Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency.

Mutations in mismatch repair (MMR) genes result in microsatellite instability (MSI) and early onset of colorectal cancer. To get mechanistic insights into the time scale, sequence and frequency of intestinal stem cell (ISC) transformation, we quantified MSI and growth characteristics of organoids of Msh2-deficient and control mice from birth until tumor formation and related them to tissue gene expression. Although in Msh2-deficient organoids MSI continuously increased from birth, growth characteristics remained stable at first. Months before tumor onset, normal Msh2-deficient tissue contained tumor precursor cells forming organoids with higher MSI, cystic growth and growth rates resembling temporarily those of tumor organoids. Consistently, Msh2-deficient tissue exhibited a tumor-like gene signature. Normal Msh2-deficient organoids showed increased inheritable transient cyst-like growth, which became independent of R-spondin. ISC transformation proceeded faster in vitro than in vivo independent of the underlying genotype but more under MMR deficiency. Transient cyst-like growth but not MSI was suppressed by aspirin. In summary, as highlighted by organoids, molecular alterations continuously proceeded long before tumor onset in MMR-deficient intestine, thus increasing its susceptibility for ISC transformation.

Use of a human skin in vitro model to investigate the influence of 'every-day' clothing and skin surface decontamination on the percutaneous penetration of organophosphates.

UNLABELLED: Organophosphates (OPs) are widely used in agriculture. Many studies have investigated the capability of personal protective equipment (PPE) to reduce chemical exposure; however, investigations into the protective effect of 'every-day' clothing are rare. The purpose of this study was to investigate the protective effect of 'every-day' clothing against dermal exposure and to measure early decontamination of skin following exposure to chlorpyrifos and dichlorvos. Using human skin in vitro, absorption of (14)C-labelled chlorpyrifos (500 ng/cm(2)), was shown to be significantly reduced when applied to clothed skin (cotton shirt), regardless of application vehicle (isopropanol (IPA) or propylene glycol (PG)). The majority of applied dose was retained within the clothing after 4 h exposure. Significant reduction in absorption of chlorpyrifos (in PG) was seen through clothed skin when supplemented with skin decontamination at 4 h, compared with clothed skin decontaminated after 24 h, however, this was not observed with IPA. Absorption of dichlorvos (5 µg/cm(2)) was greater through unclothed skin than chlorpyrifos for all vehicles (IPA, isopropyl myristate (IPM) and PG). Significant reduction in absorption was observed when decontaminating clothed skin at 30 min, compared with decontamination at 24 h (post-exposure) for all vehicles. RESULT: indicate that 'every-day' clothing is effective at reducing exposure to chemicals in contact with skin. Washing the skin surface immediately following removal of exposed clothing can further reduce exposure, depending on the properties of the chemical and vehicle applied.

Percutaneous absorption and distribution of organophosphates (chlorpyrifos and dichlorvos) following dermal exposure and decontamination scenarios using in vitro human skin model.

To date, there has been little research investigating low-level human exposure to chemicals, and so the aim of this study was to examine the percutaneous penetration of organophosphates (dichlorvos and chlorpyrifos) using low-level exposure scenarios in vitro. Dermal absorption of chlorpyrifos applied in different vehicles was measured at 0, 4, 8 and 24 h, after dose application for 4 and 24 h exposure (finite dose, 500 ng/cm(2)) in isopropanol (IPA), isopropyl myristate (IPM) and propylene glycol (PG). Dichlorvos was applied to the skin for 24 h (infinite dose, 1 mg/cm(2) and 10 mg/cm(2); finite dose, 5 µg/cm(2)) using the same vehicles. Human skin was mounted in flow through diffusion cells with minimum essential medium eagle pH 7.4 (supplemented with 2% BSA) as receptor fluid. Following exposure, the skin surface dose was removed by tissue swabbing, the stratum corneum removed by sequential tape stripping, and the skin digested prior to scintillation counting (chlorpyrifos), or GC/MS analysis (dichlorvos). The dermal absorption of chlorpyrifos was the greatest following application in PG (19.5% of dose), when compared with absorption from the IPA and IPM vehicles (10.3% and 1.9% absorbed respectively). However, dichlorvos showed greater dermal absorption than chlorpyrifos from all vehicles used, with greatest absorption from the IPA vehicle (38.6% absorbed). Although dichlorvos exhibited a short lag time (0.6 h from IPA and IP vehicles, and 0.4 h from PG), chlorpyrifos displayed greater propensity to accumulate in the stratum corneum and epidermis/dermis. These results demonstrate that prompt skin surface decontamination would be required for both dichlorvos and chlorpyrifos (and chemicals with similar properties) in the event of skin contact. The magnitude of the skin reservoir formed with chlorpyrifos was time dependent, therefore, prompt decontamination of this and similar chemicals would be required to reduce delayed systemic absorption.

