Effectiveness of ultrasound-guided transversus abdominis plane block and rectus sheath block in pain control and recovery after gynecological transumbilical single-incision laparoscopic surgery.

PURPOSE: To evaluate the effectiveness of ultrasound-guided transversus abdominis plane (TAP) and rectus sheath (RS) blocks in pain management and recovery after gynecological single-incision laparoscopic surgery (SILS). MATERIALS AND METHODS: Abilateral TAP block (Group A, n = 9), bilateral TAP and RS blocks (Group B, n = 10), and a bilateral RS block (Group C, n = 9) with 40 ml ropivacaine per patient were conducted in 28 patients undergoing SILS for ovarian tumors. A pain score and walking distance in a 6-minute walk test (6MWT) were examined. RESULTS: Pain scores were significantly lower on postoperative day (POD) 3 than on POD 1 in Groups B (p = 0.03) and C (p = 0.02). The walking distance on POD 3 was comparable with that before surgery in Group C (p = 0.75), but shorter in Groups A (p = 0.004) and B (p = 0.02). CONCLUSIONS: The RS block alone was the most effective in relieving pain and accelerating general recovery after gynecological SILS.

Is driving a car a risk for Legionnaires' disease?

Legionnaires' disease (LD) is a major cause of severe community-acquired pneumonia but the source and mode of transmission are not always apparent, especially in sporadic cases. We hypothesized that LD can be acquired from the air-conditioning systems of motor cars. Swabs were taken from the evaporator compartments of the air-conditioning system of scrapped cars. Healthy subjects who were mainly employees of regional transportation companies were tested for antibody to Legionella pneumophila serogroups 1-6; they also completed a questionnaire. Legionella species were detected in 11/22 scrapped cars by the loop-mediated isothermal amplification method. The prevalence of microplate agglutination titres > or =1:32 was significantly higher in subjects who sometimes used car air-conditioning systems. Although we did not prove a direct link between Legionella spp. in the car evaporator and LD, our findings point to a potential risk of car air-conditioning systems in LD, which needs further investigation.

Modification of the sensitivity to cisplatin with c-myc over-expression or down-regulation in colon cancer cells.

Human colon cancer SW480 cells express the c-myc gene. On the other hand, SW480DDP cell lines resistant to cisplatin exhibited decreased c-myc gene expression, but their cell growth rates remained similar to those of their parental cells. Antisense oligonucleotides to c-myc inhibited c-myc expression and induced increased resistance to cisplatin in SW480 cells. In contrast, SW480DDP cells showed increased c-myc expression and reversed sensitivity to cisplatin when these cells were transfected with c-myc cDNA from the pLNCX plasmid. Moreover, SW480DDP cells transfected with c-myc cDNA induced apoptosis when exposed to cisplatin, but not SW480 cells transfected with an antisense sequence for c-myc. Transfection either with a c-myc antisense sequence or c-myc cDNA to these cells did not change their growth rates. Thus enforced expression of c-myc in SW480 and SW620 lines sensitizes cells to cisplatin-induced apoptosis, whereas the down-regulation of c-myc in SW480DDP and SW620DDP increases cisplatin resistance when using antisense strategy.

[Laboratory system for networks in infection control].

The goals of a surveillance for nosocomial infections are to observe the magnitude and characteristics of the infections, and to plan and evaluate policies and guidelines of infection control. In clinical laboratory, although it is most important to detect their causative pathogens, further to provide the surveillance study as a multicenter. Actually, we have just started an infection control room in our hospital. Thus this room may present various informations about infection and contribute to the networks with the area community.

Autoimmune neutropenia in pregnant women causing neonatal neutropenia.

Autoimmune neutropenia (AIN) can occur during pregnancy. However, neonatal neutropenia occurring in an infant born to a mother with AIN has only rarely been documented. Recently, we have experienced two cases of AIN during pregnancy, both of which caused severe yet transient neonatal neutropenia (< 0.3 x 10(9)/l), probably as a result of transplacental maternal anti-neutrophil autoantibodies. The anti-neutrophil antibodies seemed to be against antigens other than NA1/NA2 because the autoantibodies did not bind to neutrophils of specific NA types selectively in the granulocyte indirect immunofluorescence test. Although AIN is a relatively uncommon disease, neonatal neutropenia caused by maternal AIN may not be quite as rare.

