An adult female patient with single atrium and single ventricle undergoing appendectomy: A case report.

Appendicitis is one of the common diseases, and appendectomy is one of the most commonly performed procedures. Single atrium and single ventricle are rare heart diseases, and very few patients survive to adulthood. We report a patient with single atrial and single ventricles undergoing appendectomy with transverse abdominis plane block and dexmedetomidine sedation anesthesia with smooth postoperative appendectomy.

Age-Related Pathology Associated with H1N1 A/California/07/2009 Influenza Virus Infection.

Influenza virus infection causes a spectrum of diseases, ranging from mild upper respiratory tract infection to severe lower respiratory tract infection, that can lead to diffuse alveolar damage, interstitial and airspace inflammation, or acute respiratory failure. Mechanisms instructing disease severity are not completely understood, but host, viral, and bacterial factors influence disease outcome. With age being one host factor associated with a higher risk of severe influenza, we investigated regional pulmonary distribution and severity of pneumonia after 2009 H1N1 influenza virus infection in newly weaned, adult, and aged ferrets to better understand age-dependent susceptibility and pathology. Aged ferrets exhibited greater weight loss and higher rates of mortality than adult ferrets, whereas most newly weaned ferrets did not lose weight but had a lack of weight gain. Newly weaned ferrets exhibited minimal pneumonia, whereas adult and aged ferrets had a spectrum of pneumonia severity. Influenza virus-induced pneumonia peaked earliest in adult ferrets, whereas aged ferrets had delayed presentation. Bronchial severity differed among groups, but bronchial pathology was comparable among all cohorts. Alveolar infection was strikingly different among groups. Newly weaned ferrets had little alveolar cell infection. Adult and aged ferrets had alveolar infection, but aged ferrets were unable to clear infection. These different age-related pneumonia and infection patterns suggest therapeutic strategies to treat influenza should be tailored contingent on age.

Aged Chinese-origin rhesus macaques infected with SIV develop marked viremia in absence of clinical disease, inflammation or cognitive impairment.

BACKGROUND: Damage to the central nervous system during HIV infection can lead to variable neurobehavioral dysfunction termed HIV-associated neurocognitive disorders (HAND). There is no clear consensus regarding the neuropathological or cellular basis of HAND. We sought to study the potential contribution of aging to the pathogenesis of HAND. Aged (range = 14.7-24.8 year) rhesus macaques of Chinese origin (RM-Ch) (n = 23) were trained to perform cognitive tasks. Macaques were then divided into four groups to assess the impact of SIVmac251 infection (n = 12) and combined antiretroviral therapy (CART) (5 infected; 5 mock-infected) on the execution of these tasks. RESULTS: Aged SIV-infected RM-Ch demonstrated significant plasma viremia and modest CSF viral loads but showed few clinical signs, no elevations of systemic temperature, and no changes in activity levels, platelet counts or weight. Concentrations of biomarkers of acute and chronic inflammation such as soluble CD14, CXCL10, IL-6 and TNF-α are known to be elevated following SIV infection of young adult macaques of several species, but concentrations of these biomarkers did not shift after SIV infection in aged RM-Ch and remained similar to mock-infected macaques. Neither acute nor chronic SIV infection or CART had a significant impact on accuracy, speed or percent completion in a sensorimotor test. CONCLUSIONS: Viremia in the absence of a chronic elevated inflammatory response seen in some aged RM-Ch is reminiscent of SIV infection in natural disease resistant hosts. The absence of cognitive impairment during SIV infection in aged RM-Ch might be in part attributed to diminishment of some facets of the immunological response. Additional study encompassing species and age differences is necessary to substantiate this hypothesis.

CD8-Positive T-Cell Leukoencephalitis With Astrocytopathy Clinically Presenting as Neuromyelitis Optica.

