Cytokine-induced killer cells in the treatment of patients with solid carcinomas: a systematic review and pooled analysis

Background aimsThe aim of this study was to evaluate the efficacy and safety of cytokine-induced killer (CIK) cell therapy for solid carcinomas.MethodsWe performed a computerized search of phase II/III clinical trial databases of CIK cell-based therapy using a combination of the terms ‘cytokine-induced killer cells’, ‘tumor’ and ‘cancer’.ResultsTreatment with CIK cells was associated with a significantly improved half-year survival (P = 0.003), 1-year survival (P = 0.0005), 2-year survival (P  < 0.01) and mean survival time (MST) (P  < 0.001). Patients in the CIK group showed a prolonged half-year progression-free survival (PFS) (P  < 0.01), 1-year PFS (P < 0.01) and median time to progression (MTTP) (P < 0.001). A favored disease control rate (DCR) was observed in patients receiving CIK cell therapy, while the objective response rate (ORR) was not altered (P = 0.05) compared with the non-CIK group (P = 0.007). CIK cell therapy could also reduce the adverse effects of grade III and IV leukopenia caused by chemotherapy (P = 0.002) and diminish hepatitis B virus (HBV)-DNA content (P < 0.01). However, the incidence of fever in the CIK therapy group was significantly higher than in the non-CIK group (P = 0.02). The percentage of CD3⁺, CD4⁺, CD4⁺ CD8⁺, CD3^? CD56⁺ and CD3⁺ CD56⁺ T-lymphocyte subsets in the peripheral blood of cancer patients was significantly increased, whereas the percentage of CD8⁺ T-lymphocyte cells was significantly decreased in the CIK group compared with the non-CIK group (P < 0.01).ConclusionsCIK cell therapy has demonstrated a significant superiority in prolonging the MST, PFS, DCR and quality of life (QoL) of patients.     

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Background aimsAlloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use.MethodsWe describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells.ResultsNK cells (6.0 ± 1.2 × 10⁸) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR⁺ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML.ConclusionsThe approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.     

A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies

Background aimsRetroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose.MethodsWe tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia.ResultsMedian anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24–48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10⁸, n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia.ConclusionsThe method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Effect of transcatheter renal arterial embolization combined with cryoablation on regulatory CD4+CD25+ T lymphocytes in the peripheral blood of patients with advanced renal carcinoma

ObjectiveTo analyze the effect of Argon-Helium cryosurgery (AHCS) combined with transcatheter renal arterial embolization (TRAE) on the differentiation of regulatory CD4⁺ CD25⁺ T cell (Treg) and its implication in patients with renal carcinoma.MethodsSeventy seven patients are included in the study, and divided into two groups: TRAE group (n = 45, receiving TRAE only) and TRAE + cryoablation group (n = 32, receiving cryoablation 2–3 weeks after TRAE). The percentage of Treg cells and T lymphocyte subsets (CD4⁺T, CD8⁺T, and CD4⁺T/CD8⁺T) in the peripheral blood is measured by flow cytometry previous to the therapy and 3 months after therapy. Meanwhile, the extent of tumor necrosis is measured by MRI or CT 1 month after therapy.ResultsThe percentages of Treg cells of patients in TRAE + cryoablation group decrease from (6.65 ± 1.22)% to (3.93 ± 1.16)%, (t = 42.768, P < 0.01), and the percentages of CD4⁺T and CD4⁺T/CD8⁺T increase significantly (P < 0.01). However, the results of patients in TRAE group show that the percentages of Treg, CD4⁺T, CD8⁺T and CD4⁺T/CD8⁺T increase slightly although the differences had no statistical significance (P > 0.05). The tumor necrosis rate of TRAE + cryoablation group is 57.5%, significantly higher than those of TRAE group, which shows 31.6% (t = 6.784, P < 0.01). The median survival duration of the TRAE + cryoablation group is 20 months, significantly longer than that of the TRAE group (χ² = 7.368, P < 0.01). The decreasing extent of Treg cells is correlated with tumor necrosis rates (r = 0.90, P < 0.01) and life time (r = 0.67, P < 0.01).ConclusionThe therapy of TRAE combined with cryoablation contributes to reduce the percentage of Treg cells and improve the immune situation of patients with renal cell carcinoma, which consequently increase tumor necrosis rate and prolong the patients‘ survival duration.     

