Confocal microscopy as a useful approach to describe gill rakers of Asian species of carp and native filter-feeding fishes of the upper Mississippi River system.

To better understand potential diet overlap among exotic Asian species of carp and native species of filter-feeding fishes of the upper Mississippi River system, microscopy was used to document morphological differences in the gill rakers. Analysing samples first with light microscopy and subsequently with confocal microscopy, the three-dimensional structure of gill rakers in Hypophthalmichthys molitrix, Hypophthalmichthys nobilis and Dorosoma cepedianum was more thoroughly described and illustrated than previous work with traditional microscopy techniques. The three-dimensional structure of gill rakers in Ictiobus cyprinellus was described and illustrated for the first time.

Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase.

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild-type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke-deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP-mediated mechanism.

Cytostructural localization of a tumor-associated antigen.

Tumor cell membrane glycoproteins may be involved in the induction of tumor immunity or in the escape of tumors from immunologic defense mechanisms. Forty-four benign and malignant breast lesions were examined for the presence of a carbohydrate precursor antigen (T antigen) of the human blood group system MN. T antigen was demonstrated by means of an immunohistochemical technique to detect tissue binding of peanut agglutinin, a plant lectin, with affinity for T antigen. Malignant breast lesions showed a pattern of T antigen expression different from that of benign breast tissues. A possible role for T antigen in the modulation of the immune response to breast carcinoma is suggested.

Results of the randomized phase IIB ADMIRE trial of FCR with or without mitoxantrone in previously untreated CLL.

ADMIRE was a multicenter, randomized-controlled, open, phase IIB superiority trial in previously untreated chronic lymphocytic leukemia. Conventional front-line therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). Initial evidence from non-randomized phase II trials suggested that the addition of mitoxantrone to FCR (FCM-R) improved remission rates. Two hundred and fifteen patients were recruited to assess the primary end point of complete remission (CR) rates according to International Workshop on Chronic Lymphocytic Leukemia criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity and safety. At final analysis, CR rates were 69.8 FCR vs 69.3% FCM-R (adjusted odds ratio (OR): 0.97; 95% confidence interval (CI): (0.53-1.79), P=0.932). MRD-negativity rates were 59.3 FCR vs 50.5% FCM-R (adjusted OR: 0.70; 95% CI: (0.39-1.26), P=0.231). During treatment, 60.0% (n=129) of participants received granulocyte colony-stimulating factor as secondary prophylaxis for neutropenia, a lower proportion on FCR compared with FCM-R (56.1 vs 63.9%). The toxicity of both regimens was acceptable. There are no significant differences between the treatment groups for PFS and OS. The trial demonstrated that the addition of mitoxantrone to FCR did not increase the depth of response. Oral FCR was well tolerated and resulted in impressive responses in terms of CR rates and MRD negativity compared with historical series with intravenous chemotherapy.

Results of the randomized phase IIB ARCTIC trial of low-dose rituximab in previously untreated CLL.

ARCTIC was a multicenter, randomized-controlled, open, phase IIB non-inferiority trial in previously untreated chronic lymphocytic leukemia (CLL). Conventional frontline therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). The trial hypothesized that including mitoxantrone with low-dose rituximab (FCM-miniR) would be non-inferior to FCR. A total of 200 patients were recruited to assess the primary end point of complete remission (CR) rates according to IWCLL criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity, safety and cost-effectiveness. The trial closed following a pre-planned interim analysis. At final analysis, CR rates were 76 FCR vs 55% FCM-miniR (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.73). MRD-negativity rates were 54 FCR vs 44% FCM-miniR. More participants experienced serious adverse reactions with FCM-miniR (49%) compared to FCR (41%). There are no significant differences between the treatment groups for PFS and OS. FCM-miniR is not expected to be cost-effective over a lifetime horizon. In summary, FCM-miniR is less well tolerated than FCR with an inferior response and MRD-negativity rate and increased toxicity, and will not be taken forward into a confirmatory trial. The trial demonstrated that oral FCR yields high response rates compared to historical series with intravenous chemotherapy.

Two monoclonal antibodies defining unique antigenic determinants on human B-lymphoma cells.

