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Background: Infants born with HIV-1 require lifelong antiretroviral therapy (ART). We aimed to assess whether very early ART in neonates might restrict HIV-1 reservoirs, an important step towards ART-free remission., Methods: IMPAACT P1115 is an ongoing, phase 1/2, proof-of-concept study in which infants were enrolled at 30 research clinics in 11 countries (Brazil, Haiti, Kenya, Malawi, South Africa, Tanzania, Thailand, Uganda, the USA, Zambia, and Zimbabwe) into two cohorts. Infants at least 34 weeks' gestational age at high risk for in-utero HIV-1 with either untreated maternal HIV-1 (cohort 1) or who were receiving pre-emptive triple antiretroviral prophylaxis outside of the study (maternal ART permissible; cohort 2) were included. All infants initiated treatment within 48 h of life. Cohort 1 initiated three-drug nevirapine-based ART, and cohort 2 initiated three-drug nevirapine-based prophylaxis then three-drug nevirapine-based ART following HIV diagnosis by age 10 days. We added twice-daily coformulated oral ritonavir 75 mg/m 2 and lopinavir 300 mg/m 2 from 14 days of life and 42 weeks postmenstrual age. We discontinued nevirapine 12 weeks after two consecutive plasma HIV-1 RNA levels below limit of detection. We tracked virological suppression, safety outcomes, and meeting a predetermined biomarker profile at age 2 years (undetectable RNA since week 48, HIV-1 antibody-negative, HIV-1 DNA not detected, and normal CD4 count and CD4 percentage) to assess qualification for analytical treatment interruption. This study is registered with ClinicalTrials.gov, NCT02140255., Findings: Between Jan 23, 2015, and Dec 14, 2017, 440 infants were included in cohort 1 and 20 were included in cohort 2. 54 of these infants (34 from cohort 1 and 20 from cohort 2) had confirmed in-utero HIV-1 and were enrolled to receive study ART. 33 (61%) of 54 infants were female and 21 (39%) were male. The estimated probability of maintaining undetectable plasma RNA through to 2 years was 33% (95% CI 17-49) in cohort 1 and 57% (28-78) in cohort 2. Among infants maintaining protocol-defined virological control criteria through to study week 108, seven of 11 (64%, 95% CI 31-89) in cohort 1 and five of seven (71%, 29-96) in cohort 2 had no detected HIV-1 DNA. Ten of 12 (83%, 52-100) in cohort 1 and all seven (100%, 59-100) in cohort 2 tested HIV-1 antibody-negative at week 108. Among 54 infants initiated on very early ART, ten (19%; six in cohort 1 and four in cohort 2) met all criteria for possible analytical treatment interruption. Reversible grade 3 or 4 adverse events occurred in 15 (44%) of 34 infants in cohort 1 and seven (35%) of 20 infants in cohort 2., Interpretation: Very early ART for in-utero HIV-1 can achieve sustained virological suppression in association with biomarkers indicating restricted HIV-1 reservoirs by age 2 years, which might enable potential ART-free remission., Funding: National Institute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute of Mental Health., Competing Interests: Declaration of interests MM receives research support from Gilead Sciences, ViiV Healthcare, and Merck. EVC serves as a consultant to Melinta Pharmaceuticals. EGC's spouse holds an equity interest in AbbVie. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola virus disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen., Methods: A cohort of 131 male EVD survivors in Liberia were enrolled in a case-case study. "Early clearers" were defined as those with 2 consecutive negative EBOV semen test results by real-time reverse-transcription polymerase chain reaction (rRT-PCR) ≥2 weeks apart within 1 year after discharge from the Ebola treatment unit or acute EVD. "Late clearers" had detectable EBOV RNA by rRT-PCR >1 year after discharge from the Ebola treatment unit or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical examinations and blood work., Results: Compared with early clearers, late clearers were older (median, 42.5 years; P < .001) and experienced fewer severe clinical symptoms (median 2, P = .006). Late clearers had more lens opacifications (odds ratio, 3.9 [95% confidence interval, 1.1-13.3]; P = .03), after accounting for age, higher total serum immunoglobulin G3 (IgG3) titers (P = .005), and increased expression of the HLA-C*03:04 allele (0.14 [.02-.70]; P = .007)., Conclusions: Older age, decreased illness severity, elevated total serum IgG3 and HLA-C*03:04 allele expression may be risk factors for the persistence of EBOV in the semen of EVD survivors. EBOV persistence in semen may also be associated with its persistence in other immunologically protected sites, such as the eye., Competing Interests: Potential conflicts of interest. A. Kofman reports support for attending meetings and/or travel from the CDC Foundation. R. S. B. reports $11 000 in grants or contracts from the University of Mississippi Medical Centre and $7500 in contracts or grants from the Federation University Centre for Health Transformation and Innovation, both unrelated to this work; a registration fee waiver ($650) in support of attending the Australian Society for Microbiology 2022 meeting; World Intellectual Property Organization patent WO2019060840, (Removing Interfering Host Nucleic Acids for Molecular Parasite Detection; granted 28 March 2019; patent royalties to the CDC Division of Parasitic Diseases and Malaria); unpaid participation on the Surveillance Cross-cutting Subgroup of the World Health Organization Neglected Tropical Disease Diagnostic Technical Advisory Group, unpaid participation as a member of the Strongyloides subgroup of the WHO Technical Advisory Group on Schistosomiasis and Transmitted Helminthiases, and unpaid participation at a WHO policy meeting with Dora Buonfrate of Ospidale Sacra Cuore (Italy) and Antonio Montresor of the WHO (Diagnostic Methods for the Control of Strongyloidiasis; 29 September 2020); and leadership or fiduciary roles with Strongyloides Australia (as vice president), the Australian Society for Parasitology Education Committee, and the Australian Society for Infectious Diseases Zoonosis Special Interest Group. S. H. H. reports consulting fees paid to the author from Leavitt Associates; support for attending meetings and/or travel from their employer (University of Nebraska Medical Center); patents planned, issued or pending (none related to this work); participation on the advisory board of the HHS National Coordinator of IT; and ownership of stock or stock options (none related to this article). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2022.)

