Subpopulations of Human Lung Alveolar Macrophages: Ultrastructural Features

Lung alveolar macrophages (LAM), obtained by bronchoalveolar lavage of healthy donors, were separated into four subfractions on discontinuous gradients of Percoll and subjected to light micro-scopic, transmission (TEM) and scanning electron microscopic (SEM) studies. Alveolar macrophage morphometric analysis was performed on cytocen-trifuged preparations. TEM of subpopulations revealed considerable morphologic heterogeneity. By SEM, cells of the most dense (D) subtraction were small, round, and, typically, the surface was highly ruffled with small membrane pseudopods. Cells of the least dense subtraction (A) showed a low degree of membrane folding or filopodia and were often totally disorganized.

In smokers, macrophages of fraction A had a greater area and perimeter compared with non-smokers, whereas the inverse relationship was observed for C and D cells. Also, the number of electron-dense inclusions and the level of acid phosphatase were higher in smokers than in non-smokers. Coupled with functional heterogeneity the morphologic differences described in this paper suggest that density-separated subpopulations of LAM may represent different stages of differentiation or maturation.     

The Intracellular Environment of Human Macrophages That Produce Nitric Oxide Promotes Growth of Mycobacteria

Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-γ), l-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-γ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.     

Ultrastructural Heterogeneity of the Alveolar Macrophages from Tobacco Smokers with Chronic Bronchitis

Alveolar macrophages recovered by bronchoalveolar lavage from 14 heavy smokers with chronic bronchitis were assessed. Ultrastructural examination revealed marked cellular heterogeneity. Three subpopulations of alveolar macrophages were readily identifiable. These have been termed "young," "mature," and "degrading," reflecting their ultrastructural features. In addition, a majority of the cells were found to be positive by TUNEL staining, indicating DNA damage, but a very small percentage tested positive for Caspase-3, suggesting that apoptosis might not account for the DNA damage in at least some of these cells. A small percentage of proliferating cells were noted.     

Behavior of Mycobacterium leprae in Human Macrophages In Vitro

Attempts have been made to cultivate Mycobacterium leprae in human macrophages in vitro. In 27 out of 55 experiments a two- to ninefold increase (mean 2.31 +/- 1.46) in acid-fast organisms were observed over a period of 1.5 to 3 months of cultivation. No such increase was observed with heat-killed bacilli (mean fold increase 0.88 +/- 0.19). Macrophages were necessary for obtaining increases. No multiplication was observed on artificial media. A close correlation between increases of acid-fast organisms and changes in viability as determined by the morphology of the bacilli (morphologic index) was found. The increases in acid-fast organisms could be inhibited by anti-leprosy drugs. It is concluded that multiplication of M. leprae may take place inside human macrophages in vitro. Multiplication appears not to be dependent on whether the macrophages are derived from lepromatous or tuberculoid patients or healthy individuals. Moreover, multiplication took place both at 33 and 37 C. The applicability of this method is at present limited by the restricted survival of human macrophages in vitro.     

The Mannose Receptor Mediates Uptake of Pathogenic and Nonpathogenic Mycobacteria and Bypasses Bactericidal Responses in Human Macrophages

The mannose receptor (MR) is involved in the phagocytosis of pathogenic microorganisms. Here we investigated its role in the bactericidal functions of human monocyte-derived macrophages (MDMs), using (i) trimannoside-bovine serum albumin (BSA)-coated latex beads and zymosan as particulate ligands of the MR, and (ii) mannan and mannose-BSA as soluble ligands. We show that phagocytosis of mannosylated latex beads did not elicit the production of O₂⁻. Zymosan, which is composed of α-mannan and β-glucan, was internalized by the MR and a β-glucan receptor, but the production of O₂⁻ was triggered only by phagocytosis through the β-glucan receptor. Activation and translocation of Hck, a Src family tyrosine kinase located on lysosomes, has previously been used as a marker of fusion between lysosomes and phagosomes in human neutrophils. In MDMs, Hck was activated and recruited to phagosomes containing zymosan later than LAMP-1 and CD63. Phagosomes containing mannosylated latex beads fused with LAMP-1 and CD63 vesicles but not with the Hck compartment, and the kinase was not activated. We also demonstrate that the MR was unable to distinguish between nonpathogenic and pathogenic mycobacteria, as they were internalized at similar rates by this receptor, indicating that this route of entry cannot be considered as a differential determinant of the intracellular fate of mycobacteria. In conclusion, MR-dependent phagocytosis is coupled neither to the activation of NADPH oxidase nor to the maturation of phagosomes until fusion with the Hck compartment and therefore constitutes a safe portal of entry for microorganisms.     

