Floating treatment wetlands for domestic wastewater treatment.

Floating islands are a form of treatment wetland characterized by a mat of synthetic matrix at the water surface into which macrophytes can be planted and through which water passes. We evaluated two matrix materials for treating domestic wastewater, recycled plastic and recycled carpet fibers, for chemical oxygen demand (COD) and nitrogen removal. These materials were compared to pea gravel or open water (control). Experiments were conducted in laboratory scale columns fed with synthetic wastewater containing COD, organic and inorganic nitrogen, and mineral salts. Columns were unplanted, naturally inoculated, and operated in batch mode with continuous recirculation and aeration. COD was efficiently removed in all systems examined (>90% removal). Ammonia was efficiently removed by nitrification. Removal of total dissolved N was ∼50% by day 28, by which time most remaining nitrogen was present as NO(3)-N. Complete removal of NO(3)-N by denitrification was accomplished by dosing columns with molasses. Microbial communities of interest were visualized with denaturing gradient gel electrophoresis (DGGE) by targeting specific functional genes. Shifts in the denitrifying community were observed post-molasses addition, when nitrate levels decreased. The conditioning time for reliable nitrification was determined to be approximately three months. These results suggest that floating treatment wetlands are a viable alternative for domestic wastewater treatment.

The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit.

The most primitive engrafting hematopoietic stem cell has been assumed to have a fixed phenotype, with changes in engraftment and renewal potential occurring in a stepwise irreversible fashion linked with differentiation. Recent work shows that in vitro cytokine stimulation of murine marrow cells induces cell cycle transit of primitive stem cells, taking 40 h for progression from G0 to mitosis and 12 h for subsequent doublings. At 48 h of culture, progenitors are expanded, but stem cell engraftment is markedly diminished. We have investigated whether this effect on engraftment was an irreversible step or a reversible plastic feature correlated with cell cycle progression. Long-term engraftment (2 and 6 mo) of male BALB/c marrow cells exposed in vitro to interleukin (IL)-3, IL-6, IL-11, and steel factor was assessed at 2-4-h intervals of culture over 24-48 h using irradiated female hosts; the engraftment phenotype showed marked fluctuations over 2-4-h intervals, with engraftment nadirs occurring in late S and early G2. These data show that early stem cell regulation is cell cycle based, and have critical implications for strategies for stem cell expansion and engraftment or gene therapy, since position in cell cycle will determine whether effective engraftment occurs in either setting.

Probability of establishing chimeric plasmids in natural populations of bacteria.

Formulas for estimating the probability that chimeric plasmids carried by disarmed hosts will become established in natural populations of bacteria are presented and their use illustrated with a series of realistic numerical examples. The implications of these a priori probability estimates for the problem of containment for recombinant DNA research is discussed.

Clinical impact of chemotherapy dose escalation in patients with hematologic malignancies and solid tumors.

PURPOSE: To review published controlled clinical trials examining the benefit of escalated chemotherapy in patients with hematologic and solid malignancies. METHODS: Studies were obtained by searching Medline and CancerLit and by review of bibliographies of published trials. We reviewed studies that examined dose-intense (DI) chemotherapy alone, in combination with hematopoietic colony-stimulating factors (CSFs), or high-dose therapy (HDT) with autologous bone marrow support (ABMT). RESULTS: DI therapy without CSF or ABMT has not been shown to improve overall outcome in any tumor except consolidative therapy of acute myelogenous leukemia (AML). In solid tumors, many published studies suggest that less than standard-intensity chemotherapy is suboptimal, but few studies that examined higher compared with standard-dose therapy have shown a significant difference in outcome. No studies have convincingly demonstrated improved overall survival (OS) with DI therapy with CSF support. The use of HDT with ABMT has been shown to improve survival in multiple myeloma (MM), as well as relapsed intermediate- and high-grade non-Hodgkin's lymphoma (NHL). High-dose chemotherapy with ABMT is promising in patients with metastatic breast cancer (MBC), but it should not yet be considered a standard approach for these patients. CONCLUSION: DI chemotherapy is an acceptable and standard therapeutic maneuver for patients with AML in first remission, MM, and relapsed aggressive NHL. In solid tumors, the use of DI chemotherapy either alone or with cytokine support has not been shown to improve outcome and should not be considered standard therapy. Current randomized trials should provide definitive answers about the role of DI therapy in solid tumors.

Fluctuation tests: how reliable are the estimates of mutation rates?

