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Biomedical subjects

T Kohno

Publications and source records attributed to T Kohno.

At least 217 records · Page 12Linked to original sources
PubMed1997

[Reoperation for relief of valvular and supravalvular stenosis late after aortic valve replacement with Manouguian's anulus enlargement: report of a case].

Thirteen year-old boy who received aortic valve replacement with Manouguian's anulus enlargement for congenital aortic stenosis 7 years ago developed valvular and supravalvular stenosis. Pressure gradient between left ventricle and aorta was 55 mmHg. The etiology of supravalvular stenosis was extensive patch plasty of ascending aorta and severe degeneration of expanded polytetrafluoroethylene patch. Moreover, white hard mass existed just outside the patch and was one of the components of the stenosis. This mass was acellular calcification without foreign body reaction. Degenerated prosthetic patch was thoroughly resected, and redo Manouguian's procedure with autogeneous pericardium was performed. We tried to use two sizes larger 21 mm valve, but, after all, 19 mm valve was sewn into place. His postoperative course was uneventful.

Adolescent↗
PubMed1996

Breakpoint junction of interstitial homozygous deletion at chromosome 2q33 in a small cell lung carcinoma.

We have characterized the breakpoint junction of the homozygous deletion at chromosome 2q33 in a small cell lung carcinoma cell line. Cloning and sequencing of the genomic regions surrounding the breakpoint junction of the deletion revealed that the homozygous deletion was caused by a simple interstitial deletion of a 220-kb segment. An AT-dinucleotide of contributing germline sequences was overlapped at the junction. Since there were one or two nucleotide overlaps of germline sequences at breakpoint junctions in all four cases of interstitial deletions analyzed to date, this may reflect a common mechanism underlying the occurrence of chromosomal interstitial deletion.

Base Sequence↗
PubMed1996

Three-dimensional structures of the amyloid beta peptide (25-35) in membrane-mimicking environment.

The three-dimensional structure of amyloid beta peptide (25-35), which has neurotoxic activity, in lithium dodecyl sulfate micelles was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 20 converged amyloid beta peptide structures were obtained on the basis of 110 experimental constraints, including 106 distance constraints reduced from the nuclear Overhauser effect (NOE) connectivities and four torsion angle (phi) constraints. The atomic root mean square difference about averaged coordinates is 1.04 +/- 0.25 A for the backbone atoms (N, C alpha, C) and 1.39 +/- 0.27 A for all heavy atoms of the entire peptide. The molecular structure of amyloid beta peptide in membrane-mimicking environment is composed of a short alpha helix in the C terminal position. The three residues from the N-terminus are disordered, but the remaining eight C-terminal residues are well-ordered, which is supported by the RMSD values of the C-terminal region, Lys28-Leu34. In this region, the RMS differences from averaged coordinates are 0.26 +/- 0.11 A for the backbone atoms (N, C alpha, C) and 0.77 +/- 0.21 A for all heavy atoms, which is very low compared with those for the entire peptide. The four amino acid residues from the N-terminus are hydrophilic and the other seven amino acid residues in C-terminus are hydrophobic. So, our results show that the C-terminal region of amyloid beta peptide (25-35) is buried in the membrane and assumes alpha-helical structure, whereas the N-terminal region is exposed to the solvent with a flexible structure. This structure is very similar to membrane-mediated structure of substance P previously reported. The three-dimensional structure of a non-neurotoxic mutant of amyloid beta peptide (25-35), where Asn27 is replaced by Ala, in lithium dodecyl sulfate micelles was also determined. The structure is similar to that of the wild type amyloid beta peptide (25-35) in the C-terminal region, but the N-terminal flexible region is different. The structural comparison of amyloid beta peptide (25-35), its non-neurotoxic mutant and substance P gives a structural basis to understand the mechanism of neurotoxicity caused by amyloid beta peptide.

Amino Acid Sequence↗
PubMed1996

Structures of the extracellular domain of the type I tumor necrosis factor receptor.