Ultrastructural Study Across the Leprosy Spectrum

We have carried out a systematic ultrastructural study of the bacilli, the cell-mediated response in the host, and the dermal microvasculature in lepromatous (LL), borderline lepromatous (BL), and borderline tuberculoid (BT) types of active leprosy (eight cases). In the types of least resistance (LL and BL), macrophages with large cytoplasmic processes were observed; in addition, numerous peripheral vacuoles were found in BL. Mast cells were abundant and vascular alterations constant. BT macrophages showed more regular outlines and multivacuolated cytoplasms with plentiful rough endoplasmic reticulum. Giant cells were scarce. Bacilli, both isolated and in globi, were contained within the vacuoles and appeared constantly in macrophages and endothelial and Schwann cells in LL and BL. Conversely, in BT they were found singly, infrequently in the endothelial cells, and not at all in Schwann cells. Forms in the process of destruction or degradation were more common than intact forms, in which the symmetric outline of the membrane could be seen clearly.     

Ultrastructural Study Across the Leprosy Spectrum

Three cases of eosinophilic granuloma of bone exhibited intercellular attachments between histiocytes, with a pentalaminar structure identical to that seen in nearby intracellular Birbeck granules (BG). It is proposed that the term “lattice junction” be coined to describe this organelle, which seems to be expressed only by cells of monocytichistiocytic lineage. While this finding confirms the ability of the surface membrane to form such structures, it does not necessarily preclude other intracytoplasmic sites of origin for some BG. The previously suggested role of BG in membrane storage and regulation is reiterated as a supportable model for their function.     

Tumorlets of the Lung- An Ultrastructural Study

A tumorlet of the lung is a minute tumorlike lesion found in damaged lungs in close association with the bronchioles. Histochemical and ultrastructural studies identify proliferating cells in the tumorlets as Kultschitzky-type cells. However, the pathological significance of the tumorlets, whether they are hyperplastic or neoplastic, is still controversial. Previous ultrastructural studies on the tumorlets have been carried out on formalin-fixed lung tissues. The case examined in this study was of typical tumorlets found in a so-called middle lobe syndrome of the lung of a 52-year-old woman. Tumorlets were located within the bronchiolar mucosa surrounded directly by a basal lamina and by the bronchiolar nonendocrine epithelial cells. There were no signs of invasion into the surrounding connective tissues or into lymphaticlike spaces. Between the covering bronchiolar epithelial cells and the subjacent proliferating Kultschitzky cells, specific sites of cell-to-cell attachment were noted. This finding, in addition to previously reported clinicopathological characteristics, indicates that the proliferating Kultchitsky-type cells in the tumorlets might be nonneoplastic and that tumorlets are due to hyperplasia of pure Kultschitzky-type cells, thus resembling neuroepithelial bodies of the lung.     

多发性骨髓瘤相关巨噬细胞的研究及其意义

多发性骨髓瘤（multiple myeloma,MM）微环境产生的生存信号和生长因子,是MM细胞存活、生长和形成耐药的基础。MM相关性巨噬细胞是肿瘤微环境的重要组成成份,来源于外周血单核细胞,由MM细胞慕集而来,受MM细胞和间充质基质细胞激活,并释放多种生长因子、蛋白水解酶、细胞因子和炎性介质。作用机制包括以下几个方面：生成肿瘤血管、促进生长、形成耐药,从而导致疾病的进展。由于巨噬细胞对MM的生物学特性和疾病的发展过程起关键性作用,因此,MM相关性巨噬细胞可以作为靶点,用于MM的治疗。     

Ultrastructural Study of a Perineurioma

A case of soft tissue tumor in the left brachialis muscle of a 49-year-old Japanese female patient was studied by electron microscopy. The tumor was diagnosed as intramuscular myxoma by light microscopy, but electron microscopic observation revealed that the tumor almost entirely consisted of cells similar to normal perineurial cells. The tumor cells possessed long, slender cytoplasmic processes covered by well-developed but discontinuous basal laminae, clusters of pinocytotic vesicles, and infrequent intercellular junctions. Perineurial cells have also been observed in other peripheral nerve lesions: neurofibromas, nerve sheath myxomas, and localized hypertrophic neuropathies. However, the term “perineurioma” or “perineurial cell tumor” should be reserved for discrete tumorous masses that are almost entirely composed of perineurial cells without evidence of residual axons, Schwann cells, fibroblasts, or tactile corpusclelike structures. Perineurioma may represent a third category of peripheral nerve sheath tumors, ultrastruc-turally distinct from schwannomas and neurofibromas.     

Ultrastructural Study of a Perineurioma

A case of soft tissue tumor in the left brachialis muscle of a 49-year-old Japanese female patient was studied by electron microscopy. The tumor was diagnosed as intramuscular myxoma by light microscopy, but electron microscopic observation revealed that the tumor almost entirely consisted of cells similar to normal perineurial cells. The tumor cells possessed long, slender cytoplasmic processes covered by well-developed but discontinuous basal laminae, clusters of pinocytotic vesicles, and infrequent intercellular junctions. Perineurial cells have also been observed in other peripheral nerve lesions: neurofibromas, nerve sheath myxomas, and localized hypertrophic neuropathies. However, the term “perineurioma” or “perineurial cell tumor” should be reserved for discrete tumorous masses that are almost entirely composed of perineurial cells without evidence of residual axons, Schwann cells, fibroblasts, or tactile corpusclelike structures. Perineurioma may represent a third category of peripheral nerve sheath tumors, ultrastruc-turally distinct from schwannomas and neurofibromas.     

