Secretome Analysis of Host Cells Infected with Toxoplasma gondii after Treatment of Human Epidermal Growth Factor Receptor 2/4 Inhibitors

Toxoplasma gondii, a ubiquitous, intracellular parasite of the phylum Apicomplexa, infects an estimated one-third of the human population as well as a broad range of warm-blooded animals. We have observed that some tyrosine kinase inhibitors suppressed the growth of T. gondii within host ARPE-10 cells. Among them, afatinib, human epithermal growth factor receptor 2 and 4 (HER2/4) inhibitor, may be used as a therapeutic agent for inhibiting parasite growth with minimal adverse effects on host. In this report, we conducted a proteomic analysis to observe changes in host proteins that were altered via infection with T. gondii and the treatment of HER2/4 inhibitors. Secreting proteins were subjected to a procedure of micor basic reverse phase liquid chromatography, nano-liquid chromatography-mass spectrometry, and ingenuity pathway analysis serially. As a result, the expression level of heterogeneous nuclear ribonucleoprotein K, semaphorin 7A, a GPI membrane anchor, serine/threonine-protein phosphatase 2A, and calpain small subunit 1 proteins were significantly changed, and which were confirmed further by western blot analysis. Changes in various proteins, including these 4 proteins, can be used as a basis for explaining the effects of T. gondii infections and HER2/4 inhibitors.

Suppressors for Human Epidermal Growth Factor Receptor 2/4 (HER2/4): A New Family of Anti-Toxoplasmic Agents in ARPE-19 Cells

The effects of tyrosine kinase inhibitors (TKIs) were evaluated on growth inhibition of intracellular Toxoplasma gondii in host ARPE-19 cells. The number of tachyzoites per parasitophorous vacuolar membrane (PVM) was counted after treatment with TKIs. T. gondii protein expression was assessed by western blot. Immunofluorescence assay was performed using Programmed Cell Death 4 (PDCD4) and T. gondii GRA3 antibodies. The TKIs were divided into 3 groups; non-epidermal growth factor receptor (non-EGFR), anti-human EGFR 2 (anti-HER2), and anti-HER2/4 TKIs, respectively. Group I TKIs (nintedanib, AZD9291, and sunitinib) were unable to inhibit proliferation without destroying host cells. Group II TKIs (lapatinib, gefitinib, erlotinib, and AG1478) inhibited proliferation up to 98% equivalent to control pyrimethamine (5 μM) at 20 μM and higher, without affecting host cells. Group III TKIs (neratinib, dacomitinib, afatinib, and pelitinib) inhibited proliferation up to 98% equivalent to pyrimethamine at 1–5 μM, but host cells were destroyed at 10–20 μM. In Group I, TgHSP90 and SAG1 inhibitions were weak, and GRA3 expression was moderately inhibited. In Group II, TgHSP90 and SAG1 expressions seemed to be slightly enhanced, while GRA3 showed none to mild inhibition; however, AG1478 inhibited all proteins moderately. Protein expression was blocked in Group III, comparable to pyrimethamine. PDCD4 and GRA3 were well localized inside the nuclei in Group I, mildly disrupted in Group II, and were completely disrupted in Group III. This study suggests the possibility of a vital T. gondii TK having potential HER2/4 properties, thus anti-HER2/4 TKIs may inhibit intracellular parasite proliferation with minimal adverse effects on host cells.