Good clinical outcome after accidental intra-arterial injection of flunitrazepam tablets in 16 drug abusers with critical limb ischaemia.

OBJECTIVES: Inadvertent intra-arterial injection of flunitrazepam tablets intended for intravenous use by drug abusers has devastating effects. We report here on the clinical outcome of 16 drug abusers developing critical limb ischaemia after flunitrazepam injection. METHODS: Treatment combined immediate analgesia and anticoagulation, long-lasting local thrombolysis and vasodilatation, antibiotic prophylaxis, and physical mobilization. The immediate bolus injection of 5,000 IU heparin was followed by a continuous heparin infusion up to the target partial thromboplastin time. Under arteriographic control local intra-arterial infusion with alternating 4-h cycles of 5 mg recombinant tissue plasminogen activator followed by 5 µg prostaglandinE1 (PGE1) was performed for 24-48 hours. Subsequently, 60 µg PGE1 was applied once daily. RESULTS: Drug abusers, having been injected with 4-30 mg flunitrazepam, were treated 3-72 hours after the accident, with six of them not being treated until after 24 hours. All showed a high tissue ischaemia score. At the time of being discharged from hospital 13 patients had a normal extremity. In one patient, first receiving treatment 72 hours after injection, minor amputation of fingers was necessary. The life of the patient who injected 30 mg flunitrazepam in the leg was saved after hip disarticulation. One patient developed neurological dysfunction in the affected toes. CONCLUSIONS: Intensive treatment after inadvertent intra-arterial drug injection normalized the affected extremity in most drug abusers, even after the late onset of therapy.

Ca(2+)-binding protein expression in primary human thyrocytes.

We recently identified several Ca(2+)-binding proteins (CaBP) from the S100 and annexin family to be regulated by TSH in FRTL-5 cells. Here, we study the regulation of S100A4, S100A6 and ANXA2 in primary human thyrocytes (PHT) derived from surrounding tissues (ST), cold benign thyroid nodules (CTN) and autonomously functioning thyroid nodules (AFTN). We investigated the expression and regulation of CaBP and the effect of their expression on Ca(2+) and TSHR signaling. We used an approach that accounts for the potential of an individual PHT culture to proliferate or to express thyroid differentiation features by assessing the expression of FOS and TPO. We found a strong correlation between the regulation of CaBP and the proliferation-associated transcription factor gene FOS. PKA and MEK1/2 were regulators of ANXA2 expression, while PI3-K and triiodothyronine were additionally involved in S100 regulation. The modulated expression of CaBP was reflected by changes in ATP-elicited Ca(2+) signaling in PHT. S100A4 increased the ratio of subsequent Ca(2+) responses and showed a Ca(2+) buffering effect, while ANXA2 affected the first Ca(2+) response to ATP. Overexpression of S100A4 led to a reduced activation of NFAT by TSH. Using S100A4 E33Q, D63N, F72Q and Y75K mutants we found that the effects of S100A4 expression on Ca(2+) signaling are mediated by protein interaction. We present evidence that TSH has the ability to fine-tune Ca(2+) signals through the regulation of CaBP expression. This represents a novel putative cross-regulating mechanism in thyrocytes that could affect thyrocyte signaling and physiology.

