Parameters of differentiation and proliferation in adult granulosa cell tumors of the ovary.

We evaluated parameters of cell differentiation and proliferation to improve prognostication of ovarian adult granulosa cell tumors. Recurrent tumors (n = 10, REC group) and nonrecurrent tumors (n = 30, NED group) were compared in terms of cellular atypia, nuclear area, p53 overexpression, ploidy, DNA index, mitosis count, S-phase fraction, and nucleolar organizer region number and area per cell. Cellular atypia was significantly more frequent in REC than NED tumors (50% versus 13%; P = .03). Mean nuclear area was significantly larger in the REC than in the NED group (44 microm2 versus 36 microm2; P = .006). Mitotic count was significantly higher in REC than NED tumors (mean of 4.8 versus 1.7; P = .004). S-phase fraction and ploidy did not predict outcome: neither did nucleolar organizer region numbers and area per cell, or p53 overexpression. Cellular atypia and mitotic count may help in determining the prognosis of adult granulosa tumors of the ovary. The histochemical parameters evaluated did not provide additional information.

Flow cytometry provides rapid and highly accurate detection of antisperm antibodies.

OBJECTIVE: Immunobead testing (IBT), the current standard for antisperm antibody detection, is time consuming and somewhat subjective. To overcome these limitations and maintain accuracy, we studied an immunofluorescent assay using flow cytometry. DESIGN: A validation study comparing flow cytometry to IBT in the detection of serum antisperm antibodies. SETTING: Flow cytometry laboratory. PATIENTS: Sera from 37 men after vasectomy (test) and sera from 35 fertile men (control). MAIN OUTCOME MEASURE: Test serum with and without immunoglobulin (Ig)G, IgA, and IgM antisperm antibodies as defined by IBT were analyzed by flow cytometry. Sensitivity and specificity of flow cytometry was calculated by defining the IBT as the true result. RESULTS: Flow cytometry identified 22 of 22 sera that were IgG positive (100% sensitivity), 12 of 14 sera that were IgA positive (86% sensitivity), and 4 of 4 sera that were IgM positive (100% sensitivity). Overall, 22 of 37 men were positive for antisperm antibodies. The flow cytometry correctly identified 71 of 71 negative sera (100% specificity). Fluorescence intensity values from the 37 study patients significantly correlated with immunobead binding to the head region and to the entire (more than one) region. CONCLUSIONS: Detection of IgG, IgA, and IgM antisperm antibodies by flow cytometry is highly sensitive and specific. In addition, flow cytometry is able to assess thousands of sperm rapidly and accurately, reducing sampling error and technical time.

Proliferative verrucous leukoplakia. Four cases with flow cytometric analysis.

Proliferative verrucous leukoplakia is a slow-growing but highly aggressive precancerous form of leukoplakia of unknown cause. Proliferative verrucous leukoplakia is though to possess a continuous spectrum of clinical and histopathologic expression, ranging from simple hyperkeratosis to invasive squamous cell carcinoma. Early diagnosis is difficult because of an initial innocuous character, but multiple and rapid multifocal warty recurrences are common. This article reports four additional archival cases of proliferative verrucous leukoplakia to determine if flow cytometric analysis can be useful in the early diagnosis of proliferative verrucous leukoplakia. Flow cytometric analysis was performed on available formalin-fixed paraffin-embedded specimens (N = 27). Flow cytometric analysis results showed DNA aneuploid cell lines in each proliferative verrucous leukoplakia case studied (DNA index range, 1.1 to 2.6). In all four patients the abnormal cell line DNA index appeared to be maintained throughout the sampling period. The results suggest flow cytometric analysis could be a possible aid in early recognition of proliferative verrucous leukoplakia and might enable aggressive therapy at an earlier stage.

Neutrophil CD18 expression and blockade after traumatic shock and endotoxin challenge.

