[Ehrlichiosis--a disease rarely recognized in Poland]. / Ehrlichioza--choroba malo znana i rzadko rozpoznawana w Polsce.

Ehrlichioses constitute a group of acute zoonoses caused by the infection with the microorganisms belonging to the genera Ehrlichia, Anaplasma and Neorickettsia (Rickettsiaceae). Presence of human granulocytic ehrlichiosis (HGE) caused by infection with Anaplasma phagocytophila (A. phagocytophila) and transmitted by Ixodes ricinus tick has been confirmed in Poland. All the cases described so far were noted in the area of endemic prevalence of Lyme borreliosis (north-east of Poland). Lack of characteristic symptoms makes the diagnosis of HGE difficult. It should be suspected in patients exposed to tick bites during the preceding few weeks, in whom acute, febrile illness accompanied by leucopenia and thrombocytopenia develops. The course of infection may be serious, or even life-threatening, in patients more than 40 years old; consequences of co-infection with A. phagocytophila and other tick-borne pathogens remain not well known. There is probably no risk of the development of chronic HGE. As specific diagnostic methods are not widely accessible in Poland, empirical therapy with doxycycline may be of benefit in suspected HGE cases.

Clinical and laboratory work-up of patients with neutrophil shortage or dysfunction.

Neutrophils have a crucial function in the defense against bacteria and fungi. Indeed, during chronic, severe neutropenia and in case of severe neutrophil dysfunctions, the patients may suffer recurrent and sometimes life-threatening infections. This article describes the clinical symptoms, the theory behind the antimicrobial systems of neutrophils, the methods to diagnose the various aberrations, and the possibilities for treating these patients. A few of the most common causes of neutropenia and neutrophil dysfunctions are described in detail, including recent genetic information regarding the cause of these diseases.

Application of a fluorochrome-lysostaphin assay to the detection of phagocytic and bactericidal disturbances in human neutrophils and monocytes.

The evaluation of phagocytic and microbicidal activities of the blood neutrophils has been recognized as one of the important tools for investigating phagocytic dysfunctions in patients with recurrent infections. In the present study, these activities were examined in neutrophils and monocytes from healthy adults and patients affected by primary phagocytic dysfunctions by using a modified fluorochromic microbicidal assay, discriminating simultaneously the extracellular adherence, ingestion and intracellular killing of Staphylococcus aureus Cowan I. The assay employs acridine orange staining, as described in Bellinati-Pires et al. (1989) (AO assay), but was modified by the addition of an alternative leukocyte treatment with 0.5 U/ml of lysostaphin (LS) for 5 min at 37 degrees C, after phagocytosis (AO-LS assay). The LS treatment was standardized to eliminate staphylococci adhered to the outer surface of the phagocytes without affecting the determination of intracellular live or dead bacteria, as demonstrated in normal neutrophils and monocytes. Our purpose in this study was to compare AO and AO-LS assays in order to evaluate the effect of LS on the determination of actually ingested staphylococci and to provide a means for improving the fluorochromic assay for detecting phagocytic defects, as well as bactericidal disturbances. By using the AO-LS assay, decreased ingestion of staphylococci by neutrophils in Chediak-Higashi Syndrome (CHS) was demonstrated. However, increased staphylococci adherence, as well as ingestion, was observed in neutrophils or monocytes from chronic granulomatous disease (CGD) patients, comparing AO and AO-LS assays. Bactericidal defect, which is a common feature in CHS and CGD, was detected in neutrophils or monocytes in both assays. We emphasize that such alterations were deduced by comparing the patients' results with those obtained from their respective normal controls and with the normal range of values previously established for 160 healthy adults. No alteration was observed in hyper IgE syndrome phagocytes. Despite the possible penetration of LS into the leukocytes, as stated in other studies, we concluded that a short period of phagocyte incubation with this enzyme increased the sensitivity of the fluorochromic assay to detect phagocytic defect without affecting the determination of the bactericidal activity. Moreover, comparations between AO and AO-LS assays may be important in the study of the initial pathways of staphylococci phagocyte interaction, including adherence by non-phagocytic receptors.

Biochemical changes in polymorphonuclear leucocytes in diabetic patients.

A study on the functional ability of polymorphonuclear leucocytes (PMNL) indicates that the total lysosomal enzyme levels viz. Beta-glucuronidase, lysozyme, acid phosphatase and alkaline phosphatase were not altered in diabetics, compared to that in control subjects. However, the findings also reveal that the release of these lysosomal enzymes in response to a particulate stimulus is impaired in diabetics. This suggests that the bactericidal capacity of these cells, which are involved in phagocytosis, is impaired in diabetics, making them more vulnerable to infections.