Lymphocyte subset analysis for the assessment of treatment-related complications after autologous stem cell transplantation in multiple myeloma

Background aimsThe aim of this study was to investigate the correlation between infused lymphocyte populations and lymphocyte subsets at engraftment, and the early clinical implications of lymphocyte subset recovery after autologous stem cell transplantation (ASCT) in multiple myeloma (MM).MethodsWe examined the lymphocyte populations of infused autografts and the lymphocyte subsets of peripheral blood at engraftment from 50 patients using flow cytometry. Each subset was grouped as low (below median) and high (above median) to examine the correlation with mucositis of grade 3 or more and the occurrence of infections and cytomegalovirus (CMV) reactivation.ResultsUsing Spearman correlation coefficients, we found that cell doses of infused CD8⁺ (P = 0.042) and CD19⁺ cells (P = 0.044) were significantly associated with the absolute lymphocyte count (ALC) at engraftment. The dose of infused CD34⁺ cells was not associated with the change of lymphocyte subsets except for an inverse correlation with CD4⁺ cells (P = 0.006). After adjusting for potential variables in univariate analysis, multivariate analyzes revealed that the lower ratio of infused CD4⁺ to CD8⁺ cells (P = 0.030) was an independent factor for severe mucositis. Of lymphocyte subsets at engraftment, a higher frequency of CD3⁺ (P = 0.024) and a lower frequency of CD56⁺ (P = 0.020) were independent predictors for infections after engraftment. A higher frequency of CD8⁺ cells (P = 0.041) and a lower ratio of CD4⁺ to CD8⁺ (P = 0.021) were independent predictors for CMV reactivation.ConclusionsOur data suggest that lymphocyte subset analysis of infused autograft and peripheral blood at engraftment may provide new predictors for early complications after ASCT in patient with MM.     

Serum interleukin-6 concentration reflects the extent of asymptomatic left ventricular dysfunction and predicts progression to heart failure in patients with stable coronary artery disease

BackgroundLeft ventricular ejection fraction (LVEF) remains one of the strongest predictors of long-term prognosis in patients with stable coronary artery disease (CAD). Asymptomatic left ventricular systolic dysfunction (LVSD) often precedes clinically overt heart failure (HF) and is an area of extensive research nowadays. We studied the association between serum IL-6 concentrations and the extent of LV dysfunction in patients with asymptomatic LVSD. We aimed to investigate the diagnostic value of serum IL-6 concentrations in predicting the risk of progression to HF. Seventy-one patients entered the study and were divided into three groups based on LVEF: group 1 – patients with LVEF <30% (N = 7), group 2 – patients with LVEF 30–50% (N = 37) and group 3 – patients with LVEF >50% (N = 27).ResultsDemographics were similar in all three groups. IL-6 concentration was the highest in group 1 (median 8.6 pg/mL) and the lowest in group 3 (median 2.6 pg/mL), whereas IL-6 concentration in group 2 was intermediate (median 3.7 pg/mL) (P = 0.002). We found a significant, inverse correlation between IL-6 concentration and ejection fraction. During 18-month follow-up clinically overt HF developed in 71.4% of patients in group 1 and in 37.5% of patients in group 2. None of the patients in group 3 manifested HF symptoms (P < 0.001). ROC analysis revealed high diagnostic value of serum IL-6 and LVEF in predicting progression to HF. We also found a strong, inverse correlation between IL-6 and the time of progression to HF.ConclusionsThere is a strong correlation between IL-6 and the extent of asymptomatic LVSD in patients with documented CAD. Elevated IL-6 concentrations preceded progression to clinically overt HF. Moreover, the higher the IL-6 concentration the earlier the manifestation of HF symptoms.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

Regulation of IL-21 signaling by suppressor of cytokine signaling-1 (SOCS1) in CD8(+) T lymphocytes

