Clonal selection of HIV type 1 variants associated with resistance to foscarnet in vitro: confirmation by molecular evolutionary analysis.

Foscarnet (trisodium phosphonoformate, PFA) is an effective inhibitor of retroviral reverse transcriptase (RT) and is known to block the replication of human immunodeficiency virus type 1 (HIV-1). In this article we analyzed the evolutionary process in generating HIV-1 strains related to drug resistance, using PFA as a selective pressure. PFA inhibited virus replication and protected the virus-induced cell killing, but it did not completely eliminate HIV-1 during the course of 7 weeks of treatment. The nucleotide sequence of the 859-bp DNA fragment spanning the core region of the HIV-1 pol gene was determined for 51 clones obtained from genomic DNA of the HIV-1-infected cells at different time points during PFA treatment. The nucleotide sequence analysis documented the presence of a minor HIV-1 variant prior to the PFA treatment. Molecular evolutionary techniques were utilized to analyze how the minor HIV-1 clones became predominant during this evolutionary process under the selective pressure of PFA. A phylogenetic tree analysis divided these 51 HIV-1 clones into 3 groups. One of the groups consisted of the clones associated with the resistance to PFA. The clones belonging to this group became predominant over time during the course of PFA treatment. Thus, the acquisition of PFA resistance by HIV-1 was considered to be due to clonal selection. Furthermore, among the various amino acid substitutions observed, the substitution of arginine at position 172 by lysine (Arg172Lys) clearly distinguished this group from the others. Since the consistent amino acid substitution observed here has not been identified in the HIV-1 strains resistant to other RT inhibitors, PFA in combination with other RT inhibitors is considered to be a feasible candidate for a convergent combined chemotherapy against HIV-1 in the treatment of patients with AIDS and related conditions.

Coordinated expression of integrin alpha6beta1 and laminin in hepatocellular carcinoma.

The interaction between tumor cells and laminin mediated by laminin-binding integrins is critical for tumor invasion and metastasis. The aim of this study was to clarify the altered expression of laminin-binding integrins with the change of laminin deposition in hepatocellular carcinoma (HCC) in comparison with cirrhotic or normal liver by immunohistochemistry. In HCC, hepatoma cells and sinusoidal endothelial cells expressed integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta1. Integrins alpha1beta1 and alpha6beta1 were detected in a continuous pattern along the sinusoids in accordance with laminin assembly. Integrins alpha2beta1 and alpha3beta1 were detected in a discontinuous pattern at these sites. Integrin alpha6beta4 was not detected. In cirrhotic liver, although integrins alpha1beta1 and alpha6beta1 as well as laminin were detected in a continuous pattern along the sinusoids, integrins alpha2beta1, alpha3beta1, and alpha6beta4 were not detected. In normal liver, although integrin alpha1beta1 was detected in a continuous pattern along the sinusoids, neither integrins alpha2beta1, alpha3beta1, alpha6beta1, alpha6beta4, nor laminin were detected. We have clarified that, of laminin-binding integrins, the localization of integrin alpha6beta1 shows the best correspondence with the localization of laminin. These results suggest that of laminin-binding integrins, integrin alpha6beta1 is very important for cell-laminin interactions in HCC.

Pleural SC5b-9 in differential diagnosis of tuberculous, malignant, and other effusions.

A monoclonal antibody against soluble phase-terminal complement complex (SC5b-9) was used to try to differentiate pleural effusions of tuberculous vs malignant and other origin. Effusions of tuberculous origin showed a significantly higher SC5b-9 level than did plasma, suggesting activation of complement in the pleural space. All 26 patients with tuberculous effusions showed SC5b-9 levels in pleural fluid exceeding 2.0 mg/L, while 20 with malignant effusions had levels less than 2.0 mg/L. However, rheumatoid, some parapneumonic, and treated malignant effusions showed SC5b-9 levels above 2.0 mg/L. Considering a value exceeding 2.0 mg/L, the specificity and sensitivity of the SC5b-9 estimation in tuberculosis were 0.74 and 1.0, respectively. The mean values for C4d and Bb fragments of complement were significantly (p < 0.05) higher in the tuberculous than in the malignant effusions. However, the values for Bb in 16 (62 percent) of the 26 patients with tuberculous or malignant effusions were in the same range. The activity of adenosine deaminase (ADA) was higher in the tuberculous than in the malignant effusions. While 18 of 26 patients with tuberculous effusions showed an ADA value exceeding 50 mU/ml, the estimated cutoff point (sensitivity = 0.69), 35 of the 36 nontuberculous effusions showed a true negative value (specificity = 0.97). A correlation between ADA and SC5b-9 values was observed in pleural effusions. These observations suggest that the estimation of SC5b-9 in pleural fluid presents a new approach to differentiating tuberculous vs malignant effusions.