CD4 + T cells are central to long-term immunity against viruses through the functions of T helper 1 (T H 1) and T follicular helper (T FH ) cell subsets. To better understand the role of these subsets in coronavirus disease 2019 (COVID-19) immunity, we conducted a longitudinal study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific CD4 + T cell and antibody responses in convalescent individuals who seroconverted during the first wave of the pandemic in Boston, MA, USA, across a range of COVID-19 disease severities. Analyses of spike (S) and nucleocapsid (N) epitope-specific CD4 + T cells using peptide and major histocompatibility complex class II (pMHCII) tetramers demonstrated expanded populations of T cells recognizing the different SARS-CoV-2 epitopes in most individuals compared with prepandemic controls. Individuals who experienced a milder disease course not requiring hospitalization had a greater percentage of circulating T FH (cT FH ) and T H 1 cells among SARS-CoV-2-specific cells. Analysis of SARS-CoV-2-specific CD4 + T cells responses in a subset of individuals with sustained anti-S antibody responses after viral clearance also revealed an increased proportion of memory cT FH cells. Our findings indicate that efficient early disease control also predicts favorable long-term adaptive immunity.

Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is one of the most devastating of adverse drug reactions (ADRs) and was, until recently, essentially unpredictable. With the discovery of several risk alleles for drug-induced SJS/TEN and the demonstration of effectiveness of screening in reducing incidence, the stage is set for implementation of preventive strategies in populations at risk. Yet much remains to be learned about this potentially fatal complication of commonly used drugs., (© 2017 American Society for Clinical Pharmacology and Therapeutics.)

Objective: Long-acting rilpivirine is a candidate for preexposure prophylaxis (PrEP) for prevention of HIV-1 infection. However, rilpivirine resistance mutations at reverse transcriptase codon 138 (E138X) occur naturally in a minority of HIV-1-infected persons; in particular those expressing human leukocyte antigen (HLA)-B18 where reverse transcriptase-E138X arises as an immune escape mutation. We investigate the global prevalence, B18-linkage and replicative cost of reverse transcriptase-E138X and its regional implications for rilpivirine PrEP., Methods: We analyzed linked reverse transcriptase-E138X/HLA data from 7772 antiretroviral-naive patients from 16 cohorts spanning five continents and five HIV-1 subtypes, alongside unlinked global reverse transcriptase-E138X and HLA frequencies from public databases. E138X-containing HIV-1 variants were assessed for in-vitro replication as a surrogate of mutation stability following transmission., Results: Reverse transcriptase-E138X variants, where the most common were rilpivirine resistance-associated mutations E138A/G/K, were significantly enriched in HLA-B18-positive individuals globally (P = 3.5 × 10) and in all HIV-1 subtypes except A. Reverse transcriptase-E138X and B18 frequencies correlated positively in 16 cohorts with linked HIV/HLA genotypes (Spearman's R = 0.75; P = 7.6 × 10) and in unlinked HIV/HLA data from 43 countries (Spearman's R = 0.34, P = 0.02). Notably, reverse transcriptase-E138X frequencies approached (or exceeded) 10% in key epidemic regions (e.g. sub-Saharan Africa, Southeastern Europe) where B18 is more common. This, along with the observation that reverse transcriptase-E138X variants do not confer in-vitro replicative costs, supports their persistence, and ongoing accumulation in circulation over time., Conclusions: Results illustrate the potential for a natural immune-driven HIV-1 polymorphism to compromise antiretroviral-based prevention, particularly in key epidemic regions. Regional reverse transcriptase-E138X surveillance should be undertaken before use of rilpivirine PrEP.

