Chemical Ecology of Cave-Dwelling Millipedes: Defensive Secretions of the Typhloiulini (Diplopoda, Julida, Julidae).

Cave animals live under highly constant ecological conditions and in permanent darkness, and many evolutionary adaptations of cave-dwellers have been triggered by their specific environment. A similar "cave effect" leading to pronounced chemical interactions under such conditions may be assumed, but the chemoecology of troglobionts is mostly unknown. We investigated the defensive chemistry of a largely cave-dwelling julid group, the controversial tribe "Typhloiulini", and we included some cave-dwelling and some endogean representatives. While chemical defense in juliform diplopods is known to be highly uniform, and mainly based on methyl- and methoxy-substituted benzoquinones, the defensive secretions of typhloiulines contained ethyl-benzoquinones and related compounds. Interestingly, ethyl-benzoquinones were found in some, but not all cave-dwelling typhloiulines, and some non-cave dwellers also contained these compounds. On the other hand, ethyl-benzoquinones were not detected in troglobiont nor in endogean typhloiuline outgroups. In order to explain the taxonomic pattern of ethyl-benzoquinone occurrence, and to unravel whether a cave-effect triggered ethyl-benzoquinone evolution, we classed the "Typhloiulini" investigated here within a phylogenetic framework of julid taxa, and traced the evolutionary history of ethyl-benzoquinones in typhloiulines in relation to cave-dwelling. The results indicated a cave-independent evolution of ethyl-substituted benzoquinones, indicating the absence of a "cave effect" on the secretions of troglobiont Typhloiulini. Ethyl-benzoquinones probably evolved early in an epi- or endogean ancestor of a clade including several, but not all Typhloiulus (basically comprising a taxonomic entity known as "Typhloiulus sensu stricto") and Serboiulus. Ethyl-benzoquinones are proposed as novel and valuable chemical characters for julid systematics.

Chemical composition, cytotoxic and antioxidative activities of ethanolic extracts of propolis on HCT-116 cell line.

BACKGROUND: Propolis is a complex resinous sticky substance that honeybees collect from buds and exudates of various plants. Owing to its versatile biological and pharmacological activities, propolis is widely used in medicines, cosmetics and foods. The aim of this study was to evaluate the cytotoxic and antioxidative effects of various ethanolic extracts of propolis (EEPs) on human colon cancer cell line HCT-116 and compare them with their composition determined by HPLC-DAD. RESULTS: The most abundant flavonoids in all samples were chrysin, pinocembrin and galangin (12.697-40.811 µg mg⁻¹), while the main phenolic acids were caffeic acid, ferulic acid and isoferulic acid. Dose- and time-dependent inhibition of growth of HCT-116 cells was observed for all propolis samples, with IC50 values ranging from 26.33 to 143.09 µg mL⁻¹. Differences in cytotoxic activity of propolis samples were associated with differences in their composition. All EEP samples reduced both superoxide anion radical and nitrite levels and also had strong DPPH-scavenging activity. CONCLUSION: All tested propolis samples had pronounced cytotoxic and antioxidative activities.

A stable isotope-mass spectrometric method for measuring human spermatogenesis kinetics in vivo.

PURPOSE: Currently it is thought to take 60 to 70 days to produce and ejaculate human sperm. This estimate is derived mainly from a single older, descriptive, kinetic analysis of spermatogenesis. We developed a noninvasive method to assess germ cell turnover time accurately in vivo using stable isotope labeling and gas chromatography/mass spectrometry analyses. We confirmed the postulated length of a normal cycle of spermatogenesis. MATERIALS AND METHODS: A total of 11 men with normal sperm concentrations ingested (2)H(2)O daily for 3 weeks. Semen samples were collected every 2 weeks for up to 90 days. Label incorporation into sperm DNA was quantified by gas chromatography/mass spectrometry, allowing calculation of the percent of new cells present. A cycle of sperm production was determined as the lag time until labeled sperm appeared in the ejaculate. RESULTS: Labeled sperm were detected after a mean +/- SD of 64 +/- 8 days (range 42 to 76). In 1 subject the time lag was 42 days but it was at least 60 in all other subjects. In most subjects plateau labeling in sperm was not attained. In 2 subjects the rise and fall of the labeling curve was steep and reached greater than 85% new cells, suggesting rapid washout of old sperm in the epididymal reservoir. CONCLUSIONS: This direct kinetic assessment confirms a course of spermatogenesis that is on the shorter side of traditional estimates based on prior analyses. In addition, the variability observed in healthy men suggests that characteristics such as the epididymal reservoir effect may influence the modeling of in vivo spermatogenesis.