An inhibitory compound against the interaction between Galpha(s) and the third intracellular loop region of serotonin receptor subtype 6 (5-HT(6)) disrupts the signaling pathway of 5-HT(6)

Serotonin receptor subtype 6 (5-HT(6)) is a neurotransmitter receptor, which is involved in various brain functions such as memory and mood. It mediates signaling via the interaction with a stimulatory G-protein. Especially, the third intracellular loop (iL3) of 5-HT(6) and the alpha subunit of stimulatory G protein (Galpha(s)) are responsible for the signaling process of 5-HT(6). Chemical compounds that could inhibit the interaction between the iL3 region of 5-HT(6) and Galpha(s) were screened from a chemical library consisted of 5,600 synthetic compounds. One of the identified compounds bound to Galpha(s) and effectively blocked the interaction between Galpha(s) and the iL3 region of 5-HT(6). The identified compound was further shown to reduce the serotonin-induced accumulation of cAMP in 293T cells transformed with 5-HT(6) cDNA. It also lowered the Ca2+ efflux induced by serotonin in cells expressing 5-HT(6) and chimeric Galpha(s5/q). These results indicate that the interaction between the iL3 of 5-HT(6) and Galpha(s) can be exploited for screening of regulatory compounds against the signaling pathway of 5-HT(6).

A Case of Polypoid Angiolipoma of the Distal Descending Colon as Cause of Hematochezia / 대한소화기내시경학회지

Angiolipoma is a benign tumor that is mainly observed in the subcutaneous tissue and is composed of mature adipose tissue and proliferative blood vessels. However, the condition is rare in the gastrointestinal tract including the colon. There was a case report of angiolipoma of the proximal ileum but there are no reports of angiolipoma of the colon in Korea. A 47-year-old man, who presented with intermittent left lower quadrant pain and hematochezia, underwent contrast enhancement CT, which revealed a huge mass with inhomogeneous density in the distal descending colon. The colonoscopy viewed a large polypoid mass with vascular engorgement, and a laparotomy was performed urgently due to the persistent abdominal pain, intussusception and hematochezia. The histology examination disclosed a benign angiolipoma. We report this case of symptomatic angiolipoma of the distal descending colon.

Peritonitis due to spontaneous rupture of the urinary bladder in an end stage renal disease patient with anuria / 대한내과학회지

Patients with nonfunctioning bladders, such as found in anuric renal failure, are prone to a severe form of urinary tract infection called pyocystis. Pyocystis is the accumulation of pus in the bladder. Anuric patients are often believed to be incapable of acquiring a urinary pathogen; thus, the urinary tract may be mistakenly discounted as a possible source of sepsis and bladder rupture. Although bladder rupture is normally associated with trauma, spontaneous bladder rupture may occur, especially in patients with mental disabilities, alcohol abuse and urinary tract infection. We report a rare case of a spontaneous intraperitoneal bladder rupture, probably precipitated by pyocystis. Symptoms and signs were atypical and included pain and vomiting with fever, abdominal distension and ascites. An abdominal CT and retrograde cystography showed a large quantity of intraperitoneal fluid and tearing in the dome of the bladder. Repair of the rupture site of the bladder after laparotomy was performed. However,, the patient expired due to peritonitis and pulmonary infection.

A Case of Pseudohypoparathyroidism Type I / 대한내분비학회지

Pseudohypoparathyroidism (PHP) is a rare clinical type of hypoparathyroidism. The patients with PHP show classic clinical and biochemical features of hypoparathyroidism, but elevated serum level of parathyroid hormone (PTH) and characteristic physical appearances termed 'Albright's hereditary osteodystrophy' (AHO). PHP is classified into types Ia, Ib, Ic and II according to the presence of AHO phenotype and the mechanism of PTH resistance. We experienced a case of PHP in a 12 year-old girl with carpopedal spasm, syncope and partial AHO. She showed very low serum calcium level (1.4 mmol/L), high phosphorus level (3.62 mmol/L) and high immunoreactive PTH level (186.6 ng/L). In the Ellsworth-Howard test, urinary cyclic adenosine monophosphate and phosphorus levels after an exogenous PTH injection remained unchanged. Therefore, we were able to classify the patient as either PHP type Ia or Ic. After the patient had been treated with daily calcium carbonate (1 g), cholecalciferol (250 IU) and calcitriol (0.5 microgram), her neurological signs and symptoms as well as her biochemical abnormalities of hypocalcemia and hyperphosphatemia were improved.

