Defects in neutrophil granule mobilization and bactericidal activity in familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) syndrome caused by STXBP2/Munc18-2 mutations.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by genetic defects in cytotoxic granule components or their fusion machinery, leading to impaired natural killer cell and/or T lymphocyte degranulation and/or cytotoxicity. This may accumulate into a life-threatening condition known as macrophage activation syndrome. STXBP2, also known as MUNC18-2, has recently been identified as the disease-causing gene in FHL type 5 (FHL-5). A role for STXBP2 in neutrophils, and for neutrophils in FHL in general, has not been documented thus far. Here, we report that FHL-5 neutrophils have a profound defect in granule mobilization, resulting in inadequate bacterial killing, in particular, of gram-negative Escherichia coli, but not of Staphylococcus aureus, which rather depends on intact reduced NAD phosphate oxidase activity. This impairment of bacterial killing may contribute to the apparent susceptibility to gastrointestinal tract inflammation in patients with FHL-5.

Neutrophil extracellular traps: double-edged swords of innate immunity.

Spectacular images of neutrophils ejecting nuclear chromatin and bactericidal proteins, in response to microbes, were first reported in 2004. As externalized chromatin could entangle bacteria, these structures were named neutrophil extracellular traps (NETs). Subsequent studies identified microorganisms and sterile conditions that stimulate NETs, as well as additional cell types that release extracellular chromatin. The release of NETs is the most dramatic stage in a cell death process called NETosis. Experimental evidence suggests that NETs participate in pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis, and vascular disorders. Exaggerated NETosis or diminished NET clearance likely increases risk of autoreactivity to NET components. The biological significance of NETs is just beginning to be explored. A more complete integration of NETosis within immunology and pathophysiology will require better understanding of NET properties associated with specific disease states and microbial infections. This may lead to the identification of important therapeutic targets.

Pyogranulomatous lesion causing neurological signs localised to the sacral region in a horse.

This case report describes neurological signs associated with a pyogranulomatous lesion within the sacral vertebral canal of a horse. The clinical findings included urinary overflow incontinence and reduced anal, perianal and tail tone. The horse failed to respond to medical management and a guarded prognosis for return to athletic performance initiated the decision for euthanasia.

Botryomycosis.

Botryomycosis is a chronic, granulomatous, infectious disease caused by several genera of bacteria with the formation of grains. The factors involved in its development are low virulence, an intermediate inoculum, and the immunologic status of the host. The pathogenesis of the disease is not well established, but the Splendore-Hoeppli phenomenon, which explains the formation of grains and the antigen-antibody reaction that characterizes the disease, is involved. Diagnosing botryomycosis includes clinical suspicion and microbiologic studies. Isolation of the causative agent and susceptibility tests are essential to provide appropriate treatment.

Rhesus macaque θ-defensin isoforms: expression, antimicrobial activities, and demonstration of a prominent role in neutrophil granule microbicidal activities.

Mammalian defensins are cationic, antimicrobial peptides that play a central role in innate immunity. The peptides are composed of three structural subfamilies: α-, ß-, and θ-defensins. θ-defensins are macrocyclic octadecapeptides expressed only in Old World monkeys and orangutans and are produced by the pair-wise, head-to-tail splicing of nonapeptides derived from their respective precursors. The existence of three active θ-defensin genes predicts that six different RTDs (1-6) are produced in this species. In this study, we isolated and quantified RTDs 1-6 from the neutrophils of 10 rhesus monkeys. RTD-1 was the most abundant θ-defensin, constituting ~50% of the RTD content; total RTD content varied by as much as threefold between animals. All peptides tested were microbicidal at ∼1 µM concentrations. The contribution of θ-defensins to macaque neutrophil antimicrobial activity was assessed by analyzing the microbicidal properties of neutrophil granule extracts after neutralizing θ-defensin content with a specific antibody. θ-defensin neutralization markedly reduced microbicidal activities of the corresponding extracts. Macaque neutrophil granule extracts had significantly greater microbicidal activity than those of human neutrophils, which lack θ-defensins. Supplementation of human granule extracts with RTD-1 markedly increased the microbicidal activity of these preparations, further demonstrating a prominent microbicidal role for θ-defensins.

Combined actinomycotic and pseudoactinomycotic radiate granules in the female genital tract: description of a series of cases.

