[A terrible diagnosis and hard to swallow!...].
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Actinomycosis, Cervicofacial↗
Biomedical subjects
M Bernier
Publications and source records attributed to M Bernier.
Identification of natural killer cell receptor phenotypes associated with leukemia.
Natural killer (NK) cells play a key role in defense against tumor cells that have the capacity to downregulate human leukocyte antigen (HLA) class I expression. It has been reported that leukemic cells can have downregulated expression of HLA class I molecules. The polymorphic nature of NK cell receptor (NKR) genes generates diverse repertoires in the human population, which display specificity in the innate immune response. In the present study, 11 KIR and two CD94/NKG2 receptors were genotyped by PCR-SSP in 96 leukemic patients and 148 healthy Caucasians. Here, we report a significant increased frequency of the more inhibitory AB killer cell immunoglobulin-like receptor (KIR) phenotype in leukemic patients compared to the controls (31.1% in healthy controls vs 51.0% in leukemic patients, Pc=0.002), which is related to the high prevalence of the inhibitory KIR2DL2 in this population (Pc=0.007). Moreover, two specific KIR phenotypes AB1 and AB9, including all inhibitory KIRs, were significantly associated with leukemic patients. Our study suggests that an important percentage of leukemic patients express a KIR phenotype in favor of escape from NK cell immunity.
Acute Disease↗
New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci.
Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was approximately 25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.
Bacterial Proteins↗
Immunological classification of acute myeloblastic leukemias: relevance to patient outcome.
Immunophenotyping is a major tool to assign acute leukemia blast cells to the myeloid lineage. However, because of the large heterogeneity of myeloid-related lineages, no clinically relevant immunological classification of acute myeloblastic leukemia (AML) has been devised so far. To attempt at formulating such a classification, we analyzed the pattern of expression of selected antigens, on blast cells collected at AML diagnosis. Patients were eligible if they had a first diagnosis of de novo AML and a sufficient number of blast cells for proper immunophenotyping. The relative expression of CD7, CD13, CD14, CD15, CD33, CD34, CD35, CD36, CD65, CD117, and HLA-DR were analyzed by cytometry in a test series of 176 consecutive AML cases. Statistical tools of clusterization allowed to remove antigens with overlapping distribution, leading us to propose an AML classification that was validated in a second AML cohort of 733 patients. We identified five AML subsets (MA to ME) based on the expression of seven antigens within four groups (CD13/CD33/CD117, CD7, CD35/CD36, CD15).-MA and MB-AML have exclusively myeloid features with seldom extramedullary disease and rare expression of lymphoid antigens. No cases of acute promyelocytic leukemia (APL) were observed within MB AML. MC AML have either myeloid or erythroblastic features. MD AML have more frequently high WBC counts than other subsets, which were related to the expression of CD35/CD36 and CD14 and to monoblastic differentiation. ME AML lack CD13, CD33, and CD117 but display signs of terminal myeloid differentiation. Specific independent prognostic factors were related to poor overall survival in each immunological subset: CD34+ (P<3 x 10(-4)) in MA AML, CD7+ in MB AML, non-APL cases (P<0.03) in MC AML, CD34+ (P<0.002) and CD14+ (P<0.03) in MD AML, CD14+ in ME AML (P<0.01). The inclusion of seven key markers in the immunophenotyping of AML allows a stratification into clinically relevant subsets with individual prognostic factors, which should be considered to define high-risk AML populations.
Adolescent↗
Ex vivo expansion of neutrophil precursor cells from fresh and cryopreserved cord blood cells.
