Temporal expression of inflammatory mediators in brain basilar artery vasculitis and cerebrospinal fluid of rabbits with coccidioidal meningitis.

Strokes due to transmural vasculitis associated with coccidioidal meningitis result in significant morbidity and mortality. The immunological and inflammatory processes responsible are poorly understood. To determine the inflammatory mediators, i.e. cytokines, chemokines, iNOS, matrix metalloproteinase-9 (MMP-9), that possibly contribute to vasculitis, temporal mRNA expression in brain basilar artery samples and MMP-9 protein in the CSF of male NZW rabbits infected intracisternally with 6.5 x 10(4) arthroconidia of Coccidioides immitis were assessed. Five infected and 3 sham-injected rabbits at each time point were euthanized 4, 9, 14 and 20 days post infection. All infected rabbits had neurological abnormalities and severe vasculitis in the basilar arteries on days 9-20. In basilar arteries of infected animals versus controls, mRNAs encoding for IL-6, iNOS, IFN-gamma, IL-2, MCP-1, IL-1beta, IL-10, TNF-alpha, CCR-1, MMP-9, TGF-beta, as well as MMP-9 protein in CSF, were found to be significantly up-regulated. Thus, this study identified inflammatory mediators associated with CNS vasculitis and meningitis due to C. immitis infection. Assessment of the individual contribution of each mediator to vasculitis may offer novel approaches to the treatment of coccidioidal CNS infection. This study also provides unique methodology for immunology studies in a rabbit model.

[Recent knowledge allowing diagnosis and treatment of deep-seated trichosporonosis].

Deep-seated trichosporonosis is a lethal opportunistic infection in immunocompromised patients. For the rapid diagnosis of this condition, we developed a novel nested-PCR assay that detects DNA specific for clinically important strains of Trichosporon in serum of patients with disseminated trichosporonosis. In this assay, two sets of oligonucleotide primers were derived from the sequence of 26 S ribosomal RNA genes of T. asahii. The specific fragment was amplified from T. asahii and T. mucoides but not from other microorganisms. In a retrospective study using serum samples of patients with disseminated trichosporonosis, the specific fragment was detected in 64% (7 of 11). To treat this infection, we studied the efficacy of rhG-CSF alone and in combination with antifungal agents against disseminated trichosporonosis in neutropenic mice. The results suggested that rhG-CSF might be a useful immunomodulator against Trichosporon infections and the therapeutic outcome might be better when used in combination with antifungal agents.

Experimental model of progressive disseminated trichosporonosis in mice with latent trichosporonemia.

Trichosporon asahii and Trichosporon mucoides are the most common strains of fungi that cause disseminated trichosporonosis, a severe opportunistic infection in immunocompromised hosts. We have previously established a nested PCR assay using serum samples for detection of both strains. Here we describe a new experimental animal model for investigating the underlying mechanisms of disseminated trichosporonosis. T. asahii (OMU239, a clinical isolate from a patient with acute myelogenous leukemia) and 8-week-old ICR male mice were used in all experiments. A suspension of T. asahii (3 x 10(6) CFU/animal) was injected into the caudal vein of each mouse after immunosuppression with cyclophosphamide (200 mg/kg of body weight/day for 2 days) and prednisolone (30 mg/kg/day for 1 day). Mice were then divided into four subgroups (R0, R1, R2, and R3) based on the time of reimmunosuppression. The latter was performed using the same drugs 1 week (group R1), 2 weeks (group R2), and 3 weeks (group R3) after fungal infection. Reimmunosuppression was not performed in group R0. The 5-week-survival rates of mice after T. asahii infection were 0% for group R1, 50% for group R2, 80% for group R3, and 80% for group R0. There was a significant difference in the survival rates between group R1 and either group R0 or R3 (P < 0.05). Fungal clearance in peripheral blood and various organs of group R1 and R2 was delayed relative to that of group R0 but was similar to the control in group R3 in spite of reimmunosuppression. Our results suggest that the critical period for the development of disseminated trichosporonosis in our model is shorter than 3 weeks after T. asahii infection. We concluded that mice during this critical period were in a state of latent trichosporonemia. Comparison of the survival rates suggests that the nested PCR assay was more useful than blood culture and glucuronoxylomannan antigen assay in the detection of this latent trichosporonemia.

