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PubMed2000

Diagnostic criteria for pseudo-Class III malocclusion.

The aim of this study is to identify the diagnostic criteria for pseudo-Class III malocclusion and compare it with Class I malocclusion in the southern Chinese population. Sixty-seven patients (mean age, 10.9 +/- 1.8 years) were included in this study; 36 patients represented pseudo-Class III malocclusion. Selection criteria included the following: (1) anterior crossbite (at least 2 incisors with negative overjet and overbite); (2) mandibular displacement; (3) all patients were southern Chinese who had been followed after the growth spurt, none had developed a skeletal Class III malocclusion; (4) the patients were treated for an average of 7 months to procline upper incisors and retrocline lower incisors. None of the cases received any treatment that might affect skeletal growth. Thirty-one patients with Class I malocclusion were included in the Class I malocclusion group for the comparison of dentoskeletal characteristics with the pseudo-Class III malocclusion group. Selection criteria included the following: (1) skeletal Class I malocclusion with normal overjet and overbite, (2) mild to moderate crowding with Class I molar relationship, (3) straight facial profile. The following were included in the assessment of pseudo-Class III malocclusion cases: (1) family history, (2) molar and canine relationships at habitual occlusion and centric relation, and (3) dentoskeletal morphology. The results were that 72% of the examined cases in the pseudo-Class III malocclusion group showed no family history and 75% showed Class I molar relationship at habitual occlusion. Compared with the Class I malocclusion group, subjects in the pseudo-Class III malocclusion group showed a significantly decreased midface length, increased maxillary-mandibular difference, more retroclined upper incisors, and a retrusive upper lip. In conclusion, a pseudo-Class III malocclusion is characterized by retroclined upper incisors, retrusive upper lip, decreased midface length, and increased maxillary-mandibular difference. Findings of this study showed that patients with a pseudo-Class III malocclusion exhibit certain morphologic, dental, and skeletal characteristics that should be of aid in the diagnosis of pseudo-Class III malocclusion.

Centric Relation↗

PubMed2000

Analysis of mRNA levels for the MHC class I-like molecules CD1 and FcRn in preimplantation mouse embryos.

PROBLEM: Major histocompatibility complex (MHC) antigens expressed on preimplantation embryos are important for the control of development, reproduction, and allo-recognition of the embryo by the mother. Four types of MHC class I and MHC class I-like antigens have recently been defined: class Ia, class Ib, class Ic, and class Id, based on their similar three-dimensional protein structures. Class Ia and class Ib antigens are encoded in the MHC, whereas class Ic and class Id antigens are encoded by genes on other chromosomes. Both class Ia and class Ib MHC antigens are expressed on preimplantation mouse embryos. The function of the class Ia antigens on embryos is unknown, but the function of one class Ib antigen, Qa-2, the product of the Ped gene, has been found to control the rate of early cleavage division and subsequent embryo survival. The expression of class Ic and class Id antigens on preimplantation embryos has not yet been evaluated. In the present study, we report the analysis of mRNA expression of two class Id genes, CD1 and FcRn, in preimplantation mouse embryos. METHOD OF STUDY: A reverse transcription-polymerase chain reaction (RT-PCR) assay was performed to analyze mRNA levels for CD1 and FcRn in 1-cell, 2-cell, 8-cell, and blastocyst stage embryos from C57BL/6 mice. RESULTS: No expression of CD1 mRNA was found in any of the preimplantation embryos tested. As a by-product of this study, we found a mistake in the published sequence of the mouse CD1 gene: nucleotide 746 in the cDNA is a G not a C. This base change is in a site recognized by the restriction enzyme PstI, thereby eliminating a PstI cleavage site. Expression of mRNA for FcRn was found in all preimplantation stages tested. Higher levels of mRNA for FcRn were detectable in 2-cell and 8-cell embryos compared to 1-cell and blastocyst stage embryos. CONCLUSION: This study shows that mRNA for FcRn but not for CD1 is found in preimplantation mouse embryos.

Animals↗

PubMed1992

Regulation of MHC class I synthesis and expression by human neutrophils.

