Detection of Aspergillus DNA in cerebrospinal fluid from patients with cerebral aspergillosis by a nested PCR assay.

Invasive aspergillosis (IA), a complication with high mortality rates, especially in disseminated IA with cerebral involvement, is difficult to diagnose. Biopsy of cerebral lesions is often not feasible, and culture of Aspergillus spp. from cerebrospinal fluid (CSF) is frequently negative. New molecular methods have emerged for diagnosing IA. So far, there are only few reports of Aspergillus DNA detection in CSF. After modifying the DNA extraction protocol, we detected Aspergillus DNA in CSF samples by a previously described nested PCR assay. In six patients with hematologic malignancy and cerebral aspergillosis, CSF samples were investigated for Aspergillus DNA. IA was classified according to the EORTC/MSG 2002 criteria. Two patients each had proven, probable, and possible IA. Thirty-five CSF samples were investigated for Aspergillus DNA by nested PCR. Samples with positive results in the nested PCR assay were quantified by LightCycler PCR assay. Fourteen CSF samples showed positive results in the nested PCR assay. Of these, six samples gave positive results in real-time PCR. The range of CFU per ml was 2,154 to 63,100,000. The highest number of CFU per ml was found in a CSF sample of a patient with acute lymphocytic leukemia and probable cerebral aspergillosis. Detection of Aspergillus DNA in CSF samples is thus possible and has the potential to improve diagnosis of cerebral aspergillosis. Further prospective studies with larger numbers of patients must be performed to evaluate the clinical significance of Aspergillus PCR with CSF samples.

Immunolocalization of inducible and constitutive nitric oxide synthases in human bladder cancer.

OBJECTIVES: Nitric oxide (NO) is synthesized by the enzyme family of NO synthases (NOS) and plays an important role in tumor growth and angiogenesis. NO generation by inducible NOS (iNOS) also influences the cytotoxicity of macrophages and tumor-induced immunosuppression. Before now, the expression of iNOS and constitutive NOS in bladder carcinoma tissue had not been determined. METHODS: Bladder carcinoma tissue specimens were procured from 18 patients (mean age 69.7 years) undergoing transurethral resection. In every patient, tumor biopsies were compared with biopsies of benign bladder regions. Histochemical NADPH-diaphorase staining and NOS immunohistochemistry were performed on all tissue specimens. RESULTS: Positive NADPH-diaphorase staining was detected in all sections from bladder carcinoma tissue. NOS immunohistochemistry showed a different pattern. The malignant epithelial cells were highly iNOS positive. Specimens of bladder mucosa outside of the malignant regions showed only a weak positive iNOS immunostaining. The endothelial cells of abundant precapillary vessels in the stroma of bladder tumors showed a highly positive endothelial NOS (eNOS) immunostaining compared with the stroma of nonmalignant bladder tissue. Neuronal NOS immunoreactivity was only found in nitrinergic fibers in the fibromuscular stroma. CONCLUSIONS: Bladder carcinoma tissue had a high iNOS content; benign tissue did not. NO generation from iNOS in the malignant epithelium and from eNOS in tumor stroma may play different roles in tumor angiogenesis and tumor-induced immunosuppression.

Localization of constitutive nitric oxide synthase isoforms and the nitric oxide target enzyme soluble guanylyl cyclase in the human bladder.

PURPOSE: Nitric oxide is a free radical gas synthesized from L-arginine by a family of isoenzymes called nitric oxide synthase that has an important role in smooth muscle relaxation. L-arginine, the substrate for nitric oxide synthase, may be beneficial under pathophysiological conditions in the bladder, as in interstitial cystitis. We determined the localization of nitric oxide synthase and the target enzyme of NO, soluble guanylyl cyclase, in the human bladder. MATERIALS AND METHODS: Benign bladder tissues were obtained from 18 patients with localized superficial bladder tumors undergoing transurethral bladder resection. Histochemical nicotinamide adenine dinucleotide phosphate-diaphorase staining, nitric oxide synthase immunohistochemical testing and soluble guanylyl cyclase immunoreactivity studies were performed in all benign tissue specimens. RESULTS: A different pattern of nitric oxide synthase expression was confirmed by nicotinamide adenine dinucleotide phosphate-diaphorase staining and immunohistochemical testing for endothelial and neuronal nitric oxide synthase. In addition to endothelial nitric oxide synthase expression, detrusor smooth muscle was recognized as an important location of endothelial nitric oxide synthase, while the urothelium had only small endothelial nitric oxide synthase positive cell clusters. Neuronal nitric oxide synthase expression was only found in nitrinergic fibers of the submucosal surface and between muscle cells. Detrusor and vascular smooth muscle as well as interstitial cells, nerve fibers and transitional epithelium were recognized as targets of nitric oxide, as shown by soluble guanylyl cyclase expression. CONCLUSIONS: The distribution of constitutive nitric oxide synthase isoforms and soluble guanylyl cyclase provides evidence of nitric oxide-cyclic guanosine monophosphate mediated regulation of detrusor smooth muscle relaxation, neurotransmission and blood flow. Furthermore, the urothelium may also be a target of nitric oxide.