Laxity measurement of internal knee rotation after primary anterior cruciate ligament rupture versus rerupture.

PURPOSE: The aim of the current study was to objectify the rotational laxity after primary anterior cruciate ligament (ACL) rupture and rerupture after ACL reconstruction by instrumented measurement. It was hypothesized that knees with recurrent instability feature a higher internal rotation laxity as compared to knees with a primary rupture of the native ACL. STUDY DESIGN: Cross-sectional study, Level of evidence III. METHODS: In a clinical cross-sectional study successive patients with primary ACL rupture and rerupture after ACL reconstruction were evaluated clinically and by instrumented measurement of the rotational and antero-posterior laxity with a validated instrument and the KT1000®, respectively. Clinical examination comprised IKDC 2000 forms, Lysholm Score, and Tegner Activity Scale. Power calculation and statistical analysis were performed (p value < 0.05). RESULTS: 24 patients with primary ACL rupture and 23 patients with ACL rerupture were included. There was no significant side-to-side difference in anterior translation. A side-to side difference of internal rotational laxity ≥ 10° was found significantly more frequent in reruptures (53.6%) compared to primary ruptures (19.4%; p < 0.001). A highly significant relationship between the extent of the pivot-shift phenomenon and side-to-side difference of internal rotation laxity could be demonstrated (p < 0.001). IKDC 2000 subjective revealed significantly better scores in patients with primary ACL tear compared to patients with ACL rerupture (56.4 ± 7.8 vs. 50.8 ± 6.2; p = 0.01). Patients with primary ACL tears scored significantly better on the Tegner Activity Scale (p = 0.02). No significant differences were seen in the Lysholm Score (p = 0.78). CONCLUSION: Patients with ACL rerupture feature significantly higher internal rotation laxity of the knee compared to primary ACL rupture. The extend of rotational laxity can be quantified by instrumented measurements. This can be valuable data for the indication of an anterolateral ligament reconstruction in ACL revision surgery.

Radiological Outcome Measures Indicate Advantages of Precontoured Locking Compression Plates in Elderly Patients With Split-Depression Fractures to the Lateral Tibial Plateau (AO41B3).

BACKGROUND: Split-depression fractures to the lateral tibial plateau (AO41B3) often feature severe joint surface destructions. Precontoured locking compression plates (LCPs) are designed for optimum support of the reduced joint surface and have especially been emphasized in reduced bone quality. A lack of evidence still inhibits their broad utilization in elderly patients. Thus, aim of the present study was to investigate the implant-specific radiological outcomes of AO41B3-fractures in young versus elderly patients. METHODS: The hospital's database was screened for isolated AO41B3-factures, open reduction and internal fixation (ORIF), and radiological follow-up ≥12 months. CT-scans, radiographs, and patients' records were analyzed. Patients were attributed as young (18-49) or elderly (≥50 years). Additional subgrouping was carried out into precontoured LCP and conventional implants. The Rasmussen Radiological Score (RRS) after 12 months was set as primary outcome parameter. The RRS postoperatively and the medial proximal tibial angle (MPTA) postoperatively and after 12 months were secondary outcome parameters. RESULTS: Fifty nine consecutive patients were included (26 young, 38.2 ± 7.8 years; 33 elderly, 61.3 ± 9.4 years). There were no significant differences regarding mean size and depression depth of the lateral joint surface fragments. Prior to implant-specific subgrouping, the radiological outcome measures revealed no significant differences between young (RRS = 7.7 ± 1.7; MPTA = 90.3 ± 2.3°) and elderly (RRS = 7.2 ± 1.7; MPTA = 90.5 ± 3.3°). After implant-specific subgrouping, the radiological outcome revealed significantly impaired results in young patients with conventional implants (RRS(C) = 6.9 ± 1.6, RRS(LCP) = 8.5 ± 1.5, P = .015; MPTA(C) = 91.5 ± 1.9°, MPTA(LCP) = 89.1 ± 2.1°, P = .01). The effect was even more pronounced in elderly patients, with highly significant deterioration of the radiological outcome measures for conventional implants compared to precontoured LCP (RRS(C) = 5.7 ± 1.6, RRS(LCP) = 8.2 ± .8, P < .001; MPTA(C) = 92.6 ± 4.2°, MPTA(LCP) = 89.2 ± 1.4°, P = .002). CONCLUSION: Utilizing precontoured LCP in the treatment of AO41B3-fractures is associated with improved radiological outcomes. This effect is significant in young but even more pronounced in elderly patients. Consequently, precontoured LCP should closely be considered in any AO41B3-fracture, but especially in elderly patients.

