Intraoperative evaluation of blood flow for soft tissues in orthopaedic surgery using indocyanine green fluorescence angiography: A pilot study.

OBJECTIVES: Indocyanine green (ICG) fluorescence angiography is an emerging technique that can provide detailed anatomical information during surgery. The purpose of this study is to determine whether ICG fluorescence angiography can be used to evaluate the blood flow of the rotator cuff tendon in the clinical setting. METHODS: Twenty-six patients were evaluated from October 2016 to December 2017. The participants were categorized into three groups based on their diagnoses: the rotator cuff tear group; normal rotator cuff group; and adhesive capsulitis group. After establishing a posterior standard viewing portal, intravenous administration of ICG at 0.2 mg/kg body weight was performed, and fluorescence images were recorded. The time from injection of the drug to the beginning of enhancement of the observed area was measured. The hypovascular area in the rotator cuff was evaluated, and the ratio of the hypovascular area to the anterolateral area of the rotator cuff tendon was calculated (hypovascular area ratio). RESULTS: ICG fluorescence angiography allowed for visualization of blood flow in the rotator cuff in all groups. The adhesive capsulitis group showed significantly earlier enhancement than the other groups. Furthermore, the adhesive capsulitis group had a significantly smaller hypovascular area ratio than the other groups. CONCLUSION: ICG fluorescence angiography allowed for evaluation of real-time blood flow of the rotator cuff in arthroscopic shoulder surgery. The techniques of ICG fluorescence angiography are simple and easy to observe, observer reliability is high, and it has utility for evaluating blood flow during surgery.Cite this article: N. Doi, T. Izaki, S. Miyake, T. Shibata, T. Ishimatsu, Y. Shibata, T. Yamamoto. Intraoperative evaluation of blood flow for soft tissues in orthopaedic surgery using indocyanine green fluorescence angiography: A pilot study. Bone Joint Res 2019;8:118-125. DOI: 10.1302/2046-3758.83.BJR-2018-0151.R1.

An artificially constructed radiation-responsive promoter is activated by doxorubicin.

We previously developed an artificially constructed promoter that was activated in response to X-ray irradiation in LNCap, a prostate cancer cell line. Anticancer drugs were examined to see whether some of them could stimulate the activity of the promoter. It was found that doxorubicin (Dox) treatment to LNCap transfected with a gene cassette of the luciferase gene under control of the promoter-enhanced luciferase activity in a dose-dependent manner, indicating that the promoter could be controlled by Dox. When the luciferase gene was replaced with the fcy::fur gene whose product facilitates conversion of 5-fluorocytosine into 5-fluorouracil that is highly toxic, Dox stimulated the expression of the gene product, resulting in facilitation of cell killing effect in the presence of 5-fluorocytosine. These results suggest that therapeutic gene expression controlled with an anticancer drug may lead to a more effective cancer therapy with less hazardous side effects.

Lipid microsphere formulation containing rifampicin targets alveolar macrophages.

The study demonstrated that lipid microspheres (LM) containing rifampicin (LM-RFP) could deliver the drug to alveolar macrophages in vitro and in vivo, and that intranasal administration to animals could achieve preferential accumulation in the lungs with less effect on the liver. The LM-RFP particles had a mean diameter of 247.2 +/- 75.7 nm, and their size remained stable when stored at 4 degrees C or 25 degrees C for at least 4 weeks. In vitro uptake of [(3)H]LM-RFP by alveolar macrophages was over 4 times higher than that of unencapsulated [(3)H]RFP, whereas the in vivo uptake was 30 times higher. Flow cytometric analysis and confocal laser scanning microscopy confirmed that LM could deliver the encapsulated drug effectively to alveolar macrophages in vitro and in vivo. Intranasal administration of [(3)H]LM-RFP to normal mice resulted in preferential pulmonary uptake of the drug and lower levels in the blood and liver compared with administration of unencapsulated [(3)H]RFP. In conclusion, LM-RFP could be a promising preparation for delivery via the respiratory tract to tuberculosis (TB) and TB/HIV patients.