A Case of Pseudohypoparathyroidism Type I / 대한내분비학회지

Pseudohypoparathyroidism (PHP) is a rare clinical type of hypoparathyroidism. The patients with PHP show classic clinical and biochemical features of hypoparathyroidism, but elevated serum level of parathyroid hormone (PTH) and characteristic physical appearances termed 'Albright's hereditary osteodystrophy' (AHO). PHP is classified into types Ia, Ib, Ic and II according to the presence of AHO phenotype and the mechanism of PTH resistance. We experienced a case of PHP in a 12 year-old girl with carpopedal spasm, syncope and partial AHO. She showed very low serum calcium level (1.4 mmol/L), high phosphorus level (3.62 mmol/L) and high immunoreactive PTH level (186.6 ng/L). In the Ellsworth-Howard test, urinary cyclic adenosine monophosphate and phosphorus levels after an exogenous PTH injection remained unchanged. Therefore, we were able to classify the patient as either PHP type Ia or Ic. After the patient had been treated with daily calcium carbonate (1 g), cholecalciferol (250 IU) and calcitriol (0.5 microgram), her neurological signs and symptoms as well as her biochemical abnormalities of hypocalcemia and hyperphosphatemia were improved.

Increased Expression of Tenascin on Photodamaged Vitiligo Skin / 대한해부학회지

Tenascin is suggested the one of cause of vitiligo by interfering melanocyte adhesion and migration. The distribution and expression levels of tenascin were examined by semiquantitative immunohistochemistry on skin biopsies from vitiligo patients with varying area of photodamage. The level of tenascin on adult skin is severely restricted but we had observed the increase on vitiligo skin lesions. Although it is uncertain that the increased tenascin expression is the cause or the result of disease, vitiligo and increased tenascin expression is thought to be related each other. This study has shown that tenascin increased on photodamaged vitiligo skin lesions. So we shoud consider choosing phototherapy to vitiligo.

Nip2 Expression of Postnatal, Aged and Transient Focal Cerebral Ischemic Rat / 대한해부학회지

Nip2 mRNA expression of hippocampus was clarified in the postnatal, adult and aging rat. We observed the change of Nip2 mRNA expression in the ischemic rat hippocampus at 3, 6, 24 and 72 hours after reperfusion. And we investigated the relation between Nip2 and apoptosis after ischemic insults. The Rats were killed 2, 4, 7 days after birth and normal adult rats and aged rat were killed. Male F344 rats were subjected to transient middle cerebral artery occlusion. Reperfusion was achieved by withdrawing the filament after 90 min minutes, and rats were sacrificed 3, 6, 24, 72 hours after reperfusion. Hippocampal sections were stained for TUNEL using ApopTag kit following the protocol provided by the manufacturer, and stained with 2, 3, 5 -triphenyl tetrazolium chloride and cresyl violet We used quantitative reverse transcription and polymerase chain reaction to characterise changes in the mRNA expression of Nip2 in the rat models of transient focal ischemia and postnatal, adult and aged rat. Nip2 mRNA expression were increased in the rat of postnatal development and aging more than these of adult. After reperfusion, marked increase of Nip2 mRNA was observed after 3 and 6 hours. After that time mRNA expression of Nip2 was decreased gradually. The TUNEL staining detected DNA fragmentation in neurons of the entorhinal cortex, forcep major corpus callosum and secondary visual cortex at 24 hours. And TTC staining results showed the whitish infact changes of hippocampal CA1 region and lateral habenular nucleus. We hypothesize that the overexpression of Nip2 is concerned with sensitivity to the ischemic insult at postnatal period and aging period. And, early apoptotic events after cerebral ischemic insults relate to Nip2 mRNA overexpression.

