Local delivery system of cytotoxic agents to tumors by focused sonoporation.

Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as 'sonoporation' has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. The cytotoxicity of bleomycin (BLM) was investigated using flow-cytometric analysis and Hoechst's staining in vitro assay systems. We found that the delivery of BLM by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude KSN/slc mice treated with a low dose of BLM followed by sonoporation during the 4-week experimental period. Histological analysis revealed that the cytotoxic effect was mainly apoptosis. We previously reported that the cytolethal distending toxin B (cdtB) from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, is responsible for cell cycle arrest and apoptosis in vitro. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporation-induced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy.

Rat genomic structure of amidophosphoribosyltransferase, cDNA sequence of aminoimidazole ribonucleotide carboxylase, and cell cycle-dependent expression of these two physically linked genes.

Genomic structure of rat amidophosphoribosyltransferase (ATase; EC 2.4.2.14), which catalyzes the first committed step in de novo purine nucleotide synthesis, was determined by polymerase chain reaction (PCR)-based methods. There are 11 exons and all exon-intron boundaries were conserved among rat, human, and chicken ATase genes. A rat aminoimidazole ribonucleotide carboxylase (AIRC) cDNA encoding a bifunctional enzyme of AIRC (EC 4.1.1.21) at step 6 and SAICAR synthetase (EC 6.3.2.6) at step 7 in de novo purine nucleotide synthesis was cloned and sequenced. The size of the cloned rat AIRC cDNA was 1329 bp, and amino acid identity with human and chicken AIRC was 96 and 85%, respectively. The intergenic sequence using a phage clone and the PCR product disclosed that ATase and AIRC genes are physically linked with the 736 bp sequence between the translation start sites, and the determination of the transcriptional start sites by the primer extension assay for these genes disclosed that distance between the two major transcriptional start sites is 585 bp. The amount of mRNAs of both genes showed approx. 5-6-fold increase in G1/S phase of the cell cycle over those in G0 phase in synchronized rat 3Y1 fibroblasts.

Effect of surface roughness on proliferation and alkaline phosphatase expression of rat calvarial cells cultured on polystyrene.

Rough-surfaced substrates made by a variety of methods have been shown to influence osteoblast proliferation and differentiation. The purpose of this study is to confirm the role of surface roughness in promoting osteoblastic differentiation using tissue culture polystyrene as substrate, by excluding factors other than roughness. Immature osteogenic cells derived from fetal rat calvariae were cultured on the plastic cover strips having varied degrees of roughness created by treatment with four kinds of grinding paper of different particle sizes. The proliferation and gene expression of alkaline phosphatase (ALP) and osteocalcin of the calvarial cells increased on the rough-surfaced cover strips. These levels increased in response to the increase in the degree of surface roughness up to 0.8 microm of average roughness and then decreased to the level observed for the smooth surface. These results demonstrate that the surface roughness itself caused increases in osteoblastic proliferation and differentiation in cell cultures.

SC-19220, a prostaglandin E2 antagonist, inhibits osteoclast formation by 1,25-dihydroxyvitamin D3 in cell cultures.

1,25 Dihydroxy vitamin D3 (1,25(OH)2D3), prostaglandin (PG) E2 and parathyroid hormone (PTH) induce osteoclast formation in cell cultures. Previously, we have shown that SC-19220, an antagonist of the EP1 subtype of PGE receptors, inhibited tartrate-resistant acid phosphatase (TRAP)-positive cell formation by PGE2 and PTH in adherent cell cultures taken from neonatal rats. Since 1,25(OH)2D3 has been shown to induce osteoclast formation through PGE2 synthesis, in this study we have examined the effect of SC-19220 on osteoclast formation induced by 1,25(OH)2D3 in cell cultures by measuring bone resorption as well as TRAP-positive cell formation. SC-19220 inhibited osteoclast formation by 1,25(OH)2D3 as well as by PGE2 in cell cultures. The addition of SC-19220 to the later half but not to the earlier half of the culture inhibited 1,25(OH)2D3-induced formation. In the culture in which hydroxyurea was added in the later half period, SC-19220 inhibited osteoclast formation by 1, 25(OH)2D3. Under these conditions, 17-phenyl PGE1, an EP1 agonist, induced osteoclast formation. Thus, SC-19220 inhibits certain reactions in the later processes of osteoclast formation induced by 1,25(OH)2D3. In addition, SC-19220 also inhibited osteoclast formation induced by interleukin (IL)-11 and IL-6 as well as by PTH. It is suggested that the SC-19220 inhibiting reactions are shared by all the inducers including 1,25(OH)2D3 and are essential for osteoclast formation.

T-cassette ligation: a method for direct sequencing and cloning of PCR-amplified DNA fragments.

