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HLA class II antigens and DNA restriction fragment length polymorphism in myasthenia gravis in Japan.

Human leukocyte phenotypes and genes in the HLA class II regions were studied in 46 Japanese patients with myasthenia gravis. When the HLA phenotypes of the patients with myasthenia gravis were compared with the controls, an increased frequency of HLA-DRw53 was observed in females less than 30 years of age. The genomic DNAs of the HLA-DRw53-positive patients and DRw53-positive controls were analyzed by using four complementary DNA probes for HLA class II genes. With DQB complementary DNA as the probe, a higher incidence of the 6.5-kb or 8.2-kb BamHI fragment was observed in the patients (76.0%) compared with the controls (19.0%). In contrast, no significant difference was observed between patients and controls when complementary DNAs for DRB, DQA, and DPB were used as probes. These results indicate that the genetic background of Japanese females with early-onset myasthenia gravis is different from other patients with myasthenia gravis, and that DQB genes can greatly influence the onset of myasthenia gravis.

Adolescent↗

HLA class II polymorphism in Thai patients with non-Hodgkin's lymphoma.

The distribution of HLA-DRB1 alleles and DQB1 alleles in 100 Thai patients with non-Hodgkin's lymphoma (NHL) was analysed using the polymerase chain reaction with sequence-specific primer (PCR-SSP) method, and the association between the disease and the presence of certain HLA class II alleles was investigated. The frequencies of HLA-DRB1*1502 and DRB1*09012 were increased while those of DRB1*0404, DRB1*0803 and DRB1*1106 were decreased. On the other hand, the incidence of HLA-DQB1 alleles was similar to that in the normal population. Interestingly, only HLA-DRB1*1502 showed a significant positive association with NHL, especially in patients < or / = 45 years and in male patients. It is concluded that the DRB1*1502 allele may contribute to NHL susceptibility in the Thai population. However, further studies on the functional roles of the HLA class II alleles are necessary to elucidate NHL susceptibility.

Adult↗

A recombinant bispecific single-chain Fv antibody against HLA class II and FcgammaRIII (CD16) triggers effective lysis of lymphoma cells.

Bispecific antibodies offer the possibility of improving effector-cell recruitment for antibody therapy. For this purpose, a recombinant bispecific single-chain Fv antibody (bsscFv), directed against FcgammaRIII (CD16) and human leucocyte antigen (HLA) class II, was constructed and tested in functional assays. RNA from the hybridomas 3G8 and F3.3, reacting with CD16 and HLA class II, respectively, was used to generate phage display libraries. From these libraries, reactive phages were isolated and the bsscFv was constructed by connecting both single-chain Fv components through a 20 amino acid flexible linker. After expression in SF21 insect cells and chromatographic purification, the bsscFv bound specifically and simultaneously to both antigens. The affinities of the anti-CD16 and the anti-HLA class II scFv components of the bsscFv were 8.6 x 10(-8) mol/l and 13.7 x 10(-8) mol/l, respectively, which was approximately sevenfold lower than the F(ab) fragments of the parental antibodies. In antibody-dependent cellular cytotoxicity experiments with human mononuclear cells as effectors, the bsscFv-mediated specific lysis of both HLA class II-positive, malignant human B-lymphoid cell lines and primary cells from patients with chronic B-cell lymphocytic leukaemia. Optimal lysis was obtained at bsscFv concentrations of approximately 400 ng/ml, similar to the concentration required for maximum lysis by the corresponding chemically linked bispecific antibody. Thus, this recombinant bsscFv-antibody is an efficient molecule for effector-cell mediated lysis of malignant human B-lymphoid cells.

Animals↗

Class-II HLA restriction of antigen-specific human T-lymphocyte clones. Evidence of restriction elements on both DR and MT molecules.

Blast-enriched suspensions of T cells primed for Chlamydia trachomatis antigen were cloned by a limiting dilution technique. The class-II HLA restriction of T-lymphocyte clones (TLC) was studied by using allogeneic antigen-presenting cells (APC) carrying foreign class-II HLA antigens. Most of the TLC were restricted by one or the other of the D/DR determinants of the T-cell donor; that is, they did not respond when antigen was presented by APC expressing foreign D/DR determinants. Furthermore, heterogeneity of the DR4-expressing molecule could be demonstrated by T-cell clones from one person; APC from family members expressing DR4 gave high proliferative responses, whereas no proliferation was observed with most APC from unrelated persons expressing DR4. This heterogeneity of DR4 was confirmed by mixed lymphocyte culture (MLC) experiments, indicating a close relationship between restriction epitopes and those that activate allogeneic T cells. Other clones seemed to be restricted by other class-II HLA determinants, most probably MT determinants of the T-cell donor. The restriction specificities were confirmed by subcloning experiments.

