Rokitamycin: bacterial resistance to a 16-membered ring macrolide differs from that to 14- and 15-membered ring macrolides

Rokitamycin is the latest semi-synthetic 16-membered ring macrolide introduced into clinical practice. It is characterized by greater hydrophobicity, better bacterial uptake and a slower release, more cohesive ribosomal binding, and a longer post-antibiotic-effect (PAE) than can be observed with other available 14-, 15- and 16-membered ring macrolides. Rokitamycin exerts its activity on strains that harbor inducible erm genes or the efflux gene, mef(A). It has also been reported to be more active in vitro than other 16-membered ring macrolides. However, these recognized features are not fully exploited yet because current automated test procedures used in many microbiological laboratories determine susceptibility only to erythromycin or clarithromycin. Resistance to 16-membered ring macrolides cannot be predicted solely on the basis of known resistance to erythromycin or clarithromycin as revealed by an automated susceptibility assay. At least equally important is the knowledge of the bacterial resistance phenotype. This is underlined by the existence of Gram-positive coccal strains resistant to erythromycin and other 14-,15-membered ring macrolides but susceptible to 16-membered ring macrolides. Since the local prevalence of erythromycin phenotypes is generally unknown but might determine the outcome of treatment, the procedure for identifying the phenotypes in erythromycin-resistant strains (which can be easily and cheaply performed using the two- or three-disk assay) should become routine, at least in the countries in which 16-membered ring macrolides are used. This approach should help to optimize the use of macrolides, improve our knowledge of the local prevalence of phenotypes resistant to erythromycin, and offer the possibility of treating infections caused by certain types of erythromycin-resistant pathogens.     

Maturation and differentiation of B16 melanoma cells induced by theophylline treatment.

Recent studies suggested that 3',5'-cyclic AMP (CAMP) may be involved in the regulation of cell proliferation and differentiation. Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, elevated intracellular cAMP. A melanotic clone of the B16 melanoma was treated with theophylline and studied in vitro and in vivo. With 12 hours after 1.0 mM theophylline was added to growing cultures, the number of cells incorporating tritiated thymidine (3-H-TDR) and the rate of uptake of 3-H-TDR into DNA were significantly reduced. After 7 days, the number of cells in the control cultures increased twenty-four times, whereas theophylline-treated cells increased only sixfold. Compared to the controls, the theophylline-treated cells contained ten times the melanin and an elevated cAMP content. Stimulation of melanogenesis and inhibition of proliferation increased progressively with duration of exposure to theophylline. After 5 days of culture with theophylline, cells were assayed for plating efficiency in theophylline-free medium. Although the number of colony-forming cells was unaffected by previous exposure to theophylline, the colonies were composed of fewer cells inoculated into syngeneic hosts were less tumorigenic than untreated cells. However, theophylline treatment of hosts bearing B16 tumors failed to reduce the tumor growth rate, and theophylline did not potentiate the growth inhibition resulting from treatment with the synthetic polyribonucleotide, polyinosinic-polycytidylic acid.     

Effects of theophylline treatment on mouse B-16 melanoma cells in vitro

The effects of theophylline treatment on mouse B-16 melanoma cell growth, metabolism, and membrane antigen expression in vitro were studied. Theophylline treatment inhibited DNA synthesis and the cell growth rate, and caused an elevation of intracellular cAMP levels. Cells treated with theophylline became elongated and assumed a normal fibroblast-like morphology. Theophylline treatment of B-16 cells also reduced the levels of tumor specific antigen and H-2 antigen detectable on the cell membrane.     

In vitro assessment of rokitamycin against problem gram-positive cocci.

Rokitamycin was more active than erythromycin against erythromycin-sensitive strains of Staphylococcus aureus and enterococci, but somewhat less active against coagulase-negative staphylococci. Strains with inducible resistance to erythromycin were uniformly resistant to erythromycin, while rokitamycin was active against such strains. Strains with constitutive resistance to erythromycin were also uniformly resistant to erythromycin, and most were also resistant to rokitamycin. However, 5 of 21 coagulase-negative staphylococci and 2 of 20 enterococci remained sensitive to rokitamycin. This is a novel finding, perhaps suggesting a new mechanism of macrolide resistance.     

