Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.     

Ventrolateral medullary neurons projecting to the medial preoptic/anterior hypothalamic area through the medial forebrain bundle: an electrophysiological study in the rat

Summary A total of 152 ventrolateral medullary neurons was antidromically stimulated from both the medial preoptic/anterior hypothalamic area (MPOAH) and the medial forebrain bundle (MFB) in urethane anesthetized rats. These neurons were located primarily dorsal to the lateral reticular nucleus and could be readily classified in at least two groups, type I and type II cells on the basis of electrophysiological properties. The action potentials of type I cells had a shorter duration, and their conduction velocities ranged from 0.45 to 3.1 m/s. By contrast, type II cells, most predominantly observed, were characterized by a longer duration and an unusual shape of their action potential, and the antidromic propagation into the somatodendritic complex was often blocked. The conduction velocity (mean = 0.21 m/s) and absolute refractory period (mean = 2.63 ms) of type II cells are consistent with them having fine non-myelinated axons. Injection of 6-hydroxydopamine (6-OHDA), but not 5,7-dihydroxytryptamine, directly into the MFB blocked antidromic responses of 57% of type II cells tested. The residual type II cells whose antidromic responses were not affected by 6-OHDA were located significantly rostral to the 6-OHDA sensitive cells. Neither antidromic response of type I cells tested, on the other hand, was affected by 6-OHDA. The majority of type I cells were dramatically activated by noxious pinches of the tail, whereas the noxious stimuli produced no detectable change in the firing of type II cells. These data demonstrate that ventrolateral medullary neurons projecting to the MPOAH through the MFB are comprised of at least three distinct populations: 6-OHDA resistant fast conducting cells with somatic afferents, 6-OHDA sensitive and resistant slow conducting cells.     

Development of early apopotosis and changes in lymphocyte subsets in lymphoid organs of mice orally inoculated with nivalenol

Development of early apoptosis and changes in lymphocyte subsets were examined in lymphoid organs of female BALB/c mice after oral administration of 15 mg/kg b.w. of nivalenol (NIV), the major type B trichothecene mycotoxin, by FACS analysis. Judging from the results of viable cell count and apoptotic cell index, NIV attacked Peyer's patches first and thymus most severely. In thymus, selective damage in CD4(+)CD8(+) cells was observed at 12 and 24 h after inoculation (HAI), following the peak of apoptosis at 9 HAI. CD4(+) cells were clearly suppressed at 3 HAI in Peyer's patches, at and after 9 HAI in mesenteric lymph nodes, and 3 to 12 HAI in spleen, respectively. CD8(+) cells were also suppressed at 24 HAI in mesenteric lymph nodes and at 12 HAI in spleen, respectively. As to changes in B cell subsets, IgG(+) cells significantly decreased from 3 to 12 HAI and all B cell subsets at 24 HAI in mesenteric lymph nodes. In spleen, IgM(+) cells were suppressed at 9 HAI. On the other hand, in Peyer's patches, following clear decrease in the numbers of pan-T and pan-B cells and viable cells at 3 HAI, all B cell subsets, especially IgA(+) cells, showed a significant increase in their numbers at 9 HAI, and the numbers of IgA(+) and IgM(+) cells remained higher values than controls thereafter. Taken together, in the course of recovery from NIV-induced prominent damage in Peyer's patches at 3 HAI, interaction of NIV with Peyer's patches might result in in vivo stimulation of interleukin production at this site and result in increased proliferation and differentiation of IgA-secreting B cells at and after 9 HAI.     

Immunohistochemical characteristics of duodenal adenomas in familial adenomatous polyposis with special reference to cell kinetics

The duodenum is the second most frequent site of cancer in patients with familial adenomatous polyposis (FAP). The main objective of this study was to evaluate the cell kinetics in duodenal and ampullary adenomas in FAP. The endoscopic and biopsy findings of duodenal adenomas in 22 FAP subjects and 18 non-FAP subjects were compared. Adenomas in FAP included 15 ampullary adenomas and 17 nonampullary adenomas. The cell kinetics was evaluated by immunohistochemistry for Ki-67, p53, bcl-2, and cyclooxygenase 2 (COX2), and the apoptotic index (AI) as determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method. Any correlations between the indices for cell kinetics and the endoscopic findings were identified. All 50 adenomas were histologically verified to be tubular adenoma with low-grade dysplasia. Neither the expression of Ki-67, p53, bcl-2, and COX2 nor the AI differed substantially between FAP and non-FAP subjects. In patients with FAP, duodenal adenoma tended to have a higher Ki-67-labeling index than the ampullary adenoma (54.3 +/- 11.3 versus 46.8 +/- 12.7; .05 < P < .1). In addition, the Ki-67-labeling index in endoscopically normal or slightly enlarged ampullary adenoma was significantly higher than that in markedly enlarged ampullary adenoma (51.8 +/- 11.4 versus 39.4 +/- 11.3; P < .05). Duodenal adenoma in FAP subjects was not found to have a higher proliferative activity or a smaller degree of apoptosis compared with those in non-FAP subjects. The smaller proliferative activity in larger ampullary adenoma may thus be related to the static nature of ampullary adenoma in FAP.     

