Discordance between immunochemistry of mismatch repair proteins and molecular testing of microsatellite instability in colorectal cancer.

BACKGROUND: DNA mismatch repair system deficiency (dMMR) is found in 15% of colorectal cancers (CRCs). Two methods are used to determine dMMR, immunohistochemistry (IHC) of MMR proteins and molecular testing of microsatellite instability (MSI). Only studies with a low number of patients have reported rates of discordance between these two methods, ranging from 1% to 10%. MATERIALS AND METHODS: Overall, 3228 consecutive patients with CRCs from two centers were included. Molecular testing was carried out using the Pentaplex panel and IHC evaluated four (MLH1, MSH2, MSH6, and PMS2; cohort 1; n = 1085) or two MMR proteins (MLH1 and MSH2; cohort 2; n = 2143). The primary endpoint was the rate of discordance between MSI and MMR IHC tests. RESULTS: Fifty-one discordant cases (1.6%) were initially observed. Twenty-nine out of 51 discordant cases were related to IHC misclassifications. In cohort 1, after re-reading IHC and/or carrying out new IHC, 16 discordant cases were reclassified as nondiscordant. In cohort 2, after the addition of MSH6/PMS2 IHC and re-examination, 13 were reclassified as nondiscordant. In addition, 10 misclassifications of molecular tests were identified. Finally, only 12 discordant cases (0.4%) remained: 5 were proficient MMR/MSI and 7 were dMMR/microsatellite stable. CONCLUSIONS: Our study confirmed the high degree of concordance between MSI and MMR IHC tests. Discordant cases must be reviewed, and if needed, tests must be repeated and analyzed by an expert team.

[Very preterm births in French Polynesia: update and proposal for follow-up]. / La grande prématurité en Polynésie française : évaluation et perspectives de prise en charge.

INTRODUCTION: The care of premature infants in French Polynesia is complicated by this country's geographic isolation. We undertook an evaluation of the medical care of very premature infants (VPIs) to find local solutions to this problem. OBJECTIVES: The objectives were to determine the incidence, mortality, and the short- and long-term outcome of very preterm infants in French Polynesia. METHODS: We retrospectively reviewed the medical charts of all infants born alive at<32 gestational age (GA) and>24 GA from January 2007 to December 2011. Perinatal characteristics and outcomes were examined by univariate and multivariate analysis. RESULTS: In total, 204 VPIs were born during the 5-year study period, comprising 0.9% of all births. Infants less than 28 GA comprised 0.1% of all births. Sixty-two percent of mothers were of extreme age including 43% less than 25 years old. Prematurity was attributed to spontaneous preterm labor in 63% of cases and preeclampsia in 29%. Spontaneous multiple pregnancies comprised 15% of the cases. Alcohol and tobacco consumption were frequently noted (>8% and 26% mothers, respectively). Seventy-eight percent of VPIs had received prenatal steroids. Intrauterine growth was normal in 89%. Mortality occurred in 9.3% (19 patients). Mortality was higher with lower gestational age (P<0.05) and absence of prenatal steroids (P<0.05) in univariate and multivariate analysis. The primary cause of death was sepsis. Hyaline membrane disease occurred in 44% of patients, 80% of whom received surfactant therapy. In total, 16.2% newborns developed bronchodysplasia, 3.4% necrotizing enterocolitis, 3% cerebral hemorrhage, and 1.5% leukomalacia. Long-term outcome was marked by 52% of the patients lost to follow-up by 2 years of age, mostly because of geographic isolation. For the 72 patients followed-up, four developed asthma and three cerebral palsy; 70% were attending school by 3 years of age. CONCLUSIONS: The incidence, mortality, and morbidity of very preterm birth in French Polynesia are comparable to reports from metropolitan centers in France. Conversely, nearly one-half of the patients were lost to follow-up, precluding meaningful information on intellectual development and other outcomes. We recommend organizing a long-term follow-up network to detect cognitive sequelae and adapting such a system to the geographical residence of French Polynesian families.

