Low prevalence of Babesia hongkongensis infection in community and privately-owned cats in Hong Kong.

Domestic cats are susceptible to infection with at least 11 species of Babesia. In Hong Kong, where dogs are commonly infected with B. gibsoni, a single infection in a cat by a novel species, B. hongkongensis, was reported previously. The aim of this study was to investigate the frequency of Babesia spp. detection in cats in Hong Kong. Residual blood-derived DNA from healthy free-roaming community cats (n = 239), and privately-owned cats with and without anaemia undergoing diagnostic investigations (n = 125) was tested for Babesia spp. DNA using a pan-Babesia PCR targeting mitochondrial Cytochrome B, and a B. hongkongensis specific PCR targeting 18S rRNA. Positive samples were confirmed by sequencing and comparative sequence analysis against the GenBank nucleotide database. Babesia hongkongensis was detected in 4/239 (1.7 %) community cats, and 0/125 (0.0 %) privately-owned cats. Babesia gibsoni was detected in 0/239 community cats and 1/125 (0.8 %) privately-owned cats. Cats infected with B. hongkongensis were clinically healthy at the time of sampling. The B. gibsoni-infected cat was anaemic and thrombocytopenic. Cats in Hong Kong can be infected with B. hongkongensis and B. gibsoni, albeit at low frequency. The tick vector for B. hongkongensis is yet to be identified.

Foxp4 is essential in maintenance of Purkinje cell dendritic arborization in the mouse cerebellum.

Purkinje cells (PCs) are one of the principal neurons in the cerebellar cortex that play a central role in the coordination of fine-tuning body movement and balance. To acquire normal cerebellum function, PCs develop extensive dendritic arbors that establish synaptic connections with the parallel fibers of granule cells to form the proper neuronal circuitry. Therefore, dendritic arborization of PCs is an important developmental step to construct the mature neural network in the cerebellum. However, the genetic control of this process is not fully understood. In this study, Foxp4, a forkhead transcription factor that is expressed specifically in migrating and mature PCs of cerebellum from embryonic stages to adulthood, was knocked down by small interfering RNA (siRNA) in organotypic cerebellar slice culture. When Foxp4 expression was knocked down at postnatal day 5 (P5), no abnormalities for early dendritic remodeling of PCs were observed. However, when Foxp4 was knocked down in P10 cerebellar slices, the organization of PC dendritic arbors was highly impaired, leaving hypoplastic but non-apoptotic cell bodies. The radial alignment of Bergmann glial fibers that associated with PC dendrites was also lost. These results suggest that Foxp4 is dispensable for the early PC dendrite outgrowth, but is essential for the maintenance of PC dendritic arborization and subsequent association with Bergmann glial fibers.

Genetic restoration of aldose reductase to the collecting tubules restores maturation of the urine concentrating mechanism.

To investigate the underlying causes for aldose reductase deficiency-induced diabetes insipidus, we carried out studies with three genotypic groups of mice. These included wild-type mice, knockout mice, and a newly created bitransgenic line that was homozygous for both the aldose reductase null mutation and an aldose reductase knockin transgene driven by the kidney-specific cadherin promoter to direct transgene expression in the collecting tubule epithelial cells. We found that from early renal developmental stages onward, urine osmolality did not exceed 1,000 mosmol/kgH2O in aldose reductase-deficient mice. The functional defects were correlated with significant renal cellular and structural abnormalities that included cell shrinkage, apoptosis, disorganized tubular and vascular structures, and segmental atrophy. In contrast, the transgenic aldose reductase expression in the bitransgenic mice largely but incompletely rescued urine concentrating capacity and significantly improved renal cell survival, cellular morphology, and renal structures. Together, these results suggest that aldose reductase not only plays important roles in osmoregulation and medullary cell survival but may also be essential for the full maturation of the urine concentrating mechanism.

Universal topological properties of two-dimensional trivalent cellular patterns.

Universal topological properties of two-dimensional trivalent cellular patterns are found from shell analysis of soap froth and computer-generated Voronoi diagrams. We introduce a cluster analysis based on the shell model and find the universal relation ln(a/mu(2)) = A+Bln(mu(2)), with the generalized Aboav parameter a and second moment of the number of cell edge distribution mu(2). For the second, third, and fourth shells of the cluster, A and B are the same for all samples. Furthermore, A is increasing with shell number while B is a universal number, -0.90. For the first shell, the slope B is the same for soap froths, but slightly different from Voronoi graphs.

Amelogenin-cytokeratin 14 interaction in ameloblasts during enamel formation.