Human peripheral blood neutrophils (PMN) treated with granulocyte-macrophage CSF (GM-CSF) increase the amount of class I 42-kDa H chain and 12-kDa L chain, beta 2-microglobulin (beta 2m), that they synthesize by 2.1- and 2.6-fold, respectively. To determine whether the increase in translation was associated with an increase in levels of class I H chain transcript, RNA blot analysis was performed on PMN that had been cultured in the presence of GM-CSF. Under no conditions were there increased levels of class I H chain transcript when class I heterodimer protein synthesis was increased. In addition, there was neither an increase in the synthesis of H chain mRNA, as measured by transcription assay, nor an alteration in the degradation rates of class I H chain transcript in PMN cultured with GM-CSF. In situ hybridization demonstrated that both the percentage of PMN that expressed class I transcript and the relative amounts of transcript per cell in GM-CSF-cultured PMN were the same as those in control PMN. Although there is increased translation of class I heterodimer in PMN treated with GM-CSF, there is no increase in its expression on the plasma membrane. The maintenance of constant levels of class I on the plasma membrane is dependent on continued protein synthesis and is maintained by release of class I heterodimer and free beta 2m into the medium. Heterodimer is released in the context of plasma membrane-derived vesicles, whereas beta 2m is released as a soluble protein. Maintenance of constant levels of class I heterodimer on the plasma membrane is also regulated by constitutive internalization. Up to 30% of class I molecules bearing 125I-Fab-labeled mAb to class I are internalized over 2 h at 37 degrees C. Therefore, inducible synthesis of class I by PMN is likely a consequence of post-transcriptional regulation, whereas the continued synthesis of class I heterodimer is required for maintenance of its expression. Furthermore, there is no increase in class I expression, in spite of increased synthesis, due to the release of class I heterodimer and beta 2m and the internalization of class I heterodimer from the plasma membrane. Thus, PMN are capable of post-transcriptional regulation of protein synthesis and are able to modulate the expression of plasma membrane proteins by regulated expression, release, and internalization.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1977

Relay cell classes in the lateral geniculate nucleus of the cat and the effects of visual deprivation.

This study presents evidence that the X- and Y-cells described physiologically in the A laminae of the cat's dorsal lateral geniculate nucleus (LGN) are two morphologically distinct cell types recognizable in Golgi preparations. It is shown firstly that the three cell types seen in Golgi preparations of the A laminae (large and medium-sized principal cells and small interneurons-types 1,2 and 3 in the classification of Guillery, '66) may be identified in 1-mum Epon sections of osmicated material. While cell-diameter histograms prepared from serial 1-mum sections show a unimodal distribution of cell sizes, three populations can be distinguished if attention is paid to the presence or absence of large cytoplasmic inclusions (laminar bodies). These three populations consist of large cells lacking laminar bodies (Class I), medium-sized cells possessing laminar bodies (Class II) and small cells lacking them (Class III). That these three classes correspond to the three morphological types has been shown by (i) size comparisons, and (ii) direct demonstration of laminar bodies in the Golgi-impregnated cell bodies of Guillery's type 2 cells. Histograms prepared in this way for samples taken at various positions in the LGN show that the numbers of class II cells decline from the representation of the area centralis to the monocular segment. This decline is compensated by a corresponding rise in the numbers of class I cells. This pattern of distribution is similar to the physiologically observed distribution of X- and Y-cells, indicating that X-cells are likely to be class II cells and Y-cells class I cells. The cortical projections of the various cell types have been examined by the horseradish peroxidase method. Class II cells project to area 17 only. Most class I cells also project to area 17 only, but a few very large class I cells project to area 18. From our results, it appears that very few if any cells in the A laminae have branching axons supplying both 17 and 18. The class III cells do not project to the visual cortex, a finding consistent with their identification as interneurons. Class I and II cells are also found in lamina C and in the MIN. In both these regions there is a predominance of very large class I cells, which project to area 18. Laminae Cl-C3 contain small cells lacking laminar bodies. These cells may project to both areas 17 and 18 with branching axons. They are likely to correspond to Guillery's type 4 cells (small relay cells confined to the C laminae) and to the physiologically described W-cells. Long-term monocular deprivation causes cell shrinkage which is much more severe for class I than for class II cells. There is in addition a decrease in the relative numbers of class I cells. This decrease is found in binocular deprivation also. These observations provide an anatomical basis for the reported loss of Y-cells from deprived laminae of the LGN. It is suggested that the effects of deprivation on Y-cells may be accounted for in terms of competition for synaptic space.

