HLA- and genotype-based risk assessment model to identify infantile onset pompe disease patients at high-risk of developing significant anti-drug antibodies (ADA).

In Pompe disease, anti-drug antibodies (ADA) to acid alpha-glucosidase (GAA) enzyme replacement therapy contribute to early mortality. Assessing individual risk for ADA development is notoriously difficult in (CRIM-positive) patients expressing endogenous GAA. The individualized T cell epitope measure (iTEM) scoring method predicts patient-specific risk of developing ADA against therapeutic recombinant human GAA (rhGAA) using individualized HLA-binding predictions and GAA genotype. CRIM-negative patients were six times more likely to develop high ADA titers than CRIM-positive patients in this retrospective study, whereas patients with high GAA-iTEM scores were 50 times more likely to develop high ADA titers than patients with low GAA-iTEM scores. This approach identifies high-risk IOPD patients requiring immune tolerance induction therapy to prevent significant ADA response to rhGAA leading to a poor clinical outcome and can assess ADA risk in patients receiving replacement therapy for other enzyme or blood factor deficiency disorders.

Assessment of the Brain's Macro- and Micro-Circulatory Blood Flow Responses to CO2 via Transfer Function Analysis.

OBJECTIVES: At present, there is no standard bedside method for assessing cerebral autoregulation (CA) with high temporal resolution. We combined the two methods most commonly used for this purpose, transcranial Doppler sonography (TCD, macro-circulation level), and near-infrared spectroscopy (NIRS, micro-circulation level), in an attempt to identify the most promising approach. METHODS: In eight healthy subjects (5 women; mean age, 38 ± 10 years), CA disturbance was achieved by adding carbon dioxide (CO2) to the breathing air. We simultaneously recorded end-tidal CO2 (ETCO2), blood pressure (BP; non-invasively at the fingertip), and cerebral blood flow velocity (CBFV) in both middle cerebral arteries using TCD and determined oxygenated and deoxygenated hemoglobin levels using NIRS. For the analysis, we used transfer function calculations in the low-frequency band (0.07-0.15 Hz) to compare BP-CBFV, BP-oxygenated hemoglobin (OxHb), BP-tissue oxygenation index (TOI), CBFV-OxHb, and CBFV-TOI. RESULTS: ETCO2 increased from 37 ± 2 to 44 ± 3 mmHg. The CO2-induced CBFV increase significantly correlated with the OxHb increase (R (2) = 0.526, p < 0.001). Compared with baseline, the mean CO2 administration phase shift (in radians) significantly increased (p < 0.005) from -0.67 ± 0.20 to -0.51 ± 0.25 in the BP-CBFV system, and decreased from 1.21 ± 0.81 to -0.05 ± 0.91 in the CBFV-OxHb system, and from 0.94 ± 1.22 to -0.24 ± 1.0 in the CBFV-TOI system; no change was observed for BP-OxHb (0.38 ± 1.17 to 0.41 ± 1.42). Gain changed significantly only in the BP-CBFV system. The correlation between the ETCO2 change and phase change was higher in the CBFV-OxHb system [r = -0.60; 95% confidence interval (CI): -0.16, -0.84; p < 0.01] than in the BP-CBFV system (r = 0.52; 95% CI: 0.03, 0.08; p < 0.05). CONCLUSION: The transfer function characterizes the blood flow transition from macro- to micro-circulation by time delay only. The CBFV-OxHb system response with a broader phase shift distribution offers the prospect of a more detailed grading of CA responses. Whether this is of clinical relevance needs further studies in different patient populations.

A luciferase transgenic mouse model: visualization of prostate development and its androgen responsiveness in live animals.

