Idiopathic infantile hypercalcaemia in 5-month old girl.

Idiopathic infantile hypercalcaemia (IIH) is a mineral metabolism disorder of unknown origin. It is characterized by high levels of serum calcium resulting in parathyroid hormone (PTH) suppression, muscle hypotonia, thirst, anorexia, failure to thrive, psychomotor retardation, constipation, nephrocalcinosis. Treatment consists of low calcium diet, glucocorticoids, furosemide. We present a case of 5-month old girl with IIH, where total calcaemia peaked to 4.25 mmol/l. The leading symptoms were failure to thrive, constipation, muscle hypotonia, dehydration. Rehydration, low calcium diet, and application of glucocorticoids and furosemide resulted in a drop in calcaemia to normal values and an overall clinical improvement within two weeks. Williams-Beuren syndrome (WBS), benign familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT), Jansen's metaphyseal dysplasia, primary hyperparathyroidism, vitamin D intoxication, granulomatous diseases, thyroid disease, malignancy were all ruled out. In conclusion, infants with failure to thrive should have their serum levels of minerals, especially, calcium, checked. In case of hypercalcaemia, treatment with corticosteroids and furosemide should be initiated, together with further diagnostic steps in order to elucidate its origin.

[Tropism of hepatitis C virus for leukocytes-- importance of the analysis of viral E1 and E2 envelope glycoprotein genes by sequencing]. / Tropisme leucocytaire du virus de l'hépatite C - intérêt de l'analyse des séquences des gènes des glycoprotéines d'enveloppe virales E1 et E2.

The ability of hepatitis C virus (HCV) to infect leukocytes could favour HCV pathogenesis. Although viral infection of these immunocompetent cells is poorly (or not) productive, the impact on their immunomodulatory functions could be important. Viral envelope glycoproteins E1 and E2, because of their crucial role in the recognition of viral receptors on permissive cells, could contribute to viral leukocytic tropism and, as a consequence, to the pathophysiology of HCV chronic infection.

Atypical symptoms in patients with herpesvirus DNA detected by PCR in cerebrospinal fluid.

BACKGROUND: Polymerase chain reaction (PCR) detection of herpesvirus DNA in cerebrospinal fluid (CSF) is an important tool in the diagnosis of central nervous system (CNS) syndromes. The corresponding viral infections present with diverse clinical signs, which are often classical although no sign can be considered as specific. This retrospective study aims to describe atypical symptoms in patients with herpesvirus DNA detected in CSF by PCR. A total of 3452 cerebrospinal fluid samples from patients with suspected herpesvirus infection of the CNS were investigated between 1998 and 2003 in our clinical virology laboratory. "In-house" PCRs for each herpesvirus [herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein Barr virus (EBV), or human herpes virus 6 (HHV6)] were used until 2001 and a commercially available "Herpes Consensus PCR" was used thereafter. One of the five herpesviruses investigated in this study was found in 71 (2.1%) of CSF samples (37 HSV, 14 VZV, 1 CMV, 9 EBV and 10 HHV6). These samples were obtained from 62 patients whose clinical findings were generally consistent with the PCR data. However, some little known features of herpesvirus-related symptoms, such as partial seizure associated with HSV infection, and unusual VZV or HHV6-related myelitis were also observed.

Oxygen concentration-dependent metabolism of leukotriene B4 by hepatocyte monolayers.

Leukotriene B4 was found to be metabolized by rat hepatocyte monolayers at a rate that was linear with increasing substrate concentration from 74 to 740 nM leukotriene B4. The rates of metabolism were dependent on the O2 concentration and were 315, 213, 80, and 36 pmol leukotriene B4 per min per nmol cytochrome P-450 at 20% (212 microM), 4% (42.5 microM), 2% (21.2 microM), and 1% (10.6 microM) O2, respectively. The metabolic rate was not linear with respect to O2 concentration; however, half maximal rate occurred at 4% O2, and O2 concentration found in the pericentral region of normally oxygenated liver. These results suggest that in vivo conditions of hypoxia or ischemia that lead to blood O2 concentrations less than 4% may drastically decrease hepatic clearance of leukotriene B4.

