Association of immune parameters with clinical outcome in stage III colon cancer: results of Southwest Oncology Group Protocol 9009

Levamisole (LMS), utilized in the adjuvant treatment of patients with stage III colon cancer, is immunomodulatory. To determine whether alterations in immune parameters before, during and after 12 months of 5FU/LMS therapy correlate with disease-free survival, 38 patients enrolled on Southwest Oncology Group (SWOG) protocol 8899 received extensive lymphocyte phenotypic analysis prior to therapy and 3, 6, 12 and 15 months after treatment initiation. The median follow-up of patients is 41 months. Significant increases in the proportion and total number of CD56⁺ natural killer cells were seen, starting at 3 months and continuing until 15 months (P < 0.001). Increases in the total numbers of cells expressing CD25 (interleukin-2 receptor), VLA4 and the combinations of CD4: CD45RA and CD4:CDw29 were not evident during therapy but were seen at 15 months (P < 0.05: CD25, CD4:CDw29, CD4:CD45RA; P < 0.001: VLA4). Low levels of CD8⁺ cells prior to treatment initiation and after 3 months of therapy correlated with early relapse within the first year of 5FU/LMS treatment. Patients who have remained disease-free (n = 22, median follow-up 45 months) demonstrated increases in the total numbers of CD8⁺, CD25⁺, CD56⁺, VLA4⁺, CD4: CDw29 and CD4:CD45RA cells, primarily at 15 months. In contrast, patients who relapsed had decreased numbers of CD8⁺, CD4:CDw29, CD4: CD45RA and VLA4⁺ cells and minimal increases in CD56⁺ and CD25⁺ cells. Statistically significant differences between the late-relapse group and the group remaining disease-free were seen for CD25⁺, CD4: CD45RA and CD4:CDw29 cells at the 15-month assay time (P = 0.0276, P = 0.0349, P = 0.0178 respectively). In conclusion, multiple alterations in lymphocyte phenotype, with increases in the proportion and total number of cells involved in cell-mediated immune responses, were seen during and especially following completion of therapy with 5FU/LMS. Many of these changes are significantly associated with clinical outcome and may be useful for risk stratification of stage III colon cancer patients following completion of adjuvant therapy. Received: 9 July 1999 / Accepted: 11 August 1999     

In vivo modulation of natural killer cell activity by tamoxifen in patients with bilateral primary breast cancer

Natural killer (NK) cell activity, the autologous mixed lymphocyte reaction (AMLR) and proportions of T cell subpopulations (CD3⁺/CD4⁺ and CD3⁺/CD8⁺) and NK cells (CD16⁺) were studied in 21 patients with bilateral primary breast cancer (BBC), 10 patients with single-breast cancer (SBC) and 20 healthy controls. All patients studied had no evidence of disease and had been off radiotherapy and/or chemotherapy for at least 1 year. Ten patients with BBC were also treated with tamoxifen. Patients with SBC had NK cell activity, AMLR responses and T cell subpopulations that were comparable to those of normal controls. In patients with BBC, a significant (P<0.01) increase in NK activity compared to that in normal controls (42±13% versus 21±10%, effector-to-target cell ratio, 251) and a significant (P<0.05) decrease in CD4⁺ T cell proportions (30±15% versus 49±13%) and absolute numbers (472±82/mm³ versus 953±131/mm³) were found. However, the proliferative response of BBC patients' T lymphocytes in AMLR was in the range of the normal controls. Lymphocytes derived from 10 BBC patients treated with tamoxifen exhibited NK cell activity that was comparable to that of normal controls and patients with SBC, and was significantly (P<0.01) reduced compared to the pretreatment period. BBC patients who received tamoxifen also show a reduction in the proportion of CD4⁺ T cells and in AMLR proliferative responses, which decreased compared to levels in normal controls. Taken together, these results indicate that long-term tamoxifen treatment modulates immune responses in BBC patients.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Inhibition of Dual/Mixed Tropic HIV-1 Isolates by CCR5-Inhibitors in Primary Lymphocytes and Macrophages

Background

Dual/mixed-tropic HIV-1 strains are predominant in a significant proportion of patients, though little information is available regarding their replication-capacity and susceptibility against CCR5-antagonists in-vitro. The aim of the study was to analyze the replication-capacity and susceptibility to maraviroc of HIV-1 clinical isolates with different tropism characteristics in primary monocyte-derived-macrophages (MDM), peripheral-blood-mononuclear-cells (PBMC), and CD4⁺T-lymphocytes.

