A new schematic for poly(3-alkylthiophene) in an amorphous film studied using a novel structural index in infrared spectroscopy.

The molecular structure of poly(3-alkylthiophene-2,5-diyl) in an amorphous film reveals that the short axis of the thiophene ring is kept highly oriented parallel to the substrate, whereas the long axis along the polymer chain is largely disordered. This is unveiled by infrared p-polarized multiple-angle incidence resolution spectroscopy (pMAIRS), achieved by analyzing the orientation angles of three mutually orthogonal vibrational modes localized on the thiophene ring with the aid of a newly developed structural index. This new analytical technique is useful irrespective of the crystallinity of the thin film. As a result, the intrinsic chemical parameters controlling the molecular orientation are understood in a unified manner, and the reason that the hexyl group gives the best results for a photovoltaic cell is also revealed.

Characterization of new monoclonal antibodies against porcine lymphocytes: molecular characterization of clone 7G3, an antibody reactive with the constant region of the T-cell receptor delta-chains.

A battery of mouse monoclonal antibodies (mAbs) reactive with porcine peripheral blood (PB) leukocytes was generated. Among the mAbs, 6F10 was found to react probably with cluster of differentiation (CD)8 alpha-chain, while 7G3 and 3E12 were found to recognize gammadelta T-cells, as revealed by two-color flow cytometric and immunoprecipitation studies. 7G3 was shown to react with the constant (C) region of the T-cell receptor (TCR) delta-chain by the following facts: (1) 7G3 immunoprecipitated full-length TCR delta-chain protein fused with glutathione S-transferase (GST) produced by Esherichia coli and (2) 7G3 reacted with TCR delta-chain expressing Cos-7 cells transfected with either full-length or N-terminal deleted mutant cDNA, but did not react with Cos-7 cells transfected with C-terminal deleted mutant TCR delta-chain cDNA. All three mAbs produced high-quality immunostaining results on frozen sections, revealing a distinct distribution of gammadelta T-cells and CD8(+) cells. This report precisely characterizes mAbs against porcine TCR for the first time, facilitating molecular biological investigations of the porcine immune system.

Influence of castration on development of the thymus in neonatal male rats.

The influence of castration on the development of the thymus in neonatal rats was studied to elucidate when after birth the thymus comes under inhibitory regulation by the testis in rats. The relative and absolute weights of the thymus were measured five days after castration these cases. No excessive changes in the weights of the thymus with castration were observed by 31 days after birth. Significant changes in the thymus appeared in the relative weight at 36-day-castration. The absolute weight of the thymus was also significantly increased after 41-day-castration. These findings suggest that in rats the inhibitory regulation of the thymus by testis development does not appear before at least 31 days of age.

Pretreatment of human keratinocyte sheets with laminin 5 improves their grafting efficiency.

Laminin 5 is essential in epithelial attachment to stromal tissues, suggesting that it might improve keratinocyte attachment in a variety of clinical situations. In this study, we examined the effect of exogenous laminin 5 upon the efficiency of transplantation of keratinocyte sheets in animal models. Keratinocyte sheets were prepared according to the method of Rheinwald & Green (1975). Purified laminin 5 was added to the sheets of group 1 (1.0 microg per cm2), Dulbecco's modified Eagle's medium alone was added to group 2. The sheets were grafted to the panniculus carnosus of nude mice (BALB/C nu/nu) (n = 12) and nude rats (Fisher 344) (n = 15). The take rate was assessed by measurement of the area of surviving epithelium at 7 d postgrafting. Laminin 5 bound the keratinocyte sheets of group 1. At 7 d postgrafting, the area of epithelialization of group 1 was significantly larger than that of group 2. Immunohistochemistry staining showed that collagen IV, laminin 5, and collagen VII stained more strongly at the dermal-epidermal junction in group 1 than in group 2. Integrin chains alpha6 and beta4 were similar in both groups. Electron microscopy at day 3 after grafting, showed the lamina densa of group 1 to be more continuous than in group 2. Pretreatment of cultured human keratinocyte sheets with laminin 5 improved the extent of epithelial coverage and increased the rate of neobasement membrane formation. The results suggest that laminin 5 promotes epithelial attachment by increasing the rate of basement membrane assembly.

