A Single Point Mutation in the Gene Encoding Gb3/CD77 Synthase Causes a Rare Inherited Polyagglutination Syndrome

Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR_int, and NOR2, in which Gal(α1–4), GalNAc(β1–3)Gal(α1–4), and Gal(α1–4)GalNAc(β1–3)Gal(α1–4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1–4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1–4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1–4)Gal and Gal(α1–4)GalNAc moieties.     

Effect of Lumican on the Migration of Human Mesenchymal Stem Cells and Endothelial Progenitor Cells: Involvement of Matrix Metalloproteinase-14

Background

Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC).

Methodology/Principal Findings

Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression.

Conclusion/Significance

Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.     

ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFNγ, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response.     

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.     

Change in appropriate referrals to nephrologists after the introduction of automatic reporting of the estimated glomerular filtration rate

Background:

Use of the serum creatinine concentration, the most widely used marker of kidney function, has been associated with under-reporting of chronic kidney disease and late referral to nephrologists, especially among women and elderly people. To improve appropriateness of referrals, automatic reporting of the estimated glomerular filtration rate (eGFR) by laboratories was introduced in the province of Ontario, Canada, in March 2006. We hypothesized that such reporting, along with an ad hoc educational component for primary care physicians, would increase the number of appropriate referrals.

Methods:

We conducted a population-based before–after study with interrupted time-series analysis at a tertiary care centre. All referrals to nephrologists received at the centre during the year before and the year after automatic reporting of the eGFR was introduced were eligible for inclusion. We used regression analysis with autoregressive errors to evaluate whether such reporting by laboratories, along with ad hoc educational activities for primary care physicians, had an impact on the number and appropriateness of referrals to nephrologists.

Results:

A total of 2672 patients were included in the study. In the year after automatic reporting began, the number of referrals from primary care physicians increased by 80.6% (95% confidence interval [CI] 74.8% to 86.9%). The number of appropriate referrals increased by 43.2% (95% CI 38.0% to 48.2%). There was no significant change in the proportion of appropriate referrals between the two periods (−2.8%, 95% CI −26.4% to 43.4%). The proportion of elderly and female patients who were referred increased after reporting was introduced.

Interpretation:

The total number of referrals increased after automatic reporting of the eGFR began, especially among women and elderly people. The number of appropriate referrals also increased, but the proportion of appropriate referrals did not change significantly. Future research should be directed to understanding the reasons for inappropriate referral and to develop novel interventions for improving the referral process.Until recently, the serum creatinine concentration was used universally as an index of the glomerular filtration rate (GFR) to identify and monitor chronic kidney disease.¹ The serum creatinine concentration depends on several factors, the most important being muscle mass.¹ Women as compared with men, and elderly people as compared with young adults, tend to have lower muscle mass for the same degree of kidney function and thus have lower serum creatinine concentrations.^2,3 Consequently, the use of the serum creatinine concentration is associated with underrecognition of chronic kidney disease, delayed workup for chronic kidney disease and late referral to nephrologists, particularly among women and elderly people. Late referral has been associated with increased mortality among patients receiving dialysis.^3–11In 1999, the Modification of Diet in Renal Disease formula was introduced to calculate the estimated GFR (eGFR).^12,13 This formula uses the patient’s serum creatinine concentration, age, sex and race (whether the patient is black or not). All of these variables are easily available to laboratories except race. Laboratories report the eGFR for non-black people, with advice to practitioners to multiply the result by 1.21 if their patient is black. Given that reporting of the eGFR markedly improves detection of chronic kidney disease,^14,15 several national organizations recommended that laboratories automatically calculate and report the eGFR when the serum creatinine concentration is requested.^16–19 These organizations also provided guidelines on appropriate referral to nephrology based on the value.Although several studies have reported increases in referrals to nephrologists after automatic reporting of the eGFR was introduced,^20–26 there is limited evidence on the impact that such reporting has had on the appropriateness of referrals. An increase in the number of inappropriate referrals would affect health care delivery, diverting scarce resources to the evaluation of relatively mild kidney disease. It also would likely increase wait times for all nephrology referrals and have a financial impact on the system because specialist care is more costly than primary care.We conducted a study to evaluate whether the introduction of automatic reporting of the eGFR by laboratories, along with ad hoc educational activities for primary care physicians, had an impact on the number and appropriateness of referrals to nephrologists.     

A non-functioning pancreatic neuroendocrine tumour: a case report

We present the diagnostic and therapeutic difficulties encountered in a patient with a clinically advanced pancreatic neuroendocrine tumour. The report concerns a 60-year-old female patient with the diagnosis of non-functioning pancreatic neuroendocrine tumour (NET G1) with liver, peripancreatic lymph node and mediastinal metastases. Due to the presence of advanced disease (inoperable pancreatic tumour, presence of multiple metastases) the patient was considered ineligible for surgical treatment on two occasions. Tissue samples for histopathology were collected during an exploratory laparotomy, which made it possible to establish the diagnosis. As somatostatin receptor scintigraphy was positive, the patient was started on somatostatin analogues and radionuclide therapy was initiated, resulting in satisfactory response in the form of complete remission of liver metastases and the decreased size of the primary tumour in the pancreas. The use of somatostatin analogues in the case of an inoperable neuroendocrine tumour which was assessed as clinically advanced, yet possessing a low proliferative potential, is a promising therapeutic option.     