Mephenytoin-type polymorphism of drug oxidation: purification and characterization of a human liver cytochrome P-450 isozyme catalyzing microsomal mephenytoin hydroxylation.

A genetic polymorphism causing deficient metabolism of the anticonvulsant drug mephenytoin occurs in 5% of the Caucasian and 23% of the Japanese population. By monitoring the activities of the two major oxidative pathways of mephenytoin metabolism in the column eluates, we have purified from human livers a cytochrome P-450 isozyme, P-450 meph, which exclusively and stereoselectively catalyzes the 4-hydroxylation of (S)-mephenytoin, the major pathway affected by the polymorphism, whereas P-450 meph was virtually devoid of catalytic activity for N-demethylation of mephenytoin, the pathway remaining unaffected by the genetic deficiency. P-450 meph had an apparent Mr of 55 000 and a lambda max in the reduced CO-binding spectrum of 450 nm. Polyclonal rabbit antibodies against purified human P-450 meph almost completely inhibited the 4-hydroxylation of mephenytoin but had little effect on N-demethylation in human liver microsomes. In microsomes of liver biopsies of two subjects characterized in vivo as 'poor metabolizers' of mephenytoin, immunocrossreactive and immunoinhibitable material was observed with similar or identical properties to those of P-450 meph. There was no difference in the extent of the immunochemical reaction between microsomes of in vivo phenotyped poor metabolizers and extensive metabolizers of mephenytoin. These data suggest that P-450 meph is the target of the genetic deficiency and support the concept that a functionally altered variant form of P-450 meph causes this polymorphism.

Lipid peroxidation decreases the rotational mobility of cytochrome P-450 in rat liver microsomes.

Phenobarbital-induced rat liver microsomes were subjected to NADPH- and Fe2+-catalyzed lipid peroxidation. The formation of approx. 95 nmol malondialdehyde/mg protein during 18 min peroxidation at 37 degrees C was observed. Membrane rigidity measured by means of the steady-state fluorescence anisotropy rs of diphenylhexatriene increased in parallel with the malondialdehyde formation. Both the amount of malondialdehyde and rs remained constant thereafter during incubation of the peroxidized membranes for 2 h. The aminopyrine demethylase activity decreased by about 60% upon lipid peroxidation for 18 min, whereas no significant loss of benzphetamine demethylase activity within the same time range was observed. A time-dependent formation of protein complexes of high molecular weight, comprising most of the microsomal polypeptides, upon lipid peroxidation was observed in SDS-polyacrylamide gel electrophoresis. The effect of microsomal lipid peroxidation on protein-protein interactions was examined by measuring the rotational mobility of intact cytochrome P-450. Rotational diffusion was measured by observing the decay of flash-induced absorption anisotropy r(t) of the P-450 X CO complex. Analysis was based on a 'rotation-about-membrane normal' model with the equation r(t) = r1exp(-t/phi 1) + r2exp(-t/phi 2). In control microsomes, two classes (rapid and slow) of rotating populations of cytochrome P-450 were observed with phi 1 approximately equal to 150 microseconds, fraction r1/(r1 + r2) approximately equal to 40% and phi 2 approximately equal to 2 ms, fraction r2/(r1 + r2) approximately equal to 60%. A relatively small decrease in the rotational mobility of P-450 was observed by a 18-min lipid peroxidation, while a subsequent incubation of peroxidized microsomes for 2 h at 37 degrees C resulted in a dramatic immobilization of P-450 by the increase of both r2/(r1 + r2) approximately equal to 75% and phi 2 approximately equal to 10-25 ms. The decrease in the P-450 mobility during 18-min lipid peroxidation would be due to the rigidification of the lipid bilayer. However, because the lipid fluidity remained unchanged thereafter, the significant immobilization of P-450 by the subsequent 2-h incubation is deduced to be due to formation of protein aggregates.

Mechanisms of halothane toxicity: novel insights.

