Quantitative lymph imaging for assessment of lymph function using indocyanine green fluorescence lymphography.

OBJECTIVES: A new diagnostic imaging technique that can assess lymph function is needed as a screening test in daily practice. This study assessed the use of indocyanine green (ICG) fluorescence lymphography in subjects without leg oedema. METHODS: 0.3ml of ICG (0.5 %) was injected subcutaneously at the dorsum of the foot. Subsequently, the movement of ICG dye from the injection site to the groin was traced by visualizing its fluorescence signal with an infrared light camera. The time for the dye to reach the knee and groin were measured (Transit time to knee: TT(K), Transit time to groin: TT(G)). TT(G) was measured while standing, lying at a supine position, standing with massage, and sitting while using a cycle ergometer exercise at an intensity of 50W at 50rpm in ten healthy volunteers at intervals of 14 days. RESULTS: Mean TT(G) during standing was 357+/-289 and 653+/-564 seconds for the right and left legs respectively. Compared to TT(G) in the standing position, all other conditions shortened TT(G). In another seventeen subjects without leg oedema, we compared transit time obtained with ICG fluorescence lymphography to that with dynamic lymphoscintigraphy. A significant correlation between transit time measured with ICG lymphography and dynamic lymphoscintigraphy was identified (r(2)=0.64, p<0.01). CONCLUSIONS: ICG fluorescence lymphography has the potential to become an alternative lymphatic imaging technique to assess lymph function.

Indocyanine green fluorescence angiography for intraoperative assessment of blood flow: a feasibility study.

OBJECTIVE: To introduce our preliminary experience with indocyanine green (ICG) fluorescence angiography for the assessment of lower leg bypasses. METHODS: 1ml of 0.5% indocyanine green was intravenously injected in 9 patients with PAD who underwent paramalleolar artery bypass using saphenous vein grafts. A newly developed near-infrared camera system (PDE; Hamamatsu Photonics K.K. Hamamatsu, Japan) was used for this study. RESULTS: ICG fluorescence angiography was performed without any adverse events. Fluorescence images of ICG angiography could be viewed as real-time images of the angiography in eight patients, while one patient underwent graft revision with the absence of fluorescence in ICG angiography. CONCLUSION: ICG fluorescence angiography is clinically feasible and may help surgeons assess the quality of lower leg bypasses.

Intraoperative monitoring of penile and buttock blood flow during endovascular abdominal aortic aneurysm repair.

OBJECTIVE: The purpose of this study was to assess the pelvic circulation during endovascular abdominal aortic aneurysm repair (EVAR) with a new monitoring system measuring penile and buttock blood flow. METHODS: We measured penile brachial pressure index (PBI) during EVAR by pulse-volume-plethysmography (form PWV/ABItrade mark). We also measured bilateral gluteal tissue oxygen metabolism with near-infrared spectroscopy to provide a gluteal tissue oxygenation index (TOI). Twenty-two men who underwent aortouni-iliac stentgraft with crossover bypass for exclusion of abdominal aortic aneurysm were studied. Twelve patients underwent aorto-uni-common iliac artery stentgraft (CIA) and ten underwent aorto-uni-external iliac artery stentgraft (EIA). RESULTS: In all patients, there was an immediate reduction in PBI during the EVAR procedure. After revascularization of the ipsilateral limb of the stent graft, the recovery of PBI was significantly less in EIA group. After the completion of crossover bypass, PBI in both groups recovered to the baseline values. In both groups there was a bilateral reduction in gluteal TOI during malperfusion of the internal iliac artery. After revascularization of ipsilateral limb of the stent graft, the ipsilateral TOI recovered to the baseline level in CIA patients, but recovery was incomplete in EIA patients. In contrast, contra-lateral TOI remained low in both groups after revascularization of ipsilateral limb of the stent graft. Only after completion of crossover bypass did the contra-lateral TOI recover to baseline level in both groups. CONCLUSIONS: Both TOI at the buttocks and PBI are a sensitive reflection of pelvic haemodynamics. Penile blood flow and bilateral gluteal blood flow are supplied via different circulations and both should be monitored for full assessment of the pelvic circulation.

