Biomedical subjects

I Azuma

Publications and source records attributed to I Azuma.

At least 19 recordsLinked to original sources

PubMed2001

Effect of Topically Applied Iganidipine Dihydrochloride, a Novel Ca(2+) Antagonist, on Optic Nerve Head Circulation in Rabbits.

Purpose: We studied the effect of topically applied iganidipine dihydrochloride, a novel water-soluble calcium channel blocker on the blood flow of optic nerve head (ONH), intraocular pressure, and blood pressure in rabbits.Methods: (1) 0.1% iganidipine (20 &mgr;l) was instilled into a normal eye. The change in blood flow in the ONH was measured using a hydrogen gas clearance flowmeter. (2) Iganidipine (0.0001%-0.1%) was instilled into a circulation-disordered eye before or after the intravitreal injection of endothelin-1, and change in the blood flow in the ONH was measured. (3) Changes in intraocular pressure and blood pressure after instillation of 0.1% iganidipine were measured. In all experiments, physiological saline was instilled in each contralateral eye as a control.Results: (1) Instillation of iganidipine significantly increased the blood flow in the ONH by 40% at 45 minutes after instillation. (2) Pre-instillation of 0.01 and 0.1% iganidipine almost completely inhibited the decrease of blood flow in the ONH in the circulation-disordered model. The decrease of blood flow in the ONH was corrected with post-instillation of 0.1% iganidipine. These effects were continuous. (3) Instillation of 0.1% iganidipine did not change either intraocular pressure or blood pressure.Conclusion: It was shown that instillation of iganidipine continuously increased and maintained the blood flow in the ONH in normal and circulation-disordered rabbit eye models.

Journal Article↗

PubMed2001

Cellular and humoral adjuvant activity of lectins isolated from Korean mistletoe (Viscum album colaratum).

The adjuvant effect of lectins (KML-C) isolated from Korean mistletoe (Viscum album coloratum) on induction of humoral and cellular immune responses against keyhole limpet hemocyanine (KLH) was examined. When mice were immunized subcutaneously (s.c.) with KLH (20 micrograms/mouse) admixed with or without 50 ng/mouse of KML-C (KLH + KML-C), mice immunized with KLH + KML-C showed significantly higher antibody titers against KLH than those immunized with KLH alone, showing the highest titer 5 weeks after immunization. Furthermore, boost immunization with KLH + KML-C at 2-week interval elicited much higher activity than single immunization to enhance antibody responses against KLH. The assay for determining isotypes of antibodies revealed that KML-C augmented KLH-specific antibody titers of IgG1, IgG2a and IgG2b. The culture supernatants obtained from the splenocytes of mice treated with KLH + KML-C also showed a higher level of both KLH-specific Th-1 (IL-2 and IFN-gamma) and Th-2 type cytokine (IL-4). In an in vitro analysis of T lymphocyte proliferation to KLH on week 4, the splenocytes of mice treated with KLH + KML-C showed a significantly higher proliferating activity than those treated with KLH alone. In addition, mice immunized twice with KLH + KML-C and followed by intrafootpad (i.f.) injection of KLH (50 micrograms/site) 14 weeks after the primary immunization induced a higher delayed-type hypersensitivity (DTH) reaction than mice treated with KLH alone. These results suggest that KML-C is a potent immunoadjuvant to enhance cellular and humoral immune responses.

Adjuvants, Immunologic↗

PubMed2001

Development of immunoadjuvants for immunotherapy of cancer.

Previously, we have reported that cell-wall skeleton (CWS) fraction was the major adjuvant-active principle of mycobacterial cells which were used in Freund's complete adjuvant (FCA). We have described the biochemical and immunological properties of CWS of mycobacteria and related bacteria, especially the CWS of Mycobacterium bovis BCG strain (BCG-CWS) in detail. The effectiveness of BCG-CWS for the cancer immunotherapy in patients was shown in several clinical trials. On the action mechanism of BCG-CWS on host immune cells, we have suggested that dendritic cells and macrophages express two sorts of receptors, Toll-like receptors, TLR-2 and TLR-4, and a putative binding receptor for BCG-CWS, whose signaling pathways lead to a sufficient antigen-presenting state in the activation of the innate immune system. We have also reported the usefulness of synthetic immunoadjuvants such as muramyldipeptide (MDP) derivatives, trehalose-dimycolates (TDM) and DNA fraction for the application for the cancer and infectious diseases in experimental systems and cancer patients.