The non-Hodgkin's lymphomas are a clinically, morphologically, and immunologically heterogeneous group of diseases. Why lymphoma cells are unresponsive to normal regulatory growth controls and how they differ from normal lymphocytes are not well understood. In order to address these questions we have raised monoclonal antibodies to neoplastic B-cells. Two of these, LM-26 and LM-155, show a high degree of specificity for B-cell lymphomas. When tested by fluorescence activated cell sorter analysis, LM-26 reacted with 80% (18 of 23) of B-cell lymphomas freshly explanted from patients and LM-155 reacted with 20% (5 of 23). The antigenic determinant detected by LM-26 was also found to be present on four of seven neoplastic large cell B-lymphoma lines. LM-155 detected a determinant present on all seven of these lines. For neither monoclonal antibody was there any association between antibody reactivity and the morphological subtype of lymphoma examined or the type of cell surface immunoglobulin expressed. LM-155 reacted with one case of B-cell acute lymphoblastic leukemia. Neither antibody reacted with normal B-cell blasts, normal peripheral blood mononuclear or marrow cells, T-cell leukemias or lymphomas, or chronic lymphocytic leukemia cells. Lymphocytes from reactive lymphoid hyperplasias involving lymph nodes, spleen, peripheral blood, and lung were also negative for LM-26 and LM-155 binding or showed only a small percentage of cells positive (4-8%). Both monoclonals were unreactive with non-B-lymphoid neoplastic cell lines, nine of ten Epstein-Barr virus transformed B-cell lines, and cells freshly explanted from patients with cancers of diverse cellular origins. Fluorescence activated cell sorter analysis of the expression of the antigens defined by LM-26 and LM-155 on lymphoma cells and normal B-cell blasts suggests that they are not normal differentiation antigens associated with lymphocyte activation or proliferation. The highly restricted expression of detectable levels of antigens reactive with monoclonal antibodies LM-26 and LM-155 on non-Hodgkin's lymphoma cells suggests a possible relation to their neoplastic properties. From a practical viewpoint these monoclonals may prove useful in the diagnosis, classification, detection of residual disease, and treatment of the non-Hodgkin's lymphomas.

Monoclonal antibodies to HLA-DP, DQ, and DR determinants: functional effects on the activation and proliferation of normal and EBV-transformed B cells.

To investigate the function of HLA class II molecules in B-cell activation, we generated three new anti-HLA class II monoclonal antibodies with differing specificities for DP, DQ, and DR determinants. These were tested for their ability to inhibit various B- and T-lymphocyte responses. Each of these antibodies (NB-29, DH-84, and DH-224) immunoprecipitates a heterodimer of approximately 35,000 and 28,000 mol wt from 125I-surface-labeled B-lymphoma cells, as shown by SDS-PAGE. NB-29 (IgG1) detects a polymorphic DQ determinant, while DH-224 (IgG1) is reactive with monomorphic DR determinants, and DH-84 (IgG2a) has specificity for DP, DQ, and DR. Both DH-224 and DH-84, but not NB-29, were found to inhibit significantly the stimulation of peripheral blood mononuclear cells (PBMC) by anti-mu (70%-90% inhibition) and by lipopolysaccharide (80%-90% inhibition), as measured by incorporation of tritiated thymidine. When added to highly purified populations of peripheral blood B cells, none of these anti-class II monoclonal antibodies inhibited anti-mu-induced stimulation. This suggests that the inhibitory effect that DH-224 and DH-84 have on the stimulation of unfractionated PBMC may be due to their ability to interfere with the action of accessory cells. Epstein-Barr-virus (EBV)-transformed B-cell lines, in contrast, showed substantial inhibition of growth when cultured in the presence of any of the three antibodies. With respect to T cells, DH-84 and DH-224 strongly inhibited the mixed lymphocyte response; NB-29 did not. None of these antibodies inhibited stimulation of PBMC by phytohemagglutinin (PHA). These findings suggest that DQ and DR HLA class II molecules have differing roles in B-cell activation and document a direct antiproliferative effect of anti-HLA class II monoclonal antibodies on the growth of EBV-transformed cell lines.

Pharmacokinetics of the active metabolite (MDL 74,156) of dolasetron mesylate after oral or intravenous administration to anesthetized children.

