Development and validation of a minimally invasive and image-guided tape stripping method to sample atopic skin in children.

BACKGROUND: Molecular skin profiling techniques, typically performed on skin samples taken by punch biopsy, have enhanced the understanding of the pathophysiology of atopic dermatitis (AD), thereby enabling the development of novel targeted therapeutics. However, punch biopsies are not always feasible or desirable, and novel minimally invasive methods such as skin tape stripping have been developed. AIM: To develop, optimize and validate a novel tape stripping method guided by noninvasive in vivo skin imaging to sample atopic skin in children. METHODS: Skin tape stripping-based procedures were compared and optimized using data from 30 healthy controls (HCs: 5 adults, 25 children) and 39 atopic children. Evaluations were guided by high-resolution photography, reflectance confocal microscopy, optical coherence tomography and transepidermal water loss measurements. We assessed and compared adverse events (AEs), the time needed to perform the sampling and the cDNA levels obtained from the tapes. RESULTS: Tape stripping methods based on previously described protocols resulted in erosions in all participants and required a median time of 65 min to perform (range 60-70 min), but provided good cDNA yield. Shorter durations appeared less invasive but provided lower cDNA yield. The final optimized tape stripping protocol, using 11 tapes of 22 mm in diameter, each applied twice for 5 s with 90° rotation, did not produce significant AEs, was completed within a median time of 7 min (range 5-15 min) and provided good cDNA yield both in HCs and atopic children. CONCLUSION: Our minimally invasive method is safe and reliable, and provides reproducible acquisition of cDNA in atopic children. In addition, it enables rapid sample collection, a crucial factor in clinical practice.

[Atypical femoral fractures in bisphosphonate therapy]. / Atypische Femurfrakturen unter Bisphosphonat-Therapie.

Bisphosphonates (BP) play an important role in the therapy of osteoporosis as they effectively reduce the risk of fractures. Atypical femoral fractures (AFF) have recently been described as an adverse effect of BP treatment. We present 3 women under therapy with BP, who had five AFF, among these three complete and two incomplete fractures. The incidence of AFF under therapy with BP is low, and the benefit regarding reduction of spinal fractures and fractures of the proximal femur is much higher than the risk for an AFF.

PLAG1 activation in lipoblastoma coinciding with low-level amplification of a derivative chromosome 8 with a deletion del(8)(q13q21.2).

Lipoblastoma is a benign uncommon soft-tissue-tumor resembling fetal adipose tissue affecting mainly children under three years of age. In lipoblastoma, the typical cytogenetic changes are clonal rearrangements involving chromosomal region 8q11-->q13. The oncogene PLAG1 (pleomorphic adenoma gene 1) is located within this chromosomal region on band 8q12. Recent reports have demonstrated that in lipoblastoma, the PLAG1 gene is activated by 'promoter-swapping'. Herein, we demonstrate that in lipoblastoma, the PLAG1 gene may also be activated by low-level amplification. We report on a lipoblastoma with the karyotype 48 approximately 50,XX,del(8)(q13q21.2),+del(8)(q13q21.2)x4[cp12]. Subsequent FISH analysis on uncultured tumor cells confirmed this result and demonstrated a low-level amplification of the chromosomal region 8pter-->8q13 and 8q21.2-->8qter. A partial monosomy was seen for the chromosomal region 8q13-->8q21.2. No other gains or losses were observed by CGH analysis. RT-PCR analysis showed that the PLAG1 gene is activated in the tumor sample of the lipoblastoma analyzed, in contrast to normal fatty tissue without PLAG1 expression. In conclusion, our results demonstrate that low-level amplification is a further mechanism of PLAG1 activation in lipoblastomas.

Rb-loss is associated with high malignancy in chondrosarcoma.

