Factors that influence the postoperative upper eyelid position following surgery for involutional blepharoptosis.

External levator advancement is commonly performed for involutional blepharoptosis repair; however, it is difficult to predict the postoperative upper eyelid position (UEP) accurately in blepharoptosis surgery. The purpose of this study was to determine the factors that influence postoperative UEP following surgery for involutional blepharoptosis. We retrospectively studied 40 Japanese women (80 eyelids) who underwent bilateral external levator advancement surgery. We used digital analysis software to measure the UEP and the eyebrow position from straight-gaze view photographs. Statistical analysis was performed to determine the correlation between postoperative UEP and related factors, including age, levator function, amount of levator advancement, anatomical fixed position, and preoperative and intraoperative UEP. We also compared UEP changes in mild, moderate, and severe ptosis groups. Levator function affected both preoperative and postoperative UEP. The amount of levator advancement and the anatomical fixation position on the aponeurosis did not affect the postoperative UEP. However, both preoperative (r = 0.49) and intraoperative (r = 0.55) UEPs affected the postoperative UEP. In cases of severe ptosis, there was significant re-drooping after surgery, while in cases with mild ptosis, the intraoperative eyelid position was maintained or slightly elevated. In involutional blepharoptosis, the degree of preoperative and intraoperative UEP contributed to the postoperative eyelid position. These data suggested that the levator muscle function is a major contributing factor in the pathogenesis of involutional blepharoptosis.

Effect of Serial Passage on the Pathogenicity and Immunogenicity of Vaccinia Virus LC16m8 Strain.

The phenotype of an attenuated live vaccine depends on gene mutation achieved by, for example, many passages in cultured cells. Viral clones with preferable phenotypes are selected and the causative genetic mutation(s) are later identified. LC16m8 is an example of a highly attenuated smallpox vaccine that was developed and licensed in Japan in the 1970s. LC16m8 was obtained by the passaging of Lister strain, with indicators of small plaque formation and temperature sensitivity as virus phenotypes. This strain can replicate in mammalian cells and provides robust cellular and humoral immunity, as well as long-term immune memory. Recent studies using proteome-wide antigen arrays have revealed that antibody production against LC16m8 and other VACVs differs largely among individuals. Moreover, associations between SNPs in immune-related genes and immune outcomes have been increasingly found. These results lead to predicting adverse events of a vaccine, which is a purpose of vaccinomics. Studies on VACV will continue to contribute to the understanding of host-pathogen interactions and to development of a vaccine for other infectious and non-infectious diseases. Here, we review studies of VACV, including our recent research on LC16m8, with a focus on the phenotype and genotype, and we discuss future research directions.

Profiling of the antibody response to attenuated LC16m8 smallpox vaccine using protein array analysis.

Concerns about bioterrorism and outbreaks of zoonotic orthopoxvirus require safe and efficacious smallpox vaccines. We previously reported the clinical efficacy and safety profiles of LC16m8, a live, attenuated, cell culture-derived, smallpox vaccine, examined in over 3000 healthy Japanese adults with various vaccination histories. In this study, serum of approximately 200 subjects pre and post LC16m8 vaccination were subjected to a vaccinia virus-specific protein array to evaluate the proteome-wide immunogenicity. The relationships between antigen-specific antibodies and plaque reduction neutralization titers were analyzed. LC16m8 induced antibodies to multiple vaccinia antigens in primary-vaccinated individuals and yielded effective booster responses in previously vaccinated individuals, demonstrating similar antibody profiles to those reported for other vaccinia virus strains. Several immunodominant antigens were indicated to be important for neutralization of the intracellular mature virion. The similarity of antibody profiles between LC16m8 and other smallpox vaccine strains supports the immunogenicity and protective efficacy of LC16m8.

Rezoning Free Muscle-Sparing Transverse Rectus Abdominis Myocutaneous Flaps Based on Perforasome Groupings and a New Understanding of the Vascular Architecture of the Deep Inferior Epigastric Artery-Based Flaps.