[Ischemic preconditioning in a rat adipocutaneous flap model]. / Der epigastrisch adipokutane Lappen am Tiermodell zur Evaluierung der ischämischen Präkonditionierung.

INTRODUCTION: Flap necrosis in ear, nose, and throat surgery, especially in high-risk groups, is not rare, but not all of the individual pathophysiological processes are known. The objective of this study was to establish an animal model to determine whether acute ischemic preconditioning, which has been reported to be successful in organ transplantation, will result in enhanced flap survival. METHODS AND MATERIALS: Forty-two Wistar rats were divided into three experimental groups. An epigastric adipocutaneous flap, based on both superficial epigastric arteries and veins, was raised. The flap was either raised (control), clamped for 2 h (ischemic), or subjected to ischemia of 30 min, followed by 30 min of reperfusion and another 2 h of induced ischemia (IP). The mean flap necrosis area was assessed in all groups on the 5th postoperative day. RESULTS: All animals were doing well on the final day. The average necrosis in the ischemic group was significantly greater than in the control group. No significant superiority in the IP group was demonstrated. CONCLUSION: The data show that the experimental animal model is practicable and that additional approaches to ischemic preconditioning should be verified.

From single cells to tissue architecture-a bottom-up approach to modelling the spatio-temporal organisation of complex multi-cellular systems.

Collective phenomena in multi-cellular assemblies can be approached on different levels of complexity. Here, we discuss a number of mathematical models which consider the dynamics of each individual cell, so-called agent-based or individual-based models (IBMs). As a special feature, these models allow to account for intracellular decision processes which are triggered by biomechanical cell-cell or cell-matrix interactions. We discuss their impact on the growth and homeostasis of multi-cellular systems as simulated by lattice-free models. Our results demonstrate that cell polarisation subsequent to cell-cell contact formation can be a source of stability in epithelial monolayers. Stroma contact-dependent regulation of tumour cell proliferation and migration is shown to result in invasion dynamics in accordance with the migrating cancer stem cell hypothesis. However, we demonstrate that different regulation mechanisms can equally well comply with present experimental results. Thus, we suggest a panel of experimental studies for the in-depth validation of the model assumptions.

Individual cell-based models of the spatial-temporal organization of multicellular systems--achievements and limitations.

Computational approaches of multicellular assemblies have reached a stage where they may contribute to unveil the processes that underlie the organization of tissues and multicellular aggregates. In this article, we briefly review and present some new results on a number of 3D lattice free individual cell-based mathematical models of epithelial cell populations. The models we consider here are parameterized by bio-physical and cell-biological quantities on the level of an individual cell. Eventually, they aim at predicting the dynamics of the biological processes on the tissue level. We focus on a number of systems, the growth of cell populations in vitro, and the spatial-temporal organization of regenerative tissues. For selected examples we compare different model approaches and show that the qualitative results are robust with respect to many model details. Hence, for the qualitative features and largely for the quantitative features many model details do not matter as long as characteristic biological features and mechanisms are correctly represented. For a quantitative prediction, the control of the bio-physical and cell-biological parameters on the molecular scale has to be known. At this point, slide-based cytometry may contribute. It permits to track the fate of cells and other tissue subunits in time and validated the organization processes predicted by the mathematical models.

Graves' disease and Hashimoto's thyroiditis in monozygotic twins: case study as well as transcriptomic and immunohistological analysis of thyroid tissues.

