Detection of viral genome in non-neural tissues of cattle experimentally infected with bovine herpesvirus 1

Various bone disorders become manifest as cystic lesions. The differential diagnosis must include benign and malignant tumors and also non-tumorous lesions, such as osteomyelitis. The most important and most frequent types of genuine bone cyst are juvenile bone cyst and aneurysmal bone cyst. When juvenile bone cysts occur in adults they are called solitary bone cysts. Despite intensive research the pathogenesis of bone cysts is still unknown to this day, so that successful causal therapy is impossible. The main problem in the treatment of bone cysts is their high rate of recurrence, rates ranging between 20% and 50% having been cited in the international literature. A critical review of the literature reveals few publications with helpful follow-up results. Most of the publications are case reports, and they frequently merely describe various forms of treatment. More recent reports are mainly concerned with such methods as curettage, steroid injections, and continuous decompression with perforated screws. Until the early 1980s, segmental bone resection was the treatment of choice. Because of its high complication rate it has since been abandoned. In the last analysis, the only well-established method for which long-term results obtained in studies of any size have been published, is curettage of the cyst and grafting with cancellous bone from the iliac crest. In our series, 41 patients were treated with this method, and we recorded a recurrence rate of 17.1%. Complications were rare. The risk of recurrence depended on the age of the patient. A higher recurrence rate must be expected in children under the age of 10 years. For this reason, operative treatment should not be performed until after that age if possible. Newer methods, such as steorid injections and continuous decompression by means of perforated screws, had better results in some studies, but only according to a few authors. Further research is needed to show whether our method will yield good results in the long term when applied in larger patient collectives.     

Detection of Ureaplasma urealyticum by PCR and biovar determination by liquid hybridization

An assay which combines the direct detection of Ureaplasma urealyticum with biovar determination was developed and applied to 618 urogenital specimens. U. urealyticum was detected by inhibitor-controlled PCR. A 429-bp fragment of the urease gene was amplified. The amplicons were labelled with digoxigenin during PCR. Biovar determination was performed by liquid hybridization with biotin-labelled biovar-specific probes, and the hybrids were detected with peroxidase-conjugated sheep anti-digoxigenin immunoglobulin G Fab fragments. Results of PCR and culture for 453 urogenital specimens from women and 105 urethral specimens from men could be compared. Among the specimens from women, 63% were PCR positive as well as culture positive, 0.9% were positive only by PCR, and 4% were positive only by culture. Among the specimens from men, 15% were PCR positive as well as culture positive, 1% were positive only by PCR, and 9% were positive only by culture. By using culture as the reference method, the PCR had a sensitivity of 94% and a specificity of 98% when applied to specimens from women and a sensitivity of 64% and a specificity of 99% when applied to specimens from men. Overall, 80% of the PCR-positive specimens contained biovar 1,13.5% contained biovar 2, and 6.5% contained both biovars.     

Non-radioactive detection of K-ras mutations by nested allele specific PCR and oligonucleotide hybridization

PURPOSE: The aim of the study was to evaluate prospectively urinary alpha 1-microglobulin as a marker of proximal tubular damage following acute pyelonephritis and outflow disease of the upper urinary tract in a urological population with minimal exclusion criteria. We also measured the urinary gamma-glutamyltransferase activity, urinary albumin, urinary and serum creatinine, serum IgA and serum alpha 1-microglobulin. PATIENTS AND METHODS: We studied 483 urological patients (age: 1 to 92 years, 297 men, 186 women) excluding patients receiving nephrotoxic drugs, or suffering from type 1 diabetes or renal diseases. There were 141 patients with urinary tract infection but no fever, 36 patients with high fever of non-renal origin, 51 patients with acute pyelonephritis and 156 patients with outflow disease of the upper tract, and 99 patients were included in the reference population. RESULTS: For acute pyelonephritis, vesico-ureteral reflux, and ureteral obstruction, urinary alpha 1-microglobulin had a sensitivity of 94%, 90% and 63% respectively and a specificity of 67%, 77% and 76%. The area under the curve of the receiver operator characteristic curve was significantly (p < 0.001) higher for urinary alpha 1-microglobulin than for albumin or gamma-glutamyltransferase activity. Unexpected positive results were found in acute prostatitis. The urinary alpha 1-microglobulin was the only parameter which differentiated between acute prostatitis and pyelonephritis (p < 0.001). Creatinine clearance or age had little and gender had no influence on the urinary excretion of alpha 1-microglobulin. Urine production rate significantly increases the urinary alpha 1-microglobulin/creatinine ratio. CONCLUSION: Our results suggest that the urinary alpha 1-microglobulin/creatinine ratio is a diagnostically useful marker of tubular damage in acute pyelonephritis and vesico-ureteral reflux in the urological population. Following renal colic and chronic ureteral obstruction, a significant increase in urinary alpha 1-microglobulin excretion was observed.     