Antigens, Bacterial↗

Identification of an association between HLA class II alleles and low antibody levels after measles immunization.

This is the first large cohort study to report a genetic association between humoral antibody level after measles vaccine and the HLA class II genes. The WHO goal to eradicate measles world-wide magnifies the importance of data relating to the influence of immunogenetics on measles vaccine-induced antibody responses. We present here the analysis of 242 individuals who received one dose of measles-mumps-rubella-II (MMR-II) vaccine at the age of 15 months and were genotyped for HLA class II alleles. These subjects fit into one of three categories; 72 were classified as seronegative, 93 were seropositive and 77 were serohyperpositive. HLA-DRB1*03 (odds ratio (OR), 2.22) and HLA-DPA1*0201 (OR, 1.71) were significantly associated with measles vaccine seronegativity, while additional alleles provided suggestive evidence of association with seronegativity: DQA1*0201, DQB1*0201, and DQA1*0501. The alleles DRB1*03 and DQA1*0201 remained statistically significant after accounting for the effects of other alleles. These findings are crucial in designing both measles eradication by the use of vaccine, and future vaccines to be used in genetically heterozygous populations.

Alleles↗

Analysis of soluble HLA class II antigenic material in patients with immunological diseases using monoclonal antibodies.

A quantitative assay for soluble human HLA Class II antigenic material is described. The method is a double-determinant ELISA using 2 monoclonal antibodies which recognize different epitopes on the antigen. The first rat monoclonal antibody captures antigen onto the microplate where it is then recognized by a second mouse monoclonal antibody. Bound mouse immunoglobulin is then detected by an enzyme-linked rat polyclonal antibody. The assay has been used to measure soluble HLA Class II antigen in the sera from healthy individuals and patients with leukaemia, and has shown raised levels in acute lymphoblastic but not in chronic lymphocytic leukaemia. Similar material has been identified in synovial fluid of patients with active rheumatoid arthritis. The assay is specific and simple to perform and should be applicable to probing the nature of the soluble material and its mechanism of release.

Antibodies, Monoclonal↗

HLA class II genotypic frequencies in atopic asthma: association of DRB1*01-DQB1*0501 genotype with Artemisia vulgaris allergic asthma.

Different human leukocyte antigen (HLA) class II alleles have been associated with the development of atopic asthma. To determine whether HLA class II alleles are associated with atopic asthma in a population from southeast Spain (Murcia region), 213 atopic asthmatic patients and 150 controls were selected for HLA typing. Significant association of the DRB1*01 and DQB1*0501 alleles was found in Artemisia vulgaris allergic patients (p(c) = 0.00052 and p(c) = 0.00023, respectively). No significant correlation was found in other atopic patients allergic to pollens (Phleum pratense, Olea europaea, and Salsola kali), house dust mites (Dermatophagoides pteronyssinus, D. farinae), molds (Alternaria alternata, Cladosporium herbarum), or animal danders (dog, cat). The results reveal that the DRB1*01-DQB1*0501 genotype is strongly associated with a positive response to Artemisia vulgaris in the population studied.

Alleles↗

Gestational diabetes mellitus and HLA class II (-DQ, -DR) association: The Digest Study.