Mice lacking the gene for suppressor of cytokine signaling 1 (SOCS1) show defective homeostasis of T lymphocytes due to accumulation of CD8⁺ T cells, resulting at least partly from dysregulated IL-15 signaling. IL-15 alone does not stimulate proliferation of naïve CD8 T cells, but can synergize with IL-21 to induce proliferation, suggesting a potential role for IL-21 in the defective homeostasis of CD8⁺ T lymphocytes in SOCS1^−/− mice. Since IL-21 strongly induced SOCS1 mRNA in CD8⁺ T cells, we investigated whether SOCS1 regulates their response to IL-21. CD8⁺ T cells isolated from SOCS1-deficient mice proliferated vigorously in response to IL-21 + IL-15. In CD8⁺ T lymphocytes expressing transgenic TCR, IL-21 + IL-7 provided a stronger stimulus to naïve cells whereas IL-15 + IL-21 potently stimulated memory cells. Compared to truly naïve or memory cells, SOCS1^−/− H-Y TCR⁺ CD8⁺ T cells displayed CD44^loLy6C^hiCD122^intCD127^lo partial memory phenotype and exhibited stronger response to IL-15 + IL-21 than truly naïve cells. In SOCS1^−/− CD8⁺ T cells, IL-21 caused greater reduction in IL-15 threshold for activation in a dose-dependent manner. SOCS1 deficiency did not modulate IL-21Rα expression or sensitivity to IL-21, but delayed the loss of IL-21-induced phospho-STAT3 signal. These results show that SOCS1 is a critical regulator of IL-21 signaling in CD8⁺ T cells, and support the notion that sustained IL-21 signaling might also contribute to the aberrant T cell homeostasis in SOCS1-deficient mice.     

Effect of Metformin Therapy on Vitamin D and Vitamin B12 Levels in Patients with Type 2 Diabetes Mellitus

ObjectiveTo determine the effect of metformin on 25-hydroxyvitamin D [25(OH)D] and vitamin B₁₂ levels in patients with type 2 diabetes mellitus.MethodsWe performed a retrospective review of medical records of patients treated between 2003 and 2009 at Loyola University Medical Center, Maywood, Illinois, in both ambulatory primary care and endocrinology clinics. The study cohort consisted of 706 patients with type 2 diabetes mellitus who were 20 to 93 years old (mean age, 63 ± 13) and had a mean body mass index of 33.1 kg/m². Of these patients, 42% were treated with metformin, and 34% had been diagnosed with osteoporosis or osteopenia.ResultsPatients taking metformin had statistically significant lower vitamin B₁₂ levels than those not receiving metformin (P < .0001; 95% confidence interval [CI] = − 220 to − 84 pg/mL). No statistically significant difference was found between users and nonusers of metformin in regard to 25(OH)D levels when adjusted for variables (P = .297; 95% CI for mean difference = − 0.7 to 2.2 ng/mL). Metformin use did not adversely affect successful treatment of vitamin D deficiency in this patient population as a whole, nor did it affect the subgroup with osteoporosis (P = .956). The patients with osteoporosis had statistically significant lower baseline 25(OH)D levels in comparison with those without osteoporosis, when adjustments were made for all variables (P = .003; 95% CI = 0.7 to 3.5 ng/ mL).ConclusionThis study confirms the higher prevalence of vitamin B₁₂ deficiency in metformin-treated patients with type 2 diabetes than in those not treated with metformin. This study also suggests that vitamin D deficiency is not a clinical concern among metformin-treated patients with type 2 diabetes and that metformin does not negatively affect treatment of vitamin D deficiency in these patients. (Endocr Pract. 2012;18:179–184)     

The mitochondrial Ca(2+)-activated K(+) channel contributes to cardioprotection by limb remote ischemic preconditioning in rat

