BOS is associated with decreased HDAC2 from steroid resistant lymphocytes in the small airways.

Immunosuppression therapies including corticosteroids fail to prevent bronchiolitis obliterans syndrome (BOS), primarily a disease of the small airways, following lung transplantation. We reported increases in steroid-resistant proinflammatory lymphocytes and their loss of histone deacetylase 2 (HDAC2), an important mediator of steroid action, in the blood of stable lung transplant recipients. We noted similar increases in the steroid-resistant lymphocytes in both the blood and small airways in BOS compared with the large airways. We hypothesized that these small airway cells would also exhibit a loss of HDAC2, and that these changes could be reversed by treatment with theophylline (HDAC2 activator). Blood, bronchoalveolar lavage and large and small airway brushings were collected from lung transplant patients with BOS (n = 12) or stable lung function (n = 18) and healthy aged-matched controls (n = 13). Intracellular proinflammatory cytokines [interferon (IFN-γ) and tumour necrosis factor (TNF)-α and HDAC2 were measured in CD8+ T, natural killer (NK) T-like and NK cells from cultured small airway brushings ± 5 mg/l theophylline ± 1 µM prednisolone using flow cytometry. Increased small airway CD8 T, NK T-like and NK cells were identified in BOS versus stable transplant and controls. In BOS, these cells exhibited increased IFN-γ/TNF-α and a loss of HDAC2. HDAC2 expression by small airway CD8+ T cells correlated with forced expiratory volume in 1 s (FEV1 ) (R = 0·880, P = 0·031). Theophylline and prednisolone synergistically up-regulated HDAC2 in CD8+ T cells. BOS is associated with loss of HDAC2 from steroid-resistant proinflammatory CD8+ T, NK T-like and NK cells in the small airways. Therapeutically increasing HDAC2 in these lymphocytes may reduce steroid resistance and improve graft survival.

Bronchiolitis obliterans syndrome is associated with increased p-glycoprotein expression and loss of glucocorticoid receptor from steroid-resistant proinflammatory CD8+ T cells.

Immunosuppressive therapy fails to suppress the production of proinflammatory cytokines, particularly by CD8+ T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and interferon (IFN)-γ/tumour necrosis factor (TNF)-α proinflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (± prednisolone ± Pgp inhibitor, cyclosporin A ± GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFN-γ/TNF-α proinflammatory cytokines. Pgp expression was increased in CD8+ Pgp+ T cells and correlated with IFN-γ/TNF-α expression and BOS grade. Reduced GCR was observed in CD8+ Pgp- T, natural killer (NK) T-like and NK cells from stable patients compared with controls, and reduced further in CD8+ Pgp- T cells in BOS. The addition of 2·5 ng/ml cyclosporin A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp+ T cells from BOS patients. The addition of 10 µM Compound A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid-resistant proinflammatory CD8+ T cells. Treatments that inhibit Pgp and up-regulate GCR in CD8+ T cells may improve graft survival.

Blood cytotoxic/inflammatory mediators in non-eosinophilic asthma.

BACKGROUND: Non-eosinophilic asthma (NEA) is a distinct, often corticosteroid-resistant inflammatory asthma phenotype. NK and NKT-like cells are effector lymphocytes that we have shown, like CD28null T cells, to be relatively resistant to steroids and major sources of pro-inflammatory/cytotoxic mediators. We hypothesized that these cells and mediators would be increased in peripheral blood in NEA. METHODS: Adults with severe asthma and variable airflow obstruction, poorly controlled despite maintenance therapy with inhaled glucocorticosteroids and long-acting bronchodilators, were recruited. Blood was assessed in those with eosinophilic asthma (n = 12), NEA (n = 25) and healthy non-smoking controls (n = 30). We applied flow cytometry to measure T, CD28null, NK and NKT-like cells and their expression of granzyme B, perforin, and killer inhibitory/activating receptors CD94(Kp43), CD158b and CD107A. Intracellular pro-inflammatory cytokine production (IFN-γ and TNF-α) was assessed in 18 controls and 10 patients with asthma/group. RESULTS: In NEA, there was increased expression of granzyme B by CD8+ T cells vs. CONTROLS: There was increased expression of granzyme B and CD158 and decreased CD94 on NK cells, vs. healthy controls and those with eosinophilic asthma. IFN-γ production by NK cells and TNF-α production by NKT-like cells in NEA were significantly increased vs. CONTROLS: In both eosinophilic and NEA phenotypes, there were significant increases in CD4+28null T cells (72% and 81% increases, respectively, vs. controls) and their expression of pro-inflammatory cytokines. Significant correlations were noted between blood CD4+28null T cells and neutrophil numbers in induced sputum, and between corticosteroid dose and blood NKT-like cells, and their production of granzyme B and TNF-α and NK IFN-γ. CONCLUSION AND CLINICAL RELEVANCE: In poorly controlled asthma, altered expression of cytotoxic/pro-inflammatory mediators can be seen on a variety of lymphocyte subsets in the peripheral blood; these changes are most apparent in NEA. Whether this pattern of expression is a marker of treatment responsiveness and future risk of exacerbations remains to be determined.

