Petri Lehenkari

Acute homing of bone marrow-derived mononuclear cells in intramyocardial vs. intracoronary transplantation

Objectives. Cell homing optimisation after transplantation is critical in myocardial infarction (MI) cell therapy. Design. Eight pigs were randomized to receiving autologous purified 111indium-labeled bone marrow mononuclear cells (BMMCs) (108 cells/2 ml) by intramyocardial (IM) (n=4) or by intracoronary (IC) (n=4) transplantation after 90 minutes occlusion of the CX-coronary artery. Dual isotope SPECT imaging was performed 2 and 24 hours postoperatively. Two animals were additionally analyzed on the sixth postoperative day. Tissue samples from the major organs were analyzed. Results. In SPECT imaging revealed that BMMCs administered using IM injection remained in the injured area. In contrast, minor proportion of IC transplanted cells remained in the myocardium, as most of the cells showed homing in the lungs. Analysis of the biopsies showed a seven-fold greater number of cells in the myocardium for the IM method and a 10-fold greater number of cells in the lungs in the IC group (p < 0.001). Conclusions. In producing persistently high cell homing at the infarction site, the IM transplantation is superior to the IC transplantation. However, the IC administration might be more specific in targeting injured capillaries and epithelial cells within the infarcted myocardium.

MRI-guided percutaneous retrograde drilling of osteochondritis dissecans of the knee.

ObjectiveThe purpose of this study was to evaluate the feasibility of a new method for osteochondritis dissecans (OCD) treatment.Materials and methodsTen OCD lesions of the knee unresponsive to conservative management were treated with MRI-guided percutaneous retrograde drilling to reduce symptoms and promote ossification of the lesion. All lesions were located in distal femoral condyles. Only stable OCD lesions were included (preprocedural MRI grade I or II). Five lesions were of juvenile type and five lesions were of adult type OCD. All the patients had severe limitation of activity due to the OCD-related pain.By using a 0.23xa0T open MRI scanner and spinal anesthesia, percutaneous retrograde drilling of the OCD lesions was performed (3xa0mm cylindrical drill, one to three channels). Optical tracking and MRI imaging were used to guide instruments during the procedure. Mean postprocedural clinical follow-up time was 3xa0years. Eight patients had a post-procedural follow-up MRI within 1xa0year.ResultsAll the OCD lesions were located and drilled using the 0.23xa0T open MRI scanner without procedural complications. All the patients had pain relief, mean visual analog score (VAS) declined from 6 to 2. Follow-up MRI showed ossification in all lesions. Eight patients could return to normal physical activity with no or minor effect on function (Hughston score 3–4). Treatment failed in two cases where the continuation of symptoms led to arthroscopy and transchondral fixation.ConlusionMR-guided retrograde OCD lesion drilling is an accurate, feasible, and effective cartilage-sparing techique in OCD management.

Tumor infiltrating CD8+ T lymphocyte count is independent of tumor TLR9 status in treatment naïve triple negative breast cancer and renal cell carcinoma

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor of the innate immune system that is widely expressed in cancers. We demonstrated that low tumor TLR9 expression predicts poor disease-specific survival in triple negative breast cancer (TNBC) and renal cell carcinoma (RCC). We hypothesized that this is because TLR9 expression affects tumor immunophenotype. To begin to test this, we compared the number of tumor infiltrating CD8+ T lymphocytes with TLR9 expression in treatment naïve breast cancer (n = 197) and RCC (n = 94) cohorts with known TLR9 expression status. CD8+ T lymphocyte counts were assayed with image analysis after immunohistochemistry (IHC). Tumor TLR9 expression was not correlated with CD8+ T cell counts in breast cancer or RCC. CD8+ T cell counts were significantly associated with tumor proliferation index in TNBC, but not in non-TNBC. CD8+ T cell counts were also significantly associated with tumor grade in non-TNBC, but not in TNBC. In RCC, CD8+ T cell counts were significantly associated with tumor stage. CD8+ T cell counts were significantly associated with prognosis in TNBC and RCC, but the presence of CD8+ T cells in these tumors had opposite effects on disease-specific survival: High CD8+ counts were associated with better prognosis in TNBC and worse prognosis in RCC. Among TNBC patients, those with low tumor TLR9 and low CD8+ T cell counts had the poorest prognosis (log-rank p = 0.0002 vs. high tumor TLR9 and high CD8+ T cell count). In conclusion, pre-treatment tumor TLR9 status is not associated with tumor infiltrating CD8+ T lymphocytes in TNBC or RCC. The combination of TLR9 and CD8+ TIL count might be a novel composite prognostic marker in TNBC.