Evaluation of the degree of cross-linking in UV irradiated porcine valves

A porcine heart valve was irradiated by Ultraviolet (UV) rays (10 W, 254 nm) for 2, 4, 8 and 24 hours at 4 degrees C to cross-link the structural collagen matrix. The degree of cross-linking was evaluated by assaying the released amount of hydroxyproline (Hyp) from the matrix, and comparing it with the positive controls of valves treated by glutaraldehyde (GA) solution (0.625 wt%) and the negative controls of non-treated fresh valves. The undigested weight ratio of the specimens increased by increasing the UV irradiation time. The undigested weight of the leaflets, tunica interna and tunica externa of the fresh, GA-treated and UV-irradiated specimens after collagenase digestion was compared. As UV irradiation increased, the amount of released hydroxyproline was gradually decreased until 8 hours of irradiation, after which the released hydroxyproline-reduction occurred slightly until 24 hours of irradiation time in this system. A total 47.68% of the hydroxyproline in the valve was cross-linked by UV irradiation after 24 hours, while 73.74% of the hydroxyproline in the positive control was crossed-linked. Light microscopic observation revealed that the typical crimp pattern of collagen fibers decreased and was rearranged into a dense flattened pattern as the UV irradiation induced interfibrilar cross-linking. GA-treated valves demonstrated a denser matrix pattern than the UV-irradiated specimens. Cross-linked collagenous tissue prepared by UV irradiation would be useful for improving durability and reducing the disadvantages related to using a chemical cross-linking agent.

Influence of Cortex mori on the Capsaicin-induced Neurogenic Inflammation / 대한해부학회지

The root bark of mulberry (Morus alba L. Cortex Mori and other plant of the genus Morus) has been used as an antiphlogistic, diuretic, and expectorant in herbal medicine, and the crude drug is known as "Sangbaikpi" in Korea. Capsaicin, a neurotoxin extracted from a red peppers, has selective effects on peptide-containing C-fiber and induces the release of neuropeptides from sensory nerve ending. But, any reports about the influences of Cortex mori (CM) on the capsaicin-induced neurogenic inflammation were not found, and the precise mechanism of capsaicin-induced neurogenic inflammation remains incompletely understood. The purpose of this study was to determine whether CM could inhibit the capsaicin-induced cutaneous reaction, and to find out its action mechanism. For this purpose, influences of CM on the capsaicin-induced cutaneous reaction (vascular permeability) in back skin of Sprague-Dawley rat, capsaicin-induced histamine release, calcium influx and the change of intracellular cAMP level in rat peritoneal mast cell were estimated. Results obtained were as follows; 1. Pretreatment of CM inhibited remarkably the capsaicin-induced cutaneous reaction. 2. The capsaicin-induced histamine release was inhibited by pretreatment with CM. 3. Capsaicin-induced calcium influx into mast cell was also inhibited by pretreatment with CM. 4. Capsaicin-induced decrease of cAMP level in the mast cell was inhibited significantly by pretreatment with CM. These results suggest that CM contains some substances with an anti-neurogenic inflammatory activity which inhibit the capsaicin-induced cutaneous reaction through the mechanism of mast cell stabilization.

Immunohistochemical Study on the Nitric Oxide Synthase Neurons in the Brain Stem of Rats / 대한해부학회지

Nitric oxide (NO), a free radical that has been postulated to act as a neurotransmitter, neuromodulator, or second messenger molecule in nervous system, is synthesized from L-arginine by nitric oxide synthase (NOS). The NADPH-diaphorase (NADPH-d) histochemical techenique provides a simple and robust method to stain the selected populations of NOS neurons in the brain. This study was aimed to clarify the distribution of NOS neurons in the brain stem of rats. To verify the distribution of NOS neurons in the brain stem, the neurons were stained by the NOS immunohis-tochemistry and NADPH-d histochemistry. Image analyzer-assisted densitometry and cell counting method have been used to quantitatively characterize groups of neuronal cells. Double labeling of NOS immunohistochemistry and NADPH-d histochemistry showed the coexistence of NOS and NADPH-d in same neurons. Neuronal cell bodies exhibiting NOS/NADPH-d staining were found in particular nuclei throughout the brain stem. The number of NOS/NADPH-d neurons were variable in brain stem nuclei. The NADPH-d neurons exhibited different intensities of reaction product. Some groups, including paradorsal raphe nucleus, laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus were extremely heavily stained. Other neurons such as those in the interpeduncular nucleus, central gray, substantia nigra lateralis, nucleus solitarius and raphe obscurus nucleus were moderately stained, while other neurons such supragenual nucleus, lateral paragigantocellular nucleus and prepositus hypoglossal nucleus were weakly stained. The present study describes the many locations within the brain stem in which NADPH-d occurs. Since NADPH-d activity colocalizes with NOS, the results indicate the likely involvement of nitric oxide in the neuronal functions of many brain stem nuclei of rats.