Lung cancer is associated with decreased expression of perforin, granzyme B and interferon (IFN)-γ by infiltrating lung tissue T cells, natural killer (NK) T-like and NK cells.

There is a limited understanding how of lung cancer cells evade cytotoxic attack. Previously, we have shown reduced production of the cytotoxic mediator granzyme B by CD8(+) T cells in lung cancer tissue. We hypothesized that lung cancer would be further associated with decreased production of granzyme B, perforin and proinflammatory cytokines by other cytotoxic lymphocytes, natural killer (NK) T-like and NK cells, and that this would result from soluble mediators released by the cancer cells. Lung cancer and non-cancer tissue from five patients was identified by experienced pathologists. Tumour necrosis factor (TNF)-α, interferon (IFN)-γ, granzyme B and perforin were measured in CD4 and CD8(+) T, NK T-like cells and NK cells by flow cytometry. Correlation between cancer stage and granzyme B was analysed retrospectively for 21 patients. The effects of soluble factors released by lung cancer cells on production of cytotoxic mediators and cytokines was assessed, and the role of prostaglandin E2 (PGE)2 /COX investigated using indomethacin inhibition. There were significantly decreased percentages of T, NK T-like and NK cells expressing perforin, TNF-α and IFN-γ in cancer versus non-cancer tissue, and of CD8(+) T cells and CD8(+) NK T-like cells expressing granzyme B (e.g. NK T-like cells: non-cancer 30% ± 7 versus cancer 6% ± 2·5). Cancer cells released soluble factors that inhibited granzyme B, perforin and IFN-γ production that was partially associated with the PGE2 /COX2 pathway. Thus, lung cancer is associated with decreased expression of granzyme B, perforin and IFN-γ by infiltrating T cells, NK T-like and NK cells, possibly as a result of soluble factors produced by the cancer cells including PGE2 . This may be an important immune evasion mechanism.

Up-regulation of alternate co-stimulatory molecules on proinflammatory CD28null T cells in bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is associated with lack of immunosuppression of T cell proinflammatory cytokines and increased T cell granzyme B. Repeated antigen-driven proliferation down-regulates T cell CD28. We hypothesized that down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules (CD134, CD137, CD152 and CD154) on T cells may be associated with BOS. Co-stimulatory molecules, granzyme B, perforin and intracellular cytokines were measured by flow cytometry on T cells from stable lung transplant patients (n = 38), patients with BOS (n = 20) and healthy controls (n = 10). There was a significant increase in the percentage of CD4/28(null) and CD8/28(null) T cells producing granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in BOS compared with stable patients. Down-regulation of CD28 was associated with steroid resistance and up-regulation of CD134, CD137, CD152 and CD154 on CD4(+) T cells and CD137 and CD152 on CD8(+) T cells. There was a significant correlation between increased CD28(null) /CD137 T cells producing IFN-γ, TNF-α with BOS grade (r = 0·861, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8) and time post-transplant (r = 0·698, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8). BOS is associated with down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules on steroid-resistant peripheral blood proinflammatory CD4(+) and CD8(+) T cells. Therapeutic targeting of alternate co-stimulatory molecules on peripheral blood CD28(null) T cells and monitoring response using these assays may help in the management of patients with BOS.

Role of increased CD8/CD28(null) T cells and alternative co-stimulatory molecules in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8(+) T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28(null) cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28(null) T cells in blood, lung tissue and airway. CD8/CD28(null) cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28(+) T cells. There were no changes in CD4/CD28(null) T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28(null) T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28(null) T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.

Increased T regulatory cells and decreased Th1 pro-inflammatory cytokines correlate with culture-positive infection in febrile neutropenia childhood oncology patients.