We describe a novel disease entity with the clinical and radiologic presentation of neuromyelitis optica (NMO) and widespread CD8-positive T-cell leukoencephalitis and astrocytopathy. The 59-year-old female patient had a complex 2-year neurological history that included early changes in cognition and memory, progressive lower extremity motor dysfunction, and multimodal sensory involvement. MRI of the spinal cord showed increased T2 signal in the central cord extending from C2 through T4. MRI of the brain showed symmetric radial enhancement in periventricular deep white matter without evidence of demyelinating lesions. The constellation of findings met clinical criteria for NMO. Steroid treatment was initiated with subjective improvement but she developed urosepsis and died at age 61 years. At autopsy, the spinal cord showed typical NMO findings but no evidence of complement deposition or neutrophil infiltration. There was diffuse CD8-positive T-cell infiltration and CD68-positive macrophage activation throughout subcortical white matter, optic chiasm, brainstem, and spinal cord. This was accompanied by marked astrocytopathy in all areas. Serum was negative for aquaporin-4 autoantibodies suggesting a nonhumoral basis of astrocyte damage. This first example of CD8-positive T-cell leukoencephalitis in a patient with a clinical presentation of NMO may explain the recalcitrance of some patients to therapies targeting humoral immunity.

Ultrastructure of Diaschisis Lesions after Traumatic Brain Injury.

We used controlled cortical impact in mice to model human traumatic brain injury (TBI). Local injury was accompanied by distal diaschisis lesions that developed within brain regions anatomically connected to the injured cortex. At 7 days after injury, histochemistry documented broadly distributed lesions, particularly in the contralateral cortex and ipsilateral thalamus and striatum. Reactive astrocytosis and microgliosis were noted in multiple neural pathways that also showed silver-stained cell processes and bodies. Wisteria floribunda agglutinin (WFA) staining, a marker of perineuronal nets, was substantially diminished in the ipsilateral, but less so in the contralateral cortex. Contralateral cortical silver positive diaschisis lesions showed loss of both phosphorylated and unphosphorylated neurofilament staining, but overall preservation of microtubule-associated protein (MAP)-2 staining. Thalamic lesions showed substantial loss of MAP-2 and unphosphorylated neurofilaments in addition to moderate loss of phosphorylated neurofilament. One animal demonstrated contralateral cerebellar degeneration at 7 days post-injury. After 21 days, the gliosis had quelled, however persistent silver staining was noted. Using a novel serial section technique, we were able to perform electron microscopy on regions fully characterized at the light microscopy level. Cell bodies and processes that were silver positive at the light microscopy level showed hydropic disintegration consisting of: loss of nuclear heterochromatin; dilated somal and neuritic processes with a paucity of filaments, tubules, and mitochondria; and increased numbers of electron-dense membranous structures. Importantly the cell membrane itself was still intact 3 weeks after injury. Although the full biochemical nature of these lesions remains to be deciphered, the morphological preservation of damaged neurons and processes raises the question of whether this is a reversible process.

Human Parechovirus 3 Meningitis and Fatal Leukoencephalopathy.

Human parechovirus 3 (HPeV3) is a picornavirus associated with neurologic disease in neonates. Human parechovirus 3 infection of preterm and term infants is associated with seizures and destructive periventricular white matter lesions. Despite unremarkable cerebrospinal fluid (CSF), HPeV3 RNA can be amplified from CSF and nasopharyngeal and rectal swabs. We report pathologic findings in 2 autopsy cases of infants with active HPeV3 infection. Both children were born approximately 1 month premature and were neurologically intact but, after a few weeks, developed seizures and radiologic evidence of white matter lesions. Neuropathologic examination demonstrated classic severe periventricular leukomalacia in the absence of an immune response. Human parechovirus 3 sequences were identified in RNA extracted from CSF, sera, and tissues. Human parechovirus 3 in situ hybridization detection of infected cells was limited to meninges and associated blood vessels in addition to smooth muscle of pulmonary vessels. Ultrastructural evaluation of meninges demonstrated dense core structures compatible with picornavirus virions. These findings suggest that encephalopathic changes are secondary to infection of meninges and potential compromise of vascular perfusion. Thus, parechovirus infection of vascular smooth muscle may be a more general pathogenic process.

Role for mammalian chitinase 3-like protein 1 in traumatic brain injury.