Fifty one years ago, Luria and Delbrück published in Genetics a paper that was to become a classic. In it they proved, beyond all reasonable doubt, that bacteria were mutating to phage resistance long before they could have encountered any bacteriophage. Luria and Delbrück also showed how the same experimental data could be used to estimate bacterial mutation rates. Since that time and in many different contexts the methods that they introduced have been used to estimate mutation rates. However, little seems to be known about the errors to be expected in such estimates. In what follows I examine how much uncertainty in the estimates is to be expected merely on the basis of the stochastic variability inherent in the sampling process. On the basis of this examination I question a few traditional ideas and conclude with some practical suggestions. The results were obtained by stimulation. It is my hope that they may inspire others to provide a rigorous theoretical basis for such calculations.

The population biology of bacterial plasmids: a priori conditions for the existence of mobilizable nonconjugative factors.

A mathematical model for the population dynamics of nonconjugative plasmids that can be mobilized by conjugative factors is presented. In the analysis of the properties of this model, primary consideration is given to the conditions under which these nonself-transmissible extrachromosomal elements could become established and would be maintained in bacterial populations. The results of this analysis demonstrate the existence of conditions where, as a consequence of infectious transmission via mobilization, nonconjugative plasmids could become established and be maintained even when the bacteria carrying them have lower reproductive fitnesses than plasmid-free members of the population. However, these existence conditions are stringent and suggest therefore, that it is highly unlikely that plasmids of this type would become established and maintained without some direct selection favoring their carriage. The general implications of these results and limitations of the model are discussed. Brief consideration is also given to the implications of these theoretical findings to the problems of the spread of multiple antibiotic resistance plasmids (R-factors) and the risk of contaminating natural populations of bacteria with chimeric plasmids produced by work with recombinant DNA.

The Population Biology of Bacterial Plasmids: A PRIORI Conditions for the Existence of Conjugationally Transmitted Factors.

A mathematical model for the population dynamics of conjugationally transmitted plasmids in bacterial populations is presented and its properties analyzed. Consideration is given to nonbacteriocinogenic factors that are incapable of incorporation into the chromosome of their host cells, and to bacterial populations maintained in either continuous (chemostat) or discrete (serial transfer) culture. The conditions for the establishment and maintenance of these infectious extrachromosomal elements and equilibrium frequencies of cells carrying them are presented for different values of the biological parameters: population growth functions, conjugational transfer and segregation rate constants. With these parameters in a biologically realistic range, the theory predicts a broad set of physical conditions, resource concentrations and dilution rates, where conjugationally transmitted plasmids can become established and where cells carrying them will maintain high frequencies in bacterial populations. This can occur even when plasmid-bearing cells are much less fit (i.e., have substantially lower growth rates) than cells free of these factors. The implications of these results and the reality and limitations of the model are discussed and the values of its parameters in natural populations speculated upon.

Fluctuation analysis: the probability distribution of the number of mutants under different conditions.

In the 47 years since fluctuation analysis was introduced by Luria and Delbrück, it has been widely used to calculate mutation rates. Up to now, in spite of the importance of such calculations, the probability distribution of the number of mutants that will appear in a fluctuation experiment has been known only under the restrictive, and possibly unrealistic, assumptions: (1) that the mutation rate is exactly proportional to the growth rate and (2) that all mutants grow at a rate that is a constant multiple of the growth rate of the original cells. In this paper, we approach the distribution of the number of mutants from a new point of view that will enable researchers to calculate the distribution to be expected using assumptions that they believe to be closer to biological reality. The new idea is to classify mutations according to the number of observable mutants that derive from the mutation when the culture is selectively plated. This approach also simplifies the calculations in situations where two, or many, kinds of mutation may occur in a single culture.

Regions of stable equilibria for models of differential selection in the two sexes under random mating.

The equilibrium structure of models of differential selection in the sexes is investigated. It is shown that opposing additive selection leads to stable polymorphic equilibria for only a restricted set of selection intensities, and that for weak selection intensities must be of approximately the same magnitude in the sexes. General models of opposing directional selection, with arbitrary dominance, are investigated by considering simultaneously the stability properties of the trivial equilibria and the curve along which multiple roots appear. Numerical calculations lead us to infer that the average degree of dominance determines the equilibrium characteristics of models of opposing selection. It appears that if the favored alleles are, on the average, recessive, there may be multiple polymorphic equilibria, whereas only a single polymorphic equilibrium can occur when the favored alleles are, on the average, dominant. The principle that the average degree of dominance controls equilibrium behavior is then extended to models allowing directional selection in one sex with overdominance in the other sex, by showing that polymorphism is maintained if and only if the average fitness in heterozygotes exceeds one.

Indications and relative indications for stem cell transplantation in non-Hodgkin's lymphoma.