OBJECTIVE: The expression of the leukocyte CD18 adhesion complex on polymorphonuclear leukocytes (PMNs) was measured, and the physiologic effects of blockade of the complex were studied after trauma and sepsis. SUMMARY BACKGROUND DATA: Margination of PMNs occurs early during inflammation and depends, in part, on expression of the CD18 adhesion complex. Blockade of this adherence complex can reduce PMN-mediated damage. This study tests the hypothesis that PMN activation after resuscitated trauma produces an occult endothelial injury that increases the vulnerability to a delayed inflammatory stimulus. METHODS: Anesthetized (fentanyl) mongrel pigs were sham injured or fluid resuscitated from soft tissue injury +35% hemorrhage. Systemic blood was collected at 24-hour intervals from awake animals. The CD18 density on circulating PMNs was determined with flow cytometry using mean channel fluorescence (MCF). The CD18 receptors were blocked with monoclonal antibodies either immediately before trauma or immediately before an endotoxin (lipopolysaccharide [LPS]) challenge that was administered to all groups 3 days after the shock episode. Bronchoscopy was performed before trauma, pre-LPS, and post-LPS, and protein content was measured in bronchoalveolar lavage (BAL). RESULTS: Mean channel fluorescence was reduced on PMNs for 48 hours in animals with trauma versus animals with sham injuries. Anti-CD18 therapy produced higher circulating PMN counts compared with nontreated sham or shock groups. The incremental rise of BAL protein after shock was prevented with anti-CD18; the increment after LPS was attenuated. Anti-CD18 was administered before trauma and reduced the fluids necessary to maintain cardiac filling pressures after LPS. CONCLUSIONS: These data suggest that PMNs are activated after resuscitation from traumatic shock and that these cells produce an endothelial injury that may increase the vulnerability to a septic challenge. The broad implication is that temporarily blocking PMN adhesiveness at the time of trauma might salvage some host tissue and reduce the incidence of septic complications in the post-trauma period.

Comparing flow cytometric analysis and nucleolar organizer region enumeration in archival oral premalignant lesions.

Flow cytometric analysis (FCA) and silver colloidal nucleolar organizer region-associated protein staining (AgNOR) have been used individually in assessing the histopathologic nature of various human tumors. However, few researchers have investigated the relationship between the two techniques in a single series. In a retrospective study, we examined 36 premalignant lesions of the oral cavity by FCA and AgNOR on formalin-fixed, paraffin-embedded tissue submitted to the University of Tennessee, Memphis, oral pathology laboratory. Three categories of epithelial dysplasia were represented (9 mild, 9 moderate, 6 severe), as well as four epithelial hyperplasias without dysplasia, three squamous cell carcinomas, and five fibrous nodules as controls. Parameters recorded for each case included age, race, gender, site, light microscopic diagnosis (LMD), DNA index (DI), total proliferative index (TPI), S-phase (S), range of nucleolar organizer regions (RNOR), and mean number of nucleolar organizer regions (MNOR). The average maximum nucleolar organizer region count (AMXNOR) for each LMD category was also calculated. The objective of the study was to determine if FCA or AgNOR aided in the subjective LMD of oral premalignant lesions and if the parameters recorded for the specimens exhibited any positive correlation. The FCA results indicated an abnormal DI in 6 of the 24 dysplastic lesions. A positive partial correlation was seen between DI and MNOR (r = 0.434; P < 0.012) and TPI and S (r = 0.774; P < 0.0001), holding gender and race constant. Additionally, the AMXNOR exhibited a slight tendency to increase for each increasing grade of dysplasia but this could not be confirmed statistically.(ABSTRACT TRUNCATED AT 250 WORDS)

Inability to detect fetal metaphases in flow-sorted lymphocyte cultures based on maternal-fetal HLA differences.

Separation of fetal cells from maternal blood could provide a means for prenatal diagnosis that would not endanger the fetus. In this pursuit, we attempted cytogenetic analysis of candidate fetal cells flow sorted on the basis of parental HLA disparity. Metaphases showing 46,XY or aneuploidy and concordant with prenatal diagnostic studies (i.e., amniocentesis, chorionic villus sampling) would presumably be fetal in origin. Blood samples were obtained from 78 pregnant women and their partners. Among 18 HLA informative cases in which metaphases were recovered, 15 involved fetuses that were 46,XY or aneuploid. From these 15 cases, 2,483 metaphases were analyzed. All metaphases were 46,XX. Cytogenetic analysis of flow-sorted fetal cells thus probably will need to emphasize not metaphase analysis but in situ hybridization with chromosome-specific probes.

Role of antigen-presenting cells in activation of human T cells by the streptococcal M protein superantigen: requirement for secreted and membrane-associated costimulatory factors.

