Successful Regulatory T Cell-Based Therapy Relies on Inhibition of T Cell Effector Function and Enrichment of FOXP3+ Cells in a Humanized Mouse Model of Skin Inflammation.

BACKGROUND: Recent clinical trials using regulatory T cells (Treg) support the therapeutic potential of Treg-based therapy in transplantation and autoinflammatory diseases. Despite these clinical successes, the effect of Treg on inflamed tissues, as well as their impact on immune effector function in vivo, is poorly understood. Therefore, we here evaluated the effect of human Treg injection on cutaneous inflammatory processes in vivo using a humanized mouse model of human skin inflammation (huPBL-SCID-huSkin). METHODS: SCID beige mice were transplanted with human skin followed by intraperitoneal (IP) injection of 20-40 × 106 allogeneic human PBMCs. This typically results in human skin inflammation as indicated by epidermal thickening (hyperkeratosis) and changes in dermal inflammatory markers such as the antimicrobial peptide hBD2 and epidermal barrier cytokeratins K10 and K16, as well as T cell infiltration in the dermis. Ex vivo-expanded human Treg were infused intraperitoneally. Human cutaneous inflammation and systemic immune responses were analysed by immunohistochemistry and flow cytometry. RESULTS: We confirmed that human Treg injection inhibits skin inflammation and the influx of effector T cells. As a novel finding, we demonstrate that human Treg injection led to a reduction of IL-17-secreting cells while promoting a relative increase in immunosuppressive FOXP3+ Treg in the human skin, indicating active immune regulation in controlling the local proinflammatory response. Consistent with the local control (skin), systemically (splenocytes), we observed that Treg injection led to lower frequencies of IFNγ and IL-17A-expressing human T cells, while a trend towards enrichment of FOXP3+ Treg was observed. CONCLUSION: Taken together, we demonstrate that inhibition of skin inflammation by Treg infusion, next to a reduction of infiltrating effector T cells, is mediated by restoring both the local and systemic balance between cytokine-producing effector T cells and immunoregulatory T cells. This work furthers our understanding of Treg-based immunotherapy.

Elastase-mediated fibrinogenolysis by chemoattractant-stimulated neutrophils occurs in the presence of physiologic concentrations of antiproteinases.

Plasma levels of the HNE-derived fibrinopeptide A alpha 1-21 reflect in vivo enzyme activity. To provide a possible explanation for the presence of circulating A alpha 1-21 in individuals with normal plasma antiproteinase concentrations we investigated whether PMN-associated HNE is more resistant to inhibition than the free enzyme. PMN were stimulated to migrate across 125I-fibrinogen-coated nitrocellulose filters in response to 10(-7) M FMLP, and the extent of fibrinogenolysis was determined by measuring release of A alpha 1-21 and 125I-labeled fibrinogen degradation products. The fibrinogenolytic activity of migrating PMN was then compared with that of free HNE present in PMN lysates or secreted by PMN stimulated with FMLP. Whereas the fibrinogenolytic activity of soluble HNE was completely inhibited by low concentrations (1%) of plasma or serum and macromolecular antiproteinase (alpha 1 proteinase-inhibitor and soybean trypsin-inhibitor), even in the presence of undiluted plasma or serum the activity of the migrating PMN was incompletely blocked (81-85%). Further, concentrations of alpha 1 proteinase-inhibitor and soybean trypsin-inhibitor that totally inhibited free HNE activity also incompletely blocked (88-89%) the fibrinogenolytic activity of migrating PMN, indicating that FMLP-stimulated PMN demonstrate significant fibrinogenolytic activity in the presence of antiproteinases as small as 20,000 mol wt. A specific low molecular weight HNE inhibitor (MeO-Suc-Ala2-Pro-ValCH2Cl), however, totally blocked PMN-mediated fibrinogenolysis without affecting intracellular HNE activity, HNE secretion from PMN, or PMN migration in response to FMLP. These findings support the hypothesis that PMN migrating on a fibrinogen-coated surface form zones of close contact with fibrinogen, thus preventing access of plasma antiproteinases to HNE released at the cell-substrate interface. The occurrence of this phenomenon in vivo would explain the presence of circulating A alpha 1-21 in individuals with normal antiproteinase concentrations.

