Search PubMed⌕ Search

Biomedical subjects

S Müller

Publications and source records attributed to S Müller.

At least 217 records · Page 12Linked to original sources
PubMed1998

Stimulation of HIV-1-neutralizing antibodies in simian HIV-IIIB-infected macaques.

Previously we have discovered a public idiotope, designated 1F7, that is expressed on antibodies against HIV type 1 (HIV-1) in human and nonhuman primates. To test the potential of mouse monoclonal antibody (mAb) 1F7 as a therapeutic anti-clonotypic antibody in HIV-1-infected patients, we used the simian HIV-IIIB macaque infection model, which mimics several immunological and pathological characteristics of HIV-1 infection in humans. Four healthy simian HIV-infected rhesus monkeys (Macaca mulatta) expressing the 1F7 marker on anti-gp120 antibodies were selected for this study. Three monkeys of this group were immunized several times with the murine mAb 1F7 i.v., and one monkey received as control an isotype-matched antibody, TEPC183. No serious side effect or allergic reaction was encountered. Blood collected before and during the immunization and over several months afterward were analyzed for neutralizing antibodies. Significant increases in breadth and potency of HIV-1-neutralizing antibody titers to one or more virus strains were detected in all three of the 1F7-immunized monkeys, but not in the control monkey immunized with TEPC183. These results show that an antibody, recognizing a public idiotope associated with anti-HIV-1 antibodies can function in chronically infected primates as an anti-clonotypic immunogen to boost antibodies that neutralize homologous and heterologous virus strains. This study represents a first step toward the preclinical evaluation of 1F7 as a therapeutic AIDS vaccine.

AIDS Vaccines↗
PubMed1998

Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus.

The PML protein, identified first as part of the oncogenic PML-RARalpha chimera in acute promyelocytic leukemia (APL), concentrates within discrete subnuclear structures, corresponding to some types of nuclear bodies. These structures are disrupted in APL cells, and retinoic acid (RA) can trigger their reorganization, correlating with its therapeutic effect in this type of leukemia. Recently, arsenic trioxide (As2O3) was identified as a potent antileukemic agent which, similarly to RA, induces complete remissions in APL patients. Here we show that, in APL cells, As2O3 triggers rapid degradation of PML-RARalpha and provokes the restoration of intact nuclear bodies. In non-APL cells, the ubiquitin-like protein SUMO-1 is covalently attached to a subset of wild-type PML in a reversible and phosphorylation-dependent manner. The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO-1-polymodified forms of PML are compartmentalized exclusively in the PML nuclear bodies. As2O3 administration strikingly increases the pool of SUMO-1-PML conjugates that, subsequently, accumulate in enlarged nuclear bodies. In contrast to PML-RARalpha, the overall amount of PML seems to remain unaltered up to 36 h following As2O3 treatment. These findings indicate that the conjugation of PML with SUMO-1 modulates its intracellular localization and suggest that post-translational modification by SUMO-1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures. They provide additional evidence that the role of 'ubiquitin-like' post-translational modification is not limited to a degradation signal.

Arsenic Trioxide↗
PubMed1998

Rat beta-adrenergic receptor kinases 1 and 2 in mouse neuroblastoma X rat glioma NG 108-15 hybrid cells.

Beta-adrenergic receptor kinase (betaARK, EC 2.7.1.-) has been implicated in the phosphorylation of G protein-coupled receptors, including opioid receptors. Since delta-opioid receptors of mouse neuroblastoma x rat glioma hybrid cells (NG 108-15) desensitize upon activation, this investigation was designed to find out whether NG 108-15 cells contain betaARK activity. Using the reverse transcription polymerase chain reaction technique, we identified two mRNAs, one coding for rat betaARK1 and the other for rat betaARK2. No hint was found for the presence of mouse betaARK. Examining the cytosolic betaARK activity in these hybrid cells using rhodopsin as substrate, we found a strict functional dependence on the presence of exogenous G protein subunit Gbetagamma. This relationship reflects a characteristic for betaARK1 and 2 out of the known G protein-coupled receptor kinases. Finally, highly purified recombinant betaARK1 proved active to phosphorylate enriched delta-opioid receptor preparations in an opioid agonist-dependent manner. The results reported here provide the basis to study more closely the molecular function of G protein-coupled receptor kinases in a cell line (NG 108-15) most frequently used to investigate acute and chronic opioid actions.

Animals↗
PubMed1998

Investigation of two yeast genes encoding putative isoenzymes of phosphoglycerate mutase.