The enamel protein amelogenin binds to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661). The GMp motif is found in the N-terminal region of CK14, a differentiation marker for ameloblasts. The binding affinity of CK14 and amelogenin was confirmed by dosimetric binding of CK14 to recombinant amelogenin (rM179), and to the tyrosine-rich amelogenin polypeptide. The specific binding site for CK14 was identified in the amelogenin trityrosyl motif peptide (ATMP) of tyrosine-rich amelogenin polypeptide and specific interaction between CK14 and [(3)H]ATMP was confirmed by Scatchard analysis. Blocking rM179 with GlcNAc, GMp, or CK14 with ATMP abrogates the CK14-amelogenin interaction. CK14 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Morphometry of developing teeth distinguished three phases of enamel formation; growth initiation phase (days 0-1), prolific growth phase (days 1-7), and growth cessation phase (post-day 7). Confocal microscopy revealed co-assembly of CK14/amelogenin in the perinuclear region of ameloblasts on day 0, migration of the co-assembled CK14/amelogenin to the apical region of the ameloblasts from day 1, reaching a peak on days 3-5, and a collapse of the co-assembly. Autoradiography with [(3)H]ATMP and [(3)H]GMp corroborated the dissociation of the co-assembly at the ameloblast Tomes' process. It is proposed that CK14 play a chaperon role for nascent amelogenin polypeptide during amelogenesis.

Interleukin-2 binds to ganglioside GD(1b).

We have developed a solid matrix immunoassay to determine the binding of interleukin-2 (IL-2) to specific gangliosides. The assay establishes that recombinant human IL-2 binds to ganglioside GD(1b) but not to any other gangliosides (GM(1), GM(2), GM(3), GD(1a), GD(2), GD(3), and GT(1b)). The binding varies with the ratio of GD1b and IL-2. This assay enables distinguishing the nature of the sugar moiety of the ganglioside recognized by IL-2 and establishes the dosimetry of the ganglioside-IL-2 interaction. Since rIL-2 is administered systematically into stage IV melanoma patients, we have examined 45 tumor biopsies for GD(1b) content. The incidence of GD(1b) in tumor biopsies is 51%. We postulate that GD(1b) associated on the tumor or in the circulation of cancer patients may bind to rIL-2 and prevent the availability of rIL-2 to augment antitumor-immune response.

Does human melanoma express carcinoembryonic antigen?

BACKGROUND: Several investigators have proposed that carcinoembryonic antigen (CEA), an immunogenic antigen expressed by colon carcinoma, may also be expressed by human melanoma. Because sialyl Lewisx (sLex), the carbohydrate moiety of CEA, has been identified in melanoma, we compared CEA and sLex levels in colon carcinoma cells and melanoma cells. METHODS: CEA levels were assessed for expression on the cell surface and in cell lysates of cutaneous melanoma cell lines by two different kinds of ELISA, and by Western blot analysis of immunoprecipitated CEA using monoclonal antibodies (Mabs) T84-66 and COL-1, which have defined specificities for CEA. Colon carcinoma cells and purified CEA were positive controls. RESULTS: Both Mabs reacted strongly with cell surface and cell lysates of colon cancer. Mab T84-66 reacted well with cell surface but not cell lysates of melanoma. COL-1 reacted poorly with cell surface but its binding increased with the density of melanoma cell lysates. Both Mabs intensely stained the blots of purified CEA and colon carcinoma lysates immunoprecipitated with the respective Mabs, but failed to stain the immunoprecipitates of melanoma cell lysates. Both Mabs bound to lysates immunoprecipitated with anti-sLex Mab in colon carcinoma, but not in melanoma. Cell-surface expression of CEA and sLex was significantly correlated (r2: 0.88) in colon cancer cells but not in melanoma. CONCLUSION: Our results confirm the presence of CEA in colon carcinoma but not in human cutaneous melanoma cell lines.

The enamel protein amelogenin binds to the N-acetyl-D-glucosamine-mimicking peptide motif of cytokeratins.

Amelogenins bind to GlcNAc of the dentine-enamel matrix proteins (Ravindranath, R. M. H., Moradian-Oldak, J., Fincham, A. G. (1999) J. Biol. Chem. 274, 2464-2471). The hypothesis that amelogenins may interact with the peptides that mimic GlcNAc is tested. GlcNAc-mimicking peptide (SFGSGFGGGY) but not its variants with single amino acid substitution at serine, tyrosine, or phenylalanine residues inhibited hemagglutination of amelogenins and the terminal tyrosine-rich amelogenin polypeptide (TRAP). The binding affinity of SFGSGFGGGY to amelogenins was confirmed by dosimetric binding of amelogenins or TRAP with [(3)H]peptide, specific binding in varying concentrations of the peptide, Scatchard plot analysis, and competitive inhibition with the unlabeled peptide. The ability of the peptide or GlcNAc to stoichiometrically inhibit TRAP binding of [(14)C]GlcNAc or [(3)H]peptide indicated that both the peptide and GlcNAc compete for a single binding site. Using different fragments of amelogenins, we have identified the peptide-binding motif in amelogenin to be the same as the GlcNAc-binding "amelogenin trityrosyl motif peptide." The GlcNAc-mimicking peptide failed to bind to the amelogenin trityrosyl motif peptide when the tyrosyl residues were substituted with phenylalanine or when the third proline was replaced with threonine, as in some cases of human X-linked amelogenesis imperfecta. This study documents that molecular mimicry may play a role in stability and organization of amelogenin during amelogenesis.