Nucleic acid-sensing toll-like receptors 3, 7 and 8 in esophageal epithelium, barrett’s esophagus, dysplasia and adenocarcinoma

ABSTRACT Toll-like receptors (TLRs) are immunological receptors recognizing various microbial and endogenous ligands, such as DNA, RNA, and other microbial and host components thus activating immunological responses. The expression of TLRs in esophageal adenocarcinoma is not well known. The aim of this study was to evaluate expression patterns of those TLRs that sense nucleic acids in Barrett’s esophagus with and without dysplasia and in esophageal adenocarcinoma. TLRs 3, 7 and 8 were stained immunohistochemically and evaluated in a cohort of patients with esophageal adenocarcinoma or dysplasia. Specimens with normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37) and esophageal adenocarcinoma (n = 99) were studied. We used immunofluorescence to confirm the subcellular localization of TLRs. We found abundant expression of TLR3, 7 and 8 in esophageal squamous epithelium, columnar metaplasia, dysplasia and adenocarcinoma. Cytoplasmic expression of TLR3, TLR7 or TLR8 did not associate to clinicopathological parameters or prognosis in esophageal cancer. High nuclear expression of TLR8, confirmed with immunofluorescence, in cancer cells was observed in tumors of high T-stage (p < 0.01) and in tumors with organ metastasis (p < 0.001). High nuclear TLR8 expression was associated with poor prognosis (p < 0.001). The expression of TLR3, TLR7 and TLR8 increased toward dysplasia and adenocarcinoma. We demonstrated nuclear localization of TLR8, which associates with metastasis and poor prognosis. TLR3 and TLR7 do not seem to have prognostic significance in esophageal adenocarcinoma.

Local Binary Patterns to Evaluate Trabecular Bone Structure from Micro-CT Data: Application to Studies of Human Osteoarthritis

Osteoarthritis (OA) causes progressive degeneration of articular cartilage and pathological changes in subchondral bone. These changes can be assessed volumetrically using micro-computed tomography ((mu )CT) imaging. The local descriptor, i.e. local binary pattern (LBP), is a new alternative solution to perform analysis of local bone structures from (mu )CT scans. In this study, different trabecular bone samples were prepared from patients diagnosed with OA and treated with total knee arthroplasty. The LBP descriptor was applied to correlate the distribution of local patterns with the severity of the disease. The results obtained suggest the appearance and disappearance of specific oriented patterns with OA, as an adaptation of the bone to the decrease of cartilage thickness. The experimental results suggest that the LBP descriptor can be used to assess the changes in the trabecular bone due to OA.

MRI-guidance in percutaneous core decompression of osteonecrosis of the femoral head

AbstractObjectivesThe purpose of this study was to evaluate the usefulness of MRI-guidance for core decompression of avascular necrosis of the femoral head.Materials and methodsTwelve MRI-guided core decompressions were performed on patients with different stages of avascular necrosis of the femoral head. The patients were asked to evaluate their pain and their ability to function before and after the procedure and imaging findings were reviewed respectively.ResultsTechnical success in reaching the target was 100xa0% without complications. Mean duration of the procedure itself was 54xa0min. All patients with ARCO stage 1 osteonecrosis experienced clinical benefit and pathological MRI findings were seen to diminish. Patients with more advanced disease gained less, if any, benefit and total hip arthroplasty was eventually performed on four patients.ConclusionsMRI-guidance seems technically feasible, accurate and safe for core decompression of avascular necrosis of the femoral head. Patients with early stage osteonecrosis may benefit from the procedure.Key Points• MRI is a useful guidance method for minimally invasive musculoskeletal interventions.n • Bone drilling seems beneficial at early stages of avascular necrosis.n • MRI-guidance is safe and accurate for bone drilling.

High number of transplanted stem cells improves myocardial recovery after AMI in a porcine model

Abstract Objective. The clinical data considering the bone marrow mononuclear cell (BMMNC) therapy in treatment for acute myocardial infarction (AMI) are controversial and the mechanisms remain unknown. Our objective was to study the cardiac function and changes in cytokine levels after administration of BMMNC in experimental AMI model. Design. Unlabeled or Super-Paramagnetic-Iron-Oxide-labeled BMMNCs or saline was injected into myocardium of 31 pigs after circumflex artery occlusion. Ejection fraction (EF) was measured preoperatively, postoperatively and at 21 days by echocardiography. Cardiac MRI was performed postoperatively and after 21 days in 7 BMMNC animals. Serum cytokine levels were measured at baseline, 24 h and 21 days. Cellular homing was evaluated comparing MRI and histology. Results. From baseline to 21 days EF decreased less in BMMNC group (EF mean control -19 SD 12 vs. BMMNC -4 SD 15 percentage points p = 0.02). Cytokine concentrations showed high variability between the animals. MRI correlated with histology in cell detection and revealed BMMNCs in the infarction area. By MRI, EF improved 11 percentage points. The improvement in EF was associated with the number of transplanted BMMNCs detected in the myocardium. Conclusion. BMMNC injection after AMI improved cardiac function. Quantity of transplanted BMMNCs correlated with the improvement in cardiac function after AMI.

