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upplementary Data Legend PDF file - 94K, Supplementary data legend

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PDF file - 1008KB, mRNA and protein expression of Api5 in TC-1/P0 and TC-1/P3 A17 cells (S1). Api5 expression in murine tumors and their immune resistance (S2). In vitro Generation and Characterization of KKM MART1-specific CTL clone (S3). Characterization of antigen presenting capacity of A375/no insert and A375/API5 cells in vitro (S4). API5 activates FGFR1 signaling pathway (S5).

Supplementary Figure S1 PDF file - 29K, Fold expansion of NK cells following co-cultivation of PBMC in IL-2 with irradiated solid tumor (A549, MCF7, Chang, Hela, HT29, MDA-MB-31, SK-BR-3), liquid tumor (CEM, K562, U937, KL-1) and PBMC (allogeneic and autologous) targets are shown

Supplementary Figure S6 PDF file - 23K, KL-1-mediated NK cell expansion from PBMC of healthy and patient donors was performed as described in Materials and Methods section of manuscript. PBMCs was co-cultivated with irradiated KL-1 at a ratio of 1:1 and supplemented with IL-2 500 U/ml every 3 days. On Day 14, the absolute number of NK cells was calculated by multiplying the total viable number of cells, as determined by manual counting with a hemacytometer, by the percentage of CD56+CD3- cells as determined by flow cytometry. Results are displayed as a scattergram. The mean number of NK cells expanded is shown as straight line. Although there is a trend towards increased expansion among healthy donors compared to patient donors, this linearity was not preserved and no statistically significant difference is noted between healthy donors and patients with cancer

Supplementary Figure S2 PDF file - 39K, A standard 51Cr release assay was performed using K562, U937 or KL-1 as target cell lines, as described in the "Materials and Methods" section. These targets were labeled with 51Cr (Perkin Elmer, Boston, MA, USA) at 50 ��Ci/5 �~ 105 cells and incubated at 5 �~ 103 cells/well with resting human NK cells at E:T ratios of 30:1, 10:1 and 3:1. Percent specific lysis was calculated as follows: 100 �~ (experimental release-spontaneous release)/(maximum release-spontaneous release)

Supplementary Figure S5 PDF file - 27K, Patient year of birth, disease type, stage and sites of metastasis where applicable, for samples which were evaluated in Figure 6 for KL-1 mediated NK cell expansion are shown

Supplementary Figure S4 PDF file - 1618K, Twenty-eight surface markers associated with activation, cytotoxicity, and differentiation were analyzed by flow cytometry on the gated NK population from the PBMC of patients with cancer on Day 0 (rest, pre-activation) and Day 11 (activation, post-expansion)

Supplementary Figure S3 PDF file - 1079K, Twenty-eight surface markers associated with activation, cytotoxicity, and differentiation were analyzed by flow cytometry on the gated NK population from the PBMC of healthy donors on Day 0 (rest, pre-activation) and Day 11 (activation, post-expansion)

The identification of circulating tumor cells (CTCs) is clinically important for diagnosing cancer. We have previously developed a size-based filtration platform followed by epithelial cell adhesion molecule immunofluorescence staining for detecting CTCs. To characterize CTCs independently of cell surface protein expression, we incorporated a chromosomal fluorescence in situ hybridization (FISH) assay to detect abnormal copy numbers of chromosomes in cells collected from peripheral blood samples by the size-based filtration platform. Aneuploid cells were detected in the peripheral blood of patients with lung cancer. Unexpectedly, aneuploid cells were also detected in the control group, which consisted of peripheral blood samples from patients with benign lung diseases, such as empyema necessitatis and non-tuberculous mycobacterial lung disease. These findings suggest that chromosomal abnormalities are observed not only in tumor cells, but also in benign infectious diseases. Thus, our findings present new considerations and bring into light the possibility of false positives when using FISH for cancer diagnosis.