Analysis of Yq microdeletions in infertile males by PCR and DNA hybridization techniques

Defects in spermatogenesis have been found associated with deletions of different portions of Y chromosome long arm (Yq), suggesting the presence of the azoospermia factor in the control of spermatogenesis. We studied 67 men with idiopathic azoospermia and severe oligozoospermia, cytogenetically normal, for the presence of microdeletions on Yq chromosome. By using polymerase chain reaction (PCR) and Southern blotting techniques we analysed the AZFa, AZFb and AZFc loci on Yq, where deletions have been associated with defects in spermatogenesis. Deletions of a portion of the Y chromosome were detected in five patients. Four of these patients shared deletions in distal Yq11 interval 6, including the DAZ gene, while one patient lacked loci in the proximal Yq11. Testicular histology of two patients bearing distal Yq11 deletions showed two different spermatogenic defects including Sertoli cell-only (SCO) syndrome and maturation arrest, while the patient with microdeletions in the proximal Yq11 showed a SCO phenotype.     

Detection of viral DNA in neonatal herpes encephalitis autopsy tissues by solution-phase PCR: comparison with pathology and immunohistochemistry

To detect DNA sequences of herpes simplex virus (HSV) in neural and non-neural tissue sections in disseminated human neonatal HSV infection, a solution polymerase chain reaction (PCR) protocol was developed which amplified HSV thymidine kinase and host genomic DNA sequences that were hybridized with sequence-specific probes in Southern blots. Serial sections of formalin-fixed, paraffin embedded autopsy tissues were tested by PCR and compared to histology and HSV antigen detection. The sensitivity, specificity and reproducibility of this PCR protocol were determined on uninfected and HSV-infected mouse tissues and on HSV DNA from infected tissue culture cells. Samples estimated to contain as few as 60 copies of preserved HSV DNA target sequence gave a positive PCR result. In nine neonates that died during acute HSV infection, all non-neural tissues and a minority of neural tissues with histological lesions had HSV antigen; when DNA could be amplified, HSV DNA sequences were detected by PCR. Together, these findings indicate a direct role for virus in the pathogenesis of these lesions. In the same cases, some or all brain samples were negative for HSV antigen, but nevertheless had HSV DNA sequences detected by PCR. The possible explanations for this finding are discussed. In one neonate dying seven weeks after birth, HSV sequences were found in brain lesions in the absence of HSV antigen; neither HSV DNA nor antigen were found in non-neural tissues, suggesting a latent HSV infection in brain. It is practical to apply PCR methods to detect minute quantities of viral DNA in formalin-fixed, paraffin embedded autopsy tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.     

Use of asymmetric PCR to generate long primers and single-stranded DNA for incorporating cross-linking analogs into specific sites in a DNA probe

Photoactivatable DNA analogs have been incorporated enzymatically into DNA and used to map the locations of polypeptides in protein complexes bound to DNA. We have developed a procedure for generating long primers from short oligodeoxyribonucleotides (oligos) to incorporate DNA cross-linkers at specific sites within either strand of DNA probes of < or = 206 bp. Single-stranded DNA molecules of 52-206 nucleotides in length were generated by asymmetric polymerase chain reactions (aPCR), using an excess of one short sense-strand primer to be extended and a limiting amount of each short antisense primer that is complementary to and defines the 3' end of the long primer to be generated. The noncross-linking strand of the DNA probe was also generated by aPCR from the DNA sequence of interest. The long primers were annealed to the full-length noncross-linking DNA strand to form a partially double-stranded DNA. Cross-linking analogs and radioactive deoxyribonucleotides (dNTPs), followed by normal dNTPs, were enzymatically incorporated onto the long primers to form the double-stranded DNA cross-linking probes. This method is reproducible and avoids many of the difficulties encountered by other published methods.     

