The impact of COVID-19 on medical students' practical skills and hygiene behavior regarding venipuncture: a case control study.

BACKGROUND: Despite their importance to current and future patient care, medical students' hygiene behaviors and acquisition of practical skills have rarely been studied in previous observational study. Thus, the aim of this study was to investigate the potential impact of the COVID-19 pandemic on medical student's hygiene and practical skills. METHODS: This case-control study assessed the effect of the COVID-19 pandemic on hygiene behavior by contrasting the practical skills and hygiene adherence of 371 medical students post the pandemic associated lockdown in March 2020 with that of 355 medical students prior to the SARS-CoV-2 outbreak. Students' skills were assessed using an objective structured clinical examination (OSCE). Their skills were then compared based on their results in hygienic venipuncture and the total OSCE score. RESULTS: During the SARS-CoV-2 pandemic, medical students demonstrated an increased level of compliance regarding hand hygiene before (prior COVID-19: 83.7%; during COVID-19: 94.9%; p < 0.001) and after patient contact (prior COVID-19: 19.4%; during COVID-19: 57.2%; p = 0.000) as well as disinfecting the puncture site correctly (prior COVID-19: 83.4%; during COVID-19: 92.7%; p < 0.001). Prior to the pandemic, students were more proficient in practical skills, such as initial venipuncture (prior COVID-19: 47.6%; during COVID-19: 38%; p < 0.041), patient communication (prior COVID-19: 85.9%; during COVID-19: 74.1%; p < 0.001) and structuring their work process (prior COVID-19: 74.4%; during COVID-19: 67.4%; p < 0.024). CONCLUSION: Overall, the COVID-19 pandemic sensitized medical students' attention and adherence to hygiene requirements, while simultaneously reducing the amount of practice opportunities, thus negatively affecting their practical skills. The latter development may have to be addressed by providing additional practice opportunities for students as soon as the pandemic situation allows.

Deterioration in hygiene behavior among fifth-year medical students during the placement of intravenous catheters: a prospective cohort comparison of practical skills.

BACKGROUND: The American Association of Medical Colleges has defined peripheral intravenous cannulation as one of the eight practical skills that a medical student should possess upon graduation. Since following a standard hygiene protocol can reduce the rate of complications such as bloodstream infections, the medical student's compliance to hygienic standards is highly relevant. METHODS: This unicentric longitudinal cohort study included 177 medical students undergoing OSCE 1 in the winter semesters 2016/2017 and 2017/2018 as well as OSCE 2 during the winter semesters 2018/2019 and 2019/2020 at the University of Cologne. Their performance in peripheral intravenous cannulation was rated by trained student supervisors using a scaled 13-item questionnaire and compared between OSCE 1 and OSCE 2. RESULTS: Overall, a decline in the correct placement of peripheral intravenous catheters was observed among advanced medical students during OSCE 2 (mean total score: 6.27 ± 1.84) in comparison to their results in OSCE 1 (mean total score: 7.67 ± 1.7). During OSCE 2, the students were more negligent in regard to hygienic behavior, such as disinfection of the puncture site as well as hand disinfection before and after venipuncture. Their patients were also less likely to be informed about the procedure as compared to OSCE 1. CONCLUSIONS: An unsatisfying performance in regard to peripheral intravenous cannulation was observed in medical students with hygiene compliance deteriorating between the third and fifth year of their study. Thus, we promote an extension of practical hygiene and stress management training in medical school to reduce complications associated with intravenous catheters, such as bloodstream infections.

The cartilage-specific lectin C-type lectin domain family 3 member A (CLEC3A) enhances tissue plasminogen activator-mediated plasminogen activation.

C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins. Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator (tPA) thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown. Here, we recombinantly expressed murine and human full-length CLEC3As as well as truncated forms of CLEC3A in HEK-293 Epstein-Barr nuclear antigen (EBNA) cells. We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy. We found that CLEC3A mainly occurs as a monomer, but also forms dimers and trimers, potentially via a coiled-coil α-helix. We also noted that CLEC3A can be modified with chondroitin/dermatan sulfate side chains and tends to oligomerize to form higher aggregates. We show that CLEC3A is present in resting, proliferating, and hypertrophic growth-plate cartilage and assembles into an extended extracellular network in cultures of rat chondrosarcoma cells. Further, we found that CLEC3A specifically binds to plasminogen and enhances tPA-mediated plasminogen activation. In summary, we have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation.

Identification of antibodies against extracellular matrix proteins in human osteoarthritis.

