Flow cytometric comparison of haemocytes from three species of bivalve molluscs.

Haemocyte subpopulations from three bivalve species (the clams Ruditapes philippinarum and Mercenaria mercenaria and the oyster, Crassostrea virginica) were characterised using light-scatter flow cytometry and a standard set of methods. Two parameter (forward and side scatter) plots for the three species were very similar and resembled plots for mammalian white blood cells. Two haemocyte groups (granulocytes and agranulocytes) were found in both the haemolymph and the extrapallial fluid of the clams while those two groups and an additional third group were found in the haemolymph of the oyster. All subpopulations were sorted on to glass slides, identified, photographed, and measured microscopically. Sorting of the bivalve granulocyte and agranulocyte groups indicated varying degrees of heterogeneity within each population in terms of either size or granularity, or both. However, subsorting of selected regions within the major groupings produced highly pure haemocyte populations. The comparison showed both similarities and differences among species. For instance, a distinct subpopulation of small granulocytes was present only in oysters and a subpopulation of spindle-shaped haemocytes, only in M. mercenaria. The haemocyte subpopulations delineated by light-scatter flow cytometry underscore questions about cell lineages, but the instrument also offers a powerful technique for answering them.

Fatal hypertrophic cardiomyopathy in the fetus of a woman with diabetes.

BACKGROUND: Hypertrophic cardiomyopathy is recognized in infants of diabetic mothers, and when it occurs it is generally benign and transient. We describe a case of fetal cardiac death caused by hypertrophic cardiomyopathy in an infant of a diabetic mother. CASE: Hydrops fetalis caused by hypertrophic cardiomyopathy resulted in the death of a macrosomic male fetus of a young woman who had well-controlled diabetes mellitus and was treated with insulin therapy during pregnancy. CONCLUSION: It is important to monitor fetal heart function in macrosomic infants of diabetic mothers. Hypertrophic cardiomyopathy might explain otherwise unexplained fetal deaths in women with diabetes.

Haemocyte parameters associated with resistance to brown ring disease in Ruditapes spp. clams.

Brown ring disease (BRD) is a shell disease caused by Vibrio tapetis. This pathogen disturbs the periostracal lamina causing the appearance of a brown conchiolin deposit on the inner face of the shell, within the extrapallial space. Although differences in resistance to BRD have been documented, their relationship to possible defense functions has never been investigated. In this study, flow cytometry was used to analyze cellular parameters in asymptomatic and experimentally infected Ruditapes philippinarum from France and the west coast of the USA. Parallel analyses were made on Ruditapes decussatus, the native European clam, which is highly resistant to BRD. In the haemolymph and extrapallial fluid of animals without BRD, total haemocyte counts, the percentage of granulocytes, and the phagocytic activity against latex beads or V. tapetis by the haemocytes were significantly higher in American R. philippinarum than in French R. philippinarum. In most cases, levels in R. decussatus were the highest of all three groups. Four weeks following challenge with V. tapetis, BRD prevalence reached 52 in American clams and 100% in French specimens, but only 37% in R. decussatus. In symptomatic animals, phagocytosis of V. tapetis increased significantly in the resistant species of clam, R. decussatus, was unchanged in US clams, and decreased significantly in FR specimens when compared to asymptomatic individuals from each population. Ingestion of V. tapetis by haemocytes in the extrapallial fluid, which is in contact with the periostracal lamina, could be the main defense mechanism used to counter the pathogen. Our results suggest that resistance to BRD may well be related to the concentration of granular haemocytes and the phagocytic activity of haemocytes.

Epizootiology and pathology of juvenile oyster disease in the Eastern oyster, Crassostrea virginica.