Detection of M. leprae by gene amplification; combined ethidium-bromide staining and probe hybridization

Human and animal studies have clearly demonstrated the advantageous effects of sensorially enriched rearing environments. Nevertheless, little work has been done concerning the long-lasting persistence of all these behavioral modifications. To undertake this question, a very early enrichment animal model was used. From days 10 to 24 after birth, 28 male albino rats were exposed to a multisensory stimulated environment, while other 28 littermates constituted the control group. At 3 and 6 months old two cognitive abilities were analyzed; the spatial working memory (short term memory) and the latent learning capacity (long term memory). The results evidenced an improved working memory in both 3 and 6 months old rats exposed to the early enriched environment. Moreover, the adult early stimulated group performed as well as younger subjects both on error scores and speed to solve this test. Only in the adult group of rats a superior latent learning capacity of stimulated subjects was evidenced. To conclude, the early enriched environment induced: a) persistent cognitive benefits in the adult rat and b) a more relevant influence on the subsequent behavior of older rather than younger subjects.     

Titration of varicella-zoster virus DNA in throat swabs from varicella patients by combined use of PCR and microplate hybridization

We devised a simple procedure for titration of varicella-zoster virus (VZV) DNA in throat swabs from varicella patients. DNA which was extracted from throat swabs, together with known copy numbers of a cloned VZV DNA fragment, were 10-fold serially diluted and used as template in PCR. The PCR products, after heat denaturation, again serially diluted in 1.5 M NaCl and adsorbed to microplate wells. Then, biotin-labeled DNA probes were hybridized with the immobilized DNA. The hybridization signal was produced by streptavidin-conjugated beta-galactosidase and a fluorogenic enzyme substrate. By comparing the titration curves of a clinical specimen with those of the cloned fragment, of which detection limit was about 10 copies, we estimated the copy numbers of VZV DNA in the specimen. With this technique, we evaluated the degree of potential contagiousness of the patient along the course of infection: we found that varicella patients possessed highest quantity of VZV DNA in the throat on the first day of illness.     

Detection of Leptospira DNA in patients with aseptic meningitis by PCR

Samples of cerebrospinal fluid from 103 patients with aseptic meningitis were tested by PCR for detection of leptospires, and the results were compared with those of the microscopic agglutination test (MAT) and an enzyme-linked immunosorbent assay for detection of immunoglobulin M (ELISA-IgM). Of these samples, 39.80% were positive by PCR and 8.74 and 3.88% were positive by MAT and ELISA-IgM, respectively.     

PCR and DNA hybridization indicate the absence of animal filariae from vectors of Onchocerca volvulus in Uganda

In order to identify Onchocerca volvulus larvae from vectors, DNA of filaria larvae from dissected blackflies was isolated, and a 150-bp long tandemly repeated DNA sequence (0-150), which occurs in many Onchocerca species, was amplified using polymerase chain reaction (PCR). Subsequently, the PCR product was blotted onto a nylon membrane and hybridized with DNA probes specific for O. volvulus or Onchocerca ochengi. Filaria larvae from 395 infected Simulium neavei were examined and 259 samples produced detectable PCR products. Among these samples, 239 (92%) reacted with an O. volvulus-specific oligonucleotide. A sample of 69 PCR products was tested using an O. ochengi DNA probe, but all failed to hybridize. Filaria larvae from 64 infected Simulium damnosum, presumably of the cytotypes "Nyamagasani" and "Nkusi" were studied and 0-150 was amplified from 38 samples. From these samples, 35 (92%) hybridized specifically with an O. volvulus probe but none with the O. ochengi-specific DNA sequence. Nonamplified samples were obtained mainly from blackflies that contained only 1 or 2 filaria larvae, and therefore, an insufficient DNA extraction was assumed. It can be concluded that few, if any, filaria species of animal origin were transmitted by S. neavei and S. damnosum s.l. in Kabarole and Kasese districts in Uganda.     

Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.     

Identification of H. pylori in saliva by a nested PCR assay derived from a newly cloned DNA probe

A novel probe was developed from genomic DNA of Helicobacter pylori ATCC type strain 43629. It hybridized with all 73 H. pylori clinical isolates tested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41 different HaeIII-digestion patterns from 57 H. pylori strains tested. Based on the sequence of the probe, a nested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximately equivalent to one cell. No PCR products were amplified from any of 21 non-H. pylori strains tested. Using this nested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patients with gastric H. pylori infection. These data suggest that the probe is useful for typing H. pylori and that the nested PCR is a valuable tool for detecting H. pylori DNA in saliva.     

