analysis. Antiviral properties that were induced by HHV-6 transactivation were studied in virus-naive A549 cells challenged by infection with influenza-A (H1N1) or HSV-1. Mitochondria were fragmented and 1-carbon metabolism, dUTPase, and thymidylate synthase were strongly induced by HHV-6 reactivation, whereas superoxide dismutase 2 and proteins required for mitochondrial oxidation of fatty acid, amino acid, and glucose metabolism, including pyruvate dehydrogenase, were strongly inhibited. Adoptive transfer of U2-OS cell supernatants after reactivation of HHV-6A led to an antiviral state in A549 cells that prevented superinfection with influenza-A and HSV-1. Adoptive transfer of serum from 10 patients with ME/CFS produced a similar fragmentation of mitochondria and the associated antiviral state in the A549 cell assay. In conclusion, HHV-6 reactivation in ME/CFS patients activates a multisystem, proinflammatory, cell danger response that protects against certain RNA and DNA virus infections but comes at the cost of mitochondrial fragmentation and severely compromised energy metabolism.

Distinct roles for H4 and H2A.Z acetylation in RNA transcription in African trypanosomes.

Despite histone H2A variants and acetylation of histones occurring in almost every eukaryotic organism, it has been difficult to establish direct functional links between canonical histones or H2A variant acetylation, deposition of H2A variants and transcription. To disentangle these complex interdependent processes, we devised a highly sensitive strategy for quantifying histone acetylation levels at specific genomic loci. Taking advantage of the unusual genome organization in Trypanosoma brucei, we identified 58 histone modifications enriched at transcription start sites (TSSs). Furthermore, we found TSS-associated H4 and H2A.Z acetylation to be mediated by two different histone acetyltransferases, HAT2 and HAT1, respectively. Whereas depletion of HAT2 decreases H2A.Z deposition and shifts the site of transcription initiation, depletion of HAT1 does not affect H2A.Z deposition but reduces total mRNA levels by 50%. Thus, specifically reducing H4 or H2A.Z acetylation levels enabled us to reveal distinct roles for these modifications in H2A.Z deposition and RNA transcription.

Mass-Encoded Reporters Reporting Proteolytic Activity from within the Extracellular Matrix.

Reporting matrix metalloproteinase (MMP) activity directly from the extracellular matrix (ECM) may provide critical insights to better characterize 2D and 3D cell culture model systems of inflammatory diseases and potentially leverage in vivo diagnosis. In this proof-of-concept study, we designed MMP-sensors, which were covalently linked onto the ECM by co-administration of the activated transglutaminase factor XIIIa (FXIIIa). Elements of the featured MMP-sensors are the D-domain of insulin-like growth factor I (IGF-I) through which co-administered FXIIIa covalently links the sensor to the ECM followed by an MMP sensitive peptide sequence and locally reporting on MMP activity, an isotopically labeled mass tag encoding for protease activity, and an affinity tag facilitating purification from fluids. All sensors come in identical pairs, other than the MMP sensitive peptide sequence, which is synthesized with l-amino acids or d-amino acids, the latter serving as internal standard. As a proof of concept for multiplexing, we successfully profiled two MMP-sensors with different MMP sensitive peptide sequences reporting MMP activity directly from an engineered 3D ECM. Future use may include covalently ECM bound diagnostic depots reporting MMP activity from inflamed tissues.

Effect of a Controlled Ankle Motion Walking Boot on Syndesmotic Instability During Weightbearing: A Cadaveric Study.

