Diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters (DGONC) - a molecularly defined glioneuronal CNS tumour class displaying recurrent monosomy 14.

AIMS: DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. PATIENTS AND METHODS: DNA methylation profiling was performed using the Infinium MethylationEPIC or 450 k BeadChip arrays (Illumina) and analysed using the 'conumee' package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. RESULTS: Genome-wide DNA methylation data from >25 000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. CONCLUSIONS: DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters.

Differential effects of CXCR4-CXCL12- and CXCR7-CXCL12-mediated immune reactions on murine P0106-125 -induced experimental autoimmune neuritis.

AIM: The role of chemokines and their receptors, which regulate trafficking and homing of leucocytes to inflamed organs in human or murine autoimmune neuritis, has not yet been elucidated in detail, Therefore, the role of the chemokine receptors CXCR4 and CXCR7 and their ligand CXCL12 was studied in autoimmune-mediated inflammation of the peripheral nervous system. METHODS: CXCL12/CXCR4 and/or CXCL12/CXCR7 interactions were specifically inhibited by the compounds AMD3100 or CCX771, respectively, in experimental autoimmune neuritis (EAN) of C57BL/6J mice immunized with P0106-125 peptide. RESULTS: Disease activity was significantly suppressed by blocking CXCR7 while antagonization of CXCR4 enhanced disease activity. Enhanced disease activity was accompanied by significantly increased transcription of IFN-γ, IL-12 and TNF-α mRNA in regional lymph nodes and spleen as well as by increased serum levels of IFN-γ. Furthermore, by blocking CXCR4, expression of the cell adhesion molecules ICAM-1 and VCAM-1 was upregulated on vascular endothelial cells of the sciatic nerve, which coincided with significantly increased infiltration of the sciatic nerve by CD4+ T cells and macrophages. Remarkably, combined antagonization of both CXCR4 and CXCR7 significantly suppressed disease activity. This was accompanied by increased frequencies of activated and highly IFN-γ-expressing, P0106-125 -specific T cells in regional lymph nodes and spleen; however, these cells were unable to infiltrate the sciatic nerve. CONCLUSION: These data suggest differential and hierarchically ordered roles for CXCR4/CXCL12- vs. CXCR7/CXCL12-dependent effects during EAN: CXCR7/CXCL12 interaction is a gatekeeper for pathogenic cells, regardless of their CXCR4/CXCL12-dependent state of activation.

[Differential diagnosis of lymphoid infiltrates in the central nervous system: experience of the Network Lymphomas and Lymphomatoid Lesions in the Nervous System]. / Differenzialdiagnose lymphoider Infiltrate im Zentralnervensystem : Erfahrungen des Netzwerks Lymphome und lymphomatoide Läsionen des Nervensystems.

The differential diagnosis of lymphoid lesions in the central nervous system covers a broad spectrum of neoplastic and inflammatory disorders. Complex cases benefit from the combined expertise in the fields of hematopoietic and neuroepithelial tumors as well as neuroimmunology. The Network Lymphomas and Lymphomatoid Lesions in the Nervous System (NLLLN) recommends performing a biopsy prior to any therapeutic intervention as a precise diagnosis was impossible in approximately 50 % of patients pretreated with corticosteroids. This is based on the analysis of approximately 1,000 cases in the past 4 years. In addition to total NLLLN experiences the characteristics, pathogenesis and differential diagnosis of primary lymphoma of the central nervous system are discussed.

Clonal expansions of CD8(+) T cells dominate the T cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction.

Clonal composition and T cell receptor (TCR) repertoire of CD4(+) and CD8(+) T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells. Individual CD4(+) or CD8(+) T cells were isolated from frozen sections of lesional tissue by micromanipulation and subjected to single target amplification of TCR-beta gene rearrangements. This strategy allows the assignment of a TCR variable region (V region) sequence to the particular T cell from which it was amplified. Sequence analysis revealed that in both cases investigated, the majority of CD8(+) T cells belonged to few clones. One of these clones accounted for 35% of CD8(+) T cells in case 1. V region sequence comparison revealed signs of selection for common peptide specificities for some of the CD8(+) T cells in case 1. In both cases, the CD4(+) T cell population was more heterogeneous. Most CD4(+) and CD8(+) clones were represented in perivascular infiltrates as well as among parenchymal T cells. In case 2, two of the CD8(+) clones identified in brain tissue were also detected in peripheral blood. Investigation of the antigenic specificities of expanded clones may help to elucidate their functional properties.