Loss of function of the human retinoblastoma gene (Rb) is a frequent genetic abnormality in human malignancies and causes a disturbance in the cell cycle and loss of normal proliferation and differentiation. We studied the loss of heterozygosity (LOH) of the Rb gene in 31 formalin-fixed, paraffin-embedded cartilaginous tumors using polymerase chain reaction. The tumors were subdivided into 8 cases of dedifferentiated (DD) chondrosarcoma, 17 cases of conventional chondrosarcoma (nine grade 1, seven grade 2 and one grade 3), 4 enchondromas and 2 chondroblastomas. Both components of DD chondrosarcoma, the low-grade and anaplastic components, were separated by a microdissection approach. The genetic data were correlated with the expression of the Rb protein examined by Rb immunohistochemistry. We found Rb-LOH in one grade 3 chondrosarcoma, and in the anaplastic component in 7 of 8 cases of DD chondrosarcoma (89% of all high-grade chondrosarcomas). All tumors with Rb-LOH were immunohistochemically Rb-negative. The only case of DD chondrosarcoma negative for Rb-LOH in both components of the tumor also showed weak expression of the Rb protein in the anaplastic component. All benign cartilaginous tumors, low-grade chondrosarcomas and low-grade tumor components of DD chondrosarcomas were negative regarding Rb-LOH but positive in Rb immunohistostaining. We concluded that Rb-LOH predominantly occurs in high-grade chondrosarcomas. However, it is not a marker for identifying low-grade tumors with a tendency towards progression or local recurrence.

Genetic and epigenetic alterations in tumor progression in a dedifferentiated chondrosarcoma.

In this case of a dedifferentiated chondrosarcoma, we searched for genetic or epigenetic alterations in both components of the tumor, the low grade chondroblastic component, and the high grade osteosacomatouscomponent. To date, only little is known about aberrant patterns of DNA methylation in chondrosarcomas. Microdissection was used as a valuable method for clearly separating the tissues. We examined CpG island methylation of 8 tumor suppressor genes and candidate tumor suppressor genes, which are involved in different pathways: cell cycle (p21WAF1, p16INK4, p14ARF), apoptosis (DAPK, FHIT), DNA repair (hMLH1), and cell adherence (E-Cadherin). We found p16INK4 and E-cadherin promotor methylation in the low grade chondroid compartment of the dedifferentiated chondrosarcoma. P16INK4, FHIT, and E-cadherin were methylated in the highly malignant osteosarcomatous compartment of the tumor. Earlier investigations of this chondrosarcoma showed p53 mutation and p53-LOH in the anaplastic component. As shown in this case, it was accompanied by Rb-LOH. Early methylation of p16IK4 and E-cadherin in the chondroid compartment could point to the monoclonal origin of demonstrated dedifferentiated chondrosarcoma. Further alterations, as shown in p53, Rb and FHIT, are responsible for the "switch" to a high grade anaplastic sarcoma.

A new technique of arthroscopic capsular shift in anterior shoulder instability.

We describe a new arthroscopic technique to reinforce the torn inferior glenohumeral ligament (IGHL) and the elongated capsule to the glenoid rim. The arthroscope is inserted over the superior portal and, after the insertion of a suture anchor, both limbs are pulled out over the posterior portal. The IGHL is grasped and pulled upward onto the glenoid rim using a suture retriever clamp inserted over the posterior portal. A 45 degrees curved blunt clamp (Sidewinder; Arthrex, Naples, FL) coming from the anterior penetrates the IGHL, and 1 end of the suture limb is given into the branches of the clamp and pulled out anteriorly. After a second perforation of the capsule, a horizontal suture creating a neolabrum can be placed. This technique allows a suitable reinforcement of the capsule without intraoperative complications. In cases of capsular elongation, especially a torn IGHL, the capsular instability can be addressed by the described Sidewinder technique. More sophisticated arthroscopic techniques such as this will increase the indication for arthroscopic shoulder stabilization.

[Multicentric extra-abdominal fibromatosis: a rare case]. / Multizentrische extraabdominale Fibromatose -- ein seltener Fall.

Extra-abdominal aggressive fibromatosis is a benign fibroblastic neoplasia with an infiltrative nature and a high tendency of local recurrence. Here, we report on a very rare case of multicentric fibromatosis. Low complaints led to considerable size of the tumours. The aim of the multimodal treatment was a limb salvage procedure. Adjuvant radiation therapy and chemotherapy was necessary because of the renunciation of wide resections in favour of the functionality of the limb.

[Anterior cruciate ligament reconstruction - graft options and fixation techniques]. / Die vordere Kreuzbandplastik - Transplantatwahl und Verankerungstechniken.

