Specific Immunity to Cytomegalovirus in Pediatric Cardiac Transplantation.

BACKGROUND: Cytomegalovirus (CMV) infection is implicated in endothelial dysfunction and graft damage after pediatric heart transplantation. CMV-specific immune responses are thought to be necessary for CMV viral control but there is little data in pediatric heart transplantation. METHODS: We studied 28 consecutive pediatric heart transplant recipients for 1 year posttransplant. CMV T-cell expressing IFN-γ, TNF-α, and IL-2 in response to ex vivo stimulation with CMV lysates or peptides were measured. Circulating cytokines were measured in plasma. Generalized Additive Models were applied to the data to model changes of cell population dynamics over time. RESULTS: CMV-specific T cell-mediated responses were impaired in the first 8 weeks posttransplant. During this period, 25% of patients had CMV viremia, of which those with VLs of 10 000 or more CMV deoxyribonucleic acid copies/mL were given ganciclovir. In this group, the frequency of CD4+ and CD8+ T cells producing IFN-γ and the CD8+CD57+ granzyme B+ T-cell population increased at 12 to 24 weeks and remained elevated for the duration of the study. CONCLUSIONS: We have shown that CMV viremia is associated with CMV-specific immune responses and increased CD8+CD57+ granzyme B+ cells at 1 year posttransplant; however, early responses were not predictive of impending CMV viremia. It remains to be seen if the early CMV immune response detected is associated with endothelial and allograft damage, in light of previous studies demonstrating increased vasculopathy in pediatric patients with CMV viremia.

Activation of the Large-Conductance, Voltage, and Ca2+- Activated K+ (BK) Channel in Acute Spinal Cord Injury in the Wistar Rat Is Neuroprotective.

Context/Objectives: Spinal cord injury (SCI) results in significant neuronal and glial cell death resulting in impaired neurological and motor function. Uncontrolled Ca2+ entry results in excitotoxicity and cell death. In this study, we examine the use of a BK channel activator, Isopimaric acid (ISO), as a neuroprotective agent post-SCI as this channel is involved in regulating Ca2+ entry. Design:By using a 25-g clip compression at the T6 level, we generated a SCI event in wistar rats. At 1 h post-injury we administered ISO (BK channel activator), the BK channel inhibitor iberiotoxin (IbTx), or a vehicle control for 4 weeks via mini osmotic pump (pump capacity). For 8 weeks post-injury, gait analysis of motor function was performed. At the end of 8 weeks, the extent of myelination in the spinal cord was assessed in addition to the electrophysiological profile. Results:Our immunohistological data suggests that ISO treatment leads to an increase or preservation of myelinated axonal tracts. This was further supported by our electrophysiological studies which demonstrate higher compound action potential amplitudes and speed of transmission in ISO-treated animals compared to inj-non-treated. Finally, treatment with ISO significantly improved motor function in our test model. Conclusion: In conclusion, activation of the BK channel during acute SCI may be a novel therapeutic target for acute SCI.

B-cell development and pneumococcal immunity in vertically acquired HIV infection.

OBJECTIVES: Many children with HIV infection now survive into adulthood. This study explored the impact of vertically acquired HIV in the era of antiretroviral therapy on the development of humoral immunity. DESIGN: Natural and vaccine-related immunity to pneumococcus and B-cell phenotype was characterized and compared in three groups of young adults: those with vertically-acquired infection, those with horizontally acquired infection and healthy controls. METHODS: Serotype-specific pneumococcal (Pnc) immunoglobulin M and G concentrations before and up to 1 year post-Pnc polysaccharide (Pneumovax) immunization were determined, and opsonophagocytic activity was analysed. B-cell subpopulations and dynamic markers of B-cell signalling, turnover and susceptibility to apoptosis were evaluated by flow cytometry. RESULTS: HIV-infected patients showed impaired natural Pnc immunity and reduced humoral responses to immunization with Pneumovax; this was greatest in those viraemic at time of the study. Early-life viral control before the age of 10 years diminished these changes. Expanded populations of abnormally activated and immature B-cells were seen in both HIV-infected cohorts. Vertically infected patients were particularly vulnerable to reductions in marginal zone and switched memory populations. These aberrations were reduced in patients with early-life viral control. CONCLUSION: In children with HIV, damage to B-cell memory populations and impaired natural and vaccine immunity to pneumococcus is evident in early adult life. Sustained viral control from early childhood may help to limit this effect and optimize humoral immunity in adult life.

The immunological and virological consequences of planned treatment interruptions in children with HIV infection.

