Scott W. McPherson

Assessment of Technical Skills of Orthopaedic Surgery Residents Performing Open Carpal Tunnel Release Surgery

BACKGROUND Motor skills assessment is an important part of validating surgical competency. The need to test surgical skills competency has gained acceptance; however, assessment methods have not yet been defined or validated. The purpose of the present study was to evaluate the reliability and validity of four testing measures for the integrated assessment of orthopaedic surgery residents with regard to their competence in performing carpal tunnel release. METHODS Twenty-eight orthopaedic residents representing six levels of surgical training were tested for competence in performing carpal tunnel release on cadaver specimens. Four measures were used to assess competency. First, a web-based knowledge test of surgical anatomy, surgical indications, surgical steps, operative report dictation, and surgical complications was administered. Second, residents participated in an Objective Structured Assessment of Technical Skills; each resident performed surgery on a cadaver specimen. All residents were evaluated independently by two board-certified orthopaedic surgeons with a subspecialty certificate in hand surgery with use of a detailed checklist score, a global rating scale, and a pass/fail assessment. The time for completion of the surgery was also recorded. Each assessment tool was correlated with the others as well as with the residents level of training. RESULTS Significant differences were found between year of training and knowledge test scores (F = 7.913, p < 0.001), year of training and detailed checklist scores (F = 5.734, p = 0.002), year of training and global rating scale (F = 2.835, p = 0.040), and year of training and percentage pass rate (F = 26.3, p < 0.001). No significant differences were found between year of training and time to completion of the carpal tunnel release (F = 2.482, p < 0.063). CONCLUSIONS The results of the present study suggest that both knowledge and cadaver testing discriminate between novice and accomplished residents. However, although failure of the knowledge test can predict failure on technical skills testing, the presence of knowledge does not necessarily ensure successful performance of technical skills, as cognitive testing and technical skills testing are separate domains.

Molecular cloning and characterization of the DNA of two papiilomaviruses from monkeys

Abstract Benign and malignant lesions from monkeys were analyzed for the presence of papillomavirus (PV) DNA. By hybridization with different PV DNA probes under conditions of lowered stringency, two tumors were found to contain PV-specific DNA sequences: (1) a cutaneous papilloma from a Colobus monkey; and, (2) a lymph node metastasis of a squamous cell carcinoma of the penis from a Rhesus monkey. Analysis of the DNA of the papilloma from the Colobus monkey indicated the presence of extrachromosal DNA whereas analysis of DNA from the Rhesus tumor suggested the presence of integrated viral DNA. The physical size (7.8 and 8.1 kb), colinear alignment to HPV-5, and cross-hybridization with other PV types under low stringency indicate that the two genomic DNA clones represent new PV types that have been tentatively designated as Rhesus papillomavirus type 1 (RhPV 1) and Colobus guereza papillomavirus type 2 (CgPV 2). A putative viral-host DNA junction fragment was also isolated from the Rhesus genomic library. Nucleotide sequences very closely related to RhPV 1 were observed by in situ hybridization in a laryngeal carcinoma from the Colobus guereza monkey. This report communicates the finding of novel papillomaviruses associated with a benign cutaneous tumor and genital and laryngeal malignancies in non-human primates which may have significance as a putative system for the study of papillomavirus-induced genital and laryngeal malignancies in humans.

Dendritic cells are early responders to retinal injury

The presence and activity of dendritic cells (DC) in retina is controversial, as these cells are difficult to identify in retina due to limited markers and sparse numbers. Transgenic mice that express green fluorescent protein (GFP) on the CD11c promoter to label DC allowed the visualization and quantification of retinal DC. Two retina injury models, the optic nerve crush (ONC) and light injury, were used to study their injury response. Many GFP(+) DC were tightly associated with retinal ganglion cell nerve fibers following ONC, while very few microglia (GFP(-)CD11b(+) cells) were found in close contact. The GFP(+) cells were greatly elevated in the outer plexiform layer following photic injury. All of the GFP(+) DC were CD11b(+), suggesting a myeloid origin. In addition, the GFP(+) DC upregulated expression of MHC class II after injury, while the GFP(-)CD11b(+) microglia did not. This study shows that DC were found in the retina and that they rapidly responded to neural injuries. We propose that they are a previously overlooked population, distinct from microglia, and may be important in the injury response.

