Comprehensive proteomic study identifies serpin and cystatin antiproteases as novel correlates of HIV-1 resistance in the cervicovaginal mucosa of female sex workers.

Not all individuals exposed to HIV-1 become infected, and evidence from HIV-1 highly exposed seronegative women (HIV-1-resistant) suggests that mucosal factors in the female genital tract, the first site of contact for the virus, are playing a role. To better understand factors mediating protection from HIV-1, we performed a large clinical study using the tools of systems biology to fully characterize the cervicovaginal mucosa proteome in HIV-1-resistant women. Cervicovaginal lavage fluid was collected from 293 HIV-1-resistant, uninfected, and infected sex workers and analyzed by 2D-LC LTQ-FT-MS. Of the more than 360 unique proteins identified, 41 were differentially abundant (>3-fold cutoff) in HIV-1-resistant women. The majority of over-abundant proteins were antiproteases (>40%), some with described anti-inflammatory and anti-HIV-1 activity. Quantification of specific anti-HIV-1 antiproteases Serpin A1, Serpin A3, and Cystatin B and an epithelial antiprotease A2ML1 found them to be significantly over-abundant in HIV-1-resistant women (p = 0.004; p = 0.046; p = 0.0003; and p = 0.04, respectively). Expression levels were not correlated to sexual practices or other epidemiological factors. Mucosal antiprotease levels correlated with pro-inflammatory cytokine concentration (p = <0.0001), but independently of pro-inflammatory cytokine levels in HIV-1-resistant women including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, and IL-8. This comprehensive systems biology approach identifies mucosal serpins and cystatins as novel correlates of HIV-1-resistance. This represents the first study characterizing these factors in the female genital tract.

High resolution sequence-based DPB1 typing identified two novel DPB1 alleles, DPB1*9401 and DPB1*9501, from a Kenyan population.

Two novel DPB1 alleles, DPB1*9401 and DPB1*9501, were identified from a Kenyan population during sequence-based HLA-DPB1 typing. Molecular cloning and sequencing of multiple clones confirmed that one of the new DPB1 alleles is identical to DPB1*0402 at exon 2 except for a single nucleotide substitution (CGG -->TGG), changing codon 70 from Arg to Trp. The new allele has been named DPB1*9401. This is the first report of polymorphism at codon 70 of HLA-DPB1 alleles. New codon combinations have been identified in another novel DPB1 allele named DPB1*9501. The extensive diversity at DPB1 locus of this East African population is being revealed by high resolution sequence-based DPB1 typing.

HLA-A and HLA-B in Kenya, Africa: allele frequencies and identification of HLA-B*1567 and HLA-B*4426.

HLA-A and HLA-B alleles of a population from Kenya, Africa were examined by sequencing exon 2 and exon 3 DNA and typing using a Taxonomy-based Sequence-analysis (TBSA) method. Extensive diversities were observed at both HLA-A and HLA-B loci in this population. Forty-one HLA-A alleles were identified from 159 unrelated individuals. The most frequently observed alleles were A*6802 (11.64%), A*02011/09 (9.75%), A*7401/02 (9.43%), A*3001 (7.86%), A*3002 (7.23%) and A*3601 (6.6%). Forty-nine HLA-B alleles were identified in 161 unrelated individuals, including two novel alleles, B*1567 and B*4426. The most frequently observed HLA-B alleles were B*5301 (9.01%), B*5801 (8.38%), B*4201 (7.76%), B*1503 (7.14%), B*1801 (6.21%), and B*5802 (5.90%). The most frequently observed HLA-A-B haplotypes were A*3601-B*5301 (3.55%) and A*3001-B*4201 (3.19%), followed by A*7401/02-B*5801 (2.84%), A*7401/02-B*5802 (2.84%) and A*02011/09-B*1503 (2.13%). Linkage disequilibrium and chi2 analysis showed the association of these HLA-A-B haplotypes at the antigen level to be significant. The frequencies of HLA-A and HLA-B alleles from the Kenyan population were compared with that of a population from Cameroon. The difference in allele and haplotype frequency distributions partly reflected the different ethnic composition of these two African populations.

Effect of human immunodeficiency virus (HIV) type 1 viral genotype on mother-to-child transmission of HIV-1.

The objective of this study was to determine whether the maternal infecting human immunodeficiency virus (HIV) type 1 clade affects mother-to-child transmission frequency. Mothers in the mother-to-child HIV-1 transmission study in Nairobi, Kenya, were grouped by HIV-1 status of their first enrolled child: uninfected, perinatally infected, or postnatally infected. Restriction fragment length polymorphism (RFLP) analysis was used to determine HIV-1 viral clades of nested polymerase chain reaction products from HIV-1 protease or p24 genes. When inconclusive, sequencing determined the clade. Clade distributions within the groups were compared. The 3 groups displayed a uniform clade distribution. The predominant clades were A (59%) and D (20%). Clades B, C, F, mixed, and recombinant infections comprised the remainder (21%). No significant association was seen between clades A and D and either frequency or mode of vertical transmission. RFLP analysis revealed 2 clade B infections, 9 mixed, and 5 p24/protease recombinant infections in the study population.

Risk factors for postnatal mother-child transmission of HIV-1.

