Balloon dilatation of pediatric subglottic laryngeal stenosis during the artificial apneic pause: experience in 5 children.

INTRODUCTION: Balloon dilatation is a method of choice for treatment of laryngeal stenosis in children. The aim of procedure in apneic pause is to avoid new insertion of tracheostomy cannula. PATIENTS AND METHODS: The authors performed balloon dilatation of subglottic laryngeal strictures (SGS) in 5 children (3 girls and 2 boys) without tracheotomy. Two of them with traumatic and inflammatory SGS had a tracheal cannula removed in the past. The other 3 children with postintubation SGS had never had a tracheostomy before. The need for tracheostomy due to worsening stridor was imminent for all of them. RESULTS: The total of seven laryngeal dilatations by balloon esophagoplasty catheter in apneic pause was performed in the 5 children. The procedure averted the need for tracheostomy placement in 4 of them (80%). Failure of dilatation in girl with traumatic stenosis and concomitant severe obstructive lung disease led to repeated tracheostomy. CONCLUSION: Balloon dilatation of laryngeal stricture could be done in the absence of tracheostomy in apneic pause. Dilatation averted threatening tracheostomy in all except one case. Early complication after the procedure seems to be a negative prognostic factor for the outcome of balloon dilatation.

Monitoring of humans and animals for the presence of various Rickettsiae and Coxiella burnetii by serological methods.

Serological examination of humans in Slovakia suspected of having rickettsial infections revealed the presence of antibodies to spotted fever group rickettsiae (R. conorii, R. slovaca, and R. typhi). Of interest is the finding of serological positivity to the newly recognized "IRS" agent. Antibodies to these rickettsiae and to C. burnetii were demonstrated also in domestic and hunting dogs and pet animals. These results confirm the occurrence and possible circulation of these rickettsiae and C. burnetii in the Slovak Republic.

Protein A/G dependent ELISA a promising diagnostic tool in Lyme disease seroprevalence in game animals and hunting dogs.

One of the major problems in serodiagnosis in wild animals is unavailability of specific antiglobulin conjugate. Our study focuses on validation of Protein A/G dependent ELISA in game animals like deer and mouflons as well as in hunting dogs. Binding ability of Protein A/G-conjugate to antibodies was the highest in dogs followed by fallow deer and mouflons. Three different whole cell Borrelia antigens were used to evaluate antigen dependent variation. In new Protein A/G-ELISA the highest sensitivities (90.50%, deer; 85.37%, mouflon & 94.29%, dog) were obtained by B. garinii antigen, with no statistically significant variation (chi(2), P>0.05) among all other antigens used. Average seroprevalences observed in deer, mouflons and dogs were 44.90%, 29.41% and 30.43%, respectively. Marked influence of age on seroprevalence was noticed. Protein A/G-ELISA proved to be sensitive and promising diagnostic tool in serodiagnosis of Lyme disease in game ungulates and it can be used effectively for serosurvey in different wild mammals.

Immunoglobulin G antibodies to Borrelia burgdorferi in game animals and small mammals in eastern Slovakia.

In a survey of game animals and small mammals, the sera of 185 animals were examined for the presence of immunoglobulin G antibodies to the spirochete Borrelia burgdorferi sensu lato, which causes Lyme borreliosis. These animals comprised 59 fallow deer (Dama dama), 56 mouflons (Ovis musimon) and 70 small mammals of six different species. The sera of the fallow deer and the mouflons were examined by indirect haemagglutination assay. The sera of the small mammals were examined by modified enzyme-linked immunosorbent assay, which is available in a commercial kit. The sera of the 59 fallow deer demonstrated positivity of 40.77% (titres 1:40-1:80). The 56 mouflons demonstrated seropositivity of 17.8% (1:40-1:80). The sera of the small mammals were highly positive in the yellow-necked field mouse (Apodemus flavicollis) at 42.1% (titres 1:200-1:1,600), followed by the bank vole (Clethrionomys glareolus) at 14.3% (1:400-1:800), the common vole (Microtus arvalis) at 12.5% (1:200) and the black-striped field mouse (A. agrarius) at 10.0% (1:200-1:400-800). The authors also report the rate of infestation of these small mammals by the tick Ixodes ricinus, as these mammal species are potential reservoirs for this vector. The study focuses on the relationship between the possibility of infestation by I. ricinus and the reservoir competence of the different species under study, as well as the possible spread of disease. The detected rate of seroprevalence indicates that all the investigated animals have had contact with infected ticks.

Chlamydial infection of cats and human health.