Animals↗

PubMed1995

The vertebrate alcohol dehydrogenase system: variable class II type form elucidates separate stages of enzymogenesis.

A mixed-class alcohol dehydrogenase has been characterized from avian liver. Its functional properties resemble the classical class I type enzyme in livers of humans and animals by exhibiting low Km and kcat values with alcohols (Km = 0.7 mM with ethanol) and low Ki values with 4-methylpyrazole (4 microM). These values are markedly different from corresponding parameters of class II and III enzymes. In contrast, the primary structure of this avian liver alcohol dehydrogenase reveals an overall relationship closer to class II and to some extent class III (69 and 65% residue identities, respectively) than to class I or the other classes of the human alcohol dehydrogenases (52-61%), the presence of an insertion (four positions in a segment close to position 120) as in class II but in no other class of the human enzymes, and the presence of several active site residues considered typical of the class II enzyme. Hence, the avian enzyme has mixed-class properties, being functionally similar to class I, yet structurally similar to class II, with which it also clusters in phylogenetic trees of characterized vertebrate alcohol dehydrogenases. Comparisons reveal that the class II enzyme is approximately 25% more variable than the "variable" class I enzyme, which itself is more variable than the "constant" class III enzyme. The overall extreme, and the unusual chromatographic behavior may explain why the class II enzyme has previously not been found outside mammals. The properties define a consistent pattern with apparently repeated generation of novel enzyme activities after separate gene duplications.

Alcohol Dehydrogenase↗

PubMed1998

Increasing incidence of childhood class V lupus nephritis.

OBJECTIVE: Class V lupus nephritis (LN) is reported to occur in 0-23% of patients with LN. To better characterize pediatric LN, we determined at a single center (1) the relative frequency of Class V LN on first and serial biopsies, (2) the frequency of transformation between LN classes on serial biopsies, (3) types of treatment received and outcome to date of different classes of LN. METHODS: All pediatric renal biopsies from 1985 to the present performed for diagnosis and classification of suspected LN were reviewed. Biopsy results were grouped into 2-3 year time intervals to assess trends in the distribution of WHO class diagnoses over time. RESULTS: Sixty patients underwent 97 renal biopsies. Class V LN was present in 28% (17/60) of patients on first biopsy, and in 37% (22/60) on most recent biopsy. Class V LN on first biopsy increased from 17% (8/46) before 1995 to 64% (9/14) after 1995 (p < 0.001). Age at presentation, age at biopsy, time to biopsy, and types of treatment did not differ before and after 1995. Transformation to Class V LN occurred in 19% (5/27) of patients having repeat biopsies. No transformation from Class V LN occurred on repeat biopsy. Renal outcome was available in 48 patients with followup of 4.7 +/- 3.2 years for Class V LN, and 5.2 +/- 2.4 years for non-Class V LN. Five percent (1/20) of Class V LN patients had renal dysfunction or had died compared to 21% (6/28) of non-Class V LN patients (p = NS). CONCLUSION: We found (1) a greater frequency of Class V LN than has been reported, (2) a recent increase in the incidence of Class V LN at our institution, (3) frequent transformation between classes on serial biopsies, and (4) no regression of Class V lesions in patients who had repeat biopsies.

Adolescent↗

PubMed1999

Human parainfluenza virus type 3 up-regulates major histocompatibility complex class I and II expression on respiratory epithelial cells: involvement of a STAT1- and CIITA-independent pathway.

