Ultrastructural localization of the binding fragment of tetanus toxin in putative gamma-aminobutyric acidergic terminals in the intermediolateral cell column: a potential basis for sympathetic dysfunction in generalized tetanus.

Tetanus toxin (TeTx) causes sympathetic hyperactivity, a major cause of mortality in generalized tetanus, apparently by obstructing the inhibition of sympathetic preganglionic neurons (SPNs). Neuroanatomic tracing and immunohistochemistry were used to investigate whether axon terminals in the intermediolateral cell column (IML) that synapse on SPNs and use the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) may be infected transsynaptically with TeTx. The binding fragment of TeTx (TTC; an atoxic surrogate of TeTx) and the cholera toxin B subunit (CTB; a retrograde tracer) were injected into the rat superior cervical ganglion and, over 16-48 hours, were transported to the ipsilateral IML in the caudal half of the last cervical and first three thoracic spinal cord segments. With light microscopy, diffuse CTB immunolabeling extended throughout SPN perikarya and dendrites. Punctate TTC and GABA immunolabeling were accumulated densely in the neuropil between and surrounding SPN processes. With electron microscopy, 54% of the axon terminals in the IML (n = 1,337 terminals) were TTC immunolabeled (TTC(+)), and 25% contained putative neurotransmitter levels of GABA immunolabeling (GABA(+)). On average, GABA(+) terminals had a 76% chance of also being TTC(+) and a 62% greater chance of being TTC(+) than GABA(-) terminals (P < 0.000001). Axon terminals were just as likely to be TTC(+) and/or GABA(+) regardless of whether the dendrites they synapsed on were large (>1 microM) or small in cross-sectional area or were labeled retrogradely. Sympathetic hyperactivity in tetanus may involve 1) retrograde and transsynaptic transport of TeTx by SPNs and 2) at least in part, an infection of GABAergic terminals in the IML.

Analysis of nonspecific DNA synthesis during in situ PCR and solution-phase PCR.

In this study we examine the factors that lead to nonspecific DNA synthesis during in situ PCR and solution-phase PCR. It was shown that primer-independent DNA synthesis can produce an intense signal during in situ PCR. This primer-independent pathway was apparently the result of the repair of DNA gaps induced by the heat treatment of the paraffin embedded tissue sections. This non-specific signal could be eliminated by blocking gap repair with dideoxy-TTP, avoiding heat treatment, or DNase pretreatment. The primer-independent signal was also influenced by the length and mode of fixation and the sample tissue itself. Elimination of the primer-independent signal and the use of viral primers in tissues that did not contain the virus showed that nonspecific DNA synthesis could be eliminated by the hot start modification. Primer oligomerization did not produce a signal during in situ PCR, even when it occurred robustly in the amplifying solution. Generation of the primer-independent signal in solution-phase PCR with purified DNA required a cross-linking fixative, heating, the addition of bovine serum albumin, and intact protein-DNA cross-links.

In situ detection of polymerase chain reaction-amplified HIV-1 nucleic acids and tumor necrosis factor-alpha RNA in the central nervous system.

This study determined the distribution of in situ polymerase chain reaction (PCR)-amplified HIV-1 nucleic acids in the central nervous system (CNS). Amplified viral DNA was detected in each of the seven HIV-1-positive cases and in none of the seven negative controls. HIV-1 DNA was rarely detected with standard in situ hybridization, consistent with low levels of proviral DNA. In patients with minimal clinical and pathological CNS involvement, only rare HIV-1 DNA-positive perivascular microglial cells were noted. In patients with dementia, many infected neurons and astrocytes as well as microglial cells were detected. Severe disease was also characterized by the detection of tumor necrosis factor-alpha (TNF-alpha) mRNA and viral RNA by reverse transcription (RT) in situ PCR. These results suggest that HIV-1 commonly exists in the CNS in the asymptomatic patient and that progression is marked by a dramatic increase of the number of cells with HIV-1 DNA, including neurons and astrocytes, and a concomitant upregulation of both viral and TNF-alpha transcription.