Hif-2alpha mediates UV-induced apoptosis through a novel ATF3-dependent death pathway.

In this study, we describe a novel activating transcription factor 3 (ATF3)-dependent death pathway triggered by ultraviolet (UV) irradiation. We demonstrate that ATF3 contributes to UV-induced apoptosis through the regulation of hypoxia inducible factor (Hif)-2alpha expression, which in turn induces the expression of proapoptotic genes, such as Caspase7 or TRAIL (tumor necrosis factor (ligand) superfamily, member 10). Gain of function of Hif-2alpha as well as ATF3 is sufficient to trigger cell death, whereas loss of function of both proteins drastically inhibits UV-induced apoptosis. Repression of Hif-2alpha strongly impairs ATF3-mediated death, providing evidences that Hif-2alpha is the major death effector of ATF3. In addition, Hif-1alpha, already known as a proapoptotic gene, upon UV irradiation, is not able to compensate for the lack of Hif-2alpha expression, thereby confirming the major contribution of Hif-2alpha in UV-mediated cell death. We further demonstrate that this cascade of gene activation depends on p38 and c-Jun N-terminal kinase (JNK) activity. Impairment of such a pathway is likely to contribute to oncogenesis by promoting survival of cells that could accumulate severe chromosomal alterations.

High-dose methotrexate combined with procarbazine and CCNU for primary CNS lymphoma in the elderly: results of a prospective pilot and phase II study.

BACKGROUND: To improve survival of elderly patients with primary central nervous system lymphoma (PCNSL), we conducted a phase II study with high-dose methotrexate (MTX) combined with procarbazine and CCNU. To reduce neurotoxicity, whole-brain irradiation was reserved for patients not responding to chemotherapy. PATIENTS AND METHODS: High-dose MTX was applied on days 1, 15, and 30, procarbazine on days 1-10, and CCNU on day 1. Study treatment comprised up to three 45-day cycles. There was no lower limit of Karnofsky performance status (KPS). RESULTS: Thirty patients with PCNSL (n = 29) or primary ocular lymphoma (n = 1) were included (median age 70 years, range 57-79 years). The median initial KPS was 60% (range 30%-90%). Best documented response in 27 assessable patients were 12 of 27 (44.4%) complete remissions, 7 of 27 (25.9%) partial remissions, and 8 of 27 (29.6%) disease progressions. Two patients died of probable treatment-related causes. With a median follow-up of 78 months (range 34-105), the 5-year overall survival is 33%. Eight of 30 patients (26.7%) are currently alive and well, six without signs of leukoencephalopathy. CONCLUSION: The combination of high-dose MTX with procarbazine and CCNU is feasible and effective and results in a low rate of leukoencephalopathy. Comorbidity and toxicity remain of concern when treating PCNSL in elderly patients.

Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases.

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells.

Persistent toxoplasma bradyzoite cysts in the brain: incidental finding in an immunocompetent patient without evidence of a toxoplasmosis.

We report on a 72-year-old patient in whom autopsy demonstrated incidentally intracerebral Toxoplasma gondii cysts, locally restricted in the occipital lobe, in association with only a few CD4+ and CD8+ T cells and a mild microglial activation. The patient was HIV-negative. Serologically, there was no evidence for an active inflammation (Toxoplasma gondii specific antibody IgG-positive, IgM-negative). This unusual observation may indicate that in patients with sepsis, who may yield to a state of immunodysbalance, a focal reactivation of parasites may ensue in the absence of conditions predisposing for opportunistic infection.

Cancer cell-derived long pentraxin 3 (PTX3) promotes melanoma migration through a toll-like receptor 4 (TLR4)/NF-κB signaling pathway.