We investigated the presence of autoantibodies against the extracellular matrix proteins thrombospondin-4 (TSP-4), cartilage oligomeric matrix protein (COMP), C-type lectin domain family 3 member A (CLEC3A), collagen II, collagen VI, matrilin-3, and fibrillin-2 in the serum of osteoarthritis (OA) patients. We compared those results with the presence of such antibodies in rheumatoid arthritis (RA) patients and in healthy donors (HD). Our study examines whether antibodies against extracellular proteins can be used as potential biomarkers to support the clinical diagnosis of OA. 10 OA, 10 RA patients and 10 HD were enrolled in this explorative cross-sectional study. SDS-PAGE and immunoblot were used to investigate the presence of antibodies against extracellular matrix proteins. The serum of 5/10 OA patients but 0/10 HD exhibited TSP-4 IgG isotype antibodies (P = 0.033). The serum of 8/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4 or COMP (P = 0.005). The serum of 9/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4, COMP or CLEC3A (P = 0.005). We found strong evidence for the presence of IgG isotype autoantibodies against the cartilage extracellular matrix proteins TSP-4, COMP and CLEC3A in OA. The detection of IgG isotype autoantibodies against TSP-4, COMP and CLEC3A may support the clinical diagnosis of OA. OA with autoantibodies against cartilage extracellular matrix proteins defines a new OA subgroup suggesting that patients with high concentrations of autoantibodies may benefit from an immune suppressive therapy.

B-cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice.

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis.

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.

COMP does not directly modify the expression of genes involved in cartilage homeostasis in contrast to several other cartilage matrix proteins.

OBJECTIVE: We investigated whether COMP may modify cartilage metabolism and play a role as an endogenous disease aggravating factor in OA. MATERIALS AND METHODS: Full-length and momomeric COMP was recombinantly expressed in human embryonic kidney cells and purified it via affinity chromatography. Purified COMP was used to stimulate either primary human chondrocytes or cartilage explants. Changes in the expression profiles of inflammatory genes, differentiation markers and growth factors were examined by immunoassay and by quantitative real-time reverse-transcription polymerase chain reaction. RESULTS: Incubation of primary human chondrocytes or cartilage explants in the presence of COMP did not induce statistically significant changes in the expression of IL-6, MMP1, MMP13, collagen I, collagen II, collagen X, TGF-ß1 and BMP-2. CONCLUSIONS: In contrast to collagen II and matrilin-3, COMP lacks the ability to trigger a proinflammatory response in chondrocytes, although it carries an RGD motif and can bind to integrins. COMP is a well-accepted biomarker for osteoarthritis but increased COMP levels do not necessarily correlate with inflammation.

Triazole-Bridged Peptides with Enhanced Antimicrobial Activity and Potency against Pathogenic Bacteria.

There are still no linear antimicrobial peptides (AMPs) available as a treatment option against bacterial infections. This is caused by several drawbacks that come with AMPs such as limited proteolytic stability and low selectivity against human cells. In this work, we screened a small library of rationally designed new peptides based on the cell-penetrating peptide sC18* toward their antimicrobial activity. We identified several effective novel AMPs and chose one out of this group to further increase its potency. Therefore, we introduced a triazole bridge at different positions to provide a preformed helical structure, assuming that this modification would improve (i) proteolytic stability and (ii) membrane activity. Indeed, placing the triazole bridge within the hydrophilic part of the linear analogue highly increased membrane activity as well as stability against enzymatic digestion. The new peptides, 8A and 8B, demonstrated high activity against several bacterial species tested including pathogenic N. gonorrhoeae and methicillin-resistant S. aureus. Since they exhibited significantly good tolerability against human fibroblast and blood cells, these novel peptides offer true alternatives for future clinical applications and are worth studying in more detail.

Modified CLEC3A-Derived Antimicrobial Peptides Lead to Enhanced Antimicrobial Activity against Drug-Resistant Bacteria.

Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics. Sequence changes can significantly improve the therapeutic properties of antimicrobial peptides. In our study, we apply different sequence modifications to enhance the performance of the CLEC3A-derived AMPs HT-16 and HT-47. We truncated their sequences, inserting a triple-glycine linker, adding an N-terminal tryptophan residue, and generating a D-amino acid variant, resulting in the generation of seven new peptides. We investigated their antimicrobial activity against gram-positive and gram-negative bacteria, their cytotoxicity to murine cells, and the biostability of the modified peptides in serum. We identified a novel antimicrobial peptide, WRK-30, with enhanced antimicrobial potency against S. aureus and MRSA. Additionally, WRK-30 was less cytotoxic to eukaryotic cells, allowing its application in higher concentrations in an in vivo setting. In conclusion, we identified a novel CLEC3A-derived antimicrobial peptide WRK-30 with significantly improved therapeutic properties and the potential to widen the repertoire of conventional antibiotics.