BACKGROUND: Tumor necrosis factor (TNF) is a powerful cytokine that is involved in immune and pro-inflammatory responses. Two TNF receptors that belong to the cysteine-rich low affinity nerve growth factor receptor family (TNF-R1 and TNF-R2) are the sole mediators of TNF signalling. Signalling is thought to occur when a trimer of TNF binds to the extracellular domains of two or three receptor molecules, which permits aggregation and activation of the cytoplasmic domains. The complex is then internalized within an endocytic vesicle, whereupon it dissociates at low pH. Structure of the soluble extracellular domain of the receptor (sTNF-R1) both in the unliganded and TNF-bound state have previously been determined. In both instances, the fourth subdomain of the receptor was found to be partly disordered. In the unliganded state at pH 7.5, the extracellular domain forms two distinct types of dimer, parallel and antiparallel; the antiparallel dimer occludes the TNF-binding. RESULTS: We have determined the structure of sTNF-R1 in two crystal forms in high salt at pH 3.7. The orthorhombic crystals diffract to 1.85 ånd the entire polypeptide is well ordered. In contrast, the C-terminal 32 residues are disordered in the hexagonal crystals. In the orthorhombic form, these residues exhibit a topology and disulphide connectivity that differs from the other three cysteine-rich domains in the molecule. In both forms, the interface is considerably more extensive than that used in complex formation with LTalpha. This 'low pH' dimer is different from both of the dimers observed in crystals grown at pH 7.5. CONCLUSIONS: The occurrence of the antiparallel dimers in both low pH crystal forms suggest that they are not an artefact of crystal packing. Such dimers may form in the low pH environment of the endosome. Because the dimer contact surface occludes the TNF-binding site, formation of this dimer would dissociate the TNF-receptor complex within the endosome. Three of the four cysteine-rich domains of TNF-R1 are constructed from two distinct structural modules, termed A1 and B2. The fourth subdomain comprises an A1 module followed by an unusual C2 module. Although the orientation of these modules with respect to each other is sensitive to crystal packing, ligand binding, pH and ionic strength, the modules are structurally well conserved between and within the known sTNF-R1 structures.

Antigens, CD↗
PubMed1996

Relation of oxidative stress and glutathione synthesis to CD95(Fas/APO-1)-mediated apoptosis of adult T cell leukemia cells.

An IL-2 dependent adult T cell leukemia cell line (SO4) has been established that is sensitive to CD95-mediated apoptosis as well as a subline (R-SO4) that is resistant. Incubating SO4 cells with anti-CD95 IgM mAb caused concentration-dependent cell death. On the contrary, R-SO4 cells did not die even at 1000 ng/ml of anti-CD95 IgM mAb. The levels of CD95 expression on R-SO4 cells were one-third of those on SO4 cells. However a blocking Ab, anti-CD95 IgG mAb, did not induce complete resistance of SO4 cells to anti-CD95 IgM mAb as R-SO4 cells. As CD95 and TNF receptor are similar, and TNF/TNF receptor binding induces oxygen radicals, the involvement of oxidant and antioxidant systems in CD95-mediated apoptosis has been examined. The addition of anti-CD95 IgM mAb resulted in formation of intracellular oxygen radical species in the SO4 cells as measured using 2',5',-dichlorofluorescein as substrate. The oxygen radical production induced DNA damage as determined by formation of 8-hydroxydeoxyguanosine. No increase in the formation of oxygen radicals was observed in R-SO4 cells. Concentrations of the intracellular antioxidant, glutathione, and the key enzyme for its synthesis, gamma-glutamylcysteine synthetase, were 150% increased in R-SO4 cells in comparison with that of SO4 cells. Moreover, glutathione ester decreased the formation of 8-hydroxydeoxyguanosine. These results suggested that apoptosis mediated by CD95 in ATL cells is related to the production of oxygen radical species and cellular antioxidant systems, especially, glutathione synthesis.

Apoptosis↗
PubMed1996

A newly developed procedure for monitoring of extracellular proteins using a push-pull microdialysis.

