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Biomedical subjects

A Mizuno

Publications and source records attributed to A Mizuno.

At least 19 recordsLinked to original sources

Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2.

We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.

Adenoviridae↗

Continuous antegrade blood cardioplegia: cold vs. tepid.

BACKGROUND: Continuous antegrade blood cardioplegia (CABCP) is used at different temperatures. We investigated the consequences of CABCP at 6 degrees C (COLD) vs. 28 degrees C (TEPID). METHODS: Anesthetized open-chest pigs (25 +/- 2 kg) were placed on cardiopulmonary bypass (CPB). The hearts were arrested for 30 min by 6 degrees C cold or 28 degrees C tepid CABCP (n = 8 each). After an initial 3 min antegrade application of high potassium (20 mEq) cold (6 degrees C) blood cardioplegia, the hearts were arrested for a subsequent 27 min by normokalemic blood delivered antegrade at either 6 degrees C or 28 degrees C. After this, the hearts underwent perfusion with warm systemic blood for an additional 30 min on CPB. Biochemical cardiac data (MVO2 [ml/min/100 g], release of creatine kinase [CK U/min/100 g] and lactate [mg/min/100 g]) were measured during CPB. Total tissue water content (%) and left ventricular stroke work index (SWI g x m/kg) were determined 30 min after discontinuation of CPB and compared to pre-CPB controls. RESULTS: Cold CABCP kept all hearts continuously arrested. The COLD hearts showed no biochemical or functional disturbance. The TEPID hearts intermittently fibrillated and required additional high potassium BCP shots. The TEPID hearts showed a marked CK leakage (2.6 +/- 0.4 vs. 0.7 +/- 0.4), lactate production (4.0 +/- 1.6 vs. extraction from the COLD group) despite the non-ischemic protocol, an impaired initial oxygen consumption (4.2 +/- 1.3 vs. 7.1 +/- 1.6) at the end of cardiac arrest, the formation of myocardial edema (79.5 +/- 1.0 vs. 77.0 +/- 0.8), and a depressed recovery of SWI (0.69 +/- 0.15 degrees vs. 1.41 +/- 0.13). *p < 0.05 for comparison of TEPID vs. COLD hearts using Student's t-test for unpaired data; degrees p < 0.05 for intergroup-comparison of TEPID vs. COLD vs. controls using ANOVA adjusted for repeated measures. CONCLUSIONS: Uninterrupted cardioplegia can be safely performed with cold normokalemic CABCP. In contrast, tepid normokalemic CABCP leads to fibrillation, jeopardizes the heart, and should be avoided.UND

Animals↗

Syntheses and biological evaluations of alpha-D-mannosyl [60]fullerenols.

[60]Fullerenols carrying mono- and bis-alpha-D-mannosyl linkages on the surface were prepared via a [3+2]-cycloaddition reaction between 2-azidoethyl alpha-D-mannoside and C(60) followed by polyhydroxylation with aqueous NaOH. Their biological activity was evaluated in terms of binding affinity to lectins by hemagglutination assay and surface plasmon resonance. [60]Fullerenols without the mannosyl linkage caused aggregation of erythrocytes and binding to a beta-D-galactopyranoside specific lectin (RCA(120)). In contrast, mono- and bis-mannosyl fullerenols were found to decrease the activity for both aggregating erythrocytes and binding to RCA(120), and mono-mannosyl fullerenols turned to binding to alpha-D-mannose specific lectin (Con A).

Carbon↗

Immunological study on circulating murine osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF): possible role of OPG/OCIF in the prevention of osteoporosis in pregnancy.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble member of the tumor necrosis factor receptor family and plays a crucial role in the negative regulation of osteoclastic bone resorption. We have immunized OPG/OCIF knockout mice with murine rOPG/rOCIF and established a panel of hybridomas producing monoclonal antibodies (mAbs) to murine rOPG/rOCIF. Utilizing the mAbs, we developed enzyme-linked immunosorbent assay (ELISA) systems: one detecting both homodimeric and monomeric forms of murine OPG/OCIF and the other detecting only dimeric form of murine OPG/OCIF. With the aid of these ELISA systems we showed that OPG/OCIF is present mainly as a monomer in murine blood. The concentration of OPG/OCIF in normal mouse sera was approximately 500 pg/ml and there was no statistical difference in the serum concentration of OPG/OCIF among genders, age, and strains. Interestingly, the concentration of circulating OPG/OCIF in mouse markedly increased during pregnancy. The result indicated that circulating OPG/OCIF plays an important role in the protection of bone from excess resorption during pregnancy in mammals.