BACKGROUND: Dolasetron mesylate is a selective 5-HT3 receptor antagonist under investigation as an antiemetic in children. Published studies indicate that its antiemetic activity results from the active metabolite (MDL 74,156), which is produced within 10 minutes of administration of dolasetron mesylate. METHODS: The pharmacokinetics of MDL 74,156 and the safety and tolerability of dolasetron mesylate were studied after a single oral or intravenous dose of 1.2 mg.kg-1 dolasetron mesylate to healthy children from 2 to 12 years of age. Oral dolasetron was administered to 12 children 1 to 2 hours before anesthesia. Intravenous dolasteron was administered to 18 children at induction of anesthesia. Serial blood samples were collected for 24 hours after dosing to measure the plasma concentration of MDL 74,156. Indexes of liver and kidney function were determined, and electrocardiograms and adverse events were recorded.

Acetylcholinesterase inhibition by zifrosilone: pharmacokinetics and pharmacodynamics.

OBJECTIVE: To determine the pharmacokinetics, pharmacodynamics and safety of the acetylcholinesterase inhibitor zifrosilone in healthy male volunteers. METHODS: Pharmacokinetics, pharmacodynamics, and tolerance of zifrosilone were studied in a double-blind, sequential, single-escalating-dose, randomized panel design. Each panel consisted of six subjects, with four subjects receiving zifrosilone (10, 30, 60, 90, 120, 150, 200, 250, and 300 mg orally) and two subjects receiving matching placebo. Serial blood samples were obtained for zifrosilone plasma concentrations and red blood cell acetylcholinesterase and butyrylcholinesterase activities. Participating subjects (n = 54) were men between the ages of 18 and 45 years. Each subject had a normal physical examination, electrocardiogram, serum chemistries, hematology, urinalysis, and test for human immunodeficiency virus at screening. RESULTS: A greater than proportionate increase in mean plasma concentration values for area under the curve from time zero to infinity was observed over the 200 to 300 mg dose range groups. Red blood cell acetylcholinesterase showed a dose-inhibition relationship, with a mean maximum inhibition of 20.9% at 10 mg that increased to 62.1% at 300 mg. Butyrylcholinesterase activity was relatively unaffected by zifrosilone (< 20% inhibition at 300 mg). For doses > or = 200 mg, an Emax pharmacodynamic model was used to describe the relationship between zifrosilone plasma concentration and red blood cell acetylcholinesterase inhibition (Emax = 83.8%; EC50 = 0.65 ng/ml). CONCLUSIONS: Zifrosilone showed dose-dependent pharmacokinetics after oral administration and was effective in causing selective inhibition of red blood cell acetylcholinesterase.

Time course of 5-HT2A receptor occupancy in the human brain after a single oral dose of the putative antipsychotic drug MDL 100,907 measured by positron emission tomography.

MDL 100,907 is a potent and selective antagonist of 5-HT2A serotonin receptors. Animals studies suggest that MDL 100,907 may behave as an atypical antipsychotic drug. Positron emission tomograph (PET) using [11C]NMSP as the radiotracer was used to define the time course of 5-HT2 receptor occupancy in the human frontal cerebral cortex after a single oral dose of MDL 100,907 (10 or 20 mg) in nine healthy subjects. After the baseline scan each subject was studied three times post dosing at various time points. 5-HT2 occupancies were in the range of 70 and 90% after each dose. While the occupancy remains in this range over 24 hours after 20 mg MDL 100,907, it decreases by about 20% at 24 hours compared to the timepoint at 8 hours, when only 10 mg are administered (p < 0.05). Our results should allow determination of the appropriate dosing regimen for future trials in schizophrenic patients.

Comparative prevalence of rabies antibodies among household and unclaimed/stray dogs as determined by the immune adherence haemagglutination assay.

The immune adherence haemagglutination assay (IAHA), widely used for human viral disease diagnosis, has been adapted for detection of rabies virus antibodies in dog sera. Rabies virus antibody titres obtained by the IAHA correlated well with those obtained by the currently accepted test for rabies antibody determination, the rapid-fluorescent-focus-inhibition test (RFFIT). Although it is not known if the antibodies detected in IAHA test represent neutralizing antibodies against rabies, IAHA has several advantages over the RFFIT: the IAHA is rapid, requiring about seven hours for results to be available; it is relatively inexpensive and easy to perform; uses reagents commonly available in any routine virology laboratory; and uses inactivated rabies virus, thus eliminating hazards associated with the use of live virus in RFFIT. Using this test we found that rabies antibody titres were significantly higher, and at the same time more prevalent, among household dogs than among the unclaimed/stray dogs. The results re-emphasize the increased hazard associated with unclaimed/stray dogs and the need for vaccination of all dogs.