Use of c-myb antisense oligonucleotides to increase the sensitivity of human colon cancer cells to cisplatin.

Human colon cancer SW480DDP and SW620DDP cells resistant to cisplatin exhibited stronger c-myb gene expression than the parent SW480 and SW620 cells. However, cell growth rates in the cisplatin-resistant cell lines remained similar to those of the parent cells. Antisense oligonucleotides to c-myb inhibited c-myb expression and induced increased sensitivity to cisplatin in SW480DDP and SW620DDP cells, but this did not occur with the control sense oligonucleotides. In contrast, the parent cell lines were not affected by antisense oligonucleotides to c-myb. These results indicate that the c-myb gene in human colon cancer is one of the factors related to cisplatin resistance, and support the need to develop anti-cancer therapeutics based on oncogene-targeted antisense oligonucleotide technology.

Increased sensitivity to cisplatin in gastric cancer by antisense inhibition of the her-2/neu (c-erbB-2) gene.

BACKGROUND: The c-erbB-2 oncogene encodes a transmembrane tyrosine kinase receptor and its abnormal expression may be related to the prognosis of gastric cancer. Gastric cancer is relatively resistant to various drugs, including cisplatin. Cisplatin is widely used in cancer chemotherapy, but the mechanisms of drug resistance are not yet known. METHODS: We used the human gastric cancer cell lines MKN-7 and KATO-III, which express the c-erbB-2 oncogene, as a model for relative resistance to cisplatin. We investigated whether inhibition with antisense oligonucleotides against c-erbB-2 increased the sensitivity of MKN-7 and KATO-III cells to cisplatin. RESULTS: Antisense oligonucleotides for c-erbB-2 inhibited the expression of c-erbB-2 mRNA and protein and increased sensitivity to cisplatin, but not to other drugs, in MKN-7 and KATO-III cells. Cell growth was also inhibited by c-erbB-2 antisense oligonucleotides but not sense oligonucleotides. CONCLUSION: These findings indicate that c-erbB-2 expression in gastric cancer is one of the factors related to cisplatin sensitivity, and that anti-c-erbB-2 antisense oligonucleotides induced increased sensitivity to cisplatin.

Childhood T-cell acute lymphoblastic leukemia with four distinct immunophenotypes representing different stages of T-cell development.

The authors report on a 14-year-old boy who developed T-cell acute lymphoblastic leukemia (FAB:L1) displaying 4 immunophenotypically distinct leukemic cell populations by 3-color immunofluorescence staining. Cytogenetic analysis at diagnosis showed 46,XY,add(4)(p16)[12]/46,XY[2]. A single rearrangement of the T-cell antigen receptor beta- and gamma-chain genes in these cells indicated monoclonality of the leukemic cells. These findings suggest that leukemic blast cells of monoclonal origin in this case were divided into 4 immunophenotypic populations, representing various stages of differentiation.

Quantitative evaluation of cytomegalovirus DNA in infantile hepatitis.

We used a PCR method to develop a diagnostic assay for the detection of cytomegalovirus (CMV) DNA in infantile hepatitis, which has been suggested to be associated with CMV infection. CMV DNA was detected in 25 (58.1%) of 43 patients with elevated serum alanine aminotransferase (ALT) levels but no jaundice, and no hepatitis B or C as assessed by conventional PCR. None of the samples from 97 healthy infants tested positive for CMV DNA. We assayed CMV DNA quantitatively in blood using a real-time PCR system that allowed reproducible detection of at least 10 copies of CMV DNA. When 1 microg of DNA from each blood sample was used in this system, a good correlation was obtained between the calculated and measured copy numbers of CMV DNA. This system detected CMV DNA in 29 patients (67.4%) with liver dysfunction. Serial studies in patients with liver dysfunction revealed that CMV DNA copy number decreased, ultimately to below 10, as the ALT levels normalized. In contrast, no CMV DNA copies were detectable by the real-time system in any of the samples from control subjects. These results highlight the usefulness of detecting CMV DNA in the diagnosis of infantile hepatitis and indicate that the real-time quantitative PCR assay may be a valuable tool for monitoring CMV-associated infantile hepatitis.

