Elena Angelici

Employment of terbinafine against Pneumocystis carinii infection in rat models

The anti‐Pneumocystis carinii response of terbinafine together with that of three other compounds, trimethoprim sulphamethoxazole (TMP‐SMX), atovaquone (ATQ) and albendazole (ALB), has been investigated in immunosuppressed Sprague‐Dawley rats with established pneumocystosis. Drugs were administered orally (terbinafine in dosages of 40 and 80mg/kg per day, TMP 12·5 mg/kg per day plus SMX 62·5 mg/kg per day, ATQ 100 mg/kg per day and ALB 600 mg/kg per day) to six rat groups except one which served as a control, P. carinii pneumonia (PCP) was identified post‐mortem in nine (90%) of the control rats which exhibited a marked P. carinii burden, and mean lung weights were higher with respect to the other treatment groups. During treatment, five rats in the control group died, whereas between 11 and 13 rats in all treatment groups survived. In the terbinafine groups (40 mg and 80 mg/kg per day), a mild P, carinii infection developed in three and two rats (27·2 and 18%), respectively, and almost the same infectivity score was obtained for those treated with 40 mg and 80 mg/kg per day. Histological changes in the lungs in animals receiving terbinafine treatment were minimal. Among the remaining compounds the rate of infection was seven (58·3%) for the ALB treatment group and five (45‐4%) for the ATQ group (mean score 19‐4 ±7‐1 and 23 ± 2·1, respectively). In the TMP‐SMX treatment group, there were 13 surviving rats and P. carinii organisms were found in two (15·3%, mean infection score 8 ± 1·1).

Production of plasminogen activator and plasminogen activator inhibitors by alveolar macrophages in control subjects and AIDS patients.

ObjectiveTo reveal a possible impairment of the plasminogen activator system in the pulmonary infections of AIDS patients. DesignTo test the plasminogen activator system functionality in alveolar macrophages and bronchoalveolar lavage fluid (BALF) in control subjects and AIDS patients. Procedures were designed to defect the presence of imbalance in plasminogen activator activity and to ascertain if this imbalance is due to a direct effect of the HIV virus on macrophages or to superimposed opportunistic infection. MethodsAlveolar macrophages obtained by bronchoalveolar lavage (BAL) were either lysed with Triton X-100 or cultured for 24 h. Plasminogen activators and plasminogen activator inhibitors (PAI) were measured by chromogenic substrate assay and binding to 12l-urokinase followed by 10% sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PACE), respectively. ResultsPlasminogen activator activity in BALF and in alveolar macrophages from AIDS patients was decreased. This reduction was independent of the presence of an infectious pulmonary process. In contrast, free PAI was increased in AIDS patients with Pneumocystis carinii infection. This increase is possibly caused by a different glycosylated form of PAI-2. ConclusionsOur data support the view that the pulmonary fibrogenic response is in part secondary to an imbalance within the plasminogen activator system and provide the basis for clarifying the role of these alterations in the pathophysiology of AIDS-related pulmonary infections.

Carriage of Pneumocystis carinii in children with chronic lung diseases.