Effects of the novel mitochondrial protein mimitin in insulin-secreting cells

Mimitin, a novel mitochondrial protein, has been shown to act as a molecular chaperone for the mitochondrial complex I and to regulate ATP synthesis. During Type 1 diabetes development, pro-inflammatory cytokines induce mitochondrial damage in pancreatic β-cells, inhibit ATP synthesis and reduce glucose-induced insulin secretion. Mimitin was expressed in rat pancreatic islets including β-cells and decreased by cytokines. In the ob/ob mouse, a model of insulin resistance and obesity, mimitin expression was down-regulated in liver and brain, up-regulated in heart and kidney, but not affected in islets. To further analyse the impact of mimitin on β-cell function, two β-cell lines, one with a low (INS1E) and another with a higher (MIN6) mimitin expression were studied. Mimitin overexpression protected INS1E cells against cytokine-induced caspase 3 activation, mitochondrial membrane potential reduction and ATP production inhibition, independently from the NF-κB (nuclear factor κB)-iNOS (inducible NO synthase) pathway. Mimitin overexpression increased basal and glucose-induced insulin secretion and prevented cytokine-mediated suppression of insulin secretion. Mimitin knockdown in MIN6 cells had opposite effects to those observed after overexpression. Thus mimitin has the capacity to modulate pancreatic islet function and to reduce cytokine toxicity.     

Expansion of CD14(+)CD16(+) monocytes producing TNF-α in complication-free diabetes type 1 juvenile onset patients

We concentrated on the complication-free phase of juvenile onset type 1 diabetes mellitus (T1DM) searching for associations between concentration of inflammatory factors TNF-α, CRP and VEGF and two monocyte subsets the CD14(++)CD16(-) and CD14(+)CD16(+). We analysed a randomly selected group of 150 patients without complications (disease duration 2.74±2.51years) at the start of the project and 5years later. They were compared with 24 patients with retinopathy (6.53±3.39years of disease) and 30 healthy volunteers. Our results indicate that in the complication-free period the concentration of TNF-α significantly increased and continued to increase after retinopathy was established. After 5years the percentage and absolute number of CD14(+)CD16(+) monocytes doubled in complication-free patients. Our study indicates that the size of CD14(+)CD16(+) monocyte subset may be used alternatively to CRP values as an indicator of inflammation grade. Our results imply the necessity of trials using anti-TNF-α therapy in the complication-free phase of the disease.     

Heterophil/lymphocyte ratios predict the magnitude of humoral immune response to a novel antigen in great tits (Parus major)

Animals display remarkable individual variation in their capacity to mount immune responses against novel antigens. According to the life-history theory, this variation is caused by the costs of immune responses to the hosts. We studied one of such potential costs, depletion of somatic resources in wintering wild-caught captive passerines, the great tits (Parus major) by immune challenging the birds with a novel antigen, killed Brucella abortus (BA) suspension. We found that despite mild temperature conditions in captivity and ad libitum availability of food, immune challenge depleted somatic resources (as indicated by a body mass loss) and elevated relative proportion of heterophils to lymphocytes (H/L ratio) in the peripheral blood of birds. However, body mass loss did not covary with an increase in H/L ratios between two sampling events, which indicates that these two markers of health state describe different aspects of individual physiological condition. Antibody titres were not associated with the extent of body mass loss during the development of immune response, which shows that the somatic cost of immune response was not proportional to the amount of antibody produced. Birds with high pre-immunisation H/L ratios mounted weaker antibody response, which is indicative of stress-induced suppression of humoral immune response and is consistent with the concept of an antagonistic cross-regulation between different components of the immune system. The latter finding suggests a novel diagnostic value of H/L ratios, which reinforces the utility of this simple haematological index for prediction of the outcomes of complicated immune processes.     

Membrane permeability and micro- and macroelement accumulation in spring wheat cultivars during the short-term effect of salinity- and PEG-induced water stress

The comparative responses of ten spring wheat cultivars to water stress were investigated. Wheat plants were cultured under hydroponics conditions (Hoagland nutrient) to the stage of three-leaf seedlings. Then, the water medium was supplemented with PEG (drought) or NaCl (salinity) to obtain a water status equal to −1.5 MPa. After a 2-day treatment, the changes in the following parameters were determined: fresh and dry weight, macro- and microelement accumulation, membrane injury (electrolyte leakage, lipid peroxidation) and fatty acid content of the phospholipid fraction of plasmalemma (in comparison to plants not stressed, taken as a control). Generally, the plants were more significantly influenced by water stress stimulated by PEG than by NaCl treatment, as compared to the plants cultivated in the control media. The results of the decrease in water content in leaves and electrolyte leakage from cells corresponded well with the intensity of lipid peroxidation (determined by malondialdehyde—MDA-content) and were chosen for the selection of investigated genotypes for tolerance to both stresses. The more tolerant genotypes exhibited the opposite changes in phospholipid fatty acid unsaturation for two applied stresses i.e. NaCl treatment caused a decrease in unsaturation whereas in PEG-treated plants an increase in unsaturation was observed. These changes were reversed for less tolerant plants, i.e. NaCl treatment influenced an increase in fatty acid unsaturation whereas in PEG-treated plants a decrease in unsaturation was measured. The ratio of U/S (unsaturated to saturated fatty acids) correlated with the total amount of accumulated macroelements. The content of Mg, Ca and S in leaves of plants undergoing both stress factors (NaCl and PEG) dropped whereas the K and P content increased in leaves of wheat seedlings cultured on media containing NaCl only. For microelements, a decrease in the accumulation of these nutrients was detected in all investigated seedlings. However, a greater reduction in the level of these elements occurred in seedlings grown on media with PEG in comparison to those grown on NaCl containing media.