Numerous mouse models of prostate carcinogenesis have been developed, but hitherto there has been no model in which the prostate gland could be imaged in live animals. The transgenic model generated here targeted mouse prostate gland using a firefly luciferase enzyme under the control of a small but highly active and specific supra prostate-specific antigen (sPSA) promoter. We evaluated postnatal prostate development, involution and androgen-induced restoration of prostate growth in adult transgenic mice using bioluminescence imaging. Results of our study showed that: (i) the prostate gland of male offspring did not yield a significant bioluminescence signal until after sexual maturity. Luciferase was detected in the luminal epithelial cells of the ventral and dorsolateral lobes of the prostate gland and caput epididymis, with little or no activity in 18 other organs evaluated. (ii) While a constant high level of bioluminescence was detected in the mouse prostate from 5 to 35 weeks of age, a slight drop in bioluminescence was detected at 36 to 54 weeks. (iii) Upon castration, the luciferase activity signal associated with mouse prostate detected by a cooled charge-coupled device camera was dramatically reduced. This signal could be rapidly restored to pre-castration levels after androgen administration. Androgen-induced luciferase activity subsided to nearly basal levels 5 days following the last injection. These data demonstrate that a bioluminescent mouse model with luciferase activity restricted to the prostate gland under the control of a (sPSA) promoter can be used on a real-time basis in live animals to investigate the development and responsiveness of the prostate gland to exogenously administered androgen. This model can be extended to detect the responsiveness of the prostate gland to therapy and used as a founder strain to visualize tumors in hosts with different genetic backgrounds.

Chromosomal location of murine disabled-2 gene and structural comparison with its human ortholog.

Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The three spliced forms, p96, p93, and p67 of murine Dab2 cDNAs were first isolated as phosphoproteins functioning in the macrophage CSF-1 signal transduction pathway. Subsequently, the involvement of Dab2 in ovarian cancer development has been investigated: Dab2 expression is lost or greatly diminished in breast and ovarian cancers, and gene deletions have been found. Regulation of Disabled-2 expression is also found to be important in development and physiological functions. Structural information of the murine Dab2 gene is essential for studies of transcription regulation and gene function in mouse models. In this study, the mouse Dab2 gene coding sequence was identified and sequenced from three lambda phage clones containing the gene. Two BAC clones of mouse genomic DNA were also used to identify the sequences of the non-coding first exon and promoter. The first exon is separated from the second exon by a large (15 kb) intron. The mouse gene is about 40 kb in size and consists of 15 exons, producing a 3.6 kb message. The translation initiation site resides in the middle of the second exon. The mouse Dab2 gene structure is very similar to that of its human ortholog in exon/intron sizes and promoter sequences. The chromosomal localization of mouse Dab2 was mapped by FISH to chromosome 15A2, a site of syntax with the human 5p12 where human Dab2 gene resides. The information on the mouse Dab2 gene structure and promoter will be invaluable in studies of the involvement of Dab2 gene in cancer, expression, physiological function, and development in mouse models.

Carcinoma of the nasopharynx in young patients.

PURPOSE: To provide an analysis of eighteen cases of adolescent nasopharyngeal carcinoma treated between 1971 and 1989. METHODS AND MATERIALS: Between 1971 and 1989, 48 cases of nasopharyngeal carcinoma were evaluated at the Medical College of Georgia Hospital and Clinics. Eighteen patients between the ages of 9 and 29 years were treated at the Georgia Radiation Therapy Center. All patients presented for treatment with (AJCC) Stage IV disease. Fifteen patients with lymphoepithelioma and three with squamous cell carcinoma histologies received definitive radiation therapy to a median dose of 64.8 Gy. Males outnumbered females by more than 2:1 and the majority of patients (67%) were black. Nine patients received multiagent adjuvant chemotherapy. RESULTS: Thirteen patients are alive from 7 to 166 months (median 32 months) including three with disease at 17, 24, and 132 months. Overall and disease-free survival at 5 and 10 years were 63% and 54%, respectively. Five patients died from disease; four patients had pulmonary metastases while one had CNS metastasis. Eighty percent of relapses occurred within the first 2 years following treatment. Acute and chronic toxicities were limited, consisting primarily of mucositis and xerostomia. Radiation doses of 65 Gy or more (p = 0.049) and age greater than 20 years (p = 0.005) were positive prognosticators for survival. Adjuvant chemotherapy, race, and sex were not found to be of prognostic value. Disparities in the distribution of patients with lymphoepithelioma and squamous cell histologies and the presentation of advanced regional disease precluded analysis for prognostic significance of histology and nodal status in this series. CONCLUSION: The results of the present series compare favorably with those published from other institutions. High doses of radiation and a high systemic failure rate continue to be the fundamental obstacles to effective management and enhanced survival for patients with nasopharyngeal carcinoma.

