Altered autophagy induction by sera from pregnant women with pre-eclampsia: a case-control study.

OBJECTIVE: Mechanisms leading to pre-eclampsia remain incompletely defined. Autophagy is a conserved process necessary for cell survival under adverse conditions. We hypothesised that sera from women with healthy pregnancies and women with pre-eclampsia differed in autophagy induction. DESIGN: A case-control study. SETTING: Weill Cornell Medical College. POPULATION: Twenty-four normotensive pregnant women and 20 women with pre-eclampsia. METHODS: Sera were incubated with peripheral blood mononuclear cells (PBMCs) from female donors. After 48 hours the PBMCs were lysed and the intracellular concentration of p62 was determined by enzyme-linked immunosorbent assay (ELISA). Its concentration is inversely proportional to the extent of autophagy induction. Serum endoglin, interleukin 13 (IL-13), insulin-like growth factor 1 (IGF-1), and transforming growth factor ß1 (TGF-ß1) levels were quantitated by ELISA. MAIN OUTCOME MEASURES: Differences in autophagy induction and serum mediator levels in the two groups. RESULTS: Autophagy induction increased with gestational age in sera from normotensive women (P = 0.0045), but not in women with pre-eclampsia. In the presence of an autophagy inducer, the capacity for autophagy induction decreased with gestational age in sera from women with pre-eclampsia (P = 0.0235), but not from controls. Endoglin concentrations were positively associated with the extent of autophagy induction in controls only (P = 0.0141). There was no association between autophagy and serum IL-13, IGF-1, or TGF-ß1 levels. CONCLUSIONS: Sera from women with pre-eclampsia differ from normotensive women by their inability to induce autophagy as a function of gestational age.

Virulence of Streptococcus pneumoniae may be determined independently of capsular polysaccharide.

Mice were inoculated intranasally with Streptococcus pneumoniae isolates of serotype 14 with different genetic backgrounds (14R, 14DW) and a capsular switch of 14R, strain 9VR (serotype 9V). Inoculation of the mice with 14R and 9VR resulted in 60% mortality. All the mice survived 14DW inoculation. No differences in lungs' bacterial loads were found 3 h following inoculation. Bacterial clearance of 5 logs was observed 48 h after inoculation with 14DW versus within 1 log 48 h after inoculation with 14R and 9VR. No significant differences in bacterial size or the capsular amount could be found between 14R and 14DW. We conclude that factor(s) in addition to the capsule, contribute to disease outcome.

Differential activation of the immune system by virulent Streptococcus pneumoniae strains determines recovery or death of the host.

Streptococcus pneumoniae infection may result in asymptomatic carriage, mucosal or invasive disease. We hypothesize that self-limiting or fatal disease outcome follows infection with S. pneumoniae differential activation of the host immune response. BALB/c and C57BL/6 mice were inoculated intranasally with S. pneumoniae serotype 3 strain WU2 and serotype 14 strain DW14 and mortality, bacterial load, pathological changes in the lungs and cytokines mRNA levels in the spleen were analysed. No differences between the C57BL/6 and the BALB/c inbred mice were observed except for the severity of their lung pathology and IL-4 expression. Infection of the two mouse strains with S. pneumoniae WU2 resulted in sepsis and death that occurred within 4 days post-inoculation. This death was preceded, in both mouse strains, in an increase over time of the lung bacterial load and bacteraemia. The lung pathology was characterized by diffuse pneumonia with marked congestion of the lungs. Analysis of mRNA expression of cytokines in the spleen revealed no alterations in tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, interleukin (IL)-12 and interferon (IFN)-gamma and induction of IL-10 and IL-4. The two strains of mice survived infection with S. pneumoniae DW14. This was accompanied by a reduction over time of lung bacterial load and bacteraemia. The lung pathology was characterized by focal lymphocyte infiltration and preserved architecture of the organ. Analysis of mRNA expression of cytokines in the spleen revealed a significant decrease in the levels of TNF-alpha, TGF-beta, IL-12 and IFN-gamma mRNA expression, which usually precedes cytokine protein expression. Interestingly, a significant increase in the levels of IL-4 mRNA expression was found in BALB/c mice only. This study suggests that differential activation or evasion of cytokine expression by S. pneumoniae virulent strains determines disease outcome regardless of the host's immunogenetic background.