The requirements for T-cell activation by the streptococcal superantigen (SAg), pepsin-extracted M protein from type 5 streptococci (pep M5), were studied by monitoring Ca2+ influx and cell proliferation. Cells from a pep M5-specific T-cell line showed no change in intracellular Ca2+ levels in response to pep M5 when added alone or with freshly isolated autologous antigen-presenting cells (APC). However, after being incubated with pep M5 overnight, the APC secreted soluble factors that together with pep M5 induced a marked increase in intracellular Ca2+ levels in pep M5-specific T cells or freshly isolated, purified T cells. Removal of the SAg from the overnight APC-derived supernatants resulted in loss of the Ca(2+)-mobilizing activity, which was restored within seconds of addition of SAg, suggesting that both the SAg and the soluble factors synergize to induce the Ca2+ influx. Induction of cell proliferation required additional signals inasmuch as the activated APC-derived supernatant failed to synergize with pep M5 to induce the proliferation of purified T cells and required the presence of phorbol myristate acetate for this activity. Metabolically inactive, fixed APC were impaired in their ability to present pep M5 to T cells. Presentation of pep M5 by fixed APC was, however, restored when the APC-derived soluble costimulatory factors were added to the culture. Our data suggest that pep M5-induced activation of T cells is dependent on APC-derived soluble factors and an APC membrane-associated costimulatory molecule(s). These interactions may be important in regulating the in vivo responses to M proteins, could contribute to the severity or progression of infections with Streptococcus pyogenes, and may influence the susceptibility of individuals to its associated nonsuppurative autoimmune sequelae.

Resistance to N-benzyladriamycin-14-valerate in mouse J774.2 cells: P-glycoprotein expression without reduced N-benzyladriamycin-14-valerate accumulation.

N-Benzyladriamycin-14-valerate (AD 198) is a highly lipophilic analogue of Adriamycin with novel cytotoxic mechanisms, greater in vivo antitumor activity, and the ability to circumvent multidrug resistance due to P-glycoprotein-mediated drug efflux or decreased topoisomerase II activity. To identify the mechanism(s) which may confer AD 198 resistance, J774.2 mouse macrophage-like cells were selected for growth in cytotoxic levels of AD 198 (AD 198R). AD 198R cells exhibited over-expression of the mdr1b (P-glycoprotein) gene, cross-resistance to Adriamycin and vinblastine, and potentiation of drug cytotoxicity by verapamil. However, net intracellular accumulation of AD 198 in AD 198R cells was unchanged compared to parental cells, while Adriamycin and vinblastine accumulations were reduced 40% and 95%, respectively. AD 198 was localized in the perinuclear region of the cytoplasm in both parental and AD 198R cells, with additional vesicular compartmentalization in AD 198R cells. Verapamil-induced reversal of AD 198 resistance coincided with some drug redistribution from cytoplasmic vesicles, but without redistribution of AD 198 into the nucleus. These results suggest that AD 198 resistance was not conferred through a P-glycoprotein-mediated reduction in intracellular drug accumulation but through other cytoplasmic mechanisms, including, but not limited to, drug compartmentalization.

Flow cytometer analysis of oral premalignant lesions: a pilot study and review.

In a retrospective study, we examined 34 premalignant lesions of the oral cavity by flow cytometer analysis on formalin-fixed, paraffin-embedded tissue submitted to the University of Tennessee, Memphis, Oral Pathology Laboratory. Three categories of oral epithelial dysplasia were represented (eight mild, seven moderate, nine severe), as well as five epithelial hyperplasias without dysplasia and five fibrous nodules as controls. The DNA index and total proliferative index of each case were calculated. The objective of the study was to determine the amount of epithelial dysplasia necessary in oral lesions before DNA aneuploidy or high proliferative index is detectable and thus determine if flow cytometric analysis can be a diagnostic adjunct for oral premalignant lesions. The results showed that some cases in both the control and dysplastic categories exhibited a high total proliferative index (control = 1, no dysplasia = 1, mild dysplasia = 3, moderate dysplasia = 2, severe dysplasia = 2), whereas only the dysplastic lesions had an abnormal DNA index [8 of 24 (33%)]. The results indicate that flow cytometric analysis may have some limited potential as a diagnostic adjunct in oral premalignant lesions.

External Na+ is not required for Ca2+ mobilization in platelets stimulated with rattlesnake lectin or alpha-thrombin.

