Vascularization in vivo caused by the controlled release of fibroblast growth factor-2 from an injectable chitosan/non-anticoagulant heparin hydrogel

Addition of various heparinoids to the lactose-introduced, water-soluble chitosan (CH-LA) aqueous solution produces an injectable chitosan/heparinoid hydrogel. In the present work, we examined the capability of the chitosan/non-anticoagulant heparin (periodate-oxidized (IO(4)-) heparin) hydrogel to immobilize fibroblast growth factor (FGF)-2, as well as the controlled release of FGF-2 molecules from the hydrogel in vitro and in vivo. The hydrogel was biodegraded in about 20 days after subcutaneous injection into the back of a mouse. When the FGF-2-incorporated hydrogel was subcutaneously injected into the back of both mice and rats, a significant neovascularization and fibrous tissue formation were induced near the injected site. These results indicate that the controlled release of biologically active FGF-2 molecules is caused by biodegradation of the hydrogel, and that subsequent induction of the vascularization occurs.     

Mechanisms Involved in the Binding of Thymocytes to Rat Thymic Dendritic Cells

The effects of monoclonal antibodies (mAbs) to cell-surface molecules, divalent cations, and various cell-signaling and metabolic inhibitors on the binding of thymocytes to rat thymic dendritic cells (TDC) were studied using a rosette assay. It was found that TDC/thymocyte adhesion was stronger and faster at 37°C than at 4°C. Flow cytometric analysis demonstrated that bound thymocytes were predominantly CD4⁺CD8⁺ and CD4⁺CD8^-, but in comparison to the phenotype of whole thymocytes, they were enriched in the mature TCRαβ^hi subset. The binding of thymocytes to TDC at 37°C was almost completely dependent on Ca²⁺ and Mg²⁺ and partly on an intact cytoskeleton and calmodulin-dependent protein kinase. The adhesion was independent of new protein synthesis and the activities of protein kinases A and C, tyrosine kinases, as well as phosphotyrosine protein phosphatases. The TDC/thymocyte adhesion at 37°C was partly blocked by anti-LFA-1 (WT.1), anti-CD18 (WT.3), and anti-ICAM-1 (1A29) mAb. MAbs to class II MHC (OX-3 and OX-6), CD4 (W3/25), CD8 (OX-8), and αβTCR (R73) stimulated the adhesion via an LFA-1-dependent pathway, whereas an anti-CD45 mAb (G3C5) stimulated the rosette formation independently of LFA-1. MAbs to CD2 (OX-34), CD11b (ED7), CD11b/c (OX-42), and class I MHC (OX-18) were without significant effects on the adhesion process.     

Induction of protective immunity to monoclonal-antibody-defined Plasmodium falciparum antigens requires strong adjuvant in Aotus monkeys.

Monoclonal antibodies to the major Plasmodium falciparum merozoite surface coat and rhoptry antigens were produced. A combination of the affinity-purified polypeptides with Freund complete adjuvant which was given three times completely protected an Aotus lemurinus azure (karotype VI) monkey against homologous challenge; however, immunization with the same polypeptides with a muramyl dipeptide derivative [MDP-Lys(L18)] did not protect a second Aotus monkey, even though comparable high antibody titers were induced.     

An invasion-independent pathway of blood-borne metastasis: a new murine mammary tumor model

It is generally believed that active invasion by cancer cells is essential to the metastatic process. In this report, we describe a murine mammary tumor (MCH66) model of metastasis that does not require invasion into the vascular wall of both the primary tumor and the target organ, in this case, the lung. The process involves intravasation of tumor nests surrounded by sinusoidal blood vessels, followed by intravascular tumor growth in the lung, without penetration of the vascular wall during the process. Comparative studies using a nonmetastatic MCH66 clone (MCH66C8) and another highly invasive metastatic cell line (MCH416) suggested that high angiogenic activity and sinusoidal remodeling of tumor blood vessels were prerequisites for MCH66 metastasis. Differential cDNA analysis identified several genes that were overexpressed by MCH66, including genes for the angiogenesis factor pleiotrophin, and extracellular matrix-associated molecules that may modulate the microenvironment toward neovascularization. Our analyses suggest that tumor angiogenesis plays a role in the induction of invasion-independent metastasis. This model should prove useful in screening and development of new therapeutic agents for cancer metastasis.     

Selection of higher molecular weight genomic DNA for molecular diagnosis from formalin-fixed material.