Gestational diabetes mellitus (GDM) and impaired glucose tolerance during pregnancy (IGT) are associated with an increased risk of perinatal morbidity and then further development of diabetes among 30-50% of affected women. This is a real public health problem that deserves investigation of phenotypic and genotypic predisposing markers. However, the involvement of genetic background in GDM and IGT remains unclear. In particular, association with HLA class II polymorphism has been poorly studied and has produced conflicting results. In attempt to clarify these discrepancies, we investigated HLA class II polymorphism in 95 GDM and 95 IGT women from the north of France using DNA amplification followed by restriction enzyme digestion (PCR-RFLP). Ninety-five pregnant women with normal glucose tolerance (NGT) were chosen as a control reference group. The distribution of HLA class II polymorphism was not found to be significantly different between GDM, IGT and NGT samples. In particular, we did not find any significant variation of DRB1*03 and DRB1*04 allele frequencies between these three groups. These data provide further evidence that insulin-dependent diabetes mellitus (IDDM) HLA class II susceptibility alleles cannot serve as genetic markers for susceptibility to glucose intolerance during pregnancy. However, GDM and IGT were not equivalent to the NGT control group and presented particular HLA patterns. In particular, we observed an increase of the DRB1*0701-DQA1*0201-DQB1*02 haplotype in GDM women (P = 0.02; Pc not significant) and an increase of DRB1*0101-DQA1*0101-DQB1*0501 and DRB1*1302-DQA1*0102-DQB1*0604 haplotypes in the IGT group (P = 0.02 and 8 x 10(-3), respectively; Pc not significant). In contrast, we found a decrease in the DRB1*1101 allele in IGT samples (P = 0.03; Pc not significant) and a decrease of DRB1*1103-*1104 alleles in the GDM group (P = 9 x 10(-3); Pc not significant). Although these findings are only descriptive, it points out the genetic heterogeneity of glucose intolerance during pregnancy.

Alleles↗

Testicular carcinoma and HLA Class II genes.

BACKGROUND: The association with histocompatibility antigens (HLA), in particular Class II genes (DQB1, DRB1), has recently been suggested to be one of the genetic factors involved in testicular germ cell tumor (TGCT) development. The current study, which uses genotyping of microsatellite markers, was designed to replicate previous associations. METHODS: In 151 patients, along with controls comprising parents or spouses, the HLA region (particularly Class II) on chromosome 6p21 was genotyped for a set of 15 closely linked microsatellite markers. RESULTS: In both patients and controls, strong linkage disequilibrium was observed in the genotyped region, indicating that similar haplotypes are likely to be identical by descent. However, association analysis and the transmission disequilibrium test did not show significant results. Haplotype sharing statistics, a haplotype method that derives extra information from phase and single marker tests, did not show differences in haplotype sharing between patients and controls. CONCLUSION: The current genotyping study did not confirm the previously reported association between HLA Class II genes and TGCT. As the HLA alleles for which associations were reported are also prevalent in the Dutch populations, these associations are likely to be nonexistent or much weaker than previously reported.

Adolescent↗

HLA class II alleles in systemic sclerosis patients with anti-RNA polymerase I/III antibody: associations with subunit reactivities.

OBJECTIVE: To examine HLA class II gene associations with anti-RNA polymerase (RNAP) I/III antibody responses in patients with systemic sclerosis (SSc). METHODS: HLA-DRB1, DRB3, DRB4, and DQB1 alleles were determined using polymerase chain reaction-based methods in 257 SSc patients (129 Japanese and 128 Caucasians) and 271 race-matched regional controls (138 Japanese and 133 Caucasians). Anti-RNAP I/III antibodies were identified by immunoprecipitation assay, and reactivities to individual RNAP subunits were determined by immunoblots using affinity-purified RNAP I, II, and III. RESULTS: Serum anti-RNAP I/III antibody was detected in 10 (8%) Japanese and 24 (19%) Caucasian patients with SSc. The presence of anti-RNAP I/III antibodies was associated with DRB1*0405, DRB4*01, and DQB1*0401 in Japanese, and with DRB3*02 in Caucasians, but these associations were weak and inconsistent between these 2 ethnic groups. When anti-RNAP I/III-positive SSc patients were divided into 2 groups based on the presence or absence of reactivities to individual RNAP subunit proteins, significant associations of anti-IIa/IIo reactivity with DRB3*02, anti-Ia reactivity with DRB1*04, anti-43-kDa subunit reactivity with DRB4*01, and anti-34-kDa subunit reactivity with DRB1*15 were detected. These HLA associations with subunit reactivities were generally shared by Japanese and Caucasian patients with SSc. CONCLUSION: Our results suggest that in patients with SSc, anti-RNAP I/III antibodies are composed of subsets defined by combinations of reactivities to individual RNAP subunits having specific HLA class II correlations.