Pneiimocystis carini i pneumonia (PCP) is a major opportunistic infection which occurs in AIDS patients and in i m m u n o c o m p r o m i s e d h o s t s . Prev ious ly repor ted communications, however, suggest that P. carinii may also be found in HIV negative and in healthy subjects with no underlying condition that impairs the immune function [3, lo]. Recently, P . carinii carriers states have been found among HIV negative patients suffering from chronic obstructive lung diseases with worsening of their pulmonary disorder [2, 81. The present study was carried out to detect P . carinii in nasopharingeal aspirate (NPA) specimens from immunocompetent children with chronic lung diseases (CLD). NPA has been shown to be an useful sample among HIV infected and leukaemic infants [6, 91 which provides a ready, safe and economic alternative to other invasive procedures in children. MATERIALS AND METHODS. Between 1992-1997 we investigated the occurrence of P. carinii on either NPA or blood (peripheral blood mononuclear cells PBMC) specimens obtained horn 71 CLD children (mean age 7 years) found to be normal for B and T lymphocytes count, T-cell subsets, total immunoglobulins according the age and who were not under steroid treatment (group A); thirty-one HIV infected infants with either PCP (no=15, group B) or kee of P. corinii infection or acute symptoms of pneumonia (no=16, group C) and 30 healthy immunocoinpetent subjects (group D). Specimens were assayed by nested-PCR using proteinase K digestion followed by analysis with two primer sets based on the chromosomal rRNA sequence of rat-derived P. carinii [7 ] . PCR products were evaluated by Southern hybridisation to confirm positive findings. Routine immunofluorescence with anti?. carinii monoclonal antibodies (Diagnostic Pasteur) and Gomori silver stain were also performed. A portion of each NPA was also sent for microbiological investigation. PBMCs were isolated by centrifugation on a density gradient (Lymphorep) within 7, h after collection and DNA was extracted by phenolchloroform followed by ethanol precipitation. Positive controls included purified P . carinii cysts extracted from autoptic human lung, BALs from AIDS patients with PCP. Several negative controls such as bacteria, fungi. mycoplasmas. Mycobacteria were included in each run. Statistical significance of both PCR and standard technique results was calculated with the chi-square test, Student i test and by Fishers exact test. RESULTS AND DISCUSSION. Diagnostic values for both PCR and microscopy, based upon clinical diagnosis, are given in Table 1. No specimen from healthy subjects showed evidence for P . carinii by PCR or conventional techniques (specificity 100%).

Cytomegalovirus in bronchoalveolar lavage specimens from patients with AIDS : comparison with antigenaemia and viraemia

Pulmonary infection with cytomegalovirus (CMV) is a well recognised complication of AIDS. It is often possible to detect CMV-infected cells in bronchoalveolar lavage (BAL) specimens with monoclonal antibodies, but the clinical significance of their presence remains unclear. To investigate this, 24 AIDS patients were tested for CMV antigenaemia and viraemia, in addition to CMV detection in BAL. CMV was detected in the BAL of nine patients (38%), five with clinical and laboratory evidence of pulmonary infection and four without pulmonary involvement. Blood samples positive for CMV antigen were observed in two patients with CMV-positive BAL specimens and, in both cases, antigenaemia resolved without therapy. No case of viraemia was detected. Pneumocystis carinii was detected concomitantly with CMV in the BAL of four of the patients with pulmonary involvement and in one without signs of pulmonary infection. These data suggest that CMV-positive BAL results are of limited significance in the diagnosis of CMV pneumonia in AIDS patients, unless associated with high levels of antigenaemia or viraemia and compatible clinical symptoms.

Plasminogen Activator Production in a Rat Model of Pneumocystis carinii Pneumonia

Several studies have indicated that the serine protease urokinase‐plasminogen‐activator (uPA) is an important factor in host defense against pulmonary pathogens. To gain a better insight into the role of uPA in Pneumocystis carinii (P. carinii) pneumonia (PCP), we evaluated PA production in alveolar macrophages (AMs) obtained from rats with steroid‐induced PCP. Treatment with cortisone acetate favored PCP in 91% of rats. In the bronchoalveolar lavage (BAL) samples of immunosuppressed rats both with and without PCP, we observed a decrease in uPA activity as well as a decrease in cell number. Urokinase‐PA production by AMs was reduced in rats treated with cortisone alone. However, an increase in cell‐associated uPA was observed in rats with PCP. This increase appears to be produced in response to P. carinii infection. In fact, when AMs obtained from untreated healthy or immunosuppressed uninfected rats were challenged with P. carinii, a significant increase in PA activity in cell lysates was observed, though a lower response was obtained in cortisone‐treated animals. Our results suggest that healthy AMs respond to the presence of P. carinii with an increase in uPA production and that this response in immunodepressed rat‐AMs is partially impaired.

