Early Onset Rapidly Progressive Osteoarthritis of the Hip in a Young Athlete.

We report and discuss a very rare case of early-stage rapidly progressive osteoarthritis (RPO) in a 33-year-old female athlete. The etiopathology of RPO remained unclear, although in this case mechanical overloading due to constant joint overuse appeared to be the only significant contributing factor to the very early development of RPO. Key words: rapidly progressive osteoarthritis, rapid destructive arthrosis, hip arthrosis, total hip arthroplasty, athlete, osteoarthritis.

Discrimination between healthy and degenerated bovine articular cartilage with a fiber Bragg grating based microindenter.

OBJECTIVE: In this study we aim to show that an optical fiber Bragg grating-based microindentation system, which has the potential to be deployed arthroscopically, can differentiate between healthy and degenerated articular cartilage, which represents an important challenge in minimally-invasive surgery. DESIGN: Twenty bovine osteochondral cylinders, extracted from the patellar groove of ten 24 months old animals were subjected to stepwise in vitro stress-relaxation indentation measurements. The indentation procedure comprised 15 indentation steps of 20 µm each, reaching a total depth of 300 µm. Ten samples remained untreated and served as a control group for healthy cartilage. A second group of ten samples was treated for 12 h with an aqueous trypsin solution (concentration 2.5%) to deplete the proteoglycans. For both groups and all indentation depths deeper than 100 µm, the step response functions of a two elements Maxwell-Wiechert model fitted well to the measured relaxation curves. RESULTS: The standard deviations of the identified stiffness parameters within each group were much smaller than the difference of the average stiffness values between both groups. Based on the measured stiffness values, the system was capable to discriminate between healthy and degenerated cartilage with a high level of significance (p < 0.001). The experimental results are also discussed in terms of the biomechanical changes of cartilage under the action of trypsin. CONCLUSION: The fiber Bragg grating microindentation system showed the capability to differentiate intact and proteoglycan depleted cartilage with high significance.

Are surface antigens suited to verify the redifferentiation potential and culture purity of human chondrocytes in cell-based implants.

Cell expansion in vitro is a prequisite to obtain a sufficient quantity of cells for cell-based cartilage repair of articular cartilage lesions. During this process verification of redifferentiation potential of highly expanded chondrocytes is required. Furthermore, cellular impurities of chondrocyte cultures have to be excluded. For this purpose, redifferentiation of expanded human chondrocytes in passage 3 or 5 was initiated in bioresorbable polyglycolic acid-fibrin (PGA-fibrin) scaffolds and selected potential markers were analysed during the process of cell expansion and redifferentiation. Chondrocyte expansion was accompanied by a decrease of collagen type II and COMP and an increase of collagen type I expression indicating cell dedifferentiation. Redifferentiation of chondrocytes in PGA-fibrin scaffolds was accompanied by an increase of collagen II/I ratio. Flow cytometric analyses revealed that in contrast to CD44 and CD49e, CD63 and CD166 showed significant changes in the number of positive cells during redifferentiation. CD14 and CD45 are not expressed by chondrocytes and are therefore possible candidates to detect specifically monocytes or haematopoetic cells in chondrocyte cultures. Characterization of surface antigen expression revealed two promising candidates (CD63 and CD166) to describe the process of redifferentiation, while CD14 and CD45 are suitable markers to exclude impurities by monocytes or haematopoetic cells.

[The bovine cartilage punch model: a tool for the in vitro analysis of biomaterials and cartilage regeneration]. / Das bovine Knorpelstanzenmodell : Ein Werkzeug zur In-vitro-Analyse von Biomaterialien und Knorpelregeneration.

BACKGROUND: The limited regeneration capacity of hyaline articular cartilage requires detailed studies concerning the tissue integration of cartilage transplants with meaningful but time and/or resource-consuming and in part ethically problematic animal models or, alternatively, with in vitro test systems for implant materials. MATERIAL AND METHODS: The present study describes a regeneration model with bovine cartilage rings (outer Ø 6 mm, central defect Ø 2 mm) for insertion, cultivation and biomechanical or histological testing of cartilage replacement materials (HE and safranin O staining). In this study, resorbable polymers composed of polyglycolic acid (PGA) were analyzed. RESULTS: Biomechanical testing showed a continuous decrease of the push-out force for the PGA inserts from the cartilage rings, probably due to the resorbability of the material. Histologically, clear immigration of cells into cell-free PGA was observed even after 4 weeks of culture, but in particular after 10 weeks. In addition, storage of proteoglycans was interpreted as an initial sign of the formation of new matrix. CONCLUSION: Thus, the new regeneration model is in principle suitable for the testing of biomaterials, but shows limitations in assessing the "lateral bonding" of resorbable materials.

