High activation and skewed T cell differentiation are associated with low IL-17A levels in a hu-PBL-NSG-SGM3 mouse model of HIV infection.

The humanized NOD/SCID/IL-2 receptor γ-chainnull (NSG) mouse model has been widely used for the study of HIV pathogenesis. Here, NSG mice with transgenic expression of human stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 (NSG-SGM3) were injected with peripheral blood leukocytes (PBL mice) from two HIV-infected (HIV+ ) patients who were under anti-retroviral therapy (ART; referred as HIV+ mice) or one HIV-seronegative healthy volunteer (HIV- ). Such mice are either hu-PBL-NSG-SGM3 HIV+ or HIV- mice, depending on the source of PBL. The kinetics of HIV replication and T cell responses following engraftment were evaluated in peripheral blood and secondary lymphoid tissues. High HIV replication and low CD4 : CD8 ratios were observed in HIV+ mice in the absence of anti-retroviral therapy (ART). Consistent with high activation and skewed differentiation of T cells from the HIV-infected donor, HIV+ mice exhibited a higher T cell co-expression of human leukocyte antigen D-related (HLA-DR) and CD38 than HIV- mice, as well as a shifted differentiation to a CCR7- CD45RA+ terminal effector profile, even in the presence of ART. In addition, HIV replication and the activation/differentiation disturbances of T cells were associated with decreased plasma levels of IL-17A. Thus, this hu-PBL-NSG-SGM3 mouse model recapitulates some immune disturbances occurring in HIV-infected patients, underlying its potential use for studying pathogenic events during this infection.

Apoptosis as a mechanism of natural resistance to HIV-1 infection in an exposed but uninfected population.

BACKGROUND: Apoptosis, also known as programmed cell death, has been reported not only as a pathogenic mechanism, but also as a mechanism of resistance and control of a variety of infections. Particularly during HIV-1 infection, apoptosis is the main mechanism by which infected and uninfected CD4+ lymphocytes are eliminated. However, apoptosis as a mechanism of natural resistance to HIV infection has this far not been explored. OBJECTIVE: To determine whether apoptosis could explain, at least in part, the natural resistance to HIV infection observed in some exposed but uninfected individuals (ESN). RESULTS: Our data shows that peripheral blood monocytes in the ESN group has a predisposition to undergo spontaneous apoptosis, as well as apoptosis induced by HIV infection in vitro, compared with monocyte population from the control group at low risk of HIV infection. CONCLUSIONS: These findings suggest that, in some ESN individuals, monocytes could play an important role in the control of HIV infection by undergoing apoptosis. However, since the variability among individuals is large, studies with larger cohorts focusing in monocyte apoptosis as pathogenic mechanisms are required.

Evidence for the presence of multilineage chimerism and progenitors of donor dendritic cells in the peripheral blood of bone marrow-augmented organ transplant recipients.

We have postulated that the donor leukocyte microchimerism plays a seminal role in the acceptance of allografts by inducing and perpetuating variable degree of donor-specific nonreactivity in long-surviving organ recipients. Limited information is available, however, concerning the phenotype and function of these chimeric cells in humans. The unequivocal presence of donor dendritic cells (DCs), a prominent lineage in the microchimerism observed in rodents and clinical organ recipients, was difficult to demonstrate in bone marrow (BM)-augmented organ transplant recipients. This enigma was resolved by the recent description of a method for propagating circulating human DCs from their progenitors by culture in a medium enriched with granulocyte-macrophage colony-stimulating factor and interleukin 4, a condition known to inhibit outgrowth of monocytes, thus providing a selective growth advantage to committed progenitors of the myeloid lineage. Cells from BM-augmented organ recipients and normal control subjects harvested from 12- to 14-day cultures exhibited dendritic morphology and potent allostimulatory capacity. Using appropriate primers, the presence of donor DNA was verified by polymerase chain reaction within the lineage(null)/class II(bright) sorted DC. Phenotypic analysis of cultured DCs from BM-augmented patients, unlike that of controls, exhibited a marked down-regulation of B7-1 (CD80) while retaining normal levels of expression of B7-2 (CD86) cell surface molecules. The presence of donor DNA was also confirmed by polymerase chain reaction in individually sorted lineage+ (T, B, and NK) cells and macrophages, suggesting that the chimerism in BM-augmented patients is multilineage. The presence of progenitors of donor DCs in the peripheral blood of BM-augmented patients further substantiates the already convincing evidence of stem cell engraftment.

Molecular characterization of the CCR 5 gene in seronegative individuals exposed to human immunodeficiency virus (HIV).

