**6. Diagnosis of HLH and AAHS/MAS**

In 1991 the HLH Study Group of the Histiocyte Society published the first diagnostic guidelines for HLH which were later updated in 2004 (Henter et al., 1991c, 2007). According to the current guidelines (HLH-2004), five of the following eight criteria must be fulfilled for the diagnosis of HLH: (1) fever, (2) splenomegaly, (3) cytopenias affecting two or three lineages (Hb <90 g/l; PLT <100 × 109/l; neutrophils <1.0 × 109/l), (4) hypertriglyceridemia (fasting triglycerides >3.0 mmol/l) and/or hypofibrinogenemia (<1.5 g/l), (5) hemophagocytosis in bone marrow, spleen, or lymph nodes, (6) hyperferritinemia (>500 µg/l), (7) low or absent NK-cell activity, (8) elevated level of sIL-2R (sCD25) >2400 U/ml. The last three HLH criteria were introduced in the revised diagnostic guidelines for HLH in 2004 (Henter et al., 2007).

There are no validated diagnostic criteria addressed exclusively for AAHS/MAS, and early diagnosis is often difficult (Filipovich et al., 2010; Fukaya et al., 2008; Grom & Mellins, 2010). In general, in a patient with persistently active underlying rheumatologic disease, a fall in the ESR and platelet count, particularly in combination with persistently high CRP and increasing levels of serum D-dimer and ferritin, should raise a suspicion of impeding MAS (Grom & Mellins, 2010). According to Janka, a C-reactive protein >100 mg/l, increased granulopoiesis with left shift in the bone marrow and peripheral blood, and s-ferritin concentration >10,000 μg/L (if EBV infection has been excluded) are features strongly suggestive of MAS (Janka, 2009). The diagnosis of MAS is usually confirmed by demonstration of hemophagocytosis in the bone marrow, liver, lymph nodes, etc. However, false negative results may occur owing to sampling errors, particularly at the early stages of the syndrome (Grom & Mellins, 2010; Janka 2009). In some patients, subsequent biopsies may reveal hemophagocytic macrophages. In patients with negative bone marrow biopsies, assessment of the levels of sIL-2R and sCD163 in serum may help with the timely diagnosis of MAS (Grom & Mellins, 2010; Komp et al., 1989; Schaer et al., 2005).

In particular, application of the HLH diagnostic criteria to sJIA patients with suspected MAS is problematic. Some of the HLH markers such as lymphadenopathy, splenomegaly, and hyperferritinemia are common features of active sJIA itself and therefore do not distinguish MAS from a conventional systemic JIA flare (Davi et al., 2011; Grom & Mellins, 2010). Other HLH criteria, such as cytopenias and hypofibrinogenemia, become evident only at the late stages. This is related to the fact that sJIA patients often have increased white blood cell and platelet counts and elevated s-fibrinogen as a part of the inflammatory response in sJIA. Therefore, when they develop MAS, they demonstrate cytopenias and hypofibrinogenemia to the extent seen in HLH only at the later stage of MAS, when its management becomes challenging (Davi et al., 2011; Grom & Mellins, 2010). Diagnosis of MAS is even more problematic in SLE patients with autoimmune cytopenias, which are difficult to distinguish from cytopenias caused by MAS (Carvalheiras et al., 2010; Grom & Mellins, 2010; Parodi et al., 2009). In these patients, the presence of extreme hyperferritinemia and elevated LDH should raise suspicion of MAS (Parodi et al., 2009). Attempts to modify the HLH criteria to increase their sensitivity and specificity for the diagnosis of MAS in rheumatic conditions have been initiated and continue today (Ravelli et al., 2005; Davi et al., 2011).

#### **7. Case presentations**

84 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

suppressive acitivity of TNF-α, INF-γ, and the heavy unit of ferritin on hematopoiesis;

supressive action of increased levels of

high levels of plasminogen activator secreted by macrophages stimulate plasmine and in consequence lead to

hemophagocytosis

hiperfibrynolisis

TNF-α on lipoprotein lipase

histiocytes/macrophages

organ infiltrations with activated

lymphocytes and histiocytes/macrophages

**HLH symptom/sign Causative factors**

Fever IL-1; IL-6, TNF-α

High concentration of ferritin in serum released by activated

sIL-2R in blood secreted by activated T lymphocytes

HLH – hemophagocytic lymphohistiocytosis; IL – interleukin; TNF – tumor necrosis factor; sIL-2R –

autoimmune/autoinflamatory diseases and MAS fulfill at the beginning diagnostic criteria for HLH (Janka, 2009). In patients, who already have signs of inflammation such as high leukocytosis, elevated platelet count, and elevated levels of fibrinogen, a decline in these parameters, without reaching pathological values, may herald MAS (Ravelli et al., 2005). MAS as a first symptom of sJIA may be indistinguishable from other cases of HLH when arthritis is missing. A high interleukin-1β concentration in blood may also suggest MAS rather than classic HLH (Janka, 2009; Henter et al., 1996). Although mild elevation of sIL-2R has been reported in many rheumatic diseases including JIA and SLE, a several-fold increase in the levels of sIL-2R in these diseases is highly suggestive of MAS (Grom & Mellins, 2010). Importantly, other clinical entities associated with high levels of sIL-2R include malignancies and some viral infections, such as viral hepatitis, and so these conditions should be considered in the differential diagnosis. Nevertheless, sIL-2R receptor and sCD163 are now increasingly recognized as important biomarkers of AAHS/MAS

In 1991 the HLH Study Group of the Histiocyte Society published the first diagnostic guidelines for HLH which were later updated in 2004 (Henter et al., 1991c, 2007). According to the current guidelines (HLH-2004), five of the following eight criteria must be fulfilled for the diagnosis of HLH: (1) fever, (2) splenomegaly, (3) cytopenias affecting two or three

Cytopenia in peripheral blood

blood

High concentration of triglycerides in

Low concentration of fibrinogen in blood

High concentration of the α chain of the

Increased liver transaminases and

soluble IL-2 receptor (also named sCD25).

(Filipovich, 2009; Grom & Mellins, 2010).

**6. Diagnosis of HLH and AAHS/MAS**

Table 2. Signs and symptoms of HLH and their causes

Neurological abnormalities

Hepatosplenomegaly

bilirubin in blood

Here we present three illustrative cases of patients with different autoimmune diseases developing severe AAHS/MAS in the course of their autoimmune disorder.

#### **7.1 MAS complicating juvenile arthritis and ankylosing spondylitis**

A 31-year-old male was referred from community hospital to the University Hospital (The Second Chair of Internal Medicine, Collegium Medicum, Jagiellonian University, Krakow,

Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 87

Hemoglobin g/l 120–170 99 White blood cells × 109/l 4.0–10 1.0 Lymphocytes % 20–40 64 Neutrophils % 58–66 29 Platelets × 109/l 150–400 90 Reticulocytes ‰ 3–15 2

AspAT U/l 17–59 263 AlAT U/l 21–72 116 LDH U/l 313–618 7,700 CRP mg/l <5 56.1

INR 0.85–1.15 1.30 aPTT sec 25–31.5 55.60 Fibrinogen g/l 1.8–3.5 undetectable Fibrinogen (nephelometry) g/l 1.8–3.5 0.7 Trombin time sec 14–21 60.1 D-dimer ng/ml <500 >10,000 Factor II % 70–120 45.9 Factor V % 70–120 106.5 Factor VII % 70–120 109.7 Factor VIII % 50–120 39.8 Factor IX % 70–120 94.6 Factor X % 70–120 76.2 Factor XI % 70–120 110.9 Factor XII % 70–120 81.5

Because of a rapidly progressive thrombocytopenia and neutropenia, bone marrow biopsy was reinterpreted towards a diagnosis of hemophagocytic syndrome. Multiple macrophages (10–15% of nucleated bone marrow cells) together with several hemophagocytes were found

A diagnosis of macrophage activation syndrome complicating recent adalimumab treatment was established. Six days after the patient's admission, treatment with etoposide, dexamethasone, and cyclosporine A was instituted as recommended by the modified HLH-2004 protocol (Fig. 3). Body temperature normalized at day 3 of therapy. Etoposide-related

Table 3. The patient's laboratory results on admission

**Biochemistry**

**Coagulation tests**

(Fig. 2).

**Hematological parameters Reference range Patient's result**

Poland) because of persistent fever and progressive haemostatic abnormalities (skin bruises, undetectable fibrinogen level, prolonged aPTT and INR, markedly elevated FDP and Ddimer levels). The patient was admitted to a community hospital 3 weeks earlier with signs of a possible upper respiratory tract infection (fever, dry cough, sore throat) accompanied by herpes labialis. Broad spectrum antibiotic and acyclovir treatment were both ineffective. Because of a progressing bi-cytopenia (thrombocytopenia with neutropenia; Fig. 1) a fine needle bone marrow biopsy was performed showing no signs of significant primary bone marrow suppression.

Past medical history revealed that the patient suffered from a polyarticular seropositive juvenile arthritis since the age of 8 years. In the following years the patient was hospitalized several times because of disease exacerbations. Non-steroidal inflammatory drugs, gold salts, azathioprine, methotrexate and systemic corticosteroids together with physiotherapy were administered at various time periods; synoviectomy was performed twice. At the age of 29 a diagnosis of ankylosing spondylitis was established (sacroilitis, presence of HLA-B27). Approximately 5 months before admission to our Center the patient's immunosuppressive treatment was modified because of poor disease control, and he received a TNF-α inhibitor (adalimumab) with good clinical response.

On admission the patient complained of pain localized to the right subcostal region and persistent fever. On physical examination body temperature was 39.2°C, and slight hepatomegaly and skin ecchymoses with mucosal bleeding were present. Selected laboratory results and hematologic parameters at admission are shown in Table 3.

Differential diagnosis included DIC with sepsis (blood cultures later showed negative results), neutropenic fever (neutrophils 0.29 × 109/l), and opportunistic infections (HBV, HCV, EBV, CMV, and HIV infection were excluded). Adalimumab side effects were also taken into consideration. Filgrastim, ganciclovir and intravenous gammaglobulins were instituted with no improvement. Concomitant medications included ceftazidime, vancomycin, amikacin, sulfamethoxazole/trimethoprim, linezolide, tranexamic acid, fresh frozen plasma and cryoprecipitate.

