Colorimetric detection of herpes simplex virus by DNA in situ sandwich hybridization: a rapid, formamide-free, random oligomer-enhanced method.
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Base Sequence↗
Biomedical subjects
K T Montone
Publications and source records attributed to K T Montone.
A recent decrease in the time to development of monomorphous and polymorphous posttransplant lymphoproliferative disorder.
We have noted a decrease in the time to development of posttransplant lymphoproliferative disorder (PTLD) over the last two and one-half years in our multiorgan transplant program. From February 1965 until December 1990, 1622 transplants were performed including 1489 kidneys (KTxp), 87 livers (LTxp), and 46 pancreata. Between February 1965 and July 1988 (group 1), there were 1260 transplants performed and nine cases of either monomorphous PTLD (M-PTLD, n = 8) or polymorphous PTLD (P-PTLD, n = 1) were diagnosed. The mean time to development of PTLD was 163 +/- 128 weeks, all after KTxp. Five of these nine patients received haploidentical living-related grafts. All patients had presented with advanced disease, none had transplant nephrectomy, and all died of their disease. Between July 1988 and December 1990 (group 2), 362 transplants were performed, and four cases of M-PTLD and three cases of P-PTLD were recognized. Of the seven cases of PTLD in group 2, six developed within 90 days posttransplant (early PTLD). The mean time to development of PTLD was 11 +/- 16 weeks. This was significantly earlier than group 1 (P less than .01). Four of the five cases after KTxp had a 1 or 2 DR-matched donor. Five of these seven patients had serological evidence of recent Epstein-Barr Virus infection, and four of these five had received OKT3 and then developed early PTLD. In group 2, three patients are alive 7-15 months after KTxp nephrectomy, the remaining four have died. We hypothesize that risk factors for the development of PTLD may include heavy immunosuppression, including the use of OKT3, good DR matching, and active EBV infection. Treatment should include graft removal, if applicable, and reduction or cessation of immuno-suppression.
Herpesvirus 4, Human↗
In situ hybridization for Epstein-Barr virus NotI repeats in posttransplant lymphoproliferative disorder.
Epstein-Barr Virus-associated posttransplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. In PTLD, B-cell expansions range from reactive hyperplasias to large cell lymphomas and are often associated with active Epstein-Barr virus (EBV) infection. The lymphoproliferations may infiltrate transplant allografts and therefore may need to be distinguished from acute cellular rejection (ACR). A total of 36 tissue specimens from 11 transplant patients (six kidney, two heart, three liver) with PTLD were studied for EBV content by automated in situ hybridization (ISH) on formalin-fixed or Bouin's-fixed, paraffin-embedded tissue using a synthetic 3' terminally biotin-labeled oligonucleotide DNA probe from the EBV NotI tandem repeat region. The NotI repeat is abundantly transcribed during productive EBV infection and may encode an EBV early antigen. EBV serologies from the 11 patients showed seven primary acute infections and one acute reactivation. Two serologic studies indicated infection of indeterminant onset, and serology was not performed on one patient. Histologically, seven patients presented with polymorphous infiltrates in transplant allograft biopsies, three of which progressed to disseminated monomorphous cell populations and death within 3 to 6 wk. Tissues examined by ISH from all 11 patients showed nuclear staining for EBV in the atypical lymphoid infiltrates (34/36 specimens). The nuclear signal ranged from a stippled pattern of positivity to homogeneous nuclear staining and was localized predominantly in follicular center cells and immunoblasts, although some smaller lymphocytes also contained the viral genome. ISHs performed on 31 allograft biopsies with ACR from 24 transplant patients (six kidney, five heart, 13 liver) without clinical evidence of PTLD and with serological evidence of past EBV infection were negative for the virus. Cell lines containing EBV in the productive state (EB3 and P3HR1) were positive with ISH for NotI, while a latently infected cell line (Raji) was negative. These data indicate that ISH with the NotI probe identifies amplified genome in EBV infections and is useful in discriminating the atypical infiltrate of EBV-associated PTLD from that seen in ACR.
Adolescent↗
Demonstration of passenger leukocytes in a case of Epstein-Barr virus posttransplant lymphoproliferative disorder using restriction fragment length polymorphism analysis.
