Cultured cell-derived decellularized matrices: a review towards the next decade.

In the past decade, many cell-derived decellularized matrices have been prepared for various purposes. Cell-derived decellularized matrices have many advantages for inducing specific cell functions although there are still many problems that need to be solved. This review summarizes the points that need to be considered for the preparation of cell-derived decellularized matrices and provides examples of these matrices inducing cellular functions. Moreover, the problems that need to be solved in the next decade for the further application of cell-derived decellularized matrix technology have been summarized.

Effective delivery with enhanced translational activity synergistically accelerates mRNA-based transfection.

mRNA instead of DNA provides a new and attractive approach for gene therapy and genetic vaccination. Current technologies for mRNA delivery are based on cationic lipids with DOTAP being the most efficient one. We previously reported on the synthesis of an inorganic-organic hybrid carrier by embedding inorganic nano-particles of carbonate apatite onto liposomal carrier DOTAP and demonstrated its high transfection potency of luciferase mRNA both in mitotic and non-mitotic cells. Here we show that in addition to the carrier design for effective endocytosis and release of mRNA to the cytoplasm, enhancement of mRNA translation efficiency is a prerequisite for maximum protein expression. We used the modified cap analog (ARCA) during in vitro transcription of luciferase DNA for proper cap orientation and demonstrated that transfection with ARCA-mRNA resulted in higher protein expression than the mRNA with usual cap structure for both DOTAP and DOTAP-apatite complex. Secondly, exogenous poly(A) was co-delivered with mRNA by the DOTAP-apatite, resulting in very significant expression compared to mRNA delivery only. Finally, when combined both of the effects of smart carrier and the modifications at mRNA translational level, a notable enhancement (100 times) was achieved as compared to the existing DOTAP-based liposome technology. Our findings, therefore, unveiled a novel approach that an effective delivery system can be developed by the improvement of the gene expression level in combination with the enhancement of the carrier potency.

Galactose-carrying polymers as extracellular matrices for liver tissue engineering.

Extracellular matrix (ECM) plays important roles in tissue engineering because cellular growth and differentiation, in the two-dimensional cell culture as well as in the three-dimensional space of the developing organism, require ECM with which the cells can interact. Especially, the bioartificial liver-assist device or regeneration of the liver-tissue substitutes for liver tissue engineering requires a suitable ECM for hepatocyte culture because hepatocytes are anchorage-dependent cells and are highly sensitive to the ECM milieu for the maintenance of their viability and differentiated functions. Galactose-carrying synthetic ECMs derived from synthetic polymers and natural polymers bind hepatocytes through a receptor-mediated mechanism, resulting in enhanced hepatocyte functions. Attachment and functions of hepatocytes were affected by physico-chemical properties including ECM geometry as well as the type, density and orientation of galactose. Also, cellular environment, medium composition and dynamic culture system influenced liver-specific functions of hepatocytes beside ECM.

Role of E-cadherin molecules in spheroid formation of hepatocytes adhered on galactose-carrying polymer as an artificial asialoglycoprotein model.

The role of E-cadherin in the spheroid formation of hepatocytes adhered on the poly(N-p-vinylbenzyl-D-lactonamide) (PVLA) as a model ligand for asialoglycoprotein receptors (ASGP-R) of hepatocytes was studied. Expression of E-cadherin was increased in round hepatocytes adhered on a high-coating density of PVLA (100 microg/ml), and also in flat ones adhered on a low-coating density of PVLA (1 microg/ml) in the presence of epidermal growth factor (EGF). Hepatocyte spheroids formed on the high-coating density of PVLA in the presence of EGF after 48 h were inhibited by an anti-E-cadherin monoclonal antibody (ECCD-1). From immunofluorescence and confocal laser microscopy, E-cadherin was localized in the intercellular boundaries and concentrated at the inside surface of aggregated cells. As a result, E-cadherin could play an important role in hepatocyte assembly.

A new nasal acoustic reflection technique to estimate pharyngeal cross-sectional area during sleep.

The conventional acoustic reflection technique in which acoustic waves are launched through the mouth cannot be applied during sleep, nor can it be applied to the nasopharynx, which is the major site of occlusion in patients with obstructive sleep apnea syndrome. We propose a new technique of nasal acoustic reflection to measure pharyngeal cross-sectional areas including the nasopharynx. The acoustic waves are introduced simultaneously to both nostrils during spontaneous nasal breathing. A new algorithm takes into account the nasal septum with asymmetric nasal cavities on both sides and assumes prior knowledge of the cross-sectional area of the nasal cavities and the position of the nasal septum. This method was tested on an airway model with a septum and on healthy human subjects. The conventional technique gave inaccurate measurements for pharyngeal cross-sectional areas for an airway model with asymmetric branching, whereas the new technique measured them almost perfectly. The oro- and hypopharyngeal cross-sectional area measurements acquired by the new method were not different from those obtained by the conventional method in normal subjects. This new method can be used as a monitor of upper airway dimensions in nocturnal polysomnography.

