Role of myeloid-derived suppressor cells in mouse pre-sensitized cardiac transplant model

Harness of sensitized transplantation remains a clinical challenge particularly in parallel with prolonged cold ischemia time (PCI)-mediated injury. Our present study was to test the role of myeloid-derived suppressor cells (MDSCs) in mouse pre-sensitized transplantation. Our findings revealed that CD11b + Gr1^low MDSC was shown to have strong suppressive activity. MDSCs subsets from the tolerated mice exhibited higher suppressive capacities compared with counterparts from naive (untreated) mice. Depletion of Tregs could not affect splenic CD11b + Gr1^-low MDSC frequency, but increase peripheral and intragraft CD11b + Gr1^-low frequency. Intriguingly, boost of Tregs remarkably caused an increase of CD11b + Gr1^-low frequency in the graft, peripheral blood, and spleen. Furthermore, peripheral CD11b + Gr1^-low cells were massively accumulated at the early stage when allogeneic immune response was enhanced. Taken together, MDSCs could prevent grafts from PCI-mediated injury independent on Tregs in the pre-sensitized transplant recipients. Utilization of MDSC subset particularly CD11b + Gr1^-low might provide a novel insight into improving graft outcome under such clinical scenarios.     

Combined T regulatory cell and Th2 expression profile identifies children with cow's milk allergy

The role of T regulatory cells in spontaneous recovery from cow's milk allergy (CMA) is unclear. We investigated the mRNA expression of 12 T-cell markers and the protein expression of CD4, CD25, CD127, FoxP3 after in vitro β-lactoglobulin stimulation of peripheral blood mononuclear cells from children with persisting CMA (n = 16), early recovery (n = 20) or no atopy (n = 21). Artificial neural networks with exhaustive search for all marker combinations revealed that markers FoxP3, Nfat-C2, IL-16 and GATA-3 distinguished patients with persisting CMA most accurately from other study groups. FoxP3 mRNA expression following β-lactoglobulin stimulation was highest in children with persisting CMA. Also the FoxP3 intensity in CD4⁺ CD25^highCD127^low cells was higher in children with CMA compared with non-atopic children. The expression profile of both Th2- and T regulatory cell-related genes thus reflects the clinical activity of CMA. Tolerance, in contrast, is not characterized by activation of circulating T regulatory cells.     

Expression of cancer stem cell markers in basal and penta-negative breast carcinomas – A study of a series of triple-negative tumors

Purpose

Breast cancer is a heterogeneous disease. Immunohistochemistry has given rise to triple-negative carcinoma (TNC). Concomitantly, biological origins of neoplasia and its heterogeneity has been strongly debated in cancer stem cells (CSC) theme. This study investigates the prevalence of basal (BCC) and penta-negative carcinomas (5NC) in TNC and establishes associations with CSC (CD44CD24).

Materials and methods

94 TNC were tested for CK5/6, HER1, CD44 and CD24, evaluated by a simple immunohistochemistry score and correlated with clinicopathological and survival data.

Results

BCC had higher tumor grades than 5NC (p = 0.004). CD44 negativity (p = 0.007) and CD44⁻CD24⁺ phenotype (p = 0.013) were associated with less vascular invasion amongst TNC. CD44 expression was associated with BCC (p = 0.007). CD44⁻CD24^−/low phenotype was associated with 5NC. None of the variables were associated with clinical outcome.

Conclusion

BCC and 5NC are closely related tumor subtypes. CD44⁻CD24^−/low phenotype was associated with 5NC and CD44⁻CD24⁺ phenotype was associated with vascular invasion. These results require histogenetic confirmation in larger studies.     