Ultrastructural Study of Rasmussen Encephalitis

Rasmussen encephalitis (RE) is a well-known cause of seizures in children, manifesting histopathology of chronic inflammation of the brain. Its etiology remains unknown. Many reports have suggested the possibility of etiological heterogeneity of this disorder, including the possibility that it is caused by viral infection of the central nervous system, autoimmune phenomena, or a combination of both. The authors report ultrastructural study of 12 cases of RE. They identified most of the features of RE that have been described at the light microscopic level, but did not find any evidence of viral particles, immune complex deposits, or disruption of the blood-brain barrier except in 3 cases: intranuclear (intraneuronal) measle virus-like particles were seen in 1 patient, an electron-dense deposit in the basement membrane of the capillary of another, and a tubuloreticular body in the endothelium of a brain parenchymal capillary in a third (the first 2 rather exceptional findings were previously reported). At present, these appear to be rare findings and not reliable features of RE ultrastructure. The authors also investigated evidence of programmed cell death (apoptosis) of neurons, but only nonspecific degenerative changes of the neurons were found. At present, despite interesting light microscopic and ultrastructural features of RE, its pathogenesis remains cryptogenic.     

Factor Structure of the Eyberg Child Behavior Inventory: A Parent Rating Scale of Oppositional Defiant Behavior Toward Adults,Inattentive Behavior,and Conduct Problem Behavior

Used the Eyberg Child Behavior Inventory (ECBI) to measure disruptive behavior problems in children and adolescents. A controversy exists, however, on the dimensional structure of the ECBI. To evaluate this issue, an exploratory factor analysis was first performed on a sample of 1,263 children and adolescents. This analysis identified 3 meaningful factors (i.e., Oppositional Defiant Behavior Toward Adults, Inattentive Behavior, and Conduct Problem Behavior) and a fourth, poorly defined factor. A confirmatory factor analysis (CFA) evaluated the fit of the 3 meaningful factors in a second sample of 1,264 children and adolescents. The 3-factor model with 2 correlated errors provided a excellent fit. This 3-factor model also provided a significantly better fit than 2- and 1-factor models. Multiple group CFA indicated that the factor pattern, item-factor loadings, factor correlations, and correlated errors were equivalent across the samples. The CFA on sex yielded similar results. Initial normative information is presented for boys (n = 1,322) and girls (n = 1,205) within 4 age ranges (i.e., 2-5, 6-9, 10-13, 14-17) for the 3 factors. The use of these 3 factors, especially Oppositional Defiant Behavior and Conduct Problem Behavior, should make the ECBI more useful as a screening and outcome measure.     

Ultrastructural Study of Symmetrical Acral Keratoderma

Abstract

Background: Symmetrical acral keratoderma is characterized by symmetrical brown hyperkeratotic patches on the acral extremities. However, no studies about its electron microscopic examination have been documented.

Objective: Our study was performed to further characterize the histopathology of symmetrical acral keratoderma.

Methods: A biopsy was taken from brown hyperkeratotic patches on the wrists. Investigative studies included light and electron microscopy.

Results: Light microscopy showed epidermal basket-weave hyperkeratosis and acanthosis. Ultrastructurally, the epidermis was thickened by acanthosis and compact stratum corneum. The horny cell layers were remarkably thicker in clinical affected skin than in adjacent clinically unaffected and healthy skin. The keratin filaments were remarkably clumped or aggregated and irregularly distributed in the horny, spinous, granular and basal cell layers. The tonofilaments formed tight clumps or aggregated at the perinuclear cytoplasm.

Conclusion: The main ultrastructural features of symmetrical acral keratoderma were epidermal hyperkeratosis and abnormalities of the keratin filaments and tonofilaments.     

Rapid Identification of Mycobacteria to the Species Level Using INNO-LiPA Mycobacteria, a Reverse Hybridization Assay

INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification of Mycobacterium species in culture and identifies the Mycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacterium species: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with the Mycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species- or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA as M. avium (n = 18) or M. intracellulare (n = 7) or as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex (n = 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.     

Evaluation of MALDI Biotyper Mycobacteria Library v3.0 for Identification of Nontuberculous Mycobacteria

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has demonstrated its ability to promptly identify nontuberculous mycobacteria using the Mycobacteria Library v2.0. However, some species are particularly difficult to identify reliably using this database, providing a low log(score). In this study, the identification power of an updated Mycobacteria Library (v3.0) has been evaluated. Overall, 109 NTM isolates were analyzed with both databases. The v3.0 database allowed a high-level confidence in the identification [log(score) value, ≥1.8] of 91.7% of the isolates versus 83.5% with the v2.0 version (P < 0.01).