Roxithromycin and clarithromycin, 14-membered ring macrolides, potentiate the antitumor activity of cytotoxic agents against mouse B16 melanoma cells

We previously reported antiangiogenic activity of roxithromycin and clarithromycin, 14-membered ring macrolide antibiotics. In the present study, we examined the antitumor effects of roxithromycin and clarithromycin, alone and in combination with several cytotoxic drugs, on mouse B16BL6 melanoma cells in vivo and in vitro. Both roxithromycin and clarithromycin potentiated the inhibition of tumor growth induced by cyclophosphamide, cis-diamminedichloroplatinum(II), Adriamycin and vindesine in vivo. However, neither roxithromycin nor clarithromycin, altered the cytotoxicity of 4-hydroperoxycyclophosphamide, cis-diamminedichloroplatinum(II), Adriamycin or vindesine in an in vitro cell proliferation assay. These results suggest that the antiangiogenic activity of roxithromycin and clarithromycin may provide beneficial effects in combination with cytotoxic therapies against solid tumors.     

In vitro and ex vivo effects of recent and new macrolide antibiotics on chemotaxis of human polymorphonuclear leukocytes.

The effects of five macrolide antibiotics: erythromycin, josamycin, miokamycin, roxithromycin and rokitamycin, on human polymorphonuclear leukocyte (PMN) chemotaxis was studied in vitro and ex vivo. At therapeutic concentrations none of the antibiotics tested affected in vitro PMN chemotaxis. In vitro, erythromycin, josamycin, miokamycin, roxithromycin and rokitamycin decreased PMN chemotaxis significantly only at the concentration of 10 mg/l, which is not usually reached in vivo. Ex vivo studies after the ingestion of therapeutic doses of erythromycin, josamycin, miokamycin and roxithromycin by five volunteers showed a significant effect on PMN chemotaxis. However, further studies are needed to confirm and better evaluate the clinical significance of recent and novel macrolides on PMN chemotaxis.     

Impact of treatment on the outcome of acute myeloid leukemia with inversion 16: a single institution's experience.

To identify treatment factors that may affect the survival of children with inv(16)(p13.1q22), we compared the outcomes of 19 patients with this genetic feature treated at our institution during two treatment eras. Nine patients were treated during era 1 (1980 to 1987), and 10 were treated during era 2 (1988 to 1996). All entered complete remission (CR) with induction therapy. Eight of the nine children treated in era 1 died, seven of relapsed leukemia. In contrast, three of 10 patients treated during era 2 have died, all of non-disease-related causes. Event-free survival (EFS) estimates were significantly higher for patients treated during era 2 than for those treated during era 1 (P = 0.03); the 6-year estimates were 70 +/- 15% (s.e.) and 11 +/- 7%, respectively. Era 2 treatment protocols differed from those of era 1 in their use of higher doses of cytarabine and etoposide during induction and consolidation chemotherapy and in their use of 2-chlorodeoxyadenosine (2-CDA). These results suggest that dose intensification of cytarabine benefits children with AML and inv(16), as is the case in adults. They also suggest that dose intensification of etoposide and addition of 2-CDA may also offer an advantage. This study underscores the dependence of the prognostic impact of cytogenetic features on the efficacy of treatment.     

Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients.

PURPOSE AND EXPERIMENTAL DESIGN: Using real-time quantitative methylation-specific PCR (RTQ-MSP), we quantified methylated p16INK4a sequences and determined the fractional concentrations of circulating tumor DNA in plasma, serum, and peripheral blood cells collected preoperatively, intraoperatively, and postoperatively from 49 patients with hepatocellular carcinoma (HCC). RESULTS: RTQ-MSP was sufficiently sensitive to detect down to 10 genome-equivalents of methylated p16INK4a sequences. Quantitative MSP data were expressed in terms of the methylation index, which was the percentage of bisulfite converted unmethylated and methylated p16INK4a sequences that consisted of methylated p16INK4a sequences. Quantities of methylated p16INK4a sequences were detected in peripheral circulation of 80% (23 of 29) of HCC patients. No significant difference was seen in the detectability and concentrations of methylated p16INK4a sequences (range: 10-4046 genome-equivalents/ml) between preoperative plasma and serum samples from HCC patients. Preoperatively, the p16INK4a methylation indices ranged from 0.2 to 100% and from 0.012 to 0.075% in the patients' plasma and buffy coat samples, respectively. After surgical resection, the median p16INK4a methylation indices in plasma and buffy coat concordantly decreased 12- and 15-fold, respectively. These results demonstrated the clinical usefulness and effectiveness of peripheral blood RTQ-MSP for detecting and monitoring HCC after treatment. Furthermore, none of the intraoperative plasma samples and only two of the intraoperative buffy coat samples were p16INK4a methylation positive. CONCLUSIONS: Quantification of epigenetic changes in peripheral blood by RTQ-MSP is useful for the detection and monitoring of HCC.     