Increased neutrophil elastase activity in cigarette smokers.

We compared plasma levels of the neutrophil elastase-derived fibrinopeptide A-alpha-1-21 in healthy cigarette smokers with those in nonsmokers. The mean A-alpha-1-21 concentration was fivefold higher (95% confidence interval [CI], 3.0 to 9.6) in ten cigarette smokers than in 20 healthy nonsmokers (2.0 nmol/L compared with 0.4 nmol/L; p less than 0.0001). To evaluate the acute effect of smoking on enzyme activity, a second group of ten smokers was studied. After refraining from smoking for 12 hours, each person smoked three cigarettes. The mean A-alpha-1-21 level in the second group of smokers was not different from that in the first group of smokers (1.8 nmol/L compared with 2.0 nmol/L) but was fivefold higher (95% CI, 2.6 to 8.7) than that in the nonsmokers (1.8 nmol/L compared with 0.4 nmol/L; p less than 0.0001). After smoking three cigarettes, subjects had a twofold elevation (95% CI, 1.6 to 3.5) in the mean A-alpha-1-21 concentration (from 1.8 nmol/L to 4.1 nmol/L; p = 0.002). Our data show that cigarette smoking perturbs the in-vivo elastase-antielastase balance and thus may produce lung disease through this mechanism.

Development of an assay for in vivo human neutrophil elastase activity. Increased elastase activity in patients with alpha 1-proteinase inhibitor deficiency.

Leukocyte extracts contain enzymes that digest fibrinogen and release a fibrinopeptide A-containing fragment. This study was undertaken to identify the responsible proteinase and to characterize the fibrinopeptide A-containing fragment so that it could be used as an index of enzyme activity. Both the fibrinogenolytic activity and the release of the fibrinopeptide A-containing fragment mediated by the leukocyte extracts were shown to be due to human neutrophil elastase (HNE) by the following criteria: activity was completely blocked by a specific HNE inhibitor or by adsorbing HNE from the extracts with a monospecific antibody and reconstitution with purified HNE restored the ability to release the fibrinopeptide A-containing fragment. This fragment was not released by a variety of other proteinases or by HNE-inhibitor complexes indicating that, at least with respect to the enzymes tested, it is a specific product of HNE and its release requires the free enzyme. By separating the products of HNE digestion of fibrinogen using high performance liquid chromatography, identifying the immunoreactive fractions and subjecting them to amino acid analysis, the fragment was identified as A alpha 1-21, indicating an HNE cleavage site at the Val(A alpha 21)-Glu(A alpha 22) bond. The mean plasma A alpha 1-21 level was markedly higher in patients with alpha 1-proteinase inhibitor deficiency as compared to healthy controls (0.2 nM vs. 7.9 nM; P less than 0.0001), consistent with increased in vivo HNE activity in these individuals.

Development of a radioimmunoassay for the fibrinogen-derived peptide B beta 1-42.

The peptide B beta 1-42 is the initial cleavage product of plasmin-mediated proteolysis of the NH2-terminal region of fibrinogen or fibrin I, while beta 15-42 is the major fragment released by plasmin degradation of fibrin II. Numerous studies have described the measurement of plasma B beta 1-42 levels as an index of plasmin activity. Previous assays were indirect and included quantitation of thrombin-increasable fibrinopeptide B immunoreactivity (TIFPB) or measurement of beta 15-42 with an antiserum (132) which cross-reacted with B beta 1-42. We report on a new antiserum (R142) directed against B beta 1-42 which does not cross-react with beta 15-42 or fibrinopeptide B. Employing this antiserum, a specific assay for B beta 1-42 was developed. This assay was used to measure plasmin-mediated B beta 1-42 release from fibrinogen and its subsequent proteolysis by thrombin. The selectivity constant (Kcat/Km) for thrombin cleavage of the B beta 14-15 bond of B beta 1-42 was 10(5)-fold less than that for proteolysis of the same bond in the intact fibrinogen molecule, thus explaining the stability of this peptide in the presence of thrombin activity in the blood. Similarly, the selectivity constant for plasmin cleavage of the B beta 21-22 bond of B beta 1-42 was 140-fold less than that for proteolysis of the B beta 42-43 bond of fibrinogen, indicating that secondary plasmin attack of the B beta 1-42 molecule is not physiologically important. The specific B beta 1-42 assay provided excellent recovery of peptide added to blood or plasma. Comparison of B beta 1-42 levels with TIFPB values in 37 patient samples yielded good correlation over a wide range of levels (r2 = 0.91). The median plasma B beta 1-42 level in 15 normal individuals was 1.2 pmol/mL. This is similar to the previously reported normal range for TIFPB (1 to 4 pmol/mL) but is higher than the normal level of 0.4 pmol/mL reported with the assay employing antiserum 132. This discrepancy reflects rapid removal of Arg (B beta 42) by plasma carboxypeptidase activity resulting in 50% loss of B beta 1-42 immunoreactivity with antiserum 132 but no loss with R142. To circumvent this problem, we have developed a beta 15-42 antiserum (R154) which, like antiserum 132, cross-reacts with B beta 1-42. However, B beta 1-42/beta 15-42 immunoreactivity with R154 is stable in the presence of carboxypeptidase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiographic features of malignant fibrous histiocytomas.