Chlamydia psittaci var. felis is considered as a primary and important agent in the etiology of infectious diseases of the upper respiratory tract and eyes in cats, having zoonotic potential. We investigated 13 cats aged between 2 months and 7 years, in which conjunctivitis, rhinitis, laryngotracheitis, bronchopneumonia and lymph adenopathy was clinically diagnosed. To detect the antigen of C. psittaci the Clearview Chlamydia Direct Test was used for the first time. The presence of C psittaci in 10 of 13 investigated cats and in 8 nasal mucosa smears from 10 investigated cats were confirmed in conjunctiva. Using bacteriological examination different species of the genus Staphylococcus in conjunctiva from all 13 investigated animals were also confirmed. Serological investigation using complement fixation test was negative in all animals.

Serological response of cattle to Chlamydophila abortus in Slovakia in 1996-2000.

In the Slovak Republic, in 1966-2000, 37,275 blood sera of cattle were investigated for the presence of antibodies against Chlamydophila abortus using the method of complement fixation. The antibody occurrence had following tendency: in 1996--3.72%; 1997--10.02%; 1998--9.15%; 1999--15.99%; 2000--9.51% of the tested sera contained the antibodies. In most cases, antibodies in low titres, 1:32-1:64, were detected. Positive serological reactions at such serum dilutions are not indicative of the clinical disease of cattle; they reflect an immune response of the host organism following contact with the Chlamydophila abortus antigen. The chlamydial antibody titres of 1:256, which were confirmed in 1998-1999, indicate the chlamydial infections.

Serological evidence for Borrelia burgdorferi infection associated with clinical signs in dairy cattle in Slovakia.

In the course of epizootological research on Lyme borreliosis in domestic and farm animals, the serological evidence for the occurrence of this zoonosis in cattle was screened. An ELISA showed that 25.2% of cattle from seven geographical areas in Slovakia were positive for anti-Borrelia IgG antibodies. In particular localities, the seroprevalence ranged from 0.6% to 34.3%. Of 33 cases with clinical signs, 20 ELISA-positive samples were also confirmed in Western blots. The most frequent clinical signs were lameness and swollen joints, but most of the cases were asymptomatic. The occurrence of Borrelia burgdorferi antibodies and suspected clinical signs in cattle of Slovak regions indicates that veterinarians should pay attention to this disease in their clinical practice and include it within the differential diagnosis.

Prevalence of antibodies to Encephalitozoon cuniculi (microsporidia) in angora goats--a potential risk of infection for breeders.

The presence of antibodies against Encephalitozoon cuniculi in Angora goats was detected by the method of indirect immunofluorescence (IFAT). The animals reacting at the titre 1: 64 and more were considered positive. Of the total number of 48 sera examined, 4 were positive at the titre 1: 32 and 2 were positive at the titre 1: 64. The occurrence of antibodies against E. cuniculi indicates that one of the causes of disorders in the reproductive cycle in Angora goats may be microsporidia Encephalitozoon cuniculi, and that these animals may be potential sources of infection for people.

Isolation, biochemical characterization and crystallization of the p15gag proteinase of myeloblastosis associated virus expressed in E. coli.

1. The p15gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E. coli expression system. The massive expression of the intentionally truncated precursor (Pr25lac-delta gag) was accompanied by its structurally correct processing. 2. Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed. 3. The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N-terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates. The identity of both enzymes was shown. 3. Contrary to reported data, the amino acid sequence of the p15gag proteinase differs from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing. 4. Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well as 2.3 A without deterioration.

[A skin allergy test in chlamydial abortion in sheep]. / Kozný alergický test pri chlamýdiovom potrate oviec.

46 head of pregnant sheep, the Tsigaya breed, were subjected to the skin allergy test and subsequently divided into two groups. Sheep were in the 3rd month of gravidity and were a part of a flock consisting of 300 head, in which chlamydia-induced abortion was recorded in sheep. The skin allergic test was done by the Rodolakis et al. (1977) method, modified by us, to indicate the level of cell-mediated immune response. Simultaneously with it, serological examinations (complement fixation test--CFT) were performed to find out the levels of antibody against Ch. psittaci. The results of skin allergy test (SAT) and serological examination in sheep after bivalent vaccine administration are given in Tab. II. Of the total number of sheep ranked to vaccinated group, 18 head responded positively on SAT. After vaccination, 12 head responded positively though previously responded negatively. In vaccinated group one abort recorded in the sheep. No. 12 which was on the 0 day slightly positive in the skin test. High levels of antibody were found after abortion and the skin test was highly positive. The results of SAT and serological examination in sheep, when placebo was administered, are given in Tab. III. 6 sheep aborted in the group, placebo was administered, are given in Tab. III. 6 sheep aborted in the group, of which 5 were negative and one was slightly positive on the day 0 in SAT. In 4 sheep abort was accompanied with significant increase in humoral antibody against Ch. psittaci. In sheep which aborted and were negative in SAT on the day 0, a marked positivity has been indicated in the replicated SAT test.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity studies on retroviral proteinase from myeloblastosis-associated virus.

