Citations

Abstract Structural changes on the surface of the class I Ag binding domain resulting from point mutations localized inside the Ag binding cleft of the H-2Kb and Kf glycoproteins were revealed using mAb. Both the loss and gain of antibody binding sites found among naturally occurring K glycoproteins resulted from single amino acid substitutions at a variety of different positions buried within the Ag binding groove. Each of the amino acid replacements analyzed represented naturally occurring diversity known to exist among the functional class I Ag-presenting molecules of the mouse. The binding of the affected mAb was not significantly altered in Kb molecules expressed by transfected T2 cells. Because T2 cells have been shown to express Kb molecules that are either largely devoid of bound peptides or bind a vastly different set of low affinity peptides, it is unlikely that the detected structural changes were caused by alterations in the spectrum of peptides bound by the class I variant glycoproteins. Similarly, a class I point mutant, Kb-97R, that also has been shown previously to bind a very different set of peptides in comparison to the parental Kb molecule also displays normal antibody binding properties. We conclude from these studies that structural diversity within the Ag binding cleft indirectly influences the external surface of the Ag-presenting domain of the class I H chain. Significantly, this surface is the interface between the T cell receptor and MHC molecules and may make contributions to the fine specificity of allorecognition.

Abstract Twenty-three class I DNA sequences, representing alleles of the H-2K, D, and L loci, were analyzed to assess patterns of nucleotide and amino acid diversity. Comparisons of the allelic and nonallelic sequences revealed locus specificity in regions encoding the leader peptides and the carboxyl-terminal segments of the Ag presenting molecules. Analyses focusing on the sequences that determine the Ag binding domains revealed weak or insignificant allelic associations, a finding that is in sharp contrast to previously observed relationships among the homologous human sequences. The amino acid positions exhibiting high diversity in the encoded glycoproteins in both mice and humans are localized primarily to the Ag binding site. In the mouse, diverse amino acids were positioned similarly in the K and D/L glycoproteins, although in humans, the A and B glycoproteins exhibit distinctive differences in their locations within the Ag binding site. The absence of locus specificity among the sequences that determine the Ag binding domains of the mouse is consistent with the hypothesis that ectopic gene conversion leads to interlocus exchange of class I sequences. Comparable interlocus exchanges among human class I genes have not played a similar role in shaping human A and B sequences. The basis of this difference between mice and humans is not clear. The nature of amino acid substitutions distinguishing class I loci in mice and humans are comparable, and the role of natural selection in determining diversity appears to be similar in the two species.

Abstract The ability of OVA-specific H-2Kb-restricted CTL to recognize the defined OVA258-276 peptide in the context of the Kbm mutants and variants of these mutants was examined to determine how specific variations in the Ag recognition site-influenced peptide presentation to these CTL. L cells expressing Kb or Kbm10 were equally capable of presenting the OVA peptide to Kb-restricted, OVA-specific bulk CTL, whereas L cell clones expressing Kbm8 or Kbm1 showed little to no capacity to present this peptide. L cell transfectants expressing Kbm3 and Kbm23 consistently demonstrated an intermediate to low level of presentation to bulk OVA-specific CTL. Dissection of the Kbm8 mutant revealed that cells expressing Kbm8-22 (Tyr----Phe) and/or Kbm8-24 (Glu----Ser) presented the OVA peptide significantly less well than the Kb-presenting molecule. Presentation of OVA by cells expressing Kbm8-23,30 (Met----Ile) (Asp----Asn), Kbm8-23 (Met----Ile), and Kbm8-30 (Asp----Asn) was equivalent to Kb presentation. Another mutation designated as Kbm5, that has a substitution at position 116 (Tyr----Phe), demonstrated an intermediate to high ability to present OVA258-276 to an OVA-specific CTL line. The Kbm3, Kbm11, and Kbm23 mutants were unable to present the OVA peptide to this same CTL line. Dissection of these mutants showed that the substitution at position 77 (Asp----Ser), which is shared by all three mutants, was responsible for their inability to present the peptide. A second Kb-restricted CTL line was able to recognize OVA in the context of the Asp----Ser substitution at position 77. The results of this analysis suggest that the OVA258-276 peptide interacts with multiple regions within the Ag recognition site of the Kb class I protein.