Lymphocytic populations of the posttransplant lymphoproliferative disorder in a completely matched renal allograft and a recipient's regional lymph node were examined, using restriction fragment length polymorphism analysis with probe YNH24 to the variable number tandem repeat D2S44. The donor's DNA was found in lymphocytes extracted from both the grafted kidney and the recipient's lymph node 26 days after engraftment. In both these organs, the results of in situ hybridization with a terminally biotin-labeled oligonucleotide probe to the Not I tandem repeat region of the Epstein-Barr virus (EBV) genome, as well as those of Southern blot hybridization to the EBV nuclear antigen region of the EBV genome, were positive. These findings confirmed the presence of the donor's lymphocytes ("passenger leukocytes") in the host nodal tissue in human renal transplantation and implicated EBV as playing a role in the development of posttransplant lymphoproliferative disorder. It is speculated that the EBV proliferative stimulus contributed to the recipient and the donor lymphocyte expansions. Alternatively, the proliferation of both lymphocyte populations could result from a mutual stimulation by minor histocompatibility or other antigens.
Cell Division↗
Fetal akinesia deformation sequence (Pena-Shokeir phenotype) associated with acquired intrauterine brain damage.
An infant with Pena-Shokeir phenotype was born to a cocaine-using mother. The pathologic findings included polyhydramnios, facial anomalies, arthrogryposis, camptodactyly, pulmonary hypoplasia, and tetralogy of Fallot. The neuropathologic findings were diffuse brainstem and spinal cord neuronal degeneration and focal cerebral infarction, consistent with acquired intrauterine ischemic damage.
Abnormalities, Multiple↗
Detection of Epstein-Barr virus genomes by in situ DNA hybridization with a terminally biotin-labeled synthetic oligonucleotide probe from the EBV Not I and Pst I tandem repeat regions.
We describe a case of acute, disseminated Epstein-Barr virus (EBV) infection which was analyzed for the cellular distribution of viral replication by automated, colorimetric in situ DNA hybridization using a single, synthetic, terminally biotin-labeled oligonucleotide probe composed of 23 consecutive nucleotides selected from the EBV Not I region. The GC-rich, Not I region is a 125-base pair sequence that is repeated in tandem an average of 12.6 times in the EBV genome. The synthetic sequence had 91% base homology with another EBV genomic tandem repeat, the 102-base pair Pst I region, which is also GC-rich, has an overall 70% homology with the Not I region, and is reiterated about 25 times in the viral DNA. Disseminated EBV infection was detected in nuclei of atypical lymphocytes in several organs, including lung, bronchus, trachea, spleen, liver, and stomach, with the probes. In addition, the synthetic oligomer compared favorably with a significantly more expensive, nick translated, biotinylated probe cloned from the BAM HI-V (W), large internal repeat region. This 3.0-kilobase pair (kbp) sequence is repeated an average of 11 times in EBV. Although both probes identified regions repeated multiple times in the virus, and each confirmed an identical tissue distribution for the infection, the signal obtained with the Not I/Pst I probe was more intense and confined to the nuclei of fewer lymphocytes than the general, more weakly distributed signal obtained with the probe from the large internal repeat region. Consistent positive cellular staining was obtained with the Not I/Pst I probe in both EBV-infected control tissue culture cells and in formalin-fixed, paraffin-embedded tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Serotonergic and nonserotonergic projections from the rat interpeduncular nucleus to the septum, hippocampal formation and raphe: a combined immunocytochemical and fluorescent retrograde labelling study of neurons in the apical subnucleus.
This study examined the subnuclear distribution and transmitter content of neurons in the interpeduncular nucleus (IPN) that projected to the septum, dorsal hippocampal formation, and/or raphe. Following the injection of fast blue into the medial septum/diagonal band nucleus and rhodamine-conjugated microspheres into the dorsal hippocampal formation (or vice versa), retrogradely-labelled cells were found throughout the apical subnucleus of the IPN. Incubation of these sections with 5-hydroxytryptamine antiserum indicated that a small number of fast blue- or rhodamine-positive cells also contained serotonin. Occasional apical cells contained both fast blue and rhodamine, indicating a dual projection via collaterals to both the septum and hippocampus. Injection of either dye into the raphe also retrogradely labelled cells in the apical subnucleus, none of which contained serotonin. These results suggest that the IPN may function to integrate the activity within subcortical limbic nuclei via widespread serotonergic and non-serotonergic projections.
Animals↗
Anatomic viral detection is automated: the application of a robotic molecular pathology system for the detection of DNA viruses in anatomic pathology substrates, using immunocytochemical and nucleic acid hybridization techniques.
This paper presents the first automated system for simultaneously detecting human papilloma, herpes simplex, adenovirus, or cytomegalovirus viral antigens and gene sequences in standard formalin-fixed, paraffin-embedded tissue substrates and tissue culture. These viruses can be detected by colorimetric in situ nucleic acid hybridization, using biotinylated DNA probes, or by indirect immunoperoxidase techniques, using polyclonal or monoclonal antibodies, in a 2.0-hour assay performed at a single automated robotic workstation.
Antigens, Viral↗