Susceptibility to pulmonary disease due to Mycobacterium avium–intracellulare complex may reflect low IL-17 and high IL-10 responses rather than Th1 deficiency

It remains unclear why some individuals and not others are susceptible to non-tuberculous mycobacterial lung disease (NTMLD). To determine whether NTMLD is associated with defects or biases in Th1/Th2/Th17 immunity, blood leukocytes from NTM patients with nodular bronchiectasis, their adult offspring, and healthy population controls were stimulated with staphylococcal enterotoxin B (SEB), tuberculin and sensitin to measure cytokine production. In response to SEB, NTM patients exhibited higher frequencies of IFNγ-producing CD4⁺ T cells than population controls (P < 0.001). In supernatant, levels of IL-17 were lower in patients than adult offspring. Sensitin elicited higher IFNγ responses from patients than controls (P < 0.05). Patients also produced more IL-10 in supernatant than controls after culture with tuberculin (P < 0.01) or sensitin (P < 0.05), but IL-10-producing CD4⁺ T cells were undetectable. NTMLD is not associated with deficient IFNγ production, but may be associated with reduced Th17 immunity and/or a predisposition towards IL-10 production from non-CD4⁺ T cells.     

CD4 T-cell activation and reduced regulatory T-cell populations are associated with early development of cataracts among HIV-infected adults in Uganda

Background

Cataracts contribute 12% of visual loss among HIV-infected adults in Uganda. Immuno-pathogenesis of cataracts may differ among HIV-infected individuals; thus the need for innovative therapeutic interventions among HIV-infected adults.

Methods

In a laboratory based case-control study, nested in a clinical/surgical community outreach camp, 50 adults with cataracts eligible for surgery were selected consecutively. HIV testing was done for individuals with unknown HIV sero-status. Peripheral Blood Mononuclear Cells (PBMC) were collected from all HIV-positive-adults-with-cataracts (cases) and HIV-negative-adults-with-cataracts (comparative group) and age-matched HIV-negative and HIV-positive-adults-without-cataracts (comparative group). Treg were measured as CD3 + CD4 + FoxP3 + CD25+^Bright and immune activation as CD3 + CD4 + CD38 + HALDR+ using a Facs Canto II flowcytometer. Mann Whitney test was used to compare expression among the four groups.

Results

Of 50 adults operated for cataracts, 24 (48%) were female, 25 (50%) were HIV-positive. HIV-positive-individuals had cataracts earlier [median; Inter-quartile Range (IQR); 49 (44–53) years] than HIV-negative [70 (IQR 59–75) years]; p = 0.0005. Treg were lower among individuals with cataracts irrespective of HIV status; p = 0.001; but comparable among younger HIV-positive and elderly HIV-negative with cataracts; p = 0.301. Immune activation levels were comparable among HIV-positive and HIV-negative individuals with cataracts. However, HIV-positive-individuals with cataracts expressed higher levels of immune activation than HIV-positive-individuals without cataracts; p = 0.012 and HIV-negative-individuals-with-cataracts expressed higher levels of immune activation that HIV-negative-without-cataracts; p < 0.0001.

Conclusion

CD4 T-cell activation and reduced regulatory T-cell populations were associated with cataracts among adults aging with HIV. We recommend studies on clinical relevance of immune modulation in the prevention of early development of cataracts among adults aging with HIV in Africa.     

Specific overexpression of tumour necrosis factor‐α‐induced protein (TNFAIP)9 in CD14+CD16− monocytes in patients with rheumatoid arthritis: comparative analysis with TNFAIP3