Exposure of individuals to halothane causes, in 20% of patients, a mild form of hepatotoxicity. In contrast, a very small subset of individuals only develops halothane hepatitis, which is thought to have an immunological basis. Sera of halothane hepatitis patients contain antibodies directed against some discrete liver trifluoroacetyl (TFA)-protein adducts, which arise upon oxidative biotransformation of halothane and include protein disulfide isomerase, microsomal carboxylesterase, calreticulin, ERp72, GRP 78 and ERp99. No immune response occurs in the majority of human individuals, although evidence suggests that TFA-protein adducts arise in all halothane-exposed individuals. The lack of immunological responsiveness of individuals might be due to tolerance, induced by a presumed repertoire of self-peptides that molecularly mimic TFA-protein adducts. Thus, constitutively expressed proteins of 52 and 64 kDa have been identified that confer molecular mimicry of TFA-protein adducts. The 64 kDa protein corresponds to the E2 subunit of the mitochondrial pyruvate dehydrogenase complex. Lipoic acid, the prosthetic group of the E2 subunit, is involved in the molecular mimicry process. A fraction of halothane hepatitis patients exhibit irregularities in the expression levels of the 52 kDa protein and the E2 subunit protein. Molecular mimicry of TFA-protein adducts by the 52 kDa protein and the E2 subunit protein might play a role in the susceptibility of individuals to development of halothane hepatitis.

[Rational use of viral serology in the child]. / Utilisation rationnelle des sérologies virales chez l'enfant.

Very sensitive and allowing discrimination between IgG and IgM, the enzyme immunoassays are the most commonly used to carry out viral serologies. The presence of IgG corresponding to a natural or postvaccination immune status or to a latent or occult infection is a reliable information to assess the protection against some opportunistic or teratogenic infections respectively in children or pregnant women. The search of IgM by immunocapture is suitable to early diagnose the majority of common viral diseases in childhood, specially when presenting in an atypical form, and to attest a congenital rubella infection. Moreover, IgG avidity proved to be useful in dating an infection and confirming positive IgM results. However, viral infections localized at an entry site of the host, such as respiratory and enteric diseases, as well as syndromes of herpes simplex and enteroviral origin, do not represent indications of serological diagnosis. Provided its use with discernment, the serological diagnosis of viral infections have to be considered as a high performance and of low cost biological tool.

Evidence of feline immunodeficiency virus replication in cultured Kupffer cells.

OBJECTIVE: To determine if cultured feline Kupffer cells (KC) are as permissive for feline immunodeficiency virus (FIV) as cultured human liver macrophages are for HIV. Two types of infection likely to be relevant to the in vivo situation were used. KC were infected with either free virus or autologous infected peripheral blood mononuclear cells (PBMC). METHODS: Feline KC were isolated by centrifugal elutriation from collagenase-perfused liver; cultured cells were characterized by their morphological appearance and their erythrophagocytotic properties. After infection, viral replication was measured by enzyme-linked immunosorbent assay, reverse transcriptase activity, immunofluorescence assay, in situ hybridization and electron microscopic observations. RESULTS: Three days after isolation, 85% of cultured KC were able to internalize red blood cells; 45% were CD4-positive and 65% expressed a 24 kD protein thought to be a receptor for FIV (CD9). After the addition of autologous infected PBMC or cell-free supernatant of chronically infected IRC4 cells to KC cultures, a peak of viral replication was detected at day 28. Antigen revealed by immunofluorescence assay was present in only 0.4%, and viral RNA was detected by in situ hybridization in 2% of the infected cells. CONCLUSIONS: FIV can replicate in cultured feline KC without inducing any cytopathic effect, which suggests that these cells may play a role in the physiopathology of FIV infection.

Properties of enzymes in hepatocytes that convert 5-HPETE or LTA4 into LTB4.

