Major Immunological Determinant Research Articles

Pretransplant Numbers of CD16+ Monocytes as a Novel Biomarker to Predict Acute Rejection after Kidney Transplantation

Acute rejection is one of the major immunological determinants of kidney graft function and survival. Early biomarkers to predict rejection are lacking. Emerging evidence reveals a crucial role for the monocyte-macrophage lineage cells in the pathogenesis of rejection. We hypothesized that higher pre-transplant numbers of proinflammatory CD16+ monocytes can predict rejection. The study cohort consisted of 104 kidney transplant recipients (58 non-rejectors and 46 biopsy-proven rejectors), and 33 healthy individuals. Posttransplant median ± IQR follow up time was 14.7 (0.3-34) months. Pretransplantation blood samples were analyzed by flow cytometry for monocyte immunophenotypes. Groups were compared by Cox regression models for the occurrence of acute rejection. We documented a significantly increased absolute number of pretransplant CD16+ monocytes in patients who developed biopsy proven rejection after transplantation compared to non-rejectors and healthy individuals (Hazard Ratio [HR], 1.60; 95% Confidential Interval [CI], 1.28 to 2.00; p < 0,001 and HR, 1.47; CI, 1.18 to 1.82, p < 0,001). In parallel, significantly less absolute numbers of CD16- monocytes were observed at pretransplant time point in rejectors vs non-rejectors (HR, 0.74; CI, 0.58 to 0.94; p < 0,014). A higher pre-transplant number of CD16+ monocytes is significantly associated with a higher risk of acute rejection after kidney transplantation.

Pretransplant Numbers of CD16+ Monocytes as a Novel Biomarker to Predict Acute Rejection After Kidney Transplantation: A Pilot Study.

Acute rejection is one of the major immunological determinants of kidney graft function and survival. Early biomarkers to predict rejection are lacking. Emerging evidence reveals a crucial role for the monocyte/macrophage lineage cells in the pathogenesis of rejection. We hypothesized that higher pretransplant numbers of proinflammatory CD16+ monocytes can predict rejection. The study cohort consisted of 104 kidney transplant recipients (58 with no rejection and 46 with biopsy-proven rejection) and 33 healthy persons. Posttransplant median follow-up time was 14.7 mo (interquartile range 0.3-34 mo). Pretransplantation blood samples were analyzed by flow cytometry for monocyte immunophenotypes. Groups were compared by Cox regression models for the occurrence of acute rejection. We documented a significantly increased absolute number of pretransplant CD16+ monocytes in patients who developed biopsy-proven rejection after transplantation compared with those with no rejection (hazard ratio [HR] 1.60, 95% CI 1.28-2.00, p < 0.001) and healthy persons (HR 1.47, 95% CI 1.18-1.82, p < 0.001). In parallel, significantly fewer absolute numbers of CD16- monocytes were observed at pretransplant time points in rejectors versus nonrejectors (HR 0.74, 95% CI 0.58-0.94, p < 0,014). A higher pretransplant number of CD16+ monocytes is significantly associated with a higher risk of acute rejection after kidney transplantation.

M1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza.

In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues.

Immunisation of Sheep with Bovine Viral Diarrhoea Virus, E2 Protein Using a Freeze-Dried Hollow Silica Mesoporous Nanoparticle Formulation.

Bovine viral diarrhoea virus 1 (BVDV-1) is arguably the most important viral disease of cattle. It is associated with reproductive, respiratory and chronic diseases in cattle across the world. In this study we have investigated the capacity of the major immunological determinant of BVDV-1, the E2 protein combined with hollow type mesoporous silica nanoparticles with surface amino functionalisation (HMSA), to stimulate immune responses in sheep. The current work also investigated the immunogenicity of the E2 nanoformulation before and after freeze-drying processes. The optimal excipient formulation for freeze-drying of the E2 nanoformulation was determined to be 5% trehalose and 1% glycine. This excipient formulation preserved both the E2 protein integrity and HMSA particle structure. Sheep were immunised three times at three week intervals by subcutaneous injection with 500 μg E2 adsorbed to 6.2 mg HMSA as either a non-freeze-dried or freeze-dried nanoformulation. The capacity of both nanovaccine formulations to generate humoral (antibody) and cell-mediated responses in sheep were compared to the responses in sheep immunisation with Opti-E2 (500 μg) together with the conventional adjuvant Quil-A (1 mg), a saponin from the Molina tree (Quillaja saponira). The level of the antibody responses detected to both the non-freeze-dried and freeze-dried Opti-E2/HMSA nanoformulations were similar to those obtained for Opti-E2 plus Quil-A, demonstrating the E2 nanoformulations were immunogenic in a large animal, and freeze-drying did not affect the immunogenicity of the E2 antigen. Importantly, it was demonstrated that the long term cell-mediated immune responses were detectable up to four months after immunisation. The cell-mediated immune responses were consistently high in all sheep immunised with the freeze-dried Opti-E2/HMSA nanovaccine formulation (>2,290 SFU/million cells) compared to the non-freeze-dried nanovaccine formulation (213–500 SFU/million cells). This study is the first to demonstrate that a freeze-dried silica mesoporous nanovaccine formulation gives balanced immune responses in a production animal.

Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever.

BackgroundLassa fever is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute Lassa fever infections has successfully utilized intravenous administration of ribavirin, a nucleotide analogue drug, but this is not an approved use; efficacy of oral administration has not been demonstrated. To date, several potential new vaccine platforms have been explored, but none have progressed toward clinical trials and commercialization. Therefore, the development of a robust vaccine platform that could be generated in sufficient quantities and at a low cost per dose could herald a subcontinent-wide vaccination program. This would move Lassa endemic areas toward the control and reduction of major outbreaks and endemic infections. To this end, we have employed efficient mammalian expression systems to generate a Lassa virus (LASV)-like particle (VLP)-based modular vaccine platform.ResultsA mammalian expression system that generated large quantities of LASV VLP in human cells at small scale settings was developed. These VLP contained the major immunological determinants of the virus: glycoprotein complex, nucleoprotein, and Z matrix protein, with known post-translational modifications. The viral proteins packaged into LASV VLP were characterized, including glycosylation profiles of glycoprotein subunits GP1 and GP2, and structural compartmentalization of each polypeptide. The host cell protein component of LASV VLP was also partially analyzed, namely glycoprotein incorporation, though the identity of these proteins remain unknown. All combinations of LASV Z, GPC, and NP proteins that generated VLP did not incorporate host cell ribosomes, a known component of native arenaviral particles, despite detection of small RNA species packaged into pseudoparticles. Although VLP did not contain the same host cell components as the native virion, electron microscopy analysis demonstrated that LASV VLP appeared structurally similar to native virions, with pleiomorphic distribution in size and shape. LASV VLP that displayed GPC or GPC+NP were immunogenic in mice, and generated a significant IgG response to individual viral proteins over the course of three immunizations, in the absence of adjuvants. Furthermore, sera from convalescent Lassa fever patients recognized VLP in ELISA format, thus affirming the presence of native epitopes displayed by the recombinant pseudoparticles.ConclusionsThese results established that modular LASV VLP can be generated displaying high levels of immunogenic viral proteins, and that small laboratory scale mammalian expression systems are capable of producing multi-milligram quantities of pseudoparticles. These VLP are structurally and morphologically similar to native LASV virions, but lack replicative functions, and thus can be safely generated in low biosafety level settings. LASV VLP were immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks after the first immunization. These studies highlight the relevance of a VLP platform for designing an optimal vaccine candidate against Lassa hemorrhagic fever, and warrant further investigation in lethal challenge animal models to establish their protective potential.

Interferon (IFN)–γ, Tumor Necrosis Factor–α, Interleukin‐6, and IFN‐γ Receptor 1 Are the Major Immunological Determinants Associated with Post–Kala Azar Dermal Leishmaniasis

Semiquantitative reverse-transcription polymerase chain reaction was used to analyze intralesional cytokine gene expression in 28 patients with post-kala azar dermal leishmaniasis (PKDL) and 14 patients with kala azar (KA). The data revealed mixed T helper cell type 1 (Th1) and T helper cell type 2 (Th2) responses, as reflected by elevated expression of interferon (IFN)-gamma , tumor necrosis factor (TNF)-alpha , transforming growth factor (TGF)-beta , interleukin (IL)-10, IL-6, and IL-4 mRNA, with minimal expression of IFN-gamma receptor 1 (IFN-gamma R1) mRNA in PKDL lesions, compared with that in normal skin tissue. In comparison with those in KA lesions, mRNA levels for IFN-gamma , TNF-alpha , and IL-6 were found to be significantly elevated in PKDL lesions, implying that these cytokines play an important role in PKDL pathogenesis. In the presence of elevated levels of IFN-gamma and TNF-alpha , interference with type 1 effector activity in PKDL may be due to minimal expression of the IFN-gamma R1 gene or the simultaneous presence of elevated levels of IL-10, IL-6, and TGF-beta , which have counteracting effects. After treatment, the restoration of IFN-gamma R1 at both mRNA and protein levels, coupled with down-regulation of counteracting cytokines, may facilitate the action of signals associated with IFN-gamma , yielding parasite clearance. Therefore, unfavorable clinical evolution in PKDL may not be due to the absence of an intralesional Th1 response but rather may be due to the presence of counteracting cytokines along with the down-modulation of IFN-gamma R1.