Fig. 1. Changes in neutrophils and lymphocyte counts before the admission

Poland) because of persistent fever and progressive haemostatic abnormalities (skin bruises, undetectable fibrinogen level, prolonged aPTT and INR, markedly elevated FDP and Ddimer levels). The patient was admitted to a community hospital 3 weeks earlier with signs of a possible upper respiratory tract infection (fever, dry cough, sore throat) accompanied by herpes labialis. Broad spectrum antibiotic and acyclovir treatment were both ineffective. Because of a progressing bi-cytopenia (thrombocytopenia with neutropenia; Fig. 1) a fine needle bone marrow biopsy was performed showing no signs of significant primary bone

Past medical history revealed that the patient suffered from a polyarticular seropositive juvenile arthritis since the age of 8 years. In the following years the patient was hospitalized several times because of disease exacerbations. Non-steroidal inflammatory drugs, gold salts, azathioprine, methotrexate and systemic corticosteroids together with physiotherapy were administered at various time periods; synoviectomy was performed twice. At the age of 29 a diagnosis of ankylosing spondylitis was established (sacroilitis, presence of HLA-B27). Approximately 5 months before admission to our Center the patient's immunosuppressive treatment was modified because of poor disease control, and he

On admission the patient complained of pain localized to the right subcostal region and persistent fever. On physical examination body temperature was 39.2°C, and slight hepatomegaly and skin ecchymoses with mucosal bleeding were present. Selected

Differential diagnosis included DIC with sepsis (blood cultures later showed negative results), neutropenic fever (neutrophils 0.29 × 109/l), and opportunistic infections (HBV, HCV, EBV, CMV, and HIV infection were excluded). Adalimumab side effects were also taken into consideration. Filgrastim, ganciclovir and intravenous gammaglobulins were instituted with no improvement. Concomitant medications included ceftazidime, vancomycin, amikacin, sulfamethoxazole/trimethoprim, linezolide, tranexamic acid, fresh

Days to admission

laboratory results and hematologic parameters at admission are shown in Table 3.

 Lymphocytes Neutrophils

Fig. 1. Changes in neutrophils and lymphocyte counts before the admission

received a TNF-α inhibitor (adalimumab) with good clinical response.

marrow suppression.

frozen plasma and cryoprecipitate.

%

Table 3. The patient's laboratory results on admission

Because of a rapidly progressive thrombocytopenia and neutropenia, bone marrow biopsy was reinterpreted towards a diagnosis of hemophagocytic syndrome. Multiple macrophages (10–15% of nucleated bone marrow cells) together with several hemophagocytes were found (Fig. 2).

A diagnosis of macrophage activation syndrome complicating recent adalimumab treatment was established. Six days after the patient's admission, treatment with etoposide, dexamethasone, and cyclosporine A was instituted as recommended by the modified HLH-2004 protocol (Fig. 3). Body temperature normalized at day 3 of therapy. Etoposide-related

Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 89

Fever (°C) 39.2 35.8 Splenomegaly slight - Hepatomegaly + - Hemoglobin (g/l) 85 137 Platelets (× 109/l) 14 260 Neutrophils (× 109/l) 0.29 5.3 Triglycerides (mmol/l) 6.5 2.84 Fibrinogen (g/l) undetectable 4.8 Ferritin (µg/l) >20,000 233.2 AlAT (U/l) 116 25 Bilirubin (mol/l) 19 15 LDH (U/l) 7,687 624 Hemophagocytosis + -

Table 4. Changes in selected laboratory and clinical parameters characteristic for MAS

A 58-years-old patient, suffering from rheumatoid arthritis for 12 years, about 13 months after a course of TNF-α inhibitor (etanercept) treatment was admitted to hospital in another country due to progressive weakness, weight loss, intensive ankle joint pain and fever reaching 40°C. RA flare and infection were excluded and etanercept-related bone marrow dysfunction was suspected due to agranulocytosis, leukopenia, and thrombocytopenia. The patient had decided to leave the hospital and sought consultation in our University Hospital (The Second Chair of Internal Medicine, Collegium Medicum of the Jagiellonian University, Krakow, Poland). On admission his general condition was satisfactory; physical examination having revealed *livedo reticularis* on the lower limbs, swelling and redness of the left ankle joint, slight splenomegaly and caries of teeth 11 and 24. The most important laboratory abnormalities are shown in Table 5. Rheumatoid arthritis with Felty syndrome was diagnosed 12 years ago based on the presence of 5 ACR criteria, positive rheumatoid factor (RF) and elevated anti-cyclic citrullinated peptide antibodies (anti-CCP). About a year ago etanercept was started due to the ineffectiveness

After admission the patient received three injections of filgrastim (48 mln. units each) and antimicrobial treatment was implemented (ceftazidim, amikacin, fluconazole). Teeth 11 and 24 were extracted. The patient was seen by a hematologist and a fine needle bone marrow

A macrophage activation syndrome associated with rheumatic disease (RA) was diagnosed. On day 8 of hospitalization, dexamethasone (20 mg qd) and cyclosporine A (dose adjusted to trough blood levels) were initiated. Treatment led to a quick decrease in body

temperature and little later to normalization of the peripheral blood picture (Table 6).

biopsy was performed showing numerous, typical hemophagocytes (Figure 4).

during the applied therapy (HLH-2004)

of corticosteroids and methotrexate.

**7.2 MAS complicating rheumatoid arthritis**

**(nadir/zenith) After treatment**

**Parameter (units) Baseline**

nadir occurred at day 20 (platelets 14 × 109/l; leukocytes 0.42 × 109/l) (Table 4). One dose of etoposide was omitted and filgrastim (G-CSF) was administered (48 mln units b.i.d) with a beneficial effect. Etoposide was then continued for the next 9 months.

Fig. 2. Bone marrow aspirate smears showing the centrally placed macrophage laden with erythroblasts. Normally developed myeloid cells are present nearby. Wright's stain, lower (× 400) and higher (× 1000) magnification

Fig. 3. The patient's treatment scheme with a HLH-2004 protocol

Control examination of bone marrow with a fine needle biopsy performed at week 40 of treatment confirmed the disappearance of activated macrophages. The patient felt well and continues treatment with cyclosporine and low-dose corticosteroids.

Table 4. Changes in selected laboratory and clinical parameters characteristic for MAS during the applied therapy (HLH-2004)

#### **7.2 MAS complicating rheumatoid arthritis**

88 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

nadir occurred at day 20 (platelets 14 × 109/l; leukocytes 0.42 × 109/l) (Table 4). One dose of etoposide was omitted and filgrastim (G-CSF) was administered (48 mln units b.i.d) with a

Fig. 2. Bone marrow aspirate smears showing the centrally placed macrophage laden with erythroblasts. Normally developed myeloid cells are present nearby. Wright's stain, lower

beneficial effect. Etoposide was then continued for the next 9 months.

(× 400) and higher (× 1000) magnification

Fig. 3. The patient's treatment scheme with a HLH-2004 protocol

continues treatment with cyclosporine and low-dose corticosteroids.

Control examination of bone marrow with a fine needle biopsy performed at week 40 of treatment confirmed the disappearance of activated macrophages. The patient felt well and A 58-years-old patient, suffering from rheumatoid arthritis for 12 years, about 13 months after a course of TNF-α inhibitor (etanercept) treatment was admitted to hospital in another country due to progressive weakness, weight loss, intensive ankle joint pain and fever reaching 40°C. RA flare and infection were excluded and etanercept-related bone marrow dysfunction was suspected due to agranulocytosis, leukopenia, and thrombocytopenia. The patient had decided to leave the hospital and sought consultation in our University Hospital (The Second Chair of Internal Medicine, Collegium Medicum of the Jagiellonian University, Krakow, Poland). On admission his general condition was satisfactory; physical examination having revealed *livedo reticularis* on the lower limbs, swelling and redness of the left ankle joint, slight splenomegaly and caries of teeth 11 and 24. The most important laboratory abnormalities are shown in Table 5. Rheumatoid arthritis with Felty syndrome was diagnosed 12 years ago based on the presence of 5 ACR criteria, positive rheumatoid factor (RF) and elevated anti-cyclic citrullinated peptide antibodies (anti-CCP). About a year ago etanercept was started due to the ineffectiveness of corticosteroids and methotrexate.

After admission the patient received three injections of filgrastim (48 mln. units each) and antimicrobial treatment was implemented (ceftazidim, amikacin, fluconazole). Teeth 11 and 24 were extracted. The patient was seen by a hematologist and a fine needle bone marrow biopsy was performed showing numerous, typical hemophagocytes (Figure 4).

A macrophage activation syndrome associated with rheumatic disease (RA) was diagnosed. On day 8 of hospitalization, dexamethasone (20 mg qd) and cyclosporine A (dose adjusted to trough blood levels) were initiated. Treatment led to a quick decrease in body temperature and little later to normalization of the peripheral blood picture (Table 6).

Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 91

Fig. 4. Bone marrow aspirate smears. The centrally located macrophage shows phagocytosis of erythrocytes, erythroblasts and myeloid cells. Wright's stain, lower (× 400) and higher (×

A 46-year-old man was admitted to the University Hospital (The Second Chair of Internal Medicine, Collegium Medicum of the Jagiellonian University, Krakow, Poland) in 2010 because of persistent fever and pulmonary nodules and consolidations. He has been treated for the last 10 years for systemic lupus erythematosus (SLE) with renal involvement. His SLE was diagnosed in 1991 based on the presence of several ARA criteria (fever, arthralgias, pleuritis and pericarditis, proteinuria, anemia, and the presence of ANA in high titer as well as ds-DNA antibodies) and typical results of renal biopsy. His medical history revealed arterial hypertension, peripherial artery occlusive disease and deep vein thrombosis of both lower limbs. Initially SLE was treated with corticosteroids and oral cyclophosphamide, then treatment was frequently modified according to the disease activity (azathioprine,

**Hematological parameters Reference range Patient's result**

Hemoglobin g/l 120–150 134 White blood cells × 109/l 4.0–10 9.0 Lymphocytes % 20–40 12.2 Neutrophils % 58–66 83.0 Platelets × 109/l 150–400 57 Reticulocytes ‰ 3–15 10

AspAT U/l 21–72 38 AlAT U/L 21–72 50 Bilirubin mol/l 3–22 11 LDH U/l 313–618 1,114 CRP mg/l <5 75.7

INR 0.85–1.15 0.94 aPTT sec 25–31.5 25.9 Fibrinogen g/l 1.8–3.5 5.0

Table 7. The patient's laboratory results on admission to the University Hospital

**7.3 MAS complicating systemic lupus erythematosus**

cyclosporine A, mycophenolate mofetil and plasmapheresis).