The angiographic findings in 2 cases of metastatic fibrous histiocytoma are discussed. A lesion in the brain was completely avascular and displaced the surrounding vessels, whereas the renal lesions were hypovascular, usually well demarcated, sometimes exhibited beak formation, and demonstrated fine, corkscrew-like tumor vessels with delayed emptying but no tumor blush or early draining veins.

Kinetics and imaging characteristics of 99mTC-labled complexes used for bone imaging.

Activity levels of 99TC-labeled compounds, 18F, and 85Sr were obtained at 1, 3, and 5 hr. postinjection in normal and healing fractured bone and in soft-tissue rat specimens. Serial diagnostic bone images and blood and urine kinetics were obtained in patients with each of the TC-labeled compounds. Computer-processed images were used to evaluate in vivo kinetics. 99mTC pyrophosphate provides the best overall characteristics for bone imaging. Improved quality and bioassay procedures are required, however, before any one agent can be designated the radiopharmaceutical of choice for diagnostic bone imaging.

[The radiological manifestations of fibroxanthosarcomas (author's transl)]. / Die Röntgenmanifestation des Fibroxanthosarkoms

The radiological findings in eight patients with fibroxanthosarcomas are described. In seven patients the primary tumour was in the soft tissues of the extremities or of the trunk. Infiltration of the primary tumour into a neighbouring bone, producing a "motheaten" appearance on the radiograph was seen in one case. A primary tumour in the mediastinum (one case cannot be distinguished radiologically from malignant lymphoma. Pulmonary metastases were found radiologically in five patients; in two they were solitary and in three patients there were multiple round foci measuring up to 8 cm. in diameter. Metastases in the lung hila and mediastinum were found only in the presence of pulmonary metastases (three cases). Pleural effusions developed in an advanced stage of the disease only (three cases). A solitary, purely osteolytic bone deposit was seen in two patients. Multiple metastases in the kidneys were hypo- or avascular, but some showed fine corkscrew vessels. On intravenous urography they could be demonstrated after reaching a considerable size (one case). A deposit in the jejunum presented as a small polyp. Two fibroxanthosarcomas developed as a second tumour within the operative field of a previous malignancy; one of these had been irradiated with 4,500 rads. All primary and secondary tumours showed extremely rapid growth. The palliative effect of radiation and chemotherapy was very variable.

Bronchial brush biopsy and primary lung carcinoma.

Bronchial brush biopsy was performed on 100 patients with pulmonary lesions suspected of being malignant. Of 61 cases of proved carcinoma of the lung, in 44 or 72-1% brush biopsy yielded positive results. The technique used is described and an analysis of the results is presented.

Comparison of bronchial brushing and percutaneous needle aspiration biopsy in the diagnosis of malignant lung lesions.

The results of 100 bronchial brushings and 80 percutaneous needle aspiration biopsies of the lung performed in the same department were analyzed for their accuracy in the diagnosis of pulmonary neoplasms. While both procedures have a high degree of reliability, the aspiration needle biopsy appears to be the procedure of choice in smaller, more peripheral lesions, in Pancoast tumors, and whenever a metastatic neoplasm is suspected.