The tumour necrosis factor (TNF)-α-induced proteins (TNFAIP)9 and TNFAIP3 play an important pathogenic role in murine arthritis. To clarify their pathophysiological roles in patients with rheumatoid arthritis (RA), we examined their expression and localization in peripheral blood mononuclear cells (PBMC). TNFAIP9 and TNFAIP3 mRNA expression was determined in PBMC of RA patients and healthy subjects (control). Flow cytometry was used to analyse the main TNFAIP9- and TNFAIP3-expressing cell populations. TNFAIP9 and TNFAIP3 mRNA expression levels were examined in vitro on CD14⁺ cells stimulated with TNF-α and lipopolysaccharide (LPS). The expression levels of TNFAIP9 and TNFAIP3 mRNA were also measured before and 12 weeks after treatment with tocilizumab and abatacept. TNFAIP9 expression was significantly higher, while TNFAIP3 expression was lower in PBMC of RA (n = 36) than the control (n = 24) (each P < 0·05). TNFAIP9 was expressed on CD14⁺ cells, especially in human leucocyte antigen D-related (HLA-DR)⁺CD14^brightCD16⁻cells, while TNFAIP3 was expressed mainly on CD3⁺ T cells. TNF-α and LPS induced TNFAIP9 and TNFAIP3 in human CD14⁺monocytes in vitro. Treatment with tocilizumab (n = 13), but not abatacept (n = 11), significantly reduced TNFAIP9 mRNA expression in PBMC, which was associated with reduction in the number of circulating CD14^bright monocytes. The expression of TNFAIP9 in CD14⁺ cells was specifically elevated in patients with RA, regulated by TNF-α and LPS, and suppressed by tocilizumab, while TNFAIP3 in PBMC showed different localization and induction patterns.     

Frequency analysis of HLA class I alleles in Iranian patients with progressive and non-progressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a malignant disorder of B cell origin, with low incidence in Asian populations. In this study we investigated the HLA-class I A and B allele frequencies in 87 Iranian CLL patients and 64 healthy controls using sequence specific primer-polymerase chain reaction (SSP-PCR) technique. Our results showed increased frequencies of HLA-A11:01 (p = 0.02) and HLA-B35:01 (p = 0.002) alleles and HLA-A11:01/B35:01 haplotype (p = 0.036) and decreased frequencies of HLA-A01:01 (p = 0.02), HLA-A26:01 (p = 0.03), HLA-B65:01 (p = 0.03) and HLA-B53:01 (p < 0.00001) alleles in CLL patients compared to the control group. Classification of the patients into non-progressive and progressive groups did not reveal significant differences for the frequency of any of the HLA-A and -B alleles or haplotypes between these two subtypes. Comparison between patients with immunoglobulin heavy chain variable region genes (IGHV) mutated (n = 56) and unmutated (n = 31) subtypes showed a significant increase in HLA-A32:01 (p = 0.05) and HLA-A33:01 (p = 0.05) alleles in IGHV unmutated patients compared to IGHV mutated patients. Similarly, a higher frequency of HLA-B52:01 (p = 0.037) alleles was observed in CD38⁺ compared with CD38⁻ patients. Our results obtained from an Iranian population indicate that CLL is associated with distinct HLA class I alleles and haplotypes some of which are linked to disease prognostic factors.     

Regulatory function of cytomegalovirus-specific CD4CD27CD28 T cells

CMV infection is characterized by high of frequencies of CD27⁻CD28⁻ T cells. Here we demonstrate that CMV-specific CD4⁺CD27⁻CD28⁻ cells are regulatory T cells (T_R). CD4⁺CD27⁻CD28⁻ cells sorted from CMV-stimulated PBMC of CMV-seropositive donors inhibited de novo CMV-specific proliferation of autologous PBMC in a dose-dependent fashion. Compared with the entire CMV-stimulated CD4⁺ T-cell population, higher proportions of CD4⁺CD27⁻CD28⁻ T_R expressed FoxP3, TGFβ, granzyme B, perforin, GITR and PD-1, lower proportions expressed CD127 and PD1-L and similar proportions expressed CD25, CTLA4, Fas-L and GITR-L. CMV-CD4⁺CD27⁻CD28⁻ T_R expanded in response to IL-2, but not to CMV antigenic restimulation. The anti-proliferative effect of CMV-CD4⁺CD27⁻CD28⁻ T_R significantly decreased after granzyme B or TGFβ inhibition. The CMV-CD4⁺CD27⁻CD28⁻ T_R of HIV-infected and uninfected donors had similar phenotypes and anti-proliferative potency, but HIV-infected individuals had higher proportions of CMV-CD4⁺CD27⁻CD28⁻ T_R. The CMV-CD4⁺CD27⁻CD28⁻ T_R may contribute to the downregulation of CMV-specific and nonspecific immune responses of CMV-infected individuals.     