[RNA-interference, induced by transient and continuous expression of hairpin RNA in cells from Drosophila and mammals]. / RNK-interferentsiia, indutsirovannaia vremennoi i postoiannoi ékspressiei shpilechnoi RNK, v kletkakh Drosophila i mlekopitaiushchikh.

RNA interference (RNAi) may be induced by a plasmid with an inverted repeat (IR) sequence directing transcription of hairpin-type double-stranded RNA (dsRNA). This study examines the effects of changing various parameters of IR constructs on Drosophila and mammalian RNAi, using the dual luciferase system, RNAi activity was found to vary depending on IR length ass well as the length and sequence of the internal loop separating sense and antisense sequences. Both transient and stable RNAi occurred in Drosophila cultured cells. Although transient DNA-mediated RNAi was noted in most mammalian cells, no mammalian cells stably possessing IR sequences and hence RNAi activity could be obtained. In Drosophila, DNA-mediated RNAi was considerably weaker than long-dsRNA-mediated RNAi. The cytological data indicated that this was most probably caused by abortive processing of hairpin RNA produced within cells. DNA-mediated RNAi was examined at the level of Drosophila individuals using extramacrochaetae as a model gene, and the presence of an intron sequence in the single-stranded loop region was shown to be essential for effective RNAi.

Analysis of the cell-dependent replication potentials of human immunodeficiency virus type 1 vif mutants.

Eleven in-frame vif gene mutants of HIV type 1 produced in non-permissive cells were examined for their replication potentials in various CD4-positive and -negative cell lines. Virus replication for each mutant was monitored by using several single- and multiple-cycle infectivity assays. Except for a mutant with wild-type phenotype, most mutants were severely defective for replication in all the cell lines as expected from the producer cell-dependent functioning of Vif so far reported. In contrast, two mutants, which have mutations in the hydrophilic or effector regions of Vif were found to have target cell-dependent replication potentials. These results demonstrate the presence of a novel category of the vif mutants important for elucidation of the Vif function.

Genotype-phenotype linkage for directed evolution and screening of combinatorial protein libraries.

The technologies for screening peptide and protein libraries for studies in the fields of directed protein evolution and functional genomics have advanced with astonishing speed. For screening of functional proteins, three technologies are required: (i) the construction of a gene library (genotype), (ii) the establishment of a linkage between each protein (phenotype) and its encoding gene (genotype), and (iii) the selection of desired proteins (phenotype) from the library. This review highlights the genotype-phenotype linkage technologies, which can be classified into three types; that is, cell-type linkage, virus-type linkage, and array-type linkage methods. These methods are summarized, and their advantages and disadvantages are discussed.

[CHF arising after low dose THP-COP chemotherapy in an elderly patient with malignant lymphoma].

A 76-year-old woman was admitted with a one-month history of low grade fever and dizziness. She had a palpable right supraclavicular lymph node. Abdominal ultrasonography showed swollen lymph nodes around the abdominal aorta. A specimen from the right supraclavicular lymph node showed malignant lymphoma (diffuse large B cell type). We started chemotherapy according to the low-dose THP-COP protocol (pirarubicin, cyclophosphamide, vincristine and prednisolone) on the 31st hospital day. Since no adverse effects were detected after two low-dose cycles, the patient received a third course with standard doses on the 87th hospital day. The total dose of pirarubicin was 72 mg/m2. Two days after the third course started, she suffered from dyspnea caused by congestive heart failure. A chest X-ray showed advanced cardiomegaly, severe congestion and bilateral pleural effusion. These conditions improved with transvenous administration of diuretics, a vasodilator and phosphodiesterase inhibitor. In this case, congestive heart failure developed even though the total dose of pirarubicin was lower than in previous reports of this complication. When the THP-COP protocol is indicated in elderly patients, cardiotoxicity should be monitored even if the total dose of pirarubicin is very low.

Postsurgical evaluation of idiopathic vitreomacular traction syndrome by optical coherence tomography.

