CD8+CD28-CD127loCD39+ regulatory T-cell expansion: A new possible pathogenic mechanism for HIV infection?

BACKGROUND: HIV-associated immunodeficiency is related to loss of CD4+ T cells. This mechanism does not explain certain manifestations of HIV disease, such as immunodeficiency events in patients with greater than 500 CD4+ T cells/µL. CD8+CD28-CD127loCD39+ T cells are regulatory T (Treg) lymphocytes that are highly concentrated within the tumor microenvironment and never analyzed in the circulation of HIV-infected patients. OBJECTIVES: We sought to analyze the frequency of CD8+CD28-CD127loCD39+ Treg cells in the circulation of HIV-infected patients. METHODS: The frequency of circulating CD8+CD28-CD127loCD39+ Treg cells was analyzed and correlated with viral load and CD4+ T-cell counts/percentages in 93 HIV-1-infected patients subdivided as follows: naive (n = 63), elite controllers (n = 19), long-term nonprogressors (n = 7), and HIV-infected patients affected by tumor (n = 4). The same analyses were performed in HIV-negative patients with cancer (n = 53), hepatitis C virus-infected patients (n = 17), and healthy donors (n = 173). RESULTS: HIV-infected patients had increased circulating levels of functional CD8+CD28-CD127loCD39+ Treg cells. These cells showed antigen specificity against HIV proteins. Their frequency after antiretroviral therapy (ART) correlated with HIV viremia, CD4+ T-cell counts, and immune activation markers, suggesting their pathogenic involvement in AIDS- or non-AIDS-related complications. Their increase after initiation of ART heralded a lack of virologic or clinical response, and hence their monitoring is clinically relevant. CONCLUSION: HIV infection induces remarkable expansion of CD8+CD28-CD127loCD39+ Treg cells, the frequency of which correlates with both clinical disease and signs of chronic immune cell activation. Monitoring their frequency in the circulation is a new marker of response to ART when effects on viremia and clinical response are not met.

Her2 assessment using quantitative reverse transcriptase polymerase chain reaction reliably identifies Her2 overexpression without amplification in breast cancer cases.

BACKGROUND: Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination. PATIENTS AND METHODS: We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting. RESULTS: The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results. SIGNIFICANCE: qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination.

Phenotypic Alterations Involved in CD8+ Treg Impairment in Systemic Sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis, vasculopathy, and autoimmunity. Although the exact pathogenetic mechanisms behind SSc remain to be fully elucidated, a great deal of evidence suggests the existence of an unbalanced ratio between the effector and regulatory arms of the immune system. With regard to the T regulatory (Treg) compartment, we observed that CD8+ Treg subsets display functional defects in SSc-affected patients. Since CD127 down-modulation and CD39 upregulation have been observed on Treg subsets, the phenotypic expression of these molecules was analyzed on the CD8+CD28- Treg precursors and on CD8+ Treg cells generated in vitro through interleukin-10 commitment. Immunophenotypic data from SSc patients were compared to those obtained from healthy subjects. The analyses performed on ex vivo-isolated CD8+CD28- Treg precursors did not show any significant differences in CD39 or CD127 expression as compared to values obtained from healthy donors. On the contrary, in vitro-generated CD8+ Tregs obtained from SSc patients displayed reduced expression of the CD39 molecule as compared to controls. Moreover, the percentage of CD127+ cells was significantly higher in in vitro-generated CD8+ Tregs from SSc patients compared to CD8+ Tregs obtained from healthy donors. Taken together, these findings may indicate an impairment of maturation processes affecting CD8+ Treg cells in SSc patients. This impairment of maturation involves phenotypic alterations that are mainly characterized by a deficient CD39 upregulation and a lack of down-modulation of the CD127 molecule.

Reduced Expression of SMAD4 Is Associated with Poor Survival in Colon Cancer.

PURPOSE: SMAD4 loss is associated with the development of metastases and poor prognosis. We evaluated expression of SMAD4 protein and its association with tumor characteristics, including biomarkers and outcome in terms of relapse-free survival and overall survival. EXPERIMENTAL DESIGN: We used 1,564 stage II/III colon cancer samples from PETACC-3 to evaluate SMAD4 expression by immunohistochemistry. SMAD4 protein expression was validated by assessing mRNA expression using available expression array data. SMAD4 expression was also studied on 34 adenomas and 10 colon cancer liver metastases with their primaries. Loss of SMAD4 immunoreactivity was defined as focal or diffuse. Cases without SMAD4 loss were subdivided into those with strong and weak expression. RESULTS: SMAD4 protein expression was informative in 1,381/1,564 cases. SMAD4 loss was found in 293/1,381 (21%) cases. Of 1,088 cases without SMAD4 loss (79%), 530 showed weak and 558 strong expression. SMAD4 loss occurred also in adenomas, but less extensively than in carcinomas. Liver metastases followed mostly the expression pattern of the primary tumor. SMAD4 loss, including weak expression, identified patients with poor survival in stage II as well as III and in both treatment arms. SMAD4 loss was less frequent in tumors with microsatellite instability and more frequent in those with loss of heterozygosity of 18q. CONCLUSIONS: We conclude that clonal loss of SMAD4 expression in adenomas, carcinomas, and liver metastases increases with disease progression. SMAD4 loss, and to a lesser extent weak expression, is strongly associated with poor survival regardless of stage. Clin Cancer Res; 22(12); 3037-47. ©2016 AACR.