The ratio of serum LL-37 levels to blood leucocyte count correlates with COVID-19 severity.

Beneficial effects of vitamin D on COVID-19 progression have been discussed in several studies. Vitamin D stimulates the expression of the antimicrobial peptide LL-37, and evidence shows that LL-37 can antagonize SARS-CoV-2. Therefore, we investigated the association between LL-37 and vitamin D serum levels and the severity of COVID-19. To this end, 78 COVID-19 patients were divided into 5 groups according to disease severity. We determined serum levels of LL-37, vitamin D, and routine laboratory parameters. We demonstrated a correlation of CRP, IL-6, PCT, leukocyte count, and LDH with the severity of COVID-19. Our study did not demonstrate a direct relationship between serum levels of LL-37 and vitamin D and the severity of COVID-19. LL-37 is produced by granulocytes and released at the site of inflammation. Therefore, the analysis of LL-37 in broncho-alvelolar lavage rather than in patient serum seems critical. However, since LL-37 is produced by granulocytes, we determined serum LL-37 levels as a function of leukocyte count. The LL-37/leukocyte count ratio correlates highly significantly inversely proportional with COVID-19 severity. Our results indicate that the LL-37/leukocyte count ratio could be used to assess the risk of COVID-19 progression as early as hospital admission.

Analysis of IgM, IgA, and IgG isotype antibodies Directed against SARS-CoV-2 spike glycoprotein and ORF8 in the course of COVID-19.

Immunoassays are a standard diagnostic tool that assesses immunity in severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection. However, immunoassays do not provide information about contaminating antigens or cross-reactions and might exhibit inaccurately high sensitivity and low specificity. We aimed to gain insight into the serological immune response of SARS-CoV-2 patients by immunoblot analysis. We analyzed serum immunoglobulins IgM, -A, and -G directed against SARS-CoV-2 proteins by immunoblot analysis from 12 infected patients. We determined IgG isotype antibodies by commercially available ELISA and assessed the clinical parameters of inflammation status and kidney and liver injury. Unexpectedly, we found no correlation between the presence of antibodies and the future course of the disease. However, attention should be paid to the parameters CRP, IL-6, and LDH. We found evidence of antibody cross-reactivity, which questions the reliability of results for serum samples that tested negative for anti-SARS-CoV-2 antibodies when assessed by immunoassays. Nevertheless, for the detection of IgG anti-SARS-CoV-2 antibodies, our data suggest that the use of the spike glycoprotein in immunoassays should be sufficient to identify positive patients. Using a combination of the spike glycoprotein and the open reading frame 8 protein could prove to be the best way of detecting anti-SARS-CoV-2 IgM antibodies.

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.

TAK1 mediates the collagen-II-dependent induction of the COX-2 gene and PGE2 release in primary human chondrocytes.

We investigated the role of transforming growth factor-beta activated kinase 1 (TAK1) in collagen II signaling in primary human chondrocytes (PHCs). We asked whether TAK1 acts as a modulator of collagen II signaling with respect to collagen-II-dependent induction of cyclooxigenase-2 (COX-2) in PHCs and release of PGE2 from PHCs. Therefore, PHCs were incubated with collagen II, and cells were then analyzed by RT-PCR for the expression of COX-2. ELISA was used to quantify PGE2 release. To examine the influence of TAK1 on these events, TAK1 gene silencing was performed by RNAi in PHCs prior to collagen II treatment. Results indicated that COX-2 gene expression and PGE2 release are specific outcomes of collagen II signaling and that both depend on TAK1 mediation. These findings are promising in that therapeutic inhibition of TAK1 might be used to reduce pain and relieve inflammatory symptoms that are common in osteoarthritis.

Discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes.