High dose chemotherapy with or without total body irradiation and autologous stem cell rescue has proven to be effective treatment to cure patients with relapsed intermediate grade and high grade non-Hodgkin's lymphoma. Important factors for selection of candidates most likely to do well with these approaches include patients whose disease is responsive to conventional therapy and those who have minimal disease volume at the time of transplant. The treatment-related mortality of autologous stem cell transplantation has diminished from 20% to less than 5% with improved supportive care and selection of patients with less advanced disease. Although the treatment-related mortality of allogeneic stem cell transplantation may be as high as 20-40%, a graft versus lymphoma effect may decrease relapse with the result that overall survival is not substantially different between autologous and allogeneic transplantation. The definitive indications for stem cell transplantation include patients who have relapsed with intermediate or high grade NHL. Relative indications include intermediate/high grade non-Hodgkin's lymphoma patients, "high risk" first complete remission (CR), resistant relapse; low grade non-Hodgkin's lymphoma in sensitive or resistant relapse, advanced disease (sensitive or resistant relapse, transformation), first CR (younger patients). Relative contraindications include specific patient profiles such as bulky high grade lymphoma which progresses on appropriate conventional therapy, poor performance status, active serious infection, serious cardiac, renal, pulmonary or liver dysfunction, active, central nervous system (CNS) disease unresponsive to cranial irradiation/intrathecal therapy. For patients with previous marrow involvement or active marrow involvement at the time of harvest or transplant, "purged" autografts, peripheral blood stem cell transplantation and allografts have been used successfully.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) priming with successive concomitant low-dose Ara-C for elderly patients with secondary/refractory acute myeloid leukemia or advanced myelodysplastic syndrome.

Patients with advanced MDS and secondary AML respond poorly to chemotherapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can stimulate proliferation of leukemic blasts and sensitize these cells to the cytotoxic effects of S-phase-specific drugs. This is the first report of safety and efficacy of GM-CSF prior to and during cytarabine in a low-dose, intermittent regimen for elderly patients with poor risk acute myelogenous leukemia or myelodysplastic syndrome. Twenty patients, age 68 to 86 years, each received 250 microg/m2 of GM-CSF (Sargramostatin; Immunex, Seattle, WA, USA) subcutaneously (s.c.) or intravenously (i.v.) for 3 days followed by GM-CSF at the same dose and cytarabine 100 mg/m2 i.v. for 3 days. GM-CSF and cytarabine were both administered for 3 days during weeks 2 and 3 followed by a 3-week rest period. Rates of CR and PR were 20% and 40%, respectively. These included clinically significant resolution of cytopenias and transfusion requirements. Many of the responding patients had been heavily pretreated prior to enrollment. One- and 2-year survival estimates are 44% and 19%, respectively. Myelosuppression was the most significant toxicity. Our findings suggest that this novel combination of GM-CSF with sequential and concomitant low-dose cytarabine can benefit patients with poor risk myeloid malignancies.

Murine marrow cellularity and the concept of stem cell competition: geographic and quantitative determinants in stem cell biology.

In unperturbed mice, the marrow cell numbers correlate with the stem cell numbers. High levels of long-term marrow engraftment are obtained with infusion of high levels of marrow cells in untreated mice. To address the issue of stem cell competition vs 'opening space', knowledge of total murine marrow cellularity and distribution of stem and progenitor cells are necessary. We determined these parameters in different mouse strains. Total cellularity in BALB/c mice was 530+/-20 million cells; stable from 8 weeks to 1 year of age. C57BL/6J mice had 466+/-48 million marrow cells. Using these data, theoretical models of infused marrow (40 million cells) replacing or adding to host marrow give chimerism values of 7.5 and 7.0%, respectively; the observed 8-week engraftment of 40 million male BALB/c marrow cells into female hosts (72 mice) gave a value of 6.91+/-0.4%. This indicates that syngeneic engraftment is determined by stem cell competition. Our studies demonstrate that most marrow cells, progenitors and engraftable stem cells are in the spine. There was increased concentration of progenitors in the spine. Total marrow harvest for stem cell purification and other experimental purposes was both mouse and cost efficient with over a four-fold decrease in animal use and a financial saving.

Acute nonlymphocytic leukemia and acute aplastic anemia. The simultaneous occurrence in marital partners.

A young husband and wife couple presented simultaneously with aplastic anemia and acute myelomonocytic leukemia, respectively. No etiologic agent or exposure was identified. With appropriate therapy, the wife achieved a complete remission and the husband has had a good response. Potential etiologic environmental factors known to cause both disorders are discussed. A single causative agent may induce stem-cell changes in genetically dissimilar persons and produce a spectrum of pathologic features ranging from aplastic anemia to acute leukemia.