We describe a method to ligate a PCR-amplified DNA fragment with T-protruding cassettes, which have multiple sites for endonuclease, promoter sequences of T3 and T7, and annealing sites for the universal M13 forward and reverse primers. This method, which we named T-cassette ligation, substantially facilitated direct sequencing and subcloning of PCR products. Two T-cassettes with a protruding T at their 3' ends were obtained by annealing adaptor oligomers to XbaI- or XhoI-digested pBluescript. A DNA fragment amplified by the first PCR was ligated separately to one of the two T-cassettes. The second PCR was performed using a primer complementary to one of the two T-cassettes and one of the two primers used for the first PCR to amplify the ligation product in low abundance. The resultant two DNA fragments had the T3 and T7 promoter, and an annealing site for the universal M13 forward and reverse primer, respectively. These DNA fragments were applicable to direct sequencing. For the purpose of subcloning, the third PCR was carried out with two primers each complementary to each of two T-cassettes and two PCR-amplified DNAs by the second PCR, as templates. The PCR product of the third PCR had multiple sites of the pBluescript polycloning site at both ends, facilitating its subcloning into pBluescript.

Streptococcus sobrinus antigens that react to salivary antibodies induced by tonsillar application of formalin-killed S. sobrinus in rabbits.

We previously found that tonsillar application of antigen induces a strong antibody response to Streptococcus sobrinus in saliva and blood plasma. Rabbits immunized against S. sobrinus by tonsillar application were highly resistant to experimental dental caries triggered by oral inoculation of living S. sobrinus organisms with sucrose. In the present study, we examined the reaction of S. sobrinus antigens to the antibodies induced by the tonsillar application of S. sobrinus AHT-k in rabbits and compared them to those antibodies induced by intramuscular injection. In an enzyme-linked immunosorbent assay using ultrasonic fragments from mutans group streptococci, the saliva and blood plasma selectively reacted to S. sobrinus AHT-k (serotype g) and serologically related streptococci (serotypes a, d, and h) in the sixth week after tonsillar application, whereas the blood plasma in the sixth week after intramuscular injection reacted to the unrelated streptococci (serotypes b, c, e, and f) in addition to the aforementioned streptococci. The antibody reactivity induced after tonsillar application was not lost after treatment of the antigen with heat or proteinase digestion, whereas these treatments resulted in a 70% decrease of the antibody reactivity induced by intramuscular injection. The inhibition by haptenic sugars and the decrease in immunoreactivity by heat treatment and proteinase digestion suggested that 80% of the antibodies induced by tonsillar application reacted to saccharides. These saccharide antigens appeared to be involved in a specific reaction with S. sobrinus-specific streptococci and a selective reaction with serologically related streptococci. These antigens are probably involved in anticaries reactions in experimental dental caries.

Tonsillar application of killed Streptococcus mutans induces specific antibodies in rabbit saliva and blood plasma without inducing a cross-reacting antibody to human cardiac muscle.

When Streptococcus mutans cells are injected into the skeletal muscle of rabbits, an antibody against human cardiac muscle, as well as an anti-S. mutans antibody, is induced in blood plasma. Our previous study showed that when sheep erythrocytes are applied to palatine tonsils, an antibody against the applied cells is induced both in blood plasma and saliva. This antibody has no activity against cardiac muscle. It is not clear, however, if S. mutans application to the tonsils evokes an antibody response against cardiac muscle. In this study, we immunized rabbits against S. mutans or Streptococcus sobrinus by tonsillar application or by intramuscular injection every 3 days for 6 weeks. Tonsillar applications of formalin-killed cells of S. mutans induced saliva immunoglobulin A (IgA) and blood plasma IgG to the applied cells. In contrast, intramuscular injection of such cells induced only blood plasma IgG. When the route of immunization was intramuscular injection, antibodies in blood plasma cross-reacted with cardiac muscle. By enzyme-immunohistochemistry and Ouchterlony immunodiffusion tests, no cross-reaction to cardiac muscle was observed with the antibody in saliva or in blood plasma after the tonsillar applications. Western blotting of the S. mutans antigen showed that blood plasma from rabbits injected with S. mutans reacted with antigens of 46, 52, 62, and 85 kDa, while that from rabbits subjected to tonsillar application of S. mutans did not react with these bands. Similar results were obtained for S. sobrinus applications. Thus, tonsillar applications of mutants group streptococci induce antibodies differing in antigen specificity and do not induce any cross-reacting antibody to cardiac muscle.

Tonsillar application of formalin-killed cells of Streptococcus sobrinus reduces experimental dental caries in rabbits.