Human parainfluenza virus type 3 (HPIV3) infection causes severe damage to the lung epithelium, leading to bronchiolitis, pneumonia, and croup in newborns and infants. Cellular immunity that plays a vital role in normal antiviral action appears to be involved, possibly because of inappropriate activation, in the infection-related damage to the lung epithelium. In this study, we investigated the expression of major histocompatibility complex (MHC) class I and II molecules on human lung epithelial (A549) and epithelium-like (HT1080) cells following HPIV3 infection. MHC class I was induced by HPIV3 in these cells at levels similar to those observed with natural inducers such as beta and gamma interferon (IFN-beta and -gamma). MHC class II was also efficiently induced by HPIV3 in these cells. UV-irradiated culture supernatants from infected cells were able to induce MHC class I but not MHC class II, suggesting involvement of released factors for the induction of MHC class I. Quantitation of IFN types I and II in the culture supernatant showed the presence of IFN-beta as the major cytokine, while IFN-gamma was undetectable. Anti-IFN-beta, however, blocked the HPIV3-mediated induction of MHC class I only partially, indicating that viral antigens, besides IFN-beta, are directly involved in the induction process. The induction of MHC class I and class II directed by the viral antigens was confirmed by using cells lacking STAT1, an essential intermediate of the IFN signaling pathways. HPIV3 induced both MHC class I and class II molecules in STAT1-null cells. Furthermore, MHC class II was also induced by HPIV3 in cells defective in class II transactivator, an important intermediate of the IFN-gamma-mediated MHC class II induction pathway. Together, these data indicate that the HPIV3 gene product(s) is directly involved in the induction of MHC class I and II molecules. The induction of MHC class I and II expression by HPIV3 suggests that it plays a role in the infection-related immunity and pathogenesis.

Animals↗

PubMed1999

Social class, coronary risk factors and undernutrition, a double burden of diseases, in women during transition, in five Indian cities.

OBJECTIVE: To find out the association between social class and coronary risk factors in women. DESIGN AND SETTING: Cross-sectional surveys were conducted in six-twelve urban streets in each of five cities from various regions of India following a common study protocol and criteria of diagnosis. SUBJECTS AND METHODS: We randomly selected 3257 women, aged 25-64 years inclusive, from the cities of Moradabad (n=902), Trivandrum (n=760) Calcutta (n=410), Nagpur (n=405) and Bombay (n=780). Evaluation was by questionnaires validated at Moradabad. All subjects, after pooling of data, were divided into social class 1 (n=985), social class 2 (n=790), social class 3 (n=674), social class 4 (n=602) and social class 5 (n=206), based on various attributes of socioeconomic status. RESULTS: The prevalence of hypertension, diabetes mellitus, family history of coronary disease, obesity, central obesity and sedentary lifestyle were significantly associated with higher social classes and tobacco consumption was not associated with social class. Oral contraceptive intake and postmenopausal status were also more common among higher social classes, which may be due to more education and a longer lifespan among the higher social classes, respectively. Mean total cholesterol, high density lipoprotein cholesterol, systolic and diastolic blood pressure, mean body mass index and waist-hip ratio showed significant association with higher social classes. Mean age, body weight, body mass index, waist-hip ratio, systolic and diastolic blood pressure, total cholesterol and 2-h blood glucose were significantly positively correlated with social class, as assessed by Spearman's rank correlation. Higher social classes 1-3 were more common in Trivandrum and Bombay than in Moradabad. The prevalence of hypertension, diabetes mellitus and being overweight (body mass index >25 kg/m2) were also more common in Trivandrum and Bombay compared to Moradabad. Undernutrition was negatively associated with higher social classes and was more common in Moradabad and Nagpur than Trivandrum. CONCLUSIONS: Higher social classes among Indian urban women have a higher prevalence of coronary risk factors, hypertension, diabetes mellitus, being overweight, central obesity, sedentary lifestyle, family history of coronary disease, oral contraceptive intake and postmenopausal status. Mean concentrations of total and high density lipoprotein cholesterol were also significantly associated with higher social classes.

Adult↗

PubMed1999

Class I MHC molecules: assembly and antigen presentation.