A Method for the Evaluation of Early Osseointegration of Implant Materials Ex Vivo: Human Bone Organ Model.

In the present work, an ex vivo organ model using human bone (explant) was developed for the evaluation of the initial osseointegration behavior of implant materials. The model was tested with additive manufactured Ti6Al4V test substrates with different 3D geometries. Explants were obtained from patients who underwent total knee replacement surgery. The tibial plateaus were used within 24 h after surgery to harvest bone cylinders (BC) from the anterior side using hollow burrs. The BCs were brought into contact with the test substrate and inserted into an agarose mold, then covered with cell culture media and subjected to the external load of 500 g. Incubation was performed for 28 days. After 28d the test substrate was removed for further analysis. Cells grown out BC onto substrate were immunostained with DAPI and with an antibody against Collagen-I and alkaline phosphatase (ALP) for visualization and cell counting. We show that cells stayed alive for up to 28d in our organ model. The geometry of test substrates influences the number of cells grown onto substrate from BCs. The model presented here can be used for testing implant materials as an alternative for in vitro tests and animal models.

Anteversion Angle Measurement in Suspected Torsional Malalignment of the Femur in 3-Dimensional EOS vs Computed Tomography-A Validation Study.

BACKGROUND: Computed tomography (CT) scan is the standard for assessment of femoral torsion. This observational study was conducted to evaluate the comparability of the EOS radiation dose scanning system (EOS imaging, Paris, France) and the CT scan in patients with suspected torsional malalignment of the femur. METHODS: Patients with suspected torsional malalignment of the femur were included in a study for surgical planning. The primary endpoint was to compare the 3-dimensional radiological (EOS) imaging system with the CT scan to determine femoral anteversion (AV) angle. Three independent raters performed measurements. Comparability of CT scan and EOS values was assessed by Pearson correlation, t test, interobserver reliability, and intraobserver reliability (Cronbach alpha). RESULTS: About 34 femora were examined. Interobserver reliability/intraobserver reliability was 0.911 of 0.955 for EOS and 0.934 of 0.934 for CT scan. EOS system revealed an AV angle of 12.2° ± 10.0° (-15.0° to 32.0°). CT examinations showed an AV angle of 12.6° ± 9.2° (-3.2° to 35.6°). About 11 hips featured physiological AV, 14 hips showed decreased AV (<10°) or retroversion (<0°), and 9 hips showed increased AV (>20°). Overall, a strong Pearson correlation of τ = 0.855 and a highly significant correlation in the t test for both methods was seen. In patients with decreased AV, retroversion, or increased AV, Pearson correlation only resulted in a moderate/low correlation of τ = 0.495 and τ = 0.292. The t test showed no significant correlation at malrotation. CONCLUSION: In torsional malalignment, EOS does not have correlation with CT measurements. In contrast to CT scan, EOS allows femoral torsion measurement independent of legs' positioning.

Histomorphological Alterations of Human Anterior Cruciate Ligament Grafts During Mid-Term and Long-Term Remodeling.