The effects of prostacyclin analog OP-41483 on normothermic liver ischemia and reperfusion injury in rats.

To estimate the effects of the prostacyclin analog (OP-41483) on normothermic liver ischemia and reperfusion injury, saline (Group 1, N = 8), heparin (group 2, N = 8, 100 u/kg) or OP-41483 (group 3, N = 8, 400 ng/kg/min) was infused intravenously for 30 min before and after liver ischemia in rats. There were no significant differences in survival, or transaminase at 30 min after reperfusion among the three groups. Hepatic vessel flow and tissue flow were measured for the first 30 min after reperfusion. Hepatic tissue flow increased for the first 30 min after reperfusion in the group 3 rats, but not in the groups 2 and 3 rats. There were significant differences in hepatic tissue flow between the groups 1 and 3 rats at 20 min (p < 0.05), as well as significant differences between the groups 1 and 3 rats (p < 0.01) and the groups 1 and 2 rats (p < 0.05) at 30 min after reperfusion. There were no significant differences in total hepatic inflow among the three groups. Our data suggest that OP-41483 exerts beneficial effects by improving the microcirculation and increasing the effective hepatic blood flow in the ischemically injured liver after reperfusion.

Different site mutation of the K-ras gene in a patient with metachronous double lung cancers.

We present a case with metachronous double lung cancers proved by molecular analysis. A male patient first had lung cancer discovered when he was 67 years old, and a second lung cancer was found when he was 71 years old. Pathologically, the first cancer was moderately to well differentiated adenocarcinoma and the second cancer was well differentiated adenocarcinoma. These findings suggested that the second cancer was not metastasis from the first lung cancer but a second primary lung cancer. According to the PCR and oligonucleotide mutation specific dot blot hybridization, the first lung cancer had a GGT to GTT mutation, while the second lung cancer had a GGT to TGT mutation at codon 12 of the K-ras gene. These results thus prove that these tumors were metachronous double cancers.

Analysis of environmental carcinogens associated with the incidence of lung cancer.

It is presumed that carcinogens present in human lungs contribute to the incidence of lung cancer. Most of the carcinogens are inhaled in lung alveoli with particulate matter through the respiratory tract. On the basis of chemical analysis of 256 lung specimens with carcinomas resected in the period 1991 1996, the concentration of 1-nitropyrene (1-NP) was 19.7+/-10.5 pg/g of dry weight, and that of the dinitropyrenes (DNP) was 3.50+/-0.14-6.26+/-1.76. In addition, 2-nitrofluoranthene (NF) and 3-NF were detected at a higher level of 38.6+/-17.2 and 39.1+/-14.2, respectively, pg/g of dry weight. Concentrations of benzo[a]pyrene, benzo[e]pyrene, benzo[k]fluoranthene and benzo[ghi]perylene in 37 specimens collected in the period 1991 1996, were in the range of 138+/-82-399+/-220 pg/g of dry weight. No difference was found in the concentration of chemicals deposited in lung specimens from patients with lung cancer and tuberculosis as a control. By following the prognosis of 112 patients with carcinomas, we found that the deposition of 1-NP, 1,3-DNP, and 3-NF in lung tissues influenced their 5-year-survival after determination of chemicals. Lung specimens were divided into two groups of higher and lower chemical concentrations at the levels of 18 pg/g for 1-NP, 15 for 1,3-DNP, and 35 for 3-NF, and the findings were statistically analyzed by adjusting for age, gender, smoking status and cell type. The 5-year-survival of patients was markedly lower in the higher concentration group than the lower group.