[Gait analysis after minimally invasive total hip arthroplasty]. / Ganganalyse nach minimal-invasiver Hüftprothesenimplantation.

Upon implantation of a hip prosthesis by total hip arthroplasty (THA), clinical criteria are not always sufficient for an objective assessment of the functional outcome. Thus, functional improvement of gait behavior was comparatively validated by instrumented 3D gait analysis for a current, minimally invasive surgical approach (MIS; anterolateral approach) and a conventional, transgluteal approach (KONV). In selected cases, disturbed motion sequences were registered by measuring the muscle activity via high-resolution, monopolar surface electromyography (S-EMG) above the operation area. Despite continuous and significant improvement of practically all analyzed kinematic and kinetic gait parameters for both surgical approaches already after 5 weeks but in particular after 6 and 12 months, no significant differences were detected between the 2 procedures for any parameter or time point. The S-EMG demonstrated non-physiological muscle activation on the operated, but also on the non-operated side, even at 6 months after surgery. Advantages of the MIS approach thus seem primarily restricted to early, post-operative results, such as more rapid pain reduction and rehabilitation.

Clinical impact of radiolabeled anti-CD4 antibodies in the diagnosis of rheumatoid arthritis.

Human rheumatoid arthritis (RA) is characterized by severe chronic synovitis with abundance of CD4-positive T-cells and macrophages in the inflamed synovial tissue. These cells likely play a central pathogenetic role in RA and experimental models of arthritis. CD4 is a surface molecule present on the helper/inducer subset of T lymphocytes and macrophages, although with a lower density on the latter. CD4+ T-cells/macrophages and their cytokine products, therefore, represent potential therapeutic and diagnostic targets in RA. CD4, a 55 kDa monomeric glycoprotein, binds as a T-cell coreceptor to conserved areas of the major histocompatibility complex II on antigen-presenting cells, and thereby participates in the formation of the immunological synapse and the provision of the so-called "second signal" required for full activation of T-helper cells. A specific diagnostic or therapeutic approach is the direct targeting of CD4+ T-cells by anti-CD4 monoclonal antibodies (mAbs). In addition to therapeutic clinical trials with anti-CD4 mAbs in RA, which have yielded only ambiguous results, anti-CD4 mAbs have also been developed and applied for diagnostic purposes. The studies thus far conducted in RA have focused on the following aspects: 1) comparison of anti-CD4 mAb imaging to the established early methylene diphosphonate (MDP) scan; 2) biodistribution/ pharmacokinetics studies; and 3) specificity of joint imaging with anti-CD4 mAbs in comparison to control immunoglobulins with irrelevant specificity. The available results in RA and arthritis models show that 99mTc-anti-CD4 mAbs are well-suited to actively image diseased joints, and clearly allow more specific imaging than 99mTc-MDP or control immunoglobulins. Because effective treatment is known to reduce the density of CD4+ cells in the inflamed synovial membrane, diagnostic methods targeted to CD4 warrant further attention, also for early diagnosis of clinically silent joints, precise description of the cellular infiltrates, and monitoring of anti-rheumatic therapy.

CD11c as a transcriptional biomarker to predict response to anti-TNF monotherapy with adalimumab in patients with rheumatoid arthritis.

We performed transcription profiling using monocytes to identify predictive markers of response to anti-tumor necrosis factor (anti-TNF) therapy in patients with rheumatoid arthritis (RA). Several potential predictors of response were identified, including CD11c. Validation in samples from independent cohorts (total of n = 27 patients) using reverse transcription-PCR confirmed increased expression of CD11c in responders to adalimumab (100% sensitivity; 91.7% specificity, power 99.6%; alpha = 0.01). Pretherapy CD11c levels significantly correlated with the response criteria as defined by the American College of Rheumatology (ACR) (r = 0.656, P < 0.0001). However, CD11c was neither predictive of response to methotrexate (MTX) alone (n = 34) nor to MTX in combination with adalimumab (n = 16). Clinical responders revealed a reset to a normal expression pattern of resident/inflammatory monocyte markers, which was absent in nonresponders. Therefore, an analysis of key cell types identifies potentially predictive biomarkers that may help to restrict the use of adalimumab to therapy responders. Larger studies, including studies of monotherapy with other drugs, are now needed to confirm and validate the specificity of CD11c for anti-TNF biologics.