Adjuvants, Immunologic↗

PubMed2001

Interferon gamma-producing ability in blood lymphocytes of patients with lung cancer through activation of the innate immune system by BCG cell wall skeleton.

An in vitro assay system was developed to assess the potency of the human innate immune system by measurement of IL-12, IL-18, IL-10 and IFNgamma in the supernatants of bacillus Calmette-Guerin cell wall skeleton (BCG-CWS)-stimulated blood samples. BCG-CWS is a ligand for Toll-like receptor (TLR) 2 and 4, and activates monocytes to macrophages (Mphi), and immature dendritic cells to mature antigen-presenting cells (APC). This system was found to allow the discrimination of immune suppressive states in patients with lung cancer from normal immune states in light of the cytokine profile. The following results were deduced from analyses of BCG-CWS-stimulated blood samples of lung cancer patients with reference to normal subjects. (1) The levels of production of IFNgamma and IL-10 by lymphocytes were decreased. (2) IL-12 p40 production by monocytes/Mphi was upregulated, while that of IL-10 was downregulated. (3) IL-18 was detected in all patients in a range similar to normal subjects. (4) Responses of lymphocytes to IL-2 and IL- 18 in terms of IFNgamma production were diminished. (5) The upregulated IL-12 levels were recovered to within the normal range in most patients after tumor resection. (6) Male patients showed more severe suppression of IL-12/IL-18-mediated IFNgamma production than female patients. Thus, the lesser IFNgamma production observed in patients' blood with high IL-12 p40 levels in response to BCG-CWS may reflect the production of p40 dimers or IL-23 instead of p70, or the presence of some unknown pathways to prohibit the interface between the innate and acquired immune systems. BCG-CWS-mediated Toll signaling may participate in IFNgamma induction for lymphocytes through Mphi/APC IL-12/I-18 modulation.

Adjuvants, Immunologic↗

PubMed2001

Two receptor theory in innate immune activation: studies on the receptors for bacillus Culmet Guillen-cell wall skeleton.

Activation of the innate immune system is a prerequisite for the maturation of dendritic cells (DC) and macrophages (Mphi) followed by clonal expansion of the lymphocytes, targeting cells expressing "non-self' antigens. Microbes usually have a component competent to activate DC/Mphi for antigen presentation. This component has been called adjuvant, but recently renamed "pathogen-associated molecular pattern" (PAMP) or modulin based on its molecular identification. Here, we propose the hypothesis that DC/Mphi express two sorts of receptors for PAMP, whose signaling pathways lead to a sufficient antigen (Ag)-presenting state. In bacterial infection, a Toll-like receptor (TLR) and an uptake receptor participate in DC maturation and Mphi activation. Likewise, with a number of viruses, two of the receptors, with short consensus repeats (SCR), immunoglobulin-like domains or chemokine receptor-like motifs etc. induce functional modulation of DC/Mphi. In immune therapy for cancer, primary activation of the innate system would be essential for tumor Ag-specific T cell augmentation. Cancer cells express tumor-associated Ag but barely co-express PAMP, which situation does not allow for the activation of innate immune responses. Supplementing tumor-associated Ag with PAMP may be an effective therapy for patients with cancer. Here, we discuss the possibility of an innate immune therapy for cancer with reference to bacillus Culmet Guillen cell-wall skeleton (BCG-CWS).

Adjuvants, Immunologic↗

PubMed2000

Novel mutations in the myocilin gene in Japanese glaucoma patients.