Our previous data indicated that GPM1 encodes the only functional phosphoglycerate mutase in yeast. However, in the course of the yeast genome sequencing project, two homologous sequences, designated GPM2 and GPM3, were detected. They have been further investigated in this work. Key residues in the deduced amino acid sequence, shown to be involved in catalysis for Gpm1 (i.e. His8, Arg59, His181) are conserved in both enzymes. Overexpression of the genes under control of their own promoters in a gpm1 deletion mutant did not complement for any of the phenotypes. This could in part be attributed to a lack of expression due to their weak promoters. Higher level expression under the control of the yeast PFK2 promoter partially complemented the gpm1 defects, without restoring detectable enzymatic activity. Nevertheless, deletion of either GPM2 or GPM3, or the two deletions in concert, did not produce any obvious lesions for growth on a variety of different carbon sources, nor did they change the levels of key intermediary metabolites. We conclude that both genes evolved from duplication events and that they probably constitute non-functional homologues in yeast.

Base Sequence↗
PubMed1998

Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica.

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4.5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 alpha and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters. Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

Base Sequence↗
PubMed1998

Monocyte chemoattractant protein 1 (MCP-1) gene expression in dilated cardiomyopathy.

The cytotoxic action of leukocytes may be a most probable cause of cardiac myocyte damage seen in chronic myocarditis and dilated cardiomyopathy (DCM). The migration and tissue infiltration of leukocytes is regulated by chemotactic cytokines. Recently, the presence of monocyte chemoattractant protein 1 (MCP-1) messenger RNA has been demonstrated in endomyocardial biopsy tissue obtained from patients with DCM. This chemokine could contribute to enhanced leukocyte recruitment and activation resulting in chronic damage of cardiomyocytes. Accordingly, we sought to determine whether the severity of left ventricular dysfunction in DCM is associated with quantitative alterations of MCP-1 messenger RNA and MCP-1 protein in endomyocardial biopsy tissue. A group of DCM patients with low to moderate impairment of left ventricular function (ejection fraction 45.3+/-2.3%, n=7) was compared to patients with severe left ventricular dysfunction (ejection fraction 25.5+/-3.1%, n=7). MCP-1 messenger RNA expression was determined by quantitative polymerase chain reaction. MCP-1 protein and the presence of infiltrating inflammatory cells were detected by immunohistochemistry. DCM patients with severe left ventricular dysfunction showed a 2.35 fold higher MCP-1 messenger RNA expression when compared to DCM patients with less severe dysfunction (P=0.0229). Positive immunohistochemical staining for MCP-1 was found in all seven patients with severe left ventricular dysfunction and was particularly distinct within the cardiac interstitum. In five of seven patients with less severe systolic dysfunction, MCP-1 protein was found, but was less pronounced and distributed in patchy interstitial areas, close to intramyocardial vessels. Furthermore, there was a consistent trend toward a higher infiltration of inflammatory cells in DCM patients with lower ejection fraction. In conclusion, MCP-1 is dynamically regulated in DCM related deterioration of left ventricular function. This mechanism might contribute to myocyte damage via infiltrated and activated monocytes.

Adult↗
PubMed1998

[Improved structure identification with tissue Doppler echocardiography].

