The course and mortality of a hematopoietic neoplasm in the soft-shell clam,Mya arenaria

Results from two experiments containing approximately 280 Mya arenaria indicated that significantly higher (P < 0.05) mortality occurred within the neoplastic clam population than in the nonneoplastic clam population. Using an in vivo blood cytological technique, five levels of severity were established. The levels were based on the number of neoplastic cells in the circulation with level 1 as the lowest severity and level 5 as the highest severity. Neoplastic clams that were diagnosed as the lowest level had higher survival rates (60%) than those clams diagnosed as the highest level (0%). The hematopoietic neoplasm in M. arenaria followed one of three courses: (1) in approximately 50% of the neoplastic clams the disease progressed to a higher severity and resulted in death; (2) in approximately 40% of the neoplastic clams the disease was chronic, i.e., remained at a stable level; and (3) in approximately 10% of the neoplastic clams the disease diminished in severity or disappeared entirely. In addition, 10% of the clams diagnosed as nonneoplastic at the beginning of the experiments were neoplastic by the termination of the experiment. The contraction of the disease may have been de novo or by stimulation of latent infections.The prevalence of the neoplasm in clams collected from an epizootic area followed a biphasic seasonal pattern. The highest prevalences occurred in October, November, and in May. The hematopoietic neoplasm in M. arenaria was also age and species specific.     

5-bromodeoxyuridine induction of hematopoietic neoplasia and retrovirus activation in the soft-shell clam, Mya arenaria

Hematopoietic neoplasia and virus replication were induced in shoft-shell clams, Mya arenaria, by 5-bromodeoxyuridine (5-BrdUrd). Eighty clams were found to be nonneoplastic by the in vivo bleeding method. These clams were randomly distributed into four aquaria and maintained in seawater at 1°C. 5-BrdUrd was added to aquaria in the following concentrations: 0, 20, 100, and 200 μg/ml. After 4 days of treatment, the aquaria were drained, rinsed, and fresh sea water without 5-BrdUrd was added. Sea water was changed on a weekly basis thereafter. The clams were diagnosed for neoplasia by the in vivo method at days 4, 9, 16, and 23 of the experiment. Results showed that on day 23, neoplasia was not found in the aquarium without 5-BrdUrd, but in the aquaria containing 20, 100, and 200 μg/ml of 5-BrdUrd the incidences of neoplasia were 37, 79, and 35%, respectively. 5-BrdUrd-induced neoplastic tissue was homogenized and subjected to differential ultracentrifugation. Retrovirus-like particles resembling ones previously shown to induce neoplasia were isolated from neoplastic clams which were induced by 5-BrdUrd. When these particles were inoculated into healthy clams, the clams developed neoplasia. Neoplastic hemocytes, cultured in sea water containing 50 μg/ml of 5-BrdUrd, formed pseudopodia after 4 days. Pseudopod formation is a trait of normal hemocytes. This indicates that differentiation of neoplastic hemocytes may be induced by the drug.     

Ultrastructural study of neoplastic cells in Macoma balthica (Bivalvia) from the Gulf of Gdansk (Poland)

In the Baltic clam Macoma balthica from the Gulf of Gdansk, neoplasia is considered as a serious epizootic linked to bad environmental conditions and high levels of pollutants. Previous research was focused on the diagnosis, prevalence, seasonality and histopathological characteristic of the cancer. This study is focused on electron microscopy analyses describing cell ultrastructure abnormalities related to neoplasia. Examinations using the electron microscopy highlighted changes confined to anatomic ultrastructures, shapes and functions of neoplastic cells. The lobulated appearance of the nucleus, changes in cellular matrix and the occurrence of large granular cells with hyperchromatic nuclei, atypical Golgi structures and deterioration of rough endoplasmic reticulum manifested the disease. The presence of atypical mitochondria, free ribosomes and hypertrophic nuclei suggests the adaptation of neoplastic cells to increased mitotic activity, while the observed modification of cellular membranes may reflect functional changes connected to increased pinocytotic activity or intercellular transport. The cancer cells were found to appear in two types, abnormal round-shaped cells and spindle-shaped cells, both with increased frequencies of cell division. Round-shaped cells typical for disseminated neoplasia were observed in all affected bivalves, in a few cases co-occurring with abnormal spherical cells. Spindle-shaped cells containing some intracytoplasmic filaments, and with a tendency of the nuclei to be orientated as in a palisade were interpreted as myofibroblasts-like cells and were observed in five out of eleven clams diagnosed as neoplastic. This finding represents the first demonstration of phenotypic differences in the cell types co-occurring in animals diagnosed as neoplastic and by that suggests coexistence of two types of bivalve cancer, disseminated neoplasia and probable fibrosarcoma.     