Alleles↗

Common epitope in human immunodeficiency virus (HIV) I-GP41 and HLA class II elicits immunosuppressive autoantibodies capable of contributing to immune dysfunction in HIV I-infected individuals.

We previously reported the identification of highly conserved homologous regions located in the carboxy terminus of the HIV I gp41-envelope (aa 837-844), and the amino-terminal of the beta chain of all human HLA class II antigens (aa 19-25). Murine monoclonal antibodies, raised against synthetic peptides from these homologous regions, bound not only to the isolated peptides, but also to the native gp160 and class II molecules. In this study one-third of sera from HIV I-infected individuals, at different disease stages, were found to react with both the gp41 and class II-derived peptides. These sera also reacted with "native" HLA class II molecules. The potential affects of such autoantibodies on normal immune functions were examined. It was found that in the presence of class II-cross-reactive (but not control) sera, the proliferative responses of normal CD4+ T cells to tetanus toxoid and allogeneic stimuli were markedly decreased. In addition, these sera could eliminate class II-bearing cells by antibody dependent cellular cytotoxicity. Similar affects were seen with affinity-purified IgG antibodies from patients' sera. Thus, the "molecular mimicry" between HIV I and HLA class II antigens, may lead to the generation of autoantibodies in HIV I-infected individuals that may contribute to the early functional impairment of CD4+ T cell observed in many HIV I-infected individuals.

Acquired Immunodeficiency Syndrome↗

Antibody reactivity to the HRES-1 endogenous retroviral element identifies a subset of patients with systemic lupus erythematosus and overlap syndromes. Correlation with antinuclear antibodies and HLA class II alleles.

OBJECTIVE: To evaluate the correlation between the presence of antibodies to an endogenous retroviral element-encoded nuclear protein autoantigen, HRES-1, and the presence of other antinuclear antibodies and HLA class II alleles in patients with systemic lupus erythematosus (SLE) and overlap syndromes. METHODS: Antibody reactivities to native and recombinant proteins and synthetic peptides were assessed by counterimmunoelectrophoresis, enzyme-linked immunosorbent assay, and Western blotting. HLA class II alleles were determined by oligonucleotide typing. RESULTS: Forty-eight percent of the 153 patients with autoimmune disease, and 52% of the subgroup with SLE, had HRES-1 antibodies. In contrast, 3.6% of 111 normal donors, and none of 42 patients with the acquired immunodeficiency syndrome or 50 asymptomatic human immunodeficiency virus 1-infected patients, had HRES-1 antibodies. Chi-square analyses revealed a significant association between anti-HRES-1 and anti-RNP and an inverse correlation between HRES-1 and Ro/La autoantibodies in patients with SLE or overlap syndromes. Antigenic epitopes of HRES-1 and the retroviral gag-related region of the 70-kd protein component of U1 small nuclear RNP, which share 3 consecutive highly charged amino acids (Arg-Arg-Glu), an additional Arg, and functionally similar Arg/Lys residues, represent cross-reactive epitopes between the two proteins. Selective removal of HRES-1 antibodies from sera of HRES-1-seropositive/RNP-seropositive patients by absorption on recombinant HRES-1/glutathione-S-transferase-conjugated agarose beads had no effect on anti-RNP reactivities. A comparative multivariate analysis of HLA class II genes revealed a differential segregation of DQB1 alleles in HRES-1-seropositive versus HRES-1-seronegative patients (P = 0.04). While a relative increase of DQB1*0402 among HRES-1-seropositive patients was noted across ethnic groups (P = 0.02), a decrease of DQB1*0201 and DQB1*0301 was found in white HRES-1-seropositive patients (P = 0.04). CONCLUSION: Autoantibodies to HRES-1 are detectable in a distinct subset of patients with autoimmune disease, primarily in those who do not have antibodies to Ro and La. Anti-HRES-1 and anti-RNP reactivities are mediated by cross-reactive but separate antibody molecules. HLA-DQB genes, rather than HLA-DRB or DQA genes, may have a more significant influence on generation of these antinuclear autoantibodies.

Alleles↗

5' insulin gene polymorphism confers risk to IDDM independently of HLA class II susceptibility.