Methods

Twenty-three HIV-1 isolates were phenotipically and genotipically characterized as R5, X4 or dual (discriminated as R5⁺/X4, R5/X4, R5/X4⁺). Phenotypic-tropism was evaluated by multiple-cycles-assay on U87MG-CD4⁺-CCR5⁺−/CXCR4⁺-expressing cells. Genotypic-tropism prediction was obtained using Geno2Pheno-algorithm (false-positive-rate [FPR] = 10%). Replication-capacity and susceptibility to maraviroc were investigated in human-primary MDM, PBMC and CD4⁺T-cells. AMD3100 was used as CXCR4-inhibitor. Infectivity of R5/Dual/X4-viruses in presence/absence of maraviroc was assessed also by total HIV-DNA, quantified by real-time polymerase-chain-reaction.

Results

Among 23 HIV-1 clinical isolates, phenotypic-tropism-assay distinguished 4, 17 and 2 viruses with R5-tropic, dual/mixed-, and X4-tropic characteristics, respectively. Overall, viruses defined as R5⁺/X4-tropic were found with the highest prevalence (10/23, 43.5%). The majority of isolates efficiently replicated in both PBMC and CD4⁺T-cells, regardless of their tropism, while MDM mainly sustained replication of R5- or R5⁺/X4-tropic isolates; strong correlation between viral-replication and genotypic-FPR-values was observed in MDM (rho = 0.710;p-value = 1.4e-4). In all primary cells, maraviroc inhibited viral-replication of isolates not only with pure R5- but also with dual/mixed tropism (mainly R5⁺/X4 and, to a lesser extent R5/X4 and R5/X4⁺). Finally, no main differences by comparing the total HIV-DNA with the p24-production in presence/absence of maraviroc were found.

Conclusions

Maraviroc is effective in-vitro against viruses with dual-characteristics in both MDM and lymphocytes, despite the potential X4-mediated escape. This suggests that the concept of HIV-entry through one of the two coreceptors “separately” may require revision, and that the use of CCR5-antagonists in patients with dual/mixed-tropic viruses may be a therapeutic-option that deserves further investigations in different clinical settings.     

Modulation of HERG channel inactivation by external cations

We investigated the effect of external cations on the permeability characteristics and gating kinetics of the human ether-à-go-go-related gene (HERG) current using the whole-cell patch-clamp technique. Inward HERG currents were recorded on hyperpolarization in 140 mM external Cs⁺ and Rb⁺, as well as K⁺. The permeability ratios of Rb⁺ and Cs⁺ relative to K⁺ were 1.25 and 0.56, respectively. Biphasic outward currents were recorded on depolarization in 140 mM Cs⁺ and in Rb⁺ with much smaller amplitude. The voltage dependence of inactivation was affected by external cations, such that the half-inactivation voltage shifted from –69.4±3.7 mV in K⁺ to –30.7±1.6 mV in Cs⁺ and to –35.8±1.9 mV in Rb⁺ (n=5). The time constants of inactivation were also changed significantly by external cations; of inactivation at +40 mV was 16.4±2.2 ms in 140 mM K⁺, 181±20.3 ms in Cs⁺, and 94.1±7.6 ms in Rb⁺ (n=5). Voltage dependence of activation was not altered significantly. The inhibition of the rapid inactivation mechanism by large cations may suggest that the foot-in-the-door model of gating is involved in HERG channel inactivation.     