The Antigen-Presenting Activity of Fresh, Adult Parenchymal Microglia and Perivascular Cells from Retina

Although several observations show local T cell recognition of retinal Ag, there has been no direct demonstration that the APC were retinal derived, rather than recruited. In this study, CD45+ cells isolated from immunologically quiescent murine retina were tested in vitro for functional evidence of Ag presentation to naive and Ag-experienced CD4 T cells specific for β-galactosidase. Because CD45+ cells from brain have been reported to be efficient APC, they were included for comparison. Measures of activation included changes in CD4, CD25, CD44, CD45RB, CD62L, CD69, caspase-3 activation, CFSE dilution, size, number of cells recovered, and cytokine production. Retinal CD45+ cells gave no evidence of Ag-dependent TCR ligation in naive T cells, unlike splenic APC and CD45+ cells from brain, which supported potent responses. Instead, addition of retinal CD45+ cells to cocultures of naive 3E9 T cells plus splenic APC reduced the yield of activated T cells and cytokine production by limiting T cell activation at early time points. Ag-experienced T cells responded weakly to Ag presented by retinal CD45+ cells. Activating the retinal cells with IFN-γ, anti-CD40, or LPS incrementally increased their APC activity. Addition of neutralizing Abs to TGF-β did not reveal suppressed retinal APC activity. Because retina lacks tissue equivalents of meninges and choroid plexus, rich sources of dendritic cells in brain, cells from retina may better represent the APC activity of fresh, adult CNS parenchymal and perivascular cells. The activity of the retinal CD45+ cells appears to be directed to limiting T cell responses.

Resting CD8 T cells recognize β-galactosidase expressed in the immune-privileged retina and mediate autoimmune disease when activated

Although the expression of class II major histocompatibility complex (MHC) in retina is extremely low, it is an established fact that activated CD4 T cells, specific for retinal antigens (Ags), mediate experimental autoimmune uveoretinitis (EAU). Conversely, CD8 T cells have not been shown to recognize Ag in the retina. This study investigated whether retinal‐specific Ags are detected by class I MHC‐restricted CD8 T cells. Using a CD8 T‐cell clone (β3) specific for an immunodominant epitope of β‐galactosidase (β‐gal), local Ag recognition was shown by transfer of activated β3 cells into β‐gal transgenic (Tg) mice expressing β‐gal in the retina (hi‐arr‐β‐gal mice), or in the brain and eye (GFAP‐β‐gal mice). β‐gal‐positive photoreceptor cells were damaged in the retina of hi‐arr‐β‐gal mice, and anterior segment disease was found in the eyes of GFAP‐β‐gal mice. Ag recognition by resting CD8 T cells was also evaluated. Recovery of 5(6)‐carboxyfluorescein diacetate N‐succinimidyl ester (CFSE)‐labelled β3 cells from hi‐arr‐β‐gal mice was slightly decreased compared to recovery from B10.A mice, while recovery from GFAP‐β‐gal mice was transiently increased. Conversely, recovery of CFSE− cells increased in hi‐arr‐β‐gal mice, consistent with an Ag‐dependent response. The CFSE content of the CFSE+ population was unchanged relative to β3 cells recovered from controls. Intracellular cytokine responses of β3 cells recovered from hi‐arr‐β‐gal and GFAP‐β‐gal mice correlated with the number of cells recovered, regardless of CFSE content. Even though their production of interferon‐γ and tumour necrosis factor‐α was affected little by transfer into hi‐arr‐β‐gal recipients, the ability of β3 cells to mediate delayed‐type hypersesitivity was inhibited in hi‐arr‐β‐gal mice. These results show that resting CD8 T cells are affected by the presence of Ag that originates in retina and, when activated prior to transfer, mediate pathogenic autoimmunity against retinal and other ocular targets.