Seizure-Related Change of NADPH-diaphorase and Calbindin D28k Positive Neurons in the Cerebral Cortex of Rats / 대한해부학회지

Administration of kainate (KA) results in the induction of epileptiform activity and limbic motor seizures. Nitric oxide (NO) is a gaseous messenger that plays a role in neural transmission, long term potentiation, depression and cerebral blood flow. NO is formed by NO synthase (NOS) from arginine. NO mediates the increase in cerebral blood flow during seizure activity. However, the production site of NO has not been clearly defined. Recent report showed that constitutive NOS may be induced under certain conditions. Therefore, the present study was aimed to investigate the change of NOS and calbindin D28k in the rat cerebral cortex following seizure. Rats were injected with KA and killed at 6 hours, 1, 3, 6 and 12 days after seizure. Expressional change of nNOS and calbindin D28k was assessed by histochemistry, immunohistochemistry and RT-PCR in the rat brain. Induced NADPH-d positive neurons were observed in the cerebral cortex of 1, 3, 6 and 12 days after seizure and found in specific cortical areas, such as motor cortex, somatosensory cortex, auditory cortex, visual cortex, ectorhinal cortex and perirhinal cortex. The level of nNOS mRNA increased at 1, 3, 6 and 12 days after seizure compared with control group. Induced calbindin D28k positive neurons were observed in motor cortex and somatosensory cortex 1 and 3 days after seizure. The level of calbindin D28k mRNA in the cerebral cortex was slightly decreased at 1 day after seizure. Therefore, in this study, the induced NADPH-d, calbindin D28k positive neurons and upregulated NADPH-d positive neurons may influence the cerebral blood flow and neuronal activity in the cerebral cortex during post-seizure period.

Age-related Changes of Mac1-positive Cell Distribution in the Thymus, Spleen and Popliteal Lymph Node in C57BL/6 Mice / 체질인류학회지

The following experiment was performed to clarify distributional change of the Mac1-positive cells at 1, 4, 8 ,12 and 40 weeks after birth. Thymuses, spleens and lymph nodes were removed and immunocytochemical staining using the specific Mac1 antibody was performed The results obtained from the present experiment were as follows : 1. Mac1-positive cell was absent at 1 week, they were increased by 4 weeks and decreased at 40 weeks in the thymus after birth. 2. There was no Mac1-positive cell in the marginal zone of the spleen at 4, 40 weeks, but they were shown at 8 weeks. 3. Mac1-positive cells were increased at 4 weeks in the subfollicular area of lymph nodes. There was no change of distribution in the lymph nodes. From the above results, we suggest that the age-related decrease of the immunity might be caused by decrease of Mac1-positive cells in the antigen-presenting area.

Effects of exogenous ATP on calcium mobilization and cell proliferation in C6 glioma cell

To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ((Ca2+)i) mobilization and cell proliferation were investigated. ATP-induced (Ca2+)i increased in a dose-dependent manner (10-7 M apprx 10-3 M). ATP-induced (Ca2+)i increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced (Ca2+)i increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative P2Y receptor antagonist, dose-dependently weakened ATP-induced (Ca2+)i mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.

