Value of assessing cryptococcal antigen in bronchoalveolar lavage and sputum specimens from patients with AIDS

Cryptococcus neoformans has become a significant opportunistic pathogen, accounting for 8-10% of infectious complications in patients with AIDS. When encapsulated yeast cells are observed in Giemsa-stained smears of bronchoalveolar washings (BAL), or induced sputum specimens, confirmation as C. neoformans is germane to definitive therapy. We therefore studied 30 BAL and 9 induced sputum specimens for cryptococcal antigen. Of the 30 BAL, 3 specimens were positive for cryptococcal antigen, ranging in titer from 1:4 to 1:256, and 2 of 9 sputum samples were also smear, culture and antigen positive (titer 1:2) for C. neoformans. Of the 34 negative specimens, none of the seven containing Candida species or the one containing H. capsulatum or the one containing P. carinii cross-reacted with cryptococcal anticapsular antibody. Our results indicate that when yeast forms suggestive of C. neoformans are visualized on direct smears of BAL or sputum samples, rapid confirmation as C. neoformans may be achieved by assessment for cryptococcal antigen. A correlation may also exist between antigen titer in respiratory specimens and extent of cryptococcal infection.     

Respiratory symptoms, lung function tests, airway responsiveness, and bronchoalveolar lymphocyte subsets in B-chronic lymphocytic leukemia

A respiratory questionnaire was completed and spirometry, tests for lung volumes, diffusion capacity for CO, and methacholine bronchial challenge were performed in 24 outpatients with B-chronic lymphocytic leukemia (B-CLL), aged 44-79, presenting in different stages of their disease. In 10 patients, bronchoalveolar lavage (BAL) fluid was also obtained. Ten of twenty-four patients had symptoms consistent with chronic bronchitis, unrelated both to smoking history and to the clinical stage. Abnormal values (< 2 SD) were found in 4 patients for total lung capacity (TLC), in 9 for vital capacity (VC), 8 for forced expiratory volume in 1 sec (FEV1), 11 for MEF50, 15 for MEF25 and in 7 for diffusing capacity for carbon monoxide. Seven of nineteen patients had PD20FEV1 at less than 1,600 micrograms of methacholine chloride. There was a significantly negative correlation between white blood cell count and VC (r = 0.41, P < 0.05). A positive correlation was found between PD20FEV1 and FEV1/VC (r = 0.61, P < 0.01). The mean and SEM for BAL cells/ml was 463 (71.8) x 10(3). No leukemic cells but a marked increase in T lymphocytes (32.5 +/- 7.8%) were found in BAL fluid. There were significantly negative correlations between the number of BAL CD3+ T lymphocytes and PD20FEV1 (r = 0.61, P < 0.05), and between the number of BAL CD8+ T lymphocytes and PD20FEV1 (r = 0.84, P < 0.01). In conclusion, patients with B-CLL have a high prevalence of respiratory symptoms, small airway dysfunction and CD8 "alveolitis" related to airway responsiveness; despite the well-known lung interstitial lymphocyte infiltration in B-CLL, leukemic cells are not found in BAL fluid.     

Repeatability of cellular and soluble markers of inflammation in induced sputum from patients with asthma

Sputum induced by inhalation of nebulized hypertonic saline is increasingly used to monitor airways inflammation in asthma. The aim of this study was to assess the repeatability of measuring cellular and soluble markers of inflammation in whole sputum samples as obtained by sputum induction in patients both with mild and moderate-to-severe asthma. Twelve patients with mild, atopic asthma without inhaled steroid treatment and nine patients with moderate-to-severe, atopic asthma treated with inhaled steroids were studied on two separate days at least 2 days apart. Whole sputum samples, induced by inhalation of hypertonic (4.5%) saline, were homogenized, and analysed for differential cell counts and for concentrations of albumin, fibrinogen, interleukin-8 (IL-8), and eosinophil cationic protein (ECP). Repeatability was expressed as intraclass correlation coefficient (Ri), and as coefficient of repeatability (CR) in percentage cells or in doubling concentration. Samples from two patients with mild asthma contained more than 80% squamous cells and were excluded from analysis. The repeatability for cell differential counts in both groups combined was: for neutrophils, Ri = 0.57 and CR = 31.0; for eosinophils, Ri = 0.85 and CR = 12.4; and for lymphocytes, Ri = 0.76 and CR = 6.9. The repeatability of the fluid phase measurements was: for albumin, Ri = 0.71 and CR = 3.2; for fibrinogen, Ri = 0.88 and CR = 2.8; for IL-8, Ri = 0.66 and CR = 2.2; and for ECP, Ri = 0.82 and CR = 1.1. We conclude that the repeatability of cellular and soluble markers of inflammation in induced sputum from patients with mild and moderate-to-severe asthma is satisfactory. Hence, induced sputum, processed by using the whole expectorated sample, seems to be a valuable method to monitor airway inflammation in asthma.     