History and implications of the neutrophil glycoprotein deficiencies.

This review focuses on the initial clinical descriptions and subsequent investigation of the syndrome of recurrent infections associated with neutrophil membrane glycoprotein deficiencies. Characterization of the missing group of three glycoprotein heterodimers and their role in adhesion-related neutrophil function is summarized. Study of the clinical consequences of these genetically determined membrane glycoprotein defects has also contributed to the understanding of the role of normal neutrophils in both host defense and host tissue damage.

Evaluation of a fluorochrome assay for assessing the bactericidal activity of neutrophils in human phagocyte dysfunctions.

We evaluated a method for the assessment of the phagocytic and bactericidal activity of human peripheral neutrophils against Staphylococcus aureus Cowan I, which is a modified version of the acridine orange staining technique originally described by Smith and Rommel (1977). The modification consisted of the use of free leukocyte suspensions rather than coverglass adhered leukocytes in order to avoid two main problems: the inefficient neutrophil adherence to glass that can be observed in specimens from patients with certain functional phagocyte defects, and the risk of selecting among neutrophils. An additional advantage of the modified procedure is that it permits a uniform bacteria: phagocyte ratio in different cell samples. The method was tested on 25 healthy adults and on four children with functional phagocytic defects (chronic granulomatous disease of infancy, Chediak-Higashi syndrome, and Rothmund-Thomson syndrome associated to persistent neutropenia and low chemotactic response). The neutrophils of all four patients showed a low bactericidal activity, with percent values of intracellular killed bacteria below the mean +/- 2 SD range observed in the healthy population at all incubation times tested (5, 15 and 30 min). A significant reduction in phagocytosis index and in % killed unopsonized S. aureus was observed in relation to bacteria treated with a pool of normal human serum. These results demonstrate the high sensitivity of the method, which could be used to determine intrinsic and extrinsic functional alterations in human neutrophils.

Evaluation of the patient with a possible phagocytic disorder.

This article presents an outline of a rational approach to the patient with recurrent infections. The different categories of phagocytic defects are reviewed, and a brief discussion of the various tests of phagocyte function is presented.

Metabolism of immunoglobulin E in patients with markedly elevated serum immunoglobulin E levels.

The metabolism of human IgE was studied in normals, severe atopics, and patients with the hyperimmunoglobulin E-recurrent infection (HIE; Job's) syndrome to determine whether IgE metabolism is altered in patients with marked elevation of serum IgE. Purified polyclonal 125I-IgE was administered intravenously and serial plasma and urine samples were obtained. After analysis, the metabolic data support previously published evidence that IgE (at concentrations found in normal individuals) is catabolized at a higher fractional rate than other immunoglobulins and is catabolized by both an intravascular and an extravascular pathway. In addition, the data show that the fractional catabolic rate for IgE is significantly less for the atopic patients (mean +/- SEM = 0.20 +/- 0.01) and for the HIE patients (0.15 +/- 0.02) than for the normal volunteers (0.52 +/- 0.06; P less than 0.01) and is inversely related (r = -0.851; P less than 0.001) to the serum IgE concentration. These findings have specific importance in showing that decreased fractional catabolic rate contributes substantially to elevation of IgE in atopic and HIE patients. In addition, the findings have general significance in that they lead to a unifying hypothesis of immunoglobulin catabolism.

Impaired bacterial degradation by monocytes and macrophages from a patient with treated Whipple's disease.

A patient with Whipple's disease is described, and multiparameter flow cytometric examinations of several of the patient's phagocyte functions 3 and 9 mo after the start of oxytetracycline therapy are reported. Almost no intracellular degradation of Escherichia coli or Streptococcus pyogenes proteins and DNA occurred after ingestion by the patient's monocytes and macrophages. In addition, only minor digestion of phagocytized zymosan particles was detected. The mononuclear intracellular degradation was equally impaired 3 and 9 mo after the start of therapy. The monocyte and macrophage phagocytosis and intracellular killing, and all granulocyte phagocyte functions tested, were normal. The impaired mononuclear degradation of ingested material that was measured is consistent with the accumulation of periodic acid-Schiff-positive bacterial degradation products seen in macrophages of affected tissues in vivo, and suggests a key role of macrophage dysfunction in the pathogenesis of Whipple's disease.

[Anomalies of the membrane receptors of blood phagocytic cells in nosocomial pulmonary infections]. / Anomalies des récepteurs membranaires des cellules phagocytaires sanguines au cours d'infections pulmonaires nosocomiales.