Direct energy deposition (DED) technology has gained increasing attention as a new implant surface technology that replicates the porous structure of natural bones facilitating osteoblast colonization and bone ingrowth. However, concerns have arisen over osteolysis or chronic inflammation that could be caused by Cobalt-chrome (CoCr) alloy and Titanium (Ti) nanoparticles produced during the fabrication process. Here, we evaluated whether a DED Ti-coated on CoCr alloy could improve osteoblast colonization and osseointegration in vitro and in vivo without causing any significant side effects. Three types of implant CoCr surfaces (smooth, sand-blasted and DED Ti-coated) were tested and compared. Three cell proliferation markers and six inflammatory cytokine markers were measured using SaOS2 osteoblast cells. Subsequently, X-ray and bone histomorphometric analyses were performed after implantation into rabbit femur. There were no differences between the DED group and positive control in cytokine assays. However, in the 5-bromo-2&prime, deoxyuridine (BrdU) assay the DED group exhibited even higher values than the positive control. For bone histomorphometry, DED was significantly superior within the 1000 &micro, m bone area. The results suggest that DED Ti-coated metal printing does not affect the osteoblast viability or impair osseointegration in vitro and in vivo. Thus, this technology is biocompatible for coating the surfaces of cementless total knee arthroplasty (TKA) implants.

Because of the recent technological advances, the cementless total knee arthroplasty (TKA) implant showed satisfactory implant survival rate. Newly developed 3D printing direct energy deposition (DED) has superior resistance to abrasion as compared to traditional methods. However, there is still concern about the mechanical stability and the risk of osteolysis by the titanium (Ti) nanoparticles. Therefore, in this work, we investigated whether DED Ti-coated cobalt-chrome (CoCr) alloys induce chronic inflammation reactions through in vitro and in vivo models. We studied three types of implant surfaces (smooth, sand-blasted, and DED Ti-coated) to compare their inflammatory reaction. We conducted the in vitro effect of specimens using the cell counting kit-8 (CCK-8) assay and an inflammatory cytokine assay. Subsequently, in vivo analysis of the immune profiling, cytokine assay, and histomorphometric evaluation using C57BL/6 mice were performed. There were no significant differences in the CCK-8 assay, the cytokine assay, and the immune profiling assay. Moreover, there were no difference for semi-quantitative histomorphometry analysis at 4 and 8 weeks among the sham, smooth, and DED Ti-coated samples. These results suggest that DED Ti-coated printing technique do not induce chronic inflammation both in vitro and in vivo. It has biocompatibility for being used as a surface coating of TKA implant.

Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific T cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5low cells conferred immune resistance. Mechanistic investigations revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCδ/ERK effector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCδ, or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies. Cancer Res; 74(13); 3556–66. ©2014 AACR.

The graft-healing potential of mesenchymal stromal cells (MSCs) derived from the remnants of ruptured anterior cruciate ligaments (ACLs) after ACL reconstruction may depend on the chronicity of the injury. The aim of this study was to assess the quantitative and phenotypic differences between MSCs isolated from ACL remnants in patients with (sub)acute and chronic tearing.Torn ACL remnants were harvested during ACL reconstruction from 41 patients, 24 with (sub)acute ACL (6 months from injury to surgery) and 17 with chronic ACL (time interval6 months) tears. MSCs isolated from these samples were assessed for quantitative and phenotypic differences, and the correlation between the proportion of MSCs and the chronicity of ACL tear was evaluated.At passage 0, the mean proportion of MSCs (CD34(-), CD44(+), CD90(+) and CD105(+)) was higher in (sub)acute than in chronic ACL tear samples (20.69% ± 7.82% versus 9.85% ± 8.01%, P 0.001). At passages 1 and 2, however, MSC proportions did not differ significantly in the two groups. Time interval showed a negative correlation with MSC proportion only at passage 0 (r = -0.505, P 0.001). The optimal cutoff value for time from injury to surgery yielding 10% freshly isolated ACL-MSCs, a percentage expected to have low tissue healing potential, was 23.5 months.The proportion of freshly isolated MSCs was higher in samples from patients with (sub)acute tearing than in chronic ACL tearing and negatively correlated with the time interval between trauma and surgery.

