Lakshmi K. Gaur

Influence of HLA supertypes on susceptibility and resistance to human immunodeficiency virus type 1 infection

Certain human leukocyte antigens, by presenting conserved immunogenic epitopes for T cell recognition, may, in part, account for the observed differences in human immunodeficiency virus type 1 (HIV-1) susceptibility. To determine whether HLA polymorphism influences HIV-1 susceptibility, a longitudinal cohort of highly HIV-1-exposed female sex workers based in Nairobi, Kenya, was prospectively analyzed. Decreased HIV-1 infection risk was strongly associated with possession of a cluster of closely related HLA alleles (A2/6802 supertype; incidence rate ratio [IRR], 0.45; 95% confidence interval [CI], 0.27-0.72; P=.0003). The alleles in this supertype are known in some cases to present the same peptide epitopes for T cell recognition. In addition, resistance to HIV-1 infection was independently associated with HLA DRB1*01 (IRR, 0.22; 95% CI, 0.06-0.60; P=.0003), which suggests that anti-HIV-1 class II restricted CD4 effector mechanisms may play an important role in protecting against viral challenge. These data provide further evidence that resistance to HIV-1 infection in this cohort of sex workers is immunologically mediated.

HLA-DQB1*0201/0302 is associated with severe retinopathy in patients with IDDM

Summary Some insulin-dependent diabetic (IDDM) patients develop severe forms of retinopathy. Putative risk factors such as hypertension, poor metabolic control, nephropathy and growth hormone levels do not fully explain the progress of retinopathy in these patients. It has been discussed whether there is a genetic marker, since some diabetic patients without any known predisposing risk factors develop severe retinopathy and others do not. In the present study, HLA-DR and DQ were compared in two patient groups with IDDM. One group consisted of patients with early-onset diabetes, with severe non-proliferative or proliferative retinopathy; the other group had no or only mild signs of retinopathy. High resolution HLA typing was carried out by polymerase chain reaction (PCR) and hybridization with allele specific probes. Alleles on the DR3-DQ2 haplotype, DRB1*0301, DQA1*0501 and DQB1*0201, were more frequent in patients with severe retinopathy. A difference was seen when combining certain alleles in the genotypes of DQA1*03/0501 (p > 0.05) and DQB1*0201/0302 (p < 0.01). The findings of the present study suggest that DQB1*0201/0302 is the strongest genetic marker for severe retinopathy and DRB1*0301/0401 only has a secondary influence when combined with this genotype. It seems as if IDDM patients who are positive for the genotype DR3-DQ2/DR4-DQ8 (DRB1*0301-DQA1*0501-DQB1*0201/DRB1*0401 -DQA1*03-DQB1*0302) are at greater risk of developing severe retinopathy. [Diabetologia (1996) 39: 1313–1317]

In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells.

In utero transplantation of hematopoietic stem cells is a promising treatment for immune and hematologic diseases of fetuses and newborns. Unfortunately, there are limited data from nonhuman primates and humans describing optimal transplantation conditions. The purpose of this investigation was to determine the effect of T‐cell number on engraftment and the level of chimerism after in utero transplantation in nonhuman primates. CD34+ allogeneic adult bone marrow cells, obtained from the sire after G‐CSF and stem cell factor administration, were transplanted into female fetal recipients. The average CD34+ cell dose was 3.0 × 109/kg (range, 9.9 × 108 to 4.4 × 109) and the T‐cell dose ranged from 2.6 × 105 to 1.1 × 108/kg. Chimerism was determined in peripheral blood subsets (CD2, CD13, and CD20) and in progenitor cell populations by using polymerase chain reaction. Chimerism was noted in seven of eight live‐born animals. The level of chimerism in the progenitor population was related to the fetal T‐cell dose (r = 0.64, p < 0.02). At the lowest T‐cell dose (2.6 × 105/kg), no chimerism was detected. As the T‐cell dose increased to 106–7/kg, the level of chimerism increased. Adjusting the T‐cell dose to 1.1 × 108/kg resulted in fatal graft‐versus‐host disease (GVHD). The results of this study emphasize the importance of T cells in facilitating donor cell engraftment and in producing GVHD in fetal nonhuman primates. Some animals achieved levels of chimerism in the marrow hematopoietic progenitor cell population that would likely have clinical relevance. However, the levels of chimerism in peripheral blood were too low for therapeutic benefit. Further studies are needed to test methods that are likely to enhance donor cell engraftment and peripheral blood levels of donor cells.