Expression of cyclooxygenase-2 in fine-needle aspirates from breast carcinoma and benign breast diseases.

Numerous studies have demonstrated that the levels of cyclooxygenase (COX), the enzyme that catalyzes the conversion of arachidonic acid to prostaglandin H(2), and of prostaglandins are higher in various tumors and cells during inflammation than in normal tissues. The aim of the present study was to analyze whether COX-2 isoform expression was noticeably higher in fine-needle aspirates (FNA) from breast carcinoma than in FNA from fibroadenoma and fibrocystic breast tissue. COX-2 expression was detected by immunocytochemical (IC) staining and was analyzed by microscopic scoring and computer gray-scale analysis. Evaluation of COX-2 IC positivity in FNA from three groups of patients (nine with breast carcinoma, nine with fibroadenoma, eight with fibrocystic breasts) revealed high COX-2 IC positivity in the majority of patients with breast carcinoma and low or absent COX-2 IC positivity in patients with fibrocystic breast changes. In addition, low or medium COX-2 IC positivity was found in the majority of patients with fibroadenoma, only three of these patients having high COX-2 IC positivity.

Long-term results of conventional-dose salvage chemotherapy in patients with refractory and relapsed Hodgkin's disease (Croatian experience).

BACKGROUND: The aim of this study was to analyze outcome of patients with Hodgkin's disease (HD) in whom first-line chemotherapy with mustine/vincristine/procarbazine/prednisone (MOPP) had failed. PATIENTS AND METHODS: From January 1982 to December 1989 among 210 patients treated with MOPP and radiotherapy to initial bulky sites, 65 patients were primary refractory to or relapsed after initial treatment. RESULTS: Twenty-nine of 65 patients (44%) were primary refractory to initial chemotherapy, 20 relapsed within 12 months after complete remission (CR) and 16 relapsed after CR that lasted more than 12 months. Patients with primary refractory HD and early relapse (<12 months after CR) were treated with doxorubicin/bleomycin/vinblastine/darcarbazine. In patients with late relapse (>12 months after CR) MOPP was repeated. The median follow-up for all patients was 115 months. The overall response rate was 63%. Thirty-three patients (51%) achieved a second CR and eight patients (12%) partial response. Remission rate was greatest in patients with late relapse (CR >12 months) (75 versus 55% for early relapse versus 35% for primary refractory HD) (P <0.01). At 10 years, overall and failure-free survival rates were 21 and 16%, respectively. Patients who were in first remission longer than 12 months had a superior overall survival (37 versus 18% for early relapse) and failure-free survival (24 versus 10% for early relapse). No patient with primary refractory HD was alive beyond 52 months after initial treatment failure (P <0.01). Main prognostic factors were duration of the first remission and tumor bulk at relapse. CONCLUSIONS: Our results confirm previous observations that a significant proportion of patients with HD who experience induction treatment failure cannot be cured with conventional treatment and probably need more aggressive therapy.

Does weight loss improve incontinence in moderately obese women?

The aim of this study was to evaluate the effect of weight reduction on urinary incontinence in moderately obese women. This prospective cohort study enrolled moderately obese women experiencing four or more incontinence episodes per week. BMI and a 7-day urinary diary were collected at baseline and on the completion of weight reduction. The study included 10 women with a mean (+/-SD) baseline BMI of 38.3 (+/-10.1) kg/m2 and 13 (+/-10) incontinent episodes per week. Participants had a mean BMI reduction of 5.3 (+/-6.2) kg/ m2 (P < 0.03). Among women achieving a weight loss of > or = 5%, 6/6 had > or = 50% reduction in incontinence frequency compared to 1 in 4 women with < 5% weight loss (P < 0.03). Incontinence episodes decreased to 8 (+/-10) per week following weight reduction (P < 0.07). The study demonstrated an association between weight reduction and improved urinary incontinence. Weight reduction should be considered for moderately obese women as part of non-surgical therapy for incontinence.

