The implication of serum HLA-G in angiogenesis of multiple myeloma.

BACKGROUND: Despite the advances of therapies, multiple myeloma (MM) remains an incurable hematological cancer that most patients experience relapse. Tumor angiogenesis is strongly correlated with cancer relapse. Human leukocyte antigen G (HLA-G) has been known as a molecule to suppress angiogenesis. We aimed to investigate whether soluble HLA-G (sHLA-G) was involved in the relapse of MM. METHODS: We first investigated the dynamics of serum sHLA-G, vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6) in 57 successfully treated MM patients undergoing remission and relapse. The interactions among these angiogenesis-related targets (sHLA-G, VEGF and IL-6) were examined in vitro. Their expression at different oxygen concentrations was investigated using a xenograft animal model by intra-bone marrow and skin grafts with myeloma cells. RESULTS: We found that HLA-G protein degradation augmented angiogenesis. Soluble HLA-G directly inhibited vasculature formation in vitro. Mechanistically, HLA-G expression was regulated by hypoxia-inducible factor-1α (HIF-1α) in MM cells under hypoxia. We thus developed two mouse models of myeloma xenografts in intra-bone marrow (BM) and underneath the skin, and found a strong correlation between HLA-G and HIF-1α expressions in hypoxic BM, but not in oxygenated tissues. Yet when stimulated with IL-6, both HLA-G and HIF-1α could be targeted to ubiquitin-mediated degradation via PARKIN. CONCLUSION: These results highlight the importance of sHLA-G in angiogenesis at different phases of multiple myeloma. The experimental evidence that sHLA-G as an angiogenesis suppressor in MM may be useful for future development of novel therapies to prevent relapse.

[Correlation between Serum Interleukin-33, ß2-Microglobulin Levels and DS Stage in Patients with Multiple Myeloma].

OBJECTIVE: To investigate the correlation between serum interleukin-33 (IL-33), ß2microglobulin (ß2-MG) levels and Durie-Salmon (DS) stage in patients with multiple myeloma (MM). METHODS: 100 MM patients admitted to the First Affiliated Hospital of Fujian Medical University from March 2019 to January 2021 were selected and divided into stage I, stage II and stage III groups according to the DS staging system. A baseline data questionnaire of patients was designed, then the relevant baseline data and laboratory test results of patients were recorded. The levels of serum IL-33 and ß2-MG of all patients were detected, and the correlation between serum IL-33, ß2-MG levels and DS stage of MM patients was analyzed. RESULTS: Among the 100 patients with MM, there were 32 cases in stage I, 39 cases in stage II and 29 cases in stage III. The levels of serum CRP and ß2-MG of patients in stage III were significantly higher than those of patients in stage I and II, and the levels of serum CRP and ß2-MG of patients in stage II were significantly higher than those of patients in stage I, the differences were statistically significant (P <0.05). The level of serum IL-33 of patients in stage III was significantly lower than that of patients in stage I and II, and the level of serum IL-33 of patients in stage II was significantly lower than that of patients in stage I, the differences were statistically significant (P <0.05). There was no statistical significant difference in other data between groups (P >0.05). Kendall's tau-b correlation analysis showed that the levels of serum CRP and ß2-MG were positively correlated with DS stage in MM patients (r =0.534, 0.776), the level of serum IL-33 was negatively correlated with DS stage in MM patients (r =-0.759). Ordered logistic regression analysis and forest plot showed that the low level of serum IL-33 and the high level of ß2-MG were the influencing factors of high DS stage in MM patients (P <0.05 ). CONCLUSION: DS stage of MM patients is closely related to the levels of serum IL-33 and ß2-MG, that is, the lower the serum IL-33 level and the higher the ß2-MG level, and the higher the DS stage of MM patients.

Soluble HLA-G levels in heart transplant recipients: Dynamics and correlation with clinical outcomes.

