Myelodysplasia, hypophosphataemia, vitamin D and iron deficiency in an alpaca.

A pregnant 2-year-old alpaca was presented for evaluation of progressive weight loss, decreased appetite and lethargy that developed in winter. Haematologic and serum biochemical analyses revealed marked anaemia, leukopenia, severe hypophosphataemia and mild hypocalcaemia. Evaluation of bone marrow core biopsies and aspirates revealed an increased proportion of immature haematopoietic cells, without sufficient numbers of blast cells to be termed an acute myeloid leukaemia (AML). 1 The haematological and bone marrow findings were suggestive of myelodysplastic syndrome (MDS). The anaemia, leukopenia, lethargy and weight loss remained refractory to medical therapy and the alpaca was euthanased on humane grounds.

Serum soluble Fas (CD95) and Fas ligand profiles in chronic kidney failure.

Apoptosis, or programmed cell death, is an active form of cell death that is initiated by a number of stimuli and is intricately regulated. Apoptosis in both excessive and reduced amounts has pathophysiologic implications. Accelerated programmed cell death has been observed in leukocytes among patients with chronic renal failure (CRF). This has been ascribed in part to the retention of uremic toxins. The Fas/Fas ligand (FasL) system is a key regulatory apoptotic pathway. Membrane-bound Fas is a cell-surface receptor that transduces apoptosis after interaction with membrane-bound or soluble FasL (sFasL). By contrast, soluble Fas (sFas) binds sFasL and inhibits its activity. In an attempt to examine the balance between these soluble factors in uremia, we measured soluble sFas and sFasL levels in the serum of healthy control subjects and patients with various degrees of CRF and examined the distribution of the various molecular mass fractions of these proteins in uremic serum. In brief, serum was obtained from 15 healthy volunteers, 17 patients with CRF, 11 patients undergoing maintenance hemodialysis (HD), and 7 patients undergoing peritoneal dialysis (PD). Serum sFas and sFasL were measured by enzyme-linked immunosorbent assay, and their molecular distribution was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot. Compared with results in healthy control subjects, sFas levels were significantly higher in patients with CRF and in patients undergoing dialysis. There was a significant inverse correlation between sFas levels and creatinine clearance. Serum sFasL levels were not different among the four groups. However, the sFas-to-sFasL ratio was significantly lower in healthy control subjects as compared with patients with CRF and patients undergoing dialysis. Immunoblots and densitometric analyses of sFas and sFasL depicted a known 48-kd sFas, a known 27-kd sFasL, and a 60-kd sFas-sFasL protein aggregate signal. In conclusion, serum sFas levels are increased in patients with various degrees of CRF and may bind circulating sFasL, thereby minimizing mediation of cellular apoptosis.

Autosomal dominant polycystic kidney disease decreases anion exchanger activity.

Liver cysts, the most common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD), derive from the intrahepatic biliary epithelium (IBE) and are found in 60-75% of ADPKD patients on dialysis. Secretin-induced secretion by the normal IBE is rich in HCO3-, whereas intact ADPKD liver cysts secrete primarily Cl- in response to secretin. To evaluate the mechanisms of decreased HCO3- secretion by ADPKD liver cysts, we utilized SV40 large T antigen-immortalized normal IBE and ADPKD liver cyst-derived epithelia (LCDE) cell lines that we created. These cell lines express biliary but not hepatocyte markers. Anion exchanger (AE) function was assessed by the response of intracellular pH (pHi) to acute Cl- removal. 2',7'-Bis(carboxyethyl)-5-(6)-carboxyfluorescein-loaded monolayers were continuously perfused with physiological HCO3- buffer containing Cl- or gluconate. In IBE cell line H75 (n = 6), acute Cl- removal alkalinized pHi at a rate of 0.04 +/- 0.01 min-1. AE function was significantly decreased in LCDE cell line CL3 (n = 6) to a rate of 0.01 +/- 0.01 min-1 after Cl- removal. Northern blot analysis demonstrated equivalent levels of AE2 mRNA in both cell lines. AE1 mRNA was undetectable. Immunoblot analysis demonstrated the AE2 polypeptide in both cell lines, but the level of mature glycosylated AE2 polypeptide was reduced in LCDE cells. Immunofluorescence microscopy demonstrated decreased membrane-localized AE2 in LCDE cells. These findings suggest that decreased plasmalemmal AE2 may account for decreased AE function in LCDE cells and suggest a possible explanation for decreased secretion of HCO3- by ADPKD liver cysts.