The possible beneficial effects of chronic low-dose irradiation (LDR) and its mechanism of action in a variety of pathophysiological processes such as cancer are a subject of intense investigation. While animal studies involving long-term exposure to LDR have yielded encouraging results, the influence of LDR at the cellular level has been less well defined. We reasoned that since natural killer (NK) cells constitute an early responder to exogenous stress, NK cells may reveal sentinel alterations in function upon exposure to LDR. When purified NK cells received LDR at 4.2 mGy/h for a total of 0.2 Gy in vitro, no significant difference in cell viability was observed. Likewise, no functional changes were detected in LDR-exposed NK cells, demonstrating that LDR alone was insufficient to generate changes at the cellular level. Nonetheless, significant augmentation of cytotoxic, but not proliferative, function was detected when NK cells were stimulated with low-dose IL-2 prior to irradiation. This enhancement of NK cytotoxicity was not due to alterations in NK-activating receptors, NK1.1, NKG2D, CD69 and 2B4, or changes in the rate of early or late apoptosis. Therefore, LDR, in the presence of suboptimal cytokine levels, can facilitate anti-tumor cytotoxicity of NK cells without influencing cellular proliferation or apoptosis. Whether these results translate to in vivo consequences remains to be seen; however, our data provide initial evidence that exposure to LDR can lead to subtle immune-enhancing effects on NK cells and may explain, in part, the functional basis underlying, diverse beneficial effects seen in the animals chronically exposed to LDR.

A novel cell isolation and release platform using electrospun polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers is presented. Ethanol treatment of PS-PSMA nanofibers, employed for the purpose of sterilization, significantly increases their inter-fiber space for both antibody conjugation and subsequent cell separation. For the selective isolation of CD4+ T cells from heterogeneous mixtures of mouse lymph nodes, capture efficiencies of up to 100% are achieved while maintaining cellular integrity and viability. Once captured, CD4+ T lymphocytes can also be released from the NF scaffolds, further demonstrating its potential functionality as an immune cell-supporting and releasing matrix. This is the first demonstration of lymphocyte-culture scaffolds enabling selective isolation, accommodation, and sustained release of CD4+ T cells for the purpose of cell therapies.

The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte–macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.

The detection of circulating tumor cells (CTCs) is limited by the rarity of these cells in the peripheral blood of patients with cancer. Understanding tumor biology may be useful in the development of novel therapeutic strategies for patients with lung cancer. The present study evaluated a novel size-based filtration platform for enriching CTCs from patients with lung cancer. Blood samples were obtained from 82 patients with lung cancer for CTC analysis. CTC enrichment by size-based filtration was performed on 5-ml blood samples. The collected cells were detected by immunofluorescence using monoclonal anti-human antibodies against protein tyrosine phosphatase, receptor type C (CD45) and epithelial cell adhesion molecule (EpCAM; an epithelial cell marker), as well as a DAPI nucleic acid stain. CTCs were detected in 57 patients (69.5%) using the size-based filtration platform. The mean CTC counts, defined as the number of cells with DAPI-positive, CD45-negative and EpCAM-positive staining, were 1.48±1.71 per 5 ml blood for the 66 stage I-III patients and 8.00±9.95 per 5 ml blood for the 16 stage IV patients. The presence of ≥1 CTCs per 5-ml blood sample was significantly associated with pathological stage (stage IV vs. stage I-III, P=0.009), but not with patient age or gender, tumor histology, tumor size or lymphovascular invasion. The mean CTC count of healthy donors was 0.25±0.55 per 5 ml blood. In summary, CTCs from the blood of patients with lung cancer were enriched using a size-based filtration platform and immunofluorescent staining with DAPI, CD45 and EpCAM. The CTC counts of patients with stage IV cancer were higher than those of patients with stages I-III cancer. These results suggest that this novel platform may be a useful tool for determining the prognosis of patients with lung cancer.