BACKGROUND: Neutropenia following cord blood (CB) transplantation may be abrogated by infusion of granulopoietic progenitor cells. The purpose of this study was to determine whether myeloid progenitors can be obtained by ex vivo expansion of cryopreserved cord blood aliquots, and whether these progenitors present the morphologic, biologic and functional properties of myeloid progenitors at various stages of differentiation. METHODS: The cells, plated for 7 days in serum-free medium with SCF, IL-3, G-CSF, Flt3-ligand and thrombopoietin in various combinations were assessed for the expression of CD34, CD38 and CD13. Maturation of cells into the myeloid lineage was evaluated by the expression of CD15, CD11b and CD16 and by the presence of primary (myeloperoxidase) and secondary granules (lactoferrin). The capacity of cells to phagocyte latex beads was evaluated to assess their functionality. RESULTS: We have shown that a). CD34+ cells isolated from thawed samples were able to produce expansions similar to fresh samples. b). The best combination for the expansion of neutrophil precursor cells was S3FG; c). in these conditions, all stages of myeloid progenitors were represented, but few mature cells were observed. d). However, when the cells were plated on a BM stroma to try to reproduce conditions occurring during transplant, they acquired rapidly the characteristics of mature segmented cells. e). The ex vivo generated granulocytes were able to phagocyte latex beads. DISCUSSION: In conclusion, it seems reasonable to systematically aliquot CB samples before cryopreservation. Some aliquots can then be thawed, enriched in CD34+ cells and ex vivo differentiated into myeloid lineage, while the other aliquots are conserved to be infused without manipulation.
Antigens, CD34↗
[Identification of plasmid-encoded cephalosporinase ACC-1 among various enterobacteria (Klebsiella pneumoniae, Proteus mirabilis, Salmonella) isolated from a Tunisian hospital (Sfax 997-2000)].
Because a multiresistant K. pneumoniae outbreak detected in an intensive care unit of a parisian hospital, combined to the production of the plasmid-encoded cephalosporinase ACC-1, a probable importation via a patient was suggested from another country (Tunisia). The investigation was conducted to examine 35 clinical strains of enterobacteria resistant to ceftazidime without synergy towards Augmentin. Other test of synergy with two inhibitors, BRL 42715, Ro 48-5545 was performed by diffusion method and deposit of 10 micrograms of inhibitor on disks containing ceftazidime, cefoxitin and cefotetan. Synergies were obtained suggesting a probable production of ACC-1 type among six isolates of K. pneumoniae (two), Proteus mirabilis (one) and Salmonella (three) issued from different units. The isoelectric focusing on gel revealed at least one band of beta-lactamase activity at 7.8 but also demonstrated the simultaneous production of several probable beta-lactamases including TEM-type, SHV-2 and ACC-1 among S. enterica ser. Livingstone. The PCR of the gene blaacc-1 was positive. The sequencing (1160 pb) of two products showed high identity (99-100%) with the gene blaacc-1 deposited in 1999. Finally the ACC-1 type reported in Tunisia was probably imported in France via a patient. Because a simultaneous synthesis of ESBL and ACC-1 type, its presence may be invisible and need more investigation.
Cephalosporinase↗
Early induction of apoptosis in B-chronic lymphocytic leukaemia cells by hydroxychloroquine: activation of caspase-3 and no protection by survival factors.
We have investigated the effect of hydroxychloroquine (HCQ), an anti-rheumatic drug, on malignant B cells from 20 patients with B-chronic lymphocytic leukaemia (B-CLL). HCQ induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 was 32 +/- 7 microg/ml (range, 10-75 microg/ml) for 24 h of exposure. This cytotoxic effect was owing to apoptosis, as demonstrated by morphological changes, annexin V binding capacity and DNA fragmentation (28 +/- 4% of apoptotic cells as early as 5 h post incubation, increasing to 82 +/- 4% at 18 h post treatment). The apoptosis was associated with caspase-3 activation because the cleavage and activity of caspase-3 were increased by HCQ. The amount of bcl-2 protein was reduced during apoptosis, evidenced using quantitative flow cytometry. As early as 1 h post-HCQ treatment, a reduction of the mitochondrial transmembrane potential was measured by 3,3'-dihexyloxacarbocyanine iodide. Interestingly, the HCQ effect was not affected by exposure to interleukin-4 or co-culture with bone marrow stromal cells. Our observations suggest that HCQ may offer a new therapeutic tool in the treatment of B-CLL patients.
Aged↗
Acute myeloid leukaemia M0: haematological, immunophenotypic and cytogenetic characteristics and their prognostic significance: an analysis in 241 patients.