Radical hysterectomy: An anatomic evaluation of parametrial dissection.

OBJECTIVES: This study was designed to demonstrate a reduction in the amount of blood loss for vesicouterine ligament dissection and to investigate the intrapelvic autonomic nerve pathway and its preservation by means of anatomic analysis. METHODS: The anchoring mechanism of the pelvic viscera to the pelvic wall was divided into a supporting system facing laterally and a suspensory system facing dorsoventrally. An operative procedure was designed in which both systems were separated and dissected independently. RESULTS: Between the two systems, an artificial space was developed, which required a new dissection method for the parametrium and revealed a new anatomic pathway for the ureter and autonomic nerve. The amount of blood loss (mean +/- SD) during dissection of the vesicouterine ligament was ultimately 260.1 +/- 114.8 ml. Postoperatively, the maximum capacity of the bladder was 393.9 +/- 40.4 ml, maximum detrusor pressure 6.3 +/- 4.1 cm H(2)O, mean compliance >10 ml/cm H(2)O, residual urine 23.8 +/- 9.4 ml, and maximum flow rate 25. +/- 8 2.2 ml/s, respectively. CONCLUSION: A new classification for the parametrium and its dissection method have been established. Development of this new operative procedure has also contributed to a decrease in blood loss and preservation of bladder function.

[Decreasing seropositivity of cytomegalovirus of pregnant women in Japan].

We examined seroprevalence of cytomegalovirus (CMV) of pregnant women for 18 years since 1980 with complement-fixing antibody (CF) and specific IgG antibody. CF seropositive rate decreased gradually from 93.2% to 66.7%. CMV-IgG Seropositive rates were 87.4% in 1985, and 75.2% in 1996 to 1997. Age and parity of pregnant women associated with the immune status; 35.6% of recent young primipara were susceptible.

A new proposal for radical hysterectomy.

OBJECTIVE: The authors revised the surgical procedure for radical hysterectomy utilizing detailed observation of the venous system, connective fascial sheets, and neural pathways within the uterine supports. STUDY DESIGN: The anterior, middle, and posterior uterine supports were reclassified into two systems, supporting or fascial and drainage or areoral. Supporting system consisted of the superficial layer of the vesicouterine ligament, fascial part of the Mackenrodt ligament, sacrouterine ligament, and rectovaginal ligament, whereas drainage system consisted of the deep layer of the vesicouterine ligament, vascular part of the Mackenrodt ligament, and the so-called mesoureter. The operative procedure was planned according to the continuity of these ligaments and executed first by excising the fascia and then dissecting the denuded areoral tissue. RESULTS: Among the 15 patients who underwent surgery for uterine cancer during a 2-year period the mean (SD) time required for the operation was 305.5 +/- 30.5 min and the mean (SD) total volume of blood loss 592.0 +/- 238.2 ml. A mean (SD) period of 14.3 +/- 3.8 postoperative days was required until the volume of the residual urine decreased to less than 50 mL. CONCLUSION: The present operation has been structured more three-dimensionally and systematically than before. Further, safety of the operation was significantly improved including prevention of hemorrhage and preservation of bladder function.

[Simplified avidity assay of rubella IgG antibody in rubella virus infection].

Simplified avidity assay of rubella IgG antibody with urea was evaluated to distinguish primary rubella from reinfection. In this method urea washing was done once for 10 minutes. The avidity index (AI) was calculated as the optical density percentage for the urea-washed well when compared to that of the non-treated well. We examined 292 sera from 50 patients with primary infection collected 6 to 2,259 days after the rash appeared, 29 sera from 11 patients with rubella reinfection and 69 sera from 68 pregnant women without fetal infection and having a high hemagglutination inhibition (HI) antibody. In primary infection AI increased gradually from 0%, and reached a plateau of about 60% four months after the rash appeared, whereas the mean AIs of patients after reinfection and with high HI antibody were as high as 87.1% and 89.9%, respectively. These results indicate that the simplified avidity assay in rubella IgG antibody is also valuable in diagnosing recent primary rubella in pregnant women with a high HI antibody.

[Avidity of rubella IgG antibody in rubella virus infection].

