Granulomatous Diseases - Infectious Disease and Antimicrobial Agents

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Granulomas were first observed over 200 years ago during the autopsy of a tuberculous lung. The various current definitions of the

granuloma are still based on histopathologic observations. The lesion is defined as a focal, chronic inflammatory tissue response

composed mostly of macrophages and their derivatives the epithelioid cells, evoked by persistent, poorly degradable substances

(1,3,9). In addition various granulomas may contain lymphocytes, sometimes plasma cells, giant cells, neutrophils, eosinophils, mast

cells and fibroblasts. Based on the causative agent(s) granulomas have a diverse etiology. They are classified into 4 groups: 1) T

cell-mediated immune granulomas formed to infectious agents. 2) Granulomas with unknown etiology but with a T lymphocyte-

mediated profile. 3) Foreign body granulomas induced by inanimate substances. 4) Granulomas associated with malignant tumors

(Table 1 ).

Table 1 -4 shows a wide range of causative agents which induce chronic inflammations by their persistent irritation and/or immune

stimulation.

FUNCTION OF THE GRANULOMA

The granuloma has a significant protective function. The replicating or inanimate agents cause chronic tissue irritations and evoke a

programmed tissue inflammatory response that isolates and walls off the offender. This is especially useful in the case of replicating

intracellular invaders ( Mycobacteria, Listeria ) that can disseminate throughout the body. The macrophages that converge at the site

of bacterial invasion ingest the bacteria and intracellularly kill them. The compact structure of the granuloma and the efficient

intracellular bactericidal activity successfully prevent the dissemination of the microorganisms. This is well illustrated by miliary

tuberculosis (TB) where deficient granuloma formation allows the systemic spread of the bacilli. A negative facet of the granuloma is

that the lesion may harbor within its burnt-out or calcified structure residual viable Mycobacteria, Histoplasma . These latent

organisms may cause the flare-up of the disease decades later. In the helminthic infection schistosomiasis the liver granulomas that

form around the parasite eggs shield the liver parenchyma cells against the secreted toxic substances.

MORPHOLOGY AND CELLULAR COMPOSITION OF THE GRANULOMAS

Immune granulomas are composed of tightly packed mononuclear cells which can be delineated from neighboring tissues. Previously

dermatologists emphasized the presence of palissading epithelioid cells as the hallmark of the hypersensitivity granulomas. These

cells are large with ill-defined cytoplasmic borders containing eosinophilic cytoplasm and large pale ovoid nuclei. They derive frommacrophages that had lost their phagocytic capacity. They may be present in T cell-mediated as well as in foreign body granulomas.

An additional characteristic feature of the granulomas is the multinucleated giant cells generated by the fusion of activated

macrophages. Giant cells may contain 20 or more nuclei arranged either peripherally (Langhans type) in immune or disordered

(foreign body type) lesions. Whereas epithelioid cells are non-phagocytic but secretory, giant cells retain their phagocytic capacity

and also produce and secrete cytokines. There is no functional difference between the Langhans or foreign body types of giant cells.

In the prototypical T helper 1 (Th1) lymphocyte-mediated TB granulomas the macrophages, epithelioid cells are surrounded by T

lymphocytes of the CD4+ and CD8+ subsets as well as /+ T cells. In the mature granulomas fibroblasts embedded within collagen

fibers are found at the periphery ( 3). The prototypical T helper 2 (Th2) lymphocyte-mediated usually helminthic granulomas contain in

addition to the macrophages, epithelioid cells, CD4+ and CD8+ T lymphocytes, also B lymphocytes at the periphery, mast cells and

numerous eosinophils. The granulomas are also encased in fibrous capsules. Granulomas that form around foreign body irritants

also contain macrophages, epithelioid and giant cells and pending the nature of the irritant some T lymphocytes, or in the case of the

silica granuloma also plasma cells.

MECHANISMS OF GRANULOMA FORMATIONGranulomas are complex multicellular tissue reactions formed after a cascade of interactive systems that encompass adhesion

molecule activity, capillary permeability, and chemokine, cytokine action. The exact nature and extent of the various components of

this cascade are still being explored. Observations have been pieced together from animal models of granulomatous inflammations,

biopsied material and bronchial lavage fluids of infected individuals. Historically, the prototype of the lung granuloma is the tubercle

induced in infected individuals by M. tuberculosis bacilli. Inhalation of the bacilli triggers the non-specific innate immune response

expressed chiefly by ingestion (phagocytosis) of the bacilli by alveolar macrophages and dendritic cells ( 28 ). Recognition of the alien

bacterial surface is carried out by the ancient pattern recognition system that utilizes a number of receptors notably the Toll-like

receptors (TLRs) that interact with mycobacterial lipoproteins( 32 ). After the mannose receptors-mediated phagocytosis a cascade of

intracellular signaling ensues and the key nuclear factor kappa B (NFB) is generated. This transfer factor is essential for t he

pro-inflammatory cytokines production. Cytokines are short peptides that serve as signaling agents between cells. The major

cytokines include tumor necrosis factor alpha (TNF), interleukin-1 (IL-1), interleukin-12 (IL-12). Transmembrane TNF inhibits the

intracellular growth of the bacilli ( 52 ) and induces CCL5 (RANTES) chemokine (chemoattractant peptide) secretion in macrophages

which attracts the Th1 subset of lymphocytes to the site of the invasion. Additional chemokines MCP-1, MIP-1 attract blood

monocytes from the circulation. This results in the accumulation of maturing macrophages bound by hyaluronic acid (kernel of thegranuloma). Such macrophages are capable to exert limited microbicidal effect on ingested bacilli. Neighboring innate defense

receptored T cells and natural killer (NK) T cells also contribute to the inflammatory cytokine, chemokine cascade. The dendritic cells

that had engulfed the bacilli upregulate their chemokine receptors and under the influence of CCL19, CCL20 and CCL21 chemokines

secreted by the lymph node stroma and the high endothelial cells in the venules migrate to the regional lymph node. En route, the

ingested bacilli are killed degraded and the bacillary peptide fragments are displayed on the membrane of the dendritic cells within

Granulomatous Diseases

Authors: Dov L. Boros, Ph.D. , Sanjay G. Revankar, M.D.