Haematological, immunophenotypic and cytogenetic characteristics were analysed in 241 patients with acute myeloid leukaemia (AML) M0, including 58 children. Children < 3 years and adults between 60 and 70 years of age were most frequently affected. Immunophenotyping showed a heterogeneous phenotype. Anti-myeloperoxidase was positive in about half of the patients. Cytogenetic data were available from 129 (54%) patients. A normal karyotype was found in only 24%. Most of the abnormalities were unbalanced and the chromosomes 5, 7, 8 and 11 were the most frequently affected. Survival data were available from 152 treated patients (63%). The median overall survival for all patients was 10 months, 20 months for children (n = 36), 10 months for the young adult group (n = 50) and 7 months for the elderly patients (n = 66) (P = 0.09). Karyotype was not a prognostic factor influencing survival. AML M0 shows the immunological characteristics of early progenitor cells, but the expression of the different markers and cytogenetic abnormalities is heterogeneous. The prognosis is poor compared with other de novo AML and similar to that of AML with multilineage dysplasia or AML following myelodysplastic syndromes.
Adolescent↗
Insertion of an N-terminal 6-aminohexanoic acid after the 7 amino acid position of glucagon-like peptide-1 produces a long-acting hypoglycemic agent.
The use of glucagon-like peptide-1 (GLP-1) as a routine treatment for type 2 diabetes mellitus is undermined by its short biological half-life. A cause of degradation is its cleavage at the N-terminal HAE sequence by the enzyme dipeptidyl peptidase IV (DPP IV). To protect from DPP IV, we have studied the biological activity of a GLP-1 analog in which 6-aminohexanoic acid (Aha) is inserted between histidine and alanine at positions 7 and 8. We have compared the biological activity of this new compound, GLP-1 Aha(8), with the previously described GLP-1 8-glycine (GLP-1 Gly(8)) analog. GLP-1 Aha(8) (10 nM) was equipotent with GLP-1 (10 nM) in stimulating insulin secretion in RIN 1046-38 cells. As with GLP-1 Gly(8), the binding affinity of GLP-1 Aha(8) for the GLP-1 receptor in intact Chinese hamster ovary (CHO) cells expressing the human GLP-1 receptor (CHO/GLP-1R cells) was reduced (IC(50): GLP-1, 3.7 +/- 0.2 nM; GLP-1 Gly(8), 41 +/- 9 nM; GLP-1 Aha(8), 22 +/- 7 nM). GLP-1 Aha(8) was also shown to stimulate intracellular cAMP production 4-fold above basal at concentrations as low as 0.5 nM. However, it exhibited a higher ED(50) when compared to GLP-1 and GLP-1 Gly(8) (ED(50): GLP-1, 0.036 +/- 0.002 nM, GLP-1 Gly(8), 0.13 +/- 0.02 nM, GLP-1 Aha(8), 0.58 +/- 0.03 nM). A series of D-amino acid-substituted GLP-1 compounds were also examined to assess the importance of putative peptidase-sensitive cleavage sites present in the GLP-1 molecule. They had poor binding affinity for the GLP-1 receptor, and none of these compounds stimulated the production of intracellular cAMP in CHO/GLP-1R cells or insulin secretion in RIN 1046-38 cells. GLP-1 Aha(8) (24 nmol/kg) administered sc to fasted Zucker (fa/fa) rats (mean blood glucose, 195 +/- 32 mg/dl) lowered blood glucose levels to a nadir of 109 +/- 3 mg/dl, and it remained significantly lower for 8 h. Matrix-assisted linear desorption ionization-time of flight mass spectrometry of GLP-1 Aha(8) incubated with DPP IV (37 C, 2 h) did not exhibit an N-terminal degradation product. Taken together, these results show that insertion of Aha after the 7 position in GLP-1 produces an effective, long-acting GLP-1 analog, which may be useful in the treatment of type 2 diabetes mellitus.
Amino Acid Sequence↗
Insulin regulation of a novel WD-40 repeat protein in adipocytes.
A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.
Adipocytes↗
Endocrine regulation of G-protein subunit production in an animal model of type 2 diabetes mellitus.