OBJECTIVE: To report on the rare simultaneous occurrence of Graves' disease (GD) and Hashimoto's thyroiditis (HT) in monozygotic twins. DESIGN: We compared the pattern of thyroid tissue-derived cDNAs to gain insight into previous and ongoing immune destruction and reconstruction processes using microarrays. The results were confirmed by immunohistology and real-time PCR. RESULTS: Destruction of thyroid tissue in HT reduced levels of thyrocyte-related cDNAs and cDNAs encoding extracellular matrix components, but increased levels of proteases involved in extracellular matrix degradation compared with GD. Lymphocytic infiltrates forming ectopic follicles replaced the thyroid tissue almost completely in HT. Thus, lymphocyte-related cDNA levels were higher in HT than in GD. The same was true for many chemokines and their receptors, which not only enable migration towards the thyroid but also maintain the lymphocytic infiltrate. HT also showed increased levels of cDNAs encoding molecules related to apoptosis than did GD. Surprisingly, the Th1- and Th2-specific cytokine profiles suggested for HT and GD respectively could not be confirmed. cDNAs encoding factors and receptors involved in angiogenesis were increased in GD compared with HT. CONCLUSIONS: Comparison of gene expression reflects the cellular differences between the two types of autoimmune thyroid disease in twins with identical genetic and similar environmental background.

Bovine eotaxin (CCL11)--an unusual member of the eotaxin group--attracts eosinophils in vitro but is not responsible for eosinophilia in the ovary.

Under physiological conditions normally characterised by low tissue infiltration of eosinophils, a conspicuous number of these cells are attracted into the human and ruminant ovary. Eosinophils suddenly increase in the thecal layer of the preovulatory follicle and corpus luteum at very early development. Currently, we only have a limited understanding of the mechanism for the recruitment of the ovarian eosinophils. Eotaxin (CCL11) may be one of the chemoattractants involved in stimulating eosinophils to migrate selectively into ovary. As a prerequisite for the analysis of eotaxin expression in the bovine ovary, we determined the complete bovine eotaxin mRNA sequence since it was not available from databases. The bovine eotaxin is the first member of the monocyte chemoattractant protein (MCP)/eotaxin subfamily with two mRNA isoforms varying in length in the untranslated 3'-untranslated region. The unusual amino-acid sequence of bovine eotaxin contains structural features that are so far known to be characteristic for MCP, but not eotaxin. In our microchemotaxis assays, recombinant bovine eotaxin showed a functional pattern orthologous to known eotaxins. Thus, the chimeric structure of bovine eotaxin did not affect the favoured chemotactic activity on eosinophils. Semiquantitative RT-PCR was used to investigate the expression of eotaxin in different regions of the bovine ovary. We only detected faint eotaxin mRNA signals that did not indicate physiological significance even in stimulated granulosa cell cultures, follicle-derived macrophages or fibroblasts. Taken together, bovine eotaxin attracts eosinophils in vitro but is not responsible for eosinophilia in the ovary. Its unusual chimeric structure confirms the unity of the MCP/eotaxin subfamily of CC chemokines and distinguishes it from other CC chemokine subfamilies.

CXCR6 within T-helper (Th) and T-cytotoxic (Tc) type 1 lymphocytes in Graves' disease (GD).

OBJECTIVE: In Graves' disease (GD), stimulating anti-TSH receptor antibodies are responsible for hyperthyroidism. T-helper 2 (Th2) cells were expected to be involved in the underlying immune mechanism, although this is still controversial. The aim of this study was to examine the expression of CXCR6, a chemokine receptor that marks functionally specialized T-cells within the Th1 and T-cytotoxic 1 (Tc1) cell pool, to gain new insights into the running immune processes. METHODS: CXCR6 expression was examined on peripheral blood lymphocytes (PBLs) and thyroid-derived lymphocytes (TLs) of GD patients in flow cytometry. CXCR6 cDNA was quantified in thyroid tissues affected by GD (n = 16), Hashimoto's thyroiditis (HT; n = 2) and thyroid autonomy (TA; n = 11) using real-time reverse transcriptase PCR. RESULTS: The percentages of peripheral CXCR6(+) PBLs did not differ between GD and normal subjects. CXCR6 was expressed by small subsets of circulating T-cells and natural killer (NK) cells. CXCR6(+) cells were enriched in thyroid-derived T-cells compared with peripheral CD4(+) and CD8(+) T-cells in GD. The increase was evident within the Th1 (CD4(+) interferon-gamma(+) (IFN-gamma(+))) and Tc1 (CD8(+)IFN-gamma(+)) subpopulation and CD8(+) granzyme A(+) T-cells (cytotoxic effector type). Thyroid-derived fibro-blasts and thyrocytes were CXCR6(-). There was no significant difference between the CXCR6 mRNA levels in GD compared with HT and normal TA tissues. The lowest CXCR6 mRNA levels were obtained from thyroid nodules from TA patients and GD patients with low thyroid peroxidase autoantibody levels. CONCLUSIONS: CXCR6 was overexpressed in Th1 and Tc1 TLs compared with PBLs in GD. CXCR6 could be a marker for lymphocytes that have migrated into the thyroid and assist in the thyroid, independently of the bias of the underlying disease.