BACKGROUND: We compare the vascular territory of free muscle-sparing transverse rectus abdominis myocutaneous (MS-TRAM) flaps, deep inferior epigastric perforator (DIEP) flaps, and crossover anastomosis (CA) flaps using intraoperative ex vivo angiography. We also use ex vivo angiography to analyze the vascular architecture of the MS-TRAM flap. METHODS: Our study includes 84 lower abdominal free flaps: MS-TRAM, DIEP-1 (1 perforator), DIEP-2 (2 perforators), and CA. We compare the arterial perfusion area and vascular territory pattern in each group. We also analyze the vascular architecture in MS-TRAM flaps and determine the number and location of their dominant perforators and the direction of the axial arteries connecting them. RESULTS: The CA's arterial perfusion area is the largest, and the DIEP-1's, the smallest of our groups; there is no statistically significant difference between MS-TRAM and DIEP-2. In all groups, average arterial perfusion area in the vascular pedicle's ipsilateral side is larger than in its contralateral side. The MS-TRAM and DIEP-2 flaps have homologous perfusion patterns and the same arterial perfusion areas. The DIEP-1 perfusion pattern varies with perforator location. Ex vivo angiograms show the MS-TRAM flap's axial arteries heading laterally to be larger and longer than those heading medially. CONCLUSIONS: Two dominant perforators are preferable in DIEP flap breast reconstruction. Lateral perforators play a more important role in flap perfusion than do medial ones. Crossover anastomosis is an effective technology for increasing arterial perfusion areas. Our rezoning shows which areas are better for surgery and which have a high risk of complications-valuable information for a surgeon designing a flap for breast reconstruction.

National Registry of Designated Intractable Diseases in Japan: Present Status and Future Prospects.

Japan promotes research related to intractable diseases and financially supports patients with these diseases. Intractable diseases are designated as those that fulfill the following criteria: (1) rarity (affecting less than 0.1% of the population in Japan), (2) unknown etiology, (3) lack of effective treatment, (4) necessity of long-term treatment, and (5) existence of objective diagnostic criteria and not necessarily equal to rare diseases in other countries. The construction of a national database is required to promote research to clarify the pathogenesis of these diseases and to develop pharmaceutical products and medical devices. The Ministry of Health, Labor, and Welfare launched an online registration system in 2001, but many problems associated with gathering and utilizing information on patients with intractable diseases remain. In this paper, we describe the present status of the national registry of designated intractable diseases in Japan and discuss future prospects.

Recent advances in the study of live attenuated cell-cultured smallpox vaccine LC16m8.

LC16m8 is a live, attenuated, cell-cultured smallpox vaccine that was developed and licensed in Japan in the 1970s, but was not used in the campaign to eradicate smallpox. In the early 2000s, the potential threat of bioterrorism led to reconsideration of the need for a smallpox vaccine. Subsequently, LC16m8 production was restarted in Japan in 2002, requiring re-evaluation of its safety and efficacy. Approximately 50,000 children in the 1970s and about 3500 healthy adults in the 2000s were vaccinated with LC16m8 in Japan, and 153 adults have been vaccinated with LC16m8 or Dryvax in phase I/II clinical trials in the USA. These studies confirmed the safety and efficacy of LC16m8, while several studies in animal models have shown that LC16m8 protects the host against viral challenge. The World Health Organization Strategic Advisory Group of Experts on Immunization recommended LC16m8, together with ACAM2000, as a stockpile vaccine in 2013. In addition, LC16m8 is expected to be a viable alternative to first-generation smallpox vaccines to prevent human monkeypox.

A new digital image analysis system for measuring blepharoptosis patients' upper eyelid and eyebrow positions.

PURPOSE: To accurately assess eyelid and eyebrow position, we have developed a new measurement method using a digital image analysis system. MATERIALS AND METHODS: (1) Plotting the corneal limbus by transferring to the software digital images of the patient's eyes in the primary gaze position; (2) determining the corneal ring, center, and longitudinal diameter; and (3) measuring the upper eyelid and eyebrow position. The method was tested on 24 patients with senile blepharoptosis and 45 young healthy eyelids without ptosis. RESULTS: The upper eyelid position of patients with blepharoptosis was significantly lower, and their eyebrow position is significantly higher than those of healthy subjects. The upper eyelid and eyebrow position of the patients with blepharoptosis corelated, but no correlation was observed in the healthy subjects. CONCLUSION: Our measuring system is a simpler, easier, and more accurate way of measuring both upper eyelid and eyebrow position than are currently used techniques.

Vascular augmentation of an extended latissimus dorsi myocutaneous flap through an intercostal vessel: a preliminary report.

The extended latissimus dorsi (LD) flap, which includes adipofascial tissue around the posterior iliac crest, is a good option to reconstruct a large breast. However, poor vascularity in the extended part may cause partial fat necrosis. To minimise this problem, vascular augmentation of the extended adipofascial part was performed. When dissecting under the LD muscle, a single perforator from the 11th intercostal vessels was secured and traced upwards along the rib. After the flap was transferred to the chest, the intercostal vessels were anastomosed to the serratus branches of thoracodorsal vessels. This vascular supercharged extended LD flap technique was applied for selected patients. Intraoperative angiography showed that the contrast medium injected into the intercostal artery spread across the lumbar adipofascial part of the flap. This implies that vascular supercharge through the 11th intercostal vessel promotes the vascularity of the extended LD flap and may help to reduce the flap complication rate.