Lens-specific expression of a major histocompatibility complex class I molecule disrupts normal lens development and induces cataracts in transgenic mice.

PURPOSE: Lens epithelial tissue does not normally express major histocompatibility complex (MHC) class I molecules. In addition, the mechanism of self-tolerance to intraocular antigens is unknown. To study the effect of class I expression in the lens, transgenic mice were produced that express an allo-MHC class I molecule under the alpha A-crystallin proximal promoter. METHODS: p alpha Dd was generated by fusion of the H-2Dd structural gene to the alpha A-crystallin proximal promoter. Transgenic mice were produced, and founder lines were identified by Southern blot hybridization. Eyes from transgenic mice were cryostat sectioned and stained for Dd expression or fixed in paraformaldehyde and stained for histologic analysis. Lens RNA was isolated by acid phenol extraction, and transgene expression was analyzed by nuclease protection. RESULTS: The transgenic mice demonstrated dose-dependent, nonimmunologic lens defects consistent within a given line. In the highest expressing lines, ocular defects, including microphthalmia and cataract formation, were observed. Many adult mice from these lines demonstrated lens capsule rupture and a Dd-specific inflammatory response. Inflammation did not occur in mice with intact lens capsules. CONCLUSIONS: Overexpression of H-2Dd in the lens had serious nonimmunologic consequences on lens development and cataract formation. In addition, the high copy number mice revealed at least a partial loss of immunologic tolerance on lens capsule rupture. The lack of an inflammatory response in transgenic mice with intact lens capsules suggests that the physical barrier of the lens capsule is one mechanism of maintaining immune privilege.

Transgenic expression of IFN-gamma in the murine lens results in multiple ocular abnormalities and an early but self-limited inflammatory response.

The anterior chamber of the eye is known to be an immune privileged site, due to both local and systemic effects on the immune response. Injection of IFN-gamma into the anterior chamber (AC) overcomes the suppression of antigen-specific delayed hypersensitivity responses normally seen in the eye. Transgenic mice expressing increased IFN-gamma in the lens under the alpha A-crystallin promoter were produced to determine whether the proinflammatory effects of IFN-gamma would abolish immune privilege and promote loss of tolerance as has been seen in non-immune privileged tissues. Two alpha C/IFN-gamma transgenic lines are described which demonstrate multiple ocular and lenticular abnormalities some of which are developmental in origin and others that may be secondary to the inflammatory effects of IFN-gamma. A significant inflammatory cell infiltrate which is observed in the AC and vitreous from birth to 4 weeks of age, consists initially of macrophage and polymorphonuclear leukocytes and then CD4+ T lymphocytes. However, the infiltrate is essentially resolved by 6 weeks of age. Therefore, although lens-specific expression of IFN-gamma results in early loss of immune privilege, chronic uveitis does not occur probably due to the lack of continued IFN-gamma expression.

Effects of chronic centrifugation on skeletal muscle fibers in young developing rats.

Three groups of 30-d old male and female rats were centrifuged for 2, 4, 8, and 16 weeks, after which their soleus and plantaris muscles were analysed for changes in proportion of muscle fiber types. The groups were: earth control, maintained at earth gravity without rotation; rotation control, subjected to a gravitational force of 1.05 G and 28 rpm; and rotation experimental, subjected to a gravitational force of 2 G and 28 rpm. Muscle fibers were classified into four fiber types on the basis of actomyosin ATPase activity as slow oxidative, fast oxidative glycolytic, and either fast glycolytic (plantaris) or intermediate (soleus). Hypergravity resulted in an increase in slow oxidative fibers in soleus and the deep region of plantaris. Chronic rotation of males resulted in a decreased proportion of slow oxidative fibers in soleus relative to the earth control, but not of females treated similarly. The relationship of body weight to the changes in proportion of slow oxidative fibers is discussed.