Table of ContentsCollapse All | Expand All

Function of the Granuloma

Morphology and Cellular Composition of theGranulomas

Mechanisms of GranulomaFormation

Pathogenesis of theGranulomas

Pathology of the Granuloma

Differential Diagnosis

Infectious Causes

Non-Infectious

Causes Approach toDeterminingEtiology

Laboratory Diagnosis

Cultures

Stains

Immunohistochemistry,Immunofluorescence

Stains for Non-Cellular Tissue Elements

Molecular Methods

Use of Specific Antibodies and TCell Function

Assays

Histopathology

Clinical Clues in Assessing Etiology

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ulomatous Diseases - Infectious Disease and Antimicrobial Agents http://www.antimicrobe.org/new/e37.asp

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the grooves of the major histocompatibility complex II (MHC II) molecules. Within the lymph node dendritic cell and T cell are in close

contact by means of adhesion molecules, and the process of antigen presentation to the clonally selected T cell / receptors

ensues. Continued secretion of IL-12 by the dendritic cell induces the differentiation of the T cell clones to the T h1 subset. Having

undergone the process of sensitization the Th1 cell secretes IL-2 cytokine that promotes T cell survival and proliferation. T he

sensitized Th1 cells are attracted by a gradient of a number of chemokines such as CXCL8,-9,-10,11 to the site of the focally

aggregated macrophages which contain the ingested bacilli. Focally secreted chemokines CCL2,-3,-4,-5 recruit fresh monocytes from

the circulation and by the membrane-bound TNF signal adhesion molecule expression on the converging cells is increased and the

early granuloma now organizes into a characteristic structure( 5,34 ). The CD4+ and CD8+ T cells arrange at the periphery. Some of

the CD4+ cells are interspersed with macrophages which contain ingested bacilli ( 40 ). Thus, at the first step the invaders are

sequestered by granuloma macrophages. Fibroblasts at the periphery of the compact lesion produce collagen bundles. The fibrotic

capsule delineates the lesion and ensures that the bacteria are walled off from the surrounding tissue. The mature granuloma alsoshows neovascularization. The antigenically and IL-12 stimulated CD4+ Th1 type lymphocytes secrete IFN that activates the

macrophages to acquire the microbicidal function. Such activated cells produce oxygen (hydrogen peroxide H2O2, hydroxyl radical

HO, superoxide anion O2-, singlet oxygen O2) and nitrogen (NO nitrogen oxide) breakdown products (ROS and RNS reactive

oxygen and nitrogen species respectively) that are strongly oxidative. Currently NO is considered to be the principal agent that kills

the M. tuberculosis . Continued activation of the macrophages is also provided by TNF secreted by both macrophages and Th1

cells. The CD8+ T cells also participate in protection by killing the bacilli-overloaded macrophages and releasing bacilli to be

ingested by fresh activated macrophages ( 15 , 49 , 50 , 51 , 56 ).

The hallmark of the mature TB and other granulomas is the epithelioid cells that occupy the center of the lesions. These cells are

transformed macrophages that had lost their phagocytic function but retained their secretory activity producing TNF, IL-10 and the

regulatory TGF- cytokines. Activated macrophages occasionally fuse together to create multinucleated giant cells. Such fusion was

shown experimentally to be induced by IL-1,-3,-4,-6 and GM-CSF cytokines all of which may be produced by the granulomas. Giant

cells within the granuloma retain their phagocytic and microbicidal activity and secrete IL-1 , TNF and TGF- cytokines contributing

to the granuloma cellular dynamics ( 26 ).

The prototypic T helper 2 (Th2) lymphocyte-mediated granuloma is induced by Schistosoma mansoni worm eggs. These eggs are

produced by female worms that live in the mesenteric venous plexus of the infected host. The eggs disseminate and lodge within the

microcapillaries in the liver and intestines. The eggs are ~ 150 m in length cannot be phagocytized. By secreted antigens they

induce a specific immune T helper cell response that generates the granulomatous reaction around each egg. Granulomas that form

around the eggs also serve a protective function, because the cellular layers shield the hepatic cells against the toxic effect of some

egg components. It appears that glycoconjugates of the worm eggs interact with host dendritic cells and by triggering their Toll-Like

Receptors (TLRs) can induce a biased Th2 response ( 19 ). This response generates the production of cytokines, chemokines which

recruit and activate inflammatory cells different from those present in the TB type granuloma. The resultant egg granuloma is

composed of macrophages, B cells, CD4+ CD8+ T cells, fibroblasts and many eosinophils. Information on the mechanism of

granuloma formation derives from murine experiments. During lung granuloma formation around injected egg antigen-coated beads

the innate response expressed IL-1, TNF, GM-CSF, IL-3, IL-6 cytokines and numerous chemokines both from the CCL (MCP-1, 2,

3, MIP-2, , ) and CXCL 2,5,9,10,11 families needed for cellular recruitment. The evolving adaptive immune response showed an

initial Th0 profile with IL-2, IFN, IL-4, IL-5 and IL-10 expression. By day 16 this response was polarized to the Th2 response with

elevated IL-5, IL-13 expresssion ( 12 ). Studies with injected live eggs showed that after an early Th1 profile expressing IFN-, TNF-,IL-4 and IL-2 cytokines a shift to the Th2 response occurs with IL-4, IL-5, IL-6, IL-13 expression ( 57 ). Just as histopathology may not

differentiate between foreign body and T cell-mediated lesions, the molecular mechanisms that generate the two types of lesions may

not basically differ from one another. In both responses recruitment from the circulation of monocytes or other inflammatory cells and

their attraction to the site of irritation/infection is mediated by chemokines and cytokines. The magnitude and duration of the

inflammatory response depends on the surface characteristics of the foreign body. Large non-phagocytizable particles such as

surgical sutures, thorns, graphite, plastic beads are usually more inert and will evoke transient small granulomas. Ingested particles

especially silica are powerful irritants within macrophages.They induce tissue-damaging reactive oxygen species (ROS) and reactive

nitrogen species (RNS).The pathogenic potential of the particles depend on the varieties of t he silica surface ( 47 ). Inhalation of

quartz or other silica particles may lead to silicosis, a chronic granulomatous disease accompanied by pulmonary fibrosis and

pulmonary hypertension ( 22 ). It may sometimes lead to the development of autoimmune responses due to the adjuvant action of silica

particles on the immune system. Regardless of the surface properties of the foreign irritant tissue macrophages secrete several

chemokines among them MCP-1, MIP-1, MIP-1, MIP-2 and RANTES that attract blood monocytes and lymphocytes to the site.

Concurrently, IL-1 and TNF cytokines are produced which amplify chemokine production, vascular permeability and macrophage

aggregation. Some macrophages transform to epithelioid cells and fuse to create giant cells. The cellular response usually peaks

during the early days of granuloma formation and subsides usually leaving behind a fibrous capsule. When large amounts of collagen

fibers are deposited within the granuloma a dense fibrous tissue forms and cells die out.