Nonbronchoscopic bronchoalveolar lavage in mechanically ventilated infants: technique, efficacy, and applications

Bronchoalveolar lavage with the fiberoptic bronchoscope is commonly used for the diagnosis of pulmonary infections in mechanically ventilated adults and children. However, its use for intubated infants is precluded because the small artificial airway does not permit the passage of the bronchoscope. We have developed a technique for nonbronchoscopic bronchoalveolar lavage, performed via a sterile, disposable feeding tube. We have used this technique in 15 infants with diffuse interstitial disease and/or atelectasis, while they were intubated and mechanically ventilated. The volume of the lavage effluent averaged 70.3% of the volume instilled. Specific diagnosis on the basis of the cytologic evaluation and/or culture of the lavage fluid was possible in 9 (60%) patients. Two patients with atelectasis showed radiographic evidence of improvement following the procedure. There were no complications. We conclude that nonbronchoscopic bronchoalveolar lavage is well tolerated, and clinically useful in small, mechanically ventilated infants with respiratory failure due to diffuse pulmonary disease. This technique provides a lower risk alternative to more invasive, and costly procedures.     

Pathology of symptomatic microsporidial (Encephalitozoon hellem) bronchiolitis in the acquired immunodeficiency syndrome: a new respiratory pathogen diagnosed from lung biopsy, bronchoalveolar lavage, sputum, and tissue culture

Encephalitozoon hellem is a recently described microsporidian associated with an expanding spectrum of clinical presentations in patients with the acquired immunodeficiency syndrome (AIDS). It is morphologically similar to Encephalitozoon cuniculi, a microsporidian infection of mammals and some avians, and their differentiation rests on biochemical and antigenic analyses. This report describes a patient previously diagnosed with keratoconjunctivitis due to E hellem who subsequently was found to have respiratory tract microsporidiosis by sputum cytology. He subsequently developed pulmonary symptoms and a left lower lobe interstitial infiltrate. A bronchoalveolar lavage and transbronchial biopsy revealed microsporidial bronchiolitis, and the etiologic agent was identified as E hellem using an immunofluorescent antibody technique. Lavage fluid was successfully cultured in monkey kidney cells, and cultivated E hellem organisms were studied using immunohistochemistry as well as scanning and transmission electron microscopy. The pathologic features of this newly described cause of protozoal bronchiolitis, the role of immunofluorescent antibody examination and in vitro tissue culture for species-specific diagnosis, and the significance of microsporidial pulmonary infections in AIDS patients are discussed.     

FIV vaccine studies. II. Clinical findings, hematological changes and kinetics of blood lymphocyte subsets

Five groups of cats were vaccinated with different recombinant feline immunodeficiency virus (FIV) SU vaccines expressed either in Escherichia coli or in the Baculovirus system. In Part I of this series, we described the humoral immune response and outcome of intraperitoneal FIV challenge exposure. Additionally, all cats were monitored for clinical and hematological changes and the course of blood lymphocyte subsets. These results are described in this present paper. A great increase of antibodies was found after vaccination with different recombinant FIV antigens, which did not protect the cats from intraperitoneal FIV challenge infection. This observation was paralleled by an increase of eosinophils during vaccination which was even more pronounced after challenge infection. After FIV challenge, infection lymphadenopathy, gingivitis, pharyngitis, changes in total leukocytes and neutrophils and a decrease in the CD4+:CD8+ ratio were found in cats of all groups and were considered as a sign of the FIV infection taking place, independent of vaccination. The following observations suggest that in these cats a TH2-like immune response was elicited: the high counts of eosinophils, the nature of antigen and adjuvant (aluminium hydroxide) and the high amounts of antigens used for immunization. Clearly, this type of immune response did not protect the animals from intraperitoneal FIV challenge infection.     