**Biochemistry**

1000) magnification

**Coagulation tests**

Table 5. The patient's laboratory results on admission to the University Hospital

Table 6. Changes of selected laboratory and clinical parameters of MAS during the treatment with modified HLH-2004 protocol

Fig. 4. Bone marrow aspirate smears. The centrally located macrophage shows phagocytosis of erythrocytes, erythroblasts and myeloid cells. Wright's stain, lower (× 400) and higher (× 1000) magnification

#### **7.3 MAS complicating systemic lupus erythematosus**

90 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

Hemoglobin g/l 120–170 108 White blood cells × 109/l 4.0–10 0.62 Lymphocytes % 20–40 80 Neutrophils % 58–66 13 Platelets × 109/l 150–400 56 Reticulocytes ‰ 3–15 43

AspAT U/l 21–72 16 AlAT U/l 21–72 11 Bilirubin mol/l 3–22 12 LDH U/l 313–618 420 CRP mg/l <5 54.3

INR 0.85–1.15 1.08 aPTT sec 25–31.5 31 Fibrinogen g/l 1.8–3.5 1.5

**(nadir/zenith) After treatment**

Table 5. The patient's laboratory results on admission to the University Hospital

Fever (°C) 37.2 36.6 Splenomegaly + - Hepatomegaly - - Hemoglobin (g/l) 108 133 Platelets (× 109/l) 25.2 173 Neutrophils (× 109/l) 0.6 3.43 Triglycerides (mmol/l) 2.83 2.7 Fibrinogen (g/l) 1.40 5.2 Ferritin (µg/l) 1,855 717.5 AlAT (U/l) 16 52

Bilirubin (mol/l) 12 not determined LDH (U/l) 420 not determined Hemophagocytosis + not determined

Table 6. Changes of selected laboratory and clinical parameters of MAS during the treatment

**Parameter (units) Baseline**

**Biochemistry**

**Coagulation tests**

with modified HLH-2004 protocol

**Hematological parameters Reference range Patient's**

**result**

A 46-year-old man was admitted to the University Hospital (The Second Chair of Internal Medicine, Collegium Medicum of the Jagiellonian University, Krakow, Poland) in 2010 because of persistent fever and pulmonary nodules and consolidations. He has been treated for the last 10 years for systemic lupus erythematosus (SLE) with renal involvement. His SLE was diagnosed in 1991 based on the presence of several ARA criteria (fever, arthralgias, pleuritis and pericarditis, proteinuria, anemia, and the presence of ANA in high titer as well as ds-DNA antibodies) and typical results of renal biopsy. His medical history revealed arterial hypertension, peripherial artery occlusive disease and deep vein thrombosis of both lower limbs. Initially SLE was treated with corticosteroids and oral cyclophosphamide, then treatment was frequently modified according to the disease activity (azathioprine, cyclosporine A, mycophenolate mofetil and plasmapheresis).

Table 7. The patient's laboratory results on admission to the University Hospital

Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 93

Macrophage activation syndrome was diagnosed and one month after the patient's admission treatment with dexamethasone, cyclosporine A, and etoposide was started resulting in the normalization of body temperature and peripherial blood morphology. Etoposide-related nadir occurred at day 14 (platelets 18 × 109/l, leukocytes 1.0 × 109/l). Two etoposide doses were omitted and filgrastim was administered twice (48 mln units qd). Intravenous pulses of etoposide were given for the next 2 months, followed by oral administration. A control bone marrow examination performed on week 40 of treatment showed disappearance of activated macrophages. The patient has continued treatment with cyclosporine A in combination with low-dose corticosteroids with continuous improvement

Although significant progress in understanding the genetics and pathophysiology of primary HLH has been achieved during recent years, the pathogenesis of acquired forms of HLH is still not fully understood. An exaggerated immune response is the final common pathway of HLH, however, there are multiple roads leading to it (Arceci, 2008; Janka, 2009). The immune response is often triggered by different stimulants (e.g., infection) and the underlying inherited or acquired immune defect. It has been proposed that the clinical presentation of HLH is due to uncontrolled activation of immune cells, macrophages and CD8+ T lymphocytes (cytotoxic), leading to a massive release of various mediators of inflammation such as TNF-α (tumor necrosis factor α), interleukin(IL)-6, IL-8, IL-10, IL-12, IL-18, interferon γ, macrophage inflammatory protein (MIP 1-α), and hematopoietic growth factors (e.g., GM-CSF) (Filipovich, 2009; Henter et al., 1991a, 1996, 2007; Janka & Schneider, 2004; Osugi et al., 1997). IL-10 with its anti-inflammatory properties plays many important roles in the regulation of autoimmune inflammatory responses, particularly of systemic autoimmune disorders such as HLH/MAS. The role of IL-10 as part of an important regulatory mechanism involved in HLH has long been proposed (Behrens et al., 2011; Benveniste et al., 2000; Osugi et al., 1997). Recently, the roles of T regulatory cells in HLH have also been discussed (Verbsky & Grossman, 2006). Low or absent NK-cell function is present in many HLH patients and results in difficulties in termination of the exaggerated

There are two major subtypes of genetic causes of HLH. First are those genetic defects, grouped under the term FHL, that present with HLH as the primary and only manifestation of disease (Gupta & Weitzman, 2010; Henter et al., 2007). A second group of genetic disorders include HLH as only one, although often fatal, manifestation of the disease (Gupta & Weitzman, 2010; Janka, 2009). All known genetic abnormalities causing FHL involve genes that regulate proteins important in the secretory cytolytic pathway of NK-cells and CD8+ T lymphocytes. In 1999, the first FHL-inked locus was discovered on chromosome 9q21.3–22 in several Pakistani families and was later defined as the FHL1 subtype (Ohadi et al., 1999). Shortly thereafter, mutations in the perforin gene *PRF1* were discovered on chromosome 10q21 in a group of patients with FHL (FHL2 subtype) (Stepp et al., 1999). Furthermore, mutations in genes *UNC13D* (located on chromosome 17q25; FHL3 subtype)*, STX11* (located on chromosome 6q24; FHL4 subtype), and most recently *STXBP2* (located on chromosome 19p13; FHL5 subtype) were described (Feldmann 2003; zur Stadt et al., 2005, 2009). In view of the remarkable progress since the discovery of the first genetic defect in

of his clinical and laboratory parameters (Table 8).

immune response (Filipovich, 2009; Henter et al., 2007).

**8. Pathophysiologic and molecular mechanism of HLH**

The patient's general condition on admission was satisfactory. His body temperature was 38.4°C, physical examination revealed neither hepatosplenomegaly nor lymphadenopathy. Selected laboratory results and hematologic parameters on admission are shown in Table 7. Microbial analysis of bronchoalveolar lavage obtained at bronchoscopy revealed a group I Mycobacteria-other-than-tuberculosis (MOTT). Targeted therapy was instituted (rifampicin 450 mg qd, isoniazide 250 mg qd, ethambutol 750 mg qd and clarithromycin 1000 mg bid) with no effect on the fever. Because of the persistent fever, bronchoscopy was repeated one month later, but MOTT's were no longer detectable. Due to the progressive anemia and thrombocytopenia a fine needle bone marrow biopsy was performed showing typical hemophagocytes, with some of these forming cell conglomerates (Fig. 5).

Fig. 5. Wright's stain of bone marrow aspirate smears. The centrally placed large cell conglomerate consists of activated macrophages presenting hemophagocytosis of erythroblasts and myeloid cells. Lower (× 200) and higher (× 400) magnification

Table 8. Changes of selected laboratory and clinical parameters typical for MAS during treatment (HLH-2004)

The patient's general condition on admission was satisfactory. His body temperature was 38.4°C, physical examination revealed neither hepatosplenomegaly nor lymphadenopathy. Selected laboratory results and hematologic parameters on admission are shown in Table 7. Microbial analysis of bronchoalveolar lavage obtained at bronchoscopy revealed a group I Mycobacteria-other-than-tuberculosis (MOTT). Targeted therapy was instituted (rifampicin 450 mg qd, isoniazide 250 mg qd, ethambutol 750 mg qd and clarithromycin 1000 mg bid) with no effect on the fever. Because of the persistent fever, bronchoscopy was repeated one month later, but MOTT's were no longer detectable. Due to the progressive anemia and thrombocytopenia a fine needle bone marrow biopsy was performed showing typical

hemophagocytes, with some of these forming cell conglomerates (Fig. 5).

Fig. 5. Wright's stain of bone marrow aspirate smears. The centrally placed large cell conglomerate consists of activated macrophages presenting hemophagocytosis of erythroblasts and myeloid cells. Lower (× 200) and higher (× 400) magnification

Fever (°C) 40.0 36.5 Splenomegaly - - Hepatomegaly - - Hemoglobin (g/l) 120–170 85 153 Platelets (× 109/l) 150–400 42.2 118.2 Neutrophils (× 109/l ) 6,100 3,500 Triglycerides (mmol/l) 0.3–2.26 2.63 1.07 Fibrinogen (g/l) 1.8–3.5 3.60 3.1 Ferritin (µg/l) 13–400 1,387 105 AlAT (U/L) 21–72 142 57 Bilirubin (mol/l) 3–22 12 11 LDH (U/L) 313–618 804 452 Hemophagocytosis ++ single cells Table 8. Changes of selected laboratory and clinical parameters typical for MAS during

**Baseline**

**(nadir/zenith) After treatment**

**range**

**Parameter (units) Reference**

treatment (HLH-2004)

Macrophage activation syndrome was diagnosed and one month after the patient's admission treatment with dexamethasone, cyclosporine A, and etoposide was started resulting in the normalization of body temperature and peripherial blood morphology. Etoposide-related nadir occurred at day 14 (platelets 18 × 109/l, leukocytes 1.0 × 109/l). Two etoposide doses were omitted and filgrastim was administered twice (48 mln units qd). Intravenous pulses of etoposide were given for the next 2 months, followed by oral administration. A control bone marrow examination performed on week 40 of treatment showed disappearance of activated macrophages. The patient has continued treatment with cyclosporine A in combination with low-dose corticosteroids with continuous improvement of his clinical and laboratory parameters (Table 8).