BACKGROUND: Both clinical and laboratory evidence in exposed seronegative (ESN) individuals to human HIV-1 has suggested the existence of mechanisms of natural resistance to the infection. A 32 base-pair deletion in the gene that codes for the CCR5, which is the main coreceptor for HIV-1, confers a high degree of resistance to HIV-1 infection. However, the genotype Delta32/Delta32 is present only in 2-4% of Caucasoid ESN individuals suggesting the existence of other mechanisms of protection. Mutations different from Delta32 have also been proposed as playing a role in resistance/susceptibility to this infection. OBJECTIVE: To screen for different mutations along the entire coding region of the ccr5 gene that can potentially explain the persistent seronegativity in a group of ESN individuals. STUDY DESIGN: Of a total of 86 individuals analyzed for Delta32 mutation by the PCR technique, 36 scored HIV seropositive (SP) and 50 were ESN. The entire group of ESN individuals was screened for other mutations in the ccr5 gene by single strand conformational polymorphism (SSCP) and DNA sequencing. RESULTS: The frequency of the mutant allele Delta32 was 4% (4/100) for ESN individuals and 4.2% (3/72) for SP individuals. The homozygous mutant genotype (Delta32/Delta32) was found in only 2% (1/50) of ESN individuals, but in no SP individuals. The heterozygous genotype was found in 8.3% (3/36) of SP individuals and in 4% (2/50) of ESN individuals. The differences in the allelic and genotypic frequencies among the groups were not statistically significant. A comparison between the observed and the expected genotypic frequencies showed that they were significantly different for the ESN group, suggesting a protective, yet indirect effect of the mutant genotype. CONCLUSIONS: The screening of the entire coding region of the ccr5 gene in all ESN did not revealed no other mutations that could account for resistance to HIV-1 infection. Although the CCR5 molecule is the most important coreceptor for HIV-1, mutations in this gene do not account for most of the cases of natural resistance to this virus that have so far been reported.

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes.

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

[A prospective study of neurodevelopment of uninfected children born to human immunodeficiency virus type 1 positive mothers]. / Estudio prospectivo comparativo sobre el desarrollo psicomotor de niños, nacidos de madres positivas para el virus de inmunodeficiencia humana tipo 1 no infectados.

INTRODUCTION: The human immunodeficiency virus type 1 (HIV-1) has tropism for the immune and central nervous systems (CNS). Intrauterine exposure to HIV-1 induces immunological alterations, independent of infection that might affect the development of the CNS. Similarly, the intrauterine exposure to antiretrovirals might also affect the neurodevelopment. AIM: To evaluate the neurodevelopment of babies born to HIV-1 positive mothers (exposed) and compare with babies born to HIV-1 negative mothers (unexposed). SUBJECTS AND METHODS: We carried-out an observational prospective study of neurodevelopment of 23 exposed and 20 unexposed children using the infant development scale Bayley-II, and the Denver-II test, neurological examination and anthropometric measurements during the first two years of life. RESULTS: None of the exposed babies acquired the infection. At one month of age the exposed babies exhibit normal but statistically lower values in the head circumference, compared to unexposed neonates. No differences were found in the psychomotor development index between both studied groups and exposed babies exhibited a lower mental development index but only at six months of age. The exposed babies exhibited a higher number of alterations during the neurological and Denver-II tests without reaching significant differences. CONCLUSIONS: The results suggest that intrauterine exposure to HIV-1 and to antiretrovirals in uninfected children born to HIV-1 positive mothers does not induce alterations in the neurodevelopment, at least during the first two years of life.

Depression of humoral responses and phagocytic functions in vivo and in vitro by fish oil and eicosapentanoic acid.

Previous studies have demonstrated that eicosapentanoic acid (EPA) has anti-inflammatory properties in both humans and experimental animals and may also depress humoral immunity in experimental animals. Our investigations showed that the addition of eicosapentanoic acid to human peripheral blood mononuclear cell cultures inhibited B cell responses to mitogenic stimulation and depressed the expression of interleukin 2 receptors in pokeweed mitogen-stimulated lymphocytes. Neutrophils were also affected in their ability to release the contents of primary and secondary granules, particularly when stimulated with antigen-antibody complexes. Similar depressions of B cell responses and neutrophil functions were observed in a normal volunteer who ingested 6 g/day of a commercially available fish oil extract (equivalent to 2.1 g of EPA/day) during a 6-week period. Phagocytosis, enzymatic release, circulating immunoglobulin levels, and the response to tetanus toxoid both in vivo and in vitro were depressed during ingestion of fish oil. Most parameters showed a trend toward normalization 6 weeks after the suspension of fish oil supplementation. These effects of fish oil extracts and EPA on phagocytosis and humoral responses may be advantageously used in the therapy of chronic inflammatory diseases and autoimmune diseases but could be a cause for concern when these compounds are used for longer periods of time and with minimal medical supervision for the prophylaxis of atherosclerosis.

The interaction of CD2 with its LFA-3 ligand expressed by autologous erythrocytes results in enhancement of B cell responses.