Detection of apoptosis in keloids and a comparative study on apoptosis between keloids, hypertrophic scars, normal healed flat scars, and dermatofibroma.

Recent studies have suggested that the regulation of apoptosis during wound healing is important in scar establishment and the development of pathological scarring. In this study, we demonstrate that keloid fibroblasts can be identified as apoptotic cells because of their highly condensed chromatin and discrete nuclear fragments. To further reveal the phenomenon of apoptosis, we quantified the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in surgically resected tissues of keloids (N = 10), hypertrophic scars (N = 10), normal healed flat scars (N = 10), and dermatofibroma (N = 10). The number of TUNEL-positive cells was relatively low, but was significantly higher for the keloid group compared with the normally healed flat scar group (p = 0.004), suggesting reduced cell survival and increased apoptotic cell death in a subpopulation of keloid fibroblasts. Furthermore, the number of TUNEL-positive cells was significantly higher for the keloid group compared with the dermatofibroma group (p = 0.044), suggesting that a subpopulation of keloid fibroblasts may suppress tumorgenicity at a greater rate than dermatofibroma by undergoing cell death. Hypertrophic scars had significantly higher levels of apoptosis than normally healed flat scars (p = 0.033). Therefore, these results suggest that selected fibroblasts in keloids and hypertrophic scars undergo apoptosis, which may play a role in the process of pathological scarring.

Highly increased plasma concentrations of the nicked form of beta(2) glycoprotein I in patients with leukemia and with lupus anticoagulant: measurement with a monoclonal antibody specific for a nicked form of domain V.

beta(2)-Glycoprotein I (beta(2)GPI) consists of five tandem repeated domains (I, II, III, IV, and V). The nicked form of beta(2)GPI (N-beta(2)GPI ) which was cleaved by plasmin in vitro at Lys 317-Thr 318 in domain V, showed reduced affinity for the negatively charged phospholipids, especially cardiolipin (CL). Recently, the N-beta(2)GPI was detected in the plasma of patients with disseminated intravascular coagulation syndrome (DIC) by an immunological method. In the present study, we prepared monoclonal antibodies for the nicked form, and demonstrated that the concentrations of this form of beta(2)GPI, which were analyzed by a sandwich ELISA using two specially prepared monoclonal antibodies, were significantly increased in the plasma of patients with leukemia (n = 51, mean +/- SD: 162.0 +/- 118.3 ng/ml) and with lupus anticoagulant (LA) (n =40, mean +/- SD: 3,041.5 +/- 16,579.7 ng/ml), compared to the normals (n = 33, mean +/- SD: 1.04 +/- 1.54 ng/ml). We found a significant correlation between the concentrations of N-beta(2)GPI and those of typical molecular markers for a fibrinolytic state such as plasmin-alpha(2) plasmin inhibitor complex (PIC) and D-dimer in patients with leukemia, but not in patients with LA. These results suggested that the generation of N-beta(2)GPI was caused by plasmin in the patients with leukemia, and by unknown proteases in the patients with LA. In the patients with LA, the levels of N-beta(2)GPI tended to be higher in those without thrombosis than in those with thrombosis.

Significance of coexpression of urokinase-type plasminogen activator, and matrix metalloproteinase 3 (stromelysin) and 9 (gelatinase B) in colorectal carcinoma.

Our previous study showed that proMMP-9 was activated by MMP-3 directly, and that proMMP-3 was activated by plasmin. It was postulated that the proMMP-9 activation mechanism through the protease-protease cascade existed even in vivo. The purpose of the present study was to clarify the clinical significance of the combined expression of MMP-9, MMP-3, and urokinase-type plasminogen activator (uPA) in colorectal cancer, and the role of MMP-3 or uPA expression as an activator for MMP-9. The expression of both MMP-9 and uPA was found to be correlated with liver metastasis, and with survival rate. The coexpression of MMP-9 and uPA by tumor cells was also significantly correlated with postoperative hepatic recurrence and survival rate. MMP-9 tended to be coexpressed with uPA, and was consistently associated with MMP-3 localized at the tumor-invasive front with inflammatory cells such as monocyte-macrophages. In gelatin zymography, the MMP-9 active form tended to be identified in the tumors that coexpressed both MMP-9 and uPA. We concluded that coexpression of MMP-9 and uPA in tumor tissues might be a useful predictive factor for postoperative survival and hepatic metastasis. The following activation mechanism for proteinase might occur: uPA coexpressed with MMP-9 activated plasminogen, and plasmin activated proMMP-3, which was secreted depending upon inflammatory infiltration, and then MMP-3 activated proMMP-9, resulting in colorectal cancer progression and metastasis.