Expression of ADAM15 in lung carcinomas.

ADAM15, a member of the ADAM (a disintegrin and metalloprotease) family, is a membrane protein containing both protease and adhesion domains and may, thus, be involved in tumor invasion and metastasis. The aim of this study was to analyze the expression of ADAM15 and its potential ligand, integrin alpha(v)beta3 (CD51/CD61), in lung carcinoma cell lines and tissues. Most small cell lung carcinomas (SCLCs) and non-SCLC cell lines were ADAM15, alpha(v) and beta3 integrin mRNA positive. Half of the cell lines expressed ADAM15, and three expressed the alpha(v)beta3 heterodimer at the cell surface as shown using flow cytometry. Paraffin sections of pulmonary epithelial tumors, including SCLCs (n=26), squamous cell cancer (SCCs, n=27) and adenocarcinomas (ACs, n=17) were stained with antibodies to the ectosolic and cytosolic domain of ADAM15 and alpha(v)beta3 integrin complex. The results were scored (0-12, according to Remmele's score). Normal epithelial cells of the lung were negative or slightly positive for ADAM15 (score<2). The score was always significantly higher for tumor cells. ACs showed the strongest staining (tumor center; ADAM15ecto; mean+/-SEM; 5.47+/-1.04), whereas SCLCs only showed weak ADAM15 expression (2.67+/-0.42; SCCs: 3.62+/-0.62). Frequently, significantly stronger ADAM15 expression has been shown in tumor cells located at the front of invasion compared with those within solid formations. Overall analysis of all tumor specimens and each tumor type revealed no significant correlation between tumor stage or degree of differentiation and ADAM15 ectosolic or cytosolic domain expression in tumor cells. Both molecules are often co-localized in the same tumor cells in ADAM15- and alpha(v)beta3 integrin-positive carcinomas. In summary, lung carcinoma cell lines and tissues were frequently ADAM15 positive.

[Two-tier screening process (TEOAE/AABR) reduces recall rates in newborn hearing screening]. / Sequenzielles Neugeborenen-Hörscreening (TEOAE/AABR) reduziert Recall-Rate. Erfahrungen in einem Berliner Perinatalzentrum.

BACKGROUND: 1-2/1,000 newborns are affected by connatal permanent hearing impairment. Clinical diagnosis is often delayed. This demands newborn hearing screening (NHS). Some questions regarding the optimal method remain unsolved. METHODS: The newborns in the obstetrical department (low-risk group) are tested by automated transitory evoked otoacustic emissions (TEOAE). TEOAE-fail is followed by automated auditory brainstem response (AABR) examination. All sick newborns admitted to the pediatric department (high-risk group) are primarily tested using AABR. Pathological AABR-testing leads to pedaudiological diagnostic work-up. RESULTS: In the low-risk group, 82 out of 1,584 newborns failed TEOAE-testing (recall 5.18%). Only 5 of these patients failed consecutive AABR examination (recall 0.32%). Permanent hearing loss was finally confirmed in 3 children (0.13%). 10 out of 755 newborns in the high-risk group failed AABR-testing (1.32%). In 6 of these children, hearing loss was confirmed (0.79%). CONCLUSION: A two-tier screening process as described is able to reduce recall rate, overall expenses and parental anxiety.

