Role of Bone Marrow Maturity, Insulin-Like Growth Factor 1 Receptor, and Forkhead Box Protein N1 in Thymic Involution and Rejuvenation.

Thymic involution is associated with age-related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC-inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin-like growth factor (IGF) and forkhead-box protein-N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF-1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF-1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model.

Our recent studies in an inbred swine model demonstrated that both peripheral and intra-graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor-matched kidney. Here, we have asked whether both peripheral and intra-graft regulatory elements are required for adoptive transfer of tolerance when only a long-term tolerant (LTT) kidney is transplanted. Nine highly-inbred swine underwent a tolerance-inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor-matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance-inducing and/or tolerance-maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor-matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

High incidence of xenogenic bone marrow engraftment in pig-to-baboon intra-bone bone marrow transplantation.

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3-13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

Abrogation of renal allograft tolerance in MGH miniature swine: the role of intra-graft and peripheral factors in long-term tolerance.

We have previously demonstrated that long-term tolerance (LTT) of an MHC class-I mismatched renal allograft can be achieved with a short course of cyclosporine. In order to examine regulatory mechanisms underlying tolerance in this model, we assessed the contributions of factors within the graft and in the peripheral blood for their relative roles in the maintenance of stable tolerance. Twelve LTT recipients of MHC class-I mismatched primary kidneys were subjected to a treatment consisting of donor-specific transfusion followed by leukapheresis, in order to remove peripheral leukocytes, including putative regulatory T cells (Tregs). Following treatment, 2 controls were followed clinically and 10 animals had the primary graft removed and received a second, donor-MHC-matched kidney. Neither control animal showed evidence of rejection, while 8 of 10 retransplanted animals developed either rejection crisis or full rejection of the second transplant. In vitro assays confirmed that the removed leukocytes were suppressive and that CD4(+) Foxp3(+) Treg reconstitution in blood and kidney grafts correlated with return to normal renal function in animals experiencing transient rejection crises. These data indicate that components of accepted kidney grafts as well as peripheral regulatory components both contribute to the tolerogenic environment required for tolerance of MHC class-I mismatched allotransplants.

The induction of tolerance of renal allografts by adoptive transfer in miniature swine.

Our previous in vitro data have demonstrated that regulatory mechanisms are involved in tolerance of class I-mismatched renal allografts in miniature swine treated with 12 days of high dose Cyclsporin A. In this study, we attempted to induce tolerance of class I-mismatched kidneys by adoptive transfer of cells and/or kidneys from long-term tolerant animals. Fifteen SLA(dd) miniature swine received 1.5 Gy whole body irradiation and class I-mismatched (SLA(gg) ) kidneys from naïve pigs with or without cotransplanted kidneys and/or adoptively transferred cells from long-term tolerant (LTT) SLA(dd) recipients of SLA(gg) grafts. In addition, three SLA(dd) miniature swine received class I mismatched kidney with adoptively transferred cells from LTT SLA(dd) recipients. Naïve kidneys transplanted without a LTT kidney were rejected within 9 days. All recipients of naive kidneys along with cells and kidney grafts from LTT animals showed markedly prolonged survival of the naive renal grafts (day 28, >150 and >150 days). These studies suggest that (1) tolerated kidneys have potent regulatory effects and (2) cells from LTT animals infused in conjunction with kidney grafts augment these regulatory effects. To our knowledge, these studies represent the first demonstration of successful adoptive transfer of tolerance in large animals.

Iatrogenic subfoveal diamond particle after macular hole repair surgery.

PURPOSE: To discuss surgical options and visual outcome when faced with a diamond intraoperative foreign body during macular hole surgery. METHODS: Case study of an iatrogenic in-the-macular-hole diamond particle noted during macular hole surgery. No attempt to retrieve the diamond from the macular hole was made during surgery. PATIENT: Forty-seven-year-old female patient with a 483-µm macular hole. RESULTS: The patient's macular hole closed over the diamond particle, making it subfoveal. The visual acuity improved from 20/120 to 20/40 with resolution of metamorphopsia. CONCLUSION: It is likely that the inert nature and small size of diamond particles do not significantly affect visual acuity or hole closure and do not cause retinal toxicity. The authors discourage aggressive attempts to remove such particles during surgery.

Vitreous hemorrhage after whole-body vibration training.

PURPOSE: The purpose of this study was to describe spontaneous vitreous hemorrhage after a session of whole-body vibration training. METHOD: This is a case report of a 52-year-old man with no ophthalmic history who presented with a uniocular drop in vision. RESULTS: Examination showed two areas of vitreous condensations associated with vitreous hemorrhage. Localized posterior vitreous detachment was confirmed on B-scan ultrasound. No retinal breaks were identified. CONCLUSION: The effects of whole-body vibration training have been well documented previously and parallels can be drawn with the effects of pneumatic drilling. Only one previous case of vitreous hemorrhage after whole-body vibration has been reported, and the authors urge vigilance in reporting this suspected association.