AimsTo investigate the role of the mitochondrial Ca²⁺-activated K⁺ channel in cardioprotection induced by limb remote ischemic preconditioning.Main methodsMale Sprague–Dawley rats (250–300 g) were randomized into control, ischemia/reperfusion (I/R), remote ischemic preconditioning (RPC), NS1619 (a specific mitochondrial Ca²⁺-activated K⁺ channel opener), and RPC + paxilline (a specific mitochondrial Ca²⁺-activated K⁺ channel inhibitor) groups. RPC was induced by 4 cycles of 5 min of ligation followed by 5 min of reperfusion of the left femoral artery. Myocardial I/R was achieved by ligation of the left anterior descending coronary artery for 30 min, followed by 120 min of reperfusion. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining, the hemodynamics were monitored, and lactate dehydrogenase (LDH) levels in the coronary effluent, manganese superoxide dismutase (Mn-SOD) content in mitochondria and mitochondrial membrane potential were measured spectrophotometrically. The ultrastructure of cardiomyocyte mitochondria was assessed by electron microscopy.Key findingsNS1619 (10 μM) improved heart function, decreased infarct size, reduced LDH release, maintained mitochondrial structural integrity and mitochondrial membrane potential, and increased the mitochondrial content of Mn-SOD to the same degree as RPC treatment. However, paxilline (1 μM) eliminated the cardioprotective effect conferred by RPC.SignificanceThe mitochondrial Ca²⁺-activated K⁺ channel participates in the myocardial protection by limb remote ischemic preconditioning.     

Influence of related donor age on outcomes after peripheral blood stem cell transplantation

Background aimsThe rising use of allogeneic transplantation in older recipients necessitates considering older related donors. The effect of related donor age for peripheral blood stem cell allografts (PBSC) on graft maintenance and outcomes, independent of CD34⁺cell dose, has not been well-characterized.MethodsHLA-related donors (98% siblings) underwent a uniform filgrastim-based mobilization regimen aiming to collect and infuse 5 × 10⁶ CD34⁺ cells/recipient kg. Donor and recipient age were modeled in multiple ways to account for the correlation, and outcomes reported by decade of donor age.ResultsThe median donor and recipient ages were 52 years and 54 years, respectively. The mean CD34⁺ cell dose infused was 5.6 × 10⁶ CD34⁺/kg and 75% of patients received a narrow range between 4.4 and 6.6 × 10⁶ CD34⁺ cells/kg. Neither better PBSC mobilization nor higher CD34⁺ content of allografts was significantly associated with engraftment or transplant outcomes. After adjusting for recipient age and other prognostic factors, older donor age by decade conferred a lower risk of non-relapse mortality (NRM) [hazard ratio (HR) = 0.64, 95% confidence interval (CI) 0.45–0.91, P = 0.013] and borderline improvement in overall survival (OS) (HR = 0.76, 95% CI 0.58–0.99, P = 0.045) without altering progression-free survival (PFS) (HR = 0.85, 95% CI 0.66–1.07, P = 0.18).ConclusionsOlder donor age does not worsen outcome after matched related donor PBSC transplantation in patients receiving a narrow range CD34⁺ cells. The relatively small sample size mandates that the finding of similar to improved outcomes for older related donor age must be confirmed in larger studies.     

The frog skin host-defense peptide frenatin 2.1S enhances recruitment,activation and tumoricidal capacity of NK cells

Frog skin is a source of peptides with various biological properties. Frenatin 2.1S, derived from norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus, exhibits immunostimulatory effects as demonstrated by the promotion of proinflammatory phenotypes of mononuclear cells in mouse peritoneal cavity and spleen. The aim of this study was to identify the populations of host cells sensitive to the action of frenatin 2.1S in vivo and to study its effects on their functional antitumor capacity. A single injection of frenatin 2.1S (100 μg) in BALB/c mice increased the presence of peritoneal CD11c⁺ dendritic cells and CD3⁺ T cells 24 h after administration and there was a significant increase in the number of IL-17 and CXCR3 expressing inflammatory T cells. Frenatin 2.1S treatment also increased the number of TNF-α expressing F4/80⁺ proinflammatory M1 macrophages. The most striking finding of the study is the marked increase of the number of peritoneal natural killer (NK) cells following frenatin 2.1S injection. Further, frenatin 2.1S administration led to activation of NK cells as evaluated by increased expression of NKG2D, FasL, CD69 and CD107a. The increased ratio of interferon-γ vs. IL-10 producing NK cells is further indication of the proinflammatory action of frenatin 2.1S. Peptide treatment enhanced the tumoricidal action of peritoneal NK cells on 4T1 mouse mammary carcinoma cells as revealed by the real-time automated monitoring of cell status. Our data demonstrate that frenatin 2.1S promotes activation and cytotoxic capacity of NK cells and should be regarded as a candidate for antitumor immunotherapy.     