Ramp rate of blood pressure changes does not affect aortic afferent sensitivity in anesthetized rats

To investigate whether the rate of change in blood pressure affects the sensitivity of the aortic baroreceptor afferent response, the change in aortic nerve activity (ANA) to two different rates of ramp increase in mean blood pressure (MBP), elicited by phenylephrine administration, was determined in the rat under urethane (1.5 g kg⁻¹) anesthesia. The sensitivity of the increase in ANA following a rapid (average ramp rate, 9.14 ± 0.60 mmHg s⁻¹, n = 11) or gradual (1.78 ± 0.24 mmHg s⁻¹, n = 11) increase in MBP was 2.03 ± 0.14% and 1.81 ± 0.20% of baseline mmHg⁻¹, respectively. These values were not significantly different from each other (P = 0.16). Furthermore, we found no correlation between the rate of ramp increase in MBP and the sensitivity of the increase in ANA (r = 0.24, P = 0.29, n = 22). These results suggest that, at least within the normal physiological range of MBP, the rate of the ramp change in blood pressure does not affect aortic baroreceptor afferent sensitivity in the anesthetized rat.     

Escherichia coli-induced immune paralysis is not exacerbated during chronic filarial infection

Sepsis initially starts with a systemic inflammatory response (SIRS phase) and is followed by a compensatory anti-inflammatory response syndrome (CARS) that causes impaired adaptive T-cell immunity, immune paralysis and an increased susceptibility to secondary infections. In contrast, parasitic filariae release thousands of microfilariae into the peripheral blood without triggering inflammation, as they induce regulatory, anti-inflammatory host responses. Hence, we investigated the impact of chronic filarial infection on adaptive T-cell responses during the SIRS and CARS phases of a systemic bacterial infection and analysed the development of T-cell paralysis following a subsequent adenovirus challenge in BALB/c mice. Chronic filarial infection impaired adenovirus-specific CD8⁺ T-cell cytotoxicity and interferon-γ responses in the absence of a bacterial challenge and led to higher numbers of splenic CTLA-4⁺ CD4⁺ T cells, whereas splenic T-cell expression of CD69 and CD62 ligand, serum cytokine levels and regulatory T-cell frequencies were comparable to naive controls. Irrespective of filarial infection, the SIRS phase dominated 6–24 hr after intravenous Escherichia coli challenge with increased T-cell activation and pro-inflammatory cytokine production, whereas the CARS phase occurred 6 days post E. coli challenge and correlated with high levels of transforming growth factor-β and increased CD62 ligand T-cell expression. Escherichia coli-induced impairment of adenovirus-specific CD8⁺ T-cell cytotoxicity and interferon-γ production was not additionally impaired by chronic filarial infection. This suggests that filarial immunoregulation does not exacerbate E. coli-induced T-cell paralysis.     

Educational group visits for the management of chronic health conditions: A systematic review

Objective

Review the effectiveness of group visits (appointments of multiple patients) on quality of life, function, self-efficacy, utilization, and biophysical outcomes in randomized controlled trials of patients with chronic conditions.

Methods

We searched MEDLINE^®, Cochrane, CINAHL, and PsycINFO to January 2013 for English-language trials of educational group visits led by non-prescribing facilitators (e.g., peer educators).

Results

We report on 80 arthritis/falls (n = 22), asthma/COPD (n = 10), CHF/hypertension (n = 12), diabetes (n = 29), multiple conditions (n = 4), and pain (n = 4) studies. We found moderate evidence of improved short-term self-efficacy in patients with arthritis (10 studies) and diabetes (10 studies). We found no consistent evidence of improved quality of life; however a moderately strong body of evidence suggests peer-led community-based programs might improve quality of life and utilization in patients with multiple chronic conditions. Meta-analyses found short- (14 studies; mean change HbA1c = −0.27, CI = −0.44, 0.11) and long-term (10 studies; mean change HbA1c = −0.23, CI = −0.44, −0.02) glycemic improvement.