Bilayer reconstruction for parry-romberg syndrome: using a free circumflex scapular artery-based adipofascial flap for both the buccal fat pad and subcutaneous fat.

PURPOSE: When using a free flap to reconstruct a facial deformity caused by Romberg's disease, it is important to prevent the flap from sagging after the operation. We report a new method of reconstructive surgery using a free subscapular adipofascial flap to prevent this problem. PATIENTS AND METHODS: Three female patients (ages 27, 28, and 34 years) with Parry-Romberg syndrome underwent microsurgical free scapular flap transfer for buccal defects. This operation requires making a gingivobuccal sulcus incision and forming a pocket for buccal fat reconstruction by dissecting over the periosteum of the maxillary bone. Preauricular and submandibular incisions are made to create a subcutaneous pocket for flap transfer. After the subscapular flap is elevated, we use its angiogram to observe its vascular pattern. The flap is separated to preserve the main blood vessels horizontal lower branches. The subcutaneous adipose tissue layer uses the horizontal branch, and the buccal fat pad layer the lower branch. METHODS: After the operations, the adipofascial flaps were in good condition and without postoperative complications. A half year after the first operation, revisional surgery was performed for one patient. No cases showed no sagging of the cheek, and in every case the overall appearance of the buccal region improved significantly. DISCUSSION AND CONCLUSION: Reconstruction of the buccal fat pad and subcutaneous adipose tissue using a free bilobed adipofascial flap nourished by the circumflex scapular artery, returned the adiposal tissue to its normal position, assuring more natural facial contours.

Proteolytic conversion of STAT3alpha to STAT3gamma in human neutrophils: role of granule-derived serine proteases.

Four isoforms (alpha, beta, gamma, and delta) have been identified for signal transducer and activator of transcription 3 (STAT3). It has been reported that STAT3gamma, which is derived from STAT3alpha by limited proteolysis during granulocytic differentiation, is a major STAT3 isoform expressed in human neutrophils. We confirmed that STAT3gamma was a major STAT3 isoform detected in human neutrophil lysates prepared with the conventional lysis buffer. The enzymes capable of converting STAT3alpha to STAT3gamma in vitro were localized in neutrophil granule fraction and were released into the medium upon ionomycin stimulation. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, CuSO(4), and ONO-5046 (a specific inhibitor of neutrophil elastase), but not by aprotinin, leupeptin, benzamidine, and EDTA. STAT3gamma was effectively generated in vitro from STAT3alpha by limited proteolysis with human neutrophil elastase or proteinase 3 but not cathepsin G. The converting activity in neutrophil lysates was reduced by immunodepletion of elastase but not proteinase 3. Unexpectedly, STAT3gamma was undetected in the lysates of neutrophil-derived cytoplasts, which lack granules, and the cytosol fraction prepared by nitrogen cavitation. The STAT3 isoform detected in these preparations was primarily STAT3alpha. STAT3gamma was also undetected in the lysates of PMSF-pretreated neutrophils and was markedly decreased in the lysates of ionomycin-pretreated neutrophils. These findings indicate that, in contrast to the previous reports, STAT3alpha, but not STAT3gamma, is primarily expressed in human neutrophils, and STAT3gamma is rapidly generated from STAT3alpha by limited proteolysis with granule-derived serine proteases during preparation of neutrophil lysates with the conventional lysis buffer.

Oral streptococci exhibit diverse susceptibility to human beta-defensin-2: antimicrobial effects of hBD-2 on oral streptococci.

We examined the antimicrobial effects of human beta-defensin-2 (hBD-2) on 17 species of oral streptococci to investigate the involvement of antimicrobial peptide activity in oral microflora development and the clinical use of the antimicrobial peptide for oral microflora control. Oral streptococci exhibit diverse levels of susceptibility to human beta-defensin-2 (hBD-2). Two major cariogenic bacterial species, Streptococcus mutans ( S. mutans) and S. sobrinus, were found to be susceptible to the peptide, indicating that it is a potential therapeutic agent for preventing dental caries. S. mitis exhibited the lowest susceptibility to the peptide. S. mitis is a major indigenous bacterium in the oral microflora, and our results suggest that it might possess a certain resistance mechanism against hBD-2.

Selective activation of STAT3 in human monocytes stimulated by G-CSF: implication in inhibition of LPS-induced TNF-alpha production.