The polymorphic variable number of tandem repeats in the 5' upstream region of the human insulin gene is a well-known non-human leukocyte antigen locus contributing to genetic susceptibility to IDDM. Controversy exists about the question as to whether INS susceptibility haplotypes are or are not preferentially inherited together with HLA-DR4 haplotypes. We investigated whether genetic interaction between INS and the HLA complex can be better defined using DQ genotypic and phenotypic markers in addition to DR serology. The 5' INS 1/1 genotype was positively associated with IDDM both in non-DR4 subjects (relative risk = 4.3; 95% confidence interval, 1.6-11.5) and DR4 subjects (relative risk = 4.2; 95% confidence interval, 1.9-9.0). Further subdivision of IDDM patients and matched control subjects according to HLA-DQA1 and HLA-DQB1 genotype or phenotype also failed to show any association between 5' INS and HLA class II genes in diabetic patients. The 5' INS and HLA class II polymorphisms therefore provide independent risk markers, which may both contribute to the genetic screening of a high-risk population among nondiabetic individuals.

Adolescent↗

Expression of Fas-Fas ligand antigens and apoptotic marker APO2.7 by the human conjunctival epithelium. Positive correlation with class II HLA DR expression in inflammatory ocular surface disorders.

Fas antigen (CD95) is a membrane receptor that plays a major role in induction of apoptosis. In surface conjunctival epithelial cells the expressions of Fas, Fas ligand, the apoptotic marker APO2.7 and of HLA DR class II antigen, a membrane marker known to be expressed in inflammatory conditions were investigated. Impression cytology specimens were collected in 65 patients: 20 normal ones, 15 contact lens wearers, 20 receiving chronic topical antiglaucoma treatment and 10 with nonspecific chronic conjunctivitis. Cells were processed for flow cytometry, using monoclonal antibodies to Fas, Fas ligand, APO2.7, HLA DR antigens and a negative isotypic control. Percentages of positive cells were recorded and levels of fluorescence quantified using fluorescent beads at standardized fluorescence intensities. In addition, a human conjunctival cell line was incubated with anti-Fas stimulating antibodies in order to test Fas-induced apoptosis in vitro. Fas was found in all specimens in most of the conjunctival cells, but quantitation of levels of fluorescence showed a significantly higher expression in pathologic eyes than in normal ones. Fas ligand and APO2.7 were variably expressed by conjunctival cells, but in a significantly higher percentage of cells in pathological eyes than in normal ones. In these eyes a strong expression of HLA DR was also observed, whereas normal eyes showed lowest levels. Highly significant correlations were found between Fas, Fas ligand, APO2.7 and HLA DR levels. Anti-Fas antibodies in vitro induced strong apoptosis in epithelial cells as confirmed by APO2.7 expression and DAPI staining. This study confirms that conjunctival epithelial cells normally express Fas antigen, and more inconstantly its ligand, as do corneal ones or keratinocytes. Fluorescence quantitation by flow cytometry showed much higher expression in inflammatory eyes than in normal ones, and demonstrated a strong correlation between apoptotic and inflammatory pathways in the ocular surface.

Adult↗

HLA class II genes associated with REM sleep behavior disorder.

Twenty-five white men with rapid eye movements (REM) sleep behavior disorder, but without narcolepsy, underwent HLA class II antigen typing: 84% (N=21) were DQwl (DQB1*05,06) positive (28% [N=7] were DR2 positive); DQB1*0501 (N=9) and DQB1*0602 (N=7) were the most common phenotypes. The 84% DQwl rate in men with REM sleep behavior disorder was significantly greater (p=0.015) than the 56% DQwl rate found in a local white comparison group (N=66), and was greater than the 39 to 66% DQwl rates published for 12 white groups (N=40-418/group). Thus, another disorder of REM sleep dysregulation (besides narcolepsy) appears to be strongly associated with specific HLA class II genes.

Electromyography↗

Results of Expedicion Humana. I. Analysis of HLA class II (DRB1-DQA1-DPB1) alleles and DR-DQ haplotypes in nine Amerindian populations from Colombia.