Abstract The functional properties of two amino acid substitutions, characteristic of the bm3 mutation, in the Kb class I glycoprotein were analyzed in light of the HLA-A2 crystal model. The model predicts that amino acid residues extending into the proposed ligand-binding site or projecting up from the alpha-helices are functional with respect to peptide Ag presentation; whereas those residues pointing away from the site are silent. L cell clones expressing Kb, Kbm3, and derivatives of Kbm3, Kbm3-77 (Asp----Ser "ligand-binding") and Kbm3-89 (Lys----Ala "silent"), were generated for the analysis. Serologic characterization of this panel of cells by using the mAb B8-24-3, EH-144, 20-8-4, K9-136, and Y-25 (Kb but not Kbm3 specific) revealed the loss of the epitopes recognized by these mAb in the Kbm3-89 clone and the retention of these epitopes in the Kbm3-77 clone. Analysis of the L cell clones by using B6 anti-bm3 CTL demonstrated that L cell clones expressing Kbm3 or Kbm3-77 were lysed by these CTL, whereas clones expressing Kb, Kbm3-89, and Ld were not lysed. In reciprocal experiments, bm3 anti-B6 CTL lysed L cell clones expressing Kb or Kbm3-89 but were unable to lyse clones expressing Kbm3, Kbm3-77, and Ld. The results indicate that the substitution at amino acid 89 determines the Kbm3 serologic phenotype, whereas the Kbm3 alloreactive phenotype is primarily determined by the substitution at amino acid 77. These findings are in good agreement with the predictions derived from the x-ray crystal model of the HLA-A2 molecule.

Abstract We have analyzed the functional significance of the four amino acid differences between the parental H-2Kb and mutant H-2Kbm8 glycoproteins. Six bm8 variants including single substitutions at residues 22, 23, 24, and 30 as well as paired substitutions at residues 23, 30 and 22, 24 were generated and transfected into L cells. Surface expression of these H-2Kb variants was analyzed using monoclonal antibodies which bind to well-defined H-2Kb epitopes. No alterations introduced into the conformational structure of H-2Kb by the amino acid substitutions were detected. The effect of these substitutions on CTL recognition was initially analyzed using the following bulk CTL: either H-2Kb anti-H-2Kbm8, H-2Kbm8 anti-H-2Kb, or third party anti-H-2Kb. The alloreactivity between H-2Kb and H-2Kbm8 is dominated by the amino acid substitution at residue 24 (Glu----Ser). The complete bm8 phenotype, however, also requires the additional substitution at residue 22 (Tyr----Phe). The H-2Kbm8 anti-Kb bulk CTL reacted with both variant H-2Kbm8 molecules containing single substitutions at amino acid positions 22 or 24 but not the variant molecule containing both substitutions. Further analysis using three individual H-2Kbm8 anti-Kb CTL clones indicated the complexity of the self Kbm8 phenotype. Clone 8B1.20 did not react to changes in residues 22 or 24. The 8B1.32 clone reacted with the change at residue 22 but not with the change at residue 24, although the 8B1.54 clone reacted with the change at residue 24 but not with the change at residue 22. The changes in residues 23 (Met----Ile) and/or 30 (Asp----Asn) did not impact significantly on the alloantigenic properties of Kbm8 as determined by both the bulk and cloned CTL populations. According to the three-dimensional class I structure the substitution at amino acid 24 is inaccessible to the TCR. The location of this substitution within the Ag recognition site implies that altered peptide binding, and not a disruption of MHC residues that interact with the TCR, is responsible for the alloreactivity between H-2Kb and H-2Kbm8.