The role of CXCR5 and its ligand CXCL13 in the compartmentalization of lymphocytes in thyroids affected by autoimmune thyroid diseases.

OBJECTIVE: Graves' disease (GD) and Hashimoto's thyroiditis (HT) are characterized by lymphocytic infiltrates partly resembling secondary lymphoid follicles in the thyroid. CXCR5 and its ligand CXCL13 regulate compartmentalization of B- and T-cells in secondary lymphoid organs. The aim of the study was to elucidate the role of this chemokine receptor-ligand pair in thyroid autoimmunity. METHODS: Peripheral blood and thyroid-derived lymphocyte subpopulations were examined by flow cytometry for CXCR5. CXCR5 and CXCL13 cDNA were quantified in thyroid tissues by real-time RT-PCR. RESULTS: We found no differences between the percentages of peripheral blood CXCR5+ T- and B-cells in GD patients (n=10) and healthy controls (n=10). In GD patients, the number of memory CD4+ cells expressing CXCR5 which are functionally characterized as follicular B helper T-cells is higher in thyroid-derived (18+/-3%) compared with peripheral blood T-lymphocytes (8+/-2%). The highest CXCL13 mRNA levels were found in HT (n=2, 86.1+/-1.2 zmol (10(-21) mol) cDNA/PCR) followed by GD tissues (n=16, 9.6+/-3.5). Only low amounts were determined in thyroid autonomy (TA) (n=11) thyroid tissues, irrespective of whether the autonomous nodule (0.5+/-0.1) or the surrounding normal tissue (1.8+/-0.7) had been analyzed. The same differences were found for CXCR5 (HT: 179.1+/-6.8; GD: 17.4+/-10.6; TA(nodule): 0.8+/-0.5; TA(normal): 4.4+/-3.6). In GD, there is a correlation between CXCL13 and CXCR5 mRNA levels and the number of focal lymphocytic infiltrates and germinal centers as well as anti-thyroperoxidase but not anti-TSH receptor autoantibodies. CONCLUSIONS: CXCR5 and CXCL13 play an essential role in maintaining B- and T-cells in lymphocytic infiltrates and ectopic follicles in thyroid tissue from patients affected by autoimmunity.

Differential expression and activity status of MMP-1, MMP-2 and MMP-9 in tumor and stromal cells of squamous cell carcinomas of the lung.

Matrix metalloproteinases (MMPs) play a key role in cancer progression. Interstitial collagenase (MMP-1) and type IV collagenases (MMP-2, MMP-9) are involved in the initial breakdown of collagen and basement membrane components during tumor growth and invasion. Besides tumor cells, fibroblasts are especially involved in MMP production. The aim of this study was to quantify MMP-1, MMP-2 and MMP-9 within tumor cells and tumor-surrounding fibroblasts compared to normal lung epithelial cells to gain an insight into the function of these MMPs in squamous cell carcinomas of the lung. The expression and activity of MMP-1, MMP-2 and MMP-9 were analyzed in 30 squamous cell carcinomas and in normal lung tissue from the same patients by immunohistology and gelatin zymography. The majority of tumor cells were positive for MMP-1 (mean +/- SD: 67.3 +/- 26.7%) and MMP-9 (64.7 +/- 22.8%), whereas a significantly lower percentage of normal bronchoepithelial cells (47.3 +/- 25.4 and 40.3 +/- 24.2%, respectively; p < 0.01) and fibroblasts located in the tumor-surrounding tissue (39.7 +/- 14.3 and 38.1 +/- 24.1%, respectively; p < 0.01) expressed these MMPs. Only a few tumor cells showed any immunoreactivity for MMP-2 (4.4 +/- 6.7%), whereas a higher percentage of fibroblasts tested positive for this enzyme (8.6 +/- 13.1%; p < 0.01). Using gelatin zymography, we could demonstrate that MMP-2 is activated in the tumor only, not in normal lung tissue. The coordinated expression of MMP-1, MMP-2 and MMP-9 in tumor cells and/or their induction in tumor-surrounding fibroblasts and further activation in the tumor tissue may be involved in the high invasive and metastatic potential of squamous cell carcinomas of the lung. Comparing the results from immunohistology and zymography can give indications for distribution and activity of proteinases, especially certain MMPs such as MMP-2.