The characteristics of anterior cruciate ligament (ACL)-derived mesenchymal stem cells (MSCs), such as proportion and multilineage potential, can be affected by donor age. However, the qualitative and quantitative features of ACL MSCs isolated from younger and older individuals have not yet been compared directly. This study assessed the phenotypic and functional differences in ACL-MSCs isolated from younger and older donors and evaluated the correlation between ACL-MSC proportion and donor age. Torn ACL remnants were harvested from 36 patients undergoing ACL reconstruction (young: 29.67 +/- 10.92 years) and 33 undergoing TKA (old: 67.96 +/- 5.22 years) and the proportion of their MSCs were measured. The mean proportion of MSCs was slightly higher in older ACL samples of the TKA group than of the younger ACL reconstruction group (19.69 +/- 8.57% vs. 15.33 +/- 7.49%, p = 0.024), but the proportions of MSCs at passages 1 and 2 were similar. MSCs from both groups possessed comparable multilineage potentiality, as they could be differentiated into adipocytes, osteocytes, and chondrocytes at similar level. No significant correlations were observed between patient age and MSC proportions at passages 0-2 or between age and MSC proportion in both the ACL reconstruction and TKA groups. Multiple linear regression analysis found no significant predictor of MSC proportion including donor age for each passage. Microarray analysis identified several genes that were differentially regulated in ACLMSCs from old TKA patients compared to young ACL reconstruction patients. Genes of interest encode components of the extracellular matrix (ECM) and may thus play a crucial role in modulating tissue homeostasis, remodeling, and repair in response to damage or disease. In conclusion, the proportion of freshly isolated ACL-MSC was higher in elderly TKA patients than in younger patients with ACL tears, but their phenotypic and multilineage potential were comparable.

TGF-beta1 has been known to suppress the growth of gastric cancer cells. Interestingly, TGF-beta1 treatment increased the proliferation of human gastric cancer cell line, SNU-216 cells, while it reduced the proliferation of other tumor cells including SNU-620 cells. TGF-beta1-mediated down-regulation of c-Myc and induction of p21CIP1 were observed in SNU-620, but there was no change in SNU-216 in response to TGF-beta1. Similarly, TGF-beta1 receptors were upregulated by TGF-beta1 treatment in SNU-620, but they were not responded in SNU-216. By a single strand conformation polymorphism analysis, a repeated insertion of 37 nucleotides in the exon 8 of Smad4, resulting in premature termination at codon 362, was found in SNU-216. Furthermore, this truncated Smad4 functioned as a dominant negative form in TGF-beta1-mediated reporter activity and TGF-beta1 receptor expression. However, the proliferation of tumor cells was not affected by Smad4 mutation, but it was modulated by PD98059. Taken together, a mutation in Smad4 in addition to mitogen-activated protein kinase altered the TGF-beta1-mediated signaling, which is one of key events of gastric tumorigenesis.

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I–negative or –mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1–mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1–lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation. Cancer Res; 73(8); 2598–607. ©2012 AACR.

Ethnopharmacological relevance Historical records reveal that in traditional medicine, a disease similar to diabetes was treated with ginseng. Korean red ginseng has been considered beneficial as a dietary supplement for its anti-diabetic potential. Aim This study was designed to investigate the prophylactic potential of Korean red ginseng (KRG) extract ( Panax ginseng C.A. Meyer Radix Rubra) in a well-established mouse model of Type 1 diabetes (T1D). Materials and methods The prophylactic effect of KRG extract was evaluated in mice fed with KRG extract for two weeks prior to induction of diabetes by streptozotocin (STZ) administration. Glucose levels and glucose challenge test results of KRG-treated diabetic mice were compared to those of untreated diabetic mice and healthy control mice. Examination of the immune compartments in lymphoid organs and immunohistochemical staining of pancreas for islet cell morphology and insulin producing beta cells were performed. Results KRG extract significantly lowered blood glucose levels to an average of 250 mg/dl from 350 mg/dl and improved glucose challenge testing when applied as prophylaxis. Histological findings indicated that KRG extract protected against STZ-induced destruction of pancreatic tissue and restored insulin secretion. Strikingly, this effect was accompanied by restoration of lymphocytes in secondary lymphoid organs, suggesting that KRG extract facilitated immune homeostasis. Conclusion This is the first report to demonstrate the prophylactic function of KRG extract in ameliorating the hyperglycemia of T1D. Immune compartments of diabetic mice were found to be preserved in KRG-treated mice suggesting that Korean red ginseng may benefit T1D patients, not only for its hypoglycemic but also for its immunomodulatory effects.