Determination of grepafloxacin in plasma and urine by a simple and rapid high-performance liquid chromatographic method.

A rapid, specific, sensitive and economical method has been developed and validated for the determination of grepafloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with UV detection. Plasma proteins were removed by a fast and efficient procedure that has eliminated the need for costly extraction and evaporation. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a reversed-phase TSK gel column with an isocratic mobile system. The method had a quantification limit of 0.05 microg/ml in plasma and 0.5 microg/ml in urine. The coefficients of variation (C.V.) were less than 4% for within- and between-day analyses. The method was successfully applied to a pharmacokinetic study, and was proved to be simple, fast and reproducible.

Determination of sparfloxacin in plasma and urine by a simple and rapid liquid chromatographic method.

A simple, specific, sensitive, and rapid method has been developed and validated for the determination of sparfloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with ultraviolet detection. Plasma proteins were efficiently removed by precipitation with perchloric acid after the addition of grepafloxacin as an internal standard. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a C18 reversed-phase column. The quantification limit was 0.025 mg/L in plasma and 0.5 mg/L in urine. The coefficients of variation (CV) were less than 10% for intra-day and inter-day analyses. The recovery of sparfloxacin added to plasma and urine ranged from 96.7% to 97.9%. The method has been successfully applied to pharmacokinetic studies.

Simultaneous determination of grepafloxacin, ciprofloxacin, and theophylline in human plasma and urine by HPLC.

A specific and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of grepafloxacin, ciprofloxacin, and theophylline in human plasma and urine. This assay allows these drugs to elute and be resolved in a single chromatogram at 280 nm, using a linear gradient. The procedure involves liquid-liquid extraction. Separation was achieved on a C18 reversed-phase column. The quantification limits were 0.05 mg/L in plasma and 0.5 mg/L in urine for grepafloxacin and ciprofloxacin and 0.5 mg/L in plasma and urine for theophylline. Standard curves were linear (correlation coefficients >0.999) over the ranges 0.05 to 5 mg/L for grepafloxacin and ciprofloxacin in plasma, from 0.5 to 20 mg/L for theophylline in plasma, and from 0.5 to 500 mg/L for the three drugs in urine. The coefficients of variation for the three drugs were less than 10% for within- and between-day analyses. The recoveries averaged 94.5% for theophylline, 93% for ciprofloxacin, 93.7% for grepafloxacin, and 95.1% for the internal standard (IS). The assay can be used for pharmacokinetic studies of these drugs, to investigate the interaction of grepafloxacin and ciprofloxacin with theophylline, or for routine simultaneous monitoring of theophylline, grepafloxacin, and ciprofloxacin.

Eosinophilia associated with adult T-cell leukemia: role of interleukin 5 and granulocyte-macrophage colony-stimulating factor.

To clarify the mechanism of eosinophilia in adult T-cell leukemia (ATL), we studied three ATL patients having marked eosinophilia. Eosinophil-predominant colony-stimulating activity was detected in the serum of one patient and in the conditioned media (CM) from cultured ATL cells from two patients. Soluble interleukin 5 (IL-5), but no interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was detected in sera from all patients. On the other hand, GM-CSF was produced in vitro by ATL cells from all cases, whereas detectable IL-3 and IL-5 was produced by cells from only one, suggesting that in the other two cases, the serum IL-5 was produced by the normal reacting lymphocytes. The fact that no patient showed marked neutrophilia supports the possibility that IL-5 may have a leading role in the development of eosinophilia, with GM-CSF produced by ATL cells playing a complementary role.