A microdialysis technique combined with a push-pull pump was applied for monitoring protein dynamics in the liver. A newly developed probe has a 0.34 x 10-mm (membrane thickness, 0.05 mm) dialysis membrane of polysulfon and can allow the passage of molecules of up to approximately a few hundred kilodaltons in molecular weight. The probe was inserted in the liver of a rat under pentobarbital anesthesia. Perfusion medium (phosphate-buffered saline) was pumped through the microdialysis probe and collected every 15 min. Effect of ischemic treatment of the protein constitution and the activity of lactate dehydrogenase (LDH) in dialysate were determined to confirm the accuracy of the present technique. The protein constitute in preischemic dialysate differed from those obtained in the serum and hepatic homogenate, showing that the dialysate reflected extracellular protein. LDH activity was high immediately after insertion of the probe, decreased constantly, and then reached a plateau of a relatively low level. When transit ischemic treatment (for 15 min) was performed by ligation of both hepatic artery and portal vein, LDH activity increased significantly, which continued for over 5 h. The concentration of albumin in the dialysate increased immediately after the ischemia. Such changes in LDH activity and albumin concentration reflected ischemic change, and the newly developed technique may be useful for the monitoring of extracellular dynamics of proteins with molecular weight less than 200 kDa.

Albumins↗
PubMed1996

Real time monitoring of biphasic glutamate release using dialysis electrode in rat acute brain ischemia.

Glutamate has been proposed to play a critical role in acute ischemic pathophysiology in the brain. In this study, glutamate was monitored by the dialysis electrode technique, in which glutamate is oxidized by glutamate oxidase producing hydrogen peroxide which is then amperometrically detected on a platinum electrode set at +650 mV vs Ag/AgCl. A dialysis electrode, which consists of a microdialysis probe with a built-in platinum electrode, provides a continuous glutamate oxidase perfusion inside of the probe. Perfusion with this solution allows real-time monitoring of glutamate dynamics in the extracellular space during ischemia. This study was designed to collect detailed information on rapid changes in the extracellular glutamate concentration of the rat striatum and demonstrated two distinct phases of glutamate release during early severe brain ischemia.

Amino Acid Oxidoreductases↗
PubMed1996

Major identity determinants in the "augmented D helix" of tRNA(Glu) from Escherichia coli.

By a kinetic analysis of 59 variant transcripts of Escherichia coli tRNA(Glu) with glutamyl-tRNA synthetase (GluRS), the U11.A24 base-pair, the U13.G22..A46 base-triple, and the lack of residue 47 (delta47) were found to serve as major determinants for tRNA(Glu) identity. This is the first system for which major identity determinants are reported to be clustered in the "augmented D helix", consisting of the D stem with some neighboring residues and the variable loop. Other identity determinants are U34, U35, C36 and A37 in the anticodon loop, and G1.C72 and U2.A71 in the acceptor stem. Phosphate-group protection by GluRS from ethylnitrosourea was observed most strongly for the minor groove side of D-stem helix, indicating that GluRs tightly binds to the D stem for recognition, on the minor groove side, of the potent identity-determinant groups of the U11.A24 and U13.G22 base-pairs. A46 is not involved in direct recognition by GluRS; the U13.G22..A46 base-triple is required probably for formation of the structural features that are recognized by GluRS. In this context, the essential role of characteristic delta47 in tRNA(Glu) identity may be to maintain the U13.G22..A46 base-triple.

Anticodon↗
PubMed1996

High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein.

Ricin is a cytotoxic plant protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Recent studies showed that a four-nucleotide loop, GAGA, can function as a minimum substrate for ricin (the first adenosine corresponds to the site of depurination). We previously clarified the solution structure of this loop by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678]. To elucidate further details of the structural basis for recognition of its substrate by ricin, we studied the properties of a synthetic dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an RNA hairpin structure with a GdAGA loop and in which the site of depurination is changed from adenosine to 2'-deoxyadenosine. The N-glycosidase activity against the GdAGA loop of the A-chain of ricin was 26 times higher than that against the GAGA loop. NMR studies indicated that the overall structure of the GdAGA loop was similar to that of the GAGA loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it appears that the 2'-hydroxyl group of adenosine at the depurination site (6A) does not participate in the recognition by ricin of the substrate. Since the 2'-hydroxyl group can potentially destabilize the developing positive charge of the putative transition state intermediate, an oxycarbonium ion, the electronic effect may explain, at least in part, the faster rate of depurination of the GdAGA loop compared to that of GAGA loop. We also show that the amino group of 7G is essential for substrate recognition the ricin A-chain.