BACKGROUND: Both actinomycotic granules and pseudoactinomycotic radiate granules (PAMRAGs) occur in the female genital tract, most commonly in the endometrium. It is important to distinguish between these since the former may result in pelvic inflammatory disease and require antibiotic treatment while the latter is non-infectious and does not require specific treatment. AIMS: To investigate the coexistence of actinomyces-like organisms and PAMRAGs in the same granules, and describe the presence of PAMRAGs in the cervix and the vulva. METHODS: Six cases with actinomyces-like organisms and PAMRAGs in the same granules (four in the endometrium, one in a tubo-ovarian abscess, and one in both the endometrium and a tubo-ovarian abscess) are reported as well as seven examples of PAMRAGs in the cervix and one in a vulval abscess. RESULTS: The combined granules consisted of central basophilic Gram and silver positive filamentous organisms consistent with actinomyces surrounded by radiating eosinophilic club-like formations which were Gram and silver negative, the latter consistent with PAMRAGs. The PAMRAGs in the cervix and vulva consisted entirely of Gram and silver negative radiating eosinophilic club-like formations. CONCLUSIONS: Although actinomycotic granules and PAMRAGs are distinct lesions which should be distinguished for patient management, they may coexist in the same granules. It is likely in such cases that the PAMRAGs form around the bacterial colonies which act as a nidus. The presence of radiating eosinophilic club-like formations characteristic of PAMRAGs does not preclude the presence of actinomyces. Careful morphological examination plus supportive Gram and silver stains, if necessary, allows the diagnosis of these combined granules. PAMRAGs also occur in the cervix, where it is likely that they form secondary to encrustation of inspissated mucus, and in the vulva.

Neutrophil granule proteins tune monocytic cell function.

Polymorphonuclear leukocytes (PMNs) release the contents of granules during their migration to inflammatory sites. On liberation from the first leukocyte to enter injured tissue, the granule proteins play a central role in the early inflammatory response. In particular, mononuclear phagocytes interact intimately with PMNs and their secretion products. PMN granule proteins enhance the adhesion of monocytes to the endothelium and stimulate subsequent extravasation of inflammatory monocytes. At the site of inflammation, PMN granule proteins activate macrophages to produce and release cytokines and to phagocytose IgG-opsonized bacteria. Furthermore, by direct cell-cell contacts, PMNs activate monocyte-derived dendritic cells, thereby enhancing antigen presentation. Efforts in this field might lead to the development of drugs for specific modulation of innate immune functions.

Mucocutaneous Splendore-Hoeppli phenomenon.

Splendore-Hoeppli phenomenon (asteroid bodies) is the in vivo formation of intensely eosinophilic material (radiate, star-like, asteroid or club-shaped configurations) around microorganisms (fungi, bacteria and parasites) or biologically inert substances. This study presents a literature review concerning Splendore-Hoeppli reaction in the mucocutaneous diseases. It examines the histopathological features, nature and differential diagnosis of this reaction. It also discusses the mucocutaneous infections and the non-infective diseases associated with it. Available studies indicate that several mucocutaneous infections can generate Splendore-Hoeppli reaction. The fungal infections include sporotrichosis, pityrosporum folliculitis, zygomycosis, candidiasis, aspergillosis and blastomycosis. The bacterial infections include botryomycosis, nocardiosis and actinomycosis. The parasitic conditions include orbital pythiosis, strongyloidiasis, schistosomiasis and cutaneous larva migrans. In addition, Splendore-Hoeppli reaction may be seen with non-infective pathology such as hypereosinophilic syndrome and allergic conjunctival granulomas. The Splendore-Hoeppli reaction material comprises antigen-antibody complex, tissue debris and fibrin. Although the exact nature of this reaction is unknown, it is thought to be a localized immunological response to an antigen-antibody precipitate related to fungi, parasites, bacteria or inert materials. The characteristic formation of the peribacterial or perifungal Splendore-Hoeppli reaction probably prevents phagocytosis and intracellular killing of the insulting agent leading to chronicity of infection. To conclude, Splendore-Hoeppli reaction is a tell tale of a spectrum of infections and reactive conditions. The molecular pathways involved in the development of this reaction are open for future investigations.

The antimicrobial peptides piscidins are stored in the granules of professional phagocytic granulocytes of fish and are delivered to the bacteria-containing phagosome upon phagocytosis.