**2.3. Common mechanisms of injury**

The common causes of geriatric trauma include falls, motor vehicle collisions, pedestrian injuries and thermal injuries and elder abuse (**Figure 1**).

**Falls** remain the leading cause of geriatric trauma and affect approximately 30% of persons aged ≥65 years each year [17]. Approximately 50% of people living in long-term care institutions fall each year, and 40% of them experienced recurrent falls [18]. Women experience significantly more fall-related injuries than men (35.7 vs.24.6%, respectively) [19]. Falls account for 40% of all injury-associated deaths [20]. Predisposing risk factors include age-related changes in muscle strength, gait and balance, poor vision and home hazards. İn addition, drugs and alcohol may contribute to falls. Anticoagulants usage are frequent in elderly and it may cause potentially lethal injuries even with minor traumas. Osteoporosis and the tendency to fall increase the risk of hip fractures. Also, falls are the most common cause of traumatic brain injury in the elderly. Even when those injuries are minor, they seriously affect older

**Organ system** Respiratory system

**Age-related alterations**

Elastin component of the lung matrix

type III collagen [5].

Pulmonary compliance

Osteoporosis

Stiffness of the thoracic cage

Outward recoil

Kyphosis [6].

Chest wall compliance

Thickening of the alveolar basement membrane [7].

Diffusion capacity

Gas change

Muscle atrophy

Respiratory muscle weakness [8].

Vascular stiffness

Elevated baseline blood pressure

Atherosclerosis of coronary vessel

Risk for cardiac ischemia

Increased risk of dysrhythmias

Impaired cardiac reserve

Cardiac index

Lack of classic response to hypovolemia

Left ventricle (LV) wall thickness

[10].

Retarded early diastolic cardiac filling and

LV diastolic function

Afterload

Left atrial size

myocyte mass with

deposition of amyloid and collagen

Deterioration of the cardiac conduction

Decreased sensitivity to catecholamines

Maximal heart rate

Maximum tachycardia response [11].

Geriatric Trauma

117

http://dx.doi.org/10.5772/intechopen.77151

Circulatory system

**Clinical consequences**

Risk for pneumonia [5]

Poor tolerance to rib fractures

Work of breathing

Risk for respiratory failure [8].

Forced expiratory volume in one second (FEV1)

Forced vital capacity (FVC)

FEV1/FVC [9].

Functional residual capacity (FRC)

Residual volume (RV)

Vital capacity (VC)

Geriatric trauma patients are less likely to be injured than younger people; however, they are more likely to have fatal outcomes. Death rates for Americans have decreased in the last century. Although there is a dramatic decline in deaths from cardiovascular diseseas, heart diseases remain the leading cause of deaths in the elderly. Also, trauma became the more common cause of death. According to the National Center for Health Statistics 2015 report,

Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death [3]. This multifactorial and extremely complex process results in significant anatomic and functional changes in all major organ systems.

**Airway**: The anatomy and physiology of the airway are affected with the aging process. Tooth decay which is common in elderly may cause loose, dislodged and subsequently aspiration of the teeth during emergency procedures such as endotracheal intubation (ET). Esthetic operations and loss of teeth interfere with achieving a good face-mask seal. Pharynx becomes more dry and fragile and care must be taken to prevent profuse bleeding while using laryngoscope. Oral cavity tumors and macroglossia may limit visualization of the vocal cords. Usage of Miller blade can be considered [4]. Also, cervical osteoarthritis increases the risk for spinal

The common causes of geriatric trauma include falls, motor vehicle collisions, pedestrian inju-

**Falls** remain the leading cause of geriatric trauma and affect approximately 30% of persons aged ≥65 years each year [17]. Approximately 50% of people living in long-term care institutions fall each year, and 40% of them experienced recurrent falls [18]. Women experience significantly more fall-related injuries than men (35.7 vs.24.6%, respectively) [19]. Falls account for 40% of all injury-associated deaths [20]. Predisposing risk factors include age-related changes in muscle strength, gait and balance, poor vision and home hazards. İn addition, drugs and alcohol may contribute to falls. Anticoagulants usage are frequent in elderly and it may cause potentially lethal injuries even with minor traumas. Osteoporosis and the tendency to fall increase the risk of hip fractures. Also, falls are the most common cause of traumatic brain injury in the elderly. Even when those injuries are minor, they seriously affect older

unintentional injuries became the seventh common cause of death in the elderly [2].

Most important systems which are affected are seen in **Table 1**.

cord injury. Excessive movement of the neck should be avoided.

**Age-related alterations:** See **Table 1.**

**2.3. Common mechanisms of injury**

ries and thermal injuries and elder abuse (**Figure 1**).

**2.2. Age-related alterations and clinical consequences**

**2. Pathophysiology**

**2.1. What is aging?**

116 Trauma Surgery

**Table 1.** Age-related alterations and their clinical consequences.

adults' quality of life by inducing a fear of falling, which can lead to self-imposed activity

Geriatric Trauma

119

http://dx.doi.org/10.5772/intechopen.77151

[64].

**Motor vehicle collision** involving elderly continue to increase. Age-related changes that include vision and hearing impairment, decreased night vision and glare resistance are the prominent factors on the incidence of injury and death. Additionally, medical conditions and medications may distort the reaction time, attention and judgment which increase the risk for

**Pedestrian injuries:** according to the 2015 pedestrian data, 19% of all pedestrian fatalities and an estimated 13% of all pedestrians injured were people aged 65 and older in the United States, and pedestrian-motor vehicle collisions are one of the most lethal mechanisms of

**Thermal injuries:** There is a direct relationship between age and burn mortality, as evidenced by the traditionally taught BauxScore. The empiric formula is clearly the simplest, whereby the sum of the patient's age and burn size predict mortality. Based on the data from the American Burn Association (ABA) National Burn Repository (NBR) from 2000 to 2009, overall

restrictions, anxiety, social withdraw and depression [21].

**Figure 1.** Common causes of falls in the elderly

injury in this age group with a 53% case fatality rate [22].

the collision.

**Figure 1.** Common causes of falls in the elderly [64].

**Organ system** Musculoskeletal system

Spontaneous rupture

Joint stability

ineffective repair of cartilage tissue

Osteoarthritis (cervical, temporomandibular)

Bone volume-mass

Muscle size-number [12].

Osteoporosis

Sarcopenia

Nutrition and metabolism

Taste acuity, smell and appetite decrease

Food intake [13].

Less contusions

Clinical signs may manifest late

More subdural hematoma

Vision and auditory functions [16].

Reaction time

Attention span

Less epidural hematoma

Risk for spine and spinal cord injury

Poor dentition

Inability to eat independently

Brain volume decreases

Replaced by cerebrospinal fluid [14].

Protection against contusions

Blood can be collected

Parasagittal bridging veins stretch

More prone to tear injury

Demyelination

Peripheral conduction velocity slows

Dura adheres to the skull more tightly

Cerebral blood flow

Cerebral oxygen consumption [15].

Degeneration of vertebras, intervertebral disks and facet joints.

**Table 1.**

Age-related alterations and their clinical consequences.

Central nervous system

**Age-related alterations**

Stiffening of structural instruments (tendons, ligaments, cartilage)

**Clinical consequences**

118 Trauma Surgery

Risk of injury

Risk of fracture

Difficulty for oral intubation

Risk of falls

Mobility

adults' quality of life by inducing a fear of falling, which can lead to self-imposed activity restrictions, anxiety, social withdraw and depression [21].

**Motor vehicle collision** involving elderly continue to increase. Age-related changes that include vision and hearing impairment, decreased night vision and glare resistance are the prominent factors on the incidence of injury and death. Additionally, medical conditions and medications may distort the reaction time, attention and judgment which increase the risk for the collision.

**Pedestrian injuries:** according to the 2015 pedestrian data, 19% of all pedestrian fatalities and an estimated 13% of all pedestrians injured were people aged 65 and older in the United States, and pedestrian-motor vehicle collisions are one of the most lethal mechanisms of injury in this age group with a 53% case fatality rate [22].

**Thermal injuries:** There is a direct relationship between age and burn mortality, as evidenced by the traditionally taught BauxScore. The empiric formula is clearly the simplest, whereby the sum of the patient's age and burn size predict mortality. Based on the data from the American Burn Association (ABA) National Burn Repository (NBR) from 2000 to 2009, overall mortality was 4% in all age groups and 17% in seniors [23]. Moreover, for seniors there is a greater increase in mortality risk for every 1% increase in burn size and 1-year increase in age than among adults [23].


**6. Numerical simulation for thixotropic plastic forming of composites**

To investigate thixoforming process with numerical simulation method, which is a nonlinear system, some assumptions are taken as follow: (1) The semi-solid material is assumed as a continuous and incompressible one. (2) The solid grains in semi-solid metal are uniformly distributed in liquid phase, and because of the large deformation in forming, the semi-solid material is considered as an isotropy uniform medium. According to the above assumptions, the material deformation in thixoforming is supposed as a rigid viscoplastic one.

The material adopted in this paper was SiCp/AZ61 composite, and the simulations were performed in thixo-forging and forging. The flow stress model of SiCp/AZ61 composite in thixo-forging is expressed as follow [Yan &Wang, 2011].