A major challenge for the development of an effective vaccine against tuberculosis (TB) is that the attributes of protective CD4 + T cell responses are still elusive for human TB. Infection with HIV type 1 is a major risk factor for TB, and a better understanding of HIV-induced alterations of Mycobacterium tuberculosis -specific CD4 + T cells that leads to failed host resistance may provide insight into protective T cell immunity to TB. A total of 86 participants from a TB-endemic setting, either HIV-infected or uninfected and with latent or active TB (aTB), were screened using M. tuberculosis -specific MHC class II tetramers. We examined the phenotype as well as function of ex vivo M. tuberculosis -specific tetramer + CD4 + T cells using flow cytometry. The numbers of M. tuberculosis -specific tetramer + CD4 + T cells were relatively well maintained in HIV-infected persons with aTB, despite severe immunodeficiency. However, although HIV-uninfected persons with latent TB infection exhibited ex vivo M. tuberculosis -specific CD4 + T cells predominantly of a CXCR3 + CCR6 + CCR4 - (Th1*) phenotype, aTB or HIV infection was associated with a contraction of this subset. Nevertheless, in individuals with aTB and/or HIV infection, circulating ex vivo M. tuberculosis -specific CD4 + T cells did not display defects in exhaustion or polyfunctionality compared with healthy HIV-uninfected individuals with latent TB infection. Collectively, these data suggest that increased susceptibility to TB disease could be related to a loss of circulating Th1* CD4 + T cells rather than major changes in the number or function of circulating CD4 + T cells., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Previous genome-wide association studies (GWAS) of HIV-1-infected populations have been underpowered to detect common variants with moderate impact on disease outcome and have not assessed the phenotypic variance explained by genome-wide additive effects. By combining the majority of available genome-wide genotyping data in HIV-infected populations, we tested for association between ∼8 million variants and viral load (HIV RNA copies per milliliter of plasma) in 6,315 individuals of European ancestry. The strongest signal of association was observed in the HLA class I region that was fully explained by independent effects mapping to five variable amino acid positions in the peptide binding grooves of the HLA-B and HLA-A proteins. We observed a second genome-wide significant association signal in the chemokine (C-C motif) receptor (CCR) gene cluster on chromosome 3. Conditional analysis showed that this signal could not be fully attributed to the known protective CCR5Δ32 allele and the risk P1 haplotype, suggesting further causal variants in this region. Heritability analysis demonstrated that common human genetic variation-mostly in the HLA and CCR5 regions-explains 25% of the variability in viral load. This study suggests that analyses in non-European populations and of variant classes not assessed by GWAS should be priorities for the field going forward.

Genome-wide association studies of the related chronic inflammatory bowel diseases (IBD) known as Crohn's disease and ulcerative colitis have shown strong evidence of association to the major histocompatibility complex (MHC). This region encodes a large number of immunological candidates, including the antigen-presenting classical human leukocyte antigen (HLA) molecules. Studies in IBD have indicated that multiple independent associations exist at HLA and non-HLA genes, but they have lacked the statistical power to define the architecture of association and causal alleles. To address this, we performed high-density SNP typing of the MHC in >32,000 individuals with IBD, implicating multiple HLA alleles, with a primary role for HLA-DRB1*01:03 in both Crohn's disease and ulcerative colitis. Noteworthy differences were observed between these diseases, including a predominant role for class II HLA variants and heterozygous advantage observed in ulcerative colitis, suggesting an important role of the adaptive immune response in the colonic environment in the pathogenesis of IBD.