PATHOGENESIS OF THE GRANULOMAS

Two factors contribute to the pathogenesis of the granulomatous process. 1) When the inducer agent is a pathogen/invader or

foreign body the intrinsic toxicity of the agents can damage the tissues. 2) The vigorous immune-inflammatory T cell-mediated

response evoked by the pathogen recruits macrophages and other cells that during the activated stage and phagocytosis secrete

tissue-damaging substances. Examples for the former are found in M. tuberculosis that has a waxy lipid cell wall constituent the cord

factor (trehalose 6, 6 -dimycolate) which regulates in vitro the rope-like growth of the bacteria. This factor directly damages

macrophages or lung cells and when injected into animals causes granulomatous inflammations ( 29 ). An additional component is

muramyl dipeptide that is also granulomagenic. Both components trigger the innate immune response with cytokine (TNF, IL-1,

IL-6, IL-10, IFN and chemokine CCL2 production. Mycobacterium leprae the causative organism of leprosy lives intracellularly in the

skin, nasal mucosa and Schwann cells of the peripheral nerves. As a consequence there is gross thickening of the facial skin,

hypopigmentation and loss of sensation to heat, cold and pain ( 2,6,30 ). T he Gram- bacillus Brucella rapidly multiplies in the lymphnode and causes probably by the Lipid A component of its endotoxin destruction of the lymphoreticular organs. When it colonizes the

heart tissue it can cause fatal endocarditis . The Gram+ Listeria secretes the exotoxin Listeriolysin O a hemolysin that destroys red

cells, neutrophils and monocytes. The fungus Histoplasma has tropism for the mucous membrane in the mouth where it causes

lesions. All the quoted pathogens can sustain their intra-macrophage survival by subverting the killing machinery of the cells (arrest

of the phagolysosomal fusion, disruption of signaling pathways) thereby assuring the chronicity of the infection. It should be pointed


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out that because of the intimate relationship between host and invading pathogen it is difficult to clearly isolate the pathogen or

host-derived factors in the pathogenesis of the diseases.

As will be discussed in the next section the vigorous T cell-mediated response that develops after the antigenic stimulation is a major

contributor to the pathogenesis of the disease. The CD4+ Th1 lymphocytes secrete IFN that activates macrophages. The

acquisition of the microbicidal capacity induces the release of ROS and NOS breakdown products as well as the release of

proteolytic enzymes which greatly contribute to innocent bystander cell destruction and collateral damage.

An example for the foreign body-induced pathogenesis is inhaled silica (mostly quartz) particles that are strongly irritating and

cytotoxic to macrophages that engulf them.

PATHOLOGY OF THE GRANULOMA

In the immune granulomatous inflammation the strong sustained T lymphocyte-mediated inflammatory response is largely responsible

for the ensuing pathology. This response recruits and activates inflammatory leukocytes that converge around the site of the invasion

and phagocytize the invader pathogen.

The pathology of the mature granuloma is caused by several factors: 1) It is a space-occupying lesion that physically impinges on the

neighboring tissue. 2) Leakage from activated macrophages of cytokines (TNF), oxygen (H2O2, O2, OH, O2) and nitrogen (NO)

derivatives and proteolytic enzymes that damage the neighboring tissue. 3) Caseous necrosis. In some, especially the M.

tuberculosis -induced granulomas the centrally located macrophages die by a variety of causes which comprise the toxic effect of M.

tuberculosis products, loss of vascularization, lack of oxygen, the CD8+ T cell-mediated lysis of bacilli-laden macrophages and

TNF-induced apoptosis (programmed cell death) ( 15 ,49 ). The necrotic center is devoid of M. tuberculosis . Apparently the vigorous

macrophage activity achieved its goal ( 56 ). Such necrosis is never observed in the sarcoid lesions. If the outer wall of the granuloma

is well-vascularized the lesion involutes. In contrast low vascularization promotes cavity formation and severe tissue pathology

(56 ). 4) In progressive disease the caseous necrosis of the M. tuberculosis granuloma proceeds due to proteolytic activity to

liquefactive necrosis, the surrounding lung tissue lyzes and cavities form. Further erosion of the neighboring blood vessels and

airways facilitates the spread of the bacilli. Cavitary tuberculosis is dangerous, it abrogates the protective function of the granuloma,

the bacilli spread within the host and by cough and droplet infection disseminate to the outside environment ( 15 ). 5) As a

consequence of intragranulomatous cytokine (T GF-, TNF, for Th1 lesions, IL-4, IL-13 for Th2 lesions) activity fibroblasts or

myofibroblasts get activated and lay down collagen fibers which coalesce to a dense fibrotic tissue. With the elimination of the

pathogen or the antigenic stimulus most granulomas burn out by fibrosis. When the healing process is normal minimal residual

scarring is left behind. Locally secreted TGF- also induces in macrophages or fibroblasts the production of TIMPs (tissue inhibitors

of metalloproteases) that block the degradation of the deposited collagen fibers ( 7). The burnt-out foci of the M. tuberculosis tubercle

then undergo calcification which can be observed in chest x rays. Unlike the TB granulomas, the sarcoid or schistosome worm

egg-induced granulomas never calcify ( Table 2 ). Overt fibrosis that occurs in the granulomatous lungs or livers then derange organ

function. Excessive tissue fibrosis can cause pulmonary or portal hypertension and death in sarcoidosis,

or schistosomiasis respectively. Suppuration at the center of the granulomas also occurs in deep fungal infections

(blastomycosis ,coccidioidomycosis ) and lymphogranuloma venereum . Granuloma annulare a benign dermal inflammation shows

necrobiotic degeneration of dermal collagen, surrounded by palissading inflammatory cells ( 35 ,55 ). In humans the schistosome egg

induced immunopathology is manifested by postgranulomatous liver and intestinal fibrosis ( 14 ,41 ). Here the balance between theTh1-Th2 responses comes into play. Most infected individuals suffer from a milder intestinal form of the disease. However about 5-10

percent of the patients develop the hepatosplenic manifestations with severe hepatic (Symmers ) fibrosis, portal hypertension, ascites

fluid formation, gastrointestinal bleedings and occasional death. In several studies the complexity of various contributory factors to

immunopathology emerged. Hepatosplenic disease in Kenya was associated with high IFN-, TNF- and low IL-5 cytokine

production. Such profile is observed in the Th1 type TB granuloma. Thus results indicated that inability of a patient to develop a

strong Th2 response leads to disease ( 43 ).Further research revealed that periportal fibrosis is age and gender dependent and

induced by high TNF- production ( 8). This conclusion remains controversial because a recent Brazilian study found that hepatic

fibrosis at the early hepatosplenic stage is associated with high Th2 type cytokine chiefly IL-5, IL-10 and IL-13 production ( 14 ). This

latter observation is in accordance with murine experiments that clearly showed impaired granuloma formation and liver fibrosis in

mice defective in type 2 IL-4, IL-13 cytokine production. Thus in the clear cut murine system the Th2 response appears to dampen

the early tissue-destructive IFN- mediated inflammation ( 54 ), yet it contributes to the pathology by IL-4, IL-13 cytokine-mediated liver

fibrosis ( 60 ).

DIFFERENTIAL DIAGNOSISGranulomas are caused by an extremely broad range of disease processes. These can be conveniently divided into infectious and

non-infectious causes. One of the difficulties in arriving at a diagnosis is that many etiologies have similar clinical syndromes.

However, specific tests may help in distinguishing the two types of granulomas.

Infectious Causes

Although many infections are associated with granuloma formation, relatively few microorganisms cause the majority of

cases. Mycobacteria and fungi are commonly associated with granulomatous infection, and in particular, tuberculosis is the most

common cause of granulomas worldwide. However, all mycobacteria can be associated with granulomas. Most clinically important

fungi are also associated with granuloma formation, including Aspergillus , Cryptococcus ,Candida , and Histoplasma to name a few.

