Mechanisms responsible for the thermal sensitivity of adrenal microsomal monooxygenases.

Studies were done to determine the mechanism(s) responsible for the thermal lability of adrenal microsomal monooxygenases. Preincubation of guinea pig adrenal microsomal suspensions at 37 degrees C caused large time-dependent declines in benzo(a)pyrene (BP) hydroxylase and benzphetamine (BZ) demethylase activities. Similar preincubations with hepatic microsomes had little effect on enzyme activities. The decreases in adrenal enzyme activities were completely prevented by co-incubation of microsomes with cytosol, but were not diminished by reduced glutathione, ascorbic acid, or bovine serum albumin. Partial protection was afforded by EDTA, suggesting that lipid peroxidation might be involved, but malonaldehyde production was not demonstrable and MnCl2, a potent inhibitor of lipid peroxidation, did not affect the decline in enzyme activities. The decreases in the rates of BP and BZ metabolism were prevented by including NADPH or NADP+ in the preincubation medium. The preincubation conditions causing losses of adrenal enzyme activities did not affect cytochrome P-450 concentrations or substrate binding to cytochromes P-450, as indicated by type I difference spectra. NADH-cytochrome c reductase activity also was not affected, but there were decreases in NADPH-cytochrome c reductase activity that were proportionately similar to the declines in drug-metabolizing activities. Direct assessment of NADPH-cytochrome P-450 reductase revealed similarly large decreases in enzyme activity resulting from preincubation of adrenal microsomes. The results demonstrate a need for extra caution when doing preincubation experiments with adrenal microsomal preparations, and suggest that the thermal lability of adrenal monooxygenases is attributable to effects at the active site of NADPH-cytochrome P-450 reductase.     

Lung biopsy in chronic lymphocytic leukemia

Nine patients with chronic lymphocytic leukemia (CLL), with pulmonary involvement confirmed by biopsy, presented with progressive cough and/or shortness of breath and had interstitial infiltrates on chest radiographs. Biopsies showed a dense lymphocytic infiltrate that followed bronchovascular bundles. We considered CLL the predominant finding, and the cause of the patient's pulmonary disease, in eight cases; in one, a histologically nonspecific organizing pneumonia was the main lesion and CLL was an incidental finding. Culture results were available in six cases and were negative except in one case with presumed contaminants. A granulomatous reaction was present in five cases and was necrotizing in two, although culture results were negative. The only case with a recognizable organism had noninvasive fungal hyphae growing in many of the small airways. All of the patients' respiratory symptoms improved after chemotherapy and/or steroid therapy, and the chest radiographs also showed clearing.     

Inhibition of voltage-gated K channels in synaptosomes by sn-1,2-dioctanoylglycerol, an activator of protein kinase C

Tracer efflux studies were used to determine the effect of activation of protein kinase C on K channel function in rat brain synaptosomes. Hippocampal synaptosomes were treated with sn-1,2-dioctanoylglycerol (diC8), a synthetic diacylglycerol (DG) analog that activates protein kinase C. DiC8 inhibited depolarization-induced 86Rb efflux through voltage-gated K channels but did not affect the component of efflux corresponding to Ca-activated K channels. In time-course experiments, diC8 inhibited two components of 86Rb efflux: efflux through a rapidly inactivating, voltage-gated K channel (responsible for the "A" current) and that through a slowly inactivating, voltage-gated K channel (believed to be the "delayed rectifier"). Experiments with specific blockers of these voltage-gated K channels supported this observation. Inhibition of K-stimulated 86Rb efflux by diC8 was time dependent: at least 15 sec of preincubation was required before the effect could be observed. The effect of diC8 was concentration dependent: 50 microM diC8 produced a half-maximal inhibition of K-stimulated 86Rb efflux. The idea that the inhibition of synaptosome K channels by diC8 resulted from activation of C kinase was supported by pharmacological evidence. The action of diC8 was mimicked by 1-oleoyl-2-acetylglycerol, another DG analog that activates protein kinase C, but not by deoxy-diC8, a DG analog that does not activate C kinase. Inhibition of C kinase by sphingosine or H-7 prevented the diC8 effect. These studies demonstrate that synaptosomes are a good model in which to study modulation of mammalian CNS K channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonmyeloablative bone marrow transplantation: Infectious complications in 65 recipients of HLA-identical and mismatched transplants.