Age - related Changes of T cells,B cells Distribution in C57BL/6 Mice Thymus,Spleen and Popliteal Lymph Node / 체질인류학회지

The following experiment was performed to clarify distributional changes of the Ly1, L3T4, Ly2 positive T cells and IgM positive B cells 1, 4, 8, 12 and 40 weeks after birth. Thymus, spleen and lymph node were removed and immunohistochemical staining such as avidin -biotin -peroxidase complex method was used. The results obtained from above epxperiment were as follows; 1. Ly1 positive T cells were decreased after 8 weeks in the thymus and spleen. these were decreased after 40 weeks in the lymph node. 2. There was no difference L3T4 positive T cells in the thymus, but in the spleen decrease of cell number was shown after 40 weeks. 3. Ly2 postive T cells were decreased after 4 weeks in the thymus, after 40 weeks in the spleen. There was no change of distribution in the lymph node. 4. There was no difference distribution of IgM positive B cells. The results suggest that the age related decrease of the immunity is caused by decrease of cellular immunity related to T cell depletion.

Normal Apical Pleural Tenting: CT Findings

PURPOSE: To define the anatomy of apical pleural tenting commonly seen in computed tomography(CT) of the upper posterior thorax. MATERIALS & METHODS: Chest CTs of 393 patients with no pleural disease clinically and radiographically were analyzed. GE-9800 Quick and Toshiba-900S were used, employing the usual contrast enhanced CTtechnique. CT findings of focal pleural tenting on the inner side of the upper posterior thorax(apical pleural tenting) were evalvated and analysed in terms of location and shape. The CT findings were compared with the gross findings of the inner aspect of the posterior cadaveric thorax. RESULTS: Apical pleural tenting was formed by the upper border of the subcostal muscle. It's incidence was 44%(n=171), with bilaterality in 29%(n=49), and unilaterality in 71% of cases(n=122). This tenting was most frequently found between the third rib and the fourth intercostal space(81%), and seen in the outer third(42%) or central third(41%) part of the posterior costalpleura. In fifteen cases(7%), it was directed obliquely and had changed its location from the inner to the centralor the central to the outer part. The shapes of the tenting were classified as follows : type 1(convex innerborder with sharp apex, 62%) ; type 2(convex inner border with broad apex, 23%) ; type 3(undulated contour ofapex, 13%) ; and type 4(two-spike apices, 1%). CONCLUSION: Apical pleura tenting is a normal CT finding probably demonstrated by the upper border of the subcostal muscle. Misdiagnosis of pleural disease can be avoided by recognition of the location and type of this tenting.

MR Imaging of the Dural Ligaments' Cadaveric and Clinical Study

OBJECTIVE: The purpose of this study was to describe the MR imaging appearance of the dural ligaments which is connected between the anterior dura mater and the posterior longitudinal ligament. MATERIALS AND METHODS: In 10 cadavers with formalin fixation, the dural ligaments were evaluated and photographed after removal of the posterior element of the spinal canal. MR studies of 12 patients who had distinct dural ligaments were evaluated. Spin-echo MR images were obtained on a 1. 5T unit. RESULTS: In seven of the ten cadavers, the midline dural ligament was observed; the lateral dural ligament was observed in 3 cadavers. On MR, the dural ligaments were imaged as thin lines of low signal intensity between the anterior dural margin and the posterior longitudinal ligament. Dural ligaments were well-visualized at the level of the lower lumbar spine which had a more prominent anterior epidural space than that of the upper level of the spine. Eight of 12 cases had well developed midline dural ligaments. Two cases showed distinct lateral dural ligaments and the remaining 2 cases had distinct midline and lateral dural ligaments. CONCLUSION: MR images of the dural ligament demonstrate a thin low signal line at the anterior epidural space.

Distribution of anti-TNP forming cells after TNP-LPS injection in mice popliteal lymph node and spleen / 대한해부학회지

No abstract available.

Immunohistological study of lymph follicles and germinal centers in the politeal lymph nodes of developing mice after injection of mitogen and adjuvant / 대한해부학회지

No abstract available.