PURPOSE: To describe the evolution of the serum levels of soluble HLA-G (s-HLA-G) during the first 12 months after heart transplantation (HT) and to correlate it with clinical outcomes. METHODS: Observational study based in a single-center cohort of 59 patients who underwent HT between December-2003 and March-2010. Soluble HLA-G levels were measured from serum samples extracted before HT, and 1, 3, 6 and 12 months after HT. The cumulative burden of s-HLA-G expression during the first post-transplant year was assessed by means of the area under the curve (AUC) of s-HLA-G levels over time and correlated with the acute rejection burden -as assessed by a rejection score-, the presence of coronary allograft vasculopathy (CAV) grade ≥ 1 and infections during the first post-transplant year; as well as with long-term patient and graft survival. Mean follow-up was 12.4 years. RESULTS: Soluble HLA-G levels decreased over the first post-transplant year (p = 0.020). The AUC of s-HLA-G levels during the first post-transplant year was higher among patients with infections vs. those without infections (p = 0.006). No association was found between the AUC of s-HLA-G levels and the burden of acute rejection or the development of CAV. Overall long-term survival, long-term survival free of late graft failure and cancer-free survival were not significantly different in patients with an AUC of s-HLA-G levels higher or lower than the median of the study population. CONCLUSIONS: Soluble HLA-G levels decreased over the first year after HT. Higher HLA-G expression was associated with a higher frequency of infections, but not with the burden of acute rejection or the development of CAV, neither with long-term patient or graft survival.

Correlation between Serum Interleukin-33, β2-Microglobulin Levels and DS Stage in Patients with Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the correlation between serum interleukin-33 (IL-33), β2microglobulin (β2-MG) levels and Durie-Salmon (DS) stage in patients with multiple myeloma (MM).@*METHODS@#100 MM patients admitted to the First Affiliated Hospital of Fujian Medical University from March 2019 to January 2021 were selected and divided into stage I, stage II and stage III groups according to the DS staging system. A baseline data questionnaire of patients was designed, then the relevant baseline data and laboratory test results of patients were recorded. The levels of serum IL-33 and β2-MG of all patients were detected, and the correlation between serum IL-33, β2-MG levels and DS stage of MM patients was analyzed.@*RESULTS@#Among the 100 patients with MM, there were 32 cases in stage I, 39 cases in stage II and 29 cases in stage III. The levels of serum CRP and β2-MG of patients in stage III were significantly higher than those of patients in stage I and II, and the levels of serum CRP and β2-MG of patients in stage II were significantly higher than those of patients in stage I, the differences were statistically significant (P <0.05). The level of serum IL-33 of patients in stage III was significantly lower than that of patients in stage I and II, and the level of serum IL-33 of patients in stage II was significantly lower than that of patients in stage I, the differences were statistically significant (P <0.05). There was no statistical significant difference in other data between groups (P >0.05). Kendall's tau-b correlation analysis showed that the levels of serum CRP and β2-MG were positively correlated with DS stage in MM patients (r =0.534, 0.776), the level of serum IL-33 was negatively correlated with DS stage in MM patients (r =-0.759). Ordered logistic regression analysis and forest plot showed that the low level of serum IL-33 and the high level of β2-MG were the influencing factors of high DS stage in MM patients (P <0.05 ).@*CONCLUSION@#DS stage of MM patients is closely related to the levels of serum IL-33 and β2-MG, that is, the lower the serum IL-33 level and the higher the β2-MG level, and the higher the DS stage of MM patients.

Longitudinal profile of sHLA-G during pregnancy and its association with small for gestational age births in North Indian pregnant females: A nested case-control study.

