The effect of platelet-rich fibrin on autologous osteochondral transplantation: An in vivo porcine model.

BACKGROUND: This work aimed to evaluate the efficacy of cartilage transplantation to the medial femoral condyle±platelet-rich fibrin (PRF) augmentation in a porcine model. The hypothesis of the study was that PRF may act as a bioactive cell scaffold to fill defects and enhance cartilage regeneration. METHODS: Thirty-two knees of 16 miniature pigs were randomly assigned to four groups. The critical-size osteochondral defects (8x5mm) in femoral condyle of both knees were treated with one of the following: group 1－untreated controls; group 2－cartilage fragments alone; group 3－PRF alone; group 4－PRFT+cartilage fragments. After completion of the surgical implantation, the periosteal patch harvested from the proximal tibia was sutured onto the cartilage of the medial condyle to cover the implanted defects. Animals were sacrificed at six months after treatment. The regenerated cartilages were assessed by gross inspection and histological examination. RESULTS: The best results were obtained with the repair tissue being hyaline-like cartilage (group 4). The grading score of histological evaluation demonstrated that group 4 had better matrix, cell distribution and cartilage mineralization than group 2 and group 3. PRF showed a positive effect on the cartilage repair; the procedure was more effective when PRF was combined with autologous chondrocytes. CONCLUSIONS: This approach may provide a successfully employed technique to target cartilage defects in vivo. Larger groups and longer periods of study may provide more definitive and meaningful support for using this therapeutic approach as a new way of cartilage regeneration.

Protein disulfide isomerase a4 acts as a novel regulator of cancer growth through the procaspase pathway.

Protein disulfide isomerase a4 (PDIA4) is implicated in the growth and death of tumor cells; however, its molecular mechanism and therapeutic potential in cancer are unclear. Here, we found that PDIA4 expression was upregulated in a variety of tumor cell lines and human lung adenocarcinoma tissues. Knockdown and overexpression of PDIA4 in tumor cells showed that PDIA4 facilitated cell growth via the reduction of caspases 3 and 7 activity. Consistently, Lewis lung carcinoma cells overexpressing PDIA4 grew faster than did parental cells in tumor-bearing mice, as shown by a reduced survival rate, increased tumor size and metastasis, and decreased cell death and caspases 3 and 7 activity. PDIA4 knockdown resulted in opposite outcomes. Moreover, results obtained in mice with spontaneous hepatoma indicated that PDIA4 deficiency significantly reduced hepatic tumorigenesis and cyst formation and increased mouse survival, tumor death, and caspases 3 and 7 activity. Mechanistic studies illustrated that PDIA4 negatively regulated tumor cell death by inhibiting degradation and activation of procaspases 3 and 7 via their mutual interaction in a CGHC-dependent manner. Finally, we found that 1,2-dihydroxytrideca-5,7,9,11-tetrayne, a PDIA4 inhibitor, reduced tumor development via enhancement of caspase-mediated cell death in TSA tumor-bearing mice. These findings characterize PDIA4 as a negative regulator of cancer cell apoptosis and suggest that PDIA4 is a potential therapeutic target for cancer.

Toward an ideal animal model to trace donor cell fates after stem cell therapy: production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene.

The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical cancers.

Infection with high-risk types (type 16 or type 18) of human papillomaviruses (HPVs) increases a patient's risk of cervical cancer. Given the importance of the cervix and the severe side effects resulting from traditional cancer therapies, this study aimed to achieve targeted inhibition of viral oncogenes in tumor cells using small interfering RNAs (siRNA). To accomplish this, we developed nine siRNAs against either the E6 or E7 genes of HPV-16 or HPV-18 in several combinations, yielding siRNAs targeting 16E6, 16E7, 18E6 and 18E7. We measured the effectiveness of the siRNAs by examining E6 or E7 mRNA expression after transfection of the siRNAs into HPV-positive CaSki (HPV-16) or HeLa (HPV-18) cell lines. We found that the HPV-siRNAs significantly reduced cell growth and colony formation in both cell lines. Flow cytometry analysis revealed a significant increase in apoptosis. The siRNAs had no effect on cell growth, colony formation or apoptosis in HPV-negative C33A cells, demonstrating a lack of off-target effects. In addition, an in vivo xenograft study showed that intra-tumoral injection of the siRNAs reduced tumor growth in BALB/c nude mice. In conclusion, we have developed highly specific and potent HPV-siRNAs that successfully suppress tumor growth and induce apoptosis in HPV-positive cervical cancer cells. siRNA treatment has potential for further development as an adjuvant therapy for cervical cancer.

Chitosan/gelatin hydrogel prolonged the function of insulinoma/agarose microspheres in vivo during xenogenic transplantation.