The addition of autologous erythrocytes to unfractionated human mononuclear cell cultures results in enhancement of B cell responses to antigens and mitogens. This costimulating effect of red cells is abrogated by their preincubation with anti-LFA-3 monoclonal antibody. Preincubation of mononuclear cells with anti-CD2 monoclonal antibodies (anti-Leu 5b, OKT11, used singly) has a down-regulating effect on B cell activation and no enhancement of B cell responses is seen when red cells are added to anti-CD2-treated cultures. These results demonstrate a functional effect on B cells of the interaction between the CD2 molecule on T lymphocytes and its natural ligand, LFA-3. The precise mechanism by which this costimulating effect on B lymphocytes takes place is unclear. The study of T cell populations and T cell activation markers shows that the addition of erythrocytes causes a small but reproducible increase in the number of cells expressing the IL-2 receptor and the addition of IL-2 enhances the response of mononuclear cells to antigenic stimulation in the presence of erythrocytes. However, the supernatants of mononuclear cell cultures stimulated with pokeweed mitogen in the presence of autologous erythrocytes show decreased levels of IL-2, compared to supernatants of cells stimulated with pokeweed mitogen alone. The same supernatants show increased levels of interferon-gamma, but the addition of this lymphokine to cultures stimulated with pokeweed mitogen has no potentiating effect. It is possible that the effect of erythrocytes is mediated by other growth and/or differentiation factors, and additional studies will be required to clarify this point.

Autologous red blood cells potentiate antibody synthesis by unfractionated human mononuclear cell cultures.

We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear cells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH, and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (1:50 ratio of PBMNC/RBC). The cultures were harvested on day 11; immunoglobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for calibration. While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-TT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in the presence of autologous RBC. Similarly, anti-KLH responses were easier to induce with PBMNC from an immune donor; maximal response was observed after stimulation with PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pretreatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-gamma). However, addition of IFN-gamma in different doses and at different times to PWM-stimulated PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody.

Cell microchimerism in patients with recurrent spontaneous abortion: preliminary results.

The goal of this study was to determine the prevalence of non-host male cell microchimerism in a group of women with a history of recurrent spontaneous abortion (RSA). The detection of male cell microchimerism was based upon amplification of a fragment of Y chromosome DNA obtained from peripheral blood mononuclear cells from the mother. The amplification products were electrophoresed, transferred onto nylon membranes and hybridized with a specific 32P-labelled probe. The products were visualized by autoradiography. Seventy-seven patients with RSA were studied. Some patients (42.8%) had received immunotherapy for RSA using live mononuclear cells from male donors. Of the 77 patients 46 (59.7%) were positive for the selected Y chromosome sequence, 22 (28.6%) had no evidence of Y chromosome DNA and in nine (11.7%) cases the chimeric status could not be defined since the amplified band was too faint to be clearly assigned as positive. Twenty patients were pregnant at the time of sampling. There were no statistically significant differences among the different variables studied: age of the mother, number of previous pregnancies, number of previous immunotherapeutic inoculations or period of time between the last inoculation and sampling. Male cell microchimerism has been reported in some but not all women who have given birth to male children. The dynamics for the establishment of this chimeric status and its persistence have not been defined. We found that most patients with RSA (59.7%) were positive for microchimerism but that this could not be correlated with abortion, current pregnancy or leukocyte immunotherapy. A prospective study is being undertaken to determine if there is a subset of patients negative for chimerism who become positive after alloimmunotherapy with male lymphocytes and have an improved prognosis for successful pregnancy.

Frequency of CCR5 delta-32 mutation in human immunodeficiency virus (HIV)-seropositive and HIV-exposed seronegative individuals and in general population of Medellin, Colombia.

Repeated exposure to human immunodeficiency virus (HIV) does not always result in seroconversion. Modifications in coreceptors for HIV entrance to target cells are one of the factors that block the infection. We studied the frequency of Delta-32 mutation in ccr5 gene in Medellin, Colombia. Two hundred and eighteen individuals distributed in three different groups were analyzed for Delta-32 mutation in ccr5 gene by polymerase chain reaction (PCR): 29 HIV seropositive (SP), 39 exposed seronegative (ESN) and 150 individuals as a general population sample (GPS). The frequency of the Delta-32 mutant allele was 3.8% for ESN, 2.7% for GPS and 1.7% for SP. Only one homozygous mutant genotype (Delta-32/Delta-32) was found among the ESN (2.6%). The heterozygous genotype (ccr5/Delta-32) was found in eight GPS (5.3%), in one SP (3.4%) and in one ESN (2.6%). The differences in the allelic and genotypic frequencies among the three groups were not statistically significant. A comparison between the expected and the observed genotypic frequencies showed that these frequencies were significantly different for the ESN group, which indirectly suggests a protective effect of the mutant genotype (Delta-32/Delta-32). Since this mutant genotype explained the resistance of infection in only one of our ESN persons, different mechanisms of protection must be playing a more important role in this population.

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.