Evaluation of PCR for detection of DNA specific for Aspergillus species in sera of patients with various forms of pulmonary aspergillosis.

Pulmonary aspergillosis is classified into invasive, saprophytic, and allergic forms. In this study, we evaluated the usefulness of PCR for differentiating between different forms of aspergillosis or in monitoring disease activity during treatment by detecting DNA specific for Aspergillus species in the serum. Nested PCR was used to detect Aspergillus DNA in the sera of 30 patients with various forms of pulmonary aspergillosis. The results were compared with those of latex agglutination tests for detecting galactomannan antigen. We also examined the serial changes in the results of nested PCR during and after treatment of a subgroup of patients with invasive pulmonary aspergillosis with amphotericin B. The highest proportion of positive nested PCR results were in patients with invasive aspergillosis (10 of 12; 83%), while patients with pulmonary aspergilloma had the lowest frequency of positive tests (1 of 9; 11%). These results suggested that the sensitivity of the nested PCR depends on the extent of invasion by Aspergillus species. Serial assays showed that the results of nested PCR became negative shortly after commencement of antifungal treatment and that such changes did not correlate with clinical responsiveness to treatment. Our results indicate the potential usefulness of nested PCR with serum samples for the diagnosis of invasive aspergillosis and the detection of a shift in the status of infection from a noninvasive type to invasive aspergillosis. However, the results of the nested PCR did not correlate with the response to antifungal treatment.

Comparison of PCR, (1-->3)-beta-D-glucan and galactomannan assays in sera of rats with experimental invasive aspergillosis.

We compared PCR, galactomannan detection assay using a latex agglutination test and (1-->3)-beta-D-glucan detection assay in detecting infection in rats experimentally infected with Aspergillus fumigatus. On day 2 after inoculation, (1-->3)-beta-D-glucan and nested PCR were positive for 80%, while galactomannan detection assay was positive for 60%. In addition, the positive result of nested PCR (87.5%) was higher than those of galactomannan detection assay (75%) and (1-->3)-beta-D-glucan (71.4%) on day 3 after inoculation. The sensitivity of nested PCR was superior to those of galactomannan detection assay and (1-->3)-beta-D-glucan detection assay. The three diagnostic tests were compared with histopathological findings, and the sensitivity of three diagnostic tests was correlated with histopathological changes. In addition, the elevated levels of (1-->3)-beta-D-glucan paralleled the development and progression of pulmonary aspergillosis. Our results indicate that a combination of two or three of these tests seems to provide a rapid diagnosis of invasive aspergillosis and assist in the evaluation of the development and severity of invasive aspergillosis.

Efficacy of amphotericin B and azoles alone and in combination against disseminated trichosporonosis in neutropenic mice.

The activities of amphotericin B, miconazole, fluconazole, and itraconazole against Trichosporon beigelii were assessed in a mouse model of disseminated infection. Cyclophosphamide plus prednisolone-immunosuppressed ICR mice, intravenously challenged with a lethal inoculum of (6 x 10(6) CFU/ mouse), were assigned to receive 7 days of therapy with amphotericin B (0.5 or 2 mg/kg/day), miconazole (10 or 40 mg/kg/day), fluconazole (10 or 40 mg/kg/day), or itraconazole (10 and 40 mg/kg/day). The efficacy of a combination of amphotericin B (1 mg/kg/day) with that fluconazole (10 mg/kg/ day) or itraconazole (20 mg/kg/day) with that of each agent alone was also compared. Both amphotericin B and azoles improved survival and reduced the fungal counts in kidneys of infected mice in a dose-dependent pattern. In general, fluconazole was superior to amphotericin B and the other azoles, whereas the latter two drugs were as effective as amphotericin B. The activity of amphotericin B combined with fluconazole appeared to be superior to that of each agent alone, especially in reducing the organ fungal burden. The other combination (amphotericin B plus itraconazole) had a weaker effect, but no antagonism was observed. In conclusion, azoles may be an alternative to amphotericin B for the treatment of T. beigelii infection. Furthermore, their combination with amphotericin B may improve the poor outcome seen in profoundly neutropenic patients with disseminated trichosporonosis.