The role of the chest roentgenogram in febrile neutropenic patients.

In a retrospective review of patients with neutropenia and fever, we sought to determine how often roentgenograms detected pulmonary disease, especially pneumonia, not suggested by signs and symptoms. Further, we sought to determine how often therapy was changed as a result of roentgenographic findings. Overall, 41 (22%) of 187 chest roentgenograms obtained during initial febrile episodes, recurrent fevers, or persistent fevers were abnormal. While most patients had signs and symptoms suggesting the presence of pulmonary disease, 17% had roentgenographic abnormalities detected in the absence of such findings. During initial febrile episodes, therapy was not changed in response to findings on the chest roentgenogram. However, during episodes of persistent or recurrent fever, findings on chest roentgenograms led to changes in therapy in eight (61%) of 13 episodes of which six (40%) resulted in clinical improvement. Chest roentgenograms were therefore found to be an important diagnostic tool in evaluating recurrent or persistent fever in the neutropenic patient but of little use during initial febrile episodes.

A survey of dental school applicants' career intentions and the balance with family life.

PURPOSE: To investigate the career plans of prospective dental students and how they foresee their work life balance. METHOD: Applicants to Dundee and Manchester dental schools completed anonymous questionnaires when they attended for interview. RESULTS: The useable response rate was 94% (n=436). The majority of the respondents (91.3%) intended working full time when they enter the workforce, with no significant variation detected between males and females. The cohort anticipated their mean salary to be just over 28,000 UK pounds, five years into their career, although the males felt they would be earning 5,000 UK pounds more than the females. Individuals of Pakistani and Indian origin thought they would earn most, and Asians least. Sixty-five per cent would enter general dental practice and, of these, only 2.8% expected to work exclusively within the NHS. Fifteen per cent intended to go into the hospital dental service, with orthodontics the most popular choice of subspecialty (43.7%), followed by oral surgery (31.1%). Significant variation was seen between ethnic groups, with the hospital and community dental services being more popular with those who identified themselves as of non-white ethnic origin, although the majority would still plan on entering general dental practice. Almost half (44.5%) would take time out of their career to concentrate on childcare when children were of pre-school age, with a further 11% taking longer. Ninety per cent of females and 70% of males anticipated taking time out, of a varying duration. Half of the respondents indicated that they felt a child would affect their career to a moderate extent. CONCLUSIONS: The dental profession will be severely affected if both males and females take time out of their careers in the future. As well as a work force shortage, the problems of accessibility to NHS dental services will be exacerbated if fewer dentists choose to provide NHS care.

Synergistic interactions between hematopoietic growth factors as detected by in vitro mouse bone marrow colony formation.

We have investigated the proliferative effects of several combinations of hematopoietic growth factors in agar cultures of murine bone marrow cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) synergized with granulocyte colony-stimulating factor (G-CSF), while G-CSF also synergized with macrophage colony-stimulating factor (CSF-1) and interleukin 3 (IL3), resulting in colony numbers greater than the sum of the numbers of colonies formed with each factor alone. In addition, these combinations resulted in increased colony sizes, with the formation of day-14 colonies with diameters greater than 0.5 mm. The combination of GM-CSF plus IL3 showed an increase in numbers of colonies that approximated the sum of that seen with each factor alone, however, the size of the colonies was increased with a number of day-14 and day-21 colonies having diameters greater than 0.5 mm. These data add to the list of hematopoietic factors known to synergistically stimulate myeloid progenitors and suggest that some of these interactions may be on early progenitor cells with high proliferative potentials.

Progenitor cell numbers (CFU-GM, CFU-D, and CFU-Mix) and hemopoietic recovery following autologous marrow transplantation.