Age-dependent preference in human antibody responses to Streptococcus pneumoniae polypeptide antigens.

Vulnerability to Streptococcus pneumoniae is most pronounced in children. The microbial virulence factors and the features of the host immune response contributing to this phenomenon are not completely understood. In the current study, the humoral immune response to separated Strep. pneumoniae surface proteins and the ability to interfere with Strep. pneumoniae adhesion to cultured epithelial cells were analysed in adults and in children. Sera collected from healthy adults recognized Strep. pneumoniae separated lectin and nonlectin surface proteins in Western blot analysis and inhibited on average 80% of Strep. pneumoniae adhesion to epithelial cells in a concentration-dependent manner. However, sera longitudinally collected from healthy children attending day care centres from 18 months of age and over the course of the following 2 years revealed: (a) development of antibodies to previously unrecognized Strep. pneumoniae surface proteins with age; (b) a quantitative increase in antibody responses, measured by densitometry, towards separated Strep. pneumoniae surface proteins with age; and (c) inhibition of Strep. pneumoniae adhesion to epithelial cells, which was 50% on average at 18 months of age, increased significantly to an average level of 80% inhibition at 42 months of age equalling adult sera inhibitory values. The results obtained in the current study, from the longitudinally collected sera from healthy children with documented repeated Strep. pneumoniae colonization, show that repeated exposures are insufficient to elicit an immune response to Strep. pneumoniae proteins at 18 months of age. This inability to recognize Strep. pneumoniae surface proteins may stem from the inefficiency of T-cell-dependent B-cell responses at this age and/or from the low immunogenicity of the proteins.

Apoptosis (programmed cell death) in colonic cells: from normal to transformed stage.

We determined apoptosis in whole rat colonic tissue and in isolated colonocytes from the various rat crypt regions in preneoplastic stages up to frank neoplasia following administration of the procarcinogen, dimethylhydrazine (DMH). Apoptotic cells were determined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-method, by evaluating sections stained with hematoxylin and eosin, and caspase-1 immunostaining. Apoptotic cells in whole colonic tissue from untreated rats were confined to the upper crypt while, in DMH-treated rats apoptotic and caspase-1 positive cells were located in the crypt proliferative regions. Numerous apoptotic and caspase-1-positive cells were found in sections from early tumors while in the delayed tumors, apoptotic-positive cells were absent and number of caspase-1-positive cells was negligible. A marked reduction in the apoptotic index along the crypt was observed in isolated transformed colonic cells, this was not the case for caspase-1-positive cells. We conclude that: (i) in colorectal tumors at progressive stage apoptosis is altered, (ii) the mechanistic alteration in apoptosis may be located between caspase-1-protease activity and the fragmentation process of DNA.

Intraoperative lymphatic mapping in cervix cancer patients undergoing radical hysterectomy: A pilot study.

OBJECTIVE: Intraoperative lymphatic mapping and sentinel lymph node identification (SLN) have been increasingly evaluated in the treatment of a variety of solid tumors, particularly breast cancer and melanoma. We sought to evaluate the feasibility of these procedures in patients undergoing radical hysterectomy with pelvic lymphadenectomy for treatment of early cervical cancer. METHODS: Twenty patients with normal-appearing lymph nodes underwent intracervical injection of isosulfan blue dye (lymphazurin 1%) at the time of planned radical hysterectomy and bilateral pelvic/low paraortic lymphadenectomy (40 nodal basins). Regional lymphatic tissue was inspected for dye uptake into lymphatic channels and lymph nodes. Tumor characteristics, surgical findings, and specific locations of lymphatic dye uptake were recorded and correlated with final pathology results. RESULTS: Sentinel lymph nodes were identified in 12 of 20 (60%) patients. A total of 23 sentinel nodes were identified in 17 of 40 (43%) nodal basins dissected (range: 0-2 per basin). Successful SLN identification was less likely in patients with tumors >4 cm compared with those with tumors

Initial steps in Streptococcus pneumoniae interaction with and pathogenicity to the host.