BACKGROUND: Paediatric oncology patients with febrile neutropenia are usually hospitalised and treated with empirical broad-spectrum antibiotic therapy to counter the risk of infection. However, there is currently no method available to rapidly identify bacteremia in these patients. T-helper-type-1 (Th1) cytokines are required for effective immune response to many pathogenic organisms and T regulatory cells are known suppressors of Th1 cells. We hypothesized that characterization of reduced intracellular Th1 cytokines and increased T regulatory cells (Tregs) may prove useful in identifying infection in childhood oncology patients with febrile neutropenia. METHODS: Intracellular Th 1 cytokines and Tregs were enumerated in peripheral blood from a group of childhood oncology patients with febrile neutropenia using multiparameter flow cytometry. RESULTS: There was a significant increase in the percentage of CD25(+) CD127(-) CD8(-) CD3(+) Tregs and a significant decrease in Th1 intracellular cytokines IFNγ, IL-2 and TNFα in the blood of culture positive patients compared with culture negative patients. CONCLUSIONS: Enumeration of Tregs and intracellular Th1 cytokines may provide a sensitive, specific test for determining infection in childhood oncology patients before blood culture results become available.

Time post-lung transplant correlates with increasing peripheral blood T cell granzyme B and proinflammatory cytokines.

Immunosuppression therapy following lung transplant fails to prevent chronic rejection/bronchiolitis obliterans syndrome, which we have shown is associated with lack of suppression of peripheral blood T cell granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. We hypothesized that these proinflammatory mediators may increase with time post-transplant in otherwise stable patients before clinical signs of declining lung function, and patients experiencing declining lung function would show a further increase in these mediators. Intracellular cytokine profiles and granzyme B were investigated in T cells in whole blood and airways from lung transplant patients using flow cytometry. There was a significant negative correlation between forced expiratory volume in 1 s (FEV(1) ), drug dose and time post-transplant. A significant correlation between increased granzyme B, IFN-γ, interleukin (IL)-2 and TNF-α and time post-transplant was noted in peripheral blood T cells but not lung T cells from stable patients. Patients with similar drug dose but experiencing declining FEV(1) showed a further increase in peripheral blood T cell IFN-γ, IL-2 and TNF-α. Time post-lung transplant correlates with increasing peripheral blood T cell granzyme B and proinflammatory cytokines. Declining FEV(1) is associated with a further increase in these proinflammatory mediators. Drugs that reduce these inflammatory mediators effectively may reduce the incidence of chronic graft rejection.

Posttransplant bronchiolitis obliterans syndrome is associated with bronchial epithelial to mesenchymal transition.

Bronchiolitis obliterans syndrome (BOS) compromises lung transplant outcomes and is characterised by airway epithelial damage and fibrosis. The process whereby the normal epithelial configuration is replaced by fibroblastic scar tissue is poorly understood, but recent studies have implicated epithelial mesenchymal transition (EMT). The primary aim of this study was to assess the utility of flow cytometry in detecting and quantifying EMT in bronchial epithelial cells. Large airway brushings were obtained at 33 bronchoscopies in 16 BOS-free and 6 BOS grade 1-3 patients at 2-120 months posttransplant. Flow cytometry was used to assess expression of the mesenchymal markers alphaSMA, S100A4 and ED-A FN and HLA-DR. TGF beta 1 and HGF were measured in Bronchoalveolar lavage (BAL). Expression of all three mesenchymal markers was increased in BOS, as was HLA-DR. BAL HGF, but not TGF beta 1 was increased in BOS. Longitudinal investigation of one patient revealed a 100% increase in EMT markers concurrent with a 6-fold increase in BAL TGF beta 1 and the diagnosis of BOS at 17 months posttransplant. Flow cytometric evaluation of bronchial epithelium may provide a novel and rapid means to assess lung allografts at risk of BOS.

Increased levels of T cell granzyme b in bronchiolitis obliterans syndrome are not suppressed adequately by current immunosuppressive regimens.