Antimicrobial peptides (AMPs) are increasingly recognized as a critical first line of defence against many pathogens. The genes encoding these peptides are expressed in numerous tissue and cell types from a wide variety of different species including mammals, amphibians, fish, and insects. In this study, we report that the AMPs called piscidins were primarily present in the mast cells (MCs) of fish and were only identified in fish belonging to the Order Perciformes. It is striking that histamine was seen to have a similar evolutionary history, since the only piscine MCs endowed with this molecule are in the Perciformes. We also show that both MCs and professional phagocytic granulocytes were armed with different piscidin molecules. In contrast, macrophages were devoid of these AMPs. More importantly, we found by immunoelectron microscopy that piscidins were delivered to the bacteria-containing phagosome of granulocytes upon phagocytosis, suggesting a role for these AMPs in the killing of both extracellular and intracellular pathogenic bacteria.

Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens.

A key target of many intracellular pathogens is the macrophage. Although macrophages can generate antimicrobial activity, neutrophils have been shown to have a key role in host defense, presumably by their preformed granules containing antimicrobial agents. Yet the mechanism by which neutrophils can mediate antimicrobial activity against intracellular pathogens such as Mycobacterium tuberculosis has been a long-standing enigma. We demonstrate that apoptotic neutrophils and purified granules inhibit the growth of extracellular mycobacteria. Phagocytosis of apoptotic neutrophils by macrophages results in decreased viability of intracellular M. tuberculosis. Concomitant with uptake of apoptotic neutrophils, granule contents traffic to early endosomes, and colocalize with mycobacteria. Uptake of purified granules alone decreased growth of intracellular mycobacteria. Therefore, the transfer of antimicrobial peptides from neutrophils to macrophages provides a cooperative defense strategy between innate immune cells against intracellular pathogens and may complement other pathways that involve delivery of antimicrobial peptides to macrophages.

Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes.

We recently reported that the human pathogen Streptococcus pyogenes of the M1 serotype survives and replicates intracellularly after being phagocytosed by human neutrophils. These data raised the possibility that the generation of reactive oxygen metabolites by neutrophils, and the release of microbicidal molecules from their azurophilic and specific granules into phagosomes, can be modulated by S. pyogenes bacteria expressing surface-associated M and/or M-like proteins. We now demonstrate, using flow cytometry, immunofluorescence microscopy and transmission electron microscopy, that live wild-type S. pyogenes, after internalization by human neutrophils, inhibits the fusion of azurophilic granules with phagosomes. In contrast, azurophilic granule-content is efficiently delivered to phagosomes containing bacteria not expressing M and/or M-like proteins. Also, when heat-killed wild-type bacteria are used as the phagocytic prey, fusion of azurophilic granules with phagosomes is observed. The inhibition caused by live wild-type S. pyogenes is specific for azurophilic granule-phagosome fusion, because the mobilization of specific granules and the production of reactive oxygen species are induced to a similar extent by all strains tested. In conclusion, our results demonstrate that viable S. pyogenes bacteria expressing M and M-like proteins selectively prevent the fusion of azurophilic granules with phagosomes.

Pseudoactinomycotic radiate granules of the gynaecological tract: review of a diagnostic pitfall.

The filamentous bacterium actinomyces can cause serious gynaecological tract infections, including pelvic inflammatory disease (PID) and tubo-ovarian abscess. Thus, definitive diagnosis of actinomycotic granules (AMGs) in gynaecological specimens is clinically important. Non-infectious pseudoactinomycotic radiate granules (PAMRAGs) can mimic the microscopic appearance of AMGs. PAMRAGs may be more common than actinomycotic infections in specimens from patients using intrauterine devices and may be seen in patients with PID. Although the composition and aetiology of PAMRAGs is unclear and variable, a panel of histochemical stains can aid in diagnosis. On haematoxylin and eosin (H&E) stained sections, AMGs show as distinct granules with basophilic peripheral radiating filaments and a dense central eosinophilic core, whereas H&E stained sections of PAMRAGs feature refractile granules with irregular club-like peripheral projections and no central dense core. The filaments of AMGs are Gram positive on Brown and Brenn (B&B) stain and are highlighted with Gomori methenamine silver stain (GMS). They stain negatively with a modified acid fast bacillus (AFB) stain, aiding in the distinction of actinomyces from nocardia. PAMRAGs show negative or non-specific staining with B&B, GMS, and AFB stains. Therefore, knowledge of these staining properties and the distinguishing characteristics of PAMRAGs and AMGs enables recognition of this important diagnostic pitfall.

Identification of a human HLA-E-restricted CD8+ T cell subset in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine.