Streptococcus pneumoniae (Pnc) is one of the leading pathogens in the world. Attachment to respiratory mucosal and lung surfaces is presumed to be involved in carriage, in disease and in the interaction with macrophages initiating innate immune responses. We hypothesized that bacterial adhesins mediate Pnc adhesion and host cell invasiveness. Initial studies have focused on the purification of cell wall and membrane proteins using fetuin affinity chromatography, SDS PAGE and western blot analysis probed with pooled healthy human sera. Using a Pnc clinical isolate, and a gpt mutant we have detected 10-lectin proteins isolated from the cell wall and adherent to the affinity column and 15 lectins isolated from membrane extracts. The fetuin-captured lectins agglutinated rabbit erythrocytes. 15 proteins in the cell wall and 18 proteins in the membrane that failed to bind to the fetuin column did not agglutinate rabbit erythrocytes. Further purification of the cell wall and membrane fetuin-separated fractions was achieved via anion exchange FPLC, was verified by SDS PAGE. These proteins maintained their agglutinating activity, and were subsequently tested for their ability to interfere with Pnc adhesion and invasion of epithelial cells in culture. Additional biochemical, immunological and molecular techniques are being used in attempt to identify relevant proteins.

Extensive apoptotic death of rat colonic cells deprived of crypt habitat.

Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell-to-cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD-CMK) inhibitor of interleukin-1beta-converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium-orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic-labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD-CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE-mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype.

Pathology slide review in gynecologic oncology.

OBJECTIVE: To analyze the diagnostic accuracy and alteration in treatment planning from interinstitution (different institution) pathologic consultation. METHODS: We reviewed pathologic reports from 720 referred patients. The diagnosis rendered from a gynecologic pathologist was compared with the original diagnosis. Discrepancies were coded as none, minor, or major. A discrepancy was major if it led to treatment alteration. A discrepancy was minor if it did not lead to treatment alteration. The judgment to declare a discrepancy was made by a gynecologic pathologist, a gynecologist, and three gynecologic oncologists. The review cost was $150 per case. The Cochran-Mantel-Haenszel test evaluated any systematic pattern in discrepancies. RESULTS: Seven hundred twenty specimens consisted of 113 vulvar, 170 uterine, 289 cervical, 105 ovarian, and 43 vaginal tissues. Six hundred one (84%) pathologic diagnoses showed no discrepancy. There were 104 (14%) minor and 15 (2%) major discrepancies. After reviewing 15 major discrepancies, six surgeries were canceled, two surgeries were modified, one adjuvant radiation treatment was added, one chemotherapy treatment was modified, and five adjuvant chemotherapy treatments were cancelled. No systematic error was identified with regard to the sources (tissue origin) or methods of obtaining the specimen (P = .675). The cost of reviewing 720 specimens was $108,000. The cost of identifying each major discrepancy was $7200. CONCLUSION: Reviewing pathology slides before definitive treatment reveals notable discrepancies in diagnoses. The cost of pathology review is globally expensive but has consequential impact on proper treatment planning for the individual patient.

Remission of refractory gestational trophoblastic disease in the brain with ifosfamide, carboplatin, and etoposide (ICE): first report and review of literature.

Gestational trophoblastic disease (GTD) metastatic to the brain has a very poor prognosis with a survival rate of less than 25%, especially for patients in whom brain metastases develop while on or after chemotherapy. Cure can be achieved by chemotherapy alone. The regimen of etoposide, methotrexate, actinomycin-D, vincristine, and cyclophosphamide has shown encouraging results and is considered to be standard first-line treatment for high risk patients. For patients in whom this regimen fails, a salvage chemotherapy regimen is used. The combination of ifosfamide, carboplatin, and etoposide (ICE) has synergistic activity in preclinical studies. This regimen has shown activity in metastatic breast cancer and non-small-cell lung cancer as well as platinum-resistant germ-cell tumors and metastatic GTD. This is the first report of a patient with a highly refractory GTD in whom brain metastasis developed while on chemotherapy, and whose brain metastasis went into remission with a low dose ICE regimen. Accordingly, ICE may be considered for patients with chemotherapy refractory GTD metastatic to the brain.