With the introduction of arthroscopy most of the surgeon have changed their technique in ACL-reconstruction from open to arthroscopically performed techniques. Several new techniques have been developed in the past, including new fixation devices and different grafts. The cruciate ligament reconstruction comprises a composition of both, the graft and the fixation to the bone. Well accepted autografts are the patellar midthird, the semitendinosus/gracilis or the quadriceps tendon. In special cases allografts are alternatives. The fixation can cause failure due to overstrain or creep during the postoperative period of healing. Considering their biomechanical properties, cruciate ligament reconstruction should aim for a graft behavior comparable with the native cruciate ligament. Results gained from basic science and clinical studies are reviewed in the following article.

[Clinical outcome of arthroscopic posterior cruciate ligament-plasty]. / Klinische Ergebnisse nach arthroskopischer hinterer Kreuzbandplastik.

Twenty-one patients were evaluated with an average follow up of 30 month after arthroscopical reconstruction of the posterior cruciate ligament using the IKDC-Evaluation Document, Lysholm-Score, KT 1000 and X-ray Complex ligament injuries and chronic instability of the knee were the indications for operative treatment. Semitendinosus--or patella ligament autografts were used. The Lysholm-Score significantly improved preoperatively to postoperatively (p < 0.01). The IKDC revealed 11 patients for group B, 7 patients for group C and 3 patients for group D. The KT 1000 measurement showed a postoperative drawer of 3.6 mm compared with the nonoperated side. Thirteen of the 21 patients showed a posterior drawer of less than 3 mm. The lateral posterior stress view of the X-ray revealed a 4.1 mm (+/- 2.9 mm) side to side difference postoperatively in comparison to 9.5 mm (+/- 3.5 mm) preoperatively. Three patients possessed radiological signs of osteoarthritis grad I by Ahlbäck. Our results recommend in patients with complex PCL-injuries as well as in cases of chronic posterior instability a complex reconstruction of the ligaments.

Ligation of MHC class I molecules on peripheral blood T lymphocytes induces new phenotypes and functions.

Microgram concentrations of immobilized anti-MHC class I (MHC-I) Ab induced proliferation of resting CD3+ T cells from peripheral blood. In contrast, soluble Ab did not activate T cells. Exposure of T cells to immobilized anti-MHC-I Ab for only 24 h was followed by proliferation and development of T cell-mediated cytotoxicity. Immediately following MHC-I ligation, the T cells responded with increased protein tyrosine phosphorylation, with new bands appearing in the SDS-PAGE. Exposure of T cells to immobilized anti-MHC-I Ab for 24 h induced an increased surface expression of the TCR/CD3 and CD28 molecules. MHC-I-induced proliferation of purified T cells was dependent on cellular interactions with non-T cells. Under certain conditions, in which MHC-I was ligated by picogram concentrations of immobilized anti-MHC-I Ab, anti-TCR/CD3 Ab-induced proliferation of T cells was strongly inhibited. These data clearly demonstrate that ligation of the MHC-I complex on T cells may induce both positive and negative signals. Since the physiologic ligands for MHC-I molecules are TCR and the CD8 molecules, our data may suggest that MHC-I molecules are instrumental in cellular interactions between T cells.

Ion transport mechanisms in native rabbit nasal airway epithelium.

Net ion transports in rabbit nasal airway epithelium (RNAE) were estimated from unidirectional fluxes of 22Na+, 36Cl-, and 86Rb+ (K+ tracer), short-circuit current (Isc), and epithelial conductance (Gt) under short-circuit conditions in excised parallel RNAE from the two sides of the nasal septum mounted in Ussing chambers at 37 degrees C. Net Na+ absorption (JNa; 76 nmol.min-1.cm-2) was nearly equal to the net charge flux (Jnet), equivalent to the control Isc of 137 microA/cm2 (Jnet = Isc/zF = 85 neq.min-1.cm-2). Secretions of Cl- (9 nmol.min-1.cm-2) and K+ (Rb+) (1.2 nmol.min-1.cm-2) were small. Intra-animal variations between right- and left-side Isc values were small compared with large interanimal variations, suggesting long-term regulation of JNa. Serosal ouabain (10(-4) M) abolished Isc. Mucosal amiloride (10(-4) M) maximally inhibited Isc by 68% and JNa by 78%, abolished K+ (Rb+) secretion, increased Cl- secretion slightly (to 16 nmol.min-1.cm-2), and decreased control Gt (13.4 mS/cm2) by 26%. Amiloride-insensitive JNa was inhibited approximately 50% by mucosal hydrochlorothiazide (10(-4) M) but not by mucosal bumetanide, phloridzine, or ethoxzolamide. The Cl- secretion was abolished by serosal bumetanide (10(-4) M) and also by mucosal diphenylamine-2-carboxylate (2.5.10(-4) M) or bumetanide (10(-4) M). Serosal Ba2+ (2 mM) inhibited JNa and increased K+ (Rb+) secretion. The latter was blocked by mucosal Ba2+. Passive Cl- (but not Na+) fluxes varied proportionally with Gt and were approximately four times higher than passive Na+ fluxes, suggesting 1) significant anion selectivity of a low-resistance paracellular pathway and 2) separate routes for paracellular Cl- and Na+ fluxes. We conclude that RNAE is a suitable model organ for studies of regulation of JNa in native human airway epithelia.