Tissue Doppler echocardiography (TDE) has been shown to be of particular value in patients with impaired myocardial function. Recently, the technique was successfully employed to localize the ventricular insertion of accessory atrioventricular pathways. The identification of abnormal cardiac structures is coming up now as a new field of clinical interest. The purpose of this study was to differentiate anomalous cardiac and aortic from native structures by physical properties of tissue motion using transesophageal TDE. Characteristic motion patterns of anomalous structures have not been described in detail and tissue Doppler findings have not been associated with clinical features up to now. Forty consecutive patients were included after anomalous cardiac or vascular structures had been detected by conventional transesophageal echocardiography (TEE). A control group consisted of 20 subjects. Rapidity of diagnosis in anomalous structures was divided into 3 categories, and TDE signals were related to particular pathology by a blinded, 2nd observer. Three different motion patterns could be defined: incoherent motion due to free oscillation of an anomalous structure which is independent of the surrounding tissue (Figure 1b); coherent motion with a phase difference meaning that motion depends on the motion of the surrounding tissue but is out of phase (Figure 2); concordant motion showing no difference in direction, velocity, or phase of motion compared with the surrounding tissue. Incoherent motion was present in endocarditic vegetations, 4th degree aortic plaques, Chiari network, valvular prolapse, intracavitary tumors, and freely oscillating thrombi as well as in normal valve leaflets and papillary muscles. Especially if endocarditic vegetations are present its incoherent motion facilitates to recognize these small structures. The colorcode of this motion pattern demarcates the vegetation reliably from the surrounding tissue (Figure 1b). Within 15 seconds vegetations could be detected in 9 (82%) vs 2 (18%) patients employing only conventional imaging. Using conventional echocardiographic approaches detection of vegetations is frequently hindered by their small size and minor echo intensity (Figure 1a). In contrast, size and echo intensity do not affect the tissue Doppler signal. Normal papillary muscles and distal portions of the mitral and tricuspid valves were demonstrated to regularly meet the criterion of incoherent tissue motion in the control group. In part, this was also observed with respect to the aortic and pulmonary valves. In valvular tissue incoherent motion was caused by passive floating, whereas papillary muscles show an active inverse motion for short time intervals. Nevertheless, physiologic incoherent motion did not lead to any false differential diagnosis. The phase difference of coherent motion results from damped oscillation. This phenomenon was visualized by tissue Doppler M-mode in 5 thrombi of the left atrial appendage (LAA) (100%) and in 1 ventricular thrombus (50% of all clots). Concordant motion was shown in 3rd degree aortic plaques and postrheumatic and calcified vegetations. These structures were found to be completely embedded or closely attached, so that their passive motion corresponded to the motion of the surrounding regular tissue. Detection and assessment of anomalous structures are based on their motion patterns which can be synchronous or asynchronous in comparison with the surrounding tissue. Another goal of this investigation was to test if the sensitivity of TEE to spontaneous echo contrast can be improved using TDE. In 21 patients presenting with left atrial dilation (left atrial diameter > 44 mm) due to mitral stenosis (n = 8), mitral regurge (n = 5), arterial hypertension (n = 5) and multiple valvular disease (n = 3) fundamental multiplane TEE and transesophageal TDE were performed with standardized gain setting. The control group consisted of 20 randomized individuals with normal left

Aorta, Thoracic↗
PubMed1998

Mutations in the gene for the common gamma chain (gammac) in X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (XSCID) constitutes a disorder of the immune system caused by mutations in the gene encoding the common gamma chain (gammac), a subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors, which are necessary for lymphocyte development and function. In this study the IL2RG gene of 31 patients with severe combined immunodeficiency (SCID) was examined by nonradioactive single-strand conformation polymorphism and sequence analysis. Among the 11 patients with XSCID, ten different mutations were identified in the IL2RG gene, including eight novel mutations. Ninety percent of the mothers of the XSCID patients are carriers of the mutated allele. One patient showed low numbers of B-cells, a striking deviation from the classical B-cell-positive and T-cell-negative phenotype.

B-Lymphocytes↗
PubMed1998

Trk A, B, and C are commonly expressed in human astrocytes and astrocytic gliomas but not by human oligodendrocytes and oligodendroglioma.

Neurotrophins regulate the proliferation and differentiation of neurons in the central nervous system via a family of specialized receptors, including TrkA, TrkB, and TrkC. As little is known about their expression or potential role in human glial tissues and glial tumors, we undertook an immunohistochemical analysis of human glia, glioma tissues and cell cultures of glial tumors to characterize the expression of Trk family members (full-length TrkA, TrkB, the truncated form of TrkB, and TrkC). In normal human brain Trk A, B, and C immunoreactivity was found in neurons and some weak staining was also seen astrocytes. No Trk expression was seen on oligodendrocytes. Strong reactivity was seen in reactive astrocytes in a glial scar. In a total of 34 glioma tissue specimens, which included 16 astrocytic tumors (4 low-grade astrocytomas and 12 glioblastomas multiforme) and 15 oligodendrogliomas (8 low-grade and 7 anaplastic) as well as 3 oligoastrocytomas (WHO grade II), TrkA, B, and C immunoreactivity was observed exclusively in specimens from astrocytic gliomas (16/16), but not in any of the oligodendrocytic gliomas (0/15). In the oligoastrocytomas, staining was restricted to the astrocytic component. In the astrocytoma and oligodendroglioma specimens, Trk A, B, and C immunoreactivity was also seen in the surrounding reactive astrocytes. Trk expression was independent of age, sex or histological grade of the investigated tumors. In six primary cell cultures, one derived from human astrocytes and five established from malignant astrocytomas, only TrkA immunoreactivity could be detected, while TrkB (both full-length and truncated isoforms) and TrkC were absent. The TrkA expression in primary cell cultures decreased with continuous cell passaging, and no Trk could be detected in established cell lines derived from glioblastoma. In conclusion, our data suggest that in human glial tissues Trk A, B, and C may be expressed in a lineage-restricted manner, thereby distinguishing between astrocytes and oligodendrocytes in a marker-like fashion. Trk expression, like GFAP expression appears to be increased in activated (reactive)/neoplastic astrocytes.

