Infrared microspectroscopy of individual human cervical cancer (HeLa) cells

We report infrared (IR) spectra observed for individual, cultured human cervical cancer (HeLa) cells. Spectra were collected microscopically, in reflection/absorption modes, from cells deposited and dried on microscope slides or from cells grown directly on slides. Within the spectra of the dried cells, significant spectral heterogeneity exists that was previously attributed to different stages of the cell cycle [Boydston-White. S., et al., Biospectroscopy 1999, 5, 219-227; Holman, H. Y., et al., Biopolymers Biospectrosc 2000, 57, 329-335; Tobin, M. J., et al., in First BASIE Workshop, Karlsruhe, Germany, 2003]. The results reported here confirm earlier findings and present the possibility of determining the abundance of cells within each stage of the cycle from the IR spectra. In an accompanying paper, we show that the spectra of cells in suspension exhibit spectral intensity distributions that are different from that of the dried cells. This result has far-reaching implications for the use of infrared microspectroscopy to screen dried cell preparations for the presence of abnormal cells.     

Mechanisms for the transport of unconjugated bilirubin in human trophoblastic BeWo cells

To evaluate mechanisms that mediate passage of unconjugated bilirubin (UCB) across placenta, the transport of [3H]UCB was studied in the human trophoblastic, BeWo cell line. When plotted against the unbound UCB concentration [Bf], uptake exhibited saturative kinetics with a similar apparent Km ( approximately 30 nM) for BeWo cells grown either in polarized (Transwell) or non-polarized fashion (dish). UCB release from cells, but not uptake, was inhibited by sulfobromophthalein but not by taurocholate, and almost abolished by MK571, a specific inhibitor of the activity of multidrug resistance-associated proteins (MRPs). MRP1 and MRP5 were both present in BeWo cells and the expression of MRP1, but not MRP5, was markedly higher in polarized cells. These data indicate that UCB is taken up from the fetal circulation by a still undefined, saturative process not shared by other organic anions and is then excreted to maternal circulation by proteins of the MRP family.     

Low-density lipoprotein inhibits secretion of phospholipid transfer protein in human trophoblastic BeWo cells

Human plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. In this study, we investigated the effects of lipoproteins on the secretion of PLTP in cultured BeWo choriocarcinoma cells. Low-density lipoproteins (LDLs) decreased PLTP secretion in a dose- and time-dependent manner, whereas very low density lipoproteins and high-density lipoproteins (HDLs) had little effect. LDL suppression of PLTP secretion was not altered by the inhibition of both LDL receptor and LDL receptor-related protein with receptor-associated protein. Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, U0126, could abolish the LDL-mediated inhibition of PLTP secretion. Furthermore, LDL, but not HDL, could stimulate the expression of MAPK phosphatase-1 (MKP-1) in BeWo cells that resulted in the inactivation of p44/p42 extracellular signal-regulated kinase (ERK) 1 and 2, the family members of MAPKs. These results support the conclusion that LDL-mediated suppression of PLTP secretion in BeWo cells is through a LDL receptor-independent MAPK signaling pathway.     

Neospora caninum infection in a free-ranging raccoon (Procyon lotor) with concurrent canine distemper virus infection

During a canine distemper virus (CDV) outbreak in raccoons (Procyon lotor) from Cook County, Illinois, a juvenile female suffering from seizures was killed and necropsied. Gross and histologic findings of necrotizing encephalitis and proliferative bronchopneumonia were attributed to CDV infection and considered the cause of clinical signs. A section of cerebellum stained immunohistochemically for Neospora caninum revealed an approximately 40 microm diameter, round to oval cyst with a 2- to 3-microm-thick wall and filled with 1-2 microm diameter, round to oval bradyzoites. Polymerase chain reaction (PCR) results were positive for N. caninum using DNA extracted from the brain. Specific PCR for the closely related organisms Toxoplasma gondii and Hammondia heydorni yielded negative results. This case report provides histologic, immunohistochemical, and molecular evidence that raccoons are a naturally occurring intermediate host of N. caninum.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

First report of Neospora caninum infection in adult alpacas (Vicugna pacos) and llamas (Lama glama)