PROBLEM: Small for gestational age (SGA) neonates are vulnerable to various long and short-term adverse health consequences. The expression of HLA-G in the placenta is crucial for establishment and maintenance of pregnancy. Its aberrant expression could lead to perturbed immunological interactions in the placenta which could be associated with SGA births. The objective of this study was to assess the difference in the trajectories of soluble HLA-G in maternal sera during pregnancy between women delivering SGA and appropriate for gestational age (AGA) neonates. METHOD OF STUDY: Soluble HLA-G was estimated in the maternal sera collected at different time points in pregnancy of North-Indian pregnant females delivering SGA (N = 23) or AGA (N = 17) neonates using sandwich ELISA. Linear mixed models were built and compared to study the association between sHLA-G levels during pregnancy and SGA births. RESULTS: No significant difference was observed in the sHLA-G trajectories during pregnancy in mothers delivering SGA as compared to those delivering AGA (P-value = .5677). A trend towards higher sHLA-G levels at the first trimester of pregnancy (< 14 weeks of gestation) was observed in mothers delivering SGA neonates (Median = 41.71, IQR = 21.31-71.38) as compared to those delivering AGA neonates (Median = 37.58, IQR = 19.05-73.57). CONCLUSION: During pregnancy, sHLA-G trajectories do not differ significantly between mothers delivering SGA and those delivering AGA neonates. However, sHLA-G trends towards higher levels during early pregnancy in mothers delivering SGA neonates.

Association of reduced maternal sHLA-G5 isoform levels and elevated TNF-α/IL-4 cytokine ratio with Recurrent Pregnancy Loss: A study on South Indian women.

Inflammation is of critical importance in successful implantation during pregnancy. However, the establishment of maternal immune tolerance towards semi-allograft foetus is more exigent and is achieved predominantly by human leukocyte antigen-G (HLA-G) isoforms with a special emphasis on soluble HLA-G5 (sHLA-G5). Constant inflammation and lack of resolution by anti-inflammatory milieu, due to aberrant expression of critical immunoregulatory molecules such as sHLA-G5 and dysfunctional T helper cells 1 and 2 (Th1-Th2) cytokine shift, can lead to adverse pregnancy outcomes including recurrent pregnancy loss (RPL). Serum samples of 270 pregnant women (135 healthy parous and 135 with a history of RPL) were evaluated for the concentrations of sHLA-G5, interleukin-4 (IL-4) and tumour necrosis factor-alpha (TNF-α) using sandwich enzyme-linked immunosorbent assay (ELISA) and found elevated levels of sHLA-G5 and IL-4 in controls and higher TNF-α levels and TNF-α:IL-4 ratio in patients (P < .05). Stratified data analysis based on the time of sample collection, that is the first and second trimesters exhibited higher sHLA-G5 and IL-4 in both first and second trimesters in controls than patients, while they displayed lower levels concerning TNF-α and TNF-α:IL-4 ratio (P < .05). However, within patients and controls in the first or second trimesters, there was a significant variation concerning sHLA-G5 alone. Further, the outcome of pregnancies studied in the present investigation revealed a significant elevation in sHLA-G5 levels among women with successful pregnancies compared with women who experienced pregnancy loss, therefore, concluding the potential application of sHLA-G5 isoform as a marker in assisting improved pregnancy outcomes.

Increased sHLA-G Is Associated with Improved COVID-19 Outcome and Reduced Neutrophil Adhesion.

Human leukocyte antigen (HLA) is a group of molecules involved in inflammatory and infective responses. We evaluated blood sHLA-E and sHLA-G levels in hospitalized COVID-19 patients with respiratory failure and their relationship with clinical evolution, changes in endothelial activation biomarker profile, and neutrophil adhesion. sHLA-E, sHLA-G, and endothelial activation biomarkers were quantified by ELISA assay in plasma samples. Neutrophil adhesion to endothelium was assessed in the presence/absence of patients' plasma samples. At admission, plasma levels of sHLA-G and sHLA-E were significantly higher in COVID-19 patients with respiratory failure compared to controls. COVID-19 clinical improvement was associated with increased sHLA-G plasma levels. In COVID-19, but not in control patients, an inverse correlation was found between serum sICAM-1 and E-selectin levels and plasma sHLA-G values. The in vitro analysis of activated endothelial cells confirmed the ability of HLA-G molecules to control sICAM-1 and sE-selectin expression via CD160 interaction and FGF2 induction and consequently neutrophil adhesion. We suggest a potential role for sHLA-G in improving COVID-19 patients' clinical condition related to the control of neutrophil adhesion to activated endothelium.

Circulating HLA-G and its association with cardiovascular markers in pregnancy.