Traumatic brain injury (TBI) is accompanied by inflammatory infiltrates and CNS tissue response. The astrocytosis associated with TBI has been proposed to have both beneficial and detrimental effects on surviving neural tissue. We recently observed prominent astrocytic expression of YKL-40/chitinase 3-like protein 1 (CHI3L1) associated with severity of brain injury. The physiological role of CHI3L1 in the CNS is unknown; however, its distribution at the perimeter of contusions and temporal course of expression suggested that in TBI it might be an important component of the astrocytic response to modulate CNS inflammation. To address this hypothesis, we used serially sectioned brains to quantitatively compare the neuropathological outcomes of TBI produced by controlled cortical impact in wild type (WT) and chi3l1 knockout (KO) mice where the murine YKL-40 homologue, breast regression protein 39 (BRP-39/CHI3l1), had been homozygously disrupted. At 21 days post-injury, chi3l1 KO mice displayed greater astrocytosis (increased GFAP staining) in the hemispheres ipsilateral and contralateral to impact compared with WT mice. Similarly, Iba1 expression as a measure of microglial/macrophage response was significantly increased in chi3l1 KO compared with WT in the hemisphere contralateral to impact. We conclude that astrocytic expression of CHI3L1 limits the extent of both astrocytic and microglial/macrophage facets of neuroinflammation and suggests a novel potential therapeutic target for modulating neuroinflammation.

Coxsackievirus B4 myocarditis and meningoencephalitis in newborn twins.

Coxsackievirus B4 (CB4) is a picornavirus associated with a variety of human diseases, including neonatal meningoencephalitis, myocarditis and type 1 diabetes. We report the pathological findings in twin newborns who died during an acute infection. The twins were born 1 month premature but were well and neurologically intact at birth. After a week they developed acute lethal neonatal sepsis and seizures. Histopathology demonstrated meningoencephalitis and severe myocarditis, as well as pancreatitis, adrenal medullitis and nephritis. Abundant CB4 sequences were identified in nucleic acid extracted from the brain and heart. In situ hybridization with probes to CB4 demonstrated infection of neurons, myocardiocytes, endocrine pancreas and adrenal medulla. The distribution of infected cells and immune response is consistent with reported clinical symptomatology where systemic and neurological diseases are the result of CB4 infection of select target cells.

H1N1, but not H3N2, influenza A virus infection protects ferrets from H5N1 encephalitis.

UNLABELLED: Seasonal influenza causes substantial morbidity and mortality because of efficient human-to-human spread. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. We studied systemic viral spread after intranasal infection with highly pathogenic avian influenza virus (H5N1 [A/Viet Nam/1203/2004]) in ferrets with or without prior pandemic H1N1pdm09 (A/Mexico/4108/2009) or H3N2 (A/Victoria/361/2011) infection. After intranasal challenge with H5N1 influenza virus, naive ferrets rapidly succumbed to systemic infection. Animals challenged with H5N1 influenza virus greater than 3 months after recovering from an initial H1N1pdm09 infection survived H5N1 virus challenge and cleared virus from the respiratory tract 4 days after infection. However, a prolonged low-level infection of hematopoietic elements in the small bowel lamina propria, liver, and spleen was present for greater than 2 weeks postinfection, raising the potential for reassortment of influenza genes in a host infected with multiple strains of influenza. Animals previously infected with an H3N2 influenza virus succumbed to systemic disease and encephalitis after H5N1 virus challenge. These results indicate prior infection with different seasonal influenza strains leads to radically different protection from H5N1 challenge and fatal encephalitis. IMPORTANCE: Seasonal influenza is efficiently transmitted from human to human, causing substantial morbidity and mortality. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. Infection of naive ferrets with H5N1 avian influenza virus causes a rapid and lethal systemic disease. We studied systemic H5N1 viral spread after infection of ferrets with or without prior exposure to either of two seasonal influenza virus strains, H1N1 and H3N2. Ferrets previously infected with H1N1 survive H5N1 challenge while those previously infected with H3N2 die of encephalitis. However ferrets protected from lethal H5N1 infection develop persistent low-level infection of the small intestine, liver, or spleen, providing a nidus for future viral strain recombination. The mechanism by which prior infection with specific strains of seasonal influenza virus protect from lethal H5N1 challenge needs to be elucidated in order to design effective immunization and treatments.

Exacerbation of experimental autoimmune encephalomyelitis in the absence of breast regression protein 39/chitinase 3-like 1.