Base Sequence↗
PubMed1996

Deletion mapping of chromosome 2 in human lung carcinoma.

Sixty-three non-small cell lung carcinomas (NSCLCs) and 20 small cell lung carcinomas (SCLCs) were examined for loss of heterozygosity (LOH) on chromosome 2. Fifteen highly polymorphic dinucleotide markers spanning both the short and long arms of chromosome 2 were selected for a polymerase chain reaction (PCR)-based fine mapping. They included a DNA marker localized in the homozygously deleted region at 2q33, which we previously identified in an SCLC cell line. LOH on chromosome arm 2q was detected in 23/63 (37%) of NSCLC and 6/20 (30%) of SCLC, while LOH on 2p was observed in 14/56 (25%) and 4/17 (24%), respectively. There were two commonly deleted regions mapped to 2q32-q37 and 2p16-pter, and the homozygously deleted region at 2q33 was in the commonly deleted region on 2q. In NSCLC, the incidence of LOH on 2p and 2q was significantly higher in brain metastases than in primary tumors (P = 0.005 and 0.001, respectively). In addition, LOH on chromosome arm 2q occurred more frequently in moderately/poorly differentiated tumors than in well-differentiated tumors (P = 0.046). These results suggested that inactivation of tumor suppressor genes on chromosome 2 is involved in the phenotypic alterations of NSCLC cells into more aggressive ones.

Brain Neoplasms↗
PubMed1996

Comparative allelotype of early and advanced stage non-small cell lung carcinomas.

To identify chromosomal loci of tumor suppressor genes involved in the genesis and progression of non-small cell lung carcinoma (NSCLC), comparative allelotype analysis was performed in 23 stage I primary lung tumors and in 22 metastatic lung tumors to the brain. In total, 84 loci on all 22 autosomal chromosomes were examined for loss of heterozygosity (LOH) by restriction fragment length polymorphism (RFLP) analysis with 40 polymorphic DNA probes and polymerase chain reaction (PCR)-LOH analysis of 44 polymorphic loci. LOH on chromosome arms 3p, 13q, and 17p was detected frequently (> 60%) in both stage I primary lung tumors and brain metastases, whereas the incidence of LOH on chromosome arms 2q, 5q, 9p, 12q, 18q, and 22q was more than 60% only in brain metastases. In particular, the incidence of LOH on chromosome arms 2q, 9p, 18q, and 22q in brain metastases was significantly higher than that in stage I primary lung tumors (P < 0.05). These results indicate that tumor suppressor genes on chromosome arms 3p, 13q, and 17p are involved in the genesis of NSCLC, whereas those on several chromosome arms, especially on 2q, 9p, 18q and 22q, play an important role in the progression of NSCLC.

Brain Neoplasms↗
PubMed1996

A noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for salmon calcitonin: determination of the bioavailability of subcutaneous salmon calcitonin and its correlation with the hypocalcemic activity in rats.

A noncompetitive enzyme immunoassay method (hetero-two-site enzyme immunoassay) for salmon calcitonin (SCT) and its usability for the pharmacokinetic study are described. The method in brief proceeds as follows: centrifugal filtration through a polysaccharide membrane to remove plasma proteins, biotinylation, trapping onto an anti-SCT IgG-coated polystyrene ball, acid elution, coupling with affinity-purified anti-SCT Fab'-peroxidase conjugate, final trapping onto streptavidin-coated polystyrene balls, and measurement of peroxidase activity bound to the balls by fluorometry. The practical detection limit of SCT was 0.1 pg (30 amol)/assay and 2 pg/ml as the assay sample's concentration, which was at least fivefold lower than those previously reported by competitive radioimmunoassays. The application of this method has enabled us to 1) directly estimate the bioavailability of SCT dosed subcutaneously at the therapeutic levels (1.2 and 4.7 micrograms/kg) for its antiosteoporotic effect as compared to an intravenous dose (1.2 micrograms/kg) and 2) search for the relationship between blood level and the hypocalcemic activity of SCT. The pharmacokinetic parameters of subcutaneous SCT (1.2 and 4.7 micrograms/kg) thus estimated were as follows: the area under the blood concentration-time curve (AUC) = 89 and 550 pg.hr/ml, and mean residence time (MRT) = 44 and 65 minutes, respectively, when the AUC for an intravenous SCT (1.2 micrograms/kg) = 160 pg.hr/ml and the MRT = 10 minutes.

