Inhibition of Human Mammary Cell Line Proliferation by Membrane Lectin-Mediated Uptake of Ha-ras Antisense Oligodeoxynucleotide

The Ha-ras oncogene promotes cell proliferation. Antisense oligonucleotides complementary to the ras gene sequence encompassing a mutated codon 12 selectively induce a cell proliferation inhibition. However, the concentration required to reach an effective inhibition is high due to the low efficiency of the oligonucleotide crossing through cell membranes, leading to a low concentration in the cytosol and/or the nucleoplasm. In the present paper, we show that anti-ras oligonucleotides linked to a glycosylated carrier, serum albumin bearing mannose 6-phosphate residues, are more efficient than free oligonucleotides or oligonucleotides bound to an unglycosylated carrier at inhibiting proliferation of a human tumor mammary cell line expressing the mutated Ha-ras. Using fluorescein-labeled neoglycoproteins and fluorescein-labeled oligonucleotides bound to neoglycoproteins, flow cytometry and confocal microscopy revealed that (i) these tumor cells express a membrane lectin specific for mannose 6-phosphate-bearing proteins, (ii) the membrane lectin actively mediates the uptake of macromolecules substituted with mannose 6-phosphate, and (iii) the fluorescein-labeled oligonucleotides bound to the neoglycoprotein accumulate in intracellular vesicles. Furthermore, with antisense oligonucleotides carried by the neoglycoproteins, the concentration required to inhibit cell proliferation is lower than that of the carrier-free antisense oligonucleotides.     

A Pilot Study for Targeted Surveillance of Bovine Spongiform Encephalopathy in Nigeria

Bovine spongiform encephalopathy (BSE), popularly known as ‘mad cow disease’, led to an epidemic in Europe that peaked in the mid‐1990s. Its impact on developing countries, such as Nigeria, has not been fully established as information on livestock and surveillance has eluded those in charge of this task. The BSE risk to Nigeria’s cattle population currently remains undetermined, which has resulted in international trade restrictions on commodities from the cattle population. This is mainly because of a lack of updated BSE risk assessments and disease surveillance data. To evaluate the feasibility of BSE surveillance in Nigeria, we carried out a pilot study targeting cattle that were presented for emergency or casualty slaughter. In total, 1551 cattle of local breeds, aged 24 months and above were clinically examined. Ataxia, recumbency and other neurological signs were topmost on our list of criteria. A total of 96 cattle, which correspond to 6.2%, presented clinical signs that supported a suspect of BSE. The caudal brainstem tissues of these animals were collected post‐mortem and analysed for the disease‐specific form of the prion protein using a rapid test approved by the International Animal Health Organization (OIE). None of the samples were positive for BSE. Although our findings do not exclude the presence of BSE in Nigeria, they do demonstrate that targeted sampling of clinically suspected cases of BSE is feasible in developing countries. In addition, these findings point to the possibility of implementing clinical monitoring schemes for BSE and potentially other diseases with grave economic and public health consequences.     

Renal sarcoidosis with normal serum vitamin D and refractory hypercalcemia

Introduction

Sarcoidosis is a multi-system disorder characterized by non-caseating epithelioid granulomas in multiple organs. Renal involvement may usually occur as granulomatous interstitial nephritis, but renal failure is uncommon. We report a case of renal-limited sarcoidosis diagnosed by renal biopsy, associated with abnormal calcium metabolism.

Case presentation

A 30-year-old Caucasian male presented with unexplained renal function impairment and hypercalcemia. The patient did not have any history of kidney disease, cough, skin rash, dysuria, hematuria or any other symptoms. Physical examination was unremarkable. Serum creatinine was 2.2 mg/dl and serum calcium was 11.5 mg/dl. Serum intact parathyroid hormone level (12 pg/mL) was decreased. Serum angiotensin-converting enzyme (ACE), 1,25-dihydroxyvitamin D (1,alpha-25 vit D) and pre-proparathyroid hormone (PTHrP) levels and urinary calcium excretion were all in normal range. The renal biopsy showed severe interstitial nephritis with non-caseating granuloma. The patient was treated with prednisone with starting dose of 1 mg/kg. After 2 months of prednisone therapy, serum creatinine decreased. However, because of continued of hypercalcemia unresponsive to low calcium diet and prednisone, chloroquine was prescribed. Six months after the onset, the patient's serum creatinine is stable at 1.30 mg/dl, serum calcium is 10.8 mg/dl, and serum ACE and 1,alpha-25 vit D levels are in normal range. He does not have any signs of extra-renal relapse.