Abstract Early response to therapy, including assessment of MRD is a powerful prognostic factor in childhood ALL. However, there are many other known prognostic factors, and the precise relationship among these and MRD has been incompletely explored. In COG protocol P9900, patients were assigned to one of 3 risk-directed therapies based on NCI risk group, as well as TEL-AML1 translocations (TEL) and simultaneous trisomies of chromosomes 4 and 10 (DT). Infants and Ph+ patients were excluded. Good risk patients (9904) comprised NCI SR patients with either TEL or DT without CNS disease. Poor risk patients (9906) included a subset of NCI HR patients with particularly unfavorable risk factors based largely on age and white count, while 9905 had both NCI SR and HR patients not eligible for 9904 or 9906, including NCI HR patients with TEL or DT. MRD was satisfactorily measured with at least .01% sensitivity by 4-color flow cytometry in the marrow at end of induction (D29) or in blood at Day 8 (D8). D29 MRD was associated with poor outcome in all studies, and in both HR and SR patients on 9905 as shown: Study 4 y EFS ± SE (n) p value MRD <.01% MRD >.01% 9904 Overall 94±1%(668) 75±6%(105) <.0001 TEL-AML1 94±2%(367) 68±12%(38) <.0001 DT 95±1%(316) 80±7%(69) <.0001 9905 NCI-Standard Risk 89±2%(462) 63±6%(119) <.0001 9905 NCI-High Risk 83±4%(292) 49±9%(73) <.0001 9906 74±6%(156) 39±8%(84) <.0001 The proportion of D29 MRD positive DT(17.9%) patients was higher than that of TEL patients (9.4%) but MRD was also prognostic when each group was considered separately; similar findings were seen in HR TEL and DT patients. TEL or DT was associated with a significantly better prognosis in both HR and SR patients who were MRD negative, but were not favorable factors in MRD positive patients. D8 blood MRD was also prognostic in all studies, with higher MRD levels generally associated with poorer outcome. Patients who were both D29 and D8 MRD negative had a good outcome overall (93% 4 y EFS); combining all favorable risk factors, including TEL, DT, NCI risk group, D8 and D29 MRD identified a group of patients (about 13% of B-precursor ALL patients) with 98± 1% 4 y EFS. In multivariate analysis, D29 MRD was the most important prognostic factor. NCI Risk group, DT and D8 blood were also significant, but TEL and day 8 morphologic assessment of marrow were not. We conclude that MRD is a major prognostic factor in childhood ALL even accounting for other risk factors. The prognostic significance of TEL and DT depends on MRD status. The combination of good risk clinical and genetic factors, and absence of MRD in both D8 blood and D29 marrow identifies a group of patients with outstanding outcome on limited therapy.

ABSTRACTIntroductionFinding a peripheral tissue biopsy site to diagnose early PD would be of value for clinical care, biomarker validation, and as research enrollment criteria. Whereas autopsy and advanced PD studies suggest that the submandibular gland is an important biopsy site, there are no studies in early PD. The aim of this study was to determine whether needle biopsy of the submandibular gland reveals Lewy type alpha‐synucleinopathy in early PD.MethodsTwenty‐five early PD (duration < 5 years) and 10 controls underwent transcutaneous needle core biopsies of the submandibular gland. Tissue was stained for phosphorylated alpha‐synuclein, reviewed blind to clinical diagnosis, and only nerve element staining was considered positive.ResultsMean (standard deviation) age was 69.5 (8.3) for the PD group, 64.8 (8.0) years for controls, and disease duration 2.6 (1.1) years. Six PD and 1 control subject had inadequate glandular tissue. Positive staining was found in 14 of 19 (74%) PD and 2 of 9 (22%) control subjects. PD‐positive and ‐negative cases did not differ clinically. Adverse events (mainly swelling and bruising) were common (77% of cases), but were minor and transient.ConclusionsSubmandibular gland needle biopsies identified phosphorylated alpha‐synuclein staining in 74% of early PD subjects. False positives may be true false positives or may represent prodromal PD. If confirmed in larger studies with eventual autopsy confirmation, the potential value of submandibular gland biopsies for early PD may be to aid in clinical trial inclusion/exclusion and eventually serve as a gold standard for biomarker studies short of autopsy confirmation. © 2016 International Parkinson and Movement Disorder Society

The Brain and Body Donation Program (BBDP) at Banner Sun Health Research Institute (http://www.brainandbodydonationprogram.org) started in 1987 with brain‐only donations and currently has banked more than 1600 brains. More than 430 whole‐body donations have been received since this service was commenced in 2005. The collective academic output of the BBDP is now described as the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND). Most BBDP subjects are enrolled as cognitively normal volunteers residing in the retirement communities of metropolitan Phoenix, Arizona. Specific recruitment efforts are also directed at subjects with Alzheimer's disease, Parkinson's disease and cancer. The median age at death is 82. Subjects receive standardized general medical, neurological, neuropsychological and movement disorders assessments during life and more than 90% receive full pathological examinations by medically licensed pathologists after death. The Program has been funded through a combination of internal, federal and state of Arizona grants as well as user fees and pharmaceutical industry collaborations. Subsets of the Program are utilized by the US National Institute on Aging Arizona Alzheimer's Disease Core Center and the US National Institute of Neurological Disorders and Stroke National Brain and Tissue Resource for Parkinson's Disease and Related Disorders. Substantial funding has also been received from the Michael J. Fox Foundation for Parkinson's Research. The Program has made rapid autopsy a priority, with a 3.0‐hour median post‐mortem interval for the entire collection. The median RNA Integrity Number (RIN) for frozen brain and body tissue is 8.9 and 7.4, respectively. More than 2500 tissue requests have been served and currently about 200 are served annually. These requests have been made by more than 400 investigators located in 32 US states and 15 countries. Tissue from the BBDP has contributed to more than 350 publications and more than 200 grant‐funded projects.