270 Mechanical Engineering

Fig.18 is the real stress test - a true strain curves and regression curve of the results of the comparison, Solid line is the experimental curve, dotted line is the calculation of one. The results calculated by multiple non-linear regression method are good agreement with experimental ones. So the proposed constitutive model has the higher forecast precision and

(a) 3vol.% SiCP/AZ61,570℃ (b) 6vol.% SiCP/AZ61,560℃

(c) 9vol.% SiCP/AZ61,530℃ Fig. 18. A comparison between true strain—stress curves of the test and regression curves

To investigate thixoforming process with numerical simulation method, which is a nonlinear system, some assumptions are taken as follow: (1) The semi-solid material is assumed as a continuous and incompressible one. (2) The solid grains in semi-solid metal are uniformly distributed in liquid phase, and because of the large deformation in forming, the semi-solid material is considered as an isotropy uniform medium. According to the above assumptions, the material deformation in thixoforming is supposed as a rigid

The material adopted in this paper was SiCp/AZ61 composite, and the simulations were performed in thixo-forging and forging. The flow stress model of SiCp/AZ61 composite in

thixo-forging is expressed as follow [Yan &Wang, 2011].

**6. Numerical simulation for thixotropic plastic forming of composites**

practical significance.

viscoplastic one.

$$
\sigma = \exp(-8.27366 + 50.158f\_p - 296.55f\_p^{\
u} + 14253.359 \, / \, T)
$$

$$
\text{or } 0.053 \cdot \frac{1}{\varepsilon} \ge 0.242 \cdot \left(1 - \beta f\_L\right) 2.316 \cdot \left[1 + \left(2.1 \times 10^4 \, \varepsilon\right) 0.242 \, f\_p\right] \text{-0.505}\tag{12}
$$

where σ is the stress; ε the strain; z . the strain rate; T temperature; β constant(β=1.5); *pf* is Volume fraction of SiC particle; *Lf* is liquid volume fraction

For establishing material modal of SiCp/AZ61 composite in forging, true stress-strain curves at various temperature and strain rates were performed by mean of isothermal compression experiments.

In this study, the workpiece is formed by the close-forge method. The experiment set-up was shown in Fig.19. Fig.20 shows the workpiece, whose structure and flow character are complicated. Comparisons between forging and thixo-forging of the workpiece will be done and predicted in advance using numerical simulation. This is an effective method to instruct application of semi-solid forming technology into its practice production.

The same simulated parameters are used to analyze the differences of mechanics properties and flow rule between forging and thixo-forging processes. The materials are normal and semi-solid SiCp/AZ61 composite respectively. Environment temperature is 20℃, warm-up temperature of the die is 320℃. The friction model is constant shearing stress model, whose coefficient is 0.25. Billet size is ø50×18.5mm, which is meshed to 50000 tetrahedron elements. Stroke of up-die is 14mm.

Fig. 19. Experiment set-up

Fig. 20. SiCp/AZ61 composite workpiece

Study on Thixotropic Plastic Forming of Magnesium Matrix Composites 273

Fig. 24. shows the effective stress distributions at different volume fraction of SiC particle in thixo-forging process. The effective stress was increased with the increasing of volume

(a) Vol.3% SiCP/AZ61, 560℃ (b) Vol.6% SiCP/AZ61, 560℃ Fig. 24. Effective stress distributions at different volume fraction of SiC particle in thixo-

Fig. 25 shows temperature distributions at different volume fraction of SiC particle in thixoforging process. When the volume fraction of SiC particle was 3%, the fluctuation period of temperature was 558~561℃, whose changed value was small. When the volume fraction of SiC particle was 6%, the fluctuation period of temperature was 558~572℃, whose changed value was more greater than that of the former. It could be gained that the temperature

(a) Vol.3% SiCP/AZ61, 560℃ (b) Vol.6% SiCP/AZ61, 560℃

Fig.26 shows the traditional forging and thixo-forging workpieces of SiCp/AZ61 composite. The thixo-forging has better fill effect and surface finish quality of workpiece than the traditional forging, which could achieve near-end deforming with high quality of workpiece in the former. Those coincide with the simulation results, which indicate that semi-solid

Fig. 25. Temperature distributions at different volume fraction of SiC particle in thixo-

distribution in the latter was worse than that in the former.

fraction of SiC particle.

forging process

forging process

Fig.21 and Fig.22 give the filling stages simulated results in forging and thixo-forging processes respectively. Compared with the two kind of forming processes, it can be concluded that both had the basically identical deformation processes. In the initial stage, the hexagon hole in central section of workpiece was extruded and the rest moved in the rigid motion shown in Fig.21a, Fig.22a. As the stroke increased, metal deformation entered into the second stage, in which metal flowed from central to around in the extrusion pressure, and the cetral protruded and bottom platforms were formed (Seen Fig.21b, Fig.22b). In the last stage, the metal could be filled up claw easily in thixo-forging process, and could not be filled up claw in forging process (Seen Fig.21c, Fig.22c). Therefore, forging was more difficult in filling cavity than thixo-forging.

Fig. 21. Filling stages simulated results in thixo-forging process

Fig. 22. Filling stages simulated results in forging process

Fig. 23. shows the effective stress distributions at different temperatures in thixo-forging process. The effective stress distribution was more uniform and its value was smaller with the increasing of forming temperature, which was contributed from the excellent fluidity of semi-solid composite.

Fig. 23. Effective stress distributions at different temperatures in thixo-forging process

Fig.21 and Fig.22 give the filling stages simulated results in forging and thixo-forging processes respectively. Compared with the two kind of forming processes, it can be concluded that both had the basically identical deformation processes. In the initial stage, the hexagon hole in central section of workpiece was extruded and the rest moved in the rigid motion shown in Fig.21a, Fig.22a. As the stroke increased, metal deformation entered into the second stage, in which metal flowed from central to around in the extrusion pressure, and the cetral protruded and bottom platforms were formed (Seen Fig.21b, Fig.22b). In the last stage, the metal could be filled up claw easily in thixo-forging process, and could not be filled up claw in forging process (Seen Fig.21c, Fig.22c). Therefore, forging

Fig. 23. shows the effective stress distributions at different temperatures in thixo-forging process. The effective stress distribution was more uniform and its value was smaller with the increasing of forming temperature, which was contributed from the excellent fluidity of

(a) 530℃ (b) 560℃

Fig. 23. Effective stress distributions at different temperatures in thixo-forging process

was more difficult in filling cavity than thixo-forging.

Fig. 21. Filling stages simulated results in thixo-forging process

Fig. 22. Filling stages simulated results in forging process

semi-solid composite.

Fig. 24. shows the effective stress distributions at different volume fraction of SiC particle in thixo-forging process. The effective stress was increased with the increasing of volume fraction of SiC particle.

Fig. 24. Effective stress distributions at different volume fraction of SiC particle in thixoforging process

Fig. 25 shows temperature distributions at different volume fraction of SiC particle in thixoforging process. When the volume fraction of SiC particle was 3%, the fluctuation period of temperature was 558~561℃, whose changed value was small. When the volume fraction of SiC particle was 6%, the fluctuation period of temperature was 558~572℃, whose changed value was more greater than that of the former. It could be gained that the temperature distribution in the latter was worse than that in the former.

Fig. 25. Temperature distributions at different volume fraction of SiC particle in thixoforging process

Fig.26 shows the traditional forging and thixo-forging workpieces of SiCp/AZ61 composite. The thixo-forging has better fill effect and surface finish quality of workpiece than the traditional forging, which could achieve near-end deforming with high quality of workpiece in the former. Those coincide with the simulation results, which indicate that semi-solid

Study on Thixotropic Plastic Forming of Magnesium Matrix Composites 275

deformation process was proposed. The constitutive equation of SiCp/AZ61 composites was obtained with the multiple nonlinear regression method based on data of thixotropic compression test. The calculated results were good agreement with the experimental ones. It

Numerical simulation can provide a help for the analysis of thixoforging process, and behavior of metal flow has been obtained. The effective stress distribution was more uniform and its value was more smaller with the increasing of forming temperature. The effective stress was increased with the increasing of volume fraction of SiC particle. The temperature distrubition was worse with the increasing of volume fraction of SiC particle. The differences between traditional forging and thixo-forging processes were analyzed. Results indicated that thixo-forging was better in filling cavity than forging. So the complicated workpiece can be done once in thixo-forging. Numerical simulation results are

This research was supported jointly by grant # 50465003, # 50765005 and # 51165032 from the National Natural Science Foundation of China, Innovative Group of Science and Technology of College of Jiangxi Province and the Jiangxi Province Education Commission

Hai, Z. Y., Xing, Y. L. (2004). Review of recent studies in magnesium matrix composites.

Zhou, W., Xu, Z. M. (1997). Casting of SiC reinforced metal matrix composites. *Journal of*

Kang, C. G., Choi, J. S., Kim, K. H. (1999). The effect of strain rate on macroscopic behavior

Zhang, X. Q., Wang, H. W., Liao, L. H. (2004). In situ synthesis method and damping

Mordike, B. L. (2002). Development of highly creep resistant magnesium alloys. *Journal of*

Flemings, M. C. (1991). Behavior of metal alloy in the semi-solid state. *Metall Trans A,* Vol.22,

Yan, H., Xia, J. C. (2005). Theoretical analysis of plastic forming process for semi-solid

Yan, H., Zhang, F. Y. (2005). Structure evolution of AZ61 magnesium alloy in SIMA process. *Transactions of Nonferrous Metals Society of China*, Vol.15, No.3, 560-564 Yan, H., Zhang, F. Y. (2006). Microstructural evolution of semi-solid AZ61 magnesium alloy during reheating process. *Solid State Phenomena*, Vol.116-117, 275-278 Yan, H., Zhou, B. F. (2006). Thixotrpic deformation behavior of semi-solid AZ61 magnesium

alloy during compression process, *Materials Science and Engineering B*, Vol.132,

in the compression forming of semi-solid aluminum alloy. *Materials Processing*

characterization of magnesium matrix composites. *Composites Science and*

is used to guide composites thixotropic plastic deformation process.