Conclusion

The mechanisms of abnormal calcium metabolism in this patient with renal-limited sarcoidosis are unclear.     

The effect of interleukin-10 on acute pancreatitis induced by cerulein in a rat experimental model.

To investigate whether interleukin-10, a potent anti-inflammatory cytokine, could have a therapeutic effect on rats on that were made pancreatitis by cerulein. Thirty Wistar Albino rats were randomized into sham, pancreatitis, and therapy groups (n = 10 in each). Nothing was applied to the sham group; pancreatitis by inject-ing cerulein (50 micro/g/kg/h) was induced in the pancreatitis and therapy groups. Interleukin-10 (10.000 U) was injected at 1 and 4 h after pancreatitis inductions in the therapy group. The rats were sacrificed at postoperative hour 24. The following parameters were investigated: the leukocyte count, blood glucose, amylase, lipase and tumor necrosis factor-alpha levels in the blood samples; histopathological search, and wet/dry weight ratios of the pancreas tissues. The ratio of wet/dry pancreatic tissue weight, serum tumor necrosis factor-alpha, amylase and lipase lev-els, and histologic damage scores in the pancreatitis and therapy groups were significantly higher when they were compared with the sham group(p < .01). However, all of these values were significantly lower in the therapy groups than in the pancreatitis group (p < .01). Interleukin-10 decreases pancreatic tissue injury induced by cerulein-induced pancreatitis in rats. Nevertheless, more experimental studies are needed to compare endogenous interleukin-10 with exogenous interleukin-10 effects before clinical usage of this drug.     

The importance of aprotinin and pentoxifylline in preventing leukocyte sequestration and lung injury caused by protamine at the end of cardiopulmonary bypass surgery

BACKGROUND: Protamine has adverse effects on pulmonary gas exchange during the postoperative period. The objective of this study was to investigate the importance of aprotinin and pentoxifylline in preventing the leukocyte sequestration and lung injury caused by protamine administered after the termination of cardiopulmonary bypass (CPB). METHODS: Participants (n = 39) were allocated into three groups at the termination of CPB: Group 1, (control group, n = 16); Group 2 (aprotinin group, n = 12), who received protamine + aprotinin (15,000 IU/kg); and Group 3 (Pentoxifylline group, n = 11), who received protamine + pentoxifylline (10 mg/kg). Leukocyte counts in pulmonary and radial arteries were determined after the termination of CPB and before any drug was given (t1), and 5 minutes (t2), 2 hours (t3), 6 hours (t4) and 12 hours (t5) after the administration of protamine. Alveolar-arterial O2 gradient (A-aO2) and dynamic pulmonary compliance were measured at t1, t2 and t3. RESULTS: In the control group, an increase in pulmonary leukocyte sequestration was observed 5 minutes and 2 hours after protamine administration, after which this difference disappeared. No significant degree of pulmonary sequestration was detected in any measurements after protamine was administered in the aprotinin and pentoxifylline (PTX) groups. Dynamic lung compliance was 50.1, 45.2 and 47.2 ml/cm H2O in the control group, 49.2, 61.1 and 56.3 ml/cm H2O in the aprotinin group, and 49.5, 54.5 and 50.4 ml/cm H2O in the PTX group. The A-aO2 gradient was 212.2, 263.3 and 254.3 mm Hg in the control group, 209.4, 257.1 and 217.3 mm Hg in the aprotinin group, and 211.3, 260.8 and 219.2 mm Hg in the PTX group. CONCLUSION: Aprotinin and PTX treatments have favourable effects on lung function by reducing protamine-induced leukocyte sequestration into lungs at the end of CPB.     

Design considerations and effects of LNA in PCR primers

The effects of comprehensive LNA substitution in PCR primers for amplification of human genomic DNA targets are presented in this report. Previous research with LNA in other applications has shown interesting properties for molecular hybridization including enhanced specificity in allele-specific PCR. Here we systematically modified PCR primers and conditions for the human genomic DNA targets APOB and PAH, along with a beta-globin amplification control, to study whether the number and position of LNA residues improves or diminishes amplification sensitivity and specificity. It was observed that the design rules for LNA substitution in PCR primers are complex and depend upon number, position and sequence context. Technical advantages were seen when compared to DNA controls for the best LNA primer designs, which were typically one to a few centrally located LNA residues. LNA advantages include increased maximum annealing temperature (Tmax) and increased signal with limiting primer or Taq DNA polymerase. Several well-characterized designs exhibited different efficiencies with different brands of hot-start enzymes. Many shorter LNA primers were found to be functional compared to same-length non-functional native DNA controls. These results show that LNA-substituted PCR primers have potential for use in difficult PCR techniques, such as multiplex amplification at higher Tmax, once firm LNA primer design rules are established.     