Infections are a common complication of allogeneic bone marrow transplantation and the leading cause of transplantation-related mortality. It had been hypothesized that transplantation following nonmyeloablative preparative regimens would result in fewer infections by causing less mucosal injury, less graft-versus-host disease, and allowing earlier immune reconstitution. We have retrospectively reviewed the infectious complications of 65 consecutive patients with advanced hematologic malignancies who underwent bone marrow transplantation using a novel preparative regimen consisting of cyclophosphamide, thymic irradiation, and in vivo T-cell depletion. Cytomegalovirus (CMV) infection occurred in 52% of cases in which the donor or recipient had evidence of prior CMV exposure. Using a strategy of preemptive therapy and secondary prophylaxis with ganciclovir, no CMV disease occurred. Infections with gram-positive bacteria predominated over the first 100 days after bone marrow transplantation. Thereafter, the relative proportion of gram-negative infections increased without a significant increase in episodes of neutropenia. The rate of bacterial infections was not influenced by relapse of the underlying malignancy. Seven patients developed infections with Aspergillus species, which was the most common infectious cause of death in these patients. Infections with viruses other than CMV (n=10) and with protozoan organisms (n=2) also occurred. The use of HLA-mismatched donors, the occurrence of grade II-IV acute graft-versus-host disease, and treatment with corticosteroids did not influence the risk of CMV or bacterial or fungal infections in patients who underwent transplantation following this preparative regimen. Overall, the incidence and spectrum of infections in this series was similar to the reported incidence of infections following conventional myeloablative allogeneic stem cell transplantation. We conclude that a quantitative T-cell deficiency in these extensively T-cell depleted patients may be a risk factor for infection, even in the absence of graft-versus-host disease.     

Detection of Leishmania infantum kinetoplast DNA in laryngeal tissue from an immunocompetent patient

Mucosal leishmaniasis of the upper respiratory tract is usually associated with the visceral form or is found in immunosuppressed individuals. This report presents a case of isolated mucosal leishmaniasis in an immunocompetent patient, whose diagnosis mainly rested on histology and positive polymerase chain reaction result for Leishmania donovani in the laryngeal tissue. A 59-year-old man, who never lived outside Italy, showed a subglottic mucosal polypoid-like lesion. The typical morphological picture and positive polymerase chain reaction result for L donovani by DNA extracted from laryngeal biopsy specimens allowed the diagnosis of mucosal leishmaniasis. Specific amphotericin B therapy was started, resulting in clinical and endoscopic improvement. Increased knowledge about the histological and molecular tissue analysis of Leishmania enhances the diagnostic testing for mucosal leishmaniasis, as primary mucosal leishmaniasis may occur in both immunosuppresed and immunocompetent patients who travel to or reside in areas endemic for Leishmania.     

Current status of small peripheral adenocarcinomas of the lung and their importance to pathologists

There has been a large amount of work done recently on small peripheral stage I adenocarcinomas that come to resection. Radiological (including proportion of ground glass opacity) and pathological features of these lesions (predominant bronchioloalveolar component, central scar with or without invasion <0.5 cm) have been shown to be prognostically favorable with cure rate approaching 100% in some series. Most of these studies emanate from Japan. The relevance of these studies to other parts of the world, particularly North America, is discussed in light of the fact that some recent chemotherapeutic studies with gefitinib have shown increased response in individuals of Asian origin, suggesting that some genetic differences may be significant. The relevance of these findings to pathologists and the pathological study of small peripheral adenocarcinomas from elsewhere in the world are discussed.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions

Metalloproteinases and their inhibitors are known to play an important role in the extracellular matrix remodeling associated with preinvasive lesions and invasive carcinomas; however, little is known about their role in early lung carcinoma. Immunohistochemical studies were made of the reactivity of bronchial squamous preneoplastic lesions from cigarette smokers, including basal cell hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma for matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen in 13 patients. Staining for type IV collagen disclosed discontinuities in basement membranes from basal cell hyperplasia to dysplasia, progressing to destruction in carcinoma in situ and invasive carcinoma. Reactivity for MMP-9 was mild in basal cell hyperplasia and squamous metaplasia, increasing in carcinoma in situ and invasive carcinoma. In contrast, reactivity for MMP-1 was strong in basal cell hyperplasia and squamous metaplasia, decreasing in carcinoma in situ and invasive carcinoma. Some neoplastic cells in carcinoma in situ and invasive carcinoma were MMP-3 positive. Staining for MMP-2 and TIMP-1 was moderate to strong in all squamous preinvasive lesions. Confocal microscopy showed MMP-9-positive cells passing through fragmented basement membranes in which type IV collagen and MMP-9 were colocalized. Type IV collagen colocalized with MMP-2 in all lesions and with TIMP-1 in basal cell hyperplasia and squamous metaplasia. The inverse relationships between the reactivity for MMP-1 and MMP-9 with progression of bronchial squamous preinvasive lesions suggest important roles for these MMPs in basement membrane remodeling in these lesions.     