OBJECTIVES: To evaluate the immunological and viral consequences of planned treatment interruptions (PTI) in children with HIV. DESIGN: This was an immunological and virological sub-study of the Paediatric European Network for Treatment of AIDS (PENTA) 11 trial, which compared CD4-guided PTI of antiretroviral therapy (ART) with continuous therapy (CT) in children. METHODS: HIV-1 RNA and lymphocyte subsets, including CD4 and CD8 cells, were quantified on fresh samples collected during the study; CD45RA, CD45RO and CD31 subpopulations were evaluated in some centres. For 36 (18 PTI, 18 CT) children, immunophenotyping was performed and cell-associated HIV-1 DNA analysed on stored samples to 48 weeks. RESULTS: In the PTI group, CD4 cell count fell rapidly in the first 12 weeks off ART, with decreases in both naïve and memory cells. However, the proportion of CD4 cells expressing CD45RA and CD45RO remained constant in both groups. The increase in CD8 cells in the first 12 weeks off ART in the PTI group was predominantly due to increases in RO-expressing cells. PTI was associated with a rapid and sustained increase in CD4 cells expressing Ki67 and HLA-DR, and increased levels of HIV-1 DNA. CONCLUSIONS: PTI in children is associated with rapid changes in CD4 and CD8 cells, likely due to increased cell turnover and immune activation. However, children off treatment may be able to maintain stable levels of naïve CD4 cells, at least in proportion to the memory cell pool, which may in part explain the observed excellent CD4 cell recovery with re-introduction of ART.

Lentiviral gene therapy against human immunodeficiency virus type 1, using a novel human TRIM21-cyclophilin A restriction factor.

TRIM5α (tripartite motif-containing protein-5, isoform α)-cyclophilin A fusion proteins are anti-human immunodeficiency virus (HIV) restriction factors that have evolved in certain nonhuman primates over millions of years and protect against HIV and related viruses. Restriction by TRIM5αCypA is potent and highly resistant to viral escape by mutation and, in combination with a suitable gene delivery platform, offers the possibility of novel therapeutic approaches against HIV. Here we report that lentiviral vector delivery of human mimics of TRIM5α-cyclophilin A (TRIM5CypA) fusion proteins afforded robust and durable protection against HIV-1, but resulted in downregulation of host cell antiviral responses mediated by endogenous TRIM5α. We found that substitution of TRIM5α RING, B-box, and coiled-coil domains with similar domains from a related TRIM protein, TRIM21, produced a novel and equally potent inhibitor of HIV-1. Both TRIM5CypA and TRIM21CypA inhibited transduction by HIV-1-derived viral vectors and prevented propagation of replication-competent HIV-1 in human cell lines and in primary human T cells. Restriction factor-modified T cells exhibited preferential survival in the presence of wild-type HIV. Restriction was dependent on proteasomal degradation and was reversed in the presence of the cyclophilin inhibitor cyclosporin. Importantly, TRIM21CypA did not disturb endogenous TRIM5α-mediated restriction of gammaretroviral infection. Furthermore, endogenous TRIM21 antiviral activity was assessed by measuring inhibition of adenovirus-antibody complexes and was found to be preserved in all TRIMCypA-modified groups. We conclude that lentivirus-mediated expression of the novel chimeric restriction factor TRIM21CypA provides highly potent protection against HIV-1 without loss of normal innate immune TRIM activity.

Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved.

The melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R-MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and α-melanocyte stimulating hormone (α-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSH analogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (PKB), adenosine 5' monophosphate activated protein kinase (AMPK) and Jun-amino-terminal kinase (JNK) in MCR mediated lipolysis were studied. Interestingly, results obtained in 3T3-L1 cells suggest that lipolysis stimulated by α-MSH, NDP-α-MSH, MT-II, SHU9119 and PG-901 is mediated through MC5R in a cAMP independent manner. Finally, we identify essential differences in MCR mediated lipolysis when using 3T3-L1 cells compared to primary adipocytes.

Adherence to the Mediterranean diet is associated with lower abdominal adiposity in European men and women.