Bone tunnel enlargement after anterior cruciate ligament reconstruction with semitendinosus tendon using Endobutton fixation on the femoral side.

We evaluated 29 knees with a minimum follow-up of 2 years after anterior cruciate ligament (ACL) reconstruction using doubled autogenous semitendinosus tendons. On the femoral side, a 5-mm Mersilene tape (Ethicon, Norderstedt, Germany) with an Endobutton (Acufex Microsurgical, Mansfield, MA) was used. The tendon was fixed on the tibial side with two staples. Regarding the IKDC score, 66% of the patients were graded as normal or nearly normal. The anterior laxity side-to-side difference (KT 1000, man-max-drawer) was under 3 mm in 55% and under 5 mm in 90%. Radiographs taken in the lateral and anteroposterior projections of the knee showed sclerotic bone tunnel margins. The diameter of the bone tunnels were measured, corrected for magnification, then compared with the original reamed diameter to determine any change in size. Enlargement of at least 2 mm was identified in 72% of the femoral tunnels and 38% of the tibial tunnels. No correlation was found concerning the enlargement of the tunnel and the IKDC score or the residual joint laxity. We conclude that using an Endobutton-Mersilene construct in ACL reconstruction leads to femoral and tibial bone tunnel enlargement at follow-up of 2 years.

T cell-mediated cytotoxicity against p53-protein derived peptides in bulk and limiting dilution cultures of healthy donors.

The p53 tumour suppressor gene product plays an important role in the development of most human cancers. Point mutations in the p53 gene are common in malignant states and results in over-expression of wild type and mutant determinants of the p53 protein. This process might generate MHC-I restricted epitopes for T cell recognition and p53-derived peptides have been suggested as targets for tumour-specific cytotoxic T lymphocytes (CTL). Our primary aim was to estimate the frequencies of p53-peptide reactive CTL precursors (CTLp) in peripheral blood from healthy young individuals. We selected wild type and mutated peptides derived from the p53 sequence with a binding motif for HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from healthy HLA-A2 donors were stimulated in vitro in bulk cultures as well as in limiting dilution cultures using autologous cells pulsed with p53 peptides as stimulator cells. T cell reactivity was observed towards both wild type and mutated p53 peptide epitopes with CTL precursor frequencies varying from 1:2 x 10(4) to 1:1.5 x 10(5). These results might suggest the presence of an ongoing immune response in normal individuals against cells expressing increased levels of p53 protein.

An improved method for the detection of peptide-induced upregulation of HLA-A2 molecules on TAP-deficient T2 cells.

Peptide-induced upregulation of MHC class I on TAP-deficient T2 cells is used to monitor peptide binding to class I molecules. We present a temperature-dependent modification of the T2 assay which increases the sensitivity of peptide binding to MHC class I with several orders of magnitude.

Rotationplasty as a salvage procedure in revision of tumor endoprosthesis of the distal femur--a report of two cases.

We performed two rotationplasties (type A1 and A2) as salvage procedures in cases of custom-made endoprosthetic replacements of the distal femur and knee joint. The patients suffered from malignant bone and soft-tissue tumors of the knee, respectively. One of them developed chronic osteomyelitis around both stems of the prosthesis, and three local revisions turned out to be ineffective. The other patient, suffering from parossal osteosarcoma of the distal femur, developed a local recurrence following endoprosthetic reconstruction. The functional abilities following the operation of these two patients suggested that rotationplasty was a viable alternative to above-the-knee amputation in failed or severely complicated total arthroplasty of the knee with massive bone loss.