Animals↗
PubMed1996

The ultrastructure of parapapillary chorioretinal atrophy in eyes with secondary angle closure glaucoma.

BACKGROUND: The present study was performed to investigate the ultrastructure of deep retinal layers and choroid corresponding to the parapapillary chorioretinal atrophy in eyes with secondary angle-closure glaucoma. METHODS: The glaucomatous eyes included two eyes enucleated due to iris ring melanoma with high intraocular pressure and one eye with neovascular glaucoma enucleated due to ocular pain. The control eyes included one eye enucleated due to choroidal malignant melanoma with normal intraocular pressure and one eye enucleated during surgery for supramandibular carcinoma. These eyes were studied with light and electron microscopy. RESULTS: In the region of parapapillary chorioretinal atrophy of glaucomatous eyes, the retinal pigment epithelial cells showed degenerative changes, such as loss of basal infoldings and microvilli, degenerated mitochondria, vacuolar degeneration and irregular distribution of melanin granules. The photoreceptors were decreased in number in this area of glaucomatous eyes. The lumen of the choriocapillary vessels adjacent to the optic nerve was collapsed. CONCLUSION: These results elucidate the fine structures of deep retina and choroid in the region of parapapillary chorioretinal atrophy of glaucomatous eyes, and suggest that the reduced choroidal perfusion might be the pathogenetic mechanism of glaucomatous parapapillary chorioretinal atrophy.

Adult↗
PubMed1996

Detection of amplified genomic sequences in human small-cell lung carcinoma cells by arbitrarily primed-PCR genomic fingerprinting.

The arbitrarily primed-PCR (AP-PCR) genomic fingerprinting method was applied to evaluate its effectiveness in detecting and characterizing amplified DNA fragments in two small-cell lung carcinoma (SCLC) cell lines, NCI-H69 and NCI-H82. Of the 2428 DNA fragments detected by AP-PCR using 62 arbitrary primers, 2 (0.08%) DNA fragments were amplified in NCI-H69 and 6 (0.25%) DNA fragments were amplified in NCI-H82. Based on these results, we estimate the total size of the amplified genomic regions in these cell lines to be 3000 megabase pairs (Mb) x 0.0008 = 2.4 Mb in NCI-H69 and 3000 Mb x 0.0025 = 7.5 Mb in NCI-H82. The 2 amplified fragments in NCI-H69 were mapped to chromosome 2, and all 6 amplified fragments in NCI-H82 were mapped to chromosome 8. This strongly suggests that restricted chromosomal regions are specifically amplified in these SCLC cell lines. Since the N-myc gene at 2p24 is amplified in NCI-H69 and the c-myc gene at 8q24 is amplified in NCI-H82, it is possible that these DNA fragments are co-amplified with N-myc or c-myc in these cell lines. However, since the 2 amplified fragments in NCI-H69 were not amplified in 42 other human cancer cell lines including 11 cell lines carrying amplified N-myc genes, it is also possible that there are amplified regions on chromosome 2 other than the N-myc locus at 2p24 in NCI-H69. In contrast, all 6 amplified fragments in NCI-H82 were amplified in several other human cancer cell lines carrying amplified c-myc genes. This result further indicates that these fragments were derived from an amplification unit that includes the c-myc gene. Our results show the ability of the AP-PCR method to analyze the fraction of the genome with amplification in human cancer cells.

Base Sequence↗
PubMed1996

Excellent survival in a subgroup of patients with intrapulmonary metastasis of lung cancer.