Correlation of viral infection, histology, and mortality in immunocompromised patients with pneumonia. Analysis by in situ hybridization and the polymerase chain reaction.

The purpose of this study was to determine the occurrence and histologic correlates of viral infections in immunocompromised patients with pneumonia. Of the 44 immunocompromised patients studied, 37 had AIDS. Lung tissue from these patients, including 34 with pneumocystis pneumonia, was evaluated by in situ hybridization for the presence of cytomegalovirus (CMV), adenovirus, Epstein-Barr virus, and herpes simplex virus. Fifteen of the 44 patients were positive for at least one virus (34%); CMV (13 cases) was the most common. In an additional seven cases, CMV DNA was detected using the polymerase chain reaction (PCR), for an overall viral detection rate of 22 of 44 (50%). Histologic features were diagnostic of a viral infection in nine of 15 cases (60%) of the in situ positive cases and in nine of 22 (41%) of the tissues where viral DNA was detected by PCR. Mortality rate was significantly correlated with viral detection: 77% for the viral-positive cases and 27% for the viral-negative cases (p < 0.05). We concluded that in immunocompromised patients with pneumonitis, the detection of viral DNA is strongly correlated with survival and that histologic features of the inflamed lung tissue are a specific but insensitive means of diagnosing viral presence.

Importance of different variables for enhancing in situ detection of PCR-amplified DNA.

This study determined the effects of several variables on the in situ signal after PCR amplification in fixed cells. A signal was evident in all human peripheral blood monocytes fixed in buffered formalin using primers for the human proto-oncogene bcl-2 with in situ PCR only after prolonged fixation and protease digestion. A much lower detection rate was noted after ethanol or acetone fixation due to loss of amplified product out of the nucleus into amplifying solution. This observation demonstrates the importance of cross-linking fixatives for retention of amplified DNA at the site of origin. The increased amount of target-specific DNA synthesis evident with the manual hot start modification to in situ PCR was also seen with chemical hot start mediated by the Escherichia coli single-stranded DNA-binding protein. The manual hot start method strongly suppressed in situ unwanted DNA synthesis dictated by nonsense primers; residual nonspecific synthesis was influenced by annealing temperatures and post-fixation protease digestion conditions.

Detection of amplified HPV 6 and 11 DNA in vulvar lesions by hot start PCR in situ hybridization.

We analyzed the distribution pattern of human papillomavirus (HPV) 6 and 11 DNA in vulvar lesions by in situ hybridization after amplification by the "hot start" polymerase chain reaction (PCR). HPV DNA was routinely detected in granular layer cells showing perinuclear halos and nuclear atypia by in situ hybridization with or without PCR. Cells that lack these changes rarely exhibited HPV DNA with standard in situ hybridization. After amplification, in situ analysis showed that many of the cells that lacked halos and atypia did contain HPV DNA and that the hybridization signal often localized to areas where there was a thickened granular layer. HPV DNA was not noted in the basal cells. The one copy of HPV 16 in SiHa cells was detectable after PCR with a single primer pair by in situ analysis only if the hot start modification was employed. Prior reports describing the PCR in situ methodology noted the need for from five to seven primer pairs. The hot start technique, which may be done by withholding the DNA polymerase until the temperature is sufficiently high to disfavor nontarget specific pathways, allowed the use of a single primer pair and showed that the degree of target-specific amplification, and not the size of the amplified product, determines the success of the PCR in situ technique.

An improved technique for the in situ detection of DNA after polymerase chain reaction amplification.

In situ detection of polymerase chain reaction (PCR)-amplified DNA in cell and tissue preparations previously required 5 to 7 primer pairs designed to generate a long (greater than 1,000 base pair) product. The authors describe a nonisotopic PCR in situ technique, employing a single primer pair and target sequences as short as 115 base pairs, that can detect one target molecule per cell. The essential procedural change is to withhold the DNA polymerase or primers until the reaction temperature approaches 80 degrees C. The Hot-Start method greatly increased amplification specificity which, more than product size, appears to determine success of in situ PCR. The marked improvement in specificity may permit target detection by direct incorporation of labeled nucleotides.