Continuous intra-arterial 5-FU chemotherapy in a patient with a repeated recurrence of rectal cancer: report of a case.

PURPOSE: We report a patient with a recurrent pelvic tumor after abdominoperineal resection of a rectal carcinoma who was treated sufficiently by repeated intra-arterial infusions of 5-fluorouracil. METHODS: A continuous, 24-hour 5-fluorouracil administration was made through the bilateral internal iliac artery at a dosage of 250 mg/m2/day by the subcutaneous reservoir located at both upper legs using a Baxter infusor. RESULTS: In this patient pain in the hip and pelvis was relieved. A complete regression in the infused field of pelvic tumor was observed not only with computed tomography and magnetic resonance imaging but also confirmed by operative findings at the seventh month after the intra-arterial infusion. The abnormal serum level of carcinoembryonic antigen and carbohydrate antigen 19-9 was decreased to within the normal range at the 19th and 3rd week respectively. When the repeated recurrence was suspected in follow-up, normalization of the re-elevated carcinoembryonic antigen and carbohydrate antigen 19-9 levels was also obtained by repeating the same treatment. The side effects and complications were tolerable, consisting of local skin erosion on the hips and lower extremity neuropathy caused by the 5-fluorouracil. CONCLUSIONS: Clinical local regression of a pelvic recurrence was observed in a patient with rectal recurrent tumor who received continuous intra-arterial chemotherapy. Local recurrence of rectal cancer may be controlled effectively and safely by repeating long-term, continuous, intra-arterial 5-fluorouracil infusion.

Enhanced expression of caspase-3 in hypertrophic scars and keloid: induction of caspase-3 and apoptosis in keloid fibroblasts in vitro.

Recent studies have suggested that the regulation of apoptosis during wound healing is important in scar establishment and development of pathological scarring. To examine the phenomenon of apoptosis and its involvement in the process of pathological scarring, we immunohistochemically quantified differential levels of expression of caspase-3 and -2, which are activated during apoptosis in vitro, in surgical resected scar tissues. We divided 33 cases of normally healed flat scars and 18 cases of pathological scars (15 cases of hypertrophic scars and 3 cases of keloid) into three groups (S1 = <10 months' duration; S2 = 10 to 40 months' duration; and S3 = >40 months' duration) according to the duration of scar. In all three groups examined, the semiquantitative scores for caspase-3 staining were significantly higher for the combination of hypertrophic scars and keloid as a group compared with normally healed flat scars, suggesting reduced cell survival and increased apoptotic cell death in hypertrophic scars and keloid. Apoptosis and caspase proteolytic activities were examined in vitro using two flat scar-derived fibroblast lines (FSFB-1 and -2) and two keloid-derived fibroblast lines (KFB-1 and -2). After 24 hours of serum deprivation, apoptotic cells were significantly increased in both KFB lines, whereas serum deprivation of FSFB-1 cells did not result in a significant increase in apoptotic cell number. After serum deprivation, significant increases in caspase-3 proteolytic activities were detected in both KFB lines compared with both FSFB lines. In contrast, no significant differences with caspase-8 activity were observed between similarly treated KFB and FSFB lines. Furthermore, serum deprivation-induced apoptosis of KFB-2 cells was significantly inhibited by the caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-FMK), indicating that caspase-3 is important for serum deprivation-induced apoptosis in KFB-2 cells. Considering the role of caspase-3 as a key effector molecule in the execution of apoptotic stimuli, our results suggested that enhanced expression of caspase-3 in hypertrophic scars and keloid induces apoptosis of fibroblasts, which may play a role in the process of pathological scarring.

[Qualitative study of municipalities' maternal and child health promotion planning].