Remission of refractory gestational trophoblastic disease with high-dose paclitaxel.

High-risk metastatic gestational trophoblastic disease (GTD) in patients who have failed primary chemotherapy has a very poor prognosis. About 25% of women with high-risk metastatic disease become refractory to EMA-CO (etoposide, methotrexate, actinomycin-D, cyclophosphamide and vincristine) and fall to achieve a complete remission. Currently, there is no standard salvage chemotherapeutic regime for EMA-CO failure. Paclitaxel, a taxane analog extracted from the bark of the western yew (Taxus brevlfolla), has shown antitumor activity in a variety of cancer cell lines. High in vivo efficacy was confirmed in phase II trials, especially for breast and epithelial ovarian cancer patients. Recently, two in vitro studies have shown that paclitaxel is a highly effective antineoplastic agent in choriocarcinoma cell lines. We present the first clinical report of a serologic remission with high-dose paclitaxel (250 mg/m2 i.v. infusion over 24 h every 3 weeks) of a highly refractory GTD in a patient who developed brain metastasis after multiple combined chemotherapeutic regimens. The patient tolerated paclitaxel with granulocyte colony stimulating factor support very well. The remission with paclitaxel in this patient confirms its preclinical activity in high-risk, refractory GTD.

Nikkomycin production in pellets of Streptomyces tendae.

In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.

Biotechnological applications of image analysis: present and future prospects.

The current and potential biotechnological applications of image analysis and image processing systems are reviewed. Image analysis systems have proven to be highly versatile and efficient tools for assisting academic biotechnological research. It is expected that image analysis systems will allow more rapid and accurate quantification of numerous biotechnological analyses. There is, therefore, much scope for the implementation of image analysis/processing systems in a large variety of industrial and clinical applications.

Cisplatin based combination chemotherapy in the treatment of stage III ovarian epithelial tumors of low malignant potential.

Eleven patients diagnosed with Stage III epithelial ovarian tumors of low malignant potential were treated with a combination of cisplatin and cyslophosphamide with or without doxorubicin following primary cytoreductive surgery. Residual disease after initial cytoreduction was macroscopic in nine patients and not observable in two. Eight of the nine patients with macroscopic residual were assessed by second look laparotomy (one refused the procedure), seven showed tumor and one was found to be a complete responder. Of the two patients with no residual disease, one refused second look operation and one had a negative second look. Our findings plus those of other authors suggest that cisplatin based combination chemotherapy is of limited value in the treatment of metastatic ovarian epithelial tumors of low malignant potential.

Pellet formation and cellular aggregation in Streptomyces tendae.

In submerged cultures, Streptomyces tendae tended to form fluffy spherical pellets of the noncoagulative type. An increase in the average pellet size could be attained by decreasing any of the following: shear rate, pH, temperature, or inoculum size. Conditions leading to oxygen limitation tended to reduce the average pellet size and induced pulpy growth, whereas oxygen sufficiency seemed to induce pellet formation. Factors inducing pellet formation simultaneously increased cell wall hydrophobicity. It is therefore proposed that the main forces inducing cellular aggregation in S. tendae are hydrophobic interactions of cell walls, and these interactions are controlled by availability of dissolved oxygen.

Therapy of stage III (optimal) epithelial carcinoma of the ovary with melphalan or melphalan plus Corynebacterium parvum (a Gynecologic Oncology Group Study).