Lipopolysaccharide (LPS) induced tumor necrosis factor (TNF)-alpha production in human monocytes, which was dependent on activation of extracellular signal-regulated kinase (ERK), p38, c-Jun NH(2)-terminal kinase (JNK), and nuclear factor (NF)-kappa B. LPS-induced TNF-alpha production was inhibited by granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-10. G-CSF, like IL-10, exerted the inhibitory effect even when simultaneously added with LPS. Among the signaling pathways, signal transducer and activator of transcription 3 (STAT3) was selectively activated in monocytes stimulated by G-CSF or IL-10. G-CSF-mediated inhibition of LPS-induced TNF-alpha production as well as G-CSF-induced STAT3 phosphorylation and suppressor of cytokine signaling 3 mRNA expression were prevented by pretreatment of monocytes with AG-490, an inhibitor of Janus kinase 2. G-CSF did not affect LPS-induced activation of ERK, p38, JNK, and NF-kappa B, indicating that G-CSF affects the pathway downstream or independently of these signaling molecules. G-CSF-induced, but not IL-10-induced, STAT3 phosphorylation was attenuated in the presence of LPS. These findings suggest that G-CSF, like IL-10, inhibits LPS-induced TNF-alpha production in human monocytes through selective activation of STAT3, and the immunomodulation observed in vivo by G-CSF administration may be partly ascribed to the direct effect of G-CSF on monocyte functions.

Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans.

S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-kappaB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2(DeltaS40-I64) (a TLR2 mutant with a deletion of the region of Ser(40) to Ile(64)) failed to activate NF-kappaB in response to FSL-1. The deletion mutant TLR2(DeltaC30-S39) induced NF-kappaB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu(178) to Ala (TLR2(E178A)), TLR2(E180A), TLR2(E190A), and TLR2(L132E) induced NF-kappaB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2(L107E), TLR2(L112E) (a TLR2 point mutant with a substitution of Leu(112) to Glu), and TLR2(L115E) failed to induce NF-kappaB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2(L115E), TLR2(L112E), and TLR2(DeltaS40-I64) were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2(E190A) were. In addition, these mutants, except for TLR2(E180A), functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser(40)-Ile(64) and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.

IkappaB-zeta, a new anti-inflammatory nuclear protein induced by lipopolysaccharide, is a negative regulator for nuclear factor-kappaB.

Activation of nuclear factor-kappaB (NF-kappaB), a prominent cellular response to bacterial endotoxin or other microbial products, must be strictly regulated because excessive activation leads to overproduction of cytotoxic cytokines that culminates in septic shock. During screening for genes up-regulated upon inflammation, we identified a new member of the IkappaB family proteins with the ankyrin-repeats. This protein, designated IkappaB-zeta, is hardly detectable in resting cells, but is strongly induced upon stimulation by lipopolysaccharide, which stimulates cells through the Toll-like receptor 4. Interleukin-1beta stimulation also results in the strong induction of IkappaB-zeta, but tumor necrosis factor-alpha does not. In contrast to IkappaB-alpha or IkappaB-beta, IkappaB-zeta localizes in the nucleus, where it inhibits NF-kappaB activity. NF-kappaB activity is essential for the induction of IkappaB-zeta, but is not sufficient. Thus, this protein is a new anti-inflammatory protein, which is specifically induced upon inflammation to regulate NF-kappaB activity.

Essential roles for NF-kappa B and a Toll/IL-1 receptor domain-specific signal(s) in the induction of I kappa B-zeta.

I kappa B-zeta, a new negative-regulator of nuclear factor-kappa B (NF-kappa B), is strongly induced by lipopolysaccharide or interleukin-1 beta stimulation, but not by tumor necrosis factor-alpha. Here, we analyzed the mechanisms for transcriptional induction of I kappa B-zeta. I kappa B-zeta mRNA was induced by overexpression of MyD88 or TRAF6, but not TRAF2. Stimulation of macrophages with peptidoglycan or CpG DNA, which activated Toll-like receptor 2 or 9, respectively, also resulted in I kappa B-zeta induction. Thus, activation of the MyD88-dependent signaling pathway, commonly found downstream of different Toll/interleukin-1 receptor (TIR) domains, is sufficient for I kappa B-zeta induction. The induction was inhibited by treatment with various inhibitors of NF-kappa B activation or by overexpressing I kappa B-alpha or beta, indicating essential roles for NF-kappa B in I kappa B-zeta induction. However, overexpression of the NF-kappa B subunits induced I kappa B-alpha, but not I kappa B-zeta. These results indicate the existence of another signal essential for I kappa B-zeta induction, which is specifically mediated by the TIR domain-mediated signaling pathway.