Increased sensitivity to cytosine arabinoside in human leukemia by c-raf-1 antisense oligonucleotides.

c-raf-1, a cytoplasmic serine/threonine protein kinase, plays an important role in mitogen- and damage-responsive cellular signal transduction pathways. Expression of c-raf-1 modifies cell growth, proliferation and survival. Although expression of c-raf-1 has been studied in several tumors, the role of c-raf-1 in leukemia is so far unclear. We examined the expression of c-raf-1 in the human leukemia cell lines U937 and K562, and in a cytosine arabinoside (Ara-C)-resistant cell line (K562AC) derived from K562. Expression of c-raf-1 was increased in U937 and in Ara-C-resistant K562AC cells compared with the parental cells. We then investigated whether inhibition of c-raf-1 expression by antisense oligonucleotides increases the sensitivity to Ara-C in U937 and K562AC cells. Antisense oligonucleotides for c-raf-1 inhibited expression of c-raf-1 mRNA, but did not affect cell growth and increased sensitivity to Ara-C but not to other drugs such as adriamycin, VP-16 or vincristine. These results suggest that c-raf-1 is one of the factors involved in Ara-C resistance in leukemia and lend weight to the case for development of anti-cancer therapeutics involving oncogene-targeted antisense oligonucleotides.

Quantitative PCR determination of human cytomegalovirus in blood cells.

We evaluated a rapid and sensitive method to determine human cytomegalovirus (CMV) DNA levels in blood cells using a quantitative polymerase chain reaction (PCR) technique. This method is based on real-time detection of PCR using a dual fluorescence-labeled probe and a sequence detector. Ten copies of CMV DNA were detected, when 1 microg of DNA from blood samples was used with this method, and a good correlation was obtained between increased concentrations of copy numbers calculated and measured copy numbers of CMV DNA (r = 0.999). Forty normal subjects exhibited no copies of CMV DNA. On the other hand, a 6-month-old girl tested positive for increased levels 4 weeks after liver transplant. This method is simple, accurate, and sensitive for the quantitative detection of CMV DNA in vivo, indicating possible applications for the diagnosis and monitoring of CMV infection.

[A case of Miller-Dieker syndrome associated with satellite on chromosome 17p].

In this report, we describe a one-year-old girl of the Miller-Dieker syndrome(MDS) with lissencephaly, seizures, microcephaly and mental disorders. Cytogenetic studies of this patient confirmed the presence of a 46,XX, 17ps+ chromosome karyotype, but it could not find the microdeletion of 17p13.3. Fluorescence in situ hybridization(FISH) studies confirmed a terminal deletion in the patient using the LIS1 gene probe which mapped to 17p13.3. Further it was also found the satellite on 17p13(17ps) in the patient who was rare associated with MDS. These findings suggest that FISH analysis may be useful method to detect microdeletion of LIS1 gene as 17-specific probe in the investigation of MDS patients.

Measurement of tumor necrosis factor-alpha messenger RNA in synovial fibroblasts by real-time quantitative reverse transcriptase-polymerase chain reaction.

We applied a real-time quantitative assay to determine the expression of tumor necrosis factor-alpha (TNF-alpha) messenger RNA (mRNA) in tissue samples. This method is based on the real-time monitoring of reverse transcriptase-polymerase chain reaction (RT-PCR) with a dual-fluorescence-labeled probe and a sequence detector. A linear correlation existed between assay measurements and input target (r = 0.999). TNF-alpha mRNA was detected in the synovial cells of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and also in the U937, THP1, and HL60 cell lines. Stimulation with interleukin-1alpha (IL-1alpha) caused an immediate increase in the intracellular level of TNF-alpha mRNA. In particular, the relative copy number of TNF-alpha mRNA increased dramatically from 15 to 3554 in synovial cells from RA patients. This method is simple, accurate, and sensitive for the quantitative detection of TNF-alpha mRNA. The real-time quantitative RT-PCR assay may be valuable for measuring TNF-alpha mRNA expression in clinical samples.

Clonal evolution from trisomy into tetrasomy of chromosome 8 associated with the development of acute myeloid leukemia from myelodysplastic syndrome.