We previously reported that YKL-40, the human analog of mouse breast regression protein 39 ([BRP-39] chitinase 3like 1), is elevated in the cerebrospinal fluid of patients with a variety of neuroinflammatory conditions, such as multiple sclerosis and traumatic brain injury. Expression of YKL-40 in the CNS was predominantly associated with reactive astrocytes in the vicinity of inflammatory lesions. Because previous studies have shown that reactive astrocytes play a critical role in limiting immune infiltration in the mouse model of experimental autoimmune encephalomyelitis, we explored the role of BRP-39 in regulatingneuroinflammation in experimental autoimmune encephalomyelitis. Using BRP-39--deficient (BRP-39(-/-)) mice, we demonstrate the importance of BRP-39 in modulating the severity of clinical experimentalautoimmune encephalomyelitis and CNS neuroinflammation. At disease onset, absence of BRP-39 had little effect on clinical disease orlymphocytic infiltrate, but by 14 days after immunization, differences in clinical scores were evident. By 28 days after immunization, BRP-39(-/-) mice showed more severe and persistent clinical disease than BRP-39(+/+) controls. Histopathological evaluation showed that BRP-39(-/-) mice had more marked lymphocytic and macrophage infiltrates and gliosis versus BRP-39(+/+) mice. These findings support the role of BRP-39 expression in limiting immune cell infiltration into the CNS and offer a new target to modulate neuroinflammation.

Acute murine H5N1 influenza A encephalitis.

Avian influenza A virus H5N1 has the proven capacity to infect humans through cross-species transmission, but to date, efficient human-to-human transmission is limited. In natural avian hosts, animal models and sporadic human outbreaks, H5N1 infection has been associated with neurological disease. We infected BALB/c mice intranasally with H5N1 influenza A/Viet Nam/1203/2004 to study the immune response during acute encephalitis. Using immunohistochemistry and in situ hybridization, we compared the time course of viral infection with activation of immunity. By 5 days postinfection (DPI), mice had lost substantial body weight and required sacrifice by 7 DPI. H5N1 influenza was detected in the lung as early as 1 DPI, whereas infected neurons were not observed until 4 DPI. H5N1 infection of BALB/c mice developed into severe acute panencephalitis. Infected neurons lacked evidence of a perineuronal net and exhibited signs of apoptosis. Whereas lung influenza infection was associated with an early type I interferon (IFN) response followed by a reduction in viral burden concordant with appearance of IFN-γ, the central nervous system environment exhibited a blunted type I IFN response.

Astrocyte and macrophage regulation of YKL-40 expression and cellular response in neuroinflammation.

Numerous inflammatory conditions are associated with elevated YKL-40 expression by infiltrating macrophages. Thus, we were surprised to observe minimal macrophage and abundant astrocyte expression of YKL-40 in neuroinflammatory conditions. The aims of the current study were to better delineate this discrepancy, characterize the factors that regulate YKL-40 expression in macrophages and astrocytes and study whether YKL-40 expression correlates with cell morphology and/or activation state. In vitro, macrophages expressed high levels of YKL-40 that was induced by classical activation and inhibited by alternative activation. Cytokines released from macrophages induced YKL-40 transcription in astrocytes that was accompanied by morphological changes and altered astrocytic motility. Because coculturing of astrocytes and macrophages did not reverse this in vitro expression pattern, additional components of the in vivo central nervous system (CNS) milieu must be required to suppress macrophage and induce astrocyte expression of YKL-40.

In vivo CHI3L1 (YKL-40) expression in astrocytes in acute and chronic neurological diseases.

BACKGROUND: CHI3L1 (YKL-40) is up-regulated in a variety of inflammatory conditions and cancers. We have previously reported elevated CHI3L1 concentration in the cerebrospinal fluid (CSF) of human and non-human primates with lentiviral encephalitis and using immunohistochemistry showed that CHI3L1 was associated with astrocytes. METHODS: In the current study CHI3L1 transcription and expression were evaluated in a variety of acute and chronic human neurological diseases. RESULTS: ELISA revealed significant elevation of CHI3L1 in the CSF of multiple sclerosis (MS) patients as well as mild elevation with aging. In situ hybridization (ISH) showed CHI3L1 transcription mostly associated with reactive astrocytes, that was more pronounced in inflammatory conditions like lentiviral encephalitis and MS. Comparison of CHI3L1 expression in different stages of brain infarction showed that YKL40 was abundantly expressed in astrocytes during acute phases and diminished to low levels in chronic infarcts. CONCLUSIONS: Taken together, these findings demonstrate that CHI3L1 is induced in astrocytes in a variety of neurological diseases but that it is most abundantly associated with astrocytes in regions of inflammatory cells.