Gelatin-based haemostyptic Spongostan as a possible three-dimensional scaffold for a chondrocyte matrix?: an experimental study with bovine chondrocytes.

The gelatin-based haemostyptic compound Spongostan was tested as a three-dimensional (3D) chondrocyte matrix in an in vitro model for autologous chondrocyte transplantation using cells harvested from bovine knees. In a control experiment of monolayer cultures, the proliferation or de-differentiation of bovine chondrocytes was either not or only marginally influenced by the presence of Spongostan (0.3 mg/ml). In monolayers and 3-D Minusheet culture chambers, the cartilage-specific differentiation markers aggrecan and type-II collagen were ubiquitously present in a cell-associated fashion and in the pericellular matrix. The Minusheet cultures usually showed a markedly higher mRNA expression than monolayer cultures irrespective of whether Spongostan had been present or not during culture. Although the de-differentiation marker type-I collagen was also present, the ratio of type-I to type-II collagen or aggrecan to type-I collagen remained higher in Minusheet 3-D cultures than in monolayer cultures irrespective of whether Spongostan had been included in or excluded from the monolayer cultures. The concentration of GAG in Minusheet cultures reached its maximum after 14 days with a mean of 0.83 +/- 0.8 microg/10(6) cells; mean +/-, SEM, but remained considerably lower than in monolayer cultures with/without Spongostan. Our results suggest that Spongostan is in principle suitable as a 3-D chondrocyte matrix, as demonstrated in Minusheet chambers, in particular for a culture period of 14 days. Clinically, differentiating effects on chondrocytes, simple handling and optimal formability may render Spongostan an attractive 3-D scaffold for autologous chondrocyte transplantation.

Synovitis score: discrimination between chronic low-grade and high-grade synovitis.

AIMS: To standardize the histopathological assessment of synovial membrane specimens in order to contribute to the diagnostics of rheumatic and non-rheumatic joint diseases. METHODS AND RESULTS: Three features of chronic synovitis (enlargement of lining cell layer, cellular density of synovial stroma, leukocytic infiltrate) were semiquantitatively evaluated (from 0, absent to 3, strong) and each feature was graded separately. The sum provided the synovitis score, which was interpreted as follows: 0-1, no synovitis; 2-4, low-grade synovitis; 5-9, high-grade synovitis. Five hundred and fifty-nine synovectomy specimens were graded by two independent observers. Clinical diagnoses were osteoarthrosis (n=212), post-traumatic arthritis (n=21), rheumatoid arthritis (n=246), psoriatic arthritis (n=22), reactive arthritis (n=9), as well as controls (n=49) from autopsies of patients without joint damage. Median synovitis scores when correlated with clinical diagnoses were: controls 1.0, osteoarthritis 2.0, post-traumatic arthritis 2.0, psoriatic arthritis 3.5, reactive arthritis 5.0 and rheumatoid arthritis 5.0. The scores differed significantly between most disease groups, especially between degenerative and rheumatic diseases. A high-grade synovitis was strongly associated with rheumatic joint diseases (P<0.001, sensitivity 61.7%, specificity 96.1%). The correlation between the two observers was high (r=0.941). CONCLUSION: The proposed synovitis score is based on well-defined, reproducible histopathological criteria and may contribute to diagnosis in rheumatic and non-rheumatic joint diseases.

Anti-CD4 monoclonal antibody treatment in acute and early chronic antigen induced arthritis: influence on macrophage activation.