Significance of urinary fibrin/fibrinogen degradation products (FDP) D-dimer measured by a highly sensitive ELISA method with a new monoclonal antibody (D-D E72) in various renal diseases.

To diagnose the abnormalities of coagulation-fibrinolysis in various renal diseases, we developed a new monoclonal antibody (D-D E72) against fibrin/fibrinogen degradation products D-dimer (FDP D-dimer) and established a highly sensitive enzyme-linked immunosorbent assay (ELISA) for its measurement. FDP D-dimer was assessed in 102 patients with various renal diseases, and the following results were obtained: 1. The mean level of urinary FPD D-dimer in 32 normal controls was 0.69 +/- 0.60 ng/ml (mean +/- SD). 2. The level of urinary FDP D-dimer was significantly higher in primary nephrotic syndrome group (NS), chronic renal failure group (CRF) and in the group of diabetic nephropathy (DM) than in the control group. However, no difference was observed in the level of urinary FDP D-dimer between non-nephrotic chronic glomerulonephritis group (CGN) and control group. 3. No significant correlation was revealed between D-dimer and urinary protein in CGN and NS groups. These results suggest that in addition to plasma filtration the urinary FDP D-dimer in NS, CRF and DM may be also related to abnormalities of secondary fibrinolysis in intra-glomerular fibrin deposits.

Trichosporon beigelii pneumonia in patients with hematologic malignancies.

Trichosporon beigelii is a causative agent of hypersensitivity pneumonia in immunocompetent individuals and of invasive pneumonia in immunocompromised patients. The actual incidence and clinical manifestations of T beigelii pneumonia are obscure because the diagnosis is sometimes difficult. We studied eight patients with T beigelii pneumonia diagnosed by immunohistochemical investigation of lung tissue sections and/or isolation of the organism from the lung, sputum, or blood. All patients had underlying hematologic malignancies for which they had received cytotoxic chemotherapy, resulting in profound neutropenia. The clinical manifestations were persistent fever unresponsive to broad-spectrum antibiotic therapy, cough, bloody sputum, and rapidly progressive dyspnea. The chest radiographs showed diffuse alveolar infiltrates in four patients, diffuse interstitial infiltrates in one, patchy reticulonodular infiltrates in one, and focal alveolar infiltrates in two. Histopathologic examination demonstrated numerous centrally necrotic foci with minimal cellular inflammatory reaction, intra-alveolar hemorrhage, and edema. Trichosporon beigelii consisting of both yeast and hyphal forms was located predominantly in the alveolar vessels. In neutropenic patients with hematologic malignancies, this fungus appears to enter the lung not only through the airways but also via the hematogenous route. In vitro susceptibility testing indicated borderline susceptibility to amphotericin B and showed that some azoles were active against T beigelii at safely achievable serum concentrations.

Disseminated Trichosporon beigelii infection in patients with malignant diseases: immunohistochemical study and review.

Trichosporon beigelii is a causative agent of opportunistic infection and summer-type hypersensitivity pneumonitis in Japan. However, as the diagnosis of Trichosporon beigelii infection is sometimes difficult, the actual incidence of this disease may be underestimated. Of 203 autopsy patients with malignant disease, seven (7.7%) were diagnosed with disseminated Trichosporon beigelii infection by immunohistochemical investigation of formalin-fixed, paraffin-embedded tissue sections. Including these seven, a total of 43 patients with Trichosporon beigelii infection have been reported in Japan. The majority of them had underlying hematologic malignancies, for which they received cytotoxic chemotherapy resulting in neutropenia. This study indicates that the immunohistochemical method, which can be applied to biopsy specimens, is an excellent tool for specific diagnosis of Trichosporon beigelii infection, which is an emerging fatal mycosis in immunocompromised patients with profound neutropenia.