Adipocyte beta-adrenergic sensitivity is compromised in animal models of obesity and type 2 diabetes. Although changes in the membrane concentrations of G-protein alpha subunits (Galpha) have been implicated, it remains to be determined how these changes are affected by insulin resistance in the different animal models. Because previous studies used young animals, we measured the concentrations of Galpha and Gbeta subunits in epididymal fat from aged (48 weeks old) db/db mice and from their lean littermates to more closely reproduce the model of type 2 diabetes mellitus. Levels of immunoreactive Galphas, Galphai(1/2), Galphao and Galphaq/11 were all significantly greater in adipocyte membranes from the db/db mice than in membranes from their lean non-diabetic littermate controls. Levels of Galphai(1) and Galphai(2) were also individually determined and although they appeared to be slightly higher in db/db membranes, these differences were not significant. Although the levels of both Galphas isoforms were elevated, levels of the 42 and 46 kDa proteins rose by approximately 42% and 20% respectively, indicating differential protein processing of Galphas. By contrast, levels of Galphai3 were similar in the two groups. The levels of common Gbeta and Gbeta2 were also elevated in db/db mice, whereas Gbeta1 and Gbeta4 levels were not different. To determine whether these changes were due to insulin resistance per se or to elevated glucocorticoid production, G-protein subunit levels were quantified in whole cell lysates from 3T3-L1 adipocytes that were stimulated with different concentrations of either insulin or corticosterone. Although none of the subunit levels was affected by insulin, the levels of both Galphas isoforms were increased equally by corticosterone in a concentration-dependent manner. Since glucocorticoids are known regulators of Galphas gene expression in many cell types and in adipocytes from diabetic rodents, the results presented herein appear to more accurately reflect diabetic pathophysiology than do those of previous studies which report a decrease in Galphas levels. Taken together, these results indicate that most of the selective changes in G-protein subunit production in adipocytes from this animal model of type 2 diabetes may not be due to diminished insulin sensitivity, but may be due to other endocrine or metabolic abnormalities associated with the diabetic phenotype.
Adipocytes↗
[Low cost telepathology].
Low cost, high-quality consumer-type digital cameras are now available on the market to be used for taking macrophotographs and microphotographs by simply fixing the camera over the eyepiece of a conventional light microscope using an adaptator. The quality of the images obtained is as good as obtained with more expensive materials using Tri CCD cameras. Using the JPEG format for compression, the image file size is approximately 180 Ko. We present a low cost approach we have tested for one year in our pathology department.
Computers↗
[Colonic actinomycosis. Clinical, endoscopic and histological aspects in one patient].
Colonic location of actinomycosis must be distinguished from infected colonic carcinoma and other colonic inflammatory pseudotumors. The diagnosis is often made by histological examination of the tumor. Initial treatment consists in surgery, followed by an antibiotic treatment. We report the case of a 32-year old woman with a right colonic actinomycosis.
Actinomycosis↗
Akt-dependent antiapoptotic action of insulin is sensitive to farnesyltransferase inhibitor.
CHO cells expressing the human insulin receptors (IR) were used to evaluate the effect of the potent farnesyltransferase inhibitor, manumycin, on insulin antiapoptotic function. Cell treatment with manumycin blocked insulin's ability to suppress pro-apoptotic caspase-3 activity which led to time-dependent proteolytic cleavage of two nuclear target proteins. The Raf-1/MEK/ERK cascade and the serine/threonine protein kinase Akt are two survival pathways that may be activated in response to insulin. We tested the hypothesis that inhibition of farnesylated Ras was causally related to manumycin-induced apoptosis and showed that the response to manumycin was found to be independent of K-Ras function because membrane association and activation of endogenous K-Ras proteins in terms of GTP loading and ERK activation were unabated following treatment with manumycin. Moreover, blocking p21Ras/Raf-1/MEK/ERK cascade by the expression of a transdominant inhibitory mSOS1 mutant in CHO-IR cells kept cells sensitive to the antiapoptotic action of insulin. Insulin-dependent activation of Akt was blocked by 4 h treatment with manumycin (P < 0.01), a kinetic too rapid to be explained by Ras inhibition. This study suggests that the depletion of short-lived farnesylated proteins by manumycin suppresses the antiapoptotic action of insulin at least in part by disrupting Akt activation but not that of the K-Ras/Raf-1/ERK-dependent cascade.