OBJECTIVE: The aim of the present paper is to analyze mid-term and long-term alterations of human anterior cruciate ligament (ACL) grafts during the remodeling process with special regards to cellularity, α-smooth muscle protein (αSMP) expression, and crimp length in comparison to the native ACL. METHODS: A total of 34 patients were included (23 male and 11 female). Biopsies of 13 semitendinosus tendon and 14 patellar tendon autografts were obtained during surgical revision secondary to an ACL reconstruction. According to the interval between the index procedure and sample collection, the patients were divided into four groups: 4-12 months, 13-60 months, 61-108 months, and >108 months. Seven samples of native ruptured ACL tissue obtained during surgical intervention served as control. All biopsies were taken from the intraligamentous part of the ACL or the graft. Histomorphological and immunohistochemical analyses were conducted after samples were stained using hematoxylin-eosin, Giemsa, and αSMP enzyme-labeled antibodies. The total cell density, the numbers of fibroblasts and fibrocytes, the fibroblast/fibrocyte ratio, the number of αSMP+ cell nuclei, and the percentage of αSMP+ cells per fibroblast as well as the crimp lengths were determined using light microscopy. RESULTS: In the early phase of remodeling, the grafts featured extensively high total cell counts (1021.2 ± 327.8, P = 0.001), with high numbers of fibroblasts (841.4 ± 245.2, P = 0.002), fibrocytes (174.5 ± 113.0, P = 0.04), and αSMP+ cells (78.3 ± 95.0, P = 0.02) compared to controls (390.1 ± 141.7, 304.5 ± 160.8, 65.6 ± 31.4 and 2.3 ± 2.6, respectively). Thereafter, the numbers of all cell entities decreased. After more than 108 months, the percentage of αSMP+ cells per fibroblast reached physiological values (ratio 1.3 ± 1.0, P = 0.41; control 0.8 ± 0.8), while the total cell count (834.3 ± 183.7, P = 0.001) as well as the numbers of fibroblasts (663.5 ± 192.6, P = 0.006) and fibrocytes (134.1 ± 73.0, P = 0.049) remained significantly high. The fibroblast/fibrocyte ratio showed no significant alterations over the course of time compared to the controls. The collagen crimp lengths were elongated by tendency in the early phase (28.8 ± 12.9 mm, P = 0.15; control 20.7 ± 2.2 mm) and significantly shortened over time, with the lowest values in the long term (14.8 ± 2.0 mm, P = 0.001). The comparison of biopsies from semitendinosus tendon and patellar tendon autografts revealed no significant differences for any of the histomorphological parameters investigated. CONCLUSION: This study reveals distinctive mid-term and long-term immunomorphological alterations during human ACL graft remodeling. These data clearly indicate that the remodeling is a process that continues for 9 years or more. Furthermore, it seems to be a process of adaptation rather than full restoration. Even in the long run, several biological properties of the native ACL are not completely reestablished.

Editorial Commentary: Revision of Failed Double-Bundle Anterior Cruciate Ligament Reconstruction.

Double-bundle (DB) reconstruction of the anterior cruciate ligament was favored for several years. However, recent studies increasingly show that this technique does not provide a clear advantage over the less-invasive single-bundle technique. Unfortunately, the graft fails relatively often after ACL reconstruction. Postoperative communication of the bone tunnels through bone tunnel widening is possible. Since 2 drill channels are created in the DB technique, femoral as well as tibial, it is assumed that this technique may cause problems during revision. So, in part, revision may require a 2-step procedure with bone graft filling of the tunnels as the first step. It is important that surgeons with experience using DB publish their revision strategies and experiences.

Immunohistochemical examination in arthrofibrosis of the knee joint.

