New phenotypic, functional and electrophysiological characteristics of KG-1 cells

Myeloid dendritic cells (DC) are representatives of a rare and phenotypically diverse population of professional antigen presenting cells possessing high functional heterogeneity and flexibility. Here we studied the phenotypic, functional and electrophysiological characteristics of KG-1 cells, an erythroleukemia model cell line, which shares morphological and physiological similarities with immature and mature myeloid DC. We compared the expression of internalizing receptors and other cell surface molecules, antigen uptake and migration of unstimulated and activated KG-1 cells with the characteristics of immature and mature DC. Unstimulated KG-1 cells were less potent in capturing extracellular materials than immature DC. In contrast to monocyte-derived DC KG-1 cells stimulated by PMA and ionomycin ceased to migrate along the MIP-3beta chemokine gradient despite their high expression of CCR7 chemokine receptor and MDR, a transporter implicated in DC migration. Moreover, we determined the ion channel repertoire of KG-1 cells before and after treatment with PMA and ionomycin by using the patch-clamp technique. We found that both unstimulated and activated KG-1 cells expressed time- and voltage-independent, ChTx sensitive intracellular Ca(2+)-gated potassium conductance suggesting the presence of K(Ca) channels in their membranes. Based on our results we propose that KG-1 cells resemble myeloid DC but also possess unique phenotypic, functional and electrophysiological characteristics.     

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell linesin vitro andin vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-ras ^H, v-ras ^Ki, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determinedin vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection withras, despite similarras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-ras ^H + SV40T and v -ras ^H + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.     

Smad4 overexpression causes germ cell ablation and leydig cell hyperplasia in transgenic mice

Members of the transforming growth factor-beta (TGF-beta) superfamily play a variety of important roles in testicular development and function. The tumor suppressor gene, Smad4, is a common mediator of TGF-beta, activin, and bone morphogenetic protein-mediated signaling pathways. To investigate the role of the Smad4 gene during testicular development and function, transgenic mice were generated using a Flag-tagged Smad4 gene driven by 180-bp fragment of the Mullerian inhibiting substance upstream promoter sequence. Three Smad4 transgenic founders (A, B, and G) were detected by Southern blot analysis; line B showed the highest expression of the Smad4 transgene and was further studied. The fertility in F1 generation (B) and F2 generation (BB) of the Smad4 transgenic mice was not impaired. However, in the F3 generation (B2x) all animals were impacted by the overexpression of the Smad4 transgene and two kinds of phenotypes were observed. In one group animals were completely infertile, while in the other group animals were fertile and sired the normal number of pups/litter. These groups are designated as infertile and fertile in the text. Histological evaluation of the testes from the infertile group showed variable degrees of Leydig cell hyperplasia, apoptosis of germ cells, spermatogenic arrest, seminiferous tubule degeneration, and infertility. In the fertile group, there was no apparent change in the histology of the testis except for a slight increase in the number of Leydig cells. Serum follicle-stimulating hormone levels in the adult animals of both groups of Smad4 transgenic male mice were not significantly different from normal littermates; however, testosterone levels in both groups were significantly (P < 0.05) increased. These results suggest that overexpression of Smad4 leads to testicular abnormalities and infertility supporting the hypothesis that the TGF-beta signaling pathways are carefully orchestrated during testicular development. In the absence of normal levels of Smad4 testicular function is compromised.     

Immunoglobulins in the egg,embryo and young chick

Current knowledge of the immunoglobulin classes identified in some avian species is reviewed. The distribution and fate of passively acquired immunoglobulins or specific antibodies in compartments of the egg and of the developing embryo and in the newly hatched chick are described, together with the ontogeny of active Ig biosynthesis.     