PURPOSE: To report a case of idiopathic vitreomacular traction syndrome with preoperative and postoperative evaluation by optical coherence tomography. DESIGN: Interventional case report. METHODS: A 62-year-old woman presented with blurred vision in the left eye because of idiopathic vitreomacular traction syndrome, and she underwent a pars plana vitrectomy. Optical coherence tomography was performed before and after surgery. RESULTS: Preoperative optical coherence tomography, right eye, revealed residual adhesion of incomplete posterior vitreous detachment and edematous, thickened outer retina in the macula. A successful vitrectomy relieved vitreoretinal traction with nearly complete resolution of cystoid macular edema within 1 month after surgery, followed in subsequent months by gradual foveal depression resembling a lamellar macular hole. Resolution of subretinal serous fluid was delayed with complete disappearance, some 12 months after surgery, which correlated with a gradual improvement in visual acuity. CONCLUSION: Optical coherence tomography provides a sensitive anatomical evaluation of vitreomacular traction syndrome. Reorganization of retinal tissue after surgical intervention for vitreoretinal traction may be slower than is apparent from conventional examinations.

Effective methylprednisolone dose in experimental crescentic glomerulonephritis.

Pulse methylprednisolone (MP) therapy improves the prognosis of crescentic glomerulonephritis, but the optimal dose is uncertain. We reported previously that treatment with MP at a dose of 30 mg/kg reduces glomerular crescents and infiltrating mononuclear cells and ameliorates the clinical abnormalities in an animal model of crescentic glomerulonephritis. In the present study, we assessed MP dose requirement for these beneficial effects in correlation with the effect on gene expression of chemokines, potential molecules responsible for recruitment and activation of leukocytes. Animals were treated with MP, 5 to 30 mg/kg/d, for 4 consecutive days after cellular crescents had been formed diffusely. The level of crescents and numbers of glomerular and interstitial monocytes/macrophages and T lymphocytes were reduced significantly by 5 mg/kg of MP, but maximal effect was obtained by 30 mg/kg of MP. Urinary protein was reduced significantly in a 30-mg/kg group but not in other groups. The gene expression of chemokines, MCP-1, MCP-3, TCA3, MIP-1alpha, MIP-1ss, RANTES, and lymphotactin, was enhanced in this model and was inhibited strongly by 5 mg/kg of MP. These results indicate that MP reduces the number of infiltrating mononuclear cells and crescents in the rat model in a dose-dependent fashion and that, despite the strong inhibition of chemokine expression at a lower dose, the beneficial effect of MP is maximal at a dose of 30 mg/kg.

Effects of the endogenous opioid peptide, endomorphin 1, on supraoptic nucleus oxytocin and vasopressin neurones in vivo and in vitro.

We investigated the actions of the endogenous opioid tetra-peptide endomorphin 1, a selective mu-opioid receptor agonist, on oxytocin and vasopressin cell activity in vivo and in vitro. The activity of antidromically-identified supraoptic nucleus cells were recorded from urethane-anaesthetized female rats. The firing rates of both oxytocin and vasopressin cells were reduced by intracerebroventricular endomorphin 1 (5 - 100 pmol); this inhibition was prevented by intravenous naloxone (5 mg kg(-1)). A second group of rats was infused intracerebroventricularly with endomorphin 1 (27 pmol min(-1)) over 5 days. The firing rates of oxytocin and vasopressin cells in endomorphin 1 pre-treated rats were similar to those of endomorphin 1 naïve rats, indicating tolerance to the inhibitory effects of endomorphin 1. Intravenous naloxone induced similar modest and transient increases in the firing rate of oxytocin cells in endomorphin 1 pre-treated rats and endomorphin 1 naïve rats, indicating that endomorphin 1, unlike the mu-opioid alkaloid agonist, morphine, does not induce mu-opioid dependence in these cells. In vitro, whole-cell current clamp recordings were made from supraoptic nucleus cells in superfused coronal hypothalamic slices from young female rats. Endomorphin 1 (100 nM) inhibited the firing rate of oxytocin cells but had no significant effect on vasopressin cells at up to 10 microM. Inhibition of oxytocin cells was reversed by naloxone, and remained when synaptic transmission was blocked by superfusion with low Ca(2+)/Co(2+)-containing medium. Thus, endomorphin 1 directly inhibits oxytocin cells but inhibits vasopressin cells by indirect actions. Chronic endomorphin 1 administration induces mu-opioid tolerance in oxytocin and vasopressin cells but not mu-opioid dependence in oxytocin cells.