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: I. Dynamics of QPX disease

Quahog Parasite Unknown (QPX) causes disease and mortality in hard clams, Mercenaria mercenaria. Seasonality of QPX disease prevalence in the field and changes in QPX growth and survival in vitro suggest a role of temperature in the hard clam-QPX interaction and disease development. This study specifically examined the effect of temperature on QPX disease development and dynamics. Naturally and experimentally infected clams were separately maintained in the laboratory at 13 °C, 21 °C, or 27 °C for 4 months. Following this initial treatment, temperature was adjusted to 21 °C for 5 additional months to simulate seasonal changes of temperature in the field and to investigate the effect of temperature variations on QPX disease dynamics. Mortality was continuously monitored during the experiment and clams were sampled at 2, 4 and 9 months for the assessment of QPX disease prevalence and intensity using our standard histological and quantitative PCR techniques. Results demonstrated significantly higher QPX disease prevalence and intensity, as well as higher mortality, in naturally-infected clams maintained at 13 °C as compared to those held at 21 °C or 27 °C. Similarly, disease development was significantly higher in experimentally infected clams maintained at the colder temperature (70% prevalence after 4 months) as compared to those maintained under warmer conditions (<10%). Additionally, our results demonstrated an improvement in the condition of clams initially maintained at 13 °C for 4 months after transfer to 21 °C for 5 additional months, with a significant reduction of QPX prevalence (down to 19%). Interestingly, disease development or healing in clams maintained at different temperatures exhibited a strong relationship with clam defense status (jointly submitted paper) and highlighted the impact of temperature on clam activity and QPX disease dynamics. These findings should be taken into account for the timing of activities involving the monitoring, movement (e.g. relays, transplants) or grow out (e.g. commercial culture, municipal enhancement) of hard clams in enzootic areas.     

Isolation of a viral agent causing hematopoietic neoplasia in the soft-shell clam,Mya arenaria

A virus from neoplastic soft-shell clams, Mya arenaria, has been isolated. Its morphological and physical properties are similar to those of B-type retroviruses. It is enveloped, pleomorphic, averaging 120 nm in size with an eccentric nucleoid. Possible A-type particles have also been observed. The bouyant density of the particle ranged from 1.17 to 1.18 g/cm³ and the ultraviolet absorption $\text{260}\text{280}\text{-}\text{nm}$ ratio was 1.23. The virus has been shown to cause a hematopoietic neoplasm in Mya. Purified virus from neoplastic clams was innoculated into nonneoplastic clams. Most of these clams developed tumors within 2 months. Virus isolated from inoculated clams which developed neoplasia was inoculated into nonneoplastic clams. Most of these clams also developed neoplasia thus completing Koch's postulates.     

Accuracy of blood cytological screening techniques for the diagnosis of a possible hematopoietic neoplasm in the bivalve mollusc,Mya arenaria

The two in vivo bleeding techniques currently in use in our laboratory to diagnose a hematopoietic neoplasm in Mya arenaria are: (1) phase-contrast microscopy with fresh unstained hemocytes, and (2) bright-field microscopy with Giemsa-stained hemocytes. All in vivo diagnoses were checked by histopathological studies on tissues of the same mollusc. For both methods the correct diagnosis (true + or true ?) was made in 94 out of 100 clams examined. A gradation of tissue involvement was observed in the diseased clams and the accuracy of the in vivo diagnosis is related to the disease severity. There is a positive correlation between the degree of tissue involvement and the number of circulating neoplastic cells. For this reason the more extensive the neoplasm the better is the ability to diagnose the neoplasm by the in vivo bleeding techniques. Depending on the percentage of neoplastic cells present in the hemolymph, the neoplasm was graded from level 1 to 5, with 5 being the most severe. In general, at level 1, the accuracy of a single in vivo diagnosis varied from 66 to 71% and at level 2, the accuracy of diagnosis varied from 76 to 93%, while at all other levels the accuracy was 100%. The percentage of diseased clams detected by the in vivo bleeding technique was 89–91% and the percentage of nondiseased clams detected was 95%. These values can be further improved by combining the two tests and/or through multiple bleedings. Between the two types of in vivo tests, the Giemsa-stained hemocytes provided better precision of diagnosis than the fresh unstained cells, although the differences were slight.     