The specificity of the p15 proteinase of myeloblastosis-associated virus (MAV) was tested with nonviral high molecular weight substrates and with synthetic peptides. Peptides with sequences spanning known cleavage sites in viral polyproteins of Rous sarcoma virus (RSV) and avian leukemia viruses, as well as in BSA and HSA, were synthesized, and the rate of their cleavage by the MAV proteinase was compared. Synthetic peptides require for successful cleavage at least 4 residues at the N-terminal side and 3 residues at the C-terminal side. The proteinase shows a preference for hydrophobic residues with bulky side chains (Met, Tyr, Phe) in P3, although Arg and Gln can also be accepted. Small hydrophobic residues are required in P2 and P2', and large hydrophobic residues (Tyr, Met, Phe/p-nitro-Phe) are preferred in both P1 and P1'. The difference between the specificity of the p15 proteinase and that of the HIV-1 proteinase mostly pertains to position P2' of the substrate, where bulkier side chains are accepted by the HIV-1 proteinase (Richards et al., 1990). A good chromogenic substrate for the MAV and RSV proteinases was developed and used to further characterize the MAV proteinase activity with respect to ionic strength and pH. The activity of the proteinase is strongly dependent on ionic strength and pH. Both the kcat and Km values contribute to a higher cleavage efficiency at higher salt concentrations and show a bell-shaped pH dependence curve with a sharp maximum at pH 5.5 (kcat) and 6.5 (Km).

Inhibition of avian myeloblastosis virus reverse transcriptase by diphosphates of acyclic phosphonylmethyl nucleotide analogues.

Diphosphates of N-(2-phosphonylmethoxyethyl) derivatives of heterocyclic bases were studied in the endogenous oligo(dT)12-18 primed reaction of reverse transcriptase from detergent-disrupted AMV(MAV) retrovirions. These diphosphates (analogues of nucleotide 5'-triphosphates) exhibited an inhibitory activity towards reverse transcriptase. This inhibitory activity was dependent on the character of the heterocyclic base and decreased in the order: 2-aminoadenine greater than adenine greater than guanine much greater than cytosine much greater than thymine greater than uracil. The 2-aminoadenine derivative was more potent than either AZT-TP or ddTTP, while PMEApp had approximately the same potency as the two reference compounds (IC50 approximately 1 microM at 20 microM competing substrate). This finding is consistent with the antiviral activity of the parent nucleotide analogues against retroviruses (including HIV).

[Detection of Chlamydia trachomatis in clinical material 1985-1988]. / Dôkaz Chlamydia trachomatis z klinického materiálu za roky 1985-1988.

Investigation of 529 smears from the uterine cervix of women before delivery, during the puerperium and before induced abortion revealed positive findings in 25.2% of women before delivery and in 19.3% of women before induced abortion. Perinatal infection was confirmed by a positive finding of Ch. t. in 12.8% neonates with conjunctivitis and 15.4% from the nasopharynx. The direct immunofluorescent method was used. In another group of women examined by the serological method ELISA, from a total of 182 sera in 41.7% the presence of IgG antibodies was detected. In cervical smears from 10 seropositive women in seven Ch. t. was positive.

[Immunologic status in cattle naturally infected with the microorganisms Chlamydia trachomatis and Chlamydia psittaci]. / Imunologický status u hovädzieho dobytka prirodzene infikovaného mikroorganizmami Chlamydia trachomatis a Chlamydia psittaci.

In the present paper there is a description of immunological reactions in 12 to 14 months old bullocks, naturally infected by the microorganisms Chlamydia (Ch.) psittaci and Chlamydia trachomatis. In the course of infection by the above-mentioned microorganisms (chlamydia isolated from semen) without any clinical symptoms the activity of leucocytes and polymorphonuclear cells (PMN) in the peripheral blood is variable and the concentrations of serum immunoglobulins (IG) decrease. It has been demonstrated that the infection of bullocks by Ch. trachomatis increases the bacteria absorbing capacity of PMN cells (from 14.2 to 20.1), percent of phagocytic cells in the peripheral blood (from 13.0 to 26.5%). NBT reduction activity both in a spontaneous test (from 3 to 6) and in a stimulated test (from 8 to 12), and also myeloperoxidase activity (from 0.29 to 0.42). In these animals there were also recorded decreases (although not statistically significant) in the concentrations of serum IgG1, IgG2, IgA by about 10% and by about 30% in IgM. In Ch. psittaci-infected bulls leucocyte migration in the peripheral blood decreased (from 4.5 to 2.7 mm). PMN cell adherence (from 50 to 32), lysosome amount (from 0.63 to 0.35 mg per l) also decreased. In these bullocks decreases in the concentrations of serum Ig from 12.2 to 8.2 g per l in IgG1 and from 3.8 to 1.7 g per l in IgM were also proved.