Cutaneous melanoma is one of the most aggressive cancers characterized by a high plasticity, a propensity for metastasis, and drug resistance. Melanomas are composed of phenotypically diverse subpopulations of tumor cells with heterogeneous molecular profiles that reflect intrinsic invasive abilities. In an attempt to identify novel factors of the melanoma invasive cell state, we previously investigated the nature of the invasive secretome by using a comparative proteomic approach. Here, we have extended this analysis to show that PTX3, an acute phase inflammatory glycoprotein, is one such factor secreted by invasive melanoma to promote tumor cell invasiveness. Elevated PTX3 production was observed in the population of MITFlow invasive cells but not in the population of MITFhigh differentiated melanoma cells. Consistently, MITF knockdown increased PTX3 expression in MITFhigh proliferative and poorly invasive cells. High levels of PTX3 were found in tissues and blood of metastatic melanoma patients, and in BRAF inhibitor-resistant melanoma cells displaying a mesenchymal invasive MITFlow phenotype. Genetic silencing of PTX3 in invasive melanoma cells dramatically impaired migration and invasion in vitro and in experimental lung extravasation assay in xenografted mice. In contrast, addition of melanoma-derived or recombinant PTX3, or expression of PTX3 enhanced motility of low migratory cells. Mechanistically, autocrine production of PTX3 by melanoma cells triggered an IKK/NFκB signaling pathway that promotes migration, invasion, and expression of the EMT factor TWIST1. Finally, we found that TLR4 and MYD88 knockdown inhibited PTX3-induced melanoma cell migration, suggesting that PTX3 functions through a TLR4-dependent pathway. Our work reveals that tumor-derived PTX3 contributes to melanoma cell invasion via targetable inflammation-related pathways. In addition to providing new insights into the biology of melanoma invasive behavior, this study underscores the notion that secreted PTX3 represents a potential biomarker and therapeutic target in a subpopulation of MITFlow invasive and/or refractory melanoma.

Homeostatic chemokines increase survival of B-chronic lymphocytic leukemia cells through inactivation of transcription factor FOXO3a.

B-chronic lymphocytic leukemia (B-CLL) cell is characterized by the accumulation of long-lived CD5+ B lymphocytes, whose survival in vivo is in part dependent on exogenous factors such as cytokines and/or extracellular matrix proteins. Homeostatic chemokines are critical mediators of lymphoid cell trafficking. However, how they function in cell signaling and survival remains ill-defined. In this study, we have investigated the role of the homeostatic chemokines, CXCL12, CCL21, CCL19 and CXCL13, in B-CLL cell survival. Using primary leukemic cells isolated from 26 patients, we observed that each chemokine enhances cell survival. Chemokines induced the phosphorylation of ERK1/2 and p90RSK, and of Akt and its effectors GSK3 and FOXO3a. Consistently, inhibitors against mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase inhibited chemokine-induced survival. Moreover, using a constitutively active mutated form of FOXO3a or siRNAs against FOXO3a in transfection experiments performed in primary B-CLL cells, we directly demonstrated the critical role of FOXO3a in both spontaneous and chemokine-induced B-CLL cell survival. Overall, our data support the notion that homeostatic chemokines contribute to B-CLL resistance to cell death through inactivation of the transcription factor FOXO3a, which may represent a novel therapeutic target in this hematopoietic malignancy.

Id3 is a novel regulator of p27kip1 mRNA in early G1 phase and is required for cell-cycle progression.

P27kip is a key inhibitory protein of the cell-cycle progression, which is rapidly downregulated in early G1 phase by a post-translational mechanism involving the proteosomal degradation. In this study, using a wounding model that induces cell-cycle entry of human dermal fibroblasts, we demonstrate that p27mRNA is downregulated when cells progress into the G1 phase, and then it returns to its basal level when cells approach the S phase. By using a quantitative polymerase chain reaction screening we identified inhibitors of differentiation (Id3), a bHLH transcriptional repressor, as a candidate mediator accounting for p27 mRNA decrease. Id3 silencing, using an small interfering RNA approach, reversed the injury mediated p27 downregulation demonstrating that Id3 is involved in the transcriptional repression of p27. Reporter gene experiments and a chromatin immunoprecipitation assay showed that Id3 likely exerts its repressive action through ELK1 inhibition. By inhibiting early p27 downregulation, Id3 depletion blocked (i) the G1-phase progression as assessed by the inhibition of pRb phosphorylation and p130 degradation and (ii) the G1/S transition as observed by the inhibition of cyclin A induction, demonstrating that p27 mRNA decrease is required for cell proliferation. Apart from its effect on the early p27 diminution, Id3 appears also involved in the control of the steady-state level of p27 at the G1/S boundary. In conclusion, this study identifies a novel mechanism of p27 regulation which besides p27 protein degradation also implicates a transcriptional mechanism mediated by Id3.

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.

Vav-Rac1-mediated activation of the c-Jun N-terminal kinase/c-Jun/AP-1 pathway plays a major role in stimulation of the distal NFAT site in the interleukin-2 gene promoter.