INTRODUCTION: Arthrofibrosis (AF) is the result of increased cell proliferation and synthesis of matrix proteins (collagen I, III, and VI). Especially after invasive knee surgery, e.g., ligament reconstruction or knee replacement, abnormal fibroblast proliferation with pathological periarticular fibrosis can be observed leading to severely limited joint motion. The pathogenesis of AF is currently not fully understood. The present work aims to determine pathogenic factors. MATERIALS AND METHODS: A descriptive, histological and immunohistochemical comparative study was performed on tissue samples of 14 consecutive patients undergoing arthrolysis for joint stiffness due to AF. Seven human autopsy specimens served as control. Samples were stained for expression of relevant markers such as CD68, α-smooth muscle actin (ASMA), beta-catenin, BMP-2 and examined for the histological grade of AF (cell-rich versus cell-poor) and compared to a control. Furthermore, a microscopic evaluation of the samples for cell differentiation and number was performed. RESULTS: Tissue sections of cell-rich fibrosis showed a significantly higher expression of CD68 compared to the control with less than 10% of CD68 positive cells (p = 0.002). In cell-poor fibrosis no statistically significant difference was obvious (p = 0.228). Expression of ASMA in synovia, vessels, cell-rich and cell-poor fibrosis showed median values of 2.00 in the AF group and 1.75 in the control. Both groups differed significantly (p = 0.003). AF tissue showed a significantly difference in expression of ß-catenin (p < 0.001) compared to the control. The overall difference between AF and control group in expression of BMP-2 was also statistically significant (p = 0.002). CONCLUSIONS: Expression of CD68, ASMA, beta-catenin and BMP-2 is significantly increased in AF tissue samples. Based on presented findings, histological evaluation and immunohistochemical assessment of CD68, ASMA, ß-catenin and BMP-2 expression may proof useful to diagnose AF and to analyze AF activity.

Significant increase of pathogen detection rate by dry arthroscopic biopsies at suspected low-grade infection following total knee arthroplasty: a prospective observational study.

INTRODUCTION: The differentiation between stiff-knee and low-grade periprosthetic joint infection (PJI) is the current diagnostic challenge in total knee (TKA) revision arthroplasty. The aim of this study was to investigate the additional value of dry biopsies, compared to wet biopsies, in patients presenting with knee stiffness following primary TKA. MATERIALS AND METHODS: Single center, prospective observational study. Consecutive patients with joint stiffness of unknown origin following primary TKA were enrolled. Patient assessment followed the diagnostic standard algorithm. During diagnostic arthroscopy, synovial fluid (synovial WBC, PMN%) and five dry biopsies (dry) were collected. Then fluid was infused and another five microbiology (wet) and five histological biopsies gathered, all from identical locations. The primary outcome parameter was the difference between the pathogens in wet and dry biopsies. RESULTS: 71 patients (61% females, 67 ± 10 years) were eligible. Preoperative blood serology mean CRP (0.7 ± 1.5 mg/dl; p = 0.852), WBC (6.6 ± 1.7 G/l; p = 0.056), and synovial fluid mean WBC (1639 ± 2111; p = 0.602), PMN% (38 ± 28; p = 0.738) did not differ between patients with negative, positive wet or dry biopsies. The histology was in 11% positive (p = 0.058). In 32% at least one pathogen was detected, 48% from wet, 44% from dry biopsies. An inhomogeneous distribution was found. Cutibacterium acnes (100%) was solely found in wet, Micrococcus luteus (75%), Staphylococcus capitis (67%), and Micrococcus lylae (100%) were predominantly found in dry biopsies. Additional dry biopsies increased the pathogen detection rate by 49%. CONCLUSION: The addition of dry biopsies to the current standard diagnostic algorithm for PJI increased the pathogen detection rate by 49%.

Is loss of fixation following locked plating of proximal humeral fractures related to the number of screws and their positions in the humeral head?

The aim of the study was to examine the correlation between the chosen position of screws and the complications observed in patients who underwent locked plating of proximal humeral fractures. We evaluated radiographs of 367 patients treated by locked-plating for proximal humeral fractures. Radiographs were taken at one day, 6 weeks, 3 months and 6 months after surgery, and were analyzed for secondary fracture displacement, loss of fixation, cutting out of screws and necrosis of the humeral head. Secondary loss of fixation occurred in 58 cases (15.8%) and among those cutting out of screws was observed in 25 cases (6.8%). In cases of secondary loss of fixation a mean of 6.7 screws were used to fix the fracture (vs 6.6, P=0.425). There was neither significant correlation between position of screws and the occurrence of postoperative loss of fixation in Spearman correlation nor relationship from backward logistic regression analysis. Loss of fixation following locked plating of proximal humeral fractures does not relate to the number of screws and their positions in the humeral head. In consequence, anatomic fracture reduction and restoration of the humeral head-shaft angle are still important factors and should not be disregarded.