Histodifferentiation of hair follicles in grafting of cell aggregates obtained by rotation culture of embryonic rat skin.

We have previously reported reconstruction of hair follicles from a single cell suspension of rat fetal upper lip by a two-step culture method consisting of rotation and flotation cultures. Rotation sorted out the cells and flotation facilitated histodifferentiation. In the present study, we added grafting procedures to the previous method to see whether cell aggregates obtained this way were graftable, and whether the grafting promoted histodifferentiation. The aggregates before and after flotation were grafted, and differentiation of hair follicles comparable to those in vivo was confirmed 10 days after grafting. There was no difference in the degree of differentiation between the two kinds of grafts. The grafting procedure therefore resulted in an appreciable increase in histodifferentiation even when aggregates obtained after flotation were grafted.

In vivo bioactivities and kinetic parameters of rat luteinizing hormone components: discrepancy between in vitro and in vivo assays.

Rat pituitary LH shows an electrical charge heterogeneity owing to their carbohydrate moiety. The biological potencies of these isoelectric components increase with increasing pI when examined by in vitro testosterone production in isolated rat Leydig cells. The present study was carried out to clarify if this tendency was also true with of in vivo action and to estimate their kinetic parameters. Seven isoelectric components of LH, i.e. X (pI 7.9), A (8.4), B (8.8), C (9.1), D (9.3), E (9.6) and F (9.8), were administered to cannulated adult male rats. Blood samples were serially collected over a 5 h period from the conscious animal, and rat LH and testosterone were assayed by RIA. The doses of a single component showed a linear relationship with the areas under the curve (AUC) of the blood LH. The testosterone AUC also correlated well with LH AUC. It was found that the components with lower pIs had longer half-lives and larger LH AUC, and smaller total body clearance rates with highly significant correlation coefficients. The in vivo bioactivities of the components expressed as testosterone AUC increased with decreasing pIs, indicating that the in vivo bioactivity was much influenced by the total body clearance rate. The intrinsic activities of the components were calculated as ratios of testosterone AUC to LH AUC where the effect of the clearance rate (or biological half-life) was eliminated. The intrinsic activities of components were not significantly different among the components except component X which showed a smaller activity. These data indicated that, with the exception of component X, an LH component with a lower pI has a longer biological half-life, resulting in a higher in vivo potency, quite contrary to the tendency of in vitro potency.

Effect of a collagen matrix containing epidermal growth factor on wound contraction.

Excessive wound contraction is known to lead to pathological wound contracture. Using a rabbit model, we applied a bovine type I collagen matrix sponge as a dermal substitute and human epidermal growth factor to full-thickness excisional wounds. Wound contraction was assessed 14 and 28 days after wounding. It was found that both collagen matrix and epidermal growth factor significantly inhibited wound contraction (p < 0.001) in all wounds treated with collagen matrix alone or treated with 0.1 and 1 microg of epidermal growth factor 28 days after wounding. Interestingly, the combination of collagen matrix with epidermal growth factor strongly inhibited wound contraction over matrix alone (p < 0.01 on day 28). Histological analyses showed a regular horizontal arrangement of collagen fibers in the dermis under wounds treated with these substances but not under untreated wounds. Furthermore, using a fibroblast-populated collagen gel, the direct inhibitory effect of epidermal growth factor on gel contraction by fibroblasts was also observed. Collagen gels without stimulation contracted to 29.5 +/- 0. 6% of their original size, as determined 6 days after culturing. At 3 days or more, epidermal growth factor inhibited collagen gel contraction by fibroblasts (after 6 days: 34.2 +/- 1.8%, p > 0.05; 36.5 +/- 2.8%, p < 0.05; and 39.8 +/- 2.1%, p < 0.001 at 1, 10, and 100 ng/ml of epidermal growth factor, respectively). In conclusion, collagen matrix and epidermal growth factor, particularly in combination, may be useful in the prevention of wound contracture.