Other important causes of granulomas are parasitic infections ( schistosomiasis , leishmaniasis , dirofilariasis , etc.) and rarely, viral

infections caused by cytomegalovirus , Epstein-Barr virus and measles (Table 1 -4). Relatively few bacterial infections typically cause

granulomas during infection, including brucellosis , Q-fever , cat-scratch disease ( 33 ) (Bartonella ), melioidosis , Whipple s

disease (20 ), nocardiosis and actinomycosis . Tuberculosis can often be distinguished by its tendency to produce caseation necrosis

within granulomas, though the other infectious etiologies are almost impossible to differentiate from each other based solely on

histologic appearance ( Table 3 ). One common thread among these disparate infectious syndromes is their general chronicity, many

typically associated with weeks, even months of clinical symptoms before diagnosis is made. In addition, therapy is usually

prolonged, for months to years before clinical resolution.


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Non-Infectious Causes

Whereas infections can often be confirmed with bacterial cultures of affected tissues, non-infectious causes of granulomas may be

difficult to diagnose, and often remain a diagnosis of exclusion. Berylliosis is a T lymphocyte-mediated non-necrotizing

granulomatous disorder that develops in beryllium metal-exposed workers( 39 ). Sarcoidosis is one of the most common non-infectious

granulomatous diseases, characterized by non-necrotizing epithelioid granulomas with giant cells in multiple organ systems, primarily

the lungs( 4,18 ,30 ). Over the years mycobacterial and propionibacterial organisms have been proposed as etiological agents of the

disease. A recent study detected M. tuberculosis catalase-peroxidase protein within sarcoidosis tissues providing further indication

for the infectious etiology of the disease ( 42 ). Crohn s disease is characterized by non-caseating ileal and colonic granulomas ( Table

3). Mucosal permeability to luminal pathogens among them M. paratuberculosis have been proposed as pathogenic

factors( 48 ,59 ). Another group of diseases are the vasculitis syndromes ( 46 ), which include Wegener s granulomatosis, Churg-Strauss disease, and Takayasu s arteritis to name a few. These usually cause necrotizing granulomas ( 18 ).Rheumatoid pulmonary

nodules are often associated with granulomas. In the liver, autoimmune diseases such as hepatitis and primary biliary cirrhosis are

often characterized by granulomatous pathology ( 17 ).

Foreign bodies such as talc, silicone, surgical sutures may also elicit granulomatous reactions with less complex histological

appearance. Often the clinical presentation is organ specific such as renal granulomas caused by antibiotics , anti-inflammatory

agents,diuretics( 31 ).In the lung, aspiration of vegetable matter that remains under-graded can evoke granulomatous response.

Approach to Determining Etiology

As discussed above, the differential diagnosis of granuloma formation is very broad, and it can be difficult to arrive at a specific

etiology. This is critical, as therapeutic pathways diverge from infectious to non-infectious causes, with almost all non-infectious

causes requiring prolonged use of high dose steroids, whereas infections require specific long-term antimicrobial therapy for months

and even years.

LABORATORY DIAGNOSIS

Laboratory diagnosis is an indispensable aid in the diagnosis of the granuloma etiology. Presently, a wide range of laboratory

methodologies is available for the correct diagnosis of the granulomatous condition. This includes the histopathological examination

of the granulomatous tissue, intragranulomatous localization and identification by staining, immunohistochemistry,

immunofluoroscence of the putative pathogen and its in vitro cultivation. Recent advances in molecular biology, proteomics ( 42 )

provided further sensitive and rapid tests for the correct diagnosis. The immune etiology of the granulomatous diseases can now be

confirmed by testing patient s serum or T lymphocyte responses to the suspected granulomagenic organisms. The novel

methodologies require expensive diagnostics, reagents, sophisticated instrumentation and expertise. In the following sections the

methodologies will be described ( Table 4 ).

Cultures

To establish the infectious etiology of the granuloma, cultures are prepared from homogenized biopsied tissues.Care must be

exercised to rigorously exclude an accidental contaminant in the sample. Growth of the pathogens(bacteria, fungi ) on selectiveand/or differential solid medium provides information on the Gram stainin g properties: purple for Gram+,pink for Gram- of the

pathogenic bacteria, as well as the morphology ,color of the colonies, biochemical activity and antibiotic resistance of the organisms.

Final identification of the pathogens taken from the isolated colonies can be made by specific antibodies. Using enriched media:

sheep blood agar, chocolate agar, brain heart infusion agar many organisms can be grown overnight. However several classica l

granulomagenic organisms such as Mycobacteria, Brucella, Listeria or Histoplasma grow slowly and final diagnosis may be made

only after 7-30 days. This is a disadvantage when there is an urgency for a quick diagnosis. The actual choice of the biopsied tissue

taken is also decisive for success. T he highest percentage of M. tuberculosis positive cultures was obtained from samples with

necrotic granuloma centers, whereas poorly formed granulomas yielded much lower positive cultures. Burnt-ot fibrotic lesions were

usually culture negative.

Culture of Mycobacterium = TuberculosisFor primary isolation of the bacteria special media are required. Many laboratories prefer the egg-based Lowenstein-Jensen solid

medium on which bacteria may take 2-4 weeks to grow. The American Trudeau Society medium and the Middlebrook 7 H10 solid or

liquid media can also be used. The latter may yield a more rapid growth of the bacteria ( 24 ). Many laboratories prefer the

commercial automated BACTEC MGIT (Mycobacteria Growth Indicator Tube) 960 system in which bacterial growth in hundreds of inoculated tubes can be monitored. Growth of acid fast bacteria alone does not provide the final identification for M. tuberculosis .

This is accomplished by using molecular biology ( 10 ), chromatography for cell wall lipids and serotyping. M. leprae does not grow on

artificial media and one must rely on clinical findings and consistent histopathology for the diagnosis ( 6).

Culture of BrucellaBrucella is a Gram-coccobacil lus. Isolation from blood or tissues is done on bacterial media under aerobic or anaerobic condition.

The organism is fastidious, grows on enriched media such as trypticase soy agar supplemented with 5 percent sheep blood, brain

heart infusion agar or chocolate agar under 8-10 percent CO2.Growth is slow may take 7 or more days, but it is recommended to

hold cultures for 3 weeks. Additional flora is sometimes suppressed by added bacitracin,polymyxin inhibitors. On positive plates

small non-hemolytic colonies appear. Biochemical activity such as catalase, urease, oxidase, H2S production and sensitivity to basic

fuchsin aid in the correct identification ( 37 ).

Culture of ListeriaListeria is a Gram+ coccobacillus. It can be grown on sheep blood agar where it shows hemolytic action, and brain heart infus ion

agar with added glucose. It can break down esculin by its glucosidase enzyme and exhibits phospholipase activ ity as virulencefactor. Those properties are utilized in a selective chromogenic medium where glucosidase activity on a chromogenic substrate is

observed. Within 24-48 hr blue turquoise colonies appear and phospholipase action generates a white precipitate around the

colonies ( 37 ).