Prognostic significance of the carcinoma area in the thickest part of the lymph node.

BACKGROUND/AIMS: The carcinoma volume in each metastatic lymph node varies widely. Our aim was to define the meaning of carcinoma volume in lymph nodes as a prognostic factor. METHODOLOGY: One hundred and five patients with preoperatively untreated esophageal carcinoma who underwent surgery were enrolled as subjects. In the present study, the carcinoma area within lymph nodes at their thickest level was substituted for carcinoma volume in lymph nodes for measurement and analysis. A total of 3,703 lymph nodes were isolated and the area of the carcinoma in the thickest part of the lymph node (CALN) was measured. Univariate and multivariate analysis of prognostic significance were made for the factors age, sex, cancer location, cell differentiation, pT, conventional classification of lymph nodes for surgical dissection (n), number of metastatic lymph nodes (MLN number), and CALN. RESULTS: In all 105 cases, n was the best prognostic factor and CALN was more useful than MLN number. In the cases in which absolute curative resection was successful, CALN was the best prognostic factor. CONCLUSIONS: The carcinoma area in the thickest part of the lymph node is a meaningful prognostic factor.

[Heritability of human skeletal morphology].

The shape of human skeleton is determined by both genetic and environmental factors. In this report, I discussed the relative contribution of these two components in the osteometric traits by investigating 6 skeletal series affiliated to different social ranks. They were composed of 4 pedigrees, the Kanaseki family, the Tokugawa family (Shogun), the Makino family (Daimyo) and the Kuse family (Karo), and two control samples of unrelated individuals from the Edo period and recent times. The three familial samples, except the Kanaseki family, were the Japanese aristocrats of the Edo period. To estimate the family resemblance in skeletal morphology, I calculated Q-mode correlation coefficients (Q) and standardized Euclidean distances (D) for skeletal measurements of the relatives from 4 pedigrees and control samples of recent Japanese. Comparing Q and D between related and unrelated pairs, strong resemblances were detected among the related one. Family resemblance tended to reveal remarkable similarities in neuro-cranium, humerus, femur and tibia. Then, to determine the genetic contribution to these family resemblances, I estimated heritabilities (h2) for each measurement according to Falconer and Sjøvold. High heritabilities were seen in skull measurements as mentioned by Sjøvold. However, high heritabilities were also demonstrated in the epiphyseal part of the extremity bones. Environmental factors affecting the shape of human skeleton were analyzed by using canonical discriminant analysis for the measurements of the 6 skeletal series. Two environmental factors, social rank and the period that populations belong to, were detected from these analyses. Social rank seemed to be related with mechanical stress on the bone and the period with living condition such as nutrition and medical care. The influence by mechanical stress was remarkably recognized in such high mobility areas as facial cranium, forearm and leg. On the other hand, the influence on bones by living condition was more remarkable in the lower extremity than in the upper one.

An extremely large solitary primary paraganglioma of the lung: report of a case.

We present herein the case of a 38-year-old woman found to have an extremely large solitary primary paraganglioma of the lung. The patient presented with chest pain on exertion and a mass was discovered in the left lower lobe of the lung by chest X-rays and computed tomography (CT). As no other neoplasms were detected elsewhere, a left lower lobectomy was performed. The patient has remained well without any evidence of recurrence for 5 years since her operation. The tumor, measuring 13 x 12 x 7 cm, was composed of ovoid cells (Zellballen), which were positive for Fontana-Masson and Grimelius stains, and sustentacular cells. Immunohistochemically, the ovoid cells were positive for neuron-specific enolase, S-100, CAM5.2, Leu7, and chromogranin A, and negative for carcinoembryonic antigen and epithelial membrane antigen. The sustentacular cells were positive for S-100 protein and CAM5.2, and negative for glial fibrillary acid protein. Therefore, the tumor was diagnosed as a paraganglioma. The tumor from our patient is the largest of the 17 solitary primary pulmonary paragangliomas reported thus far in the English-language literature.