Recent studies suggest that innate immune responses by natural killer (NK) cells play a significant role in restricting human immunodeficiency virus type-1 (HIV-1) pathogenesis. Our aim was to characterize changes in NK cells associated with HIV-1 clade C disease progression. Here we used multiparametric flow cytometry (LSRII) to quantify phenotype and function of NK cells in a cross-sectional analysis of cryopreserved blood samples from a cohort of 41 chronically HIV-1-infected, treatment-naive adult South Africans. These individuals ranged in disease severity from early (CD4 count >500) to advanced HIV-1 disease (CD4 count <50). We found that the frequency of NK cells expressing KIR2DL1, an inhibitory receptor, and/or KIR2DS1, an activating receptor, tended to decrease with increasing HIV-1 viral load. We also discovered a significant increase (p < 0.05) in overall NK cell degranulation with disease progression. We found that acutely activated NK cells (CD69(pos)) were deficient in NKp46 expression ex vivo. In conclusion, we observed that with viremia and advanced HIV-1 disease, activated NK cells lack NKp46 expression, and KIR2DS1(pos) and/ or KIR2DL1(pos) NK cells are reduced in frequency. These findings suggest that modulation of receptor expression on NK cells may play a role in HIV-1 pathogenesis, and provide new insights on immunological changes in advanced HIV-1 disease.

CCR5 is one of the primary coreceptors for Env-mediated fusion between cells and human immunodeficiency virus type 1 (HIV-1). Analyses of CCR5 variants in cohorts of HIV-1 high-risk individuals led to the identification of multiple single amino-acid substitutions, which may have functional consequences. This study focused on eight naturally occurring allelic variants located between amino-acid residues 60 and 334 of CCR5. All studied allelic variants were highly expressed on the cell surface of HEK-293 cells and permissive for HIV-1 infection. Variant G301V showed 3.5-fold increase in 50% effective concentration (EC(50)) for CCL4 (MIP 1beta) in a competitive binding assay. There was also a significant reduction in CCL5 (RANTES) EC(50) for the R223Q, A335V and Y339F variants. The most unexpected functional abnormality was exhibited by the R60S variant that exhibited a loss of ligand-induced desensitization in chemotaxis assays, but showed normal CCL4 and CCL5 binding avidity. This mutation is located in the first intracellular loop, a domain that has not previously been shown to be involved in receptor desensitization. In conclusion, our results support earlier studies showing that these naturally occurring point mutations do not limit HIV-1 infection, and indicated that single amino-acid changes can have unexpected functional consequences., (Genes and Immunity (2005) 6, 609-619. doi:10.1038/sj.gene.6364247; published online 14 July 2005.)

Objective: To assess the association between human leukocyte antigen (HLA) haplotypes and the incidence rates of CD4 decline to < 20% and to AIDS., Design: Retrospective cohort study of 95 HIV-1-infected hemophilic sibling pairs., Methods: HLA haplotype-sharing between siblings was assigned on the basis of serologic typing of HLA class I alleles and molecular typing of HLA class II alleles. Concordance of time to CD4 decline to < 20% and to AIDS within and between sibling pairs was assessed by analysis of variance models and calculations of intraclass correlation coefficients. The age-adjusted relative risks of these two endpoints for unique class II haplotypes were determined from proportional hazards models., Results: Sibling pairs sharing one or two haplotypes were significantly concordant in CD4 decline and AIDS status within 5 years of seroconversion. No concordance was found in pairs sharing zero haplotypes. At 6-10 years after seroconversion, significant concordance of these two endpoints was also observed in the pairs sharing one haplotype. The concordant results were not explained by the use of zidovudine within the pairs. Among the individuals in this cohort, the relative hazards for CD4 decline to < 20% and for AIDS were significantly elevated for one class II haplotype (DQB1*0501, DQA1*0101, DRB1*0101). In addition, the risk for AIDS was significantly increased for two other class II haplotypes (DQB1*0603, DQA1*0103, DRB1*1300, DRB3*0202 and DQB1*0301, DQA1*0501, DRB1*1400, DRB3*0202) and significantly decreased for one haplotype (DQB1*0302, DQA1*0301, DRB1*0401, DRB4*0101)., Conclusions: These data demonstrate that HIV-1 disease progression is associated with the genes in the major histocompatibility complex that regulate the host's immune response.