Culture of Fungi


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Most clinically important fungi can be isolated on Sabouraud s dextrose agar with specific modifications (Emmons) that favor growth

of common clinical species. Cultures are usually positive within 3-7 days, though certain fungi may take weeks to grow. Such

is Histoplasma capsulatum a fastidious dimorphic fungus that grows in the yeast form in the lungs. Granulomas can be isolated by

bronchoscopic biopsy. The fungus can be grown on brain heart infusion agar with added gentamicin, vancomycin chloramphenicol

antibiotics to prevent the growth of contaminating bacteria. At 30-37oC it grows slowly (2-4 wks) as budding

yeasts. Nocardia and Actinomyces will grow on routine media taking up to 2 weeks to yield a positive culture.

Stains

Multiple differential stains are available to the pathologist for identification of potential infectious causes of granuloma. T he tissue

sample has to be properly fixed to preserve the cellular and acellular elements of the biopsied material. The paraffin-embedded

tissue is sliced to 5-6m thick sections and stained by a variety of stains that reveal the various cellular and tissue elements.

Haematoxylin-Eosin (H&E) is the most widely used stain for the microscopic diagnosis of granuloma etiology. The lesions are usually

well-delineated from the surrounding tissue. The stained slide will show cellular nuclei stained purple and cytoplasm stained light

rose or red. Such stain can distinguish by morphology lymphocytes, macrophages, epithelioid cells, granulocytes and fibroblasts.

Within the granuloma the characteristic palissading arrangement of the epithelioid cells interspersed with lymphocytes or the

peripheral lymphoid cells is seen. Central caseous necrosis, giant cells with numerous nuclei provide clues for the architecture and

/or the etiology of the lesion. Eosinophils and mast cells present in helminthic granulomas may be degranulated and need special

tissue stains for identification ( 16 ).Some microorganisms can be seen and identified within the granuloma by the H&E stain.

Dominici stain is used for the identification of tissue eosinophils which stains the granules bright red. A more specific identification

uses immunofluorescence microscopy. A primary antibody prepared against the cationic major basic protein (MBP) within the

eosinophilic granules binds to the protein. Then a fluoresceinated secondary antibody that recognizes the primary antibody is

applied. After the proper washes and controls the preparation is identified under the microscope where the granules show bright

green fluorescence.

Acidified Toluidin blue stain is used for the identification of tissue mast cells. The sulfated acid mucopolysaccharides within the

granules stain red to purple. A more specific immunohistochemistry method uses a specific antibody prepared to the tryptase enzyme

within the granules of the mast cells. The bound primary antibody couples with the secondary antibody-biotin-streptavidin-

horseradish peroxidase complex which stains the granules reddish brown. Non-specific esterase stain is used for the detection of

tissue macrophages. The substrates used are naphtyl acetate or butyrate. The cytoplasmic enzyme stains red.

Immunohistochemistry, Immunofluorescence

Advances in the identification of specific surface markers of T and B lymphocyte and the different T lymphocyte phenotypes made

the preparation of specific antibodies possible. Presently an array of commercially available antibodies can identify T lymphocytes

(anti- CD3 marker), T helper lymphocytes (anti-CD4 marker), cytotoxic T cells (anti-CD8 marker), B lymphocytes (anti-CD20 marker),

and macrophages(ant i-CD68 marker). Application of such antisera to the granuloma tissue with the avidin-biotin-horseradish

peroxidase complex can reveal the exact identity and location of the granuloma cells.An elegant refinement uses double

immunofluoresence for further analysis of the T helper lymphocyte subset. The first antibody tagged with one fluorochrome will

identify the CD4+ T cell while a second antibody specific for a certain cytokine(IFN,IL-4,IL-10 etc) with a different fluorochrome tag

penetrates the cell membrane rendered permeable by a chemical and will stain the intracellular cytokine. The intracellular cytokine

content either IFN or IL-4 will identify the T helper1 (Th1)or T helper 2 (Th2) phenotypes respectively of the CD4+ T cell,which in

turn characterizes the granuloma and predicts its efficacy in the elimination of an intracellular invader such as M. tuberculosis .

Application to the granuloma of a specific stain for the identification of an invader can provide the final evidence for the etiology of the

lesion. This sometimes is fraught with difficulties. A very efficient granuloma should show a paucity of the invader having had

eliminated the majority of it by cellular mirobiocidal activity. In fact scrutiny of the biopsied material from patients with confirmed

clinical TB reveals only a low percentage of acid fast bacilli (AFB) positive lesions. Nevertheless the gold standard for the diagnosis

of TB is the presence of AFB in the granuloma tissue. The standard stain is the Ziehl-Neelsen acid fast sta in. An alternative stain

for M. tuberculosis detection is the auramine-rhodamine fluorescent stain that has affinity for the mycolic acid cell wall component of

the bacilli. In a dark field bacilli are bright yellow. Such stain enhances the sensitivity of detection( 11 ).However the presence of AFB

within the granulomas does not prove that the pathogen is M. tuberculosis , because acid-fast M. avium or other mycobacteria may

also be present.

Treponema pall idum is also difficult to detect by regular stains. Needle-aspirated regional lymph node tissue can be stained with

fluorescein tagged anti-treponema antibody and under dark field the stained bacilli fluoresce bright green.

Localization by immunohistochemistry within sarcoid-like granuloma macrophages of inhaled pigeon antigen helps to confirm the

diagnosis of hypersensitivity pneumonitis. Fungi are detected in tissues with Gomori s methenamine silver stain that stains the cell

wall brown to black. The procedure may generate artifacts, therefore the morphology of the fungus has to be ascertained. The

periodic acid Schiff (PAS) reagent stains red the cell wall of Candida . The Calcofluor white (fluorescent) staining may also be used

to stain fungi. Specialized stains such as the Fontana-Masson to detect melanin in fungi and the mucicarmine stain for cryptococcal

cell-wall polysaccharide may further assist in determining the fungal etiology. Despite the use of specific stains, in many instances

organisms due to their paucity in the histopathologic specimen can not be demonstrated and cultures are needed for identification.

Stains for Non-Cellular Tissue Elements

Collagen appears in many granulomas especially at the healing phase of the lesion. The routinely used stain is Mallory s trichrome

which stains the collagen fibers blue, elastin fibrils pink or yellow and muscle fibers red.

The periodic acid Schiff (PAS) reagent stains glycogen as well as mucopolysaccharides in the tissues.

Molecular Methods

Staining of tissue sections and culture of viable pathogens can provide the most definitive diagnosis for the etiology of the


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granuloma. As mentioned in the previous sections the paucity of the pathogen such as AFB and low sensitivity of the staining method

can prevent the correct diagnosis. Though by means of laser capture microdissection granulomas can be obtained free from the

surrounding tissue ( 53 ) growth of pathogens ( M. tuberculosis , fungi) may take weeks, delaying diagnosis. The advent of molecular

methods especially the polymerase chain reaction (PCR) as a diagnostic tool made the detection of uncultivable organisms possible.