Animals↗

Lactosylceramide is essential for the osteoclastogenesis mediated by macrophage-colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand.

Glycosphingolipids and their metabolites play important roles in a variety of biological processes. Several signal molecules are localized in a glycolipid-enriched microdomain on the cell surface, and their signals are regulated by the glycolipid composition. However, the function of glycolipids in osteoclastogenesis has not been clearly understood. We found that D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glucosylceramide synthase inhibitor, completely inhibits the osteoclast formation induced by macrophage-colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand (RANKL) in a dose-dependent manner. Expression of RANK, the receptor of RANKL, induced by macrophage colony-stimulating factor, was reduced markedly in D-PDMP-treated cells. d-PDMP also inhibited the phosphorylation of the inhibitor of nuclear factor-kappa B and extracellular signal-regulated kinase 1/2 induced by RANKL. In several experiments with the addition of glycolipids to D-PDMP-treated purified bone marrow cells, lactosylceramide (LacCer) strongly affected the differentiation into tartrate-resistant acid phosphatase mononucleated cells, but not positive multinucleated cells. GM3 and GM1 also recovered, but less effectively compared with LacCer. Moreover, exogenous LacCer recovered the reduced expression of RANK and the phosphorylation of inhibitor of NF-kappa B and extracellular signal-regulated kinase 1/2 after stimulation by RANKL at the same level of cells without D-PDMP treatment. Our data suggest that glycosphingolipids, especially LacCer, are necessary for the initiation step of RANKL-induced osteoclastogenesis.

Animals↗

Real-time observation of a single DNA digestion by lambda exonuclease under a fluorescence microscope field.

A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA-protein interactions, such as the DNA digestion reaction by lambda exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual lambda exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of lambda exonuclease, a stained lambda DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by lambda exonuclease revealed that the DNA digestion rate was approximately 1000 bases/s and also demonstrated high processivity.

Avidin↗

Effect of leptin on insulin sensitivity in the Otsuka Long-Evans Tokushima Fatty rat.

Leptin has been proposed to be a sensor of energy storage in adipose tissues, and is capable of mediating a feedback signal to the hypothalamus, which is involved in the regulation of energy homeostasis and body weight. In order to investigate the issue of whether resistance to the activity of leptin on insulin sensitivity is observed in young Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 8 weeks of age, leptin (50 nmol/kg/h) was administered intravenously for 16 h to OLETF and Long-Evans Tokushima Otsuka (LETO) (lean controls) rats, followed by a measurement of insulin-stimulated glucose uptake in hindlimb muscles during hyperinsulinemic euglycemic clamp technique. In the case of LETO rats, the administration of leptin significantly decreased plasma insulin levels prior to the clamp test, but did not change plasma glucose levels. Furthermore, leptin led to an increase in insulin-stimulated glucose uptake in hindlimb muscles. However, in the case of OLETF rats, leptin administration changed neither plasma insulin levels nor insulin-stimulated glucose uptake. These data demonstrate that OLETF rats at 8 weeks of age have already become resistant to high concentration of peripheral leptin.

Animals↗

Atomic force microscopy of RecA--DNA complexes using a carbon nanotube tip.

We report high resolution images of RecA-double stranded (ds) DNA complexes obtained by atomic force microscopy (AFM). When a carbon nanotube (CNT) tip was used, AFM images visualized the 10-nm pitch of RecA-dsDNA complexes and RecA filaments as three-dimensional surface topography without reconstruction analysis. The depth of the notch between two pitches was less than 1 nm. When adsorbed on a soft surface covered with proteins, naked DNA, RecA monomers, RecA hexamers, and short RecA filaments were all clearly resolved in one image. The high resolution images with a CNT tip provided valuable information on the initiation process of RecA-dsDNA complex formation.

DNA↗

Indirect micromanipulation of single molecules in water-in-oil emulsion.