Progressively developed myocardial apoptotic cell death during late phase of reperfusion

Myocardial apoptosis is primarily triggered during reperfusion (R). The aim of this study was to test the hypothesis that R-induced apoptosis develops progressively during the late phase of R, and that R-induced apoptosis is associated with changes in expression of anti- and pro-apoptotic proteins and infiltrated inflammatory cells. Thirty-one dogs were subjected to 60 min of left anterior descending coronary occlusion followed by 6, 24, 48, and 72 h R, respectively. There was no group difference in collateral blood flow, measured by colored microspheres during ischemia. Necrotic cell death (TTC staining) was significantly increased during R, starting at 27 ± 2% at 6 h R and increasing to 41 ± 2% at 24 h R. There was no further change at 48 (37 ± 3%) and 72 (36 ± 6%) h R, respectively. TUNEL positive cells (% total normal nuclei) in the peri-necrotic zone progressively increased from 6 (26 ± 2^*) to 24 (38 ± 1^*), 48 (48 ± 3^*) and 72 (59 ± 4^*) h R, respectively. The number of detected TUNEL positive cells at these time points was consistent with an increased intensity of DNA ladders, identified by agarose gel electrophoresis. Compared with normal tissue, western blot analysis showed persistent reduction in expression of anti-apoptotic protein Bcl-2 from 6 (16 ± 0.8%^*) to 72 h R (78 ± 2%^*), and increase in expression of pro-apoptotic proteins including Bax from 6 (30 ± 3%^*) to 72 h R (66 ± 3%^*), and p53 from 6 (12 ± 1%^*) to 72 h R (91 ± 2%^*), respectively. Immunohistochemical staining revealed that infiltrated neutrophils (mm² myocardium) were significantly correlated with development of necrotic and apoptotic cell death from 6 to 24 h R, respectively (P < 0.05), while large macrophage infiltration seen during 48 to 72 h R were correlated with apoptotic cell death (P < 0.05). These results indicate that 1) necrosis peaked at 24 h R when apoptosis was still progressively developing during later R; 2) changes in Bcl-2 family and p53 proteins may participate in R-induced myocardial apoptosis; 3) inflammatory cells may play a role in triggering cell death during R. ^* P < 0.05 vs. normal nuclei and tissue; P < 0.01 vs. 6 h R.     

Interferon β enhances the natural killer activity of patients with bladder carcinoma

We have investigated the effect of interferon (INFß) on the natural killer (NK) cytotoxic activity of peripheral blood mononuclear cells (PBMC) from patients with superficial and infiltrative transitional-cell carcinoma of the bladder (TCC) against both NK-sensitive and NK-resistant target cells. The normal NK activity found in PBMC from these patients can be significantly enhanced by short-term incubation (18 h) with INFß (P<0.05). The depressed NK cytotoxic activity found in PBMC from patients with infiltrative TCC can also be significantly enhanced, but not normalized, by short-term incubation with INFß (P<0.05). In kinetic studies we found that the maximal levels of the INFß-promoted cytotoxic activity against NK-sensitive and against NK-resistant target cells in PBMC from TCC patients were reached after 18 h of culture. Short-term-INFß-incubated PBMC from patients with TCC of the bladder also showed marked cytotoxic activity against NK-resistant target cells. The effector cells of the INFß-induced cytotoxic activity in PBMC from patients with TCC were CD16⁺ CD3^– NK cells. This cytotoxic inducer effect of INFß synergized with that of interleukin-2. In conclusion, INFß can enhance the NK activity of PMBC from patients with TCC of the bladder.     

Increased urinary zinc excretion in cancer patients is linked to immune activation and renal tubular cell dysfunction

Urinary zinc excretion is known to be increased in cancer patients, but the pathogenesis of this phenomenon remains uncertain. Both skeletal muscle catabolism and renal tubular cell dysfunction have been proposed to explain this observation. We have investigated urinary zinc and N-acetyl--d-glucosaminidase (NAG), an indicator of renal tubular cell dysfunction, as well as serum neopterin, an index of systemic immune activation, in 22 patients with cancer and seven controls. Both serum neopterin and urinary zinc were significantly elevated in cancer patients (15.8 ± 12.7 versus 7.3 ± 2.3 nmol l^–1 and 1.77 ± 0.80 versus 1.21 ± 0.41 mmol mol^–1 creatinine, P < 0 and P < 0.05, respectively), while NAG was similar in cancer patients and the controls (13.58 ± 13.80 versus 13.68 ± 12.19 kat mol^–1 creatinine). A significant correlation was observed between serum neopterin and urine zinc (rs = 0.5119, P < 0.02), serum neopterin and urine NAG (rs = 0.6761, P < 0.002), and urinary zinc and NAG (rs = 0.6348, P < 0.002). In conclusion, the present data indicate a link between urinary zinc excretion and immune activation as well as renal tubular cell dysfunction. In addition, renal tubular cell dysfunction appears to be linked to immune activation.     