MAM-6 antigen, a new serum marker for breast cancer monitoring

Almost all carcinomas contain a cell surface antigen, MAM-6, which has been defined by several monoclonal antibodies, including 115D8 (Hilkens et al., Int. J. Cancer, 34: 197-206, 1984). A quantitative sandwich radioimmunoassay, using 115D8 as catcher and as tracer antibody, has been developed to detect MAM-6 in serum. To quantitate the MAM-6 level, pooled human milk was used as a standard, and arbitrary units were chosen. Less than 5% of the sera of apparently healthy individuals contained more than 5 units/ml. In sera of patients with benign breast lesions, the same low levels were detected. However, concentrations over 5 units/ml were found in 24, 21, 43, and 79% of the sera of patients with pathological Stages I, II, III, and IV breast cancer, respectively. MAM-6 levels were also increased in almost all sera tested from patients with advanced stages of ovarian carcinoma, but in a low percentage of sera from patients with other advanced cancers. A longitudinal study was carried out to test the MAM-6 assay as clinical marker to monitor the therapeutic response of breast cancer. Increasing or decreasing MAM-6 serum levels correlated in 93% of the cases with breast cancer progression or regression, indicating that the assay can be used to monitor the course of the disease during therapy. In some breast cancer patients, elevated MAM-6 levels were observed prior to any clinical indication of tumor recurrence.     

Tumor regression of B16F10 melanoma in vivo by prevention of neovascularization: study on theophylline

The aim of this study was to determine the effect of theophylline on neovascularization and tumor regression in murine B16F10 melanoma. Theophylline had no direct toxicity to host and significantly reduced (p < 0.001) tumor volume and neovascularization in B16F10 melanoma implanted murine model. The effect of theophylline on neovascularization was observed distinctly in histologic analysis. This effect is mediated, in part by blocking endothelial cell proliferation, thereby preventing neovascularization of the tumor. Further investigations with theophylline can elucidate the exact mechanism of action which characterize neovascularization activity.     

Clinical evaluation of a new serum tumour marker CA 242 in pancreatic carcinoma.

The aim of this study was to evaluate the new monoclonal tumour marker CA 242 in the diagnosis of pancreatic carcinoma and to compare it with the established markers CA 50 and CEA. Serum concentrations were determined in 113 patients with jaundice, in 20 patients with laboratory values suggesting cholestasis, and in 60 patients with a suspicion to have chronic pancreatitis. Twenty-four of these 193 patients had pancreatic carcinoma and two patients had carcinoma of papilla of Vater. The sensitivities of CA 242, CA 50 and CEA were 80.7%, 96.1%, and 92.3%, respectively. The specificities were 79.0%, 58.0%, and 59.2%. The sensitivities of combinations of CA 50 and CEA with CA 242 did not exceed the sensitivity of CA 50 alone. The specificity of CA 242 was improved by combining it with CEA (92.2%). The serum marker CA 242 seems to be less sensitive than CEA and CA 50 in the detection of pancreatic carcinoma, but it may prove useful because of its high specificity.     

Etidocaine, a new local anesthetic.

Different local anesthetics have different physicochemical properties that result in different biological effects. Etidocaine (Duranest) is said to be a long-acting anesthetic with a rapid onset of action, properties that are particularly useful in busy office practices in which lengthy operative procedures are performed. Clinical testing confirmed that this anesthetic does, indeed, work in this manner, which makes it a good addition to the varieties of local anesthetics available.     