Our previous studies in volunteers immunized with Salmonella enterica serovar Typhi (S. Typhi) have suggested an important role for CD8+ T cells in host defense. In this study we describe a novel subset of nonclassical human HLA-E-restricted S. Typhi-specific CD8+ T cells derived from PBMC of Ty21a typhoid vaccinees. CD3+CD8+CD4-CD56- T cells effectively killed S. Typhi-infected targets regardless of whether they share classical HLA class I molecules with them, by a FAS-independent, granule-dependent mechanism, as evidenced by induction of granzyme B release and the blocking effects of concanamycin and strontium ions. The expression of HLA-E Ags, but not CD1-a, -b, or -c, on the membrane of S. Typhi-infected targets rendered them susceptible to lysis. Moreover, anti-HLA-E Abs partially blocked these responses. We also demonstrated that presentation of S. Typhi Ags via HLA-E could stimulate IFN-gamma production. Increases in the net frequency of IFN-gamma spot-forming cells were observed in the presence of targets coated with peptides that contain S. Typhi GroEL HLA-E binding motifs. These results demonstrate that HLA-E binds nonamer peptides derived from bacterial proteins and trigger CD8+-mediated lysis and IFN-gamma production when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections.

Human NKT cells express granulysin and exhibit antimycobacterial activity.

Human NKT cells are a unique subset of T cells that express an invariant V alpha 24 TCR that recognizes the nonclassical Ag-presenting molecule CD1d. Activation of NKT cells is greatly augmented by the marine sponge-derived glycolipid alpha-galactosylceramide (alpha GalCer). Because human monocyte-derived cells express CD1d and can harbor the intracellular pathogen Mycobacterium tuberculosis, we asked whether the addition of alpha GalCer could be used to induce effector functions of NKT cells against infected monocytes, macrophages, and monocyte-derived dendritic cells. NKT cells secreted IFN-gamma, proliferated, and exerted lytic activity in response to alpha GalCer-pulsed monocyte-derived cells. Importantly, alpha GalCer-activated NKT cells restricted the growth of intracellular M. tuberculosis in a CD1d-dependent manner. NKT cells that exhibited antimycobacterial activity also expressed granulysin, an antimicrobial peptide shown to mediate an antimycobacterial activity through perturbation of the mycobacterial surface. Degranulation of NKT cells resulted in depletion of granulysin and abrogation of antimycobacterial activity. The detection of CD1d in granulomas of tuberculosis patients supports the potential interaction of NKT cells with CD1d-expressing cells at the site of disease activity. These studies provide evidence that alpha Gal Cer-activated CD1d-restricted T cells can participate in human host defense against M. tuberculosis infection.

The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry.

The immunodominant 120 kDa protein (p120) of Ehrlichia chaffeensis was demonstrated to be exposed on the surface of purified whole ehrlichial cells examined by immunoelectron microscopy with a rabbit antibody against a portion of the domain containing tandem repeat units. In the intracellular location, the 120 kDa protein was detected by immunoelectron microscopy in the outer membrane of the cell wall of dense-core forms of the ehrlichiae in infected canine macrophage-like cells and as a component of the intramorular fibrillary matrix. No 120 kDa protein was detected in the cell wall of ehrlichial reticulate cells. Recombinant Escherichia coli with a plasmid containing the entire 120 kDa protein gene, but no bacteria with non-recombinant plasmid, attached to the surface of HeLa cells as visualized by electron microscopy. Some of the recombinant 120 kDa protein expressing E. coli invaded the HeLa cells as determined by gentamicin protection assays and by intravacuolar localization ultrastructurally.

Survival of Mycobacterium avium and Mycobacterium tuberculosis in acidified vacuoles of murine macrophages.

Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the phagosome and inhibition of phagosome-lysosome fusion. In contrast to mycobacteria, Coxiella burnetii, the etiologic agent of Q fever, inhabits a spacious acidified intracellular vacuole which is prone to fusion with other vacuoles of the host cell, including phagosomes containing mycobacteria. The Coxiella-infected cell thus provides a unique model for investigating the survival of mycobacteria in an acidified phagosome-like compartment. In the present study, murine bone marrow-derived macrophages were infected with either Mycobacterium avium or Mycobacterium tuberculosis and then coinfected with C. burnetii. We observed that the majority of phagocytosed mycobacteria colocalized to the C. burnetii-containing vacuole, which maintained its acidic properties. In coinfected macrophages, the growth of M. avium was not impaired following fusion with the acidified vacuole. In contrast, the growth rate of M. tuberculosis was reduced in acidified vacuoles. These results suggest that although both species of mycobacteria inhibit phagosome-lysosome fusion, they may be differentially susceptible to the toxic effects of the acidic environment in the mature phagolysosome.