Human Leukocyte Antigen-G (HLA-G) prevents the activity of immune cells and is decreased in women with preeclampsia. We aimed to investigate the associations between circulating soluble HLA-G (sHLA-G) and 92 cardiovascular disease-related biomarkers from a previously published multiplex study in women with preeclampsia and controls. We found 15 markers significantly associated with circulating sHLA-G in univariate analyses. After multivariable adjusted regression, only proto-oncogene tyrosine-protein kinase Src (SRC) and vascular endothelial growth factor D were significantly associated with sHLA-G. Low SRC, previously observed in the circulation of preeclamptic women, may be regulated by low sHLA-G, and reflect decreased trophoblast differentiation and syncytical formation.

Cytokines and Soluble HLA-G Levels in the Acute and Recovery Phases of Arbovirus-Infected Brazilian Patients Exhibiting Neurological Complications.

Severe neurological complications following arbovirus infections have been a major concern in seasonal outbreaks, as reported in the Northeast region of Brazil, where the same mosquito transmitted Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) viruses. In this study, we evaluated the levels of 36 soluble markers, including cytokines, chemokines, growth factors, and soluble HLA-G (Luminex and ELISA) in: i) serum and cerebrospinal fluid (CSF), during the acute phase and two years after the infection (recovery phase, only serum), ii) the relationship among all soluble molecules in serum and CSF, and iii) serum of infected patients without neurological complications, during the acute infection. Ten markers (sHLA-G, IL-10, IL-22, IL-8, MIP-1α, MIP-1ß, MCP-1, HGF, VEGF, and IL-1RA) exhibited differential levels between the acute and recovery phases, with pronounced increases in MIP-1α (P<0.0001), MCP-1 (P<0.0001), HGF (P= 0.0001), and VEGF (P<0.0001) in the acute phase. Fourteen molecules (IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-15, IL-17A, IFN-α, TNF, and G-CSF) exhibited distinct levels between arbovirus patients presenting or not neurological complications. IL-8, EGF, IL-6, and MCP-1 levels were increased in CSF, while RANTES and Eotaxin levels were higher in serum. Soluble serum (IL-22, RANTES, Eotaxin) and CSF (IL-8, EGF, IL-3) mediators may discriminate putative risks for neurological complications following arbovirus infections. Neurological complications were associated with the presence of a predominant inflammatory profile, whereas in non-complicated patients an anti-inflammatory profile may predominate. Mediators associated with neuroregeneration (EGF and IL-3) may be induced in response to neurological damage. Broad spectrum immune checkpoint molecules (sHLA-G) interact with cytokines, chemokines, and growth factors. The identification of soluble markers may be useful to monitor neurological complications and may aid in the development of novel therapies against neuroinflammation.

Endothelial Progenitor Cell CD34+ and CD133+ Concentrations and Soluble HLA-G Concentrations During Pregnancy and in Cord Blood in Undifferentiated Connective Tissue Diseases Compared to Controls.

The objective of this study is to evaluate endothelial progenitor cells (EPCs) CD34+ CD133- and CD34+ CD133+ and soluble HLA-G (sHLA-G) concentrations among undifferentiated connective tissue disease (UCTD) subjects, compared to controls, during pregnancy and in cord blood. This is a case-control study including 29 controls and 29 UCTDs. CD34+ CD133-, CD34+ CD133+, and sHLA-G concentrations were detected in maternal plasma and in cord blood. This study was approved by the Medical-Ethical Committee of our Institution (Current Research Project N. 901-rcr2017i-23 of IRCCS Foundation Policlinico San Matteo of Pavia). Circulating CD34+ CD133- and CD34+ CD133+ counts and sHLA-G (soluble human leucocyte antigen G) concentrations in maternal peripherical blood were higher in UCTD compared to those in controls in first and third trimester of pregnancy and at delivery (p < 0.001). Maternal CD34+ CD133- and CD34+ CD133+ counts were strongly and significantly correlated in UCTD (Spearman's rho = 0.79, p < 0.0001) but not in controls (Spearman's rho = 0.10, p = 0.35). Cord blood CD34+ CD133- and CD34+ CD133+ median counts and median sHLA-G concentrations were higher among UCTD subjects than in controls (p < 0.001). Cord blood CD34+ and CD133+ counts were inversely and significantly correlated with sHLA-G concentrations among UCTDs, but not in controls. Early UCTD is characterized by increased EPC levels in maternal plasma and in cord blood and higher levels of sHLA-G, compared to controls. Data suggest that fetoplacental unit plays an independent role in the EPC response to a systemic autoimmune disease.