Bystander killing of neurons by cytotoxic T cells specific for a glial antigen

To explore pathways to neuron loss in inflammatory diseases, transgenic mice expressing β‐galactosidase (β‐gal) in either astrocytes or photoreceptor cells, or both, were inoculated with activated, β‐gal‐specific cytotoxic CD8 T lymphocytes (CTLs). β‐gal‐positive astrocytes in brain were rapidly attacked, with particular damage in cerebellum. Substantial loss of cerebellar granule cells was found, even though these neurons did not express β‐gal. The small number of β‐gal‐positive retinal astrocytes present in these mice was also rapidly destroyed by transferred CTLs, but without detectable consequences for retinal neurons. However, in mice with photoreceptor cell‐specific β‐gal expression, near‐total destruction of photoreceptor cells was produced by CTL transfer. Attack on photoreceptor cells displayed minimal inflammation, and onset was a week later than onset of astrocyte‐directed disease. CTL transfer into F1 mice expressing β‐gal in both astrocytes and photoreceptor cells confirmed that pathogenesis directed against antigen expressed in glia versus neurons proceeded in two distinct, independent phases. β‐gal‐positive retinal astrocytes were severely affected by 5 days post‐transfer, followed by rapid resolution. Photoreceptor cells in the same retina were unaffected until 12 days post‐transfer. The susceptibility of photoreceptor cells was not enhanced by the prior CTL attack on β‐gal‐expressing retinal astrocytes. The results demonstrate that extensive bystander killing of neurons can occur in vivo as a result of direct CTL attack on surrounding astrocytes. Antigen‐expressing retinal neurons were also efficiently killed by CTLs, but by a mechanism that was substantially delayed and dissociated from the killing of retinal astrocytes.

Peripheral Induction of Tolerance by Retinal Antigen Expression

The contribution of peripheral expression of tissue-specific CNS Ags to the generation of tolerance is uncertain. To study this question, we examined mice transgenic (Tg) for expression of β-galactosidase (βgal) on the retinal photoreceptor cell arrestin promoter, in conjunction with TCR Tg mice producing CD4+ T cells specific for βgal (βgalTCR). Several strategies were used to test the hypothesis that βgal expressed in the retina supported thymus-independent tolerance and regulatory T cell development. Retinal expression generated an immunoregulatory response that depressed development of immune responses to βgal following systemic immunization with βgal. This regulation was transferable to naive mice by CD3+4+25+ T cells from naive retinal βgal+ donors. Experiments that removed the βgal+ retina by enucleation showed that subsequent development of a regulatory response was lost. Adoptive transfer of CD25− βgalTCR T cells into retinal βgal Tg mice on the Rag−/− background led to regulatory activity that limited lymphopenia-induced proliferation of βgalTCR T cells in mice with retinal expression of βgal and inhibited the ear-swelling assay for delayed type hypersensitivity. These results show that retinal expression of very small amounts of a tissue-specific Ag can generate tolerance that includes regulatory T cells.

Local Activation of Dendritic Cells Alters the Pathogenesis of Autoimmune Disease in the Retina

Interest in the identities, properties, functions, and origins of local APC in CNS tissues is growing. We recently reported that dendritic cells (DC) distinct from microglia were present in quiescent retina and rapidly responded to injured neurons. In this study, the disease-promoting and regulatory contributions of these APC in experimental autoimmune uveoretinitis (EAU) were examined. Local delivery of purified, exogenous DC or monocytes from bone marrow substantially increased the incidence and severity of EAU induced by adoptive transfer of activated, autoreactive CD4 or CD8 T cells that was limited to the manipulated eye. In vitro assays of APC activity of DC from quiescent retina showed that they promoted generation of Foxp3+ T cells and inhibited activation of naive T cells by splenic DC and Ag. Conversely, in vitro assays of DC purified from injured retina demonstrated an enhanced ability to activate T cells and reduced induction of Foxp3+ T cells. These findings were supported by the observation that in situ activation of DC before adoptive transfer of β-galactosidase–specific T cells dramatically increased severity and incidence of EAU. Recruitment of T cells into retina by local delivery of Ag in vivo showed that quiescent retina promoted development of parenchymal Foxp3+ T cells, but assays of preinjured retina did not. Together, these results demonstrated that local conditions in the retina determined APC function and affected the pathogenesis of EAU by both CD4 and CD8 T cells.

Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells

BackgroundWe previously reported that the peripheral regulatory T cells (pTregs) generated ‘on-demand’ in the retina were crucial to retinal immune privilege, and in vitro analysis of retinal dendritic cells (DC) showed they possessed antigen presenting cell (APC) activity that promoted development of the Tregs and effector T cells (Teffs). Here, we expanded these findings by examining whether locally generated, locally acting pTregs were protective against spontaneous autoimmunity and autoimmunity mediated by interphotoreceptor retinoid-binding protein (IRBP). We also examined the APC capacity of retinal DC in vivo.MethodsTransgenic (Tg) mice expressing diphtheria toxin receptor (DTR) and/or green fluorescent protein (GFP) under control of the endogenous FoxP3 promoter (GFP only in FG mice, GFP and DTR in FDG mice) or the CD11c promoter (GFP and DTR in CDG mice) were used in conjunction with Tg mice expressing beta-galactosidase (βgal) as retinal neo-self antigen and βgal-specific TCR Tg mice (BG2). Retinal T cell responses were assayed by flow cytometry and retinal autoimmune disease assessed by histological examination.ResultsLocal depletion of the Tregs enhanced actively induced experimental autoimmune uveoretinitis to the highly expressed retinal self-antigen IRBP in FDG mice and spontaneous autoimmunity in βgal-FDG-BG2 mice, but not in mice lacking autoreactive T cells or their target antigen in the retina. The presence of retinal βgal downregulated the generation of antigen-specific Teffs and pTregs within the retina in response to local βgal challenge. Retinal DC depletion prevented generation of Tregs and Teffs within retina after βgal injection. Microglia remaining after DC depletion did not make up for loss of DC-dependent antigen presentation.ConclusionsOur results suggest that local retinal Tregs protect against spontaneous organ-specific autoimmunity and that T cell responses within the retina require the presence of local DC.

Lymphopenia-induced Proliferation is a Potent Activator for CD4+ T Cell Mediated Autoimmune Disease in the Retina

To study retinal immunity in a defined system, a CD4+ TCR transgenic mouse line (βgalTCR) specific for β-galactosidase (βgal) was created and used with transgenic mice that expressed βgal in retinal photoreceptor cells (arrβgal mice). Adoptive transfer of resting βgalTCR T cells, whether naive or Ag-experienced, into arrβgal mice did not induce retinal autoimmune disease (experimental autoimmune uveoretinitis, EAU) and gave no evidence of Ag recognition. Generation of βgalTCR T cells in arrβgal mice by use of bone marrow grafts, or double-transgenic mice, also gave no retinal disease or signs of Ag recognition. Arrβgal mice were also resistant to EAU induction by adoptive transfer of in vitro-activated βgalTCR T cells, even though the T cells were pathogenic if the βgal was expressed elsewhere. In vitro manipulations to increase T cell pathogenicity before transfer did not result in EAU. The only strategy that induced a high frequency of severe EAU was transfer of naive, CD25-depleted, βgalTCR T cells into lymphopenic arrβgal recipients, implicating regulatory T cells in the T cell inoculum, as well as in the recipients, in the resistance to EAU. Surprisingly, activation of the CD25-depleted βgalTCR T cells before transfer into the lymphopenic recipients reduced EAU. Taken together, the results suggest that endogenous regulatory mechanisms, as well as peripheral induction of regulatory T cells, play a role in the protection from EAU.