The patterns of DNA degradation in frozen, methanol-fixed, and formalin-fixed tissues were investigated by high-performance liquid chromatography (HPLC). The chromatograms all yielded one major peak with or without several extra minor peaks representing molecular weights of preserved genomic DNA. The most characteristic differences were in the retention times of the major peaks, with the earliest major peak occurring in the formalin-fixed tissues, and followed by the methanol-fixed, and frozen tissue samples, in that order. This means that the molecular weight of the DNA from formalin-fixed tissue is much shortened than that recovered from methanol-fixed tissue and frozen tissue. The results also indicated that a small amount of higher molecular weight DNA is still preserved in formalin-fixed tissues. To improve the amplification efficiency of polymerase chain reaction (PCR) analysis of formalin-fixed material, we isolated the higher molecular weight DNA from formalin-fixed, paraffin-embedded tissue from four different organs and compared the amplification efficiencies with those of the crude DNA extract. We used eight sets of oligonucleotide primers producing 262 to 989 base pair (bp) fragments of beta-globin. The results showed that the PCR amplification analyses were more efficient with the isolated higher molecular weight DNA than with the crude DNA extract. Our study demonstrated that not all the DNA in formalin-fixed, paraffin-embedded tissue samples is totally degraded but that a small amount of higher molecular weight DNA persists. The feasibility of molecular diagnosis using formalin-fixed material can be improved by isolating the preserved higher molecular weight DNA by HPLC.     

Enhancement of lipopolysaccharide-stimulated cyclooxygenase-2 mRNA expression and prostaglandin E2 production in gingival fibroblasts from individuals with Down syndrome

It is well known that Down syndrome (DS) is a premature ageing syndrome. Periodontal disease in individuals with DS develops rapidly and extensively in a relatively younger age bracket compared with that in healthy controls. The mechanisms involved in the periodontal inflammatory processes in DS patients are not fully understood. In the present study, the non-inflamed gingival fibroblasts isolated from seven patients with DS (DGF) and seven healthy controls (NDGF) were stimulated with lipopolysaccharide (LPS) derived from Actinobacillus actinomycetemcomitans (A. a.). We measured the level of prostaglandin E2 (PGE2) production by DGF and NDGF by radioimmunoassay, and also measured the mRNA expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) by using the real-time PCR method. We found the higher levels of LPS-stimulated COX-2 mRNA expression and PGE2 production in DGF when compared with those in NDGF. This study may indicate that overexpression of LPS-stimulated COX-2 induced a greater ability of DGF to produce PGE2, and that these phenomena may be responsible for the severer periodontal disease in DS patients.     

Preparation of a protein micro-array using a photo-reactive polymer for a cell-adhesion assay

A protein micro-array, called a "cell chip" was constructed by using a photo-reactive polymer for a cell-adhesion assay. Various amounts of albumin or fibronectin were covalently immobilized on a polystyrene dish using a micro-spotter with a dip pen. First, poly(acrylic acid) carrying azidophenyl groups was synthesized as the photo-reactive polymer. Secondly, the aqueous solution of a photo-reactive polymer (several nanoliters) was cast using the dip pen of the micro-spotter and dried in air. Subsequently, aqueous solutions of proteins were cast on the same place using the micro-spotter. After drying, the dish was irradiated with ultraviolet light. Finally, the immobilization was confirmed by staining with a dye. The immobilization was stable even after washing with Tween-20. The protein-immobilized area depended on the manipulation of the micro-spotter and the size of the dip pen. Subsequently, cell adhesion on the photo-immobilized protein micro-array was investigated. The adhesion behavior of cells depended on the kind of immobilized proteins and the kind of cells. The protein micro-array will be useful for cell diagnosis and for the selection of biomaterials to regulate cell behavior.     

Proliferative funiculitis with a prominent infiltration of mast cells

A surgical case of proliferative funiculitis (pseudosarcomatous myofibroblastic proliferation of the spermatic cord) with a prominent mast cell infiltration is reported. A 67-year-old man with a history of right inguinal herniorrhaphy 7 years earlier was operated on for diffuse swelling of the inguinal region and scrotum. A large lipoma was found in the inguinal region, and a nodular lesion, measuring 2.7 cm in maximal dimension, was firmly attached to the right spermatic cord. The nodular lesion showed diffuse proliferation of fibroblasts and myofibroblasts associated with the deposition of collagen. A diffuse infiltration of numerous mast cells was found throughout the lesion. Lesions that belong to the group of inflammatory pseudotumors are rarely accompanied by a prominent mast cell infiltration, and the differentiation from mast cell neoplasms is often problematic in such cases. The present case is the first example of proliferative funiculitis associated with this rare phenomenon.     

Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis

The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.