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The stromal vascular fraction

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.

Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigencomplexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n ‫؍‬ 367), non-NPC head and neck cancer patients (n ‫؍‬ 43), and biopsy-proven NPC patients (n ‫؍‬ 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.

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.

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 5 0.001). Ab_EBV positivity was a risk factor for all lymphomas combined (odds ratio [OR] 5 1.42, 95% confidence interval [CI]51.15-1.74), and specifically for chronic lymphocytic leukaemia (OR 5 2.96, 95%CI 5 2.22-3.95). Lower levels of ab_EBV were observed for follicular lymphoma (OR 5 0.38, 95%CI 5 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 preceeding or paralleling possible EBV associated oncogenic events. ' 2007 Wiley-Liss, Inc.

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.

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