Bronchiolitis obliterans syndrome (BOS) is characterized by persistent alloreactive, infective and non-specific epithelial injury, loss of epithelial integrity and dysregulated repair. We have reported increased apoptosis of epithelial cells collected from the large airway in lung transplant recipients. As part of the alloreactive response, T cells induce apoptosis of target epithelial cells by secreting granzyme b. We hypothesized that granzyme b would be increased in lung transplant patients with acute rejection and BOS and that commonly used immunosuppressive agents would fail to suppress this serine protease adequately. We investigated intracellular T cell granzyme b in blood, bronchoalveolar lavage (BAL) and large airway brushing (23 controls, 29 stable transplant, 23 BOS, 28 acute rejection, 31 infection) using flow cytometry and assessed the effect of clinically relevant concentrations of cyclosporin A, tacrolimus, methylprednisolone and a protease inhibitor, gabexate mesilate, on in vitro granzyme b production. Granzyme b was increased significantly in all compartments of all transplant groups compared to controls. Surprisingly, granzyme b was even higher in patients with BOS than in patients with acute rejection. In longitudinal analysis in three patients, blood granzyme b increased prior to or at the onset of BOS. In vitro, methylprednisolone and gabexate mesilate had no effect and cyclosporin A and tacrolimus only a moderate effect on production of granzyme b by CD8(+) T cells. Increased T cell granzyme b production may contribute to BOS pathogenesis and is not curtailed by current immunosuppressants. Longitudinal investigation of granzyme b in blood may provide an adjunctive non-invasive method for predicting BOS/OB.

Airway infection in stable lung transplant patients is associated with decreased intracellular T-helper type 1 pro-inflammatory cytokines in bronchoalveolar lavage T-cell subsets.

Current immunosuppression protocols to prevent lung transplant rejection reduce pro-inflammatory and T-helper type 1 (Th1) cytokines. However, Th1 T-cell pro-inflammatory cytokine production is important in host defense against bacterial infection in the lungs. Excessive immunosuppression of Th1 T-cell pro-inflammatory cytokines leaves patients susceptible to infection. To investigate whether pulmonary infection in lung transplant recipients is associated with reduced Th1 T-cell pro-inflammatory cytokines, whole blood and bronchoalveolar lavage (BAL) fluid from 13 stable lung transplant patients with 'culture-negative' BAL and 13 patients with 'culture-positive' BAL was stimulated in vitro, and cytokine production by CD8+ and CD4+ T-cell subsets was determined using multiparameter flow cytometry. In BAL samples, there was a significant decrease in interleukin-2 (IL2) in CD3+ T cells and tumor necrosis factor-alpha (TNF-alpha) in CD8+ T cells (but not CD4+) in 'culture-positive' compared with 'culture-negative' transplant patients. There was no difference in blood Th1 T-cell cytokines between 'culture-positive' compared with 'culture-negative' transplant patients. A decrease in Th1 cytokines IL-2 and TNF-alpha in BAL T-cell subsets is associated with isolation of potentially pathogenic organisms in the lungs in stable lung transplant patients. Excessive immunosuppression of these Th1 T-cell pro-inflammatory cytokines in stable transplant patients may leave them susceptible to infection. Modifying immunosuppression by monitoring intracellular Th1 pro-inflammatory cytokines in BAL T cells may help to improve morbidity and infection rates in stable lung transplant patients.

Increased Peripheral Blood Pro-Inflammatory/Cytotoxic Lymphocytes in Children with Bronchiectasis.

OBJECTIVE: Bronchiectasis (BE) in children is common in some communities including Indigenous children in Australia. Relatively little is known about the nature of systemic inflammation in these children, especially the contribution of specific pro-inflammatory and cytotoxic lymphocyte subsets: T-cells, natural killer (NK) cells and NKT-like cells. We have shown that these cells produce increased cytotoxic (granzyme b and perforin) and inflammatory (IFNγ and TNFα) mediators in several adult chronic lung diseases and hypothesised that similar changes would be evident in children with BE. METHODS: Intracellular cytotoxic mediators perforin and granzyme b and pro-inflammatory cytokines were measured in T cell subsets, NKT-like and NK cells from blood and bronchoalveolar samples from 12 children with BE and 10 aged-matched control children using flow cytometry. RESULTS: There was a significant increase in the percentage of CD8+ T cells and T and NKT-like subsets expressing perforin/granzyme and IFNγ and TNFα in blood in BE compared with controls. There was a further increase in the percentage of pro-inflammatory cytotoxic T cells in Indigenous compared with non-Indigenous children. There was no change in any of these mediators in BAL. CONCLUSIONS: Childhood bronchiectasis is associated with increased systemic pro-inflammatory/cytotoxic lymphocytes in the peripheral blood. Future studies need to examine the extent to which elevated levels of pro-inflammatory cytotoxic cells predict future co-morbidities.

Optimal storage conditions for preserving granulocyte viability as monitored by Annexin V binding in whole blood.