Conclusions

Group visits may improve self-efficacy and glycemic control. There was little consistent evidence of improved quality of life, functional status, or utilization.

Practice implications

Group visits represent a reasonable alternative for educating patients with chronic illness, though varied participation/retention suggests they should not be the sole alternative.     

Detection of T cells reactive to intestinal alkaline phosphatase, an autoantigen in acute bacterial infections, and discrimination between autoantigen-specific CD5+ and CD5− B cells

Autoantibodies of the IgG isotype, specifically directed against intestinal alkaline phosphatase (IAP), occur transiently in the majority of sera from patients with acute bacterial infections. Sometimes they are observed in autoimmune diseases. Using a T cell proliferation assay, it was found that isolated peripheral blood mononuclear cells (PBMC) from IAP autoantibody (IAPA)-positive patients (n=18) responded significantly to IAP, whereas proliferation could not be induced in PBMC from healthy donors (n=11). Significant stimulation of PBMC from patients (n=11) was not obtained by use of transferrin, a common autoantigen in humans, indicating the specificity of stimulation of IAP-reactive T cells. Furthermore, T cell proliferation was observed when a highly purified IAP fragment (CNBr 21) spanning amino acids 334–462 of the primary structure of IAP was used as antigen. Thus, it was shown that an immunodominant T cell epitope resides within the CNBr 21 fragment which also contains a discontinuous B cell epitope as evaluated previously. Double immunocytochemical staining of T cell-depleted PBMC with IAP and an anti-human CD5 antibody allowed the detection of CD5⁺ B lymphocytes, which probably produce natural IAPA (nIAPA). These nIAPA-specific CD5⁺ B cells occurred with approximately the same frequency among T cell-depleted PBMC from healthy donors and those from patients. In contrast, IAPA-producing CD5⁻ B cells were found in B cell-enriched preparations from patients, but not in those from healthy individuals.     

Interleukin‐17A deficiency ameliorates streptozotocin‐induced diabetes

Interleukin-17 (IL-17) is a cytokine with critical functions in multiple autoimmune diseases. However, its roles in type I diabetes and the underlying mechanisms remain to be fully elucidated. In the current study, we investigated the impact of IL-17 deficiency on streptozotocin (STZ) -induced diabetes. Il-17^−/− mice exhibited attenuated hyperglycaemia and insulitis after STZ treatment compared with control mice. The Il-17^−/− mice had fewer CD8⁺ cells infiltrating the pancreas than wild-type controls after STZ injection. Wild-type mice showed increased percentage and number of splenic CD8⁺ cells and decreased Gr1⁺ CD11b⁺ myeloid-derived suppressor cells (MDSC) after STZ treatment, but Il-17^−/− mice maintained the percentages and numbers of splenic CD8⁺ cells and MDSC, suggesting that IL-17 is implicated in STZ-induced cellular immune responses in the spleen. We further purified the MDSC from spleens of STZ-treated mice. Il-17^−/− MDSC showed increased ability to suppress CD8⁺ cell proliferation in vitro compared with wild-type MDSC. Transfer of MDSC to diabetic mice showed that MDSC from Il-17^−/− mice could ameliorate hyperglycaemia. Moreover, recipients with MDSC from Il-17^−/− mice had a decreased percentage of CD8⁺ cell in the spleen compared with recipients with MDSC from wild-type mice. These data suggest that IL-17 is required in splenic MDSC function after STZ delivery. In summary, our study has revealed a pathogenic role of IL-17 in an STZ-induced diabetes model with important implications for our understanding of IL-17 function in autoimmune diseases.     

Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens

Regulatory T cells (Tregs) are defined as CD4⁺CD25⁺ cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4⁺CD25⁻ cells in Treg-depleted birds. The CD4⁺CD25⁺ cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4⁺CD25⁺ cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4⁺CD25⁺ cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4⁺CD25⁻ cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4⁺CD25⁺ cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4⁺CD25⁻ cells at 12 d post injection.     