A novel approach to DNA probe hybridization and heteroduplex analysis, termed directed heteroduplex analysis (DHDA) is presented here to illustrate its utility in simplification of human lymphocyte antigen (HLA)-typing. By strategic labeling of single-stranded probe sequences, DHDA allows the identification of specific heteroduplex structures that contribute to the differentiation of DQA1 and DQB1 alleles. Because of the high degree of polymorphism among major histocompatibility complex class II second exon sequences, this analysis of 50 different heteroduplex molecules provides evidence of the importance of unpaired bases and mismatched base pairs and their effect on heteroduplex electrophoretic-mobility differences. This strategy is further used to genotype accurately a family for DQA1 which was previously analyzed by sequence specific oligonucleotide (SSO) probe hybridization. To differentiate by SSO-typing among the DQA1 and DQB1 alleles analyzed in this study requires the use of 23 different probes. Equivalent results are obtained by DHDA using only three probes. Therefore, this study suggests that accurate HLA-typing can be simplified by DHDA. Additionally, DHDA may be useful for differentiation of DNA sequence polymorphisms in other genetic systems.

Melanoma cell lines treated with or without interferon-gamma were tested for the presence of DR alpha mRNA and protein. The six lines examined fell into three general categories: two that expressed high levels of DR alpha mRNA and protein before and after interferon-gamma treatment, one that expressed very low levels before treatment with interferon-gamma, but was induced to express high levels after interferon-gamma treatment, and three that expressed very low levels before treatment, and were only slightly inducible after treatment with interferon-gamma. The presence of DR-alpha protein on the melanoma cell surface was always positively correlated with the presence of DR alpha mRNA in the cells. Furthermore, in the cell line that was interferon-gamma-inducible, the time at which DR alpha mRNA and protein appeared and the doses of interferon-gamma needed to induce this appearance were directly correlated. Methylation patterns of the DR alpha gene in these cell lines were also studied in order to determine whether the degree of DR alpha gene methylation among the lines correlated with expression of the gene. Digestion of DNA with the restriction enzyme MspI, which recognizes the sequence 5'CCGG3' and 5'CmCGG3', led to the appearance of a 3.1 kb band from all lines tested. Hpa II digestion, which recognizes 5'CCGG3', but not 5'CmCGG3', led to the appearance of 3.1, 4.4, and 6.7 kb bands in all lines tested except for DUMEL 8, which showed only the 3.1 kb band. Interestingly, DUMEL 8 expressed very low levels of DR alpha mRNA and protein before and after interferon-gamma treatment. We conclude that interferon-gamma has a regulatory effect on DR alpha genes of various melanoma cell lines to varying degrees. This may reflect an effect of interferon-gamma on certain subpopulations of melanocytes in vivo. Our data also indicate that partial methylation of the DR alpha gene does not inhibit its expression. Furthermore, interferon-gamma does not appear to induce expression of the DR gene by altering methylation patterns within the region recognized by our probe.

We examined the possibility that one mechanism for controlling HLA-DR alpha gene expression involves the alteration of chromatin structure. Chromatin structure was analyzed by measuring the susceptibility of DR alpha genes in intact nuclei to nuclease treatment. We first examined a somatic cell hybrid of a T-lymphoblastoid cell line (LCL) and a B-LCL, since the DR alpha gene, which is inactive in the T-LCL parent, is expressed in the hybrid, thus providing a system to study DR alpha gene induction. The hybrid line 174 X CEM.T1 contains and expresses solely the DR alpha gene from the T-LCL parent, since the DR alpha gene from the B-LCL parent, 174, is deleted. Using cytoplasmic dot blot analysis and RNA-DNA Northern hybridization, we detected DR alpha-specific transcripts in the hybrid, but not in the parental lines, indicating activation of the DR alpha gene in the hybrid. The transcribed DR alpha gene from the hybrid was compared with the untranscribed gene from the T-LCL parental line, and an association between DR alpha gene expression and increased sensitivity to DNase I was observed. A switch in the chromatin structure of the DR alpha gene from a closed to an open configuration apparently occurred in this hybrid. Such a change is associated with DR alpha gene expression. Comparison of a DR-positive B-LCL and an isogenic DR-negative T-LCL also showed that the chromatin of the former is more sensitive to DNase I digestion. There were no restriction enzyme fragment length differences between the DR alpha genes from 174 X CEM.T1 and CEMR, indicating that the process of somatic cell hybridization did not result in DNA rearrangement or translocation.