[Intestinal fistula: an unusual complication of necrotizing enterocolitis in the preterm infant]. / La fistule digestive : une complication inhabituelle de l'entérocolite ulcéronécrosante du nouveau-né prématuré

Intestinal fistula is a rare complication in necrotizing enterocolitis (NEC) and is typically associated with a colonic stricture. We report the case of a preterm infant with severe NEC, who developed an ileocolic fistula followed by the appearance of a colonic stricture after surgical treatment. This report shows how complex the outcome of NEC can be: a contrast enema should be done in NEC when the clinical or biological outcome is not favorable, in order not to delay the diagnosis of intestinal fistula.

[Revelation of an acute lymphoblastic leukemia in the delivery room]. / Leucémie aiguë lymphoblastique néonatale: une affection rare à révélation immédiate en salle de naissance.

Acute leukemia is uncommon in neonates and has a much poorer prognosis than in older children. We report on a case of acute lymphoblastic leukemia observed in a neonate who had bleeding and hepatosplenomegaly at birth, which justified intensive care during the first postnatal week. Despite early appropriate treatment, the patient died at 7 months of age. We present here physical and laboratory findings, which indicate a grim prognosis. These criteria should be considered carefully in order to ensure a realistic information for the parents and appropriate decisions.

Reproductive cytotoxicity is predicted by magnetic resonance microscopy and confirmed by ubiquitin-proteasome immunohistochemistry in a theophylline-induced model of rat testicular and epididymal toxicity.

This study investigated the testicular changes in the rat induced by the nonspecific phosphodiesterase inhibitor, theophylline using magnetic resonance microscopy (MRM) and ubiquitin immunostaining techniques. In vivo T1- and T2-weighted images were acquired at 2 T under anesthesia. Increased signal observed in the theophylline-treated rats suggests that leakage of MRM contrast was occurring. In vivo MRM results indicate that day 16 testis displayed an increased T1-weighted water signal in the area of the seminiferous tubule that decreased by day 32. These findings were validated by histopathology, suggesting that in vivo MRM has the sensitivity to predict changes in testis and epididymal tissues. The participation of the ubiquitin system was investigated, using probes for various markers of the ubiquitin-proteasome pathway. MRM can be used to detect subtle changes in the vascular perfusion of organ systems, and the up-regulation/mobilization of ubiquitin-proteasome pathway may be one of the mechanisms used in theophylline-treated epididymis to remove damaged cells before storage in the cauda epididymis. The combined use of in vivo MRM and subsequent tissue or seminal analysis for the presence of ubiquitin in longitudinal studies may become an important biomarker for assessing testis toxicities drug studies.

[Use of human recombinant erythropoietin in children with cancer]. / Utilisation de l'érythropoïétine recombinante humaine chez l'enfant atteint de cancer.

Eighty percent of children with cancer suffer from anemia at the time of diagnosis. The physiopathology of anemia is complex. Although anemia can be life threatening, its consequences on the physical, psychological and social state of the child are often minimized. Blood transfusion is the main treatment of anemia: its efficacy is immediate but shortlasting, and it involves infectious and hemolytic risks. The human recombinant erythropoietin has been used for more than 25-years, and is often prescribed to adults with cancer and anemia. The human recombinant erythropoietin rHuEPO is nowadays used when blood transfusion is contra-indicated because of religious or cultural considerations, although several promising studies have been conducted about rHuEPO and children with cancer since 1996: it might be soon the preferential alternative treatment to anemia in children with cancer.

High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1.

Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from Abc1 knockout (-/-) mice revealed an approximately 70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/- and -/- compared with their littermate controls. The inactivation of the Abc1 gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of Abc1-/- mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that Abc1-/- mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.

Functional and structural diversity of the human Dickkopf gene family.

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.

Incidence of intracranial bullet fragment migration.