*Journal of Materials Science*, Vol. 39: 6153-6171

*Technology*, Vol.88, No.1-3: 159-168

*Technology*, Vol. 67:720-727

No.5: 957-981

No.1-2, 179-182

*Materials Processing Technology*, Vol. 63 , No.3: 358-363

*Materials Processing Technology*, Vol. 117, No.3: 391-394

material. *Materials Science Forum*, Vol.488-489, 389-392

accorded with experimantal ones.

**8. Acknowledgement**

Foundation.

**9. References**

SiCp/AZ61 composite has good flow property, and can be used to form complicated workpiece.

(a) Traditional forging workpiece (b) Thixo-forging workpiece

Fig. 26. Traditional forging and thixo-forging workpieces of composite


2.5.2. Trypan Blue Assay

The MTT assay detects viable cells but does not take into consideration cell loss caused by cell death; thus, the percentage of viable cells was additionally determined using a dye exclusion test with trypan blue (TB) dye, which is based on the principle that living cells possess intact cell membranes that exclude TB, whereas dead cells do not. The TB test was performed as previously described [41]. Briefly, an aliquot of cell suspension was centrifuged (950 rpm during 5 min, Eppendorf Centrifuge 5804, Hamburg, Germany), and the pellet was resuspended in PBS or serum-free complete medium. Then, 10 μL of cell suspension corresponding to each experimental condition was mixed with 10 μL of TB (0.4%). After incubating the mixture for 3 min at room temperature, the cells were examined using an automated cell counter (TC10TM, Hercules, CA, USA, BioRad).

### *2.6. Expression of Main Genes Related to Early and Late Di*ff*erentiation*

To analyze the expression of early and late genes, cells were collected at the ID and FD time-points, respectively. Total RNA was extracted from cells di fferentiated in sterile six-well plates (at a cellular density of 8 × 104 cell/well) using the extraction kit PureLink ™ RNA Mini Kit (Waltham, MA USA, ThermoFisher Scientific), according to the manufacturer's instructions. The extracted RNA was verified and quantified spectrophotometrically using NanoDrop (Thermo Scientific). Complementary DNA (cDNA) was synthesized from 1 μg of RNA using the RevertAid H Minus First-Strand cDNA synthesis kit (Fisher Scientific), according to the manufacturer's protocol. Gene expression was assessed using quantitative real-time PCR (qRT-PCR) in a LightCycler 480 II thermocycler with Fast Sybr Green Master Mix (Waltham, MA USA, Applied Biosystem). β-Actin was used as an endogenous control. The primer sequences used for amplification are presented in Table A1 (Appendix A).

The reaction mixtures were incubated for an initial denaturation at 95 ◦C for 10 min, followed by 45 PCR cycles (95 ◦C for 15 s, 60 ◦C for 1 min, 95 ◦C for 15 s, and 60 ◦C for 1 min). The ΔΔCT method was used to measure relative quantification, and the levels of transcripts were normalized to that of β-actin. The levels of each mRNA were calculated as relative expression to the basal condition (designated as 1). 3T3-L1 cells treated for 48 h with the AC, without CCT, were considered representative of the basal condition. Three independent experiments were performed, each in duplicate.

Calibrated ΔCt values from undi fferentiated and control di fferentiated cells were used to evaluate the expression of genes. Fold changes in gene expression were calculated using the 2−ΔΔCt method [28]. Expression of the *aP2-1* gene was used as a specific adipocyte marker. *aP2* is widely used as a marker of di fferentiated adipocytes [42].


**4.2.3 Summary**

86 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

Initial level

For PES Membrane For PA membrane For PSF Membrane

Fig. 12. *Total* bilirubin (TBIL), *direct* bilirubin (DBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) level changes after 4 h hemodialysis. Data are expressed

1234

TBIL DBIL ALT AST

We speculated that the high-flux dialysis membrane might possibly let some biocompatibility markers enter dialysis solution so that the plasma levels of these markers could provide biased information. The plasma levels of biocompatibility markers may have also been influenced by adsorption to the membrane surface (Benz et al., 2007; Gotz et al., 2008). The adsorption to the membrane was not determined in our study. However, the protein adsorption capacity was investigated, and no difference was observed. On the basis of our results, we concluded that the designed modifications of the new high-flux PES dialyzer resultED in its higher middle molecule clearance efficacy, and had an effect on thrombogenicity as assessed by platelet behavior and fibrinolysis. Although coagulation system judged by one of the evaluated parameters was slightly higher compared with the other dialyzers, it was still within the biocompatible dialyzer range. In terms of complement activation and changes in leukocyte count, the new dialyzer is also comparable with the other biocompatible dialyzers. Besides the thrombogenicity, complement activation, and WBC count changes, other issues must be considered when evaluating bio(in)compatibility

One further aspect merits consideration is that the PES membrane dialyzer series exhibits a higher permeability and thus, cytokine-inducing substances, possibly present in the dialysis fluid, might gain access to the blood stream through internal filtration (backfiltration). Therefore, investigations on the pyrogen permeability of PES membranes have been performed to the studies on the inflammatory response of the membrane. In the study, the dialysate compartment was deliberately contaminated with purified lipopolysaccharides (LPS) from Escherichia coli, as well as with LPS derived from Stenotrophomonas (Sten)

as the meansSD, n =3

0

50

50

0

100

100

150

150

Normalized value (%

Normalized value (%)

200

200

250

250

300

(Krieter et al., 2007; Klingel et al., 2004)

The PES hollow fiber membrane hemodialyzer was effective and safe in the therapy for uremic patients. The PES hollow fiber hemodialysis membrane could effectively remove water and waste products including not only small molecular weight solutes such as urea and creatinine but also "middle" molecular solute as 2-microglobulin. Slight neutropenia and platelet adhesion were observed at the initial stage of the hemodialysis and no significant difference was found in electrolyte or blood biochemistry before or after the treatment. The data indicated that the performances of PES, PSF and PA hemodialyzers in the clinical setting were comparable and the PES hemodialyzer might be better than the others. The results indicated that PES hollow fiber membrane had a potentially wide application for hemodialysis.


**2. Immune interventions promoting active responses against tumor cells**

Therapeutic strategies that target immune activation have shown significantly increased survival and quality of life for cancer patients [17]. Cancer immunotherapy comprises a variety of treatment approaches and combinations, incorporating the specificity of the adaptive immune response (T cells and antibodies) as well as the diverse and potent cytotoxic weaponry of both adaptive and innate immunity [18]. In this section, we provide an overview of key immunotherapeutic approaches.

Some of these strategies involve the application of soluble antibody molecules that specifically recognize and bind TAAs, resulting in blocked receptor signaling and/or passive immunotherapy. In particular, targeting tumor cells by engaging surface antigens differentially expressed in cancers has been widely used. For example, rituximab targets CD20 in non-Hodgkin B cell lymphoma. At least nine monoclonal antibodies (mAbs) targeting six TAAs (HER2/Neu, EGFR, VEGF, CD20, CD52 and CD33) are approved for the treatment of solid and hematological malignancies [19].

they are infused in the patient [31]. One of the most advanced ACTs in clinical use is called CAR (chimeric antigen receptor) T cell therapy, which involves genetic modification of the patient's T cells to enhance their ability to recognize and attack cancer cells [32]. CAR-T cells have been engineered to express multiple CARs that recognize several tumor antigens. This technology has been successfully applied in clinical trials for hematological malignancies, with durable and complete remission in acute lymphoblastic leukemia [33], chronic lymphocytic leukemia [34] and B-cell lymphomas [35]. Another interesting application is the introduction of CARs targeting negative regulatory receptors, such as PD-1, resulting in reversal

Gene-based Interventions for Cancer Immunotherapy http://dx.doi.org/10.5772/intechopen.80386

While cancer immunotherapies continue to evolve, the recurring role for gene transfer as a fundamental component of many of these approaches is quite evident. Here, we explore several immunotherapy approaches that rely on some aspects of gene transfer, highlighting both clinical and technological advances, especially as related to virotherapy, suicide genes,

Genetic instability intrinsic to cancer generates innumerable missense mutations in tumor cells and thus generates specific targets for T cell immunity [37]. Since these neoantigens are not expressed in normal somatic cells, they are inviting targets for the development of cancer

Although the term vaccine initially referred to the use of prophylactic immunizations for bacterial or viral infections, there are vaccines for therapeutic purposes, especially when we refer to cancer. This strategy has been gaining prominence lately as it offers the opportunity for a lasting effector response and with far fewer side effects than established traditional treatments, such as chemotherapy. In general terms, cancer vaccines seek to restore the ability of the immune system to recognize and eliminate neoplastic cells. In addition, the possibility of generating memory T cells favors long-lasting protective effects, including the prevention of metastasis after primary remission, which would greatly increase the survival and quality of life of these patients.

One of the earliest reports of cancer immunotherapy was conducted by William B. Coley. After observing that established tumors associated with fever or infection generally had higher rates of spontaneous regression, Streptococcus (Coley's toxin) was injected into an inoperable bone tumor. Despite generating data with difficult interpretation, it sparked a debate and numerous other fronts of investigation [39]. Corroborating this hypothesis, Lamm et al. demonstrated that Bacillus Calmette-Guerin (BCG) could be used to activate the immune system and thus enable the treatment of bladder cancer. This therapy, approved by the FDA, is still

In both of the pioneering works described above, bacterial components having immunostimulatory properties were used. It is now clear that the formulation of vaccines should include

vaccines and rational combinations of immunotherapies [38].

of immunosuppression in the tumor [36].

vaccines and CAR-T cells.

**3. Cancer vaccines**

in clinical use [40].

**3.1. Improving vaccine efficacy**

Approved by the Food and Drug Administration (FDA) in 2011, ipilimumab is a mAb against cytotoxic T lymphocyte–associated protein 4 (CTLA-4), a negative checkpoint of T cell function. Thus, checkpoint blockade with ipilimumab releases the brakes of the immune system, promoting T cells to combat cancer cells, and has already benefited thousands of patients with advanced melanoma, a disease that typically kills in less than a year [20]. Additional targets of immune checkpoint therapy include programmed cell death protein 1 (PD1) and its ligand PD-L1, which are even more effective and have fewer side effects as compared to anti-CTLA4 [21]. Moreover, checkpoint inhibitors may be used in combination with each other or with other therapies resulting in the induction of sustained antitumor responses in a wide variety of tumors [22–25]. Checkpoint blockade has undoubtedly been one of the most impressive advancements in cancer therapeutics in recent years, prolonging and saving the lives of many cancer patients. Even so, this approach does not directly induce a *de novo* immune response but releases experienced T cells from inhibitory signaling.