Fibrogenesis in acute liver injuries.

We describe changes in the hepatic extracellular matrix (ECM) and its producing cells in acute liver injuries, primarily referring to transforming growth factor-beta 1 (TGF-beta), which is the most important cytokine involved in fibrogenesis. In addition, we describe the relationship between vitronectin (VN) and plasminogen activator inhibitor 1(PAI-1) in the self regulating mechanism of TGF-beta action for fibrogenesis in acute viral hepatitis. In the very early stage of acute liver injury, following aggregation of platelets, immunolocalization of TGF-beta is observed in injured areas. And at the cell migration stage, the infiltration pattern of inflammatory cells was characterized by an ordered progression of inflammatory cells, beginning with platelets and followed by polymorphonuclear leukocytes and macrophages. Following recruitment of inflammatory cells to the necrotic area, it appears that TGF-beta could be produced and activated by these inflammatory cells, resulting in the intensification of active TGF-beta distributions in the injured area. At the fibrosis stage, TGF-beta could also be produced by Ito cells, endothelial cells and hepatocytes at the periphery of the necrotic area, and may play important roles in the promotion of production and accumulation of ECM components in injured regions. In addition, the consistent localization of PAI-1 with VN in ECM near the necroinflammatory areas suggests that PAI-1 and VN could be involved in the modulation of fibrogenesis in acute liver injuries. In many kinds of acute liver injuries, fibrogenesis is usually considered to be transient, and injured liver tissues able to nearly recover in order.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of fibrous capsule formation surrounding hepatocellular carcinoma. Immunohistochemical study.

In 14 cases of hepatocellular carcinoma with capsule, we studied the mechanism of capsule formation by the immunoperoxidase technique using antibodies to types I, III, and IV collagen, antilaminin antibody, and anti-prolyl hydroxylase antibody. Marked round cell infiltration was observed in the noncancerous side of the capsule and around compressed hepatocytes near the capsule. Thin capsules were composed primarily of type III collagen produced by an increased number of fibroblasts, transitional Ito cells, and hepatocytes near the capsule. In thickened capsules, the noncancerous side consisted primarily of type III collagen and the cancerous side of types I and III collagen. Type I as well as type III collagen was produced by fibroblasts, transitional Ito cells, and hepatocytes. The capsule thus formed is suggested to be part of the defense mechanisms against the growth of hepatocellular carcinoma.

Urinalysis of body packers in Japan.

Urinalysis was performed on nine body packers/smugglers who were referred to the emergency room of a hospital near the New Tokyo International Airport between September 1994 and February 1996. This analysis had rarely been used on suspected body packers in Japan. Only one of the nine body packers was a female. Foreign bodies were detected in the gastrointestinal tracts of the body packers by plain x-ray photography or computerized tomography, and the suspected drugs were cocaine (five cases), heroin (two cases), opiate (one case), and marijuana (one case). The results of urinalysis and confessions of the smugglers corresponded well for the latter three drugs (four cases). In two of the suspicious cocaine cases, opiates were detected along with cocaine by urinalysis, and the metabolites were confirmed by gas chromatography-mass spectrometry. The urinary screening tests of another two suspicious cocaine cases were negative. Opiates (morphine and codeine), but no cocaine metabolites, were detected in the urine of the subject who confessed to smuggling in cocaine only. It may be inferred from these results that urinalysis on body packers is beneficial to both the patient (body packer) and the physician in preventing the disastrous outcome of drug intoxication. It can also be concluded that there is a need for the prompt establishment of a protocol that includes urinalysis upon admission to the hospital for the management of body packers in Japan.

Long-term follow-up of interferon-treated chronic hepatitis C and serum hepatic fibrosis markers.

