Brenda Roberts

Selective Glial Vulnerability following Transient Global Ischemia in Rat Brain

Global cerebral ischemia selectively damages neurons, but its contribution to glial cell death is uncertain. Accordingly, adult male. rats were sacrificed by perfusion fixation at 1, 2, 3, 5, and 14 days following 10 minutes of global ischemia. This insult produces CA1 hippocampal neuronal death at post-ischemic (PI) day 3, but minor or no damage to neurons in other regions. In situ end labeling (ISEL) and immunohistochemistry identified fragmented DNA of dead or dying glia and distinguished glial subtypes. Rare ISEL-positive oligodendroglia, astrocytes, and microglia were present in control brain. Apoptotic bodies and ISEL-positive glia significantly increased at PI day 1 in cortex and thalamus (p <0.05) but were similar to controls in other regions and at other PI intervals. Most were oligodendroglia, although ISEL-positive microglia and astrocytes were also observed. These results show that oligodendroglia die rapidly after brief global ischemia and are more sensitive than neurons in certain brain regions. Their selective vulnerability to ischemia may be responsible for the delayed white matter damage following anoxia or CO poisoning or that associated with white matter arteriopathies. Glial apoptosis could contribute to the DNA ladders of apoptotic oligonucleosomes that have been found in post-ischemic brain.

DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Parameters evaluated in the CA1 region of the hippocampus included morphology, in situ end labeling (ISEL) of fragmented DNA, and expression of p53. Neurons were indistinguishable from controls at postischemic day 1 but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appeared normal. In situ end labeling was absent. At days 3 and 5, approximately 40 to 60% of CA1 neurons had shrunken eosinophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL. By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL. Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis. These results show that DNA fragmentation occurs after the development of delayed neuronal death in CA1 neurons subjected to 10 minutes of global ischemia. They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CA1 neurons in this model.

CD4+ and CD8+ cells accumulate in the brains of acquired immunodeficiency syndrome patients with human immunodeficiency virus encephalitis.

To test the hypothesis that CD4+ T lymphocytes accumulate in brains of end-stage acquired immunodeficiency syndrome (AIDS) patients, we examined T-lymphocyte subsets in the CA1, CA3, and CA4 regions of the hippocampus of AIDS patients with (n = 10) and without (n = 11) human immunodeficiency virus encephalitis (HIVE) plus controls (n = 7). HIV p24 antigen was common in monocytic cells and rare in activated/memory CD45RO+ lymphocytes. Hippocampal activated/memory CD45RO+ T lymphocytes significantly increased (P < .001) in seven of the eight hippocampal subregions with hippocampal HIVE (1.14 ± 1.4 T cells/high-power field [hpf]), but AIDS hippocampus without HIVE were similar to controls (0.03 ± 0.07 T cells/hpf and 0.03 ± 0.09 T cells/hpf, respectively). CD45RO+ and CD3+ lymphocytes were similar in numbers and distribution, whereas CD4+ and CD8+ lymphocytes were weakly immunoreactive and less frequent. All four lymphocyte subtypes were present in perivascular spaces and microglial nodules of HIVE, and had direct contact with neurons. Monocytes, microglia, and multinucleated giant cells were immunoreactive for CD4 in AIDS cases with hippocampal HIVE but microglia in remaining AIDS cases and controls were CD4−. CD68+ macrophages significantly increased in hippocampus of HIVE patients (P < .05) and were predominately perivascular in the absence of local HIVE. These studies show that CD4+ T lymphocytes, as well as CD8+ T lymphocytes, participate in the local inflammatory response of HIVE in end-stage AIDS patients, and suggest that their recruitment requires local HIV infection. The perineuronal location of CD4+ cells provides the potential for lymphocyte-mediated neuronal injury or trans-receptor-mediated neuronal infection.

HIV infection of choroid plexus in AIDS and asymptomatic HIV-infected patients suggests that the choroid plexus may be a reservoir of productive infection

The choroid plexus (CPx) may be an important site of viral dissemination since monocytes and dendritic cells in its stroma are infected with HIV in AIDS patients and since the ratio of CPx to brain infection is more than 2 : 1. In order to see if CPx infection also develops in asymptomatic (ASY) HIV-infected patients, we examined archival formalin-fixed brain and CPx from 14 AIDS and seven ASY cases, using routine histology, immunohistochemistry for HIV gp41, and DNA extraction and gene amplification for HIV DNA. Eight of 14 AIDS (57%) had HIV-positive cells in the CPx and four (29%) had HIV encephalitis. Two of seven ASY cases (29%) had HIV-positive cells in the CPx but none had HIV encephalitis. Extracted DNA from brain, CPx and systemic organs of five ASY cases was amplified by nested PCR with or without Southern blotting for HIV env gene. It was positive in systemic organs in five cases; in CPx in four cases; and in brain in one case. This study shows that the CPx is a site of HIV infection in ASY patients and that the frequency of CPx infection is higher than seen in brain in both AIDS and ASY cases. The results are consistent with the hypothesis that the CPx may be a site for hematogeneous spread and a reservoir for HIV infection during the period of clinical latency.