Vaccines are strategies to activate effector immune cells upon stimulation with tumor antigens, promoting the patient's own immune system to mount an immune response against neoplastic cells. Numerous vaccine approaches have been attempted and share the goal of providing effective target antigens while reverting, perhaps, the immunosuppressive tumor microenvironment and activating the ability of DCs to present these antigens. One example is GVAX (Cell Genesys, Inc., South San Francisco, CA), a polyvalent vaccine derived from a cultured cancer cell line expressing a plurality of shared tumor antigens. In addition, the cells have been genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), an immune-modulatory cytokine that can activate antigen-presenting cells (APCs) locally at the vaccine site. Indeed, autologous and/or allogeneic GM-CSF-secreting tumor cell vaccines have demonstrated evidence of immunologic responses in patients with various types of cancers, for example, chronic myeloid leukemia [26], melanoma [27], pancreatic adenocarcinoma [28] and prostate cancer [29].

 OVs act as immunotherapies, promoting antitumor responses due to the viral infection of tumor cells and their acute lysis. An example of this therapy is an intralesional injection with talimogene laherparepvec (Imlygic, T-VEC, Amgen, Thousand Oaks, CA), a genetically engineered oncolytic HSV (herpes simplex virus), with mutations in infectious cell proteins (ICPs) 34.5 and 47, and expressing US11 and GM-CSF [30].

Alternatively, the patient's own T cells or NK cells may be used as a therapeutic agent. Such adoptive cell therapy (ACT) involves the recovery and *ex vivo* expansion of the patient's cells, providing the opportunity for selection and activation of tumor-specific populations, before they are infused in the patient [31]. One of the most advanced ACTs in clinical use is called CAR (chimeric antigen receptor) T cell therapy, which involves genetic modification of the patient's T cells to enhance their ability to recognize and attack cancer cells [32]. CAR-T cells have been engineered to express multiple CARs that recognize several tumor antigens. This technology has been successfully applied in clinical trials for hematological malignancies, with durable and complete remission in acute lymphoblastic leukemia [33], chronic lymphocytic leukemia [34] and B-cell lymphomas [35]. Another interesting application is the introduction of CARs targeting negative regulatory receptors, such as PD-1, resulting in reversal of immunosuppression in the tumor [36].

While cancer immunotherapies continue to evolve, the recurring role for gene transfer as a fundamental component of many of these approaches is quite evident. Here, we explore several immunotherapy approaches that rely on some aspects of gene transfer, highlighting both clinical and technological advances, especially as related to virotherapy, suicide genes, vaccines and CAR-T cells.


**5. Conclusions**

A higher TST level (≥13 ng/dL) was associated with a favorable prognosis in Enza- and/or Abi-treated patients. A TST level of 13 ng/dL may predict a favorable response to novel AR-targeted drugs in CRPC patients.

*J. Clin. Med.* **2019**, *8*, 489

**Supplementary Materials:** The supplementary materials are available online at http://www.mdpi.com/2077- 0383/8/4/489/s1.

**Author Contributions:** S.S. designed the study, collected the data, and completed the manuscript. M.X. and M.M. performed the statistical analysis. H.A.U. supervised the statistical analysis. S.K., Y.Y., H.K., H.M. (Hiroaki Matsumoto), N.T., K.H., M.N., H.N., H.M. (Hideyasu Matsuyama) collected the data. A.K., K.A., T.I. supervised the manuscript. All authors have read and approved the final version of the manuscript and agree with the order of presentation of the authors.

**Funding:** The present work was supported by a Grant-in-Aid for Scientific Research(C) (grant #25462469) to S. Sakamoto.

**Acknowledgments:** The authors are grateful to all members of Chiba University Hospital and the four related institutions for collecting and analyzing the patients' data. The authors particularly appreciate the help of Hisayo Karahi, a research assistant, for the documentation work.

**Conflicts of Interest:** The authors declare no conflict of interest.


**4. The spatial layer of the WS stack**

This chapter presents the integration process of spatial technologies and the Semantic Web technologies at the backdrop of Industrial Archaeology, and its associated tool called the spatial facilitator which is a query and rule engine. The technologies discussed in previous chapters are used and adjusted for processing the spatial knowledge through knowledge technologies within the Semantic Web framework in the research works. This chapter attempts to outline the methods and the processes of these adjustments and how they return the results through knowledge tools as SWRL and SPARQL.

The discussions of the last two chapters aim at laying a background on the concepts of integration process. The discussions on Semantic Web and its underlying technologies and the spatial technology in GIS in the last two chapters have clearly pointed out that the technical advancements toward semantic technologies are integrating every data structures so it will integrate spatial data structure in future. However, for now it is still a topic of

178 Semantics – Advances in Theories and Mathematical Models

based on Semantic Web technology to identify the objects and define them in the ontology. These objects once defined, performs as common schemas between data sources to achieve a sense of data interoperability. The definitions of objects add semantics to the objects and thus adding knowledge about the objects. Knowledge techniques based on Description Logics (DLs) exploit these semantics to manipulate implicit knowledge within the knowledge base. Inference engines utilize the definition of DLs to infer the knowledge base through Horn based rules. The knowledge base stored in OWL syntactic structure is inferred through SWRL to infer the rules. This inference is complimented through querying

Carrying the discussion from last chapter, this research attempts to use the Semantic Web techniques to perform spatial analysis in form of spatial SPARQL and spatial SWRL. The spatial analysis through Semantic Web can only be possible through providing spatial signatures to the defined objects in the ontology. This will allow the knowledge techniques to process spatial solutions. The spatial integration is carried out through OWL/RDF again and the spatial management is carried out again through tools as SWRL and SPARQL. This simplistic yet but effective approach of spatial integration into Semantic Web technologies

The Semantic Web stack shown in figure 5 and 6 can adjust a layer of spatial information into it. The research proposes such an arrangement in the stack. A layer of spatial data mixing seamlessly with the semantic proposition in the layer Ontology through its OWL/RDF based syntax can be envisaged. This layer since uses the standard syntax of OWL/RDF can perform spatial queries through SPARQL or infer rules through standards as SWRL. The next chapter discusses this integration process of spatial technology and Semantic Web technology which is undertaken by defining spatial FILTERs for SPARQL queries and spatial Built-ins for SWRL rules. Ideally the layer should be the top most layer of knowledge level but spatial layer does not yet possess any standards that are standardized by W3C so could not be placed there. It is hence placed as the bottom layer in the certificate level. The next chapter discusses this adjustment in stack in detail and how to

This chapter presents the integration process of spatial technologies and the Semantic Web technologies at the backdrop of Industrial Archaeology, and its associated tool called the spatial facilitator which is a query and rule engine. The technologies discussed in previous chapters are used and adjusted for processing the spatial knowledge through knowledge technologies within the Semantic Web framework in the research works. This chapter attempts to outline the methods and the processes of these adjustments and how they return

The discussions of the last two chapters aim at laying a background on the concepts of integration process. The discussions on Semantic Web and its underlying technologies and the spatial technology in GIS in the last two chapters have clearly pointed out that the technical advancements toward semantic technologies are integrating every data structures so it will integrate spatial data structure in future. However, for now it is still a topic of

provides the possibility to include different modes of data into its framework.

apply spatial queries and rules on any existing ontology.

the results through knowledge tools as SWRL and SPARQL.

**4. The spatial layer of the WS stack**

with SPARQL.

research. It could be conceived from earlier discussions that the integration process requires adjustments of the spatial components within the ontological framework. This chapter is dedicated to discuss the steps and process of this adjustment. The spatial signature of objects plays an important role in determining them. The identification of objects is the process of signing these spatial signatures on them. These signatures should be integrated within the semantics of the objects seamlessly in order to process the spatial knowledge through the knowledge technology. It should be noted however that the Semantic Web technologies are in the maturation process and hence there exists certain processing problems within especially for the non-conventional data type as that of spatial data. Thus, it needs to be sorted out through the existing tested techniques. The research in GIS systems uses the capabilities of existing RDBMS to process the spatial data through spatial operations and functions and use the results of these processes.

The Semantic Web stack discussed in the previous chapter can be updated to address the inclusion of a spatial component. Every tangible object has its spatial signature and thus it becomes indispensable to address the spatial component within its semantic framework. The Semantic Web technologies and its architecture are mostly influenced by the nature of information available on the Internet. Hence, these levels deals mostly with managing the semantic based information through knowledge technologies. However, in recent years there has been huge surge of other forms of information on Web platform and they need to be managed as well. With the advancement in spatial technologies, the trend of disseminating spatial information through Web based environment is rapidly growing. This has raised the issue of the integration of spatial component into the Semantic Web framework.

A layer representing geospatial data in the Semantic Web stack can be placed just above the knowledge layers as could be seen in figure 7. As the technologies within knowledge level are standardized by W3C, the geospatial layer needs to be above the level. However, the technologies within knowledge level needs to blend spatial components seamlessly both syntactically and semantically to maintain the satisfiability required for the consistency of the ontology. This integration procedure should be adjusted within the knowledge tool within the knowledge level of the stack. This approach thus uses the knowledge techniques through adding the spatial structures within them and implementing the spatial knowledge processing along with semantic knowledge processing. The first Semantic Web tool that comes direct in contact with the integration procedure is the structural schema of the knowledge base which is termed as top level ontology in general sense. The top level ontology is the structural schema that represents the nature of knowledge the ontology possesses. It should include the components to adjust the behavior of the knowledge base. Hence the initial task that needs to be adjusted within any top level ontology to perform spatial knowledge processing is to include spatial components within it.