Endocrinology of pregnancy: Consequences for the diagnosis and treatment of pregnancy disorders

Placental human chorionic gonadotropin and corpus luteum secretion of progesterone and oestradiol are the main endocrine events at the beginning of pregnancy, whilst the luteo-placental shift is an important step during the later stages. Progesterone not only affects decidualisation, but is the major immunological determinant and controls uterine contractibility and cervical competence. These properties all contribute considerably towards the correct development of pregnancy and delivery at term.

Expression of membrane-bound HLA-G at the maternal-fetal interface is not associated with pregnancy maintenance among patients with idiopathic recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) has many known aetiologies. However, using current diagnostic testing, a large fraction of recurrent pregnancy losses remain unexplained. Many of these may have immune underpinnings. HLA-G is a non-classical MHC class I product whose fairly restricted expression at the maternal-fetal interface suggests a role in successful embryonic implantation and/or subsequent pregnancy maintenance. The study of immune-mediated RPL should be enhanced by comparing groups of idiopathic RPL patients with normal fetal chromosomes to RPL patients with known chromosomal abnormalities in their index pregnancies. We hypothesized that if alteration of HLA-G expression at the maternal-fetal interface were associated with immune-mediated RPL, such changes might be detectable using these comparisons. HLA-G protein expression at the maternal-fetal interface in maternal and gestational age-matched women with history of idiopathic RPL and normal male fetuses were compared with expression in RPL patients with known fetal trisomy 16 in their index pregnancy. We detected no significant quantitative differences in the levels of HLA-G between these groups of RPL patients. Within the limitations of this study, we conclude that HLA-G expression is not a major immunological determinant of pregnancy maintenance among patients with idiopathic RPL.

Characterization of a Novel Baboon Virus Closely Resembling Human T-Cell Leukemia Virus

We report the isolation of a virus from a baboon imported from Kenya and the analysis of the nucleotide sequence of theenvgene. Comparison of the complete nucleotide sequence of theenvgene of different HTLV-1 strains and the baboon T-cell leukemia virus (designated BTLV) indicated similarities ranging from 92.5 to 97.4%. In contrast, only 89.1% similarity was observed between the BTLVenvsequence and that of simian T-cell leukemia virus (PtM3). The sequences corresponding to the glycosylation sites, endoproteolytic processing site, and major immunological determinants were strictly conserved between BTLV and HTLV-1. To characterize the expressed protein we used a vaccinia expression system, which indicated that a protein of 62 kDa is encoded by the envelope gene. The protein acquired mostly high mannose modifications and was localized predominantly in the endoplasmic reticulum. A fraction of the protein was expressed at the cell surface, where it could induce membrane fusion of target cells. The existence of HTLV-1-like viruses in baboons indicates the potential risk of transmission of such virus from these nonhuman primates to humans, thus highlighting the need for specific screening for such viruses during xenotransplantation.

Structure of a novel phosphocholine-containing glycoglycerolipid from Mycoplasma fermentans

Mycoplasma fermentans is thought to be a pathogen of rheumatoid arthritis or cofactor of AIDS. A novel phosphocholine-containing glycoglycerophospholipid named GGPL-I was isolated from a M. fermentans-infected human helper T-cell culture. It was revealed that GGPL-I is a lipid component of the M. fermentans and a major immunological determinant. The GGPL-I was purified by DEAE-Sephadex column chromatography and repeated Iatrobeads column chromatography. The purified glycophospholipid was subjected to structural characterization by thin-layer chromatography, Fourier-transform infrared spectrometry, liquid secondary ion mass spectrometry, and nuclear magnetic resonance spectroscopy. Its structure was determined to be as follows: 6'-O-phosphocholine-alpha-glucopyranosyl-(1'-3)-1,2-diacyl-sn-glycerol. This glycoglycerophospholipid is unique in containing phosphocholine, which is attached to C-6 of glucose. The stereospecific numbering (sn) of naturally occurring GGPL-I was determined through comparison with chemically synthesized compounds.