Neospora caninum is a cyst-forming coccidian that mainly affects bovines, although Neospora infection has also been described in other domestic and wild ruminant species. Serum samples from 78 alpacas (Vicugna pacos) and 73 llamas (Lama glama) at a unique dilution of 1:50 tested by indirect fluorescent antibody test (IFAT) were further analyzed serologically by IFAT and Western blot in both ruminant species to avoid cross-reactions with closely related coccidian parasites and to confirm the existence of N. caninum-specific antibodies. IFAT titers ranging between 1:50 and 1:800 were found. When using Western blot, N. caninum tachyzoite-specific immunodominant antigens with apparent molecular weights of 17-18, 34-35, 37, and 60-62 kDa were also recognized, although some sera with 1:50 IFAT titers proved not to have N. caninum-specific antibodies. As expected, higher IFAT titers were associated with higher anti-N. caninum reactivity in Western blot. This report documents for the first time the presence of N. caninum infection in adult alpacas and llamas from Peru.     

Differential susceptibility to human immunodeficiency virus type 1 infection of myeloid and plasmacytoid dendritic cells

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) plays an important role in HIV-1 transmission and pathogenesis. Here, we studied the susceptibility of ex vivo-isolated CD11c+ myeloid DCs (MDCs) and CD123+ plasmacytoid DCs (PDCs) to HIV-1 infection and the function of these cells early after infection. Both DC subsets were susceptible to CCR5- and CXCR4-using HIV-1 isolates (BaL and IIIB, respectively). However, MDCs were more susceptible to HIV-1(BaL) infection than donor-matched PDCs. In addition, HIV-1(BaL) infected MDCs more efficiently than HIV-1(IIIB), whereas PDCs were equally susceptible to both isolates. While exposure to HIV-1 alone resulted in only weak maturation of DCs, Toll-like receptor 7/8 ligation induced full maturation in both infected and uninfected DCs. Maturation did not increase HIV-1 replication in infected DCs, and infected DCs retained their ability to produce tumor necrosis factor alpha after stimulation. Both HIV-1 isolates induced alpha interferon production exclusively in PDCs, irrespective of productive infection. In conclusion, PDCs and MDCs were susceptible to HIV-1 infection, but neither displayed functional defects as a consequence of infection. The difference in susceptibility of PDCs and MDCs to HIV-1 may have implications for HIV-1 transmission and DC-mediated transfer of HIV-1 to T cells.     

Coyotes (Canis latrans) are definitive hosts of Neospora caninum

Four captive-raised coyote pups consumed tissues from Neospora caninum-infected calves. Faeces were examined from 4 days before to 28 days after infection. One pup shed N. caninum-like oocysts, which tested positive for N. caninum and negative for Hammondia heydorni using PCR tests. Coyotes are the second discovered definitive host of N. caninum, after dogs. In North America, the expanding coyote ranges and population increase the probability of contact with domestic livestock. To reduce the risk of transmission of N. caninum to intensively farmed cattle, we recommend protection of feedstuffs using canid-proof fences, and careful disposal of dead stock.     

High prevalence of antibodies to Neospora caninum in white-tailed deer (Odocoileus virginianus)

Serum samples of 400 white-tailed deer (Odocoileus virginianus) from 16 preserves in northeastern Illinois were tested for Neospora caninum antibodies in the N. caninum agglutination test using mouse-derived N. caninum tachyzoites and mercaptoethanol. Antibodies were found in 162 deer with titres of 1:40 (47 deer), 1:80 (32 deer), 1:160 (17 deer), 1:200 (eight deer), 1:400 (19 deer), 1:800 (17 deer) and > or = 1:1600 (22 deer). There were no significant differences in prevalence between age or sex of the deer. The high prevalence of N. caninum infection in deer is consistent with a sylvatic cycle of N. caninum.     

Infrared microspectroscopy of individual human cervical cancer (HeLa) cells suspended in growth medium

We report for the first time the infrared spectra of individual human cervical cancer (HeLa) cells suspended in buffer or cell culture medium. Although we did not establish whether these cells were viable at the time of spectral data acquisition, we believe that the methodology used is applicable to the study of live cells. Data were collected either from entire cells, using 25- to 40-microm apertures, or via an imaging approach, where pixels measuring 6.25 x 6.25 microm were assembled to form a map of a cell in suspension. Measurements were carried out both in reflection/absorption and in transmission modes. The results reported here might have far-reaching implications for the use of infrared microspectroscopy to monitor cell proliferation, drug response, and other cell biological parameters in live cells.     