An HLA class II-positive melanoma cell line, DU-Mel 17, expressing both DR and DQ was shown to be contaminated with Mycoplasma hyorhinis. After treatment of the cell line with anti-mycoplasma drugs (a tetracycline derivative and a pleuromutilin), both DR and DQ expression on the cell surface, as well as DR alpha mRNA, were greatly decreased and the cells were rendered mycoplasma-free. Interestingly, drug-treated cells became resistant to geneticin, an unrelated drug. To test whether drug treatment or elimination of mycoplasma caused the abolition of class II expression, contaminated DU-Mel 17 cells were inoculated into peritoneal cavities of nude mice and later removed upon growth of a solid tumor. The cells were shown to be mycoplasma-free, and they expressed DR alpha mRNA, as well as DR and DQ proteins on their surfaces. Although it appears that the drugs were responsible for abating class II molecule expression in DU-Mel 17 cells, the identical drug-treatment regimen on a class II-positive, mycoplasma-free B cell line did not alter DR alpha mRNA or class II protein levels. Based on restriction fragment lengths, the DR alpha gene appeared to be grossly intact after drug treatment, and alterations in methylation patterns were not observed. These studies indicate that anti-mycoplasma treatment of DU-Mel 17 cells resulted in selection of a class II-negative, drug-resistant clone(s). Such treatment may result in a phenotype that is divergent from the parental cell line in one or more characteristics.

We examined the possibility that one mechanism for controlling HLA-DR alpha gene expression occurs through DNA hypomethylation. We employed the restriction enzyme Hpa II, which recognizes the sequence 5'CCGG3' but not 5'CmCGG3', to study DNA methylation. We first compared a DR-positive B lymphoblastoid cell line (LCL) with an isogenic DR-negative T-LCL. Using a genomic probe for the DR alpha gene, we showed that an Hpa II digestion of DNA from the B-LCL resulted in bands of lower molecular weight than that of the T-LCL. This indicates that the B-LCL DR gene is hypomethylated relative to the T-LCL gene. Demethylation of the gene from the B-LCL is incomplete, suggesting that complete demethylation is not required for its expression. We also examined somatic cell hybrids of T-LCL and B-LCL since the DR alpha gene, which is inactive in the T-LCL, is expressed in the hybrids, providing a system to study DR alpha gene induction. We examined the hybrid line 174 X CEM.T1, which contains and expresses solely the DR alpha gene from the T-LCL parent since both copies of the DR alpha gene from the B-LCL parent, 174, are deleted. The expressed DR alpha gene from the hybrid was compared with the unexpressed gene from the T-LCL parental line, and again an association between DR alpha gene expression and DNA hypomethylation was observed. In contrast to the DR alpha gene from B-LCL, which is not completely demethylated, the DR alpha gene in this hybrid line is not methylated at either of the Msp I sites covered by our probe. This suggests that different regulatory mechanisms operating through DNA methylation may be involved in the expression of DR alpha genes from T-LCL and B-LCL. Examination of another hybrid line which has DR alpha genes from both parental lines supports this contention. The implications of these findings are discussed.

An HLA class II-positive melanoma cell line, DU-Mel 17, was treated with three compounds known to induce differentiation in various cell lines. Neither retinoic acid nor dibutyryl cAMP altered levels of DR alpha mRNA, but 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) significantly decreased the level of DR alpha mRNA 48 h after treatment. Optimal effect of the hormone on DR alpha mRNA was reached by 72 h. DU-Mel 17 cells were responsive to 1,25(OH)2D3 in a dose-dependent manner, and a reduction in DR alpha mRNA was seen at concentrations as low as 5 x 10(-13) M. The action of 1,25(OH)2D3 on DR alpha mRNA levels was dependent on protein synthesis as evidenced by inhibition of its effect upon addition of cycloheximide. Both DR and DQ protein levels on the surface of DU-Mel 17 were beginning to decline by 72 h after 1,25(OH)2D3 treatment, and by 96 h these proteins were decreased by 65%. 1,25(OH)2D3 was not capable of altering expression of class II molecules on three different class II-positive B lymphoblastoid cell lines, although one of these lines was shown to express the receptor for 1,25(OH)2D3. These findings are important because 1) there is no known physiologic regulator that actively down-regulates class II molecules that are present in and/or on cells, 2) levels of mRNA derived from a very limited number of genes are known to be altered by 1,25(OH)2D3, and 3) they support the contention that 1,25(OH)2D3 may alter the differentiation state of these cells and the activity of the normal counterpart of these cells in an immune response.