Insertional gene fusion technology.

The classical 'end to end' gene fusion technique has widely been used for monitoring gene expression, biological screening and purification of recombinant proteins. Recent progress with the 'insertional' gene fusion approach, on the other hand, has demonstrated that this technique can be utilized for membrane protein topology analysis, display of randomized protein libraries and design of biosensor proteins. In this review, we describe examples of insertional gene fusion and compare the old and new gene fusion techniques.

STABLE: protein-DNA fusion system for screening of combinatorial protein libraries in vitro.

We have developed a new method that permits the complete in vitro construction and selection of peptide or protein libraries. This method relies on an in vitro transcription/translation reaction compartmentalized in water in oil emulsions. In each emulsion compartment, streptavidin (STA)-fused polypeptides are synthesized and attached to the encoding DNA via its biotin label. The resulting protein-DNA fusion molecules recovered from the emulsion can be subjected to affinity selection based on the properties of the peptide portion, whose sequence can be determined from that of its DNA-tag. This method, named 'STABLE' (STA-biotin linkage in emulsions), should be useful for rapid in vitro evolution of proteins and for ligand-based selection of cDNA libraries.

Complications associated with vortex vein damage in scleral buckling surgery for rhegmatogenous retinal detachment.

PURPOSE: To further understand postoperative complications after vortex vein damage during scleral buckling surgery. METHODS: The records of 34 patients (34 eyes) with vortex vein damage during scleral buckling surgery for rhegmatogenous retinal detachment were reviewed and compared with the records of 410 eyes undergoing similar surgery without vortex vein damage. RESULTS: Postoperative complications were noted in 16 eyes (47%) of the damaged vortex vein group. The incidence of choroidal detachment, vitreous opacities, intraocular pressure elevation, and vitreous hemorrhage were 27%, 18%, 9%, and 6%, respectively, with a higher incidence than in the group without vortex vein damage. Other complications included development of epiretinal membrane (9%), subretinal hemorrhage (3%), and anterior segment ischemia (3%). Serous choroidal detachment occurred in the early postoperative days and subsided within 3 weeks. Vitreous opacification became marked in the later periods and continued for 2 months or longer. The incidence of postoperative choroidal detachment in the vortex vein damage group was related to the patient's age (P = .002) and the cutting of the vortex veins (P = .048), but was not related to preoperative conditions of retinal detachment or the number of vortex veins damaged. All the eyes except one achieved retinal reattachment after initial surgery. CONCLUSIONS: Choroidal detachment and vitreous opacity are common after scleral buckling surgery with vortex vein damage. Although intervention of the vortex veins during scleral buckling surgery is acceptable when performing otherwise difficult to achieve ample scleral indentation, it should be minimized to avoid increased incidence of postoperative complications.

Increased plasma levels of adrenomedullin in patients with systemic inflammatory response syndrome.

We measured the plasma levels of adrenomedullin (AM), a novel vasodilating peptide, in 89 patients with various forms of systemic inflammatory response syndrome (SIRS) and 13 healthy volunteers serving as controls. Plasma levels of AM in SIRS (burns: 20.5 +/- 3. 2 fmol/ml [mean +/- SEM]; pancreatitis: 13.8 +/- 3.8 fmol/ml; trauma: 14.9 +/- 2.5 fmol/ml; traumatic shock: 41.1 +/- 7.8 fmol/ml; severe sepsis: 59.9 +/- 11.2 fmol/ml; septic shock: 193.5 +/- 30.1 fmol/ml) were significantly increased over those of controls (5.1 +/- 0.2 fmol/ml). The patients with traumatic shock or septic shock especially had higher levels of plasma AM than those with trauma or severe sepsis, respectively. These data showed that in patients with SIRS, plasma AM levels increased in proportion to the severity of illness. Subsequently, we measured the plasma levels of mediators such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-8, plasminogen activator inhibitor (PAI)-1, and thrombomodulin (TM) in patients with traumatic shock and septic shock. A significant correlation was observed between plasma AM and TNF-alpha levels in patients with septic shock, suggesting an important role for AM as well as of TNF-alpha in the pathophysiology of inflammation. Plasma AM and IL-8 levels correlated positively with Acute Physiology and Chronic Health Evaluation (APACHE) II score, peak multiple organ failure (MOF) score during the first month and prognosis in patients with septic shock, as did plasma IL-6 levels in patients with traumatic shock. The plasma AM level might serve as a useful marker for evaluating the severity of disease and as an early predictor of subsequent organ failure and outcome in septic shock.