Based on real-time observation and micromanipulation, analytical methods for single DNA molecules have been under development for some time. Precise manipulation, however, is still difficult because single molecules are too small for conventional techniques. We have developed a chemical reaction system that uses water droplets in oil as containers of materials. The water droplets can be manipulated by optical force. The manipulation of the water droplets permits the fusion of two selected droplets. This process corresponds to mixing of different samples. We designate this system as "w/o (water-in-oil emulsion) microreactor system", and each droplet can be thought of as a "microreactor". In this system, single molecules can be manipulated readily, as a molecule can be contained in a microm-sized microreactor. The microreactor utilizes extremely small quantities of samples, therefore, reactions are rapid, as diffusion times in the microreactor are very short. The manipulation technique of the microreactors based on optical force has been applied to induce fusion between microreactors loaded with DNA and YOYO, a fluorescent dye that binds to DNA. This fusion induced a rapid binding of YOYO.

Benzoxazoles↗

Cold continuous antegrade blood cardioplegia: high versus low hematocrit.

OBJECTIVE: Cold continuous antegrade blood cardioplegia (CCABCP) is used with different hematocrit values. We investigated the consequences of CCABCP with low hematocrit (LH: 20-25%) versus high hematocrit (HH: 40-45%). METHODS: Anesthetized open chest pigs (25 kg) were placed on cardiopulmonary bypass (CPB). The hearts were arrested for 30 min by 6 degrees C CCABCP with either LH or HH (n=8, each): After an initial 3 min application of high potassium (20 mEq) BCP the hearts were arrested for subsequent 27 min by normokalemic 6 degrees C cold blood delivered continuously antegradely. Thereafter the hearts underwent perfusion with warm systemic blood for an additional 30 min on CPB. Biochemical cardiac data (MVO(2) (ml min(-1)100 g(-1)), release of creatine kinase (CK; units min(-1)100 g(-1))) and lactate (mg min(-1)100 g(-1))) and the coronary vascular resistance index (CVRI (mmHg ml(-1)ming)) were measured during CPB. Total tissue water content (%) and left and right ventricular stroke work indices (LV-and RV-SWI (g m kg(-1))) were assessed 30 min after discontinuation of CPB and compared to pre-CPB controls. RESULTS: The hearts of the LH group had no biochemical or functional disturbance. The HH group showed marked CK leakage (0.6+/-0.2* vs. 0.1+/-0.1, *P<0.05 for comparison of LH vs. HH with Student's t-test for unpaired data), impaired initial oxygen consumption (4+/-1* vs. 7+/-1) after cardiac arrest, an increased CVRI (82+/-12* vs. 50+/-8), the formation of myocardial edema (81.0+/-1.3* vs. 77.5+/-1.2), and poor functional recovery (LVSWI 0.2+/-0.1* vs. 1.0+/-0.1; RVSWI 0.1+/-0.1* vs. 0.5+/-0.1). The absence of lactate production in both groups was in accord with the non-ischemic protocol. CONCLUSIONS: CCABCP with a low hematocrit of 20-25% is cardioprotective. In contrast, CCABCP with a high hematocrit of 40-45% jeopardizes the heart despite avoiding ischemic periods, and should be avoided.

Animals↗

Severe dental open bite malocclusion with tongue reduction after orthodontic treatment.

We treated a 21-year-old woman with a severe open bite and macroglossia with a standard edgewise appliance and without partial glossectomy. This was followed by retention using a Begg-type plate retainer for the upper dental arch and a fixed canine-to-canine for the lower arch. A crib was added to the upper plate retainer for suppression of a tongue thrust. The lower arch relapsed during the retention period, with a widening of the intermolar distance, flaring of the anterior teeth, and increased mobility of the teeth. We chose tongue reduction to resolve these problems and one-third of the middle dorsal part of the tongue was excised. After the tongue reduction, the patient experienced no functional problem in mastication, swallowing, and gustation, but she complained of mild speech difficulty and slight pain on the dorsal portion of her tongue. These symptoms disappeared 6 months after surgery. At this time, the mandibular dental arch was markedly improved. The flared lower dental arch had returned to an upright position and the tooth mobility reduced to normal. No appliance was used after surgery. Most of the recovery changes occurred within 4 months. This case highlights the importance of the teeth tending to move toward a balance between the tongue pressure from the inside and labio-buccal pressure from the outside.