Rat hepatocyte homogenates convert 5-hydroperoxyeicosatetraenoic acid (5-HPETE) into biologically active leukotriene B4 (LTB4) as well as less active all-trans-LTB4 (i.e., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Here, we present a hypothesis of the reaction mechanism and the minimal structural requirements of the active enzyme based on the following experimental evidence: The ED50 of the inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,6-dehydro-eicosatetraenoic acid was approximately 100-fold higher than for 5-lipoxygenase. Propanethiol and O2 were strong inhibitors of LTB4 formation, whereas butylated hydroxytoluene, nordihydroguaiaretic acid, metyrapone, Desferal and CO had no effect. Cytochrome c, catalase, hematin, and a Fe3+/Fe2+ couple, but not iron-free protoporphyrin IX, catalyzed the formation of only all-trans-LTB4. LTB4 formation in hepatocyte homogenates was heat- and trypsin-sensitive whereas all-trans-LTB4 formation was not. We propose that a ferric heme iron forms a ferryl-hydroxo complex upon homolytic scission of the oxygen-oxygen bond in 5-HPETE and the resulting 5,6-trans-epoxide radical is oxidized by the ferryl-hydroxo complex to yield LTA4. A mechanism for hydrolysis of LTA4 is described that results in formation of LTB4 (less than 1% yield) rather than all-trans-LTB4.

Quantification of 5- and 15-HPETE's by direct chemical ionization mass spectrometry.

This report describes the application of direct chemical ionization mass spectrometry (DCIMS) to the identification and quantification of 5- and 15-HPETEs. A unique feature of the method is use of a polyimide-coated fused silica fiber that allows vaporization of the hydroperoxides, with very low excess energy, into the plume of the chemical ionization reagent gas plasma. Mass spectra are obtained that allow identification of the nonreduced and nonderivatized free acid forms of 5- and 15-HPETE as well as their quantification from 1 microgram to 100 picograms.

Debrisoquine-type polymorphism of drug oxidation: purification from human liver of a cytochrome P450 isozyme with high activity for bufuralol hydroxylation.

Indirect evidence suggests that the genetically defective metabolism of drugs such as debrisoquine and bufuralol observed in up to 10% of the population (poor metabolizers) is caused by the absence or functional deficiency of a cytochrome P450 isozyme. Using bufuralol-1'-hydroxylation to carbinol to optimize the procedure, 3 cytochrome P450 isozymes (P450A, P450buf, P450C) were purified to apparent electrophoretic homogeneity from human liver microsomes. P450buf had a specific activity of 20.3 nmol carbinol X nmol P450-1 X 15 min-1 as compared to microsomes (10.0 nmol carbinol X nmol P450(-1) X 15 min-1) when (+)-bufuralol was used as substrate. The stereoselective metabolism of (-)- and (+)-bufuralol to carbinol by purified P450buf [(-)/(+) ratio: 0.13] was strikingly different from that in the microsomes of either an extensive [(-)/(+) ratio: 0.4] or poor metabolizer [(-)/(+) ratio: 0.83] of bufuralol. We propose that this isozyme is the major bufuralol and debrisoquine hydroxylating species and is the target of the genetic deficiency.

Isolation and characterization of a lectin from Sarcocystis gigantea.

A hydrophobic, galactose-specific lectin was isolated by means of affinity chromatography from Sarcocystis gigantea. Adsorbents with different spacer lengths were tested. S. gigantea lectin differs from sheep muscle lectin in the spacer length needed for adsorption. Sodium dodecyl sulfate-gel electrophoresis of the S. gigantea lectin revealed a subunit size about 19 kDa and the presence of disulfide cross-linked dimers. The lectin is present in high concentration in cystozoites, cyst fluid and cyst wall material.

Isolation, sequence and molecular karyotype analysis of the actin gene of Cryptosporidium parvum.

Actin is an ubiquitous and highly conserved microfilament protein which is hypothesized to play a mechanical, force-generating role in the unusual gliding motility of sporozoan zoites and in their active penetration of host cells. We have identified and isolated an actin gene from a Cryptosporidium parvum genomic DNA library using a chicken beta-actin cDNA as an hybridization probe. The nucleotide sequences of two overlapping recombinant clones were identical and the amino acid sequence deduced from the single open reading frame was 85 % identical to the P. falciparum actin I and human gamma-actin proteins. The predicted 42 106-Da Cryptosporidium actin contains 376 amino acids and is encoded by a single-copy gene which contains no introns. The nucleic acid coding sequence is 72% biased to the use of A or T in the third position of codons. Chromosome-sized DNA released from intact C. parvum oocysts was resolved by OFAGE into 5 discrete ethidium bromide-staining DNAs ranging in size from 900 to 1400 kb; the cloned C. parvum actin gene hybridized to a single chromosomal DNA of approximately 1200 kb.

Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase-thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum.

We have determined the nucleic acid sequences of a gene encoding the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) from bovine and human AIDS isolates of Cryptosporidium parvum. THe DHFR-TS gene was isolated from genomic DNA libraries by hybridization with a probe amplified from C. parvum genomic DNA using generic TS primers in the polymerase chain reaction. Genomic Southern and electrophoretic karyotype analyses reveal C. parvum DHFR-TS is a single-copy gene on a 1200-kb chromosome. The DHFR-TS nucleic acid sequence contains no introns and the single 1563-bp open reading frame encodes a 179 residue N-terminal DHFR domain connected by a 55 amino acid junction peptide to a 287 residue C-terminal TS domain. The sequences of the DHFR-TS gene from the bovine and human C. parvum isolates differ at two positions in the 5'-flanking sequence and at 38 positions in the encoding sequence. These DNA sequence polymorphisms will provide a powerful probe to examine the genotypic diversity and genetic population structure of C. parvum. The two sequences encode identical TS domains which share all except one of the phylogenetically conserved amino acid residues identified among reported TS sequences. The predicted DHFR domain sequences contain nine amino acid differences; these polymorphisms all map to non-active site, surface locations in known DHFR structures. The C. parvum DHFR active site contains novel residues at several positions analogous to those at which point mutations have been shown to produce antifolate resistance in other DHFRs. Thus C. parvum DHFR may be intrinsically resistant ti inhibition by some antifolate DHFR inhibitors which may explain why cryptosporidiosis is refractory to treatment with the clinically common antibacterial and antiprotozoal antifolates.

A novel multi-domain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion.

Cryptosporidium parvum is a protozoan parasite which produces self-limited disease in immunocompetent hosts and devastating, persistent diarrhea in immunocompromised individuals. There is no effective treatment for cryptosporidiosis and little is known about the basic biology of the organism. Cloning and sequence analysis of the gene encoding GP900, a previously identified > 900 kDa glycoprotein, predicts a mucin-like glycoprotein composed of distal cysteine-rich domains separated by polythreonine domains and a large membrane proximal N-glycosylated core region. A trinucleotide repeat composed predominantly of the triplet ACA encodes the threonine domains. GP900 is stored in micronemes prior to appearance on the surface of invasive forms. The concentration of native GP900 which inhibits 50% (IC50) of invasion in vitro is low picomolar; the IC50 for a recombinant cysteine rich-domain is low nanomolar. These observations indicate that GP900 is a parasite ligand for a host receptor involved in attachment/invasion and suggest that immunotherapy or chemotherapy directed against GP900 may be feasible.

Protection of SIVmac-infected macaque monkeys against superinfection by a simian immunodeficiency virus expressing envelope glycoproteins of HIV type 1.

The infection of macaque monkeys by attenuated simian immunodeficiency virus can vaccinate against pathogenic molecular clones and isolates of the same virus. The correlates of this potent protective immunity are not fully understood but may be the key to an effective AIDS vaccine for humans. Aiming to determine whether host immune responses to envelope glycoprotein are an essential component of the immunity to primate lentiviruses, we have tried to superinfect SIVmac-infected macaque monkeys with SHIVsbg, a chimeric primate lentivirus constructed from the SIVmac239 genome with the env, rev, tat, and vpu genes from HIV-1 Lai. After inoculation of a large dose of SHIVsbg, the chimeric virus was isolated by coculture of mononuclear blood cells from four of five SIV-infected monkeys, but three animals were protected from extracellular SHIV viremia and did not seroconvert to HIV-1 glycoproteins. In the two SIV-infected monkeys that did develop SHIV viremia, cell-associated viral load was reduced at least 100-fold. These data indicate that an antiviral response capable of effectively controlling primate lentivirus replication might not necessarily involve the envelope glycoprotein.