BACKGROUND: Syndesmotic injuries can lead to long-term complications; hence, they require careful management. Conservative treatment is adequate when 1 syndesmotic ligament is injured, but surgery is often necessary to achieve articular congruity when 3 syndesmotic ligaments are ruptured. However, there is some controversy over the best treatment for 2-ligament injuries. PURPOSE: To evaluate the effect of a controlled ankle motion (CAM) walking boot on syndesmotic instability following iatrogenic isolated anterior inferior tibiofibular ligament (AiTFL) injury and combined AiTFL/interosseous ligament (IOL) injuries in a cadaveric simulated weightbearing model. STUDY DESIGN: Controlled laboratory study. METHODS: Ten cadaveric specimens were dissected to expose the tibial plateau and syndesmosis. The specimens were fitted to a custom-made device, and a reproducible axial load of 750 N was applied. Iatrogenic rupture of the syndesmotic ligaments (AiTFL + IOL) was done sequentially. Uninjured syndesmoses, isolated AiTFL rupture, and combined AiTFL/IOL rupture were compared with and without axial loading (AL) and CAM boot. The distal tibiofibular relationship was evaluated using a previously validated computed tomography scan measurement system. Wilcoxon tests for paired samples and nonparametric data were used. RESULTS: The only difference noted in the distal tibiofibular relationship during AL was an increase in the external rotation of the fibula when using the CAM boot. This was observed with AiTFL rupture (8.40° vs 11.17°; P = .009) and combined AiTFL/IOL rupture (8.81° vs 11.97°; P = .005). CONCLUSION: AL did not cause a significant displacement between the tibia and fibula, even when 2 ligaments were ruptured. However, the CAM boot produced a significant external rotation with 1 or 2 injured ligaments. CLINICAL RELEVANCE: Further studies are needed to assess the capacity of the CAM walking boot to prevent malreduction when external rotation forces are applied to the ankle. Moreover, special care should be taken during the fitting of the CAM boot to avoid overinflation of the cushions.

Epidemiology of syndesmotic fixations in a pediatric center: A 12-year retrospective review.

Syndesmosis injuries need to be accurately diagnosed and managed to avoid chronic pain, early arthritis, and instability. To this end, the present study aimed to analyze the epidemiology of syndesmotic injuries in a pediatric ankle fracture cohort and identify patient and surgery-related characteristics.A retrospective review of all the ankle fractures during a 12-year period at a single pediatric referral center was conducted. Inclusion criteria were: a fractured ankle that underwent a surgical fixation, at least 1 radiograph available for review before fixation, available information regarding surgery, including operative report and fluoroscopic images, and younger than 18 years at the time of surgery. Demographic information, trauma, radiographs, surgical details, clinical examination, follow up, outcomes, and physeal status (skeletally immature, transitional, or mature) were recorded. Finally, patients were divided in 2 groups: with or without syndesmotic fixation. Statistical analysis included descriptive statistics, Mann-Whitney test for nonparametric data to compare continuous parameters, and χ test for categorical parameters.A total of 128 patients were included with a mean age of 14.1 years. There were 80 boys and 48 girls. There were 51 skeletally immature patients, 23 with transitional fractures, and 54 that were skeletally mature. The main finding of this study is that only 11 patients from the mature group underwent syndesmotic fixation. There were no cases of syndesmotic fixation in the skeletally immature and transitional groups.This is the first retrospective study to focus specifically on syndesmotic injuries in a pediatric population who underwent ankle fracture fixation. Only 11 skeletally mature patients underwent syndesmotic fixation out of 128 patients in this cohort. This result raises the question of whether there are accurate diagnostic tools to evaluate syndesmosis in children.

Validation of the French version of the KOOS-child questionnaire.

PURPOSE: The Knee Injury Osteoarthritis Outcome Score (KOOS) questionnaire is one of the frequently used outcome scores in pediatric studies. However, a recent study demonstrated that the pediatric population had a limited understanding of some of its questions. Therefore, the KOOS-Child questionnaire was developed specifically for this population. Our team produced a French adaptation based on the English version. The objective of the current study was to validate the French adaptation of the KOOS-Child questionnaire. METHODS: After ethic board approval, the questionnaire was translated from English to French by two French speaking orthopedic surgeons. Following consensus, the translated version was retranslated to English by a professional translator. A group of experts compared the original and back translated version and decided on a final adapted questionnaire version. Ninety-nine 8-16 year-old patients were prospectively recruited from our pediatric orthopedic surgery clinic. Twenty-one control participants and 78 patients suffering from knee pain were recruited. The participants were asked to answer the translated French version of the KOOS-Child questionnaire and two validated French pediatric quality of life surveys. RESULTS: Statistical analysis demonstrated no statistically significant demographic difference between the control population and the patients suffering from a knee pathology. The mean for the five different domains of the KOOS-Child questionnaire showed statistical differences (p < 0.001) between the two groups. Construct validity was demonstrated through testing of previously validated hypothesis of correlation. Internal consistency was also confirmed in injured patients. CONCLUSIONS: In conclusion, the current study results demonstrate good to excellent internal consistency, good construct validity and inconclusive discriminant capacity of the French adaptation of the KOOS-Child questionnaire. LEVEL OF EVIDENCE: II.

Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve.

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.

Production of biologically active recombinant human factor H in Physcomitrella.

The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma.

Ectodomain shedding generates Neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.

As a type II transmembrane protein in basal keratinocytes, collagen XVII provides stable adhesion between epidermis and dermis in the skin. Its ectodomain can be shed from the cell surface, and autoantibodies in certain blistering diseases preferentially recognize the shed form. Major epitopes of collagen XVII are clustered within the juxtamembranous noncollagenous 16th A domain, and ectodomain shedding occurs within this region, suggesting that cleavage generates neoepitopes. However, the candidate cleavage sites have been controversial, and the mechanism of neoepitope generation is unclear. In this study, we investigated cleavage sites in the noncollagenous 16th A domain to understand the generation of neoepitopes and their pathological role. Polyclonal Abs recognizing the stretch Leu(524)-Gly(532) preferentially reacted with the shed ectodomain, but not with the full-length form, indicating that a neoepitope was localized at this site. The neoepitope-specific Ab fixed complement and induced granulocyte-dependent dermal-epidermal separation in cryosections of normal human skin. The physiological cleavage sites were identified using mass spectrometry. N termini were found at Asp(514), Leu(524), Glu(525), and Gly(526), among which Asp(514) and Glu(525) were blocked by acetylation and pyroglutaminate. In silico prediction of B cell epitopes indicated that the antigenicity of the Leu(524)-Gly(532) region increased substantially after shedding, regardless of the cleavage sites. Correspondingly, neoepitopes were found in the skin and blister fluids of patients with bullous pemphigoid, and bullous pemphigoid sera reacted with the peptide Leu(524)-Gly(532). Taken together, these data demonstrate that physiological shedding of collagen XVII generates neoepitopes, which may serve as a target of blister-inducing autoantibodies.

Quantitative proteomics analysis of detergent-resistant membranes from chemical synapses: evidence for cholesterol as spatial organizer of synaptic vesicle cycling.

Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exoendocytic cycling within the nerve terminal that at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomics analysis of detergent-resistant membranes (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography-tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exoendocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this, SV membrane and endocytic proteins displayed a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localized with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover SV proteins formed cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exoendocytic vesicle cycling at synapses.

Identification of the thrombin light chain a as the single best mass for differentiation of gastric cancer patients from individuals with dyspepsia by proteome analysis.

Gastric cancer mortality is second only to lung cancer, and its prognosis is dismal. Using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry, we previously identified a single best mass, which could separate gastric cancer from patients without cancer, with a sensitivity of 89.9% and a specificity of 90%. Using protein liquid chromatography systems with various chromatography media and MS/MS analysis, we were able to identify thrombin light chain A, a proteolytic fragment of prothrombin, as the single best mass for early detection of gastric cancer patients. These findings indicate that disturbances in the coagulation-system are early events in gastric cancer biology and that a decrease or loss of thrombin light chain A, which we termed negative serum protein profiling, may contribute to the diagnosis of cancer patients.

Overexpression of cathepsin B in gastric cancer identified by proteome analysis.

We aimed to validate an analytical approach based on proteomics on gastric cancer specimens for the identification of new putative diagnostic or prognostic markers. Primary screening was performed on gastrectomy specimens obtained from ten consecutive patients with gastric cancer. Gastric epithelial cells were obtained with an epithelial cell enrichment technique, homogenized and then separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The differential protein expression pattern was verified stepwise by Western blotting and immunohistochemistry on samples from 28 and 46 cancer patients, respectively. The putative clinical applicability and prognostic use were tested by an enzyme-linked immunoabsorbent assay on serum samples obtained from 149 cancer patients. One hundred-ninety-one differentially expressed protein spots were found by 2-D PAGE and identified by mass spectrometry, including cathepsin B, which was over-expressed in six (60%) patients. Western blotting confirmed that the active form of cathepsin B is over-expressed, while immunohistochemistry showed strong cytoplasmic staining in cancer tissues of 45 (98%) patients. The serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls (P = 0.0026) and correlated with T-category and the presence of distant metastases (P < 0.05). Serum levels above 129 pmol x L(-1) were associated with a reduced survival rate (P = 0.0297). Proteome analysis is a valuable tool for the identification of prognostic markers in gastric cancer: Increased cathepsin B serum levels are associated with advanced tumor stages and progressive disease, which enables the classification of some gastric cancer patients into a subgroup that should undergo aggressive therapy.