OBJECTIVE: To investigate the indirect effects of anti-CD4 treatment on the functions of macrophages (CD4(-) in mice) in the acute and early chronic phase of mouse antigen induced arthritis (AIA). METHODS: C57BL/6 mice with AIA were treated intraperitoneally with the anti-CD4 mAb GK1.5 or control rat IgG on days -1, 0, 1, 3, 5, and 7. Proinflammatory cytokines (IL1 beta, IL6, and TNF alpha) were quantified by sandwich ELISA in joint extracts, serum, and supernatants of ex vivo stimulated spleen/lymph node cells or peritoneal macrophages (+LPS/IFN gamma). Nitric oxide (NO) levels in supernatants of ex vivo stimulated peritoneal macrophages were measured by the Griess reaction. Proteolytic activity in joint homogenates was analysed by gelatin, casein, and elastin zymography, and substrate assays. RESULTS: Anti-CD4 treatment significantly reduced joint swelling in acute (days 3, 5) and early chronic AIA (day 7) and diminished inflammation and destruction scores in late chronic AIA (day 21). On day 3, anti-CD4 treatment significantly reduced IL6 levels in all compartments. IL1 beta was reduced in joint extracts, unaffected in serum or cells from lymphoid organs, and increased in stimulated peritoneal macrophages. TNF alpha was significantly increased in the joints, decreased in serum, and otherwise unchanged. NO production by stimulated peritoneal macrophages was significantly reduced by anti-CD4 treatment. Lower activity of matrix metalloproteinases and neutrophil elastase was seen in joint extracts of anti-CD4 treated animals than in IgG treated AIA controls. CONCLUSION: CD4(+) T cell directed treatment had strong local and systemic effects on macrophages. These indirect effects may contribute to the reduction of destructive mediators/joint destruction in AIA.

Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry.

OBJECTIVES: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (Mphi) in immunohistochemistry (IHC) and flow cytometry (FACS). METHODS: IHC was performed on cryostat sections of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes using the anti-CD68 mAbs KP1, EBM11, and PGM1, and the fibroblast (FB) markers CD90 and prolyl 4-hydroxylase. Expression of CD68 was also analysed by FACS on the monocytic cell lines THP-1 and U937, as well as on synovial fibroblasts (SFB), skin FB, and gingival FB (both surface and intracellular staining). RESULTS: In IHC, there was an overlap between CD68 (mAbs KP1 and EBM11) and the FB markers CD90/prolyl 4-hydroxylase in the lining layer, diffuse infiltrates, and stroma of RA and OA synovial membranes. In FACS analysis of THP-1 and U937 cells, the percentage of cells positive for the anti-CD68 mAbs KP1 and EBM11 progressively increased from surface staining of unfixed cells, to surface staining of pre-fixed cells, to intracellular staining of the cells. Upon intracellular FACS of different FB, nearly all cells were positive for KP1 and EBM11, but only a small percentage for PGM1. In surface staining FACS, a small percentage of FB were positive for all three anti-CD68 mAbs. CONCLUSION: An overlap between CD68 (mAbs KP1 or EBM11) and the FB markers CD90 or prolyl 4-hydroxylase may prevent unequivocal identification of Mphi in synovial tissue by IHC or in monocytic cells and FB upon intracellular FACS. This may be due to sharing of common markers by completely different cell lineages.

Anti-CD4 monoclonal antibody treatment in acute and early chronic antigen-induced arthritis: influence on T helper cell activation.

To examine the effects of anti-CD4 mAb treatment in acute and chronic antigen-induced arthritis (AIA), C57BL/6 mice were treated intraperitoneally either with the depleting anti-CD4 mAb GK1.5 or with rat-IgG (control) on Days -1, 0, 1, 3, 5, and 7. Arthritis was monitored by assessment of joint swelling and histological evaluation in the acute (Day 3) and the chronic phase (Day 21) of AIA. To determine the effects on cellular immune responses, in vivo T-cell reactivity (delayed type hypersensitivity; DTH) was measured, as well as protein levels of TH1- (IL-2, IFN-gamma) and TH2-cytokines (IL-4, IL-10) in joint extracts and supernatants of ex vivo stimulated spleen and lymph node cells. The humoral immune response was analysed by measuring serum antibodies against methylated bovine serum albumine (mBSA) and extracellular matrix proteins. Treatment with GK1.5 reduced swelling, inflammation, and destruction of the arthritic joint. Unexpectedly, the effects were even more pronounced in the acute than in the chronic phase. The anti-inflammatory effect was accompanied by a diminished DTH against the arthritogen mBSA and a decrease of TH1-cytokine production in spleen and pooled body lymph nodes, whereas the TH2-cytokine production in these organs was unchanged and the humoral immune response was only moderately reduced. There was a failure of depleting CD4+ T-cells in the joint, reflected also by unchanged local cytokine levels. Therefore, systemic rather than local effects on the TH1/TH2 balance appear to underlie the therapeutic efficacy of anti-CD4 treatment in AIA.

