A new familial sclerosing bone dysplasia.

Osteoscleroses are a heterogeneous group of bone remodeling disorders characterized by an increase in bone density. Here we report on a consanguineous Lebanese family in which two sisters, aged 39 and 36 years, exhibit a severe genu varum, a square-face appearance, high forehead, slight proptosis of the eyes, symmetric enlargement of the jaw, protruding chin, and short stature. Bone X-rays showed the presence of hyperostosis of the cranial base and vault with increased density of the orbits, hyperostosis of the bones, thickening of the cortices, diaphyseal modeling defects, cortical thickening of the medullary cavity, mild enlargement of the medullary cavity of the short long bones, short femoral necks, increased width of the ribs, and narrow interpedicular distances of the lower lumbar spine. Osteodensitometry showed values 200% to 300% above values for age. A cervical MRI revealed the presence of a diffuse osteosclerosis with calcification of the posterior vertebral ligament and a narrow canal between C2 and T2. Blood test results were unremarkable. Serum osteocalcin levels were in the normal range, whereas high values of serum C-telopeptide were noted. A bone biopsy showed only the presence of compact bone and did not allow for histomorphometric analysis. Molecular studies excluded genes known to be involved in sclerosing bone dysplasias as the cause of this condition. In vitro analysis of osteoclast function indicated that contrary to most cases of autosomal recessive osteopetrosis, osteoclasts both formed and resorbed but exhibited a small decrease in resorptive activity compared with osteoclasts generated from normal control individuals. Differential diagnoses are discussed, and the possibility that this may be a novel clinical entity is raised.

Homing properties of adipose-derived stem cells to intracerebral glioma and the effects of adenovirus infection.

The inevitable clinical recurrence of high grade gliomas after standard treatment is due to the highly diffuse infiltrating parts of these tumors, which remain after surgery and respond poorly to radiation and chemotherapy. It has been proposed to employ the homing capacity of neural stem cells (NSCs) to different types of intracerebral pathology for selective targeting of glioma cells, and delivery of transgenic expressed therapeutics. This approach has been successful in a number of preclinical experimental studies, however, a major drawback for clinical translation has been the limitation of harvesting and ex vivo expansion of NSCs in patients. Here we demonstrate that adipose derived stem cells (ASCs), which are easily harvested in relatively large quantities in humans, display the same tropism for gliomas as NSCs in vitro and in vivo. Both ipsilateral as well as contralateral injection of these cells in brains of glioma-bearing mice, led to extensive homing to the tumor by the ASCs. The potential of loading these cellular vehicles with transgenes was assessed using adenoviral vectors. ASCs could be infected with adenoviral vectors, albeit at very high MOI. Insertion of the arg-gly-asp (RGD) motif into the adenovirus fiber knob, thereby redirecting primary attachment of the virus to integrins, resulted in a striking 7000-fold increase in infection efficiency. However, in vivo migration of adenovirus-infected ASCs was not observed, most likely due to an inflammatory response to these cells which was not observed with control non-infected ASCs. These results indicate that ASCs are an interesting candidate for further development for cell-based therapy of gliomas, however adenoviruses are not appropriate vectors for delivery of transgenes in this context.

Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies.

The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 +/- 1.1%, mean +/- SEM) and hip/thigh region (1.2 +/- 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.

Epstein-Barr virus infection and risk of lymphoma: immunoblot analysis of antibody responses against EBV-related proteins in a large series of lymphoma subjects and matched controls.

Epstein-Barr Virus (EBV) is consistently associated with distinct lymphoproliferative malignancies and aberrant EBV antibody patterns are found in most EBV cancer patients. We evaluate the detection of an abnormal reactive serological pattern to EBV (ab_EBV) infection and the risk of lymphoma in a multicentric case-control study. Serum samples were collected at study entry from 1,085 incident lymphoma cases from Spain, France, Germany, Czech Republic, Italy and 1,153 age, sex and country matched controls. EBV immunoglobulin G (IgG) serostatus was evaluated through a peptide-based ELISA combining immunodominant epitopes of EBNA1 (BKRF1) and VCA-p18 (BFRF3). Further, immunoblot analysis was performed to evaluate distinct antibody diversity patterns to EBV early antigens (EA), besides EBNA1, VCA-p18, VCA-p40 (BdRF1) and Zebra (BZLF1). Patients with chronic active EBV infection and aberrant EBV activity were characterized as having an abnormal reactive pattern (ab_EBV). Ab_EBV was observed in 20.9% of 2,238 included subjects with an increased proportion of cases presenting ab_EBV as compared to the control population (23.9% vs. 18.0% p = 0.001). Ab_EBV positivity was a risk factor for all lymphomas combined (odds ratio [OR] = 1.42, 95% confidence interval [CI]=1.15-1.74), and specifically for chronic lymphocytic leukaemia (OR = 2.96, 95%CI = 2.22-3.95). Lower levels of ab_EBV were observed for follicular lymphoma (OR = 0.38, 95%CI = 0.15-0.98). EBV may be involved in a larger subset of lymphomas among clinically immunocompetent subjects than previously thought, probably explained by an underlying loss of immune control of EBV latent infection. Ab_EBV is a useful tool to explore EBV imbalances preceding or paralleling possible EBV associated oncogenic events.

Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells.

Adipose tissue contains a stromal vascular fraction (SVF) that is a rich source of adipose tissue-derived stem cells (ASCs). ASCs are multipotent and in vitro-expanded ASCs have the capacity to differentiate, into amongst others, adipocytes, chondrocytes, osteoblasts, and myocytes. For tissue engineering purposes, however, it would be advantageous to use the whole SVF, which can be transplanted without further in vitro selection or expansion steps. Because little is known about the freshly isolated ASCs in the SVF, we phenotypically characterized human freshly isolated ASCs, using flow cytometry. In addition, we investigated whether freshly isolated ASCs have functional properties comparable to cultured ASCs. For this, the differentiation potential of both freshly isolated ASCs and cultured ASCs into the osteogenic pathway was analyzed. Freshly isolated ASCs slightly differed in immunophenotype from cultured ASCs. Contrary to cultured ASCs, freshly isolated ASCs were shown to be highly positive for CD34, and positive for CD117 and HLA-DR. On the other hand, expression of CD105 and especially CD166 on the freshly isolated ASCs was relatively low. After osteogenic stimulation of freshly isolated ASCs, both Runx-2 and CollaI gene expression were significantly increased (p < 0.05). However, there was a difference in the kinetics of gene expression between freshly isolated and cultured ASCs and also between the different SVF isolates tested. There was no difference in alkaline phosphatase activity between freshly isolated ASCs and cultured ASCs. In addition, freshly isolated ASCs stained positive for osteonectin and showed matrix mineralization. We conclude that although there are minor differences in phenotype and kinetics of differentiation between freshly isolated ASCs and cultured ASCs, the use of freshly isolated ASCs for tissue engineering purposes involving bone repair is potentially applicable.

Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects.

Epstein-Barr virus (EBV)-specific immunoblot analysis was used to reveal the molecular diversity of immunoglobulin (Ig) G and IgA antibody responses against Epstein-Barr nuclear antigen (EBNA), early antigen (EA), and viral capsid antigen (VCA) in serum samples from patients with nasopharyngeal carcinoma (NPC) and control subjects, by use of immunofluorescence assay (IFA). Control donors (n=150) showed IgG responses to few EBV proteins--VCA-p18, VCA-p40, EBNA1, and Zebra--and sporadically weak IgA reactivity to EBNA1 and VCA-p18. Patients with NPC stage 1 (n=6) had similar response patterns. Patients with NPC stage 2-4 (n=132) showed significantly more diverse IgG and IgA responses to EA and VCA proteins--VCA-p18/-p40, EBNA1, Z-encoded broadly reactive activator, and EAd-p47/54, -DNAse, -thymidine kinase, and -p138. No correlation was found between IFA titers and the number of EBV proteins recognized by IgG or IgA. Our results reveal dissimilarity between EBV polypeptides recognized by IgG and IgA antibodies, which suggests independent B cell triggering events.

Antibody responses to Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) and expression of LMP1 in juvenile Hodgkin's disease.

A large group of juvenile Hodgkin's disease patients (n = 242, mean age 11.7 years, 75% [n = 181] seropositive) was evaluated for anti-Epstein-Barr virus (EBV) antibody responses and the presence of EBV-encoded EBER-RNA and latent membrane protein-1 (LMP1)-protein expression in the tumor. The molecular diversity of anti-EBV antibody responses in Hodgkin's disease patients with EBV-positive and-negative tumors was studied by enzyme-linked immunosorbent assay (ELISA) and immunoblot. Using purified recombinant LMP1 protein as antigen, the presence of antibodies to LMP1 was related to expression of LMP1 in the tumor cells and specific EBV-serological patterns. Antibodies to LMP1 were detected in 30% of the EBV-seropositive Hodgkin's disease patients. The presence of antibodies to LMP1 was not associated with a distinct anti-EBV antibody diversity profile (ELISA), but a significantly higher percentage of patients with antibodies to LMP1 had antibodies to ZEBRA and viral capsid antigen (VCA)-p18 (Immunoblot). Significantly more patients with an EBV-positive tumor had detectable antibody responses to LMP1, but the presence of antibodies to LMP1 did not reflect the expression of LMP1 protein in the tumor cells. Interestingly, all patients with the strongest antibody responses to LMP1 had EBV-negative tumors, suggesting immunological selection in vivo.