Juvenile Oyster Disease (JOD) causes mortalities of small cultured oysters, Crassostrea virginica. The present study was an intensive epizootiological and pathological investigation of JOD in eight sequentially deployed cohorts at sites on Long Island, New York. JOD symptoms and mortalities began in all groups at about the same time. Lesions on the mantle were detected histologically about 1 week before the principal symptom, a conchiolin deposit on the inner shell, appeared. Mortality began about 1 week later and reached 60-90% in oysters <25 mm. Mantle lesions were highly correlated with subsequent conchiolin-deposit prevalence and with total mortality. Larger juveniles (25-40 mm) were affected by the disease and produced conchiolin deposits, but mortalities did not exceed 30%. Mortalities were consistently related to size, but not necessarily to age or length of "exposure" in the field. There was no indication that JOD was linked to a particular broodstock or hatchery. Wild spat deployed at experimental sites showed JOD symptoms before the hatchery-produced groups did and cohorts maintained inside a hatchery experienced essentially no JOD. Histological examination of cohorts experiencing high mortalities failed to reveal an obvious etiological agent, but showed a disease pattern similar to that described for other bivalve diseases with a bacterial etiology. Similarities and differences between this and other studies of JOD suggest that one or more bacterial species is responsible for JOD, but that a trigger, probably temperature, is also involved and may vary from site to site.

Thrombotic thrombocytopenic purpura as an initial presentation of limited systemic sclerosis.

Thrombotic thrombocytopenic purpura (TTP) is a rare complication of scleroderma (systemic sclerosis, SSc). In the 5 reports documenting the association of TTP and SSc, the TTP syndrome developed on a background of well established SSc. We describe a 51-year-old woman with a 5 month history of an evolving connective tissue disease syndrome who presented initially with TTP, followed 4 months later by limited cutaneous SSc and Raynaud's phenomenon.

In vivo dynamics of the microparasite Perkinsus marinus during progression and regression of infections in eastern oysters.

We used a total body parasite burden assay for the oyster pathogen Perkinsus marinus to investigate quantitative changes in microparasite burdens and frequency distributions. Heretofore, this type of study was limited mainly to macroparasites. The apparent in vivo growth pattern of P. marinus was characterized by a "lag" phase (< 10 cells/g wet weight [g wwt], a "log" phase (10-10,000 cells/g wwt), and a "stationary" phase (> 10,000 cells/g wwt). Infections declined exponentially under conditions unfavorable to the parasite but with a lengthening half-life, suggesting that elimination of parasites became increasingly difficult at low densities. Oysters held for 11 wk at 15 C, a temperature considered more favorable for oyster hemocytes than for P. marinus, were not able to eliminate infections. Parasite distributions within host populations were aggregated; in each sample, 1 or 2 oysters contained more parasites than all other oysters in the sample combined and the variance-to-mean ratio exceeded 1:1. The few hosts with large parasite burdens may be the most important individuals for survival and transmission of parasites. They are likely to play a key role in initiating and maintaining epizootics either in situ or after introduction of infected animals into a naive population.

Congenital cystic tumors of the atrio-ventricular node: successful demonstration by an abbreviated dissection of the conduction system.

Pathologists are dissuaded from the pathological study of the conduction system by the high cost and complexity of a traditional complete study. However, a simplified, low-cost approach can produce concrete information when performed in carefully selected cases of atrioventricular block, as demonstrated in the two cases of congenital cystic tumor of the atrioventricular node described in this report.

Perkinsus marinus tissue distribution and seasonal variation in oysters Crassostrea virginica from Florida, Virginia and New York.

Perkinsus marinus infection intensity was measured in eastern oysters Crassostrea virginica collected in October and December 1993, and March, May, and July 1994 from 3 U.S. sites: Apalachicola Bay (FL), Chesapeake Bay (VA), and Oyster Bay (NY). Gill, mantle, digestive gland, adductor muscle, hemolymph, and remaining tissue (including gonadal material and rectum) were dissected from 20 oysters from each site at each collection time. Samples were separately diagnosed for P. marinus infections by incubation in Ray's Fluid Thioglycollate Medium (RFTM) and subsequent microscopic quantification of purified enlarged hypnospores. At all sampling times and sites, average P. marinus infection intensity (g wet wt tissue-1 or ml hemolymph-1) was lowest in hemolymph samples, and generally highest in the digestive gland. Perkinsus marinus prevalence was 100% at both FL and NY sites for each of the 5 collection times, and, for the VA site, was less than 100% in only 1 month (May 1994). Seasonal intensity patterns and mean total body burdens differed among the sites. Average body burden was highest in VA during October and progressively declined to a minimum in May. This decline was probably due to mortality of heavily infected oysters and diminution of parasite activity associated with colder temperatures and reduced salinities. Intensities varied little during the months of October and December at both the FL and NY sites. Minimum average intensities were observed in March in FL oysters and May in NY oysters. Relatively high P. marinus infection levels that persisted throughout the winter in NY oysters compared with VA oysters could reflect constant high salinity in Long Island Sound which favors parasite activity, and also rapid decline in temperature in the fall that may have prevented epizootic oyster mortalities.