BACKGROUND/AIMS: We investigated the clinical application of serum fibrosis markers in a long-term follow-up of patients with chronic hepatitis C treated with interferon-alpha. METHODOLOGY: This study included 52 patients treated with interferon-alpha (total: 480 MU) for 6 months. They each underwent liver biopsy before and after treatment. Twenty-eight patients who underwent liver biopsy less than 2 years after treatment were classified as group 1, and 24 patients as group 2. The two groups were subdivided into HCV RNA-negative responders and HCV RNA-positive nonresponders. Liver specimens were estimated using grading and staging scores. Serum hyaluronan, PIIIP, and type IV collagen levels were measured before and after treatment. RESULTS: In the responders of groups 1 and 2, grading score after treatment was significantly decreased compared with that before treatment. Staging score after treatment was significantly improved only in the responders of group 2. In the responders of group 2, serum hyaluronan level was significantly decreased compared with that before treatment. In group 2, the grading score was significantly correlated with serum PIIIP and type IV collagen levels, and the staging score was significantly correlated with only serum hyaluronan level. CONCLUSIONS: These findings indicate that the serum PIIIP and type IV collagen levels reflect the activity, and serum hyaluronan reflects the degree of fibrosis in liver specimens of HCV RNA-negative patients in a long-term follow-up of patients after interferon-alpha treatment.

Serum hyaluronate predicts response to interferon-alpha therapy in patients with chronic hepatitis C.

BACKGROUND/AIMS: The response to interferon therapy for chronic hepatitis is known to decrease with progression of the hepatic fibrosis. On the other hand, serum hyaluronate reflects hepatic sinusoidal capillarization or liver cirrhosis, and also serum type IV collagen, which is one of the main components of the basement membrane, rises with the progression of hepatic fibrosis. In this study, the relationship between the degree of hepatic fibrosis and the response to interferon-alpha was determined retrospectively in patients with chronic hepatitis C. In addition, whether the measurement of serum hyaluronate and type IV collagen before interferon-alpha therapy was useful for predicting the response to interferon-alpha therapy in chronic hepatitis C was determined. MATERIALS AND METHODS: Thirty-seven patients with elevated serum ALT levels for at least 6 months and histologically determined chronic hepatitis were studied. All patients were positive for anti-HCV and negative for hepatitis B surface antigen. Twenty-eight healthy adults with normal blood biochemical data, who were negative for hepatitis B antigen and HCV antibody tests, had limited alcohol intake were used as controls. The test group was given IFN-alpha by intramuscular injection for 14 days, and then were treated 3 times per week for 24 weeks. RESULTS: The extent of hepatic fibrosis, particularly, perisinusoidal fibrosis (P < 0.01) was significantly greater in nonresponders than in responders. The mean serum hyaluronate and type IV collagen levels were more elevated in nonresponders than in responders, especially, the serum hyaluronate level showed a significant difference (P < 0.01). Most of the patients having a serum hyaluronate level of more than 100 ng/ml were nonresponders who had chronic active hepatitis with bridging necrosis on liver biopsy. Serum hyaluronate and type IV collagen levels showed significant positive correlation with degree of the portal fibrosis (P < 0.01), perisinusoidal fibrosis (P < 0.001) and focal necrosis (P < 0.01) in histological findings of liver biopsy specimens. CONCLUSION: These results suggest that serum hyaluronate and type IV collagen levels reflect the extent of the hepatic fibrosis in chronic hepatitis C and also that serum hyaluronate level predicts the response to interferon-alpha therapy in patients with chronic hepatitis C.

Biological monitoring of metabolites of sarin and its by-products in human urine samples.