Matrix metalloproteinase activity in human intrahepatic biliary epithelial cell lines from patients with autosomal dominant polycystic kidney disease.

Hepatic cysts derived from intrahepatic bile ducts are the most common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD). Cyst enlargement involves cell proliferation, fluid secretion into cysts, and alterations in extracellular matrix. To study hepatic cyst formation, continuous cell lines from human normal intrahepatic biliary epithelium (IBE) and ADPKD liver cyst-derived epithelium (LCDE) were developed. Because matrix degradation and remodeling are important for cyst formation and growth, we investigated matrix modifying enzymes expressed in IBE and LCDE cell lines. Gelatin substrate zymography showed that two matrix degrading activities with characteristics of matrix metalloproteinases are secreted from these cell lines. Western immunoblotting suggests that these activities correspond to the 72 kDa (Gelatinase A) and 92 kDa (Gelatinase B) type IV collagenases. Although the level of Gelatinase A activity is comparable in both IBE and LCDE cell lines, Gelatinase B activity is substantially increased in LCDE lines.

Continuous epithelial cell lines from ADPKD liver cysts exhibit characteristics of intrahepatic biliary epithelium.

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into epithelial cells removed from the lumen of liver cysts from four female patients with autosomal dominant polycystic kidney disease (ADPKD). Liver cyst-derived epithelial (LCDE) cell lines are grown in a hormonally supplemented medium in the presence of lethally irradiated NIH/3T3 fibroblast coculture. LCDE cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratin 7 and 19 and gamma-glutamyl transpeptidase while being negative for hepatocyte markers. SV40 large T antigen is localized to the cell nucleus. LCDE cells have been grown continuously for periods exceeding 12 mo and 25 passages (170 population doublings). LCDE cells exhibit intracellular pH regulatory pathways that, with one exception, are similar to those found in normal intrahepatic biliary epithelium. These LCDE cell lines exhibit impaired alkalinization in response to Cl- substitution. This finding is suggestive of decreased function or abundance of a Cl-/HCO3- anion exchanger and could account for the failure of ADPKD hepatic cysts to secrete HCO3- in response to secretin.

Correction of the cystic fibrosis defect by gene complementation in human intrahepatic biliary epithelial cell lines.

BACKGROUND/AIMS: Hepatobiliary disease is the second most common cause of mortality in patients with cystic fibrosis (CF). In the liver, only the intrahepatic biliary epithelial (IBE) cells express cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine whether human CF-derived IBE cells can be infected with adenovirus and the CF phenotype complemented. METHODS: IBE cells were isolated from 2 patients with CF and immortalized using retrovirus transduction of SV40 large T antigen. Immortalized cells were infected with the adenovirus vector Ad2/CFTR2 and assayed 2-31 days postinfection for cyclic adenosine monophosphate (cAMP)-induced halide efflux. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe 6-methoxy-N-(3-sulfopropyl)-quinolinium. RESULTS: CF-derived IBE cell lines express biliary specific markers and express no cAMP-inducible halide efflux. Following infection with the adenovirus vector Ad2/CFTR2, a cAMP-induced halide efflux was observed for 31 days, although the number of responsive cells decreased with time. CONCLUSIONS: Human CF-IBE cells can be infected by adenovirus and the defective CFTR complemented. The loss of responsive cells with time could be due to loss of construct and/or a reduced growth of cells that are overexpressing CFTR. These CF-IBE cell lines offer an opportunity to determine the mechanisms responsible for hepatobiliary disease in the patients with CF.

Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines.