Eyelid reconstruction using a vascularized composite flap.

For reconstruction of full-thickness eyelid defects, it is optimal to use the eyelid. As a new surgical technique, we reconstructed these defects using a composite flap with vascular pedicles. A composite flap was prepared using the inferior medial palpebral artery as a nutrient vessel for reconstruction in two cases. After surgery, partial necrosis of the skin in the composite flap occurred, and ciliary loss was observed; however, the tarsoconjunctival layer could be reconstructed, and good results were obtained in terms of function and morphology.

Extended lid-splitting surgery of marginal eyelid defects.

The technique of extended lid-splitting surgery is described. This procedure was used to repair marginal defects of the lower and upper lid, and provided postoperative results that were excellent both functionally and cosmetically, principally because the technique allowed the anatomical structures to be maintained.

Unilateral multiple adenolymphomas originating in the parotid gland: a clinicopathological study.

The validity of Seifert's hypothesis about growth pattern of adenolymphomas has been evaluated in three of five patients with adenolymphomas of the parotid gland, who at the time of a parotidectomy, were found to have multiple unilateral adenolymphomas in the intraglandular lymph nodes. This evaluation, which was based on assessing the epithelial: lymphoid tissue ratio in excised adenolymphomas and the growth in tumor size, showed that there was no association between the histological changes in the epithelial:lymphoid tissue and the tumor size. Adenolymphomas seem to have no consistent growth pattern and develop independently. However, as multiple tumors were detected histopathologically in lymph nodes as small as 5 mm in diameter the importance of a detailed examination of patients with adenolymphomas is stressed, so that other tumours are not overlooked.

Unusual palpebral foreign bodies.

We encountered cases of palpebral foreign bodies, which were a bamboo chip that entered in a trauma, a gelatinoid material that formed an iatrogenic ulcer, and self-injected lipstick. Treatments and complications of these unusual palpebral foreign bodies are evaluated.

Evaluation of the median forehead flap and the nasolabial flap in nasal reconstruction.

Basal cell carcinoma, which accounts for 70%-80% of all cutaneous malignancies in the United States, has increased recently in Japan. We compared methods for reconstruction after surgery for basal cell carcinoma, which is expected to increase further in the future. Thus patients who underwent reconstruction after surgery for basal cell carcinoma of the nose using medial forehead flaps and nasolabial flaps were selected, and the effectiveness of these flaps was compared by taking the size and location of the tissue defect into consideration. As a result, possibly because of anatomical and histological differences of the face between Caucasians and Asians, better results were obtained with nasolabial flaps than with median forehead flaps.

Development of new wound dressing composed of spongy collagen sheet containing dibutyryl cyclic AMP.

Although cyclic AMP has been considered to regulate cell proliferation, the mechanism of this function is largely unknown. Recent studies suggest that cyclic AMP promotes the proliferation of skin cells in a dose-dependent manner. An ointment containing dibutyryl cyclic AMP has been used in the treatment of skin ulcers and found to be effective in promoting tissue repair. To search more efficacious wound management, the authors developed a new wound dressing composed of a spongy atelo-collagen sheet containing dibutyryl cyclic AMP. This wound dressing was evaluated in two types of animal tests. One is the application of the wound dressing to a full-thickness skin defect in order to evaluate the granulation tissue formation and the wound size reduction. The wound dressing was found to promote the granulation tissue formation and naturally reduce the wound size. The other test was the application of the wound dressing to the full-thickness skin defect, leaving behind a skin island in a central portion, in order to evaluate the epithelialization. This skin island left in a full-thickness skin defect was extremely enlarged. The enlargement of the skin island seems to be related to the epithelialization from the margin of the skin island as well as by the expansion of a skin island induced by contraction of the developed granulation tissue in the surrounding wound area. These results suggest that an atelo-collagen spongy sheet containing dibutyryl cyclic AMP is effective in promoting the granulation tissue formation and epithelialization.