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution.

Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs. At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 beta-lactamase and its inhibitory protein as our model protein-ligand system. 'Allosteric GFP biosensors' constructed by this method may be used in a wide range of applications including biochemistry and cell biology.

[Decreased plasma soluble P-selectin level in coronary sinus after successful coronary angioplasty in patients with unstable angina].

P-selectin, an adhesion molecule, is involved in the alpha-granules of platelets with several factors such as platelet factor 4 (PF-4) and in Weibel-Parade bodies of endothelial cells with von Willebrand factor. The levels of the soluble form of P-selectin increase after angina episodes in patients with unstable angina, indicating that soluble P-selectin is associated with platelet activation and thrombogenesis in the coronary circulation. To evaluate the effect of successful coronary angioplasty on platelet activation or thrombogenesis in the coronary circulation, plasma soluble P-selectin, PF-4 and von Willebrand factor antigen levels were measured in blood obtained from the coronary sinus before and after successful coronary angioplasty in 15 patients with unstable angina. Fifteen patients with normal coronary angiograms served as controls. Plasma P-selectin, PF-4 and von Willebrand factor antigen levels were determined by sandwich enzyme-linked immunosorbent assays. Increased plasma soluble P-selectin (159.7 +/- 74.5 vs 78.7 +/- 26.4 ng/ml, p < 0.01) and PF-4 (456.5 +/- 87.0 vs 118.7 +/- 62.3 IU/ml, p < 0.01) levels were found in patients with unstable angina compared with those in controls, and were significantly decreased after angioplasty (147.8 +/- 69.6 ng/ml, p < 0.05; 401.6 +/- 108.5 IU/ml, p < 0.05), whereas von Willebrand factor antigen was unchanged. The ratio of plasma soluble P-selectin levels after and before angioplasty correlated with the corresponding ratio of plasma PF-4 levels (r = 0.53, p < 0.05), but not with the ratio of plasma von Willebrand factor antigen levels. The plasma levels of soluble P-selectin, which increase in the coronary circulation in patients with unstable angina, decrease after successful coronary angioplasty. Such data indicate that soluble P-selectin is associated with platelet activation and the therapeutical procedure improves the thrombogenic state in the coronary circulation.

Possibility of pre-operative diagnosis of lymph node metastasis based on morphology.

BACKGROUND/AIMS: The accuracy of pre-operative diagnosis of lymph node metastasis is insufficient. Our aim was to define the possibility of diagnosing metastatic lymph nodes based on morphology. METHODOLOGY: One hundred and fifty-seven patients with pre-operatively untreated esophageal squamous cell carcinoma underwent resection, 5334 lymph nodes were isolated, and the short and long diameters were measured. We tried to construct a linear regression line for metastasis rate versus lymph node size (long diameter classified at intervals of 1 mm) by each location. The ratio of short diameter to long diameter (SL ratio) of metastasis-positive lymph nodes was compared with that of negative ones at each location. RESULTS: Gradient and intercept of overall regression line was 0.0213 and 0.0101, respectively, and the long diameter producing a metastasis rate of 80% (LD80) was 37.1 mm. Metastasis-positive lymph nodes larger than calculated LD80 represented no more than 9.5% of all the corresponding metastasis-positive nodes. The locations with significant difference of SL ratio between metastasis-positive and negative ones were limited to right cardiac, left gastric artery, thoracic paratracheal, bifurcation, and the highest mediastinal nodes. CONCLUSIONS: There is a low possibility that lymph node metastasis can be exactly diagnosed pre-operatively based on the size and morphology.