Mechanisms of macrolide resistance in clinical pneumococcal isolates in a university hospital, Ankara, Turkey

Macrolide resistance in Streptococcus pneumoniae is usually caused by the presence of the erm(B) or mef(A) resistance determinants. The aim of the present study was to identify the predominant macrolide resistance mechanisms among erythromycin-resistant S. pneumoniae isolated in a university hospital, Ankara, Turkey. A total of 669 S. pneumoniae strains were isolated from clinical specimens of patients admitted to the hospital between 1994--2002. The minimum inhibitory concentrations (MICs) of penicillin G, erythromycin A and clindamycin were determined by the agar dilution method according to NCCLS guidelines. Ninety-one (13.6%) isolates were resistant to erythromycin. Erythromycin-resistant isolates were examined for their macrolide resistance phenotypes by a triple disc diffusion assay. It assigned 57 (62.6%) of the 91 erythromycin-resistant pneumococci to cMLS(B) phenotype, 19 (20.9%) to iMLS(B) phenotype and 15 (16.5%) to M phenotype. All erythromycin-resistant isolates were analyzed by PCR for the presence of erm(B) and mef(A) determinants. The isolates were characterized for the underlying resistance genotype, with 83.5% having erm(B), 16.5% having the mef(A) genotypes. This study provides further evidence of the dissemination of macrolide-resistant mutants in pneumococci as the use of new, long-acting macrolides increases. This is the first article about MLS(B) resistance phenotypes and genotypes of S. pneumoniae from Turkey and it emphasizes the need for future epidemiological monitoring of macrolide-resistant pneumococci.     

In vitro responsiveness to interleukins and theophylline of CD16+, CD56- natural killer cells in a patient with chronic granular lymphocyte disorder.

We report here the case of a 55-year-old patient with chronic granular lymphocyte disorder associated with moderate neutropenia. The majority of peripheral blood lymphocytes displayed a CD3-, CD8-, CD16+, CD56(NKH1)- phenotype. The patient's cells showed high spontaneous cytotoxic activity against K562 targets and developed the ability to kill the natural killer (NK)-resistant target Daudi following activation with interleukin 2 (IL-2). Simultaneously, IL-2 induced proliferation of these cells, albeit to a low level. The effects of IL-2 are likely to be mediated through the IL-2R beta chain (p70) which is expressed on these cells in the absence of the IL-2R alpha chain (p55, Tac). IL-4 was demonstrated to be inhibitory of both the cytotoxic and proliferative effects of IL-2. Thus, despite an unusual CD56- phenotype, the expanded lymphocyte population in this patient display functional and phenotypic properties of normal, non-activated NK cells. These cells probably represent the counterpart of a minor NK cell subpopulation, present in normal individuals at a low frequency, and which has never been fully characterized functionally. In addition, we show that the cytolytic activity of this NK cell population can be blocked in vitro in the presence of a cAMP analog or of theophylline, possibly providing new means of investigating the role of NK cell cytotoxicity on the pathogenesis of associated symptoms in such patients.     

Detection of aberrant p16 methylation in the serum of colorectal cancer patients.

PURPOSE: This study was designed to detect aberrant p16 promoter methylation in the serum of patients with colorectal cancer (CRC) and to explore the possibility of using this assay in early detection or as a prognostic marker of CRC patients. EXPERIMENTAL DESIGN: Methylation-specific PCR was used to detect p16 methylation in DNA extracted from 52 CRCs and matching serum samples and control serum samples from 34 patients with adenomatous polyps and 10 healthy individuals. The association of p16 hypermethylation in serum DNA of CRC patients with clinicopathological characteristics was then analyzed. RESULTS: P16 hypermethylation was found in 20 of 52 (38%) CRCs. Among the 20 cases with aberrant methylation in the tumor tissues, similar changes were also detected in the serum of 14 (70%) cases. No methylated p16 sequences were detected in the peripheral serum of the other 32 CRC cases without these changes in the tumor, in 34 patients with adenomatous polyps, or in 10 healthy control subjects. Clinicopathological analysis revealed that p16 methylation in serum was significantly associated with later Dukes' stage (P = 0.03). CONCLUSIONS: This assay offers a potential means for the serum-based detection and/or monitoring of CRC patients.     