Binding properties of avian retroviral proteins. II. Binding of protein ASLV NC(p12) to viral RNA and proviral DNA.

Binding of the major avian retroviral nucleocapsid protein ASLV NC(p12) to the MAV-1 (myeloblastosis-associated virus) proviral dsDNA and viral ssRNA was analysed using electron microscopy. Specificity of the binding was estimated by special computer programs. The NC(p12) protein bound to MAV-1 proviral dsDNA (clone pAT153--MAV-1), but specificity of this binding was not found by computer evaluation. NC(p12) also bound to nondenatured 70S viral RNA at a rate of 25 +/- 3 molecules per RNA molecule. When this RNA was denatured either before or after the complexing, it showed no binding affinity for the protein. This result implies that preserved secondary structure of the viral RNA was required for the binding.

Binding properties of avian retroviral proteins. I. Preparation and basic characterization of ASLV NC(p12) and MA(p19).

Using SP-Sephadex column chromatography we isolated from an avian retrovirus, AMV(MAV), nucleic acid-binding proteins ASLV NC(p12) and MA(p19). As shown by several criteria, namely SDS-PAGE, PR(p15) protease activity, and nucleic acid binding assay with the use of both ss and ds DNAs, our NC(p12) and MA(p19) isolates are virtually pure proteins mutually not cross-contaminated. Rabbit anti-NC(p12) and anti-MA(p19) sera which we prepared did not cross-react mutually. We conclude that both NC(p12) and MA(p19) and antibodies against them are adequately pure preparations for investigating their nucleic acid binding specificities towards AMV(MAV) genomic RNA and MAV-1 proviral DNA using electron microscopy supported by computer analysis of electron micrographs.

Binding properties of avian retroviral proteins. III. Binding of protein ASLV MA(p19) to viral RNA and proviral DNA.

The binding of the avian retroviral matrix protein ASLV MA(p19) to homologous viral ssRNA and proviral dsDNA was analysed using electron microscopic methods combined with a special computer evaluation. No binding affinity of MA(p19) to homologous nondenatured or denatured viral RNA was found. In contrast, ASLV MA(p19) was shown to have one specific binding site on MAV-1 proviral dsDNA at position 6795 +/- 345 bp from the 5' end of the molecule. A second specific binding site was found in a cellular sequence.

Transcription of the chicken myb proto-oncogene starts within a CpG island.

The nucleotide sequence of an 8.2-kb DNA fragment from the 5' proximal part of the chicken myb proto-oncogene spanning 1761 nucleotides upstream and 6436 nucleotides downstream from a presumed c-myb initiation codon was determined. A 3.3-kb G + C-rich region found in this sequence had also other features characterizing CpG islands, i.e. no CpG underrepresentation and lack of CpG methylation. In haematopoietic tissues c-myb mRNA synthesis starts in two major regions of the CpG island, namely 98 to 108 and 143 to 145 nucleotides upstream from the c-myb initiation codon. These two regions are in or close to the 124-bp evolutionarily conserved element located in the middle part of the CpG island. No alternative splicing of the 5' end of c-myb mRNA suggested earlier (1,2) was observed. The c-myb promoter contains neither TATA nor CAAT box-like structures at the usual positions. Instead, numerous potential Sp1 factor binding sites were found both upstream and downstream from the transcription initiation sites. Moreover, consensus v-myb protein DNA-binding sites were revealed in the promoter region and in sequences downstream from it.

Supercoil-induced Z-DNA formation within 5'-end of chicken myb proto-oncogene.

We have analyzed the recently sequenced and characterized 2.9 kb fragment derived from the 5'-end of chicken myb proto-oncogene with respect to structural perturbations induced by DNA supercoiling. Within the first intron a 50 bp sequence stretch was localized, starting approximately 450 nucleotides downstream from putative ATG initiation codon, which forms a non-B-DNA structure. Fine mapping with structural probes revealed the three adjacent regions with imperfect purine-pyrimidine alternation creating together relatively long Z-forming tract, parts of which may undergo a B-Z DNA transition at different superhelical densities.

A search for c-myb gene regulatory sequences: cloning and restriction analysis of the 18-kb BamHI fragment of chicken chromosomal DNA, containing the 5' part of the c-myb gene.

The 18-kb BamHI fragment of the chicken chromosomal DNA derived from the 5' end of the myb proto-oncogene has been cloned. The 5' part of this clone represented by a 7.2-kb BamHI-EcoRI fragment has been analysed by means of restriction mapping, which revealed the existence of about 2.5 kb of the CpG dinucleotide-rich sequence within this fragment. A short probe prepared from the CpG-rich sequence hybridizes with the 3.6-kb c-myb mRNA. Based on our results and published c-myb cDNA sequence, we conclude that the cloned fragment contains c-myb promoter.