Alkyl and Aryl Transferases↗
Rapid detection of group B streptococci in pregnant women at delivery.
BACKGROUND: Group B streptococcal infections are an important cause of neonatal morbidity and mortality. A rapid method for the detection of this organism in pregnant women at the time of delivery is needed to allow early treatment of neonates. METHODS: We studied the efficacy of two polymerase-chain-reaction (PCR) assays for routine screening of pregnant women for group B streptococci at the time of delivery. We obtained anal, vaginal, and combined vaginal and anal specimens from 112 pregnant women; in 57 women, specimens were obtained before and after the rupture of the amniotic membranes. The specimens were tested for group B streptococci by culture in a standard selective broth medium, with a conventional PCR assay, and with a new fluorogenic PCR assay. RESULTS: Among the 112 women, the results of the culture of the combined vaginal and anal specimens were positive for group B streptococci in 33 women (29.5 percent). The two PCR assays detected group B streptococcal colonization in specimens from 32 of these 33 women: the one negative PCR result was in a sample obtained after the rupture of membranes. As compared with the culture results, the sensitivity of both PCR assays was 97.0 percent and the negative predictive value was 98.8 percent. Both the specificity and the positive predictive value of the two PCR assays were 100 percent. The length of time required to obtain results was 30 to 45 minutes for the new PCR assay, 100 minutes for the conventional PCR assay, and at least 36 hours for culture. CONCLUSIONS: Colonization with group B streptococci can be identified rapidly and reliably by a PCR assay in pregnant women in labor both before and after the rupture of membranes.
Anal Canal↗
Cysteine 981 of the human insulin receptor is required for covalent cross-linking between beta-subunit and a thiol-reactive membrane-associated protein.
The cytoplasmic domain of the insulin receptor (IR) beta-subunit contains cysteine (Cys) residues whose reactivity and function remain uncertain. In this study, we examined the ability of the bifunctional cross-linking reagent 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting proteins that possess reactive thiols. Transfected Chinese hamster ovary cells expressing either the wild-type human IR, C-terminally truncated receptors, or mutant receptors with Cys --> Ala substitutions and mouse 3T3-L1 adipocytes were used to compare the BMH effect. The results showed the formation of a large complex between the wild-type human receptor beta-subunit and molecule X, a thiol-reactive membrane-associated protein, in both intact and semipermeabilized cells in response to BMH. Prior cell stimulation with insulin had only a modest effect in this process. Western blot analysis revealed that the receptor alpha-subunit was not present in the beta-X complex. The BMH cross-linking did not inhibit in vitro tyrosine phosphorylation of the receptor complexed with molecule X. Both the human IR Cys981Ala mutant and murine IR, that lacks the equivalent of human Cys(981), failed to react with BMH. Finally, no covalent association between IR beta-subunit and IRS-1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treated cells expressing the wild-type human IR. These results demonstrate a striking difference in reactivity among the cytoplasmic IR beta-subunit thiols and clearly show that Cys(981) of human IR beta-subunit is in close proximity to a thiol-reactive membrane-associated protein under basal and insulin-stimulated conditions.
3T3 Cells↗
Discrete region of the insulin receptor carboxyl terminus plays key role in insulin action.
In the present study, we attempted to determine the importance of a 23-amino-acid sequence within the carboxyl terminus of the human insulin receptor (IR) molecule in modulating insulin action in Chinese hamster ovary cells. Stable expression of a minigene encoding the receptor fragment led to an increase in insulin-induced IR autophosphorylation that was 2.4-fold higher when compared to that of IR-expressing cells transfected with empty vector. Insulin-stimulated downstream signaling was also significantly elevated in cells expressing the minigene. It was found that expression of the minigene had no effect toward insulin-like growth factor I receptor kinase activity and function. These results indicate that the IR carboxyl terminus contains a motif that acts as a physiologic modulator of insulin signaling. J. Cell. Biochem. 78:160-169, 2000. Published 2000 Wiley-Liss, Inc.
Animals↗
[Analysis of diagnostic reasoning of nursing students].
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Education, Nursing, Baccalaureate↗