Effect of administration of Streptococcus salivarius K12 on the occurrence of streptococcal pharyngo-tonsillitis, scarlet fever and acute otitis media in 3 years old children.

OBJECTIVE: Streptococcus salivarius K12 (BLIS K12) is a probiotic strain strongly antagonistic to the growth of Streptococcus pyogenes, the most important bacterial cause of pharyngeal infections in humans. Shown to colonize the oral cavity and to be safe for human use, BLIS K12 has previously been reported to reduce pharyngo-tonsillitis episodes in children or adults known to have experienced recurrent streptococcal infection. The present study was focussed upon evaluating the role of BLIS K12 in the control of streptococcal disease and acute otitis media in children attending the first year of kindergarten. PATIENTS AND METHODS: By randomization, 222 enrolled children attending the first year of kindergarten were divided into a treated group (N = 111) receiving for 6 months a daily treatment with BLIS K12 (Bactoblis®) and a control group (N = 111) who were monitored as untreated controls. During the 6 months of treatment and 3 months of follow-up, the children were evaluated for treatment tolerance, and for episodes of streptococcal pharyngo-tonsillitis, scarlet fever and acute otitis media. RESULTS: During the 6-month trial (N = 111 per group) the incidence of streptococcal pharyngo-tonsillitis, scarlet fever and acute otitis media was approximately 16%, 9% and 44% respectively in the treated group and 48%, 4% and 80% in the control group. During the 3-months follow-up (N = 29 per group) the corresponding rates of infection were 15%, 0% and 12% in the treated group and 26%, 6% and 36% in the controls. No apparent side effects were detected in the treated group either during treatment or follow-up. All of the enrolled children completed the study. CONCLUSIONS: The daily administration of BLIS K12 to children attending their first year of kindergarten was associated with a significant reduction in episodes of streptococcal pharyngitis and acute otitis media. No protection against scarlet fever was detected.

A tumor suppressor locus in familial and sporadic chordoma maps to 1p36.

Previous cytogenetic/FISH data have demonstrated 1p36 deletions in a relapsing familial clivus chordoma developed by a patient who has 2 daughters, respectively affected with childhood astrocytoma and clivus chordoma. Using an approach that combined the LOH (loss of heterozygosity) study of the father chordoma and the daughter astrocytoma and a segregation analysis from parents to sibs using 17 CA-repeats spanning 1p36.32-1p36.11, we mapped the cancer susceptibility locus in this family to the 1p36 region. The LOH and haplotype information was elaborated using a pairwise linkage analysis that gave a maximum lod score of 1.2. Additional LOH data relating to 6 sporadic chordomas allowed us to define an SRO (the smallest region of overlapping loss) of about 25 cM from D1S2845 (1p36.31) to D1S2728 (1p36.13). Our overall findings converge on mapping to 1p36 a tumor-suppressor gene involved in familial and sporadic chordoma.

A recurrent 19q11-12 breakpoint suggested by cytogenetic and fluorescence in situ hybridization analysis of three glioblastoma cell lines.

Loss of genetic material at chromosome 19 is a rather frequent finding in malignant gliomas. Loss of heterozygosity at region 19q13.3 is common in oligodendrogliomas and is also present, together with other genetic alterations on the same chromosome, in glioblastoma multiforme (GBM). Here we describe the results of cytogenetic and fluorescence in situ hybridization analysis on three GBM cell lines in which a series of complex chromosomal rearrangements affecting chromosome 19 were present. These genetic alterations suggest the presence of a common breakpoint at 19q11-12 which may point to the localization of a fragile site and/or to the presence of tumor suppressor gene(s) in the pericentromeric region of chromosome 19.

First cytogenetic study of a recurrent familial chordoma of the clivus.