Comparison of different strategies for in vivo seeding of prevascularized scaffolds.

Scaffolds seeded with multipotent precursor cells were hypothesized to heal critically sized bone defects. However, the success of this concept was limited by low cell survival after transplantation due to a lack of nutrients and oxygen. In vivo prevascularization of scaffolds before cell seeding may improve cell survival, yet the best seeding technique and time point of cell application remain elusive. Thus, the aim of this study was to compare different strategies. Demineralized bone matrix scaffolds were implanted around the saphenous arteriovenous (AV) bundle in nude mice. In vivo seeding was performed 0, 5, or 21 days after implantation using enhanced green fluorescent protein (eGFP)-expressing mesenchymal stem cells (MSCs). Cells were applied either by injection or the repetitive dripping technique. In vitro seeded and subcutaneously implanted scaffolds served as controls. Fourteen days after cell application, the fluorescence intensity of transplanted cells and the extent of newly formed vessels were quantified. We found that the AV flow through model as well as cell application increased vessel formation. In vitro seeding resulted in significantly higher cell numbers than in vivo seeding. With increasing time of prevascularization, the number of cells declined dramatically. In vivo seeding by cell injection was superior to the repetitive dripping protocol. On subcutaneously implanted scaffolds, significantly, more cells were found than on axially perfused scaffolds. We conclude that in vitro seeding is more efficient compared to the two novel in vivo seeding techniques of prevascularized scaffolds. With increasing time of prevascularization, the seeding efficiency for the in vivo methods further decreases, presumably due to the ingrowth of connective tissue. Even though, the presence of MSCs and the longer period of prevascularization enhances vessel formation, this conceivable advantage is limited supposedly by the inferior seeding efficiency.

Acute fractures to the proximal fifth metatarsal bone: development of classification and treatment recommendations based on the current evidence.

Fractures to the proximal fifth metatarsal bone are among the most frequent injuries to the foot. Various classifications intend to distinguish different fracture entities in regard to prognosis and treatment. The most commonly used classification by Lawrence and Botte delineates three fracture zones and gives treatment recommendations based on retrospective case series. Aim of our study was to critically review the literature and reevaluate the classification and treatment recommendations based on the highest level of evidence available. We performed a systematic literature search in Medline, Embase and Cochrane library and identified six prospective trials either comparing the same treatment for different fracture entities or different treatment strategies for the same fracture entity. The studies reveal that all "tuberosity avulsion fractures" (Zone 1, according to Lawrence and Botte) heal well using functional treatment. Even multifragmentary, displaced and intraarticular fractures in Zone 1 give comparable good results. Treatment with a short leg cast leads to a significant delay in return to preinjury level when compared to functional treatment. "Jones' fractures" (Zone 2) also demonstrate good to excellent results and complete bone healing when treated functionally. In contrast, "diaphyseal stress fractures" (Zone 3) at the distal limit of the fourth-fifth intermetatarsal articulation and just distally feature a significantly higher rate of treatment failure when treated non-operatively in a non-weight bearing short leg cast. Early intramedullary screw fixation leads to a significantly shorter time to bone healing and return to sport. In conclusion, acute fractures to the proximal fifth metatarsal bone should be classified into two entities only: First, metaphyseal fractures not extending beyond the distal end of the fourth-fifth intermetatarsal articulation, as these fractures, regardless the number of fragments, displacement and intraarticular involvement, should be treated functionally. Second, meta-diaphyseal fractures located at the distal end of the fourth-fifth intermetatarsal articulation or just distally, as these fractures require early intramedullary screw fixation.

Long-term detection of fluorescently labeled human mesenchymal stem cell in vitro and in vivo by semi-automated microscopy.