Assessment of haemic neoplasia in different soft shell clam Mya arenaria populations from eastern Canada by flow cytometry

Diagnosis of haemic neoplasia (HN) in the soft shell clam, Mya arenaria, is often achieved by hematocytology and histology. Since neoplastic cells display tetraploid DNA contents, haemocyte cell cycle analysis was developed for use as a diagnosis tool. The aim of this study was to assess the application of a flow cytometry procedure of cell cycle analysis established for the common cockle, to clams and to evaluate different thresholds of value for the percentage of tetraploid cells for establishing HN disease status of individual clams and clam populations. HN status of six clam populations from eastern Canada was determined. Results of the present study demonstrate a flow cytometry procedure to be useful for HN diagnosis in clams. Individual clams were considered to be affected by HN when presenting at least 20% of haemocytes in S-4N phase; and negative when presenting less that 5% of haemocytes in S-4N phase. As discussed in this paper, intermediate cases represent uncertain diagnoses including either false-negative or false-positive clams, which are difficult to discriminate. At a population level, an additional threshold of 15% for the mean intensity of the disease is proposed, which means having in the population several individual clams presenting more than 20% of their haemocytes in S-4N phase. Based on these thresholds of value, only one population was considered as free of HN disease, and one population was unequivocally affected by HN. For the four other clam populations, further investigations are needed toward development and use of specific and objective biomarkers of HN.     

Impaired defense mechanisms in bay mussels, Mytilus edulis, with hemic neoplasia

Immunocompetence of bay mussels, Mytilus edulis, with hemic neoplasia was investigated with an in vitro yeast phagocytosis assay and by in vivo clearance from the blood of injected Cytophaga sp. bacteria. The yeast phagocytosis assay was conducted with hemocytes maintained in 90% plasma. Neoplastic hemocytes, characterized by enlarged nuclei and scant cytoplasm, failed to phagocytose yeast cells. In contrast, greater than 90% of hemocytes from unaffected animals and morphologically normal hemocytes from mussels with the disease phagocytosed yeast. Substitution of normal plasma with that from a mussel with advanced disease (essentially 100% neoplastic hemocytes) did not affect the phagocytic capability of normal hemocytes. Conversely, normal plasma did not enhance the phagocytic capabilities of neoplastic cells. Mussels with advanced disease showed reduced bacterial clearance; control or lightly affected mussels (less than 11% neoplastic hemocytes) cleared greater than 90% of injected bacteria in 4 hr, while mussels with advanced disease cleared 44-83%. These experiments indicate that mussels with advanced hemic neoplasia have compromised defense systems. This may account for the reported mortality in mussels and other bivalve molluscs with hemic neoplasia.     

Disseminated neoplasia in cockles Cerastoderma edule: ultrastructural characterisation and effects on haemolymph cell parameters

Disseminated neoplasia (DN) has been detected in cockles from various beds in Galicia (NW Spain). A study was performed to characterise cockle neoplastic cell ultrastructure and to evaluate the effect of this disease at different severity stages on various haemolymph cell parameters. Examination of cockle neoplastic cells with transmission electron microscopy (TEM) showed round shapes and a lack of pseudopods, a high nucleus:cytoplasm diameter ratio, Golgi complexes, abundant mitochondria, ribosomes, and numerous endoplasmic reticulum tubes and electron-lucent vesicles. Various haemolymph cell parameters (cell mortality, non-specific esterase and lysosome biovolume, reactive oxygen intermediates [ROI] production, phagocytosis ability, intracellular Ca2+ and actin levels) were compared between DN severity categories by flow cytometry; haemocyte mortality, non-specific esterase activities and lysosome biovolume were found to be higher with increasing DN severity. The phagocytic ability of neoplastic cells was sharply reduced with regard to haemocytes. The cytoplasmic-free Ca2+ level was higher and actin content lower in haemolymph cells of diseased cockles compared to unaffected ones. A significant increase in ROI production was detected in later stages of disease progression.     