The top level ontology is the structural schema that represents the nature of knowledge the ontology possesses. It should be noted that the top level ontology is syntactically presented through OWL/RDF and contains the top level concepts of the domain. Among these top level concepts, the concepts presenting the spatial components for storage, retrieval and processing of the spatial knowledge should be present. Moving down to the enrichment process, the spatial signatures are mapped to the objects within the knowledge base is again

Spatialization of the Semantic Web 181

already standardized filters and built-ins of both the technologies thus forwarding the

The top level ontology or more popularly upper ontology describes the general concept behind the knowledge domain. This ontology varies with the domain it addresses. There are efforts to come out with a universal upper ontology which addresses the requirements of every knowledge domains but they still are in the phase of researches. Every domain uses its own standard upper ontology for its purpose. This research work attempts to propose an upper level ontology for the domain of industrial archaeology. This top level ontology is the main driving force behind the ArchaeoKM framework. It represents the knowledge possessed by archaeologists in form of descriptions, observations and rules represented through different axioms within the ontology. This ontology serves as a foundational ontology to which objects can be instantiated during identification process. The axioms are the building blocks of the ontology. The integration of spatial components within the framework holds major importance and is required to be adjusted within the top level ontology of ArchaeoKM. The spatial extension of the top level ontology is discussed in the

The realization of spatial signatures of the identified objects in the knowledge base has been discussed earlier. The attachments of these spatial signatures provide a framework that could exploit the developments in spatial technology to provide the objects their spatial identity in respect to their surrounding objects. However, it is important to adjust the components of the spatial technology in the top level ontology. This section covers the

Although the impact of spatial integration is realized in the semantic level when the spatial components are integrated in the ontology, the usage of spatial features begins earlier than that. The spatial functionalities provided by database system form foundations to how they should be adjusted. A parallel structure facilitating the spatial components in different levels of the system architecture has already been presented in chapter 2 through figure 3. At the syntactic level where most of knowledge generation activities are carried out, spatial components are handled through spatially annotating the identified objects. This spatial annotation process draws a Minimum Bounding Rectangles (MBRs) around the objects and stores them as spatial data type in PostgreSQL database system. These MBRs would be used to carry out spatial rules while managing knowledge. It should be noted that the MBRs are not the optimal way of representing the objects and would constitutes some degrees of error during the analysis process. The ideal approach would be to use the boundaries of the objects for representation and analysis purpose. The algorithm to extract point cloud from the boundary is still in the domain of research and not completely matured and hence this

It is the semantic level where the most of the integration work is carried out. The domain ontology is modified to represent the spatial functions and operations within it. The research work revolves around two categories of spatial operations and the integration process takes

arguments of the process in standardizing these built-ins too.

**4.1 The top level ontology**

next section.

**4.2 The spatial top level ontology**

spatial top level ontology of the ArchaeoKM framework.

research uses MBRs to put forward the ideas.

Fig. 7. The inclusion of a Geospatial layer in the Semantic Web Stack.

encoded with OWL/RDF syntax. The methodology of this integration is discussed in later sections within this chapter.

Similarly, the spatial filters and spatial built-ins defined in this layer facilitate the spatial querying and the spatial rule definition. The layers of Rules: SWRL/RIF and Querying: SPARQL provide a base to knowledge management through processing the spatial information semantically within the knowledge base. The only adjustment that is needed is to execute the built-ins and filters in conjunction to the processing capabilities of spatial extensions within current database systems.

Putting forward the arguments on the authenticity of the layer with respect to other layers, the geospatial layer exploits the capabilities of the layers below maintaining the trend of the stack. At the time of integration, the spatial components are included within the top level ontology which stores, retrieves and processes spatial knowledge and utilizes the capabilities of the other technologies in the stack. The spatial components on the top level ontology and the mapped spatial signatures are encoded through the OWL/RDF syntactical structure thus justifying the involvement of ontologies in the stack. Then after, the capability of the SWRL language is exploited through spatial built-ins for spatial SWRL rules. Similarly, the querying capability of the SPARQL language is exploited through spatial filters for the query language. These filters and built-ins can be used with conjunction to 180 Semantics – Advances in Theories and Mathematical Models

Fig. 7. The inclusion of a Geospatial layer in the Semantic Web Stack.

sections within this chapter.

extensions within current database systems.

encoded with OWL/RDF syntax. The methodology of this integration is discussed in later

Similarly, the spatial filters and spatial built-ins defined in this layer facilitate the spatial querying and the spatial rule definition. The layers of Rules: SWRL/RIF and Querying: SPARQL provide a base to knowledge management through processing the spatial information semantically within the knowledge base. The only adjustment that is needed is to execute the built-ins and filters in conjunction to the processing capabilities of spatial

Putting forward the arguments on the authenticity of the layer with respect to other layers, the geospatial layer exploits the capabilities of the layers below maintaining the trend of the stack. At the time of integration, the spatial components are included within the top level ontology which stores, retrieves and processes spatial knowledge and utilizes the capabilities of the other technologies in the stack. The spatial components on the top level ontology and the mapped spatial signatures are encoded through the OWL/RDF syntactical structure thus justifying the involvement of ontologies in the stack. Then after, the capability of the SWRL language is exploited through spatial built-ins for spatial SWRL rules. Similarly, the querying capability of the SPARQL language is exploited through spatial filters for the query language. These filters and built-ins can be used with conjunction to already standardized filters and built-ins of both the technologies thus forwarding the arguments of the process in standardizing these built-ins too.

### **4.1 The top level ontology**

The top level ontology or more popularly upper ontology describes the general concept behind the knowledge domain. This ontology varies with the domain it addresses. There are efforts to come out with a universal upper ontology which addresses the requirements of every knowledge domains but they still are in the phase of researches. Every domain uses its own standard upper ontology for its purpose. This research work attempts to propose an upper level ontology for the domain of industrial archaeology. This top level ontology is the main driving force behind the ArchaeoKM framework. It represents the knowledge possessed by archaeologists in form of descriptions, observations and rules represented through different axioms within the ontology. This ontology serves as a foundational ontology to which objects can be instantiated during identification process. The axioms are the building blocks of the ontology. The integration of spatial components within the framework holds major importance and is required to be adjusted within the top level ontology of ArchaeoKM. The spatial extension of the top level ontology is discussed in the next section.

#### **4.2 The spatial top level ontology**

The realization of spatial signatures of the identified objects in the knowledge base has been discussed earlier. The attachments of these spatial signatures provide a framework that could exploit the developments in spatial technology to provide the objects their spatial identity in respect to their surrounding objects. However, it is important to adjust the components of the spatial technology in the top level ontology. This section covers the spatial top level ontology of the ArchaeoKM framework.

Although the impact of spatial integration is realized in the semantic level when the spatial components are integrated in the ontology, the usage of spatial features begins earlier than that. The spatial functionalities provided by database system form foundations to how they should be adjusted. A parallel structure facilitating the spatial components in different levels of the system architecture has already been presented in chapter 2 through figure 3. At the syntactic level where most of knowledge generation activities are carried out, spatial components are handled through spatially annotating the identified objects. This spatial annotation process draws a Minimum Bounding Rectangles (MBRs) around the objects and stores them as spatial data type in PostgreSQL database system. These MBRs would be used to carry out spatial rules while managing knowledge. It should be noted that the MBRs are not the optimal way of representing the objects and would constitutes some degrees of error during the analysis process. The ideal approach would be to use the boundaries of the objects for representation and analysis purpose. The algorithm to extract point cloud from the boundary is still in the domain of research and not completely matured and hence this research uses MBRs to put forward the ideas.

It is the semantic level where the most of the integration work is carried out. The domain ontology is modified to represent the spatial functions and operations within it. The research work revolves around two categories of spatial operations and the integration process takes

Spatialization of the Semantic Web 183

FILTERs can be used to compare strings and derive results. The functions like regular expression which matches plain literal with no language tag can be used to match the lexical forms of other literals by using string comparison function. In addition, SPARQL FILTER uses the relational operators as = or > or < for the comparison and restrict to the results that they return. The FILTER principle can hence be extended in order to process the geospatial

Geoprocessing functions need to be addressed through enriching the knowledge base with the spatial operations which is related to them during the execution of the query. The enrichment process should be rolled back after the results are returned into its original form iff the SELECT statement is used under the filter. The optimization of the SPATIAL\_FILTER is discussed later which highlights the management of the knowledge base during the

The following example demonstrates the syntax of geoprocessing filters in SPARQL. It could be seen that a new spatial filter through the keyword SPATIAL\_FILTER is introduced which helps the translation engine during the parsing process. The SPARQL statement with spatial filters in the example returns names of all the buildings in class feat:Building which are intersecting with the buffer of 2000 meters of the rivers in class feat:River with their

> ?feat1 feat:name ?name1 ?feat2 feat:name ?name2

> > ?feat1 rdfs:type feat:River

?feat2 rdfs:type feat:Building

SPATIAL\_FILTER [buffer (?x, 2000,?feat1)] SPATIAL\_FILTER [intersection (?y,?x,?feat2)]

In case of georelationship filter, it is straightforward as the enrichment process requires enriching the object properties imitating spatial relationship between objects through the results of the spatial operations at the database level. As with the previous case, the georelationship filter uses the keyword SPATIAL\_FILTER. This keyword parses the spatial components from the SPARQL statements. The following example illustrates the execution of SPARQL with these filters. The first feature is a feat:River which is of kind of feat:Feature, and the second feature is a feat:Building which is also of kind of feat:Feature. The

SPATIAL\_FILTER selects the rivers and buildings which are touching spatially.

**4.3.1 Spatial SPARQL queries**

functions.

**Geoprocessing FILTER**

respective rivers names.

execution of the SPARQL queries.

WHERE

{

}

**Georelationship FILTER**

SELECT ?name1 ?name2

the functions and operations within these two categories which are the georelationship functions and the geoprocessing functions. These functions are defined by the OGC consortium. The Open Geospatial Consortium, Inc. (OGC) is an international industry consortium of 404 companies, government agencies and universities participating in a consensus process to develop publicly available interface standards. OpenGIS® Standards support interoperable solutions that "geo-enable" the Web, wireless and location-based services, and mainstream IT. The standards empower technology developers to make complex spatial information and services accessible and useful with all kinds of applications.

The top level ontology should model spatial technology in terms of its spatial functions and operations. This modeling process should accommodate the spatial functions and operations and maintain their true identity.