More than 20,000 passengers of Tokyo underground trains were intoxicated with warfare toxic chemicals. Most of the patients examined had marked miosis and decreased serum cholinesterase activity. Transient increase of serum CPK activity after 3 days of the exposure was the another sign. We intensively analyzed the metabolites in the urine of 4 patients. The following analytic results indicated the exposure to sarin as well as contaminated compounds such as diisopropyl methylphosphonate (DIMP), ethyl methylphosphonate fluoridate (EMPF, or ethylsarin), diethyl methylphosphonate (DEMP), and ethyl isopropyl methylphosphonate (EIMP). (1) Isopropanol (IPA) and ethanol (EtOH) were detected of large quantities in the urine samples, and were thought to be derived from sarin and the sarin counterpart, EMPF, DIMP, DEMP and EIMP. (2) Monoalkyl methylphosphonic acids (isopropyl methylphosphonic acid (IMPA) and ethyl methylphosphonic acid (EMPA) also were excreted in large amounts with taking the similar excretion pattern of IPA and EtOH. (3) The metabolite only derived from sarin and ethylsarin is F anions whose integral output in the urine was less than the equimolar level of the excreted (IMPA + EMPA + IPA + EtOH). (4) Other corroborative findings were low lethality: of more than 5,510 patients treated, 11 were acutely dead. (5) Nine exposed males had higher sister chromatid exchange (SCE) rate (5.00 +/- 1.48/cell) than the control (3.81 +/- 0.697/cell), because dialkyl methylphosphonates seemed to have alkylating activity and producing DNA adducts. The SCE rate also increased after the in vitro exposure of lymphocytes to dialkyl methylphosphonates.

[Clinical studies on effect of cefbuperazone on surgical infections].

We conducted clinical studies on the efficacy and safety of cefbuperazone (CBPZ) on surgical infections with the following results. 1) In evaluable 32 patients, CBPZ was evaluated to be clinically effective in 23 (71.9%) and the efficacy rate was better in those who were administered 4 g/day of CBPZ (87.5%, 14/16) than in those who were given 2 g/day (57.1%, 8/14). 2) Antibacterial activity of CBPZ was evaluated in 41 isolated bacterial strains. Pathogen eradication rate by bacterial species was 75.6% (31/41). CBPZ exerted excellent antibacterial effects on Escherichia coli (100%, 8/8) and anaerobic bacteria such as Bacteroides (88.9%, 8/9). Resistant bacteria to CBPZ were Enterococcus faecalis and Pseudomonas aeruginosa. 3) No serious side effects were noted in any of the 34 patients who entered in this study. Abnormal laboratory test results were noted in 2 patients (5.9%) and they were transient elevation of transaminases and alkaline phosphatase. From the results shown above, CBPZ appears to be a highly useful antibiotic for the treatment of surgical infection.

Patent vitelline duct in an adult deceptively appeared to be acquired umbilical urachal sinus: a case report.

Here is presented a surprisingly rare case in a 40-year-old male who had patent vitelline duct by nature. However, his congenital disease appeared deceptively to be an acquired umbilical urachal sinus on the diagnostic evaluations including fistulography before surgery. The diagnosis was definitely confirmed after the successful surgical procedure. The principal reason why these diseases were indistinguishable was reviewed. The incidence of each disease and incidence of association with umbilical fistula in each disease were discussed. With regard to these incidences, we compared urachal anomalies with vitelline duct anomalies through reference of several literatures. This is the most unique event we have ever clinically experienced.

[A case of bilateral synchronous renal cell carcinoma treated with left partial nephrectomy and laparoscopic right nephrectomy].

We describe a case of bilateral synchronous renal cell carcinoma. A 70-year-old female was admitted to our department because of further examination for bilateral renal masses. Computed tomographic scanning and ultrasound examinations revealed bilateral solid enhanced renal masses, and bilateral renal cell carcinomas were suspected. First, partial left nephrectomy was performed. On the 21th day after the first operation, we confirmed the recovery of the left kidney, and performed right nephrectomy laparoscopically. The histopathological diagnosis revealed bilateral renal cell carcinomas. The patient is alive with no metastatic lesions and no recurrence at 16 months after the operations.

[Long-term effects of bicycle-ergometry training on exercise endurance and dyspnea in patients with chronic pulmonary emphysema].