Altered response of oyster hemocytes to Haplosporidium nelsoni (MSX) plasmodia treated with enzymes or metabolic inhibitors.

To avoid phagocytosis, parasites may mask themselves with host-like molecules that prevent recognition as nonself or they may produce substances that interfere with host cellular defenses. The protozoan parasite Haplosporidium nelsoni, which causes MSX disease in the eastern oyster Crassostrea virginica, is not ingested by host hemocytes. To assess potential avoidance mechanisms, oyster hemocytes were incubated with plasmodial stages of the parasite that had been pretreated with one of a variety of enzymes (proteases and carbohydrases) to alter surface molecules or with metabolic inhibitors to prevent the synthesis or active uptake of "masking" molecules, as well as the production and discharge of inhibitory substances. The maximum increase in phagocytosis resulting from treatment with carbohydrases was 12.5% (beta-galactosidase) and with proteases was 18% (Proteinase K). Inhibitors of aerobic metabolism resulted in a similar level of enhancement. In contrast, treatment of parasites with the glycolysis inhibitor iodoacetate enhanced phagocytosis by up to 66%. Thus, the process that obstructs phagocytosis involves aerobic and, especially, anaerobic pathways. The greater effect of a metabolic inhibitor compared to enzymes suggests that the mechanism involves more than just surface modification and may include the production of interference molecules.

Variability in molluscan hemocytes: a flow cytometric study.

Reported variability in numbers and relative proportions of hemocytes in marine bivalves may be related to environmental conditions and laboratory method differences. An automated identification assay, flow cytometry, removes much laboratory bias, but its usefulness is limited because the putative cell types in delineated subpopulations have never been confirmed. The present study was designed to: (1) confirm the identity of oyster hemocyte subpopulations described by flow cytometry, and (2) use flow cytometry in an experimental analysis of potential causes of variation. Light-scatter flow cytometry consistently differentiated three subpopulations in oysters from two mid-Atlantic (USA) sites. Cell sorting and microscopy identified them as granular, small granular, and agranular (hyalinocytes and apparently degranulated) hemocytes. Subpopulation proportions estimated by microscopy and by flow cytometry were significantly correlated (r(2) = 0.27 to 0.50). In a 4-week laboratory experiment, neither temperature (12 vs. 22 degrees C) nor food (fed vs. not fed) had a statistically significant effect on total or differential counts, or on hemocyte viability. Most of the variability was attributable to individual differences and was very similar to that reported for vertebrates. We hypothesize that variability in molluscan hemocytes may be more immediately linked to individual metabolic condition than to ambient changes.

Aortic dissection and use of the nicotine patch: a case involving a temporal relationship.

A 33-year-old woman presented with chest and abdominal pain shortly after first and second applications of the nicotine patch. Type A aortic dissection was diagnosed and repaired. Pathological examination revealed cystic medial necrosis, subacute and acute dissection, with no evidence of chronic aortic insufficiency. The close temporal relationship between applications of the nicotine patch and onset of symptoms compatible with dissection followed by extension raises the possibility that the nicotine patch was implicated in, or precipitated, this woman's aortic dissection.

TCDD residues in fish and shellfish from U.S. waterways.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) in the edible portion of fish and shellfish from various U.S. waterways has been monitored since 1979. Analytical results for the period 1979-1994 are reported. Extracts obtained after column chromatographic and liquid chromatographic cleanup were examined by electron capture detection-gas chromatography (GC), and final quantitation and confirmation were performed by GC/mass spectrometry with multiple ion detection. Analyses of 1623 test samples indicated that TCDD residues in fish and shellfish were not widespread but rather were localized in areas near waste sites, chlorophenol manufacturers, and pulp and paper mills. Analytical results indicated that levels in aquatic species from these sites have been declining steadily. No TCDD (limit of detection and confirmation, 1-2 ppt) has been found in recent years in aquatic species from most Atlantic, Pacific, and Gulf of Mexico sites and Great Lakes other than Lake Ontario and Saginaw Bay (Lake Huron).