Living Streptococcus sobrinus cells were orally inoculated into nonimmune rabbits and rabbits immunized with formalin-killed cells of S. sobrinus through tonsillar application to examine the anticaries potential of this method of immunization. The living S. sobrinus cell numbers and the caries areas in the rabbits immunized by tonsillar application decreased to a level one-fifth of that in nonimmune rabbits.

Simultaneous induction of specific immunoglobulin A--producing cells in major and minor salivary glands after tonsillar application of antigen in rabbits.

The immunoglobulin A (IgA)-producing cells in the stroma of major salivary glands are induced by antigenic stimulation of the mucosal immune system. Whether such cells also are induced in minor salivary glands by this stimulation remains to be determined. After application of sheep red blood cells to the palatine tonsils every 3 days for 6 weeks, anti-sheep red blood cell IgA was detected in saliva both by agglutination tests and by enzyme-linked immunosorbent assay. Using enzyme-linked immunospot assay, an increase in the number of anti-sheep red blood cell IgA-producing cells was found in minor as well as in major salivary glands of the sixth week of application; such cells constituted 4.9% to 5.9% of the total number of IgA-producing cells in these tissues. Tonsillar application of whole cells of formalin-killed Streptococcus sobrinus induced anti-S. sobrinus IgA in saliva. The number of anti-S. sobrinus IgA-producing cells in the above glands simultaneously increased over 6 weeks, and reached 5.2-5.6% of the total number of IgA-producing cells.

Evaluation of interleukin 1 as a mucosal adjuvant in immunization with Streptococcus sobrinus cells by tonsillar application in rabbits.

To evaluate interleukin 1 (IL-1) as a mucosal adjuvant in the induction of salivary antibodies to Streptococcus sobrinus, S. sobrinus together with IL-1 was applied through the palatine tonsils of rabbits. IL-1 caused approximately 50 and 100% increases in the antibodies reacting against S. sobrinus fragments in the saliva and blood plasma, respectively, compared to the antibodies in those same fluids after tonsillar applications of S. sobrinus alone. In the case of the addition of IL-1, the antibodies reacting to the protein antigens of S. sobrinus increased in each fluid, without affecting the antibodies reacting to saccharide antigens. Delayed-type hypersensitivity to S. sobrinus, characterized by ear swelling and by an increase in IFN-gamma mRNA in RT-PCR analysis, was found to be induced only in rabbits immunized with IL-1. S. sobrinus protein antigens caused ear swelling as intense as that caused by S. sobrinus fragments. Thus, IL-1 induced an antibody response and cell-mediated immunity mainly reacting to protein antigens of S. sobrinus.

Sheep red blood cell instillation at palatine tonsil effectively induces specific IgA class antibody in saliva in rabbits.

We have shown that the palatine tonsil effectively incorporates exogenous foreign substances instilled at its surface. It is not clear whether antigen-specific IgA can be induced by the instillation. Sheep red blood cells (SRBC) were instilled at the palatine tonsil every three days as the antigen, and the agglutination titer of specific IgA in saliva was examined. Nasal or intragastric administration, which have been shown to induce specific antibody in saliva, were done as control experiments. Anti-SRBC antibody in saliva from the tonsillar instillation group was detected in the second week, and the agglutination titer reached a maximum in the 6th week after instillation. The maximum titers in the tonsillar instillation group and nasal administration group were 16 (P < 0.01, n = 7) and 4 times (P < 0.01, n = 7) higher, respectively, than that in the intragastric administration group. In the tonsillar instillation group, the number of specific antibody- producing cells per 10(5) lymphocytes was the highest in the parotid glands compared with the lymphoid tissues such as the retropharyngeal lymph nodes, nasal mucosa, mesenteric lymph nodes, Peyer's patches, cervical lymph nodes, palatine tonsil and spleen. In the nasal administration group, the number of lymphocytes was the highest in the nasal mucosa. The results indicate that tonsillar instillation was more effective than nasal administration in inducing specific iIgA in saliva.

Increase in the potential of osteoblasts to support bone resorption by osteoclasts in vitro in response to roughness of bone surface.

The development of the potential of osteoblasts to support bone resorption by osteoclasts in response to roughness on bone slices was examined in the co-incubation cell system of immature osteoclasts and osteoblastic cells. The immature osteoclasts, which need alkaline phosphatase (ALP)-positive osteoblastic cells for bone resorption, were generated in mouse spleen cultures with 1, 25-dihydroxyvitamin D(3) and prostaglandin E(2). ALP-negative osteoblastic cells from mouse calvaria were incubated on rough surfaced bone slices for 3 days. The number of ALP-positive cells increased greatly on the rough surface, but little on the smooth surface. When immature osteoclasts were added and incubated for 1 more day, the resorption pit number and the total pit areas on the smooth surface were not much different from those before incubation but were approximately four times higher on the rough surface.