Patients in intensive care units have frequent and severe opportunistic bacterial pneumoniae, even if they were previously free from respiratory disease. A search was made in these patients for possible immune deficiencies. Granulocyte and monocyte phagocytic activities were studied separately in 17 patients with bacterial bronchopneumonia (male: 13, female: 4; age: 41 +/- 5 yr). The ability of three types of particles (opsonized zymosan, immunoglobulin coated and glutaraldehyde-treated sheep red cells) to trigger ingestion was measured. Cells were tested either in normal AB serum or in the presence of patient's serum. A substantial 40% decrease of the fraction of granulocytes ingesting zymosan was found in our experimental conditions (p less than 0.001). Activity with the other particles was not significantly altered. Patients' sera were at least as efficient as a pool of normal sera in opsonizing zymosan. Furthermore, no phagocytic inhibitor was found in the patients tested. Underlying mechanisms for these abnormalities remain unknown, but a better understanding of the aetiology of the altered bactericidal function of phagocytic cells is required before suggesting immunomodulating treatments.

Deficiency in C3b receptors on neutrophils of patients with chronic granulomatous disease and hyperimmunoglobulin-E recurrent infection (Job's) syndrome.

C3b receptor (CR1) expression by neutrophils (PMNs) and erythrocytes (Es) from patients with chronic granulomatous disease (CGD) or with hyper-IgE, frequent infection (Job's) syndrome was compared with that of control subjects. The control subjects consisted of one group of patients with infections and a second group of normal, healthy individuals. Three quantitative assays were used: rosette formation with C3b-coated cellular intermediates (EAC43b), binding of radiolabeled monoclonal anti-CR1 ([125I]anti-CR1) to PMN surfaces, and binding of the antibody to nonidet P-40 (NP-40) extracts of PMNs and Es in an immunoradiometric assay. Rosette formation by the PMNs of five male CGD patients was about 50% of that of paired normal control subjects, whereas the rosette formation of three female CGD patients was similar to that of the control subjects. Surface binding of [125I]anti-CR1 to PMNs of 10 CGD patients was about half that of the normal subjects (mean percent binding was 2.33% for the CGD patients vs. 3.86% for the normal subjects, giving a difference of -1.53 +/- 0.22%, P less than 0.001 by the paired-sample t test). The degree of PMN binding was similarly low for both the male and the female CGD patients. Conversely, the binding of anti-CR1 to the PMNs of 11 infected control patients appeared to be similar to that of the normal subjects (4.51% for the patient vs. 4.21% for the paired normal subjects). The infected control group originally included four Job's syndrome patients, and when this subgroup was analyzed separately, their PMNs were shown to bind significantly less anti-CR1 than did the PMNs of the normal subjects (P less than 0.01 by the paired-sample t test). In contrast, the other infected control patients showed higher-than-normal levels of anti-CR1 binding (P less than 0.05). When compared to that of the normal subjects, the total CR1 quantitated in PMN extracts was also lower than normal in CGD patients (P less than 0.01 and in the PMN extracts of eight Job's syndrome patients tested (P less than 0.01). The PMNs of the other infected control subjects were not significantly different from those of the normal subjects in total CR1 expression. Extracts of Es from Job's syndrome patients also had fewer than normal CR1 (P less than 0.02). On the other hand, CR1 levels in E extracts from the CGD patients and the other control patients were similar to those in the normal control subjects. Quantitations of C3, C4, and factor B were normal in CGD.(ABSTRACT TRUNCATED AT 400 WORDS)

Deficiency of a leukocyte surface glycoprotein (LFA-1) in two patients with Mo1 deficiency. Effects of cell activation on Mo1/LFA-1 surface expression in normal and deficient leukocytes.