Residual tumor micro-foci and overwhelming regulatory T lymphocyte infiltration are the causes of bladder cancer recurrence.

Bladder cancer has an unexplained, high recurrence rate. Causes of recurrence might include the presence of sporadic tumor micro-foci in the residual urothelial tissue after surgery associated with an inverted ratio between intratumoral effector and regulatory T cell subsets. Hence, surgical specimens of both tumors and autologous, macroscopically/histologically free-of-tumor tissues were collected from 28 and 20 patients affected by bladder or renal cancer, respectively. The frequencies of effector (IFNγ+ and IL17+ T cells) and regulatory (CD4+CD25hiCD127lo and CD8+CD28-CD127loCD39+ Treg) T cell subpopulations among tumor infiltrating lymphocytes were analyzed by immunofluorescence, while the gene expression of MAGE-A1 and MAGE-A2 tumor-associated antigens was studied by RT-PCR. The results show that both the T cell infiltrate and the frequency of MAGE-A1/A2 gene expression were comparable in tumors and in autologous free-of-tumor tissues in bladder cancer, while the autologous free-of-tumor renal tissues showed reduced T cell infiltrate and frequency of MAGE gene expression as compared to the autologous tumors. Importantly, the intra-tumor T effector/Treg cell ratio was consistently <1 in bladder cancer patients (n. 7) who relapsed within two years, while it was always >1 in patients (n. 6) without recurrence (regardless of tumor stage) (P = 0.0006, Odds ratio = 195). These unprecedented findings clarify the pathogenic mechanism of bladder cancer recurrence and suggest that microscopically undetectable micro-foci of tumor may predispose to recurrence when associated with an inverted intratumoral T effector/Treg cell ratio.

The Reliability of Endoscopic Biopsies in Assessing HER2 Status in Gastric and Gastroesophageal Junction Cancer: A Study Comparing Biopsies with Surgical Samples.

AIM: The aim of this study is to validate the accuracy of HER2 assessment on biopsies by comparing matched biopsy/surgical material from the same patients. METHODS: HER2 status was evaluated by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 103 cases of gastric and gastroesophageal junction cancers in coupled biopsy and surgical material. RESULT: Complete concordance between IHC and FISH results on biopsy versus surgical samples was noted in 80% and 95% of cases, respectively. At comprehensive comparison, including IHC and FISH data on biopsy and surgical samples, 89% of biopsies were predictive of HER2 status in surgical samples, whereas 11% showed variable inconsistencies. The majority of these (10 of 12 cases) showed IHC score 0/1+ on biopsy but were all IHC positive and amplified at surgery; in particular, three (3 of 35; 8.5%) IHC score 0 and four (4 of 16; 25%) IHC score 1+ cases were FISH amplified on biopsy material also, whereas the remaining three cases were FISH non-amplified on biopsy. The percentage of cases, which were FISH amplified with IHC score 1+ or 2+ on biopsies, were similar (25% and 33%, respectively) and they also shared a similar grade of amplification. These data suggest that both IHC score 1+ and 2+ on biopsy material represent "equivocal cases" that may merit further investigation. CONCLUSIONS: The predictive value of HER2 IHC in biopsies is high. FISH analysis should be considered for IHC score 2+ and 1+ biopsy cases. Approximately 8% of cases will not be accurately predicted by biopsy evaluation.

Basophils infiltrate human gastric mucosa at sites of Helicobacter pylori infection, and exhibit chemotaxis in response to H. pylori-derived peptide Hp(2-20).

Basophils, which are normally confined to the circulation, can migrate to sites of allergic inflammation. Using the specific mAb, BB1, we detected basophil infiltration of the gastric mucosa of Helicobacter pylori-infected patients affected by moderate and severe gastritis. Basophils were not found in H. pylori-free individuals or in subjects with mild gastritis. The H. pylori-derived peptide, Hp(2-20), was a potent basophil chemoattractant in vitro, whereas the control peptide, Hp1, was ineffective. Basophils from peripheral blood of healthy volunteers expressed mRNA for the formyl peptide receptors, N-formyl-peptide receptor (FPR), FPR-like (FPRL)1, and FPRL2. Preincubation of basophils with FMLP or Hp(2-20) caused complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with a low concentration of FMLP, which binds with high affinity to FPR, but not to FPRL1 or FPRL2, did not affect the chemotactic response to Hp(2-20). In contrast, a high concentration of FMLP, which binds to FPRL1 and FPRL2, reduced the chemotactic response to Hp(2-20). The FPR antagonist, cyclosporin H, prevented chemotaxis induced by FMLP, but not by Hp(2-20). Hp(2-20) could be responsible, at least in part, for basophil infiltration of the gastric mucosa of H. pylori-infected patients presumably through the interaction with FPRL1 and FPRL2.