Two recurrences of a familial clivus chordoma, arisen from a patient who developed the primary tumor at age of 8 years, were investigated by cytogenetic and the fluorescence in situ hybridization (FISH) approach. Of the patient's 3 daughters, 2 developed, respectively, a clivus chordoma and an astrocytoma in infancy, a familial aggregation highly suggestive of a genetic background. After a 31-year hiatus, 2 tumor recurrences, developed over 17 months, were removed surgically. Both were hypo- or nearly diploid, and had a pronounced karyotypic heterogeneity with clonal and non-clonal rearrangements affecting several chromosomes. The same rearrangement, a dic(1;9)(p36.1;p21), was shared in both tumor specimens and, in 90% of the cells, chromosome 1p appeared to be involved in unbalanced translocations with different chromosomes, leading to variable losses of 1p. Previous cytogenetic data concerning chordoma are limited to 10 sporadic tumors with an abnormal karyotype; although no tumor-specific rearrangements have been identified, chromosome 1p appears to be involved frequently.

Glycoconjugate and adhesion molecule changes during the cell cycle in a human embryonic epithelial cell line.

The changes in the expression of glycoconjugates and adhesion molecules were studied by selective lectin binding and immunocytochemical reactions in a human embryonic epithelial cell line (EUE cells), synchronized in the cell phases. The results can be summarized as follows: most of the tested lectins display a more diffuse binding for the cytoplasm in G1 than S and G2 phases; in the S and particularly in G2 phases the cytoplasm glycoconjugates are arranged around the nucleus; cells in mitosis always show a strong binding towards all tested lectins. Cellular fibronectin and its receptor beta 1 integrin are well expressed in G1, but the strongest reaction is observed in the S phase. The immunoreactions for laminin and uvomorulin (L-CAM) are poorly positive in all cell cycle phases. The immunocytochemical reaction for heparan sulfate is positive, with a stronger reaction in S and G2 than G1; on the contrary a diffuse staining with the anti-dermatan sulfate proteoglycan antibody appears unchanged during the cell cycle.

Chromosomal instability in fibroblasts and mesenchymal tumors from 2 sibs with Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive genodermatosis associated with increased risk of mesenchymal tumors. The putative gene has been provisionally assigned to chromosome 8. Using a cytogenetic-molecular approach, we studied lymphocytes, fibroblasts, osteosarcoma (OS) and malignant fibrous histiocytoma (MFH) from 2 affected fraternal twins, looking for constitutive markers of chromosome instability and tumor chromosomal changes which might reflect the common genetic background. The rate of spontaneous chromosome aberrations was not increased in lymphocytes. Conversely, karyotyping of primary fibroblasts from one sib evidenced chromosome breaks and both numerical and structural chromosome changes in 24% and 17% of the metaphases respectively. FISH of a 8q21.3 cosmid allowed us to detect trisomy of the target region on 7% of fibroblast nuclei from both sibs, 47% and 12% of OS and MFH cells. Pronounced chromosomal instability and clonal rearrangements leading to different chromosome-8 derivatives were detected in both tumors. CGH experiments showed multiple gains/losses, among which del(6q), also revealed by cytogenetics, and 7p gain were common, whereas 8q amplification was present only in OS. Chromosomal instability, observed in fibroblasts from the RTS patients studied, accounts for the increased risk of mesenchymal tumors in these patients.

Inhibition of axonal growth from sensory neurons by excess nerve growth factor.

Fifteen-day embryonic rat dorsal root ganglion (DRG) neurons were exposed to 1 to 200 ng/ml nerve growth factor (NGF). Maximal neurite outgrowth was obtained with 10 to 20 ng/ml. Neurite outgrowth was reduced to 89% of maximal by increasing NGF to 50 ng/ml, to 66% by 100 ng/ml, and to 18% by 200 ng/ml NGF. Identical effects were seen with mouse 2.5S NGF and recombinant human NGF. Neuron cell counts demonstrated that significant cell death did not occur. In time course experiments, significant inhibition, compared with control, began within 1 hour of adding 200 ng/ml and 3 hours of adding 50 ng/ml NGF. The inhibitory effect of NGF on neurite outgrowth was reversed within 3 hours when DRG were incubated with 5 ng/ml NGF after treatment with 50 or 200 ng/ml NGF medium for 12 hours. The inhibition demonstrated for neurons did not occur in PC12 cells; axonal growth was not inhibited by up to 1,000 ng/ml NGF. Excess brain-derived neurotrophic factor or neurotrophin-3 did not inhibit neurite outgrowth. We conclude that high concentrations of NGF produces specific and reversible arrest of neurite outgrowth from sensory neurons. This observation has important clinical implications, because these inhibitory concentrations have been exceeded when NGF has been administered into the central nervous system of humans and animals.