Adult↗
PubMed1998

Enhanced photodynamic effects using fractionated laser light.

Photodynamic eradication of tumour cells depends on the presence of a photosensitizer and light delivery to the cells. The present study investigated the influence of fractionated light (on-off mode) on cell killing as documented by a colony-forming assay. Photosensitizers were m-THPC (ethanol soluble, Foscan) and m-THPC-MD (water soluble, both from Scotia Pharmaceuticals, Guildford, UK). Fractionated laser light at a wavelength of 652 nm with a light duration of 0.05 s was more effective than continuous illumination at the same power density for both photosensitizers. We propose that fractionated laser light is more toxic due to short phases of recovery during the dark intervals, probably resulting in more singlet oxygen under these conditions. By use of Foscan, for example, and fractionated laser light, a similar effect is expected for the treatment of solid tumours. In this case we expect improvements in photodynamic therapy (PDT) for patients by lowering the concentrations of photosensitizer and/or by reducing the applied light dose.

Antineoplastic Agents↗
PubMed1998

Primary culture of porcine PGCs requires LIF and porcine membrane-bound stem cell factor.

We studied the effect of murine leukaemia inhibitory factor (LIF), human basic fibroblast growth factor (bFGF) and porcine stem cell factor (SCF) on the survival and/or proliferation of porcine primordial germ cells (PGCs) obtained from 27-day-old embryos in vitro. PGCs were cultured in embryonic stem cell (ESC) medium supplemented with or without either LIF (1000 IU/ml) alone or LIF together with bFGF (10 ng/ml). They were seeded on mitotically inactivated feeder cells, either STO or transfected STO cells (STO#8), expressing the membrane-bound form of porcine SCF. PGCs were identified by their alkaline phosphatase (AP) activity and counted after 1, 3 and 5 days in culture. After 1 day of culture, PGCs cultured on STO#8 cells showed significantly higher survival than PGCs cultured on STO cells (p < 0.05). The combined effect of SCF and LIF caused a significant increase in PGC number by day 3 of culture when PGCs were cultured on either STO cells (p < 0.01) or STO#8 (p < 0.001). When SCF and LIF were used together with bFGF no increase in the PGC number was observed. Our results suggest that the membrane-bound form of porcine SCF plays a pivotal role in the primary culture of porcine PGCs and that bFGF is not required in vitro.

Alkaline Phosphatase↗
PubMed1998

Stable production of human insulin-like growth factor 1 (IGF-1) in the milk of hemi- and homozygous transgenic rabbits over several generations.

One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 +/- 12 micrograms/ml (ranging from 223 +/- 61 to 484 +/- 39 micrograms/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 +/- 41 micrograms/ml (ranging from 360 +/- 15 to 678 +/- 80 micrograms/ml). Homozygous hulGF-1 females clearly showed a significantly increased production performance of the recombinant protein.

Animals↗
PubMed1998

Maintenance of locomotor abilities following Laufband (treadmill) therapy in para- and tetraplegic persons: follow-up studies.

Recent reports indicate that walking capabilities in spinal cord damaged persons significantly improve--as compared to conventional rehabilitation therapy--after intensive training of aided (Laufband) treadmill-stepping. In the present report, follow up investigations on two collectives of spinal cord injured (sci) persons are described who had undergone (Laufband) treadmill therapy either during a period of renewed rehabilitation months or years after spinal cord injury (35 chronic patients) or during their first postacute rehabilitation period (41 acute patients). Among the initially chronic patients, 20 from 25 still wheelchair-bound before the onset of (Laufband) treadmill therapy, ie not capable of raising from the wheelchair and walking without help by other persons, became independent walkers after therapy. Assessment of voluntary muscle activity in resting position before and after the period of therapy had shown only small increases in most patients, indicating the involvement of motor automatisms and better utilisation of remaining muscle function during walking. Follow-up assessments performed 6 months to 6 1/2 years after discharge from the hospital revealed that the walking capabilities achieved by (Laufband) treadmill therapy in the 35 initially chronic patients were maintained in 31 persons, in three they had further improved, in only one it was reduced. These results indicate that the improvements achieved under clinical conditions can be maintained in every day life under domestic surroundings. From 41 initially acute patients, 15 had further improved and none had reduced his walking capability 6 months to 6 years after discharge from the hospital.

Acute Disease↗
PubMed1998

Absence of association between a common mutation in the methylenetetrahydrofolate reductase gene and the risk of coronary artery disease.