Inhibition of cytokines expression in human microglia infected by virulent and non-virulent mycobacteria.

The pathogenesis of tuberculosis (TBC) meningitis is still unknown. As shown by previous studies, human microglia can be the target of mycobacteria, but no data are available about their cellular response to infection. Consequently, we studied the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-10 in human microglia pure cultures infected with the two variants of Mycobacterium avium (domed-opaque (SmD) and transparent (SmT)) and with Mycobacterium tuberculosis. Results showed that microglia was productively infected by mycobacteria which could grow inside the cells. Mycobacteria internalization was more rapid for M. avium, but M. tuberculosis infection turned out to be more efficient due to the incorporation of densely packed bacteria. TNF-alpha expression was not affected by M. avium, whereas an increase followed by a decrease was observed in M. tuberculosis. Both IL-1 and IL-10 cytokine expression was rapidly inhibited by infection with the more virulent bacteria, whereas the non-pathogenic one had almost no effect. Also, the expression of the co-stimulatory molecule CD137, a member of tumor necrosis factor receptor family, was affected by infection with virulent mycobacteria. Our results show that microglia response to mycobacterial infection is modulated in correlation with virulence, mainly toward inhibition of inflammatory response. This observation might be one of the mechanisms by which non-pathogenic mycobacteria are quickly eliminated, explaining one of the bases of virulence.

Dystrophin antisense oligonucleotides decrease expression of nNOS in human neurons.

Nitric oxide (NO) plays an important role in the pathogenesis of neurodegenerative disease. It has been shown that neuronal NO synthase (nNOS), the enzyme that constitutively produces NO in brain, is a component of the dystrophin-associated protein complex. The absence of dystrophin causes Duchenne muscular dystrophy. Thus, we attempted to study whether or not a decrease of dystrophin expression would induce a modification in nNOS expression in cultured human neurons. Human fetal neuronal cultures were treated with antisense oligonucleotides against different isoforms of dystrophin and the expression of nNOS tested by RT-PCR and immunocytochemistry. Results showed that nNOS mRNA was significantly decreased by about 35% in neurons treated with brain-specific dystrophin (brain Dp427) antisense, whereas iNOS expression was not affected. Accordingly, a decrease in immunostaining for nNOS was observed in antisense treated neurons compared to controls. Expression of neuronal markers, such as bFGF or synaptophysin, was not affected by the same antisense treatment. Astrocytes were not affected by treatment, as shown by utrophin expression, a dystrophin-like protein that was not modified in pure astrocytic cultures. Thus, we conclude that a decrease of dystrophin in human neurons is associated with a decrease of nNOS expression.

Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: modulation by FGF-2.

CD137 (ILA, 4-1BB), a member of the tumor necrosis factor receptor family, and its ligand CD137-L were assayed by RT-PCR and immunocytochemistry in cultured human brain cells. Results demonstrated that both neurons and astrocytes expressed specific RNA for CD137 and its protein, which was found both on the plasma membrane and in the cytoplasm. Surprisingly, microglia, which also expressed CD137 mRNA, showed negative immunostaining. CD137-L-specific RNA was detected only in astrocytes and neurons. When brain cells were treated with fibroblast growth factor-2 (FGF-2), upregulation of CD137 but not of its ligand was observed in neurons and astrocytes. Protein localization was also affected. In microglia, an inhibition of RNA expression was induced by treatment, whereas CD137-L remained negative. Our data are the first demonstration that human brain cells express a protein found thus far in activated immunocompetent cells and epithelia. Moreover, they suggest not only that CD137 and CD137-L might play a role in interaction among human brain cells, but also that FGF-2 might have an immunoregulatory function in brain, modulating interaction of the central nervous system with peripheral immunocompetent cells.

Developmentally regulated expression and localization of dystrophin and utrophin in the human fetal brain.

Expression of dystrophin and the dystrophin-related protein utrophin has been studied in the human fetal brain both in vivo and in vitro. Results showed that both these proteins were developmentally regulated, even if their expression followed a different pattern. Utrophin was found since very early stages of development, reached a peak between week 15-20 of gestation, declining then, so that at week 32 was barely detectable. The protein was mainly found in neuronal cell bodies, partially associated to the plasma membrane, and in astrocytes cytoplasm. On the contrary, the brain form of dystrophin was first detectable at week 12, increased up to week 15 and then remained stable. Dystrophin localization was similar but not identical to utrophin. In neurons, it was also partially associated with the plasma membrane of cell body and axon hillock. However, the most was concentrated in the cytoplasm and in the processes, where it appeared associated to neurofilaments. Astrocytes were negative for brain dystrophin, but positive for the muscle isoform. Results suggest that utrophin and dystrophin are likely to play a key, though different, role in the immature brain. They help in understanding the basic mechanism(s) underlying cognition defects frequently observed in Duchenne and Becker dystrophic patients.