Detection and identification of tumor-associated protein variants in human hepatocellular carcinomas.

The proteomic approach is a valuable tool to detect and identify proteins that are associated with cancer. In previous investigations on experimentally induced rat hepatomas, we detected aldose reductase-like protein (ARLP) as a highly significant marker protein. Our present study was intended to look for the presence of similar tumor-associated marker proteins on human hepatocellular carcinomas (HCC). We found several novel tumor-associated protein variants that represent members of the aldo-keto reductase (AKR) superfamily. Human aldose reductase-like protein-1 (hARLP-1) was the most prominent tumor-associated AKR member detected in HCC by 2-dimensional electrophoresis (2-DE) and identified by mass spectrometric fingerprinting. The enzyme was found in 4 distinct forms (hARLP-1, 36/7.4 (kd/pI); hARLP-2, 36/7.2; hARLP-3, 36/6.4; and hARLP-4, 33/7.35). In addition, a human aldose reductase-like protein (hARLP-5, 36/7.6) was identified that differed from hARLP-1 by 1 amino acid (D313N), indicating 2 allelic forms of the human aldose reductase-like gene. A novel antibody directed against common parts of the hARLPs revealed hARLP reactivity in human HCC by immunohistochemistry. Furthermore, aldose reductase (AR) was identified and characterized as a tumor-associated variant. In conclusion, in all investigated human HCCs at least one of the various types of the described tumor-associated proteins of the AKR superfamily was clearly present. Of these HCC samples, 95% were positive for hARLPs as proven by 2-DE analysis and/or by use of the antibody directed against hARLP. Thus, hARLP is a strong candidate for use as an immunohistochemical diagnostic marker of human HCC.

Proteome analysis for the identification of tumor-associated biomarkers in gastrointestinal cancer.

Gastrointestinal cancers are usually diagnosed at advanced stages, making a curative treatment difficult. Biomarkers can help to overcome this problem by allowing earlier diagnosis, and thus better therapy. Proteomics tools are novel technologies to identify such biomarkers. This review summarizes advances in biomarker detection using two-dimensional gel electrophoresis (2D-PAGE), chromatography and mass spectrometry technologies. 2D-PAGE combined with mass spectrometry has led to the identification of several differentially expressed proteins in cancer tissue. However, for serum analysis, 2D-PAGE has severe limitations. For serum-based cancer diagnosis, surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry is a promising new technology. The potential of proteins identified with this technology as novel cancer biomarkers still needs to be confirmed in clinical trials.

Link between organ-specific antigen processing by 20S proteasomes and CD8(+) T cell-mediated autoimmunity.

Adoptive transfer of cross-reactive HSP60-specific CD8(+) T cells into immunodeficient mice causes autoimmune intestinal pathology restricted to the small intestine. We wondered whether local immunopathology induced by CD8(+) T cells can be explained by tissue-specific differences in proteasome-mediated processing of major histocompatibility complex class I T cell epitopes. Our experiments demonstrate that 20S proteasomes of different organs display a characteristic composition of alpha and beta chain subunits and produce distinct peptide fragments with respect to both quality and quantity. Digests of HSP60 polypeptides by 20S proteasomes show most efficient generation of the pathology related CD8(+) T cell epitope in the small intestine. Further, we demonstrate that the organ-specific potential to produce defined T cell epitopes reflects quantities that are relevant for cytotoxic T lymphocyte recognition. We propose tissue-specific antigen processing by 20S proteasomes as a potential mechanism to control organ-specific immune responses.