Immunogenetic correlates for Chlamydia trachomatis–Associated tubal infertility ☆

OBJECTIVE To understand immunogenetic mechanisms of Chlamydia trachomatis infection and tubal scarring. METHODS We measured and compared previously significant human leukocyte antigen (HLA) class II DQ alleles, their linked DRB genes, and polymorphisms in selected cytokine genes (tumor necrosis factor α-308 promoter; transforming growth factor β1-10 and -25 codons; interleukin 10-1082, -819, and -592 promoters; interleukin 6-174 promoter; and interferon γ+874 codon 1) among Kenyan women with confirmed tubal infertility with and without C trachomatis microimmunofluorescence antibody. RESULTS Two class II alleles, HLA-DR1*1503 and DRB5*0101, were detected less commonly in C trachomatis microimmunofluorescence seropositive women than in C trachomatis microimmunofluorescence seronegative women with infertility (0% versus 20%; odds ratio [OR] 0.05; 95% confidence interval [CI] 0, 0.7, and 6% versus 26%; OR 0.2; 95% CI 0.02, 1.0, respectively). These alleles are commonly linked as a haplotype at the DRB locus. This finding could not be explained through linkage disequilibrium with the other studied HLA or cytokine genes. CONCLUSION These alleles may lead to an immunologically mediated mechanism of protection against C trachomatis infection and associated tubal damage, or alternatively increase risk for tubal scarring due to another cause.

Macrophages from High‐Risk HLA‐DQB1*0201/*0302 Type 1 Diabetes Mellitus Patients are Hypersensitive to Lipopolysaccharide Stimulation

Levels of nonantigen‐induced pro‐inflammatory cytokines and prostaglandin in macrophages isolated from human leucocyte antigen (HLA)‐matched type 1 diabetes mellitus patients, first‐degree relatives and healthy controls were determined. We hypothesize that monocytes isolated from patients are sensitized or preactivated and therefore, have an altered response to in vitro stimulus compared with control groups as measured by levels of pro‐ and anti‐inflammatory mediators. In this study, peripheral blood monocytes were differentiated to macrophages with macrophage‐colony stimulating factor (M‐CSF) to determine lipopolysaccharide (LPS)‐stimulated tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, IL‐12 and prostaglandin E‐2 (PGE‐2) secretion from hetero‐ or homozygous HLA DQB1*0201 and *0302 type 1 diabetes mellitus patients, first‐degree relatives and homozygous HLA DQB1*0602 healthy controls. LPS‐stimulated secretion of TNF‐α, IL‐1β and IL‐6 was immediate and markedly higher in the HLA‐DQB1*0201/*0302 type 1 diabetes patients compared with all other groups including HLA‐matched healthy first‐degree relatives. In DQB1*0201/*0302 diabetes patients PGE‐2 secretion was delayed but increased by LPS stimulation compared with HLA‐matched healthy relatives. IL‐12 was not detected at any condition. These data suggest that macrophages from DQB1*0201/*0302 type 1 diabetes patients are sensitized to secrete both cytokines and PGE‐2 following nonantigenic stimulation. Sensitized macrophages may be important to high‐risk DQB1*0201/*0302‐associated type 1 diabetes.

Fetal Immune Suppression as Adjunctive Therapy for In Utero Hematopoietic Stem Cell Transplantation in Nonhuman Primates

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow‐derived, CD34+‐enriched donor cells. Animals treated postnatally received either donor‐derived T cell–depleted or CD34+‐enriched marrow cells. Chimerism was determined by traditional and real‐time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune‐suppressed animals relative to controls (11.3% ± 2.7% and 5.1% ± 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence‐activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at ≥ 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune‐suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6‐month follow‐up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long‐term chimerism also was not improved with postnatal donor cell infusion.

HLA-DR Class II Associations with Rubella Vaccine-Induced Joint Manifestations

HLA class II (HLA-DR) frequencies were examined in relation to incidence of acute arthralgia or arthritis in 283 white women who had received RA27/3 rubella vaccine (n = 146) or placebo (n = 137) postpartum. Leukocyte DNA was molecularly typed for HLA-DRB1 gene expression. Univariate analysis revealed higher frequencies of DR2 (odds ratio [OR], 4.8; 95% confidence interval [CI], 1.2-18.8) and DR5 (OR, 7.5; 95% CI, 1.5-37.5) but lower frequencies of DR4 (OR, 2.3; 95% CI, 1.1-4.9) and DR6 (OR, 2.8; 95% CI, 1.4-5.8), in rubella vaccinees compared with placebo recipients with arthropathy. Logistic regression modelling of DR, treatment, age, time postpartum, and arthropathy revealed that the odds of developing arthropathy was 1.9 times greater (95% CI, 1.07-3.44) after rubella vaccine than placebo. Risk for arthropathy (regardless of rubella vaccination) was also influenced by DR interactions: odds were 8 times greater in individuals with both DR1 and DR4 (95% CI, 1.45-44.02) and 7.1 times greater with both DR4 and DR6 present (95% CI, 1.85-27.52), suggesting that coexpression of these specificities may predispose to postpartum arthropathy.