A novel inhalation allergen present in the working environment of beekeepers.

BACKGROUND: Inhalation allergies, caused by allergens from various kinds of pollen, house dust mites, animal epithelium, and mould fungi, are strongly increasing in frequency. In 2.6% of the cases the allergen source remains unidentified. The present paper describes a so far unknown inhalation allergy which was observed in the case of a patient working with hives. METHODS AND RESULTS: The allergen was characterized by immunoblotting, enzyme-linked immunosorbent assay inhibition, and isoelectrofocusing, using the serum of the patient. It is present in both the bee bodies and the larvae, has a molecular mass of 13 kDa, and an isoelectric point of 5.85. It is thermolabile and does not cross-react with allergens from birch, mugwort and timothy grass pollen, mould fungi, or bee venom. The N-terminal amino acid sequence of allergen from larvae was determined to be (2)QIEELKTRLHT(12). A similar allergen of 13 kDa was also found in Varroa mite accompanying bee populations. CONCLUSION: Honey bees (including the larva stadium) and Varroa mite contain a 13-kDa protein causing an allergic reaction. Presently, there is no evidence whether the case described is a singular phenomenon or whether this allergen is a more common inducer of allergies among subjects exposed to honey bees. However, a bee and Varroa mite allergy has to be considered for beekeepers after exclusion of known inhalation allergies.

Further characterization of IgE-binding antigens from guinea pig hair as new members of the lipocalin family.

BACKGROUND: Guinea pigs are important sources of inhalant allergens in home and working environments. However, little is known about the molecular characteristics and the relevant epitopes of guinea pig allergens. Recently, several allergens have been identified in hair extract and urine, and the major allergen Cav p 1 (20 kDa) has been characterized. OBJECTIVE: The aim of the present study was to isolate and to characterize a further major allergen from guinea pig hair with 17 kDa. METHODS: Guinea pig hair extract was fractionated using anion exchange chromatography and reverse-phase high performance liquid chromatography. Analyses were carried out by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, 2D-PAGE, immunoblotting, immunoblot inhibition, glycoprotein detection, and N-terminal amino acid sequencing. RESULTS: The nonglycosylated 17 kDa allergen, which was named Cav p 2, was purified to homogeneity. On the basis its 15 N-terminal residues, there was 69% identity with a sequence of Bos d 2, an allergenic protein from cow dander belonging to the lipocalin family. The 2D-immunoblotting analyses of guinea pig hair extract demonstrated that Cav p 2 and Cav p 1, contained several isoforms with pI values ranging from 3.6 to 5.3. The 2D-immunoblot inhibition disclosed cross-reactive IgE epitopes on the allergens Cav p 2 and Cav p 1. Furthermore, Cav p 1 can form both monomers (20 kDa) and dimers (40-42 kDa). CONCLUSION: These studies provide important information on the isoallergen character of two relevant guinea pig allergens Cav p 1 and Cav p 2 as well as on their cross-reactive properties.

Cell volume regulation: osmolytes, osmolyte transport, and signal transduction.

In recent years, it has become evident that the volume of a given cell is an important factor not only in defining its intracellular osmolality and its shape, but also in defining other cellular functions, such as transepithelial transport, cell migration, cell growth, cell death, and the regulation of intracellular metabolism. In addition, besides inorganic osmolytes, the existence of organic osmolytes in cells has been discovered. Osmolyte transport systems-channels and carriers alike-have been identified and characterized at a molecular level and also, to a certain extent, the intracellular signals regulating osmolyte movements across the plasma membrane. The current review reflects these developments and focuses on the contributions of inorganic and organic osmolytes and their transport systems in regulatory volume increase (RVI) and regulatory volume decrease (RVD) in a variety of cells. Furthermore, the current knowledge on signal transduction in volume regulation is compiled, revealing an astonishing diversity in transport systems, as well as of regulatory signals. The information available indicates the existence of intricate spatial and temporal networks that control cell volume and that we are just beginning to be able to investigate and to understand.