[In situ PCR in the diagnosis of acute leukemia]. / PCR in situ u dijagnostici akutnih leukemija.

Cytomorphologic and cytochemical bone marrow analysis is essential in the diagnosis of acute leukemia. Immunophenotyping and conventional cytogenetics, just as fluorescent in situ hybridization (FISH) are other diagnostic procedures, as well as genome analysis by PCR (polymerase chain reaction). PCR is inevitable in searching for minimal residual disease, because it may detect very small amount of malignant hematopoietic cells even when a patient is in complete remission (less than 5% malignant cells in bone marrow and disappearance from peripheral blood) which helps better monitoring of patients. By in situ hybridization (ISH) it is possible to associate specific cell type with genome alteration, but the method is not sensitive enough. By combining ISH and PCR a novel technique with increased sensitivity was developed, PCR in situ, which enables nucleic acid amplification in an intact cell. In this case report we present two patients whose bone marrow aspirates were analyzed also by PCR in situ.

Double immunoenzymatic APAAP staining for the detection of leukemia-associated immunophenotypes.

Detection of unusual or aberrant cell immunophenotype with flow cytometry is the basis for the immunologic recognition of minimal residual disease (MRD) in patients with acute leukemia (AL). In this study, we have shown that the double immunocytochemical alkaline phosphatase antialkaline phosphatase (APAAP) staining technique also makes possible the detection of leukemic cells with unusual (leukemic) combinations of antigens (ULCA) both at diagnosis and during follow-up of patients with ULCA+ AL. The applicability of double APAAP was analyzed on bone marrow (BM) samples obtained from 12 patients (8 with AML, 3 with ALL, and 1 with undifferentiated acute leukemia [AUL]) randomly chosen from a larger group of 22 ULCA+ patients treated at our center in a 3-year period (22% observed ULCA+ AL frequency). The percentages of ULCA+ BM cells before chemotherapy were in the range of 5%-60%, which dropped to 0%-7% in 10 patients who achieved remission (range 0%-7%, p < 0.01). However, these cells could also be found 60 days after the initiation of therapy, ranging from 0%-2% of all nucleated cells. In 2 of 10 patients who achieved remission, 2% ULCA+ BM cells were found on days 35 and 60 after initiation of chemotherapy, and this finding was followed by relapse on days 110 and 270. However, the other 8 patients remained in remission despite positive finding of ULCA+ BM cells ranging from 0.2%-2% on at least one occasion. In 2 patients with AML FAB-M3 and cytomorphologic remission, the finding of ULCA+ cells by double APAAP correlated with the molecular finding of PML/RARalpha junction. These results indicate that double APAAP staining can identify leukemic cells in samples with a cytomorphologic pattern consistent with remission, but its applicability in detection of MRD awaits additional studies on a larger number of patients with ULCA+ AL.

Relation between urinary cytology abnormalities and cyclosporine A therapy in bone marrow transplant recipients.

The aim of the study was to determine the relationship, if any, between abnormalities in urinary cytology and the administration of cyclosporine A in bone marrow transplant recipients. Specific attention was given to the presence of tubular cells with round inclusions (TCRI). Two bone marrow transplant recipient groups were studied: one with allogeneic bone marrow transplantation (BMT) (20 patients) who were treated with cyclosporine A, and the other with autologous BMT (12 patients) who did not receive cyclosporine A. Urinary cytology showed TCRI in 41.66% of the patients after autologous BMT and in 80% of the patients after allogeneic BMT. In the group of patients treated with allogeneic BMT, the occurrence of TCRI was associated with a high incidence of glycosuria and was followed by an increase in the blood level of cyclosporine A, an increase in the serum creatinine concentration and a decrease in the creatinine clearance. These results demonstrated that TCRI, although related to, were not found to be exclusively specific to the administration of cyclosporine A.