Given the lack of consistent evidence of the relationship between Mediterranean dietary patterns and body fat, we assessed the cross-sectional association between adherence to a modified Mediterranean diet, BMI, and waist circumference (WC). A total of 497,308 individuals (70.7% women) aged 25-70 y from 10 European countries participated in this study. Diet was assessed at baseline using detailed validated country-specific questionnaires, and anthropometrical measurements were collected using standardized procedures. The association between the degree of adherence to the modified-Mediterranean Diet Score (mMDS) (including high consumption of vegetables, legumes, fruits and nuts, cereals, fish and seafood, and unsaturated:saturated fatty acids ratio; moderate alcohol intake; and low consumption of meat and meat products and dairy products) and BMI (kg.m(-2)) or WC (cm) was modeled through mixed-effects linear regression, controlling for potential confounders. Overall, the mMDS was not significantly associated with BMI. Higher adherence to the Mediterranean diet was significantly associated with lower WC, for a given BMI, in both men (-0.09; 95% CI -0.14 to -0.04) and women (-0.06; 95% CI -0.10 to -0.01). The association was stronger in men (-0.20; 95% CI -0.23 to -0.17) and women (-0.17; 95% CI -0.21 to -0.13) from Northern European countries. Despite the observed heterogeneity among regions, results of this study suggest that adherence to a modified Mediterranean diet, high in foods of vegetable origin and unsaturated fatty acids, is associated with lower abdominal adiposity measured by WC in European men and women.

Pediatric human immunodeficiency virus infection and circulating IgD+ memory B cells.

Levels of circulating naive and memory B cells were measured in human immunodeficiency virus (HIV)-infected children and control subjects to determine whether the irreversible depletion of memory B cells described in HIV-infected adults occurs in children with HIV infection. Depletion of circulating IgD+ memory B cells was seen in HIV-infected children despite control of the HIV load with highly active antiretroviral therapy (HAART) (P =. 04). IgD+ memory B cell percentages did not correlate with CD4+ cell percentages (P =. 027) or disease duration (P =. 026). Naive/transitional and IgD- memory B cell numbers were not affected. Pediatric HIV infection is associated with selective depletion of circulating IgD+ memory B cells despite control of the HIV load with HAART.

Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display.

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.

Activation of fibroblast procollagen alpha 1(I) transcription by mechanical strain is transforming growth factor-beta-dependent and involves increased binding of CCAAT-binding factor (CBF/NF-Y) at the proximal promoter.

During normal developmental tissue growth and in a number of diseases of the cardiopulmonary system, adventitial and interstitial fibroblasts are subjected to increased mechanical strain. This leads to fibroblast activation and enhanced collagen synthesis, but the underlying mechanisms involved remain poorly understood. In this study, we have begun to identify and characterize mechanical strain-responsive elements in the rat procollagen alpha 1(I) (COL1A1) gene and show that the activity of COL1A1 promoter constructs, transiently transfected into cardiac fibroblasts, was increased between 2- and 4-fold by continuous cyclic mechanical strain. This was accompanied by an approximately 3-fold increase in the levels of total active transforming growth factor-beta (TGF-beta) released into the medium. Inclusion of a pan-specific TGF-beta neutralizing antibody inhibited strain-induced COL1A1 promoter activation. Deletion analysis revealed the presence of two potential strain response regions within the proximal promoter, one of which contains an inverted CCAAT-box overlapping a GC-rich element. Both mechanical strain and exogenously added TGF-beta1 enhanced the binding activity of CCAAT-binding factor, CBF/NF-Y, at this site. Moreover, this element was sufficient to confer strain-responsiveness to an otherwise unresponsive SV40 promoter. In summary, this study demonstrates that strain-induced COL1A1 promoter activation in cardiac fibroblasts is TGF-beta-dependent and involves increased binding of CCAAT-binding factor at the proximal promoter. Furthermore, these findings suggest a novel and potentially important TGF-beta response element in the rat COL1A1 gene.

Immunohistochemical demonstration of tumour associated antigens in carcinoma-in-situ of the testis.

Atypical germ cells or so-called carcinoma-in-situ of the testis are often found in the tissue adjacent to germ cell tumours. The present study was performed to investigate if tumour associated antigens demonstrated immunohistochemically in the tumours could also be demonstrated in the carcinoma-in-situ. Using indirect immunoperoxidase technique 39 orchidectomy specimens were examined for the presence of a series of antigens: alpha-foetoprotein (AFP), alpha1 -antitrypsin (A1 AT), human chorionic gonadotrophin (hCG). specific pregnancy ß-glycoprotein (SP1 ), human placental lactogen (hPL), carcino-embryonic antigen (CEA) and ferritin (FER). FER was demonstrated in the atypical cells in 24/29 cases of carcinoma-in-situ of the testis. HCG was demonstrated in intratubular syncytiotrophoblast-like cells in one case and in atypical germ cells in another specimen. No staining reaction was found for the other antigens investigated Normal germinal epithelium and germinal epithelium in non-malignant pathological changes of the testis were never stained. These findings which indicate that FER may be a possible marker of carcinoma-in-situ of the testis is of utmost interest, however investigation of a larger series is mandatory.