We deciphered constituent parts of a signal transduction cascade that is initiated by collagen II and results in the release of various pro-inflammatory cytokines, including interleukin-6 (IL-6), in primary human chondrocytes. This cascade represents a feed-forward mechanism whereby cartilage matrix degradation is exacerbated by the mutually inducing effect of released collagen II fragments and pro-inflammatory cytokines. We previously proposed discoidin domain receptor 2 as a central mediator in this event. Since this cascade plays a prominent role in the pathogenesis of osteoarthritis, our study further investigates the hypothesis that discoidin domain receptor 2 is a candidate receptor for collagen II, and that transcription factor NFkappaB, lipid kinase PI3K, and the MAP kinases are constituent parts of this very signal transduction cascade. To accomplish this, we selectively knocked down the molecules of interest in primary human chondrocytes, induced the specified cascade by incubating primary human chondrocytes with collagen II, and observed the outcome, specifically the changes in interleukin-6 release. Knockdown was performed by siRNA-mediated gene silencing in the case of discoidin domain receptor 2 (DDR2) or by using specific inhibitors for the remainder of the molecules. Results indicated that discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes and that MAP kinases p38, JNK and ERK, as well as transcription factor NFkappaB, are integral components of intracellular collagen II signalling. Given the detrimental role of these molecules in osteoarthritis, our findings provide new targets for more specific therapeutics, which may have fewer side effects than those currently applied.

Detection of annexin A8 antibodies in serum of patients with antiphospholipid syndrome.

INTRODUCTION: Antibodies specific for annexin A8 (AnxA8) have not been investigated in patients suffering from antiphospholipid syndrome (APS) yet. The aim of this study was to compare the presence of AnxA8 antibodies in serum of APS patients with that of age-matched healthy controls and to investigate whether AnxA8 antibodies are potential biomarkers for APS. MATERIALS AND METHODS: We enrolled 22 APS patients and 22 healthy controls in this case-control study. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to investigate the presence of AnxA8 antibodies, and we applied enzyme-linked immunosorbent assay to investigate the presence of cardiolipin (CL) and beta-2-glycoprotein I (ß2GPI) antibodies. RESULTS: The serum of 9/22 APS patients showed AnxA8 IgG isotype antibody reactivity compared to serum of 2/22 healthy controls (P = 0.034). When we also included weak immunoblot signals, 12/22 APS patients exhibited AnxA8 IgG isotype antibody reactivity compared to 3/22 healthy controls (P = 0.005). We also investigated the presence of AnxA8 IgM isotype antibodies in the serum of APS patients but found no statistically significant difference between the APS patient group and healthy control group (P = 0.500). We further investigated the presence of ß2GPI and CL IgG and IgM isotype antibodies. AnxA8 IgG isotype antibodies were present in APS patients in a similar frequency as the APS "criteria" antibody against CL (P = 0.764). CONCLUSION: We demonstrated that AnxA8 IgG isotype antibodies are potential biomarkers for the diagnosis of APS.

Metformin causes a futile intestinal-hepatic cycle which increases energy expenditure and slows down development of a type 2 diabetes-like state.

OBJECTIVE: Metformin, the first line drug for treatment of type 2 diabetes, suppresses hepatic gluconeogenesis and reduces body weight in patients, the latter by an unknown mechanism. METHODS: Mice on a high fat diet were continuously fed metformin in a therapeutically relevant dose, mimicking a retarded formulation. RESULTS: Feeding metformin in pharmacologically relevant doses to mice on a high fat diet normalized HbA1c levels and ameliorated glucose tolerance, as expected, but also considerably slowed down weight gain. This was due to increased energy expenditure, since food intake was unchanged and locomotor activity was even decreased. Metformin caused lactate accumulation in the intestinal wall and in portal venous blood but not in peripheral blood or the liver. Increased conversion of glucose-1-13C to glucose-1,6-13C under metformin strongly supports a futile cycle of lactic acid production in the intestinal wall, and usage of the produced lactate for gluconeogenesis in liver. CONCLUSIONS: The reported glucose-lactate-glucose cycle is a highly energy consuming process, explaining the beneficial effects of metformin given continuously on the development of a type 2 diabetic-like state in our mice.

TAK1 downregulation reduces IL-1beta induced expression of MMP13, MMP1 and TNF-alpha.