Neoplasia detection in Macoma balthica from the Gulf of Gdansk: comparison of flow cytometry, histology and chromosome analysis

A flow cytometry protocol was applied for the detection of neoplasia in Macoma balthica L. from the Gulf of Gdansk (Baltic Sea, Poland). A simple method, based on an osmotic shock, was used to permeabilise gill cells. The cytometric pattern of normal clams consisted of 2 peaks, a major peak B and a smaller peak C. The cytometric pattern of affected clams consisted of 2 peaks named B' and C'. Two parameters were used to define the stages of abnormalities in M. balthica clams based on the percentage of cells in peaks B, C, B' and C' and on the ratio between the fluorescence value of peaks B, C, B' and C' in all individuals. Three stages of neoplasia were clearly distinguished by flow cytometry considering peak C'. Stage 1 was characterised by a major population of cells in peak B' and more than 10% of cells in the C' peak. Stage 2 consisted of a lower percentage of cells in peak B' and more than 25% of cells in peak C'. Stage 3 of the neoplasia was characterised by a further reduction in peak B' and more than 40% of cells in peak C'. Flow cytometry allowed for objective detection of neoplasia and provided a rapid method for measuring the DNA content of thousands of cells per individual. The accuracy of flow cytometry was assessed by comparing with standard histological techniques, used here as a reference technique for the detection of neoplasia, and with chromosome analysis. All individuals were analysed in parallel using the 3 techniques. The proportion of normal and affected individuals diagnosed using flow cytometry was comparable to the proportion determined by histology and chromosome analysis.     

Survey for neoplasia in Macoma balthica from the Gulf of Gdansk by flow cytometry

Using flow cytometry, 234 Macoma balthica were examined during a survey to determine frequency of neoplasia in the Gulf of Gdansk (Poland). Clams were collected in 4 locations and DNA content in gill tissue cells was determined by flow cytometry using propidium iodide staining. Cell permeabilization was induced by osmotic shock. Prevalence of neoplasia ranged from 9.6 to 26.7% depending on location. DNA content in aneuploid cells was higher than in normal dividing cells. The fluorescence value for aneuploid cells corresponded to tetraploid/pentaploid cells. Three stages of neoplasia were defined, based on the percentage of aneuploid cells determined by flow cytometry. Histopathological and cytogenetic analyses were also carried out on the same clams for comparative study. Proportions of normal and affected clams detected using flow cytometry were similar to those identified using both methods. In the present study, no clear relationship was demonstrated between prevalence of neoplasia and pollutant detection in the different sampling sites.     

Alternate pathogenesis of systemic neoplasia in the bivalve mollusc Mytilus.

The proliferative disease systemic neoplasia, also termed hemic neoplasia or disseminated sarcoma, was studied in four Puget Sound, Washington populations of the bay mussel (Mytilus sp.). Using flow cytometric measurement of DAPI-stained cells withdrawn from the hemolymph, DNA content frequency histograms were generated for 73 individuals affected by the disease. The cells manifesting systemic neoplasia were found to exist as either of two separate types, characterized by G0G1 phase nuclear DNA contents of either approximately 4.9 x haploid (pentaploid form) or approximately 3.8 x haploid (tetraploid form). The two disease forms were found to coexist in all four mussel populations sampled, with overall relative prevalences of 66% pentaploid form, 29% tetraploid form, and 5% exhibiting both disease forms simultaneously. These findings represent the first unequivocal demonstration of multiple cell types in a bivalve neoplasia. The two forms appear to represent separate pathogenetic processes rather than sequential stages of a single pathogenesis. Two cell cycling parameters associated with proliferative activity were employed to compare the alternate forms: (i) the percentage of cells assigned to the DNA Synthesis (S) phase of the neoplastic cell cycle, and (ii) the proportion of neoplastic cell mitotic figures in hemocytological preparations. Mean values for both parameters were significantly higher for mussels with the tetraploid form of the disease, suggesting a higher rate of proliferation relative to the pentaploid form. Qualitatively, cells of the tetraploid form contained slightly lower nuclear and cytoplasmic volumes compared to those of the pentaploid form. An observed wide variation in neoplastic cell nuclear size within either disease form may reflect the distribution of cells in the G0G1, S, and G2M phases of the cell cycle. Potential etiologic relationships between the two forms are discussed.     

Incidence of AIDS and excess of mortality associated with HIV in haemophiliacs in the United Kingdom: report on behalf of the directors of haemophilia centres in the United Kingdom.

