Rapid Isolation of Functional ex vivo Human Skin Tissue-Resident Memory T Lymphocytes.

Studies in animal models have shown that skin tissue-resident memory T (TRM) cells provide enhanced and immediate effector function at the site of infection. However, analyses of skin TRM cells in humans have been hindered by the lack of an optimized isolation protocol. Here, we present a combinatorial strategy-the 6-h collagenase IV digestion and gentle tissue dissociation - for rapid and efficient isolation of skin TRM cells with skin tissue-specific immune features. In comparison with paired blood circulating memory T cells, these ex vivo isolated skin T cells express typical TRM cell markers and display higher polyfunctional properties. Moreover, these isolated cells can also be assessed for longer periods of time in ex vivo cultures. Thus, the optimized isolation protocol provides a valuable tool for further understanding of human skin TRM cells, especially for direct comparison with peripheral blood T cells at the same sample collection time.

Significant prolongation of orthotopic corneal-graft survival in FTY720-treated mice.

BACKGROUND: The novel immunomodulator, FTY720, mainly acts through sequestering of lymphocytes to secondary lymphatic tissue, thereby suppressing their infiltration into grafted organs. This study aimed to investigate its influence on corneal-graft survival. METHODS: Sixteen BALB/c mice (H-2d) received corneal transplants from C3H (H-2k) mice. Eight mice were treated with FTY720 (10 mg/kg per day) orally from day -1 to day 11, and all animals received 0.1% dexamethasone eye drops for the same time. In addition, eyes and regional lymph nodes from similarly treated animals were subjected to immunohistochemistry and proliferation assays. RESULTS: FTY720 significantly prolonged graft survival from 28+/-8.1 to 36.5+/-7.1 days (P=0.021). In treated animals, corneal infiltration by CD4+ and F4/80+ cells was reduced from 70.8+/-60.3 to 7.0+/-9.0 (P=0.004) and from 97.5+/-30.7 to 44.8+/-24.9 (P=0.01) cells, respectively, and allogeneic T-cell proliferation was decreased. CONCLUSIONS: FTY720 treatment substantially protects corneal allografts and may provide an immunomodulatory strategy in clinical corneal transplantation.

Low-dose anesthesia for corneal transplantation in mice.

BACKGROUND: Despite technical difficulties caused by the small dimensions, scientific and economic considerations stimulated activities in the murine keratoplasty model. Delicate surgery requires stable anesthesia with rapid postoperative waking. METHODS: For corneal transplantation, all animals were intraperitoneally injected with 0.08 ml of a 1:8 mixture of 2% xylazine and 2.5% esketamine hydrochloride. Thus they all received 0.18 mg of xylazine and 1.8 mg of ketamine. In other words, 4.5 mg/kg BW of xylazine and 45 mg/kg BW of ketamine were injected in a mouse weighing 25 g. The inhalation anesthetic isoflurane was used for short interventions like removing lid sutures or in the case of an animal awaking before the end of the operation. A small but effective set of apparatus comprising a glass bottle, a test tube and several interconnected syringes was developed to create a closed system and visualize the amount of isoflurane used in the animal. Air bubbles showed the amount of isoflurane escaping from the opened respiratory mask. RESULTS: The dose reduction of systemic anesthesia requires additional inhalation anesthesia with isoflurane in nearly all cases. The equipment developed is easy to handle and allows systemic anesthesia with a very low dose. CONCLUSION: This murine anesthesia model may be expected to help other surgeons performing corneal transplantations in mice.

Ipsilateral submandibular lymphadenectomy does not prolong orthotopic corneal graft survival in mice.

PURPOSE: To explore the impact of selective lymphadenectomy on the outflow from the outer eye and corneal graft survival in mice. METHODS: BALB/c mice underwent submandibular lymphadenectomy either on the left side 7 or 21 days before the experiments or bilaterally 7 days in advance. First, (99m)Tc colloidal albumin (Nanocoll) was given as a subcutaneous lower-lid, upper-lid or subconjunctival injection, and count rates were determined 24 h later in the eyes, submandibular lymph nodes, spleen, liver and blood. Second, corneal graft survival was assessed in lymphadenectomised and control mice, and IFN-gamma secretion was determined in draining lymph nodes at the time of transplant rejection. RESULTS: Following subconjunctival Nanocoll injection, count rates/min/mg tissue were significantly higher in the ipsilateral submandibular lymph node than in the other tissues or blood (<0.01). Ipsilateral lymphadenectomy prior to injection resulted in considerably increased count rates in contralateral nodes ( P<0.01), which were higher after injections into upper than into lower lids ( P=0.004). Bilateral submandibular lymphadenectomy led to enhanced count rates in the eyes, blood, spleen and liver (all P<0.01). Removal of the ipsilateral lymph node prior to corneal transplantation did not prolong allograft survival ( P>0.05) and considerably increased IFN-gamma secretion in contralateral nodes after prior removal of the ipsilateral ones paralleled transplant rejection. CONCLUSIONS: In mice, removed submandibular lymph nodes are functionally completely replaced by the contralateral nodes. These studies demonstrate for the first time lymphatic drainage crossing the midline of the body. Consequently, unilateral lymphadenectomy does not improve corneal graft survival.

Ballistic CTLA4 and IL-4 gene transfer into the lower lid prolongs orthotopic corneal graft survival in mice.