Association of Chlamydia trachomatis Heat-Shock Protein 60 Antibody and HLA Class II DQ Alleles

A total of 113 female commercial sex workers had individual alleles for HLA class II genes determined by using labeled sequence-specific oligonucleotide probes to hybridize to polymerase chain reaction products of amplified DNA. Women also had microimmunofluorescent (MIF) antibody titers to Chlamydia trachomatis elementary bodies and ELISA antibody to recombinant chlamydial heat-shock protein 60 (Chsp60) determined. Women were prospectively followed at monthly intervals over 2 years for incident C. trachomatis infection and acute pelvic inflammatory disease (PID). HLA DQA1*0401 and DQB1*0402 alleles were statistically associated with increased prevalence and amount of antibody to Chsp60 but not MIF antibody. However, these alleles did not alter the risk for chlamydial PID. The potential role that HLA DQ may play in chlamydial disease pathogenesis requires further study.

Genetic and Immunological Markers of Insulin Dependent Diabetes in Black Americans

ICA and GAD65 autoantibody profiles and HLA-DR and DQ analysis were performed on 43 Black juvenile onset IDDM patients and 34 unrelated Black controls from Tennessee, USA. 75% of patients were positive for GAD65 autoantibodies but only 53% had ICA; 39% both ICA and GAD65 antibodies. The strongest HLA association was with the DR3 haplotype DRB1*03 DQA1*0501 DQB1*0201 (63% of patients v 12% of controls RR = 13.0, p < 0.00002). DRB1*04 DQA1*0301 DQB1*0302, associated with IDDM in Caucasians but rare in Negroids, occurred in 27% of patients and 6% of controls (RR = 5.9, p < 0.04). All patients carried DQB1*0302 or DQB1*0201. DQB1*0602 was significantly reduced in patients (2.4% v 41%, RR = 0.036, p < 0.008) and DRB1*1501 was absent in patients (0% v 35%). The frequency of GAD65 autoantibodies in Black American IDDM patients is comparable to that in Caucasians; however ICA positivity is reduced. GAD65 antibodies may therefore be a more sensitive serological test to identify individuals in the Black American general population for markers associated with increased risk of developing IDDM. Current screening methods for predicting preclinical IDDM in Caucasians relies on a combination of immune and HLA markers of IDDM; studies of these markers in the Black Americans will make it possible to extend these options to additional genetically diverse populations.

Low-Dose Streptozotocin Induces Sustained Hyperglycemia in Macaca nemestrina

The potential for using macaques to create a nonhuman primate diabetic model was investigated. The significant objectives were to determine a) prognosis of STZ induced permanent beta cell destruction in nonhuman primates, and b) the potential to use STZ treated animals in a model of autoimmune diabetes by following adoptively transferred lymphocytes into MHC identical macaques. Beta cell impairment was achieved by a single intravenous, low dose (1CMH) mg/kg body weight) streptozotocin injection in a majority of pigtailed macaques (Macaco nemestrina). Multiple injections, even at low doses at close intervals affected liver and kidney functions in addition to beta cell destruction. Abnormal IVGTT were observed in all streptozo-tocin-treated animals, in some within a week to 10 days. The fasting blood glucose levels rose from <70 mg/dl in pre-STZ stage to above 400 mg/dl in severely diabetic macaques. Histological evidence suggests loss of beta cells when animals were euthanized within two to four weeks post-STZ treatment. Near complete destruction of beta cells was observed in animals maintained longer than three months on insulin. Donor T cells from STZ-treated animals were incubated overnight with 10U/ml IL-2 and 2.5 ug/ml PHA and then injected iv into a MHC-identical non-diabetic sibling. Three weeks later a second injection of donor PMBC labeled with vital dye Cell Tracker Green was given and the animal was euthanized after 24 hours. The recipient showed labeled donor T cells in the pancreas, spleen and peripheral blood, consistent with specific homing of activated lymphocytes from the diabetic donor.