FISH characterization of the Xq21 breakpoint in a translocation carrier with premature ovarian failure.

A panel of ordered YAC clones, isolated using STSs in the Xq13-Xq23 region, was used to characterize by Fluorescent In Situ Hybridization (FISH) the Xq21 breakpoint in a t(X;1)(q21;p34) translocation female with premature ovarian failure. The YAC 949E11 was found to span the breakpoint, but also to join the two non-overlapping YACs 36CB1 and 40AB3, proximal and distal, respectively, to the patient's Xq21 breakpoint.

Deletion mapping of gliomas suggest the presence of two small regions for candidate tumor-suppressor genes in a 17-cM interval on chromosome 10q.

The loss of genetic material on chromosome 10q is frequent in different tumors and particularly in malignant gliomas. We analyzed 90 of these tumors and found loss of heterozygosity (LOH) in >90% of the informative loci in glioblastoma multiforme (GBM). Initial studies restricted the common LOH region to 10q24-qter. Subsequently, the study of a pediatric GBM suggested D10S221 and D10S209, respectively, as centromeric and telomeric markers of a 4-cM LOH region. It is interesting to note that, in one subset of cells from this tumor, locus D10S209 seems involved in the allelic imbalance of a larger region, with D10S214 as telomeric marker. This 17-cM region contains the D10S587-D10S216 interval of common deletion recently defined on another set of gliomas.

In situ hybridization study of myelin protein mRNA in rats with an experimental diabetic neuropathy.

Distribution of protein zero (P0) and myelin basic protein (MBP) mRNAs in the sciatic nerve from rats with alloxan-induced diabetes was analyzed at two different time points using in situ hybridization. Some animals of each diabetic group were treated with insulin. Densitometric quantitation of silver clusters revealed that 5 weeks after diabetes induction P0 mRNA only is significantly increased, while at 14 weeks both P0 and MBP mRNA contents are markedly higher than controls. Insulin treatment normalizes glycemia levels and slightly counteracts increased P0 mRNA at both stages of diabetes. An increase in MBP mRNA is observed in chronic diabetic animals only, and is unaltered by the normoglycemic effect of insulin. The increased transcript levels of P0 and MBP suggest that Schwann cells can modulate gene expression of myelin-specific proteins in response to diabetic-induced metabolic derangement. Such a change may represent a higher turnover of myelin proteins as an attempt by the Schwann cells to repair the diabetes-induced nerve damage. The observed pattern of transcript amount is only slightly influenced by insulin treatment.

Treatments with saline solutions and DNase I have different effects on DNA content and distribution in human and in mouse chromosomes.

The aim of this work was to re-examine, on a quantitative basis, the relationship between banding pattern after Giemsa staining and the amount (and distribution) of DNA along the length of the chromatid arms. To do this, we investigated by cytofluorometric methods the occurrence of possibly different extraction of chromosome DNA after some alternative G-banding procedure, i.e. treatment of chromosomes with saline solutions, or DNasi I digestion in situ. The G-banding procedure entailing trypsin pretreatment is known to be difficult to standardize; in the present investigation, it was also found that trypsin induced a massive, although quantitatively variable, extraction of DNA from fixed metaphase chromosomes. G-banding-like patterns may be obtained, by treating chromosomes preparations with saline solutions. Both PBS and Tris-HCl treatment for the times considered induced a G-banding-like pattern after Giemsa staining, regardless of the age of chromosome preparations; no banding was observed after staining DNA with PI, nor extraction of DNA was found to occur. DNase I digestion initially induced a G-banding in both human and mouse chromosomes after Giemsa staining, with concomitant extraction of DNA (but without apparent G-banding-like pattern after PI staining); after 30 min digestion, a C-banding-like pattern was observed after both Giemsa and PI staining. Exposure to PBS or Tris-HCl buffer at room temperature may therefore be recommended as a G-banding inducing treatment, since it allows the classification of single chromosomes after Giemsa staining, without determining significant displacement of genomic DNA, which can be submitted to further analysis in situ.