The paper provides evidence that transforming growth factor-beta activated kinase 1 (TAK1, MEKK7), a downstream mediator of IL-1beta signal transduction, plays an important role in the regulation of catabolic events and inflammatory processes in the context of degenerative joint diseases. We investigated the expression of TAK1 in human articular chondrocytes and in the murine growth plate by cDNA array, quantitative RT-PCR and immunohistochemistry, respectively. The human chondrosarcoma cell line SW1353 was stimulated with the proinflammatory cytokine IL-1beta. The subsequent expression of proteolytic enzymes and proinflammatory cytokines was quantified. TAK1 specific siRNA was used to study the influence of TAK1 downregulation on the expression of MMP-13, MMP1 and TNF-alpha. As a result we demonstrated the expression of TAK1 in normal and osteoarthritic human articular cartilage. Expression of TAK1 in the hypertrophic zone of the growth plate gave us a first evidence for a catabolic function of TAK1 concerning cartilage metabolism. By gene suppression with RNAi technology we could show that TAK1 downregulation leads to a 60-70% reduced release of TNF-alpha, a 40-50% reduced release of MMP13, and a 20-30% reduction of MMP1 release. As TNF-alpha is a main player in inflammatory processes, and MMP13 is one of the major proteases involved in cartilage degradation, our results suggests that TAK1 has an important regulatory role in the context of degenerative joint diseases and thus is an attractive drug target in attempts to reduce inflammation and suppress structural changes in OA induced by IL-1beta.

Detection of multiple annexin autoantibodies in a patient with recurrent miscarriages, fulminant stroke and seronegative antiphospholipid syndrome.

Anti-phospholipid syndrome (APS) is one of the main causes for recurrent miscarriages. The diagnosis of APS is based on the occurrence of clinical symptoms such as thrombotic events or obstetric complications as well as the detection of antiphospholipid antibodies directed against ß2-glycoprotein I and cardiolipin, or a positive lupus anticoagulant assay. However, there is a subpopulation of patients with clinical symptoms of APS, but the lack of serological markers (seronegative APS). In addition, a large proportion of patients with unexplained recurrent miscarriages exist. These cases may be attributed, at least in part, to a seronegative APS.The presence of autoantibodies against annexins is potentially associated with APS. Here we used immunoassays and immunoblots to detect autoantibodies directed against annexin A1-5, and A8, respectively, in a patient with a seronegative APS and a history of six recurrent pregnancy losses and fulminant stroke. We found strong IgM isotype antibody reactivity directed against annexin A2 and annexin A8, and moderate to weak IgM isotype antibody reactivity directed against annexin A1, A3, and A5. Further studies will evaluate the diagnostic value of IgM isotype antibodies against annexin A1-A5, and A8 for seronegative APS and recurrent miscarriages.

Expression of matrilins during maturation of mouse skeletal tissues.

The matrilins are a recently discovered family of non-collagenous extracellular matrix proteins. During embryogenesis, all matrilins are expressed in skeletal tissues. Additionally, matrilin-2 and -4 are expressed in the dermis and in connective tissues of internal organs, e.g. of the lung and kidney. After birth, the expression of matrilin-1 and -3 remains specific for cartilage and bone whereas matrilin-2 and -4 display a broader tissue distribution and could be detected in epithelial, muscle, and nervous tissue as well as in loose and dense connective tissue. In epiphyseal cartilage of growing long bones, matrilin-1 and -3 are present in all cartilage regions, in contrast to matrilin-2, which is expressed in the proliferative and the upper hypertrophic zones. Similarly matrilin-4 was detected all over the epiphyseal cartilage, with the weakest expression in the hypertrophic zone. Although it was shown that matrilin-1 and -3 can form hetero-oligomers and are often co-localized in tissue, clear differences in their spatial distribution could be demonstrated by double-immunolabelling. During joint development matrilin-2 and matrilin-4 are present at the developing joint surface, while in articular cartilage of 6-week-old mice all matrilins are only weakly expressed.

Anti-glycolipid antibodies in patients with neuropathy: a diagnostic assessment.

Anti-glycolipid antibodies are associated with immune-mediated neuropathies and screening is often performed as part of the diagnostic assessment for patients presenting with peripheral neuropathy. We report our experience in testing for immunoglobulin (Ig) G and IgM anti-glycolipid (GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GQ1b, sulfatides) antibodies in 290 consecutive patients presenting with neuropathy. Anti-glycolipid antibodies were detected significantly more often (43%) in patients who were diagnosed with definite immune-mediated neuropathy than in patients without a final diagnosis of immune-mediated neuropathy (control group) (23%). With positive and negative predictive values of 22% and 90%, respectively, anti-glycolipid antibodies are not a very reliable diagnostic tool in early patient contact. Certain antibodies (IgM to GM2, GT1a and IgG to GM3, GD3 and GT1b) were equally or more prevalent in the control group; clinicians should be aware of this distribution when receiving positive screening results for these antibodies. Concomitant IgG and IgM reactivities were found for GM1, GM2, GD1b and sulfatides, and were detected more frequently in patients with definite immune-mediated neuropathies.