HLA-G 14 bp In/Del and +3142 C/G genotypes are differentially expressed between patients with grade IV gliomas and controls.

Aim: Human Leukocyte Antigen-G (HLA-G) is a non-classical class I molecule that is involved in maternal-fetal immunotolerance. In cancer, this molecule contributes to the tumor escape. The aim of this study was to evaluate the 14 bp In/Del and +3142 C > G polymorphisms of the HLA-G 3' UTR and its relation with plasma and tissue HLA-G expression in patients with grade IV (high-grade) and grade I/II (low-grade) gliomas and controls.Patients and methods: Peripheral blood and tumor biopsies were collected from 85 patients with gliomas and blood samples from 94 controls. Polymorphisms were analyzed from blood DNA. Soluble HLA-G (sHLA-G) was measured by ELISA in plasma of the subjects and the tissue expression by immunohistochemistry on patient's tissue.Results: Higher levels of sHLA-G were observed in grade IV gliomas patients than in controls (p < 0.0001). In grade IV patients, the heterozygous 14pb In/Del, +3142 C/G genotypes and Del/C*In/G haplotype were associated with higher sHLA-G levels (p < 0.0001) when compared with controls. GBM patients were stratified into high and low sHLA-G expression and an association was found between +3142 C allele and high sHLA-G plasmatic levels (p = 0.0095). Tissue HLA-G immunolabel was higher in high-grade than low-grade gliomas (p = 0.0033).Conclusion: This was the first study evaluating HLA-G 3' UTR polymorphisms and expression in patients with gliomas. The 14 bp In/Del and +3142 C/G genotypes and haplotypes showed high influence over sHLA-G expression, suggesting a heterozygous advantage in the tumor context and may contribute to a worse prognosis in glioma patients.

Pregnancy and postpartum levels of circulating maternal sHLA-G in preeclampsia.

Preeclampsia is a leading cause of maternal and offspring mortality and morbidity, and predicts increased future cardiovascular disease risk. Placental dysfunction and immune system dysregulation are likely key pathophysiological factors. Soluble human leukocyte antigen G (sHLA-G) may dampen the specific immune response towards placental trophoblasts. Previous studies have shown low sHLA-G levels in preeclampsia, but postpartum, levels are unknown. Furthermore, the relationship between sHLA-G and sFlt-1 and PlGF, placental function markers, is unknown. We hypothesized that low maternal sHLA-G during pregnancy would be associated with placental dysfunction, including preeclampsia, gestational hypertension, and dysregulated sFlt-1 and PlGF, and that sHLA-G would remain decreased following preeclampsia. We included 316 pregnant women: 58 with early-onset preeclampsia (<34 weeks' gestation), 81 with late-onset preeclampsia (≥34 weeks' gestation), 25 with gestational hypertension, and 152 normotensive controls. Postpartum (1 or 3 years), we included 321 women: 29 with early-onset preeclampsia, 98 with late-onset preeclampsia, 57 with gestational hypertension, and 137 who were normotensive during their index pregnancies. In pregnancy, plasma sHLA-G was significantly lower both in the early- and late-onset preeclampsia groups compared to controls. In women with preeclampsia or gestational hypertension, sHLA-G was inversely correlated with serum sFlt-1. Postpartum, plasma sHLA-G levels were significantly higher in women who had had early-onset preeclampsia compared to controls. Our results support that sHLA-G may be important for placental function. Unexpectedly, sHLA-G was elevated up to 3 years after early-onset preeclampsia, suggesting an excessively activated immune system following this severe preeclampsia form, potentially contributing to future cardiovascular disease risk.