The effects of extracellular sodium on platelet aggregation and calcium mobilization in platelets stimulated with either rattlesnake (Crotalus atrox) lectin (RSL) or alpha-thrombin were compared. The absence of extracellular sodium had no effect on platelet aggregation or calcium mobilization in response to all levels of RSL tested. In contrast platelet aggregation was sodium-dependent in response to less than or equal to .2 units/ml alpha-thrombin. Surprisingly, calcium mobilization occurred in platelets treated with a threshold level of alpha-thrombin in the absence of external sodium. Thus sodium-dependent platelet aggregation in response to a low dose of thrombin apparently is not the result of sodium-dependent calcium mobilization.

Evaluation of lymphocyte phenotype and phytohemagglutinin response in healthy very low birth weight infants.

Sixteen healthy very low birth weight infants (VLBWI) were studied in a serial fashion over a 3-week period. Subjects were evaluated for lymphocyte phenotype, phytohemagglutinin (PHA) response, and metabolic status including weight, blood urea nitrogen, creatinine, albumin, pH, calcium, phosphorus, and ammonia. Lymphocyte phenotype determination showed a decreased proportion of CD3+ cells (66.8 +/- 10.4 vs 75.9 +/- 6.1, P less than 0.02) in VLBWI. When subsets of the CD4+ population were examined, VLBWI had a lower proportion of CD4+/CD29+ cells (8.2 +/- 5.8% vs 23.5 +/- 8.0%, P less than 0.0001) than adults and a higher proportion of CD4+/CD45R+ cells (35.6 +/- 12.4% vs 22.2 +/- 7.4%, P less than 0.03). The CD4+ subsets in VLBWI were similar to those seen in term infants. The peak PHA response in VLBWI was greater than that of adults (P less than 0.01). There was little change in the immune measurements over the 3-week study period. There were no strong correlations between any of the immunological measurements and the metabolic measurements except that the proportion of CD8+ cells increased with birth weight. Our findings demonstrate that immune measurements in healthy VLBWI differ from values found in adults but are similar to those of full-term infants. Lower proportions of the CD4+/CD29+ cells (the helper/inducer subset for antibody production) may contribute to some of the differences in immune function reported in neonates.

T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein.

M proteins, the major virulence factor of group A streptococci, have been implicated in the pathogenesis of acute rheumatic fever (ARF) and other streptococcal related autoimmune diseases. A 22-kD fragment of M type 5 protein is a potent stimulant of human T cells and has recently been shown by our laboratory to belong to the newly designated family of superantigens. Using flow cytometry and the polymerase chain reaction, we demonstrate that this molecule reacts with subsets of human T cells expressing specific T cell receptor (TCR) V beta elements, namely V beta 2, 4, and 8. We employed similar techniques to analyze the TCR V alpha usage of pep M5-stimulated T cells. These studies revealed that the preferential usage of particular V alpha elements is not specific for the superantigen; rather, it may reflect the repertoire of the individual being tested. The expansion of a large number of T cells bearing specific TCR V beta sequences by M protein may account for its role in mediating the pathogenesis of post-streptococcal diseases. Furthermore, the preferential usage of TCR V alpha elements in certain individuals may be an important factor that predisposes them to development of self-reactivity.

Neuropeptide-Y enhances luteinizing hormone (LH)-releasing hormone-induced LH release and elevations in cytosolic Ca2+ in rat anterior pituitary cells: evidence for involvement of extracellular Ca2+ influx through voltage-sensitive channels.

The present studies were designed to investigate the mechanism by which neuropeptide-Y (NPY) augments the effect of LHRH to stimulate the release of LH from cultured rat anterior pituitary cells. Anterior pituitary cells from ovariectomized rats were enzymatically dispersed, cultured for 3 days, and then exposed to various secretagogues during 3-h incubations. As reported by this laboratory previously, NPY alone (100 nM) did not affect LH release, but significantly enhanced the LH response to 1 nM LHRH. This facilitatory action of NPY was mimicked by the dihydropyridine Ca2+ channel agonist Bay K 8644 (1 microM), and the enhancement of LHRH-induced LH release by either NPY or Bay K 8644 was prevented by the dihydropyridine antagonist nitrendipine (1 microM). Nitrendipine alone reduced the response to LHRH by approximately 25%, but did not affect basal LH release. In contrast, NPY failed to amplify the release of PRL in response to TRH, another Ca2(+)-mobilizing hormone. To test whether NPY also enhances the increase in cytosolic Ca2+ induced by LHRH, anterior pituitary cells were acutely dispersed into single cell suspensions, loaded with the fluorescent Ca2+ probe Indo-1 AM, and analyzed with a UV laser in an EPICS-753 flow cytometer at a rate of 500 cells/sec for 200 sec. The ratio of intracellular fluorescence resulting from Ca2+ bound to the Indo-1 to the fluorescence from Indo-1 alone (Indo-1 ratio), which is an index of the concentration of free cytosolic Ca2+, was determined for each cell. Approximately 7% of anterior pituitary cells responded to LHRH (1 or 10 nM) with significant increases in Indo-1 ratios, indicative of an increase in the concentration of free cytosolic Ca2+. EGTA (2.5 mM) reduced the basal Indo-1 ratios and attenuated, but did not abolish, the initial increase in response to LHRH, consistent with the initial extracellular Ca2+ influx-independent phase of the response to LHRH. NPY alone (100 nM) did not affect the Indo-1 ratios in anterior pituitary cells, but pretreatment with the peptide for 10 min before the scans significantly augmented the Indo-1 ratio response to 10 nM LHRH. This effect of NPY was also blocked by EGTA. Taken together, these biochemical and pharmacological studies suggest that NPY enhances the release of LH stimulated by LHRH by increasing extracellular Ca2+ entry, possibly by selectively affecting that component of the response involving dihydropyridine-sensitive L-type voltage-sensitive Ca2+ channels during the initial stages of the cellular response to LHRH.