Device that keeps a pierced ear hole intact while treating an infected earlobe.

The authors describe a simple, inexpensive, Teflon shaft and silicone ring device that keeps a pierced hole in an infected earlobe intact while an infection is being treated.

Cytotoxicity tests for antimicrobial agents using cultured skin substitutes fixed at interface of air and culture medium.

The present study is focused on a new cytotoxicity test using cultured dermal and epidermal sheets, which are fixed at the air and medium interface as a wound surface model. The cultured dermal sheet is composed of human fibroblasts and a collagen matrix, and the cultured epidermal sheet is composed of human keratinocytes and a collagen matrix. Each cultured sheet was fixed at the air and medium interface, over which a piece of test specimen was placed. The in vitro system created, provides a mimetic wound surface since during wound repair, fibroblasts are embedded in an extracellular matrix, while keratinocytes migrate and proliferate on provisional granulation tissue. The results thus obtained in this cytotoxicity test are useful for determining the efficacious amount of antimicrobial agent used in clinical cases.

Side-to-side earlobe variations with respect to surface area and shape: a quantitative study.

Side-to-side earlobe variations, based on the size of the earlobe surface area and a classification into earlobe types, were quantitatively analyzed in 58 female students, ranging from 20 to 22 years old. Using a new earlobe measurement technique, a closed-circuit outline of each ear was directly traced onto a clear polyethylene sheet, after which the surface area of the earlobe portion of each ear was identified, measured by a digital planimeter, and analyzed. Results revealed that side-to-side differences in the size of the earlobe surface area existed in all subjects. In contrast, based on classifying the earlobe shape into three types (i.e., tapering, square, or pendulous), differences in the mean earlobe surface area value among the three types were not significant. However, when the classification of the earlobe shape was reduced to only two types (pendulous or nonpendulous), the mean earlobe surface area value between the two types was highly significant. These results thus suggest that for a quantitative analysis of earlobe variations, classification of the earlobe shape into two types rather than into three types is the better method. This method makes it easy to distinguish earlobe differences. Further, if the earlobe differences are noted before ear piercing, a balanced ear piercing effect can be achieved.

Evaluation of an allogeneic cultured dermal substitute composed of fibroblasts within a spongy collagen matrix as a wound dressing.

The purpose of this study was to examine whether epithelialisation is promoted when an allogeneic cultured dermal substitute is used as a biological wound dressing. The dermal substitute was prepared by plating fibroblasts on to a spongy collagen matrix, and then culturing them for 7 to 10 days. A new animal wound model was designed to measure re-epithelialisation quantitatively. A full thickness skin defect was made on the dorsum of each of 33 rats; the skin was excised, leaving a layer of pannicular carnosus with an island of intact skin in the central portion of the skin defect. In the first group of rats (n = 13), a piece of cultured dermal substitute was applied to the wound, and a medicated covering material was placed over it. Re-epithelialisation from the island of intact skin was monitored over a period of 7 days. In the second group of rats (n = 10), the wound was covered with an acellular collagen matrix in conjunction with the medicated covering material, and in the third group of rats (n = 10), the wound was covered with the medicated covering material alone. Both the macroscopic and histological findings indicated that the epithelial migration of the first group of rats was far more rapid than that in the other two groups. It comes to the conclusion that the application of this new fibroblastic cultured dermal substitute provided a good environment for the promotion of wound healing.

Mixed cultures comprising syngeneic and allogeneic mouse keratinocytes as a graftable skin substitute.