Gangliosides as targets for immunotherapy for pancreatic adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma cells express gangliosides and sialyl Lewis (sLe) antigens. It is not known whether these carbohydrate antigens can be targeted by immunotherapy. The authors measured the expression of GM(2) and sLe antigens on the surface of pancreatic carcinoma cells and the serum levels of total gangliosides, GM(2), and antiganglioside antibodies in patients with pancreatic carcinoma. METHODS: Cell surface GM(2) and sLe antigens were measured by cell suspension enzyme linked immunoadsorbent assay (ELISA) in four pancreatic carcinoma cell lines. Sera from 20 pancreatic carcinoma patients and 20 age- and gender-matched healthy volunteers were analyzed for antiganglioside and anti-sLe immunoglobulin (Ig) M titers by ELISA. Serum levels of total gangliosides and GM(2) also were measured. RESULTS: All cell lines expressed GM(2) and sLe antigens. When compared with age- and gender-matched volunteers, patients had significantly higher serum levels of total gangliosides (25.6 +/- 9.0 mg/dL vs. 15.6 +/- 2.7 mg/dL; P < 0.001), GM(2) (0.278 +/- 0.415 mg/dL vs. 0.013 +/- 0.018 mg/dL; P = 0.02), ELISA units of anti-GM(2) IgM antibody (368 +/- 95 vs. 155 +/- 25; P = 0.04) and anti-GD(1b) IgM antibody (351 +/- 91 vs. 138 +/- 26; P = 0.03), but not anti-sLe(x) IgM (1389 +/- 345 vs. 1081 +/- 224; P = 0.46) or anti-sLe(a) IgM antibody (1097 +/- 253 vs. 1200 +/- 315; P = 0.80). Patients with unresectable tumors had higher serum levels of total gangliosides compared with patients with resectable tumors, and a serum level > 25 mg/dL was found to correlate significantly with poor overall survival (P < 0.02). CONCLUSIONS: Increased serum levels of total gangliosides and GM(2) may reflect shedding or release of gangliosides from the surface of tumor cells. Production of IgM antibody against GM(2) and GD(1b) indicates that these gangliosides are immunogenic antigens that may be potential targets for effective active immunotherapy.

Immunology of gangliosides.

This review discusses the immunology of gangliosides from the perspective of tumor, neuronal and general immunology. Antiganglioside antibodies in human sera are invariably IgM and are found in healthy individuals. Their titers decline with age. Persistent high titer of IgM is associated with several diseases, particularly neuropathies. Membrane-bound gangliosides are important tumor-associated antigens and targets for immune attack. Cells enriched with gangliosides can be used as cancer vaccines. Efficacy of these vaccines depends on the viability of whole cells, integrity of the cell membranes, adjuvants and topography of the tumor-associated antigens. The role of antiganglioside IgM is to eliminate the immunosuppressive gangliosides shed from tissues during ageing, degeneration of neural and extraneural tissues, and tumor growth and necrosis. In addition, in vitro observations with human and murine monoclonal antibodies suggest that they are capable of complement dependent cytotoxicity and apoptosis.

The menstrual cycle and platelet 5-HT uptake.

The association between the onset of depressive illness and the paramenstruum has been reported. In the present study, we looked at changes in platelet 5-HT uptake in six healthy, regularly menstruating subjects on days 1, 10, and 24 of three consecutive menstrual cycles to explore possible biochemical bases for mood changes associated with menstruation. Platelet 5-hydroxytryptamine (5-HT) uptake was determined by suspension of platelet pellets in 0.5-5 microM hydroxy [G-3H] tryptamine [( 3H]-5-HT) creatinine sulfate solution at 37 degrees C and measurement of radioactivity of [3H]-5-HT in the platelets using liquid scintillation counting. Mood changes were measured using the Moos Menstruation Distress Questionnaire and the Spielberger State Anxiety Scale. Analysis of variance and trend analysis revealed significant elevation of Km and Vmax on day 24 of the cycle, which was consistent across the three menstrual cycles studied. A significant linear rise of "negative affect" across each cycle peaking on day 1 was detected. "Water retention" and "behavioral change" also peaked on day 1. No significant correlation, however, was obtained between the mood scores and platelet 5-HT uptake. These findings are discussed.