A wide variety of drug delivery systems have been developed for the delivery of anticancer agents. One of the most frequently used natural biomaterials in drug delivery systems is polysaccharides; however, they are difficult to digest and to eliminate from the body after systemic administration due to their high molecular weight natures and the absence of degrading enzymes. Therefore, the development of degradable and eliminable natural biomaterials is critical for successful in vivo applications. In the present study, we report the development of self-assembled biodegradable nanoparticles based on recombinant human gelatin (rHG) modified with alpha-tocopheryl succinate (TOS). The rHG-TOS nanoparticles efficiently encapsulated 17-AAG (17-allylamino-17-demethoxygeldanamycin), a small molecular anticancer drug targeting heat shock protein 90. The formation of 17-AAG-loaded nanoparticles was confirmed using TEM and dynamic light scattering analysis and found to be within the size of 90-220 nm. The loading efficiency, sustained release pattern, and stability of 17-AAG from the rHG-TOS nanoparticles were determined using HPLC. Furthermore, the passive targeting of rHG-TOS nanoparticles to the tumor area via enhanced permeability and retention effect was examined by noninvasive live animal imaging in a tumor mouse model. Finally, the 17-AAG-loaded nanoparticles were nonimmunogenic and more efficient than free 17-AAG in manifesting an anticancer effect in the tumor model. Overall, our data demonstrate rHG-TOS as a promising tool for the delivery of 17-AAG featuring therapeutic efficacy and biocompatibility.

Synovium-derived mesenchymal stem cells (MSCs) offer a promising therapeutic option for cartilage regeneration. The conventional method of MSC isolation involves single-cell suspensions using collagenases. Recently, a nonenzymatic explant technique was developed to isolate MSCs. We compared these techniques in the isolation of functional MSCs. MSCs were isolated from human fibrous and adipose synovium of osteoarthritic patients using explants or enzymatic methods. Total cell number, percentage of MSCs, and surface marker expression of MSCs were measured following expansion. Multipotentiality was determined using a MSC functional identification kit. MSCs isolated from fibrous or adipose synovium using these two techniques expressed similar levels of the surface markers CD44, CD90, and CD105, and displayed similar multipotentiality in generating adipocytes, osteoblasts, and chondrocytes. Total cell number and number of CD34(-)CD44(+)CD90(+) MSCs after 10-day expansion were similar in each culture, regardless of the source and method used, although the percentage of MSCs was slightly higher in explant cultures. There were no correlations between MSC yield and patient age, Hospital for Special Surgery score, and degree of deformity under all culture conditions. Both the enzymatic and explant techniques yielded similar yields of MSCs with similar characteristics. Because the explant technique is simpler and less invasive, it may be preferred over enzymatic techniques for isolating MSCs from the synovium of osteoarthritic patients for cartilage regeneration.