Peanut agglutinin: a new marker for tissue histiocytes.

The histiocyte (macrophage) is a unique cellular constituent of the immune system which is involved in immune and non-immune cellular reactions, as well as in the genesis of a variety of benign and malignant neoplasms. The ability to distinguish histiocytes from similar appearing cells morphologically, is often difficult, yet may be of considerable practical and theoretical importance. The present study describes a new marker for histiocytes, applicable to routinely processed tissues. An immunoperoxidase procedure to detect binding of peanut agglutinin (PNA) was applied to 58 specimens consisting of lymph nodes, extranodal lymphoid tissues, neoplasms and reactive histiocytic lesions. Results were compared with a currently accepted histiocytic marker--cytoplasmic muramidase. Of the 58 tissues, 51 showed PNA binding to morphologically recognizable histiocytes, whereas muramidase was detected in only 44. PNA binding- appeared at least as sensitive and specific as muramidase, as a marker for histiocytes, and offers the advantage that it does not bind to cells of the granulocytic series.

Vitamin E does not modify HDL-cholesterol.

Vitamin E has been reported to increase serum high-density lipoprotein (HDL) cholesterol. Given the known inverse relationship between HDL-cholesterol and risk of developing coronary artery disease, these preliminary results require verification or refutation. In the present study, 39 volunteers were given 600 U of vitamin E daily for 30 days. Serum vitamin E, cholesterol, HDL-cholesterol, and triglycerides were measured at appropriate intervals. No significant change in serum HDL-cholesterol with vitamin E therapy could be documented.

Uterine smooth-muscle tumors of uncertain malignant potential.

OBJECTIVE: To determine whether tumors meeting the criteria of Hendrickson and Kempson for uterine smooth-muscle tumors of uncertain malignant potential have a natural history different from those of leiomyomas and leiomyosarcomas. METHODS: Tumors with five to ten mitoses per ten high-power fields and with mild or moderate cellular atypia were classified as tumors of uncertain malignant potential. Tumors with two to four mitoses per ten high-power fields and severe cellular atypia would also be classified as tumors of uncertain malignant potential, but we had no tumors that fell into this latter group. Forty-seven women with leiomyosarcoma or smooth-muscle tumors of uncertain malignant potential were identified. Paraffin-embedded blocks were recut, and hematoxylin and eosin-stained sections were studied for mitotic counts and cellular atypia. Statistical analysis used chi 2, Fisher exact test, Student t test, and Kaplan-Meier life table analysis. RESULTS: Fifteen tumors were classified as uncertain malignant potential and 32 as leiomyosarcomas. The patients with leiomyosarcoma were significantly older and more likely to present with extrauterine disease. Those with tumors of uncertain malignant potential had a 5-year disease-free survival of 66% and overall survival of 92%, compared to 28 and 40%, respectively, for leiomyosarcomas; these differences were statistically significant. Patients with tumors of uncertain malignant potential tended to have a protracted clinical course after development of recurrence, and several survived longer than 5 years with metastatic disease. CONCLUSIONS: Patients with five to ten mitoses per ten high-power fields and mild to moderate cellular atypia had a prognosis significantly better than that of patients with leiomyosarcomas. In this group, only 27% developed a recurrence, and after recurrence they tended to have a protracted course. Some of these tumors do have a very aggressive course, and the term "uncertain malignant potential" is appropriate.

Nesidioblastosis in adults. A surgical dilemma.

Nesidioblastosis is the leading cause of hyperinsulinemia in newborns and infants. To our knowledge, it has not been previously reported in adults unless associated with other diseases. Three males and three females, aged 11 to 57 years, are described. Pancreatic resections ranged from 50% to 100%. Postoperatively, three patients were normoglycemic, two were insulin-dependent diabetics, and one had recurrent hypoglycemia controlled by drugs. The histologic findings in adults were very similar to those in infants with nesidioblastosis. It appears that a minimum resection of 75% to 80% of the pancreas will benefit the majority of patients, alleviating hypoglycemia and preventing the development of diabetes.

The pathology of head and neck tumors: spindle cell lesions (sarcomatoid carcinomas, nodular fasciitis, and fibrosarcoma) of the aerodigestive tracts, Part 14.