From 1997, basic maternal and child health services have been provided through municipalities. The ministry of Health and Welfare has requested all municipalities to publish a MCH promotion plan. We conducted a qualitative analysis of important contents of the plans using an evaluation index of original dichotomous variables. Out of 3,256 municipalities, 2,873 developed the plans for MCH promotion. Most of the plans present descriptions of the significance of MCH promotion planning, goals of the plans, problems of current MCH statistics and services, and plans of providing services. The proportion was low for plans which have needs assessment of target population, indicators of evaluation of plans, description of the relationship between objectives and services, importance of objectives, action plans, specific chapter or evaluation of the plan, monitoring of the plan, and plans for informing public of the MCH plan. Therefore, the MCH promotion plans have weaknesses in evaluation and action. The MCH promotion plans of middle sized cities were more likely to have these important contents. We will continue to analyze the processes for producing of excellent MCH promotion plans to extract universal promoting factors for producing MCH promotion plans.

A monoclonal antibody specific to the granulocyte-derived elastase-fragment D species of human fibrinogen and fibrin: its application to the measurement of granulocyte-derived elastase digests in plasma.

When granulocytes are stimulated under certain clinical conditions, elastase is released therefrom and digests fibrin(ogen) independently of the plasmin system, which may also be mobilized simultaneously. Thus, discrimination of these 2 systems becomes urgent for the diagnosis and treatment of the underlying diseases. Using as immunogen a 97-kd granulocyte-elastase digest of human fibrinogen, we raised an antibody IF-123 that specifically recognizes elastase digests of human fibrin(ogen). The 97-kd elastase fragment resembles plasmic fragment D(1), and the epitope of this antibody is located on the Aalpha (196-204) residue segment. This segment appears to be masked in fibrin(ogen) but exposed when the Aalpha Leu 204-Ile 205 peptide bond is cleaved by elastase. Cathepsin G concomitantly released from granulocytes failed to expose the epitope. By an enzyme immunoassay using IF-123 as the capture antibody, the elastase digests of fibrin(ogen) can be measured in plasma samples without interference by abundantly coexisting fibrinogen. Indeed, we found that the elastase digests were mostly elevated in patients with inflammation or malignant tumors, but remained in a normal range in patients with a benign gastrointestinal tract disease such as duodenal ulcer and polyps in the gallbladder or the colon. Like the plasmic D-dimer, the elastase digests predominantly consisted of the DD/E complex and DD/E-containing high-molecular weight derivatives apparently corresponding to the phase-3 plasmic digests of cross-linked fibrin. (Blood. 2000;95:1721-1728)

The estimation of cancer incidence in Aichi Prefecture, Japan: use of degree of completeness of registration.

This study was conducted to estimate true incidence of the total number of cancer cases and of cancers at selected sites in the population of Aichi Prefecture, Japan, using the degree of completeness of registration (CR). Information on incidence (I), death (D), and cases first notified via death certificate (DCN) was based on the data from 1976 to 1993. Using this information, CR for each cancer site was calculated by gender and a 3-year period of diagnosis [CR = (1-PDCN l/D)/(1-PDCN), PDCN: proportion of DCN cases]. The true number of incident cases and incidence rates (estimated incidence rate: EIR) were then estimated from the number of cases observed and from CR. CR for all sites of cancer was 62-78 percent in males and 54-72 percent in females, and CR was higher in cancers with low I/D ratios than those with high I/D ratios. Annually, about 2,500-3,000 cases in each gender were unreported. The EIR of all sites was estimated at 252-280 in males and 213-207 in females compared with the incidence rates observed (observed incidence rate: OIR) as 157.0-218.6 in males and 115.3-149.2 in females. OIR was closer to the EIR for cancers with low I/D ratios than those with high I/D ratios. These values are important for planning and evaluating cancer control strategies in the population.

Evaluation of the death certificate follow-up method for the analysis of survival rate: data from Aichi Prefecture, Japan.