Using inbred mouse strains C3H/He and BALB/c and an anti-class I MHC monoclonal antibody, we examined the graftability of mixed cultures composed of syngeneic and allogeneic mouse keratinocytes. Keratinocytes from neonatal C3H/He (syngeneic donor) and BALB/c (allogeneic donor) mice were cultured for 7 days, both individually and at the inoculum ratio of 1:1, 1:3, or 1:15. The resulting sheets of keratinocytes were implanted subcutaneously into adult C3H/He mice and morphological evaluation was made on the 4th, 7th, and 14th days after grafting. The control cultures of 100% allogeneic keratinocytes were rejected, whereas epidermal reconstruction without typical signs of rejection took place in all grafts derived from the mixed cultures, including the 1:15 cultures. However, immunofluorescence staining showed that, within the grafts, the allogeneic keratinocytes were gradually replaced by syngeneic cells. Thus, these results suggested that graftable skin substitutes may be produced by co-culturing a small amount of autologous keratinocytes with allogeneic keratinocytes, which are readily available.

Morphological aspects of LFA-1/ICAM-1 and VLA4/VCAM-1 adhesion pathways in human lymph nodes.

Monoclonal antibodies specific for the adhesion molecules participating in lymphocyte homing, lymphocyte function associated antigen-1 (LFA-1) and very late antigen 4 (VLA4), and their respective ligands, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), were used to characterize their expression pattern in human lymph nodes by immunohistochemical and immunoelectron microscopic techniques. The location of LFA-1-positive lymphocytes and selective expression of ICAM-1 on the luminal plasma membrane of high endothelial venule endothelium suggested that the LFA-1/ICAM-1 adhesion pathway participates only in the initial step of the lymphocyte migration process. Lymphocytes passing through endothelium appear not to be influenced by this pathway. VCAM-1 was detected occasionally on the endothelium of high endothelial venules in the hyperplastic lymph nodes in the mesentery, but not in peripheral lymph nodes. VLA4-positive lymphocytes tended to be more frequently observed within high endothelial venules in mesenteric lymph nodes than in peripheral ones. Strong expression of both ligands, ICAM-1 and VCAM-1, was noted on the plasma membrane of follicular dendritic cells, and was especially prominent on their labyrinthine folding, and on the interdigitating cells in the paracortex. Furthermore, both LFA-1- and VLA4-positive lymphocytes localized around these cells. This suggests that LFA-1/ICAM-1 and VLA4/VCAM-1 adhesion pathways play an important role in the lymphocyte recognition of antigen-presenting cells.

Development of a new wound dressing with antimicrobial delivery capability.

A bilaminar wound dressing composed of an outer membrane and an inner three-dimensional matrix of a fabric or a sponge may be considered to constitute an ideal structure that promotes wound healing: the outer membrane prevents body fluid loss, controls water evaporation, and protects the wound surface from bacterial invasion, and the inner matrix encourages adherence by tissue growth into the matrix. Using this concept, we developed a biosynthetic wound dressing with a drug delivery capability. This medicated wound dressing is composed of a spongy sheet of a chitosane derivative and collagen mixture that is laminated to an antimicrobial-impregnated polyurethane membrane. In this study, a gentamycin sulfate-impregnated wound dressing was prepared and evaluated. The antimicrobial efficacy of this wound dressing was examined on an agar plate seeded with Pseudomonas aeruginosa. Also, the cytotoxicity of an antimicrobial released from this wound dressing was examined in an in vitro system with cultured skin substitutes. Both in vitro tests have shown that this wound dressing is capable of suppressing bacterial growth and minimizing cellular damage. In addition, in the treatment of wounds inflicted on rats and rabbits, this wound dressing was shown to be efficacious in covering full-thickness and split-thickness skin defects. Finally, the efficacy of this wound dressing was evaluated in a nonrandomized open-label study of 31 clinical cases. In 31 cases treated with this wound dressing, good or excellent wound healing was achieved.