Adult↗

Juvenile hyaline fibromatosis complicated with oral squamous cell carcinoma: a case report.

A 45-year-old woman was referred because of swelling of the palate, gingival hypertrophy, and multiple cutaneous tumors. She had many cutaneous tumors, which covered most of her body, and she also displayed contractures of the major joints. Maxillary and mandibular gingival hypertrophy, malposition of the teeth, and swelling of the hard palate were the oral findings. The histopathologic features of the cutaneous and gingival tumors were consistent with hyaline fibromatosis, and the swelling of the palate proved to be a squamous cell carcinoma. The carcinoma was treated with tegafur/uracil and seemed to respond to this therapy.

Antimetabolites, Antineoplastic↗

Specificity of carbohydrate structures of gangliosides in the activity to regenerate the rat axotomized hypoglossal nerve.

We previously reported that a ganglioside mixture from bovine brain could prevent neuronal death and promote regeneration in rats with hypoglossal nerve resection. In the present study, we have compared the neurotrophic effects of various glycosphingolipids including lactosyl-ceramide. The findings revealed that GT1b had the activity of neuronal death prevention equivalent to a ganglioside mixture or autograft, while other glycolipids exhibited about 60% activity. However, the capability to promote the regeneration varied among glycolipids, that is, GT1b (86%), GD1b (55%), GD1a (35%), GQ1b (34%), GM1 (20%), lactosyl-ceramide (17%) in the number of horseradish peroxidase-positive neurons as an indicator of regeneration. The experiments with oligosaccharides of GT1b or GD1b and ceramide showed that the carbohydrate moiety mainly exerts neurotrophic effects. These findings suggested that fine structures of carbohydrate moiety in gangliosides are critical in the regenerative activity in this hypoglossal nerve regeneration system.

Animals↗

Defective morphogenesis and functional maturation in fetal islet-like cell clusters from OLETF rat, a model of NIDDM.

A failure in the compensate proliferation of pancreatic beta-cells, as the primary pathogenic event, has been reported in OLETF rat, a model of NIDDM. The aim of the present study is to define whether the beta-cell defect is attributed to the fetal stage islet development, if so, whether the defect involves down regulation of PDX-1 protein expression. Morphological changes, beta-cell function, and the expression of PDX-1 protein were examined in the cultured fetal islet-like cell clusters (ICCs) from OLETF rats along with their diabetes-resistant control counterpart LETO rats in the presence of 5.5 or 11.1mM glucose for 48, 72, 96, and 120-hr, respectively. We have observed four abnormalities in the ICCs of OLETF rats. First, a defective morphogenesis was noted during the 72 to 120-hr ICC culture, a period characterized by a dramatic increase in both beta-cell and non-beta-cell (alpha, delta, and PP) populations in control rats. This defective morphogenesis was demonstrated by a growth retardation of epithelial stratification and poor development of both beta-cell and non-beta-cell masses along with a parallel decline in relevant islet hormone contents. Second, a functional defect was characterized by failure to response to glucose during the 96 to 120-hr-cultured ICCs. Third, the ultrastructural analysis revealed a significant reduction in the number of secretory granules. Four, Western blot analysis showed a significant decrease of PDX-1 protein expression in the OLETF ICCs cultured in 11.1mM glucose for 48 to 72-hr and in 5.5mM glucose for 120-hr. Therefore, we concluded that during the fetal stage of islet development, OLETF rats exhibit both morphological and functional defects.

Animals↗

Transforming growth factor beta affects osteoclast differentiation via direct and indirect actions.

Transforming growth factor beta (TGF-beta) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-alpha has also been shown. Therefore, we investigated TGF-beta effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-alpha. Both OPG and TGF-beta inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-beta also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg-/-) mice. TGF-beta strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFbeta-inhibited cocultures of opg-/- cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-beta were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-beta greatly increased osteoclast formation in recombinant RANKL- or TNF-alpha-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-alpha. These data suggest that TGF-beta may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-alpha) is essential for osteoclast formation, factors such as TGF-beta may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Animals↗

[Bacteria isolated from surgical infections and their susceptibilities to antimicrobial agents. Special references to bacteria isolated between April 1998 and March 1999].