Tetrasomy 8, though rare, is usually associated with trisomy 8, a far more common chromosomal abnormality in acute myeloid leukemia (AML). Yet the clonal relationship between trisomy 8 and tetrasomy 8 in the cases with these chromosomal abnormalities has been unclear. Here, we report a case of a 17-year-old male, diagnosed as having a myelodysplastic syndrome (MDS). Chromosome analysis showed the presence of trisomy 8. Five years later, he developed overt AML exhibiting tetrasomy 8 only. After chemotherapy, the blast cells in the bone marrow decreased to 3.4%, and the karyotype showed trisomy 8 alone. Fluorescence in situ hybridization using a probe specific for chromosome 8 showed that the percentages of cells exhibiting 2/ 3 /4 signals were 7.8/89.2/2.0 at the MDS stage, 20.5/36.1/41.0 when overt AML developed and 24.0/72.1/2.4 after chemotherapy. These results suggested that tetrasomy 8 is derived from the AML clone, possibly evolved from the MDS clone with trisomy 8. To our knowledge, this is the first detailed case report of clonal evolution from trisomy 8 into tetrasomy 8 associated with the development of AML from MDS.

[A case of 9p-syndrome identified chromosome 20 on chromosome 9p by M-FISH].

In this report, we describe one-year-old girl diagnosed with 9p-syndrome. Cytogenetic studies of this patient confirmed a karyotype of 46,XX,add(9) (p24) chromosome, but could not find the additional fragment on 9p22 in one allele. Fluorescence in situ hybridization (FISH) studies could not confirm the fragment in the patient using the LIS1 gene probe which mapped to 9p22. The more recently developed M-FISH method clearly showed that the additional fragment was 20p in this patient. These findings suggest that M-FISH analysis may be a useful method for identifying unknown additional and rearranged chromosomes.

Chemically modified ribozyme to V gene inhibits anti-DNA production and the formation of immune deposits caused by lupus lymphocytes.

A variety of autoantibodies is responsible for the tissue injury in autoimmune diseases. We have demonstrated that the human anti-DNA Ab O-81, of which Ids are commonly detected in renal glomeruli of active lupus nephritis, uses the V3-7 gene. We tried to develop a new therapy for lupus nephritis by using chemically modified ribozymes to specifically inhibit the expression of the mRNA of Ig V gene. The transfection of hammerhead ribozyme or the addition of chemically modified ribozyme against the flanking region of V3-7 caused a potent and selective inhibition of anti-DNA production in V3-7-using B cell clones, but not in irrelevant V gene-using clones in vitro. Chemically modified ribozyme was long-acting and resistant to RNase, and nonspecific cytotoxicity of the ribozyme was negligible. To know the efficacy of the ribozyme in vivo, we used a model of immune complex nephritis in SCID mice in which 5 x 10(6) PBLs from patients with active lupus nephritis (lupus PBL) were transferred twice. The injection of lupus PBL in combination with chemically modified ribozyme to increase resistance to RNase significantly reduced anti-DNA Ab levels in blood and decreased levels of urinary protein in the immune deposit models. Immunofluorescence study also revealed a marked decrease in IgG deposits at renal glomeruli in the ribozyme-treated group. These results indicate an efficacy of chemically modified ribozyme therapy for autoantibody-mediated immune diseases.

Quantitative analysis of estrogen receptor alpha and beta messenger ribonucleic acid levels in normal endometrium and ovarian endometriotic cysts using a real-time reverse transcription-polymerase chain reaction assay.