PURPOSE: A chitosan/gelatin solution with glycerol 2-phosphate disodium salt hydrate in liquid phase at room temperature becomes a hydrogel at 37 degrees C. The material can be used as an injectable cell carrier into the human body for gelation in situ. We hoped that the chitosan/gelatin hydrogel provided extra protection for insulinoma/agarose microspheres during xenogenic transplantation. MATERIALS AND METHODS: Mouse insulinoma was microencapsulated in agarose as microspheres, which were macroencapsulated in chitosan/gelatin hydrogel. Insulin secreting profiles were first demonstrated in vitro. Diabetic rats were injected subcutaneously with insulinoma/agarose microspheres or insulinoma/agarose microspheres suspended in chitosan/gelatin solution. The nonfasting blood glucose concentrations (NFBG) of diabetic rats were measured perioperatively. Rats were humanely killed 1 month postoperatively and the hydrogel was retrieved for histological examination. RESULTS: The insulinoma/agarose microspheres continually secreted insulin for 1 month when macroencapsulated in chitosan/gelatin hydrogel in vitro. The NFBG of diabetic rats injected with insulinoma/agarose microspheres decreased to euglycemic status albeit hyperglycemia was restored within 10 days. The NFBG of diabetic rats injected with chitosan/gelatin hydrogel, which contained insulinoma/agarose microspheres, was maintained at less than 200 mg/dL for 25 days. The histological section revealed immune cell infiltration and accumulation within the hydrogel and around the iusulinoma/agarose microspheres that may have contributed to the slowly increasing NFBG after day 25. CONCLUSION: This study showed that chitosan/gelatin hydrogel can be used as a cell carrier for an injectable bioartificial pancreas; the hydrogel prolonged the function of cells encapsulated in agarose microspheres during xenogenic transplantation.

Seroprevalence of canine distemper virus in cats.

A seroepidemiological survey of canine distemper virus (CDV) infection in Asian felids revealed that the prevalence of antibodies varied depending on region and, in some cases, exposure to dogs. The serologic pattern in cats with antibodies indicated that they had likely been exposed to field strains rather than typical CDV vaccine strains.

Serosurvey for selected virus infections of wild carnivores in Taiwan and Vietnam.

Serum samples from two leopard cats (Felis bengalensis) and four Formosan gem-faced civets (Paguma larvata taivana) in Taiwan, September 1995, and nine leopard cats in Vietnam, August and December 1997, were examined for the prevalence of antibodies against feline parvovirus, feline herpesvirus type 1, feline calicivirus and feline immunodeficiency virus. All civets and nine of 11 leopard cats were shown to have antibodies against feline parvovirus (FPV), and FPV's were isolated from mononuclear cells in the peripheral blood of the six leopard cats.

Feline immunodeficiency virus subtype C is prevalent in northern part of Taiwan.

The seroepidemiological survey of cats conducted in northern part of Taiwan in 1998 revealed that the positive rate of feline immunodeficiency virus (FIV)-infection was 21.9% (7/32) and the rate was much higher than those of previous reports. We succeeded in isolation of three strains of FIV from peripheral blood mononuclear cells of the blood samples. Nucleotide sequences of the env variable V3 to V5 region of the strains revealed that the isolates from distinct areas belong to subtype C. These data together with our previous report (Inada et al. 1997. Arch. Virol., 142: 1459-1467) indicate that FIV subtype C is prevalent in northern part of Taiwan.

Phylogenetic analysis of feline immunodeficiency virus isolated from cats in Taiwan.

Feline immunodeficiency virus was isolated from four cats in Taiwan. The isolates were designated TI-1, TI-2, TI-3 and TI-4. Each was isolated from PBMCs following co-cultivation of PBMCs with a feline T-lymphoblastoid cell line (MYA-1 cells). However, the Taiwanese isolates did not grow in a feline kidney cell line (CRFK cells). The nucleotide sequences of the V3-V5 region of the envelope gene of the Taiwanese isolates were determined and compared with those of previously described isolates. Phylogenetic analysis of this region indicates that Taiwanese isolates belong to subtype C.

A polysomnographic study of sleep disturbance in community elderly with self-reported environmental chemical odor intolerance.

Subjective sleep complaints and food intolerances, especially to milk products, are frequent symptoms of individuals who also report intolerance for low-level odors of various environmental chemicals. The purpose of the present study was to evaluate the objective nature of nocturnal sleep patterns during different diets, using polysomnography in community older adults with self-reported illness from chemical odors. Those high in chemical odor intolerance (n = 15) exhibited significantly lower sleep efficiency (p = .005) and lower rapid-eye-movement (REM) sleep percent (p = .04), with a trend toward longer latency to REM sleep (p = .07), than did those low in chemical intolerance (n = 15), especially on dairy-containing as compared with nondairy (soy) diets. The arousal pattern of the chemical odor intolerant group differed from the polysomnographic features of major depression, classical organophosphate toxicity, and subjective insomnia without objective findings. The findings suggest that community elderly with moderate chemical odor intolerance and minimal sleep complaints exhibit objectively poorer sleep than do their normal peers. Individual differences in underlying brain function may help generate these observations. The data support the need for similar studies in clinical populations with chemical odor intolerance, such as multiple chemical sensitivity patients and perhaps certain veterans with "Persian Gulf Syndrome."