The standard, nested, or real time (RT) PCR enjoy wide-spread use because the laboratory can utilize for the assay the formalin-

fixed, paraffin-embedded histological sections. The sensitiv ity and speed of detecting a specific message for a pathogen may

surpass that of a culture because result is obtained with in 24 hr. However, some of these assays are not standardized and caution

must be used in the interpretation because false-positive results may occur.( 13 ,27 ,38 ,44 ,48 ,53 ). Currently commercial kits with primer

probes specific for pathogen DNA are available which greatly amplify the sensitivity of the detection. For M. tuberculosis diagnosis

the insertion sequence (IS) 6110 is routinely used with great success. Compared with the efficacy of the Ziehl-Neelsen staining or

culture, laboratories report double or triple sensitivity and specificity in detection. Another commercial kit (Genprobe)for Mycobacterium tuberculosis detection targets the 16S rRNA genes with sensitivity and specificity of >95% in smear positive

cases. The usefulness of the assay in extra-pulmonary TB is unclear, particular ly when histopathologic stains are negative. The

great advantage of the PCR assay is its applicability to detect ing the DNA of any bacilli or fungi. However it is labor-intensive

because for each pathogen a specific probe has to be prepared or obtained. A more sophisticated assay uses the DNA microarray

method in which multiple DNA probes with known identities are fixed on a glass surface and molecular hybridization is carried out

with the pathogen DNA ( 36 ,45 ).This assay can simultaneously detect hundreds or thousands of genes. Such approach is very

helpful in the differentia l diagnosis when more than one pathogen has to be considered. Low density microarray can simultaneously

detect Mycobacterium spp, Yersinia spp, Bartonella and other pathogens( 45 ).Microarray has also been applied to the simultaneous

different iation of Mycobacteria species. Using the DNA gyrase B subunit (gyrB) genes as a probe Mycobacterium tuberculosis,

Mycobacterium avium, Mycobacterium intracellulare and other species could be identified ( 23 ).

Use of Specific Antibodies and T Cell Function Assays

Detection of specific antibodies remains a cornerstone of diagnosis of many granulomatous diseases of infectious, or unknown

etiology. Anti-neutrophil cytoplasmic antibodies may be the only clue to granulomatous vasculitis( 46 ), and fastidious, slow-growingpathogens are often only suspected but diagnosis is confirmed when specific antibodies are positive. This may be particularly

important for infections due to Coxiella , Brucella and Bartonella , among others. Fluorescence antibody techniques are becoming

more commonly used for specific pathogens, though are not generally available for organisms causing granulomatous infections.

Overall, the non-culture serological methods of diagnosis may be useful when traditional approaches fail to discern the cause of a

granulomatous lesion and will likely increase in use as more commercial tests become available. Examination of peripheral blood or

bronchial T cell responsiveness specific for a pathogen can provide evidence for the immune etiology of the granulomatous disease.

Thus, in vitro T cell proliferation ,or cytokine (IFN,IL-4, etc) production to a soluble antigen of a pathogen,or detection of intracellular

cytokines by immunofluorescence in laser capture microdissection- isolated granulomas serve as important corollaries in the

diagnosis. Finally, the time-honored and simple intradermal test using PPD for TB detection, or fungal antigens can indicate either a

past exposure to the pathogens or an ongoing infection. Either way it reinforces the infectious etiology of the granulomatous disease.

Histopathology

The histologic appearance in stained sections of a particular granulomatous lesion may provide information as to its etiology. The

morphology (compact organized, diffuse, encased in fibrous capsule) and the cellular composition of the lesion are helpfulguidelines( 18 ).First,the immune or foreign body characteristics have to be established. The latter can be diagnosed with some

assurance because the irritant (talc, quartz, silicon, graphite, suture) is present within the granuloma. As mentioned in previous

sections neither the presence of epithelioid nor the type of giant cells can differentiate between the two types of granulomas. The

presence within the granulomas of CD4+ or CD8+ T lymphocytes is indicative of an immune etiology. This is the case in

sarcoidosis( 4) or Crohn s disease( 25 ,59 ).

Histopathological differentiation of the various granulomatous diseases is a challenging task because often neither the morphology

nor the cellular composition can furnish a clue. The M. tuberculosis induced tubercle, the beryllium metal-induced pulmonary

granuloma ( 39 ,58 ) or the sarcoid lesion ( 4,18 ,30 ) all contain epitheliod cells and T lymphocytes. The central caseating necrosis

associated with granulomas is characteristic of tuberculous, but not the beryllium-induced, sarcoid or Crohn s disease lesions ( Table

3). An additional difficulty for the pathologist is the morphological spectrum seen in TB or leprosy ( 2). In TB tissue responses range

from sheets of foamy macrophages packed with acid fast-staining bacilli to hematogeneously spread 1-3 mm size miliary granulomas

in immunocompromised humans, to caseting or non-caseating organized epithelioid garnulomas ( 51 ). Necrotizing granulomas are

often indicative of vasculitic lesions. In the necrobiotic (collagenolytic) granulomas the cellular infiltrate contains also neutrophils and

eosinophils ( 35 ,55 ). The presence of associated pathologic findings can also be useful, such as the eosinophilic infiltrate and/or

eosinophil cationic protein in the serum in Churg-Strauss disease. Examination of stained slides does not only aid in the correct

diagnosis of the disease, but can be a valuable tool in retrospective analyses of archival material. Detection in hundreds of slides of

AFB of proven TB cases can establish a correlation between the presence of AFB and active or dormant infection. A multivariate

analysis of the presence of epithelioid granulomas with subsequent surgical bowel resection revealed that in Crohn s disease the

frequency of granuloma presence may indicate a more aggressive disease process ( 25 ).

The usual dilemma, however, is what to make of granulomatous inflammation when none of the above clues is present, and repeat

biopsies and cultures may be necessary to arrive at a final diagnosis.

Clinical Clues in Assessing Etiology

The most useful aspect of arriving at a diagnosis is the history of the illness and sometimes the occupation of the patient (berylliosis).

This can often give clues for infectious or non-infectious causes regarding exposures and course of illness. Indeed, most

granulomatous infections have chronic, indolent presentations. Exposure to tuberculosis or a positive PPD skin test result may lead

to empiric therapy if the clinical suspicion is high. Of the fungal etiologies, the endemic fungi have geographic restrictions

(histoplasmosis ) that are helpful in narrowing the differential. Candida is associated with intravenous drug use

and Aspergillus with neutropenia . Brucellosis , Q-fever and cat-scratch disease are zoonoses that have relatively specific animal

exposures associated with them. Nocardia is usually related to prior steroid use, and Actinomycosis will frequently present with a

draining sinus tract in the area of infection. Specific organ involvement is also useful, due to unique disease manifestations for that


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organ system. Renal disease might point one towards vasculitis, while lung disease would suggest a work-up for sarcoidosis. Table

4 provides serologic testing and other recommendations for diagnosis of the various infectious causes of granulomas.

Other studies that may assist in diagnosis include more sophisticated imaging, such as computed tomography (CT) of the chest to

better characterize lesions seen on chest x-ray and detect lymphadenopathy that is not apparent on clinical examination. This usually

leads to biopsy of suspicious lesions that eventually makes the diagnosis.