Several years ago, the only factor known to be necessary for the assembly and surface expression of class I MHC was beta 2m; even for beta 2m, it was unclear at what point in class I maturation its role was played. Recent experiments that employed attachment of an endoplasmic reticulum (ER) retention signal to beta 2m have shown that the point of time at which beta 2m is required is while the class I heavy chain is in the ER. Later association between beta 2m and class I is not vital in order for properly folded class I to be expressed at the cell surface. After crystallization of the first class I MHC molecule, it was realized that not only is antigen presented by class I, but that antigen is presented in the form of a peptide that stabilizes the class I structure and allows its transit to the cell surface. Class I allelic differences influence interactions with both peptide and beta 2m, with likely consequences for the ability of the class I heavy chains to present antigen through alternative pathways. Furthermore, it is now also clear that formation of appropriate disulfide bonds in the class I heavy chain is needed before class I can bind peptide antigen securely, a process that may be assisted by an ER chaperone. Many different proteins that are resident in the ER, such as calnexin, transporter associated with antigen processing (TAP), calreticulin, and tapasin, have been found to be integral to class I assembly. TAP, tapasin, and calreticulin bind preferentially to the open form of class I, which can be distinguished with the use of a monoclonal antibody specific for this form. Calreticulin and calnexin contrast in their interactions with class I, despite other similarities between these two chaperones. Overall, class I MHC assembly is now understood to involve the interplay of multiple intra- and intermolecular events in a defined chronological order which ensure continual reporting of cellular contents to cytotoxic T lymphocytes.

Alleles↗

PubMed1993

Invariant chain retains MHC class II molecules in the endocytic pathway.

During exocytosis of MHC class II, the class II alpha beta heterodimer associates with a third polypeptide termed invariant chain (Ii). Class II and Ii are coordinately processed and transported until proteolytic cleavage of Ii in an acidic compartment immediately before class II surface expression. Although the lysosomotropic agent chloroquine (CQ) prevents dissociation of class II/Ii within the cell, the ultimate fate of these complexes has not been determined. We considered two alternative possibilities. If Ii encodes an intracellular retention signal, then persistent association of Ii with class II could lead to intracellular accumulation of class II/Ii complexes. Alternatively, if Ii does not block further transport of class II, then CQ treatment should result in aberrant expression of class II/Ii complexes at the cell surface. Ltk- and EL4 cells transfected with I-A(d) alone or I-A(d) plus Ii were treated with CQ and examined for changes in surface class II and Ii expression. Anti-Ii mAb surface staining did not increase with prolonged CQ treatment, but a dramatic decrease in surface class II staining was observed. This decrease in class II was observed both with genomic Ii and p31 cDNA transfectants and was rapidly reversed upon drug removal. Accumulation of Ii and class II within treated cells was directly observed by intracellular staining. Similar effects on MHC surface expression were observed with the lysosomotropic agents primaquine and NH4Cl and the cysteine protease inhibitor leupeptin. Ii-negative cells treated in parallel displayed no effect of the lysosomotropic agents or leupeptin on class II surface staining. These results indicate that dissociation of Ii from newly synthesized class II is required for transport of the alpha beta dimer to the cell surface, and suggest that Ii serves to retain class II molecules in a post-Golgi endocytic compartment.

Ammonium Chloride↗

PubMed1997

Initiation of autoimmune diabetes in NOD/Lt mice is MHC class I-dependent.

MHC class II alleles clearly contribute a primary genetic component of susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. However, IDDM does not occur in NOD mice made MHC class I-deficient by a functionally inactivated beta2-microglobulin allele (beta2m(null)). In the present study the beta2m(null) mutation was used to examine the relative contributions of MHC class I and class II-dependent T cell responses for initiating autoimmune pancreatic beta cell destruction in NOD mice. Splenocytes from diabetic NOD donors transferred IDDM to both lymphocyte-deficient NOD-scid (class I+) and NOD-scid.beta2m(null) mice (class I-). In contrast, splenocytes from young prediabetic NOD donors only transferred IDDM to class I+, but not class I- NOD-scid recipients. However, splenocytes from prediabetic NOD donors did transfer IDDM to NOD-scid.beta2m(null) recipients previously engrafted with class I+, but not class I-, pancreatic islets. CD4+ T cell lines reactive against some syngeneic class I+ targets could be isolated from NOD.beta2m(null) mice. However, NOD.beta2m(null) T cells underwent activation-driven deletion when transferred into class I+ NOD-scid recipients. Hence, the class I autoreactive T cells present in NOD.beta2m(null) donors did not elicit IDDM when transferred into class I+ NOD-scid recipients. Collectively, these results show that autoimmune IDDM in NOD mice is initiated by MHC class I-dependent T cell responses, but this leads to the subsequent activation of additional T cell populations that can mediate pancreatic beta cell destruction in a MHC class I-independent manner.