Exercise reconditioning has long been considered an essential component of the pulmonary rehabilitation regime. To investigate the long-term effects of exercise training on exercise endurance and dyspnea in patients with chronic pulmonary emphysema (CPE), we developed a supervised exercise program using bicycle ergometers. Nineteen patients (mean age: 67) with moderate to severe airflow limitation (average FEV1/FVC, 39.8%) were enrolled in a predominantly outpatient rehabilitation program. After their lactate thresholds were measured by an incremental symptom-limited maximum test, patients engaged in exercise training 15 to 30 minutes per day in an arbitrary frequency and at a work rate at or below their lactate threshold (average: 20.1 w). No significant improvements were observed in resting pulmonary function, blood gas, nutrition, or systemic muscle volume. After long-term exercise training (average period: 33 months; average frequency: 6.5 times per month, individually), the patients demonstrated significantly improved exercise endurance and relief of their dyspnea during exercise. These findings provide a physiologic rationale for long-term exercise training by patients with CPE.

[Change in prevalence of allergic diseases in primary school children in Fukuoka City for the last fifteen years].

We have been investigating the yearly change of prevalence rate of childhood allergic diseases using same method and in same region in five primary schools in Fukuoka City for the last fifteen years. From 1981 to 1995, revised ATS-DLD questionnaire had been administered yearly to the first graders of primary school children (6-7 yrs). The total number of subjects for fifteen years were 8000. The average number of children in each year was 533. 1. The cumulative prevalence of bronchial asthma statistically increased from 5.7% (average of 1981 to 1983) to 7.7% (1993 to 1995). It was 1.7 times higher on boys. 2. The cumulative prevalence of atopic dermatitis did not change yearly and its average was 36.3%. The boys/girls ratio was 1.2. Remission rate of atopic dermatitis statistically increased from 14.3% (average of 1987 and 1988) to 19.6% (1994 and 1995). 3. The cumulative prevalence of allergic rhinitis did not change yearly and its average was 17.6%. It was 1.5 times higher on boys. 4. The cumulative prevalence of allergic conjunctivitis statistically increased from 8.4% (average of 1987 and 1988) to 11.1% (1994 and 1995).

[Sarcoidosis presenting as a diffuse micronodular shadow on a chest radiograph].

A 36-year-old man was admitted to our hospital with chest-radiographic findings of diffuse ground-glass shadows in both lungs. A chest CT scan revealed disseminated micronodules with mediastinal lymphadenopathy. Histological examination of a transbronchial lung-biopsy specimen showed non-caseous epithelioid cell granulomata. Marked lymphocytosis and an abnormally high CD 4/CD 8 ratio was found in bronchoalveolar lavage fluid. These findings suggested a diagnosis of sarcoidosis despite the atypical radiographic findings.

[Subcutaneous sarcoidosis of the extremities].

A 65-year-old man was referred to our hospital due to abnormal chest roentgenograms showing bilateral hilar enlargement with diffuse micronodular opacities. He presented with uveitis and elevated serum ACE and tested negative for tuberculin response. Transbronchial lung biopsies as well as bronchoalveolar lavage were performed and yielded a diagnosis of sarcoidosis. Several months later, the patient showed multiple subcutaneous nodules around the knee joints and elbow joints with exacerbation of intrapulmonary lesions. A skin biopsy revealed multiple foci of typical non-caseating epithelioid cell granulomata. These pulmonary and subcutaneous lesions rapidly resolved in response to the systemic administration of corticosteroids. Subcutaneous sarcoidosis may be a subacute variant of sarcoidosis associated with systemic involvement.

[Cough variant asthma among patients with chronic persistent cough and its clinical outcome].

Thirty-nine patients with chronic persistent cough continuing more than eight weeks were examined regarding to their airway responsiveness to methacholine challenge using the Astograph (TCK-6100H, Chest Corp. Japan). Of these, twenty-three individuals (59%) had airway hyperresponsiveness, leading to diagnose as having cough variant asthma (CVA). Patients with CVA had a higher percentage of eosinophils in peripheral blood and a lower forced expiratory volume in one second than those in non-CVA group. Furthermore, four patients (17%) developed the classic signs and symptoms of asthma, whereas 7 cases (30%) resolved their cough without further treatment and 12 cases (52%) continued to have CVA. These data suggested that a significant proportion of patients with chronic persistent cough had CVA and some of them may develop to classic wheezing asthma.