The annual multicenter studies on isolated bacteria from infections in general surgery and their antimicrobial susceptibility have been conducted in Japan since July 1982. This paper describes the results obtained in fiscal 1998 (from April 1998 to March 1999). The number of cases investigated as objectives was 225 for one year. A total of 429 strains (121 strains from primary infections and 308 strains from postoperative infections) were isolated from 183 cases (81.3% of total cases). In primary infections, the isolation rates of anaerobes and Escherichia coli were higher than in postoperative infections, while in postoperative infections, those of Gram-positive aerobes and Pseudomonas aeruginosa were higher than in primary infections. On the whole, among Gram-positive aerobes, the isolation rate of Enterococcus faecalis was the highest, followed by Staphylococcus aureus with high frequency in isolation from postoperative infections. Among Gram-positive anaerobes, Peptostreptococcus spp. and Streptococcus spp. were predominantly isolated. Among Gram-negative aerobes, E. coli, P. aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae were frequently isolated. Among Gram-negative anaerobes, Bacteroides fragilis group was the majority of isolates. In primary infections, the percentage of Gram-negative aerobes has gradually increased since fiscal 1995 or 1996 with these years as the turning point, while those of Gram-positive and Gram-negative anaerobes have gradually declined. In postoperative infections, the percentage of Gram-negative anaerobes has increased continuously since the mid-1980s. The percentage of MRSA among S. aureus rose to 89.7%, which was the highest level since the beginning of this study. The susceptibilities of B. fragilis, which did not show apparent changes, were recognized to have decreased against cephems in fiscal 1998. Among other bacteria in B. fragilis group, development of resistance to cephems has continued on a long-term basis since the mid-1980s. E. coli and K. pneuminiae have obviously not changed in susceptibilities, however, the susceptibilities of isolated strains in fiscal 1998 against high-generation cephems, oxacephems and monobactams have declined. We found neither vancomycin-resistant nor teicoplanin-resistant strains of S. aureus and Enterococcus spp.

Anti-Bacterial Agents↗

Genetic evidence for obesity loci involved in the regulation of body fat distribution in obese type 2 diabetes rat, OLETF.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese type 2 diabetes in human. Obesity is essential for the onset of type 2 diabetes in this rat. Our present investigation was designed to identify quantitative trait loci (QTLs) contributing to obesity by performing a whole-genome search using 214 F(2) intercross progeny between OLETF and F344 rats. We have identified six QTLs responsible for adiposity indices of fat pads on rat chromosomes 2 (Obs1 for mesenteric fat), 4 (Obs2 for retroperitoneal fat), 8 (Obs3 for mesenteric fat), 9 (Obs4 for retroperitoneal fat), and 14 (Obs5 and Obs6 for retroperitoneal fat), demonstrating that the adiposity indices of individual fat pads were under the control of different genes. As expected, the OLETF allele corresponds to increased adiposity indices for all QTLs, except for Obs3, in which the F344 allele leads to an increase in the index.

Adipose Tissue↗

Electrically stimulated induction of hsp70 gene expression in mouse astroglia and fibroblast cells.

Mouse astroglial cells, which were cultured on an electrode, were found responsive to an electric stimulation of sine wave potential in enhancing hsp70 mRNA resulting from an activation of hsp70 gene expression. On the basis of this finding, electrically responsive cells were established by transfecting mouse 3T3-L1 cells with a constructed plasmid encoding hsp70 promoter and the firefly luciferase gene. A stable cell line has been established through selection of heat-stimulated luciferase expression. A 1-h electric stimulation of the cells resulted in activation of luciferase expression, which was confirmed to produce an increase in light emission. The sequential pattern of the electrically stimulated expression of luciferase was found different from that of the heat stimulation. Furthermore, the promoter was activated depending on the potential and duration of the stimulation applied. Consequently, the electric stimulation has proven effective on activating hspP70 promoter. This cell line is feasible in expressing the gene of interest by electrical stimulation, which lead us to construct environment responsive cells in general.

3T3 Cells↗