Vav, a hematopoiesis-specific signaling protein, plays an important role in T-cell development and activation. Vav upregulates the expression of the interleukin-2 (IL-2) gene, primarily via activation of the distal NFAT site in the IL-2 gene promoter (NFAT-IL-2). However, since this site cooperatively binds NFAT and AP-1, the relative contribution of Vav to NFAT versus AP-1 activation has not been determined. Here, we studied the respective roles of the AP-1 and NFAT pathways in the T-cell receptor (TCR)-mediated, Vav-dependent activation of NFAT-IL-2. Although Vav stimulated the transcriptional activity of an NFAT-IL-2 reporter gene, it failed to stimulate the transcriptional or DNA-binding activities of an AP-1-independent NFAT site derived from the human gamma interferon gene promoter. Vav also did not stimulate detectable Ca(2+) mobilization and nuclear translocation of NFATc or NFATp. On the other hand, Vav induced the activation of Rac1 or Cdc42 and c-Jun N-terminal kinase (JNK), enhanced the transcriptional and DNA-binding activities of AP-1, and induced increased phosphorylation of c-Jun. Dominant-negative Vav and/or Rac1 mutants blocked the TCR-mediated stimulation of these events, demonstrating the physiological relevance of these effects. Vav also associated with Rac1 or Cdc42 in T cells, and anti-CD3 antibody stimulation enhanced this association. These findings indicate that a Rac1-dependent JNK/c-Jun/AP-1 pathway, rather than the Ca(2+)/NFAT pathway, plays the predominant role in NFAT-IL-2 activation by Vav.

High-dose methotrexate-based immuno-chemotherapy for elderly primary CNS lymphoma patients (PRIMAIN study).

To investigate immuno-chemotherapy for elderly immuno-competent patients (⩾65 years) with newly diagnosed primary central nervous system lymphoma, we conducted a multicentre single-arm trial. One cycle consisted of rituximab (375 mg/m2, days 1, 15, 29), high-dose methotrexate (3 g/m2 days 2, 16, 30), procarbazine (60 mg/m2 days 2-11) and lomustine (110 mg/m2, day 2)-R-MPL protocol. Owing to infectious complications, we omitted lomustine during the study and consecutive patients were treated with the R-MP protocol. Three cycles were scheduled and repeated on day 43. Subsequently, patients commenced 4 weekly maintenance treatment with procarbazine (100 mg for 5 days). Primary end point was complete remission (CR) after 3 cycles. We included 107 patients (69 treated with R-MPL and 38 with R-MP). In all, 38/107 patients achieved CR (35.5%) and 15 (14.0%) achieved partial remission. R-MP was associated with a lower CR rate (31.6%) compared with R-MPL (37.7%), but respective 2-year progression-free survival (All 37.3%; R-MP 34.9%; R-MPL 38.8%) and overall survival (All 47.0%; R-MP 47.7%; R-MPL 46.0%) rates were similar. R-MP was associated with less ⩾grade 3 toxicities compared with R-MPL (71.1% vs 87.0%). R-MP is more feasible while still associated with similar efficacy compared with R-MPL and warrants further improvement in future studies.

A 43-year-old woman with a temporal mass.

A 43-year-old woman with a past medical history of breast cancer and an acute myeloid leukemia (AML) presented with headache over a 3-week period. The clinical examination was completely unremarkable. CT and MRI scans showed a contrast enhancing lesion in the left temporal lobe. Histopathologic examination revealed a malignant, hematopoietic tumor with high mitotic activity, areas of necrosis and diffuse infiltration of the brain parenchyma. Positive staining for Chloroacetateesterase and lysozyme of tumor cells identified its myeloid lineage. The diagnosis was granulocytic sarcoma (GS)/chloroma, a metastatic manifestation of AML. Granulocytic sarcoma (GS) most often occurs in patients with AML, myelodysplastic syndromes and myeloproliferative disorders, and can involve any organ. However intracerebral manifestation of GS is a rare event. In this case histopathological features and differential diagnoses of intracerebral GS are discussed.

Long-term remission in progressive multifocal leukoencephalopathy caused by idiopathic CD4+ T lymphocytopenia: a case report.