The association of interleukin-16 gene polymorphisms with IL-16 serum levels and risk of nasopharyngeal carcinoma in a Chinese population

Interleukin (IL)-16 plays a fundamental role in inflammatory diseases, as well as in the development and progression of tumors. Genetic variation in DNA sequence of IL16 gene may lead to altered cytokine production and/or activity, and this variation may modulate an individual's susceptibility to nasopharyngeal carcinoma (NPC). To test this hypothesis, we investigated the association of IL16 gene polymorphisms and serum IL-16 levels with NPC risk in a Chinese population. We analyzed IL16 gene rs11556218 T/G, rs4778889 T/C, and rs4072111 C/T polymorphisms using PCR-RFLP and DNA sequencing, and serum IL-16 levels were measured by ELISA. The IL16 rs11556218 T/G polymorphism was significantly associated with the susceptibility to NPC patients. The TG genotype was associated with a significantly higher risk of NPC as compared with the TT genotype (OR?=?2.05, 95 % CI 1.04–4.01; p?=?0.037). Patients carrying the G allele had a significantly higher risk for developing NPC compared with individuals carrying the T allele (OR?=?1.79, 95 % CI 1.07–3.01; p?=?0.027). The serum IL-16 levels were increased in NPC patients compared with controls (p?<?0.01); the genotypes carrying the IL16 rs11556218 G variant allele were associated with increased serum IL-16 levels compared with the homozygous wild-type genotype in NPC patients (all p values <0.01). Our data suggested that IL16 rs11556218 T/G polymorphism was associated with increased susceptibility to NPC through increasing the production of serum IL-16 levels.     

Human kallikrein 6: a new potential serum biomarker for uterine serous papillary cancer.

PURPOSE: The discovery of novel biomarkers might greatly contribute to improve clinical management and outcomes in uterine serous papillary carcinoma (USPC), a highly aggressive variant of endometrial cancer. EXPERIMENTAL DESIGN: Human kallikrein 6 (hK6) gene expression levels were evaluated in 29 snap-frozen endometrial biopsies, including 13 USPC, 13 endometrioid carcinomas, and 3 normal endometrial cells by real-time PCR. Secretion of hK6 protein by 14 tumor cultures, including 3 USPC, 3 endometrioid carcinoma, 5 ovarian serous papillary carcinoma, and 3 cervical cancers, was measured using a sensitive ELISA. Finally, hK6 concentration in 79 serum and plasma samples from 22 healthy women, 20 women with benign diseases, 20 women with endometrioid carcinoma, and 17 USPC patients was studied. RESULTS: hK6 gene expression levels were significantly higher in USPC when compared with endometrioid carcinoma (mean copy number by real-time PCR, 1,927 versus 239, USPC versus endometrioid carcinoma; P < 0.01). In vitro hK6 secretion was detected in all primary USPC cell lines tested (mean, 11.5 microg/L) and the secretion levels were similar to those found in primary ovarian serous papillary carcinoma cultures (mean, 9.6 microg/L). In contrast, no hK6 secretion was detectable in primary endometrioid carcinoma and cervical cancer cultures. hK6 serum and plasma concentrations (mean +/- SE) among normal healthy females (2.7 +/- 0.2 microg/L), patients with benign diseases (2.4 +/- 0.2 microg/L), and patients with endometrioid carcinoma (2.6 +/- 0.2 microg/L) were not significantly different. In contrast, serum and plasma hK6 values in USPC patients (6.1 +/- 1.1) were significantly higher than those in the noncancer group (P = 0.006), benign group (P = 0.003), and endometrioid carcinoma patients (P = 0.005). CONCLUSIONS: hK6 is highly expressed in USPC and is released in the plasma and serum of USPC patients. hK6 may represent a novel biomarker for USPC for monitoring early disease recurrence and response to therapy.     

TRA-1-60: a new serum marker in patients with germ-cell tumors.

TRA-1-60 is a monoclonal antibody (MAb) that recognizes a mucin-like antigenic determinant expressed on the surface of embryonal carcinoma (EC) progenitor cells. In order to determine whether this antigen is released into the serum of patients with a non-seminomatous germ-cell tumor (NSGCT), we developed a sensitive 2-step immunoenzymometric assay. Of 42 EC-positive NSGCT patients tested, 32 (76%) were found to release TRA-1-60-reactive antigen into their serum, in contrast to 1 positive finding in 10 EC-negative NSGCT patients. The marker was found in 67% (10/15) of the EC-positive patients who were negative for both AFP and HCG. Sera from seminoma patients did not contain elevated levels of the TRA-1-60 antigen. Therefore, we propose that the TRA-1-60 antigen is a useful additional serum marker for following the progress of NSGCT(EC+) patients.