CLU facilitates proliferation and IFN-γ production of murine NK cells stimulated with suboptimal dose of IL-2, without affecting natural cytotoxicity. CLU is a secreted, multifunctional protein implicated in several immunologic and pathologic conditions. As the level of serum CLU was shown to be elevated during inflammatory responses, we questioned if CLU might interact with circulating lymphocytes leading to functional consequences. To assess this possibility directly, mouse splenocytes and purified NK cells were cultured with varying dose of CLU, and its effect on cell proliferation was examined. Our data showed that CLU up-regulated DNA synthesis and expansion of NK cells significantly in response to a suboptimal, but not maximal, dose of IL-2, and CLU alone did not exhibit such effects. This CLU-mediated synergy required the copresence of CLU at the onset of IL-2 stimulation and needed a continuous presence during the rest of the culture. Importantly, NK cells stimulated with CLU showed increased formation of cell clusters and a CD69 activation receptor, representing a higher cellular activation status compared with those from the control group. Furthermore, these NK cells displayed elevated IFN-γ production upon RMA/S tumor target exposures, implying that CLU regulates not only NK cell expansion but also effector function of NK cells. Collectively, our data present a previously unrecognized function of CLU as a novel regulator of NK cells via providing costimulation required for cell proliferation and IFN-γ secretion. Therefore, the role of CLU on NK cells should be taken into consideration for the previously observed, diverse functions of CLU in chronic inflammatory and autoimmune conditions.

We explored the feasibility of magnetite (Fe(3)O(4))-gold (Au) core-shell nanocrystals as a useful vehicle for biomedical application such as cell separation. Streptavidin-fluorescein isothiocyanate (STA-FITC) was conjugated to the surface of the Fe(3)O(4)-Au core-shell nanocrystals using a carbodimide activation protocol. These nanocrystals were further tested for their ability to bind CD4+ T lymphocytes, bound to biotin-labeled anti-CD4 mAbs, isolated from the spleen of C57BL/6 mice. Our data show that the Fe(3)O(4)-Au nanocrystals successfully pulled down CD4+ T lymphocytes from the whole splenocytes with high specificity. Therefore, our nanocrystals provide an efficient tool for the cell separation process and further present the dramatic potential to be applied to other areas of biomedical application including diagnosis, monitoring, and treatment of human diseases.

The hydrophobic polymer NFs could be dispersed in the aqueous solution easily by simple ethanol treatment, which increases the loading of enzyme or antibody effectively. The degree of dispersion of nanofibers was somewhat proportional to the concentration of ethanol, the loading and activity of immobilized enzymes were proportional increased to the degree of dispersion of nanofibers. Next, the feasibility of anti CD4 monoclonal antibodies conjugated on ethanol dispersed nanofibers (anti CD4 mAbs/EtOH-NF) are developed as a useful vehicle for biomedical application such as specific cell capture and separation. The anti CD4 mAbs/EtOH-NF were examined for their ability to bind CD4+ T cells, bound to anti-CD4 mAbs, isolated from the mixture of lymphocytes. The results show that the anti CD4 mAbs/EtOH-NF successfully isolated CD4+ T cells from the mixture of lymphocytes with high specificity. Therefore, biocompatible of alcohol treated nanofibers provide an efficient tool for the cell separation process and further present the potential to be applied to other areas of biomedical application.

SummaryTo the best of our knowledge, no reports have directly compared synovial fluid (SF)- and synovial membrane (SM)-derived mesenchymal stem cells (MSCs) from primary knee osteoarthritis patients in terms of MSC proportion, either immediately after isolation or during culture. Any possible correlation between SM- and SF-MSC purity and osteoarthritis severity, also remains unclear. We therefore assessed quantitative and phenotypic differences in MSCs isolated from SF and SM. We also evaluated the correlation between sample MSC purity, and disease severity, in patients with osteoarthritis. The main result of the current study was that the mean SF-MSC proportion at passage 0 was negatively correlated with Kellgren–Lawrence (KL) grade (r = −0.565, P = 0.002). In addition, KL grade was a only significant independent negative predictor of SF-MSC proportion at passage 0 (β = −0.356, P = 0.039). Conclusively, the proportion of SF-MSCs in fresh samples, evaluated at the single cell level, was inversely correlated with osteoarthritis severity.