Myocilin is a gene responsible for juvenile onset primary open angle glaucoma (POAG) mapped as the GLC1A locus and, many mutations have been reported worldwide. Some mutations were found not only in patients with juvenile onset POAG, but also in patients with late onset POAG and in patients with normal tension glaucoma. To investigate the mutation prevalence in Japan, we performed a mutation analysis in 140 unrelated Japanese patients. We have identified the 10 sequence variants, of which four were highly probable for disease-causing mutations (Arg46ter, Arg158Gln, Ile360Asn, and Ala363Thr), and six polymorphisms (Gln19His, Arg76Lys, Asp208Glu, Val439Val, Arg470His, and Ala488Ala). Thus, myocilin mutations were found at the rate of 4/140 (2.9%) probands, similar to previous reports with other ethnic populations.

Adult↗

PubMed2000

Innate immune therapy for cancer. Screen for molecules capable of activating the innate immune system.

Tumor cells usually express antigens which are distinguishable from normal "self" antigens and are thereby recognized by the host immune system. However, the host immune system barely responds to tumors in patients. Supplementation with adjuvant (such as BCG-CWS) in patients with cancer contributes to regression of intrinsically growing cancer. The adjuvant targets antigen-presenting cells, i.e. innate immunity, but not lymphocytes, and promotes up-regulation of MHC, co-stimulators and initial cytokines in antigen-presenting cells. We hypothesized that the role of the adjuvant is to provide conditions suitable for antigen-presentation where antigens are available and the lack of adjuvant-induced priming of antigen-presenting cells results in unresponsiveness to tumor antigens. Here, we report innate immune therapy applicable to cancer patients by supplementation with adjuvants for induction of potent immune responses against tumors.

Humans↗

PubMed2000

Effect of the consumption of ethanol on the microcirculation of the human optic nerve head in the acute phase

Purpose: The effect of the consumption of ethanol on the circulation of the optic nerve head (ONH) in the human eye in the acute phase and its mechanism were studied.Methods: Eleven volunteers drank a bottle of beer (633 mL) with or without ethanol (29.5 g). Normalized blur (NB), a quantitative index of blood flow velocity, was measured in the temporal site of the ONH. NB, blood pressure (BP) and pulse rate (PR) were measured before, immediately after, and every 15 minutes for 90 minutes after consumption. Intraocular pressure (IOP) and plasma ethanol concentration were measured before, and 30 and 90 minutes after consumption. Genotyping of the aldehyde dehydrogenase (ALDH) 2 gene was also performed.Results: NB in the ONH increased significantly from 15 to 45 minutes after consumption of ethanol and the maximum increase was 14% at 15 minutes. IOP was lowered at 90 minutes after consumption, but it was not significant. Mean BP was lowered significantly after 60 minutes. PR and ocular perfusion pressure did not change. A significant correlation was found between plasma ethanol concentration at 30 minutes and maximum NB. NB in the ALDH 2-deficient group was significantly larger from 15 to 45 minutes after consumption than in the proficient group.Conclusions: It appeared that the consumption of ethanol can increase the blood flow in the human ONH in the acute phase through decreased resistance in blood vessels induced by acetaldehyde, a metabolite of ethanol.

Journal Article↗

PubMed2000

Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors.

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.

Antigen Presentation↗

PubMed2000

[Long-term effect of topically applied isopropyl unoprostone on microcirculation in the choroid-retina].

PURPOSE: To study the long-term effect of topically applied 0.12% isopropyl unoprostone (unoprostone, Rescula) on microcirculation in the ocular fundus. MATERIALS AND METHODS: Using a laser speckle tissue circulation analyzer, normalized blur (NB), a quantitative index of blood flow velocity and tissue blood flow, was measured in the optic nerve head (ONH) and choroid-retina before and 4.5 hours after an instillation of placebo into both eyes in 11 normal human volunteers. Intraocular pressure (IOP), blood pressure, and pulse rate were also measured. A drop of unoprostone or the placebo was instilled into each eye in a double-blind manner twice a day for 21 days (the treated or untreated eye). RESULTS: Twenty-one days later, NB values in the ONH and the choroid-retina increased significantly and the IOP decreased significantly only in the treated eyes. Ocular perfusion pressure showed no significant changes. CONCLUSIONS: These results suggest that the increase of the blood flow in the microcirculation in the human ocular fundus following the relatively long-term topical application of unoprostone may be due to reduction in vascular resistance.