The induction of apoptosis in human cervical carcinoma (HeLa) cells by gamma-linolenic acid

A high concentration (50 μg/ml) of gamma-linolenic acid (GLA) induced morphological lesions typical of apoptosis, as well as DNA fragmentation, in HeLa cells. A lower concentration of GLA (20 μg/ml), caused an increased proliferating cell nuclear antigen (PCNA) labelling, with 92.7% cells positive, compared to 27.7% at a concentration of 50 μg/ml GLA. In correlation with these results, the number of cells with degraded DNA below the G₀/G₁ peak increased significantly in the 50 μg/ml GLA-treated cells, but increased only slightly in cells exposed to the lower level of GLA. The high levels of PCNA induced by 20 μg/ml GLA, in both G₁ and S phases, may indicate a state of DNA repair synthesis, whilst at the higher concentration of GLA, most of the cells became apoptotic. Since apoptosis is associated with the deregulation of c-Myc expression, and as the Raf-1-MAP kinase cascade activates the expression of c-Myc and c-Jun, we investigated the effects of 20 and 50 μg/ml GLA on the Raf-1, c-Myc and c-Jun levels, and on the activity of MAP kinase. The results showed that 50 μg/ml GLA lowered the activity of MAP kinase. As expected with the decreased MAP kinase activity in the cells exposed to the higher level GLA, the c-Jun levels were also lowered. The levels of c-Myc, however, were increased. It is therefore possible that the deregulated expression of c-Myc in the HeLa cells exposed to the high level of GLA (50 μg/ml) may contribute to the induction of apoptosis in HeLa cells.     

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Spain

In recent years, neosporosis has been identified as a major cause of abortion in dairy and beef cattle. Although the disease has been described worldwide, there is a Jack of information concerning the prevalence of this infection in different cattle production systems. The aim of this study was to investigate the seroprevalence of Neospora caninum infection in a representative area of beef and dairy cattle production in Spain. A cross-sectional study was undertaken in which herds constituted the initial sampling unit and two strata (dairy and beef herds) were considered. Using a 95% level of confidence and setting 5% (beef) and 5.4% (dairy) error limits, 216 beef and 143 dairy herds were randomly selected and sampled. Nine animals (> 1 year old) were randomly sampled in each herd to detect the presence of the infection. A herd was considered infected when at least one animal was seropositive. In total, serum samples from 1121 dairy and 1712 beef animals were collected and tested for specific anti-N. caninum IgG using an ELISA. Specific antibodies were detected in 55.1% (119/216) beef and 83.2% (119/143) dairy herds. Individual prevalences obtained were 17.9% (306/1712) for beef and 35.9% (402/1121) for dairy animals. Presence of N. caninum infection was higher in dairy than in beef herds and the association between infection and the cattle production system (dairy or beef) was statistically significant [(chi2)Y= 29.21, P < 0.001, OR = 4.04 (2.35-6.99)]. Herd size of dairy cattle did not appear to be associated with N. caninum infection. On the contrary, infection was associated with herd size in beef cattle (chi2 = 12.79, P < 0.01). Finally, no association was found between replacement or pasture management and infection in beef herds.     

Chickens (Gallus domesticus) are natural intermediate hosts of Neospora caninum

Neospora caninum naturally infects many mammal species, but has not previously been demonstrated in birds. We examined sera for N. caninum antibodies from 200 outdoor chickens and from 200 chickens confined indoors in the state of Bahia, Brazil. Seroprevalence was greater in outdoor chickens (23.5% versus 1.5%, P<0.001). PCR testing for N. caninum was positive in six of 10 seropositive chickens. Amplicons from two of these were sequenced and had 97-98% nucleotide identity with N. caninum. This finding extends the list of intermediate hosts of N. caninum to include birds and may have important epidemiological consequences.     