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into human intrahepatic biliary epithelial (IBE) cells from three different normal individuals. These IBE cell lines grow in a hormone-supplemented medium in the presence of NIH/3T3 fibroblast coculture. These cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratins 7 and 19 and gamma-glutamyl transpeptidase while being negative for the hepatocyte markers albumin and asialoglycoprotein receptor. To evaluate ion transport pathways in IBE cell lines, we utilized intracellular pH (pHi) measurements obtained using the intracellular fluorescent indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In the absence of HCO3(-)-CO2, an amiloride-sensitive Na(+)-H+ exchanger participated in the regulation of basal pHi. In the presence of HCO3(-)-CO2, a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive, Na-, Cl-, and HCO3(-)-dependent acid extrusion mechanism accounted for approximately 60% of pHi recovery from acidic pHi; this mechanism is most consistent with the presence of a Na-dependent Cl-HCO3- exchanger (Na+HCO3(-)-Cl-H+). Under basal conditions, Cl- depletion revealed a DIDS-sensitive alkalinization consistent with a Na-independent Cl(-)-HCO3- exchanger. These model systems will allow the opportunity to study the normal mechanisms of IBE function and to study the pathobiology of IBE processes in disease states.

Vocal quality of decannulated children following laryngeal reconstruction.

Laryngotracheal reconstruction with expansion surgery is a reliable method for treatment of laryngotracheal stenosis in children. It allows for decannulation of the patient and restoration of vocal communication. Previous information regarding vocal quality in these patients has been scant. Sixteen children who underwent successful laryngotracheal reconstruction were evaluated using a standardized voice assessment during each of the following: phoneme prolongation, counting, storytelling, and spontaneous conversation. Interjudge ratings were obtained to establish reliability. The location and severity of the laryngotracheal stenosis and the method of treatment were reviewed. Twelve children have a functional voice compared to six preoperatively. Overall, vocal parameters suggested a particular pattern consisting of a lower than optimal pitch and a restricted pitch range. Laryngotracheal reconstruction does make oral communication possible for children who would otherwise have persistent tracheotomy. However, vocal quality appears to be disturbed in most of the patients.

Butyrate induces an ectoMg2(+)-ATPase activity in Li-7A human hepatoma cells.

The human hepatoma cell line (Li-7A) possesses a high concentration of epidermal growth factor (EGF) receptors and exhibits ectoATPase activity in the presence of either MgATP or CaATP (Knowles: J. Cell. Physiol., 134:109-116, 1988). Growth for 96 hours in the presence of both EGF and cholera toxin or another cyclic AMP elevating agent induced an ectoATPase activity which was more active with CaATP and resistant to inhibition by the sulfydryl reagent, p-chloromercuriphenylsulfonate (pCMPS) (Knowles: Arch. Biochem. Biophys., 263: 264-271, 1988). In contrast, treatment of cells with butyrate, a short chain organic acid which can be derived from the analogue, dibutyryl cyclic AMP, resulted in a 4-7-fold increase of an ectoATPase which was more active with MgATP and highly sensitive to pCMPS inhibition. Maximal induction by butyrate required 48 hours and was dependent on butyrate concentration, but was independent of EGF and cyclic AMP elevating agents. Of six organic acids tested, butyrate was most effective in the induction of the ectoMg2(+)-ATPase. The increase in the ectoMg2(+)-ATPase activity could be prevented with actinomycin D and cycloheximide, indicating that both transcription and translation were necessary for induction. In addition to the induction of the ectoMg2(+)-ATPase, butyrate induced alkaline phosphatase activity, but had no effect on a third ectoenzyme 5'-nucleotidase. These data further support our proposal that two distinct ectoATPases exist in the plasma membrane of Li-7A hepatoma cells.

Albumin inhibition of the antileukemic activity of hydroxylated phenothiazines.

Trifluoperazine (TFP) shows cytotoxic activity against human acute lymphatic leukemia (ALL) in vitro. This activity is inhibited by increasing serum concentration and by albumin. Despite its in vitro activity, the drug is inactive in vivo. To determine if increased phenothiazine hydrophilicity could protect against albumin inhibition of antileukemic activity, we compared ALL cytotoxic median effective dose concentrations of a series of hydroxylated phenothiazines in 5% fetal bovine serum (FBS) and in 5% FBS supplemented with albumin. Albumin inhibits the activity of all drugs. A representative derivative 7,8-dihydroxychlorpromazine, although active in vitro, is inactive against L1210 and P388 murine leukemias in vivo.