High plasma soluble levels of the immune checkpoint HLA-G molecule among bodybuilders.

INTRODUCTION: Studies report that intense physical activity influences the down-regulation of immune function in athletes as well as the interaction between adipose tissue and the immune system. AIM: This study aimed to compare the plasma soluble levels of the immune checkpoint HLA-G (sHLA-G) molecule with the fat mass and muscle mass index among 77 bodybuilders and 64 controls. RESULTS: The comparisons of the percentage of body fat (%BF) revealed that the groups of male and female bodybuilders showed a statistically significant reduction in the percentage of body fat when compared to their control group, (P <0.0001, for both comparisons). Regarding sHLA-G levels, the comparisons showed that the group of male bodybuilders had significantly higher sHLA-G levels compared to the group of female bodybuilders (P = 0.0011). CONCLUSION: Our results showed that in bodybuilders with less body fat, the systemic levels of soluble HLA-G, an immunological molecule with recognized immunosuppressive function, are significantly higher and suggest that this immune mechanism may corroborate the immunosuppressive state in athletes undergoing intense and prolonged physical training.

Soluble HLA-G and HLA-G Bearing Extracellular Vesicles Affect ILT-2 Positive and ILT-2 Negative CD8 T Cells Complementary.

Tumor immune escape is associated with both, the expression of immune checkpoint molecules on peripheral immune cells and soluble forms of the human leukocyte antigen-G (HLA-G) in the blood, which are consequently discussed as clinical biomarker for disease status and outcome of cancer patients. HLA-G preferentially interacts with the inhibitory receptor immunoglobulin-like transcript (ILT) receptor-2 in the blood and can be secreted as free soluble molecules (sHLA-G) or via extracellular vesicles (EV). To investigate the contribution of these two forms to the expression of checkpoint molecules in peripheral blood, we primed peripheral blood mononuclear cells with purified soluble sHLA-G1 protein, or EV preparations derived from SUM149 cells transfected with membrane-bound HLA-G1 or control vector prior to anti-CD3/CD28 T cell activation. Our study demonstrated that priming of PBMC with sHLA-G1 protein prior to 48 h activation resulted in enhanced frequencies of ILT-2 expressing CD8+ T cells, and in an upregulation of immune checkpoint molecules CTLA-4, PD-1, TIM-3, and CD95 exclusively on ILT-2 positive CD8+ T cells. In contrast, when PBMC were primed with EV (containing HLA-G1 or not) upregulation of CTLA-4, PD-1, TIM-3, and CD95 occurred exclusively on ILT-2 negative CD8+ T cells. Taken together, our data suggest that priming with sHLA-G forms induces a pronounced immunosuppressive/exhausted phenotype and that priming with sHLA-G1 protein or EV derived from HLA-G1 positive or negative SUM149 cells affects CD8+ T cells complementary by targeting either the ILT-2 positive or negative subpopulation, respectively, after T cell activation.

Integrated analysis of DNA methylation profile of HLA-G gene and imaging in coronary heart disease: Pilot study.

AIMS: Immune endothelial inflammation, underlying coronary heart disease (CHD) related phenotypes, could provide new insight into the pathobiology of the disease. We investigated DNA methylation level of the unique CpG island of HLA-G gene in CHD patients and evaluated the correlation with cardiac computed tomography angiography (CCTA) features. METHODS: Thirty-two patients that underwent CCTA for suspected CHD were enrolled for this study. Obstructive CHD group included fourteen patients, in which there was a stenosis greater than or equal to 50% in one or more of the major coronary arteries detected; whereas subjects with Calcium (Ca) Score = 0, uninjured coronaries and with no obstructive CHD (no critical stenosis, NCS) were considered as control subjects (n = 18). For both groups, DNA methylation profile of the whole 5'UTR-CpG island of HLA-G was measured. The plasma soluble HLA-G (sHLA-G) levels were detected in all subjects by specific ELISA assay. Statistical analysis was performed using R software. RESULTS: For the first time, our study reported that 1) a significant hypomethylation characterized three specific fragments (B, C and F) of the 5'UTR-CpG island (p = 0.05) of HLA-G gene in CHD patients compared to control group; 2) the hypomethylation level of one specific fragment of 161bp (+616/+777) positively correlated with coronary Ca score, a relevant parameter of CCTA (p<0.05) between two groups evaluated and was predictive for disease severity. CONCLUSIONS: Reduced levels of circulating HLA-G molecules could derive from epigenetic marks. Epigenetics phenomena induce hypomethylation of specific regions into 5'UTR-CpG island of HLA-G gene in CHD patients with obstructive non critical stenosis vs coronary stenosis individuals.