HLA class II variation was analyzed in nine Native American populations of Colombia using PCR/SSOP typing methods. Under the auspices of the Expedition Humana, approximately 30 unrelated native Colombia Indian samples each from the Tule (NW Pacific Coast), Kogui (Sierra Nevada). Ijka (Sierra Nevada), Ingano (Amazonas), Coreguaje (Amazonas), Nukak (Amazonas), Waunana (Pacific), Embera (Pacific) and Sikuani (Northeastern Plains) were collected and analyzed at the DRBI, DQA1, DQB1 and DPB1 loci. The number of different DRB1, DQA1, DQB1 and DPB1 alleles in the Colombian Indians is markedly reduced in comparison with neighboring African Colombian populations, which exhibit a very high degree of class II variability, as discussed in an accompanying paper. In the Colombian Amerindian groups, DR2 (DRB1*1602), DR4 (DRB1*0407, *0404, *0403 AND *0411), DR6 (DRB1*1402) and DR8 (DRB1*0802) comprise > 95% of all DRB1 alleles. We also found an absence of DR3 in all populations, and DR1, DR7 and DR9 allelic groups were either very rare or absent. Each Colombian Amerindian population has a predominant DRB1 allele (f = approximately 0.22-0.65) and DRB1-DQA1-DQB1 haplotype. Several novel DR-DQ haplotypes were also found. At the DPB1 locus, DPB1*0402 (f = 0.28-0.82), *1401 (f = 0.03-0.45), and *3501 (f = 0.03-0.27), were the three most prevalent alleles, each population maintaining one of these three alleles as the predominant (f > 0.26) DPB1 allele. The reduction of diversity for the HLA class II alleles in the Colombian Indians is suggestive of a population bottleneck during the colonization of the Americans, with little to no subsequent admixture with neighboring African Colombian populations in the last approximately 300 years.

Colombia↗

Indirect allorecognition of mismatched donor HLA class II peptides in lung transplant recipients with bronchiolitis obliterans syndrome.

A correlation between indirect allorecognition of mismatched donor HLA class I peptides and development of bronchiolitis obliterans syndrome (BOS) after lung transplantation has been previously observed. The aim of this study was to determine whether there was a correlation between indirect allorecognition of mismatched donor HLA class II peptides and development of BOS after lung transplantation. Peripheral blood mononuclear cells from nine BOS+ and nine BOS-lung transplant recipients were cultured with synthetic peptides corresponding to the beta-chain hypervariable region of a mismatched donor HLA-DR molecule. Then, proliferative alloreactivity as well as frequency of alloreactive T cells were determined. In addition, the immunodominant epitopes from the donor HLA-DR molecules were identified in selected patients. T cells from BOS+ patients showed a dose-dependent proliferative alloreactivity against donor HLA-DR peptides that was significantly higher than that observed in BOS- patients (p=0.001). Similarly, the frequency of HLA-DR alloreactive T cells was significantly higher in BOS+ patients than in BOS- patients (p=0.001). This T-cell alloreactivity was directed against a single immunodominant HLA-DR peptide. These results suggest that indirect alloreactivity to donor HLA class II molecules may play a role in the pathogenesis of BOS after lung transplantation.

Amino Acid Sequence↗

HLA class II alleles are not a general susceptibility factor in Guillain-Barré syndrome.

OBJECTIVE: To assess whether human leukocyte antigen (HLA)-DRB1 and HLA-DQB1 alleles confer susceptibility to Guillain-Barre syndrome (GBS) or are related to specific clinical or serologic subgroups of GBS. METHODS: The HLA-DRB1 and HLA-DQB1 loci were genotyped by PCR amplification with sequence-specific primers in 164 well-documented Dutch patients with GBS and 207 healthy Dutch control subjects. Patients with GBS were divided into subgroups based on clinical features, severity of disease, antecedent infection, and anti-ganglioside antibodies. Data were compared with those of all case-control HLA studies in GBS performed previously. RESULTS: In this case-control study, HLA-DRB1 and HLA-DQB1 alleles did not differ between GBS patients and control subjects. The frequency of HLA-DRB1*01 was increased in patients who needed mechanical ventilation (odds ratio 4.2; 95% CI 1.9 to 9.6; p(c) = 0.02). Multivariate logistic regression analysis showed that this association was independent of the severity of paresis and the presence of cranial nerve involvement (all p < 0.05). There was a tendency toward an association between certain HLA alleles and several anti-ganglioside antibodies. CONCLUSIONS: Human leukocyte antigen (HLA) class II antigens are not a general susceptibility factor in Guillain-Barre syndrome (GBS). However, HLA class II alleles may be a determinant in distinct subgroups of GBS, indicating the need for further exploration in large-scale studies.

Adolescent↗