17β-Estradiol induces the proliferation of the in vitro cultured human urothelium

Objective. The genitourinary tract is considered to be a target for the actions of sex steroid hormones. Decreased ovarian function and lack of estrogen after menopause are associated with lower genitourinary tract symptoms as well as bladder dysfunctions such as incontinence. Estrogen may also affect urothelial cells. The estrogen receptors (ERs) are found in the mucosa of the urinary tract. The purpose of this study was to culture human urothelial cells (HUCs) originating from urothelial tissue biopsies and to use them as a reproducible test platform to evaluate the effect of 17β-estradiol (E2). Material and methods. Urothelial tissue biopsies were obtained from 95 patients undergoing gynaecological open surgery for urinary incontinence, paediatric vesicoureteral reflux or transurethral resection of the prostate (TURP) for benign prostatic hyperplasia. HUCs originating from biopsies were cultured in vitro in the absence or in the presence of 0.1 nmol, 0.01 µmol and 1 µmol of E2. ER expression of the cultured HUCs was examined by Western analysis and immunofluorescence microscopy, which was also used for HUC characterization. The effect of E2 in the proliferation of the HUCs was determined by tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-assay. Results. HUCs were cultured successfully in four to six passages but there was variation between samples. The cultured cells showed expression of β4-integrin, E-cadherin and cytokeratins 7, 8, 9 and 19, indicating the epithelial origin of the cells. Both types of ERs, ERα and ERβ, were found in the in vitro cultured HUCs. E2 treatment of HUCs did not affect remarkably the expression of ERα but cell proliferation was induced. However, no concentration-dependent effect was seen. Conclusions. This study indicates that HUCs originating from small tissue biopsies can be cultured in several passages in vitro and could have potential in repairing or restoring urinary tract tissue by tissue engineering therapy. HUCs serve as a good in vitro test platform, as shown by analysing E2-treated HUCs. E2 induced the proliferation of cultured HUCs even though concentration dependency was not observed. The findings of this study may have relevance in determining the mechanisms of estrogen therapy in postmenopausal urinary tract symptoms and in the future development of tissue engineering technology.

Breast cancer carcinoma-associated fibroblasts differ from breast fibroblasts in immunological and extracellular matrix regulating pathways

Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix -related pathways were over-represented in CAFs when compared to NFs (p<0.001). Under hypoxic conditions, the expression levels of pyruvate dehydrogenase kinase-1 (PDK1) and pyruvate dehydrogenase kinase-4 (PDK4) were lower in CAFs when compared to NFs (fold changes 0.6 and 0.4, respectively). In normoxia, when compared to NFs, CAFs displayed increased expression of glucose transporter 1 (GLUT-1) and PDK1 (fold changes 1.5 and 1.3, respectively). With respect to the assessed surface markers, only CD105 was expressed differently in MSCs when compared to fibroblasts, being more often expressed on MSCs. Cells with myofibroblast features were present in both NF and CAF samples. We conclude, that CAFs differ distinctly from NFs at the gene expression level, this hypothesis was also tested in silico for other available gene expression data.

Mitochondrial DNA Depletion and Deletions in Paediatric Patients with Neuromuscular Diseases: Novel Phenotypes

OBJECTIVEnTo study the clinical manifestations and occurrence of mtDNA depletion and deletions in paediatric patients with neuromuscular diseases and to identify novel clinical phenotypes associated with mtDNA depletion or deletions.nnnMETHODSnMuscle DNA samples from patients presenting with undefined encephalomyopathies or myopathies were analysed for mtDNA content by quantitative real-time PCR and for deletions by long-range PCR. Direct sequencing of mtDNA maintenance genes and whole-exome sequencing were used to study the genetic aetiologies of the diseases. Clinical and laboratory findings were collected.nnnRESULTSnMuscle samples were obtained from 104 paediatric patients with neuromuscular diseases. mtDNA depletion was found in three patients with severe early-onset encephalomyopathy or myopathy. Two of these patients presented with novel types of mitochondrial DNA depletion syndromes associated with increased serum creatine kinase (CK) and multiorgan disease without mutations in any of the known mtDNA maintenance genes; one patient had pathologic endoplasmic reticulum (ER) membranes in muscle. The third patient with mtDNA depletion was diagnosed with merosine-deficient muscular dystrophy caused by a homozygous mutation in the LAMA2 gene. Two patients with an early-onset Kearns-Sayre/Pearson-like phenotype harboured a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content.nnnCONCLUSIONSnNovel encephalomyopathic mtDNA depletion syndrome with structural alterations in muscle ER was identified. mtDNA depletion may also refer to secondary mitochondrial changes related to muscular dystrophy. We suggest that a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content associated with Kearns-Sayre/Pearson syndromes may be secondary changes caused by mutations in an unknown nuclear gene.