Plasma levels of soluble HLA-G and cytokines in papillary thyroid carcinoma before and after thyroidectomy.

BACKGROUND: Increasing evidence shows that chronic inflammation plays an important role in thyroid tumorigenesis. Cytokines as central mediators in inflammatory microenvironment can present both pro-tumour and anti-tumour effects and cytokine release may be influenced by soluble HLA-G (sHLA-G), an immune checkpoint molecule whose expression can also be induced by certain cytokines. AIM: To understand the role of these soluble factors in papillary thyroid cancer (PTC). METHODS: We evaluated plasma levels of sHLA-G and of 13 cytokines using ELISA and flow cytometry, respectively, in PTC patients at two time points: pre- and post-thyroidectomy; and control subjects. RESULTS: Compared with controls, IL-6 levels were increased, while IL-1ß, IFN-α and TGF-ß1 levels were decreased in pre-thyroidectomy PTC patients. IFN-α and TGF-ß1 efficiently discriminated patients from controls and were associated with extrathyroidal extension and lymph node metastasis, respectively. In addition, TNF and IL-13 were associated with male gender, lymph node metastasis and Hashimoto thyroiditis, and sHLA-G with tumour invasion. Compared with pre-thyroidectomy, IL-4, IL-10, TNF, IFN-α and TGF-ß1 levels were increased in post-thyroidectomy. CONCLUSION: There are significant changes in the cytokine profile after surgical removal of the thyroid tumour, and IFN-α e TGF-ß1 showed to be promising cytokines for discriminating PTC patients from controls. We also found that different cytokines are associated with clinicohistopathological characteristics of PTC related to poor prognosis, suggesting that cytokines seem to play an important role in PTC development and management.

Soluble HLA-G expression levels and HLA-G/irinotecan association in metastatic colorectal cancer treated with irinotecan-based strategy.

We here explore the soluble Human Leukocyte Antigen-G (sHLA-G) expression level as clinical biomarker in metastatic colorectal cancer (mCRC). To this aim the sHLA-G protein was measured in plasma samples of 40 patients with mCRC treated with the FOLFIRI (irinotecan (CPT-11) plus 5-fluorouracil (5-FU) and leucovorin (LV)) regimen. The results suggest a link between HLA-G levels and irinotecan (CPT-11) pharmacokinetic, leading to hypothesize a molecular interaction between sHLA-G and CPT-11. This interaction was confirmed experimentally by fluorescence spectroscopy. HLA-G is known to exist in a number of polymorphs that affect both the protein expression levels and its peptide-binding cleft. The interaction between HLA-G polymorphs and CPT-11 was explored by means of computational modelling, confirming the hypothesis that CPT-11 could actually target the peptide binding cleft of the most common HLA-G polymorphs.

Evaluation of Plasma Levels of Soluble HLA-G and HLA-G Genotypes in Kidney Transplant Recipients.