Characterization of the small subunit ribosomal RNA gene of the oyster parasite Haplosporidium costale.

The small subunit ribosomal RNA (SSU rRNA) gene of the oyster parasite Haplosporidium costale was characterized from spore DNA by polymerase chain reaction (PCR) and molecular cloning. Sequence analysis showed that identical clones were obtained from separate batches of spore samples. The gene is 1791 nucleotides in size. It has 84.5% sequence similarity to that of a related oyster parasite, Haplosporidium nelsoni, 71.8% similarity to that of its oyster host, Crassostrea virginica, and 75.4% similarity to that of another oyster parasite, Perkinsus marinus. Among the variable regions of these SSU rRNA genes, H. costale-specific primers were designed and used to confirm parasite identity by the PCR technique. A common 150 base pair amplification product was obtained from DNA samples of H. costale spore DNA, DNA prepared from tissue sections of oysters infected with H. costale plasmodia, and the H. costale SSU rRNA clone. There was no detectable product from DNA samples isolated from tissue sections of oysters infected with H. nelsoni plasmodia.

The cardiovascular pathology of phospholipid antibodies: An illustrative case and review of the literature.

There is some confusion in the literature regarding the pathology associated with phospholipid antibodies. These antibodies, formed to a number of negatively charged phospholipids, are associated with an increased tendency to both arterial and venous thrombosis and embolism and may be manifest in a primary syndrome, a syndrome secondary to systemic lupus erythematosis, or as an isolated phenomenon, which may or may not be associated with thromboembolism. The pathogenesis of thrombosis in these syndromes has not yet been elucidated. Indeed it is not even clearly established that the antibodies have a pathogenic role, as opposed to existing as an epiphenomenon or arising secondary to some form of vascular damage. Clarification of the reported pathologic literature is needed as a basis for studying the pathogenesis of thrombosis in these syndromes, and therefore we present a review of papers that have reported pathology in association with phospholipid antibodies. In order to illustrate the review, we present a typical case of primary antiphospholipid antibody syndrome.

Clinicopathologic correlations of antiphospholipid antibodies. An autopsy study.

The clinicopathologic correlations of antiphospholipid antibodies (aPLs) have so far only been examined in case reports and highly selected series. This study assessed the incidence of aPLs in 156 consecutive, unselected autopsies and correlated the pathological findings with the clinical histories. Elevations of aPLs were found in 20.5% of the autopsy population, compared with 9.6% of age- and sex-matched controls and 2% of healthy normal subjects. There was a higher incidence of thromboembolic disease in patients with elevated aPL levels compared with those without, but the histology of thrombi was similar in both groups, with no evidence of vasculitis in the aPL-positive individuals. Patients with transient ischemic attacks and cardiac valve lesions had a high incidence of aPLs, as reported previously. Five cases that fit the designation of primary antiphospolipid antibody syndrome were noted. The study concludes that aPLs are relatively common in a hospital autopsy population and are commonly associated with thromboembolic events, that the thromboemboli are not associated with vasculitis, and that primary aPL syndrome is more common than generally appreciated.

Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin.

Small subunit ribosomal RNA gene sequence of the oyster parasite Perkinsus marinus.

The small subunit rRNA gene of the oyster pathogen Perkinsus marinus was characterized from cells of infected oyster hemolymph by polymerase chain reaction and molecular cloning. The gene, 1,793 nucleotides in size, has 77.2% sequence similarity to that of its host, the eastern oyster Crassostrea virginica. The sequence was confirmed using recently available in vitro cultures of P. marinus. DNA from pure P. marinus culture was amplified with specific primers synthesized according to the sequence from infected oyster hemolymph, and predicted size fragments were obtained. Furthermore, restriction digests yielded fragments of expected size in amplified rDNA from in vitro cultures. The P. marinus sequence has 97.5% similarity to the Perkinsus sp. sequence from the Australian mollusc Anadara trapezia.