Effect of Postmortem Interval on In Situ End-Labeling of DNA Oligonucleosomes

Abstract. In situ end-labeling (ISEL) of DNA oligonucleosomes facilitates detection of apoptosis in tissue sections by binding labeled nucleotides to the oligonucleosomal fragments. Although ISEL is used in postmortem material, the effect of autolysis is not specifically known. Accordingly, normal rat brain and intestine were immersed in formalin after postmortem intervals (PMI) from 0 to 72 hours (b) or were fixed by perfusion with ethanol or paraformaldehyde-glutaraldehyde (PF-G). Omission of the binding enzymes or pretreatment with DNAase served as negative and positive controls. With DNA polymerase, material fixed by perfusion or by formalin immersion with PMI of O contained ISEL-positive nuclei only in apical intestinal cells and rare intravascular blood cells in brain. Postmortem intervals from 8 to 72 h did not alter this staining pattern although false-positive ISEL developed at section edges as a result of tissue drying. Terminal deoxynucleotidyl transferase gave similar results in the formalin-fixed material with PMI from 0-48 h but nonspecific nuclear labeling was increased with a PMI of 72 h and was widespread in the PF-G and ethanol-perfused material. This study shows that PMI of at least 72 h do not affect the sensitivity or the specificity of ISEL and confirm the reliability of this procedure in postmortem material. The results also indicate that false-positive ISEL occurs if the tissue is allowed to dry or if certain combinations of fixatives and binding enzymes are used.

Glutathione-linked detoxification pathway in normal and malignant human bladder tissue

This study compares the levels of glutathione (GSH) and GSH-dependent detoxification enzymes, which have been implicated in anti-cancer drug resistance, in paired normal and malignant human bladder tissues, a tumor with high incidence of inherent drug resistance. Although the mean GSH transferase (GST) activity did not differ significantly in normal and neoplastic bladder tissues, this enzyme activity was relatively higher in tumor tissues of five out of ten patients as compared with corresponding normal tissues. Similarly, the mean GSH content and GSH reductase activity did not differ significantly between normal and neoplastic bladder tissues. On the other hand, the mean GSH peroxidase activity towards cumene hydroperoxide and catalase activity in bladder tumors was higher by about 1.5 and 1.4 times, respectively (P < 0.05), compared with those of normal tissues. GST isoenzymes corresponding to the three major classes (alpha, mu and pi) were expressed in every normal bladder tissue examined in the present study. Overexpression of GST pi was observed in 60% of the bladder tumors, whereas alpha and mu type GST proteins in tumor tissues were lower at frequencies of 62.5% and 37.5%, respectively, compared with the corresponding normal tissues. These results suggest that (a) elevated levels of GSH peroxidase, catalase and GST pi in human bladder tumors may contribute, at least in part, to the intrinsic drug resistance of this neoplasm and (b) anti-oxidative enzymes GSH peroxidase and/or catalase may represent markers for this neoplasia, although a large number of tissue specimens must be analyzed to validate this hypothesis.