Caecal water and electrolyte absorption and the effects of acetate and glucose, in dehydrated, low-NaCl diet hens

Summary In vivo electrolyte transport and water absorption from the caeca of dehydrated, low-NaCl diet hens are reported. In the absence of luminal glucose or acetate, net electrolyte transport rates and water absorption are small. When physiological concentrations of acetate (40 mM) are included in the perfusate, Na⁺ transport and water absorption increase significantly (P<0.01): 38±7 eqNa⁺/caecum kg·h and 256±33 l H₂O/caecum · kg · h.A similar increase in water absorption occurs with the inclusion of 15 mM glucose in the perfusate (219±30 l H₂O/caecum · kg · h), however both net Na⁺ and Cl^– absorption increase: 28±6 eq Na⁺/caecum · kg · h and 21±5 eq Cl^–/caecum kg · h.These pronounced increases in electrolyte and water absorption are not accompanied by any significant increase in transmural potential difference.The data presented establish caeca as important sites in the recuperation of water and electrolytes in dehydrated, low-NaCl diet hens.Abbreviations ECPD electrochemical potential difference - PD (transmural) potential difference - PEG polyethylene glycol     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3<Superscript>+</Superscript>, CD14<Superscript>+</Superscript> and CD19<Superscript>+</Superscript>

Background

Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3⁺, CD14⁺ and CD19⁺. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.

Methods

PBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3⁺, CD14⁺, CD19⁺ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.

Results

Negative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3⁺ cells then in 8/26 (30.8%) of CD14⁺ and CD19⁺ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3⁺ (2.4%), then in CD19⁺ (1.2%), and much lower in CD14⁺ (0.4%) cells.

Conclusions

Our results indicate that CD3⁺ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

     

Effect of Recombinant Interferon-α2b (Laferon) on Transport of Sodium Ions in Human Neuroblastoma Cells

To elucidate molecular mechanisms of neurotropic action of a recombinant interferon, IFN-2b (laferon), its effect on transport of ²²Na⁺ through the membrane of cultured human neuroblastoma cells (line IMR 32) was investigated. Within the first minutes after treatment with IFN-2b, the influx of ²²Na⁺ ions was reduced by 20%, as compared with the control. Depolarization of the plasma membrane by a mixture of veratrine and scorpion (Leiurus quinquestriatus) toxin (200 and 10 g/ml, respectively) increased this flux by 50% in the control and by 70% in the IFN-2b-treated cells. A blocker of voltage-operated sodium channels, tetrodotoxin (TTX, 4 · 10^-7 M), suppressed the inward flux of ²²Na⁺ ions (completely in the control cells and by 75% in the IFN-2b-treated cells). The influx of ²²Na⁺ ions into neuroblastoma cells depended on the concentration of IFN-2b in the incubation medium, reaching a maximum at concentrations of 600-1000 IU/ml. This allows us to suggest that entry of Na⁺ ions into neuroblastoma cells caused by IFN-2b is basically performed through voltage-operated TTX-sensitive sodium channels.     

Plasma hormonal and electrolyte alterations in cycling buffaloes (Bubalus bubalis) during hot summer months

Plasma levels of progesterone, prolactin, luteinizing hormone, and electrolytes were monitored by radioimmunoassay in ten cycling buffaloes maintained at Punjab Agricultural University, Ludhiana during the hot summer months of June–July. The plasma progesterone concentration ranged from 0.28±0.04 to 3.09±0.03 ng/ml at various stages of the oestrous cycle. Prolactin values ranged from 319±23 to 371±25 ng/ml and LH levels from 0.95±0.05 to 1.35±0.08 ng/ml. Concentrations differed significantly (P0.05) at various stages of the cycle. Levels of electrolytes, viz. Ca^{+ +}, Na⁺ and K⁺, were well within the normal range. The high levels of prolactin, progesterone and LH during the hot summer were assessed in relation to poor reproductive efficiency in buffaloes.     