Clinical-grade varicella zoster virus-specific T cells produced for adoptive immunotherapy in hemopoietic stem cell transplant recipients

Background aimsVaricella zoster virus (VZV) causes life-long latent infection in healthy individuals, which reactivates in 10–68% of stem cell transplant patients. Reconstituting immunity through adoptive transfer of T cells specific for VZV may aid in the prophylaxis and treatment of VZV infections. The potential for generating T cells specific for VZV using a clinically approved VZV vaccine strain was investigated.MethodsThe Varivax^® vaccine was used to stimulate peripheral blood mononuclear cells from healthy donors. Only reagents approved for clinical manufacture were used. Monocyte-derived dendritic cells pulsed with Varivax (R) were used to stimulate autologous mononuclear cells at a responder to stimulator ratio of 10:1. On day 7, a second stimulation was performed; 20 U/mL interleukin (IL)-2 were added from day 7 and 50 U/mL IL-2 from day 14 onwards. Cell phenotype and functionality were assessed after 21 days of culture.ResultsA mean increase of 11-fold in cell number was observed (n = 18). Cultures were mainly T cells (mean CD3 ⁺ 89.7%, CD4 ⁺ 54.2%, CD8 ⁺ 28.7%) with effector and central memory phenotypes. Cells produced one or more T helper (Th)1 cytokine (interferon-γ, tumor necrosis factor-α and IL-2), and CD4 ⁺ (but not CD8 ⁺) cells expressed the cytoxicity marker CD107 when restimulated with VZV antigens.ConclusionsWe have demonstrated a clinically applicable method that yields high numbers of highly reactive T cells specific for VZV. We propose that reconstructing host immunity through adoptive transfer of VZV-specific T cells will reduce the frequency of clinical VZV infection in the period of severe immune suppression that follows allogeneic stem cell transplantation.     

Action of ANP on the nongenomic dose-dependent biphasic effect of aldosterone on NHE1 in proximal S3 segment

The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na⁺/H⁺ exchanger isoform), after the acid load induced by NH₄Cl, and on the cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15 ± 0.008 and the basal pHirr was 0.195 ± 0.012 pH units/min (number of tubules/number of tubular areas = 16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10^?12 M) increases the pHirr to approximately 59% of control value, and ALDO (10^?6 M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10^?6 M) or BAPTA (5 × 10^?5 M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca²⁺]_i was 104 ± 3 nM (15), and ALDO (10^?12 or 10^?6 M) increased the basal [Ca²⁺]_i to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca²⁺]_i and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca²⁺]_i that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.     

Exonuclease 1 (EXO1) gene variation and melanoma risk

ObjectiveDNA repair pathway genes play an important role in maintaining genomic integrity and protecting against cancer development. This study aimed to identify novel SNPs in the DNA repair-related genes associated with melanoma risk from a genome-wide association study (GWAS).MethodsA total of 8422 SNPs from the 165 DNA repair-related genes were extracted from a GWAS of melanoma risk, including 494 cases and 5628 controls from the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS). We further replicated the top SNPs in a GWAS of melanoma risk from the MD Anderson Cancer Center (1804 cases and 1026 controls).ResultsA total of 3 SNPs with P value <0.001 were selected for in silico replication. One SNP was replicated: rs3902093 [A] in EXO1 promoter region (P_discovery = 6.6 × 10^?4, P_replication = 0.039, P_joint = 2.5 × 10^?4; OR_joint = 0.80, 95% CI: 0.71, 0.90). This SNP was associated with the expression of the EXO1; carriers of the A allele showed lower expression (P = 0.002).ConclusionOur study found that a promoter region SNP in the editing and processing nucleases gene EXO1 was associated with decreased expression of EXO1 and decreased melanoma risk. Further studies are warranted to validate this association and to investigate the potential mechanisms.     