Calcitonin inhibits thyrotropin-releasing hormone-induced increases in cytosolic Ca2+ in isolated rat anterior pituitary cells.

Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min. Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH. In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.

Calcium mobilization in human platelets using indo-1 and flow cytometry.

Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha-thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.

Mitogenic stimulation of human B lymphocytes by the mannose-specific adhesin on Escherichia coli type 1 fimbriae.

Escherichia coli type 1 fimbriae contain in association with the major structural protein a lectin-like adhesin moiety that mediates attachment of E. coli to mannose-containing receptors on the surface of host cells. We have investigated the lymphocyte mitogenic activity of this mannose-specific adhesin by comparing the ability of purified wild type type 1 fimbriae containing the adhesin and mutant type 1 fimbriae lacking the adhesin to stimulate proliferation in human lymphocytes. Both fimbriae stimulated a peak of proliferation at 8 days whereas only the wild type fimbriae stimulated an additional peak of proliferation occurring at 3 days. Proliferation at 3 days but not at 8 days could be blocked by the addition of alpha-methyl-D-mannoside. Neonatal lymphocytes from umbilical cord blood responded to both wild type and mutant fimbriae in a fashion similar to adult cells. Stimulation of separated T and non-T cell populations indicated that the proliferation seen at 3 days was solely due to non-T cells whereas the 8-day response was due to T cell proliferation. The addition of gamma-irradiated T cells did not appear to enhance the 3-day response of the non-T cells. However, the 8-day response by T cells was dependent on the presence of gamma-irradiated non-T cells. In cultures of unseparated cells, wild type fimbriae stimulated more than 75% of the B cells to enter the S and G2 phase at 3 days whereas at 8 days cycling T cells were present in both wild type and mutant fimbriae-stimulated cultures. Taken together, our observations suggest that the adhesin molecule stimulates a polyclonal mitogenic response in B cells that peaks at 3 days, and other structural components of the fimbriae are responsible for evoking an 8-day (probably immune) response in T cells.

Fibrinogen binding and resistance to phagocytosis of Streptococcus sanguis expressing cloned M protein of Streptococcus pyogenes.

The biological properties of Streptococcus pyogenes M protein cloned and expressed in S. sanguis were investigated. The spm-5 gene previously cloned into Escherichia coli was subcloned into the E. coli-S. sanguis shuttle plasmid pVA838 to produce a newly constructed plasmid, pBK100. Cells of S. sanguis transformed with pBK100 expressed 53-, 55-, and 58-kilodalton polypeptides reacting with type 5 M protein antiserum in immunoblots. The M protein was expressed on the surface of S. sanguis cells as shown by the capacity of the intact cells to (i) inhibit the reactivity of anti-type 5 antibodies with purified M protein as demonstrated by enzyme-linked immunosorbent assay; (ii) inhibit the opsonization by M5 antisera of type 5 S. pyogenes; (iii) express M-protein-like fibrils on the surface of the organisms that react with M5 antisera as revealed by immunoelectron microscopy; (iv) bind plasma fibrinogen and, as a consequence, resist phagocytosis by human blood neutrophils; and (v) be rendered susceptible to phagocytosis by opsonic M5 antisera. These results provide additional evidence that streptococcal M proteins bind host proteins as a ploy to evade host defense mechanisms.