Administration, Topical↗

PubMed2000

[Effect of topically applied iganidipine dihydrochloride, a novel Ca2+ antagonist, on optic nerve head circulation in rabbits].

PURPOSE: We studied the effect of topically applied iganidipine dihydrochloride, a novel water-soluble calcium channel blocker on the blood flow of optic nerve head (ONH), intraocular pressure, and blood pressure in rabbits. METHODS: 1. 0.1% iganidipine (20 microliters) was instilled into a normal eye. The change in blood flow in the ONH was measured using a hydrogen gas clearance flowmeter. 2. Iganidipine (0.0001%-0.1%) was instilled into a circulation-disordered eye before or after the intravitreal injection of endothelin-1, and change in the blood flow in the ONH was measured. 3. Changes in intraocular pressure and blood pressure after instillation of 0.1% iganidipine were measured. In all experiments, physiological saline was instilled in each contralateral eye as a control. RESULTS: 1. Instillation of iganidipine significantly increased the blood flow in the ONH by 40% at 45 minutes after instillation. 2. Pre-instillation of 0.01 and 0.1% iganidipine almost completely inhibited the decrease of blood flow in the ONH in the circulation-disordered model. The decrease of blood flow in the ONH was corrected with post-instillation of 0.1% iganidipine. These effects were continuous. 3. Instillation of 0.1% iganidipine did not change either intraocular pressure or blood pressure. CONCLUSION: It was shown that instillation of iganidipine continuously increased and maintained the blood flow in the ONH in normal and circulation-disordered rabbit eye models.

Administration, Topical↗

PubMed1999

Inhibitory effect of trehalose dimycolate (TDM) and its stereoisometric derivatives, trehalose dicorynomycolates (TDCMs), with low toxicity on lung metastasis of tumour cells in mice.

We examined the effect of semisynthetic trehalose-6,6 '-dimycolate (TDM) and its synthetic stereoisomeric derivatives (trehalose 6,6'-dicorynomycolates; TDCMs) prepared in oil-in-water (o/w) emulsion on inhibition of lung metastasis produced by highly metastatic murine tumour cells, colon 26-M3.1 carcinoma and B16-BL6 melanoma cells, using experimental and spontaneous metastasis models. Intravenous (i.v.) administration of TDM (100 microg/mouse) 1, 3 or 8 days before tumour inoculation significantly inhibited lung metastasis of colon 26-M3.1 cells, in a dose-dependent manner. Single administration of TDM 1 day after tumour inoculation also showed the therapeutic effect on experimental lung metastasis of colon 26-M3.1 cells. Similarly, multiple administrations of TDM after tumour inoculation resulted in a significant inhibition of spontaneous lung metastasis of B16-BL6 cells (on day 35), although it showed no effect on suppression of tumour growth (on day 21). In comparison of toxicity in vivo among TDM and four TDCMs such as TDCM(2R,3R), TDCM(2S,3R), TDCM(2R,3S) and TDCM(2S,3S), all of the TDCMs appeared to be less toxic than TDM itself. Furthermore, all of the TDCMs were prophylactically as well as therapeutically active for inhibition of lung metastasis of both colon 26-M3.1 and B16-BL6 tumour cells, showing higher inhibitory activity than that of TDM. In particular, TDCMs induced a marked suppression of the growth of B16-BL6 tumour cells in vivo. These results suggest that systemic administration of TDM as well as TDCMs led to inhibition of tumour metastasis and TDCMs are more potential to suppress tumour growth and inhibit tumour metastasis than TDM.

Adjuvants, Immunologic↗

PubMed1999

Human MD-1 homologue is a BCG-regulated gene product in monocytes: its identification by differential display.