Correlation between expression of selectins and migration of eosinophils into the bovine ovary during the periovulatory period.

Leukocytes enter specific ovarian areas at a precisely defined moment, influencing cyclically changing structures such as follicles and corpora lutea. As yet, no studies have been published on the trafficking mechanisms involving the interaction between adhesion molecules on endothelial cells (ECs) and those on leukocytes. First, antibodies against human adhesion molecules were examined by flow cytometry with the aim of identifying the same bovine antigen. Western blot analysis and immunoprecipitation revealed that the molecules had the same molecular weight as their human counterparts. Afterwards, we investigated the distribution of these antigens in various ovarian stages using immunohistology. Among the molecules, P-selectin (CD62P) and L-selectin (CD62L) showed stage-dependent expression, and were thus examined further. In the preovulatory follicle, microvascular ECs were negative for CD62P. Few of the leukocytes expressed CD62L. In a freshly ruptured follicle, CD62P expression was found in the dilated vessels of the former thecal layer. Simultaneously, a large proportion of the rapidly increased numbers of leukocytes, mainly eosinophils, located around the microvessels of the outer thecal layer expressed CD62L. In the early corpus luteum development stage, CD62L showed peak expression with 70%-80% positive cells compared to leukocytes. In the secretory stage, the septal venules showed a consistent, but now weak, staining for CD62P. Few leukocytes expressed CD62L. During regression, the total number of leukocytes, now representing macrophages, increased significantly, but the proportion of CD62L-positive cells remained constant. In summary, we found a strong correlation of CD62P expression on activated ECs and the appearance of CD62L-positive leukocytes in the early corpus luteum development stage, suggesting the participation of both selectins in the migration of eosinophils under physiological conditions.

Graves' disease is associated with an altered CXCR3 and CCR5 expression in thyroid-derived compared to peripheral blood lymphocytes.

The mechanisms by which T cells accumulate in the thyroid and support the autoimmune process in patients with Graves' disease (GD) are poorly understood. Chemokines and their receptors may be involved in this process. We have analysed the expression of CXCR3 and CCR5 as Th1-specific chemokine receptors, CCR3 as a marker for Th2 cells, CXCR4 (expressed on unprimed, naive T cells) and CCR2 (known to be involved in autoimmunity) on peripheral blood (PBL) and thyroid-derived lymphocytes (TL) using flow cytometry. Chemokine receptor expression on PBL of GD patients (n = 16) did not differ from that of normal controls (n = 10). In GD, CXCR3+ (67.3 +/- 4.0% versus 45.7 +/- 2.1%) and CCR5+ T cells (42.5 +/- 3.4% versus 18.8 +/- 2.1%) showed a significant enrichment in the TL compared to PBL. The positive cells were contributed mainly by the CD4+CD45R0+ subset. TL are mostly primed CD45R0+ T cells, but surprisingly, they had significantly higher levels of CXCR4+ cells among TL (96.2 +/- 1.0%) compared to PBL (66.8 +/- 4.2%). However, CXCR4 has been induced during in vitro isolation of TL. There was no correlation between chemokine receptors and the level of TSH-receptor and thyroid peroxidase autoantibodies. CCR3+ and CCR2+ cells remained unchanged in TL compared to PBL. We could confirm the results using RT PCR and immunohistology. In summary, TL showed a different chemokine receptor pattern compared to PBL from the same patient. This indicates a role for CXCR3 and CCR5 in the recruitment of T cells to the thyroid in GD.

Regulation of sodium-iodide-symporter gene expression in human thyrocytes measured by real-time polymerase chain reaction.