Time course of change in soleus muscle fibers of rats subjected to chronic centrifugation.

Soleus muscles from three groups of rats were analysed for changes in muscle fiber populations and diameters after being subjected to chronic centrifugation. The experimental groups were: earth control, raised at earth gravity with no rotation; rotation control, subjected to a gravitational force of 1.03 g and 30 rpm; and rotation experimental, subjected to a gravitational force of 2 g and 30 rpm. One male and one female of each group were killed at 7, 14, 21, 35, 42, 45, 49, 56 and 60 d after initiation of the experiment. Hypergravity resulted in an increase in the proportion of slow oxidative fibers. The time course of change in their muscle fiber populations is plotted. The time course of change in the ratio of body weight to muscle fiber diameter in the rotation control animals differed from that of the earth control and rotation experimental animals.

A comparison of dynamic cerebral autoregulation across changes in cerebral blood flow velocity for 200 s.

OBJECTIVES: The dynamic interaction between blood pressure (BP) and cerebral blood flow velocity (CBFV) is not fully understood, especially for CBFV changes lasting longer than 50 s. The interaction between BP and CBFV is relatively well characterized for periods <50 s using transfer function (TF) estimations of phase, gain, and coherence. We used TF estimations to compare the phase and gain for periods >50 s with those for periods <50 s. MATERIALS AND METHODS: BP and CBFV (of the middle cerebral artery) were simultaneously recorded in 23 healthy subjects (10 men, 13 women, mean age 35 ± 10 years) under normo- and hypocapnia (induced by hyperventilation). TF and coherence estimations were based on Welch's periodogram method with a windowing of 200 s (frequency resolution, 0.005 Hz, corresponding to a period of 200 s). Means of the phase, gain, and coherence were calculated over frequency periods of 0.005-0.02 Hz (sVLF), 0.02-0.07 Hz (VLF), 0.07-0.15 Hz (LF), and 0.15-0.40 Hz (HF) and analyzed using the t-test and Pearson correlation. RESULTS: Compared with the VLF range, normo- and hypocapnia phases were slightly but significantly lower in sVLF, while gain and coherence were not different. Hypocapnia induced significant (mostly p < 0.01) phase increases and gain decreases as well as coherence decreases in all frequency ranges. The phase and gain correlated significantly (-0.87 < r > -0.99) (p < 0.001) and inversely in all frequency ranges <0.15 Hz under both respiratory conditions. In some instances, the phase indicated disturbed autoregulation. CONCLUSION: In the frequency range <0.15 Hz, the phase and gain correlate highly and linearly with high consistency. The phase, gain, and coherence were similar in sVLF and VLF ranges. The phase was slightly lower in the sVLF range than in the VLF range. Notably, the data suggest that autoregulatory failure may occur in healthy persons.

A study of the effect of pregnancy on muscle fibers of the rectus abdominis muscle of the rat.

Samples of the rectus abdominis muscle were taken from Sprague-Dawley rats at 0, 3, 6, 6, 12, 15, 18, and 21 days of pregnancy, and at 1, 3, 6, 9, 12, and 15 days of postpartum. Sections were incubated for actomyosin adenosine triphosphatase activity following preincubation at a basic pH. Muscle fibers within a unit area of each sample were identified as to fiber type according to their enzyme activity, and the population of each type counted. The proportion of each fiber type was calculated and the diameter of 24 fibers of each type measured. No changes were noted in the muscle fiber proportions through the course of the experiment. Differential changes in muscle fiber diameters were noted in each of the three muscle fiber types. Slow oxidative fibers underwent an increase in diameter through the last half of pregnancy. The diameter was further increased as stretch of the muscle was released after birth, and did not decrease in the postpartum period. Fast glycolytic fibers decreased in diameter during the last half of pregnancy, but returned to the prepregnancy diameter in the first postpartum day. The diameter of the fast oxidative glycolytic fibers remained unchanged through the course of pregnacy and in the postpartum period.