Migration of retained bullets or bullet fragments may present as a complication of gunshot wounds to the head. This phenomenon has been reported in cases of abscess formation or retained copper fragments. Management of such migratory fragments is controversial. The purpose of this study is to determine the incidence of fragment migration in a population of neurosurgical patients treated for gunshot wounds to the head. Two-hundred and thirteen cases treated at Detroit Receiving Hospital between 1985 and 1987 were reviewed. Each patient treated had initial and one week follow-up imaging studies. Nine cases of documented migratory intracranial bullet fragments were identified. Thus, the incidence in this population is 4.2%. The fragments in eight cases were composed of copper, and in the remaining case, lead. No case was associated with an abscess. Fragments in the anterior fossa were found to migrate towards the sella turcica, while those of the middle fossa and posterior hemispheres migrate towards the confluence of sinuses (Torcula Herophili). Fragment migration was documented as early as 36 h post-injury. Based on this study, we recommend serial imaging studies to look for migrating bullet fragments and surgical removal aided by intra-operative ultrasound to localize the fragment when possible.

Development of a gas chromatography-mass spectrometry method for the analysis of aminoglycoside antibiotics using experimental design for the optimisation of the derivatisation reactions.

A packed column GC-electron-capture detection method for the analysis of the aminoglycoside antibiotics kanamycin and gentamicin was adapted to capillary GC-MS. The analytes were derivatised using a two-step procedure involving trimethylsilylation of the hydroxyl groups with trimethylsilylimidazole and acylation of the amino groups with heptafluorobutyrylimidazole. Electron impact mass spectra of the resulting derivatives of kanamycin A and gentamicins C1, C1a and C2 are given and interpreted. The derivatisation procedure was optimised using experimental design. This chemometrical approach considers main effects as well as interactions of the influential parameters, thus conducting a more thorough investigation of the method than the common step-by-step approach. Optimisation using fractional factorial and Box Behnken Designs produced a derivatisation method featuring better yield than previously published methods while in many cases requiring less reagents and shorter reaction times.

Structure-property relationships of trimetazidine derivatives and model compounds as potential antioxidants.

Twenty-five compounds (trimetazidine derivatives and other compounds, mostly having a free phenolic group) were examined for their radical scavenging and antioxidant properties. Their reaction with DPPH (2,2-diphenyl-1-picrylhydrazyl) as a measure of radical scavenging capacity was assessed by two parameters, namely EC50 (the concentration of antioxidant decreasing DPPH by 50%), and log Z, a kinetic parameter proposed here and derived from initial second-order rate constants and antioxidant/DPPH ratios. Antioxidant activities were determined by the inhibition of lipid peroxidation and albumin oxidation. The most active compounds were derivatives having a trolox or hydroquinone moiety. Physicochemical and structural properties were determined by molecular modeling as lipophilicity (virtual log P calculations) and H-Surf (solvent-accessible surface of hydroxyl hydrogen) and by quantum mechanical calculations (deltaH(ox) = oxidation enthalpy; deltaH(abs) = enthalpy of hydrogen abstraction). QSAR models were derived to identify molecular mechanisms responsible for the reactivity toward the DPPH radical and for the inhibition of lipid peroxidation. A useful prediction of antioxidant capacity could be achieved from calculated molecular properties and the kinetic parameter developed here.

Co-stimulation of human peripheral blood mononuclear cells with IL-2 and anti-CD3 monoclonal antibodies induces phosphorylation of CREB.

Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated. The absence of P-CREB following CD3 cross-linking was unexpected, as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes. Therefore, P-CREB was evaluated following co-stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 did not further augment phosphorylation of CREB following CD2 cross-linking. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed. Phosphorylation was not associated with the induction of cAMP, and inhibition of PKA signaling had no effect on P-CREB. Consistent with signal transduction through p56lck or p59fyn, inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulated cells not pretreated with a PKC inhibitor. In contrast to previous studies performed in the absence of exogenous IL-2, no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay. These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co-operative stimulus promoting phosphorylation of CREB following receptor cross-linking.

Curcumin and curcumin derivatives inhibit Tat-mediated transactivation of type 1 human immunodeficiency virus long terminal repeat.