Volume-stimulated,Cl−-dependent K+ efflux is highly expressed in young human red cells containing normal hemoglobin or HbS

Summary We report here that a Cl^–-dependent K^– (KCl) efflux, which is stimulated by N-ethylmaleimide, (NEM) and by increased red cell volume, exists in young red cells of individuals with normal hemoglobin A (AA) and in those homozygous for hemoglobin S (SS). We have investigated this KCl efflux in several density-defined red cell fractions obtained from Percoll-Stractan continuous density gradients. We found high activity of the NEM-stimulated KCl transport in reticulocytes and young red cells from nine sickle cell (SS) patients (43±27 mean±sd mmol K⁺/liter of cells/hr=flux units (FU)) and in the young cell fraction of three AA individuals with high reticulocytosis recuperating from nutritional anemias (41.7±10 FU). In addition, we observed significant interindividual variation of this KCl efflux in the discocyte fraction of SS blood. Cell swelling markedly stimulated the KCl efflux, in SS whole blood (9.8±7.4 FU, in SS young cells (13±13 FU), and in AA young cells (21.4±11 FU). The activity of the Na–K–Cl cotransport, as estimated by the bumetanide sensitive K⁺ efflux was not found to be cell-age dependent in either AA or SS cells.Measurements of red cell density by isopycnic gradients indicated that 27% of the young cells reduce their volume by a Cl^–-dependent process in hypotonic or low pH-induced swelling.The large volume-stimulated KCl efflux in AA young cells raises the possibility that these fluxes may be involved in the maturation of erythropoietic precursors. The high activity in the red cells of sickle cell anemia patients and its interindividual variation may have pathophysiological consequences since it reverses the decrease in the intracellular concentration of hemoglobin which occurs in response to low pH or osmolarity, an unwelcome pro-sickling event.     

Na+/H+ exchange is increased in sickle cell anemia and young normal red cells

Summary Red cell volume regulation is important in sickle cell anemia because the rate and extent of HbS polymerization are strongly dependent on initial hemoglobin concentration. We have demonstrated that volume-sensitive K:Cl cotransport is highly active in SS whole blood and is capable of increasing MCHC. We now report that Na⁺/H⁺ exchange (Na/H EXC), which is capable of decreasing the MCHC of erythrocytes with pH_i<7.2, is also very active in the blood of patients homozygous for HbS. The activity of Na/H EXC (maximum rate) was determined by measuring net Na⁺ influx (mmol/liter cell·hr=FU) driven by an outward H⁺ gradient in oxygenated, acidloaded (pH_i 6.0), DIDS-treated SS cells. The Na/H EXC activity was 33±3 FU (mean±se) (n=19) in AA whites, 37±8 FU (n=8) in AA blacks, and 85±15 FU (n=14) in SS patients (P<0.005). Separation of SS cells into four density-defined fractions by density gradient revealed mean values of Na/H EXC four to five times higher in reticulocytes (SS1), discocytes (SS2) and dense discocytes (SS3), than in the fraction containing irreversibly sickled cells and dense discocytes (SS4). In contrast to K:Cl cotransport, which dramatically decreases after reticulocyte maturation, Na/H EXC persists well after reticulocyte maturation. In density-defined, normal AA red cells, Na/H EXC decreased monotonically as cell density increased. In SS and AA red cells, the magnitude of stimulation of Na/H EXC by cell shrinkage varied from individual to individual. We conclude that Na/H EXC is highly expressed in SS and AA young red cells and decays slowly after reticulocyte maturation.     

Efficacy of adjuvant immunotherapy with cytokine-induced killer cells in patients with locally advanced gastric cancer

Purpose

To determine the long-term efficacy of adjuvant immunotherapy with autologous cytokine-induced killer (CIK) cells for locally advanced gastric cancer patients.

Experimental design

One hundred and fifty-one patients with stage III/IV gastric cancer who had undergone gastrectomy were enrolled, assigned to two groups (immunotherapy group versus no immunotherapy group/or control group), and followed.