Differential susceptibility to nitric oxide-evoked apoptosis in human inflammatory cells

Apoptosis of neutrophils and their subsequent phagocytosis is critical to the successful resolution of inflammation. During inflammation, activated inflammatory cells generate reactive oxygen and nitrogen species, including nitric oxide (NO) and superoxide anion (O₂^??), which rapidly combine to generate peroxynitrite (ONOO^?). NO and ONOO^? are proapoptotic in human neutrophils. This study examines the effects of NO and ONOO^? on caspase activation and mitochondrial permeability in human neutrophils and determines the ability of these species to evoke apoptosis in human monocyte-derived macrophages (MDMs). NO or ONOO^? release from donor compounds was characterized by electrochemistry and electron paramagnetic resonance. Neutrophils and MDMs isolated from the peripheral blood of healthy volunteers were exposed to NO or ONOO^? before analysis of apoptosis by caspase activation, mitochondrial permeability, and annexin V binding. Both NO and ONOO^? induced apoptosis via rapid activation of caspases 2 and 3 in neutrophils. In contrast, only ONOO^? promoted apoptosis in MDMs, whereas a variety of NO donors were ineffective at inducing apoptosis in this cell type. We propose that human macrophages are refractory to NO-stimulated apoptosis in order that they persist long enough within the inflammatory focus to phagocytose apoptotic neutrophils, thereby ensuring successful resolution of inflammation.     

In vitro development of Neospora caninum (Protozoa: Apicomplexa) from dogs

The development of Neospora caninum isolated from naturally infected dogs was examined in mammalian cell cultures. Tachyzoites developed by endodyogeny when inoculated onto bovine monocyte or bovine cardiopulmonary artery endothelial cell cultures. Tachyzoites were 5.0 by 2.0 microns and had a posteriorly located nucleus. Cytopathogenic effects of parasite development consisted of the formation of holes in the cell monolayer associated with the rupture of infected host cells. Serial passage of tachyzoites was achieved by subinoculation of tachyzoites onto non-infected bovine monocyte cell cultures. It appears that N. caninum can be continuously grown in cell cultures.     

Functional activation of T cells by dendritic cells and macrophages exposed to the intracellular parasite Neospora caninum

Neospora caninum is an intracellular protozoan pathogen that causes abortion in cattle. We studied how the interaction between murine conventional dendritic cells or macrophages and N. caninum influences the generation of cell-mediated immunity against the parasite. We first explored the invasion and survival ability of N. caninum in dendritic cells and macrophages. We observed that protozoa rapidly invaded and proliferated into these two cell populations. We then investigated how Neospora-exposed macrophages or dendritic cells distinguish between viable and non-viable (heat-killed tachyzoites and antigenic extract) parasites. Viable tachyzoites and antigenic extract, but not killed parasites, altered the phenotype of immature dendritic cells. Dendritic cells infected with viable parasites down-regulated the expression of MHC-II, CD40, CD80 and CD86 whereas dendritic cells exposed to N. caninum antigenic extract up-regulated the expression of MHC-II and CD40 and down-regulated CD80 and CD86 expression. Moreover, only viable tachyzoites and antigenic extract induced IL-12 synthesis by dendritic cells. MHC-II expression was up-regulated and CD86 expression was down-regulated at the surface of macrophages, regardless of the parasitic form was encountered. However, IL-12 secretion by macrophages was only observed under conditions using viable and heat-killed parasite. We then analysed how macrophages and dendritic cells were involved in inducing T-cell responses. T lymphocyte IFN-γ-secretion in correlation with IL-12 production occurred after interactions between T cells and dendritic cells exposed to viable tachyzoites or antigenic extract. By contrast, for macrophages IFN-γ production was IL-12-independent and only occurred after interactions between T cells and macrophages exposed to antigenic extract. Thus, N. caninum-induced activation of murine dendritic cells, but not that of macrophages, was associated with T cell IFN-γ production after IL-12 secretion.     

Differential susceptibility to TRAIL of normal versus malignant human urothelial cells

Comparing normal human urothelial (NHU) cells to a panel of six representative urothelial cell carcinoma (UCC)-derived cell lines, we showed that while TRAIL receptor expression patterns were similar, susceptibility to soluble recombinant crosslinked TRAIL fell into three categories. 4/6 carcinoma lines were sensitive, undergoing rapid and extensive death; NHU and 253J cells were partially resistant and HT1376 cells, like normal fibroblasts, were refractory. Both normal and malignant urothelial cells underwent apoptosis via the same caspase-8/9-mediated mechanism. Rapid receptor downregulation was a mechanism for evasion by some UCC cells. TRAIL resistance in malignant urothelial cells was partially dependent on FLIP(L) and was differentially mediated by p38(MAPK), whereas in normal cells, resistance was mediated by NF-kappaB. Importantly, extensive killing of UCC cells could be induced using noncrosslinked TRAIL after prolonged exposure, with no damage to their homologous, normal urothelial cell counterparts.