BCG-CWS is a therapeutically potent immune activator which improves the prognosis of cancer patients. However, the targeting effector cells and molecules for BCG-CWS in the human immune system have not been determined. Here, we found that BCG-CWS activates human monocytes and concomitantly down-regulates expression of a human homologue of chicken MD-1 in the activated monocytes by differential display. According to a previous study, MD-1 forms a complex with the Toll family protein RP-105 on murine B cell lines to facilitate its stable expression. Thus, MD-1 may participate in regulation of innate immune activation on human monocytes. Our results, taken together with these recent findings regarding Toll family proteins, suggest that BCG-CWS acts on monocytes to modulate the human innate immune system via regulation of Toll family proteins.

Amino Acid Sequence↗

PubMed1999

Lectins isolated from Korean mistletoe (Viscum album coloratum) induce apoptosis in tumor cells.

Cytotoxic lectins (KML-C) were isolated from an extract of Korean mistletoe [Viscum album C. (coloratum)] by affinity chromatography on a hydrolysed Sepharose 4B column, and the chemical and biological properties of KML-C were examined, partly by comparing them with a lectin (EML-1) from European mistletoe[Viscum album L. (loranthaceae)]. The hemagglutinating activity of KML-C was inhibited by N-acetyl-D-galactosamine and D-galactose at the minimum concentrations of 6.3 and 12.5 microM/ml, respectively. Further biochemical analyses indicated that KML-C consists of four chains (Mr = 27.5, 30, 31 and 32.5 kDa) which, in some of the molecules, are disulfide-linked, and that the chains of KML-C are distributed over a broad range of isoelectric points (pI), 8.0 to 9.0, whereas the range for EML-1 is 6.6-7.0. A difference was also observed between the N-terminal sequences of KML-C and EML-1. The isolated lectins showed strong cytotoxicity against various human and murine tumor cells, and the cytotoxic activity of KML-C was higher than that of EML-1. Tumor cells treated with KML-C exhibited typical patterns of apoptotic cell death, such as apparent morphological changes and DNA fragmentation, and its apoptosis-inducing activity was blocked by addition of Zn2+, an inhibitor of Ca2+/Mg2+ -dependent endonucleases, in a dose-dependent manner. These results suggest that KML-C is a novel lectin related to the cytotoxicity of Korean mistletoe, and that its cytotoxic activity against tumor cells is due to apoptosis mediated by Ca2+/Mg2+ -dependent endonucleases.

Animals↗

PubMed1999

Mechanisms underlying the neurogenic relaxation of isolated porcine sphincter pupillae.

Mechanisms of relaxation induced by nerve stimulation were examined in isolated porcine iris sphincter muscle in reference to norepinephrine, nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) and the functional interaction of inhibitory and excitatory nerves. Changes in isometric tension were recorded in strips of the sphincter pupillae, which were stimulated by transmurally applied electrical pulses. The presence of neurons containing acetylcholinesterase and tyrosine hydroxylase (TH) was determined histochemically. Transmural electrical stimulation (0.5-20 Hz) produced a frequency-related contraction, which was reversed to a relaxation by atropine in prostaglandin F2alpha-contracted strips. The relaxant response was abolished by timolol and suppressed by metoprolol, a beta1-adrenoceptor antagonist, but was not influenced by butoxamine, a beta2-receptor antagonist. Norepinephrine-induced relaxations were also attenuated only by timolol and metoprolol. Treatment with NG-nitro-L-arginine, a NO synthase inhibitor, and [D-p-Cl-Phe6,Leu17]VIP, a VIP receptor antagonist, did not inhibit the neurogenic relaxation. Contractions induced by nerve stimulation were potentiated by timolol and physostigmine but not by the NO synthase inhibitor. In the sphincter muscle, cholinesterase- and TH-positive nerve fibers and bundles were histologically detected. It is concluded that porcine iris sphincter is innervated by cholinergic excitatory and adrenergic inhibitory nerves. The neurogenic relaxation is associated solely with activation of beta1 adrenoceptors by norepinephrine but is not mediated by NO or VIP.

Acetylcholinesterase↗

PubMed1999

Effect of lomerizine, a new Ca(2+)channel blocker, on the microcirculation in the optic nerve head in conscious rabbits: a study using a laser speckle technique.