In the field of transplantation, expression of HLA-G, a nonclassical HLA molecule with immunosuppressive functions and limited gene polymorphism, is considered beneficial for graft acceptance; various studies have aimed to demonstrate this role in transplantation. Recently, in other clinical conditions, it has been observed that insulin resistance was associated with HLA-G14bpins/del polymorphism, the most studied regulatory polymorphism of this molecule. In the present study, plasma levels of the soluble form of HLA-G (sHLA-G) were analyzed in kidney transplant recipients (n = 103) with different HLA-G14bpins/del genotypes. In a group of 26 recipients, sHLA-G was detected before and after transplantation (1 year) to evaluate early variations. In 77 recipients, sHLA-G was detected after transplantation (3-24 years) and correlated with occurrence of long-term post-transplant morbidity (diabetes mellitus, hyperlipidemia, hypertension, obesity, etc.). METHODS: Levels of sHLA-G were measured in plasma with an enzyme-linked immunosorbent assay; HLA-G14bpins/del and HLA-G+3142C>G genotypes were assessed using direct polymerase chain reaction. RESULTS: Plasma levels of sHLA-G significantly decreased during the first year after transplantation (P = .019); no significant correlations were found with genotypes or early post-transplant events. Lower levels of sHLA-G were found in recipients with post-transplant diabetes mellitus or obesity carrying the HLA-G14bpins/ins (P = .006 and P = .003, respectively) or HLA-G+3142G/G genotypes. CONCLUSIONS: A complex modulation of HLA-G, which includes both immunologic and metabolic effects, could affect the risk for long-term post-transplant morbidity in kidney transplant recipients. Associations of HLA-G, diabetes, and obesity deserve to be investigated by deeply exploring HLA-G regulatory variants.

Serum sHLA-G: Significant diagnostic biomarker with respect to therapy and immunosuppressive mediators in Head and Neck Squamous Cell Carcinoma.

Head & Neck Squamous Cell Carcinoma is one of the highest mortality factors in the world due to the lack of potential biomarker for early detection of disease. There is an urgent need for molecular marker involved in disease progression which remains suppressed normally, required for specificity. HLA-G is highly expressed in cancers and creates immune-suppressive microenvironment. Cancerous cells secrete inflammatory cytokines like IL-10,IFN-γ which increase expression of immunosuppressive molecules, such as HLA-G. We evaluated sHLA-G protein level in serum of 120 HNSCC patients at diagnosis and after therapy and compared with 99 individuals by SPR, ELISA and determined its mRNA level by qRT-PCR. sHLA-G was correlated with serum IL-10 and IFN-γ of the patients. Significant elevated levels of sHLA-G were observed in patients (8.25 ± 1.74 ng/µl) than control (6.45 ± 1.31 ng/µl). Levels were declined in (8.09 ± 1.79 ng/µl to 6.64 ± 1.33 ng/µl) patients in response to therapy. sHLA-G levels with tumor burden (8.16 ± 1.91 to 6.63 ± 1.32 ng/µl), node (8.62 ± 1.45 to 6.66 ± 1.26 ng/µl), PDSCC (8.14 ± 0.62 to 5.65 ± 0.27 ng/µl) and oropharynx (7.90 ± 1.24 to 6.10 ± 1.33 ng/µl) showed a positive and significant response to therapy. Findings indicate that sHLA-G can be a potential diagnostic serum protein marker for HNSCC due to its suppressive function and over expression in diseased condition with the influence of cytokines.

Soluble HLA-G pre-transplant levels to identify the risk for development of infection in heart transplant recipients.

Infection is still a leading cause of death during the first year after heart transplantation. We evaluated the pre-transplant levels of HLA (Human Leukocyte antigen) - G molecules as a means of identifying heart recipients at risk of serious infections. We prospectively analyzed 122 adult heart transplant (HT) recipients. Serum samples were collected beforetransplantation and analyzed for sHLA-G levels by ELISA assay. The clinical follow-up period lasted 5 years. Clinical outcomes were bacterial infections requiring intravenous anti-microbial agents, cytomegalovirus (CMV) disease, and fungal infections requiring therapy. We found that 39 patients (32%) developed at least 1 serious bacterial infection. Higher pre-transplant sHLA-G levels were a risk factor for serious infection (above median value 5.4 ng/ml; relative risk 3.70; 95% confidence interval 1.03-12.64; p = 0.043). Patients with high levels of pre-transplant sHLA-G are also characterized by a lower overall survival at 5 years (p = 0.017), with microbial infections as major causes of death. No association was observed with the development rejection episode. Early monitoring of sHLA-G molecules proved useful for the identification of heart recipients who are at risk of serious infections.