OBJECTIVE: To identify factors affecting HIV-1 breastfeeding transmission. DESIGN: Longitudinal observational cohort study. METHODS: HIV-1 seropositive pregnant women and seronegative controls were enrolled at a maternity hospital in Nairobi. Women and their children were followed from birth, and data on HIV-1 transmission, breastfeeding, clinical illness, and growth were collected. Specimens for HIV-1 serology and/or polymerase chain reaction were obtained at birth, 2, 6, and 14 weeks, 6, 9, 12, and 18 months, and every 6 months thereafter. Children were classified as HIV-1 uninfected, perinatally, or postnatally infected. Potentially breastfeeding transmission related risk factors were compared between postnatally infected and uninfected children. RESULTS: Among children born to seropositive or seroconverting mothers, 317 were uninfected, 51 infected perinatally and 42 infected postnatally. Identified risk factors for postnatal transmission were maternal nipple lesions (OR = 2.3, CI 95% 1.1-5.0), mastitis (OR = 2.7, CI 95% 1.1-6.7), maternal CD4 cell count < 400 mm3 (OR = 4.4, CI 95% 1.9-9.9), maternal seroconversion while breastfeeding (OR = 6.0, CI 95% 1.8-19.8), infant oral thrush at < 6 months of age (OR = 2.8, CI 95% 1.3-6.2) and breastfeeding longer than 15 months (OR = 2.4, CI 95% 1.2-5.1). All factors, except maternal seroconversion due to its rarity, were independently associated with an increased postnatal transmission risk by multivariate logistic regression analysis. CONCLUSION: In addition perinatal antiretroviral therapies, public health strategies should address: (i) prevention of maternal nipple lesions, mastitis and infant thrush; (ii) reduction of breastfeeding duration by all HIV-1-infected mothers; (iii) absolute avoidance of breastfeeding by those at high risk, and (iv) prevention of HIV-1 transmission to breastfeeding mothers.

Interleukin-10 response abnormalities in systemic lupus erythematosus.

It has been previously reported that the production of interleukin-6 (IL-6) is often enhanced in systemic lupus erythematosus (SLE). The authors examined the secretion of IL-6, tumour necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor, IL-1 alpha and IL-4 by B cells and monocytes from lupus patients and compared this to the production in normal controls and in rheumatoid arthritis patients. IL-6 production was increased an average of 3.4-fold compared to that in normal subjects and 8.4-fold compared to rheumatoid arthritis patients. In SLE, a strongly positive correlation was found between the levels of IL-6 and TNF-alpha (R = 0.8987, P = 0.002). Since production of both IL-6 and TNF-alpha is regulated by IL-10, the enhancement of the production of these cytokines could reflect a defect in either IL-10 production or responsiveness. However, spontaneous production of IL-10 was enhanced in cultures of B cells and monocytes from lupus patients, compared to normal controls, the levels being increased 3.1- to 6-fold for monocytes and B cells, respectively. The finding of increased secretion of these cytokines implies an abnormality in IL-10-mediated suppression in SLE. To assess this possibility, the authors examined recombinant human IL-10-mediated suppression of IL-6 production by monocytes and B cells from lupus patients, compared to normal controls, and found that whereas IL-10 caused a concentration-dependent suppression of IL-6 production in normal B cells and monocytes, this suppression was deficient in B cells and monocytes from lupus patients. In SLE, it therefore appears that there may be an intrinsic defect in IL-10-induced suppression of cytokine synthesis. This could explain the increased levels of IL-10 and IL-6 found in this condition, and may also be responsible for the characteristic polyclonal B-cell activation that is seen.

Normal human cord blood B cells can produce high affinity IgG antibodies to dsDNA that are recognized by cord blood-derived anti-idiotypic antibodies.

It is possible to identify, in Epstein-Barr virus-transformed normal human cord blood B cell populations, cells present at a low frequency that produce IgG antibodies specific for dsDNA. By cloning out these B cells as immortalized monoclonal cell lines, it could be shown that the antibodies were the products of CD5 positive B cells. Two monoclonal anti-dsDNA antibodies were derived from cell lines T52 and A7 and these were further characterized as anionic (pI approximately 6.4) IgG4 kappa antibodies that bound with affinities of 7.18 x 10(9) l/mol and 3.28 x 10(9) l/mol, respectively, to dsDNA but did not bind to ssDNA. These affinities were similar to those of polyclonal IgG anti-dsDNA antibodies from lupus patients, which ranged from 1 x 10(9) -8.9 x 10(10) l/mol. Both T52 and A7 monoclonal anti-dsDNA antibodies were recognized by cord blood-derived IgM antibodies. These IgM antibodies were not rheumatoid factors but bound to the F(ab')2 of A7 and T52 while failing to recognize T50, which is an autologous IgG4 kappa monoclonal antibody without specificity for dsDNA. A cloned B cell line A24 generated from the same cord blood sample as A7 produced an IgM monoclonal antibody that bound to the heavy chains of T52 and A7, but not T50 on Western blot and inhibited the binding of these antibodies to dsDNA. A7 and T52 competitively inhibited each other in their binding to the anti-idiotype A24, and A24 inhibited the binding to dsDNA of some polyclonal IgG anti-dsDNA antibodies purified from sera of lupus patients. The level of inhibition of binding of these antibodies to dsDNA was directly proportional to the levels of expression of the idiotype recognized by A24 on these antibodies. The normal human cord blood, therefore, may contain cells that form an idiotype/anti-idiotype network in which the idiotype is expressed on IgG antibodies with specificity for dsDNA and the anti-idiotype is an IgM antibody that binds to a heavy chain idiotope in such a way as to interfere with its interaction with dsDNA. The presence of a similar idiotype on some polyclonal anti-dsDNA antibodies in lupus that are similarly inhibitable by the cord blood-derived anti-idiotype raises the possibility that this network may persist in later life and perhaps become dysfunctional in systemic lupus erythematosus.