Detection of T lymphocytes and T lymphocyte subsets in lichen planus: in situ and in peripheral blood

BACKGROUND: Abnormal immune mechanisms are thought to be important in the pathogenesis of lichen planus (LP). This is a study to clarify the changes that occur in T lymphocytes and T lymphocyte subsets, both in situ and in peripheral blood. METHODS: A group of 100 patients with LP were included in this study. T lymphocytes and T lymphocyte subsets were detected in lesional skin by immunoperoxidase cell surface staining using monoclonal antibodies. Peripheral T lymphocytes and T lymphocyte subsets were also detected by indirect immunofluorescence using monoclonal antibodies. A group of 10 normal healthy subjects were used as controls. RESULTS: The study of the lesional T lymphocytes and T lymphocyte subsets demonstrated that helper T cells was the predominant subset in LP lesions in most of the patients. This predominance was evident irrespective of the duration of the disease and was more evident in late than in early lesions. The percentage of both total T lymphocytes and helper T cells in peripheral blood was decreased significantly in patients compared with controls. A significant decrease in helper T cells and the helper/cytotoxic T cell ratio was detected in patients with a longer duration of the disease. CONCLUSION: Activation of helper T lymphocytes that were found to be the predominant subsets in LP lesions may be responsible for epidermotropic cellular infiltrates leading to damage and destruction of epidermal cells.     

Phenotypic characterization of T cells in bronchoalveolar lavage fluid (BALF) and peripheral blood of patients with diffuse panbronchiolitis; the importance of cytotoxic T cells

Abnormal cholesterol fraction is an essential risk factor for atherosclerosis of large cerebral arteries in young Asians. In order to reduce the medical cost and social resource for cholesterol electrophoresis, especially in undeveloped and developing Asian countries, we evaluated the validity of Nanji's GUT score for predicting TC: HDLC ratio in this population. Results showed that GUT score only predicted 71% of them. We also tested the predictive power of CUT index, and predicting rate was 81%. Therefore, Nanji's GUT score is not an ideal surrogate for cholesterol electrophoresis. We recommend CUT index to screen for high-risk subjects till a new method can satisfy the economic pattern in Asian countries.     

Chronic and acute prenatal and postnatal ethanol exposure on lymphocyte subsets from offspring thymic, splenic, and intestinal intraepithelial sources

The overall objective of this study was to analyze the effects of a combined prenatal and postnatal (entire gestational human chronic drinking model) ethanol exposure on T-cell development in mice. Specifically, this study evaluated the effects of chronic exposure to prenatal ethanol on lymphocyte makeup and proliferative capabilities of postnatal offspring's (4 and 12 weeks) peripheral lymphoid tissues. Chronic exposure regimens were conducted over the entire gestational period and through postnatal day 14 or 21. Thymus, spleen, and intestinal intraepithelial lymphocytes were harvested and analyzed by flow cytometry for percentages of T-cell subsets. Splenic lymphocytes were also analyzed for their ability to proliferate in response to a T-cell mitogen. Limited effects of chronic ethanol exposure were seen.     

Interpreting data on lymphocyte subsets in the blood of HIV patients - organ distribution, proliferation and migration kinetics are critical factors

Lymphocytes stay in the blood only a short time before migrating to lymphoid and nonlymphoid organs. They represent only about 2% of all lymphocytes in the body. The ratio of CD4+/CD8+ lymphocytes in the blood depends on age and genetic influences. In HIV infection not only relative but also absolute numbers of lymphocyte subsets should be determined. The different effects of proliferation and apoptosis on lymphocytes in HIV infection have to be considered. Lymphocyte levels in the blood of HIV patients do not mirror alterations in the lamina propria of the gut or lymph nodes. The dynamic aspects of lymphocyte life span and migration during HIV infection and the progression to AIDS as well as the effects of treatment have to be taken into consideration to enable a meaningful interpretation of experimental data. More data from animal models of HIV infection are needed to study these kinetic aspects.     