Rapid clearance of simian immunodeficiency virus particles from plasma of rhesus macaques

Perturbation of the equilibrium between human immunodeficiency virus type 1 (HIV-1) and the infected host by administering antiretroviral agents has revealed the rapid turnover of both viral particles and productively infected cells. In this study, we used the infusion of simian immunodeficiency virus (SIV) particles into rhesus macaques to obtain a more accurate estimate of viral clearance in vivo. Consistently, exogenously infused virions were cleared from plasma with an extremely short half-life, on the order of minutes (a mean of 3.3 min). This new estimate is approximately 100-fold lower than the upper bound of 6 h previously reported for HIV-1 in infected humans. In select animals, multiple tissues were collected at the completion of each experiment to track the potential sites of virion clearance. Detectable levels of SIV RNA were found in lymph nodes, spleen, lungs, and liver, but not in other tissues examined. However, only approximately 1 to 10% or less of the infused virions were accounted for by the thorough tissue sampling, indicating that the vast majority of the infused particles must have been degraded over a short period of time. Should the rapid clearance of virions described here be applicable to infected patients, then HIV-1 production and thus the number of productively infected CD4(+) T lymphocytes or the viral burst size must be proportionally higher than previous minimal estimates.     

Antibody-secreting cells specific for simian immunodeficiency virus antigens in lymphoid and mucosal tissues of immunized macaques

OBJECTIVES: To examine whether the route of immunization affects the induction of antibody-secreting cells (ASC) in the circulation of macaques. The distribution of ASC in the rectal mucosa and lymphoid tissues following challenge with simian immunodeficiency virus (SIV) was investigated. DESIGN: Macaques were immunized with recombinant SIV gp120 and p27 antigens by the targeted iliac lymph node (TILN) route of immunization or the nasal and rectal route, augmented by intramuscular immunization [naso-rectal intramuscular (NRI)]. The macaques were challenged with live SIV by the rectal route and ASC were assayed in the circulation before and after SIV challenge, and in the tissues removed at post-mortem. METHODS: ASC were examined in the circulation by Elispot assay. Mononuclear cells were prepared from peripheral blood, iliac and axillary lymph nodes and spleen. Rectal tissue was treated by enzyme digestion to elute mononuclear cells. RESULTS: TILN and NRI immunization induced circulating IgA and IgG ASC to both gp120 and p27. Following rectal challenge with SIV, TILN macaques were protected from infection whereas NRI route-immunized and unimmunized controls became infected. IgA ASC to p27 were increased significantly in the iliac lymph nodes of the TILN immunized macaques compared with unimmunized controls (P < 0.05). Only IgA ASC were found in the rectal mucosa of the immunized protected macaques but both IgA and IgG ASC were detected in the unimmunized infected macaques. Overall the number of IgG ASC specific for p27 was significantly higher in the infected NRI and control macaques than in the protected macaques (P < 0.02). A progressive increase in IgG but not IgA ASC was detected in the peripheral blood mononuclear cells of the unimmunized infected macaques. CONCLUSIONS: The results suggest that cells secreting IgA antibodies to p27 in the iliac lymph nodes of the TILN immunized macaques correlate significantly with protection from infection. The unimmunized infected macaques showed a progressive increase in IgG ASC in the peripheral blood after SIV challenge; this was found in the iliac and axillary lymph nodes and also in the spleen, suggesting that it is an immune response to the SIV infection.     

Detection of Naegleria fowleri cysts in environmental samples by using a DNA probe

In this study we tried to detect DNA Naegleria fowleri in artificially contaminated environmental samples, with or without sediments, containing 10(4) cysts of this pathogenic amoeba. We used two assays to extract DNA from samples: first, direct DNA extraction, which gave positive results only for water samples without sediment; second, DNA extraction after sample incubation on agar plates, which allowed us to remove amoeba growing out of the sediments, and which gave positive results for all samples, even those initially with sediments (5, 500 or 500 mg). Thus, this molecular identification appears as a powerful tool to investigate N. fowleri growth in environmental samples.     

In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production. METHODS AND RESULTS: The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion). CONCLUSIONS: Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.