Enterovirus in sudden unexpected deaths in infants.

BACKGROUND: Conventional approaches to virus detection failed to provide convincing evidence of a viral etiology in sudden unexplained deaths in infants (SUDI). Many viruses may not have been detected by the routinely used methods; among them enteroviruses (EV) have seldom been found in SUDI. METHODS: In this study EV were sought directly in stools, in pharyngeal and tracheal samples and in myocardial and lung tissues, by using a nested PCR; they were also sought indirectly by detecting IgM antibodies with a new capture immunoassay. Twenty-four SUDI cases were divided into two groups: Group I, certainly associated with; or Group II, not associated with clinical, biologic or histologic signs of viral infection. RESULTS: EV were found in stools but their prevalence was not significantly different between Group I and Group II (20 and 22.2%, respectively). On the contrary EV were detected in respiratory tract and/or lung samples in 53.8% of infants of Group I and in none of Group II. Anti-EV IgM antibodies were detected in 55.5% of infants of Group I and in none of Group II. CONCLUSIONS: These results indicate that EV infection may be specifically associated with the subgroup of SUDI with viral signs, raising the question of its role in this condition.

Molecular epidemiology of ocular isolates of adenovirus 8 obtained over nine years.

Twenty nine strains of adenovirus 8 have been isolated over nine years in Strasbourg, France, 22 of which were from one private ophthalmologist. To assess a possible relation between these strains, the DNA of adenovirus was analysed by restriction fragment length polymorphism using eight different enzymes. Among these, three proved discriminant (Xba I, Bgl II, Eco RI) and made it possible to define 13 genotypes differing from each other by one to three DNA bands. Seven genotypes were unique isolates, while three, representing 16 strains, were isolated over five to eight years. All the genotypes but one were closely related, with 87% homology. All 13 differed from an adenovirus 8 strain from Lyon (homology 68-76%). This study confirmed the stability of adenovirus 8 in a given population.

Molecular mimicry in halothane hepatitis: biochemical and structural characterization of lipoylated autoantigens.

Exposure of human individuals to halothane causes, in about 20% of all cases, a mild transient form of hepatotoxicity. A small subset of exposed individuals, however, develops a potentially severe and life-threatening form of hepatic damage, coined halothane hepatitis. Halothane hepatitis is thought to have an immunological basis. Sera of afflicted individuals contain a wide variety of autoantibodies against hepatic proteins, in both trifluoroacetylated form (CF3CO-proteins) and, at least in part, in native form. CF3CO-proteins are elicited in the course of oxidative biotransformation of halothane, and include the trifluoroacetylated forms of protein disulfide isomerase, microsomal carboxylesterase, calreticulin, ERp72, GRP 78, and ERp99. Current evidence suggests that CF3CO-proteins arise in all halothane-exposed individuals; however, the vast majority of individuals appear to immunochemically tolerate CF3CO-proteins. The lack of immunological responsiveness of these individuals towards CF3CO-proteins might be due to tolerance, induced through the occurrence of structures in the repertoire of self-determinants, which immunochemically and structurally mimic CF3CO-proteins very closely. In fact, lipoic acid, the prosthetic group of the constitutively expressed E2 subunits of the family of mammalian 2-oxoacid dehydrogenase complexes and of protein X, was shown by immunochemical and molecular modelling analysis to be a perfect structural mimic of N6-trifluoroacetyl-L-lysine (CF3 CO-Lys), the major haptenic group of CF3CO-proteins. As a consequence of molecular mimicry, autoantibodies in patients' sera not only recognize CF3CO-proteins, but also the E2 subunit proteins of the 2-oxoacid dehydrogenase complexes and protein X, as autoantigens associated with halothane hepatitis. Furthermore, a fraction of patients with halothane hepatitis exhibit irregularities in the hepatic expression levels of these native, not trifluoroacetylated autoantigens. Collectively, these data suggest that molecular mimicry of CF3CO-Lys by lipoic acid, or the impairment thereof, might play a role in the susceptibility of individuals for the development of halothane hepatitis.