PURPOSE: To explore outflow from the eye and to determine and modulate the influence of lymphatic drainage on corneal graft survival in mice. METHODS: Tracer experiments were conducted in BALB/c mice using the (99m)Tc colloidal albumin Nanocoll. Count rates were determined in the eyes, submandibular lymph nodes, spleen, liver and blood 24 h after subconjunctival, intracorneal, intracameral (anterior chamber), intravenous and subcutaneous lower-lid or upper-lid injections ( n=6 each). Four groups of BALB/c mice ( n=8) received corneal transplants from C3H mice; two of them were treated ballistically with vector CTLA4+IL-4 onto the leg or the lower lid, one group was untreated and the other control group was treated with an empty minimalistic, immunologically defined, gene expression (MIDGE) vector. RESULTS: Radioactivity was detected in the liver, spleen and ipsilateral submandibular lymph node after intracameral injection as follows: 91.9%, 6.6% and 1.2% respectively. Radioactivity uptake of the ipsilateral submandibular lymph node was also low after intravenous injection (0.1%) but high after intracorneal (33.8%), lower-lid (62.0%) and subconjunctival (71.2%) injection. Vector CTLA4+IL-4 treatment of the lower lid but not of the leg prolonged graft survival ( P=0.004). CONCLUSION: These tracer studies confirmed for the first time identical lymphatic drainage from the cornea and the lower lid. Logically, lymphatic drainage could be manipulated and graft survival improved by gene transfer to the lower lid.

Influence of ballistic gene transfer on antigen-presenting cells in murine corneas.

PURPOSE: The outcome of corneal transplantation may depend on passenger cells in corneal epithelium and stroma. Their presence in normal corneas is controversial. This study aimed at examining this question and elucidating the still unknown influence of ballistic gene transfer. METHODS: Central 2.5 mm discs of epithelial flatmounts and frozen stromal sections cut parallel to the outer corneal surface were stained for F4/80+ and MHC II+ cells. Corneas were immunohistologically examined in an untreated state and after gene gun treatment using minimalistic immunologically defined gene expression (MIDGE) vector DNA of IL-4 and CTLA4 ( n=6), or untreated/gene-gun-treated donor corneas (C3H mice) were orthotopically grafted into gene-gun-treated eyes (BALB/c mice), and their survival was investigated ( n=8). RESULTS: Untreated control corneas contained 115.7+/-33.7 F4/80+ and 106.8+/-46.2 MHC II+ cells in the epithelium, and 48.9+/-13.2 versus 7.3+/-5.5 in the stromal layer. Ballistic gene transfer caused migration of F4/80+ cells into the corneal stroma ( P<0.01). Graft survival (27.4+/-16.8 days) was not prolonged by gene gun transfection of donor and recipient corneas but increased significantly to 64+/-28 days ( P<0.01) after treating only the recipient. CONCLUSIONS: A multiplicity of F4/80+ and MHC II+ cells in normal murine corneas diminishes the immune privilege of the eye. Ballistic gene transfer impedes graft survival by triplicating these passenger cells in the stromal layer. However, ballistic transfer of MIDGE vector DNA of IL-4 and CTLA4 markedly improves graft survival when treating only the recipient eye.

[Analysis of patient's satisfaction with the mandibular implant-borne overdentures].

OBJECTIVE: To compare the subjective attitudes of fully edentulous patients in mandible toward the implant-borne overdentures and toward the previous complete dentures. METHODS: 16 fully edentulous patients in mandible were consecutively included in this study and the attitudes toward the implant-borne overdentures and the previous complete dentures were surveyed through questionnaires at the first visit and after the implant prosthesis. RESULTS: There exists significant difference regarding to the comfort, function and fixedness between implant-borne overdentures and the previous complete denture. CONCLUSION: It was revealed that mandibular bar-clip retention implant-borne overdentures can highly fulfill patient's demands.

Minimizing side effects of ballistic gene transfer into the murine corneal epithelium.

BACKGROUND: The beneficial effect of modulating an allospecific immune response by ballistic IL-4 and CTLA4 gene transfer to deliver minimalistic immunologically defined gene expression (MIDGE) vectors into the corneal epithelium was demonstrated in corneal transplantation. However, side effects reduced graft survival in control animals after ballistic transfer without DNA. METHODS: An adapter was constructed for the gene gun apparatus to enlarge and keep constant the distance between the gun and the cornea. Mice were treated by ballistic transfer of luciferase- or IL-10 -encoding MIDGE vectors using gold particles different in quantity, size and size uniformity. Levels of protein expression were determined. Treated corneas were observed under the scanning electron microscope and immunohistologically. Three groups of Balb/c (H-2d) mice received a C3H (H-2 k) corneal graft and two of them had gold particles delivered into the corneal epithelium by gene gun. RESULTS: Using the gene gun and the distance piece, scanning electron microscopy did not reveal morphological differences of the corneal surface compared with untreated corneas on day 2 and 5. Sagittal histological sections of the central cornea did not show an invasion of macrophages 24 h after treatment. The expression of luciferase and IL-10 was not reduced when a smaller amount of gold (0.1 mg instead of 0.5 mg) was employed. Ballistic gold treatment did not reduce graft survival. CONCLUSION: Ballistic gene transfer into the corneal epithelium allows high cytokine expression in the cornea without measurable side effects if an apparatus is used that is adapted for this specific purpose.