Whole-blood culture is a valid low-cost method to measure monocytic cytokines — A comparison of cytokine production in cultures of human whole-blood, mononuclear cells and monocytes

Whole-blood and peripheral blood mononuclear cell (PBMC) cultures are used as non-validated surrogate measures of monocytic cytokine production. The aim of this investigation was to compare ex vivo cytokine production from human whole-blood and PBMC with that from isolated monocytes. We also assessed the intra- and inter-individual variation in cytokine production. In 64 healthy men (age 19-40 years) IL-6, TNF and IL-10 were measured by enzyme-linked immunosorbent assay in supernatants from whole-blood, PBMC and monocytes cultured 24 h with lipopolysaccharide (LPS) or UV-killed L. acidophilus. Cytokines produced from whole-blood was found to be more strongly correlated with monocytic cytokines than cytokines from PBMC, particularly after LPS-stimulation: r = 0.57, P < 0.001 versus r = 0.33, P = 0.01 for IL-6 and r = 0.43, P < 0.001 versus r = 0.30, P = 0.02 for TNF-α. Adjustment for a preceding 8-week dietary fatty acid-intervention did not change any of the associations. Based on measurements at three time-points 8 weeks apart the intra-individual variation was ≥ 50% smaller than the inter-individual variation (P < 0.05) in most whole-blood cytokine responses and LPS-stimulated IL-6 from PBMC. We conclude that whole-blood cultures are well-suited low-cost proxy-measures of monocytic cytokine production. Moreover, large inter-individual variation in cytokine production was demonstrated whereas the individual responses in whole-blood were reproducible even over long time-periods.     

CD45RA expression on HCMV-specific effector memory CD8+ T cells is associated with the duration and intensity of HCMV replication after transplantation

In this cross-sectional study on 42 solid organ transplant recipients, the association of kinetics of human cytomegalovirus (HCMV) replication and EMRA HCMV-specific CD8+ T cells was investigated. Correlation was observed between the duration of HCMV replication after transplantation and CD45RA+CD27− (r = 0.609; p = 0.004), CD45RA+ CD28− (r = 0.579; p = 0.008) or CD45RA+CCR7− (r = 0.488; p = 0.029) HCMV-specific CD8+ T cells percentages. In the multivariate regression analyses, CD45RA+CD27−, CD45RA+CD28− or CD45RA+CCR7− HCMV-specific CD8+ T cells percentages increased 5.58% (p = 0.001), 5.35% (p = 0.001) or 4.49% (p = 0.012), respectively, with every 10-day increase in the duration of HCMV replication. Moreover, CD45RA+CD27− or CD45RA+CD28− frequencies increased 4.16% (p = 0.024) or 3.58% (p = 0.049), respectively, with every unity increase in log₁₀ genomes/mL. These observations support the major association between the frequency of EMRA HCMV-specific CD8+ T cells and the duration of post-transplant HCMV replication episodes in solid organ transplantation recipients.     

Distinctive Pattern of Cytokine Production and Adhesion Molecule Expression in Peripheral Blood Memory CD4+ T Cells from Patients with Active Crohn’s Disease

An expansion of both circulating and intestinal lamina propria CD4⁺CD45RO⁺ T cells has been described in patients with Crohn’s disease. We studied both the cytokine profile and the expression of adhesion molecules on this T-cell subset. Peripheral blood CD4⁺CD45RO⁺ T cells from patients with Crohn’s disease (n=45) were assessed by flow cytometry and RT-PCR methods. The cytokine profile was also measured in intestinal lamina propria from seven patients. They were classified according to the CDAI and the results were compared with those of patients with ulcerative colitis (n=21) and noninflammatory intestinal conditions (n=15), and healthy controls (n=39). The mean percentage of circulating CD4⁺CD45RO⁺ T cells producing intracellular TNF was higher in active than in inactive Crohn’s disease patients (p < 0.001), active (p = 0.49) and inactive ulcerative colitis (p = 0.019), and healthy controls (p =0. 017). TNF expression correlated with CDAI (p < 0.001). An increased expression of intracellular IL-2, IL-6, and IL-10 in active Crohn’s disease patients was also found. CD62L was downregulated in active Crohn’s disease patients while no differences were observed in CD49d and CD11a expression. Lamina propria CD4⁺CD45RO⁺ T cells from active Crohn’s disease lesions showed an increased intracellular staining of TNF, IFN-γ, and IL-10. Both peripheral and intestinal mucosa CD4⁺CD45RO⁺ T cells from active Crohn’s disease patients show an increased production of TNF. In addition, the circulating CD4⁺CD45RO⁺ T-cell subset expresses a pattern of adhesion molecules that promotes homing to extranodal lymphoid tissues. This T-cell subset may play a relevant role in the immunopathogenesis of Crohn’s disease.Dr. García de Tena and Dr. Manzano are joint first authors     