Respiratory scleroma: a clinicopathologic and ultrastructural study.

Respiratory scleroma (rhinoscleroma) is a chronic granulomatous infection produced by Klebsiella rhinoscleromatis, a gram-negative aerobic coccobacillus. This disease is endemic to Africa, Central and South America, South Central and Eastern Europe, the Middle East, and China. Sporadic cases have been reported in the United States, especially in persons who migrated from the aforementioned areas. The majority of cases affect the nose, but extension to the soft and hard palate, upper lip, and maxillary sinuses also is frequent. This study comprises 11 patients (6 females and 5 males) with respiratory scleroma identified over a 6-year period in Guatemala. Their ages ranged from 16 to 60 years. Light microscopy showed a dense plasmacytic infiltrate, Mikulicz histiocytes, and Russell bodies within the plasma cells. Ultrastructural study revealed Mikulicz histiocytes, cytoplasmic vacuoles containing bacilli, and so-called A and B granules. We favor the term respiratory scleroma for this lesion because it affects not only the nose but also the upper and lower respiratory tracts as well as the mouth.

Characterization of immunorelated peptides to porcidin P1.

Porcidin P1, an antimicrobial peptide purified from the granules of porcine polymorphonuclear neutrophils (PMN) using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC), was covalently conjugated to BSA and used to generate monospecific polyclonal ascites. Antibodies raised against porcidin P1 were covalently coupled to an Affi-gel Hz affinity column and used for immunoaffinity chromatography of peptides from porcine PMN cell extract. Eleven immunorelated peptides were eluted from the column from neutrophil cell extracts and purified to homogeneity by HPLC. The molecular weights of the immunorelated peptides were determined by mass spectral analysis and ranged in size from 1.91 to 10.65 kDa. Of the 11 immunorelated peptides which were bound to the affinity column, only six peptides were recognized by the anti-porcidin antibodies after HPLC purification. Three immunoreactive peptides displayed potent antibacterial activity towards Staphylococcus aureus and Escherichia coli, reducing viability by as much as 99.9% (> 3 log reduction in CFU) when 5 mu g/mL of each purified peptide was used. The polyclonal monospecific antibodies also reacted with proteins from ovine and human PMN, illustrating possible structural relationships between small antibacterial peptides from the different species.

Pathology of infection caused by Dermatophilus-like organisms in porcine tonsils.

The authors investigated the occurrence of Dermatophilus-like organisms in sulphur granules of porcine tonsils. Light and electron microscopic studies, together with histochemical examination, were carried out to elucidate the mode of growth of the organism in the tonsils, the interaction between the organisms and host cells, and the nature of the radiating clubs around the organisms. Sulphur granules were found in about 15 and 70 per cent of market pigs and breeding pigs, respectively. Of the pigs having tonsillar granules, Dermatophilus-like organisms were observed in about 70 per cent of market pigs, and in nearly all breeding pigs. The organisms invaded tonsillar crypts to produce lesions resembling actinomycotic abscesses up to 5 mm in diameter. Dermatophilus-like organisms were demonstrated in various morphological forms ranging from filamentous to tuber-shaped or coccoid bodies. In the lesion, the bacterial cells adjacent to the host cell reaction showed distinct degenerative changes forming thick amorphous masses on the surface of the bacterial cells. The amorphous masses seemed to be derived from the bacterial cells but showed histochemical components different from those of the bacterial cells. These masses had numerous protrusions forming clubs. Phagocytic neutrophils close to the amorphous masses were presumed to play a role in deposition of the club material. Macrophages also appeared to participate in the inflammation leading to a granulomatous lesion. These findings suggested that the clubs might be formed by an interaction between the organisms and host cell reaction.

Splendore-Hoeppli phenomenon in Pityrosporum folliculitis (pseudoactinomycosis of the skin).

Actinomyces-like granules showing the Splendore-Hoeppli phenomenon have been demonstrated in histologic material, e.g. uterine curettings, in various conditions unrelated to genuine actinomycotic infection. The exact nature of the granules and the mechanism of their formation is open to speculation. We report two patients with a follicular skin eruption (Pityrosporum folliculitis) where pseudoactinomycotic granules were found in the skin biopsies. To the best of our knowledge, such findings have not previously been reported, but supposedly they are not rare. In order to avoid an incorrect diagnosis of actinomycosis, and unnecessary therapy, it is important to be familiar with the phenomenon.