To evaluate the use, for studies on survival, of death certificates from population-based cancer registries in Japan, we compared 5-year survival rates by two different methods: passive, derived from death certificates, and active, using family register systems. Registered cancer cases from Aichi Prefectural Cancer Registry were used as a model. The study subjects comprised 9244 cancer patients (3830 males and 5414 females) newly diagnosed at the Aichi Cancer Center Hospital and recorded in the Aichi Prefectural Cancer Registry between 1983 and 1991. The passive follow-up method, using death certificates, identified 87-89% of deaths among the study subjects. The proportion of deaths which were not identified by the passive follow-up method did not vary greatly with age or gender, but was higher among patients in the earlier rather than the later stages of cancer. Overall, the absolute and relative effects of unregistered deaths on apparent survival rate vary with the absolute survival rate. The absolute and relative differences between the survival rates calculated by the two methods are explained more clearly when data are analyzed by cancer site. The results of the present study provide useful information for interpreting the survival rate following diagnosis of cancer estimated by the passive follow-up method, i.e. using death certificates from a population-based cancer registry.

eaeA genes in Escherichia coli derived from Japanese patients with sporadic diarrhea.

To investigate the prevalence of attaching and effacing Escherichia coli, we examined 364 strains isolated from the feces of 9,684 patients with diarrhea at the Anjo Kosei Hospital in Japan for the presence of eaeA. Twenty-nine (8%) of the strains were eaeA positive. Of enteropathogenic E. coli (EPEC), 11 of the 87 (13%) strains were for the positive eaeA gene. The serotypes and the numbers of eaeA-positive strains among the strains tested were as follows: O26:H-(2/3), O55:H7 (4/4), O55:H-(2/ 2) and O128:H2(3/3). Two enterohemorrhagic E. coli (EHEC) strains (Verotoxin positive O157:H7) were also eaeA positive. Among 260 non-EPEC strains that were not categorized as diarrheagenic E. coli, 16 (6%) were eaeA positive. Those serotypes were as follows: O15:H2, O20:H6, O28:H28, O63:H6. O153:H7, O28:H6, O153:H19 and O157:H45. EPEC strains including O18:H7 and six other serotypes, enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC) were all eaeA negative.

Selective expansions of T cells expressing V beta 8 and V beta 13 in skin lesions of patients with chronic cutaneous lupus erythematosus.

There are several clinical types of cutaneous lupus erythematosus (LE), including acute cutaneous LE (ACLE), which occurs in 50-60% of patients with systemic LE (SLE), chronic cutaneous LE (CCLE), which is almost the same as discoid LE (DLE), and subacute cutaneous LE (SCLE). Although several important hypotheses have been proposed to explain cutaneous LE, the pathomechanisms still remain complicated and obscure. Of special interest is whether and how the T cell receptor (TCR) repertoire of infiltrating lymphocytes is involved in the development of the different types. To address this issue, we immunohistochemically examined the V beta usage of infiltrating T cells in skin lesions, as well as in peripheral blood mononuclear cells (PBMC) of patients with cutaneous LE. The number of V beta 3.1 CD3+ cells in the PBMC of patients with ACLE and CCLE was significantly lower than in controls. In contrast, the number of V beta 3.1 CD3+ cells was elevated in the skin lesion of CCLE over that in psoriasis vulgaris or atopic dermatitis. Furthermore, skin lesions in CCLE patients showed a higher incidence of V beta 8.1 CD3+ and V beta 13.3 CD3+ cells than did those in ACLE patients. These results suggest that skin lesions of CCLE are oligoclonally associated with selective expansions of TCR V beta chains and may be induced by antigen stimuli, including superantigens.

A monoclonal antibody that recognizes a neo-antigen exposed in the E domain of fibrin monomer complexed with fibrinogen or its derivatives: its application to the measurement of soluble fibrin in plasma.