Capsule contraction syndrome with haptic deformation and flexion.

We report a case series of capsule contraction syndrome in 5 eyes of 4 patients and describe a previously unreported complication: full flexion of the haptics onto the anterior surface of the optic. Haptics have been reported to slide anterior to the optic while remaining in their original coronal plane. As surgeons move to the use of preloaded injectable IOLs, it is important to scrutinize haptic-optic junction design and IOL material in the light of this complication.

Incidence of blindness due to diabetic eye disease in Fife 1990-9.

AIMS: In the light of goals for reducing blindness due to diabetes, published in the St Vincent Declaration, 1989, the aim of this study was to find the incidence and prevalence of blindness in the diabetic population of Fife. METHODS: All blind registrations for the period 1990-9 were studied. Those with diabetes as the first or main diagnosis were included as new diabetic blind. The prevalence of diabetes was studied in a large sample population and extrapolated to the estimated population of Fife. RESULTS: The incidence of blindness due to diabetes was 64 (SD 24, 95% CI 49-79) per 100 000 diabetic population/year. The point prevalence of blindness due to diabetes on 31 December 1999 was 210 per 100 000 diabetic population. CONCLUSION: The incidence of blindness due to diabetes, in a diabetic population, is now known. Without this benchmark it is impossible to assess the implementation of the St Vincent Declaration.

Quantitative structure-activity relationship (QSAR) analysis of surfactants influencing attachment of a Mycobacterium sp. to cellulose acetate and aromatic polyamide reverse osmosis membranes.

A series of 23 neutral, anionic, and zwitterionic surfactants were tested at a concentration of 0.1% wt/vol for their influence on attachment of a Mycobacterium sp. to cellulose acetate (CA) and polyamide (PA) reverse osmosis (RO) membranes. Four cell attachment bioassays were used: (1) semiconcurrent addition of surfactant and bacteria to RO coupons (standard assay); (2) surfactant pretreatment of RO membranes (membrane pretreatment assay); (3) surfactant treatment of adsorbed cells (detachment assay); and (4) surfactant pretreatment of mycobacteria (cell pretreatment assay). Seventeen surfactants inhibited attachment to PA membranes, whereas 15 inhibited attachment to CA in standard assays and, in 13 cases, the same surfactant inhibited attachment to both PA and CA. Despite greater cell attachment to PA than CA, surfactants were typically more effective in the former membrane system. More surfactants were effective in impairing cell attachment than in promoting detachment and a number enhanced attachment in membrane pretreatment assays, suggesting surface modification of RO membranes. Cell pretreatment inhibited attachment to CA membranes, suggesting the bacterial surface was also a target for detergent activity. Multivariate regression and cluster analyses indicated that critical micellar concentration (CMC) was positively correlated with Mycobacterium attachment in CA and PA standard assays. Surfactant dipole moment and octanol/water partitioning (LogP) also contributed to detergent activity in the PA system, whereas dipole moment, molecular topology (i.e., connectivity indices), and charge properties influenced activity in the CA system. Influential variables in membrane pretreatment assays included the LogP, topology indices, and charge properties, whereas CMC played a diminished role. Surfactant dipole moment was most influential in CA membrane detachment assays. Increasing system ionic strength by LiBr addition strengthened inhibition of cell attachment to CA membranes by dodecylbenzene sulfonic acid (DBSA) and promoted DBSA adsorption to CA surfaces as indicated by attenuated total reflection Fourier-transform infrared spectrometry. Results indicate that inhibition of bacterial attachment to RO membranes may be maximized by manipulating surfactant molecular structure to optimize surface adsorption behavior.

Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism.

We have studied the mechanism of inhibition of the recombinant Rhodococcus proteasome by four different chemical classes of active site-directed small molecule inhibitors. Clasto-lactacystin beta-lactone is a time-dependent inhibitor of the Rhodococcus proteasome's ability to hydrolyze Suc-Leu-Leu-Val-Tyr-AMC, a substrate for this proteasome's single type of active site, and proceeds with a kinact/[I] of 1,700 M-1 s-1. Using peptide mapping of tryptic digests, LC/MS, and amino acid sequence analysis, we have established that the Ogamma of the hydroxyl group on the N-terminal threonine of the beta-subunit is the sole modification made by the beta-lactone. Active site titrations of the Rhodococcus proteasome with reversible peptide aldehydes show the expected stoichiometry of one inhibitor molecule per beta-subunit. Prior modification with beta-lactone completely abrogates the binding of peptidyl boronic acid inhibitors, suggesting that these inhibitors also inactivate the enzyme by reacting with the Ogamma moiety on Thr1. High performance liquid chromatography analysis of peptidyl vinyl sulfone-modified intact Rhodococcus proteasome beta-subunit and its tryptic peptides suggests that the peptidyl vinyl sulfone modifies a residue in the N-terminal 20 amino acids. This modification is also blocked by prior treatment with beta-lactone.