Evaluation of the performance of LNA and MGB probes in 5'-nuclease PCR assays

The aim of this study was to evaluate the use of Locked Nucleic Acids (LNA) probes in 5'-nuclease PCR, by comparison with Minor Groove Binder (MGB) probes routinely practiced in laboratories on ABI Prism 7700. The comparison was made using a collection of Staphylococcus aureus strains that have already been characterized by MGB 5'-nuclease PCR assays in a previous study [Mol Cell Probes, submitted for publication]. The sensitivity and specificity of 5'-nuclease PCR assays targeting the Staphylococcal enterotoxin genes sea to see were compared and showed that the LNA and MGB methods were equivalent. In conclusion, the LNA 5'-nuclease PCR assays developed in this work provide a specific and sensitive alternative to the well-established MGB 5'-nuclease PCR assays used for the rapid detection of bacterial pathogens genes on ABI Prism 7700.     

The importance of pulmonary artery circulation during cardiopulmonary bypass

This study sought to determine changes in transpulmonary difference in blood cells and alveolar-arterial oxygen (A-aO2) gradient when pulmonary artery circulation was obstructed in patients undergoing coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB). Eighteen patients were divided into group A (control group; X-clamp placed on aorta, n = 9) and group B (pulmonary ischaemia group; X-clamp placed on aorta and pulmonary artery, n = 9). Haematological parameters were compared before CPB and up to 90 min after declamping. A-aO2 gradient differences were compared before and 2 h and 6 h after declamping. A transpulmonary increase in leucocyte levels normalized after 60 min in group A but remained higher in group B. A transpulmonary increase in neutrophils normalized after 60 min in group A and 90 min in group B. Increased lymphocyte levels normalized after 30 min in group A and 90 min in group B. A-aO2 gradient was determined as: group A (294.8 +/- 74.3) and group B (321.2 +/- 73.3) before X-clamping; group A (132.7 +/- 22.7) and group B (236.6 +/- 41.5) 2 h after declamping; and group A (72.2 +/- 22.7) and group B (189.4 +/- 88.9) 6 h after declamping. When pulmonary artery circulation was obstructed during the X-clamping period, leucocyte, neutrophil and lymphocyte sequestration within both lungs increased, and an increased A-aO2 gradient was observed because of tissue damage. To prevent post-operative complications, precautions to maintain normal pulmonary artery circulation are recommended.     

Genome-wide scans for diabetic nephropathy and albuminuria in multiethnic populations: the family investigation of nephropathy and diabetes (FIND)

The Family Investigation of Nephropathy and Diabetes (FIND) was initiated to map genes underlying susceptibility to diabetic nephropathy. A total of 11 centers participated under a single collection protocol to recruit large numbers of diabetic sibling pairs concordant and discordant for diabetic nephropathy. We report the findings from the first-phase genetic analyses in 1,227 participants from 378 pedigrees of European-American, African-American, Mexican-American, and American Indian descent recruited from eight centers. Model-free linkage analyses, using a dichotomous definition for diabetic nephropathy in 397 sibling pairs, as well as the quantitative trait urinary albumin-to-creatinine ratio (ACR), were performed using the Haseman-Elston linkage test on 404 microsatellite markers. The strongest evidence of linkage to the diabetic nephropathy trait was on chromosomes 7q21.3, 10p15.3, 14q23.1, and 18q22.3. In ACR (883 diabetic sibling pairs), the strongest linkage signals were on chromosomes 2q14.1, 7q21.1, and 15q26.3. These results confirm regions of linkage to diabetic nephropathy on chromosomes 7q, 10p, and 18q from prior reports, making it important that genes underlying these peaks be evaluated for their contribution to nephropathy susceptibility. Large family collections consisting of multiple members with diabetes and advanced nephropathy are likely to accelerate the identification of genes causing diabetic nephropathy, a life-threatening complication of diabetes.