Variant lines of mouse kidney cells transformed by an SV40tsA mutant with growth properties of wild-type transformed cells at nonpermissive temperature.

MKSA207 cells, a BALB/c mouse kidney line transformed by a tsA mutant of SV40, are temperature-dependent for the expression of the 'standard transformed phenotype'. At the permissive temperature (33.5 degrees C), the mKSA207 cells resembled wild-type (wt) SV40 transformants; they contained the intranuclear SV40 T antigen, grew to high saturation density in monolayer culture in either 10% or 0.5% serum, and also in methylcellulose suspension culture and became multinucleate in cytochalasin B. At the nonpermissive temperature (39.8 degrees C), the mKSA207 cells lost some of their transformed properties; they grew only to low density in 10% serum, hardly grew at all in 0.5% serum or in methylcellulose suspension culture, and remained mono- or binucleate in cytochalasin B. At 40 degrees C in low serum, mKSA207 cells lost the intranuclear T antigen and when fed 10% serum at 39.8 degrees C, accumulated large amounts of T antigen in the cytoplasm. Derivatives of mKSA207 have been selected at 39.8 degrees C in liquid medium and methylcellulose suspension culture. The heat adapted lines, like wt SV40 transformants, exhibited the standard transformed phenotype at both 33.5 and 39.8 degrees C. It is unlikely that acquisition of temperature-independence for the transformed phenotype was due to reversion of the tsA gene to wild-type because the heat-adapted cell lines displayed the cytoplasmic T antigen at 39.8 degrees C, characteristic of the parental mKSA207 cells and SV40 rescued from one of the heat-adapted lines was temperature sensitive for growth. The T antigen levels (complement fixation units per 10(6) cells) of heat-adapted lines grown at 39.8 degrees C were comparable to those of mKSA207 cells grown at 33.5 or 39.8 degrees C.     

Mapping thymidine kinase-deficient mutants of vaccinia virus by marker rescue with hybrid plasmid DNAs containing portions of the HindIII-J fragment of virus DNA

Five hybrid plasmids were constructed, each containing a portion of the vaccinia virus DNA HindIII-J fragment. These plasmid DNAs were used in marker rescue experiments to map the mutations in the thymidine kinase (TK) gene of three TK- vaccinia virus mutants. The TK gene of each of the three mutants was rescued by DNA from plasmid pPJ701, which contained about one-half of the HindIII-J fragment. Two mutants, 1004B and 1017-1, but not the third, 1016-1, were rescued by DNA of two plasmids, pPJ702 and pPJ703, which contained 16 and 18%, respectively, of one end of the J fragment. Mutant 1016-1 could be rescued by plasmid pPJ705 containing a 1.69-kb fragment of the HindIII-J fragment. The J fragment DNA in plasmid pPJ705 is located adjacent to that and separated by an EcoRI site from pPJ703 in the vaccinia virus genome. These results indicate that the mutation site in the TK gene of 1016-1 differs from that in 1004B or 1017-1 and suggests that the structural gene for the vaccinia virus TK lies near one end of the HindIII-J fragment and spans the EcoRI site.     

Astroglial responses against Abeta initially occur in cerebral primary cortical cultures: species differences between rat and cynomolgus monkey.

In the present study, we investigated how amyloid beta (Abeta) peptides initially affect neuronal cells in primary cerebral cortical cultures from rat and cynomolgus monkey. In these cultures, complicated interactions between glial and neuronal cells occur; moreover, synaptic interactions similar to those observed in vivo also occur between neuronal cells in these cultures. In this study, we applied low concentrations of Abeta to these well-characterized primary cultures to investigate how Abeta initially affects neurons or astroglial cells. In both rat and monkey cortical cultures, treatment with low concentrations of Abeta failed to drastically change or damage of neurons. Abeta treatment, however, significantly activated astrocytes, resulting in increased apolipoprotein E (ApoE) production. Rat astrocytes were more sensitive to Abeta than monkey astrocytes, and responded to Abeta via a different mechanism. In monkey astrocyte cultures, only direct treatment with Abeta increased ApoE production. In rat astrocyte cultures, however, treatment with conditioned media from cortical cultures grown with Abeta increased ApoE production, indicating that some sort of neuron-derived soluble factor(s) was also involved in activating rat astrocytes. These species differences suggest that monkey cortical cultures would be more useful as an in vitro model system to understand the details of how Abeta accumulates in the human brain, since monkeys are phylogenetically more similar to humans.     

A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides

A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [³H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Galβ1,3[3OMeGalβ1,4GlcNAcβ1,6]αBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.