Collagen type I increases bone remodelling around hydroxyapatite implants in the rat tibia

The early interface reaction of cancellous bone to a nanocrystalline hydroxyapatite (HA) cement containing 3 wt% collagen type I (HA/Coll) with a setting under physiological temperature and pH was observed using immunohistochemical techniques. Pure HA served as a control. Cylinders with a diameter of 2 mm were implanted into the proximal tibia of 72 adult Wistar rats. Histological sections of 6 animals were prepared after 1, 2, 4, 6, 14 and 28 days. First, osteoblast-like cells as well as a marked reaction for osteonectin, osteopontin and its ligand CD44 were observed as early as 2 days after implantation at the interface around HA/Coll implants. Further, reactivity for ED1 and cathepsin D, both markers for phagocytotic cells, appeared earlier and stronger around HA/Coll. In cell counts, a significantly higher average number of ED1- and cathepsin D-positive phagocytotic cells was observed around the HA/Coll implants on days 6 (p < 0.01), 14 and 28 (p < 0.05). The number of osteopontin-positive cells was significantly higher around HA/Coll implants at days 6 and 14 (p < 0.05). Two weeks after the implantation, first islands of newly formed woven bone were observed around the HA/Coll implant, but not around the control implant. The amount of direct bone contact after 28 days averaged 28% around pure HA and 51% around HA/Coll implants (p < 0.05). While both implants displayed a good osteoconductivity, a higher bone remodelling activity was observed around collagen-containing HA implants compared to pure HA implants. It appears that the addition of collagen to HA implants can enhance both phagocytotic and osteogenic processes. This may result in an earlier acceptance and better osseointegration of the HA/Coll implants into the surrounding tissue.     

Single-Step Multiplex PCR Assay for Characterization of New World Leishmania Complexes

We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmania parasites, particularly where different Leishmania complexes are found in the same geographical area.     

Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas

We compared hyperplastic-like polyps that preceded microsatellite-unstable adenocarcinomas to incidental hyperplastic polyps to identify distinguishing morphologic criteria. The study group included 106 hyperplastic-like, nonadenomatous, serrated polyps, most from the ascending colon in 91 patients; the control group included 106 rectosigmoid hyperplastic polyps from 106 patients in whom adenocarcinoma did not develop. Study group polyps had an expanded crypt proliferative zone, a serrated architectural outline that became apparent in the basilar crypt regions, basilar crypt dilation, inverted crypts, and a predominance of dysmaturational crypts (crypts with minimal cell maturation). In contrast, control group polyps had a proliferative zone confined to the basal crypt region, serrated architecture that became apparent in the superficial crypt region, rare to no basilar crypt dilation, and rare or no dysmaturational crypts. Hyperplastic-like polyps that preceded microsatellite-unstable adenocarcinomas had a distinctive constellation of morphologic features related to altered and decreased cell function and control that resulted in dysmaturational crypts. Dysmaturation constitutes a range of morphologic alterations, some of which overlap with incidental-type innocuous hyperplastic polyps. The morphologic features described herein provide initial guidelines to identify this potentially important subset of premalignant serrated-like polyps.     

Monoaminergic Fluorescence in Frog Skin

The Falck-Hillarp fluorescence technique was employed in an attempt to determine the distribution of sympathetic innervation in frog skin. No evidence was found of a direct monoaminergic nerve supply to the cells of the non-glandular epithelium in the epidermis. Instead, specific fluorescence was mainly confined to the vicinity of the skin glands. Fluorescent fibers were observed surrounding the mucous type of gland. The secretory content of this gland was not fluorescent. In the granular type of gland the main source of fluorescence was the secretory granules filling the lumen. These developed a fluorescence in the spectral range of 5-hydroxytryptamine. The brightness of the fluorescence indicated a very high content of this amine. Fluorimetric analysis showed that no catecholamines were present in the secretion. In glands devoid of secretory granules there were some indications of a monoaminergic innervation of the secretory epithelium, but this was hard to determine because of the abundant nonspecific fluorescence. Sparse dots of specific fluorescence were found close to the surrounding smooth muscle cells. — These findings rule out the possibility of a direct sympathetic nervous control of the non-glandular epithelium in frog skin but indicate that this is instead confined to the skin glands.