The use of seeded scaffolds in regenerative medicine is limited by the low survival of transplanted mesenchymal stem cells (MSC). Current approaches aim at improving cell viability but require an adequate long-term detection of the transplanted cells. Unfortunately, commonly performed labeling techniques have not been validated for this purpose, and studies often reveal inconclusive results. Consequently, we intended to identify the most suitable method for long-term detection of human MSC (hMSC) in vitro and in vivo. hMSC were labeled using the vital stainings PKH26 and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as well as enhanced green fluorescent protein (eGFP) transduction. Metabolic activity and relative fluorescence intensity (RFI) were quantified in vitro over 21 days at 8 time points using standardized semi-automated microscopy and flow cytometry. In vivo, cell seeded scaffolds were subcutaneously implanted in nude mice, and RFI was analyzed over 42 days at 5 time points. In vitro, PKH26 and CFDA-SE significantly reduced metabolic activity. RFI of both stainings significantly decreased after 1 day and further faded to <1% after 7 days. In contrast, labeling with eGFP showed no metabolic effect on hMSC, and no significant reduction of RFI over the total period of 21 days. In vivo, RFI of eGFP labeled cells reached a plateau phase after 21 days and displayed a 3.8-fold higher RFI compared with PKH26 and CFDA-SE on day 42 evaluated in 280 field of views per scaffold using three scaffolds for each labeling technique and time point. We conclude that PKH26 and CFDA-SE are unsuitable for long-term detection of hMSC. eGFP transduction, in turn, allows long-term detection of hMSC in vitro and in vivo. Our results suggest that eGFP is currently the best option among the fluorescent labeling techniques to follow the fate of transplanted hMSC.

Differential gene expression of bone marrow-derived CD34+ cells is associated with survival of patients suffering from myelodysplastic syndrome.

One feature of the molecular pathology of myelodysplastic syndromes (MDS) is aberrant gene expression. Such aberrations may be related to patient survival, and may indicate to novel diagnostic and therapeutic targets. Therefore, we aimed at identifying aberrant gene expression that is associated with MDS and patient survival. Bone marrow-derived CD34+ hematopoietic progenitor cells from six healthy persons and 16 patients with MDS were analyzed on cDNA macroarrays comprising 1,185 genes. Thereafter, our patients were followed-up for 54 months. We found differential expression of genes that were hitherto unrecognized in the context of MDS. Differential expression of 10 genes was confirmed by quantitative real-time RT-PCR. Hierarchical cluster analysis facilitated the separation of CD34+ cells of normal donors from patients with MDS. More importantly, it also distinguished MDS-patients with short and long survival. Scrutinizing our cDNA macroarray data for genes that are associated with short survival, we found, among others, increased expression of six different genes that encode the proteasome subunits. On the other hand, the most differentially down-regulated gene was IEX-1, which encodes an anti-apoptotic protein. We confirmed its decreased expression on RNA and protein level in an independent validation set of patient samples. The presented data broadens our notion about the molecular pathology of MDS and may lend itself to better identify patients with short survival. Furthermore, our findings may help to define new molecular targets for drug development and therapeutic approaches for patients with poor prognosis.

Age-related transcription levels of KU70, MGST1 and BIK in CD34+ hematopoietic stem and progenitor cells.

Despite the known longevity of human hematopoietic stem and progenitor cells (HSC), numerous functional impairments of these cells can be observed in an age-dependent manner. However, the molecular alterations associated with aging of HSC are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from newborn, young and old healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and evaluated by FACS analysis. We performed cDNA macroarray analyses on a first set of CD34+ samples (n=13). We found the genes encoding KU-antigen 70 kD (KU70), microsomal glutathione S-transferase 1 (MGST1) and BCL2-interacting killer (BIK) to possess age-related mRNA expression levels. KU70 is a DNA repair gene and part of the DNA-dependent protein kinase (DNA-PK) complex. Its expression was negatively correlated with donor age showing highest expression levels in newborn, 2.6-fold lower levels in young and 6.3-fold lower levels in old donors. The transcription levels of MGST1, a gene protecting against oxidative stress, progressively increased with age. Expression was lowest in newborn, 2.6-fold higher in young and 4.3-fold higher in old donors. BIK is a proapoptotic gene and its expression was positively correlated with donor age: lowest in newborn, 1.8-fold higher in young and 4.1-fold higher in old donors. These findings were confirmed with an independent, second set of CD34+ samples (n=16) by means of quantitative real-time RT-PCR. Elucidation of age-dependent molecular alterations in healthy HSC facilitate a better understanding of functional impairments in hematopoiesis and may become valuable for anti-aging drug development and the emerging field of regenerative medicine.