The Wistar Furth rat: an animal model of hereditary macrothrombocytopenia.

The mechanisms that determine and regulate platelet size are unknown. By phase microscopy, we observed that Wistar Furth (WF) rats had macrothrombocytopenia. In this study, we have characterized and compared platelets and megakaryocytes of WF rats with those of Wistar, Long-Evans hooded (LE), and Sprague-Dawley rats. In addition, we have examined the mode of inheritance of this WF rat platelet abnormality. The average platelet count of WF rats was only one-third that of the other three rat strains. In contrast, the mean platelet volume (MPV) of adult WF rats was twice that of the other rat strains; however, the average megakaryocyte diameter and DNA content distribution of WF rats were not significantly different from those of LE rats. The average megakaryocyte concentration was 30% lower in the WF strain compared with that of LE rats. Mazelike membrane formations were observed in WF platelets and megakaryocytes by electron microscopy. Reciprocal crosses of WF and LE rats resulted in offspring with MPVs and platelet counts like those of LE rats, indicating that the macrothrombocytopenic trait is recessive in its inheritance. Reciprocal marrow transplants between the WF and LE strains resulted in MPVs like those of the donor strain, demonstrating that the macrothrombocytopenia is an intrinsic marrow abnormality of the WF strain. Splenectomy did not alter the MPV of WF rats. The response of WF megakaryocytes and platelets to severe, acute thrombocytopenia was similar to that of LE rats except that the shift to higher megakaryocyte DNA contents was muted and platelet recovery was slower in the WF rats. In summary, the WF rat has a hereditary macrothrombocytopenia that is recessive in nature and not due to differences in megakaryocyte size or DNA content. These results suggest that the macrothrombocytopenia of WF rats results from the formation of fewer platelets per megakaryocyte, possibly resulting from a qualitative or quantitative defect in some component necessary for proper subdivision of megakaryocyte cytoplasm into platelets.

Membrane skeletal alterations during in vivo mouse red cell aging. Increase in the band 4.1a:4.1b ratio.

We have examined membrane protein profiles for alterations during red blood cell aging. To obtain populations of in vivo-aged red cells, we maintained mice in a state of continuous erythropoietic suppression for up to 8 wk using serial hypertransfusion. The circulating t1/2 of red cells from mice which had been erythropoietically suppressed for 8 wk was less than 1 d compared with a t1/2 of 15 d for red cells from normal animals. The most obvious alteration in membrane proteins was an increase in the ratio of the membrane skeletal components 4.1a:4.1b from 0.3 for the normal red cell population to greater than 1 for these old cells. The 4.1a:4.1b ratio thus appears to be a useful index of red cell age. Analyses of the density profile of cells aged in the hypertransfused mice disclosed that these old cells had a density range similar to that of controls, suggesting that cell density does not increase significantly with red cell age in the mouse.

Isolation and characterization of lactose-binding lectins from the venoms of the snakes Lachesis muta and Dendroaspis jamesonii.

Two lectins have been isolated: one from the venom of Lachesis muta (bushmaster lectin) and one from Dendroaspis jamesonii venom (Jameson's mamba lectin). The lectin from bushmaster venom (BML) is similar to the lactose-binding lectins previously isolated from snake venoms (Gartner et al. (1980) FEBS Lett. 117, 13-16; Gartner & Ogilvie (1984) Biochem. J. 224, 301-307) in that it is calcium-dependent, lactose inhibitable, and is a dimer of molecular weight 28,000. In contrast, the lactose-blockable lectin from Jameson's mamba venom (JML) has an apparent molecular weight of 26,000 and agglutinates erythrocytes in the presence of EDTA. The absorption spectra of BML were affected by the binding of calcium, or calcium and lactose to the lectin. However, JML spectra were not affected by these conditions. While the hemagglutination activity of each of the previously described lactose-binding snake venom lectins is inhibited by reducing agent, the activities of BML and JML are not affected by reducing agent. Antiserum against bushmaster lectin cross-reacts with thrombolectin, cottonmouth lectin (CML), rattlesnake lectin (RSL), and copperhead lectin (CuHL) but not lectin from Jameson's mamba venom. This evidence plus a comparison of atomic absorption spectra, isoelectric points and amino acid analyses of the lectins demonstrate that JML and BML are different from thrombolectin, CML, RSL, and CuHL.