The transcription of HIV1 provirus is regulated by both cellular and viral factors. Various evidence suggests that Tat protein secreted by HIV1-infected cells may have additional action in the pathogenesis of AIDS because of its ability to also be taken up by non-infected cells. Curcumin [diferuloylmethane or 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is the yellow pigment in turmeric Curcuma longa (Linn). It exhibits a variety of pharmacological effects including antiinflammatory and antiretroviral activities. Here, we demonstrated that curcumin used at 10 to 100 nM inhibited Tat transactivation of HIV1-LTR lacZ by 70 to 80% in HeLa cells. In order to develop more efficient curcumin derivatives, we synthesized and tested in the same experimental system the inhibitory activity of reduced curcumin (C1), which lacks the spatial structure of curcumin; allyl-curcumin (C2), which possesses a condensed allyl derivative on curcumin that plays the role of metal chelator; and tocopheryl-curcumin (C3), which enhances the antioxidant activity of the molecule. Results obtained with C1, C2 and C3 curcumin derivatives showed a significant inhibition (70 to 85%) of Tat transactivation. Despite the fact that tocopheryl-curcumin (C3) failed to scavenge O2.-, this curcumin derivative exhibited the most activity; 70% inhibition was obtained at 1 nM, while only 35% inhibition was obtained with the curcumin.

CD2 signalling induces phosphorylation of CREB in primary lymphocytes.

Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1) family. We have used a human T-lymphotropic virus type 1 (HTLV-1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV-1 transcription following CD2 but not CD3 receptor cross-linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor-mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross-linking increased cAMP detected by competitive enzyme-linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross-linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post-translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp-cAMP-sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross-linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF-1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE-mediated transcription following ligand engagement (e.g. cell-to-cell contact).

Signaling via the CD2 receptor enhances HTLV-1 replication in T lymphocytes.

Human T lymphotropic virus type 1 (HTLV-1) is considered the etiologic agent of adult T cell leukemia/lymphoma and several chronic progressive immune-mediated diseases. Approximately 1-4% of infected individuals develop disease, generally decades following infection. Increased proviral transcription, mediated by the viral 40-kDa trans-activating protein, Tax, has been implicated in the pathogenesis of HTLV-1-associated diseases. Since the HTLV-1 promoter contains sequences responsive to cyclic AMP and protein kinase C, we hypothesized that lymphocyte activation signals initiated through the TCR/CD3 complex or CD2 receptor promote viral replication in HTLV-1-infected lymphocytes. We demonstrate that mAbs directed against the CD2, but not the CD3 receptor increase viral p24 capsid protein 1.5- to 5.7-fold in CD2/CD3+ HTLV-1-infected cell culture supernatants. Northern blot analysis demonstrated a 2.5- to 4-fold increase in all species of viral mRNA following CD2 cross-linking of OSP2/4 cells, an immortalized HTLV-1 cell line. Consistent with transcriptional regulation, reporter gene activity increased approximately 11-fold in CD2-stimulated Jurkat T cells cotransfected with a Tax-expressing plasmid and a CAT reporter gene construct under control of the HTLV-1 promoter. These data suggest a possible physiologic mechanism, whereby CD2-mediated cell adhesion and lymphocyte activation may promote viral transcription in infected lymphocytes.

Application of experimental design to generate relevant information and representative calibration data.

The basic requirement for a good calibration is representative data. This paper outlines techniques for selecting samples from an existing population. The concept of factorial designs is explained, and three ways of applying experimental design to generate representative data are described. These are: to vary the experimental conditions; focus on some of the parameters of interest directly (reference values); to vary the underlying conditions which generate consistent variations in the spectra,for example production factors. Finally the paper gives an example of the use of the concept of experimental design to pick out samples from a population.

Stimulation of the CD2 receptor pathway induces apoptosis in human T lymphotropic virus type I-infected cell lines.