OBJECTIVE--To estimate the cumulative incidence of AIDS by time since seroconversion in haemophiliacs positive for HIV and to examine the evidence for excess mortality associated with HIV in those who had not yet been diagnosed as having AIDS. DESIGN--Analysis of data from ongoing national surveys. SETTING--Haemophilia centres in the United Kingdom. PATIENTS--A total of 1201 men with haemophilia who had lived in the United Kingdom during 1980-7 and were positive for HIV. INTERVENTION--None. END POINTS--Diagnosis of AIDS; death in those not diagnosed as having AIDS. MEASUREMENTS AND MAIN RESULTS--Estimation of cumulative incidence of AIDS and number of excess deaths in seropositive patients not diagnosed with AIDS. Median follow up after seroconversion was 5 years 2 months. Eight five patients developed AIDS. Cumulative incidence of AIDS five years after seroconversion was 4% among patients aged less than 25 at first test positive for HIV, 6% among those aged 25-44, and 19% among those aged greater than or equal to 45. There was little evidence that type or severity of haemophilia or type of factor VIII or IX that had caused HIV infection affected the rate of progression to AIDS. Mortality was increased among those who had not been diagnosed as having AIDS, especially among those with "AIDS related complex." Thirteen deaths were observed among 36 patients diagnosed as having AIDS related complex against 0.65 expected, and 34 deaths in 1080 other patients against 22.77 expected; both calculations were based on mortality rates observed in haemophiliacs in the United Kingdom in the late 1970s. CONCLUSIONS--Rate of progression to AIDS depended strongly on age. There is a substantial burden of fatal disease among patients positive for HIV who have not been formally diagnosed as having AIDS.     

Susceptibility of juvenile Crassostrea gigas and resistance of Panope abrupta to Mikrocytos mackini

Samples from the field and laboratory exposure to Mikrocytos mackini (a tiny protistan parasite of unknown taxonomic affiliation) confirmed that juvenile Pacific oysters (Crassostrea gigas) are susceptible to infection and the resulting disease. In the laboratory bath exposure experiment, a prevalence of infection approaching 100% and mortalities were observed in the small oysters (about 18 mm in shell length). However, in the same laboratory exposure experiment, similar aged geoduck clams (Panope abrupta, about 8mm in shell length) were resistant to infection. The main route of infection in the oysters appeared to be via the digestive tract and possibly the gills where the parasite multiplied within host cells. Other tissues such as the adductor muscle and vesicular connective tissue were subsequently colonized. Although the infection resulted in the mortality of some oysters, others appeared to overcome the disease.     

Phenotypic plasticity and priority rules for energy allocation in a freshwater clam: a field experiment

We studied resource allocation among maintenance, reproduction and growth in the freshwater clam Anodonta piscinalis. Recent theoretical and empirical studies imply that organisms with indeterminate growth may have priority rules for energy allocation. That being so, the traits involved should potentially be capable of considerable phenotypic modulation, as a mechanism to adjust allocation. We tested this hypothesis using a 1-year reciprocal transplant experiment at six sites. Experimental clams were caged at higher than natural densities in order to detect any phenotypic modulation of the traits and discover the putative priority rules in energy allocation. We recorded the survival and shell growth of clams during the experiment, and the reproductive output, somatic mass and fat content of clams at the end of the experiment. Shell growth, somatic mass, and the reproductive output of females varied more among transplant sites than among the populations of origin, suggesting a high capacity for phenotypic modulation. However, the reproductive investment, somatic mass and shell growth were also affected by origin; clams from productive habitats invested more in reproduction and were heavier. In comparison to undisturbed clams, the reproductive output of the experimental clams was similar and their fat content was higher, whereas their shell growth was considerably slower and their somatic mass lower. These results suggests that when resources are limiting (due to high density) reproductive allocation overrides allocation to somatic growth. The highest mortality during the experiment coincided with the period of reproductive stress in the spring. Additionally, the proportion of reproducing females was lower in those transplant groups where the survival rate was lowest, suggesting that maintenance allocation overrides allocation to reproduction when available resources are scarce. The results of this field experiment support theoretical predictions and results of previous laboratory experiments that suggest that there are priority rules for energy allocation in organisms with indeterminate growth.     

DGGE-based detection method for Quahog Parasite Unknown (QPX)

Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.     