OBJECTIVE: To quantify messenger RNA (mRNA) levels of the two estrogen receptor isoforms, estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) in the eutopic endometrium and ovarian endometriotic cysts. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with endometriosis and patients with uterine leiomyoma or carcinoma in situ. INTERVENTION(S): Gonadotropin-releasing hormone agonist (GnRH-a)-treated (n = 12) or untreated (n = 24) endometriotic cysts were obtained from 36 patients during laparoscopic cystectomy. Eutopic endometrium tissues were obtained from 24 patients during or immediately after surgery. MAIN OUTCOME MEASURE(S): ER-alpha and ER-beta mRNA levels, using a real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay, TaqMan RT-PCR. RESULT(S): Eutopic endometrium and ovarian endometriotic cysts showed predominantly higher levels of ER-alpha mRNA than ER-beta mRNA. Although ER-alpha and ER-beta mRNA levels in the eutopic endometrium were affected by a cyclic change in ovarian hormones, ovarian endometriotic cysts were less affected. Moreover, a long-term hypoestrogenic state induced by GnRH-a especially decreased ER-alpha mRNA levels in endometriotic cysts. Consequently, the relative ratios of ER-alpha to ER-beta mRNA levels in both GnRH-a-treated and untreated endometriotic cysts were significantly lower than those in the eutopic endometrium. CONCLUSION(S): The results suggest that the principal and regulatory effects of estrogens may be mediated mainly via ER-alpha rather than ER-beta in both the eutopic endometrium and endometriotic cysts.

[Activities of antimicrobial agents against 5,180 clinical isolates obtained from 26 medical institutions during 1998 in Japan. Levofloxacin--Surveillance Group].

The surveillance study was conducted to determine the antimicrobial activity of fluoroquinolones (ofloxacin, levofloxacin, ciprofloxacin, tosufloxacin) and other 20 antimicrobial agents against 5,180 clinical isolates obtained from 26 medical institutions during 1998 in Japan. The resistance to fluoroquinolones was remarkable in Enterococci, methicillin-resistant staphylococci and Pseudomonas aeruginosa from UTI. However, many of the common pathogens such as Streptococcus pneumoniae including penicillin-resistant isolates, methicillin-susceptible Stahylococcus aureus, Moraxella catarrhalis, the family of Enterobacteriaceae, Haemophilus influenzae including ampicillin-resistant isolates have been kept to be susceptible to fluoroquinolones. About 90% of P. aeruginosa isolates from RTI were susceptible to fluoroquinolones. In conclusion, the results from this surveillance study suggest that fluoroquinolones are useful in the treatment of various bacterial infections including respiratory infections.

Detection of monoclonal proteins by capillary electrophoresis using a zwitterion in the running buffer.

Some cases have been reported in which a small monoclonal protein (M-protein) cannot be detected by conventional cellulose acetate membrane electrophoresis (CAE) or capillary zone electrophoresis (CZE) using a short fused-silica capillary. This is probably because these methods do not have the necessary sensitivity or resolution. To overcome this problem, we improved the CZE system by using a longer capillary and adding a zwitterion to the running buffer (pH 10.0). A comparison of CZE and CAE demonstrated that with the exception of alpha(1)- and alpha(2)-globulin, the correlation was satisfactory in serum samples from 34 patients with M-proteins which had been detected by immunoelectrophoresis. In addition, a comparison of CZE electropherograms with those from CAE showed that small M-proteins that went undetected by CAE could be detected by CZE in four patients whose diseases included epipharyngeal carcinoma, solitary plasmacytoma, Crow-Fukase syndrome and macroglobulinemia. The improved resolution produced by a longer capillary may be effective for the detection of small M-proteins.

Compound heterozygous mutations (PHE53/54DEL and HIS373LEU) of the P450c17 gene result in a 17alpha-hydroxylase/17,20-lyase deficient male pseudohermaphrodite with unambiguous external genitalia.

The autosomal recessive disease 17alpha-hydroxylase/17,20-lyase deficiency is characterized by mutation of the P450c17 enzyme, which catalyzes 17alpha-hydroxylation and 17,20-lysis in the steroidogenic pathways. Although 17 mutations of this enzyme have been reported, only a few of them resulted in a completely unambiguous phenotype of female external genitalia in 46, XY individuals. We report here a Japanese patient with a 46,XY karyotype, who showed such a unambiguous female external genitalia. Nucleotide sequencing of the P450c17 gene revealed the patient to be a compound heterozygote carrying two different mutations (PHE53/54DEL in exon 1 and HIS373LEU in exon 6). As these mutations have been previously detected in unrelated Japanese patients, it is confirmed that these mutations accumulate regionally. Since these mutations could be screened by a multiple genotyping method, the method is applicable when 17alpha-hydroxylase/17, 20-lyase deficiency is suspected in Japanese patients.