Verapamil potentiation of VP-16-213 in acute lymphatic leukemia and reversal of pleiotropic drug resistance.

Verapamil, the calcium-influx-blocking agent, has previously been shown to have favorable interactions with antineoplastic drugs. Our study of human T cell acute lymphatic leukemia (ALL) GM3639 indicates that verapamil enhances the in vitro cytotoxicity of VP-16-213 against drug-sensitive ALL by reducing the concentration of VP-16-213, resulting in 50% cell viability from 104.5 +/- 26.6 nM to 46.0 +/- 2.7 nM (P less than 0.05). The addition of verapamil to VP-16-213 treatment of BDF/1 mice bearing L1210 leukemia increases their mean survival from 21.2 +/- 3.6 to 50.4 +/- 4.3 days (P less than 0.01) and the survival of CD2F/l mice bearing P388 leukemia from 27.8 +/- 3.7 to 49.1 +/- 5.0 days (P less than 0.01). The 30-day survival is significantly increased in L1210 and P388 leukemia mice, and 60-day survival is significantly increased in P388 leukemic mice by verapamil. We developed a vincristine (VCR)-resistant subline of GM3639 T cell ALL, L23, by continuous exposure of drug-sensitive cells to VCR. This subline demonstrates pleiotropic cross resistance to VP-16-213 and daunorubicin. The addition of verapamil to VCR, to VP-16-213, and to daunorubicin completely restores responsiveness to these drugs, as indicated by the normalization of the VCR and VP-16-213 concentrations required for cytotoxicity and the concentration of daunorubicin required for inhibition of thymidine incorporation.

Calcium modifies the accumulation and retention of daunorubicin by Ehrlich ascites carcinoma.

Verapamil restores daunorubicin sensitivity to daunorubicin resistant Ehrlich ascites carcinoma but is without effect when used with daunorubicin in daunorubicin sensitive parental Ehrlich ascites tumor. Energy dependent daunorubicin efflux is more active in drug resistant than in drug sensitive cells. However, daunorubicin retention decreases equivalently in drug resistant and sensitive cells with increasing calcium levels in the presence of both intact and interrupted outward transport. Therefore, (1) daunorubicin accumulation and retention in Ehrlich ascites carcinoma cells is influenced by at least two independent mechanisms and (2) it is likely that verapamil modifies daunorubicin activity in drug resistant tumor variants by mechanisms beyond calcium inhibition.

Verapamil restoration of daunorubicin responsiveness in daunorubicin-resistant Ehrlich ascites carcinoma.

We have studied the influence of verapamil hydrochloride on the in vitro and in vivo effects of daunorubicin in Ehrlich ascites carcinoma. Daunorubicin-sensitive tumor was rendered resistant to daunorubicin by the continuous treatment of sequential generations of tumor-bearing BALB/c mice. The ability of daunorubicin to inhibit [(3)H]uridine and [(3)H]thymidine incorporation and the effect of daunorubicin on the mean survival time of host animals bearing daunorubicin-sensitive and daunorubicin-resistant Ehrlich ascites carcinoma were compared. The addition of verapamil to daunorubicin in vitro reduced the concentration of daunorubicin required to inhibit 50% of DNA and RNA synthesis in the daunorubicin-resistant tumor to that required in the daunorubicin-sensitive tumor, from 6 and 4.4 mug/ml to 1.5 and 1.3 mug/ml, respectively. Verapamil also restored drug sensitivity to daunorubicin-resistant Ehrlich ascites carcinoma in vivo. The 21.7+/-0.7 d mean survival time (MST) of BALB/c mice bearing daunorubicin-resistant tumor treated with daunorubicin alone rose to 44.0+/-0.7 d when the same tumor was treated with verapamil and daunorubicin, P < 0.001. This in vivo effect is specific for daunorubicin-resistant Ehrlich ascites carcinoma, since there is no alteration in MST of BALB/c mice bearing daunorubicin-sensitive or daunorubicin-resistant tumor when they are treated with verapamil alone or when BALB/c mice bearing daunorubicin-sensitive tumor are treated with daunorubicin and verapamil.