BACKGROUND: Recently, intrapulmonary metastases in non-small cell lung cancer have been considered to have less influence on prognosis than extrapulmonary metastases. We report a subgroup found among patients with intrapulmonary metastases showing a good prognosis. METHOD: A retrospective study was performed on 236 consecutive patients with non-small cell lung cancer who underwent surgical resection of their tumors. Intrapulmonary metastases were found histopathologically in 50 of them, and their clinicopathologic features were investigated. RESULTS: Analysis of postsurgical results revealed a subgroup of patients showing excellent prognosis (n = 15). They had well-differentiated adenocarcinomas with bronchioloalveolar spread and pT1-2 N0, without vascular or lymphangitic invasion. Their actuarial 5-year survival rate was 100%, with a mean survival interval to date of 28 months. However, none of the other 35 patients survived for 5 years, with a mean survival interval to date of 11 months. CONCLUSIONS: We have clarified that patients with histopathologically diagnosed intrapulmonary metastases from non-small cell lung cancer do not constitute a homogeneous group. Pulmonary metastases with good prognosis, which are considered to be hematogenous metastases, may be benign lesions such as adenomatous or atypical adenomatous hyperplasias mimicking malignant tumors.

Adenocarcinoma↗
PubMed1996

Activity of Fgr protein-tyrosine kinase is reduced in neutrophils of patients with myelodysplastic syndromes and chronic myelogenous leukemia.

The Fgr protein-tyrosine kinase, p55(c-fgr), is specifically expressed and functions in cells of myelomonocytic lineages. We examined levels of expression and enzymatic activity of p55(c-fgr) peripheral blood neutrophils of patients with myelodysplastic syndromes (MDS) and chronic myelogenous leukemia (CML) by comparison with those of normal individuals. While neutrophils of eight normal subjects gave uniform results, the specific enzymatic activity of p55(c-fgr), a ratio of the total kinase activity versus the protein level was reduced in seven out of eight patients with MDS and all of five patients with CML. The specific kinase activity of p55(c-fgr) correlated significantly with the activity of neutrophil alkaline phosphatase (NAP) which has been considered to be a marker of neutrophil maturity (r=0.568, P<0.01). The reduced activity of this tyrosine kinase was considered to be a biological parameter for immaturity and to reflect dysfunction of neutrophils of patients with MDS and with CML.

Humans↗
PubMed1996

Diagnostic criteria for hypocellular acute leukemia: a clinical entity distinct from overt acute leukemia and myelodysplastic syndrome.

In order to establish diagnostic criteria for hypocellular acute leukemia (HL), we have reviewed 32 cases selected on the basis of hypothetical 40% or less cellularity, by focusing on morphology, immunophenotype, karyotype and response to low dose Ara-C (LDAC) regimen and compared them with 40 cases of myelodysplastic syndrome (MDS) and 66 cases of overt acute myeloid leukemia (AML). The onset age ranged from 44 to 75 years (median 67 years). Bone marrow (BM) cellularity ranged from 12.4 to 39.8% (mean 29.8%) in HL, being significantly lower than in MDS (mean 80.7%) or AML (mean 86.4%) (P < 0.001). All reviewed cases characteristically showed smoldering clinical course, bi- or pancytopenia with rare leukemic blasts in the peripheral blood (PB), proliferation of type I leukemic blasts in the BM and markedly reduced background hematopoietic cells with some dysplastic changes in 12/32 cases (37.50/6). Blast percentage (blast %) in the BM ranged from 38.2 to 93.7% (mean 57.3%) in all nucleated cells (ANC). Although a considerable number of cases had blasts with negative or very low myeloperoxidase activity, immunophenotyping revealed that the leukemic blasts in HL had only myeloid markers. Karyotyping revealed non-random chromosome abnormalities in 30% of cases analyzed, which were considerably different from those seen in MDS. With LDAC regimen, a significantly higher CR rate (13/20 cases: 65.0%) was gained in HL than in RAEB/RAEB-t (0%) and overt AML in the elderly cases (27.3%) (P < 0.05). In CR, most cases showed recovery to normocellular BM with an apparent normalization of PB parameters. However, 12 CR cases relapsed 4-12 months later; most of which again showed hypocellular BM. These results indicate that HL is a distinct subtype of AML characterized by slow but distinct proliferation of immature myeloid blasts and by unique hematological features distinct from MDS or overt AML in the elderly. We propose the following diagnostic criteria: (1) pancytopenia with rare appearance of blasts in PB; (2) less than 40% BM hypocellularity; (3) more than 30% blasts in BM-ANC; and (4) myeloid phenotypes of leukemic blasts by MPO staining and/or immunophenotyping.

Acute Disease↗