The term spindle cell lesion, or tumor, is a purely descriptive one and if applied without further qualification is meaningless as a guide to therapy and prognosis. The three lesions presented in this report--sarcomatoid carcinomas, nodular fasciitis, and fibrosarcoma--serve to illustrate this point. One, the sarcomatoid carcinoma, is an epithelial malignancy in which the majority of the sarcoma-like spindle cells are believed to be variants of the epithelial cells. Nodular fasciitis, a self-limited and benign soft tissue lesion, is composed principally of myofibroblasts. Primarily an extramucosal lesion, it presents a pseudosarcomatous microscopic appearance. Fibrosarcomas represent the other end of the spindle cell lesion spectrum in that they are soft tissue malignancies of fibroblastic origin. Clinical, pathologic, and biologic implications of these lesions when they arise in the mucosae of the upper aerodigestive tracts of the head and neck are presented.

DNA transfection of Mammalian cells using polybrene.

Many techniques have been developed to transfect mammalian cells with DNA (1-2). The most commonly used method is to expose cells to a coprecipitate of DNA and calcium phosphate (3). This technique works very well with both genomic and recombinant DNA sequences for some cell lines, e.g., mouse L-cells, but less well with other cell types such as Chinese hamster ovary (CHO) cells (2-4). Since the ability to rescue mutant phenotypes by exogenous DNA provides a means to identify gene products and to clone their genes (5), the reduced ability to transform CHO cells by the calcium phosphate technique has slowed the use of the large number of CHO cell mutants (6) in transfection and rescue experiments. In this chapter we will describe a simple, reproducible method for DNA transfection (7) that is a modification of a method originally developed for the introduction of viral sequences into chick embryo fibroblasts (8). The method involves exposing the cells to DNA in the presence of the polycation polybrene, followed by a DMSO shock. The polybrene apparently interacts with the charge on the cell and the DNA, allowing the DNA to absorb more easily to the cell membrane.

Kinetochore-staining of spermatid micronuclei: studies of mice treated with X-radiation or acrylamide.

The rodent spermatid micronucleus (MN) assay was used in conjunction with immunofluorescent techniques to distinguish kinetochores in MN following exposure of mice to X-radiation or acrylamide. After either treatment, modest increases in kinetochore-positive MN were observed. Spermatids which had been exposed during meiotic prophase to X-rays (400 cGy) had approximately 10-fold increases in MN compared to controls; up to 15% of the MN observed were kinetochore-positive. Following acrylamide treatment of meiotic prophase cells, there was a doubling of spermatid MN over baseline levels, approximately one-third of which were kinetochore-positive.

Evidence that methanol inhalation does not induce chromosome damage in mice.

Mice were exposed by inhalation to 800 or 4000 ppm methanol for 5 days, and cytogenetic effects were analyzed in blood erythrocytes, lung cells, and testicular germ cells. The results were uniformly negative; no increased frequencies of micronuclei in blood cells, of sister-chromatid exchanges, chromosome aberrations, or micronuclei in lung cells, or of synaptonemal complex damage in spermatocytes were found. From the standpoint of risk assessment, these experimental studies do not reveal any evidence of a cytogenetic hazard associated with inhalation of methanol.

The effect of the aqueous solubility of xanthine derivatives on the release mechanism from ethylcellulose matrix tablets.

Release data from ethylcellulose (EC) matrix tablets was analyzed to determine which release equation provides the best fit to the data and to observe the effect of drug solubility on the release mechanism(s). Tablets were prepared by direct compression of drug, EC, and lubricant in an appropriate mass ratio to achieve a high and a low drug loading. Theophylline, caffeine, and dyphylline were selected as non-electrolyte xanthine derivatives with solubilities from 8.3 to 330 mg/ml at 25 degrees C. Drug release studies were conducted in 37 degrees C water with UV detection at 272 nm. Several equations to characterize release mechanisms were tested with respect to the release data. Drug diffusion, polymer relaxation, and tablet erosion were the mechanisms considered. Parameters were generated and ANOVA data presented by WinNonlin Pro(R) software. The Akaike Information Criterion was also considered to ascertain the best fit equation. At high drug loading, drug was released by a diffusion mechanism with a rate constant that increased with an increase in aqueous solubility. At low drug loading, polymer relaxation also became a component of the release mechanism. However, its contribution to drug release was less pronounced as solubility decreases, becoming negligible in the case of theophylline.