The avidity assay of rubella IgG antibody with urea was evaluated to distinguish the early convalescent phase of primary rubella from the late one and reinfection. We examined 116 sera from 23 patients with primary infection collected 7 to 1,477 days after the rash appeared and 25 sera from 8 patients with rubella reinfection and 16 sera from 15 pregnant women without fetal infection having high hemagglutination inhibition (HI) antibody or weak positive rubella IgM antibody. The avidity index (AI) was calculated as the ratio of the optical density from the urea-washed well to that of the non-treated well, expressed as a percentage. In primary infection AI were lower than 30% within 30 days after the rash appeared, then gradually increased and reached a plateau of 60 to 80% three months later, whereas the AIs of patients after reinfection, with high HI antibody and weak positive IgM antibody were as high as 79.2 +/- 7.5%, 93.5 +/- 4.1% and 90.6 +/- 5.0%, respectively. These results indicate that the measurement of avidity in rubella IgG antibody is valuable in diagnosing recent primary rubella in pregnant women having a high HI antibody or IgM antibody.

Dissection of the cardinal ligament in radical hysterectomy for cervical cancer with emphasis on the lateral ligament.

Surgical experience with carcinomas of the uterus and rectum has provided new insights into the surgical anatomy of a lamina, which separates the paravesical space from the pararectal space. It has been proved that each of the lamina consists of the cardinal and lateral ligaments and pelvic splanchnic nerves, descending in the following order. The cardinal and lateral ligaments, as a connective stalk, insert into the lateral walls of the uterus and rectum extending from the inner aspect of the pelvic wall. Clarification of this structural relationship led to the development of a new procedure for the dissection of the cardinal ligament in radical hysterectomy, while still preserving the lateral ligament. This facilitated systematic dissection of the cardinal and uterosacral ligaments with posterior manipulation, leading to a reduction in blood loss and to prevention of brisk bleeding from the venous plexuses.

[Studies on the management of pregnant women having high hemagglutination inhibition (HI) antibody titer of rubella virus in the first trimester examination of rubella virus antibody in the pregnant women having high HI titer by different methods and prognosis of their infants after birth].

Several serological studies of rubella virus (RV) infection were carried out on sera of 45 pregnant women having RV hemagglutination inhibition (HI) antibody titer more than 1:512 in the first trimester, including a follow-up study of these women's pregnancies. In order to detect RV-specific IgM antibody, various procedures were employed as follows: Complement fixation test (CF), HI test after treatment of the sera with Protein-A (ProA-HI), enzyme linked immunosorbent assay (ELISA) by indirect or sandwich procedures and several tests using fractionated sera by sucrose density gradient ultracentrifugation (SDG). Positive ratios found with the CF test and ProA-HI were 26.7% and 20.0%, respectively. In indirect ELISA, 4 cases were positive for RV-specific IgM, but all sera were negative in sandwich ELISA and SDG tests. Both RV isolation from urine of some infants and detection of high RV-specific IgM antibody in cord sera failed to be positive. Of 44 pregnancies, 4 cases resulted in spontaneous abortion and all others in normal delivery. These forty normal infants had no sign of congenital rubella syndrome, but one case among them showed polysyndactyly . The results obtained here seemed to indicate that sandwich ELISA is the most valuable and convenient method among the tested procedures for clinically determined diagnosis of recent RV infection in pregnant women having a high HI antibody titer.

[Antibody response to live rubella vaccination and its clinical meanings].

Serological antibody response for over a year to rubella live virus vaccination was examined by hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) of 22 seronegative and 25 seropositive sera of young women. In the seronegative group, some HI antibody response appeared first 2 weeks after the vaccination. A positive response of ELISA-IgM was first observed at 3 weeks, showing slowly declining values within the following year. Similarly, ELISA-IgG response occurred first and was positive 3 weeks later with gradual increases in both ELISA-values and positive ratios for 5 months. In the seropositive group, responses of both ELISA-IgM and -IgG were found within a few levels of ELISA-values from 2 weeks after the vaccination. Higher ELISA-IgM values observed after the natural infections than those following in the vaccination should be cited. These possibly show different amounts of viral antigens introduced in vivo. Reinfection of high HI antibody cases may be distinguished by small changes in the ELISA-value's pattern. When the above data are taken together, single measurement of ELISA-IgM does not seem to always be definitive and characteristic changes in positive patterns may be much more important in clinically diagnosing recent rubella virus infection.

[Clinical evaluation of sulbactam/cefoperazone in the field of obstetrics and gynecology].

One or 2 grams of sulbactam/cefoperazone (SBT/CPZ) in 250 ml of 5% glucose solution was administered twice daily by drip infusion to 10 patients with female genital organ infections. The clinical effectiveness was seen in 8 patients but not in 2 patients associated with emergence of replaced organism or non-eradicated causative organism. Antibacterial activity of SBT/CPZ vs. CPZ in the 20 clinical isolates revealed that SBT/CPZ was 1 or 3 tubes superior to CPZ alone in the beta-lactamase producing organisms, but, 1 or 2 tubes inferior to CPZ in the beta-lactamase non-producing organisms. In 1 case increases in GOT and GPT, and in another increases in GOT and GPT, and a decrease in platelets were observed even though their degrees were mild.