Three years of experience with a dissection table ventilation system.

A dissection table ventilation system that draws air across the cadaver and away from the table top was designed to fit the Shandon-Lipshaw AN-52 dissection table. Each U-shaped unit consists of a pair of hollow collection arms that attach to a collecting manifold at one end. During dissection the manifold is coupled to a central ventilation system through a flexible duct. The air from the table ventilation system is exhausted after passing through a heat recovery system. The unit is raised from the table surface during dissection of the body cavities to increase the efficiency of fume/odor removal. Eight hour exposure data for formaldehyde concentrations are presented. Data were collected from detectors positioned at selected levels above the cadaver during dissection, and above a tray on the table top containing a known volume of 4% formaldehyde or the West Virginia School of Osteopathic Medicine embalming fluid under varying airflow conditions. The results demonstrate that the table ventilation system is effective in reducing exposure to formaldehyde in the dissection laboratory.

MHC class II expression in double mutant mice lacking invariant chain and DM functions.

Invariant (Ii) chain and DM functions are required at distinct stages during class II maturation to promote occupancy by diverse peptide ligands. The class II molecules expressed by mutant mouse strains lacking Ii chain or DM activities display discrete structural and functional abnormalities. The present report describes the cellular and biochemical characteristics of Ii-DM- doubly deficient mice. As for Ii chain mutants, their mature Aalphab Abetab dimers similarly exhibit reduced mobilities in SDS-PAGE, and in functional assays these molecules behave as if empty or occupied by an easily displaced peptide. Additionally, the present experiments demonstrate that the production of floppy Aalphab Abetab dimers is TAP independent. In comparison with Ii chain mutants, Ii-DM- doubly deficient cell populations exhibit increased peptide binding activities and consistently greater presentation abilities in T cell stimulation assays. These functional differences appear to reflect higher class II surface expression associated with their increased representation of B lymphocytes. We also observe defective B cell maturation in mice lacking Ii chain or DM expression, and interestingly, B cell development appears more severely compromised in Ii-DM- double mutants. These mutant mice lacking both Ii chain and DM activities should prove useful for analyzing nonconventional class II Ag presentation under normal physiological conditions in the intact animal.

Peptide dependency of alloreactive CD4+ T cell responses.

Alloreactivity, the capacity of a large number of T lymphocytes to react with foreign MHC molecules, represents the cellular basis for the rejection of tissue grafts. Although it was originally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells, studies in the MHC class I system have clearly demonstrated that MHC-bound peptides can influence this interaction. It remains unclear, however, whether peptides play an equally important role for the recognition of MHC class II molecules by alloreactive CD4+ T cells. Another issue that remains unresolved is the overall frequency of peptide-dependent versus peptide-independent alloreactive T cells. We have addressed these questions with antigen-presenting cells (APC) from H2-M mutant mice that predominantly express a single MHC class II-peptide complex, H2-Ab bound by a peptide (CLIP) derived from the class II-associated invariant chain. APC from these mice were used as targets and stimulators for alloreactive CD4+ T cells. Results demonstrated that the vast majority of CD4+ alloreactive T cells recognize MHC class II molecules in a peptide-dependent fashion.

A critical, invariant chain-independent role for H2-M in antigen presentation.

Antigen presentation by MHC class II (class II) is facilitated by the accessory molecules, invariant chain (Ii) and H2-M. Ii associates with class II during biosynthesis and promotes transport of class II to Ag-loading compartments. One function of H2-M is the removal of Ii fragments from MHC class II. We have previously demonstrated that Ii-deficient mice, unlike class II-deficient mice, are resistant to L. major infection. In the present study, we found that H2-M-deficient (H2-M0) mice were susceptible to progressive infection with L. major. The dispensability of Ii for control of L. major allowed genetic analysis of whether H2-M functions by association with or independently of Ii. In contrast to Ii-deficient (Ii0) mice, Ii0H2-M0 mice were as susceptible to L. major as H2-M0 mice. Thus, H2-M has an essential, Ii-independent function during presentation of microbial pathogens.

CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4.

Murine CD1 has been implicated in the development and function of an unusual subset of T cells, termed natural T (NT) cells, that coexpress the T cell receptor (TCR) and the natural killer cell receptor NK1.1. Activated NT cells promptly produce large amounts of IL-4, suggesting that these cells can influence the differentiation of CD4+ effector T cell subsets. We have generated mice that carry a mutant CD1d1 gene. NT cell numbers in the thymus, spleen, and liver of these mice were dramatically reduced. Activated splenocytes from mutant mice did not produce IL-4, whereas similarly treated wild-type splenocytes secreted large amounts of this cytokine. These results demonstrate a critical role for CD1 in the positive selection and function of NT cells.

H2-M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T cell repertoire selection.

H2-M is a nonconventional major histocompatibility complex (MHC) class II molecule that has been implicated in the loading of peptides onto conventional class II molecules. We generated mice with a targeted mutation in the H2-Ma gene, which encodes a subunit for H2-M. Although the mutant mice express normal class II cell surface levels, these are structurally distinct from the compact SDS-resistant complexes expressed by wild-type cells and are predominantly bound by class II-associated invariant chain peptides (CLIPs). Cells from these animals are unable to present intact protein antigens to class II-restricted T cells and show reduced capacity to present exogenous peptides. Numbers of mature CD4+ T lymphocytes in mutant mice are reduced 3- to 4-fold and exhibit altered reactivities. Overall, this phenotype establishes an important role for H2-M in regulating MHC class II function in vivo and supports the notion that self-peptides contribute to the specificity of T cell positive selection.

The alloreactive and self-restricted CD4+ T cell response directed against a single MHC class II/peptide combination.

The cellular basis for allograft rejection derives from the strong T cell response to cells bearing foreign MHC. While it was originally assumed that alloreactive T cells focus their recognition on the polymorphic residues that differ between syngeneic and allogeneic MHC molecules, studies with MHC class I-restricted CTL have shown that MHC-bound peptides play a critical role in allorecognition. It has been suggested that alloreactive T cells depend more strongly on interactions with the MHC molecule than with the associated peptide, but there is little evidence to support this idea. Here we have studied the alloreactive and self-restricted response directed against the class II H2-Ab molecule bound with a single peptide, Ep, derived from the H2-Ealpha chain. This MHC class II-peptide combination was a poor target and stimulator of alloreactive CD4+ T cell responses, indicating that MHC-bound peptides are as important for alloreactive CD4+ T cells as they are for alloreactive CTL. We also generated alloreactive T cells with exquisite specificity for the Ab/Ep complex, and compared their reactivity with self-restricted T cells specific for the same Ab/Ep complex. Our results showed that peptide-specific alloreactive T cells, as compared with self-restricted T cells, were more sensitive to peptide stimulation, but equally sensitive to amino acid substitutions in the peptide. These findings indicate that alloreactive and self-restricted T cells interact similarly with their MHC/peptide ligand.

The imprint of intrathymic self-peptides on the mature T cell receptor repertoire.

The analysis of T cell receptor alpha (TCR alpha) chains in mice transgenic for a TCR beta chain has allowed us to demonstrate a central role for self-peptides in the positive intrathymic selection of major histocompatibility complex (MHC) class II-restricted T cells. Analysis of specific V alpha-J alpha joins in mature CD4+ TCRhigh thymocytes and in peripheral CD4+ T cells revealed a limitation in amino-acid sequences. By analysis of immature thymocytes, we could show that this limited repertoire was selected from a more diverse repertoire. By analysis of the same beta chain-transgenic mice bred to H-2Ma-deficient mice that express one or a very limited number of peptides, we could demonstrate that the V alpha-J alpha join repertoire was now altered and much more limited. Together, these data provide molecular and genetic evidence that the intrathymic positive selection of the TCR repertoire is critically affected by self-peptides presented by MHC class II molecules, most likely on thymic cortical epithelial cells.