Results

The 5-year overall survival (OS) and 5-year disease-free survival (DFS) rates for immunotherapy versus control group were 32.4 versus 23.4?% (P?=?0.071) and 28.3 versus 10.4?% (P?=?0.044), respectively. For patients with intestinal-type tumors, the 5-year OS and DFS rates were significantly higher for immunotherapy (OS, 46.8 vs. 31.4?% and P?=?0.045; DFS, 42.4 vs. 15.7?% and P?=?0.023). In the immunotherapy group, the mean CD3⁺ level, CD4⁺ level, and CD4⁺/CD8⁺ ratio increased from 50.8, 26.5, and 0.9?%, respectively, at baseline to 62.6, 35.0, and 1.4?%, respectively, 1?week after the first CIK-cell treatment, returned to baseline after 2?months, and maintained a higher level (60.7?±?8.2?%, 34.2?±?7.1?%, and 1.3?±?0.3?%, respectively) 2?months after 3 cycles of immunotherapy.

Conclusions

Adjuvant immunotherapy with CIK cells prolongs DFS in patients with locally advanced gastric cancer and significantly improves OS in patients with intestinal-type tumors. Intestinal-type tumors could be selected as an important indication for CIK-cell therapy. This treatment may help improve T-lymphocyte subset distribution and improve the host??s immune functions, but multiple cycles are necessary for long-term therapeutic efficacy.     

Distinct myeloid suppressor cell subsets correlate with plasma IL-6 and IL-10 and reduced interferon-alpha signaling in CD4<Superscript>+</Superscript> T cells from patients with GI malignancy

Interferon-alpha (IFN-α) promotes anti-tumor immunity through its actions on immune cells. We hypothesized that elevated percentages of myeloid-derived suppressor cells (MDSC) and increased pro-inflammatory cytokines in peripheral blood would be associated with impaired response to IFN-α in patients with gastrointestinal (GI) malignancies. This study evaluated relationships between plasma IL-6, IL-10, circulating MDSC subsets, and IFN-α-induced signal transduction in 40 patients with GI malignancies. Plasma IL-6 and IL-10 were significantly higher in patients versus normal donors. CD33⁺HLADR⁻CD11b⁺CD15⁺ and CD33⁺HLADR^−/lowCD14⁺ MDSC subsets were also elevated in patients versus normal donors (P < 0.0001). Plasma IL-6 was correlated with CD33⁺HLADR⁻CD15⁺ MDSC (P = 0.008) and IL-10 with CD33⁺HLADR⁻CD15⁻ MDSC (P = 0.002). The percentage of CD15⁺ and CD15⁻ but not CD14⁺ MDSC subsets were inversely correlated with IFN-α-induced STAT1 phosphorylation in CD4⁺ T cells, while co-culture with in vitro generated MDSC led to reduced IFN-α responsiveness in both PBMC and the CD4⁺ subset of T cells from normal donors. Exploratory multivariable Cox proportional hazards models revealed that an increased percentage of the CD33⁺HLADR⁻CD15⁻ MDSC subset was associated with reduced overall survival (P = 0.049), while an increased percentage of the CD33⁺HLADR^−/lowCD14⁺ subset was associated with greater overall survival (P = 0.033). These data provide evidence for a unique relationship between specific cytokines, MDSC subsets, and IFN-α responsiveness in patients with GI malignancies.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

CD8 T cell activation after intravenous administration of CD3×CD19 bispecific antibody in patients with non-Hodgkin lymphoma

A bispecific antibody directed to T and B cells (CD3×CD19 bsAb) was daily infused intravenously in escalating doses from 10 g up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed at _1/2 of 10.5 h with peak levels of 200–300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon , IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1ß in serum). No gross changess in histology or number of IL-2R⁺, CD4⁺ or CD8⁺ cells were found in the lymph nodes after therapy, but one patient showed activated CD8⁺ T cells within the tumour nodules. In conclusion, after intravenously administered CD3×CD19 bsAb only moderate toxicity was found, probably due to CD8⁺ T cell activation and cytokine release, without CD4⁺ T cell activation.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).