Serum proteome profiling detects myelodysplastic syndromes and identifies CXC chemokine ligands 4 and 7 as markers for advanced disease.

Myelodysplastic syndromes (MDS) are among the most frequent hematologic malignancies. Patients have a short survival and often progress to acute myeloid leukemia. The diagnosis of MDS can be difficult; there is a paucity of molecular markers, and the pathophysiology is largely unknown. Therefore, we conducted a multicenter study investigating whether serum proteome profiling may serve as a noninvasive platform to discover novel molecular markers for MDS. We generated serum proteome profiles from 218 individuals by MS and identified a profile that distinguishes MDS from non-MDS cytopenias in a learning sample set. This profile was validated by testing its ability to predict MDS in a first independent validation set and a second, prospectively collected, independent validation set run 5 months apart. Accuracy was 80.5% in the first and 79.0% in the second validation set. Peptide mass fingerprinting and quadrupole TOF MS identified two differential proteins: CXC chemokine ligands 4 (CXCL4) and 7 (CXCL7), both of which had significantly decreased serum levels in MDS, as confirmed with independent antibody assays. Western blot analyses of platelet lysates for these two platelet-derived molecules revealed a lack of CXCL4 and CXCL7 in MDS. Subtype analyses revealed that these two proteins have decreased serum levels in advanced MDS, suggesting the possibility of a concerted disturbance of transcription or translation of these chemokines in advanced MDS.

Exisulind induces apoptosis in advanced myelodysplastic syndrome (MDS) and acute myeloid leukaemia/MDS.

The influence of Exisulind on the viability and apoptosis of CD34(+) stem cells from patients with advanced myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML)/MDS was investigated. In eight out of 10 patient samples Exisulind reduced the fraction of viable cells by inducing apoptosis. We found evidence that Exisulind-mediated apoptosis depends on c-Jun NH(2)-terminal kinase (JNK) activation. Addition of a specific JNK-inhibitor to Exisulind-treated advanced MDS and AML/MDS cells partly abrogated apoptosis. We propose that Exisulind is tested in clinical phase I/II trials for the treatment of advanced MDS and AML/MDS.

X-linked sideroblastic anemia associated with a novel ALAS2 mutation and unfortunate skewed X-chromosome inactivation patterns.

Historically X-linked sideroblastic anemia, with rare exceptions, was thought to be manifested only in males. Since the discovery of the erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) and the cloning of its gene (ALAS2) 15 years ago, mutation analysis has revealed that clinical expression of this X-linked disorder is prevalent in females as well. However, presence of the disease in both genders within affected kindreds appears to be very uncommon. We report a unique family with the disorder in three women who have had widely disparate clinical courses. The anemia is associated with a previously unrecognized ALAS2 mutation (Arg436Trp) and is unresponsive to pyridoxine. To clarify the varied clinical courses of the patients, X-chromosome inactivation patterns were examined in hematopoietic and non-hematopoietic cells. We observed inactivation patterns supporting the conclusions that one daughter has a mild phenotype at age 31 because of moderate constitutive skewed X-chromosome inactivation, another daughter with clinical onset at age 16 is severely affected due to extreme constitutive X-skewing, whereas the mother developed progressive anemia in the fifth decade as she acquired an age-related non-random X-inactivation in hematopoietic cells. In addition, we observed random X-inactivation in reticulocytes of all three women that contrasted with a markedly skewed inactivation pattern in bone marrow erythroid cells. This discordance is attributable to apoptosis of erythroid precursors derived from progenitor cells with an active X-chromosome bearing the ALAS2 mutation. The features of the disorder in this family are also instructive in regard to the differential diagnosis of sideroblastic anemias in women.

The nonsteroidal anti-inflammatory drug Exisulind selectively induces apoptosis via JNK in secondary acute myeloid leukemia after myelodysplastic syndrome.