It is important in the laboratory to develop techniques to preserve leucocyte viability in blood specimens for subsequent flow cytometric analysis. This article describes a new simple whole blood lysis method using Annexin V FITC staining which can be used to define both early and late apoptosis of granulocytes in heterogeneous cell populations, without the need for additional stains or to purify the cells (which may result in loss of the cells of interest). The differential Annexin V binding assay was in good agreement with the light microscopy reference method and showed excellent correlation with 7-aminoactinomycin D (7-AAD) staining. It was not affected by problems of morphological interpretation and artifactal changes of granulocyte deformability noted using light microscopy, or the technical difficulties encountered due to red cell contamination using the 7-AAD method. Using this new differential Annexin V staining method, we determined the optimum conditions that maintain granulocyte viability for subsequent flow cytometric analysis and are now employed in our laboratory. These conditions were lithium heparin (Hep) anticoagulated whole blood specimens kept at 4 degrees C with the addition of nutrient medium. Specimens that are anticoagulated with acid citrate dextrose (ACD) or ethyl-diacetyl-tetraacetic acid (EDTA) should also be treated similarly to preserve granulocyte viability and to overcome problems associated with identification of cell populations by flow cytometry.

Acquisition, retention, and extinction of operant discriminations in rats with nucleus basalis magnocellularis lesions.

Effects of bilateral ibotenic acid lesions of nucleus basalis magnocellularis (NBM) and scopolamine treatment on different aspects of learning and memory in an operant discrimination task were assessed. In Experiment 1, NBM lesions impaired acquisition performance. In Experiment 2, scopolamine lowered response rates but did not affect discrimination accuracy in lesioned or control rats. In Experiment 3, although pretrained rats showed transient increases in commission errors, percentage correct responding remained above chance levels after lesion. During extinction in Experiment 4, operant responding diminished more quickly in pretrained NBM-lesioned rats than in controls, but subsequent reacquisition performance was equivalent in both groups. Results suggest the NBM is importantly involved in discrimination learning, but cholinergic activity may be less critical for memory retention than for acquisition.

Dopaminergic agonists differentially affect open-field activity of rats with A10 lesions.

Dopaminergic systems appear to exert considerable control over locomotor activity. Although dopamine neurons are located in relatively close proximity within the mesencephalon, their axons project to more diffuse areas, perhaps reflecting some underlying heterogeneity in their function. The purpose of this study was to determine whether dopamine agonists differentially affect activity by acting upon distinct dopamine systems. Bilateral radio-frequency lesions of area A10 in rats failed to affect spontaneous open-field behavior over a 1-month postoperative period. When injected with 1 mg/kg of apomorphine, however, experimental rats more than doubled their activity as compared to the response of sham-operated controls. In contrast, no difference between the two groups of animals was observed in terms of increased activity following 3 mg/kg of either d-amphetamine or methylphenidate. These results are consistent with previous work indicating the involvement of ventromedial mesencephalic dopamine somata in the control of locomotor activity. The data suggest, however, that systems in addition to the dopaminergic mesolimbic projection are responsible, in part, for the hyperactivity elicited by d-amphetamine or methylphenidate.

Simple and configural association learning in rats with bilateral quisqualic acid lesions of the nucleus basalis magnocellularis.

We hypothesized that bilateral quisqualic acid lesions of the nucleus basalis magnocellularis (NBM) in rats would impair configural but not simple association learning. In experiment 1, rats were tested in a negative patterning operant discrimination where they were food-reinforced for responding to a light or a tone (L+, T+) but not for responding to the configural stimulus consisting of the light and tone presented simultaneously (LT-). Consistent with our hypothesis, NBM-lesioned rats showed a transient but significant impairment, responding normally to L+ and T+ but responding more often to LT-, in addition to responding more often during the inter-trial interval (ITI) than controls. In experiment 2, rats were tested in a simple operant discrimination where rats were food-reinforced for responding to a light (L+) but not for responding to a tone (T-). Although NBM-lesioned rats again responded normally to L+ as predicted, NBM-lesioned rats were transiently impaired, making more T- responses and more ITI responses than controls. Together, these results suggest that the NBM is involved in both configural and simple association learning but that this involvement is limited to learning to withhold responding to non-reinforced contextual or discrete stimuli. Finally, rats from experiment 2 underwent extinction trials, where results showed no difference between NBM-lesioned and control groups, suggesting that the NBM is not involved in the extinction of conditioned responding to previously reinforced stimuli.

Oat cell carcinoma of lung masquerading as prostatic carcinoma.