We examined the effect of a new Ca(2+)channel blocker, lomerizine (KB-2796), and compared it with that of nilvadipine, on the optic nerve head circulation in conscious rabbits using a laser speckle method. Lomerizine (0.03, 0.1 and 0.3 mg kg(-1), i.v.) and nilvadipine (0.003, 0.01 and 0.03 mg kg(-1), i.v.) each significantly increased the normalized blur values (an index of tissue blood velocity) in the optic nerve head in a dose-dependent manner. Neither lomerizine nor nilvadipine caused a significant change in intraocular pressure. Lomerizine produced no significant change in mean arterial blood pressure, although at 0.3 mg kg(-1), i. v. heart rate was significantly increased 5 min after its administration. In contrast, nilvadipine significantly decreased mean arterial blood pressure at 5 to 15 min after its administration and increased heart rate at 5-30 min after its administration (both effects being dose-dependent). Our results indicate that while lomerizine, like nilvadipine, increased tissue blood velocity in the optic nerve head, it did not affect mean arterial blood pressure at the doses that affected optic nerve head circulation, unlike nilvadipine. The plasma concentration of lomerizine (free base) obtained from rabbits at 15 min after administration at a dose of 0. 03 mg kg(-1)i.v., when time there was a significant increase in tissue blood velocity in the optic nerve head, was very similar to plasma concentration with healthy subjects receiving lomerizine at 10 mg (5 mgx2) day(-1), p.o., a dose that achieved a significant reduction in the frequency and mean duration of headache attacks but did not affect the blood pressure or heart rate. These results suggest that lomerizine may be clinically effective in favorably affecting the optic nerve circulation without producing systemic effects such as the hypotension seen during treatment with other Ca(2+)channel blockers.

Animals↗

PubMed1999

Immunological aspects of chitin and chitin derivatives administered to animals.

Chitosan amino groups are recognized by the immune system. Therefore, every derivative of chitin should be assayed immunologically if biomedical applications are sought. Macrophages are activated to various extents by chitin derivatives. Deacetylated chitin (30% deacetylation) and chitin sulfate stimulate the production of circulating antibodies. Accumulation of carboxymethyl chitin takes place in granulocytes and macrophages. These polysaccharides activate complement in analogy to zymosan. Intraperitoneal injection of N-acetylchitohexaose inhibits the growth of tumor cells and pathogens on a similar level as that of lentinan.

Animals↗

PubMed1999

Experimental optic cup enlargement caused by endothelin-1-induced chronic optic nerve head ischemia.

PURPOSE: Vascular insufficiency of the optic nerve head may contribute to glaucomatous optic neuropathy, especially in normal-tension glaucoma. We investigated the effect of chronic optic nerve head ischemia, created by repeated intravitreal injection of endothelin-1 (ET-1), on the morphology and function of the optic nerve. METHODS: In pigmented rabbits, we injected ET-1 (10(-6) M, 10 microL) into the posterior vitreous of one eye twice a week for 4 weeks (N = 7). The vehicle for ET-1 was injected into the contralateral eye as a control (N = 7). The subsequent observation period was set at 8 weeks. The microcirculation of the optic nerve head was noninvasively monitored with a laser speckle circulation analyzer. To evaluate the changes of visual function, visual-evoked potentials were recorded. Morphologic changes of the optic nerve head were analyzed with stereography, and the ratio of cup area (CA) to disk area (DA) was measured by calculating the number of pixels in each area with a microcomputer. RESULTS: Capillary blood flow in the optic nerve head was continuously below 80% of the baseline throughout the study. The visual-evoked potential latency was significantly delayed in ET-1-treated eyes. The CA/DA ratio was significantly increased relative to baseline in the ET-1 treated eyes. Histologic examination showed axonal loss and demyelination affecting the prelaminar portion of the optic nerve. The intraocular pressure was not significantly different from the control value. CONCLUSION: Optic nerve head ischemia could contribute to the enlargement and excavation of the disk cup independent of the intraocular pressure level.

Animals↗