Donor buffy-coat infusion and chemotherapy for leukemia in relapse after marrow transplantation.

A patient relapsing with blastic lymphoid transformation of chronic myeloid leukemia after bone marrow transplantation received donor buffy-coat infusion. Low-dose chemotherapy was added because of a rapid WBC increase. Complete hematologic and cytogenetic remission was obtained. The patient remained in complete hematologic and cytogenetic remission for four months until he died in an accident. Two patients with acute leukemia failed to respond to a similar treatment.

Correlation of morphological FAB classification and immunophenotyping: value in recognition of morphological, cytochemical and immunological characteristics of mixed leukaemias.

Correlation between the FAB classification and immunophenotype was studied in 169 consecutive adult patients with acute leukaemia (AL). The lineage of leukaemic cells could be determined in the majority of cases, whereas 3 patients (1.8%) remained unclassified. In 22 out of 71 patients (31%) with acute myeloid leukaemia (AML) FAB M1 and M2 types, and in 5 out of 16 patients (31%) with chronic myeloid leukaemia (CML) in myeloid blast crisis, leukaemic cells did not express myeloid lineage-related markers, indicating asynchronous expression of cell markers in a substantial proportion of patients. Flow cytometric two-colour immunofluorescence revealed mixed AL immunophenotype in 6 out of 169 patients (3.4%). This group included five CD2+AML (5% of AML tested) and one undifferentiated AL expressing CD10(CALLA), CDw65(VIM-2). The former group included FAB M1, M2, M3 and M4 forms of AML with a single cell population, and an AML M2 patient with both cytochemically and immunologically two separate populations of leukaemic cells. This further illustrates the heterogeneity of the target cell(s) for leukaemogenesis and the level of differentiation of AML cells. However, there was no difference in the treatment response and the remission duration between AML patients and patients with mixed phenotype AML.

Significance of proliferative epithelial changes in breast fine-needle aspiration.

The authors analyzed 6439 fine-needle aspirations (FNA). The total number of FNA performed per year at the authors' institution was found to be increasing. The authors analyzed the numbers of carcinomas and proliferative breast alterations separately. A statistically significant increase was recorded in the group of aspirate specimens that had epithelial proliferation; the increase was greater than that seen in aspirate specimens with normal epithelium (P less than 0.001). Biopsy was recommended in 151 of 6439 (2.3%) FNA and performed for 67 of the 151 (1.0%) recommendations. Cytologic findings were compared with pathohistologic findings. A histologic carcinoma was found in 14 of 67 (20.8%) patients who had biopsy performed. The authors concluded that clinicians should give particular attention to atypia in cytologic aspirate specimens.

Prognostic significance of cytochemical analysis of leukemic M2 blasts.

Cytochemical analysis of leukemic blasts from 46 patients with acute myeloblastic M2 leukemia (according to the FAB classification) was performed before and after cytostatic therapy, and compared with findings obtained in 20 age- and sex-matched control subjects. Cytochemical findings for myeloperoxidase (MPO), Sudan black B, acid phosphatase and alpha-naphthyl-acetate esterase (ANAE) were related to the achievement of the first complete remission (CR), i.e. data were compared after the patients had been divided into CR and non-CR groups. The analysis clearly showed that a high proportion of myeloperoxidase- and, to a lesser extent, Sudan black B-positive blasts before treatment may have constituted a significantly unfavourable prognostic factor.

Expression of haematopoietic progenitor cell-associated antigen BI-3C5/CD34 in leukemia.

The expression of progenitor cell-associated antigen CD34, defined with monoclonal antibody BI-3C5, was investigated in cells from 109 patients with leukaemia. No reactivity was found in chronic leukaemias, whereas 31% of acute myelogenous leukaemia (AML) and most non-T, non-B acute lymphoblastic leukaemia (ALL) expressed CD34. Examples of BI-3C5+ AML included M1 and M2 FAB types only; all but one were myeloperoxidase positive. In combination with pan-myeloid markers, BI-3C5 is useful for identification of immature myeloid cells.