Tumor necrosis factor–alfa in nonhealing venous leg ulcers

BACKGROUND Venous leg ulcers are responsible for more than half of all lower extremity ulcerations, affecting more than one million Americans annually. Studies have demonstrated alterations in levels of proinflammatory cytokines in patients with chronic wounds, including tumor necrosis factor-alfa (TNFalpha), which may be implicated in wound chronicity. OBJECTIVE To test the hypothesis that recalcitrant venous leg ulcers have increased local tissue TNFalpha as compared to normal skin. METHODS Five patients with nonhealing healing chronic venous leg ulcers were recruited. Two 4-mm punch biopsy specimens were obtained: one from the wound margin and one from noninvolved, non-sun exposed normal skin on the flexor aspect of the forearm. Tissue samples were processed using fixed with formalin stained by immunohistochemistry for TNFalpha. Qualitative and quantitative comparisons were made for the presence of TNFalpha receptor in all tissue samples, specifically comparing the presence of TNFalpha in nonhealing venous leg ulcer samples versus normal skin. RESULTS The overall staining score for nonhealing venous leg ulcers was significantly higher compared to respective normal skin samples (P = .01). In addition, immunostaining for TNFalpha was significantly less in the two nonhealing venous leg ulcers that were present for the shortest duration compared to the other ulcers of longer duration (P = .048). LIMITATIONS The small sample size may mitigate the clinical implications of findings. CONCLUSIONS Increased levels of TNFalpha in nonhealing venous leg ulcers, especially those of longer duration, implies that excessive inflammation may be causal in wound chronicity and suggests potential therapeutic alternatives.

Differential expression of glutathione S-transferase, glutathione peroxidase and glutathione reductase in normal and malignant human breast tissues

In the present study we have compared the levels of glutathione (GSH) S-transferase, GSH peroxidase and GSH reductase in human breast tumors and adjacent normal tissues obtained from the same individuals. We have also quantitated GST pi type antigen in these samples by western blotting. GST pi activity towards 1-chloro-2,4-dinitrobenzene was found to be elevated in tumors from three out of six patients (patient nos. 2, 4 and 5), whereas this activity was suppressed in tumor from patient no. 1. Results of Western blotting using antibodies raised against GST pi of human placenta were in agreement with the GST activity data. GSH peroxidase activity with cumene hydroperoxide as substrate was found to be elevated in four tumor samples (patient nos. 2, 4, 5, and 6) but suppressed in tumor from patient no. 1. On the other hand, GSH reductase activity was elevated in three samples (patients nos. 2, 4 and 5) and downregulated in the remaining three samples (patients nos. 1, 3 and 6). These results indicate that GSH-related enzymes are differentially altered in human breast tumors and GST pi type isoenzyme(s), unlike certain other human carcinomas such as colonic, are not uniformly elevated in human breast tumors.

Tumor necrosis factor-α in vitiligo: direct correlation between tissue levels and clinical parameters.

Background: Experimental evidences have shown that tumor necrosis factor (TNF)-α may play a role in the pathogenesis of nonsegmental vitiligo, and successful cases of vitiligo treated with TNF-α inhibitors have been recently reported. Materials and methods: Two cases of refractory generalized vitiligo, which showed high tissue levels of TNF-α, were commenced anti-TNF-α antibody etanercept 50 mg weekly. A retrospective study, considering chart review and immunohistochemical staining for TNF-α, was then carried out on eight additional patients affected by untreated vitiligo. Results: Etanercept achieved improvement of vitiligo in two patients at 6-month follow-up. Five out of eight specimens showed a strong cytoplasmic staining for TNF-α. Considering all 10 cases, patients with a strong TNF-α staining were characterized by a higher vitiligo disease activity score than patients with a weak staining. Discussion: These findings, albeit limited in significance by the low number of cases and the retrospective nature of the study, confirm a probable role of TNF-α in the pathogenesis of vitiligo. The intensity of TNF-α staining in vitiligo lesions may be worth to be further studied as a biomarker for potentially successful anti-TNF-α treatment of nonsegmental vitiligo in cases refractory to conventional treatment.

Immunohistochemical localization, purification, and characterization of human urinary bladder glutathione S-transferases

This study describes immunohistochemical localization, purification and characterization of glutathione S-transferase (GST) of human urinary bladder. Even though all the three major classes of isoenzymes (alpha, mu, and pi) were expressed in human bladder, more than 90% of total GST activity was accounted for by a pi class anionic form. Human bladder alpha, mu, and pi class GSTs were immunologically related to respective isoenzymes of other human tissues. GST pi was present in all 13 samples analyzed, whereas GST alpha and mu were detected in nine and eleven samples, respectively. GST alpha of human bladder appeared to be unique, because unlike this class of GSTs of other human tissues, bladder enzyme had lower affinity for GSH linked to epoxy-activated Sepharose 6B affinity resin. Immunohistochemical staining indicated localization of GST alpha in epithelial surface cells, underlying submucosa and smooth muscle, whereas mu and pi class isoenzymes were predominantly distributed in epithelial surface cells. These results suggest that human bladder GSTs may play an important role in providing protection against xenobiotics because epithelium is considered a target for several carcinogens and all the three classes of isoenzymes are expressed in these cells.