YKL-40 expression in traumatic brain injury: an initial analysis.

YKL-40 (chitinase 3-like protein 1) is expressed in a broad spectrum of inflammatory conditions and cancers. We have previously reported that YKL-40 levels are elevated in the cerebrospinal fluid (CSF) of macaques and humans with lentiviral encephalitis, as well as multiple sclerosis (MS). The current study assessed temporal CSF YKL-40 levels in subjects with severe traumatic brain injury (TBI; Glasgow Coma Scale [GCS] score

YKL-40 is directly produced by tumor cells and is inversely linked to EGFR in glioblastomas.

YKL-40 is a secreted chitinase-like molecule whose expression is associated with glioma grade. Expression is higher in astrocytomas than oligodendrogliomas and has been reported to predict shorter survival and radiation resistance in glioblastomas (GBMs). Whether YKL-40 is directly produced by glioma cells or other admixed nonneo-plastic cells, and whether it correlates with 1p/19q status or other hallmark molecular abnormalities, are unclear. A rank-order list of YKL-40 expression was determined immunohistochemically in 79 untreated high-grade adult glio-mas, including 28 anaplastic oligodendrogliomas (AOs) and 51 GBMs. Relative YKL-40 expression was compared with glioma class, key molecular alterations, and immunohistochemical markers via a series of Spearman rank correlations. YKL-40 mRNA in situ hybridization with colocalization assessment via confocal microscopy was also performed. YKL-40 mRNA was abundant in glioma cells as well as reactive astrocytes, but was low in admixed neurons and macrophages. YKL-40 expression was higher in GBMs than AOs (P < 0.0001) and among GBMs, YKL-40 expression was lower in tumors with either EGFR amplification (P = 0.005) or elevated EGFR expression (P = 0.001). Among AOs, no difference in YKL-40 expression was seen in tumors with 1p19q codeletion (P = 0.3), but loss of heterozygosity in 10q23 correlated with increased YKL-40 expression (P = 0.03). These data suggest that YKL-40 is predominantly expressed by neoplastic glial cells and is related to certain key molecular alterations.

[Observation on therapeutic effect of bee needle therapy on chronic lumbar muscle strain].

OBJECTIVE: To explore a new method for treatment of chronic lumbar muscle strain. METHODS: One hundred and fifty cases were randomly divided into 2 groups, an observation group of 78 cases and a control group of 72 cases. The observation group was treated with bee-needle therapy, with Jiaji (EX-B 2) on the loin and Shenshu (BL 23), Zhishi (BL 47), Ciliao (BL 32), Weizhong (BL 40), Ashi points selected as main; and the control group was treated with routine acupuncture at the same points as those in the observation group, in combination with fire-cupping or warm needle moxibustion. They were treated once daily, 10 sessions constituting one course. After 3 courses and a half year later, therapeutic effects were observed and followed up. RESULTS: In the observation group, 49 cases were cured, 27 improved, 2 ineffective, with a cured rate of 62.8%, and in the control group, the corresponding figures were 29, 40, 3 and 40.3%, with a significant difference between the two groups in the cured rate (P < 0.01). CONCLUSION: The therapeutic effect of the bee-needle therapy on chronic lumbar muscle strain is better than that of the routine acupuncture, which provides a better method for treatment of chronic lumbar muscle strain.

PK11195 labels activated microglia in Alzheimer's disease and in vivo in a mouse model using PET.

Activated microglia may promote neurodegeneration in Alzheimer's disease (AD) and may also help in amyloid clearance in immunization therapies. In vivo imaging of activated microglia using positron emission tomography (PET) could assist in defining the role of activated microglia during AD progression and therapeutics. We hypothesized that PK11195, a ligand that binds activated microglia, could label these cells in postmortem AD tissues and in vivo in an animal model of AD using PET. [(3)H](R)-PK11195 binding was significantly higher in AD frontal cortex compared to controls and correlated mainly with the abundance of immunohistochemically labeled activated microglia. With age, the brains of APP/PS1 transgenic mice showed progressive increase in [(3)H](R)-PK11195 binding and [(11)C](R)-PK11195 retention in vivo assessed using microPET, which correlated with the histopathological abundance of activated microglia. These results suggest that PK11195 binding in AD postmortem tissue and transgenic mice in vivo correlates with the extent of microglial activation and may help define the role of activated microglia in the pathogenesis and treatment of AD.