A stable marker chromosome with a cryptic centromere: evidence for centromeric sequences associated with an inverted duplication.

Centromere activation, an important mechanism in karyotype evolution, is occasionally observed in some human chromosome rearrangements. We report a possible occurrence of centromere activation in a marker chromosome containing an atypical centromere associated with an inverted duplication of the region 14q32 --> qter. The marker chromosome's reduced centromere lacks both the alpha and beta satellite sequences usually found at normal centromeres. In an attempt to identify the centromeric sequences, the marker chromosome was flow-sorted and amplified by a degenerate oligonucleotide primer polymerase chain reaction. Reverse chromosome painting experiments showed that the marker chromosome contains sequences that are unique to the distal region of chromosome 14, as well as a low copy number of (centromeric) sequences that are also highly represented in the centromeres of chromosomes 18 and 19. These data suggest the activation of a novel centromere in the 14q32 --> qter region, very likely consequent to the duplication of the region itself.

Hypertonic stress induces c-fos but not c-jun expression in the human embryonal EUE epithelial cell line.

Recent evidence has indicated a role for the two early response genes c-fos and c-jun in transcriptional regulation of genes acting in osmoregulation. On this basis we investigated their expression in response to hypertonic stress in the human embryonal EUE epithelial cell line. EUE cells have proven to be a useful tool for studying long-term in vitro adaptation to hypertonic stress. After culturing EUE cells in hypertonic medium a marked c-fos induction was observed, both at the mRNA and the protein level. Northern analysis of fos-mRNA showed a peak expression at 4 h, followed by a progressive decline till complete extinction at 8 h. Immunofluorescence analysis of FOS protein evidenced a similar, although slightly delayed kinetics of expression. Conversely, neither c-jun nor c-myc up-regulation could be detected. The treatment of EUE cells with cycloheximide led to superinduction of c-fos expression, (with high levels up to 12 h), and to a c-jun expression that was just detectable. Hypertonic stimulation of the transformed cell lines A549, MCF7 and JR induced both c-fos and c-jun only in JR cells. Hypertonic shock was also effective in inducing c-fos expression in fetal human diploid fibroblasts, although the response was earlier and more transient than in EUE cells. These findings indicate that c-fos is a primary response gene in hypertonic stress-activated cells, although the pattern and kinetics of its induction may differ according to the type of cell.

Cell cycle effects of hypertonic stress on various human cells in culture.

Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immunolabelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protein band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.

Nuclear phospholipids during the adaptation of human EUE cells to hypertonic stress.

The phospholipid component of interphase nuclei was analysed in EUE cells (an established cell line from embryonic human epithelium) grown in an isotonic culture medium and during the adaptation process to a hypertonic medium, using a highly specific ultracytochemical procedure, viz. labelling with the phospholipase A2 gold-complex. Within the nucleus, the phospholipids were localized in domains involved in different steps of the synthesis and processing of the RNA. These localizations did not vary at the two key steps of the adaptation process to hypertonic medium: short-term treatment (6 h) representing critical shock condition, and long-term growth (5 days) representing the adapted cells under survival conditions. On the contrary, deep changes of the labelling intensity of phospholipids at these sites occurred at the different times of hypertonic treatment and followed the same course as those observed in the ultramorphological patterns of transcription: the chromatin condensation, as evaluated by image analysis, the permanent nucleolar components, the interchromatin and the perichromatin granules. These data endorse the hypothesis that nuclear phospholipids could be involved in different steps of the transcriptional activity. They are indicative of the deep changes occurring in the EUE cells submitted to hypertonic stress.