Disaggregated osteoclasts increase in resorption activity in response to roughness of bone surface.

The roughness of the bone matrix surface affects osteoblastic differentiation. However, the effect of the roughness of the matrix surface on osteoclastic bone resorption remains to be studied. We examined the latter effect using disaggregated osteoclasts from neonatal rats. The resorption pit number and the total pit area on the rough surface were not different from those on smooth surfaces after 1 day, but they were 2 or more times higher after 3 days. The number of osteoclasts was not different on bone slices with either smooth or rough surfaces at 3 days. The alkaline phosphatase (ALP)-positive osteoblasts were relatively rare in both types of slices at first, then the number and the diameter of the enzyme-positive cells and the clusters preferentially increased on the rough bone slices. When hydroxyurea was added to the culture in order to suppress the proliferation and the subsequent differentiation of osteoblastic cells on rough surfaces, the increase in resorption on the rough surfaces was effaced; however, this agent had little affect on resorption of the smooth surfaces. The addition of ALP-positive cells to disaggregated osteoclasts increased bone resorption on the smooth surface. The results suggest that osteoblast development and subsequently bone resorption by osteoclasts is enhanced by the roughness of matrix surfaces.

Preferential degradation of osteoclasts by titanium tetrachloride.

Although titanium alloys are known to be biocompatible with bone tissue after implantation in human beings, the effect of titanium on osteoclasts remains to be studied. We examined the effect of titanium salt on the formation and survival of osteoclasts in cell culture. The addition of 10 microM titanium tetrachloride caused a decrease in the cell number of osteoclast-like cells induced in bone marrow cell cultures taken from mice. The addition of 10 microM titanium tetrachloride caused degradation of the disaggregated osteoclasts taken from neonatal rats and a decrease in bone resorption. Along with the increase in the degradation of osteoclasts, the number of apoptotic cells increased. Titanium tetrachloride dose-dependently decreased the cell number and alkaline phosphatase activity of osteoblastic cell cultures taken from rat calvaria. However, these concentrations were 30-40 times higher than those in the case of osteoclast-like cell formation. These results showed that titanium ions caused a preferential degradation of osteoclasts rather than osteoblasts, most likely by apoptosis.

Detection and rapid increase of salivary antibodies to Staphylococcus lentus, an indigenous bacterium in rabbit saliva, through a single tonsillar application of bacterial cells.

In rabbits, Staphylococcus lentus is one of the major bacteria in saliva and a minor bacteria in jejunum fluids and nasal wash. The presence and induction of naturally occurring antibodies reacting to rabbit indigenous bacteria were studied. In non-immune rabbits, the proportion of anti-S. lentus IgA antibodies to total IgA in the saliva was several times higher than that in the intestinal fluids and the nasal wash. The salivary antibodies were found to have increased 1 week after a single tonsillar application of isolated S. lentus cells but not after a single nasal application or a single intragastric instillation. In addition, the anti-S. lentus antibodies in the saliva highly increased with weekly tonsillar applications of isolated S. lentus but increased only one-fifth with weekly nasal applications of the same cells. These results strongly suggest that the palatine tonsils, which we believe had already been sensitized by S. lentus in the physiological condition, induced naturally occurring antibodies in rabbit saliva.

PCR with end trimming and cassette ligation: a rapid method to clone exon-intron boundaries and a 5'-upstream sequence of genomic DNA based on a cDNA sequence.

We described a method for PCR amplification of unknown flanking genomic DNA fragments. This method is a combination of PCR with "end-trimming method" and "cassettes and cassette-primers method". In this method, genomic DNA was digested with three different groups of restriction enzymes. DNA in group 1 was digested with BamHI, BglII, FbaI, or MboI. DNA in group 2 was digested with BlnI, NheI, SpeI, or XbaI. DNA in group 3 was digested with SalI or XhoI. Digested DNA in each group was end-trimmed with Klenow fragment of DNA polymerase I in the presence of only one dNTP; dGTP, dCTP, and dTTP for group 1, 2, and 3, respectively. The synthesized cassettes, C1, C2, and C3, had 5'protruding sequences of 5'-ATC-3',5'-TAG-3', and 5'-CGA-3', respectively. Each compatible cassette was ligated to the end-trimmed DNAs in group 1-3, respectively. Nested PCR was then performed using an end-trimmed and cassette-ligated DNA as a template. Primers annealing to known sequences and cassettes were used for the nested PCR. The amplified DNA fragments were electrophoresed on a polyacrylamide gel and purified. The sequences of the DNA fragments were determined after cloning into pBluescript.