We demonstrate that CD2 receptor engagement, but not CD3 crosslinking, induces apoptosis in lymphocytes transformed by human T-cell lymphotrophic virus type I (HTLV-I). Mitogenic pairs of anti-CD2 monoclonal antibodies inhibited [3H]thymidine incorporation from 25 to 62% in CD2+ HTLV-I-infected lymphocytes. This inhibition was associated with a 20-40% reduction in cell number and viability over a 3-day period, morphologic evidence of apoptosis, and irreversible DNA fragmentation. While cyclosporin A abrogated CD2-mediated proliferation in peripheral blood mononuclear cells, it had no effect on CD2-induced apoptosis in the HTLV-I-infected cell lines. Since HTLV-I is mitogenic to resting lymphocytes through CD2 activation pathways, these results suggest that HTLV-I-infected lymphocytes are primed for apoptosis following additional CD2 stimulation. This CD2-mediated apoptosis might be a factor in immune regulation of HTLV-I-associated diseases or might offer a novel adjunctive approach to treatment.

Enhanced human T-cell lymphotropic virus type I expression following induction of the cellular stress response.

Human T-cell lymphotropic virus type I (HTLV-I) infection is typically associated with long incubation periods between virus exposure and disease manifestation. Although viral protein expression is considered to play an important role in the pathogenesis of HTLV-I-associated diseases, limited information is known regarding host cell mechanisms that control viral gene expression. This study was designed to evaluate modulation of HTLV-I gene expression following induction of the cellular stress response in HTLV-I-infected lymphocytes. The cellular stress response was elicited by treatment with either Na arsenite or thermal stress and was monitored by demonstrating increased expression of the 72-kDa heat shock protein. Induction of the cellular stress response in HTLV-I-infected lymphocytes resulted in significantly increased HTLV-I-mediated syncytia formation due to enhanced HTLV-I envelope (gp46) expression. Intracellular viral proteins and released p24 capsid protein were increased in stressed infected lymphocytes as compared to nonstressed infected lymphocytes. Furthermore, HTLV-I-LTR reporter gene constructs had increased activity (three- to sixfold) in a transiently transfected, uninfected lymphocyte cell line following induction of the cellular stress response. Quantitation of HTLV-I RNA expression by slot blot analysis of infected lymphocytes suggested variable increases in RNA accumulation. Northern blot analysis demonstrated no qualitative changes in expression of RNA species. These data suggest a relationship between modulation of viral replication and a basic cellular response to stress and have important implications for understanding host cell control mechanisms of HTLV-I expression.

The "fallen lung with absent hilum" signs of complete bronchial transection.

A 19-year-old pedestrian who was the victim of a motor vehicle accident had a left pneumothorax with a large air leak. Despite a well-placed chest tube, a chest radiograph showed that the left lung had fallen down and away from the mediastinum and that no hilar structures were visible. This "fallen lung with an absent hilum" is considered virtually diagnostic of complete mainstem bronchus transection.

Cervical osteomyelitis due to i.v. heroin use: radiologic findings in 14 patients.

We reviewed the radiographs of 14 patients who had cervical osteomyelitis and were IV heroin users. Eleven were men and three were women. Their age range was 33-48 years (mean, 39 years). Eleven regularly used the jugular vein access, and three alternated between the jugular and femoral veins. Initial radiographs of the cervical spine in 13 patients showed destruction of two or more vertebral bodies and the adjacent intervertebral disk, as well as a prevertebral soft-tissue mass. In one patient, findings on initial radiographs were normal, but marked destruction at two contiguous intervertebral levels and a large prevertebral abscess were identified 2 weeks later. All the patients had positive results on cultures of joint aspirates or bone biopsy materials (10 patients) or blood (four patients). Ten grew Staphylococcus aureus; two, Staphylococcus epidermidis; one, Streptococcus viridans; and one, Pseudomonas aeruginosa. CT in nine patients showed inflammatory reaction adjacent to the carotid sheath resulting from the repeated jugular injections and delineated the extent of prevertebral abscess and bone destruction. Scintigrams were of minimal value in establishing the diagnosis. Advanced vertebral body destruction, disk space infection, prevertebral abscess, and anterior cervical inflammatory reaction appear to be typical findings on radiographs in heroin abusers with cervical osteomyelitis.