Adoptive Transfer↗

PubMed1991

Comparison of 'normal' TMJ-function in Class I, II, and III individuals.

Fifty-seven non-orthodontically treated young adults, judged as either clinically healthy or with only mild symptoms of TMJ-dysfunction, were divided into groups according to their sagittal molar relationship (Class I-, Class II-, and Class III-group). All underwent a standardized clinical and axiographic examination with an axiographic recording device (SAS-SYSTEM). The comparison of the lengths of the axiographical protrusive curves showed significantly higher values in the Class II group than in the Class I (P less than 0.01) and in the Class I group than in the Class III group (P less than 0.001). Analogous differences could be found in clinical protrusive measurements at the incisal point and in lateral excursions. It was concluded that the protrusive condylar movement capacity coincides with the dental sagittal relationship. In opening, significant differences were seen axiographically (Class I v. Class III: P less than 0.001; Class II v. Class III: P less than 0.01), but not clinically. Since axiographic measurements show only condyle translation, but not rotation in contrast to measurements at the incisal point, it can be concluded that the differences in opening are also translatory and not rotational. Measurements of the average change of curvature of axiographic tracings showed significantly less curved protrusive tracings in Class III than in Class I (P less than 0.001) and Class II individuals (P less than 0.01), supporting the concept that in the Class III group condyle movement is restricted to the straight posterior slope of the tubercle.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Early events in the assembly of major histocompatibility complex class II heterotrimers from their free subunits.

Endogenous antigen presentation by major histocompatibility complex class II molecules can be understood if class II alpha beta heterodimers bind peptide in the endoplasmic reticulum (ER) before they associate with the invariant chain (Ii). We have studied the assembly of class II molecules from the free alpha, beta and Ii subunits to examine the existence of a class II alpha beta heterodimer as an intermediate in the assembly of class II alpha beta Ii heterotrimers in the ER. In human kidney cell transfectants, the free class II alpha and beta subunits and the class II alpha beta heterodimer are retained in the ER by association with the chaperonin immunoglobulin binding protein (BiP) and Ii is retained through its cytoplasmic tail. Co-expression of Ii results in release of BiP from class II alpha beta complexes and exit of class II alpha beta Ii heterotrimers from the ER. We show that the cytoplasmic tail and the transmembrane region of the class II alpha and beta chain is not essential for proper assembly of the class II alpha beta heterodimer. We followed assembly of the class II alpha beta Ii heterotrimers in wild-type cells. The class II subunits assemble post-translationally. No class II alpha beta heterodimers could be isolated as intermediates in the formation of class II alpha beta Ii heterotrimers, suggesting that peptide binding by class II molecules in the ER is necessarily inefficient.

Base Sequence↗

PubMed1996

Can we subtype alcoholism? A latent class analysis of data from relatives of alcoholics in a multicenter family study of alcoholism.

We attempt to identify distinctive subtypes of alcoholics using latent class analysis with data from 2551 relatives of alcoholic probands, all participants in the Collaborative Study of the Genetics of Alcoholism. Latent class analysis is a multivariate technique using cross-classified data to identify unobserved ("latent") classes that explain the relationships among observed variables. Data on 37 life-time symptoms of alcohol dependence from 1360 female and 1191 male relatives were analyzed, with a 4 class solution selected as the best fitting among the 2 through 6 class solutions that were examined. We observed the following classes: class 1, nonproblem drinkers (39.6% male, 50% female); class 2, mild alcoholics (persistent desire to stop, tolerance, and blackouts) (31.8% male, 28.7% female); class 3, moderate alcoholics (social, health, and emotional problems) (18.9% male, 14.6% female); and class 4, severely affected alcoholics (withdrawal, inability to stop drinking, craving, health, and emotional problems) (9.7% male, 6.7% female). There was little evidence for the construct of alcohol abuse; endorsement probabilities for abuse symptoms (e.g., arrest and DWIs) were very low for all classes, whereas hazardous use was common among men in class 1. In addition to those in class 3 and class 4, a majority of men in class 2 qualified for DSM-III-R alcohol dependence, suggesting a biomodal distribution of drinkers and alcoholics, with little nondependent problem drinking among men in this high-risk sample. We conclude that, in this sample, alcoholism is not differentiated by symptom profiles but rather lies on a continuum of severity, with the possible exception of withdrawal, which characterized only class 4 individuals.