Effects of low-power 632 nm radiation (HeNe laser) on a human cell line: influence on adenylnucleotides and cytoskeletal structures.

HeNe (632 nm) irradiation (5, 15 and 30 min) of an embryonal human cell line (EUE) was used to study the short-term effects on energy charge and the rapid, energy-dependent, remodelling processes of cytoskeletal and adhesion structures. The adenosine triphosphate (ATP) concentration, tested by luminometric and high performance liquid chromatography (HPLC) procedures, is constant after 15 and 30 min of HeNe treatment; the lower phosphorylated nucleotides, i.e. adenosine diphosphate (ADP) and adenosine monophosphate (AMP), change after 30 min in opposite directions: the ADP concentration decreases by 39% whilst that of AMP increases about sixfold. The adenylate energy charge (AEC) decreases by 21.7% in treated EUE cells (AEC = 0.65) in comparison with untreated EUE cells (AEC = 0.83). In HeNe-treated cells, the remodelling of cytoskeletal and adhesion molecules becomes evident after 15 min of treatment. The following events are important: (1) modification of stress fibre assembly and increase in vinculin-containing adhesion plaques; (2) assembly and bundling of intermediate filaments; (3) increase in laminin and L-cell adhesion molecules (L-CAM) expression. The lowered energy charge in irradiated cells is related to the increase in AMP production at the expense of ADP. ATP is dynamically constant despite its requirement in short-time remodelling processes of the cytoskeletal network which are enhanced in irradiated cells.

Myelin protein transcripts increase in experimental diabetic neuropathy.

A Northern blot analysis of P0 and MBP myelin protein transcripts in the sciatic nerve from rats with alloxan-induced diabetes at two different time points is described. After 5 weeks of diabetes induction, only P0 mRNA is significantly increased by 39%, while at 14 weeks both P0 and MBP mRNA contents are markedly higher than controls. Insulin treatment normalizes glycemia levels, partially counteracts P0 mRNA increase at both stages of diabetes and delays MBP mRNA increase present only in chronic animals. We suggest that increased transcript levels of P0 and MBP in Schwann cells may represent a higher turnover of myelin sheath specific proteins in diabetic syndrome, as attempt to repair the hyperglycemia-induced nerve damage, which is partially prevented by insulin treatment.

Identification of the chromosome 14 origin of a C-negative marker associated with a 14q32 deletion by chromosome painting.

A constitutional chromosome 14 rearrangement was observed in a female with a psychodevelopmental disorder. Karyotype analysis using a variety of chromosome techniques, QFQ, GTG, CBG, Ag-NOR and DA-DAPI, showed a deletion of chromosome 14q32.1-qter region in association with a supernumerary marker chromosome. The marker, resembling a submetacentric, approximately half the size of a G group chromosome is C band and Ag-NOR negative. The heteromorphism of the satellites showed that the deleted chromosome 14 is paternal in origin. Chromosome painting using an Alu-PCR probe specific for the human chromosome 14 and fluorescent in situ hybridization (FISH) showed that the marker contains chromosome 14q32 sequences. It is likely that the marker was generated from the deleted chromosome 14 region through a complex rearrangement.

Aldose reductase is involved in long-term adaptation of EUE cells to hyperosmotic stress.

Aldose reductase has been shown to be expressed in large amount by human embryonic epithelial cells (EUE) in response to osmotic stress. This conclusion is the result of studies undertaken following the purification to homogeneity of two forms of a 35-kDa protein overexpressed in EUE cells grown in hypertonic saline culture medium as compared to EUE cells grown in isoosmotic medium. Amino-acid composition, molecular weight and partial internal amino-acid sequence showed that the above proteins are two different forms of aldose reductase. These findings were confirmed by the observation that aldose reductase activity increased about 150-fold in adapted cells and returned to basal levels in de-adapted cells.