The sodium-iodide-symporter (NIS) plays a key role in iodination, the first step in the biosynthesis of the thyroid hormones, and is thought to be critically involved in several thyroid disorders associated with altered iodine up-take. To elucidate the pathogenic role of NIS in these diseases a sensitive technique is needed to measure human NIS gene expression. We established a real time RT-PCR for accurate quantification of hNIS mRNA levels based on fluorescence-labelled hybridisation probes in the LightCycler system. Human NIS expression was investigated in primary cultures of human thyrocytes. After optimisation of PCR conditions less than 10 molecules hNIS were detected with high sensitivity, specificity and reproducibility. Under basal conditions NIS expression varied from 83 to 593 copies per 10 6 GAPDH molecules. Stimulation of thyrocytes with TSH (0.1-10 U/ml) or forskolin (0.1-15 microM) results in a dose- and time-dependent up-regulation of NIS expression reaching a maximum at 10 mU/ml TSH (2211 +/- 761 copies) or 10 microM forskolin (1663 +/- 302 copies) after 24 h. In conclusion, we here established a real-time RT-PCR combining the advantages of rapid thermocycling and online detection of NIS mRNA amplification. The sensitive quantification of human NIS mRNA expression offered by this novel technique may improve analysis of hNIS regulation and measurement of NIS mRNA expression in small biopsies.

Evidence of leptin expression in normal and polycystic human ovaries.

Leptin, the 'obese' protein, is found in cultured granulosa cells derived from human pre-ovulatory follicles. However, the occurrence of leptin has not been studied in intact ovaries, either normal or polycystic, until now. Paraffin sections from 25 human ovaries of different cycle stages and 25 wedge resections of polycystic ovaries were investigated by means of immunochemistry. Additionally, three ovaries were available for reverse transcription-polymerase chain reaction analysis. Leptin-positive cells were located in the granulosa cells of pre-antral follicles, and distinctly in the thecal layer of intact and regressing antral follicles. In the corpus luteum (CL) in the developmental stage, the former epithelioid leptin-positive thecal cells became fibroblast-like in the septum. In the CL of the secretory stage, single leptin-positive cells were detected between luteal cells. In polycystic ovaries, leptin-positive cells were noted both in the hypertrophied thecal layer and in the luteinized granulosa layer. Our findings on leptin expression at the protein level were confirmed by a positive mRNA signal for leptin in granulosa cells and in the CL. Additionally, mRNA of the full-length leptin receptor OB-R and of the short isoforms B219.1-B219.3 was identified in granulosa cells and the CL, as well as in the cortex and medulla. We conclude that leptin is produced in the ovary and may act in autocrine and paracrine ways.

GRO-alpha in normal and pathological thyroid tissues and its regulation in thyroid-derived cells.

Thyroid glands affected by Graves' disease (GD) show striking leukocytic infiltration, mainly by T-cells. The mechanisms by which the various leukocytes are maintained in the thyroid are unknown. Growth-regulated oncogene-alpha (GRO-alpha) in interaction with its receptor CXCR2 is a chemoattractant for both T-cells and neutrophils and may be one of the chemokines involved in the cell maintenance. GRO-alpha and CD18 mRNA as a marker of leukocytic infiltration were quantified in thyroid tissue using competitive RT-PCR. We found very high GRO-alpha mRNA levels in all thyroid tissues. In GD patients (n=16), the GRO-alpha mRNA did not correlate with the CD18 mRNA level or thyroid peroxidase and TSH-receptor antibodies in patients' sera. In thyroid autonomy (n=10), the GRO-alpha mRNA levels were significantly lower in autonomous single adenomas compared with the corresponding normal tissue. In order to define the cellular source of GRO-alpha mRNA and protein, we examined various thyroid-derived cells. Thyrocytes, thyroid-derived leukocytes and fibroblasts showed basal GRO-alpha mRNA and protein expression, which was remarkably upregulated by different stimuli in vitro. The expression of GRO-alpha by thyroid carcinoma cell lines confirms that thyrocytes may actually produce GRO-alpha. As shown by flow cytometry and immunohistology, CD68+ monocytes/macrophages are the only cell population strongly expressing CXCR2 in the thyroid.