REFERENCES

1. Adams DO. The granulomatous inflammatory process.A review. Am J Pathol 1976;84:164-191 [PubMed]

2.Adams LB,Krahenbuhl JL.Human leprosy.In:DL Boros ed.Granulomatous Infections and Inflammations.Cellular and Molecular Mechanisms.Washington,DC:ASM Press, 2003, pp 207-244

3.Agostini C, Semenzato G.Biology and immunology of the granuloma.In: DG James, A Zumla ed.The Granulomatous

Disorders.Cambridge UK: Cambridge University Press, 1999,pp3-16

4.Agostini C. Semenzato G. Sarcoidosis.In DL Boros ed.Granulomatous Infections and Inflammations:Cellular and Molecular

Mechanisms.Washington,DC:ASM Press, 2003,pp 265-292

5.Algood HM, Chan J, Flynn JL. Chemokines and tuberculosis. Cytokine Growth Factor Rev 2003; 14:467-471 [PubMed]

6.Anderson H,Stryjewska B,BoyantonBL,Schwartz MR.Hansen disease in the United States in the 21st century. Arch Pathol Lab Med

2007;131:982-986 [PubMed]

7.Azouz A,Razzaque MS,El-Hallak M,Taguchi T.Immunoinflammatory responses and fibrogenesis. Med Electron Microsc 2004;37:

141-148 [PubMed]

8.Booth M, Mwatha JK, Joseph S, Jones FM, Kadzo H, Ireri E, Kazibwe F, Kemijumbi J, Kariuki C, Kimani G, Ouma JH, Kabatereine

NB, Vennervald BJ, Dunne DW. Periportal fibrosis in human Schistosoma mansoni infection is associated with low IL-10, low IFN-,

high TNF- or low RANTES depending on age and gender. J Immunol 2004;172: 1295-1303 [PubMed]

9. Boros DL.Granulomatous inflammations Prog Allergy 1978;24;183-267 [PubMed]

10.Chen ES, Moller DR.Expression profiling in granulomatous lung disease. Proc Am Thorac Soc 2007; 4: 101-107 [PubMed]

11.Cheng AG, Chang A, Farwell DG, Agoff SN. Auramine orange stain with fluorescence microscopy is a rapid and sensitive

technique for the detection of cervical lymphadenitis due to mycobacterial infection using fine needle aspiration cytology: a case

series. Otolaryngol Head Neck Surg 2005; 133:381-385 [PubMed]

12.Chiu B-C, Freeman CM, Stolberg VR, Hu JS, Komuniecki E, Chensue SW. The innate pulmonary granuloma. Characterization

and demonstration of dendritic cell recruitment and function. Am J Pathol 2004; 164:1021-1030 [PubMed]

13.Cousins DV,Wilton SD,Francis BR,Gow BL.Use of polymerase chain reaction for rapid diagnosis of tuberculosis. J Clin Microbiol

1992;30:255-258 [PubMed]

14.de Jesus AR, Magalhaes A, Miranda DG, Miranda RG, AraAujo MI, de Jesus AA, Silva A, Santana LB, Pearce E, Carvalho EM.

Association of type 2 cytokines with hepatic fibrosis in human Schistosoma mansoni infection. Infect Immun 2004;

72:3391-3397 [PubMed]

15.Dheda K, Booth H, Huggett JF, Johnson MA, Zumla A, Rook GAW. Lung remodeling in pulmonary tuberculosis. J Infect Dis 2005;

192:1201-1210 [PubMed]

16.Djukanovic R, Wilson JW, Britten KM, Wilson SJ, Walls AF, Roche WR, Howarth PH, Holgate ST. Quantitation of mast cells and

eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and health control subjects using immunohistochemistry. Am

Rev Respir Dis 1990; 142:863-871 [PubMed]

17.Dourakis SP,Saramadou R,Alexopoulou A,Kafiri G,Deutsch M,Koskinas J,Archimandritis AJ. Hepatic granulomas: a 6 year

experience in a single center in Greece. Eur J Gastroenterol Hepatol 2007; 19: 101-104 [PubMed]

18.El-Zammar A, Katzenstein A-L A. Pathological diagnosis of granulomatous lung disease: a review. Histopathology 2007;

50:289-310 [PubMed]

19.Faveeuw C,Angeli V, Fontaine J,Maliszewski C,Capron A,Van Kaer L,Moser M,Capron M,Trottein F.Antigen presentation by CD1d

contributes to the amplification of Th2 response to Schistosoma mansoni glycoconjugates in mice. J Immunol 2002;169;

906-912 [PubMed]

20.Fenollar F,Peuchal X,Raoult D.Whipple s disease. New Engl J Med 2007; 356:55-66 [PubMed]

21.Frevel T, Schafer KL, Totsch M, Bocker W, Dockhorn-Dworniczak B. PCR based detection of mycobacteria in paraffin wax

embedded material routinely processed for morphological examination. Mol Pathol 1999; 52:283-288 [PubMed]

22.Fujimura N. Pathology and pathophysiology of pneumoconiosis. Curr Opin Pulm Med 2000; 6:140-144 [PubMed]

23.Fukushima M, Kakinuma K,Hayashi H,Nagai H,Ito K,Kawaguchi R.Detection and identification of Mycobacterium species isolates

by DNA microarray. J Clin Microbiol 2003;41:2605-2615 [PubMed]

24.Heifets LB,Good RC.Current laboratory methods for the diagnosis of tuberculosis.Chapter 7.In:

Tuberculosis:Pathogenesis,Protection and Control.Ed.BR Bloom.American Society for Microbiology Washington,DC 1994: pp 85-97


ri 9 21/07/2014 9:32


8/9

25.Heresbach D, Alexandre JL, Branger B, Bretagne JF, Cruchant E, Dabadie A, Dartois-Hoquin M, Girardot PM, Jouanolle H,

Kerneis J, Le Verger JC, Louvain V, Polotis, J, Richecoeur M, Robaszkiewicz M, Seyrig JA; ABERMAD (Association Bretonne d Etude

et de Recherche sur les Maladies de Appareil Digestif). Frequency and significance of granulomas in a cohort of incident cases of

Crohn s disease. Gut 2005; 54:215-222 [PubMed]

26.Hernandez-Pando R, Bornstein QL, Aguilar Leon D, Orozco EH, Madrigal VK, Martinez Cordero E. Inflammatory cytokine

production by immunological and foreign body multinucleated giant cells. Immunology 2000; 100:352-358 [PubMed]

27.Hilleman D,Galle J,Vollmer E,Richter E.Real-time PCR assay for improved detection of Mycobacterium tuberculosis complex in

paraffin-embedded tissue. Int J Tuberc Lung Dis 2006;10: 340-342 [PubMed]

28.Hirsch CS, Ellner JJ, Russel DG, Rich EA. Complement receptor-mediated uptake and tumor necrosis-alpha-mediated growthinhibition of Mycobacterium tuberculosis by human alveolar macrophages. J. Immunol 1994; 152:743-753 [PubMed]