Adolescent↗

PubMed2006

Melanosomal targeting sequences from gp100 are essential for MHC class II-restricted endogenous epitope presentation and mobilization to endosomal compartments.

CD4+ T lymphocytes play an important role in CD8+ T cell-mediated responses against tumors. Considering that approximately 20% of melanomas express MHC class II, it is plausible that concomitant presentation by MHC class I and class II shapes positive (helper T cells) or negative (regulatory T cells) antitumor responses. Interestingly, gp100, a melanoma antigen, can be presented by both MHC class I and class II when expressed endogenously, suggesting that it can reach endosomal/MHC class II compartments (MIIC). Here, we showed that gp100 putative NH2-terminal signal sequence and the last 70 residues in COOH terminus are essential for MIIC localization and MHC class II presentation. Confocal microscopy analyses confirmed that gp100 was localized in LAMP-1+/HLA-DR+ endosomal/MIIC. Gp100 targeting sequences were characterized by deleting different sections in the COOH terminus (last 70 residues). Transfection in 293T cells, expressing MHC class I and class II molecules, revealed that specific deletions in COOH terminus resulted in decreased MHC class II presentation, without effects on class I presentation, suggesting a role in MIIC trafficking for these deleted sections. Then, we used these gp100 targeting sequences to mobilize green fluorescent protein to endosomal compartments and to allow MHC class II and class I presentation of minimal endogenous epitopes. We conclude that these specific sequences are MIIC-targeting motifs, which could be included in expression cassettes for endogenously expressed tumor or viral antigens for MHC class II and class I presentation and optimize in vivo T-cell responses or as an in vitro tool for characterization of new MHC class II epitopes.

Antigen Presentation↗

PubMed1997

MHC class I and II expression in prostate carcinoma and modulation by interferon-alpha and -gamma.

BACKGROUND: Expression of Major Histocompatibility Complex (MHC) class I and II antigens are critical for the cellular immune response. Loss of MHC expression represents one mechanism by which cancer cells escape immune recognition. PURPOSE: To define MHC class I and II expression by prostate cancer (PCa) in vivo and in vitro and the ability to modulate MHC expression in vitro with IFN-alpha and -gamma. METHODS: Frozen tissue sections of 25 benign prostatic hyperplasia (BPH) and 18 PCa specimens were studied by immunohistochemistry. PCa cell lines LNCaP, PC-3, and DU-145 were studied by FACS, ELISA, and cytospin. Class I was detected by monoclonal antibody (mAb) W6/32, and class II by mAb 13.17. The effects of IFN-alpha and -gamma were assessed by testing the three cell lines in the presence or absence of varying concentrations of the cytokine for varying incubation times. RESULTS: Class I was strongly expressed by 24/25 BPH specimens; 4/18 (22%) PCa were homogeneously class I-positive, while 5/18 (28%) were heterogeneously positive and 9/18 (50%) were class I-negative. PC-3 and DU-145 expressed normal levels of class I, while LNCaP expressed only low levels. All line except LNCaP demonstrated significant up-regulation of class I with either IFN-alpha or -gamma. Class II expression was not seen in BPH epithelium nor in 17/18 PCa. Class II could be only weakly induced in the three PCa lines. CONCLUSIONS: These findings confirm prior studies demonstrating that class I expression is commonly lost or diminished in PCa. In addition, class II up-regulation by IFN-gamma appears very limited in relation to other normal or neoplastic epithelium. IMPLICATIONS: The present findings, taken together with previous studies, are most consistent with the expression of neoantigens by PCa, which are recognized and appropriately eliminated by the cellular immune system. This selective pressure favors outgrowth of cells which down-regulate or lose class I and/or class II expression. Understanding PCa immunobiology will help in the development of effective immunotherapy for this disease.

Enzyme-Linked Immunosorbent Assay↗

PubMed2003

Investigations into the role of anti-HLA class II antibodies in TRALI.