BACKGROUND: Elevated total plasma homocysteine levels are associated with an increased risk of coronary artery disease. Plasma homocysteine levels are influenced by nutritional and hereditary factors. A point mutation (cytosin to thymidine substitution; C677-->T) in the gene encoding methylenetetrahydrofolate reductase (MTHFR), has been reported to render the enzyme thermolabile and has been associated with elevations in homocysteine levels in homozygous carriers (TT genotype). METHODS: To examine the hypothesis that the T allele (coding for the thermolabile defect of MTHFR) influences the risk of coronary artery disease, we genotyped 340 patients with coronary artery disease and 105 control subjects in whom coronary artery disease was excluded by coronary angiography. Furthermore, we studied the genotype frequency in 104 age- and sex-matched healthy persons as a control group without signs of atherosclerotic disease. RESULTS: Allele frequencies for C (wild-type allele) and T allele (mutant allele) were 0.68 and 0.32 respectively in the healthy control subjects, 0.66 and 0.34 respectively in patients with angiographically excluded coronary artery disease and 0.69 and 0.31 respectively in coronary artery disease patients (P = NS). The allele frequencies of the total study population were 0.68 and 0.32. CONCLUSION: Our data show that homozygosity for the C677-->T mutation in this European population is not associated with increased risk of coronary artery disease. This finding suggests that the C677-->T mutation of the MTHFR gene does not represent a marker for increased cardiovascular risk.

Adult↗
PubMed1998

Internalin B is essential for adhesion and mediates the invasion of Listeria monocytogenes into human endothelial cells.

Listeria monocytogenes causes rhombencephalitis in humans and animals and also affects the fetus in utero, causing disseminated sepsis. In both instances, the infection occurs by the crossing of endothelial cells lining a physiological barrier, the blood-brain barrier or the transplacental barrier. In this study, the ability of L. monocytogenes wild-type EGD to invade human umbilical vein endothelial cells (HUVECs) was evaluated using wild-type bacteria and isogenic Listeria mutants. Here, we show that invasion of HUVECs by L. monocytogenes is dependent on the expression of the internalin B gene product. This was demonstrated in several ways. First, L. monocytogenes strains lacking the inlB gene did not invade HUVECs. Secondly, avid invasion was obtained when a strain deleted for inlAB was complemented with a plasmid harbouring inlB only, whereas strains expressing inlA did not enter HUVECs. Thirdly, entry of wild-type EGD could be blocked effectively with antibodies to InlB. Fourthly, cell binding assays and flow cytometry with HUVECs showed binding of purified InlB, but not InlA, suggesting a tropism of InlB for this cell type. Finally, physical association of purified native InlB with the surface of non-invasive mutants dramatically increased their ability to invade HUVECs. In laser-scanning confocal microscopy, binding of InlB was observed as focal and localized patches on the cell surface of HUVECs. Qualitative examination of the entry process by scanning electron microscopy revealed that both wild-type EGD and a recombinant strain overexpressing only InlB enter HUVECs in a similar fashion. The entry process was polarized, involved single bacteria and occurred over the entire surface of endothelial cells.

Antibodies, Bacterial↗
PubMed1998

Tree-pollen allergy is efficiently treated by short-term immunotherapy (STI) with seven preseasonal injections of molecular standardized allergens.

The efficacy and tolerance of short-term immunotherapy (STI) by seven preseasonal injections of tree-pollen allergens (ALK7 Frühblühermischung) was investigated in a double-blind, placebo-controlled, multicenter study with 111 rhinoconjunctivitis patients. Nasal and bronchial symptoms simultaneously analyzed, and nasal symptoms as a single end point, but not the overall score of nasal, bronchial, and conjunctival symptoms, showed a significantly lower increase with STI during birch-pollen exposure (both P=0.033, n=105, Mann-Whitney U-test). However, a selective analysis with patients from centers with high recruitment figures (n> or =10 patients, n=29 STI, n=32 placebo) showed a significantly lower increase of nasal, bronchial, and overall symptom score (STI 11.0 vs placebo 18.0, P=0.001, U-test). STI had equidirected effects on conjunctival, nasal, and bronchial symptoms analyzed as multiple end points, although conjunctival symptoms were not significantly different as a single end point. The seasonal increase in drug use was reduced by 62% in the STI group compared with placebo (P=0.032, t-test). Specific IgG4 increased only after STI (P<0.001); IgE was not significantly different. Eosinophil cationic protein remained unchanged with STI, but significantly increased with placebo in the pollen season (P=0.003). STI was well tolerated. In conclusion, STI was shown to be efficacious and safe for the treatment of patients with tree-pollen rhinoconjunctivitis.

Adolescent↗