The Plasminogen System and Transforming Growth Factor-β in Subjects With Obstructive Sleep Apnea Syndrome: Effects of CPAP Treatment

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) has emerged as a risk factor for cardiovascular disease. A prothrombotic state may affect coagulation and participate in the atherosclerotic process in subjects with OSAS. These alterations in coagulation seem to involve the plasminogen activation system. We evaluated the imbalances of the plasminogen activation system related to OSAS, and we assessed the effects of CPAP on the plasminogen activation system. METHODS: Thirty-nine subjects were submitted to a home-based cardiorespiratory sleep study, and 14 healthy subjects (apnea-hypopnea index < 5) were used as controls. Serum levels of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and active transforming growth factor-β (TGF-β) were measured. These molecules were reassessed in only 17 of the subjects after 1 month of CPAP. RESULTS: PAI-1 and tPA were significantly higher in the subjects with OSAS compared with the controls, whereas TGF-β and uPA levels were lower. PAI-1 showed a significant positive correlation with the apnea-hypopnea index, percentage of time spent at O2 saturation < 90%, and oxygen desaturation index, whereas TGF-β was inversely related to all 3 of these parameters. After the CPAP therapy, PAI-1 significantly decreased, whereas TGF-β showed a significant increase, although the values did not reach those of the controls. uPA and tPA did not show significant differences after the treatment. CONCLUSIONS: Our results suggest an imbalance of fibrinolysis related to OSAS and an improvement of the prothrombotic state after the CPAP treatment.

A Simplified Approach for the Estimation of the Ventilatory Compensation Point

UNLABELLED Incremental cardiopulmonary exercise test with gas exchange measurement is the gold standard for the identification of the ventilatory compensation point (VCP). It has previously been demonstrated that the change in the slope of increment of minute ventilation over HR (ΔV˙E/ΔHR) can be used alternatively to the ventilatory equivalent for CO₂ (V˙E/V˙CO₂) method for detection of VCP in healthy subjects undergoing cycle ergometer (C) incremental exercise. The same evaluation during treadmill (T) incremental exercise and comparison between C and T have not yet been performed. PURPOSE We analyzed, during both C and T incremental exercises, the V˙E/HR and the respiratory rate (RR)/HR relationships, expressed either as slope or as an absolute value. We hypothesized that changes in the slope of increment of the two relationships could represent a reliable method for VCP detection, regardless of exercise mode and protocol. METHODS Fourteen healthy male subjects (age = 31 ± 7 yr (mean ± SD)) underwent two T incremental exercises--fast (FT) and slow (ST) protocols (8 km·h⁻¹, 2% (F(T)) and 1% (S(T)) grade per minute)--and one C incremental exercise (30 W·min⁻¹). O₂ uptake (V˙O₂), V˙CO₂, V˙E, HR, and RR were measured breath by breath. RESULTS A good between-method agreement in the detection of VCP by the ΔV˙(E)/ΔV˙CO₂, ΔV˙(E)/ΔHR, and the ΔRR/ΔHR slope changes was found in both T protocols and C. No differences (C vs T and F(T) vs S(T)) were found in the slope of the ΔV˙(E)/ΔHR and ΔRR/ΔHR relationships after the VCP and in the V˙(E)/HR and RR/HR absolute values at VCP. CONCLUSIONS In healthy young males, the ΔV˙E/ΔHR and ΔRR/ΔHR relationships during T and C incremental exercises can be reliably used to detect the VCP as an alternative to the ventilatory equivalent method.

Urokinase Plasminogen Activator and TGF-β production in Immunosuppressed Patients with and without Pneumocystis carinii

Pnewnocysris carinii infection is the most frequent complication in AIDS patients and alveolar macrophages (AM) play a significant role in the clearance of P. carinii from the lung by binding. phagocytizing and degrading the offending organism [5]. Several studies indicate that mkinase plasminogen activator (uPA) and transforming growth factor p (TGF-8) are impatant factors in host defense against pulmonary pathogens. Urokinase PA is a serine protease that converts plasminogen to the active enzyme plasmin. and appears to play a role in the n m a l functicming of the immune response. In fact. using transgenic mice lacking the uPA gene it has been demonstrated that uPA is required for the pulmonary inflammatory response to P . carinii [4]. TGF-p is secreted by different cell types as an inactive complex and then activated by proteolysis with release of active TGF-P from the complex. Activated mouse peritoneal macrophages are capable of activating endogeneous latent TGF-b when stimulated with U S : this process depends u p the presence of uPA and plasmin. Active TGFp. in turn. reduces uPA production and induces PAI production [7]. Several evidences suggest a role for TGF-P in HIV replication [8] and P. carinii clearance [9]. Therefore the aim of this study was to assess the production of uPA and TGF-P in H W and immunodepressed patients with and without P. carinii.