Forty-one consecutive patients were treated with high-dose chemotherapy with or without total body irradiation followed by autologous marrow transplantation. Four treatment regimens of varying intensity were used. Every patient's harvested marrow was evaluable for nucleated cell and progenitor cell loss during the cell separation and cryopreservation process. Of the 41 patients, 38 were evaluable for peripheral blood count recovery. Multivariate analysis of colony-forming cell assays and recovery of neutrophils and platelets showed a significant association with absolute numbers of post-thaw mixed colony-forming units (CFU-Mix) infused (p less than 0.002). Prefreeze CFU-Mix also correlated with recovery to a lesser degree, as did absolute numbers of nucleated cells. The number of diffusion chamber colony-forming units (CFU-D) prefreeze, but not post-thaw infused into the patient, was associated with recovery of neutrophils (p = 0.0001), but not platelets. When the precursor cell numbers were adjusted for body weight, post-thaw CFU-Mix showed the best correlation with recovery of both platelets and neutrophils. Prefreeze CFU-D per kg was also associated with recovery of neutrophils (p = 0.02). To some extent nucleated cells per kg predicted for recovery with neutrophils and platelets (p less than 0.05). When analyzed according to treatment regimen, cyclophosphamide-BCNU-VP16 (CBV) or cyclophosphamide-total body irradiation (CY/TBI) was associated with prolonged recovery compared to cyclophosphamide-adriamycin-vinblastine (CAV) or etoposide-cyclophosphamide (EC). In this setting only CFU-D number predicted neutrophil recovery (p less than 0.002). We conclude that determination of the number of total nucleated cells, CFU-D, and CFU-Mix, before cryopreservation of the sample is important in predicting hemopoietic reconstitution in autologous bone marrow transplantation.

Stem cell transplantation in the normal nonmyeloablated host: relationship between cell dose, schedule, and engraftment.

In previous studies we have shown high rates of stable engraftment when 40 million male BALB/c cells were infused intravenously daily for 5 days (a total of 200 million cells) to normal nonmyeloablated female hosts. The present studies evaluate engraftment of male BALB/c bone marrow cells in female host marrow, spleen, and thymus 20-25 weeks after transplantation using varying cell dosages within a 5-day schedule. Engraftment in recipient mice was assessed by detection of male specific sequence in recipient DNA from each organ. When 40 million cells were given per daily injection for 1, 2, 3, 4, or 5 days, engraftment percentages in host marrow were 11 +/- 0.83, 20 +/- 2.0, 23 +/- 2.5, 32 +/- 6.3, and 39% +/- 5.7 (+/- standard error of mean), respectively, yielding engraftment percentages per million cells infused of 0.28, 0.25, 0.19, 0.20, and 0.20%, respectively. When levels of 2.5, 5, 10, 20, or 40 million cells were injected 5 times over a 5-day schedule into normal BALB/c female hosts, progressively increasing levels of engraftment from 3 +/- 0.6 to 39% +/- 5.7 were seen in host marrow. Highest levels of engraftment per million cells injected were obtained on days 1 and 2 of a 5-day schedule and with a level of 10 million cells given daily over 5 days. Engraftment profiles varied with spleen and thymus and percent engraftment was generally lower than for marrow. The present work indicates that regardless of cell level infused or number of infusions, rates of engraftment observed in marrow approached or exceeded the highest rates of engraftment estimated by theoretical calculations based on replacing host cells ("replacement model") or adding to host cells ("incremental model"). Engraftment in spleen and thymus was lower, but also at times approached or exceeded theoretical maxima. These data show extraordinary levels of engraftment in normal hosts, suggesting that rates in this competitive model are superior to those seen in irradiated hosts; alternatively, there may be selective repression of host stem cell proliferation and differentiation.

Treatment of small-cell carcinoma of the cervix with weekly combination chemotherapy.

Three patients with small-cell carcinoma of the cervix entered a pilot study of combination chemotherapy with agents that are not cross-resistant. Two patients had local disease and the third had extensive metastatic disease of the liver. The regimen consisted of weekly chemotherapy for 16 weeks with cisplatin, vincristine, methotrexate, doxorubicin, cyclophosphamide and etoposide followed by radiotherapy and/or surgery. The two patients with local disease achieved a pathological complete response, with no evidence of disease at 24 months and 15 months from diagnosis. The third patient achieved a partial response and is alive at 13 months with progressive disease. Side-effects were tolerable.

Manipulation of the stem cell as a target for hematologic malignancies.

Hematologic malignancies affect more than 80,000 patients in the United States each year. Some patients with lymphoma and leukemia are cured with conventional chemotherapy treatments. For others, autologous or allogeneic bone marrow transplantation may be the best therapeutic option. This chapter will explore novel therapies for the hematologic malignancies, using the stem cell as a target. We review work in the murine model looking at (1) the phenotype of the engrafting cells, (2) stem cell competition and host stem cells, (3) allochimerism with low-dose total body irradiation, and (4) the tolerance approach with costimulator blockade. Human data, including stem cell migration, adhesion receptor expression, and manipulations for gene therapy, are reviewed. The NOD/scid mouse model serves as a bridge between the basic bench work and human clinical trials, and we discuss applications related to umbilical cord blood and gene therapy, as well as discuss the inherent variability of this system. Finally, we address unique clinical applications in gene therapy, high-dose cell transplants, minimal myeloablation, and cellular immune therapy as approaches to treatment of for patients with hematologic malignancies.