Pulmonary atypical carcinoid: predictors of survival in 106 cases

Pulmonary neuroendocrine tumors (NE) include a spectrum of tumors from typical carcinoid (TC) to atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell carcinoma (SCLC). Little is known about prognostic predictors for AC because of its rarity. Survival analysis was performed on 106 ACs with clinical follow-up from the AFIP and the Pathology Panel of the International Association for the Study of Lung Cancer (IASLC). The tumors fulfilled the 1999 WHO/IASLC criteria for AC of a NE tumor with a mitotic rate of 2 to 10 per 2 mm(2) of viable tumor or coagulative necrosis. Multiple clinical and histologic features were analyzed by Kaplan-Meier and Cox regression analysis. Of the clinical features, higher stage (P = .003) and a tumor size of 3.5 cm or greater (P = .003) were associated with a worse prognosis. Features that were histologically unfavorable by univariate analysis were mitotic rate (P =.002), pleomorphism (P = .018), and aerogenous spread (P =.007). Histologically favorable features by univariate analysis were the presence of palisading (P = .008), papillary (P = .039), pseudoglandular (P =.026), and rosette (P = .022) patterns. Female gender showed a trend toward a poorer prognosis (P =.085) and was included in the multivariate model. Multivariate analysis stratified for stage showed mitoses (P<.001), a tumor size of 3.5 cm or greater (P =.017), and female gender (P =.012) to be the only negative independent predictors of prognosis and the presence of rosettes (P = .016) to be the only independent positive predictor. We further divided the AC into subgroups of low (2 to 5 mitoses/2 mm(2)) and high (6 to 10 mitoses/2 mm(2)) mitotic rate and compared the survival with TC and with LCNEC. Within the category of AC, the patients with a higher mitotic rate had a significantly worse survival than those with a lower mitotic rate (P<.001) stratified for stage. Five- and 10-year survival rates for AC (61% and 35%, respectively) stratified for stage were significantly worse than for TC and better than that for LCNEC and SCLC. Chemotherapy or radiation therapy was given in 12 of 52 and 14 of 52 cases, respectively, but the data were insufficient to evaluate tumor response. We conclude that AC is an aggressive neuroendocrine neoplasm with survival intermediate between TC and LCNEC and SCLC. Higher mitotic rate, tumor size of 3.5 cm or greater, female gender, and presence of rosettes are the only independent predictors of survival. Surgical resection remains the treatment of choice, and the role of chemotherapy and radiation therapy remains to be proven.     

Recognition of a Nocardia transvalensis complex by resistance to aminoglycosides, including amikacin, and PCR-restriction fragment length polymorphism analysis.

Amikacin resistance, rare among nocardiae, was observed in 58 clinical isolates of nocardiae. All of these isolates hydrolyzed hypoxanthine, and 75 to 100% utilized citrate, D-galactose, and D-trehalose as sole carbon sources. Based on utilization of I-erythritol, D-glucitol, i-myo-inositol, D-mannitol, and ribitol and susceptibility to amoxicillin-clavulanic acid, the 58 isolates were separable into four groups. One group was negative for I-erythritol and ribitol and included all the isolates belonging to Nocardia asteroides complex antibiogram type IV. The remaining three groups were positive for I-erythritol and ribitol and were grouped within Nocardia transvalensis. The group that included the type strain was designated N. transvalensis sensu stricto, and the other two groups were designated new taxons 1 and 2. PCR-restriction fragment length polymorphism (RFLP) analysis of a 439-bp segment of the 65-kDa heat shock protein gene with XhoI and HinfI produced identical patterns for 53 (91%) and 58 (100%) isolates, respectively, and differentiated them from all other Nocardia taxa. NarI- and HaeIII-derived RFLP patterns clearly differentiated each of the four biochemically defined taxa. These four groups were also distinguishable by using the chromogenic substrates in Dade MicroScan test panels. By high-performance liquid chromatography, these isolates exhibited the same unique mycolic acid-ester elution patterns that differed from those of all other clinically significant nocardiae. Gas-liquid chromatographic analysis of fatty acids also produced similar patterns for all isolates that distinguished them from all other Nocardia taxa, but did not differentiate the four taxa within the complex. We propose the designation N. transvalensis complex for these four groups of nocardiae, pending further genetic evaluation.