Using urea-solubilized human fibrin monomer as an immunogen, we raised in mice a battery of monoclonal antibodies that reacted with the immunogen but not with urea-treated or native fibrinogen. Although they all failed to react with acid-solubilized fibrin monomer (acid-FM) alone, an antibody designated as IF-43 was found to recognize acid-FM, which was bound with fibrinogen or its derivatives to form a 1:2 complex of soluble fibrin. The epitope for this antibody, thus, appears to be exposed most probably by conformation changes induced in the acid-FM molecule upon formation of the complex. Because IF-43 was able to recognize fibrin-derived plasmic fragment E treated with urea but not the thrombin- and urea-treated amino-terminal disulfide knot of fibrinogen, the presence of the A alpha (52-78) residue segment seems to be prerequiste for the epitope expression. The antibody was found to react with soluble fibrin monomer spiked to normal plasma dose-dependently up to 200 micrograms/mL. By an aggregation assay using latex beads coated with IF-43, we found that concentrations of soluble fibrin monomer in plasma derived from patients with thrombotic diseases were mostly elevated, but not necessarily correlated with those of the D-dimer, reflecting another aspects of the disease. Furthermore, the soluble fibrin monomer in plasma derived from patients with thrombotic diseases was found to be depleted solely of the A peptides, but not the B peptides, based on its subunit polypeptide compositions lacking the beta-chain on immunoblotting.

Protective factor against progression from atrophic gastritis to gastric cancer--data from a cohort study in Japan.

To investigate the association between atrophic gastritis and gastric cancer and to identify the risk and protective factors for the progression of atrophic gastritis to cancer, we conducted a prospective study on 5,373 subjects with neither cancer nor resected stomach who underwent gastroscopic examination and completed a life-style questionnaire. After an average of 6 years of follow-up, 69 gastric-cancer cases were identified, 65 from the subjects without atrophic gastritis and 4 from the subjects without atrophic gastritis. The presence of atrophic gastritis increased the risk of gastric cancer 2.19-fold, the risk trend increasing with the degree and the extension of atrophy [relative risk (RR) 1.60 for mild atrophy and 2.85 for moderate and severe atrophy]. Among the subjects with atrophic gastritis, family history of gastric cancer (RR 2.27) and a preference for spicy food (RR 1.84) increased the risk and self-administered meal controls, such as portion reduction (RR 0.44) reduction of salty food (RR 0.56) and the change to the consumption of easily digested food (RR 0.57) decreased the risk of gastric cancer. The results of this study suggest that atrophic gastritis increases the risk of gastric cancer but that dietary modification prevents the progression from atrophic gastritis to gastric cancer, regardless of pre-cancerous lesions.

Types of methicillin-resistant Staphylococcus aureus associated with high mortality in patients with bacteremia.

Forty-seven strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from 47 patients with bacteremia were analyzed by chromosomal DNA digestion pattern using pulsed-field gel electrophoresis and evaluated for serological coagulase type, enterotoxin type, and toxic shock syndrome toxin-1 production. The mortality rate was significantly higher in the older patients (> or = 51 years of age) than in the younger patients (< or = 50 years of age) (50% vs. 4%, p = 0.0007). Methicillin-resistant Staphylococcus aureus strains of serological coagulase type II were more likely to be associated with mortality in older patients than were strains of the other types (p = 0.037).

[Morphological response of urinary bacteria to ceftibuten].

UNLABELLED: The morphological response of urinary Gram-negative bacteria to ceftibuten (CETB) was investigated in four patients with urinary tract infections (one patient: acute uncomplicated cystitis, three patients: chronic complicated urinary tract infection). The daily dose of CETB was 400 mg administered orally and the durations were 3 days for the acute uncomplicated cystitis case and 5 days for the chronic complicated urinary tract infection cases. In the four patients, changes in urinary CETB concentrations, viable bacterial counts, and morphology of the bacteria were studied after the initial administration. The urinary concentrations of CETB were 7.38-60.3 micrograms/ml at one hour after the first administration. The urinary viable cell counts were 1-5 x 10(7) cells/ml before administration, but they were reduced to 1-3 x 10(3) cells/ml at one hour after the first oral administration. Morphological changes of bacteria: Under phase contrast microscopy, filamentous debris and spherical bacteria with severe body damage were observed at 30 minutes after the first administration. By transmission electron microscopy, the cell wall of the filamentous bacteria showed a number of projections with formation of vacuolar structures in the space between the cell wall and the irregularly-shaped cytoplasm. According to the Japanese UTI criteria, the efficacy rate was 100% in the 4 patients. Neither side effect nor abnormal laboratory result was observed in any patient. CONCLUSION: The morphological changes of the bacteria suggested that CETB, in vivo, bounds not only penicillin-binding protein (PBP)-3 but also PBP-1.