Mo1, a phagocyte surface glycoprotein heterodimer, is involved in a number of phagocyte adhesion functions such as binding and ingestion of serum-opsonized particles, zymosan-induced degranulation, and superoxide generation. Deficiency of this antigen in humans has been associated with increased susceptibility to recurrent bacterial infections. The beta subunit of Mo1 is shared by another surface glycoprotein named LFA-1, which is involved in lymphocyte proliferation, cytolytic T cell, and natural killing activities. Two unrelated patients with Mo1 deficiency were found to be deficient in LFA-1 as well as in the common beta subunit. Investigation of lymphocyte functions in these two patients revealed normal mixed leukocyte culture-generated cytolytic T cell and natural killing activities and significantly reduced proliferative response to phytohemagglutinin. LFA-1-deficient cells also proliferated in response to soluble antigen and different alloantigens. These responses were partially blocked by anti-LFA-1 antibody. Whereas LFA-1 was undetectable by immunofluorescence and immunoprecipitation on the patients' resting T cells, significantly reduced (approximately 5% of normal) but detectable amounts of the heterodimeric LFA-1 antigen were found on mitogen and alloantigen-activated T cells. On granulocytes, Mo1 surface expression was also dependent on the state of cellular activation. The amount of surface Mo1 present on resting normal granulocytes increased by 3-10-fold following exposure to stimuli that induced degranulation, suggesting the presence of a major intracellular pool for this antigen. Analysis of subcellular fractions from granulocytes showed that intracellular Mo1 is located primarily in the specific granule fraction. Activated granulocytes had little or no increase in their surface expression of LFA-1 antigen. Deficient granulocytes had significantly increased numbers of Mo1 antigen expressed on the surface following stimulation with calcium ionophore (1 microM). However, the amount expressed continued to be significantly reduced compared with normal cells. Quantitation of surface Mo1 on granulocytes exposed to calcium ionophore (1 microM) showed that both parents in one family but only the mother in the other family had significantly reduced levels of Mo1, suggesting heterogeneity in the inheritance of this disorder. Whereas LFA-1 deficiency on lymphocytes was associated with normal alloantigen-induced cytolytic T cell and natural killing activities in these two patients, functions which were in part dependent on small amounts of detectable LFA-1 antigen, the Mo1 deficiency state led to significant defects in phagocyte adhesion functions. Hence, the clinical symptoms associated with this combined deficiency state reflect a more profound phagocyte than lymphocyte disorder.

Subcellular localization of the large subunit of Mo1 (Mo1 alpha; formerly gp 110), a surface glycoprotein associated with neutrophil adhesion.

Mo1 alpha (formerly gp 110) is a neutrophil glycoprotein whose deficiency is associated with abnormalities in several neutrophil functions, including defects in adherence, chemotaxis, and phagocytosis. Examination of whole cells and subcellular components by the use of both immunological and electrophoretic techniques demonstrated that Mo1 alpha was located primarily in the specific granules but that a small portion was present in the plasma membrane, where it is exposed to the extracellular environment and can bind to anti-Mo1 antibody. During degranulation, Mo1 alpha is translocated from the specific granules to the plasma membrane, resulting in a 5-10-fold increase in the surface expression of this glycoprotein. These findings plus previous work suggest that plasma membrane-associated Mo1 alpha is needed for a normal interaction between neutrophils and underlying surfaces, and raise the possibility that the increase in surface adhesiveness of neutrophils that have discharged their specific granules might be due in part to the increase in the amount of Mo1 alpha in the plasma membranes of these degranulated cells.

[Neutrophil functions and interactions in the inflammatory reaction]. / Fonctions et interactions du neutrophile dans la réaction inflammatoire.

After 14 days' bone marrow maturation, neutrophil granulocytes reach the tissues where for 1-2 days they form the army whose phagocytic function was described by llya Metchnikoff in 1882. At that time, Paul Ehrlich was developing his neutrophil secretory theory which had less success until it returned with a vengeance in the last decade. Neutrophils are not only phagocytes. Above all they are cells that secrete bactericidal effectors and regulators (amplifiers and modulators) of the inflammatory focus. More and more sophisticated methods are being used to study phagocytosis, from the point of view both of the mechanism of chemotaxis and its role in inflammation and of the mediators of oxygen-dependent bactericidal action (superoxide anion, oxygenated water, hydroxyl radicals, myeloperoxidase, halogen ions and superoxide dismutase). In addition, the importance of oxygen-independent bactericidal mechanisms has been confirmed by the discovery of proteins such as BPI (Bactericidal Permeability Increasing Protein). Study of neutrophil dysfunction throws light on a number of neutrophil regulatory and effector mechanisms; it also proves useful in explaining the recurrent infections observed in some congenital disorders (chronic granulomatous disease, the "lazy leucocyte syndrome", the Chediak-Higashi syndrome, ichthyrosis , Job's syndrome...) or those associated with transitory neutrophil disorders (measles, severe bacterial infection...). Neutropenia induced by some antibiotics is easily demonstrated, but the interactions between these antibiotics and neutrophils are complex: phagocyte concentration of antibiotic, neutrophil inactivation of antibiotic, effect of antibiotic on microbe-leucocyte interaction such as an alteration in phagocytic and chemotactic response. The neutrophil is the first blood cell to arrive at the inflammatory focus; it is also at the centre of the response, next to the humoral mediators which both act upon it and which it itself secretes.

Disorders of stimulus-response coupling in neutrophils.

The initiation of both the normal and pathologic responses of human neutrophils to surface stimulation and the ensuing biochemical and physiologic events are elucidated. This knowledge has contributed to an understanding of the controlling mechanisms that may account for impaired phagocytic function in several clinical disorders associated with recurrent bacterial infections.