Cytologic evaluation of bronchoalveolar lavage fluid from horses with recurrent airway obstruction after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively

OBJECTIVE: To determine cytologic changes in horses with recurrent airway obstruction (heaves) after administration of aerosolized beclomethasone dipropionate and dexamethasone parenterally. ANIMALS: 6 horses with inducible and reversible heaves. PROCEDURE: Episodes of heaves were induced by exposure to moldy hay and straw for 7 days. Horses were assigned to treatment groups (aerosolized beclomethasone, parenterally administered dexamethasone, aerosolized propellant), and pulmonary inflammation was evaluated by serial cytologic examination of bronchoalveolar lavage (BAL) fluid samples obtained on days 0, 7, 10, 14, and 21. Total and differential cell counting and phenotypic analysis of lymphocyte subpopulations in BAL fluid were performed. RESULTS: 7 days of natural challenge induced neutrophilic inflammation. Neutrophil counts in BAL fluid were reduced in beclomethasone- and dexamethasone-treated horses on days 10 and 14 but rebounded to pretreatment values on day 21. The proportion of proinflammatory lymphocyte subpopulations (CD4+ and B+) and MHC class-II antigen expression were increased on days 14 and 21 in propellant-treated horses, compared with beclomethasone- and dexamethasone-treated horses. CONCLUSIONS: Aerosolized beclomethasone attenuated neutrophilic pulmonary inflammation and prevented alteration in lymphocyte subpopulations in horses with heaves. Results were similar to the response associated with parenterally administered dexamethasone. Short-term administration of aerosolized beclomethasone without minimizing environmental allergen exposure is not expected to provide prolonged anti-inflammatory benefit for horses with heaves.     

The oxygen effect in permeabilized and histone-depleted cells: an enhanced OER for DNA double-strand breaks, compared to single-strand breaks, is abolished by soluble scavengers

OBJECTIVES: It is generally believe that lipoprotein(a) (Lp(a)) levels remain relatively constant in the same individual, but there is a paucity of data to substantiate this belief. This study was undertaken to determine the extent of intra-individual variation in Lp(a) over a 12-month period. DESIGN AND METHODS: Lp(a) was measured monthly in duplicate over a 12-month period in 11 females and 11 males who were healthy, free-living, normal subjects by the Incstar Immunoprecipitin method using a goat antibody which was monospecific for Lp(a). RESULTS: Some subjects showed considerable month-to-month variations which were not correlated with changes in other lipid parameters or with weight. Others showed fairly constant Lp(a) levels, with a few values which were quite different from the rest. This was not attributable to methodological factors; low and high controls gave mean (mg/L), SD and CV values of 181, 8.6, 4.7 and 431, 14, 3.3, respectively. The difference between the minimum and maximum values in the same individuals ranged from a low of 14 mg/L in one subject to a high of 229 mg/L in another over the one-year period. CONCLUSIONS: Lp(a) showed greater intra-individual variations in normal subjects than is commonly believed. It is therefore recommended that Lp(a) should be measured sequentially over a few weeks to arrive at a mean value for assessing risk of coronary heart disease.     

Eicosanoids and inflammatory cells in frostbitten tissue: prostacyclin, thromboxane, polymorphonuclear leukocytes, and mast cells

The pathophysiology of cold injury is still controversial. An inflammatory process has been implicated as the underlying mechanism and certain anti-inflammatory substances such as ibuprofen and acetylsalicylic acid have been used in the clinical treatment of frostbite injury. It has been postulated that the progressive ischemic necrosis is secondary to excessive thromboxane A2 production, which upsets the normal balance between prostacyclin (prostaglandin I2) and thromboxane A2. It was aimed to clarify the pathophysiology of cold injury in this study. Twenty-one New Zealand White rabbits, each weighing 1.2 to 2.9 kg, were divided into control (n = 10) and frostbitten (n = 11) groups the randomly. The rabbit ears in the frostbitten group were subjected to cold injury, and the levels of thromboxane A2 (as thromboxane B2) and of prostaglandin I2 (as 6-keto-prostaglandin F1alpha) and the number of inflammatory cells (polymorphonuclear leukocytes and mast cells) were measured in normal and frostbitten skin of rabbit ears. The levels of 6-keto prostaglandin F1alpha and thromboxane B2, the stable metabolites of prostaglandin I2 and thromboxane A2, respectively, were increased in a statistically significant way (p < 0.002) by frostbite injury; however, thromboxane B2 increased more than 6-keto prostaglandin F1alpha. Polymorphonuclear leukocytes and mast cells, absent in normal skin, were present in the frostbitten skin. There was a statistically significant (p < 0.01) correlation between the time a rabbit ear was maintained at below -10 degrees C and skin survival and between the weights of rabbits and skin survival (p < 0.024). All these findings suggest that inflammation is involved in frostbite injury; a decrease in prostaglandin I2/thromboxane A2 ratio could be one of the factors leading to necrosis; the bigger the animal, the better its ability to counter frostbite.     

NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions

In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56+ CD7 and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class I- target K562. The CD56+ CD16+ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor, but no IFN-gamma. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.     

Stem cells from bone marrow, umbilical cord blood and peripheral blood for clinical application: current status and future application

Bone marrow transplantation (BMT) has progressed rapidly during the past two decades to that of a treatment of choice as a therapeutically effective modality for the treatment of selected patients with malignant disease and non-malignant hematological disorders. However, its use is limited by availability of human leukocyte antigens (HLA)-matched donor cells, engraftment and graft-versus-host disease (GVHD). Prevention of GVHD, improvement in the speed and quality of marrow reconstitution, and screening of new immunomodulating agents which improve engraftment and augment hemopoiesis are intense areas of investigation. To this end there has clearly been progress in purification and characterization of human stem cells from different tissue sources. Discussed in this review are: (a) stem cell purification, characterization and ex vivo expansion; (b) bone marrow stem cell transplantation; (c) cord blood stem cell transplantation; (d) peripheral blood stem cell transplantation; (e) fetal liver stem cell transplantation; (f) in utero stem cell transplantation; and (g) evaluation of the capacity of stem cells to serve as targets for gene therapy.     

Manipulating blood T cells and B cells from squirrel monkeys: some technical considerations

The squirrel monkey Saimiri sciureus is an experimental host for a range of human pathogens, and for the assessment of vaccine candidate antigens and vaccine strategies. This experimental host is thus particularly suitable for the follow-up of humoral responses. To understand some of the mechanisms that underlie the defense against experimental pathogens, there is a need of basic knowledge on cellular immune effectors also. The authors report here their experience in characterizing squirrel monkey blood T and B lymphocytes, and in studying in vitro induced activation and proliferation of T and B cells. Particular emphasis is given to the in vitro differentiation of squirrel monkey B cells into immunoglobulin secreting cells, with respect to Plasmodium falciparum antigens.     

Time-dependent changes of inflammatory mediators in the lungs of humans exposed to 0.4 ppm ozone for 2 hr: a comparison of mediators found in bronchoalveolar lavage fluid 1 and 18 hr after exposure

Acute exposure of humans to ozone results in reversible respiratory function decrements and cellular and biochemical changes leading to the production of substances which can mediate inflammation and acute lung injury. While pulmonary function decrements occur almost immediately after ozone exposure, it is not known how quickly the cellular and biochemical changes indicative of inflammation occur in humans. Increased bronchoalveolar lavage (BAL) fluid levels of neutrophils (PMNs) and prostaglandins (PGE2) have been reported in humans as early as 3 hr and as late as 18 hr after exposure. The purpose of this study was to determine whether a broad range of inflammatory mediators are elevated in BAl fluid within 1 hr of exposure. We exposed eight healthy volunteers twice: once to 0.4 ppm ozone and once to filtered air. Each exposure lasted for 2 hr during which the subjects underwent intermittent heavy exercise (66 liters/min). BAL was performed 1 hr after the exposure. Ozone induced rapid increases in PMNs, total protein, LDH, alpha-1 antitrypsin, fibronectin, PGE2, thromboxane B2, C3a, tissue factor, and clotting factor VII. In addition, there was a decrease in the recovery of total cells and alveolar macrophages, and decreased ability of alveolar macrophages to phagocytize Candida albicans. A comparison of these changes with changes observed in an earlier study in which subjects underwent BAL 18 hr after an identical exposure regimen indicates that IL-6 and PGE2 levels were higher 1 hr after exposure than 18 hr after exposure, fibronectin and tissue-plasminogen activator levels were higher 18 hr after exposure, and that PMNs, protein, and C3a were present at essentially the same levels at both times. These results indicate that (i) several inflammatory mediators are already elevated 1 hr after exposure; (ii) some mediators achieve their maximal levels in BAL fluid at different times following exposure. These data suggest that the inflammatory response is complex, depending on a cascade of timed events, and that depending on the mediator of interest one must choose an appropriate sampling time.