Primary application of PPE68 of Mycobacterium tuberculosis

PPE68 protein is absent from BCG and the attenuated strains of Mycobacterium tuberculosis (MTB). In this study, the shuttle plasmid pBudCE4.1/PPE68/OriM was constructed and transformed into BCG to obtain PPE68 recombination BCG (PPE68-rBCG), and BALB/c mice were immunized with PPE68-rBCG to evaluate the immunological characterization of PPE68-rBCG. The level of lgG2a, IFN-γ, IL-12 and IL-4 in serum of immunized mice were detected, the proliferation response of spleen lymphocyte were measured, the frequency of CD4⁺, CD8⁺ and CD4⁺/CD8⁺ were determined, and the spleen and lung tissue were prepared for pathological analysis. PPE68-rBCG was constructed successfully and could induce powerful Th1 immune response in mice. Besides, we took the purified recombination PPE68 (rPPE68) protein as diagnostic antigen to detect pulmonary tuberculosis patients (n = 252) and extrapulmonary tuberculosis patients (n = 66). We also used anti-PPE68 polyclonal antibody as coating antibody to detect specific antigen in the same serum samples. Our data provide an experimental basis for potential application of rPPE68 in the diagnosis of tuberculosis, especially for extrapulmonary tuberculosis.     

Clinicopathologic correlation of cancer stem cell markers CD44, CD24, VEGF and HIF-1α in ductal carcinoma in situ and invasive ductal carcinoma of breast: an immunohistochemistry-based pilot study

CD24^−/lowCD44⁺ cells have been identified as putative cancer stem cells (CSCs) in breast cancer. However, the expression of these markers, as well as their association with clinical parameters or tumor microenvironment of breast cancer, remains largely unknown. In the present study, we examined the expression of CD44, CD24, VEGF, and HIF-1α in human breast tumor tissues and assessed their clinicopathological correlations. We investigated tissue samples, including 117 cases of invasive ductal carcinoma (IDCa), 14 cases of ductal carcinoma in situ (DCIS), and 15 cases of intraductal hyperplasia (IDH) from breast tissues. The expression of CD44, CD24, HIF-1α, and VEGF was evaluated using immunohistochemical staining. CD24, CD44, HIF-1α, and VEGF were expressed in 49 (41.9%), 51 (43.6%), 32 (27.4%), and 97 cases (82.9%), respectively, in IDCa. CD24^−/lowCD44⁺ cells were noted in 48 (41.3%) cases. The levels of CD24 and VEGF expression correlated positively with tumor malignancy (P < 0.05). Meanwhile, the expression of CD24, CD44, and VEGF correlated significantly positively with increasing tumor grade (P < 0.05). In addition, associations between CD44 and VEGF, CD24 and VEGF, HIF-1α and VEGF, CD24^−/lowCD44⁺ and VEGF, CD24^−/lowCD44⁺ and HIF-1α were also observed (P < 0.05). The HIF-1α expression level was relatively higher in early stage breast cancer patients with CD24^−/lowCD44⁺ cells. Taken together, our results suggest that CD24 and VEGF may play important roles in breast tumorigenesis and progression, while HIF-1α may play a role in the early stage of breast carcinogenesis.