Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in S?o Paulo City,Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13–15, 17, 18, 24, 26, 29, 33, 44) have not.DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.     

Primary Immunodeficiency Syndrome in Spain: First Report of the National Registry in Children and Adults

The Spanish Registry for Primary Immunodeficiency Diseases (REDIP) was organized in 1993. One thousand sixty-nine cases of primary immunodeficiency diseases (PID) were registered in patients diagnosed between January 1980 and December 1995. PID diagnosis was made according to the World Health Organization criteria. The most frequent disorders were IgA deficiency (n = 394) and common variable immunodeficiency (n = 213), followed by severe combined immunodeficiency (n = 61), C1 inhibitor deficiency (n = 52), X-Iinked agammaglobulinemia (n = 49), IgG subclass deficiency (n = 48), and chronic granulomatous disease (n = 32). A comparative study between REDIP and data recently obtained from the European registry (ESID Report, 1995) revealed important differences between phagocytic disorders and complement deficiencies reported in both registries, 4.9 vs 8.7 and 6.0 vs 3.6, while percentages of predominantly antibody deficiencies and T cell and combined deficiencies concurred with those reported in the European registry, 69.3 vs 64.7 and 14.7 vs 20.2, respectively. The heterogeneous nature of the geographical distribution of cases submitted may indicate underdiagnosis of PID in some country areas; surprisingly, the interval between the onset of clinical symptoms and diagnosis was significant, even in immunodeficiency diseases, such as IgA deficiency, which are easy to diagnose.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Comparison of immunofluorescence with enzyme immunoassay for detection of Q fever

To evaluate enzyme immunoassay (EIA) as an alternative to indirect immunofluorescence assay (IFA) to screen for Q fever in humans, 157 serum samples from patients suspected of having the disease were tested for immunoglobulin G antibodies toCoxiella burnetii. The agreement between the tests and the sensitivity of EIA were excellent (96.8% and 98.4%, respectively) when an IFAtiter of > 1/160 was considered positive. All serum samples with a titer of > 1/320 in the IFA were also positive by the EIA. The EIA seems to be an acceptable alternative to IFA for screening for Q fever.     

Heterogeneous pattern of chromosomal breakpoints involving the MYC locus in multiple myeloma

Chromosomal rearrangements of the MYC locus, which often involve the IG loci, are recurrent events in multiple myeloma (MM) and plasma cell leukemia (PCL). We used dual-color fluorescence in situ hybridization (FISH) to characterize the breakpoint locations of chromosomal translocations/rearrangements involving the MYC locus at 8q24 found in a panel of 14 MM cell lines and 70 primary tumors (66 MM and 4 PCL). MYC locus alterations were observed in 21 cases: MYC/IG (mainly IGH@) fusions in 11 cell lines and three patients (2 MM and 1 PCL), and extra signals and/or abnormal MYC localizations in seven patients (5 MM and 2 PCL). Fourteen of these cases were investigated by FISH analyses by use of a panel of BAC clones covering about 6 Mb encompassing the MYC locus. The breakpoints were localized in a region 100-250 kb centromeric to MYC in four cases, a region 500-800 kb telomeric to the gene in four cases, and regions > or = 2 Mb centromeric or telomeric to MYC in five cases. Two different breakpoints were detected in the KMS-18 cell line, whereas the insertion of a MYC allele was found in a complex t(16;22) chromosomal translocation in the RPMI8226 cell line. Our data document a relatively high dispersion of 8q24 breakpoints in MM.     

Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis

Peritoneal fibrosis is one of the most common morphological changes observed in continuous ambulatory peritoneal dialysis (CAPD) patients. Both resident fibroblasts and new fibroblast-like cells derived from the mesothelium by epithelial-to-mesenchymal transition are the main cells involved fibrogenesis. In order to establish markers of peritoneal impairment and pathogenic clues to explain the fibrogenic process, we conducted an immunohistochemical study focused on peritoneal fibroblasts. Parietal peritoneal biopsies were collected from four patient groups: normal controls (n=15), non-CAPD uremic patients (n=17), uremic patients on CAPD (n=27) and non-renal patients with inguinal hernia (n=12). To study myofibroblastic conversion of mesothelial cells, -smooth muscle actin (SMA), desmin, cytokeratins and E-cadherin were analyzed. The expression of CD34 by fibroblasts was also analyzed. Fibroblasts from controls and non-CAPD uremic patients showed expression of CD34, but no myofibroblastic or mesothelial markers. The opposite pattern was present during CAPD-related fibrosis. Expression of cytokeratins and E-cadherin by fibroblast-like cells and -SMA by mesothelial and stromal cells supports that mesothelial-to-myofibroblast transition occurs during CAPD. Loss of CD34 expression correlated with the degree of peritoneal fibrosis. The immunophenotype of fibroblasts varies during the progression of fibrosis. Myofibroblasts seem to derive from both activation of resident fibroblasts and local conversion of mesothelial cells.Manuel López-Cabrera and Rafael Selgas contributed equally to the article.     

Polymorphisms of toll-like receptor 2 and 4 genes in rheumatoid arthritis and systemic lupus erythematosus

Human toll-like receptors (TLRs) participate in the innate response and signal the activation of adaptive immunity. Therefore, these TLRs may be important in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We investigated, by using a polymerase chain reaction restriction-fragment length polymorphism method, the possible association between the polymorphisms of TLR2 (Arg677Trp and Arg753Gln) and TLR4 (Asp299Gly and Thr399Ile) genes with the susceptibility or severity of RA and SLE. Our study population consisted of 122 patients with SLE, 224 patients with RA, and a control group of 199 healthy individuals. The TLR2 polymorphisms were very rare in our population; no individual carrying the TLR2-Arg677Trp polymorphism was observed, whereas the TLR2-Arg753Gln polymorphism was present in only 1% of the total population. We found no statistically significant differences in the TLR4-Asp299Gly and the TLR4-Thr399Ile genotype or allele distribution between SLE patients, RA patients, and control individuals. Similarly, no association was found with any of the demographic and clinical parameters tested either in RA or in SLE patients. In conclusion, a case-control study was used to analyze, for the first time, the influence of TLR2 and TLR4 gene polymorphism on the predisposition and clinical characteristics of SLE and RA but provided no evidence for association of TLR2 or TLR4 gene polymorphism with either disease in the population under study.     

Single fiber electromyography (SFEMG) in mitochondrial diseases (MD)

Conventional EMG, nerve conduction studies and SFEMG were performed in 18 patients with various phenotypes of MD. 14 cases showed findings consistent with mild myopathy, 2 patients signs of sensory-motor axonal neuropathy and 2 cases a mixture of myopathy and axonal neuropathy. Motor unit fiber density was mild increased in 8 out of 13 tested cases. Jitter was abnormal in 10 out of 18 tested patients. Jitter abnormalities were not related to myopathic or neurogenic features in the EMG study, and may be observed in muscles without clinical weakness. The results suggest the existence of neuromuscular transmission disturbances in patients with MD.     

The N-terminal domain of the vaccinia virus E3L-protein is required for neurovirulence, but not induction of a protective immune response

Encephalitis is a rare, but serious complication from vaccination against smallpox using replication competent strains of vaccinia virus. In this report we describe mutants of vaccinia virus, containing N-terminal deletions of the vaccinia virus interferon resistance gene, E3L, that are attenuated for neuropathogenesis in a mouse model system. These recombinant viruses replicated to high titers in the nasal mucosa after intra-nasal infection of C57BL/6 mice but failed to spread to the lungs or brain. These viruses demonstrated reduced pathogenicity after intra-cranial infection as well, indicating a decrease in neurovirulence. Intra-nasal inoculation or inoculation by scarification with a low dose of recombinant virus containing a deletion of the entire N-terminal domain of E3L protected against challenge with a high dose of wild-type vaccinia virus, suggesting that this replication competent, but attenuated strain of vaccinia virus may have promise as an improved vaccine for protecting against smallpox, and as a vector for inducing mucosal immunity to heterologous pathogenic organisms.     

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.