Iron localization in superficial siderosis of the central nervous system.

Originally conceived as an uncommon disorder, with the advent of MRI, CNS superficial siderosis has been observed more frequently. We present histologic, histochemical, immunohistochemical, immunofluorescent and ultrastructural evaluation of a 56-year-old woman with superficial siderosis. Iron was concentrated in macrophages, superficial astrocytes and gray matter oligodendroglia deep within the cord. While spatially associated with dystrophic glial and neuronal spheroids, iron did not colocalize with mitochondria. Neurotoxic effects were observed despite selective iron localization only within a variety of non-neuronal cell types.

The positron emission tomography ligand DAA1106 binds with high affinity to activated microglia in human neurological disorders.

Chronic microglial activation is an important component of many neurological disorders, and imaging activated microglia in vivo will enable the detection and improved treatment of neuroinflammation. 1-(2-chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox-amide (PK11195), a peripheral benzodiazepine receptor ligand, has been used to image neuroinflammation, but the extent to which PK11195 binding distinguishes activated microglia and reactive astrocytes is unclear. Moreover, PK11195 may lack sufficient sensitivity for detecting mild neuroinflammation. We hypothesized that N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl) acetamide (DAA1106), a new ligand that binds specifically to peripheral benzodiazepine receptor, binds to activated microglia in human neurological diseases with higher affinity than does PK11195. We therefore compared the pharmacological binding properties of [3H](R)-PK11195 and [3H]DAA1106 in postmortem tissues from patients with cerebral infarcts, amyotrophic lateral sclerosis, Alzheimer disease, frontotemporal dementia, and multiple sclerosis (n=10 each). In all diseases, [3H]DAA1106 showed a higher binding affinity as reflected by lower dissociation constant (KD) values than that of [3H](R)-PK11195. Moreover, specific binding of both ligands correlated with the presence of activated microglia identified by immunohistochemistry in situ. We conclude that 1) ligands that bind peripheral benzodiazepine receptor mainly label activated microglia in human neurological disorders and that 2) DAA1106 may possess binding characteristics superior to those of PK11195, which may be beneficial for in vivo positron emission tomography imaging.

Longitudinal in vivo positron emission tomography imaging of infected and activated brain macrophages in a macaque model of human immunodeficiency virus encephalitis correlates with central and peripheral markers of encephalitis and areas of synaptic degeneration.

Human immunodeficiency virus encephalitis is characterized by infiltration of the brain with infected and activated macrophages; however, it is not known why disease occurs after variable lengths of infection in 25% of immunosuppressed acquired immune deficiency syndrome patients. We determined in vivo correlates (in peripheral blood and the central nervous system) for the development and progression of lentiviral encephalitis by longitudinally following infected and activated macrophages in the brain using positron emission tomography (PET). Using human postmortem brain tissues from both lentivirus-infected encephalitic patients and cell culture systems, we showed that the PET ligand [(3)H](R)-PK11195 bound specifically to virus-infected and activated macrophages. We longitudinally imaged infected and activated brain macrophages in a cohort of macaques infected with simian immunodeficiency virus using [(11)C](R)-PK11195. [(11)C](R)-PK11195 retention in vivo in the brain correlated with viral burden in the brain and cerebrospinal fluid, and with regions of both presynaptic and postsynaptic damage. Finally, longitudinal changes in [(11)C](R)-PK11195 retention in the brain in vivo correlated with changes in circulating monocytes as well as in both natural killer and memory CD4(+) T cells in the periphery. Our results suggest that development and progression of simian immunodeficiency virus encephalitis in vivo correlates with changes in specific cell subtypes in the periphery. A combination of PET imaging and the assessment of these peripheral immune parameters may facilitate longitudinal assessment of lentiviral encephalitis in living patients as well as evaluation of therapeutic efficacies.