Treatment of patients suffering from myelodysplastic syndromes and secondary acute myeloid leukemia after MDS is often unsuccessful. Pro-apoptosis with arsenic trioxide has recently been proposed as a novel therapeutic approach. Exisulind is another potentially pro-apoptotic agent, and therefore, we investigated its influence on proliferation, differentiation, cell cycle and apoptosis in two sAML/MDS cell lines, one de-novo AML cell line and healthy CD34+ bone marrow cells. Treatment of sAML/MDS cells with Exisulind clearly inhibited colony formation in the CFU-assays. Interestingly, Exisulind did not alter the percentages of sAML/MDS cells in G1-, G2-, M- or S-phase, but reduced proliferation and induced apoptosis in this cell type. Exisulind had no effect on de-novo AML or normal CD34+ cells. We detected increased c-Jun NH2-terminal kinase activity in sAML/MDS cells treated with Exisulind. Adding a specific JNK-inhibitor to Exisulind-treated sAML/MDS cells partly abrogated apoptosis, thus proving that Exisulind-mediated apoptosis in sAML/MDS cells is dependent on JNK activation. We conclude that JNK is one mediator of apoptosis in sAML/MDS cells treated with Exisulind. Moreover, our data strongly suggests to explore the potential use of Exisulind as a novel, pro-apoptotic therapy for patients with MDS and sAML/MDS.

Optimization and evaluation of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) with reversed-phase protein arrays for protein profiling.

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry with protein arrays has facilitated the discovery of disease-specific protein profiles in serum. Such results raise hopes that protein profiles may become a powerful diagnostic tool. To this end, reliable and reproducible protein profiles need to be generated from many samples, accurate mass peak heights are necessary, and the experimental variation of the profiles must be known. We adapted the entire processing of protein arrays to a robotics system, thus improving the intra-assay coefficients of variation (CVs) from 45.1% to 27.8% (p<0.001). In addition, we assessed up to 16 technical replicates, and demonstrated that analysis of 2-4 replicates significantly increases the reliability of the protein profiles. A recent report on limited long-term reproducibility seemed to concord with our initial inter-assay CVs, which varied widely and reached up to 56.7%. However, we discovered that the inter-assay CV is strongly dependent on the drying time before application of the matrix molecule. Therefore, we devised a standardized drying process and demonstrated that our optimized SELDI procedure generates reliable and long-term reproducible protein profiles with CVs ranging from 25.7% to 32.6%, depending on the signal-to-noise ratio threshold used.

The Ets transcription factor ESE-1 mediates induction of the COX-2 gene by LPS in monocytes.

Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins that are major inflammatory agents. COX-2 production is triggered by exposure to various cytokines and to bacterial endotoxins. We present here a novel role for the Ets transcription factor ESE-1 in regulating the COX-2 gene in response to endotoxin and other pro-inflammatory stimuli. We report that the induction of COX-2 expression by lipopolysaccharide (LPS) and pro-inflammatory cytokines correlates with ESE-1 induction in monocyte/macrophages. ESE-1, in turn, binds to several E26 transformation specific (Ets) sites on the COX-2 promoter. In vitro analysis demonstrates that ESE-1 binds to and activates the COX-2 promoter to levels comparable to LPS-mediated induction. Moreover, we provide results showing that the induction of COX-2 by LPS may require ESE-1, as the mutation of the Ets sites in the COX-2 promoter or overexpression of a dominant-negative form of ESE-1 inhibits LPS-mediated COX-2 induction. The effect of ESE-1 on the COX-2 promoter is further enhanced by cooperation with other transcription factors such as nuclear factor-kappa B and nuclear factor of activated T cells. Neutralization of COX-2 is the goal of many anti-inflammatory drugs. As an activator of COX-2 induction, ESE-1 may become a target for such therapeutics as well. Together with our previous reports of the role of ESE-1 as an inducer of nitric oxide synthase in endothelial cells and as a mediator of pro-inflammatory cytokines in vascular and connective tissue cells, these results establish ESE-1 as an important player in the regulation of inflammation.