T-cell activation. VI. Inhibitory and stimulatory effects of anti-major histocompatibility complex class I antibodies in allogeneic mixed lymphocyte culture.

Murine T splenocytes stimulated in primary allogeneic mixed lymphocyte culture (MLC) were incubated with soluble anti-major histocompatibility complex (MHC) class I monoclonal antibodies. These antibodies induced inhibition in the cytotoxicity of the responding population and this inhibition was not dependent on the domain on class I molecules recognized by the antibodies. Cross-reactivity of the antibodies between the responder and stimulating cell population caused a marked reduction in the inhibitory effect compared to systems where no such cross-reactivity was present. Saturating levels of the antibodies caused a reduction in generation of T-cell cytotoxicity, whereas low concentrations stimulated the same response. These results demonstrate that the MHC class I molecules of T cells are of significant importance in antigen-induced signal transduction.

[Semitendinosus tendon vs. patellar ligament. Results of a prospective randomized study after anterior cruciate ligament reconstruction]. / Semitendinosussehne vs. Ligamentum patellae. Klinische Ergebnisse einer prospektiven randomisierten Studie nach vorderer Kreuzbandplastik.

UNLABELLED: The aim of our prospective randomised study was to evaluate the clinical results after arthroscopical reconstruction of the ACL using the midthird patellar ligament or semitendinosus tendon. METHODS: Forty patients were followed up two years postoperatively. Twenty of them received either a patellar ligament graft (BTB-group) or doubled semiteninosus tendon (SET-group). The clinical evaluation included the preoperative and two years postoperative assessment, based on the IKDC-Score, Tegener-Score and Mc-Carroll-Score. The a.p.-translation was evaluated using the KT-1000. RESULTS: Sixteen (80%) patients of the SET-group and 10 (50%) patients of the BTB-group showed good and excellent results in the over all assessment with the IKDC-Score. The mean side to side KT-1000 difference yielded 1.6 mm (-2-4 mm) in the BTB-group and 2.7 mm (0-7 mm) in the SET-group (p < 0.05). The retropatellar pain syndrome based on the Mc-Caroll-Score showed 17.4 points in the BTB-group in comparison to 19.5 points in the SET-group (p < 0.05). The level of activity using the Tegner-score showed preoperatively for both the SET- and BTB-group 6.9 points and postoperatively for the SET-group 6.7 points and for the BTB-group 5.6 points. CONCLUSIONS: Despite the inferior a.p.-stability for the patients who received doubled semitendinosus tendon grafts they presented clinically superior results compared to the BTB-group. Therefore this technique seems to be the alternative method. In order to improve the stability we recommend the usage of three or four stranded grafts and an improved fixation technique.

[Bilateral dysfunction of the quadriceps muscle after unilateral cruciate ligament rupture with concomitant injury central activation deficit]. / Bilateraler Funktionsverlust der Quadrizepsmuskulatur nach einseitiger Kreuzbandruptur mit Begleitverletzung durch zentrales Aktivierungsdefizit.

The aim of the study was to investigate the ability to activate the quadriceps femoris muscle voluntarily in patients after severe knee injuries and it's influence on muscle function. 33 male patients after ACL-rupture with concomitant injuries were investigated with a twitch-interpolation-method to determine the maximal voluntary contraction force (MVC) and the ability to activate the quadriceps muscle voluntarily. The results were compared to a previously investigated group of patients after isolated ACL-rupture and to an aged-matched control group. The patients with extensive knee injuries presented a significant higher deficit of voluntary quadriceps muscle activation on the injured (78.8 +/- 2.09%, mean +/- SEM) and equally on the uninjured side (78.9 +/- 1.91%) compared to the patients after isolated ACL-rupture and to controls. A minor MVC-deficit of the patients quadriceps muscle of the uninjured side could be explained by the voluntary activation deficit alone, the major MVC deficit of the injured side by muscle atrophy and a voluntary activation deficit. The activation deficit is probably the underlying reason for persisting muscle wasting and [not readable: see text]