Characterization of a type IIb sodium-phosphate cotransporter from zebrafish (Danio rerio) kidney.

Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent P(i) cotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number ). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.

Minimal contribution of cell-bound antibodies to the immunoscintigraphy of inflamed joints with 99mTc-anti-CD4 monoclonal antibodies.

AIM: The cellular joint infiltrate in rheumatoid arthritis patients is rich in CD4-positive T-helper lymphocytes and macrophages, rendering anti-CD4 monoclonal antibodies (mAbs) suitable for specific immunoscintigraphy of human/experimental arthritis. Following intravenous injection, however, mAbs are present both in the free form and bound to CD4-positive, circulating monocytes and T-cells. Thus, the present study aimed at analyzing the relative contribution of the free and the cell-bound component to the imaging of inflamed joints in experimental adjuvant arthritis (AA). METHODS: AA rat peritoneal macrophages or lymph node l-cells were incubated in vitro with saturating amounts of 99mTc-anti-CD4 mAb (W3/25) and injected i.v. into rats with AA. RESULTS: In vitro release of 99mTc-anti-CD4 mAb from the cells was limited (on average 1.57%/h for macrophages and 0.84%/h for T-cells). Following i.v. injection, whole body/joint scans and tissue measurements showed only negligible accumulation of radioactivity in inflamed ankle joints (tissue: 0.22 and 0.34% of the injected activity, respectively), whereas the radioactivity was concentrated in liver (tissue: 79% and 71%, respectively), kidney, and urinary bladder. Unlike macrophages, however, anti-CD4 mAb-coated T-cells significantly accumulated in lymphoid organs, the inflamed synovial membrane of the ankle joints, as well as in elbow and knee joints. CONCLUSION: While the overall contribution of cell-bound mAbs to the imaging of arthritic joints with anti-CD4 mAbs is minimal, differential accumulation of macrophages and T-cells in lymphoid organs and the inflamed synovial membrane indicates preferential migration patterns of these 2 cell populations in arthritic rats. Although only validated for 99mTc-anti-CD4 mAbs, extrapolation of the results to other anticellular mAbs with similar affinity for their antigen may be possible.

Identification of a region critically involved in the interaction of phlorizin with the rabbit sodium-D-glucose cotransporter SGLT1.

In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604-610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of alpha-methyl-D-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K(i) for phlorizin increased at least by a factor of 5 compared to the wild-type K(i) of 4.6 +/- 0.7 micromol/l; most striking changes were observed for Y604K (K(i) = 75.3 +/- 19.0 micromol/l) and C608K (K(i) = 83.6 +/- 13.9 micromol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (Km = 0.8 +/- 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside.

Systemic macrophage activation in locally-induced experimental arthritis.

Local and systemic macrophage activation was examined during the course of monoarticular murine antigen-induced arthritis (AIA), induced by systemic immunization and subsequent local induction. The levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-12p70, and nitric oxide (NO) were determined in joints, sera, and supernatants of peritoneal macrophages (the latter unstimulated or stimulated ex vivo with LPS/IFN-gamma). In comparison with normal mice, systemic immunization (day 0) was associated to significant rise of TNF-alpha in serum, IL-1beta in the joints, IL-6 in unstimulated macrophages and IL-12p70 in stimulated macrophages. Local induction led to a further significant increase of: (i) TNF-alpha, IL-1beta, and IL-6 in the joints; and (ii) IL-1beta, and IL-6 in sera and stimulated macrophages during acute and/or early chronic AIA (days 1 to 7). Unstimulated macrophages showed increased NO release (day 3), while stimulated macrophages significantly increased secretion of IL-12p70 (day 1). In late chronic AIA (day 21), cytokine/NO expression returned to immunization levels or below at all sites; solely IL-1beta in the joints remained significantly above normal levels. Therefore, the prevalently local AIA model is characterized by a mixture of local and systemic activation of the mononuclear phagocyte system (MPS). While systemic MPS activation preceding arthritis induction can be attributed to systemic immunization, further systemic activation during arthritis appears an integral pathogenetic component of AIA.