We believe this is the second case of oat cell carcinoma metastatic to the prostate to be reported. The patient was treated for prostatic carcinoma diagnosed by rectal examination. Subsequent biopsy confirmed oat cell carcinoma and led to the discovery of pulmonary carcinoma. Tumors metastatic to the prostate are rare and are identified when they result in urologic symptoms. A review of tumors metastatic to the prostate is presented.

Urachal carcinoma.

The clinical and morphologic features in 14 cases of urachal adenocarcinoma are reviewed. The disease occurred more frequently in men than in women. Seven patients (50%) remain alive and currently free of disease at periods ranging from fifteen months to ten years (median: 6 years). Neither the histologic appearance nor the differentiation of the tumor appeared to affect prognosis. It is concluded that the criteria previously recommended to establish the diagnosis of this rare malignancy were too restrictive.

Does long-term finasteride therapy affect the histologic features of benign prostatic tissue and prostate cancer on needle biopsy? PLESS Study Group. Proscar Long-Term Efficacy and Safety Study.

OBJECTIVES: Finasteride, a common agent used to treat benign prostatic hyperplasia (BPH), inhibits 5-alpha-reductase. Testosterone is converted by 5-alpha-reductase to the more potent dihydrotestosterone, which is the primary androgen in the prostate. Leuprolide is a stronger antiandrogen that is used to downstage prostate cancer before radical prostatectomy. Leuprolide induces marked atrophy of prostate carcinoma cells, which sometimes makes pathologic diagnosis of cancer difficult, although evaluation at radical prostatectomy is easier than at biopsy. It is unknown whether finasteride produces similar changes, which would result in greater diagnostic difficulty because such changes would be seen on biopsy to rule out cancer in men with suspicious clinical findings treated for BPH. The current study investigated the histologic effects of finasteride therapy on human prostate cancer and benign prostatic tissue on needle biopsy. METHODS: In blinded manner, we reviewed 53 needle biopsy specimens showing prostate carcinoma (35 treated with finasteride, 18 with placebo). Also reviewed in blinded manner were 50 benign needle biopsy specimens (25 treated with finasteride, 25 with placebo). The Gleason score, number of cores involved, percentage cancer involvement in a core, percentage of atrophic changes in cancer cells, presence of mitoses, blue-tinged mucinous secretions, prominent nucleoli, and high-grade prostatic intraepithelial neoplasia were documented for each case in the cancer group. The percentage of atrophy, basal cell hyperplasia, transitional metaplasia, chronic inflammation, and stromal proliferation was documented for each case in the benign group. RESULTS: No significant histologic differences were present in either the benign or cancer group between cases treated with finasteride and placebo. CONCLUSIONS: We conclude that finasteride treatment for BPH does not cause difficulty in the diagnosis of cancer in prostate needle specimens. It is possible that there are severely atrophic areas resulting from finasteride treatment that are undersampled. However, the conclusion that cancer seen on needle biopsy in men treated with finasteride is unaltered and readily identified as cancer remains valid.

Postnatal development of acetylcholinesterase in the caudate-putamen nucleus and substantia nigra of rats.

The postnatal development of acetylcholinesterase (AChE, EC 3.1.1.7) and NADH-diaphorase was examined in the caudate-putamen nucleus and substantia nigra of rats ranging from 3 to 90 days in age. From 3 to 15 days post partum islands of AChE and NADH-diaphorase activity were observed in the caudate-putamen nucleus. Individual neuronal somata could also be seen in AChE-stained sections up to 15 days. At later ages neuropil staining became increasingly dense, and this presumably accounted for the infrequent visualization of cell bodies in the brains of older animals. During development AChE appeared in the caudate-putamen nucleus in a lateral to medial topographic order; analogously, enzyme staining in the neostriatum reappeared in the same lateral to medial topographic order in adult rats following irreversible AChE inhibition by intramuscularly injected bis-(1-methylethyl)phosphorofluoridate (di-isopropylfluorophosphate: DFP). Furthermore, DFP treatment in mature animals revealed the presence of AChE in striatal neurons having morphologies similar to those observed in newborn rats. A similar time-course of postnatal AChE development was observed in the substantia nigra. In both the pars compacta and pars reticulata individual cell bodies, which were visible at early ages (3-10 days), became increasingly obscured at later times after birth by extra-somata staining. Between the 6th and 15th postnatal days AChE-containing fibers were seen projecting apparently from pars compacta into pars reticulata. Comparison of the present results with histochemical data of other investigators on the postnatal development of monoamines indicated the likelihood of cholinergicmonoaminergic interactions in the neostriatum and substantia nigra.