The perioperative granulocyte/lymphocyte ratio is a clinically relevant marker of surgical stress in patients with colorectal cancer

PurposeThis study was to assess the clinical relevance of the blood granulocytes to lymphocytes (G/L) ratio as an early marker of surgical stress in patients with colorectal cancer.MethodsThirty-three patients with colorectal cancer were prospectively to undergo laparoscopic-assisted (n = 12) or open (n = 21) surgical resection. Granulocyte and lymphocyte counts were used to calculate the G/L ratios in blood samples from all patients before the operation and post-operatively on days 1, 3 and 7. Additionally, serum inflammatory cytokines, interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF) and macrophage (M)-CSF were assayed as markers of surgical stress.ResultsSeven of 33 patients developed unexpected complications. Serum IL-6 (P < 0.0001), G-CSF (P = 0.0257), and M-CSF (P < 0.0001) were higher on day 1 vs before the operation. Similarly, the G/L ratios were higher on days 1–3 vs before the operation (P < 0.0001) and then gradually decreased together with the surgical stress levels. The G/L ratios and the numbers of granulocytes and lymphocytes in the blood showed no correlation with serum IL-1β or TNF-α. In contrast, the G/L ratios and the numbers of granulocytes in the blood showed significant correlation with IL-6 (Rs = 0.710, P < 0.0001, Rs = 0.653, P < 0.0001, respectively), with G-CSF (Rs = 0.626, P < 0.0001, Rs = 0.578, P < 0.0001), with M-CSF (Rs = 0.470, P < 0.0001, Rs = 0.372, P < 0.0001). However, the number of lymphocytes showed inverse correlation with IL-6 (Rs = ?0.493, P < 0.0001), G-CSF (Rs = ?0.440, P < 0.0001) and M-SCF (Rs = ?0.443, P < 0.0001).ConclusionThe G/L ratio appears to be a simple and clinically relevant parameter for the assessment of perioperative stress in patients undergoing colorectal surgery.     

Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol

Background aimsWe have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safeMethodsFive patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2–3 weeks in culture, a median of 4.65 × 10⁶ immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 daysResultsITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3⁺ CD16⁺ CD56⁺ CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10⁶ CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1–13) from the first infusion of CIK cells.ConclusionsThis study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.     

Effects of shell morphological traits on the weight traits of Manila clam (Ruditapes philippinarum)

The shell length, height, and width, live body weight, and edible tissue weight of Manila clam of 1, 2, and 3 years of age were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and live body weight or edible tissue weigh used as a dependent variable for calculating the path coefficients, correlation index and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight or edible tissue weight were all highly significant (P < 0. 01). The shell height at 1-year old clams was highly correlated with the live body weight and edible tissue weight. The shell width of 2- to 3-year-old clams was strongly associated with the live body weight, while the shell length was closely linked to the edible tissue weight. The results of coefficients of determination for the morphological traits against weight traits agreed well with the results of path analysis. The correlation indices for all morphological traits against weight traits were approximately the same as determination coefficients regardless of clam age. The correlation indices (R²) of morphological traits against the live body weight of clams of all ages and edible tissue weight of 1-year-old clams were larger than 0.85, but R² of morphological traits against the edible tissue weight of 2- and 3-year-old clams was smaller than 0.85, indicating that some other factors might be associated with the edible tissue weight of 2- and 3-year-old clams. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), shell width X₃ (cm) against live body weight Y (g), edible tissue weight Z (g): for 1-year-old clams: Y = ?4.317 + 0.18X₁ + 0.147X₂, (X₁ < 0.01, X₂ < 0.01), Z = ?1.011 + 0.095X₂, (X₂ < 0.01); for 2-year-old clams: Y = ?15.119 + 0.249X₁ + 0.176X₂ + 0.688X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?4.248 + 0.198X₁, (X₁ < 0.05, X₃ < 0.01); and for 3-year-old clams: Y = ?25.013 + 0.415X₁ + 1.184X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?7.082 + 0.119X₁ + 0.332X₃, (X₁ < 0.05, X₃ < 0.01).     

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

Background aimsThe beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC).MethodsThirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-³H)thymidine.ResultsThe results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502).ConclusionsWe propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.