Virulence of parasites in hosts under environmental stress: experiments with anoxia and starvation

Most environments periodically impose severe stress that may cause high mortality and alter population structure, for example, by removing sick and old individuals. We examined how anoxic conditions and starvation of the host affect virulence of two closely related trematode parasites, Rhipidocotyle campanula and R. fennica . These parasites differ by prevalence of infection and by exploitation rate of individual hosts (freshwater clam, Anodonta piscinalis ). Infection by R. campanula is rare (<5% prevalence of infection) and destroys on average 90% of the gonad tissue of the individual host. Infection by R. fennica is more common (20–60% prevalence of infection) and leads to on average 30% gonad destruction. In the end, both infections lead to host infertility. We predicted that R. campanula induces higher host mortality than R. fennica under host stress. In two laboratory experiments, we exposed naturally-infected and uninfected clams to anoxia and to starvation. Anoxia occasionally takes place during winter in eutrophic lakes, while some degree of starvation should occur seasonally. We found that mortality rate of clams was much higher under anoxia than under starvation, and that infection increased mortality rate under both types of host stress. As predicted, R. campanula induced higher host mortality than R. fennica . Host survival was population-specific, suggesting that clams of different origins carried different amount of energy reserves. Severe environmental perturbation may remove R. campanula infected individuals from the host population, but recolonization from the fish host is likely to prevent extinction of the parasite suprapopulation. The observed high host mortality induced by R. campanula may be one ecological explanation for the consistently lower prevalence of infection of R. campanula when compared to R. fennica .     

Cytogenetics and acute non lymphocytic leukemia

The authors report haematologic and cytogenetic data from 47 patients with ANLL, demonstrating the usefulness of cytogenetic studies for the classification as well as for the prognosis of this disorder. Chromosome studies also permitted the classification of marrow cellularity in: all diploid metaphases (NN), diploid and aneuploid metaphases (AN), and all aneuploid metaphases (AA). The remission rate for patients in whom only normal metaphases were detected (NN patients) was 83% while the remission rates were 67% and 33% respectively for patients in whom both normal and abnormal metaphases were seen (AN patients) and for those in whom only abnormal metaphases were noted (AA patients). In all FAB subgroups, complete remission was related to chromosomal abnormalities, except for M4 patients who evidenced a large number of complete remissions, although presenting more chromosomal abnormalities. The longer survival in this subgroup may be related to rearrangements of chromosome 16, which is associated with a better prognosis.     

At the tipping point: Differential influences of warming and deoxygenation on the survival,emergence, and respiration of cosmopolitan clams

Although warming and low dissolved oxygen (DO) levels are co‐occurring significant climatic stressors in the ocean, the combined effects of these stressors on marine benthic animals have not been well established. Here, we tested the effects of elevated temperatures and low dissolved oxygen levels on the survival, emerging behavior from sediment, and the respiration of juvenile cosmopolitan Manila clams (Venerupis philippinarum) by exposing them to two temperatures (20 and 23.5°C) and DO levels (3.5 and 6–7 mg/L). Although within previously described tolerable ranges of temperature and DO, this 3.5°C increase in temperature combined with a 50% decrease in DO had a devastating effect on the survival of clams (85% mortality after 8 days). The mortality of clams under normoxia at 23.5°C appeared to be higher than under the low DO condition at 20°C. On the other hand, more clams emerged from sediment under the low DO condition at 20°C than under any other conditions. Oxygen consumption rates were not significantly affected by different conditions. Our results suggest temperature elevation combined with low oxygen additively increases stress on Manila clams and that warming is at least as stressful as low DO in terms of mortality. However, low DO poses another threat as it may induce emergence from sediment, and, thus increase predation risk. This is the first evidence that a combination of warming and deoxygenation stressors should reduce population survival of clams much more so than changes in a single stressor.     

Combination chemotherapy for acute lymphoblastic leukaemia in adults.

Fifty-one adults with acute lymphoblastic leukaemia were entered into a trial of intense initial chemotherapy and early "prophylaxis" of the central nervous system (CNS). Initial treatment with OPAL (Oncovin (vincristine), prednisolone, adriamycin (doxorubicin), and L-asparaginase (colaspase)) followed by craniospinal or cranial irradiation and intrathecal methotrexate produced remission in 36 patients (71%). Seventeen of these patients relapsed three to 18 months after the start of remission; the remainder had been in remission for 12 to 52 months by the end of the study. The predicted median duration of complete remission was 18.5 months. None of the four patients who initially had clinical evidence of CNS disease, three of whom also had leukaemic cells identical to those found in Burkitt''s lymphoma, achieved remission. Those patients who initially had hepatomegaly or splenomegaly had a shorter remission than those without. The predicted median survival was 27 months in those who achieved complete remission, one month in those who did not, and 21 months overall. The addition of colaspase and doxorubicin to vincristine and prednisolone and the use of early CNS treatment clearly improved the remission rate among adults with acute lymphoblastic leukaemia, though the presence and length of remission was affected by the extent of disease at presentation. Burkitt-like leukaemia, which had a poor prognosis, is probably a separate disease and may benefit from a different therapeutic approach.