BACKGROUND: TRALI is thought to be triggered by recipient-specific anti-HLA class I or antibodies against neutrophils in donor plasma. Recently, anti-HLA class II have also been implicated. The prevalence of anti-HLA class II was investigated in normal volunteer platelet donors and in two nonfatal TRALI cases utilizing a flow-based assay. Potential target antigens also were investigated. STUDY DESIGN AND METHODS: Commercial flow cytometry-based assays (FlowPRA, One Lambda, Inc.) for anti-HLA class I and II were compared with standard lymphocytotoxicity tests. Subsequently, 151 volunteer platelet donors and two clinical cases of TRALI were screened with FlowPRA. Immunohistochemical studies were performed on lung tissue from a surgical case, an autopsy case, and a fatal TRALI case. RESULTS: The FlowPRA assays showed moderate concordance for anti-HLA class I (kappa = 0.448) and good concordance for class II antibodies (kappa = 0.801), when compared to standard lymphocytotoxicity assays. Ten and 9 percent of female platelet donors were positive for anti-HLA class I and class II, respectively. Two nonfatal cases of TRALI showed both anti-HLA class I and anti-HLA class II. Immunohistochemical analysis of a TRALI case revealed granulocyte aggregation in alveolar capillaries with activated vascular endothelial cells. HLA class II antigen expression was not present on vascular endothelium or intravascular WBCs; however, strong expression was seen on alveolar macrophages. CONCLUSION: FlowPRA assays often detect anti-HLA class I not detected by conventional lymphocytotoxicity assays. These assays reveal anti-HLA class II in normal female donor plasma and in sera implicated in TRALI. Immunohistochemical studies failed to reveal endothelial or intravascular-WBC HLA class II antigen expression in lung tissue derived from TRALI cases or controls, but demonstrated HLA class II expression on pulmonary macrophages.

Cell Adhesion↗

PubMed1995

Tap-1 and Tap-2 gene therapy selectively restores conformationally dependent HLA Class I expression in type I diabetic cells.

Genetic susceptibility to many autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM) is statistically linked to the HLA class II region of chromosome 6. However, a distinguishing feature of patients with HLA class II-linked autoimmune disease is an abnormally low density of conformationally correct, self-peptide filled HLA class I molecules on the lymphocyte cell surface. The transporters associated with antigen processing (Tap-1 and Tap-2) are essential for normal class I expression and presentation of intracellular peptides, and these genes are located within the HLA class II region. The aims of this project were to determine if Tap genes could be implicated in the defective class I expression associated with IDDM by using a novel Epstein-Barr virus (EBV)-mediated gene transfer system to introduce a cloned, normal Tap-2 or Tap-1 gene into B cell lines from normal and IDDM patients and analyzing the effect on conformationally dependent class I expression. The results show that Tap-2 gene transfer in B cells from 40% of randomly selected IDDM patients increased expression of conformationally correct, cell-surface class I molecules to levels comparable with similarly treated B cells from normal control individuals. B cells from another 40% of IDDM patients responded to Tap-1 gene transfer. These effects were specific because B cells from normal individuals did not respond to Tap-1 or Tap-2 gene transfer with increased class I expression, and B cells from IDDM patients responding to Tap-2 gene transfer did not respond to Tap-1 gene transfer and vice versa. Thus, these complementation studies identify distinct, non-overlapping subsets of IDDM patients whose class I defect in B cells can be reversed by Tap-1 or Tap-2 gene transfer. The increase in class I expression induced by Tap gene transfer is associated with a reduction in the number of peptide-empty class I molecules as demonstrated by the response to exogenous peptide loading. Furthermore, the increase in self-peptide filled class I molecules induced by Tap gene transfer into B cells from IDDM patients is associated with restored antigen presentation to autologous T cells. These studies conclude that Tap gene dysfunctions may contribute to the defect in class I phenotype and antigen presentation demonstrated by IDDM patients. Defective presentation of self-peptides by antigen presenting cells can lead to the failed T cell education and tolerance to self antigens evident in IDDM. These studies functionally identify HLA class II region genes that contribute to an immunologic defect in IDDM.

ATP Binding Cassette Transporter, Subfamily B, Mem↗