A randomized prospective therapy trial in patients with stage III optimal epithelial carcinoma of the ovary was accomplished by the Gynecologic Oncology Group. Therapy with melphalan or melphalan plus immuno-adjuvant, Corynebacterium parvum (C. parvum), was utilized as adjuvant treatment following surgical therapy. One hundred eight-five patients were eligible for evaluation with 87 patients in the melphalan group and 98 patients in the melphalan plus C. parvum group. The comparison of the treatment regimens showed no differences with respect to either progression-free interval or survival. However, it should be noted that a 50% 3-year survival was obtained. A group was identified, using four prognostic factors that had 80% survival at 3 years. Maximum size of the residual tumor, as well as performance status, was not prognostically significant. This study demonstrates a lack of efficacy of the addition of C. parvum to melphalan for this patient population.

The canine laboratory in the training of the oncology fellow.

This report deals with one fellow's experience in the dog lab established to aid in the surgical training of the gynecologic oncology fellow. The first experiment was designed to train the fellow in bowel resection and end-to-end anastomosis, comparing suture and staple techniques. A second study evaluated the physical and biological properties of polyglactin 910 (Vicryl) mesh to determine its applicability in reconstructing the pelvic floor following pelvic exenteration.

Vicryl mesh in pelvic floor reconstruction.

A new synthetic absorbable mesh made of polyglactin 910 (Vicryl) fiber was used to reconstruct the pelvic floor in seven women undergoing pelvic exenteration. The technique is described. The follow-up ranged from three to 31 months and no patient developed a bowel problem. The material seems to be appropriate for this use, is completely absorbed, and acts as a latticework for the deposition of granulation tissue. The technique can be applied in patients requiring pelvic irradiation following surgery for malignant neoplasms of the gastrointestinal or genitourinary tracts. The small bowel is effectively held out of the pelvis and the radiation field, and is spared the effects of the radiation beam.

A randomized clinical trial of adjuvant adriamycin in uterine sarcomas: a Gynecologic Oncology Group Study.

After hysterectomy, 156 evaluable patients with stage I (limited to the corpus) or stage II (limited to the corpus and cervix) uterine sarcomas were randomly assigned to adjuvant chemotherapy with Adriamycin (Adria Laboratories, Columbus, Ohio) for six months or to no further treatment. Pelvic irradiation (external or intracavitary) was optional before randomization. Of 75 patients receiving Adriamycin, 31 have suffered recurrences compared with 43 of 81 receiving no adjuvant chemotherapy. This difference is not statistically significant. Moreover, there is no difference in progression-free interval or survival. The optional radiotherapy did not influence the outcome although there was a suggestion that vaginal recurrence was decreased by pelvic radiotherapy. The recurrence rates in specific cell types (leiomyosarcoma, homologous mixed mesodermal sarcoma, or heterologous mixed mesodermal sarcoma) were not significantly different although the pattern of recurrence differed, with pulmonary metastases being more common in leiomyosarcoma and extrapulmonary recurrence being more common in mixed mesodermal sarcoma. The outcome with respect to chemotherapy was not altered even after adjusting for maldistribution of cases. Thus, we could not show a benefit for this dose schedule of Adriamycin as adjuvant treatment for uterine sarcomas.

Prevalence of papillomarvirus infection in colposcopically directed cervical biopsy specimens in 1972 and 1982.

A total of 1264 consecutive cervical biopsy specimens obtained at the Parkland Memorial Hospital Dysplasia Clinic during 1972 were reviewed. Histopathologic specimens were assessed with special reference to changes induced by human papillomavirus. In 1972, only 0.7% of biopsy specimens were reported as consistent with human papillomavirus infection. Upon review, however, 36.5% of these specimens were found to demonstrate histologic criteria for the diagnosis of human papillomavirus infection. Approximately half of biopsy specimens reclassified as human papillomavirus were originally interpreted as inflammation; the others were interpreted as cervical intraepithelial neoplasia. Patients with human papillomavirus infection were significantly younger than patients with cervical intraepithelial neoplasia (24.9 versus 30.2 years). These findings were compared with 965 cervical biopsy specimens obtained in 1982. Thirty-four percent of these biopsy specimens revealed human papillomavirus infection. These observations support the concept that human papillomavirus infection of the cervix is not a new entity but a previously unrecognized finding whose prevalence has been relatively stable over a 10-year period.