Progressive multifocal leukoencephalopathy is caused by JC virus, an opportunistic infection of the central nervous system. Antiretroviral treatment for progressive multifocal leukoencephalopathy in human immunodeficiency virus-infected patients is beneficial, but few data exist for patients who are not infected with human immunodeficiency virus. Idiopathic CD4+ T lymphocytopenia excludes human immunodeficiency virus infection. We describe a patient with progressive multifocal leukoencephalopathy with underlying idiopathic CD4+ T lymphocytopenia in whom functional recovery occurred without antiviral therapy.

The immunological synapse and Rho GTPases.

Rho GTPases are molecular switches controlling a broad range of cellular processes including lymphocyte activation. Not surprisingly, Rho GTPases are now recognized as pivotal regulators of antigen-specific T cell activation by APCs and immunological synapse formation. This review summarizes recent advances in our understanding of how Rho GTPase-dependent pathways control T lymphocyte motility, polarization and activation.

[Strabologic and neuro-ophthamologic aspects of childhood cancer]. / Strabologische und neuroophthalmologische Aspekte kindlicher Tumorerkrankungen.

Pediatric oncology has achieved major progress by continuous optimization of diagnostic and therapeutic approaches. In the interdisciplinary team, the ophthalmologist plays an important role. In the field of strabismus and neuro-ophthalmology clinical symptoms as strabismus, visual loss, anisocoria, visual field defects, and involuntary eye movements may be key indicators of childhood cancer. The appropriate diagnostic workup as well as the diagnostic and therapeutic steps during the course of the disease are important often requiring individually tailored approaches.

Differential requirements for ERK1/2 and P38 MAPK activation by thrombin in T cells. Role of P59Fyn and PKCepsilon.

Activation of the mitogen-activated protein kinase (MAPK) cascade is a well documented mechanism for the G-protein-coupled receptors. Here, we have analysed the requirements for ERKs and p38 MAPK activation by thrombin in Jurkat T cells. We show that thrombin-mediated ERKs activation requires both PTK and PKC activities, whereas p38 MAPK activation is dependent only on PTKs. Thrombin-induced ERK and p38 MAPK activation was more pronounced in p56Lck deficient cells indicating that this PTK exerts a negative control on MAPK activity. Accordingly, overexpression of p50 Csk a kinase that inactivates p56Lck induced constitutive activation of ERKs. Requirement for a Src kinase was evidenced by expression of a constitutively active form of p59Fyn in Jurkat cells. Besides its effect on tyrosine phosphorylation events, thrombin also triggered a rapid and robust redistribution of PKCepsilon and delta from the cytosol to the membrane. Expression of constitutively active and dominant negative PKCepsilon demonstrates the pivotal role of this PKC isoform in ERKs activation by thrombin. These data are consistent with a model where thrombin induces ERK activation via both PKC-dependent and independent pathways, whereas p38 MAPK activation requires only PTKs. The PKC-independent pathway requires Src kinases other than p56Lck more likely p59Fyn, while the PKC-dependent mechanism depends on PKCepsilon

Possible genetic defects in regulation of glycosaminoglycans in patients with diabetic nephropathy.

The hypothesis of genetic defects in glycosaminoglycan (GAG) regulation among patients with insulin-dependent diabetes mellitus (IDDM) and nephropathy was assessed by studies in tissue cultures of fibroblasts obtained from 7 patients with normal urinary albumin excretion, 11 patients with diabetic nephropathy, and 6 nondiabetic control subjects. The incorporation of [2H] glucosamine and [35S] sulfate into hyaluronic acid (HA), chondroitin sulfate and dermatan sulfate (CS + DS), and heparan sulfate (HS) was measured in cells, matrix, and medium and related to micrograms of tissue protein. Large interindividual variations were seen in all three groups, and the incorporation of [3H] glucosamine into HA, CS + DS, and HS and [35S] sulfate into CS + DS and HS were not significantly different between the three groups. However, the fractional incorporation of [3H]glucosamine into HS was significantly reduced in diabetic patients with nephropathy compared with control subjects. This was the case not only when related to the total amount of GAGs (P = 0.014) but also when related to HA (P = 0.014). No significant difference was seen between control subjects and normoalbuminuric diabetic patients. The degree of N-sulfation of HS was not significantly different between the experimental groups. The results suggest that patients with diabetic nephropathy may suffer from deficiencies of coordinate regulation in the biosynthesis of GAG in fibroblasts, which may lead to a reduced density of HS in the extracellular matrix. If these changes reflect alterations in the biosynthesis of GAG from endothelial, myomedial, and mesangial cells, this observation may be relevant for the pathogenesis of severe diabetic complications.