29.Hunter RL, Olsen MR, Jagannath C, Actor JK. Multiple roles of cord factor in the pathogenesis of primary, secondary and cavitary

tuberculosis, including a revised description of pathology of secondary disease. Ann Clin Lab Sci 2006; 36:371-386 [PubMed]

30.James DG,Zumla A.The Granulomatous Disorders.Cambridge:Cambridge University Press,1999

31.Javaud N,Belenfant X,Stirnemann J,LaederichJ,Ziol,M,Callard P, Ronco,P,Rondeau,E,Fain O. Renal granulomatosis:a

retrospective study of 40 cases and review of the literature. Medicine 2007;86:170-180 [PubMed]

32.Krutzik SR, Modlin RL. The role of Toll-like receptors in combating mycobacteria. Semin Immunol 2004; 16:35-41 [PubMed]

33.Lamps LW,Scott MA.Cat-scratch disease:historic,clinical,and pathologic perspectives. Am J Clin Pathol 2004;121:Suppl

S71-80 [PubMed]

34.Ly LH, Russel MI, McMurray DN. Microdissection of the cytokine milieu of pulmonary granulomas from tuberculous guinea pigs.

Cell Microbiol 2007; 9:1127-1136 [PubMed]

35.Lynch JM, Barrett TL. Collagenolytic (necrobiotic) granulomas: part 1-the blue granulomas. J Cutan Pathol 2004;

31:353-361 [PubMed]

36. Maldonado RR,Espinosa ML,Suarez CS,Beattie WG,Beattie KL.Hybridization of glass-tethered oligonucleotide probes to target

strands preannealed with labeled auxiliary oligonucleotides. Mol Biotechnol 1999:11: 1-12 [PubMed]

37. Manual of Clinical Microbiology ed PR Murray Wshington,DC:ASM Press 2003

38. Margolis B, Kuzu I, Herrmann M, Raible MD, Hsi E, Alkan S. Rapid polymerase chain reaction-based confirmation of cat scratch

disease and Bartonella henselae infection. Arch Pathol Lab Med 2003; 127:706-710 [PubMed]

39. Middleton DC.Chronic beryllium disease:Uncomon disease,less common diagnosis. Environ Health Perspect

1998;106:765-767 [PubMed]

40. Modlin RL, Hofman FM, Meyer PR, Sharma OP, Taylor CR, Rea TH. In situ demonstration of T lymphocyte subsets in

granulomatous inflammation: leprosy, rhinosclerosis and sarcoidosis. Clin Exp Immunol 1983;51:430-43 [PubMed]

41. Mohamed AR, al Karaur M, Yasawy MI Schistosomal colonic disease. Gut 1990;31: 439 442 [PubMed]

42. Moller DR.Potential etiologic agents in sarcoidosis. Proc Am Thorac Soc 2007; 4: 465-468 [PubMed]

43. Mwatha JK, Kimani G, Kamau T, Mbugua GG, Ouma JH, Mumo J, Fulford AJ, Jones FM, Butterworth AE, Roberts MB, Dunne

DW. High levels of TNF, soluble TNF receptors, soluble ICAM-1 and IFN-, but low levels of IL-5, are associated with hepatosplenic

disease in human schistosomiasis mansoni. J Immunol 1998; 160:1992-1999 [PubMed]

44. Newby DT,Hadfield TL, Roberto FF.Real time PCR detection of Brucella abortus:a comparative study of SYBR green

I,5 -exonuclease,and hybridization probe assays Appl Environ Microbiol 2003:69:4753-4759 [PubMed]

45. Odenthal M,Koenig S,Farbrother P,Drebber U,Bury Y,Dienes HP,Eichinger L. Detection of opportunistic infections by low-densitymicro-arrays: a diagnostic approach for granulomatous lymphadenitis Diagn Mol Pathol 2007;16:18-26 [PubMed]

46. Ozaki S.ANCA-associated vasculitis:diagnostic and therapeutic strategy. Allergol Intl 2007;56:87-96 [PubMed]

47.Rimal B,Greenberg AK,Rom WN. Basic pathogenic mechanisms in silicosis: current understanding Curr Opin Pulm Med

2005;11:169-173 [PubMed]

48.Romero C, Hamdi A, Valentine JF, Naser SA. Evaluation of surgical tissue from patients of Crohn s disease for the presence of

Mycobacterium avium subspecies paratuberculosis DNA by in situ hybridization and nested polymerase chain reaction. Inflamm

Bowel Dis 2005; 11:782-783 [PubMed]

49.Russel DG.Who puts the tubercle in tuberculosis? Nature Rev Microbiol 2007;5:39-47 [PubMed]

50.Salgame P. Host innate and Th1 responses and the bacterial factors that control Mycobacterium tuberculosis infection. Curr Opin

Immunol 2005;17:374-380 [PubMed]

51.Saunders BM, Britton WJ. Life and death in the granuloma: immunopathology of tuberculosis. Immunol Cell Biol 2007;

85:103-111 [PubMed]

52.Saunders BM, Tran S, Ruuls S, Sedgwick JD, Briscoe H, Britton WJ. Transmembrane TNF is sufficient to initiate cell migration

and granuloma formation and provide acute, but not long term, control of Mycobacterium tuberculosis infection. J Immunol 2005;


ri 9 21/07/2014 9:32


9/9

174:4852-4859 [PubMed]

53.Selva E, Hofman V, Berto F, Musso S, Castillo L, Santini J, Dellamonica P, Hofman P. The value of polymerase chain reaction

detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection. Pathology 2004;

36:77-81 [PubMed]

54.Stadecker MJ, Asahi H, Finger E, Hernandez HJ, Rutitzky LI, Sun J. The immunobiology of Th1 polarization in high-pathology

schistosomiasis. Immunol Rev 2004; 201:168-179 [PubMed]

55.Stefanaki K, Tsivitanidou-Kakourou T, Stefanaki C, Valari M, Argyrakos T, Konstantinidou CV, Karentzou O, Katsambas A.

Histological and immunohistochemical study of granuloma annulare and subcutaneous granuloma annulare in children. J Cutan

Pathol 2007; 34:392-396 [PubMed]

56.Ulrichs T, Kaufmann HE. New insights into the function of granulomas in human tuberculosis. J Pathol 2006;

208:261-269 [PubMed]

57.Vella AT, Pearce EJ. CD4+ Th2 response induced by Schistosoma mansoni eggs develops rapidly, through an early transient

Th0-like stage. J Immunol 1992; 148:2283-2288 [PubMed]

58.Verma DK, Ritchie AC, Shaw ML. Measurement of beryllium in lung tissue of a chronic beryllium disease case and cases with

sarcoidosis. Occup Med 2003; 53:223-227 [PubMed]

59.Weinstock JV.Crohn s Disease.In:DL Boros ed.Granulomatous Infections and Inflammations:Cellular and Molecular

Mechanisms.Washington,DC:ASM Press,2003 pp293-320

60.Wynn TA, Thompson RW, Cheever AW, Mentink-Kane MM. Immunopathogenesis of schistosomiasis. Immunol Rev 2004;

201:156-167 [PubMed]

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